Modelling and Phenotypic Screening of NAP-6 and 10-Cl-BBQ, AhR Ligands Displaying Selective Breast Cancer Cytotoxicity in Vitro

Article abstract of DOI:10.1002/cmdc.202000721

To exploit the interaction of the aryl hydrocarbon receptor (AhR) pathway in developing breast-cancer-specific cytotoxic compounds, we examined the breast cancer selectivity and the docking pose of the AhR ligands (Z)-2-(2-aminophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (NAP-6; 5) and 10-chloro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one (10-Cl-BBQ; 6). While the breast cancer selectivity of 5 in vitro is known, we discuss the SAR around this lead and, by using phenotypic cell-line screening and the MTT assay, show for the first time that 6 also presents with breast cancer selectivity, notably in the triple-negative (TN) receptor breast cancer cell line MDA-MB-468, the ER+ breast cancer cell lines T47D, ZR-75-1 and the HER2+ breast cancer cell line SKBR3 (GI₅₀ values of 0.098, 0.97, 0.13 and 0.21 μM, respectively). Indeed, 6 is 55 times more potent in MDA-MB-468 cells than normal MCF10A breast cells (GI₅₀ of 0.098 vs 5.4 μM) and more than 130 times more potent than in cell lines derived from pancreas, brain and prostate (GI₅₀ of 0.098 vs 10–13 μM). Molecular docking poses of 5 and 6 together with analogue synthesis and phenotypic screening show the importance of the naphthalene moiety, and an ortho-disposed substituent on the N-phenyl moiety for biological activity.

Full text of DOI:10.1002/cmdc.202000721

Accepted Article
Title: Modelling and phenotypic screening of NAP-6 and 10-Cl-BBQ,
AhR ligands displaying selective breast cancer cytotoxicity in vitro
Authors: Jennifer R. Baker, Brett L. Pollard, Andrew J. S. Lin, Jayne
Gilbert, Stefan Paula, Xiao Zhu, Jennette A. Sakoff, and
Adam McCluskey
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000721
Link to VoR: https://doi.org/10.1002/cmdc.202000721
01/2020

10.1002/cmdc.202000721
ChemMedChem
FULL PAPER
Modelling and phenotypic screening of NAP-6 and 10-Cl-BBQ,
AhR ligands displaying selective breast cancer cytotoxicity in
vitro
Jennifer R. Baker,[a] Brett L. Pollard,[a] Andrew J. S. Lin,[a] Jayne Gilbert,[b] Stefan Paula,[c] Xiao Zhu,[d]
Jennette A. Sakoff *[b] and Adam McCluskey *[a]
[a]
Dr JR Baker (0000-0002-9560-301X), Mr BL Pollard (0000-0002-5148-9665), Dr AJS Lin (0000-0001-5558-2261) and Prof A McCluskey (0000-0001-7125-
863X)
Chemistry, School of Environmental & Life Sciences
The University of Newcastle
University Drive, Callaghan, NSW 2308, Australia
E-mail: Adam.McCluskey@newcstle.edu.au
[b]
Dr J Gilbert (0000-0001-5034-386X) and Dr JA Sakoff (0000-00020-7009-5792)
Experimental Therapeutics Group, Department of Medical Oncology
Calvary Mater Hospital
Edith Street, Waratah NSW 2298, Australia.
[c]
Prof S Paula (0000-0003-4100-1467),
Department of Chemistry
Purdue University, 560 Oval Drive, West Lafayette Indiana USA.
Dr. X. Zhu (0000-0003-4946-2081),
Research Computing, Information Technology at Purdue (ITaP),
Purdue University, 155 South Grant Street, West Lafayette, IN (USA)
Equal author contribution
[d]
*
Supporting information for this article is given via a link at the end of the document.
Abstract: To exploit the interaction of the aryl hydrocarbon receptor
(AhR) pathway in developing breast cancer specific cytotoxic
compounds, we examined the breast cancer selectivity and the
docking pose of the AhR ligands (Z)-2-(2-aminophenyl)-1H-
benzo[de]isoquinoline-1,3(2H)-dione (NAP-6; 5) and 10-chloro-7H-
benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one (10-Cl-BBQ; 6).
While the breast cancer selectivity of 5 in vitro is known, we discuss
the SAR around this lead, and show for the first time using phenotypic
cell line screening and the MTT assay that 6 also presents with breast
cancer selectivity, notably in the triple negative (TN) receptor breast
cancer cell line MDA-MB-468, the ER+ breast cancer cell lines T47D,
ZR-75-1 and the HER2+ breast cancer cell line SKBR3 (GI50 values of
0.098, 0.97, 0.13 and 0.21 µM, respectively). Indeed, 6 is 55-fold more
potent in MDA-MB-468 cells than the normal MCF10A breast cells
(GI50 of 0.098 vs 5.4 µM) and more than 130-fold more potent than in
cell lines derived from pancreas, brain and prostate (GI50 of 0.098 vs
10-13 µM). Molecular docking poses of 5 and 6 together with
analogue synthesis and phenotypic screening show the importance of
the naphthalene moiety, and an ortho-disposed substituent on the N-
phenyl moiety for biological activity.
Herceptin is used in HER-2 (human epidermal growth factor
receptor) positive tumours [1,2]. Failure to respond to
treatment is reported in 70% of patients with HER2-positive
cancer, and resistance is noted with Tamoxifen and
anastrozole [3].
Tumours lacking the estrogen (ER),
progesterone (PR), and HER2 receptors, i.e. the triple negative
breast cancers (TN), are highly heterogenous and present a
significant therapeutic challenge [4,5].
The arylhydrocarbon receptor (AhR) belongs to the basic-
helix-loop-helix transcription factor family. The AhR forms a
complex with heatshock protein 90, prostaglandin E synthase
3, and a single molecule of the immunophilin-like protein
hepatitis B virus X–associated protein 2. Its movement within
cells is controlled by binding of the AhR nuclear translocator
(ARNT). This binding affords an AhR-ARNT heterodimer
complex, culminating in downstream activation of CYP-1A1, -
1A2, and -1B1 [6,7]. The CYP1s enact critical metabolic steps
for the deactivation and excretion of toxins (and endogenous
substrates) [8].
In humans the AhR has a major role in the modulation of
the effects of environmental toxins such as benzo[a]pyrene (
1)
and 2,3,7,8-tetrachlorodibenzo-p-dioxin ( ) (Figure 1) [6-9].
2
Introduction
The AhR also modulates the effects of endogenous ligands,
e.g. tryptophan and prostaglandins [10,11]. AhR function is
acknowledged as complex and can be activated in both the
presence and absence of endogenous and/or exogenous
ligands affecting gene transcription [10,12]. The AhR can also
induce non transcription factor actions such as Src kinase
activation and direct interaction with other receptor signalling
pathways [13].
Breast cancer is the most commonly diagnosed cancer in
women. Early detection is rewarded with good long term
survival however, metastatic disease is aggressive and
incurable with a 5 year survival of only 25% [4]. Early stage
disease is treated with surgery and radiotherapy while
aggressive disease is treated with chemotherapy, and
hormonal and targeted therapies. Hormone sensitive tumours
can be treated with Tamoxifen and anastrozole while
1
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10.1002/cmdc.202000721
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In recent work from our laboratory we have exploited the
published link between the AhR pathway and breast cancer
initiation and progression in the development of potentially
A2780 (ovarian), H460 (lung), A431 (skin), Du145 (prostate),
BE2-C (neuroblastoma), MIA-PaCa2 (pancreas) and SMA560
(spontaneous murine astrocytoma) cell lines, and in a select
therapeutic small molecules [14-21]. Notable efforts by others panel of breast cancer cell lines MCF-7, BT474, T47D, ZR-75-1,
have identified aminoflavone ( ) which progressed to clinical SKBR3 and MDA-MB-468, BT20 and MDAMB-231 together
trials [22]. Our efforts have generated the halogenated aryl with the non-cancer breast cell line MCF10A (Table 1). As
hydrocarbon (HAH) ANI-7 ( ) and the polyaryl hydrocarbon previously shown for [14] and [21]; presented with
(PAH) (Z)- 2-(2-aminophenyl)-1H-benzo[de]isoquinoline- significant selectivity towards the growth inhibition of breast
1,3(2H)-dione (NAP-6) ( ) as two small molecules displaying cancer cell lines. Indeed, was more potent in the triple
negative cell line MDA-MB-468 than or 5, with a GI50 value
of 0.098 compared with 0.23 and 0.43 µM, respectively.
Notwithstanding this, was also potent in the ER+ breast
3
4
4
5
6
5
6
high levels of specificity towards breast cancer cell lines and
activation of the AhR pathway (Figure 1).
4
6
cancer cell lines T47D, ZR-75-1 and the HER2+ breast cancer
cell line SKBR3 (GI50 values of 0.97, 0.13 and 0.21 µM,
respectively). In fact,
468 cells than the normal MCF10A breast cells (GI50 of 0.098
vs 5.4 M) and more than 130-fold more potent than in cell
lines derived from pancreas, brain and prostate (GI50 of 0.098
vs 10-13 M). This is the first time that the breast cancer
selectivity of has been identified.
The core scaffold of and , with limited synthetic handles
6 was 55-fold more potent in MDA-MB-


6
5
6
to easily introduce a range of substituents, was not readily
amenable to rapid focused library development. Thus, we
explored the in silico docking of
model to identify more accessible compounds [20] (Figure 2).
Inspection of docked within the ligand binding site of the
5 and 6 in our AhR homology
6
AhR homology model revealed a predominately hydrophobic
pocket buried in the interior of the receptor. This is consistent
with the nature of the known AhR ligands (Figure 1) [10,22,24].
The binding site is lined by a number of hydrophobic residues
including Phe21, Phe27, Leu34, Phe50, Met66, Leu79, Ala93,
Ile105 and Val107, creating a non-polar environment capable
of generating hydrophobic interactions. Evaluation of docking
Figure 1. Selected aryl hydrocarbon receptor ligands: benzo[a]pyrene (
2,3,7,8-tetrachlorodibenzo-p-dioxin ( ), aminoflavone ( ), ANI-7 ( ), (Z)-2-(2-
aminophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (NAP-6) and 10-
chloro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one (10-Cl-BBQ) ( ).
1),
2
3
4
(5)
6
results conducted with
6 suggested that the compound
Encouraged by our recent discoveries and our application
of an in-house AhR homology model in the design of these
analogues we turned our attention to 10-Cl-BBQ (6) (Figure 1).
This compound is a known ligand of AhR [23], and in essence
is a hybrid of ANI-7 and NAP-6 with the core scaffold
combining the aryl halogen of the acrylonitrile with the
polyaromatic rings of the naphthalimide. Our aim was to
formed key π-π stacking interactions from one of the
naphthalene rings to His63 and His17 within the hydrophobic
binding pocket, plus a hydrogen bond between a hydrogen on
the aromatic ring and Met66. The hydrogen bond with Met
involves an aromatic ring hydrogen atom as the donor and the
sulfur atom lone pairs of the methionine side chain as the
acceptor. This is not a standard (“canonical”) H-bond and is
explore the SAR and breast cancer selectivity of 5 and 6 for the
development of breast cancer selective agents [21].
…
analogous to C-H O interactions that are sometimes
described as “weak” hydrogen bonds. Whereas these bonds
tend to be generally weaker than standard hydrogen bonds,
their strength increases if polycyclic rings are involved, which
is the case here. Moreover, a very tight fit between ligand and
receptor was observed, which implies van-der-Waals
interactions as an additional contributor to binding.
Results and Discussion
In the first instance we compared the cytotoxicity of
with that of in a broad panel of cancer cell lines including
HT29 (colon), U87 and SJ-G2 (glioblastoma), MCF-7 (breast),
4 and
5
6
Table 1. Growth inhibition, GI50 values (M), against a broad panel of cancer cell lines and a focused panel of breast cancer cell lines by 10-Cl BBQ (6).
Data for ANI-7 (4) and NAP-6 (5) have been included for comparison. (Lower values indicate higher potency).
Broad Cancer Cell Line Panel
HT29
U87
A2780
H460
A431
Du145
BE2-C
SJ-G2
MIA-PaCa2
Tissue of
origin
colon
brain
ovary
lung
skin
prostate
neural
brain
pancreas
ANI-7 a
6.0±0.20
36±3
13±2
3.0±0.4
0.51±0.05
27±1
18±2
13±2
42±3
4
2
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NAP-6 b
5.0±0.55
5
10-Cl-BBQ
4.0±0.72
6
>50
21±4
15±7.5
10±2.0
0.25±0.12
0.82±0.20
>50
>50
>50
>50
7.6±2.0
4.1±0.5
13±0.58
5.8±0.78
13±1.5
10±0.7
Breast Cancer Cell Line Panel
MCF-7
ER+ c
BT474
ER+
T47D
ER+
ZR-75-1
ER+
SKBR3
HER2 d
MDA-MB468
BT20
TN
MDA-MB-231
MCF10A
normal f
Classification
TN e
TN
ANI-7 a
0.38±0.03
1.0±0.3
0.16±0.02
0.18±0.02
0.12±0.03
0.13±0.04
0.21±0.03
0.22±0.02
0.21±0.06
0.23±0.01
1.0±0.4
14±1.5
>10
17±4
26±3
31±1.5
5.4±1.2
4
NAP-6 b
0.70±0.12
5.8±1.1
0.43±0.07
>10
0.18±0.02
0.97±0.3
0.43±0.07
35±3
>10
5
10-Cl-BBQ
6
0.098±0.04
a Gilbert et al 2018; b Gilbert et al 2020; c estrogen receptor (ER) positive; d human epidermal growth factor positive (HER2); e triple negative for ER,
progesterone (PR) and HER2 receptors; f normal breast cell line.
As our, and Punj’s prior reports have clearly identified
ANI-7, NAP-6 and 10-Cl-BBQ all elicit their breast cancer
specificity through the AhR [15, 21, 25], we sought to
examine these compounds and the analogues produced
herein in our previously reported AhR homology model.
attractive van-der-Waals interactions. Additionally, no
substantive interaction with the carbonyl moiety was
evident. Combined, these data suggested that significant
scaffold simplification with retention of activity may be
feasible. Our scaffold simplification approach suggested
three key components that in principle could give rise to
Libraries A, B and C which could be rapidly investigated
(Figure 3).
Analysis of the docking of
6 suggested that the
benzimidazole moiety afforded no meaningful interactions
within the AhR binding site, with the possible exception of
A
B
Figure 2
.
A) Docked pose of 10-Cl-BBQ (
6
) bound in the AhR PAS-B domain homology model, illustrating the central and buried nature of the ligand binding site.
Residues are shown as wire frame and coloured by atom type.
B
) A 2D generated ligand interaction diagram of the AhR PAS-B domain, shown with 10-Cl-BBQ (6)
docked. Hydrophobic residues are shown in green; hydrophilic residues are shown in purple; the green dashed line depicts π-π and H-bond interactions.
3
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Scheme 1. Reagents and conditions: i) 2-, 3-, 4- chloroaniline (8-10), AcOH,
24h, .
From analysis of the data presented in Table 2, the scaffold
simplification (removal of one of the naphthyl phenyl rings) of
afforded no analogue with notable cytotoxicity against any
6
of the cancer cell lines evaluated. As a result we next turned
our attention to the synthesis of Library B analogues, which
most closely resemble leads 5 and 6. In this series, as with the
Figure
simplification of the core structure of NAP-6 (
in blue are those that are shared with the parent structures.
3
.
Potential focused Libraries A,
B
and
C
arising from the
) The structures
Library A analogues, the chlorophenyl moiety was retained.
Compound access was afforded by either an ethanol reflux
with catalytic trimethylamine, or heating in the room
temperature ionic liquid [BMIM][Br], from 1,8-naphthalic
5
) and 10-Cl-BBQ (
6
We have previously reported the synthesis of a wide
variety of anhydride modified analogues as clathrin, dynamin
and protein phosphatase inhibitors [26-29], the chemistry of
which permitted rapid access to focused libraries that would
enable SAR acquisition. Of the three analogue types
identified, Library A addresses the importance of the second
naphthyl aromatic moiety; Library B the importance of the N-
imide substituent, in engaging with the AhR binding pocket;
and Library C the nature of the N-imide carbonyl interactions.
Commencing with Library A, a 3-membered focused library
anhydride (14) and chloroanilines (8-10) (Scheme 2) [27]. The
2-chloro moiety was synthetically inaccessible presumably
due to a combination of electronic effects and steric clashes
with the naphthalic anhydride functionality; the more planar
structure of the naphthalimide backbone (for 15), compared
to the more flexible homophthalic moiety (for 11), resulted in
steric hindrance that significantly affected the reaction rate;
however, the 3- and 4-chloro derivatives were successfully
afforded. The initial Library B analogues comprised naphthalic
N-imides (16-17), with the screening data presented in Table
3.
retaining the chlorophenyl moiety of
treatment of homophthalic anhydride (
chloroaniline (8-10) to afford the corresponding imides 11-13
6
7
was synthesised on
) with 2-, 3-, and 4-
in good yields (see Experimental). These analogues were
screened in a broad cancer screening panel comprising HT29
(colon), U87 and SJ-G2 (glioblastoma), MCF-7 (breast), A2780
(ovarian), H460 (lung), A431 (skin), Du145 (prostate), BE2-C
(neuroblastoma), MIA-PaCa2 (pancreas) and SMA560
(spontaneous murine astrocytoma) cell lines, as well as the
normal breast cell line MCF10A. Initial screening was
conducted at 25 µM and these data are presented in Table 2.
Scheme 2. Reagents and conditions: i) 2-, 3-, 4- chloroaniline (
EtOH, 18 h, Δ; or 2-, 3-, 4- chloroaniline ( 10), [BMIM][Br], 1 h, Δ.
8-10), Et3N,
8-
Table 2. Percent cell growth inhibition in response to 25 µM of drug, against a panel of cancer cell lines by homophthalic imides 11-13. (Higher values
indicate higher potency).
MIA-
PaCa2
R
HT29
U87
MCF-7
A2780
H460
A431
Du145
BE2-C
SJ-G2
SMA560
MCF10A
10±2
<10
20±12
17±6
10±2
14±1
<10
<10
16±3
<10
nd
<10
11
12
21±2
<10
38±1
21±6
15±3
<10
<10
23±2
17±7
14±3
nd
12±6
4
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15±6
<10
14±6
27±8
<10
<10
<10
10±3
13±1
11±4
nd
<10
13
nd = not determined
Table 3. Growth inhibition, GI50 values (M), against a panel of cancer cell lines by naphthalene imides 16-30. Low values indicate higher potency. Data in italics is
percentage growth inhibition at a fixed 25 M compound concentration (higher values indicate higher potency).
R
HT29
U87
MCF-7
A2780
H460
A431
Du145
BE2-C
SJ-G2
MIA-
SMA560 MCF10A
PaCa2
4.5±0.10 19±4.2 3.3±0.71 7.5±0.60
15±1.8
5.4±0.61 15±2.3 7.8±1.4 9.2±0.67 6.9±0.18
nd
nd
6.4±0.59
16
27±7.4 30±2.0
21±9.5
<10
23±7.4
13±1
<10
32±0.0
<10
29±1.0
10±1
<10
37±4.5 24±0.0 30±0.5
31±1.0
<10
21±5.8
<10
nd
17
18
<10
<10
<10
nd
nd
nd
nd
-
<10
<10
<10
51±3
<10
-
<10
<10
18±5
63±4
21±6
-
<10
14±1
12±3
39±3
11±3
-
nd
24±5
49±10
71±6
21±3
-
10±4
34±7
42±3
14±4
-
<10
<10
<10
20±2
10±3
-
19
20
16±7
36±3
<10
11±3
38±4
<10
15±3
23±4
<10
10±3
55±0
12±4
-
nd
nd
21
22
nd
b
-
-
-
-
23
16±7
13±5
nd
nd
16±4
21±3
<10
<10
11±3
10±2
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
nd
nd
24
25
12±6
11±2.3 8.8±4.1 5.0±0.17 8.5±2.8 5.8±0.33 3.3±0.32 3.6±0.70 5.4±1.1 5.8±0.17 6.2±0.67
nd
nd
4.9±0.50
4.8±0.30
nd
26
27
11±2.4 26±2.8
6.1±2.2
5.3±1.3
11±2.6
4.3±0.96 21±8.0 4.4±1.0 12±6.8
4.9±1.8
43±3
nd
70±3
36±5
59±2
38±1
50±5
64±4
63±1
38±1
88±4
28
29
<10
nd
nd
<10
<10
<10
14±5
25±4
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
nd
nd
14±7
12±2
11±8
19±3
30
a
from Gilbert et al, 2020
b insoluble in the testing media, nd = not determined.
5
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As with the homophthalic analogues 11-13, the
chlorophenyl 16
the initial 25 M compound concentration examined (Table 3).
In comparison to , modelling analysis, after docking in our in-
-17 displayed only low levels of cytotoxicity at

6
house AhR homology model [19], revealed that these
compounds only interacted with His63 and Met66 and failed
to engage with His17 (Supplementary Information, Figure S1),
which could explain their lower activities.
Chlorine moiety removal gave 18 which showed very low
levels of cytotoxicity (Table 3), however these data and the
insights gained on analysis of the docked poses of 15-18
suggested that the introduction of H-bonding moieties
capable of interaction with Gln109, Thr15 and Gly47 may
increase AhR binding site affinity, and based on our
hypothesis, activity against the breast cancer cell line MCF-7
(Figure 2 and Figure 4). Accordingly we synthesised, as
described in Scheme 2, three series of analogues with -OH (19
-
B
21), -NH2 (22 23) and -CO2H (24 and 25) substituents on the N-
-
Figure 4. A) Docked pose of
5
in the AhR binding pocket with three types
of favourable binding interactions evident: π-π interactions between the two
rings of the aromatic system of the ligand and His63, a H-bond between a
hydrogen atom attached to the aromatic system and Met66, and another H-
bond between the 2-NH2 moiety and Gln109. Residues are shown as wire frame
imide phenyl moiety. Of these eight analogues, 4-NH2 23 was
insoluble in the assay medium. Of the remaining seven
analogues, breast cancer selectivity in MCF-7 cells was
observed. Indeed, 2-OH 19, 3-OH 20 and 4-OH 21 showed the
greatest potency in MCF-7 cells with a percentage cell death
level of 24%, 49% and 71%, respectively. Interestingly, the
amino analogue 3-NH2 22 showed none of the potency or
selectivity of the parental 2-NH2 5. This is consistent with the
predicted AhR mediated cytotoxicity against breast cancer.
We do note, however, the inherent risk in drawing hard and
fast SAR conclusions from analysis of single datapoint, with
these data only circumstantially support our modelling
hypothesis. We do note that with the active analogues their
cancer cell line response phenocopies that observed with ANI-
7, NAP-6 and 10-Cl-BBQ. Their mode of action is thus
consistent with activation of the AhR pathway.
and coloured by atom type.
generated AhR PAS-B domain, shown with
shown in green; hydrophilic residues are shown in purple; the green dashed line
depicts π-π interactions.
B
) A 2D generated ligand interaction diagram of the
5
docked. Hydrophobic residues are

Docking revealed that the compounds adopted a very
similar overall (“consensus”) orientation (Supporting
Information, Figure S2). Subtle differences in a compound’s
position determined whether certain interactions were
present or not. With 5, the 2-NH2 moiety results in the N-
phenyl ring twisting from planarity and this allows access to a
strong H-bond with Gln109; this coupled with  interactions
with the two naphthalene rings to His63 and a H-bond from
the aromatic-H to Met66 affords a strong binding site
interaction, evidenced by the increased MCF-7 cytotoxicity
and the lack of effect with the 3-NH2 moiety. Due to the
symmetry of the molecule, we occasionally observed the H-
bond with Gln109 to be substituted by one with Gly47, with all
the other interactions remaining the same. The cytotoxicity
data obtained with the carboxylate substituted 24 and 25, is
consistent with their AhR binding poses with the carboxylate
moiety failing to engage with Gln109 (Supporting
Information). From these data, it appears critical that the N-
phenyl moiety adopts a skewed conformation to allow access
to Gln109, and as such the corresponding aminobenzyl amines
(26-28) would be expected to result in a decrease in MCF-7
cytotoxicity, although the increased flexibility imparted by the
methylene spacer may off-set this somewhat permitting the
aminophenyl moiety to rotate and potentially interact with
Gln109. As shown from the cytotoxicity data, the aminobenzyl
analogues (26-27) displayed increased potency with no breast
A
cancer selectivity, while 28 displayed reduced potency but
maintained some breast selectivity, possibly due to its ability
to interact with Gly47, which is consistent with the above
modelling hypothesis.
6
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In a similar manner removal of the phenyl ring and
Interestingly, the final orientations (average of the last 20
introduction of ethanol (29) and propanol (30) moieties also ns of simulation) of both compounds deviated from their
resulted in a loss of activity (Table 3). This strongly supports docking-predicted poses (Figure 5A and B). The deviation was
this class of compounds favouring the retention of the
naphthalene moiety, and an ortho-disposed substituent on rotation by approximately 90° around an axis perpendicular to
the N-phenyl moiety. Further substantiation of the critical the plane of the central ring of the molecule. A comparison of
much larger for 19 than for 5 and could be described as a
nature of the 2-NH2 moiety was evident on examination of the snapshots from the final stages of the MD simulations that
corresponding 2-CH3 31, with the in silico analysis showing the included the roles assumed by water molecules in the binding
loss of the Gln109 H-bond, and a corresponding loss in pocket revealed two distinct patterns (Figure 5C and D). 2-
cytotoxicity activity. In a similar vein, a 2-pyridyl 32 supports Amino
5 preserved the H-bond to Gln109 and maintained
the 2-NH2 moiety participating in H-bond donation. Pyridyl 32 close contacts with Met66 as well as co-planarity with the
was inactive in our cancer screening panel. The exception to imidazole ring of His63 while also permitting the presence of
these observations was with the 2-hydroxy analogue 19
According to our docking results, and 19 exhibited the other hand, was surrounded by three water molecules, one of
same binding pose and engaged the AhR with the same set of which formed a bridged H-bond between the hydroxyl group
interactions. However, showed good potencies against the of 19 and Thr15 and His17. This prevents 19 from making a
cells lines tested whereas 19 was mostly inactive. This was critical H-bond to Gln109. Moreover, the π-π interactions with
.
two water molecules in the binding site. 2-Hydroxy 19, on the
5
5
contrary to our docking model predictions. In our previous
reports with the phenylacrylonitrile class of compounds with
His63 were absent because of a significant reorientation of the
ligand which eliminated the co-planarity of the two entities.
copious data in IC50 format (not a mix of IC50 and %-inhibition) This latter perturbation is a direct consequence of the
we noted excellent model to activity correlations [20]. To additional water molecule present in the AhR binding pocket
account for this apparent discrepancy, we subjected the
docked poses of both compounds to 100 ns of molecular
with 19
.
Presumably, the somewhat smaller size of the
hydroxyl group compared to that of the amino group along
dynamics (MD) simulations with the introduction of explicit with slight differences in H-bonding capabilities of these two
water molecules. Despite being much more demanding on functional groups allowed 19 to adopt a pose significantly
computational resources, MD – unlike docking – does not different from that of 5. This finding was not possible without
suffer from limitations due to the neglect of solvent molecules
or rigidity of the receptor and can therefore provide a more
detailed and realistic picture of the events taking place in the
binding site.
examination of the molecular dynamics and inclusion of water
molecules within this system.
A
B
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C
D
Figure 5. A) Final pose of
5
(yellow, last 20 ns of a 100 ns simulation),
B
)
19 (orange) compared to docking predicted pose (cyan); C) Binding pose of 5; and D) 19
with H-bonds and explicit solvent molecules.
As ligand binding is usually accompanied by
conformational changes of the receptor, the different binding
Our final synthetic modification accessed a single Type C
analogue based on 2-amino . Treatment of with I2 and
5
5
pose observed for 19 most likely resulted in a different overall NaBH4 afforded the carbonyl free 36 (Scheme 3). This
conformation of the AhR. This in turn could have had an effect analogue was inactive, supporting an important role for the
on the interactions between the AhR its protein binding imide carbonyls in engaging, directly or indirectly, with the
partners and thereby affect the AhR signaling pathway. This AhR binding site.
assumption was supported by a comparison of RMSD values
of the protein backbone, which showed noticeable differences
Examination of the AhR docking poses of 32
reveals that with each analogue, key binding interactions are
, 33 and 36
between the two ligand/receptor complexes throughout the lacking within the AhR binding site (Supporting Information,
duration of the simulations (Supporting Information, Figure
S3). Such a scenario would be compatible with only
Figure S4). With 2-pyridyl 32 the predicted binding pose
results in loss of part of the  interactions with H63 and of
5
activating the pathway and 19 being unable to do so, despite H-bond with Gln109; 33 sees the loss of part of the 
its ability to bind.
interactions and the H-bonds with Met66 and Gln109; and 36
, we revisited our initial the loss of H-bonds with Met66 and Gln109. In each instance,
the loss of the aforementioned binding interactions reduces
35, as the observed cytotoxicity (Table and Supporting
outlined in Scheme 1. The 3- and 4-amino analogues were Information, Table S1).
unable to be synthesised, with the ring-opened derivative Having defined the SAR for molecule
afforded in each instance. The 2-amino analogue (34 inverted light microscopy the significant differential effect of
Supporting Information, Figure S4) was found to be inactive in on the induction of cell death in MDA-MB-468 breast cancer
,
Given our findings with
homophthalic imide Type A analogues, attempting the
synthesis of the equivalent 2-, 3- and 4- amino 34
5
-
2
5
, we now show using
;
5
our cancer screening panel (Supporting Information, Table S1), cells compared with normal breast cells within 16h of
and on examination of the binding poses, while 2-amino 34 continuous exposure (Figure 6).
showed a similar twisting of the N-phenyl moiety and
participation in a H-bond with Gln109, the loss of the
aromatic-H H-bond with Met66 and only one  interaction
with His63 appears to have rendered these analogues poor
binders in the AhR binding domain.
Scheme 3. Reagents and Conditions: i) I2, NaBH4, THF, 25-60 ºC, 24 h.
A
B
8
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Experimental Section
Biology
Cell culture and stock solutions
Stock solutions were prepared as follows and stored at -20 °C: drugs
were stored as 40 mM solutions in DMSO. All cell lines were cultured in
a humidified atmosphere 5% CO2 at 37 ºC. The cancer cell lines were
maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Trace
Biosciences, Australia) supplemented with 10% foetal bovine serum, 10
mM sodium bicarbonate, penicillin (100 IU/mL), streptomycin (100
µg/mL), and glutamine (4 mM). The non-cancer MCF10A cell line was
cultured in DMEM:F12 (1:1) cell culture media, 5% heat inactivated
horse serum, supplemented with penicillin (50 IU/mL), streptomycin
(50 g/mL), 20 mM Hepes, L-glutamine (2 mM), epidermal growth
factor (20 ng/mL), hydrocortisone (500 ng/mL), cholera toxin (100
ng/mL), and insulin (10 g/mL).
C
D
Figure 6. Light microscopy images of MDA-MB-468 (A,B) and MCF10A (C,D
)
cells treated with (B,D) or without (A,C) 5 (50 µM) for 16 h.
Conclusions
In vitro growth inhibition assay
Cells in logarithmic growth were transferred to 96-well plates.
Cytotoxicity was determined by plating cells in duplicate in 100 L
medium at a density of 2500-4000 cells/well in 96 well plates. On day
0, (24 h after plating) when the cells were in logarithmic growth 100 L
medium with or without the test agent was added to each well. After
72 h drug exposure growth inhibitory effects were evaluated using the
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
assay with absorbances read at 540 nm. Percentage growth inhibition
was determined at a fixed drug concentration of 25 M. A value of
100% is indicative of complete cell growth inhibition. Those analogues
showing appreciable percentage growth inhibition underwent further
dose response analysis allowing for the calculation of a GI50 value. An
eight-point dose response curve was produced, using MS Excel
software. Each data point is the mean ±S.E.M. calculated from four to
five replicates, which were performed on separate occasions and
separate cell line passages. From these dose-response curves, the GI50
value was calculated, representing the drug concentration at which cell
growth is 50% inhibited based on the difference between the optical
density values on day 0 and those at the end of drug exposure [31, 32].
Both compounds
as AhR ligands [21, 23], and
molecule. Examination of in a broad panel of cancer cell lines
and in an expanded panel of breast cancer cell lines revealed,
for the first time, comparable breast cancer selectivity to that
5
and
6
have previously been identified
5
as a breast cancer selective
6
of 5. Relative to the broad panel of cancer cell lines, 6 was up
to 130-fold more potent in breast cancer cells, and up to 55-
fold more potent in breast cancer cells compared with normal
breast cells. The greatest potency was 100nM in MDA-MB-468
breast cancer cells. In the drug resistant BT20 and MDA-MB-
231 cell lines, the observed activity was more modest at >10
µM. Scaffold simplification approaches with 5 and 6 identified
Type A, B and C analogues as potential scaffolds for interaction
with the AhR binding site. Subsequent synthesis and screening
against a cancer cell line panel revealed that Type A analogues
were, to all intentions, devoid of noteworthy cytotoxicity.
Examination of Type
B analogues revealed potential
Morphological Assessment: Live cells were examined for
morphological alterations after 16 h exposure with and without 50µM
interactions within the AhR binding site through π-π
interactions with His63, and aromatic hydrogen bond with
5
using phase contrast microscopy (Olympus CKX41 inverted
microscope 100x magnification).
Met66 and a H-bond from the -NH2 moiety of
5 to Gln109.
Removal of one of these interactions was detrimental to
activity with the synthesis of homophthalic anhydride derived
33, 2-pyridyl 32 and the carbonyl free 36 lacking one or more
Docking of compounds into the homology model of the AhR PAS-
B domain
Development of a homology model for the AhR PAS-B domain
of these binding site interactions effected
a loss of
cytotoxicity. In each instance the loss of activity was
consistent with a subtle change in binding site pose of the
respective analogues. Further analysis of 2-hydroxy 19 via
molecular dynamic simulations demonstrated that whilst the
Homology modelling was performed with the suite MOE (Molecular
Open Environment, Chemical Computing Group, Montreal, Canada) as
described previously. Briefly, the sequence of the human AhR was
obtained from the NCBI website (https://www.ncbi.nlm.nih.gov/,
accession code NP_001612.1) and only the part corresponding to the
PAS-B domain was considered. A homology search with MOE identified
the crystal structure 4F3L (a transcriptional activator complex with a
basic helix-loop-helix PAS-domain) as the most suitable template
(24.8% identity and 48.8% similarity) [33]. Model generation was
executed at default settings with the Amber force field. Subsequent
analysis of the model’s geometry revealed no steric clashes or outliers
in backbone bond lengths as well as bond, dihedral, and rotamer angles.
same interactions were observed with the receptor as with 5,
after 100 ns of solvent-mediated MD, the pose of 19 deviated
significantly from the docking-predicted pose, resulting in the
loss of the critical interactions previously observed. This effect
was only observable via MD simulations with our docking
efforts failing to provide an explanation for the observed
activity discrepancy.
Collectively these data support the further development of
Docking of compounds into the homology model
5
and
6 as potential breast cancer specific agents targeting the
The structures of all ligands to be docked were constructed in MOE and
their conformations energy-minimized using Molecular Mechanics in
conjunction with the MMFF94x force field. The binding site for docking
AhR pathway.
9
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10.1002/cmdc.202000721
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was defined by the position of co-crystallized ligands in related proteins
with PAS-B domains, such as 3F1O [34], 3H7W [35], and 3H82 [36], after
being superimposed onto the homology model.
spectra and carbon NMR (13C NMR) spectra were acquired at 400 and
100 MHz respectively. All spectra were recorded in deuterated
dimethyl sulfoxide (DMSO-d6) or deuterated chloroform (CDCl3)
obtained from Cambridge Isotope Laboratories Inc. Chemical shifts ()
were measured in parts per million (ppm) and referenced against the
internal reference peaks. Coupling constants (J) were measured in
Hertz (Hz). Multiplicities are denoted as singlet (s), broad singlet (bs),
doublet (d), doublet of doublets (dd), triplet (t), quartet (q), triplet of
doublets (td), doublet of triplets (dt) and multiplet (m). Peaks are listed
in decreasing chemical shift in the following format: chemical shift
(integration (1H), multiplicity (1H), coupling constant (1H).
Docking was performed using MOE’s default settings, using the triangle
matcher method in combination with the London dG scoring function
for the initial placement of the ligand, followed by a refinement of the
30 top poses with rigid receptor setting and the GBVI/WSA scoring
function. Analysis and visualization of the docking output, such as
identification of hydrogen bonds, steric clashes, hydrophobic
interactions, or π-πinteractions were performed in MOE.
The Biotage® initiator+ was used to perform microwave reactions.
Molecular dynamics simulations
The stability of docking-predicted ligand poses was evaluated in
molecular dynamics (MD) simulations using the program NAMD
(version 2.10) [37]. The simulations were conducted using periodic
boundary conditions in all directions. The cut off distance for both
Coulombic and van der Waals (vdW) interactions was set at 12 Å, and
the force-based switching function was used for the vdW interactions
with a switching range of 10–12 Å. Long-range Coulombic interactions
were evaluated by the Particle Mesh Ewald method [38]. The
temperature was maintained at 303.15 K, controlled by Langevin
dynamics, while the pressure was maintained at 1 atm using the Nosé–
Hoover Langevin piston method [39]. The bonds with hydrogen atoms
were constrained to their equilibrium lengths using the RATTLE
algorithm [40]. Simulations were performed with an energy
minimization of 10000 steps, six equilibrium stages, totalling 0.75 ns,
followed by a production phase of 100 ns with a time step of 2 fs. During
the equilibration, all heavy atoms of the protein–ligand complex were
harmonically restrained to their initial positions, with an initial force
constant of 25 kcalmol-1 Å-2 that was gradually decreased at each stage.
Simulations were visualized with the program VMD [41].
2-(2-Chlorophenyl)isoquinoline-1,3(2H,4H)-dione (11)
To a mixture of homophthalic anhydride (7) (1.0 mmol, 0.162 g) and
2-chloroaniline (1.5 mmol, 0.191 mL) was added acetic acid (10 mL).
This solution was then heated, with magnetic stirring, to 125 ºC for 24
h. The ensuing mixture was cooled to room temperature, then treated
with water (30 mL). The resultant precipitate was collected by filtration
then dried in vacuo to afford the title compound as an off-white solid
(0.031 g, 11%), m.p.: 120-122 ºC [47].
1H NMR (400 MHz, CDCl3)  8.26 (dd, J = 7.9, 1.0 Hz, 1H), 7.66 (td, J
= 7.6, 1.4 Hz, 1H), 7.59 – 7.55 (m, 1H), 7.51 – 7.48 (m, 1H), 7.44 – 7.39
(m, 2H), 7.37 (d, J = 7.7 Hz, 1H), 7.30 – 7.26 (m, 1H), 4.25 (ABq, ΔνAB
32 Hz, JAB = 22.4 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 169.3, 164.4,
134.4, 134.3, 133.3, 132.8, 130.6, 130.42, 130.39, 129.7, 128.1, 127.9,
127.6, 125.3, 37.0; IR (cm-1) 1674 (C=O), 743 (C-Cl); LCMS (ESI+) m/z:
272 [M+H]+ (100%), 274 [M+H]+ (35%).
=
2-(3-Chlorophenyl)isoquinoline-1,3(2H,4H)-dione (12)
To a mixture of homophthalic anhydride (7) (1.0 mmol, 0.162 g) and
3-chloroaniline (1.5 mmol, 0.191 mL) was added acetic acid (10 mL).
This solution was then heated, with magnetic stirring, to 125 ºC for 24
h. The ensuing mixture was cooled to room temperature, then treated
with water (30 mL). The resultant precipitate was collected by filtration
then dried in vacuo to afford the title compound as an off-white solid
(0.107 g, 39%), m.p.: 150-153 ºC [47].
1H NMR (400 MHz, CDCl3)  8.24 (d, J = 7.8 Hz, 1H), 7.66 (t, J = 7.8
Hz, 1H), 7.49 (t, J = 7.8 Hz, 1H), 7.45 – 7.43 (m, 2H), 7.36 (d, J = 7.6 Hz,
1H), 7.24 (s, 1H), 7.12 – 7.11 (m, 1H), 4.23 (s, 2H); 13C NMR (101 MHz,
CDCl3) δ 169.8, 164.9, 136.2, 135.0, 134.4, 134.2, 130.4, 129.7, 129.23,
129.22, 128.2, 127.5, 127.1, 125.3, 37.1; IR (cm-1) 1677 (C=O), 742 (C-
Cl); LCMS (ESI+) m/z: 272 [M+H]+ (100%), 274 [M+H]+ (35%).
Starting from a docked ligand–receptor pose, the system was solvated,
and potassium and chloride ions were added at 150 mM to mimic
physiological conditions while preserving charge neutrality. The system
was then solvated in a cubic box of water. The dimensions of the box
were chosen such that the minimum distance between any atom of the
solute and the box walls was 12 Å. All simulations used the CHARMM36
force field for the protein [42] and the ions, and the CHARMM-modified
TIP3P model for water molecules [43–47].
The ligand parameters were generated from the CHARMM general
force field.
Chemistry
General Methods
2-(4-Chlorophenyl)isoquinoline-1,3(2H,4H)-dione (13)
All reactions were performed using standard laboratory equipment and
glassware. Solvents and reagents were purchased from Sigma Aldrich,
Alfa Aesar or AK Scientific and used as received. Organic solvents were
of bulk quality, and were distilled from glass prior to use. Organic
solvent extracts were dried with magnesium sulfate (MgSO4), and
removed under reduced pressure with either Büchi or Heidolph rotary
evaporators. Melting points were recorded in open capillaries on a
Büchi 565 Melting Point Apparatus. Where available, literature values
are provided and appropriately referenced. Electrospray mass spectra
were recorded using HPLC-grade 10% acetonitrile/H2O (with 0.1%
formic acid) as carrier solvents on an Agilent Technologies 1260 Infinity
UPLC system with a 6120 Quadrupole LC/MS in electrospray ionization
(ESI) positive and negative modes. TLC was performed on Merck silica
gel 60 F254 pre-coated aluminium plates with a thickness of 0.2 mm.
Column chromatography was performed under ‘flash’ conditions on
Merck silica gel 60 (230-400 mesh).
To a mixture of homophthalic anhydride (7) (1.0 mmol, 0.162 g) and
4-chloroaniline (1.5 mmol, 0.191 mL) was added acetic acid (10 mL).
This solution was then heated, with magnetic stirring, to 125 ºC for 24
h. The ensuing mixture was cooled to room temperature, then treated
with water (30 mL). The resultant precipitate was collected by filtration
then dried in vacuo to afford the desired compound as an off-white
solid (0.123 g, 45%), m.p.: 158-160 ºC [48].
1H NMR (400 MHz, CDCl3)  8.24 (d, J = 7.8 Hz, 1H), 7.66 (td, J = 7.8,
1.3 Hz, 1H), 7.49 (2 overlapping doublets, d, J = 8.8 Hz, 2H; other
doublet J not determined, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.15 (d, J = 8.8
Hz, 2H), 4.23 (s, 2H); 13C NMR (101 MHz, CDCl3) δ 169.9, 165.0, 134.9,
134.3, 134.2, 133.6, 130.1 (2C), 129.8 (2C), 129.7, 128.2, 127.5, 125.4,
37.1; IR (cm-1) 1666 (C=O), 743 (C-Cl); LCMS (ESI+) m/z: 272 [M+H]+
(100%), 274 [M+H]+ (35%).
Nuclear magnetic resonance (NMR) spectroscopy was performed on a
Bruker Avance III 400 MHz spectrometer, where proton NMR (1H NMR)
2-(3-Chlorophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (16)
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A solution of 1,8-naphthalic anhydride (14) (1.0 mmol, 0.198 g), 3-
chloroaniline (3.0 mmol, 0.315 mL) and triethylamine (10 drops) in 15
mL absolute ethanol was heated to reflux for 44 h, with magnetic
stirring, then allowed to cool to room temperature. The mixture was
filtered in vacuo, the yellow precipitate washed with cold ethanol (15
mL) and cold diethyl ether (15 mL), and dried in vacuo to afford the
desired compound as an off-white solid (0.132 g, 43%), m.p.: 232-234
ºC.
1H NMR (400 MHz, CDCl3)  8.65 (d, J = 7.0 Hz, 2H), 8.29 (d, J = 8.1
Hz, 2H), 7.81 (t, J = 7.6 Hz, 2H), 7.48 (m, 7.49 – 7.48, 2H), 7.35 (s, 1H),
7.24 (m, 7.24 – 7.23, 1H); 13C NMR (101 MHz, CDCl3) δ 164.3 (2C), 136.6,
135.1, 134.7 (2C), 131.9 (2C), 130.4, 129.4, 129.2, 128.7, 127.26, 127.25
(2C), 122.7 (3C); IR (cm-1) 1667 (C=O), 774 (C-Cl); LCMS (ESI+) m/z: 308
[M+H]+ (100%), 310 [M+H]+ (35%); HRMS (ESI+) m/z calculated for
C18H11ClNO2 (M+H)+ 308.0473; compound did not ionise.
122.7 (2C), 119.6, 116.2, 115.2; IR (cm-1) 3243 (O-H), 1645 (C=O), 777
(Ar-H); LRMS (ESI+) m/z: 290 [M+H]+, (100%); (ESI-) m/z: 288 [M-H]-
(100%).
2-(3-Hydroxyphenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (20)
A solution of 1,8-naphthalic anhydride (14) (1.0 mmol, 0.198 g), 3-
aminophenol (3.0 mmol, 0.327 g) and triethylamine (8 drops) in 15 mL
absolute ethanol was heated to reflux for 18 h, with magnetic stirring,
then allowed to cool to 0 ºC. The mixture was filtered in vacuo, the pale
brown precipitate washed with cold ethanol (3 mL) and cold diethyl
ether (3 mL), then dried in vacuo to afford the desired compound as a
creamy-coloured solid (0.166 g, 57%), m.p.: 296-298 ºC.
1H NMR (400 MHz, DMSO-d6)  9.64 (s, OH), 8.51 (d, J = 1.8 Hz, 2H),
8.49 (s, 2H), 7.90 (t, J = 7.8 Hz, 2H), 7.30 (t, J = 8.1 Hz, 1H), 6.87 – 6.84
(m, 1H), 6.79 - 6.78 (m, 2H); 13C NMR (101 MHz, DMSO-d6) δ 163.3 (2C),
153.4, 134.4 (2C), 131.5, 130.6 (2C), 130.3, 129.6, 127.9, 127.2 (2C),
122.9, 122.7 (2C), 119.1, 116.4; IR (cm-1) 3290 (O-H), 1650 (C=O), 760
(Ar-H); LRMS (ESI+) m/z: 290 [M+H]+, (100%); (ESI-) m/z: 288 [M-H]-
(100%); HRMS (ESI-) m/z calculated for C18H10NO3 (M-H)- 288.0666;
found: 287.9986.
2-(4-Chlorophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (17)
A solution of 1,8-naphthalic anhydride (14) (1.0 mmol, 0.198 g), 4-
chloroaniline (3.0 mmol, 0.315 mL) and triethylamine (10 drops) in 15
mL absolute ethanol was heated to reflux for 44 h, with magnetic
stirring, then allowed to cool to room temperature. The mixture was
filtered in vacuo, the yellow precipitate washed with cold ethanol (15
mL) and cold diethyl ether (15 mL), and dried in vacuo to afford the
desired compound as an off-white solid (0.139 g, 45%), m.p.: 282-284
ºC [49].
1H NMR (400 MHz, CDCl3)  8.64 (d, J = 7.7 Hz, 2H), 8.28 (d, J = 7.7
Hz, 2H), 7.80 (t, J = 7.7 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.0
Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 164.4 (2C), 134.8, 134.6, 134.0,
131.90 (2C), 131.87 (2C), 130.2 (2C), 129.8 (2C), 128.7, 127.2 (2C), 122.8
(2C); IR (cm-1) 1701 (C=O), 776 (C-Cl); LCMS (ESI+) m/z: 308 [M+H]+
(100%), 310 [M+H]+ (35%).
2-(4-Hydroxyphenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (21)
A solution of 1,8-naphthalic anhydride (14) (1.0 mmol, 0.198 g), 4-
aminophenol (3.0 mmol, 0.327 g) and triethylamine (8 drops) in 15 mL
absolute ethanol was heated to reflux for 18 h, with magnetic stirring,
then allowed to cool to 0 ºC. The mixture was filtered in vacuo, the dark
purple precipitate washed with cold ethanol (3 mL) and cold diethyl
ether (3 mL), and dried in vacuo to afford the desired compound as a
purple-grey solid (0.231 g, 80%), m.p.: 291-293 ºC [52].
1H NMR (400 MHz, DMSO-d6)  9.64 (s, 1H, OH), 8.49 (s, 2H), 8.48
(s, 2H), 7.89 (t, J = 7.8 Hz, 2H), 7.14 (dt, J = 8.8, 2.8 Hz, 2H), 6.87 (dt, J =
8.8, 2.8 Hz, 2H); 13C NMR (101 MHz, DMSO-d6) δ 163.9 (2C), 157.1,
134.3 (2C), 131.4, 130.7 (2C), 129.1 (2C), 127.8, 127.2 (2C), 126.9, 122.7
(2C), 115.4 (2C); IR (cm-1) 3280 (O-H), 1648 (C=O), 781 (Ar-H); LRMS
(ESI+) m/z: 290 [M+H]+, (100%); (ESI-) m/z: 288 [M-H]- (100%).
2-Phenyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (18)
To a mixture of 1,8-naphthalic anhydride (14) (0.56 mmol, 0.111 g)
and aniline (0.61 mmol, 0.057 g) was added 1-butyl-3-
methylimidazolium bromide (0.8 mL). This solution was then heated,
with magnetic stirring, to 140 ºC for 18 h. The ensuing mixture was
cooled to room temperature, then treated with absolute ethanol (3 mL)
before chilling to 0 ºC. The resultant precipitate was collected by
filtration in vacuo, washed with cold ethanol (4 × 7.5 mL) and cold
diethyl ether (3 × 7.5 mL), then dried in vacuo to afford the desired
compound as a tan crystalline solid (0.153 g, 44%), m.p.: 179 ºC [50].
1H NMR (400 MHz, DMSO-d6)  8.53 (d, J = 3.6 Hz, 2H), 8.51 (d, J =
2.6 Hz, 2H), 7.91 (t, J = 7.8 Hz, 2H), 7.53 (t, J = 7.3 Hz, 2H), 7.46 (t, J = 7.3
Hz, 1H), 7.40 (t, J = 4.2 Hz, 2H); 13C NMR (101 MHz, DMSO-d6)  163.7
(2C), 136.1 (2C), 134.4 (2C), 131.5, 130.7, 129.1, 128.9, 128.2 (2C),
127.9 (3C), 127.3 (2C), 122.6; IR (cm-1) 3050 (Ar-H), 1660 (C=O), 700
(Ar-H); LCMS (ESI+) m/z: 274 [M+H]+ (100%).
2-(3-Aminophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (22)
A solution of 1,8-naphthalic anhydride (14) (1.34 mmol, 0.266 g),
m-phenylenediamine (4.38 mmol, 0.474 g) and triethylamine (10
drops) in absolute ethanol (20 mL) was heated to reflux, with magnetic
stirring, for 36 h. Upon cooling to room temperature, the mixture was
filtered in vacuo, and the resultant yellow precipitate was washed with
ethanol (2 × 15 mL) cold diethyl ether (2 × 15 mL), and dried in vacuo to
afford the desired compound as a yellow solid (0.308 g, 80%), m.p.:
>250 ºC (decomp.).
1H NMR (400 MHz, DMSO-d6)  8.50 (s, 2H), 8.48 (s, 2H), 7.89 (t, J =
8.0 Hz, 2H), 7.13 (t, J = 8.0 Hz, 1H), 6.65 (d, J = 8.0 Hz, 1H), 6.51 (s, 1H),
6.48 (d, J = 12.0 Hz, 1H), 5.23 (bs, NH2); 13C NMR (101 MHz, DMSO-d6)
 163.5 (2C), 149.4, 136.6, 134.3 (2C), 131.4, 130.7 (2C), 129.1 (2C),
127.8, 127.2 (2C), 122.6, 116.0, 114.3, 113.6; IR (cm-1) 3411 (NH2), 3348
(NH2), 2971 (Ar-H), 1658 (C=O), 776 (Ar-H); LCMS (ESI+) m/z: 289
[M+H]+ (100%); HRMS (ESI+) m/z calculated for C18H13N2O2 (M+H)+
289.0972; found: 289.0973.
2-(2-Hydroxyphenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (19)
A solution of 1,8-naphthalic anhydride (14) (1.0 mmol, 0.198 g), 2-
aminophenol (3.0 mmol, 0.327 g) and triethylamine (8 drops) in 15 mL
absolute ethanol was heated to reflux for 18 h, with magnetic stirring,
then allowed to cool to 0°C. The mixture was filtered in vacuo, the pale
brown solid washed with cold ethanol (3 mL) and cold diethyl ether (3
mL), and dried in vacuo to afford the desired compound as a creamy-
coloured solid (0.167 g, 58%), m.p.: 310-312 ºC [51].
1H NMR (400 MHz, DMSO-d6)  9.64 (s, OH), 8.51 (d, J = 1.8 Hz, 2H),
8.49 (s, 2H), 7.90 (t, J = 7.8 Hz, 2H), 7.30 (t, J = 8.1 Hz, 1H), 6.87 – 6.84
(m, 1H), 6.79 - 6.78 (m, 2H); 13C NMR (101 MHz, DMSO-d6) δ 163.6 (2C),
157.8, 137.0, 134.4 (2C), 131.4, 130.7 (2C), 129.4, 127.8, 127.2 (2C),
2-(4-Aminophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (23)
A solution of 1,8-naphthalic anhydride (14) (0.99 mmol, 0.198 g), p-
phenylenediamine (3.13 mmol, 0.339 g) and triethylamine (10 drops)
in ethanol (15 mL) was heated to reflux, with magnetic stirring, for 20
h. After cooling to room temperature, the mixture was filtered in vacuo,
and the reddish-brown precipitate was washed with ethanol (20 mL)
and cold diethyl ether (20 mL), and dried in vacuo to afford the desired
11
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10.1002/cmdc.202000721
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FULL PAPER
compound as a mustard-yellow solid (0.197 g, 69%), m.p.: >250 ºC
(decomp.) [49].
116.3, 40.4; IR (cm-1) 3413 (NH2), 3344 (NH2), 1692 (C=O); LRMS (ESI+)
m/z 303 [M+H]+ (100%).
1H NMR (400 MHz, DMSO-d6)  8.49 (s, 2H), 8.47 (s, 2H), 7.88 (t, J =
8.0 Hz, 2H), 6.94 (d, J = 8.0 Hz, 2H), 6.64 (d, J = 8.0 Hz, 2H), 5.25 (s, 2H);
13C NMR (101 MHz, DMSO-d6)  164.0 (2C), 148.5, 134.2 (2C), 131.4,
130.7 (2C), 129.2 (2C), 127.4, 127.2 (2C), 123.8, 122.7 (2C), 113.7 (2C);
IR (cm-1) 3417 (NH2), 3350 (NH2), 3237 (Ar-H), 1652 (C=O), 779 (Ar-H);
LCMS (ESI+) m/z: 289 [M+H]+ (100%).
2-(3-Aminobenzyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (27)
A solution of 1,8-naphthalic anhydride (1.0 mmol, 198 mg) was
combined with 3-aminobenzylamine (3.0 mmol, 366 mg) and
triethylamine (10 drops) in ethanol (15 mL) was heated to reflux, with
magnetic stirring, for 44 h. After cooling to room temperature, the
mixture was filtered in vacuo, and the pale brown solid was washed
with cold ethanol (10 mL) and cold diethyl ether (2 x 10 mL), and dried
in vacuo to afford the desired compound as a pale tan solid (0.238 g,
79%), m.p.: 201-203 °C.
1H NMR (400 MHz, CDCl3)  8.59 (d, J = 7.8 Hz, 2H, H), 8.18 (d, J =
7.8 Hz, 2H), 7.73 (t, J = 7.8 Hz, 2H), 7.09 (t, J = 7.4 Hz, 1H), 6.95 (d, J =
7.4 Hz, 1H), 6.87 (s, 1H), 6.56 (d, J = 7.4 Hz, 1H), 5.30 (s, 2H), 3.62 (s,
2H); 13C NMR (101 MHz, CDCl3) δ 164.4 (2C), 146.6, 138.6, 134.1 (2C),
131.7, 131.5 (2C), 129.5, 128.3, 127.1 (2C), 122.8 (2C), 119.4, 115.6,
114.4, 43.6; IR (cm-1) 3431 (NH2), 3353 (NH2), 1650 (C=O); LRMS (ESI+)
m/z: 303 (M+H)+ (100%); HRMS (ESI+) m/z calculated for C19H15N2O2
(M+H)+ 303.1128; found: 303.1129.
5-(1,3-Dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)isophthalic acid (24)
A solution of 1,8-naphthalic anhydride (1.0 mmol, 198 mg) was
combined with 5-aminoisophthalic acid (1.0 mmol, 181 mg) and
triethylamine (10 drops) in ethanol (15 mL) and was heated to reflux,
with magnetic stirring, for 24 h. The mixture was filtered hot in vacuo.
The resultant off-white solid was taken up in methanol (30 mL) and
heated to boiling (100 °C) for 30 min. The mixture was filtered hot in
vacuo, washed with cold ethanol (10 mL) and cold diethyl ether (2 x 10
mL), and dried in vacuo to afford the desired compound as an off-white
solid (0.217 g, 60%), m.p.: >360 °C.
1H NMR (400 MHz, DMSO-d6)  13.41 (bs, 2H), 8.56 – 8.51 (m, 5H),
8.24 (s, 2H), 7.92 (t, J = 7.7 Hz, 2H); 13C NMR (101 MHz, DMSO-d6) 
166.1 (2C), 163.6 (2C), 136.9, 134.50 (2C), 134.45 (2C), 132.2 (2C), 131.5,
130.7 (2C), 129.7, 128.0, 127.2 (2C), 122.7 (2C); IR (cm-1) 3100-2500 (O-
H), 1695 (C=O), 1668 (C=O), 772 (Ar-H); LCMS (ESI-) m/z: 360 [M-H]-
(100%); HRMS (ESI-) m/z calculated for C20H10NO6 (M-H)- 360.0514;
found: 360.0503.
2-(4-Aminobenzyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (28)
A solution of 1,8-naphthalic anhydride (1.0 mmol, 198 mg) was
combined with 4-aminobenzylamine (3.0 mmol, 366 mg) and
triethylamine (10 drops) in ethanol (15 mL) was heated to reflux, with
magnetic stirring, for 20 h. After cooling to room temperature, the
mixture was filtered in vacuo, and the pale brown solid was washed
with cold ethanol (10 mL) and cold diethyl ether (2 x 10 mL), and dried
in vacuo to afford the desired compound as a yellow solid (0.359 g,
95%), m.p.: > 250 °C (decomp.).
1H NMR (400 MHz, DMSO-d6)  8.49 (dd, J = 1.5, 7.2 Hz, 2H), 8.43
(dd, J = 1.5, 8.4 Hz, 2H), 7.85 (dd, J = 7.2, 8.4 Hz, 2H), 7.08 (d, J = 8.4 Hz,
2H), 6.46 (d, J = 8.4 Hz, 2H), 5.06 (s, 2H), 4.96 (bs, NH2); 13C NMR (101
MHz, DMSO-d6) δ 163.5 (2C), 148.0, 134.5 (2C), 131.4, 131.0 (2C), 129.4
(2C), 127.4 (3C), 124.6, 122.1 (2C), 113.8 (2C), 42.7; IR (cm-1) 3467 (NH2),
3380 (NH2), 1684 (C=O); LRMS (ESI+) m/z: 303 (M+H)+ (100%); HRMS
(ESI+) m/z calculated for C19H15N2O2 (M+H)+ 303.1128; found: 303.1125.
5-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)isophthalic
triethylammonium salt (25)
acid,
1,8-naphthalic anhydride (1.0 mmol, 198 mg) was combined with
2-aminoterephthalic acid (1.0 mmol, 0.181 g) and triethylamine (10
drops) in 15 mL ethanol in a 10-20 mL microwave vial. A stirrer bar was
added, the vial capped and the reaction mixture irradiated at 120 °C for
75 min. The reaction was cooled to 0 °C and a pale brown solid was
isolated via vacuum filtration, washed with cold ethanol (3 mL) and cold
diethyl ether (3 mL), and dried in vacuo to afford the desired compound
as an eggshell-coloured solid (0.164 g, 35%), m.p. 215 °C (decomp.).
1H NMR (400 MHz, DMSO) δ 8.55 – 8.47 (m, 4H), 8.03 (dd, J = 26.5,
8.1 Hz, 2H), 7.94 – 7.86 (m, 3H), 2.73 (q, J = 7.1 Hz, 6H), 0.94 (t, J = 7.2
Hz, 9H); 13C NMR (101 MHz, DMSO-d6) 167.1, 166.6, 163.8 (2C), 135.6,
135.4, 134.3 (2C), 132.5, 131.5, 131.2, 131.0, 130.5 (2C), 128.8, 128.0,
127.6, 127.2 (2C), 122.8, 44.9 (3C), 8.7 (3C); IR (cm-1) 3000-2500 (O-H),
1709 (C=O), 1666 (C=O), 765 (Ar-H); LCMS (ESI-) m/z: 360 [M-H]- (100%);
HRMS (ESI-) m/z calculated for C20H10NO6 (M-H)- 360.0514; found:
360.0509.
2-(2-Hydroxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (29)
1,8-naphthalic anhydride (1.0 mmol, 198 mg) was combined with
ethanolamine (1.1 mmol, 0.084 mL) in 15 mL ethanol in a 10-20 mL
microwave vial. A stirrer bar was added, the vial capped and the
reaction mixture irradiated at 120 °C for 20 min. The reaction was
cooled to 0 °C and a crystalline tan solid was isolated via vacuum
filtration, washed with cold ethanol (3 mL) and cold diethyl ether (3
mL), and dried in vacuo to afford the desired compound as a tan
crystalline solid (0.207 g, 86%), m.p.: 174-175 °C [27].
1H NMR (400 MHz, DMSO-d6)  3.61 (m, 2H), 4.14 (t, J = 6.6 Hz, 2H),
4.81 (t, J = 6.0 Hz, OH), 7.85 (dd, J = 7.5, 8.1 Hz, 2H), 8.44 (dd, J = 0.9,
8.1 Hz, H2), 8.47 (dd, J = 0.9, 7.5 Hz, 2H); 13C NMR (101 MHz, DMSO-d6)
δ 163.5 (2C), 134.2 (2C), 131.3, 130.6 (2C), 127.4, 127.2 (2C), 122.2 (2C),
57.8, 41.8; IR (cm-1) 3300 (O-H), 2952 (C-H), 2861 (C-H), 1645 (C=O), 783
(Ar-H); LRMS (ESI+) m/z: 242 (M+H)+ (100%).
2-(2-Aminobenzyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (26)
A solution of 1,8-naphthalic anhydride (1.0 mmol, 198 mg) was
combined with 2-aminobenzylamine (3.0 mmol, 366 mg) and
triethylamine (10 drops) in ethanol (15 mL) was heated to reflux, with
magnetic stirring, for 44 h. After cooling to room temperature, the
mixture was filtered in vacuo, and the pale brown solid was washed
with cold ethanol (10 mL) and cold diethyl ether (2 x 10 mL), and dried
in vacuo to afford the desired compound as a bright yellow solid (0.250
g, 80%), m.p.: 232-233 °C [27].
1H NMR (400 MHz, CDCl3)  8.59 (d, J = 7.4 Hz, 2H), 8.17 (d, J = 8.2
Hz, 2H), 7.73 (t, J = 7.8 Hz, 2H), 7.65 (d, J = 7.4 Hz, 1H), 7.07 (t, J = 7.2
Hz, 1H), 6.71 (t, J = 7.4 Hz, 1H), 6.65 (d, J = 7.9 Hz, 1H), 5.32 (s, 2H), 4.69
(s, 2H); 13C NMR (101 MHz, CDCl3) δ 165.1 (2C), 146.3, 134.3 (2C), 133.3,
131.7 (2C), 131.6, 129.3, 128.2, 127.1 (2C), 122.7 (2C), 121.3, 118.2,
2-(3-Hydroxypropyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (30)
1,8-naphthalic anhydride (1.0 mmol, 198 mg) was combined with
3-amino-1-propanol (1.1 mmol, 0.084 mL) in 15 mL ethanol. The
reaction was heated to 100 °C (reflux) for 18 hours. The reaction was
cooled to room temperature and a pale brown solid was isolated via
vacuum filtration, washed with cold ethanol (3 mL) and cold diethyl
12
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10.1002/cmdc.202000721
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FULL PAPER
ether (3mL) and dried in vacuo to afford the desired product as an off-
white crystalline solid (0.209 g, 82%), m.p.: 121-123 °C.
2-(1H-Benzo[de]isoquinolin-2(3H)-yl)aniline (36)
To naphthalimide 22 (0.31 mmol, 0.091 g) was added sodium
borohydride (1.6 mmol, 0.063 g), followed by THF (7 mL). To this
solution was then added, dropwise, a solution of freshly sublimated
iodine (1.04 mmol, 0.132 g) in tetrahydrofuran (7 mL), at room
temperature, stirred magnetically. The mixture was then heated to 60
ºC for 18 h, at which point it was cooled to room temperature, then,
treated with cold water (20 mL). The resultant solution was chilled to 0
ºC; the precipitate collected in vacuo, washed with water (10 mL), and
dried in vacuo to afford the desired compound as an off-white solid
(0.025 g, 31%), m.p.: >178 ºC (decomp.).
1H NMR (400 MHz, DMSO-d6)  8.49 (d, J = 7.6 Hz, 2H), 8.45 (d, J =
7.6 Hz, 2H), 7.86 (t, J = 7.6 Hz, 2H), 4.49 (t, J = 5.2 Hz, 1H), 4.11 (t, J = 7.2
Hz, 2H), 3.50 (dd, J = 11.7, 6.2 Hz, 2H), 1.80 (q, J = 6.4 Hz, 2H); 13C NMR
(101 MHz, DMSO-d6) δ 163.4 (2C), 134.3 (2C), 131.3, 130.7 (2C), 127.4,
127.2 (2C), 122.1 (2C), 59.0, 37.6, 31.0; IR (cm-1) 3300 (O-H), 2952 (C-H),
2861 (C-H), 1645 (C=O), 783 (Ar-H); LRMS (ESI+) m/z: 256 (M+H)-
(100%); HRMS (ESI+) m/z calculated for C15H14NO3 (M+H)+ 256.0968;
found: compound did not ionise.
1H NMR (400 MHz, DMSO-d6
J = 8.1 Hz, 2H), 7.49 – 7.43
2-(o-Tolyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (31)
(m, 2H), 7.32 (d, J = 6.9 Hz, 2H), 7.02 (d, J = 7.8 Hz, 1H), 6.88 – 6.82 (m,
1H), 6.73 (dd, J = 7.9, 1.4 Hz, 1H), 6.56 (td, J = 7.6, 1.5 Hz, 1H), 4.73 (s,
2H), 4.37 (s, 4H); 13C NMR (101 MHz, DMSO-d6)  142.6, 137.4, 134.1
(2C), 132.7, 127.6, 125.8 (2C), 125.7 (2C), 124.3, 121.7 (2C), 119.7,
116.6, 114.5, 53.9 (2C); IR (cm-1) 3443 (N-H2), 3345 (N-H2), 1606 (N-H2),
765 (Ar-H); LCMS (ESI+) m/z: 261 [M+H]+ (100%); HRMS (ESI+) m/z
calculated for C18H17N2 (M+H)+ 261.1386; found: 261.1388.
A solution of 1,8 naphthalic anhydride (14) (0.35 mmol, 0.070 g), o-
toluidine (1.20 mmol, 0.128 g) and triethylamine (10 drops) in absolute
ethanol (10 mL) was heated at reflux for 23 h, with magnetic stirring.
The reaction was cooled to 0 ºC, and a brown solid was isolated by
vacuum filtration, washed with cold ethanol (2 × 10 mL) and cold diethyl
ether (2 × 5 mL) and dried in vacuo to afford the desired compound as
a tan crystalline solid (0.034 g, 20%), m.p.: 217 ºC.
1H NMR (400 MHz, DMSO-d6)  8.55 (d, J = 2.4 Hz, 2H), 8.53 (d, J =
1.4 Hz, 2H), 7.93 (t, J = 8.0 Hz, 2H), 7.42 – 7.33 (m, 4H), 2.07 (s, 3H); 13C
NMR (101 MHz, DMSO-d6) δ 163.3 (2C), 135.7 (2C), 135.2, 134.6 (2C),
131.5, 131.0 (2C), 130.4, 129.2, 128.5, 128.0, 127.3 (2C), 126.7, 122.3,
17.1; IR (cm-1) 1656 (C=O), 1374 (C-H3), 773 (Ar-H); LCMS (ESI+) m/z:
288 [M+H]+ (100%); HRMS (ESI+) m/z calculated for C19H14NO2 (M+H)+
288.1019; found: compound did not ionise.
Acknowledgements
JRB is the recipient of an Australia government HDR scholarship.
Funding from the Australian Cancer Research Foundation and the
Ramaciotti Foundation is gratefully acknowledged. SP is the recipient
of a Fulbright Senior Scholar Fellowship.
2-(Pyridin-2-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (32)
A solution of 1,8-naphthalic anhydride (14) (0.48 mmol, 0.097 g)
and 2-aminopyridine (1.03 mmol, 0.097 g) in ethanol (7 mL) was heated
to reflux, with magnetic stirring, for 36 h. The ensuing mixture was
chilled; the resulting precipitate was subsequently collected in vacuo,
washed with cold ethanol (10 mL) and cold diethyl ether (10 mL). The
solid was then recrystallised from ethanol to afford the desired
compound as a tan crystalline solid (0.030 g, 23%), m.p.: >250 ºC
(decomp.).
1H NMR (400 MHz, DMSO-d6)  8.66 (dd, J = 5.2, 1.5 Hz, 1H), 8.54 (t,
J = 8.3 Hz, 4H), 8.05 (td, J = 7.7, 1.9 Hz, 1H), 7.95 – 7.91 (m, 2H), 7.61
(dt, J = 7.9, 0.9 Hz, 1H), 7.55 (ddd, J = 7.5, 4.9, 1.1 Hz, 1H) 13C NMR (101
MHz, DMSO-d6)  163.5 (2C), 149.6 (2C), 149.4 (2C), 138.7, 134.8 (2C),
131.5, 130.8, 127.9, 127.3 (2C), 124.4, 124.2, 122.2; IR (cm-1) 3072 (Ar-
H), 1658 (C=O), 771 (Ar-H); LCMS (ESI+) m/z: 275 [M+H]+ (100%); HRMS
(ESI+) m/z calculated for C17H11N2O2 (M+H)+ 275.0815; found: 275.0818.
Keywords: naphthalimides • aryl hydrocarbon receptor • breast
cancer • homology modelling • molecular dynamics
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and cold diethyl ether (2 × 5 mL), and dried in vacuo to afford the
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1H NMR (400 MHz, DMSO-d6
J = 7.7 Hz, 1H), 7.51 (t, J =
7.5 Hz, 1H), 7.44 (dd, J = 6.0, 3.2 Hz, 2H), 7.38 (t, J = 7.1 Hz, 1H), 7.32 (d,
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