A reassessment of mycophenolic acid as a lead compound for the development of inhibitors of chikungunya virus replication

Article abstract of DOI:10.1016/j.tet.2017.12.053

Mycophenolic acid (MPA) has been previously reported as an inhibitor of the chikugunya virus (CHIKV) with an EC₅₀ value of 0.2 μM. We used MPA as a lead compound designing and synthesizing a series of isatins and benzolactones in a typical medicinal chemistry program. The synthesis and testing of 19 derivatives produced compounds with no desired activity which prompted us to retest the lead compound, MPA. We can reveal that MPA shows no anti-CHIKV activity and therefore needs to be reassessed as a lead compound for this target.

Full text of DOI:10.1016/j.tet.2017.12.053

Accepted Manuscript
A reassessment of mycophenolic acid as a lead compound for the development of
inhibitors of chikungunya virus replication
Adel A. Rashad, Johan Neyts, Pieter Leyssen, Paul A. Keller
PII:
S0040-4020(17)31337-6
10.1016/j.tet.2017.12.053
TET 29209
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 18 September 2017
Revised Date: 20 December 2017
Accepted Date: 27 December 2017
Please cite this article as: Rashad AA, Neyts J, Leyssen P, Keller PA, A reassessment of mycophenolic
acid as a lead compound for the development of inhibitors of chikungunya virus replication, Tetrahedron
(2018), doi: 10.1016/j.tet.2017.12.053.
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A Reassessment of Mycophenolic Acid as a
Lead Compound for the Development of
Inhibitors of Chikungunya Virus Replication
Leave this area blank for abstract info.
Adel A. Rashad, Johan Neyts, Pieter Leyssen and Paul A. Keller*
School of Chemistry, University of Wollongong, Wollongong 2522, Australia

1
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Tetrahedron
journal homepage: www.elsevier.com
A reassessment of mycophenolic acid as a lead compound for the development of
inhibitors of chikungunya virus replication
Adel A. Rashada, Johan Neytsb, Pieter Leyssenb and Paul A. Kellera,*
a School of Chemistry, University of Wollongong, Wollongong 2522, Australia
b KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and
Chemotherapy, B-3000 Leuven, Belgium
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Mycophenolic acid (MPA) has been previously reported as an inhibitor of the chikugunya virus
(CHIKV) with an EC50 value of 0.2 µM. We used MPA as a lead compound designing and
synthesizing a series of isatins and benzolactones in a typical medicinal chemistry program. The
synthesis and testing of 19 derivatives produced compounds with no desired activity which
prompted us to retest the lead compound, MPA. We can reveal that MPA shows no anti-CHIKV
activity and therefore needs to be reassessed as a lead compound for this target.
Available online
Keywords:
2009 Elsevier Ltd. All rights reserved
.
Mycophenolic acid
Chikungunya
Isatin
Benzolactone
have also been documented12, and eye infections which may
cause neuro retinitis.13,14 CHIKV-induced mortality is limited
(1:1000), with most deaths occurring in neonates, the elderly, and
patients with underlying health conditions.
1. Introduction
Chikungunya virus (CHIKV) is a pathogen belonging to the
genus Alphavirus, family Togaviridae. It is mainly transmitted
through the bite of an infected Aedes aegypti or A. albopictus
mosquito, and the infection is characterized by a typical and
devastating arthritis disease. The first report on a patient with
chikungunya fever dates from 1952.1 After five decades of only
sporadic cases, a series of large epidemics occurred, starting in la
Réunion island (2005-2006) during which almost 1:3 of the
population became infected, progressing around the Pacific
Ocean through India (1.4 to 6.5 million cases in 2006-2007), in
2009 followed by 3,000 and 42,000 cases in Malaysia and
Thailand, respectively.2,3 In Europe, as well as multiple imported
cases, the first patients with indigenous CHIKV transmission
were documented in Italy in 2007.4,5 Presently, CHIKV infections
are reported in nearly 40 countries (e.g. Singapore and Italy), in
many of which the virus has now become endemic.6 In 2008, the
virus was listed as a category C priority pathogen by the US
National Institute of Allergy and Infectious Diseases (NIAID)
due to its high morbidity rate and major health impact.7,8
Despite the vast impact of CHIKV infection, a vaccine is still
not available. Currently, the treatment options are limited to
symptomatic remedies with e.g. corticosteroids as no antiviral
drug is yet available.15,16 The development of a chemotherapeutic
strategy is considered to be as equally important as the
development of a vaccine. In the past years, CHIKV attracted
attention from the scientific community following the elucidation
of the structure of the viral protease17 and the viral envelope
proteins.18-20 These reports were followed by in silico structure-
based drug design efforts to develop small-molecule inhibitors.21-
25
However, only a few lead compounds with acceptable activity
have been reported so far.26 These include mycophenolic acid
(MPA 1),27 harringtonine 21,28 and 12-O-tetradecanoylphorbol
13-acetate (TPA, 3, Figure 1).29 TPA 3 has been reported as the
most potent compound with an EC50 = 0.0029 ꢀM and SI = 1965.
However, it is also one of the most potent tumour-promoting
agents known to date,19 and therefore is less likely to be further
developed as possible antiviral drug. Harringtonine 2 is a natural
product with a complex chemical structure, which complicates
further development, however MPA 1, is a low molecular weight
inhibitor and presents as an excellent candidate for further
exploration.
The symptoms of CHIKV infection generally start 4-7 days
after the mosquito bite. The course of infection typically has two
phases, the first acute phase lasting between 1-10 days,
characterized by a painful polyarthralgia, high fever, headache,
vomiting, rash, and myalgia. The second is the persistent or
chronic phase with disabling polyarthralgia that can last from
weeks to years.9,10 During the recent epidemics, neurological
disorders such as encephalitis, peripheral neuropathy and
myelopathy have been reported.11 Cases of multi-organ failure
MPA 1 was reported to inhibit CHIKV replication and virus-
induced cell death with an EC50 value of 0.2 µM and a selectivity
index value of 150,27 and was found to induce CHIKV apoptosis.

2
oral drug absorption occurs in the small intestine (pH ~ 5.533,34).
ACCEPTED MANUSCRIPT
Derivatives of MPA 1, in particular those for which the
metabolic drawbacks are overcome, could possibly lead to an
antiviral treatment for CHIKV infection.
MPA 1 can be considered as being composed of three
connected moieties (Figure 2): an aromatic head (the 3-oxo-1,3-
dihydroisobenzofuran moiety),
a rigid linker (the alkene
connection) and an acidic tail (the carboxylic acid group). The
aromatic head carries two major hydrogen bond acceptors (the
carbonyl group and the oxygen atom of the lactone ring). The
aromatic benzo moiety carries a hydroxyl group, a methoxy
group and a methyl group. In this study, the three components of
MPA 1 were investigated as optimizable components, starting
from simple core units such as benzoic acids and anilines; Figure
2 shows the possible access to MPA 1 analogues starting from
such derivatives. Both the acids and anilines could be converted
into the aromatic heads carrying H-bond acceptor features, which
could be connected through simple C-C coupling, to the linker
and the acidic tail. R groups on the acids and anilines were
selected to give similar environment as in 1.
Fig .1 Chemical structure of anti-CHIKV lead compounds with promising
antiviral activity.
In addition to benzolactones, isatins were also selected as
alternative possible replacements for the aromatic head, as they
are known to possess various pharmacological properties
including anticholinesterase, anticonvulsant, antiinflammatory,
antihypertensive, antihypoxic, antimicrobial, and antiviral
properties.35,36 The structure of isatin could participate in the
biological activity, with the two carbonyl groups (red circles,
Figure 2) accepting H-bonds from the target receptor residues,
and the NH donating a H-bond to acceptor residues.37,38 The
aromatic core π-system of isatin was also found to participate
through aromatic interactions with some target receptor
residues.39
It inhibits the cellular inosine monophosphate dehydrogenase
(IMPDH), an enzyme that involved in the de novo biosynthesis
of guanine nucleotide. It is well known that MPA 1 suffers from
a metabolic drawback: rapid conjugation of the C7 phenolic
hydroxyl group with glucuronic acid results in more soluble
metabolites that are more easily cleared.20,31-32 Despite this
property, MPA 1 is a relatively small molecule with numerous
positions available for modification, including the removal or
substitution of the phenolic hydroxyl group. This study describes
the exploration of MPA 1 as a starting point for the development
of compounds with putative anti-CHIKV activity.
2. Results and Discussions
Replacing the rigid linker alkene with the phenyl ring (blue
boxes, Figure 2) would be an applicable strategy for obtaining an
analogous linker between the aromatic head and the acidic tail of
MPA analogues. Utilizing cross coupling chemistry40-42 would
facilitate facile access to the bi-aromatic system. This phenyl
aromatic linker may also provide a π-electron system for
interaction within the yet unknown target site
2.1. Design of MPA analogues:
MPA 1 has good drug-like properties with a molecular weight
of 320.3, a calculated logP (clogP) value of 2.92, and a calculated
logD value of 3.56 (pH = 1-2), 0.67 (pH = 7.4) and 2.52 (pH =
5.5), indicating good blood solubility and oral bioavailability -
Fig. 2. Design of MPA 1 analogues starting from benzoic acids and aniline derivatives. The red circles indicate the HB accepting groups, the blue boxes
indicate the rigid linker and the green circle refers to the acidic tail moiety that could be either COOH or the bioisostere tetrazole.

3
The acidic tail remained either as a carboxylic group or the
ACCEPTED MANUSCRIPT
bioisostere tetrazole43 (green, Figure 2). Tetrazole rings have
ionisable acidic proton at pH 7.4. They are also planar in
structure, and the anion is 10 times44 more lipophilic than the
carboxylate anion and this can enhance drug absorption as a
result. Tetrazole rings are also resistant to many of the metabolic
reactions that occur to the carboxylic groups.44,45
2.2. Chemistry:
2.2.1. Synthesis of the aromatic heads:
The benzolactone aromatic head synthesis was achieved via
Pd catalysed chemistry46 from benzoic acids using
dibromomethane as the solvent and carbon donor (Scheme 1).
The Br substituent is prepositioned at the benzolactone C6 in
preparation for metal catalysed cross coupling.
R1
R1
O
1 mol% Pd(OAc)2, K2HPO4,
CH2Br2, 140 °C, 36 h
X
X
COOH
O
R2
R2
4, R1 = OMe, R2 = H, X = Br 7, R1 = OMe, R2 = H, X = Br, 38%
5, R1 = Me, R2 = H, X = Br
6, R1 = R2 = OMe, X = H
8, R1 = Me, R2 = H, X = Br, 55%
9, R1 = R2 = OMe, X = H, 58%
10, R1 = R2 = OMe, X = Br, 84%
(a)
(a) = Br2, acetic acid, -5 ºC - rt, 4 h, 84%
Scheme 1. The synthesis of the benzolactone aromatic heads.
The synthesis of an amino substituted benzolactone was
attempted using 2-amino-3-bromo-5-methylbenzoic acid 11,
which then cyclised to afford the aminobenzolactone 14 (Scheme
2). Unsurprisingly, the amino-protected compound 13 was not
formed, instead, the β-lactam product 12 was obtained which was
then incorporated into the project as an alternative aromatic head.
Scheme 3. The synthesis of the isatin aromatic heads.
2.2.2. Synthesis of the acid conjugates:
1. n-BuLi, THF, -78 °C
2. pivaloyl chloride,
-78 °C - rt, 3 h
O
The benzolactone-acid, isatin-acid and β-lactam-acid
conjugate syntheses were achieved through the one step C-C
cross coupling reaction with the commercially available 3-(3-
boronophenyl)propanoic acid 27 (Scheme 4). This facile one step
synthesis gave the three main components of the MPA designed
analogues, the aromatic head, the rigid linker (phenyl) and the
acidic tail (COOH).
NH2
Br
COOH
N
O
Br
47%
Me
11
Me
12
0%
2.2.3. Synthesis of the tetrazole conjugates:
O
Tetrazole conjugates were synthesised from acetonitrile
intermediates via 3-(3-boronophenyl)propanoic acid 39 in a
coupling reaction with the bromobenzolactones. Due to the high
cost of 39, the ester 40 was also synthesized by reacting 3-
iodophenol with 2-chloroacetonitrile, followed by a borylation
reaction (Scheme 5).
O
Br
NH
O
HN
Br
COOH
O
Me
Me
13
Scheme 2 The designed synthesis of the aminobenzolactone 14.
14
The benzolactone-acetonitrile intermediates 44 and 45 were
accessed via Suzuki coupling with 39 or 40 (Scheme 6).
Subsequent [2 + 3] cycloaddition reaction using NaN3/NH4Cl in
DMF afforded the tetrazoles 46 and 47 (Scheme 6).
The isatin aromatic heads 16 and 25 were formed by reaction
with oxalyl chloride (Scheme 3), followed by the insertion of the
C5 bromo substituent using either NBS, or Br2 in acetic acid
producing 17 and 26 respectively. The 7-Me substituted isatin 20
was produced from the aniline 18 with the coupling handle (Br)
already in place, using 2,2,2-trichloroethane-1,1-diol and
hydroxylamine. These derivatives were selected to demonstrate
similarity to the lead MPA 1, with the OH group masked as
methoxy groups to avoid the metabolic liability.
The modest yields (35%) of the tetrazole formation (Scheme
6) were considered insufficient to justify the synthesis of further
benzolactone-tetrazole conjugates. An alternative synthetic
strategy was devised in which the tetrazole moiety was attached
to the boronic ester, prior to the coupling reaction with the 6-
bromobenzolactones. The previously synthesized 2-(3-
iodophenoxy)acetonitrile 42 was used to access the intermediate
48 utilizing the azide tetrazole formation via mixing with NaN3
and NH4Cl in DMF containing few drops of glacial acetic acid
(Scheme 7). The tetrazole 48 was then reacted with

4
R1
R = OMe, R =AH CCEPTED MANUSCRIPT
O
7,
R1
1
2
O
17, R1 = R2 = OMe, R3 = H
20, R1 = R2 = H, R3 = Me
26, R1 = OMe, R2 = H, R3 = Me
33, R1 = R2 = R3 = H
Br
OH
Br
8, R1 = Me, R2 = H
10, R1 = R2 = OMe
28, R1 = R2 = H
B
O
O
OH
Br
R2
HOOC
N
R2
H
27
R3
O
Pd(PPh3)4, K3PO4, H2O, 85 °C, 4-8 h
Pd(PPh3)4, K3PO4, H2O, 85 °C,
DME or diglyme, 4-8 h
N
Pd(PPh3)4, K3PO4, H2O,
85 °C,1,4-dioxane, 18 h,
60%
R1
O
R1
O
Me
O
HOOC
HOOC
12
O
O
HOOC
R2
N
R2
H
R3
29, R1 = OMe, R2 = H, 62%
30, R1 = Me, R2 = H, 54%
31, R1 = R2 = OMe, 56%
32, R1 = R2 = H, 78%
O
34, R1 = R2 = OMe, R3 = H, 40%
35, R1 = R2 = H, R3 = Me, 55%
36, R1 = OMe, R2 = H, R3 = Me, 48%
37, R1 = R2 = R3 = H, 65%
N
Me
O
38
Scheme 4. The synthesis of the acid conjugates.
identifiable degradation products possibly arising both from the
reactivity of isatin core and the interference with the
cycloaddition reaction.
bis(pinacolato)diboron 43, potassium acetate and PdCl2(dppf)
catalyst in THF (Scheme 7) to afford the tetrazole salt
intermediate 49. The tetrazole conjugates 50 and 51 were
synthesized via Suzuki reaction of the tetrazole salt 49 with the
remaining 6-bromobenzolactones (Scheme 7).
Scheme 5. The synthesis of the boronic ester 40.
Scheme 7. The synthesis of the tetrazole salt intermediate 49.
Scheme 6. The synthesis of the tetrazole conjugates 46 and 47.
The isatin-acetonitrile intermediates were also accessed
through the reaction with 39 (Scheme 8), however, the
subsequent [2 + 3] cycloaddition, to form the isatin-tetrazole
conjugates, was not successful. The reaction resulted in the
disappearance of all starting materials but produced un-
Scheme 8. The synthesis of the isatin-acetonitrile intermediates and
subsequent attempt to synthesize the isatin-tetrazole conjugates.

5
targeting the CHIKV and must be reassessed as a lead
compound.
ACCEPTED MANUSCRIPT
Coupling of the β-lactam intermediate 12 with the boronic
ester 40 (Scheme 9) formed the biaryl with concurrent β-lactam
opening to give the benzoic acid derivative 59. The difference
between the reaction to give 38 (Scheme 4) and that to give 59
(Scheme 9) was the workup, with the former quenched at 0 °C
compared to room temperature in the case of 59. It is believed
that during this thermally uncontrolled procedure, the β-lactam
ring was hydrolysed to provide the COOH moiety.
2.4. Conclusions
To summarize, using MPA as a lead compound for the design
and synthesis of potential compounds targeting the CHIKV, we
synthesized a series of isatin and bezolactone derivatives.
Subsequent testing revealed no activity which led to retesting of
the lead MPA 1. We found that it did not possess any anti-
CHIKV activity and therefore we conclude that it cannot be
considered as an inhibitor of CHIKV.
Table 1.
Antiviral activity of compounds in a virus-cell-based assay
against Chikungunya virus (strain 899).
Compound
EC50 (µM)
11 ± 1
NA
CC50 (µM)
28 ± 10
ND
1
29
30
31
NA
ND
Scheme 9. The coupling reaction of the β-lactam 12 with 40.
NA
ND
2.3. Antiviral evaluation
32
34
35
36
37
38
44
45
46
47
50
51
NA
ND
All compounds were evaluated for antiviral activity in a virus-
cell-based assay against Chikungunya virus (Indian Ocean strain
899) (Table 1). Compounds for which an EC50 value could be
calculated from the antiviral dose-response curve, were subjected
to a similar experiment on treated, uninfected cells to assess the
effect of the compound on host cell metabolism and to determine
its selectivity.
NA
ND
NA
ND
NA
ND
NA
ND
NA
ND
NA
ND
Overall, few compounds elicited an antiviral effect in this
assay, and the compounds that did, only showed a limited
selectivity (EC50/CC50). Furthermore, microscopic inspection of
the assay conditions at which some inhibition of virus-induced
cell death was detected by the MTS/PMS readout method
revealed that none of the compounds matched the hit selection
criteria (100% inhibition of virus-induced cell death without any
apparent change in host cell or monolayer morphology).
Therefore, none of these compounds could be considered to be
selective inhibitors of CHIKV replication.
NA
ND
NA
ND
NA
ND
NA
ND
NA
ND
53
54
55
59
84 ± 14
NA
88 ± 18
ND
NA
ND
This surprising lack of activity for all our synthesized
analogues turned our attention back to the original lead
compound MPA 1, questioning the reported activity against
CHIKV. Therefore, in order to test the accuracy of the original
finding, we purchased and retested MPA 1, and subjected it to
the same anti-CHIKV assay. To our dismay, it also came up as
being not active (Table 1).
NA
ND
NA = not active; ND = not determined; EC50 = concentration of
compound that inhibits virus-induced cell death with 50%; CC50
concentration of compound that reduces the metabolic activity of
treated, uninfected cells with 50%; results are reported as median and
standard deviation from a series of at least 2 independent
experiments.
=
In order to reason these outcomes, we examined the assay
conditions described in the original manuscript that defined MPA
as an anti-CHIKV agent. The reported incubation time of the
assays was restricted to 48 hours – in this scenario, MPA reduces
the GTP concentration, and as CHIKV replicates much slower, it
has not had the opportunity to kill the cells at this 48 hours post-
infection timepoint. If a compound is a true antiviral, then it
should show antiviral activity even five days post-infection.
Therefore, in Chikungunya antiviral testing, it is imperative to
incubate the assay for five days to allow sufficient time to show a
time-dependency of the antiviral effect. In our five-day anti-
CHIKV assays, we have shown that there is no lasting antiviral
effect of MPA against CHIKV. Therefore, MPA is likely a
'braking' compound, applying the ‘brakes’ on the replication of
CHIKV, but not completely stopping it from multiplying.
Therefore, MPA 1 cannot be considered a lead compound
2.5. Experimental section
2.5.1. Chemical synthesis
Unless otherwise stated, chemicals were purchased from
Sigma Aldrich (Australia). (3-(Cyanomethoxy)phenyl)boronic
acid was purchased from Combi-Blocks, Inc., USA. All H, 13C
1
and spectra were recorded at 500 and 125 MHz, respectively on a
Varian Inova 500 MHz spectrometer. Chemical shifts (δ) are
reported in parts per million relative to TMS using CDCl3 as a
solvent unless otherwise noted. The solvents used for 13C NMR
1
were the same used for the H NMR unless otherwise noted.
Coupling constants (J) are reported in Hertz (Hz). Multiplicities
are reported as singlet (s), broad (br), doublet (d), doublet of
doublets (dd), doublet of doublets of doublets (ddd), doublet of

6
triplet (dt), multiplet (m), pentet (p), sextet (sex) or septet (sp).
Electron impact (EI) and electrospray (ES) mass spectra (MS)
were recorded on a Shimadzu QP-5000 spectrometer and high
resolution (HR) on a VG AutoSpec spectrometer. Electrospray
(ESI) mass spectra were recorded on a Micromass Platform LCZ
spectrometer and high resolution on a Micromass QTOF2
spectrometer. Ion mass to charge (m/z) values are stated with
their relative abundances as a percentage in parentheses. Peaks
assigned to the molecular ion are denoted by M+ or M- (when
using the ESI-). Thin Layer Chromatography (TLC) was
performed using Merck Silica Gel F254 aluminium sheets.
Column chromatography purifications were performed using
Flash Silica Gel. Infrared (IR) spectra were recorded on a
(Shimadzu FT-IR spectrometer) fitted with a Smart Omni-
Sampler germanium crystal accessory. All IR spectra were
recorded on neat samples with strong, medium and weak peaks
are assigned as s, m and w, respectively. Melting points were
NMR, δ: 170.1 (C=O), 146.1 (C3a), 139.8 (C5), 137.7 (C4),
ACCEPTED MANUSCRIPT
126.3 (C7), 124.7 (C7a), 120.6 (C6), 68.2 (CH2), 16.6 (CH3)
ppm. EI-MS m/z 228 (M+ 81Br, 54), 226 (M+ 79Br, 55), 199
(100%), 197 (99), 171 (32), 169 (30). HRMS (ESI) calcd for
C9H879BrO2 (MH+), 226.9708; found, 226.9705.
2.5.2.3. 5,7-Dimethoxyisobenzofuran-1(3H)-one4 1
(9)
Following the general procedure
1
using 2,4-
dimethoxybenzoic acid 6 (182 mg). The benzofuran 9 was
isolated after column chromatography as a white solid (112.5 mg,
58%). Spectral data of the isolated benzofuran 9 are in agreement
with that reported.41
2.5.2.4. 6-Bromo-5,7-dimethoxyisobenzofuran-
1(3H)-one (10)
determined using
a Gallenkamp (Griffin) melting point
In
a
round
bottom
flask
(50
mL),
5,7-
apparatus. Temperatures are expressed in degrees Celsius (ºC)
and are uncorrected. HPLC purity check was performed using
Waters 1525 HPLC pump, WatersTm 486 absorbance detector
(using the wavelength, 254 nm) and Nova-Pak® Silica 3.9 x 150
mm C18 column. HPLC solvents used: solvent A hexane, solvent
B isopropanol containing 0.1% TFA and solvent C isopropanol.
Retention times in minutes for the HPLC results are expressed as
Rt.
dimethoxyisobenzofuran-1(3H)-one 9 (150 mg, 0.773 mmol) was
dissolved in glacial acetic acid (20 mL) with vigorous stirring
and the flask was cooled to -5 - 0 ºC using an ice bath. A solution
of bromine (136 mg, 0.851 mmol) in glacial acetic acid (10 mL)
was added dropwise over 20 min. The reaction was allowed to
warm up slowly to room temperature and was then allowed to stir
for 4 h. The formed solid was filtered and washed with dilute
acetic acid, and was then subjected to column chromatography
(60% CH2Cl2 in hexane) to afford the benzofuran 10 (176.7 mg,
84%) as a white solid. It could also be recrystallised from 1,4-
dioxane, mp: 189-190 ºC; IR (cm-1): υ 1700 (s, C=O), 1316 (w,
C-O), 1071 (w, C-O), 1036 (w, C-Br). 1H NMR (CDCl3), δ: 6.46
(s, 1H, H4), 5.07 (s, 2H, CH2), 4.02 (s, 6H, OCH3). 13C NMR, δ:
168.4 (C=O), 162.2 (C7), 159.5 (C5), 150.5 (C3a), 107.8 (C7a),
96.2 (C6), 95.1 (C4), 69.3 (CH2), 57.1 (C7-OCH3), 56.6 (C5-
OCH3) ppm. EI-MS m/z 274 (M+ 81Br, 38), 272 (M+ 79Br, 40),
256 (18), 254 (20), 245 (32), 243 (30), 228 (100%), 226 (100),
215 (20), 213 (21). HRMS (ESI) calcd for C10H1079BrO4 (MH+),
272.9762; found, 272.9754.
2.5.2. General procedure 1: synthesis of the
isobenzofuran-1(3H)-ones (7-10)
Under a N2 atmosphere, a sealed tube was charged with the
appropriate benzoic acid (1 mmol), palladium acetate (22.4 mg,
0.1 mmol), dipotassium phosphate (K2HPO4, 522.5 mg, 3 mmol)
and dibromomethane (4 mL). The contents of the tube were
degassed by flushing N2 through a needle for 10 min. The tube
was then sealed, and heated in an oil bath at 140 ºC for 36 h. The
tube was then allowed to cool to room temperature, the content
was diluted with CH2Cl2 (20 mL), and filtered through celite. The
filtrate was washed with 1 M HCl (20 mL) and brine (2 x 15
mL), and was dried (MgSO4). The solvent was evaporated under
reduced pressure and the residue was either subjected to flash
column chromatography (10% methanol in chloroform) or
recrystallised from chloroform/hexane mixture (1:3).
2.5.2.5. 6-Bromoisobenzofuran-1(3H)-one (28)
This compound was prepared according to reported
procedures.42
2.5.2.1. 6-Bromo-7-methoxyisobenzofuran-1(3H)-
one (7)
2.5.2.6. 5-Bromo-3-methyl-7-pivaloyl-7-
azabicyclo[4.2.0]octa-1,3,5-trien-8-one (12)
Following the general procedure
1 using 3-bromo-2-
In an oven dried 50 mL flask and under a N2 atmosphere, 2-
amino-3-bromo-5-methylbenzoic acid 11 (750 mg, 3.26 mmol)
was dissolved in dry THF (20 mL), and the flask was then sealed
with a rubber cap with fitted with a N2 balloon, and cooled to -78
ºC. n-BuLi (4.08 mL of 2 M solution, 8.15 mmol) was added
dropwise through a needle and the mixture was stirred for 20 min
at -78 ºC before adding pivaloyl chloride (0.48 mL, 3.91 mmol)
dropwise. The reaction was then allowed to warm to room
temperature and stirred for 3 h. The reaction mixture was then
diluted with ethyl acetate (10 mL) and water (10 mL), the organic
layer was separated and washed with water (2 x 20 mL), brine (2
x 10 mL) and dried (MgSO4). The combined organic layers were
concentrated under reduced pressure to yield the β-lactam
derivative 12 as a colourless oil (452 mg, 47%), IR (cm-1): υ 1745
methoxybenzoic acid 4 (231 mg), the benzofuran 7 was isolated
after column chromatography, as a white solid (92 mg, 38%),
mp: 102-103 ºC; IR (cm-1): υ 1752 (s, C=O), 1288 (m, C-O),
1064 (w, C-O), 1111 (w, C-Br). 1H NMR (CDCl3), δ: 7.84 (d, J =
7.9 Hz, 1H, H5), 7.06 (d, J = 7.9 Hz, 1H, H4), 5.23 (s, 2H, CH2),
4.16 (s, 3H, OCH3). 13C NMR, δ: 167.5 (C=O), 156.3 (C7), 148.3
(C3a), 139.3 (C5), 118.6 (C4), 117.9 (C7a), 117.2 (C6), 68.7
(CH2), 62.9 (CH3) ppm. EI-MS m/z 244 (M+ 81Br, 28), 242 (M+
79Br, 30), 215 (31), 213 (33), 198 (100%), 196 (98), 185 (17),
183 (15). HRMS (ESI) calcd for C9H879BrO3 (MH+), 242.9649;
found, 242.9657.
2.5.2.2. 6-Bromo-7-methylisobenzofuran-1(3H)-one
(8)
1
(s, C=O), 1634 (s, C=O), 1465 (w, C-N), 1059 (w, C-Br). H
Following the general procedure
1 using 3-bromo-2-
NMR (CDCl3), δ: 7.94 (s, 1H, H4), 7.86 (s, 1H, H2), 2.45 (s, 3H,
CH3), 1.42 (s, 9H, pivaloyl 3xCH3). 13C NMR, δ: 168.1 (pivaloyl
C=O), 159.7 (C6), 142.3 (C=O), 140.9 (C4), 139.3 (C1), 127.4
(C2), 121.8 (C3), 117.8 (C5), 38.3 (C(CH3)3), 27.6 (pivaloyl
3xCH3), 20.9 (CH3) ppm. EI-MS m/z 297 (M+, 81Br, 33), 295
(M+, 79Br, 35), 282 (98), 280 (100%), 255 (18), 253 (20), 240
methylbenzoic acid 5 (215 mg), the benzofuran 8 was isolated
after recrystallisation, as a pale yellow solid (124 mg, 55%), mp:
91-92 ºC; IR (cm-1): υ 1739 (s, C=O), 1358 (w, C-O), 1069 (w,
C-Br). 1H NMR (CDCl3), δ: 7.79 (d, J = 8.0 Hz, 1H, H5), 7.18 (d,
J = 8.0 Hz, 1H, H4), 5.20 (s, 2H, CH2), 2.74 (s, 3H, CH3). 13C

7
(35), 238 (37). HRMS (ESI) calcd for C13H15BrNO2 (MH+),
296.0286; found, 296.0294.
2.5.2.7. 4,6-Dimethoxyindoline-2,3-dione4 3 (16)
added Raney Nickel (0.1 mol %) and the mixture was
A
CCEPTED MANUSCRIPT
vigorously stirred at room temperature until the H2 ceased to
evolve. Hydrazine monohydrate (2.0 g, 41 mmol) in methanol
(10 mL) was added through a syringe slowly over 10 min. The
mixture was then heated at reflux for 4 h. The reaction mixture
was filtered hot through celite, and the filtrate was concentrated
under reduced pressure. The obtained residue was recrystallised
from petroleum spirit to yield 5-methoxy-2-methylaniline 23 as
white solid (3.8 g, 91%). Spectral data for this aniline derivative
23 were in agreement with the reported literature.46
This compound was prepared following the reported
procedure.43 The 4,6-dimethoxyindole-2,3-dione 16 was obtained
as a yellow solid (5.2 g, 80%).
2.5.2.8. 5-Bromo-4,6-dimethoxyindoline-2,3-dione
(17)
In a 100 mL round bottom flask, a solution of 4,6-
dimethoxyindole-2,3-dione 16 (143 mg, 0.5 mmol) in acetic acid
(10 mL) was stirred at room temperature until all the isatin was
completely dissolved before being cooled to 0 ºC using an ice
bath. N-Bromosuccinimide (97.9 mg, 0.55 mmol) was added
portion wise over 15 min. After addition of the NBS, the reaction
mixture started to solidify, and the flask was removed from the
ice bath and was allowed to warm to room temperature and then
stirred for 8 h. The yellow solid was filtered from the mother
liquor to which cold water (10 mL) was added to precipitate the
remaining soluble product. The combined solids were washed
with saturated NaHCO3 solution (2 x 15 mL) and with 1:1 water
ethanol mixture (25 mL) and then vacuum dried.
Recrystallisation from 1,4-dioxane yielded the 5-bromo-4,6-
dimethoxyindoline-2,3-dione 17 as a yellow solid (118 mg,
60%), mp: >250 ºC; IR (cm-1): υ 3195 (m, NH), 1718 (s, C=O),
2.5.2.12. 4-Methoxy-7-methylindoline-2,3-dione
(25)
In a 250 mL round bottom flask, 5-methoxy-2-methylaniline
23 (2.0 g, 15 mmol) was dissolved in anhydrous benzene (20
mL), and oxalyl chloride (4.12 g, 2.8 mL, 32.4 mmol) was added
dropwise. When the HCl ceased to evolve, the reaction was
heated at reflux for 3 h. The benzene and oxalyl chloride were
then removed by distillation, and the residue was quickly
suspended in dichloroethane (20 mL) and then cooled to 0 ºC in
an ice bath. Aluminium chloride (2.1 g, 15.5 mmol) as a
suspension in dichloroethane (10 mL) was added portion wise
with vigorous stirring and the reaction mixture was allowed to
warm to room temperature over 3 h. The reaction mixture was
then heated at reflux for 40 min. After cooling to room
temperature, the mixture was poured onto crushed ice, portioned
between 0.1 M HCl and ethyl actetate. The aqueous layer was
further extracted with ethyl acetate (2 x 100 mL). The combined
organic layers were washed with brine (100 mL), dried (MgSO4)
and evaporated under reduced pressure. The resulting orange
solid was subjected to a flash column chromatography and
elution with 30% ethyl acetate in petroleum spirit yielded 4-
methoxy-7-methylindoline-2,3-dione 25 as an orange solid (0.84
g, 30%), mp: 235-236 ºC; IR (cm-1): υ 3195 (m, NH), 1635 (s,
1
1264 (m, C-O). H NMR (DMSO-d6), δ: 11.14 (s, NH), 6.36 (s,
H7), 3.99 (s, 3H, C6-OCH3), 3.94 (s, 3H, C4-OCH3). 13C NMR,
δ: 178.4 (C3), 165.5 (C2), 161.7 (C4), 160.4 (C6), 151.4 (C7a),
102.7 (C3a), 91.8 (C7), 86.2 (C5), 58.1 (C4-OCH3), 57.2 (C6-
OCH3) ppm. EI-MS m/z 287 (M+ 81Br, 40), 285 (M+ 79Br, 42),
259 (98), 257 (100%), 215 (6), 213 (7). HRMS (ESI) calcd for
C10H9N79BrNO4 (MH+), 285.9715; found, 285.9719.
2.5.2.9. 5-Bromo-7-methylindoline-2,3-dione4 4 (20)
1
C=O), 1250 (m, C-O). H NMR (DMSO-d6), δ: 10.99 (s, NH),
7.36 (d, J = 8.7, 1H, H6), 6.61 (d, J = 8.7, 1H, H5), 3.83 (s, 3H,
OCH3), 2.09 (s, 3H, CH3). 13C NMR, δ: 180.9 (C2), 160.1 (C3),
156.4 (C4), 149.1 (C7a), 141.8 (C6), 112.9 (C7), 106.5 (C5),
105.8 (C3a), 55.8 (OCH3), 14.6 (CH3) ppm. EI-MS m/z 191 (M+,
100%), 163 (50), 149 (10), 134 (7), 121 (7), 106 (93). HRMS
(ESI) calcd for C10H12NO3 (MH+), 192.0661; found, 192.0653.
4-Bromo-2-methylaniline 18 (6.88 g, 37 mmol) was added to
a 500 mL flask and was reacted with chloral hydrate (7.35 g, 44.4
mmol), hydroxylamine hydrochloride (9.25 g, 133 mmol),
sodium sulfate (42 g, 295 mmol) in water (250 mL) and 2 M HCl
(12.5 mL), by heating the mixture at 55 ºC with vigorous stirring
for 14 h. After cooling to room temperature, the aqueous layer
was decanted from the formed gummy residue which was further
washed with cold water and dried under vacuum. The flask was
then cooled to -5 - 0 ºC using ice/salt bath before adding cold
sulfuric acid (22.5 mL). The flask was allowed to warm to room
temperature before heating the reaction mixture gradually to 70
ºC where the mixture started to get darker in colour. When the
colour change became stable after almost 30 min, the reaction
was heated to 80 ºC for 10 min. After cooling to room
temperature, the mixture was poured onto crushed ice (150 mL)
and stirred for 1 h. The separated solid was then filtered and
washed with water (3 x 100 mL) to give a red solid of 5-bromo-
7-methylindoline-2,3-dione 20 (6.65 g, 75%) and was sufficiently
pure for the next step. The spectral data were consistent with the
reported literature.44
2.5.2.13. 5-Bromo-4-methoxy-7-methylindoline-2,3-
dione (26)
In
a
100 mL round bottom flask, 4-methoxy-7-
methylindoline-2,3-dione 25 (440 mg, 2.3 mmol) was dissolved
in glacial acetic acid (15 mL) and the mixture was stirred until
the solid dissolved completely. The flask was then cooled to 0 ºC
using an ice bath. A solution of bromine (405 mg, 2.5 mmol) in
glacial acetic acid (5 mL) was then added dropwise over 10 min.
The flask was allowed to warm to room temperature and was
stirred for 2 h. The resulting solid was filtered and cold water (15
mL) added to the filtrate and the resulting solid was filtered. The
combined solids were washed with saturated NaHCO3 (2 x 25
mL) and water (2 x 25 mL), and recrystallised from acetic acid to
yield 5-bromo-4-methoxy-7-methylindoline-2,3-dione 26 as an
orange solid (520.5 mg, 84%), mp: >250 ºC; IR (cm-1): υ 3162
(m, NH), 1696 (s, C=O), 1248 (w, C-O). 1H NMR (DMSO-d6), δ:
11.20 (s, NH), 7.67 (s, H6), 3.97 (s, 3H, OCH3), 2.12 (s, 3H,
CH3). 13C NMR, δ: 180.7 (C2), 159.2 (C3), 152.6 (C4), 148.9
(C7a), 142.6 (C6), 117.7 (C7), 110.1 (C3a), 108.1 (C5), 61.8
(OCH3), 14.6 (CH3) ppm. EI-MS m/z 271 (M+ 81Br, 90), 269 (M+
79Br, 92), 243 (98), 241 (100%), 228 (20), 226 (21), 185 (80).
HRMS (ESI) calcd for C10H979BrNO3 (MH+), 269.9766; found,
269.9773.
2.5.2.10. 4-Methoxy-1-methyl-2-nitrobenzene4 5 (22)
This compound was synthesized following the reported
procedure from 4-methyl-3-nitrophenol 21.45 The 4-methoxy-1-
methyl-2-nitrobenzene 22 was isolated as a white crystalline
solid (5.34 g, 80%).
2.5.2.11. 5-Methoxy-2-methylaniline4 6 (23)
To a solution of 4-methoxy-1-methyl-2-nitrobenzene 22 (5.1
g, 30.5 mmol) in methanol (35 mL) under a N2 atmosphere was

8
COOH-CH2-CH2), 2.59 (s, 3H, CH3). 13C NMR, δ: resonance
2.5.3. General procedure 2: Suzuki coupling of 6-
bromobenzolactone derivatives 7, 8, 10 and 28 with
3-(3-boronophenyl)propanoic acid 27
ACCEPTED MANUSCRIP
T
attributed to COOH not observed, 171.6 (C=O), 146.3 (phenyl
C1), 143.5 (isobenzofuran C7a), 140.5 (phenyl C3), 140.3
(isobenzofuran C4), 137.6 (phenyl C2), 135.7 (isobenzofuran
C5), 132.3 (phenyl C5), 129.5 (phenyl C6), 128.7 (isobenzofuran
C6), 127.6 (phenyl C4), 123.8 (isobenzofuran C3a), 119.1
(isobenzofuran C7), 68.5 (isobenzofuran C1), 35.7 (COOH-CH2-
CH2), 30.7 (COOH-CH2-CH2), 14.9 (CH3) ppm. EI-MS m/z 296
(M+, 100%), 278 (54), 260 (46), 250 (80), 136 (60), 223 (40).
HRMS (ESI) calcd for C18H17O4 (MH+), 297.1127; found,
297.1129. HPLC purity: 97.9%, Rt=5.571 (1% solvent B in
solvent A).
Under a N2 atmosphere, a 25 mL flask was charged with the
bromoisobenzofuran-1(3H)-one
boronophenyl)propanoic acid
(1
equiv),
(1.2
3-(3-
equiv),
27
tetrakis(triphenylphosphine)palladium(0) (5 mol%) and 1,4-
dioxane (5 mL). The flask was sealed with a rubber cap. The
reaction mixture was degassed by flushing N2 through a needle
for 10 min. The flask was heated in an oil bath at 85 ºC. When
the solid dissolved, a N2 degassed solution of potassium
phosphate (3 equiv) in water (3 mL) was added to the reaction
mixture and heating at 85 ºC continued for 4-6 h. After cooling to
room temperature, the solvent was concentrated under reduced
pressure, and 2 M HCl (15 mL) was added to the residue. The
suspended solid was either collected by filtration or extracted
with ethyl acetate (2 x 25 mL). The combined ethyl acetate
extracts were dried (MgSO4) and evaporated under reduced
pressure. The obtained residues were dissolved in ethanol and
were subjected to PLC (10% methanol in CH2Cl2, 0.5% acetic
acid).
2.5.3.3. 3-(3-(4,6-Dimethoxy-3-oxo-1,3-
dihydroisobenzofuran-5-yl)phenyl)propanoic acid
(31)
Following the general procedure 2 using 6-bromo-5,7-
dimethoxyisobenzofuran-1(3H)-one 10 (80 mg, 0.293 mmol), 3-
(3-boronophenyl)propanoic acid 27 (68.2 mg, 0.352 mmol),
tetrakis(triphenylphosphine)palladium(0) (16.9 mg, 0.015 mmol)
and potassium phosphate (186.6 mg, 0.879 mmol) and heating
for 6 h, the acid 31 was isolated as a pale yellow oil (56 mg,
56%), IR (cm-1): υ 2989 (m, OH), 1750 (s, C=O), 1739 (s, C=O),
2.5.3.1. 3-(3-(4-Methoxy-3-oxo-1,3-
dihydroisobenzofuran-5-yl)phenyl)propanoic acid
(29)
1
1208 (w, C-O). H NMR (CDCl3), δ: 7.33 (t, 1H, J = 7.9 Hz,
phenyl_5 H), 7.18 (d, 1H, J = 7.9 Hz, phenyl_4 H), 7.12-7.10 (m,
2H, phenyl_2,6 H), 6.50 (s, 1H, isobenzofuran_7 H), 4.98 (s, 2H,
isobenzofuran CH2), 4.03 (s, 3H, isobenzofuran_4 OCH3), 3.85
(s, 3H, isobenzofuran_6 OCH3), 2.97 (t, 3H, J = 7.6 Hz, COOH-
CH2-CH2), 2.66 (t, 3H, J = 7.6 Hz, COOH-CH2-CH2). 13C NMR,
δ: 177.2 (COOH), 169.0 (C=O), 162.8 (isobenzofuran C6), 159.4
(isobenzofuran C4), 149.3 (phenyl C1), 140.7 (isobenzofuran
C7a), 133.8 (phenyl C3), 129.3 (phenyl C2), 128.7 (phenyl C5),
127.7 (phenyl C4), 127.4 (phenyl C6), 117.5 (isobenzofuran
C3a), 106.1 (isobenzofuran C5), 95.5 (isobenzofuran C7), 68.3
(isobenzofuran C1), 56.3 (isobenzofuran_6 OCH3), 56.2
(isobenzofuran_4 OCH3), 35.6 (COOH-CH2-CH2), 30.7 (COOH-
CH2-CH2) ppm. EI-MS m/z 342 (M+, 100), 326 (25), 296 (26).
HRMS (ESI) calcd for C19H18O6 (MH+), 343.1182; found,
343.1190. HPLC purity: 98.7%, Rt=13.234 (5% solvent B in
solvent A).
Following the general procedure
2 using 6-bromo-7-
methoxyisobenzofuran-1(3H)-one 7 (110 mg, 0.453 mmol), 3-(3-
boronophenyl)propanoic acid 27 (105.5 mg, 0.544 mmol),
tetrakis(triphenylphosphine)palladium(0) (26.2 mg, 0.023 mmol)
and potassium phosphate (288.5 mg, 1.359 mmol) and heating
for 5 h, the acid 29 was isolated as a white solid (87.5 mg, 62%),
mp: 116-117 ºC; IR (cm-1): υ 2989 (m, OH), 1755 (s, C=O), 1718
(s, C=O), 1216 (m, C-O). 1H NMR (acetone-d6), δ: 10.54 (bs, 1H,
COOH), 7.73 (d, 1H, J = 7.5 Hz, isobenzofuran_6H), 7.45 (s, 1H,
phenyl_2H), 7.41 (d, 1H, J = 7.7 Hz, phenyl_4H), 7.40-7.37 (m,
2H, phenyl_5,6 H), 7.30 (d, 1H, J = 7.5 Hz, isobenzofuran_7 H),
5.37 (s, 2H, isobenzofuran CH2), 3.88 (s, 3H, OCH3), 2.99 (t, 2H,
J = 6.7 Hz, COOH-CH2-CH2), 2.68 (t, 2H, J = 6.7 Hz, COOH-
CH2-CH2). 13C NMR (CDCl3), δ: resonance attributed to COOH
not observed, 168.9 (C=O), 156.9 (isobenzofuran C4), 148.4
(phenyl C1), 140.5 (isobenzofuran C7a), 137.8 (phenyl C5),
137.1 (isobenzofuran C6), 135.7 (phenyl C6), 129.5 (phenyl C3),
128.7 (phenyl C2), 127.9 (isobenzofuran C5), 127.5 (phenyl C4),
118.0 (isobenzofuran C3a), 117.1 (isobenzofuran C7), 68.9
(isobenzofuran C1), 62.7 (OCH3), 47.4 (COOH-CH2-CH2), 30.8
(COOH-CH2-CH2) ppm. EI-MS m/z 312 (M+, 75), 295 (20), 266
(17), 239 (100%), 223 (50), 208 (15), 195 (51). HRMS (ESI)
calcd for C18H17O5 (MH+), 313.1076; found, 313.1078. HPLC
purity: 97.4%, Rt=8.245 (1% solvent B in solvent A).
2.5.3.4. 3-(3-(3-Oxo-1,3-dihydroisobenzofuran-5-
yl)phenyl)propanoic acid (32)
Following
the
general
procedure
2
using
6-
bromoisobenzofuran-1(3H)-one 28 (90 mg, 0.422 mmol), 3-(3-
boronophenyl)propanoic acid 27 (98.2 mg, 0.506 mmol),
tetrakis(triphenylphosphine)palladium(0) (24.4 mg, 0.021 mmol)
and potassium phosphate (268.7 mg, 1.266 mmol) and heating
for 4 h, the acid 28 was isolated as a white solid (92.8 mg, 78%),
mp: 118-119 ºC; IR (cm-1): υ 2972 (m, OH), 1743 (s, C=O), 1699
2.5.3.2. 3-(3-(4-Methyl-3-oxo-1,3-
dihydroisobenzofuran-5-yl)phenyl)propanoic acid
(30)
1
(s, C=O). H NMR (CDCl3), δ: 8.11 (s, 1H, isobenzofuran_4 H),
7.90 (d, 1H, J = 8.0 Hz, phenyl_4 H), 7.55 (d, 1H, J = 7.9 Hz,
phenyl_6 H), 7.47 (s, 1H, phenyl_2 H), 7.46 (d, 1H, J = 6.2 Hz,
isobenzofuran_6 H), 7.41 (t, 1H, J = 8.0 Hz, phenyl_5 H), 7.27
(d, 1H, J = 6.7 Hz, isobenzofuran_7 H), 5.37 (s, 2H,
isobenzofuran CH2), 3.05 (t, 2H, J = 7.6 Hz, COOH-CH2-CH2),
2.75 (t, 2H, J = 7.6 Hz, COOH-CH2-CH2). 13C NMR, δ: 178.3
(COOH), 171.4 (C=O), 145.5 (phenyl C1), 142.8 (isobenzofuran
C7a), 141.4 (phenyl C2), 139.9 (isobenzofuran C5), 133.4
(phenyl C3), 129.6 (phenyl C5), 128.3 (phenyl C6), 127.6
(isobenzofuran C6), 126.7 (isobenzofuran C3a), 125.6 (phenyl
C4), 124.3 (isobenzofuran C4), 122.7 (isobenzofuran C7), 69.9
(isobenzofuran C1), 35.6 (COOH-CH2-CH2), 30.8 (COOH-CH2-
CH2) ppm. EI-MS m/z 282 (M+, 70), 253 (20), 237 (100%), 223
(16). HRMS (ESI) calcd for C17H15O4 (MH+), 283.0970; found,
Following the general procedure
2 using 6-bromo-7-
methylisobenzofuran-1(3H)-one 8 (95 mg, 0.418 mmol), 3-(3-
boronophenyl)propanoic acid 27 (97.3 mg, 0.502 mmol),
tetrakis(triphenylphosphine)palladium(0) (24.2 mg, 0.021 mmol)
and potassium phosphate (266.2 mg, 1.254 mmol) and heating
for 4 h, the acid 30 was isolated as a white solid (66.8 mg, 54%),
mp: 110-111 ºC; IR (cm-1): υ 2989 (m, OH), 1731 (s, C=O), 1696
(s, C=O). 1H NMR (CDCl3), δ: 7.55 (d, 1H, J = 7.8 Hz,
isobenzofuran_6 H), 7.50 (d, 1H, J = 7.3 Hz, phenyl_4 H), 7.37
(t, 1H, J = 7.3 Hz, phenyl_5 H), 7.26 (d, 1H, J = 7.3 Hz,
phenyl_6 H), 7.20 (s, 1H, phenyl_2 H), 7.16 (d, 1H, J = 7.3 Hz,
isobenzofuran_7 H), 5.28 (s, 2H, isobenzofuran CH2), 3.02 (t,
2H, J = 7.3 Hz, COOH-CH2-CH2), 2.74 (t, 2H, J = 7.3 Hz,

9
283.0974. HPLC purity: 99.6%, Rt=6.285 (2% solvent B in
solvent A).
C7a), 141.7 (phenyl C3), 138.7 (phenyl C1), 137.6 (isatin C6),
ACCEPTED MANUSCRIPT
134.8 (isatin C5), 128.9 (phenyl C5), 127.4 (phenyl C6), 126.5
(phenyl C2), 123.8 (phenyl C4), 122.1 (isatin C4), 119.8 (isatin
C3a), 118.1 (isatin C7), 35.3 (COOH-CH2-CH2), 30.4 (COOH-
CH2-CH2), 15.5 (CH3) ppm. EI-MS m/z 309 (M+, 100%), 277
(25), 253 (80), 207 (79), 193 (30). HRMS (ESI) calcd for
C18H16NO4 (MH+), 310.1079; found, 310.1071. HPLC purity:
98.1%, Rt=5.339 (1% solvent B in solvent A).
2.5.4. General procedure 3: Suzuki coupling of 5-
bromoisatin derivatives 17, 20, 26 and 33 with 3-
(3-boronophenyl)propanoic acid 27
Under a N2 atmosphere, a 25 mL flask was charged with the
5-bromoisatin derivative (1 equiv), 3-(3-boronophenyl)propanoic
acid 27 (1.2 equiv), tetrakis(triphenylphosphine)palladium(0) (5
mol%) and DME (3 mL) or diglyme (3 mL), was sealed with a
rubber cap. The reaction mixture was degassed by flushing N2
through a needle for 10 min, and was then heated in an oil bath at
85 ºC. When the solid dissolved, a N2 degassed solution of
potassium phosphate (3 equiv) in water (3 mL) was added to the
reaction mixture and heating continued at 85 ºC for 4-6 h. After
cooling to room temperature, the solvent was concentrated under
reduced pressure. A solution of HCl 2 M (15 mL) was added, and
the resulting suspended solid was either collected by filtration or
extracted with ethyl acetate (2 x 25 mL). The combined organic
layers were dried (MgSO4) and evaporated under reduced
pressure. The combined residues were then dissolved in ethanol
and were subjected to PLC (10% methanol in CH2Cl2, 0.5%
acetic acid) to yield the isatin-acid conjugates 34-37.
2.5.4.3. 3-(3-(4-Methoxy-7-methyl-2,3-
dioxoindolin-5-yl)phenyl) propanoic acid (36)
Following the general procedure 3 using 5-bromo-4-methoxy-
7-methylindoline-2,3-dione 26 (100 mg, 0.370 mmol), 3-(3-
boronophenyl)propanoic acid 27 (86.1 mg, 0.444 mmol),
tetrakis(triphenylphosphine)palladium(0) (21.4 mg, 0.019 mmol)
and potassium phosphate (235.6 mg, 1.11 mmol) and heating for
5 h, the acid 36 was isolated as a red solid (60 mg, 48%), mp:
219-220 ºC; IR (cm-1): υ 3182 (m, NH), 2982 (m, OH), 1749 (s,
1
C=O), 1692 (s, C=O), 1287 (w, C-O). H NMR (DMSO-d6), δ:
11.16 (s, NH), 7.40 (s, 1H, isatin_6 H), 7.13 (t, J = 7.4 Hz, 1H,
phenyl_5 H), 7.26 (s, 1H, phenyl_2 H), 7.22 (d, J = 7.4 Hz, 1H,
pheny_6 H), 7.19 (d, J = 7.4 Hz, 1H, phenyl_4 H), 3.81 (s, 3H,
OCH3), 2.86 (t, J = 7.4 Hz, 2H, COOH-CH2-CH2), 2.56 (t, J =
7.4 Hz, 2H, COOH-CH2-CH2), 2.41 (s, 3H, CH3). 13C NMR, δ:
182.8 (COOH), 174.5 (isatin C2), 160.5 (isatin C3), 154.6 (isatin
C4), 149.3 (isatin C7a), 142.6 (phenyl C1), 141.5 (isatin C6),
137.4 (phenyl C3), 129.5 (phenyl C2), 129.1 (phenyl C5), 128.8
(isatin C7), 127.7 (phenyl C6), 127.3 (phenyl C4), 116.5 (isatin
C5), 110.3 (isatin C3a), 62.3 (OCH3), 35.9 (COOH-CH2-CH2),
31.0 (COOH-CH2-CH2), 15.6 (CH3) ppm. EI-MS m/z 339 (M+,
100%), 311 (75), 293 (10), 277 (77). HRMS (ESI) calcd for
C19H18NO5 (MH+), 340.1185; found, 340.1187. HPLC purity:
99.4%, Rt=5.579 (1% solvent B in solvent A).
2.5.4.1. 3-(3-(4,6-Dimethoxy-2,3-dioxoindolin-5-
yl)phenyl)propanoic acid (34)
Following the general procedure 3 using 5-bromo-4,6-
dimethoxyindoline-2,3-dione 17 (100 mg, 0.349 mmol), 3-(3-
boronophenyl)propanoic acid 27 (81.2 mg, 0.419 mmol),
tetrakis(triphenylphosphine)palladium(0) (20.2 mg, 0.017 mmol)
and potassium phosphate (222.2 mg, 1.047 mmol) and heating
for 6 h, the acid 34 was isolated as a yellow solid (49.5 mg,
40%), mp: 175-176 ºC; IR (cm-1): υ 3185 (m, NH), 2978 (m,
1
OH), 1718 (s, C=O), 1696 (s, C=O), 1290 (w, C-O). H NMR
2.5.4.4. 3-(3-(2,3-Dioxoindolin-5-
yl)phenyl)propanoic acid (37)
(DMSO-d6), δ: 12.09 (s, 1H, COOH), 10.36 (s, NH), 7.31 (t, J =
7.5 Hz, 1H, phenyl_5 H), 7.20 (d, J = 7.5 Hz, 1H, phenyl_4 H),
7.08-7.06 (m, 2H, phenyl_2,6 H), 6.35 (s, 1H, isatin_7 H), 3.97
(s, 3H, isatin_4 OCH3), 3.84 (s, 3H, isatin_C6 OCH3), 2.85 (t, J =
7.5 Hz, 2H, COOH-CH2-CH2), 2.58 (t, J = 7.5 Hz, 2H, COOH-
CH2-CH2). 13C NMR, δ: 178.1 (COOH), 174.0 (isatin C2), 166.6
(isatin C3), 161.5 (isatin C6), 160.1 (isatin C4), 149.2 (phenyl
C1), 141.0 (isatin C7a), 131.2 (phenyl C3), 130.3 (phenyl C5),
128.3 (double height, phenyl C4 and C6), 127.4 (phenyl C2),
108.2 (isatin C7), 100.6 (isatin C5), 90.1 (isatin C3a), 56.7
(isatin_4 OCH3), 56.3 (isatin_6 OCH3), 35.0 (COOH-CH2-CH2),
30.3 (COOH-CH2-CH2) ppm. EI-MS m/z 355 (M+, 40), 299 (4),
254 (100%), 191 (45). HRMS (ESI) calcd for C19H18NO6 (MH+),
356.1134; found, 356.1116. HPLC purity: 99.3%, Rt=35.814 (3%
solvent B in solvent A).
Following the general procedure 3 using 5-bromoindoline-2,3-
dione 33 (100 mg, 0.442 mmol), 3-(3-boronophenyl)propanoic
acid
27
(102.9
mg,
0.530
mmol),
tetrakis(triphenylphosphine)palladium(0) (25.5 mg, 0.022 mmol)
and potassium phosphate (281.5 mg, 1.326 mmol) and heating
for 4 h, the acid 37 was isolated as an orange solid (84.8 mg,
65%), mp: 182-183 ºC; IR (cm-1): υ 3185 (m, NH), 2989 (m,
1
OH), 1714 (s, C=O), 1684 (s, C=O). H NMR (DMSO-d6), δ:
11.14 (s, NH), 7.89 (dd, J = 8.2, 1.8 Hz, 1H, isatin_6 H), 7.77 (d,
J = 1.4 Hz, 1H, isatin_4 H), 7.53 (s, 1H, phenyl_2 H), 7.46 (d, J
= 7.6 Hz, 1H, phenyl_4 H), 7.35 (t, J = 7.5 Hz, 1H, phenyl_5 H),
7.21 (d, J = 7.5 Hz, 1H, phenyl_6 H), 6.99 (d, J = 7.9 Hz, 1H,
isatin_7 H), 2.88 (t, J = 7.6 Hz, 2H, COOH-CH2-CH2), 2.59 (t, J
= 7.5 Hz, 2H, COOH-CH2-CH2). 13C NMR, δ: 184.9 (COOH),
174.4 (isatin C3), 160.0 (isatin C2), 150.4 (isatin C7a), 142.2
(phenyl C3), 139.1 (phenyl C1), 136.9 (isatin C6), 135.4 (isatin
C5), 129.4 (phenyl C5), 127.9 (phenyl C6), 126.7 (phenyl C2),
124.4 (phenyl C4), 122.9 (isatin C4), 118.9 (isatin C3a), 113.1
(isatin C7), 35.7 (COOH-CH2-CH2), 30.9 (COOH-CH2-CH2)
ppm. EI-MS m/z 295 (M+, 40), 267 (100%), 250 (95), 207 (20),
193 (25). HRMS (ESI) calcd for C17H14NO4 (MH+), 296.0914;
found, 296.0912. HPLC purity: 98.2%, Rt=7.237 (1% solvent B
in solvent A).
2.5.4.2. 3-(3-(7-Methyl-2,3-dioxoindolin-5-
yl)phenyl)propanoic acid (35)
Following the general procedure
3 using 5-bromo-7-
methylindoline-2,3-dione 20 (100 mg, 0.416 mmol), 3-(3-
boronophenyl)propanoic acid 27 (96.8 mg, 0.499 mmol),
tetrakis(triphenylphosphine)palladium(0) (24 mg, 0.021 mmol)
and potassium phosphate (264.9 mg, 1.248 mmol) and heating
for 4 h, the acid 35 was isolated as a red solid (70 mg, 55%), mp:
238-239 ºC; IR (cm-1): υ 3172 (m, NH), 2989 (m, OH), 1731 (s,
1
C=O), 1696 (s, C=O). H NMR (acetone-d6), δ: 9.94 (s, NH),
2.5.4.5. 3-(3-(3-Methyl-8-oxo-7-pivaloyl-7-
azabicyclo[4.2.0]octa-1,3,5-trien-5-
yl)phenyl)propanoic acid (38)
7.81 (s, 1H, isatin_4 H), 7.66 (s, 1H, phenyl_2 H), 7.60 (s, 1H,
isatin_6 H), 7.50 (d, J = 7.6 Hz, 1H, pheny_6 H), 7.40 (t, J = 7.6
Hz, 1H, phenyl_5 H), 7.29 (d, J = 7.6 Hz, 1H, phenyl_4 H), 3.03
(t, J = 7.3 Hz, 2H, COOH-CH2-CH2), 2.71 (t, J = 7.3 Hz, 2H,
COOH-CH2-CH2), 2.41 (s, 3H, CH3). 13C NMR (DMSO-d6), δ:
184.8 (COOH), 173.9 (isatin C3), 160.1 (isatin C2), 148.5 (isatin
Under a N2 atmosphere, a 25 mL round bottom flask was
charged with the β-lactam derivative 12 (100 mg, 0.339 mmol),

10
3-(3-boronophenyl)propanoic acid 27 (72.3 mg, 0.372 mmol),
tetrakis(triphenylphosphine)palladium(0) (19.6 mg, 0.016 mmol)
and potassium phosphate (215.9 mg, 1.02 mmol). A mixture of
1,4-dioxane (10 mL) and water (3 mL) was added and the
reaction mixture was degassed by flushing N2 through a needle
for 10 min. The flask was then sealed with a rubber cap, heated at
85 ºC for 18 h. The solvent was then concentrated under reduced
pressure and the flask was cooled to 0 ºC. Chilled 1 N HCl (10
mL) was added dropwise with stirring, and the resulting solid
was extracted with ethyl acetate (2 x 15 mL). The combined
organic layers were washed with water (15 mL), brine (15 mL)
and dried (MgSO4) and concentrated under reduced pressure. The
pressure, ethyl acetate (30 mL) was added and the mixture
ACCEPTED MANUSCRIPT
was sonicated for 10 min and filtered. The filtrate was triturated
with MgSO4 and charcoal (0.5 g), and filtered through a short pad
of celite. The filtrate was then concentrated under reduced
pressure and the resulting residue was recrystallised from a
mixture of ethyl acetate/hexane (3:2) to give the boronic ester 40
as a pink solid (1.1 g, 95%), mp: 40-41 ºC; IR (cm-1): υ 2980 (m,
C-H aliphatic), 2152 (m, CN), 1128 (w, C-O). 1H NMR (CDCl3),
δ: 7.43 (d, 1H, J = 7.6 Hz, phenoxy_6 H), 7.39 (s, 1H,
phenoxy_2 H), 7.08 (t, 1H, J = 7.9 Hz, phenoxy_5 H), 6.96 (d,
1H, J = 8.0 Hz, phenoxy_4 H), 4.76 (s, 2H, CH2), 1.26 (s, 12H,
4xCH3). 13C NMR, δ: 157.1 (phenoxy C1), 132.6 (phenoxy C3),
131.4 (phenoxy C5), 124.7 (phenoxy C4), 114.8 (CN), 114.6
(phenoxy C2), 94.6 (phenoxy C6), 83.7 (dioxaborolan C4, C5),
53.9 (CH2), 25.2 (4xCH3) ppm. ESI-MS m/z 259 (M+). HRMS
(ESI) calcd for C14H19BNO3 (MH+), 260.1458; found, 260.1462.
resulting residue was subjected to
a
flash column
chromatography (50% CH2Cl2, 50% ethyl acetate and 1% acetic
acid) to afford 38 as a pale yellow oil (74.2 mg, 60%), IR (cm-1):
υ 2959 (m, OH), 1696 (s, C=O), 1662 (s, C=O), 1481 (m, C-N).
1H NMR (CDCl3), δ: 8.56 (s, 1H, COOH), 7.77 (s, 1H, H4), 7.32-
7.29 (m, 2H, phenyl_5 H, H2), 7.20 (d, 1H, J = 7.5 Hz, phenyl_6
H), 7.19 (s, 1H, phenyl_2 H), 7.15 (d, 1H, J = 7.7 Hz, phenyl_4
H), 2.97 (t, 2H, J = 7.5 Hz, COOH-CH2-CH2), 2.68 (t, 2H, J =
7.6 Hz, COOH-CH2-CH2), 2.37 (s, 3H, CH3), 1.03 (s, 9H,
pivaloyl 3xCH3). 13C NMR, δ: 178.4 (pivaloyl C=O), 176.9
(COOH), 171.9 (azabicyclo C=O), 140.4 (phenyl C1), 140.1
(azabicyclo C6), 139.8 (phenyl C3), 136.4 (phenyl C5), 135.8
(azabicyclo C4), 133.1 (azabicyclo C2), 130.9 (azabicyclo C3),
128.8 (azabicyclo C1), 128.7 (phenyl C2), 127.3 (phenyl C6),
126.8 (azabicyclo C5), 124.8 (phenyl C4), 39.3 (C(CH3)3), 35.8
(COOH-CH2-CH2), 30.9 (COOH-CH2-CH2), 27.2 (pivaloyl
3xCH3), 21.0 (CH3) ppm. EI-MS m/z 365 (M+, 60), 350 (70), 320
(50), 299 (25), 277 (100%), 235 (26), 220 (23), 193 (52). HRMS
(ESI) calcd for C22H24NO4 (MH+), 366.1705; found, 366.1717.
HPLC purity: 98.9%, Rt=27.924 (3% solvent B in solvent A).
2.5.7. General procedure 4: Suzuki coupling of 6-
bromobenzolactone derivatives 7 and 8 with (3-
(cyanomethoxy)phenyl)boronic acid 39 or ester 40
A round bottom 25 mL flask was charged with the appropriate
dihydroisobenzofuran
derivative
(1
equiv),
(3-
(cyanomethoxy)phenyl)boronic acid 39 (1 equiv) or 2-(3-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phenoxy)acetonitrile
40
(1.2
equiv),
tetrakis(triphenylphosphine)palladium(0) (5 mol%), 1,4-dioxane
(10 mL) was added and the flask was sealed with a rubber cap.
The reaction mixture was degassed by flushing N2 through a
needle for 10 min. The flask was heated in an oil bath at 85 ºC.
When all the solid was dissolved, potassium phosphate (3 mmol,
636.8 mg) dissolved in water (5 mL) was added to the reaction
via a needle. The flask was then heated at 85 ºC for 7-15 h. The
solvent was concentrated under reduced pressure, and was added
1 M HCl (10 mL), the reaction mixture turned to a white turbid
solution which was then extracted with ethyl acetate (2 x 30 mL),
washed with brine (2 x 10 mL) and dried (MgSO4). The solvent
was evaporated and the residue was subjected to flash column
chromatography (40% ethyl acetate in petroleum spirit).
2.5.5. 2-(3-Iodophenoxy)acetonitrile (42)
A solution of 3-iodophenol 41 (2.0 g, 9.09 mmol) and NaOH
(0.91 g, 22.73 mmol) in dry DMF (25 mL) was stirred vigorously
for 30 min until the solution became pale brown. 2-
Chloroacetonitrile (1.03 g, 0.86 mL, 13.64 mmol) was then
added dropwise through a syringe and the mixture was further
stirred for 18 h at room temperature. The resulting brown
solution was then poured onto crushed ice (100 mL), stirred for
30 min, and the resulting oil was extracted with ethyl acetate (2 x
25 mL). The combined organic layers were washed with water (2
x 25 mL), brine (2 x 25 mL) and dried (MgSO4). The organic
fraction was then triturated with charcoal (0.5 g) and filtered
through celite, and evaporated under reduced pressure to afford
2-(3-iodophenoxy)acetonitrile 42 as a yellow oil (1.88 g, 80%),
IR (cm-1): υ 2942 (m, C-H aliphatic), 2246 (s, CN), 1203 (w, C-
O). 1H NMR (CDCl3), δ: 7.37 (d, 1H, J = 7.7 Hz, phenoxy_6 H),
7.29 (s, 1H, phenoxy_2 H), 7.02 (t, 1H, J = 7.9 Hz, phenoxy_5
2.5.7.1. 2-(3-(4-Methoxy-3-oxo-1,3-
dihydroisobenzofuran-5-yl)phenoxy)acetonitrile
(44)
Following the general procedure
4 using 6-bromo-7-
methoxyisobenzofuran-1(3H)-one 7 (100 mg, 0.411 mmol), (3-
(cyanomethoxy)phenyl)boronic acid 39 (72 mg, 0.411 mmol),
tetrakis(triphenylphosphine)palladium(0) (23.7 mg, 0.021 mmol)
and heating for 15 h, the acetonitrile intermediate 44 was isolated
as a white solid (60.7 mg, 50%), mp: 103-104 ºC; IR (cm-1): υ
2920 (w, C-H aliphatic), 2165 (m, CN), 1759 (s, C=O), 1212 (w,
C-O). 1H NMR (CDCl3), δ: 7.74 (d, 1H, J = 7.7 Hz,
isobenzofuran_6 H), 7.43 (t, 1H, J = 8.0 Hz, phenyl_5 H), 7.25-
7.23 (m, 2H, phenyl_4 H, isobenzofuran_7 H), 7.19 (s, 1H,
phenyl_2 H), 7.02 (d, 1H, J = 8.2 Hz, phenyl_6 H), 5.30 (s, 2H,
isobenzofuran CH2), 4.83 (s, 2H, CN-CH2), 3.90 (s, 3H, OCH3).
13C NMR, δ: 168.5 (C=O), 156.7 (phenyl C1), 156.4
(isobenzofuran C4), 148.7 (isobenzofuran C7a), 138.7
(isobenzofuran C6), 137.4 (phenyl C5), 134.7 (isobenzofuran
C5), 129.8 (phenyl C4), 124.1 (phenyl C3), 117.9 (phenyl C6),
117.1 (phenyl C2), 116.1 (CN), 115.1 (isobenzofuran C3a), 114.5
(isobenzofuran C7), 68.7 (isobenzofuran C1), 62.7 (CN-CH2),
53.7 (OCH3) ppm. EI-MS m/z 295 (M+, 15), 255 (100%), 237
(13), 208 (10), 180 (16). HRMS (ESI) calcd for C17H14NO4
(MH+), 296.0914; found, 296.0913. HPLC purity: 98.3%,
Rt=16.676 (1% solvent B in solvent A).
H), 6.90 (d, 1H, J = 8.0 Hz, phenoxy_4 H), 4.68 (s, 2H, CH2). 13
C
NMR, δ: 157.1 (phenoxy C1), 132.5 (phenoxy C5), 131.6
(phenoxy C4), 124.7 (phenoxy C2), 115.3 (CN), 114.6 (phenoxy
C6), 94.9 (phenoxy C3), 54.0 (CH2) ppm. EI-MS m/z 259 (M+,
100%), 219 (27), 203 (5). HRMS (ESI) calcd for C8H7INO
(MH+), 259.9572; found, 259.9565.
2.5.6. 2-(3-(4,4,5,5-Tetramethyl-1,3,2-
dioxaborolan-2-yl)phenoxy)acetonitrile (40)
Under a N2 atmosphere, an oven dried 100 mL flask was
charged with 2-(3-iodophenoxy)acetonitrile 42 (1.16 g, 4.48
mmol), bis(pinacolato)diboron 43 (4.55 g, 17.92 mmol),
potassium acetate (1.76 g, 17.92 mmol) and [1,1′-
bis(diphenylphosphino)ferrocene]dichloropalladium(II)
(PdCl2(dppf)) (32.8 mg, 1 mol%). Dry THF (50 mL) was added
to the mixture, and the flask was heated at reflux for 18 h under
N2 atmosphere. The solvent was then evaporated under reduced

11
2.5.7.2. 2-(3-(4-Methyl-3-oxo-1,3-
dihydroisobenzofuran-5-yl)phenoxy)acetonitrile
Following the general procedure 5 using 2-(3-(4-methyl-3-
ACCEPTED MANUSCRIPT
oxo-1,3-dihydroisobenzofuran-5-yl)phenoxy)acetonitrile 45 (51.9
mg), the final tetrazole conjugate 47 was isolated as a pale yellow
gummy solid (21 mg, 35%), IR (cm-1): υ 3392 (m, NH), 2988 (w,
C-H aliphatic), 1741 (s, C=O), 1668 (m, C=N), 1212 (w, C-O).
1H NMR (CD3OD), δ: 7.58 (d, 1H, J = 7.7 Hz, isobenzofuran_5
H), 7.48 (d, 1H, J = 7.7 Hz, isobenzofuran_4 H), 7.44 (t, 1H, J =
7.9 Hz, phenyl_5 H), 7.12 (d, 1H, J = 7.9 Hz, phenyl_6 H), 7.03
(s, 1H, phenyl_2 H), 6.99 (d, 1H, J = 7.5 Hz, phenyl_4 H), 5.51
(s, 2H, isobenzofuran CH2), 5.36 (s, 2H, O-CH2), 2.55 (s, 3H,
CH3). 13C NMR (DMSO-d6), δ: 170.9 (C=O), 162.4 (phenyl C3),
157.3 (tetrazole C), 147.2 (isobenzofuran C3a), 142.1
(isobenzofuran C7), 141.1 (isobenzofuran C6), 135.7 (phenyl
C5), 135.4 (isobenzofuran C5), 129.7 (phenyl C1), 122.9
(isobenzofuran C7a), 122.7 (phenyl C6), 120.8 (isobenzofuran
C4), 115.8 (phenyl C2), 114.1 (phenyl C4), 68.5 (isobenzofuran
C3), 59.3 (tetrazole-CH2-O), 14.4 (CH3) ppm. EI-MS m/z 322
(M+, 50), 239 (75), 223 (26), 195 (60), 165 (100%). HRMS (ESI)
calcd for C17H15N4O3 (MH+), 323.1144; found, 323.1138. HPLC
purity: 98.7%, Rt=10.695 (5% solvent B in solvent A).
(45)
Following the general procedure
methoxyisobenzofuran-1(3H)-one 8 (100 mg, 0.358 mmol), 2-(3-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)
acetonitrile
4 using 6-bromo-7-
40
(111.4
mg,
0.430
mmol),
tetrakis(triphenylphosphine)palladium(0) (20.7 mg, 0.018 mmol)
and heating for 7 h, the acetonitrile intermediate 45 was isolated
as white solid (93.4 mg, 76%), mp: 99-100 ºC; IR (cm-1): υ 2972
(w, C-H aliphatic), 2169 (m, CN), 1743 (s, C=O). 1H NMR
(CDCl3), δ: 7.51 (d, 1H, J = 7.7 Hz, isobenzofuran_6 H), 7.43 (t,
1H, J = 7.9 Hz, phenyl_5 H), 7.33 (d, 1H, J = 7.7 Hz,
isobenzofuran_7 H), 7.02 (d, 2H, J = 8.0 Hz, phenyl_4,6 H), 6.93
(s, 1H, phenyl_2 H), 5.28 (s, 2H, isobenzofuran CH2), 4.83 (s,
2H, CN-CH2), 2.60 (s, 3H, CH3). 13C NMR, δ: 171.4 (C=O),
156.7 (phenyl C1), 146.7 (isobenzofuran C7a), 142.8
(isobenzofuran C4), 142.1 (isobenzofuran C5), 137.6 (phenyl
C3), 135.6 (phenyl C5), 130.1 (isobenzofuran C3a), 124.4
(phenyl C4), 124.1 (isobenzofuran C6), 119.4 (isobenzofuran
C7), 116.4 (phenyl C2), 115.3 (CN), 114.1 (phenyl C6), 68.6
(isobenzofuran C1), 53.9 (CN-CH2), 15.0 (CH3) ppm. EI-MS m/z
279 (M+, 90), 239 (100%), 223 (15), 195 (12), 165 (24). HRMS
(ESI) calcd for C17H14NO3 (MH+), 280.0974; found, 280.0966.
HPLC purity: 98.1%, Rt=11.021 (1% solvent B in solvent A).
2.5.9. 5-((3-Iodophenoxy)methyl)-1H-tetrazole (48)
In a round bottom 50 mL flask, a solution of 2-(3-
iodophenoxy)acetonitrile 42 (1.6 g, 6.18 mmol), sodium azide
(1.6 g, 24.7 mmol) and amonium chloride (1.3 g, 24.7 mmol) in
dry DMF (25 mL), containing few drops of glacial acetic acid,
was heated at reflux for 14 h under a N2 atmosphere. The solvent
was then concentrated under reduced pressure, and the resulting
residue was dried under vacuum. Ethanol (25 mL) was added to
the residue and the mixture was sonicated for 10 min, filtered and
ethanol was evaporated under reduced pressure resulting in a
white fluffy solid of the tetrazole intermediate 48 (1.5 g, 80%),
mp: 99-100 ºC; IR (cm-1): υ 2995 (w, NH), 1589 (m, C=N), 1240
(w, C-O). 1H NMR (CD3OD), δ: 7.46 (s, 1H, phenoxy_2 H), 7.39
(t, 1H, J = 7.0 Hz, phenoxy_5 H), 7.09-7.08 (m, 2H,
phenoxy_4,6 H), 5.47 (s, 2H, CH2). 13C NMR, δ: 159.7 (phenoxy
C1), 155.3 (tetrazole C), 132.4 (phenoxy C4), 132.2 (phenoxy
C5), 125.4 (phenoxy C2), 115.3 (phenoxy C6), 94.9 (phenoxy
C3), 61.1 (CH2) ppm. EI-MS m/z 302 (M+, 100%), 220 (80), 203
(10). HRMS (ESI) calcd for C8H8IN4O (MH+), 302.9743; found,
302.9734.
2.5.8. General procedure 5: synthesis of the
benzolactone-tetrazole conjugates 46 and 47 using
sodium azide method
Under a N2 atmosphere, a round bottom flask (25 mL) was
charged with the appropriate acetonitrile derivative 44-45 (0.186
mmol), sodium azide (48.4 mg, 0.744 mmol), amonium chloride
(39.4 mg, 0.744 mmol), DMF (10 mL) and a catalytic amount of
glacial acetic acid (5-10 drops), and the reaction mixture was
then heated at reflux for 24 h. DMF was concentrated under
reduced pressure and the residue was dried by vacuum. Ethanol
was added to the resulting dry residue and the mixture was
sonicated for 10 min, filtered and the ethanol solution was
concentrated and subjected to PLC (90% CH2Cl2, 10% ethanol
and 0.1% TFA) to yield the tetrazole conjugate.
2.5.8.1. 6-(3-((1H-Tetrazol-5-yl)methoxy)phenyl)-7-
methoxyisobenzofuran-1(3H)-one (46)
2.5.10. Potassium 5-((3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenoxy)methyl)tetrazol-1-ide
(49)
Following the general procedure 5 using 2-(3-(4-methoxy-3-
oxo-1,3-dihydroisobenzofuran-5-yl)phenoxy)acetonitrile 44 (55
mg), the tetrazole conjugate 46 was isolated as a pale yellow
solid (21.4 mg, 34%), mp: 130-131 ºC; IR (cm-1): υ 3404 (w,
NH), 2907 (w, C-H aliphatic), 1751 (s, C=O), 1660 (m, C=N),
Under a N2 atmosphere, an oven dried 100 mL flask was
charged with 5-((3-iodophenoxy)methyl)-1H-tetrazole 48 (960
mg, 3.179 mmol), bis(pinacolato)diboron 43 (3.2 g, 12.72 mmol),
potassium acetate (1.25 g, 12.72 mmol) and PdCl2(dppf) (23.3
mg, 1 mol%). Dry THF (30 mL) was added to the mixture, and
the flask was heated at reflux for 18 h under a N2 atmosphere.
The solvent was then concentrated under reduced pressure.
Ethanol (30 mL) was added to the resulting residue and the
mixture was further heated at reflux for 10 min, filtered hot and
concentrated under reduced pressure to afford a white solid of the
boronic ester potassium salt 49 (1.1 g, 99%), mp: >250 ºC ; IR
(cm-1): υ 2983 (s, C-H aliphatic), 1680 (m, C=N), 1233 (w, C-O).
1H NMR (CD3OD), δ: 7.38 (s, 1H, phenoxy_2 H), 7.28 (d, 1H, J
= 7.1 Hz, phenoxy_6 H), 7.04-6.99 (m, 2H, phenoxy_4,5 H),
5.25 (s, 2H, CH2), 1.90 (s, 12H, 4xCH3). 13C NMR, δ: 178.5
(phenoxy C1), 159.5 (tetrazole C), 158.3 (phenoxy C3), 130.8
(phenoxy C5), 130.2 (phenoxy C4), 124.2 (phenoxy C6), 114.2
(phenoxy C2), 93.6 (dioxaborolan C4, C5), 61.2 (CH2), 22.6
(4xCH3) ppm. ESI-MS m/z 301.1 (M+K+).
1
1134 (w, C-O). H NMR (CD3OD), δ: 7.72 (d, 1H, J = 7.7 Hz,
isobenzofuran_5 H), 7.42 (t, 1H, J = 7.9 Hz, phenyl_5 H), 7.37
(d, 1H, J = 7.7 Hz, isobenzofuran_4 H), 7.22-7.17 (m, 2H,
phenyl_2,4 H), 7.10 (d, 1H, J = 8.2 Hz, phenyl_6 H), 5.50 (s, 2H,
isobenzofuran CH2), 5.34 (s, 2H, OCH2), 3.78 (s, 3H, OCH3). 13
C
NMR, δ: 169.7 (C=O), 157.9 (phenyl C3), 156.5 (isobenzofuran
C7), 149.6 (tetrazole C), 138.7 (isobenzofuran C3a), 137.7
(isobenzofuran C5), 135.1 (phenyl C5), 129.5 (isobenzofuran
C6), 129.4 (phenyl C6), 123.0 (phenyl C1), 117.6 (phenyl C4),
117.5 (phenyl C2), 115.9 (isobenzofuran C7a), 113.9
(isobenzofuran C4), 69.3 (isobenzofuran C3), 61.7 (tetrazole-
CH2-O), 59.9 (OCH3) ppm. EI-MS m/z 338 (M+, 14), 255
(100%), 239 (10), 211 (12), 195 (11). HRMS (ESI) calcd for
C17H15N4O4 (MH+), 339.1093; found, 339.1107. HPLC purity:
96.3%, Rt=16.339 (5% solvent B in solvent A).
2.5.8.2. 6-(3-((1H-Tetrazol-5-yl)methoxy)phenyl)-7-
methylisobenzofuran-1(3H)-one (47)

12
C1 H11N4O3 (M-H+), 307.0831; found, 307.0826. HPLC
purity: 98.8%, Rt=13.175 (5% solvent B in solvent A).
2.5.11. General procedure 6: synthesis of the
benzolactone-tetrazole conjugates 50 and 51 using
the coupling method:
Under a N2 atmosphere, a mixture of the appropriate
bromoisobenzofuran-1(3H)-one derivative (1 mmol) and
ACCEPTED MANUSCRIPT
6
2.5.12. General procedure 7: Suzuki coupling of the
5-bromoisatin derivatives 17, 20, 26 and 33 with
(3-(cyanomethoxy)phenyl)boronic acid 39:
potassium
5-((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
Under a N2 atmosphere, a 25 mL flask was charged with the
yl)phenoxy)methyl)tetrazol-1-ide 49 (374 mg, 1.1 mmol),
tetrakis(triphenylphosphine)palladium(0) (80.9 mg, 7 mol%) and
potassium phosphate (636.8 mg, 3 mmol) was added to a solvent
mixture of 1,4-dioxane (25 mL) and water (10 mL) in a 50 mL
round bottom flask. The reaction mixture was degassed by
flushing N2 through a needle for 10 min, the flask was then
sealed with a rubber cap, heated at 85 ºC for 20-24 h. The solvent
was then concentrated under reduced pressure. Saturated
NaHCO3 (25 mL) was added to the resulting residues, and the
mixture sonicated for 15 min, before the turbid solution was
washed with CH2Cl2 (25 mL). The aqueous layer was filtered,
cooled to 0 ºC using an ice bath, and the filtrate was then
neutralized with 2 M HCl. The resulting white solid was
collected by vacuum filtration, washed with chilled methanol and
dried under vacuum to afford the tetrazoles.
5-bromoisatin
(cyanomethoxy)phenyl)boronic
derivative
(1
acid
equiv),
(1
(3-
equiv),
39
tetrakis(triphenylphosphine)palladium(0) (5 mol%), potassium
phosphate (3 equiv) and a mixture of diglyme (3 mL) and water
(3 mL), and the reaction mixture was degassed by flushing N2
through a needle for 10 min. The flask was then sealed and
heated at 85 ºC for 5-9 h. After cooling to room temperature, the
solvent was concentrated under reduced pressure, and 1 N HCl
(15 mL) was added. The suspended solid was either collected by
filtration or extracted with ethyl acetate (2 x 25 mL). The
combined organic layers were dried (MgSO4) and evaporated
under reduced pressure. The combined residues were adsorbed
onto silica and subjected to flash column chromatography (40%
ethyl acetate in petroleum spirit) to yield the acetonitrile
derivatives.
2.5.11.1. 6-(3-((1H-Tetrazol-5-yl)methoxy)phenyl)-
5,7-dimethoxyisobenzofuran-1(3H)-one (50)
2.5.12.1. 2-(3-(4,6-Dimethoxy-2,3-dioxoindolin-5-
yl)phenoxy)acetonitrile (52)
Following the general procedure 6 using 6-bromo-5,7-
dimethoxyisobenzofuran-1(3H)-one 10 (1 mmol, 273 mg) and
heating for 24 h, the final tetrazole conjugate 50 was isolated as a
white solid (195 mg, 53%), mp: charring; IR (cm-1): υ 3204 (w,
Following the general procedure 7 using 5-bromo-4,6-
dimethoxyindoline-2,3-dione 17 (40 mg, 0.139 mmol), (3-
(cyanomethoxy)phenyl)boronic acid 39 (24.6 mg, 0.139 mmol),
tetrakis(triphenylphosphine)palladium(0) (8 mg, 0.007 mmol),
potassium phosphate (88.5 mg, 0.417 mmol) and heating for 9 h,
the acetonitrile intermediate 52 could not be isolated. Analysis of
the crude mixture: EI-MS m/z 338 (M+, 17), 309 (7), 382 (66),
257 (100%), 225 (25). HRMS (ESI) calcd for C18H15N2O5 (MH+),
339.0981; found, 339.0980. This sample was collected from the
reaction mixture before purification, but could not be further
purified and was not sufficiently stable to perform NMR
experiments.
1
NH), 1740 (s, C=O), 1614 (m, C=N), 1218 (w, C-O). H NMR
(DMSO-d6), δ: 7.36 (t, 1H, J = 7.9 Hz, phenyl_5 H), 7.05 (d, 1H,
J = 8.0 Hz, phenyl_6 H), 7.00 (s, 1H, phenyl_2 H), 6.93 (d, 1H, J
= 7.7 Hz, phenyl_4 H), 6.82 (s, 1H, isobenzofuran_4 H), 5.49 (s,
2H, isobenzofuran CH2), 5.12 (s, 2H, OCH2), 3.90 (s, 3H,
isobenzofuran_7 OCH3), 3.89 (s, 3H, isobenzofuran_5 OCH3).
13C NMR, δ: 167.9 (C=O), 162.6 (isobenzofuran C5), 158.9
(phenyl C3), 157.4 (isobenzofuran C7), 153.6 (tetrazole C), 149.2
(isobenzofuran C3a), 134.9 (phenyl C5), 129.5 (phenyl C6),
122.8 (phenyl C4), 115.9 (phenyl C10), 115.7 (phenyl C11),
114.1 (isobenzofuran C7a), 104.7 (isobenzofuran C4), 96.2
(isobenzofuran C6), 67.8 (isobenzofuran C3), 59.2 (tetrazole-
CH2-O), 56.6 (isobenzofuran_7 OCH3), 56.1 (isobenzofuran_5
OCH3) ppm. ESI-MS m/z 367.1 (M-H+). HRMS (ESI) calcd for
C18H15N4O5 (M-H+), 367.1042; found, 367.1038. HPLC purity:
95.2%, Rt=18.896 (10% solvent B in solvent A).
2.5.12.2. 2-(3-(7-Methyl-2,3-dioxoindolin-5-
yl)phenoxy)acetonitrile (53)
Following the general procedure
7 using 5-bromo-7-
methylindoline-2,3-dione 20 (100 mg, 0.416 mmol), (3-
(cyanomethoxy)phenyl)boronic acid 39 (73.6 mg, 0.416 mmol),
tetrakis(triphenylphosphine)palladium(0) (24 mg, 0.021 mmol),
potassium phosphate (264.9 mg, 1.248 mmol) and heating for 5
h, the acetonitrile intermediate 53 was isolated as a red solid
(63.2 mg, 52%), mp: 208-209 ºC; IR (cm-1): υ 3185 (w, NH),
2365 (m, CN), 1750 (s, C=O), 1587 (s, C=O). 1H NMR (acetone-
d6), δ: 10.13 (s, NH), 7.83 (s, 1H, isatin_4 H), 7.68 (s, 1H,
isatin_C6), 7.47 (t, J = 7.8 Hz, 1H, phenoxy_5 H), 7.40-7.38 (m,
2H, phenoxy_2,4 H), 7.09 (d, J = 8.0 Hz, 1H, phenoxy_6 H),
5.21 (s, 2H, CH2), 2.39 (s, 3H, CH3). 13C NMR, δ: 185.1 (isatin
C3), 160.5 (phenoxy C1), 158.6 (isatin C2), 149.8 (isatin C7a),
142.4 (phenoxy C3), 138.9 (isatin C7), 136.4 (isatin C6), 131.3
(isatin C5), 123.3 (phenoxy C5), 121.8 (isatin C4), 121.2
(phenoxy C4), 119.5 (phenoxy C2), 116.7 (phenoxy C6), 115.3
(CN), 114.1 (isatin C3a), 54.7 (CH2), 15.8 (CH3) ppm. EI-MS
m/z 292 (M+, 93), 264 (80), 236 (100%), 196 (17), 180 (11).
HRMS (ESI) calcd for C17H13N2O3 (MH+), 293.0926; found,
293.0931. HPLC purity: 97.9%, Rt=13.070 (2.5% solvent C in
solvent A).
2.5.11.2. 6-(3-((1H-Tetrazol-5-
yl)methoxy)phenyl)isobenzofuran-1(3H)-one (51)
Following
the
general
procedure
6
using
6-
bromoisobenzofuran-1(3H)-one 28 (213 mg, 1 mmol) and
heating for 20 h, the final tetrazole conjugate 51 was isolated as a
white solid (181 mg, 59%), mp: charring; IR (cm-1): υ 3195 (w,
NH), 1751 (s, C=O), 1653 (m, C=N). 1H NMR (CD3OD), δ: 8.07
(s, 1H, isobenzofuran_7 H), 7.99 (d, 1H, J = 8.0 Hz,
isobenzofuran_5 H), 7.67 (d, 1H, J = 8.0 Hz, isobenzofuran_4
H), 7.38 (t, 1H, J = 7.9 Hz, phenyl_5 H), 7.33 (s, 1H, phenyl_2
H), 7.24 (d, 1H, J = 7.5 Hz, phenyl_6 H), 7.09 (d, 1H, J = 8.0 Hz,
phenyl_4 H), 5.42 (s, 2H, isobenzofuran CH2), 5.37 (s, 2H, O-
CH2). 13C NMR, δ: 171.9 (C=O), 159.3 (phenyl C3), 146.3
(tetrazole C), 142.4 (isobenzofuran C3a), 140.9 (isobenzofuran
C6), 133.2 (phenyl C1), 129.7 isobenzofuran C5), 125.9 (phenyl
C5), 122.9 (phenyl C6), 122.7 (isobenzofuran C7), 122.6
(isobenzofuran C7a), 119.7 (phenyl C2), 114.4 (isobenzofuran
C4), 113.7 (phenyl C4), 69.9 (isobenzofuran C3), 61.1 (tetrazole-
CH2-O) ppm. ESI-MS m/z 307.1 (M-H+). HRMS (ESI) calcd for
2.5.12.3. 2-(3-(4-Methoxy-7-methyl-2,3-
dioxoindolin-5-yl)phenoxy)acetonitrile (54)
Following the general procedure 7 using 5-bromo-4-methoxy-
7-methylindoline-2,3-dione 26 (100 mg, 0.370 mmol), (3-

13
(cyanomethoxy)phenyl)boronic acid 39 (65.5 mg, 0.370 mmol),
tetrakis(triphenylphosphine)palladium(0) (21.4 mg, 0.019 mmol),
potassium phosphate (235.6 mg, 1.11 mmol) and heating for 8 h,
the acetonitrile intermediate 54 was isolated as a red solid (41.7
mg, 35%), mp: 179-180 ºC; IR (cm-1): υ 3190 (m, NH), 2166 (w,
6.47 (s, 1H, NH), 4.73 (s, 2H, CH2), 2.28 (s, 3H, CH3), 0.97
ACCEPTED MANUSCRIPT
(s, 9H, pivaloyl 3xCH3). 13C NMR, δ: 177.7 (pivaloyl C=O),
176.7 (COOH), 173.8 (C3'), 156.4 (C2), 141.7 (C5), 137.8 (C1),
135.7 (C6'), 133.2 (C4), 130.5 (C6), 129.7 (C1'), 129.6 (C6'),
123.4 (C4'), 115.6 (CN), 115.1 (C3), 113.3 (C2'), 53.5 (CH2),
38.9 (C(CH3)3), 26.9 (pivalamido 3xCH3), 20.8 (CH3) ppm. ESI-
MS m/z 365.0 (M-H+). HRMS (ESI) calcd for C21H23N2O4
(MH+), 367.1658; found, 367.1656. HPLC purity: 99.4%,
Rt=17.599 (2% solvent B in solvent A).
1
CN), 1706 (s, C=O), 1611 (s, C=O), 1294 (w, C-O). H NMR
(CD3OD), δ: 7.40 (s, 1H, isatin_6 H), 7.38 (t, J = 8.2 Hz, 1H,
phenoxy_5 H), 7.12 (d, J = 8.2 Hz, 1H, phenoxy_4 H), 7.11 (s,
1H, phenoxy_2 H), 7.01 (d, J = 8.3 Hz, 1H, phenoxy_6 H), 5.00
(s, 2H, CH2), 3.91 (s, 3H, OCH3), 2.22 (s, 3H, CH3). 13C NMR, δ:
160.2 (isatin C3), 156.7 (phenoxy C1), 154.9 (isatin C2), 148.6
(isatin C4), 142.1 (isatin C7a), 138.9 (isatin C6), 137.8 (phenoxy
C3), 129.2 (phenoxy C5), 123.4 (isatin C5), 120.5 (isatin C7),
115.9 (phenoxy C4), 115.7 (phenoxy C6), 115.5 (phenoxy C2),
113.7 (CN), 109.5 (isatin C3a), 61.1 (CH2), 53.3 (OCH3), 13.7
(CH3) ppm. EI-MS m/z 322 (M+, 74), 294 (68), 280 (4), 254 (7),
226 (100%), 211 (11). HRMS (ESI) calcd for C18H15N2O4 (MH+),
323.1032; found, 323.1018. HPLC purity: 99.5%, Rt=11.766
(2.5% isopropanol in solvent A).
2.5.14. Antiviral assay
CHIKV Indian Ocean strain 899 (Genbank FJ959103.1) was
generously provided by Prof. S. Günther (Bernhard Nocht
Institute for Tropical Medicine, Hamburg, Germany) (Panning M
et al, Emerging Infectious Diseases 2008). BGM cells were
maintained in cell growth medium composed of minimum
essential medium (MEM Rega-3, Gibco, Belgium) supplemented
with 10% Foetal Bovine Serum (FBS, Integro, The Netherlands),
1% L-glutamine (Gibco), and 1% sodium bicarbonate (Gibco).
The antiviral assays were performed in virus growth medium
which is the respective cell growth medium supplemented with
2% (instead of 10%) FBS. Cell cultures were maintained at 37 °C
in an atmosphere of 5% CO2 and 95-99% humidity.
2.5.12.4. 2-(3-(2,3-Dioxoindolin-5-
yl)phenoxy)acetonitrile (55)
Following the general procedure 7 using 5-bromoindoline-2,3-
dione
33
(100
mg,
0.442
mmol),
(3-
BGM cells were seeded in 96-well tissue culture plates
(Becton Dickinson, Aalst, Belgium) at a density of 2.5 × 104
cells/well in 100 µl assay medium and were allowed to adhere
overnight. Next, a compound dilution series was prepared in the
medium on top of the cells after which the cultures were infected
with 0.001 MOI of CHIKV 899 inoculum in 100 µl assay
medium. On day 5 post-infection (p.i.), the plates were processed
using the MTS/PMS method as described by the manufacturer
(Promega, The Netherlands). The 50% effective concentration
(EC50), which is defined as the compound concentration that is
required to inhibit virus-induced cell death by 50%, was
(cyanomethoxy)phenyl)boronic acid 39 (78.2 mg, 0.442 mmol),
tetrakis(triphenylphosphine)palladium(0) (25.5 mg, 0.022 mmol),
potassium phosphate (281.5 mg, 1.326 mmol) and heating for 5
h, the acetonitrile intermediate 55 was isolated as a red solid
(73.7 mg, 60%), mp: 205-206 ºC; IR (cm-1): υ 3168 (w, NH),
2166 (m, CN), 1732 (s, C=O), 1621 (s, C=O). 1H NMR (acetone-
d6), δ: 10.04 (s, NH), 7.95 (d, J = 8.2, 1H, phenoxy_4 H), 7.83 (s,
1H, phenoxy_2 H), 7.47 (t, J = 8.2 Hz, 1H, phenoxy_5 H), 7.39-
7.38 (m, 2H, isatin_4,7H), 7.14 (d, J = 8.2 Hz, phenoxy_6H),
7.09 (d, J = 8.6 Hz, 1H, isatin_6H), 5.21 (s, 2H, CH2). 13C NMR,
δ: 184.1 (isatin C3), 159.6 (phenoxy C1), 157.8 (isatin C2), 150.4
(isatin C7a), 141.5 (phenoxy C3), 137.0 (phenoxy C4), 135.7
(phenoxy C2), 130.6 (phenoxy C5), 123.1 (isatin C5), 121.0
(isatin C7), 118.9 (phenoxy C6), 116.0 (CN), 114.6 (isatin C3a),
113.1 (isatin C4), 112.9 (isatin C6), 53.8 (CH2) ppm. EI-MS m/z
278 (M+, 46), 250 (100%), 207 (5), 182 (49). HRMS (ESI) calcd
for C16H11N2O3 (MH+), 279.0770; found, 279.0759. HPLC
purity: 99.7%, Rt=20.046 (2.5% solvent C in solvent A).
determined
using
logarithmic
interpolation.
Potential
cytotoxic/cytostatic effects of selected compounds were also
evaluated in uninfected cells by means of the MTS/PMS method.
The 50% cytotoxic concentration (CC50; i.e., the concentration
that reduces the overall metabolic activity of the cells by 50%)
was calculated using logarithmic interpolation. All assay wells
were checked microscopically for minor signs of virus-induced
CPE or possible alterations to the cell or monolayer morphology
caused by the compound.
2.5.13. 3'-(Cyanomethoxy)-5-methyl-2-pivalamido-
[1,1'-biphenyl] -3-carboxylic acid (59)
Acknowledgements
Under a N2 atmosphere, a 25 mL round bottom flask was
charged with the β-lactam derivative 12 (100 mg, 0.339 mmol),
the boronic ester 40 (96.6 mg, 0.373 mmol),
tetrakis(triphenylphosphine)palladium(0) (19.6 mg, 0.016 mmol)
and potassium phosphate (215.9 mg, 1.02 mmol). A mixture of
1,4-dioxane (10 mL) and water (3 mL) was added and the
reaction mixture was degassed by flushing N2 through a needle
for 10 min. The flask was then sealed with a rubber cap, heated at
85 ºC for 12 h. The solvent was then concentrated under reduced
pressure and 1 N HCl (10 mL) was added dropwise to the
resulting residue which was extracted with ethyl acetate (2 x 15
mL). The combined organic layers were washed with water (15
mL), brine (15 mL) and dried (MgSO4). The mixture was then
concentrated under reduced pressure, and the resulting residue
was subjected to a flash column chromatography and elution with
90% CH2Cl2, 10% ethanol and 0.1% TFA yielded 59 as a pale
yellow oil (59.4 mg, 48%), IR (cm-1): υ 3328 (m, NH), 2967 (m,
AAR thanks the University of Wollongong for a scholarship.
Supplementary material
1
The H and 13C NMR spectra. This material is available free
of charge via the Internet at http://pubs.acs.org.
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