Discovery and optimization of novel phenyldiazepine and pyridodiazepine based Aurora kinase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.103800

Aurora B plays critical role in the process of chromosome condensation and chromosome orientation during the regulation of mitosis. The overexpression of Aurora B has been observed in several tumor types. As a part of our ongoing effort to develop Aurora B inhibitors, herein, we described the design, synthesis and evaluation of phenyl/pyridine diazepine analogs. The diazepane aniline pyrimidine (4a) was identified as an initial hit (Aurora B IC₅₀ 6.9 μM). Molecular modeling guided SAR optimization lead to the identification of 8-fluorobenzodiazepine (6c) with single digit nM potency (Aurora B IC₅₀ 8 nM). In the antiproliferation assay 6c showed activity across the cell lines with IC₅₀ of 0.57, 0.42, and 0.69 μM for MCF-7, MDA-MB 231, and SkoV3 respectively. In the in vivo PK profile. 6c has shown higher bioavailability (73%) along with good exposure (AUC of 1360 ng.h/mL).

Full text of DOI:10.1016/j.bioorg.2020.103800

BioorganicChemistry99(2020)103800
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery and optimization of novel phenyldiazepine and pyridodiazepine
based Aurora kinase inhibitors
Natarajan Tamizharasana,b, Chandru Gajendranc, Rajendra Kristame, Suresh P. Sulochanad,
Dhanalakshmi Sivanandhanc, Ramesh Mullangid, Logesh Mathivathananf,
⁎
⁎
Gurulingappa Hallurb, , Palaniswamy Suresha,
a Supramolecular and Catalysis Lab, Department of Natural Products Chemistry, School of Chemistry, Madurai Kamaraj University, Madurai 625021, Tamil Nadu, India
b Medicinal Chemistry Department, Jubilant Biosys Ltd., Bangalore 560022, Karnataka, India
c Oncology Department, Jubilant Biosys Ltd., Bangalore 560022, Karnataka, India
d Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd., Bangalore 560022, Karnataka, India
e Computational Chemistry Department, Jubilant Biosys Ltd., Bangalore 560022, Karnataka, India
f Florida International University, 11200, SW 8th St, Miami, FL 33199, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Aurora B plays critical role in the process of chromosome condensation and chromosome orientation during the
regulation of mitosis. The overexpression of Aurora B has been observed in several tumor types. As a part of our
ongoing effort to develop Aurora B inhibitors, herein, we described the design, synthesis and evaluation of
phenyl/pyridine diazepine analogs. The diazepane aniline pyrimidine (4a) was identified as an initial hit
(Aurora B IC50 6.9 µM). Molecular modeling guided SAR optimization lead to the identification of 8-fluor-
obenzodiazepine (6c) with single digit nM potency (Aurora B IC50 8 nM). In the antiproliferation assay 6c
showed activity across the cell lines with IC50 of 0.57, 0.42, and 0.69 µM for MCF-7, MDA-MB 231, and SkoV3
respectively. In the in vivo PK profile. 6c has shown higher bioavailability (73%) along with good exposure (AUC
of 1360 ng.h/mL).
Oncology
Aurora kinase inhibitor
Phenyldiazepine
Pyridodiazepine
1. Introduction
orientation on the mitotic spindle. CPC is formed by Aur B in the inner
centromere protein (INCENP) with Borealin and Survivin [3]. Aur B
The aurora kinases (A, B and C) belong to the family of serine-
threonine kinases that are critical for regulation of cell division. They
are overexpressed in a variety of cancers including the lung, breast and
colon cancer [1]. Their overexpression levels correlate with the clinical
staging of cancers and are also responsible for poor prognosis [2]. The
isoforms aurora A, B and C display structural similarity in their kinase
domain, however each kinase plays a different role in mitosis. Aurora A
has associated with late S phase and enter into the M phase. It is re-
quired for many processes including centrosome maturation and se-
paration, chromosome alignment, and mitotic spindle formation.
Aurora B (Aur B) plays roles in M phase for chromosome-microtubule
alignment and chromosomal cytokinesis [3]. Whereas the function of
the Aurora C is not well understood, and it has few overlapping func-
tions with Aur B. Aur B is a member of chromosomal passenger complex
(CPC), it required for chromosome condensation and chromosome
protein activation requires phosphorylation of INCENP C-terminal IN-
box region of TSS motif and it is done by Aur B itself by autopho-
sphorylation of Thr 232 on the activation loop of Aur B [3]. The activity
of the Aur B is essential for maintaining the spindle assembly check-
point (SAC), ensuring the proper chromosome alignment and segrega-
tion to enable cytokinesis. Inhibition of Aur B leads to abrogation of the
SAC, thereby inducing cells to exit mitosis without dividing [4]. Al-
though both Aurora kinases A & B are being pursued as oncology tar-
gets, but more reports focusing on Aur B are found in the literature [5].
Cells appear to be sensitive to Aur B inhibition as the mutations in Aur B
found to be enough for high penetrant cell-death phenotype. Suppres-
sing Aur B kinase activity alters alignment of chromosome, cytokinesis
and spindle checkpoint function. These features make Aur B an at-
tractive validated drug target [6].
Over the past decades, extensive research has been carried out
Abbreviations: Aur B, Aurora B; DIPEA, N,N’-Diiosopropylethylamine; T3P, 1-propane phosphonic cyclic anhydride; TFA, Trifluoroacetic acid; LAH, Lithium
Aluminium Hydride; TLC, Thin layer chromatography; AcOH, Acetic acid; INCENP, Inner Centromere Protein; PBS, Phosphate buffered saline
⁎ Corresponding authors.
E-mail addresses: Gurulinappa.hallur@jubilantbiosys.com (G. Hallur), suresh.chem@mkuniversity.ac.in, ghemistry@gmail.com (P. Suresh).
https://doi.org/10.1016/j.bioorg.2020.103800
Received 16 December 2019; Received in revised form 18 March 2020; Accepted 24 March 2020
Availableonline29March2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

N. Tamizharasan, et al.
BioorganicChemistry99(2020)103800
Fig. 1. Structures of known Aurora inhibitors.
towards the identification of small molecules as Aurora kinase in-
hibitors and a handful of them have entered clinical trials [7,8].
However, most of the earlier designed Aurora kinase inhibitors that
reached clinical evaluations lack on the key features which includes
pharmacological selectivity, pharmacokinetic profile, and the potential
for the clinical assessment of pharmacodynamics biomarkers to corre-
late clinical outcome and toxicity with target engagement. Continuous
research efforts have led to the development of various inhibitors that
display selectivity to either Aur A or Aur B. Vertex and Merck has de-
veloped inhibitors VX-680 (tozasertib)/MK-0457 [9] for solid tumors.
However, its clinical development was halted due to observed safety
concerns related to QTC prolongation. Other compounds such as Aur A
inhibitor MLN8054 and its close analogue MLN8237 [10] (Fig. 1) taken
forward to the clinical trials for solid tumors and leukemia. Further-
more, quinazoline AZD-1152 (barasertib) [11] (Fig. 1) developed by
AstraZeneca for solid tumors and Acute Myeloid Leukemia conditions
and 3-aminopyrazole PHA-739358 [12] (Fig. 1) developed by Nerviano
for solid tumors and Chronic Myelogenous Leukemia are under in phase
II trials. Eli Lilly and AurKa Pharma developed Aur A inhibitor
LY3295668 (AK-01) [13] for Small cell lung cancer condition, now in
Phase I & II clinical trials. Amgen developed pan Aurora inhibitor, oral
drug AMG 900, under phase I trial for solid tumor and hematologic
cancers [14]. Despite the development of several Aurora inhibitors,
currently under clinical development, there is a need for the develop-
ment of an ideal Aurora inhibitor with better efficacy and safety profile,
since many of the clinical trial compounds suffer from one or more
other drawbacks. Concerning this, as a part of our ongoing effort to
develop an Aur B inhibitor, we designed phenyl/pyridine diazepine
analogs as Aur B inhibitor. Similar to the typical kinase inhibitors, our
present designed phenyl/pyridine diazepine analogs consist of three
parts such as a hinge binder (pyrimidine amine), a solvent accessible
group, and DFG-in loop interacting groups. The design idea for com-
pound 4a is to have the pharmacophores from Pan Aurora inhibitors
VX-680 and CCT137690 [15] and hybridize pyrimidine moiety which is
expected to have potent anticancer property [16,17], the hinge binder
and skeleton with solvent accessible group (Fig. 2). The designed series
of phenyl/pyridine diazepine are synthesized and its potential in bio-
logical system has been studied and the observed results are rationa-
lized by computational docking studies.
Fig. 2. Rational design idea to prepare Aurora Kinase inhibitors.
2. Results and discussion
[18–21] of 2,4-dichloropyrimidine with 1-methyl-1,4-diazepane gave
intermediate 3a which was coupled with 4-substituted anilines under
Buchwald condition [22] to access diazapane analogs 4a-4d. For
The general synthetic route used for preparation of diazapane
compounds 4a-4d (Scheme 1) was started from commercially available
2,4-dichloro-5-fluoropyrimidine (1). Displacement of 4-chloro group
2

N. Tamizharasan, et al.
BioorganicChemistry99(2020)103800
Scheme 1. General scheme for preparation of diazepane/diazepine series.
Scheme 2. Preparation of intermediate 2b.
Scheme 3. Preparation of intermediate 2c.
preparing compounds 5a-5k, first pyridodiazapine intermediate 2b was
synthesized from 2-fluoronicotinonitrile. 2-Fluorine displacement with
sarcosine ethyl ester hydrochloride gave intermediate 8. Further es-
terification of 8 followed by the reductive cyclization with Raney Ni in
the presence of molecular hydrogen gave pyridodiazepinone 10. The
resulting amide bond was reduced with LAH which yield pyr-
idodiazepine intermediate 2b which was used in the similar fashion to
synthesize compounds 5a–5k. In addition, we also synthesized phe-
nyldiazepine analogs 6a-6g using intermediate 8-fluorobenzodiazepine
which was synthesized from 2-amino-4-fluorobenzoic acid (11). 2-
Amino-4-fluorobenzoic acid (11) was reacted with glycine ester to form
an amide intermediate (12). This intermediate 12 was further cyclized
with acetic acid followed by the reduction with LAH to give 2c. 4-
Substituted aniline intermediates which were not commercially avail-
able, were synthesized starting from 4-fluoronitrobenzene following
previously reported methods [23–25] (Schemes 2 and 3).
Scaffold 2a-2c & 3a-3c:
3b)
3b)
5b: R1 = CH3, R2 = N, R3 = H, R4 = p-N-Acetylpiperazine (from
5c: R1 = CH3, R2 = N, R3 = H, R4 = p-Morpholine (from 3b)
5d: R1 = CH3, R2 = N, R3 = H, R4 = p-CONH2 (from 3b)
5e: R1 = CH3, R2 = N, R3 = H, R4 = N-Methylpyrazole (from 3b)
5f: R1 = CH3, R2 = N, R3 = H, R4 = p-CH2COOH (from 3b)
5 g: R1 = CH3, R2 = N, R3 = H, R4 = p-N,N-Dimethylpiperidin-4-
amine (from 3b);
5 h: R1 = CH3, R2 = N, R3 = H, R4 = p-Piperidine (from 3b)
5i: R1 = CH3, R2 = N, R3 = H, R4 = p-Piperazine (from 3b)
5j: R1 = CH3, R2 = N, R3 = H, R4 = p-COOH (from 3b)
5 k: R1 = CH3, R2 = N, R3 = H, R4 = 3,4,5-Trimethoxy (from 3b)
Library-3:
6a: R1 = H, R2 = CH, R3 = F, R4 = p-N-Acetylpiperazine (from 3c)
6b: R1 = H, R2 = CH, R3 = F, R4 = p-Morpholine (from 3c)
6c: R1 = H, R2 = CH, R3 = F, R4 = p-SO2NH2 (from 3c)
6d: R1 = H, R2 = CH, R3 = F, R4 = p-N-Methylpiperazine (from
3c)
2a, R1 = CH3
2b, R1 = CH3, R2 = N, R3 = H
2c, R1 = H, R2 = CH, R3 = F
6e: R1 = H, R2 = CH, R3 = F, R4 = 3,4,5-Trimethoxy (from 3c)
6f: R1 = H, R2 = CH, R3 = F, R4 = N-Methylpyrazole (from 3c)
6 g: R1 = H, R2 = CH, R3 = F, R4 = p-N,N-Dimethylpiperidin-4-
amine (from 3c)
3a, R1 = CH3
3b, R1 = CH3, R2 = N, R3 = H
3c, R1 = H, R2 = CH, R3 = F
Library-1:
Enzymatic activity of the prepared compounds against Aurora B
kinase was determined using the Lance Ultra kinase assay. The in-
hibitory activity was represented as the relative percentage inhibition
of the positive control PF03814735. The results of the enzymatic ac-
tivity summarized in Table 1.
4a, R1 = CH3, R4 = p-N-Methylpiperazine (from 3a)
4b, R1 = CH3, R4 = p-N-Acetylpiperazine (from 3a)
4c, R1 = CH3, R4 = p-Morpholine (from 3a)
4d, R1 = CH3, R4 = p-Piperazine (from 3a)
Library-2:
The moderate in vitro potency (6930 to 1400 nM) observed with
compounds 4a-4d prompted us to investigate their binding modes and
5a: R1 = CH3, R2 = N, R3 = H, R4 = p-N-Methylpiperazine (from
3

N. Tamizharasan, et al.
BioorganicChemistry99(2020)103800
Table 1
4a shows a couple of key interactions with residues of the hinge.
The pyrimidine nitrogen interacts with the backbone amino group of
Ala-157, while the amino linker between the pyrimidine and phenyl
rings interacts with the backbone carbonyl of Ala-157. There could be a
weak interaction between the proton on the carbon at the 5th position of
the pyrimidine ring and the backbone carbonyl of Glu-155. The phenyl
ring shows cation-π interactions with the terminal amino group of side
chains of Lys-164. The terminal piperazine group is exposed to the
solvent, while the diazepane group in compound 4a sits in a pocket
surrounded by the P-loop, the β-strands β3, β6, β7, β8 and the DFG loop.
There appeared to be quite some space in this hydrophobic pocket that
can be filled with hydrophobic groups. One of the designs that were
considered involved introduction of hydrophobic groups interaction
through aromatic conjugation on to diazapane ring. Compound 5a
having a pyridine moiety fused to the diazapane which improved the
potency by 116-fold. The docking score of 5a was –9.4. In the diazepane
group of compound 4a, the protons of carbons present at in the 3rd and
5th positions of the diazepane ring as well as those of the methyl group
substituted on the nitrogen in the 4th position of the diazepane ring
sterically clashes with the phenyl ring of Phe-88 from the P-loop.
Additionally, this methyl substituent on the nitrogen in the 4th position
of diazepane ring also sterically clashes with the sidechain of Leu-207
(Fig. 3b). In compound 5a, since the diazepane ring is fused to the
pyridine ring, the carbon in the 5th position of the diazepane ring
changes from the sp3 hybridized carbon to a sp2 hybridized carbon and
hence lack the proton projecting towards the phenyl ring of Phe-88,
thus relieving the diazepane ring of clashes with the phenyl ring
(Fig. 3c). The pyridine ring of the pyridodiazepine system picks a ca-
tion-π interaction with the terminal amino group of Lys-106 from the β3
strand. Similarly, for compounds 5b, 5c, and 5d potency was improved
by 51, 42 and 57-folds respectively. The docking scores for these
compounds were –9.8, –9.2 and –9.3 respectively. We explored various
p-substituted aniline modifications on left hand side keeping pyr-
idodiazapine constant on the right-hand side. Solubilizing groups such
as piperidines (5h and 5g), piperazines (5a, 5b and 5i) and morpholine
(5c) substituted on 4th position of aniline showed comparable potency
in the range of 33–96 nM. Introduction of the carboxylic group at the
4th position on 5j showed further enhancement in the potency (15 nM).
Isostere replacement of acid with amide (5d) was also tolerated. The
carboxylic group in compounds 5f and 5j as well as the carbonyl of
amide group in compound 5d show strong interactions with Lys-164
and Lys-85 leading to a slight boost in potency. Phenyl acetic acid group
(5f) retained potency similar to benzoic acid (5j). Trimethoxy phenyl
(5k) and N-methylpyrazole (5e) groups showed potency of 219 nM and
377 nM respectively. Inspired by the above results further, we focused
again on the RHS of the compounds to explore design changes to further
enhance the potency. The docking scores of all these analogs were in
the range of –8.1 to –9.6, while that of 5j was –10.9.
Enzymatic assay.
S. No. Compound
R1
R2
–
R3 R4
In vitro IC50 (nM)
number
1
4a
CH3
–
p-N-
Methylpiperazine
6930
1421
2
3
4
5
4b
4c
4d
5a
CH3
CH3
CH3
CH3
–
–
p-N-Acetyl piperazine 1800
499
810
802
–
–
p-Morpholine
p-Piperazine
p-N-
1400
2750
60
–
–
N
H
2.2
Methylpiperazine
p-N-Acetylpiperazine
p-Morpholine
p-CONH2
6
5b
5c
5d
5e
5f
CH3
CH3
CH3
CH3
CH3
CH3
N
N
N
N
N
N
H
H
H
H
H
H
36
33
42
377
31
68
6.4
7
5.6
8
15.2
9
N-Methylpyrazole
p-CH2COOH
p-N,N-
78.4
10
11
0.8
5g
16.6
Dimethylpiperidin-4-
amine
12
13
14
15
16
17
18
19
5h
5i
CH3
CH3
CH3
CH3
H
N
H
H
H
H
F
p-Piperidine
p-Piperazine
p-COOH
96
47
15
219
9
66.1
24.5
5.2
N
5j
N
5k
6a
6b
6c
6d
N
3,4,5-Trimethoxy
p-N-Acetylpiperazine
p-Morpholine
p-SO2NH2
11.1
CH
CH
CH
CH
2.8
1.9
2.6
0.5
H
F
11
8
H
F
H
F
p-N-
7
Methylpiperazine
3,4,5-Trimethoxy
N-Methylpyrazole
p-N,N-
20
21
22
6e
6f
H
H
H
CH
CH
CH
F
F
F
36
50
10
2.4
9.7
8.9
6g
Dimethylpiperidin-4-
amine
23
PF-
4.9
1
03814735
IC50 values are presented as the means
SD of triplicate experiments
key interactions in the ATP-binding site of Aur B and accordingly the
plan of designs to enhance the potency. Normal rigid docking was
performed using the Schrodinger/GLIDE module [26] by docking the
dataset compounds in the ATP-binding site of 4AF3.pdb and the results
were analyzed to gain insights for future designs. Not all compounds
docked in the ATP-binding site, especially, the larger compounds. One
of the larger compounds (4a), used to run Induced-Fit Docking [27,28],
in which conformational changes of residues within the receptor active
site as well as those of the ligands being docked are explored combining
the Schrodinger modules, Glide and Prime. The output of this routine is
an ensemble of receptor-ligand complexes. One of the model selected
based on the predicted binding mode of 4a and the expected interac-
tions. In this routine, the interactions with the kinase hinge were con-
strained so that all poses will show those interactions and other poses
will be discarded by the module. This model then used to perform rigid
docking again, in the ATP-binding site. The docking procedure fol-
lowed; which involves preparation of the ligands using Schrodinger/
LigPrep module, conformation analysis using Schrodinger/Macro
Model module and finally docking all the conformations [29,30]. The
forcefield used in all modules is OPLS2005 [31]. The LigPrep module
calculates and fixes charges of all atoms, explores stereoisomers of all
chiral centers and generates all possible tautomers. Though the corre-
lation between the experimental potencies and docking scores is not
significant for using the model for prediction purposes, the SAR of the
series of compounds was studied qualitatively, using this model. All the
compounds were docked with a model of Aurora kinase B in complex
with compound 4a, generated using 4AF3.pdb by induced-fit docking
(IFD) module of Schrodinger software [3b,32] as mentioned above.
Schrodinger/Glide [26] with default settings was used to predict the
binding modes. The predicted binding mode of 4a in the ATP-binding
site of Aur B kinase clearly showed that the key interactions and di-
rections of the modifications lead to the improvement of the potency
(Fig. 3a). The docking score of 4a was –9.3.
We examined the predicted binding mode of compound 5a and
observed that the pyridine nitrogen, carrying a partial negative charge
is close to the partial negative charge carrying backbone carbonyl of
Glu-204, thus leading to a slight repulsion (Fig. 3c). Additionally, we
observed that there is still some space beyond the pyridine that can be
filled beneficially. Two changes were envisaged: (1) replacement of the
pyridine with a fluoro substituted benzene to make the benzodiazepine
ring system and (2) removing the methyl substitution from the nitrogen
in the diazepine ring. In compound 6d, the phenyl group of the ben-
zodiazepine ring has a proton in the place of nitrogen, and it exhibits
weak interaction with the same backbone carbonyl of Glu-204. Ad-
ditionally, it is not having the methyl group substituted on the 4th
position nitrogen of the diazepine ring, this compound is relieved of
steric clashes with the phenyl ring of Phe-88 as well as with the side
chain of Leu-207 (Fig. 3d). The fluorine substituted on the phenyl ring
of benzodiazepine picks up additional van der Waals contacts leading to
an improvement in the potency. All these clearly reflect that 6d en-
hanced potency by about 8-folds compared to compound 5a. The
4

N. Tamizharasan, et al.
BioorganicChemistry99(2020)103800
Fig. 3. (a) The predicted binding mode of compound 4a with Aur kinase B in complex (b) The predicted binding mode of compound 4a showing the steric clashes
(orange and red-colored dashes) of the 4-methyl substituted diazepane ring and residues Phe-88 and Leu-207 (c) The predicted binding mode of compound 5a
showing the steric clashes of the methyl group on the diazepine ring with Phe-88 and Leu-207 as well as the closeness of the pyridine nitrogen to the backbone
carbonyl of Glu-204 (d) The predicted binding mode of compound 6d. (For interpretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)
docking score of 6d was –9.9.
(> 50 µM). 6d and 6g were quite stable in human liver microsomes
compared to the other compounds. In case of mouse liver microsomes,
most of the tested compounds showed > 70% disappearance of parent
compound. 5i and 6c compounds were clean (< 50% @ 20 µM) in CYP
inhibition screening for CYP3A4, 2D6 and 2C9 isoforms. Taking into
account of potency and ADME data, we selected 5i and 6c for phar-
macokinetic (PK) profiling in mice by oral and IV administration
(Table 4). Compound 6c showed relatively better PK profile with lower
Vd, higher bioavailability (73%) along with good exposure (AUC of
1360 ng h/mL) which correlated well with observed lower clearance.
On the other hand, 5i with high clearance showed moderate exposure
(AUC of 635 ng h/mL) and 44.2% bioavailability.
The 8-fluorobenzodiazepine compounds (6a, 6b, 6d-6f) showed
better potency compared to their corresponding pyridodiazepine ana-
logs (5a, 5b, 5c, 5e, 5g and 5k). Even the docking scores of the 8-
fluorobenzodiazepine series (–8.9 to –10.6) were slightly better than
those of the pyridodiazepine series. The milieu of interactions and steric
clashes as in the case of compounds 5a (60 nM) and 6d (7 nM) also
clearly separates the pairs of compounds in terms of their relative po-
tencies: 5k (219 nM) and 6e (36 nM), 5e (377 nM) and 6f (50 nM) as
well as 4d (2750 nM) and 5i (47 nM). Similarly, piperidine (6g) and
morpholine (6b) analogs showed potency of 10 nM and 11 nM re-
spectively. The same milieu of interactions and steric clashes that
contrast the potencies of compounds 4a (6930 nM), 5a (59 nM) and 6d
(7 nM) explain the relative potencies of 4b (1800 nM), 5b (36 nM) and
6a (9 nM) as well as 4c (1400 nM), 5c (33 nM) and 6b (11 nM).
Sulfonamide (6c) group at 4th position of LHS phenyl showed a potency
of 8 nM. The docking score of 6c was –10.6. Trimethoxyphenyl (6e) and
N-methylpyrazole (6f) groups showed improvement in potency with the
IC50 values of 36 nM and 50 nM respectively.
3. Conclusions
A series of novel pyridodiazepine and benzodiazepine analogs were
designed and synthesized as Aurora B kinase inhibitors and their
structure activity relationship has been investigated. Molecular mod-
eling guided modifications lead to the identification of 8-fluor-
obenzodiazepine compounds 6a, 6c and 6d with single digit nM po-
tency towards Aurora B IC50 9, 8, and 7 nM respectively. Introduction of
phenyl or pyridine conjugation with diazepine ring showed the im-
provement in the potency resulting from enhanced hydrophobic inter-
action with DFG-in loop. In antiproliferation assay 6c exhibited activity
across the cell lines with IC50 of 0.57, 0.42, and 0.69 µM for MCF-7,
MDA-MB 231, and SkoV3 respectively. In vivo PK profile of 6c showed
higher bioavailability (73%) along with good exposure (AUC of
1360 ng·h/mL). This work provides an attractive lead for further
structure optimization in the discovery of potent Aurora B kinase in-
hibitors. Aurora B inhibitors are under continuous evaluation in clinical
trials and a potent inhibitor may soon become a crucial lead in treat-
ment of cancer.
Following the assessment of all the prepared compounds for Aur B
enzymatic activity, selected compounds with sufficient activity against
Aur B were examined for their antiproliferative activity against human
cancer cell lines MCF-7, MDA-MB 231, SkoV3, A375 and A549. The
screening results of antiproliferative activity of compounds 5a, 5b, 5i,
6a, 6c, 6d, 6g are summarized in Table 2. Among the tested compound
6c showed activity across the cell lines with IC50 of 0.57, 0.42, 0.69,
3.97 & 1.53 µM for MCF-7, MDA-MB 231, SkoV3, A375 and A549 re-
spectively. Compounds 5i, 6a, 6d and 6g showed < 1 µM IC50 for MCF7
whereas compounds 6a and 6d were more potent against MDA-MB 231
with IC50 of 0.23 and 0.16 µM respectively.
Based on the promising biochemical and cellular potency, we se-
lected 5a, 5i, 6a, 6c, 6d and 6g for in vitro ADME profiling (Table 3).
Except for 6a and 6c, all the tested compounds showed good solubility
5

N. Tamizharasan, et al.
BioorganicChemistry99(2020)103800
Table 2
Cell proliferation assay.
S.
Compound No.
Structure
In vitro cellular potency IC50 (µM)
No.
MCF-7
1.69
MDA-MB 231
SkoV3
> 10
A375
2.40
A549
8.39
1
5a
5b
0.70
1.86
0.38
0.60
1.19
2.38
1.17
2
3.09
1.01
> 10
4.53
1.40
0.72
4.04
3.78
3
4
5i
0.77
0.55
0.37
0.24
0.71
0.23
0.27
0.06
> 10
1.12
1.30
1.59
0.31
0.23
3.75
1.26
0.83
0.25
6a
5
6
6c
6d
0.57
0.95
0.23
0.40
0.42
0.16
0.20
0.06
0.69
1.38
0.30
0.75
3.97
1.19
0.67
0.50
1.53
0.52
> 10
7
8
6g
0.32
0.10
0.13
1.12
0.27
0.59
0.06
> 10
0.47
1.37
0.58
0.28
0.17
1.44
0.35
0.60
0.05
PF-03814735a
0.44
0.27
IC50 values are presented as the means
SD of triplicate experiments
a
PF-03814735 suppress expression level of Aur B in cell-based assay [33].
4. Experimental section
4.1. Lance ultra kinase assay
Table 3
ADME properties of selected compounds.
Compounds
5a
5i
6a
6c
6d
6g
Solubilitya (µM)
92.3
120.2
51.96
72.22
> 20
> 20
> 20
1.5
3.6
56.5
91
To determine potencies (IC50) values of the inhibitors against
Aurora B kinase, a fluorescence resonance energy transfer (FRET)-based
Lance Ultra KinaSelectSer/Thr assay (Perkin-Elmer) was performed
under conditions recommended by the manufacturer. In brief, in a half-
area 96-well white opaque bottom plate (Perkin Elmer)
HLMb (% disappearance)
MLMc (% disappearance)
CYP 3A4 IC50 (µM)
CYP2D6 IC50 (µM)
CYP2C9 IC50 (µM)
65.69
86.21
64
29.31
32
97.92
93.75
73.41
> 20
> 20
> 20
97.14
98.20
–
–
–
–
–
–
–
–
–
–
–
–
5 µL/well of test compound in DMSO was added at a final con-
centration of 10 µM and serially diluted (3-fold dilution, 10 con-
centrations). Aurora B kinase inhibitor (PF-03814735) was used as a
positive control. 10 µL reaction mixture containing a Ulight-PLK
a
In PBS pH = 7.4 from DMSO solution.
After 30 min.
b,c
Table 4
Pharmacokinetic parameters following intravenous and oral administration in mice.
Intravenous PK Parametersa
Oral PK parametersb
Compounds
Dose (mg/Kg)
CL (mL/min/kg)
Vdss (L/kg)
T1/2 (h)
Dose (mg/Kg)
Cmax (nM)
AUC last (ng·h/mL)
F (%)
5i
1
1
116
6.53
1.93
0.80
0.41
10
10
326
635
44.2
73.3
6c
55.09
1360
2217
a
b
Formulation for IV dose: 5% DMSO, 5% solutol: absolute alcohol (1:1 v/v) and 90% normal saline.
Formulation for oral dose: 0.025% Tween-80 and 0.5% methyl cellulose in Milli-Q water.
6

N. Tamizharasan, et al.
BioorganicChemistry99(2020)103800
(Ser137) peptide substrate (50 nM) and ATP (15 µM) in assay buffer
(50 mM HEPES, pH 7.4, 1 mM EGTA, 10 mM MgCl2, 2 mMDTT and
0.01% Tween 20) was added to each well. The reaction was initiated by
adding 5 nM Aurora B enzyme (Invitrogen) at 5 µL per well. After in-
cubation for about 60 min at room temperature, phosphorylation was
detected by the addition of a europium-labeled antiphospho-antibody
(2 nM). Antibody binding produced a FRET signal that was recorded at
using EnVision reader/PheraStar which is excited at 337 nm and emits
at 620 and 665 nm. IC50 values were determined by plotting % in-
hibition against concentration of the compound using 4-parameter fit in
GraphPad PRISM 5 software.
again purged for 5 min and stirred at 100 °C for 12 h. The reaction
mixture was concentrated under high vacuum to get crude compound.
The crude residue was purified by gradient column chromatography
using methanol in dichloromethane to afford the title compound as a
grey color solid (0.03 g, 21%). 1H NMR (400 MHz, DMSO‑d6): δ 8.76 (s,
1H), 7.87 (d, J = 6.4 Hz, 1H), 7.46 (d, J = 8.8 Hz, 2H), 6.81 (d,
J = 9.6 Hz, 2H), 3.75 – 3.69 (m, 4H), 3.02 (s, 4H), 2.73 (br, 2H), 2.60
(br, 2H), 2.48 (s, 4H), 2.33 (br, 3H), 2.23 (s, 3H), 1.90 (s, 2H). 13C NMR
(100 MHz, DMSO‑d6): δ 156.13, 151.39 (JCF = 5 Hz), 145.96, 143.33
(JCF = 25 Hz), 141.09 (JCF = 242 Hz), 133.99, 119.88, 116.36, 57.82,
56.24, 55.05, 49.30, 48.02, 47.82, 46.05, 27.18; HRMS (ES+) m/z
calculated for C21H31N7F, 400.2619; found: 400.2601.
4.2. Cell proliferation assay
4.5.2. 1-(4-(4-((5-Fluoro-4-(4-methyl-1,4-diazepan-1-yl)pyrimidin-2-yl)
amino)phenyl) piperazin-1-yl)ethan-1-one (4b)
To identify compounds that have inhibitory effect on cancer cell
proliferation, the alamar blue cell viability assay was performed as per
manufacturer's instructions. Human cancer cells (5000 cells/well) were
plated overnight in full-growth medium (containing 10% FBS and 1%
penicillin and streptomycin) in 96-well black, flat-bottom polystyrene
plate (Corning) at a total volume of 100 µL/well and the plates were
incubated in a humidified 37 °C incubator with 5% CO2. The next day
compounds were added to the cells at concentrations ranging from
10 µM to 1.5 nM (final DMSO concentration 0.3%) in the culture
medium. After 72 h incubation, effect of compounds on proliferation
was determined by the addition of alamar blue (10 µL/well). The plates
were then incubated at 37 °C for an additional 3 h and fluorescence was
read on a Tecan M200 plate reader (excitation 540 nm; emission
590 nm). A reference compound (Aurora B inhibitor, PF-03814735)
was run on each plate as a screening control. GI50 values were de-
termined by plotting % inhibition against concentration of the com-
pound using 4-parameter fit in GraphPad PRISM 5 software.
The title compound was prepared according to general procedure B.
The crude residue was purified by gradient column chromatography
using methanol in dichloromethane to afford the title compound as a
pale brown color solid (0.217 g, 71.4%). 1H NMR (400 MHz, DMSO‑d6):
δ 8.78 (s, 1H), 7.86 (d, J = 7.2 Hz, 1H), 7.49 (d, J = 9.2 Hz, 2H), 6.84
(d, J = 8.8 Hz, 2H), 3.71 – 3.69 (m, 4H), 3.53 (d, J = 4.8 Hz, 4H), 3.01
(t, J = 4.4 Hz, 2H), 2.94 (t, J = 4.8 Hz, 2H), 2.62 (t, J = 4.4 Hz, 2H),
2.48 (s, 2H), 2.25 (s, 3H), 2.00 (s, 3H), 1.85 (t, J = 5.2 Hz, 2H). 13C
NMR (100 MHz, DMSO‑d6): δ 168.63, 156.11, 151.37 (JCF = 5 Hz),
145.77, 143.25 (JCF = 25 Hz), 141.10 (JCF = 241 Hz), 134.57, 119.81,
117.03, 58.03, 56.42, 50.26, 49.84, 48.52 (JCF = 6.2 Hz), 48.18
(JCF = 6.2 Hz), 46.48, 46.08, 27.66. HRMS (ES+) m/z calculated for
C22H31ON7F: 428.2569; found: 428.2551.
4.5.3. 5-Fluoro-4-(4-methyl-1,4-diazepan-1-yl)-N-(4-morpholinophenyl)
pyrimidin-2-amine (4c)
The title compound was prepared according to general procedure B.
The crude residue was purified by gradient column chromatography
using methanol in dichloromethane to afford the title compound as a
pale pink color solid (0.218 g, 88%). 1H NMR (400 MHz, DMSO‑d6): δ
8.76 (s, 1H), 7.86 (d, J = 7.2 Hz, 1H), 7.49 (d, J = 9.6 Hz, 2H), 6.82 (d,
J = 9.2 Hz, 2H), 3.71 – 3.69 (m, 8H), 2.98 (t, J = 4.8 Hz, 4H), 2.62 (t,
4.3. General information
All reagents were purchased from the commercial suppliers and
used without further purification unless otherwise noted. 1H NMR
spectra were recorded on Varian 400 MHz spectrometer with CDCl3 or
DMSO‑d6 as the solvent. 13C NMR spectra were recorded at 100 MHz.
All final compounds were purified to > 95% purity as determined
HPLC analysis using a Waters HPLC system.
J = 4.8 Hz, 2H), 2.25 (s, 3H), 1.85 (t, J = 5.6 Hz, 2H), 1.21 (s, 2H). 13
C
NMR (100 MHz, DMSO‑d6): δ 156.13, 151.37 (JCF = 5 Hz), 146.01,
143.25 (JCF = 25 Hz), 141.07 (JCF = 241 Hz), 134.28, 119.85, 116.08,
66.65, 58.03, 56.43, 49.88, 48.35, 48.15, 46.48, 27.67; HRMS (ES+)
m/z calculated for C20H28ON6F: 387.2303; found: 387.2300.
4.4. General Procedure-A
4.4.1. HCl salt of 1-(2-chloro-5-fluoropyrimidin-4-yl)-4-methyl-1,4-
diazepane (3a)
4.5.4. tert-Butyl 4-(4-((5-fluoro-4-(4-methyl-1,4-diazepan-1-yl)pyrimidin-
2-yl)amino) phenyl)piperazine-1-carboxylate (3d)
To a stirred solution of 2,4-dichloro-5-fluoropyrimidine (1.4 g,
8.38 mmol) in acetonitrile was added DIPEA (2.16 g, 16.7 mmol) and 1-
methyl-1,4-diazepane (0.957 g, 8.38 mmol), then it was stirred at 80 °C
for overnight. The reaction mixture was evaporated to give crude. The
crude residue was washed with diethyl ether (100 mL) and dried under
vacuum to afford the title compound as an off-white solid (1.2 g, 51%).
1H NMR (400 MHz, DMSO‑d6): δ 10.68 (s, 1H), 8.20 (d, J = 6.4 Hz,
1H), 4.01 (br, 1H), 3.82 (br, 1H), 3.75 (s, 3H), 3.48 (br, 2H), 3.26 (br,
1H), 2.74 (s, 3H), 2.14 (br, 2H).
The title compound was prepared according to general procedure B.
The crude residue was purified by gradient column chromatography
using methanol in dichloromethane to afford the title compound as a
pale black solid (0.38 g, 92%). 1H NMR (400 MHz, DMSO‑d6): δ 8.78 (s,
1H), 7.86 (d, J = 7.2 Hz, 1H), 7.49 (d, J = 8.8 Hz, 2H), 6.83 (d,
J = 8.8 Hz, 2H), 3.70 (s, 4H), 3.42 (s, 4H), 2.96 (s, 4H), 2.67 (s, 2H),
2.25 (s, 3H), 1.87 (s, 2H), 1.39 (s, 9H).
4.6. General procedure-C
4.5. General procedure-B
4.6.1. 5-Fluoro-4-(4-methyl-1,4-diazepan-1-yl)-N-(4-(piperazin-1-yl)
phenyl)pyrimidin-2-amine (4d)
4.5.1. 5-Fluoro-4-(4-methyl-1,4-diazepan-1-yl)-N-(4-(4-methylpiperazin-
1-yl)phenyl) pyrimidin-2-amine (4a)
To a solution of tert-butyl 4-(4-((5-fluoro-4-(4-methyl-1,4-diazepan-
1-yl)pyrimidin-2-yl)amino)phenyl)piperazine-1-carboxylate (0.1 g,
0.20 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid
(0.4 mL) at 0 °C and the resulting mixture was stirred at room tem-
perature for 12 h. The progress of the reaction was monitored by TLC.
The reaction mixture was concentrated under reduced pressure to get
crude residue. The crude residue was purified by gradient column
chromatography using methanol in dichloromethane to give the title
compound as a pale yellow solid (0.075 g, 94.9%). 1H NMR (400 MHz,
To a solution of HCl salt of 1-(2-chloro-5-fluoropyrimidin-4-yl)-4-
methyl-1,4-diazepane (0.1 g, 0.35 mmol) in tert-butanol (5 mL) was
added 4-(4-methylpiperazin-1-yl)aniline (0.054 g, 0.28 mmol) and po-
tassium carbonate (0.144 g, 1 mmol). The resulting mixture was purged
by argon gas for 15 min. Tris(dibenzylideneacetone)dipalladium(0)
(0.016 g, 0.0175 mmol) and 2-dicyclohexylphosphino-2′,4′,6′-triiso-
propylbiphenyl (0.016 g, 0.035 mmol) added into reaction mixture and
7

N. Tamizharasan, et al.
BioorganicChemistry99(2020)103800
DMSO‑d6): δ 8.73 (s, 1H), 7.85 (d, J = 7.2 Hz, 1H), 7.46 (d, J = 8.8 Hz,
2H), 6.79 (d, J = 9.2 Hz, 2H), 3.70 (s, 4H), 2.91 (s, 4H), 2.79 (s, 4H),
2.62 (s, 2H), 2.48 (s, 2H), 2.25 (s, 3H), 1.86 (s, 2H). 13C NMR
(100 MHz, DMSO‑d6): δ 156.16, 151.36 (JCF = 5 Hz), 146.78, 143.25
(JCF = 25 Hz), 141.05 (JCF = 242 Hz), 133.89, 119.88, 116.31, 58.05,
56.44, 50.84, 48.56 (JCF = 5.4 Hz), 48.18 (JCF = 5.5 Hz), 46.49, 46.14,
27.69; HRMS (ES-) m/z calculated for C20H27N7F: 384.2306; obtained:
384.2307 (Negative mode).
J = 10.4 Hz, 2H), 3.59 (t, J = 10.4 Hz, 2H), 2.97 (s, 3H).
4.6.7. 5-Fluoro-4-(1-methyl-1,2,3,5-tetrahydro-4H-pyrido[2,3-e][1,4]
diazepin-4-yl)-N-(4-(4-methylpiperazin-1-yl)phenyl)pyrimidin-2-amine
(5a)
The title compound was prepared according to general procedure B.
The crude residue was purified by gradient column chromatography
using methanol in dichloromethane to afford the title compound as a
pale yellow solid (0.080 g, 52.6%). 1H NMR (400 MHz, DMSO‑d6): δ
8.75 (s, 1H), 7.98 (d, J = 3.2 Hz, 1H), 7.86 (d, J = 6.4 Hz, 1H), 7.40 –
7.36 (m, 3H), 6.84 (d, J = 8.8 Hz, 2H), 6.64 (t, J = 11.6 Hz, 1H), 4.73
(s, 2H), 3.97 (s, 2H), 3.52 (s, 2H), 3.03 (s, 4H), 2.97 (s, 4H), 2.48 (s,
3H), 2.21 (s, 3H). 13C NMR (100 MHz, DMSO‑d6): δ 160.54, 156.18,
151.28, 146.30, 143.58 (JCF = 24.8 Hz), 241.7 (JCF = 241.7 Hz),
138.26, 133.62, 120.90, 120.49, 116.33, 114.34, 114.16, 55.14, 52.04,
49.99, 49.92, 49.42, 46.15. HRMS (ES+) m/z calculated for C24H30FN8:
449.2577; found: 449.2575.
4.6.2. N-(3-Cyanopyridin-2-yl)-N-methylglycine (8)
To a stirred solution of 2-fluoronicotinonitrile (4 g, 32.7 mmol) in
ethanol were added triethyl amine (16.51 g, 163.5 mmol) and sarcosine
ethyl ester hydrochloride (7.5 g, 49.1 mmol), then it was stirred at 80 °C
for overnight. The reaction mixture was evaporated to give crude
product of the title compound as an off-white solid (1.89 g, 87.9%).
Crude as such was taken for next step.
4.6.3. Ethyl N-(3-cyanopyridin-2-yl)-N-methylglycinate (9)
To a solution of N-(3-cyanopyridin-2-yl)-N-methylglycine (6.2 g,
32.4 mmol) in ethanol (150 mL) was added concentrated sulfuric acid
(10 mL) at room temperature dropwise for 15 min under nitrogen at-
mosphere. The resulting mixture was stirred at 80 °C for 12 h. The
reaction mixture was concentrated under reduced pressure to remove
volatiles. The crude residue was neutralized by aqueous saturated so-
lution of sodium bicarbonate and extracted with ethyl acetate. The
organic layer was washed with water, brine, dried over anhydrous so-
dium sulfate, filtered and evaporated under reduced pressure to afford
the title compound as a black solid (6.8 g, 95.7%). 1H NMR (400 MHz,
DMSO‑d6): δ 8.24 (d, J = 3.2 Hz, 1H), 7.74 (d, J = 7.2 Hz, 1H), 6.67 (q,
J = 6.8 Hz, 1H), 4.32 (s, 2H), 4.20 (q, J = 14 Hz, 2H), 3.46 (s, 3H),
1.26 (t, J = 14.4 Hz, 3H).
4.6.8. 1-(4-(4-((5-Fluoro-4-(1-methyl-1,2,3,5-tetrahydro-4H-pyrido[2,3-
e][1,4]diazepin-4-yl)pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)ethan-1-
one (5b)
The title compound was prepared according to general procedure B.
The crude residue was purified by gradient column chromatography
using methanol in dichloromethane to afford the title compound as a
pale brown solid (0.040 g, 24.6%). 1H NMR (400 MHz, DMSO‑d6): δ
8.79 (s, 1H), 7.98 (d, J = 3.2 Hz, 1H), 7.87 (d, J = 6.8 Hz, 1H), 7.42 (d,
J = 8.8 Hz, 2H), 7.38 (d, J = 7.2 Hz, 1H), 6.87 (d, J = 9.2 Hz, 2H),
6.65 (q, J = 7.2 Hz, 1H), 4.74 (s, 2H), 3.97 (s, 2H), 3.54 (t, J = 8.8 Hz,
6H), 3.04 (s, 2H), 2.97 (s, 5H), 2.01 (s, 3H). 13C NMR (100 MHz,
DMSO‑d6): δ 168.64, 160.52, 156.12, 151.34, 146.30, 146.07, 143.72
(JCF = 25 Hz), 141.10 (JCF = 242 Hz), 138.25, 134.21, 120.85, 120.40,
117.04, 114.33, 52.01, 50.26, 49.97, 49.84, 46.10, 21.63; HRMS (ES+)
m/z calculated for C25H30ON8F: 477.2521; found: 477.2498.
4.6.4. 1-Methyl-1,2,4,5-tetrahydro-3H-pyrido[2,3-e][1,4]diazepin-3-one
(10)
To a solution of ethyl N-(3-cyanopyridin-2-yl)-N-methylglycinate
(3 g, 13.6 mmol) in ethanol (100 mL) in a clean and dry hydrogenating
steel vessel, Raney Ni (0.38 g, 10%) was added, under nitrogen atmo-
sphere. The mixture was hydrogenated at 100 psi for overnight. After
TLC showed completion of reaction, the mixture was filtered through a
celite bed and the filtrate was concentrated to give title compound as
off-white solid (1.6 g, 66%). 1H NMR (400 MHz, DMSO‑d6): δ 8.07 (s,
1H), 7.99 (dd, J = 4.8 Hz, J = 5.2 Hz, 1H), 7.27 (d, J = 6 Hz, 1H), 6.53
(q, J = 7.2 Hz, 1H), 4.27 (d, J = 4.8 Hz, 2H), 4.10 (s, 2H), 3.09 (s, 3H).
4.6.9. 5-Fluoro-4-(1-methyl-1,2,3,5-tetrahydro-4H-pyrido[2,3-e][1,4]
diazepin-4-yl)-N-(4-morpholinophenyl)pyrimidin-2-amine (5c)
The title compound was prepared according to general procedure B.
The crude residue was purified by gradient column chromatography
using methanol in dichloromethane to afford the title compound as an
off-white solid (0.046 g, 31%). 1H NMR (400 MHz, DMSO‑d6): δ 8.77 (s,
1H), 7.99 (d, J = 3.6 Hz, 1H), 7.87 (d, J = 6.8 Hz, 1H), 7.41 (d,
J = 8.8 Hz, 2H), 7.38 (d, J = 7.6 Hz, 1H), 6.85 (d, J = 8.8 Hz, 2H),
6.65 (q, J = 7.2 Hz, 1H), 4.73 (s, 2H), 3.97 (s, 2H), 3.71 (t, J = 9.2 Hz,
4H), 3.52 (s, 2H), 3.00 (t, J = 9.6 Hz, 4H), 2.97 (s, 3H). 13C NMR
(100 MHz, DMSO‑d6): δ 160.52, 156.15, 151.35, 151.30, 146.30,
143.73 (JCF = 25 Hz), 141.09 (JCF = 242 Hz), 138.25, 133.93, 120.87,
120.46, 116.09, 114.33, 66.65, 52.02, 50.09, 49.99, 49.88. HRMS (ES
+) m/z calculated for C23H27ON7F: 436.2256; found: 436.2256.
4.6.5. 1-Methyl-2,3,4,5-tetrahydro-1H-pyrido[2,3-e][1,4]diazepine (2b)
To a solution of 1-methyl-1,2,4,5-tetrahydro-3H-pyrido[2,3-e][1,4]
diazepin-3-one (2.4 g, 13.5 mmol) in THF (30 mL) was added 2M so-
lution of LAH in THF (33.7 mL, 67.5 mmol) at 0 °C and stirred for 12 h
at 60 °C. The progress of the reaction was monitored by TLC. The crude
residue was quenched by ice and extracted with dichloromethane.
Combined organic layer was dried over anhydrous sodium sulfate,
concentrated to get crude residue. The crude residue was purified by
gradient column chromatography using methanol in dichloromethane
to give the title compound as a yellow liquid (1.9 g, 86.3%). 1H NMR
(400 MHz, DMSO‑d6): δ 8.01 (dd, J = 2 Hz, J = 1.6 Hz, 1H), 7.33 (dd,
J = 1.2 Hz, J = 1.6 Hz, 1H), 6.69 (dd, J = 4.8 Hz, J = 4.8 Hz, 1H),
3.64 (s, 2H), 3.05 – 3.02 (m, 2H), 2.89 (s, 5H), 2.38 (br, 1H).
4.6.10. 4-((5-Fluoro-4-(1-methyl-1,2,3,5-tetrahydro-4H-pyrido[2,3-e]
[1,4]diazepin-4-yl)pyrimidin-2-yl)amino)benzamide (5d)
The title compound was prepared according to general procedure B.
The crude residue was purified by gradient column chromatography
using methanol in dichloromethane to afford the title compound as an
off-white solid (0.015 g, 11.1%). 1H NMR (400 MHz, DMSO‑d6): δ 9.35
(s, 1H), 8.00 – 7.96 (m, 2H), 7.76 (d, J = 8.4 Hz, 2H), 7.72 (s, 1H), 7.65
(d, J = 8.8 Hz, 2H), 7.46 (d, J = 6.8 Hz, 1H), 7.06 (s, 1H), 6.68 (q,
J = 7.2 Hz, 1H), 4.80 (s, 2H), 4.01 (s, 2H), 3.58 (s, 2H), 2.98 (s, 3H).
13C NMR (100 MHz, DMSO‑d6): δ 168.07, 160.41, 155.44, 151.54,
146.39, 144.13, 143.64 (JCF = 25 Hz), 141.56 (JCF = 244 Hz), 138.17,
128.64, 126.60, 120.56, 117.40, 114.28, 51.74, 50.04, 50.03. HRMS
(ES+) m/z calculated for C20H21ON7F: 394.1786; found: 394.1778.
4.6.6. 4-(2-Chloro-5-fluoropyrimidin-4-yl)-1-methyl-2,3,4,5-tetrahydro-
1H-pyrido[2,3-e][1,4]diazepine (3b)
The title compound was prepared according to general procedure A.
The crude residue was purified by gradient column chromatography
using ethyl acetate in hexane to give the title compound as an off-white
solid (0.4 g, 94.5%). 1H NMR (400 MHz, DMSO‑d6): δ 8.12 (d,
J = 6.4 Hz, 1H), 8.00 (dd, J = 4.8 Hz, J = 4.8 Hz, 1H), 7.41 (d,
J = 7.2 Hz, 1H), 6.67 (q, J = 7.6 Hz, 1H), 4.78 (s, 2H), 3.99 (t,
4.6.11. 5-Fluoro-4-(1-methyl-1,2,3,5-tetrahydro-4H-pyrido[2,3-e][1,4]
diazepin-4-yl)-N-(1-methyl-1H-pyrazol-3-yl)pyrimidin-2-amine (5e)
The title compound was prepared according to general procedure B.
8

N. Tamizharasan, et al.
BioorganicChemistry99(2020)103800
The crude residue was purified by gradient column chromatography
using methanol in dichloromethane to afford the title compound as an
off-white solid (0.050 g, 41.6%). 1H NMR (400 MHz, DMSO‑d6): δ 9.04
(s, 1H), 8.00 (d, J = 4 Hz, 1H), 7.86 (d, J = 6.8 Hz, 1H), 7.46 (s, 2H),
6.66 (t, J = 12 Hz, 1H), 6.36 (s, 1H), 4.72 (s, 2H), 3.97 (s, 2H), 3.96 (s,
3H), 3.49 (s, 2H), 2.96 (s, 3H). 13C NMR (100 MHz, DMSO‑d6): δ
160.60, 155.60, 151.44, 151.38, 148.95, 146.32, 143.68 (JCF = 25 Hz),
141.29 (JCF = 241 Hz), 138.42, 130.93, 121.00, 114.43, 52.15, 50.15,
49.94, 38.61. HRMS (ES+) m/z calculated for C17H20N8F: 355.1789;
found: 355.1782.
4.7.3. tert-Butyl4-(4-((5-fluoro-4-(1-methyl-1,2,3,5-tetrahydro-4H-pyrido
[2,3-e][1,4]diazepin-4-yl)pyrimidin-2-yl)amino)phenyl)piperidine-1-
carboxylate (3f)
The title compound was prepared according to general procedure B.
The crude residue was purified by gradient column chromatography
using methanol in dichloromethane to give the title compound as a
pale-yellow solid (0.1 g, 55.2%). 1H NMR (400 MHz, CDCl3): δ 8.13 (d,
J = 3.6 Hz, 1H), 7.80 (d, J = 6.4 Hz, 1H), 7.39 (d, J = 8.4 Hz, 2H),
7.35 (d, J = 7.2 Hz, 1H), 7.14 (d, J = 8.4 Hz, 2H), 6.68 – 6.65 (m, 2H),
4.72 (s, 2H), 4.23 (br, 2H), 4.07 (s, 2H), 3.42 (br, 2H), 3.09 (s, 3H), 2.79
(t, J = 24.4 Hz, 2H), 2.61 (t, J = 24 Hz, 1H), 1.82 (d, J = 12.8 Hz, 2H),
1.60 (d, J = 12.8 Hz, 2H), 1.48 (s, 9H).
4.6.12. Ethyl 2-(4-((5-fluoro-4-(1-methyl-1,2,3,5-tetrahydro-4H-pyrido
[2,3-e][1,4]diazepin-4-yl)pyrimidin-2-yl)amino)phenyl)acetate (3e)
The title compound was prepared according to general procedure B.
The crude residue was purified by gradient column chromatography
using methanol in dichloromethane to afford the title compound as a
pale yellow solid (0.060 g, 20%). 1H NMR (400 MHz, CDCl3): δ 11.59
(s, 1H), 8.11 (s, 1H), 7.68 (d, J = 6.8 Hz, 1H), 7.31 (s, 4H), 7.11 (s, 1H),
6.73 (s, 1H), 4.88 (s, 2H), 4.23 (s, 2H), 4.16 (q, J = 14.4 Hz, 2H), 3.81
(s, 2H), 3.65 (s, 2H), 3.28 (s, 3H), 1.27 (t, J = 14 Hz, 3H).
4.7.4. 5-Fluoro-4-(1-methyl-1,2,3,5-tetrahydro-4H-pyrido[2,3-e][1,4]
diazepin-4-yl)-N-(4-(piperidin-4-yl)phenyl)pyrimidin-2-amine (5h)
The title compound was prepared according to general procedure C.
The crude residue was purified by flash column chromatography using
methanol in dichloromethane to give the title compound as a white
solid (0.025 g, 30.8%). 1H NMR (400 MHz, DMSO‑d6): δ 8.93 (s, 1H),
7.99 (d, J = 2.8 Hz, 1H), 7.90 (d, J = 6.8 Hz, 1H), 7.47 (d, J = 8.4 Hz,
2H), 7.39 (d, J = 7.2 Hz, 1H), 7.08 (d, J = 8.4 Hz, 2H), 6.64 (t,
J = 5.2 Hz, 1H), 4.75 (s, 2H), 3.99 (s, 2H), 3.54 (s, 2H), 3.01 (s, 2H),
2.97 (s, 3H), 2.57 (d, J = 11.6 Hz, 3H), 1.65 (d, J = 12.4 Hz, 2H), 1.50
– 1.42 (m, 2H). 13C NMR (100 MHz, DMSO‑d6): δ 160.49, 155.96,
151.41, 151.35, 146.32, 143.68 (JCF = 25 Hz), 141.23 (JCF = 245 Hz),
139.21, 138.22, 126.86, 120.77, 119.22, 114.26, 51.95, 50.03, 50.01,
46.99, 42.20, 34.70. HRMS (ES+) m/z calculated for C24H29N7F:
434.2463; found: 434.2462.
4.7. General procedure -D
4.7.1. 2-(4-((5-Fluoro-4-(1-methyl-1,2,3,5-tetrahydro-4H-pyrido[2,3-e]
[1,4]diazepin-4-yl)pyrimidin-2-yl)amino)phenyl)acetic acid (5f)
A solution of ethyl 2-(4-((5-fluoro-4-(1-methyl-1,2,3,5-tetrahydro-
4H-pyrido[2,3-e][1,4]diazepin-4-yl)pyrimidin-2-yl)amino)phenyl)
acetate (0.06 g, 0.13 mmol) in a mixture of ethanol (5 mL) and tetra-
hydrofuran (5 mL) was added 2 N sodium hydroxide solution (0.13 mL,
0.27 mmol) slowly at room temperature. The resulting mixture was
stirred at room temperature for 12 h. The reaction mixture was con-
centrated to remove volatiles. The aqueous layer was acidified with
1.5 N hydrochloric acid and extracted with ethyl acetate. The combined
organic layers were dried over anhydrous sodium sulfate, concentrated
to get crude compound. The crude residue was purified by flash column
chromatography using methanol in dichloromethane to afford the title
compound as an off white solid (0.035 g, 62%). 1H NMR (400 MHz,
DMSO‑d6): δ 12.4 (br, 1H), 9.17 (s, 1H), 7.98 (d, J = 6.8 Hz, 1H), 7.90
(d, J = 5.2 Hz, 1H), 7.62 (s, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.18 (d,
J = 8.8 Hz, 2H), 6.80 (t, J = 12.8 Hz, 1H), 4.91 (s, 2H), 4.03 (s, 2H),
3.88 (s, 2H), 3.49 (s, 2H), 3.10 (s, 3H). 13C NMR (100 MHz, DMSO‑d6):
δ 173.37, 158.75, 156.02, 154.41, 152.29, 141.33 (JCF = 25 Hz),
140.83 (JCF = 244 Hz), 139.61, 139.52, 129.91, 128.98, 123.37,
119.93, 113.68, 51.33, 49.61, 49.58, 48.70. HRMS (ES+) m/z calcu-
lated for C21H22O2N6F: 409.1783; found: 409.1789.
4.7.5. tert-Butyl 4-(4-((5-fluoro-4-(1-methyl-1,2,3,5-tetrahydro-4H-pyrido
[2,3-e][1,4]diazepin-4-yl)pyrimidin-2-yl)amino)phenyl)piperazine-1-
carboxylate (3g)
The title compound was prepared according to general procedure B.
The crude residue was purified by flash column chromatography using
methanol in dichloromethane to give the title compound as a grey color
solid (0.1 g, 36.6%). 1H NMR (400 MHz, DMSO‑d6): δ 8.83 (s, 1H), 7.99
(d, J = 3.6 Hz, 1H), 7.88 (d, J = 6.8 Hz, 1H), 7.43 – 7.37 (m, 3H), 6.86
(d, J = 8.8 Hz, 2H), 6.66 (t, J = 12 Hz, 1H), 4.74 (s, 2H), 3.97 (s, 2H),
3.52 (br, 2H), 3.43 (br, 4H), 2.99 – 2.96 (m, 7H), 1.4 (s, 9H).
4.7.6. 5-Fluoro-4-(1-methyl-1,2,3,5-tetrahydro-4H-pyrido[2,3-e][1,4]
diazepin-4-yl)-N-(4-(piperazin-1-yl)phenyl)pyrimidin-2-amine (5i)
The title compound was prepared according to general procedure C.
The crude residue was purified by flash column chromatography using
methanol in dichloromethane to give the title compound as a white
solid (0.05 g, 61.7%). 1H NMR (400 MHz, DMSO‑d6): δ 8.74 (s, 1H),
7.99 (d, J = 3.6 Hz, 1H), 7.86 (d, J = 6.8 Hz, 1H), 7.38 (d, J = 8.4 Hz,
3H), 6.82 (d, J = 8.4 Hz, 2H), 6.45 (t, J = 11.6 Hz, 1H), 4.73 (s, 2H),
3.97 (br, 2H), 3.52 (br, 2H), 2.97 (s, 3H), 2.94 (br, 4H), 2.81 (br, 4H).
13C NMR (100 MHz, DMSO‑d6): δ 160.54, 156.20, 151.28, 147.10,
146.29, 143.72 (JCF = 25 Hz), 141.07 (JCF = 241 Hz), 138.27, 133.52,
120.90, 120.56, 116.31, 114.33, 52.05, 50.82, 50.00, 49.99, 46.13.
HRMS (ES-) m/z calculated for C23H26N8F: 433.2259; found: 433.2263.
(Negative mode)
4.7.2. N-(4-(4-(Dimethylamino)piperidin-1-yl)phenyl)-5-fluoro-4-(1-
methyl-1,2,3,5-tetrahydro-4H-pyrido[2,3-e][1,4]diazepin-4-yl)pyrimidin-
2-amine (5g)
The title compound was prepared according to general procedure B.
The crude residue was purified by flash column chromatography using
methanol in dichloromethane to afford the title compound as a pale
yellow solid (0.1 g, 30.8%). 1H NMR (400 MHz, DMSO‑d6): δ 8.74 (s,
1H), 7.99 (d, J = 4.4 Hz, 1H), 7.86 (d, J = 6 Hz, 1H), 7.38 (d,
J = 8 Hz, 3H), 6.84 (d, J = 8.4 Hz, 2H), 6.64 (t, J = 11.6 Hz, 1H), 4.73
(s, 2H), 3.97 (s, 2H), 3.58 – 3.52 (m, 4H), 2.97 (s, 3H), 2.59 – 2.53 (m,
3H), 2.18 (s, 6H), 1.81 (d, J = 12.4 Hz, 2H), 1.47 (q, J = 21.6 Hz, 2H).
13C NMR (100 MHz, DMSO‑d6): δ 160.54, 156.17, 151.28, 146.50,
146.29, 143.73 (JCF = 25 Hz), 141.06 (JCF = 241 Hz), 138.26, 133.46,
120.9, 120.53, 116.88, 114.33, 61.90, 52.05, 50.00, 49.99, 49.52,
41.87, 28.37. HRMS (ES-) m/z calculated for C26H32N8F: 475.2728;
found: 475.2736 (Negative mode).
4.7.7. Ethyl 4-((5-fluoro-4-(1-methyl-1,2,3,5-tetrahydro-4H-pyrido[2,3-e]
[1,4]diazepin-4-yl) pyrimidin-2-yl)amino)benzoate (3h)
The title compound was prepared according to general procedure B.
The crude residue was purified by gradient column chromatography
using methanol in dichloromethane to give the title compound as an
off-white solid (0.15 g, 52%). 1H NMR (400 MHz, CDCl3): δ 8.15 (dd,
J = 2 Hz, J = 1.6 Hz, 1H), 7.98 (d, J = 9.2 Hz, 2H), 7.85 (d,
J = 6.8 Hz, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.43 (d, J = 6.8 Hz, 1H),
6.99 (s, 1H), 6.71 (q, J = 7.2 Hz,1H), 4.77 (s, 2H), 4.36 (q, J = 12 Hz,
2H), 4.09 (t, J = 10 Hz, 2H), 3.47 (t, J = 9.6 Hz, 2H), 3.10 (s, 3H), 1.39
(t, J = 14 Hz, 3H).
9

N. Tamizharasan, et al.
BioorganicChemistry99(2020)103800
4.7.8. 4-((5-Fluoro-4-(1-methyl-1,2,3,5-tetrahydro-4H-pyrido[2,3-e][1,4]
diazepin-4-yl) pyrimidin-2-yl)amino)benzoic acid (5j)
to give the title compound as a yellow liquid (1.05 g, 77.2%). 1H NMR
(400 MHz, DMSO‑d6): δ 7.01 (t, J = 7.2 Hz, 1H), 6.51 – 6.44 (m, 2H),
3.84 (s, 2H), 3.11 – 3.09 (m, 2H), 3.04 – 3.02 (m, 2H).
The title compound was prepared according to general procedure D.
The crude residue was purified by flash column chromatography using
methanol in dichloromethane to give the title compound as an off-white
solid (0.1 g, 71%). 1H NMR (400 MHz, DMSO‑d6): δ 9.79 (s, 1H), 8.10
(d, J = 6 Hz, 1H), 7.92 – 7.85 (m, 4H), 7.69 (d, J = 8.4 Hz, 2H), 6.89
(t, J = 6 Hz, 1H), 5.03 (s, 2H), 4.37 (br, 2H), 4.08 (s, 2H), 3.22 (s, 3H).
13C NMR (100 MHz, DMSO‑d6): δ 167.45, 154.09, 153.09, 153.02,
152.78, 143.86 (JCF = 28 Hz), 140.93 (JCF = 245 Hz), 139.12, 138.82,
136.55, 130.83, 124.32, 118.66, 113.27, 50.96, 49.50, 48.34. HRMS
(ES+) m/z calculated for C20H20O2N6F: 395.1626; found: 395.1663.
4.9.2. 4-(2-Chloro-5-fluoropyrimidin-4-yl)-1-methyl-2,3,4,5-tetrahydro-
1H-pyrido[2,3-e][1,4]diazepine (3c)
The title compound was prepared according to general procedure A.
The crude residue was purified by gradient column chromatography
using ethyl acetate in hexane to give the title compound as an off-white
solid (1.5 g, 80.2%). 1H NMR (400 MHz, DMSO‑d6): δ 8.13 (d,
J = 6.4 Hz, 1H), 7.10 (t, J = 7.2 Hz, 1H), 6.54 (d, J = 11.2 Hz, 1H),
6.47 (t, J = 8.4 Hz, 1H), 5.99 (s, 1H), 4.69 (s, 2H), 3.91 (s, 2H), 3.28 (s,
2H).
4.7.9. 5-Fluoro-4-(1-methyl-1,2,3,5-tetrahydro-4H-pyrido[2,3-e][1,4]
diazepin-4-yl)-N-(3,4,5-trimethoxyphenyl)pyrimidin-2-amine (5k)
The title compound was prepared according to general procedure B.
The crude residue was purified by flash column chromatography using
methanol in dichloromethane to give the title compound as an off-white
solid (0.1 g, 44.4%). 1H NMR (400 MHz, DMSO‑d6): δ 8.91 (s, 1H), 7.99
(d, J = 3.2 Hz, 1H), 7.91 (d, J = 7.2 Hz, 1H), 7.38 (d, J = 7.2 Hz, 1H),
7.03 (s, 2H), 6.64 (q, J = 6.8 Hz, 1H), 4.75 (s, 2H), 4.02 (br, 2H), 3.73
(s, 6H), 3.59 (s, 3H), 3.55 (d, J = 4.4 Hz, 2H), 2.97 (s, 3H). 13C NMR
(100 MHz, DMSO‑d6): δ 160.45, 155.87, 153.08, 151.39, 146.45,
143.61 (JCF = 26 Hz), 141.24 (JCF = 242.1 Hz), 138.07, 137.56,
132.51, 120.82, 114.35, 97.06, 60.58, 56.22, 51.82, 50.24, 49.78.
HRMS (ES + ) m/z calculated for C22H26N6O3F: 441.2050; found:
441.2076.
4.9.3. TFA salt of 1-(4-(4-((5-fluoro-4-(8-fluoro-1,2,3,5-tetrahydro-4H-
benzo[e][1,4]diazepin-4-yl)pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)
ethan-1-one (6a)
The title compound was prepared according to general procedure B.
The crude residue was purified by flash column chromatography using
methanol in dichloromethane. The crude material was once again
purified by Prep. HPLC (0.1% TFA in water/ACN) to give the title
compound as a pale pink solid (0.049 g, 22.2%). 1H NMR (400 MHz,
DMSO‑d6): δ 9.25 (br, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.37 (d,
J = 8.8 Hz, 2H), 6.96 (d, J = 8.4 Hz, 3H), 6.54 (dd, J = 2.8 Hz,
J = 2.4 Hz, 1H), 6.39 (t, J = 6.4 Hz, 1H), 5.97 (br, 1H), 4.68 (s, 2H),
3.94 (s, 2H), 3.56 (s, 4H), 3.28 (s, 2H), 3.11 (s, 2H), 3.04 (s, 2H), 2.02
(s, 3H). 13C NMR (100 MHz, DMSO‑d6):
δ 168.70, 162.30
(JCF = 240 Hz), 158.89 (JCF = 34 Hz), 152.90, 152.30 (JCF = 10 Hz),
152.053, 147.13, 140.25 (JCF = 244 Hz), 137.22, 132.072, 122.76,
121.86, 117.07, 104.99 (JCF = 21 Hz), 104.53 (JCF = 23 Hz), 52.46,
51.63, 49.79, 45.65, 21.61. HRMS (ES+) m/z calculated for
4.8. Ethyl(2-amino-4-fluorobenzoyl)glycinate (12)
A mixture of 2-Amino-4-fluoro benzoic acid (5 g, 32.2 mmol), gly-
cine ethyl ester HCl salt (4.95 g, 35.48 mmol), in dichloromethane
(270 mL), DIPEA (19.28 mL, 112.87 mmol) was added at 0 °C then 1-
propane phosphonic anhydride (20.51 g, 64.5 mmol) was added and
stirred at room temperature overnight. The solution was diluted with
dichloromethane, washed with saturated bicarbonate solution and
brine, dried over sodium sulfate, filtered and the solvent was removed
under vacuum. Purification of the crude product by flash chromato-
C25H28ON7F2: 480.2318; found: 480.2320.
4.9.4. TFA salt of 5-fluoro-4-(8-fluoro-1,2,3,5-tetrahydro-4H-benzo[e]
[1,4]diazepin-4-yl)-N-(4-morpholinophenyl)pyrimidin-2-amine (6b)
The title compound was prepared according to general procedure B.
The crude residue was purified by flash column chromatography using
methanol in dichloromethane. The crude material was once again
purified by Prep. HPLC (0.1% TFA in water/ACN) to give the title
compound as a white solid (0.031 g, 16.6%). 1H NMR (400 MHz,
DMSO‑d6): δ 9.33 (br, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.35 (d,
J = 8.8 Hz, 2H), 6.96 (d, J = 8.4 Hz, 2H), 6.90 (br, 1H), 6.54 (d,
J = 9.2 Hz, 1H), 6.38 (t, J = 7.2 Hz, 1H), 5.98 (br, 1H), 4.69 (s, 2H),
3.95 (s, 2H), 3.73 (s, 4H), 3.29 (s, 2H), 3.08 (s, 4H). 13C NMR
(100 MHz, DMSO‑d6): δ 162.33 (JCF = 241 Hz), 159.11, 158.76, 152.25
(JCF = 10 Hz), 151.98, 139.95 (JCF = 245 Hz), 135.39 (JCF = 21 Hz),
132.12 (JCF = 10 Hz), 130.98, 123.41, 121.46, 116.32, 104.96
(JCF = 21 Hz), 104.48 (JCF = 23 Hz), 66.42, 52.63, 51.65, 49.66,
45.30. HRMS (ES+) m/z calculated for C23H25ON6F2: 439.2052; found:
439.2032.
graphy gave the title compound as an off-white solid (5.12 g, 66%). 1
H
NMR (400 MHz, CDCl3): δ 7.40 (t, J = 2.2 Hz, 1H), 6.48 (br, 1H), 6.39
– 6.35 (m, 2H), 4.26 (q, J = 3.5 Hz, 2H), 4.17 (d, J = 1.2 Hz, 2H), 1.31
(t, J = 3.6 Hz, 3H).
4.9. 8-Fluoro-3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione (13)
To a solution of methyl (2-amino-4-fluorobenzoyl)glycinate (5.12 g,
21.3 mmol) in acetic acid (140 mL) was heated at 120 °C for 12 h. After
completion of the reaction as confirmed by TLC, AcOH was removed in
vacuo, the residue was dissolved in ethyl acetate and washed with sa-
turated sodium bicarbonate solution and brine. The organic layers were
dried over sodium sulfate and the solvent was removed under vacuum.
Purification of the crude product by flash chromatography gave the title
compound as a yellowish liquid (3.71 g, 89.8%). 1H NMR (400 MHz,
DMSO‑d6): δ 10.41 (s, 1H), 8.49 (t, J = 6 Hz, 1H), 7.79 (q, J = 6.8 Hz,
1H), 7.05 (t, J = 8.4 Hz, 1H), 6.88 (dd, J = 2.8 Hz, J = 2.8 Hz, 1H),
3.60 (d, J = 5.6 Hz, 2H).
4.9.5. TFA salt of 4-((5-fluoro-4-(8-fluoro-1,2,3,5-tetrahydro-4H-benzo[e]
[1,4]diazepin-4-yl)pyrimidin-2-yl)amino)benzenesulfonamide (6c)
The title compound was prepared according to general procedure B.
The crude residue was purified by flash column chromatography using
methanol in dichloromethane. The crude material was once again
purified by Prep. HPLC (0.1% TFA in water/ACN) to give the title
compound as a white solid (0.035 g, 19%). 1H NMR (400 MHz,
DMSO‑d6): δ 9.53 (s, 1H), 8.00 (d, J = 6.8 Hz, 1H), 7.75 (d, J = 8.8 Hz,
2H), 7.68 (d, J = 8.8 Hz, 2H), 7.13 (br, 3H), 6.55 (dd, J = 2 Hz,
J = 2 Hz, 1H), 6.44 (t, J = 8.4 Hz, 1H), 5.96 (br, 1H), 4.72 (s, 2H), 3.95
(s, 2H), 3.30 (s, 2H). 13C NMR (100 MHz, DMSO‑d6): δ 162.27
(JCF = 240.2 Hz), 158.81 (JCF = 36.4 Hz), 154.35, 152.30, 151.42,
143.95, 141.79 (JCF = 27.9 Hz), 141.51 (JCF = 244 Hz), 131.9
(JCF = 9 Hz), 126.98, 122.41, 118.26, 105.07 (JCF = 21 Hz), 104.60
(JCF = 23 Hz), 52.19, 51.49, 45.41. HRMS (ES+) m/z calculated for
4.9.1. 8-Fluoro-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine (2c)
To a solution of 8-fluoro-3,4-dihydro-1H-benzo[e][1,4]diazepine-
2,5-dione (1.6 g, 8.24 mmol) in THF (30 mL) was added 2 M solution of
LAH in THF (20.6 mL, 41.2 mmol) at 0 °C and stirred for 12 h at 60 °C.
The progress of the reaction was monitored by TLC. The crude residue
was quenched with ice and extracted with dichloromethane. The
combined organic layers were dried over anhydrous sodium sulfate,
concentrated to get crude residue. The crude residue was purified by
gradient column chromatography using methanol in dichloromethane
10

N. Tamizharasan, et al.
BioorganicChemistry99(2020)103800
C20H20N5O2F2S: 432.1306; found: 432.1297.
J = 11.6 Hz, 2H), 1.70 (t, J = 22 Hz, 2H). 13C NMR (100 MHz,
DMSO‑d6): δ 162.29 (JCF = 240 Hz), 158.88 (JCF = 34 Hz), 158.36,
152.86, 152.35, 152.11, 146.43, 140.23 (JCF = 245 Hz), 132.06,
122.66, 121.82, 117.32, 104.95 (JCF = 21 Hz), 104.53 (JCF = 23 Hz),
62.76, 52.47, 51.60, 48.71, 45.44, 34.78, 25.98; HRMS (ES-) m/z cal-
culated for C26H30N7F2: 478.2525 found: 478.2528.(Negative mode).
4.9.6. TFA salt of 5-fluoro-4-(8-fluoro-1,2,3,5-tetrahydro-4H-benzo[e]
[1,4]diazepin-4-yl)-N-(4-(4-methylpiperazin-1-yl)phenyl)pyrimidin-2-
amine (6d)
The title compound was prepared according to general procedure B.
The crude residue was purified by flash column chromatography using
methanol in dichloromethane. The crude material was once again
purified by Prep. HPLC (0.1% TFA in water/ACN) to afford the title
compound as a white solid (0.057 g, 19.9%). 1H NMR (400 MHz,
DMSO‑d6): δ 9.53 (br, 1H), 9.00 (br, 1H), 7.91 (d, J = 7.2 Hz, 1H), 7.44
(d, J = 8.8 Hz, 2H), 6.99 (br, 1H), 6.94 (d, J = 8.8 Hz, 2H), 6.55 (d,
J = 9.2 Hz, 1H), 6.40 (t, J = 6.8 Hz, 1H), 5.95 (br, 1H), 4.67 (s, 2H),
3.92 (s, 2H), 3.72 (d, J = 12.4 Hz, 2H), 3.51 (d, J = 12 Hz, 2H), 3.25
(s, 2H), 3.16 (t, J = 10.8 Hz, 2H), 2.90 (s, 5H). 13C NMR (100 MHz,
DMSO‑d6): δ 162.24 (JCF = 240 Hz), 158.71 (JCF = 34 Hz), 154.19,
152.33, 151.58, 145.53, 140.67 (JCF = 244 Hz), 133.35, 131.99,
122.38, 121.77, 117.05, 105.21 (JCF = 21 Hz), 104.60 (JCF = 23 Hz),
52.91, 52.24, 51.53, 46.86, 45.62, 42.56. HRMS (ES+) m/z calculated
for C24H28N7F2: 452.2374; found: 452.2376.
Author contributions
These authors contributed equally.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
We thank Jubilant Biosys Ltd., Bangalore for supporting and
funding in completion of this work. Dr. PS gratefully acknowledge
SERB, New Delhi, and CSIR, New Delhi for financial support.
4.9.7. TFA salt of 5-fluoro-4-(8-fluoro-1,2,3,5-tetrahydro-4H-benzo[e]
[1,4]diazepin-4-yl)-N-(3,4,5-trimethoxyphenyl)pyrimidin-2-amine (6e)
The title compound was prepared according to general procedure B.
The crude residue was purified by flash column chromatography using
methanol in dichloromethane. The crude material was once again
purified by Prep. HPLC (0.1% TFA in water/ACN) to afford the title
compound as a white solid (0.07 g, 37.2%). 1H NMR (400 MHz,
DMSO‑d6): δ 8.99 (br, 1H), 7.93 (d, J = 6.8 Hz, 1H), 7.04 (t,
J = 7.2 Hz, 1H), 6.98 (s, 2H), 6.54 (dd, J = 2.8 Hz, J = 2 Hz, 1H), 6.40
(t, J = 8 Hz, 1H), 5.93 (br, 1H), 4.68 (s, 2H), 3.96 (s, 2H), 3.72 (s, 6H),
3.59 (s, 3H), 3.27 (s, 2H). 13C NMR (100 MHz, DMSO‑d6): δ 162.27
(JCF = 241 Hz), 154.85, 153.15, 152.38 (JCF = 10 Hz), 151.33, 141.67,
141.02 (JCF = 244 Hz), 136.81, 133.06, 131.89 (JCF = 10 Hz), 122.72,
105.04 (JCF = 21 Hz), 104.60 (JCF = 23 Hz), 97.90, 60.58, 56.26,
51.82, 51.55, 45.57; HRMS (ES+) m/z calculated for C22H24O3N5F2:
444.1842 found: 444.1852.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.103800.
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