Discovery and biological evaluation of phthalazines as novel non-kinase TGFβ pathway inhibitors

Article abstract of DOI:10.1016/j.ejmech.2021.113660

TGFβ is crucial for the homeostasis of epithelial and neural tissues, wound repair, and regulating immune responses. Its dysregulation is associated with a vast number of diseases, of which modifying the tumor microenvironment is one of vital clinical interest. Despite various attempts, there is still no FDA-approved therapy to inhibit the TGFβ pathway. Major mainstream approaches involve impairment of the TGFβ pathway via inhibition of the TGFβRI kinase. With the purpose to identify non-receptor kinase-based inhibitors to impair TGFβ signaling, an in-house chemical library was enriched, through a computational study, to eliminate TGFβRI kinase activity. Selected compounds were screened against a cell line engineered with a firefly luciferase gene under TGFβ-Smad-dependent transcriptional control. Results indicated moderate potency for a molecule with phthalazine core against TGFβ-Smad signaling. A series of phthalazine compounds were synthesized and evaluated for potency. The most promising compound (10p) exhibited an IC₅₀ of 0.11 ± 0.02 μM and was confirmed to be non-cytotoxic up to 12 μM, with a selectivity index of approximately 112-fold. Simultaneously, 10p was confirmed to reduce the Smad phosphorylation using Western blot without exhibiting inhibition on the TGFβRI enzyme. This study identified a novel small-molecule scaffold that targets the TGFβ pathway via a non-receptor-kinase mechanism.

Full text of DOI:10.1016/j.ejmech.2021.113660

European Journal of Medicinal Chemistry 223 (2021) 113660
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery and biological evaluation of phthalazines as novel non-
kinase TGFb pathway inhibitors
,
c
Anupreet Kharbanda a, Lingtian Zhang a, Debasmita Saha a, Phuc Tran a, Ke Xu b
,
,
c
,
, *
Ming O. Li b d, Yuet-Kin Leung e, Brendan Frett a, Hong-yu Li a
a Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, USA
b Immunology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA
c Immunology and Microbial Pathogenesis Graduate Program, Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, NY, 10065,
USA
d Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA
e Department of Pharmacology & Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
TGFb is crucial for the homeostasis of epithelial and neural tissues, wound repair, and regulating immune
responses. Its dysregulation is associated with a vast number of diseases, of which modifying the tumor
microenvironment is one of vital clinical interest. Despite various attempts, there is still no FDA-
Received 15 May 2021
Received in revised form
15 June 2021
Accepted 16 June 2021
Available online 19 June 2021
approved therapy to inhibit the TGF
the TGF pathway via inhibition of the TGF
based inhibitors to impair TGF signaling, an in-house chemical library was enriched, through a
computational study, to eliminate TGF RI kinase activity. Selected compounds were screened against a
cell line engineered with a firefly luciferase gene under TGF -Smad-dependent transcriptional control.
Results indicated moderate potency for a molecule with phthalazine core against TGF -Smad signaling. A
series of phthalazine compounds were synthesized and evaluated for potency. The most promising
compound (10p) exhibited an IC50 of 0.11 0.02 M and was confirmed to be non-cytotoxic up to 12 M,
with a selectivity index of approximately 112-fold. Simultaneously, 10p was confirmed to reduce the
Smad phosphorylation using Western blot without exhibiting inhibition on the TGF RI enzyme. This
pathway via a non-receptor-kinase
b
pathway. Major mainstream approaches involve impairment of
b
b
RI kinase. With the purpose to identify non-receptor kinase-
b
b
Keywords:
b
TGF-
TGF-
b
b
pathway inhibitor
inhibitor
b
Pthalazine derivatives
Non-kinase inhibitor
Cytokines
m
m
b
study identified a novel small-molecule scaffold that targets the TGF
b
mechanism.
© 2021 Published by Elsevier Masson SAS.
Transforming growth factor-beta (TGFb) is a profibrogenic
promoter, which is known as the “TGFb paradox.” [6] In a meta-
cytokine, which plays a crucial role in metazoan biology. Nearly all
the cell types respond to TGF , it maintains homeostasis of
epithelial and neural tissues, aids in wound repair, and regulates
aspects of the immune system [1]. The release of TGF stimulates
the TGF receptors (TGF RI and TGF RII), which activates SMAD
proteins that modulate gene expression (Fig. 1).
Perturbation of the TGF pathway can interrupt immune-
static state, the concentration of TGF
b
increases significantly in the
is responsible for the polari-
b
tumor microenvironment [7,8]. TGF
b
zation of M1 macrophages to tumor-associated macrophages
b
(TAM) [9]. Studies involving obstruction of TGFb signaling exhibit
b
b
b
an improvement in the CD8þ T-cell mediated immune response, an
increase in cytotoxic T-cells and NK cells at metastatic sites, and
suppression of T-regs [10e12]. Further studies revealed that upre-
b
modulatory activities, which has implications in many diseases
like cancer, asthma, nephritis, cardiovascular disease, rheumatoid
gulation of TGFbRII in the tumor microenvironment correlates to
drug resistance to chemotherapeutics like cisplatin and targeted
arthritis, and multiple sclerosis [2e5]. In the context of cancer, TGF
b
therapeutics such as Erlotinib, Gefitinib, Crizotinib, Vemurafenib,
exhibits a functional switch from a tumor suppressor to a tumor
and Sorafenib [13]. Suppressing TGF
enhance the efficacy of PD-1 checkpoint inhibitors [14]. Owing to
its role in immune regulation, TGF is a highly researched cytokine
in cancer, autoimmune diseases, and infectious disease [15e22].
In the past few decades, modifying TGF signaling has been
b has also been shown to
b
* Corresponding author.
E-mail address: HLi2@uam.edu (H.-y. Li).
b
https://doi.org/10.1016/j.ejmech.2021.113660
0223-5234/© 2021 Published by Elsevier Masson SAS.

A. Kharbanda, L. Zhang, D. Saha et al.
European Journal of Medicinal Chemistry 223 (2021) 113660
Fig. 1. TGF
of TGF type-I (TGF
by phosphorylating receptor-associated Smads (R-Smads), Smad2, and Smad3. The activated R-Smads form a complex with Co-Smad and shuttles into the nucleus, binding to DNA
b
signaling pathway via SMAD- TGF
b
ligand binds to transmembrane TGF
b
type-II receptor (TGF
bRII) and induces its phosphorylation, followed by autophosphorylation
b
bRI) receptor forming a heterotetrameric receptor complex with two type I receptors and two type II receptors. Activated TGFbRI initiates TGFb/Smad signaling
at Smad-binding element in the promoter region of the TGF
AKT pathway, etc.
b targeted gene. TGFb receptor complex can also activate non-smad dependent pathways like Ras/ERK pathway, PI3K/
investigated with TGFbRI kinase inhibitors, such as 1 (LY-2157299),
2 (SB-505124), 3 (SD-208), and 4 (GW6604) (Fig. 2), and more
[23e38]. These inhibitors are ATP mimetic drugs and are designed
to target TGF
have the potential for promiscuity within the kinome as the ATP
pocket is heavily conserved [39e42]. Therefore, targeting TGF RI
possesses off-target liabilities by inadvertently blocking kinases
similar to TGF RI, such as ALK-4 and ALK-7 [24,43]. Moreover, TGF
bRI directly. However, like other kinase inhibitors, they
b
b
b
can also activate numerous non-canonical pathways, which can
play an essential role in tumor suppression, and kinase inhibition
can interfere with the activation of non-canonical TGF
generating undesired side effects [44e47]. Therefore, there is a
need to investigate therapeutic strategies that impair the TGF
b pathways
b-
Smad pathway without directly interfering with kinase activity. A
similar approach has resulted in the characterization of SIS3,
SMAD3 specific inhibitors, Asiaticoside, a SMAD 7 modulator, and
Pirfenidone, which can decrease TGF
[20,48,49]. In this study, we explored further to identify non-
receptor kinase-based inhibitors to impair TGF signaling.
In order to develop a new approach to target the TGF pathway,
we performed a phenotypic screening using HEK293 cell-line, with
a luciferase reporter under transcriptional regulation by TGF
b protein expression
b
b
b
.
Fig. 2. Small molecule inhibitors of TGFbRI.
2

A. Kharbanda, L. Zhang, D. Saha et al.
European Journal of Medicinal Chemistry 223 (2021) 113660
Using the luciferase assay, we screened a diverse, in-house library
give 14 [52]. 1-(3-(trifluoromethyl)benzyl)piperazine was then
coupled to intermediate 14 to yield 15, which were subjected to
Buchwald-Hartwig amination with 4-morpholinoaniline to give
final compound 16. To generate the isoquinoline isomer, 4-
morpholinoaniline was reacted with 14 and then subjected to a
BuchwaldeHartwig cross-coupling reaction to generate 18
(Scheme 3) [53].
that was computationally predicted to have limited-to-no TGF
inhibition. Hits from the designed experiment were confirmed not
to have direct TGF RI inhibition using an enzymatic inhibitory
assay (Fig. 3). From this approach, we uncovered a novel phthala-
zine chemotype that exhibited potent impairment of the TGF
pathway without any direct inhibition of the TGF RI kinase. We
bRI
b
b
b
extensively explored the chemotype by generating a small, focused
phthalazine library and evaluated the library in cell and enzymatic
assays. This study identified 10p as the most efficacious inhibitor of
Phenotypical Screening. To identify non-kinase inhibitor scaf-
folds that could potentially block the TGF
a computational study using our in-house library to identify scaf-
folds that possessed low potential for kinase inhibition
b pathway, we performed
the TGF
b
pathway with minimal cytotoxicity and no inhibition of
RI kinase. Additionally, inhibition of Smad phosphoryla-
a
the TGF
b
(Supplementary section 7). Computationally filtered compounds
tion was confirmed using Western blot.
were screened using HEK293 cell line with a luciferase reporter,
under transcriptional regulation by TGF
b. This cell line was engi-
neered to monitor input into the TGF -Smad signaling pathway by
b
1. Results and discussion
placing the firefly luciferase gene under Smad-dependent tran-
scriptional control. Therefore, a decrease in luminescent signal
Chemistry. Compounds 8a-r and 10a-r were synthesized ac-
cording to the synthetic route depicted in Scheme 1. Isoindoline-
1,3-dione 5 was reacted with hydrazine hydrate in ethanol to afford
2,3-dihydrophthalazine-1,4-dione, which was subsequently reac-
ted with phosphorus oxychloride (POCl3) to give 1,4-dichloroph-
thalazine 6 [50]. 1,4-dichlorophthalazine was coupled to an amine
in ethanol to yield 7 (or 9a-r), which was further reacted with a
benzyl substituted piperazine to give 8a-r (or 10a-r). To generate a
carbon-carbon bond with the phthalazine core, 7 underwent
Suzuki-Miyaura reaction with a respective boronic acid pinacolate
ester to generate final compounds 11 and 12, as shown in Scheme 2
[51].
To understand the importance of both the nitrogen, in the core,
on potency against the TGFb pathway, compounds were generated
with an isoquinoline core in place of the phthalazine. To synthesize
isoquinolines, 4-bromoisoquinoline was oxidized using m-chlor-
operbenzoic acid, which was further chlorinated using POCl3 to
correlates to a reduction in TGF
identifying strategies to impair the TGF
inhibiting kinase potency in an attempt to limit toxicity and
improve efficacy while targeting the TGF pathway.
From the initial screen, we identified a phthalazin-1-amine (8a)
with potency against the TGF pathway with an IC50 of
10.04 0.62 M as refereed by a reduction in luminescent signal.
This impairment of the TGF pathway could stem from few sig-
nificant reasons. One, 8a could possess TGF RI inhibitory activity,
b
signaling. We were interested in
b
pathway without directly
b
b
m
b
b
directly blocking the Smad-mediated activation of the firefly
luciferase gene. Two, 8a could be cytotoxic and non-specifically
impair cell viability, causing a reduction in cellular processes such
as gene transcription and protein synthesis. Three, interaction with
luciferase reporter system a cross screening was performed in
HEK293 cell line with different pathways to confirm no inhibition
against another pathway, to eliminate the possibility of luciferase
Fig. 3. Identification of non-receptor-kinase TGFb pathway inhibitors.
3

A. Kharbanda, L. Zhang, D. Saha et al.
European Journal of Medicinal Chemistry 223 (2021) 113660
Scheme 1. Synthesis of 8a-r and 10a-r: (a.i) NH2NH2, EtOH, reflux for 12 h; (a.ii) POCl3, DMF, 120 ꢀC, 2 h; (b) EtOH, 65 ꢀC, 3 h; (c) n-BuOH, overnight, 150 ꢀC.
Scheme 2. Synthesis of 11 and 12: (a) Pd(PPh3)4, Cs2CO3, DMF:H2O (4:1), 115 ꢀC, MW, 1 h.
reporter system interference (Supplementary section 8.3). On
eliminating the above three possibilities, we would support that
the compounds interfere with TGFb signaling via a novel mecha-
Considering these results, 8a appeared to block the TGF
b
pathway
without inhibiting TGF
candidate.
bRI and was pursued further as a hit
nism. To confirm this hypothesis, we performed immunoblot
analysis to observe reduced Smad2/3 phosphorylation when
treated with an inhibitor.
To eliminate the possibility of cytotoxicity, we incorporated
matched viability assays to determine a preliminary safety window.
To explore structure-activity relationships (SAR) further, struc-
ture 8a was classified into three regions (Fig. 4). Substitution on the
benzylic ring (Region A) of 8a was investigated as depicted in
Table 1 (Entries 1e18). Introduction of bromo at the meta-position
(8c, IC50 ¼ 1.32
0.23
m
M; GI50 ¼ 13.14
0.54 mM; Selectivity
When running the reporter assay, we assessed TGF
b
pathway
Index ¼ 9.98) improved the selectivity index compared to its po-
sitional isomers 8b and 8d. Even though the replacement of m-
bromo with m-fluoro (8e) or m-chloro (8f) did not exhibit
improvement in potency, inhibition improved as the size of the
halogen increased. Further SAR studies focused on modifying the
meta position with electron-withdrawing (8g-j) or donating (8k-m)
groups. Electron withdrawing groups exhibited high selectivity for
impairment (IC50) as well as cell viability (GI50) to establish a
Selectivity Index (GI50/IC50). Using this approach, we determined
compound 8a had a selectivity index of 4, indicating that at non-
cytotoxic concentrations of 45.62 2.29
impairing the TGF pathway. Next, we subjected 8a to
biochemical inhibitor assay to assess direct inhibition of the TGF
kinase, and 8a did not exhibit TGF
mM, the compound was
b
a
b
RI
b
RI inhibition (Table 4, Entry 1).
targeting the TGFb pathway compared to cell viability, and the
4

A. Kharbanda, L. Zhang, D. Saha et al.
European Journal of Medicinal Chemistry 223 (2021) 113660
Scheme 3. Synthesis of 16 and 18: (a.i)m-CPBA, DCM, rt, 3 h; (a.ii) POCl3, DMF, 120 ꢀC, 2 h; (b) n-BuOH, overnight, 150 ꢀC; (c) Pd2dba3, ( )-BINAP, Dioxane, KtBuO, 110 ꢀC, overnight;
(d) EtOH, 65 ꢀC, 3 h.
Table 1
TGFb inhibitory potency and cytotoxicity of compounds 8a-r.
Compd
R
TGF
b
pathway
HEK293 viability GI50
(
mM)
Selectivity index (GI50/IC50)
impairment IC50 (mM)
8a
8b
8c
8d
8e
8f
8g
8 h
8i
8j
8k
8l
8 m
8n
8ꢀ
8p
8q
8r
H
o-Br
m-Br
p-Br
m-F
m-Cl
m-CF3
m-NO2
m-CN
m-COOMe
m-OMe
m-OCF3
m-Me
m,p-di-Cl
m,p-di-F
o,o-di-Cl
o,p-di-F
m-Cl,p-F
10.04 0.62
5.84 0.36
1.32 0.23
11.53 0.08
7.37 0.26
1.56 0.22
0.24 0.03
1.23 0.10
1.72 0.11
0.17 0.02
5.27 0.69
5.89 0.74
1.96 0.44
5.47 0.32
4.00 0.85
9.45 0.28
3.82 0.99
7.49 0.39
45.62 2.29
12.4 0.21
13.14 0.54
18.49 1.16
35.31 1.59
14.13 1.48
16.85 0.25
9.26 0.32
14.64 0.53
7.59 0.29
10.00 0.66
98.29 0.5
13.48 1.15
36.24 3.3
12.26 0.43
14.21 0.54
11.50 0.26
11.48 2.49
4.54
2.12
9.98
1.60
4.79
9.05
69.59
7.55
8.51
44.65
1.89
16.69
6.87
6.62
3.06
1.50
3.01
1.53
highest
selectivity
index
M, GI50 ¼ 16.85
was
observed
0.25
with
M, Selectivity
8g
phthalazine-core (Table 2 (Entries 16, 18)). When the nitrogen
adjacent to morpholinoaniline was removed, an 8-fold reduction in
(IC50 ¼ 0.24
0.03
m
m
Index ¼ 69.59).
potency was observed (16, IC50
GI50 ¼ 11.24 1.14 M; Selectivity Index ¼ 5.71). Whereas, removal
of the other nitrogen caused a complete loss of inhibition (18,
0.73 2.35 M; Selectivity
¼
1.97
0.21
mM;
We then explored changing the flexible carbon-nitrogen linker
to a ridged carbon-carbon linker, as shown in Table 2 (Entries
11e12). Carbon-carbon bonds were generated with benzene (11) or
pyrazole (12), and we observed a loss of potency by 12-fold and 33-
fold compared to 8g, respectively. We then modified the phthala-
zine at Region B ring system to assess the significance of the
m
IC50 ¼ 20.10
m
M; GI50 ¼ 49.52
m
Index ¼ 2.46), which explicitly indicates the phthalazine-core is
integral for potency.
5

A. Kharbanda, L. Zhang, D. Saha et al.
European Journal of Medicinal Chemistry 223 (2021) 113660
Table 2
TGFb inhibitory potency and cytotoxicity of compounds 11, 12, 16, and 18 in HEK-293 cell line.
Compd Structure
11
TGF
b
pathway impairment IC50
(m
M) HEK293 viability GI50
11.14 0.88
(
m
M) Selectivity index (GI50/IC50
)
3.01 0.99
3.71
12
16
18
8.17 0.67
20.55 3.24
11.24 1.14
49.52 2.35
2.51
5.71
2.46
1.97 0.21
20.10 0.73
amines with aromatic amines improved potency and selectivity
profiles (Table 3, Entries 10j-10o). Which was further improved by
increasing the size of the substitution on the aromatic ring. Further
introduction of mono and di-methyl substitution on the morpho-
line ring improved the potency and selectivity (10p-r). Overall, SAR
at this position suggested bulky, para substitution on aromatic
amines is most suitable for impairing the TGF
limited cytotoxicity was observed with 10p (IC50 ¼ 0.11 0.02
GI50 ¼ 12.32 1.02 M, Selectivity Index ¼ 112.0).
Since our primary aim was to investigate TGF
b
pathway with
mM,
m
b
-Smad pathway
inhibitors without directly interfering with the receptor, we per-
formed an enzymatic assay to confirm that compounds have
Fig. 4. SAR study of the phthalazine scaffold.
limited to no inhibition at the TGF
point inhibition in an enzymatic assay for compounds 8a, 8c, 8g,
10m, 10n, and 10o at 2.0 M, and 20 M. We observed no inhibition
for the compounds at both 2.0 M, and 20 M, where the control
compound (LY-2157299) showed 73% and 100% inhibition, respec-
tively, of TGF RI (Table 4 and Supplementary Section 9). These re-
sults suggest that these compounds inhibit TGF -Smad signaling
via a non-receptor-kinase mechanism. Next, Western blot analysis
bRI kinase. We performed single
m
m
We progressed 8g as a lead candidate and further explored the
SAR on this scaffold by modifying the phthalazine substitution at
Region C (Fig. 4), depicted in Table 3(Entries 10a-r). Introduction of
saturated acyclic amines (10a-b), cyclic amines (10c-f), or a com-
bination of both (10g-i) exhibited no improvement, as compounds
were either less potent or more cytotoxic. By replacing saturated
m
m
b
b
6

A. Kharbanda, L. Zhang, D. Saha et al.
European Journal of Medicinal Chemistry 223 (2021) 113660
Table 3
TGFb inhibitory potency and cytotoxicity of compounds 10a-r.
Compd
10a
-NH-R0
TGF
b
pathway impairment IC50
(
mM)
HEK293 viability GI50
73.58 3.24
(
mM)
Selectivity index (GI50/IC50
)
19.05 0.14
3.86
10b
7.12 0.19
12.58 2.75
1.77
10c
10d
5.91 0.79
7.55 0.67
38.41 2.7
6.49
1.61
12.14 0.65
10e
3.91 0.67
10.26 0.25
2.63
10f
7.51 0.72
72.28 3.63
76.41 0.56
9.62
5.54
10g
13.78 1.00
10 h
10i
17.81 0.75
7.08 0.32
110.15 6.29
6.18
1.15
8.14 0.04
10j
10k
10l
7.16 0.68
2.36 0.315
4.54 0.28
10.39 0.12
53.81 0.17
40.07 1.37
1.45
22.81
8.83
(continued on next page)
7

A. Kharbanda, L. Zhang, D. Saha et al.
European Journal of Medicinal Chemistry 223 (2021) 113660
Table 3 (continued)
Compd
10 m
-NH-R0
TGF
b
pathway impairment IC50
(
mM)
HEK293 viability GI50
16.64 0.08
(
mM)
Selectivity index (GI50/IC50
)
0.88 0.06
18.82
10n
0.36 0.03
13.39 1.25
37.38
10ꢀ
0.47 0.12
18.01 0.29
38.09
112.0
10p
0.11 0.02
12.32 1.02
10q
10r
0.19 0.01
20.01 0.30
105.31
0.21 0.01
9.69 0.14
46.14
few kinases like CSF-1R, TRKA, FLT3, AURK-A, Nek-2 with com-
pounds 8g, 10n, and 10p; we observed almost no inhibition against
any of these kinases showing their selectivity for TGFb-Smad
pathway and also resonating with the finding that compounds are
Table 4
Inhibition of TGF
bRI at 2.0
m
M, and 20
mM.
Compd
Percent
Inhibition at 2.0
Percent
Inhibition at 20 mM
m
M
non-kinase inhibitors. (Supplementary section 10).
8a
8g
10p
10q
0%
0%
0%
0%
73%
0%
0%
0%
0%
2. Conclusions
LY-2157299
100%
The TGF
implications in cancer, autoimmune disease, and infectious disease.
However, there is not an FDA-approved therapy to target the TGF
pathway. Because most strategies targeting the TGF pathway have
relied on direct inhibition of TGF RI, we attempted to identify non-
receptor-based methods to impair TGF signaling. By completing a
b pathway is essential for immune regulation, which has
b
was performed to confirm TGF
b
pathway inhibition by
b
compounds 8g and 10p; inhibition of phosphorylation of Smad2/3
b
in isolated CD4þ T cell was observed by performing immunoblot
b
analysis at concentrations of 0.5 and 5
selectivity enzymatic screening panel was also performed against
mM (Fig. 5). Additionally, a
phenotypic screen using a cell line engineered with a firefly lucif-
erase gene under Smad-dependent transcriptional control, we
8

A. Kharbanda, L. Zhang, D. Saha et al.
European Journal of Medicinal Chemistry 223 (2021) 113660
overnight at 150 ꢀC. Next, n-butanol was concentrated and reaction
was purified by Flash chromatography on alumina. Pale yellow
solid; Yield: 57%; Rf ¼ 0.47 (50% EtOAc/Hexane on alumina);1H
NMR (400 MHz, CDCl3)
d 8.06e7.96 (m, 2H, AreH), 7.79e7.72 (m,
2H, AreH), 7.54 (dd, J ¼ 8.0, 1.1 Hz, 2H), 7.50 (d, J ¼ 8.4 Hz, 2H), 7.29
(td, J ¼ 7.5, 1.2 Hz, 1H), 7.11 (td, J ¼ 7.7, 1.7 Hz, 1H), 6.90 (d, J ¼ 9.0 Hz,
2H), 3.85 (t, J ¼ 4.0 Hz, 4H)) 3.73 (s, 2H), 3.37 (brs, 4H), 3.10 (t,
J ¼ 4.0 Hz, 4H), 2.78 (brs, 4H); 13C NMR (101 MHz, CDCl3)
d 147.38,
137.36, 132.70, 131.08, 130.81, 128.43, 127.29, 125.16, 124.60, 123.63,
122.09, 116.84, 66.95, 61.74, 53.17, 51.29, 50.11; LCMS (ESI): Calcu-
lated for [Mþ1]þ C29H31BrN6O 559.51, found 559.60.
Synthesis
of
4-(4-(3-bromobenzyl)piperazin-1-yl)-N-(4-
morpholinophenyl)phthalazin-1-amine (8c)
- 4-chloro-N-(4-
morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146 mmol)
was dissolved in n-butanol (1 mL) and 1-(3-bromobenzyl)pipera-
zine (44.6 mg, 0.175 mmol) was added and was heated overnight at
150 ꢀC. Next, n-butanol was concentrated and reaction was purified
by Flash chromatography on alumina. Yellow solid; Yield: 49%;
Rf ¼ 0.456 (50% EtOAc/Hexane on alumina); 1H NMR (400 MHz,
CDCl3)
d
8.00 (d, J ¼ 6.3 Hz, 1H), 7.95 (brs, 1H, NH), 7.73 (dd, J ¼ 6.2,
2.7 Hz, 2H), 7.52 (s, 2H), 7.49 (s, 1H), 7.38 (d, J ¼ 7.8 Hz, 1H), 7.29 (d,
J ¼ 7.3 Hz, 1H), 7.18 (t, J ¼ 7.7 Hz, 1H), 6.88 (dd, J ¼ 8.9, 2.9 Hz, 2H),
3.84 (brs, 4H), 3.57 (brs, 2H), 3.35 (brs, 4H), 3.08 (brs, 4H), 2.68 (brs,
Fig. 5. Western blot to study the inhibition of smad-phosphorylation by designed
inhibitors 8g and 10p was observed in CD4þ T cells from wild-type mice spleens which
were treated with human TGFb1 (and without for negative control).
4H). 13C NMR (101 MHz, CDCl3)
d 155.52, 149.81, 149.78, 147.26,
140.54, 132.01, 131.00, 130.96, 130.20, 129.84, 127.70, 125.06, 123.53,
122.41, 122.01, 116.79, 66.95, 62.45, 53.14, 51.21, 50.12; LCMS (ESI):
Calculated for [Mþ1]þ C29H31BrN6O 561.53, found 561.35.
identified a novel phthalazine scaffold that blocked TGF
without directly inhibiting TGF RI. By completing SAR exploration,
we identified compound 10p, which inhibited the TGF pathway at
an IC50 of 0.11 0.02 M. Compound 10p did not display cytotoxic
effects until around 12 M, and exhibited a selectivity index of
approximately 112 fold. Significantly, 10p reduced the Smad2/3
phosphorylation without inhibiting the TGF RI enzyme directly.
Therefore, we discovered a novel small-molecule scaffold that tar-
gets the TGF pathway through a non-receptor-kinase mechanism.
b signaling
b
b
Synthesis
of
4-(4-(4-bromobenzyl)piperazin-1-yl)-N-(4-
m
morpholinophenyl)phthalazin-1-amine (8d)
- 4-chloro-N-(4-
m
morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146 mmol)
was dissolved in n-butanol (1 mL) and 1-(4-bromobenzyl)pipera-
zine (44.6 mg, 0.175 mmol)) was added and was heated overnight
at 150 ꢀC. Next, n-butanol was concentrated and reaction was pu-
rified by Flash chromatography on alumina. Yellow solid; Yield:
65%; Rf ¼ 0.42 (50% EtOAc/Hexane on alumina); 1H NMR (400 MHz,
b
b
Work is being completed to identify the mechanism of action of the
phthalazine scaffold, which may uncover novel, druggable path-
CDCl3) d 8.02e7.94 (m, 2H, AreH), 7.77e7.70 (m, 2H, AreH), 7.50 (d,
ways within the TGFb signaling cascade.
J ¼ 8.7 Hz, 2H), 7.43 (d, J ¼ 8.4 Hz, 2H), 7.23 (d, J ¼ 8.5 Hz, 2H), 6.87
(d, J ¼ 9.0 Hz, 2H), 3.84 (t, J ¼ 4.0 Hz, 4H), 3.55 (s, 2H), 3.34 (brs, 4H),
3.08 (t, J ¼ 4.0 Hz, 4H), 2.67 (brs, 4H); 13C NMR (101 MHz, CDCl3)
3. Experimental section
d
147.28, 137.04, 131.35, 131.04, 130.98, 130.81, 125.05, 123.53,
Compounds 6, 7, 8a-r, 9a-r, 10a-r, and 11e18 were prepared
according to the mentioned schemes. Synthesis and characteriza-
tion are presented in the Supporting Information. The purity of all
compounds was determined to be ꢁ 95% by HPLC and LCMS system
at wavelengths of 214 and 254 nm.
122.04, 120.89, 116.78, 66.94, 62.35, 53.10, 51.20, 50.11; LCMS (ESI):
Calculated for [Mþ1]þ C29H31BrN6O 559.51, found 559.66.
Synthesis
of
4-(4-(3-fluorobenzyl)piperazin-1-yl)-N-(4-
morpholinophenyl)phthalazin-1-amine (8e)
- 4-chloro-N-(4-
morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146 mmol)
was dissolved in n-butanol (1 mL) and 1-(3-fluorobenzyl)pipera-
zine (34 mg, 0.175 mmol)) was added and was heated overnight at
150 ꢀC. Next, n-butanol was concentrated and reaction was purified
by Flash chromatography on alumina. Greenish yellow solid; Yield:
25%; Rf ¼ 0.44 (50% EtOAc/Hexane on alumina);1H NMR (400 MHz,
Synthesis
of
4-(4-benzylpiperazin-1-yl)-N-(4-
4-chloro-N-(4-
morpholinophenyl)phthalazin-1-amine (8a)
-
morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146 mmol)
was dissolved in n-butanol (1 mL) and 1-benzylpiperazine
(30.8 mg, 0.175 mmol)) was added and was heated overnight at
150 ꢀC. Next, n-butanol was concentrated and reaction was purified
by Flash chromatography on alumina. Greenish yellow solid; Yield:
28%; Rf ¼ 0.39 (50% EtOAc/Hexane on alumina); 1H NMR (400 MHz,
DMSO)
d
9.06 (d, J ¼ 7.8 Hz, 1H), 8.93 (brs, 1H, NH), 8.17 (d,
J ¼ 7.8 Hz, 1H), 8.10 (p, J ¼ 8.5 Hz, 2H), 7.65 (d, J ¼ 10.1 Hz, 1H),
7.55e7.45 (m, 2H, AreH), 7.35 (d, J ¼ 8.8 Hz, 2H), 7.27 (t, J ¼ 8.0 Hz,
1H), 7.05 (d, J ¼ 9.0 Hz, 2H), 4.42 (s, 2H), 3.72 (t, J ¼ 4.0 Hz, 4H),
3.55e3.44 (m, 4H), 3.37 (brs, 4H), 3.20e2.99 (m, 4H); 13C NMR
CDCl3) d 8.05e7.93 (m, 2H, AreH), 7.78e7.71 (m, 2H, AreH), 7.49 (d,
J ¼ 8.3 Hz, 2H), 7.40e7.30 (m, 4H, AreH), 7.27 (d, J ¼ 7.1 Hz,1H), 6.90
(d, J ¼ 9.0 Hz, 2H), 3.85 (t, J ¼ 4.0 Hz, 4H), 3.64 (s, 2H), 3.37 (brs, 4H),
3.10 (t, J ¼ 4.0 Hz, 4H), 2.72 (brs, 4H); 13C NMR (101 MHz, CDCl3)
(101 MHz, DMSO)
d 163.54, 161.11, 153.86, 150.44, 135.70, 132.64,
131.24, 126.91, 128.26, 126.91, 126.40, 126.12, 123.55, 118.80, 116.85,
116.39, 66.46, 58.24, 50.73, 48.72, 47.68; LCMS (ESI): Calculated for
[Mþ1]þ C29H31FN6O 499.26, found 499.42.
d
147.36, 137.61, 131.05, 129.29, 128.28, 127.20, 125.14, 123.60,
122.08, 116.83, 66.95, 63.09, 53.05, 51.10, 50.12; LCMS (ESI):
Calculated for [Mþ1]þ C29H32N6O 481.37, found 481.38.
Synthesis
of
4-(4-(3-chlorobenzyl)piperazin-1-yl)-N-(4-
Synthesis
of
4-(4-(2-bromobenzyl)piperazin-1-yl)-N-(4-
morpholinophenyl)phthalazin-1-amine (8f) - 4-chloro-N-(4-
morpholinophenyl)phthalazin-1-amine (8b)
-
4-chloro-N-(4-
morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146 mmol)
was dissolved in n-butanol (1 mL) and 1-(3- chlorobenzyl)pipera-
zine (36.7 mg, 0.175 mmol)) was added and was heated overnight
at 150 ꢀC. Next, n-butanol was concentrated and reaction was
morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146 mmol)
was dissolved in n-butanol (1 mL) and added 1-(2-bromobenzyl)
piperazine (44.6 mg, 0.175 mmol)) was added and was heated
9

A. Kharbanda, L. Zhang, D. Saha et al.
European Journal of Medicinal Chemistry 223 (2021) 113660
purified by Flash chromatography on alumina. Pale yellow solid;
Yield: 36%; Rf ¼ 0.46 (50% EtOAc/Hexane on alumina); 1H NMR
0.146 mmol) was dissolved in n-butanol (1 mL) and methyl 3-
(piperazin-1-ylmethyl)benzoate (41.0 mg, 0.175 mmol)) was added
and was heated overnight at 150 ꢀC. Next, n-butanol was concen-
trated and reaction was purified by Flash chromatography on
alumina. Yellow solid; Yield: 91%; Rf ¼ 0.18 (50% EtOAc/Hexane on
(400 MHz, CDCl3)
d 8.04e7.98 (m, 2H, AreH), 7.80e7.73 (m, 2H,
AreH), 7.50 (d, J ¼ 8.5 Hz, 2H), 7.37 (s, 1H, NH), 7.25 (s, 3H), 6.91 (d,
J ¼ 9.0 Hz, 2H), 3.86 (t, J ¼ 4.0 Hz, 4H), 3.59 (s, 2H), 3.37 (brs, 4H),
3.11 (t, J ¼ 4.0 Hz, 4H) 2.70 (brs, 4H); 13C NMR (101 MHz, CDCl3)
alumina); 1H NMR (400 MHz, CDCl3)
d 8.04e7.99 (m, 2H, AreH),
d
147.52, 147.38, 147.17, 140.03, 138.68, 134.16, 131.20, 131.17, 129.54,
7.98e7.94 (m, 1H, AreH), 7.95e7.91 (m, 1H, AreH), 7.78e7.71 (m,
2H, AreH), 7.61e7.57 (m, 1H, AreH), 7.53e7.46 (m, 2H, AreH), 7.40
(t, J ¼ 7.7 Hz, 1H), 6.90 (d, J ¼ 8.0 Hz, 2H), 3.90 (d, J ¼ 4.1 Hz, 3H),
3.86e3.82 (m, 4H), 3.66 (s, 2H), 3.43e3.40 (d, J ¼ 9.6 Hz, 4H),
3.11e3.07 (m, 4H), 2.75e2.64 (m, 4H); 13C NMR (101 MHz, CDCl3)
129.15, 127.34, 127.26, 125.13, 123.63, 122.24, 116.84, 66.94, 62.46,
53.06, 51.12, 50.07; LCMS (ESI): Calculated for [Mþ1]þ C29H31ClN6O
515.23, found 515.46.
Synthesis
of
N-(4-morpholinophenyl)-4-(4-(3-(tri-
fluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine (8g) -
4-chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg,
0.146 mmol) was dissolved in n-butanol (1 mL) and 1-(3- (tri-
fluoromethyl)benzyl)piperazine (42.7 mg, 0.175 mmol)) was added
and was heated overnight at 150 ꢀC. Next, n-butanol was concen-
trated and reaction was purified by Flash chromatography on
alumina. Yellow solid; Yield: 65%; Rf ¼ 0.42 (50% EtOAc/Hexane on
d 167.15, 149.81, 147.35, 138.40, 133.77, 131.33, 131.06, 131.02, 130.25,
130.14, 128.45, 128.40, 127.89, 125.11, 123.58, 122.07, 116.82, 66.94,
62.67, 53.11, 52.10, 51.18, 50.11; IR Absorption nmax/cmꢂ1 3189.21
(NH), 2840.41 (AreH), 1713.53 (C]O), 1287.13 (CeN aromatic),
1234.73 (CeN aliphatic), 1198.11 (CeO ester), 1121.27 (CeO ether);
LCMS (ESI): Calculated for [Mþ1]þ C31H34N6O3 539.21, found
539.27.
alumina); 1H NMR (400 MHz, CDCl3)
d
8.05e7.98 (m, 2H, AreH),
Synthesis of 4-(4-(3-methoxybenzyl)piperazin-1-yl)-N-(4-
7.79e7.73 (m, 2H, AreH), 7.62 (s, 1H, NH), 7.58 (d, J ¼ 7.6 Hz, 1H),
7.51 (t, J ¼ 7.6 Hz, 3H), 7.44 (t, J ¼ 7.6 Hz, 1H), 6.90 (d, J ¼ 8.9 Hz, 2H),
3.85 (t, J ¼ 4.0 Hz, 4H), 3.66 (s, 2H), 3.37 (brs, 4H), 3.10 (t, J ¼ 4.0 Hz,
morpholinophenyl)phthalazin-1-amine (8k) - 4-chloro-N-(4-
morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146 mmol)
was dissolved in n-butanol (1 mL) and 1-(3-methoxybenzyl)
piperazine (36.1 mg, 0.175 mmol)) was added and was heated
overnight at 150 ꢀC. Next, n-butanol was concentrated and reaction
was purified by Flash chromatography on alumina. Pale yellow
solid; Yield: 36%; Rf ¼ 0.46 (50% EtOAc/Hexane on alumina);1H
4H), 2.71 (brs, 4H); 13C NMR (101 MHz, CDCl3)
d 147.49, 139.11,
132.39, 131.18, 131.15, 130.76, 130.44, 128.74, 125.68, 125.11, 124.01,
123.63, 122.83, 122.21, 116.82, 66.94, 62.52, 53.11, 51.16, 50.07; IR
Absorption nmax/cmꢂ1 3314.72 (NH), 2961.11, 2920.26, 2849.86
(AreH), 1325.63 (CeN aromatic), 1112.64 (CeO aliphatic ether),
1070.03 (CeN aliphatic); LCMS (ESI): Calculated for [Mþ1]þ
C30H31F3N6O 549.25, found 549.50.
NMR (400 MHz, CDCl3)
d 8.04e7.98 (m, 2H, AreH), 7.97e7.91 (m,
2H, AreH), 7.50 (d, J ¼ 8.1 Hz, 2H), 7.26e7.20 (m, 1H, AreH), 6.94 (d,
J ¼ 7.5 Hz, 2H), 6.88 (d, J ¼ 9.0 Hz, 2H), 6.82e6.77 (m, 1H), 3.85 (t,
J ¼ 4.0 Hz, 4H), 3.80 (s, 3H), 3.60 (s, 2H), 3.36 (brs, 4H), 3.08 (t,
Synthesis of N-(4-morpholinophenyl)-4-(4-(3-nitrobenzyl)
piperazin-1-yl)phthalazin-1-amine
(8h)
-
4-chloro-N-(4-
J ¼ 4.0 Hz, 4H), 2.70 (brs, 4H); 13C NMR (101 MHz, CDCl3)
d 159.63,
morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146 mmol)
was dissolved in n-butanol (1 mL) and 1-(3-nitrobenzyl)piperazine
(38.7 mg, 0.175 mmol)) was added and was heated overnight at
150 ꢀC. Next, n-butanol was concentrated and reaction was purified
by Flash chromatography on alumina. Yellow solid; Yield: 65%;
Rf ¼ 0.42 (50% EtOAc/Hexane on alumina); 1H NMR (400 MHz,
147.24, 139.60, 131.00, 130.93, 129.17, 125.11, 123.56, 121.99, 121.51,
116.80, 114.51, 112.65, 66.95, 63.04, 55.20, 53.15, 51.24, 50.14; LCMS
(ESI): Calculated for [Mþ1]þ C30H34N6O2 511.28, found 511.38.
Synthesis
of
N-(4-morpholinophenyl)-4-(4-(3-(tri-
fluoromethoxy)benzyl)piperazin-1-yl)phthalazin-1-amine (8l) -
4-chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg,
0.146 mmol) was dissolved in n-butanol (1 mL) and 1-(3-(tri-
fluoromethoxy)benzyl)piperazine (45.5 mg, 0.175 mmol)) was
added and was heated overnight at 150 ꢀC. Next, n-butanol was
concentrated and reaction was purified by Flash chromatography
on alumina. Dark Yellow solid; Yield: 65%; Rf ¼ 0.42 (50% EtOAc/
CDCl3)
d
8.22 (s, 1H, NH), 8.10 (d, J ¼ 8.1 Hz, 1H), 8.01 (d, J ¼ 6.9 Hz,
1H), 7.96 (d, J ¼ 6.4 Hz, 1H), 7.77e7.69 (m, 3H, AreH), 7.55e7.45 (m,
3H, AreH), 6.88 (d, J ¼ 6.6 Hz, 2H), 3.84 (brs, 4H), 3.69 (s, 2H), 3.36
(brs, 4H), 3.08 (brs, 4H), 2.71 (brs, 4H); 13C NMR (101 MHz, CDCl3)
d
148.31, 147.29, 140.52, 135.11, 131.08, 131.01, 129.23, 125.01, 123.78,
123.50,122.26,122.03,116.78, 66.94, 62.16, 53.17, 51.18, 50.97, 50.11;
LCMS (ESI): Calculated for [Mþ1]þ C29H31N7O3 526.26, found
526.41.
Hexane on alumina);1H NMR (400 MHz, CDCl3)
d 8.03e7.97 (m, 2H,
AreH), 7.74 (p, J ¼ 6.9 Hz, 2H), 7.50 (d, J ¼ 8.8 Hz, 2H), 7.39 (d,
J ¼ 8.5 Hz, 2H), 7.16 (d, J ¼ 8.4 Hz, 2H), 6.88 (d, J ¼ 8.9 Hz, 2H),
3.88e3.80 (m, 4H), 3.60 (s, 2H), 3.36 (brs, 4H), 3.13e3.04 (m, 4H),
Synthesis of 3-((4-(4-((4-morpholinophenyl)amino)phthala-
zin-1-yl)piperazin-1-yl)methyl)benzonitrile (8i) - 4-chloro-N-(4-
morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146 mmol)
was dissolved in n-butanol (1 mL) and 3-(piperazin-1-ylmethyl)
benzonitrile (35.1 mg, 0.175 mmol)) was added and was heated
overnight at 150 ꢀC. Next, n-butanol was concentrated and reaction
was purified by Flash chromatography on alumina. Brown solid;
Yield: 83%; Rf ¼ 0.14 (50% EtOAc/Hexane on alumina); 1H NMR
2.69 (brs, 4H); 13C NMR (101 MHz, CDCl3)
d 148.30, 148.28, 147.38,
136.70, 131.10, 131.07, 130.34, 125.06, 123.56, 122.14, 120.76, 116.79,
66.94, 62.21, 53.10, 51.16, 50.09; IR Absorption nmax/cmꢂ1 3320.08
(NH), 2817.04 (AreH), 1254.94 (CeN aromatic), 1220.67 (CeO, alkyl
aryl ether), 1117.51 (CeO aliphatic ether), 1157.38 (CeN aliphatic);
LCMS (ESI): Calculated for [Mþ1]þ C30H31F3N6O2 565.25, found
565.45.
(400 MHz, CDCl3)
d
8.06e7.92 (m, 2H, AreH), 7.82e7.71 (m, 2H,
Synthesis of 4-(4-(3-methylbenzyl)piperazin-1-yl)-N-(4-
morpholinophenyl)phthalazin-1-amine (8m) - 4-chloro-N-(4-
morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146 mmol)
was dissolved in n-butanol (1 mL) and 1-(3-methylbenzyl)pipera-
zine (33.3 mg, 0.175 mmol)) was added and was heated overnight
at 150 ꢀC. Next, n-butanol was concentrated and reaction was pu-
rified by Flash chromatography on alumina. Yellow solid; Yield:
48%; Rf ¼ 0.46 (50% EtOAc/Hexane on alumina); 1H NMR (400 MHz,
AreH), 7.66 (s, 1H, NH), 7.61 (d, J ¼ 7.9 Hz, 1H), 7.56e7.47 (m, 3H,
AreH), 7.44e7.39 (m, 1H, AreH), 6.87 (d, J ¼ 10.4 Hz, 2H), 3.92e3.76
(m, 4H), 3.62 (s, 2H), 3.45e3.25 (m, 4H), 3.16e3.03 (m, 4H),
2.78e2.60 (m, 4H); 13C NMR (101 MHz, CDCl3)
d 155.48, 149.88,
147.36, 142.55, 139.82, 133.43, 132.45, 131.09, 131.05, 130.95, 130.88,
130.12, 129.11, 125.02, 123.53, 122.10, 118.91, 116.79, 112.34, 66.93,
62.17, 53.15, 51.16, 50.09. LCMS (ESI): Calculated for [Mþ1]þ
C30H31N7O 506.26, found 506.41.
CDCl3)
d 8.05e7.98 (m, 1H, AreH), 7.94 (brs, 1H, NH), 7.78e7.68 (m,
Synthesis of methyl 3-((4-(4-((4-morpholinophenyl)amino)
phthalazin-1-yl)piperazin-1-yl)methyl)benzoate (8j) - 4-chloro-
2H, AreH), 7.49 (d, J ¼ 7.7 Hz, 2H), 7.23e7.14 (m, 3H, AreH), 7.07 (d,
J ¼ 7.3 Hz,1H), 6.89 (d, J ¼ 9.0 Hz, 2H), 3.85 (t, J ¼ 4.7 Hz, 4H), 3.58 (s,
2H), 3.36 (brs, 4H), 3.09 (t, J ¼ 4.7 Hz, 4H), 2.70 (brs, 4H), 2.34 (s,
N-(4-morpholinophenyl)phthalazin-1-amine
(7)
(50
mg,
10

A. Kharbanda, L. Zhang, D. Saha et al.
European Journal of Medicinal Chemistry 223 (2021) 113660
3H); 13C NMR (101 MHz, CDCl3)
130.92, 130.02, 128.11, 127.87, 126.37, 125.13, 121.98, 116.82, 66.95,
63.17, 53.18, 51.20, 50.15, 21.40; LCMS (ESI): Calculated for [Mþ1]þ
C30H34N6O 495.28, found 495.44.
Synthesis of 4-(4-(3,4-dichlorobenzyl)piperazin-1-yl)-N-(4-
morpholinophenyl)phthalazin-1-amine (8n)
d
147.24, 137.85, 137.71, 130.99,
160.15, 149.97, 148.47, 132.25, 131.75, 128.06, 124.26, 116.82, 113.41,
103.67, 66.87 (2C), 54.39 (2C), 52.20 (2C), 50.58 (2C), 49.73; LCMS
(ESI): Calculated for [Mþ1]þ C29H30F2N6O 517.25, found 517.39.
Synthesis of 4-(4-(3-chloro-4-fluorobenzyl)piperazin-1-yl)-
N-(4-morpholinophenyl)phthalazin-1-amine (8r) - 4-chloro-N-
(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146 mmol)
was dissolved in n-butanol (1 mL) and 1-(3-chloro-4-fluorobenzyl)
piperazine (40 mg, 0.175 mmol)) was added and was heated
overnight at 150 ꢀC. Next, n-butanol was concentrated and reaction
was purified by Flash chromatography on alumina. Light Yellow
solid; Yield: 77%; Rf ¼ 0.40 (50% EtOAc/Hexane on alumina); 1H
- 4-chloro-N-(4-
morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146 mmol)
was dissolved in n-butanol (1 mL) and 1-(3,4-dichlorobenzyl)
piperazine (42.9 mg, 0.175 mmol)) was added and was heated
overnight at 150 ꢀC. Next, n-butanol was concentrated and reaction
was purified by Flash chromatography on alumina. Yellow solid;
Yield: 65%; Rf ¼ 0.42 (50% EtOAc/Hexane on alumina); 1H NMR
NMR (400 MHz, CDCl3)
d
7.97 (d, J ¼ 17.9 Hz, 2H), 7.80e7.70 (m, 2H,
(400 MHz, CDCl3)
d
8.14e8.08 (m, 1H, AreH), 8.03e7.98 (m, 1H,
AreH), 7.50 (d, J ¼ 8.5 Hz, 2H), 7.41 (dd, J ¼ 7.2, 2.1 Hz,1H), 7.24e7.19
(m, 1H, AreH), 7.08 (t, J ¼ 8.7 Hz, 1H), 6.89 (d, J ¼ 8.9 Hz, 2H),
3.89e3.79 (m, 4H), 3.54 (brs, 2H), 3.35 (s, 4H), 3.14e3.05 (m, 4H),
AreH), 7.81e7.73 (m, 2H, AreH), 7.56e7.43 (m, 3H, AreH), 7.39 (d,
J ¼ 8.2 Hz, 1H), 7.22 (d, J ¼ 8.2 Hz, 1H), 6.90 (d, J ¼ 8.8 Hz, 2H),
3.90e3.79 (m, 4H), 3.56 (s, 2H), 3.36 (brs, 4H), 3.16e3.04 (m, 4H),
2.67 (brs, 4H); 13C NMR (101 MHz, CDCl3)
d 158.44, 155.97, 147.33,
2.69 (brs, 4H); 13C NMR (101 MHz, CDCl3)
d
147.74, 138.30, 132.33,
135.22, 135.18, 131.07, 131.01, 130.98, 128.64, 128.57, 125.06, 123.54,
122.04, 120.76, 120.59, 116.81, 116.38, 116.17, 77.32, 77.21, 77.00,
76.68, 66.95, 61.83, 53.07, 51.18, 50.12, 15.26; LCMS (ESI): Calculated
for [Mþ1]þ C29H30ClFN6O 533.22, found 533.47.
131.46, 131.31, 131.06, 130.87, 130.26, 128.37, 125.10, 123.69, 122.72,
122.70, 122.51, 116.80, 66.92, 61.78, 52.99, 51.08, 49.97; LCMS (ESI):
Calculated for [Mþ1]þ C29H30Cl2N6O 549.20, found 549.73.
Synthesis of 4-(4-(3,4-difluorobenzyl)piperazin-1-yl)-N-(4-
Synthesis of N-(2-methoxyethyl)-4-(4-(3-(trifluoromethyl)
benzyl)piperazin-1-yl)phthalazin-1-amine(10a) - 4-chloro-N-(2-
methoxyethyl)phthalazin-1-amine (9a) (50 mg, 0.11 mmol) was
dissolved in n-butanol (1 mL) and 1-(3-(trifluoromethyl)benzyl)
piperazine (32.9 mg, 0.13 mmol) was added and was heated over-
night at 150 ꢀC. Next, n-butanol was concentrated and reaction was
purified by Flash chromatography on alumina. Dark yellow solid;
Yield: 49%; Rf ¼ 0.33 (50% EtOAc/Hexane on alumina); 1H NMR
morpholinophenyl)phthalazin-1-amine (8o)
- 4-chloro-N-(4-
morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146 mmol)
was dissolved in n-butanol (1 mL) and 1-(3,4-difluorobenzyl)
piperazine (37.1 mg, 0.175 mmol)) was added and was heated
overnight at 150 ꢀC. Next, n-butanol was concentrated and reaction
was purified by Flash chromatography on alumina. Off-white solid;
Yield: 53%; Rf ¼ 0.36 (50% EtOAc/Hexane on alumina); 1H NMR
(400 MHz, CDCl3)
d
8.02e7.94 (m, 2H, AreH), 7.78e7.72 (m, 2H,
(400 MHz, CDCl3)
d
8.01 (t, J ¼ 4.1 Hz, 1H), 7.82 (brs, 1H, NH),
AreH), 7.50 (d, J ¼ 8.5 Hz, 2H), 7.21 (dd, J ¼ 9.6, 8.1 Hz,1H), 7.11e7.04
(m, 2H, AreH), 6.90 (d, J ¼ 9.0 Hz, 2H), 3.87e3.83 (m, 4H), 3.55 (s,
2H), 3.35 (brs, 4H), 3.12e3.07 (m, 4H), 2.68 (brs, 4H); 13C NMR
7.77e7.69 (m, 2H, AreH), 7.62 (s, 1H), 7.58 (d, J ¼ 6.7 Hz,1H), 7.51 (d,
J ¼ 7.5 Hz, 1H), 7.44 (t, J ¼ 7.3 Hz, 1H), 3.85 (s, 2H), 3.71 (t, J ¼ 4.9 Hz,
2H), 3.66 (s, 2H), 3.40 (s, 3H), 3.36 (s, 4H), 2.70 (s, 4H); 13C NMR
(101 MHz, CDCl3)
d
149.03, 148.97, 148.17, 147.38, 135.24, 135.21,
(101 MHz, CDCl3) d 160.70, 155.40, 151.78, 139.13, 138.75, 132.39,
135.17, 135.15, 131.09, 131.04, 125.07, 124.72, 124.67, 124.65, 123.56,
122.08, 117.68, 116.84, 66.95 (2C), 61.96 (2C), 53.06 (2C), 51.18 (2C),
50.11; LCMS (ESI): Calculated for [Mþ1]þ C29H30F2N6O 517.25,
found 517.36.
132.17, 131.26, 130.75, 130.43, 128.81, 128.73, 125.71, 125.67, 125.53,
125.51, 125.47, 124.99, 124.22, 124.18, 124.02, 123.98, 123.36, 122.82,
121.13, 62.52, 58.86, 53.11, 51.18, 45.57, 39.89.; LCMS (ESI): Calcu-
lated for [Mþ1]þ C23H26F3N5O 446.21, found 446.26.
Synthesis of 4-(4-(2,6-dichlorobenzyl)piperazin-1-yl)-N-(4-
Synthesis of N1,N1-dimethyl-N2-(4-(4-(3-(trifluoromethyl)
benzyl)piperazin-1-yl)phthalazin-1-yl)ethane-1,2-diamine(10b)
- N1-(4-chlorophthalazin-1-yl)-N2,N2-dimethylethane-1,2-diamine
(9b) (50 mg, 0.11 mmol) was dissolved in n-butanol (1 mL) and
1-(3-(trifluoromethyl)benzyl)piperazine (32.9 mg, 0.13 mmol) was
added and was heated overnight at 150 ꢀC. Next, n-butanol was
concentrated and reaction was purified by Flash chromatography
on alumina. Yellow solid; Yield: 42%; Rf ¼ 0.07 (50% EtOAc/Hexane
morpholinophenyl)phthalazin-1-amine (8p)
- 4-chloro-N-(4-
morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146 mmol)
was dissolved in n-butanol (1 mL) and 1-(2,6-dichlorobenzyl)
piperazine (42.9 mg, 0.175 mmol)) was added and was heated
overnight at 150 ꢀC. Next, n-butanol was concentrated and reaction
was purified by Flash chromatography on alumina. Yellow solid;
Yield: 69%; Rf ¼ 0.61 (50% EtOAc/Hexane on alumina); 1H NMR
(400 MHz, CDCl3)
d
7.66 (d, J ¼ 7.6 Hz, 1H), 7.61 (d, J ¼ 7.6 Hz, 1H),
on alumina); 1H NMR (400 MHz, CDCl3)
d 8.12e8.04 (m, 1H, AreH),
7.37 (p, J ¼ 8Hz, 2H), 7.10 (d, J ¼ 8.4 Hz, 2H), 6.92 (d, J ¼ 8.0 Hz, 2H),
6.75 (t, J ¼ 8.0 Hz, 1H), 6.51 (d, J ¼ 8.7 Hz, 2H), 3.50e3.44 (m, 6H),
3.00e2.85 (brm, 4H), 2.71 (t, J ¼ 4 Hz, 4H), 2.44 (t, J ¼ 4 Hz, 4H). 13C
8.04e7.95 (m, 1H, AreH), 7.80e7.70 (m, 2H, AreH), 7.62 (s, 1H), 7.58
(d, J ¼ 7.9 Hz, 1H), 7.51 (d, J ¼ 7.7 Hz, 1H), 7.44 (t, J ¼ 7.6 Hz, 1H), 3.80
(brs, 2H), 3.66 (s, 2H), 3.35 (brs, 4H), 2.85 (s, 2H), 2.70 (brs, 4H), 2.44
NMR (101 MHz, CDCl3)
d
147.40, 137.02, 134.19, 131.11, 131.06,
(s, 6H); 13C NMR (101 MHz, CDCl3)
d 155.46, 152.07, 139.28, 132.37,
128.83, 128.35, 125.22, 123.67, 122.13, 116.85, 66.95, 56.50, 53.01,
51.24, 50.10; LCMS (ESI): Calculated for [Mþ1]þ C29H30Cl2N6O
549.20, found 549.60.
131.09, 130.57, 128.70, 125.68, 124.71, 123.95, 123.26, 122.55, 121.09,
62.57, 58.23, 53.22 (2C), 51.25 (2C), 44.80 (2C), 38.67 (2C), 29.68;
LCMS (ESI): Calculated for [Mþ1]þ C24H29F3N6 459.24, found
459.14.
Synthesis of 4-(4-(2,4-difluorobenzyl)piperazin-1-yl)-N-(4-
morpholinophenyl)phthalazin-1-amine (8q)
-
4-chloro-N-(4-
Synthesis of 1-(4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-
yl)phthalazin-1-yl)piperidin-4-ol (10c) - 1-(4-chlorophthalazin-
1-yl)piperidin-4-ol (9c) (50 mg, 0.11 mmol) was dissolved in n-
butanol (1 mL) and 1-(3-(trifluoromethyl)benzyl)piperazine
(32.9 mg, 0.13 mmol) was added and was heated overnight at
150 ꢀC. Next, n-butanol was concentrated and reaction was purified
by Flash chromatography on alumina. Orange solid; Yield: 65%;
Rf ¼ 0.12 (50% EtOAc/Hexane on alumina); 1H NMR (400 MHz,
morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146 mmol)
was dissolved in n-butanol (1 mL) and 1-(2,4-difluorobenzyl)
piperazine (37.1 mg, 0.175 mmol)) was added and was heated
overnight at 150 ꢀC. Next, n-butanol was concentrated and reaction
was purified by Flash chromatography on alumina. White solid;
Yield: 31%; Rf ¼ 0.36 (50% EtOAc/Hexane on alumina);1H NMR
(400 MHz, CDCl3)
d 8.30 (s, 1H, NH), 8.02e7.98 (m, 1H, AreH),
7.84e7.79 (m, 2H, AreH), 7.47 (d, J ¼ 8.8 Hz, 3H), 6.93 (d, J ¼ 9.0 Hz,
CDCl3) d 8.02e7.96 (m, 2H, AreH), 7.77e7.70 (m, 2H, AreH), 7.62 (s,
5H), 3.86 (t, J ¼ 8.8 Hz, 4H), 3.73 (s, 2H), 3.42 (brs, 4H), 3.13 (t,
1H), 7.59 (d, J ¼ 7.6 Hz, 1H), 7.52 (d, J ¼ 7.7 Hz, 1H), 7.44 (t, J ¼ 7.6 Hz,
J ¼ 4.0 Hz, 4H), 2.80 (brs, 4H); 13C NMR (101 MHz, CDCl3)
d 167.20,
1H), 3.97e3.90 (m, 1H), 3.75e3.67 (m, 2H), 3.46 (brs, 4H), 3.15 (t,
11

A. Kharbanda, L. Zhang, D. Saha et al.
European Journal of Medicinal Chemistry 223 (2021) 113660
J ¼ 10.4 Hz, 2H), 2.72 (brs, 4H), 2.15e2.05 (m, 2H), 1.90e1.66 (m,
on alumina. Brown solid; Yield: 53%; Rf ¼ 0.03 (50% EtOAc/Hexane
4H); 13C NMR (101 MHz, CDCl3)
d
158.34, 157.82, 139.02, 132.46,
on alumina); 1H NMR (400 MHz, DMSO‑d6)
d 8.40e8.34 (m, 1H,
130.79,130.59,128.76,125.75,125.73,125.09,124.93,124.16,124.05,
123.97, 123.74, 68.24 (2C), 62.53 (2C), 53.05 (2C), 50.91, 49.05 (2C),
34.68 (2C); LCMS (ESI): Calculated for [Mþ1]þ C25H28F3N5O 472.23,
found 472.39.
AreH), 8.08e8.02 (m, 1H, AreH), 8.01e7.96 (m, 2H, AreH),
7.89e7.81 (m, 1H, AreH), 7.78 (brs, 1H), 7.68 (d, J ¼ 8.0Hz 1H),
7.65e7.56 (m, 1H, AreH), 3.68 (s, 4H), 3.42 (s, 4H), 3.17 (s, 2H), 2.90
(s, 2H), 2.74 (brs, 2H), 2.66 (brs, 2H), 2.35e2.27 (m, 1H), 1.76 (d,
J ¼ 11.4 Hz, 3H), 1.57e1.36 (m, 3H); 13C NMR (101 MHz, DMSO‑d6)
Synthesis
fluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine (10d) -
4-chloro-N-(1-methylpiperidin-4-yl)phthalazin-1-amine (9d)
of
N-(1-methylpiperidin-4-yl)-4-(4-(3-(tri-
d
154.48, 152.41, 144.50, 140.17, 133.36, 132.98, 129.73, 125.67,
125.63, 124.91, 124.58, 124.21, 123.33, 120.64, 120.43, 66.01 (2C),
61.76 (2C), 56.68 (2C), 56.32 (2C), 53.11, 51.54, 51.23, 34.10; LCMS
(ESI): Calculated for [Mþ1]þ C27H33F3N6O 515.27, found 515.20.
Synthesis of 4-(2-((4-(4-(3-(trifluoromethyl)benzyl)piperazin-
(50 mg, 0.10 mmol) was dissolved in n-butanol (1 mL) and 1-(3-
(trifluoromethyl)benzyl)piperazine (29.3 mg, 0.12 mmol) was
added and was heated overnight at 150 ꢀC. Next, n-butanol was
concentrated and reaction was purified by Flash chromatography
on alumina. Brown solid; Yield: 82%; Rf ¼ 0.03 (50% EtOAc/Hexane
1-yl)phthalazin-1-yl)amino)ethyl)thiomorpholine
1,1-dioxide
(10 h) - 4-(2-((4-chlorophthalazin-1-yl)amino)ethyl)thiomorpho-
line 1,1-dioxide (9 h) (50 mg, 0.091 mmol) was dissolved in n-
butanol (1 mL) and 1-(3-(trifluoromethyl)benzyl)piperazine
(26.6 mg, 0.109 mmol) was added and was heated overnight at
150 ꢀC. Next, n-butanol was concentrated and reaction was purified
by Flash chromatography on alumina. Golden solid; Yield: 62%;
Rf ¼ 0.06 (50% EtOAc/Hexane on alumina); 1H NMR (400 MHz,
on alumina); 1H NMR (400 MHz, CDCl3)
d 8.02e7.95 (m, 1H, AreH),
7.78e7.65 (m, 3H, AreH), 7.59 (s, 1H, NH), 7.55 (d, J ¼ 7.5 Hz, 1H),
7.48 (d, J ¼ 7.8 Hz, 1H), 7.41 (t, J ¼ 7.7 Hz, 1H), 4.27 (brs, 1H), 3.63 (s,
2H), 3.32 (brs, 4H), 2.95e2.91 (m, 2H), 2.67 (brs, 4H), 2.33 (s, 3H),
2.24 (t, J ¼ 3 Hz, 4H), 1.68 (dd, J ¼ 22.0, 10.5 Hz, 2H); 13C NMR
(101 MHz, CDCl3)
d 155.30, 151.12, 139.28, 132.37, 132.36, 130.68,
130.66, 130.35, 128.68, 125.65, 125.54, 124.89, 124.18, 123.91, 123.17,
122.83, 121.10, 120.63, 62.54, 54.70, 53.24, 51.28, 47.46, 45.84,
32.00; LCMS (ESI): Calculated for [Mþ1]þ C26H31F3N6 485.26, found
485.36.
CDCl3) d 8.05e8.01 (m, 1H, AreH), 7.81e7.73 (m, 3H, AreH), 7.61 (s,
1H, NH), 7.57 (d, J ¼ 7.6 Hz, 1H), 7.51 (d, J ¼ 7.8 Hz, 1H), 7.44 (t,
J ¼ 7.6 Hz, 1H), 3.75 (dd, J ¼ 11.0, 5.5 Hz, 2H), 3.66 (s, 2H), 3.41e3.28
(m, 4H), 3.09 (dd, J ¼ 15.7, 6.5 Hz, 8H), 2.91 (t, J ¼ 5.9 Hz, 2H), 2.70
Synthesis of N-cyclohexyl-4-(4-(3-(trifluoromethyl)benzyl)
(brs, 4H); 13C NMR (101 MHz, CDCl3)
d 155.68,151.72,139.22,132.37,
piperazin-1-yl)phthalazin-1-amine (10e)
-
4-chloro-N-cyclo-
131.01, 130.56, 128.72, 125.67, 125.54, 124.19, 123.97, 123.18, 121.08,
120.75, 120.13, 62.55 (2C), 55.12 (2C), 53.19 (2C), 51.27 (2C), 50.66,
43.27, 38.73; LCMS (ESI): Calculated for [Mþ1]þ C26H31F3N6O2S
549.22, found 549.31.
hexylphthalazin-1-amine (9e) (50 mg, 0.106 mmol) was dissolved
in n-butanol (1 mL) and 1-(3-(trifluoromethyl)benzyl)piperazine
(31.2 mg, 0.127 mmol) was added and was heated overnight at
150 ꢀC. Next, n-butanol was concentrated and reaction was purified
by Flash chromatography on alumina. Dark yellow solid; Yield:
64%; Rf ¼ 0.82 (50% EtOAc/Hexane on alumina); 1H NMR (400 MHz,
Synthesis
fluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine (10i) -
4-chloro-N-(2-(piperidin-1-yl)ethyl)phthalazin-1-amine (9i)
of
N-(2-(piperidin-1-yl)ethyl)-4-(4-(3-(tri-
CDCl3)
d
8.01e7.90 (m, 2H, AreH), 7.77e7.67 (m, 2H, AreH), 7.60 (s,
(50 mg, 0.10 mmol) was dissolved in n-butanol (1 mL) and 1-(3-
(trifluoromethyl)benzyl)piperazine (29.4 mg, 0.12 mmol) was
added and was heated overnight at 150 ꢀC. Next, n-butanol was
concentrated and reaction was purified by Flash chromatography
on alumina. Dark yellow solid; Yield: 51%; Rf ¼ 0.08 (50% EtOAc/
1H, NH), 7.56 (d, J ¼ 7.5 Hz, 1H), 7.49 (d, J ¼ 7.6 Hz, 1H), 7.42 (t,
J ¼ 7.6 Hz, 1H), 5.39 (brs, 1H), 4.26 (s, 1H), 3.64 (s, 2H), 3.32 (s, 4H),
2.68 (s, 4H), 2.19 (d, J ¼ 10.8 Hz, 2H), 1.73 (d, J ¼ 12.4 Hz, 2H), 1.62 (d,
J ¼ 13.0 Hz, 1H), 1.45e1.29 (m, 4H), 1.24e1.16 (m, 1H); 13C NMR
(101 MHz, CDCl3)
d
154.83, 150.82, 139.10, 132.42, 131.03, 130.87,
Hexane on alumina); 1H NMR (400 MHz, CDCl3)
d 8.02e7.93 (m,
130.54, 128.72, 125.61, 125.43, 124.92, 124.18,123.97, 123.37, 121.88,
120.93, 62.50 (2C), 53.14 (2C), 51.16 (2C), 50.23 (2C), 33.22 (2C),
25.11 (2C); LCMS (ESI): Calculated for [Mþ1]þ C26H30F3N5 470.25,
found 470.35.
2H, AreH), 7.78e7.69 (m, 2H, AreH), 7.61 (s, 1H), 7.57 (d, J ¼ 7.3 Hz,
1H), 7.50 (d, J ¼ 7.5 Hz, 1H), 7.43 (t, J ¼ 7.6 Hz, 1H), 3.73 (t, J ¼ 8.0 Hz,
2H), 3.65 (s, 2H), 3.34e3.33 (m, 4H), 2.77 (t, J ¼ 6.0 Hz, 2H), 2.69
(brs, 3H), 2.55 (brs, 4H), 1.70e1.58 (m, 4H), 1.48 (brs, 2H), 1.23 (s,
Synthesis of 1-(piperidin-1-yl)-4-(4-(3-(trifluoromethyl)
benzyl)piperazin-1-yl)phthalazine (10f) - 1-chloro-4-(piperidin-
1-yl)phthalazine (9f) (50 mg, 0.11 mmol) was dissolved in n-
butanol (1 mL) and 1-(3-(trifluoromethyl)benzyl)piperazine
(32.9 mg, 0.13 mmol) was added and was heated overnight at
150 ꢀC. Next, n-butanol was concentrated and reaction was purified
by Flash chromatography on alumina. Yellow solid; Yield: 71%;
Rf ¼ 0.85 (50% EtOAc/Hexane on alumina); 1H NMR (400 MHz,
1H); 13C NMR (101 MHz, CDCl3)
d 155.25, 152.25, 139.26, 132.39,
130.90, 130.83, 130.54, 128.70, 125.69, 124.71, 124.19, 123.93, 123.21,
122.01, 121.08, 62.58 (2C), 57.14 (2C), 54.13 (2C), 53.24 (2C), 51.23,
38.00, 25.58, 24.02; LCMS (ESI): Calculated for [Mþ1]þ C27H33F3N6
499.28, found 499.23.
Synthesis of N-(1H-pyrazol-5-yl)-4-(4-(3-(trifluoromethyl)
benzyl)piperazin-1-yl)phthalazin-1-amine (10j)
- 4-chloro-N-
(1H-pyrazol-5-yl)phthalazin-1-amine (9j) (50 mg, 0.11 mmol) was
dissolved in n-butanol (1 mL) and 1-(3-(trifluoromethyl)benzyl)
piperazine (32.9 mg, 0.13 mmol) was added and was heated over-
night at 150 ꢀC. Next, n-butanol was concentrated and reaction was
purified by Flash chromatography on alumina. Bright yellow solid;
Yield: 54%; Rf ¼ 0.55 (50% EtOAc/Hexane on alumina); 1H NMR
CDCl3) d 8.03e7.96 (m, 2H, AreH), 7.75e7.69 (m, 2H, AreH), 7.62 (s,
1H, NH), 7.59 (d, J ¼ 7.6 Hz, 1H), 7.51 (d, J ¼ 7.8 Hz, 1H), 7.44 (t,
J ¼ 7.6 Hz, 1H), 3.66 (s, 2H), 3.45 (brs, 4H), 3.37e3.28 (m, 4H), 2.72
(brs, 4H), 1.85e1.75 (m, 4H), 1.72e1.64 (m, 2H); 13C NMR (101 MHz,
CDCl3)
d 159.00, 157.59, 132.48, 131.28, 130.61, 130.60, 130.58,
128.77, 125.77, 125.30, 124.81, 124.11, 124.16, 124.07, 123.75, 62.52
(2C), 53.04 (2C), 52.47 (2C), 50.86 (2C), 26.14, 24.66; LCMS (ESI):
Calculated for [Mþ1]þ C25H28F3N5 456.23, found 456.36.
(400 MHz, CDCl3)
d
8.65 (d, J ¼ 6.9 Hz, 1H), 7.88e7.81 (m, 1H,
AreH), 7.74e7.64 (m, 2H, AreH), 7.62 (brs, 1H, NH), 7.57 (d,
J ¼ 7.6 Hz, 1H), 7.52 (d, J ¼ 7.8 Hz, 1H), 7.49 (d, J ¼ 2.4 Hz, 1H), 7.44 (t,
J ¼ 7.6 Hz, 1H), 6.38 (s, 1H), 3.66 (s, 2H), 3.25 (brs, 4H), 2.68 (brs,
Synthesis of 1-(2-((4-(4-(3-(trifluoromethyl)benzyl)piper-
azin-1-yl)phthalazin-1-yl)amino)ethyl)piperidin-4-ol (10g) - 1-
4H); 13C NMR (101 MHz, CDCl3)
d 146.61, 139.14, 132.34, 131.36,
(2-((4-chlorophthalazin-1-yl)amino)ethyl)piperidin-4-ol
(9g)
131.29, 130.64, 130.48, 129.35, 128.74, 125.65, 125.53, 124.47, 124.37,
124.32, 124.08, 124.02, 123.97, 122.82, 62.50 (2C), 53.03 (2C), 50.88;
LCMS (ESI): Calculated for [Mþ1]þ C23H22F3N7 454.19, found
454.32.
(50 mg, 0.097 mmol) was dissolved in n-butanol (1 mL) and 1-(3-
(trifluoromethyl)benzyl)piperazine (28.4 mg, 0.116 mmol) was
added and was heated overnight at 150 ꢀC. Next, n-butanol was
concentrated and reaction was purified by Flash chromatography
12

A. Kharbanda, L. Zhang, D. Saha et al.
European Journal of Medicinal Chemistry 223 (2021) 113660
Synthesis of 4-((4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-
yl)phthalazin-1-yl)amino)phenol (10k) - 4-((4-chlorophthalazin-
1-yl)amino)phenol (9k) (50 mg, 0.11 mmol) was dissolved in n-
butanol (1 mL) and 1-(3-(trifluoromethyl)benzyl)piperazine
(32.9 mg, 0.13 mmol) was added and was heated overnight at
150 ꢀC. Next, n-butanol was concentrated and reaction was purified
by Flash chromatography on alumina. Yellow solid; Yield: 76%;
Rf ¼ 0.08 (50% EtOAc/Hexane on alumina); 1H NMR (400 MHz,
124.97, 124.19 (q, J ¼ 271 Hz), 123.95 (q, J ¼ 4 Hz), 123.50, 121.99,
121.85, 117.14, 62.53, 55.06, 53.17, 51.21, 49.77, 46.01; IR Absorption
nmax/cmꢂ1 3303.42 (NH), 2922.50, 2808.82 (AreH), 1325.60 (CeN,
aromatic), 1159.61 (CeF), 1091.85 (CeN aliphatic); LCMS (ESI):
Calculated for [Mþ1]þ C31H34F3N7 562.29, found 562.44.
Synthesis of N-(4-(4-ethylpiperazin-1-yl)phenyl)-4-(4-(3-
(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine
(10o) - 4-chloro-N-(4-(4-ethylpiperazin-1-yl)phenyl)phthalazin-1-
amine (9o) (50 mg, 0.087 mmol) was dissolved in n-butanol (1 mL)
CDCl3) d 8.04e7.96 (m, 1H, AreH), 7.78e7.73 (m, 1H, AreH), 7.56 (s,
2H), 7.48 (d, J ¼ 7.9 Hz, 4H), 7.41 (dd, J ¼ 14.5, 6.8 Hz, 3H), 6.88 (d,
and
1-(3-(trifluoromethyl)benzyl)piperazine
(25.5
mg,
J ¼ 8.4 Hz, 1H), 3.51 (s, 2H), 2.96e2.83 (m, 4H), 2.44 (brs, 4H); 13C
0.104 mmol) was added and was heated overnight at 150 ꢀC. Next,
n-butanol was concentrated, and reaction was purified by Flash
chromatography on alumina. Dark green solid; Yield: 56%; Rf ¼ 0.28
(50% EtOAc/Hexane on alumina); 1H NMR (400 MHz, CDCl3)
NMR (101 MHz, DMSO)
d 155.45, 152.96, 151.17, 140.18, 133.48,
132.99, 131.77, 131.49, 129.75, 129.41, 126.11, 125.65, 124.63, 124.22,
123.71, 123.40, 123.14, 122.80, 120.91, 115.20, 61.76 (2C), 53.09 (2C),
51.52; LCMS (ESI): Calculated for [Mþ1]þ C26H24F3N5O 480.20,
found 480.38.
d
8.05e7.97 (m, 1H, AreH), 7.76e7.72 (m, 2H, AreH), 7.62 (s, 1H,
NH), 7.57 (d, J ¼ 7.6 Hz, 1H), 7.51 (d, J ¼ 8.0 Hz, 3H),7.46e7.34 (m,
2H), 6.93 (d, J ¼ 9.0 Hz, 2H), 3.66 (s, 2H), 3.36 (brs, 4H), 3.23e3.15
(m, 5H), 2.75e2.65 (m, 8H), 2.52 (q, J ¼ 8.0 Hz, 4H); 13C NMR
Synthesis of N-(4-methoxyphenyl)-4-(4-(3-(trifluoromethyl)
benzyl)piperazin-1-yl)phthalazin-1-amine (10l) - 4-chloro-N-(4-
methoxyphenyl)phthalazin-1-amine (9l) (50 mg, 0.10 mmol) was
dissolved in n-butanol (1 mL) and 1-(3-(trifluoromethyl)benzyl)
piperazine (29.4 mg, 0.12 mmol) was added and was heated over-
night at 150 ꢀC. Next, n-butanol was concentrated and reaction was
purified by Flash chromatography on alumina. Yellow solid; Yield:
69%; Rf ¼ 0.82 (50% EtOAc/Hexane on alumina); 1H NMR (400 MHz,
(101 MHz, CDCl3)
d 147.21, 139.24, 132.35, 131.02, 130.93, 130.73,
130.57 (q, J ¼ 32 Hz), 128.71, 125.67 (q, J ¼ 4 Hz), 125.07, 124.18 (q,
J ¼ 271 Hz), 123.97 (q, J ¼ 4 Hz), 121.96, 118.74, 117.30, 116.15, 62.55,
53.18, 52.70, 52.34, 51.21, 49.70, 11.71; IR Absorption nmax/cmꢂ1
3309.53 (NH), 2920.83, 2850.03 (AreH), 1326.05 (CeN aromatic),
1118.73 (CeN aliphatic), 1071.14 (CeF); LCMS (ESI): Calculated for
[Mþ1]þ C32H36F3N7 576.30, found 576.41.
CDCl3)
d
8.01 (d, J ¼ 7.3 Hz, 1H), 7.96 (d, J ¼ 6.7 Hz, 1H), 7.78e7.70
(m, 2H, AreH), 7.61 (s,1H, NH), 7.57 (d, J ¼ 7.4 Hz,1H), 7.54e7.40 (m,
4H, AreH), 6.86 (d, J ¼ 7.1 Hz, 2H), 3.77 (s, 3H), 3.65 (s, 2H), 3.36
Synthesis of N-(4-(3-methylmorpholino)phenyl)-4-(4-(3-
(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine
(10p) - 4-chloro N-(4-(3-methylmorpholino)phenyl)phthalazin-1-
amine (9p) (50 mg, 0.087 mmol) was dissolved in n-butanol
(1 mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (25.5 mg,
0.104 mmol) was added and was heated overnight at 150 ꢀC. Next,
n-butanol was concentrated, and reaction was purified by Flash
chromatography on alumina. Dark green solid; Yield: 51%; Rf ¼ 0.54
(brs, 4H), 2.70 (brs, 4H); 13C NMR (101 MHz, CDCl3)
d 155.52,
149.88, 139.22, 132.37, 131.06, 131.00, 130.40, 128.72, 125.66, 125.03,
124.42, 123.97, 123.95, 123.91, 123.54, 122.47, 122.02, 114.30, 62.53,
55.52 (2C), 53.16 (2C), 51.20; LCMS (ESI): Calculated for [Mþ1]þ
C27H26F3N5O 494.21, found 494.38.
Synthesis
of
N-(4-(piperidin-1-yl)phenyl)-4-(4-(3-(tri-
fluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine (10m)
- 4-chloro-N-(4-(piperidin-1-yl)phenyl)phthalazin-1-amine (9m)
(50 mg, 0.091 mmol) was dissolved in n-butanol (1 mL) and 1-(3-
(trifluoromethyl)benzyl)piperazine (26.8 mg, 0.109 mmol) was
added and was heated overnight at 150 ꢀC. Next, n-butanol was
concentrated and reaction was purified by Flash chromatography
on alumina. Brown solid; Yield: 44%; Rf ¼ 0.84 (50% EtOAc/Hexane
(50% EtOAc/Hexane on alumina);1H NMR (400 MHz, CDCl3)
d 8.09
(brs, 1H, NH), 8.02e7.99 (m, 1H, AreH), 7.77e7.74 (m, 2H, AreH),
7.61 (s, 1H), 7.58 (d, J ¼ 7.3 Hz, 1H), 7.53e7.50 (m, 1H, AreH),
7.47e7.38 (m, 3H, AreH), 6.75 (d, J ¼ 8.8 Hz, 2H), 3.66 (s, 2H), 3.35
(m, 4H), 3.33 (s, 2H), 2.91 (m, 4H), 2.70 (m, 4H), 1.23 (s, 3H), 1.01 (d,
J ¼ 6.4 Hz, 1H); 13C NMR (101 MHz, CDCl3)
d 149.82, 149.79, 147.30,
139.07, 132.38, 131.25, 131.18, 130.60, 128.74, 125.69, 125.11, 124.02,
123.96, 123.66, 122.31, 116.87, 71.65 (2C), 62.51 (2C), 55.54 (2C),
53.09, 51.14, 19.07; IR Absorption nmax/cmꢂ1 3311.32 (NH), 2814.57
(AreH), 1325.67 (CeN aromatic), 1108.56 (CeN aliphatic), 1070.77
(CeO aliphatic ether); LCMS (ESI): Calculated for [Mþ1]þ
C31H33F3N6O 563.27, found 563.36.
on alumina); 1H NMR (400 MHz, CDCl3)
d 8.03e7.99 (m, 1H, AreH),
7.98e7.90 (m, 1H, AreH), 7.77e7.70 (m, 2H, AreH), 7.62 (s, 1H, NH),
7.58 (d, J ¼ 7.5 Hz, 1H), 7.51 (d, J ¼ 7.8 Hz, 1H), 7.47e7.38 (m, 3H,
AreH), 6.93 (d, J ¼ 9.0 Hz, 2H), 3.66 (s, 2H), 3.36 (brs, 4H), 3.14e3.03
(m, 4H), 2.70 (brs, 4H), 1.70 (dt, J ¼ 11.2, 5.7 Hz, 4H), 1.57e1.51 (m,
2H); 13C NMR (101 MHz, CDCl3)
d
160.70, 148.56, 139.21, 132.38,
Synthesis of N-(4-(2-methylmorpholino)phenyl)-4-(4-(3-
(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine
(10q) - 4-chloro-N-(4-(2-methylmorpholino)phenyl) phthalazin-1-
amine (9q) (50 mg, 0.087 mmol) was dissolved in n-butanol (1 mL)
132.37, 131.03, 130.99, 130.57 (q, J ¼ 32 Hz), 128.72, 125.67 (q,
J ¼ 4 Hz), 125.04, 124.19 (q, J ¼ 271 Hz), 123.97 (q, J ¼ 4 Hz), 123.58,
122.03, 117.68, 113.74, 62.54, 53.17, 51.49, 51.19, 25.92, 24.22; LCMS
(ESI): Calculated for [Mþ1]þ C31H33F3N6 547.28, found 547.47.
Synthesis of N-(4-(4-methylpiperazin-1-yl)phenyl)-4-(4-(3-
(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine
(10n) - 4-chloro-N-(4-(4-methylpiperazin-1-yl)phenyl)phthalazin-
1-amine (9n) (50 mg, 0.089 mmol) was dissolved in n-butanol
(1 mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (26.08 mg,
0.106 mmol) was added and was heated overnight at 150 ꢀC. Next,
n-butanol was concentrated, and reaction was purified by Flash
chromatography on alumina. Brown solid; Yield: 80%; Rf ¼ 0.196
(50% EtOAc/Hexane on alumina); 1H NMR (400 MHz, CDCl3)
and
1-(3-(trifluoromethyl)benzyl)piperazine
(25.5
mg,
0.104 mmol) was added and was heated overnight at 150 ꢀC. Next,
n-butanol was concentrated, and reaction was purified by Flash
chromatography on alumina. Dark brown solid; Yield: 76%;
Rf ¼ 0.55 (50% EtOAc/Hexane on alumina);1H NMR (400 MHz,
CDCl3) d 8.06e7.98 (m, 2H, AreH), 7.79e7.73 (m, 2H, AreH), 7.62 (s,
1H, NH), 7.60e7.56 (m, 1H, AreH), 7.53e7.43 (m, 4H, AreH), 6.90 (d,
J ¼ 8.9 Hz, 2H), 3.81 (m, 2H), 3.66 (s, 2H), 3.42e3.32 (m, 6H), 2.71
(m, 4H), 2.41e2.35 (m, 2H), 1.24 (d, J ¼ 6.3 Hz, 6H); 13C NMR
(101 MHz, CDCl3)
d 149.82, 149.79, 147.30, 139.07, 132.39, 132.38,
d
8.05e7.90 (m, 2H, AreH), 7.77e7.69 (m, 2H, AreH), 7.61 (s, 1H,
131.25, 131.18, 130.6, 128.74, 125.69, 125.11, 124.17, 124.02, 123.66,
122.31, 116.87, 71.65, 62.51 (2C), 55.54 (2C), 53.09 (2C), 51.14, 19.07
(2C). LCMS (ESI): Calculated for [Mþ1]þ C31H33F3N6O 563.27, found
563.36.
NH), 7.56 (d, J ¼ 7.4 Hz, 1H), 7.50 (d, J ¼ 7.6 Hz, 2H), 7.47e7.40 (m,
2H, AreH), 6.90 (d, J ¼ 9.0 Hz, 2H), 3.64 (s, 2H), 3.35 (s, 4H),
3.16e3.10 (m, 4H), 2.69 (s, 4H), 2.60e2.54 (m, 4H), 2.33 (s, 3H); 13C
NMR (101 MHz, CDCl3)
130.92, 130.55 (q, J ¼ 64 Hz), 128.71, 128.58, 125.65 (q, J ¼ 4 Hz),
d
150.04, 147.19, 139.22, 132.37, 131.01,
Synthesis of N-(4-(2,6-dimethylmorpholino)phenyl)-4-(4-(3-
(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine (10r) -
13

A. Kharbanda, L. Zhang, D. Saha et al.
European Journal of Medicinal Chemistry 223 (2021) 113660
4-chloro- N-(4-(2,6-dimethylmorpholino)phenyl) phthalazin-1-
amine (9r) (50 mg, 0.087 mmol) was dissolved in n-butanol
(1 mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (25.5 mg,
0.104 mmol) was added and was heated overnight at 150 ꢀC. Next,
n-butanol was concentrated, and reaction was purified by Flash
chromatography on alumina. Yellow solid; Yield: 56%; Rf ¼ 0.41
(50% EtOAc/Hexane on alumina); 1H NMR (400 MHz, CDCl3)
Synthesis
fluoromethyl)benzyl)piperazin-1-yl)isoquinolin-4-amine (16) -
In mL oven dried microwave vial 4-bromo-1-(4-(3-(tri-
of
N-(4-morpholinophenyl)-1-(4-(3-(tri-
5
fluoromethyl)benzyl)piperazin-1-yl)isoquinoline (15) (100 mg,
0.22 mmol) and 4-morpholinoaniline (59.3 mg, 0.33 mmol) was
charged in 2 mL dioxane and added potassium tert-butoxide
(74.8 mg, 0.66 mmol) The reaction mixture was vigorously
degassed with nitrogen for 10 min. Following, 0.3% of Pd2dba3 and
0.9% ( )-BINAP of was added and the reaction was heated at 110 ꢀC
for overnight. Next solvent was then removed by rotavap, and the
resulting residue dissolved in dichloromethane (30 mL) with rapid
stirring. Water (30 mL) was then added to the resulting mixture,
and the organic layer was separated. The aqueous layer was then
extracted with dichloromethane, and the combined organic layers
were dried over Na2SO4. Reaction was purified by Flash chroma-
tography on alumina. Yellow solid; Yield: 42%; Rf ¼ 0.25 (50%
d
8.05e8.00 (m, 2H, AreH), 7.79e7.74 (m, 2H, AreH), 7.62 (s, 1H,
NH), 7.59e7.57 (m, 1H, AreH), 7.53e7.48 (m, 3H, AreH), 7.45e7.40
(m,1H, AreH), 6.90 (d, J ¼ 8.0 Hz, 2H), 4.03e3.97 (m,1H), 3.83e3.75
(m, 2H), 3.66 (s, 2H), 3.56 (s, 1H), 3.38e3.35 (m, 4H), 2.81e2.75 (m,
1H), 2.74e2.63 (m, 4H), 2.46e2.40 (m, 2H), 1.23 (d, J ¼ 8.0 Hz, 2H);
13C NMR (101 MHz, CDCl3)
d 160.69, 147.43, 139.06, 138.75, 132.39,
132.16, 131.24, 131.18, 130.6, 128.80, 128.74, 125.69, 125.34, 125.11,
124.20,124.17,124.02,123.64,122.28,116.87, 71.77, 62.51 (2C), 55.54
(2C), 53.09 (2C), 51.14, 19.05 (2C); IR Absorption nmax/cmꢂ1 3329.72
(NH), 2813.99 (AreH), 1325.36 (CeN aromatic), 1235.23 (CeN
aliphatic), 1071.22 (CeO, aliphatic ether); LCMS (ESI): Calculated for
[Mþ1]þ C32H35F3N6O 577.29, found 577.31.
EtOAc/Hexane on alumina); 1H NMR (400 MHz, CDCl3)
d 8.14 (d,
J ¼ 8.1 Hz, 1H), 8.02 (s, 1H, NH), 7.88 (d, J ¼ 8.2 Hz, 1H), 7.65 (s, 1H,
NH), 7.57 (t, J ¼ 7.6 Hz, 2H), 7.52 (d, J ¼ 8.1 Hz, 2H), 7.44 (t, J ¼ 7.7 Hz,
1H), 6.80 (s, 4H), 3.83 (t, J ¼ 8.0 Hz, 4H), 3.68 (s, 2H), 3.39 (brs, 4H),
3.03 (t, J ¼ 8.0 Hz, 4H), 2.73 (brs, 4H); 13C NMR (101 MHz, CDCl3)
Synthesis
of
N-(4-morpholinophenyl)-4-(4-((3-(tri-
fluoromethyl)benzyl)amino)phenyl)phthalazin-1-amine (11) e
In mL oven dried microwave vial 4-chloro-N-(4-
morpholinophenyl)phthalazin-1-amine (7) (150 mg, 0.44 mmol)
and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-N-(3-(tri-
5
d 157.86, 145.44, 139.40, 139.34, 133.71, 133.40, 132.33, 130.59,
129.86, 129.45, 128.70, 126.24, 125.96, 125.64, 123.95, 122.38,
122.24, 121.50, 117.76, 117.64, 67.01 (2C), 62.50 (2C), 53.32 (2C),
51.44 (2C), 50.61; LCMS (ESI): Calculated for [Mþ1]þ C31H32F3N5O
548.26, found 548.31.
fluoromethyl)benzyl)aniline (249 mg, 0.66 mmol) was charged in
DMF:H2O (4:1) 2 mL and added Cesium carbonate (430.4 mg,
1.32 mmol). The reaction mixture was vigorously degassed with
nitrogen for 10 min. Following, 3.0 mol% of Pd(PPh3)4 was added and
the reaction was irradiated (Biotage 400 MW) at 115 ꢀC for 1 h. The
reaction mixture was filtered through a pad of Celite with CH2Cl2
washing. The combined filtrate was concentrated and the residue
was washed with water and CH2Cl2 followed by Flash chromatog-
raphy on alumina. Brown solid; Yield: 29%; Rf ¼ 0.42 (50% EtOAc/
Synthesis
fluoromethyl)benzyl)piperazin-1-yl)isoquinolin-1-amine (18) -
In mL oven dried microwave vial 4-bromo-N-(4-
of
N-(4-morpholinophenyl)-4-(4-(3-(tri-
5
morpholinophenyl)isoquinolin-1-amine (17) (100 mg, 0.26 mmol)
and 1-(3-(trifluoromethyl)benzyl)piperazine (95.1 mg, 0.39 mmol)
was charged in 2 mL dioxane and added potassium tert-butoxide
(87.4 mg, 0.78 mmol) The reaction mixture was vigorously
degassed with nitrogen for 10 min. Following, 0.3% of Pd2dba3 and
0.9% ( )-BINAP of was added and the reaction was heated at 110 ꢀC
for overnight. Next solvent was then removed by rotavap, and the
resulting residue dissolved in dichloromethane (30 mL) with rapid
stirring. Water (30 mL) was then added to the resulting mixture,
and the organic layer was separated. The aqueous layer was then
extracted with dichloromethane, and the combined organic layers
were dried over Na2SO4. Reaction was purified by Flash chroma-
tography on alumina. Brown solid; Yield: 18%; Rf ¼ 0.84 (50% EtOAc/
Hexane on alumina); 1H NMR (400 MHz, CDCl3)
d
8.10 (d, J ¼ 7.6 Hz,
1H), 7.97 (d, J ¼ 7.8 Hz,1H), 7.75e7.67 (m, 2H, AreH), 7.63 (s,1H, NH),
7.75e7.44 (m, 7H, AreH), 6.88 (d, J ¼ 9.0 Hz, 2H), 6.69 (d, J ¼ 8.6 Hz,
2H), 4.43 (s, 2H), 3.83 (t, J ¼ 4Hz, 4H), 3.07 (t, J ¼ 4Hz, 4H); 13C NMR
(101 MHz, CDCl3)
d 148.17, 147.42, 140.34, 131.29, 131.12, 130.93,
130.77, 129.12, 127.13, 126.83, 125.76, 124.09, 124.10, 123.99, 122.23,
116.79, 112.59, 66.93 (2C), 50.01 (2C), 47.66; LCMS (ESI): Calculated
for [Mþ1]þ C32H28F3N5O 556.23, found 556.46.
Synthesis
of
N-(4-morpholinophenyl)-4-(1-(3-(tri-
fluoromethyl)benzyl)-1H-pyrazol-4-yl)phthalazin-1-amine
(12)- In mL oven dried microwave vial 4-chloro-N-(4-
morpholinophenyl)phthalazin-1-amine (7) (150 mg, 0.44 mmol)
and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-(3-(tri-
Hexane on alumina); 1H NMR (400 MHz, CDCl3)
d
8.11 (d, J ¼ 8.3 Hz,
5
1H), 7.90 (d, J ¼ 8.4 Hz, 1H), 7.79 (s, 1H, NH), 7.67 (d, J ¼ 7.2 Hz, 1H),
7.63 (d, J ¼ 4Hz, 1H), 7.57 (brs, 1H), 7.54e7.50 (m, 2H, AreH), 7.46 (d,
J ¼ 8.8 Hz, 2H), 6.93 (d, J ¼ 8.9 Hz, 2H), 5.10 (s, 1H), 3.90e3.82 (m,
4H), 3.66 (s, 4H), 3.33 (s, 2H), 3.16e3.00 (m, 8H); LCMS (ESI):
Calculated for [Mþ1]þ C31H32F3N5O 548.26, found 548.40.
fluoromethyl)benzyl)-1H-pyrazole (232.2 mg, 0.66 mmol) was
charged in DMF:H2O (4:1) 2 mL and added Cesium carbonate
(430.4 mg, 1.32 mmol). The reaction mixture was vigorously
degassed with nitrogen for 10 min. Following, 3.0 mol% of
Pd(PPh3)4 was added and the reaction was irradiated (Biotage
400 MW) at 115 ꢀC for 1 h. The reaction mixture was filtered
through a pad of Celite with CH2Cl2 washing. The combined filtrate
was concentrated and the residue was washed with water and
CH2Cl2 followed by Flash chromatography on alumina. Orange
solid; Yield: 65%; Rf ¼ 0.28 (50% EtOAc/Hexane on alumina); 1H
Associated content
Supporting Information. The Supporting Information is avail-
able free of charge via the Internet at http://pubs.acs.org.
Synthesis and characterization of 6, 7, 8a-r, 9a-r, 10a-r, and
11e18; details of cell-based screening for TGF
ment and HEK293 viability; details and results for enzymatic
screening against TGF RI (PDF).
b pathway impair-
NMR (400 MHz, CDCl3)
d 8.19e8.14 (m, 1H, AreH), 8.09e8.04 (m,
b
1H, AreH), 8.00 (s, 1H, NH), 7.95 (s, 1H), 7.84e7.76 (m, 2H, AreH),
7.56 (t, J ¼ 6.2 Hz, 4H), 7.46 (d, J ¼ 5.9 Hz, 2H), 6.90 (d, J ¼ 8.9 Hz,
2H), 5.43 (s, 2H), 3.86e3.82 (m, 4H), 3.11e3.08 (m, 4H); 13C NMR
Molecular formula strings and some data (CSV).
(101 MHz, CDCl3)
d
147.63, 139.98, 137.05, 131.83, 131.45, 131.42,
Declaration of competing interest
131.40, 131.14, 131.13, 131.08, 130.76, 130.00, 129.48, 126.48, 125.96,
125.13, 124.54, 122.31, 121.76, 120.36, 119.28, 116.68, 66.91 (2C),
55.73 (2C), 49.95; LCMS (ESI): Calculated for [Mþ1]þ C29H25F3N6O
531.21, found 531.35.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
14

A. Kharbanda, L. Zhang, D. Saha et al.
European Journal of Medicinal Chemistry 223 (2021) 113660
the efficacy of vaccine and PD-1 checkpoint blockade immunotherapy,
Acknowledgment
OncoImmunology
2162402X.2017.1308616.
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TGF- 1 in tumor cells impacts MMP-9 in tumor microenvironment, Sci. Rep. 7
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e1308616,
https://doi.org/10.1080/
For completion of this work, H. L. was supported by the grants
NIH 1R01CA194094 and 1R01CA197178. This work was also sup-
ported by the Memorial Sloan Kettering Cancer Center Support
Grant/Core Grant (P30 CA08748). Figures for the publication are
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€
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TGF
TGF
SAR
NK
ALK
ATP
SMAD
MW
rt
b
b
transforming growth factor-beta
Transforming growth factor-beta receptor
Structure activity relationship
Natural killer
Activin Receptor-Like Kinase
Adenosine Triphospate
small mothers against decapentaplegic
Microwave
room temperature
R
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