Synthesis and biological evaluation of novel indole-pyrimidine hybrids bearing morpholine and thiomorpholine moieties

Article abstract of DOI:10.1016/j.ejmech.2017.04.011

Based on our previous screening hit compound 1, a series of novel indole-pyrimidine hybrids possessing morpholine or thiomorpholine moiety were synthesized via an efficient one-pot multistep synthetic method. The antiproliferative activities of the synthesized compounds were evaluated in?vitro against four cancer cell lines including HeLa, MDA-MB-231, MCF-7, and HCT116. The results revealed that most compounds possessed moderate to excellent potency. The IC₅₀ values of the most promising compound 15 are 0.29, 4.04, and 9.48?μM against MCF-7, HeLa, and HCT116?cell lines, respectively, which are 48.0, 4.9, and 1.8 folds more active than the lead compound 1. Moreover, fluorescence-activated cell sorting analysis revealed that compound 14 showing the highest activity against HeLa (IC₅₀?=?2.51?μM) displayed a significant effect on G₂/M cell-cycle arrest in a concentration-dependent manner in HeLa cell line. In addition, representative nine active hybrids were evaluated for tubulin polymerization inhibitory activities, and compound 15 exhibited the most potent anti-tubulin activity showing 42% inhibition at 10?μM. These preliminary results encourage a further investigation on indole-pyrimidine hybrids for the development of potent anticancer agents that inhibit tubulin polymerization.

Full text of DOI:10.1016/j.ejmech.2017.04.011

Accepted Manuscript
Synthesis and biological evaluation of novel indole-pyrimidine hybrids bearing
morpholine and thiomorpholine moieties
Peng-Cheng Diao, Qui Li, Meng-Jin Hu, Yu-Feng Ma, Wen-Wei You, Kwon Ho Hong,
Pei-Liang Zhao
PII:
S0223-5234(17)30266-0
10.1016/j.ejmech.2017.04.011
EJMECH 9358
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 December 2016
Revised Date: 5 April 2017
Accepted Date: 6 April 2017
Please cite this article as: P.-C. Diao, Q. Li, M.-J. Hu, Y.-F. Ma, W.-W. You, K.H. Hong, P.-L.
Zhao, Synthesis and biological evaluation of novel indole-pyrimidine hybrids bearing morpholine
and thiomorpholine moieties, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.04.011.
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ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel indole-pyrimidine hybrids
bearing morpholine and thiomorpholine moieties
Peng-Cheng Diaoa,#, Qui Lia,#, Meng-Jin Hua, Yu-Feng Maa, Wen-Wei Youa, Kwon Ho Hongb,*
Pei-Liang Zhaoa,*
aGuangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical
Science, Southern Medical University, Guangzhou 510515, P.R.China
bDepartment of Medicinal Chemistry and Institute for Therapeutics Discovery and
Development, College of Pharmacy, University of Minnesota, Minneapolis 55414, United States

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel indole-pyrimidine hybrids
bearing morpholine and thiomorpholine moieties
Peng-Cheng Diaoa,#, Qui Lia,#, Meng-Jin Hua, Yu-Feng Maa, Wen-Wei Youa, Kwon Ho Hongb,*
Pei-Liang Zhaoa,*
5
aGuangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical
Science, Southern Medical University, Guangzhou 510515, P.R.China
bDepartment of Medicinal Chemistry and Institute for Therapeutics Discovery and
Development, College of Pharmacy, University of Minnesota, Minneapolis 55414, United States
10
ABSTRACT: Based on our previous screening hit compound 1, a series of novel
indole-pyrimidine hybrids possessing morpholine or thiomorpholine moiety were synthesized via
an efficient one-pot multistep synthetic method. The antiproliferative activities of the synthesized
compounds were evaluated in vitro against four cancer cell lines including HeLa, MDA-MB-231,
MCF-7, and HCT116. The results revealed that most compounds possessed moderate to excellent
potency. The IC50 values of the most promising compound 15 are 0.29, 4.04, and 9.48 µM against
MCF-7, HeLa, and HCT116 cell lines, respectively, which are 48.0, 4.9, and 1.8 folds more active
than the lead compound 1. Moreover, fluorescence-activated cell sorting analysis revealed that
compound 14 showing the highest activity against HeLa (IC50 = 2.51 µM) displayed a significant
effect on G2/M cell-cycle arrest in a concentration-dependent manner in HeLa cell line. In addition,
representative nine active hybrids were evaluated for tubulin polymerization inhibitory activities,
and compound 15 exhibited the most potent anti-tubulin activity showing 42% inhibition at 10 µM.
These preliminary results encourage a further investigation on indole-pyrimidine hybrids for the
development of potent anticancer agents that inhibit tubulin polymerization.
15
20
25
Keywords: Indole-pyrimidine; Morpholine; Thiomorpholine; Antiproliferative activity; Tubulin
polymerization
30
——————————
Corresponding author. E-mail: plzhao@smu.edu.cn (P. L. Zhao), hong0207@umn.edu (K. H.
Hong)
# These authors contributed equally to this work.
1

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1. Introduction
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In recent decades, microtubules have been important molecular targets for the development of
anticancer drugs due to their crucial roles in the regulating cancer cell survival and progression
including cellular signaling, motility, cell shape maintenance, secretion, intercellular transport and
spindle formation during mitosis [1-4]. Inhibiting tubulin polymerization or interfering with
microtubule disassembly ultimately leads to cell cycle arrest or apoptosis of cancer cells [5-7].
Hence, there are commonly two major categories of anti-tubulin agents: inhibitors of the tubulin
polymerization and stabilizers of the microtubule structure.
Recently, our research group discovered indole-pyrimidine hybrid 1, having piperazine on the
pyrimidine moiety (Fig. 1), as a potent inhibitor of the polymerization of tubulin with a low toxicity
[8]. The agent shows low IC50 values ranged from 5.01 to 14.36 µM against four cancer cell lines
and does not affect the normal human embryonic kidney cells, HEK-293. Interest towards
morpholine- or thiomorpholine-containing indole or pyrimidine systems as anticancer agents has
increased due to their important chemopreventive and chemotherapeutic effects on cancer [9-14].
As shown in Fig. 1, pyrimidine derivative BKM-120 containing two morpholine groups manifests a
great antiproliferative activity against PI3K-deregulated cell lines (GI50 values against A2780,
U87MG, MCF7 and DU145 are 0.1-0.7 nM), and is currently in phase III clinical trials for the
treatment of advanced breast cancer [15-16]. In addition, Kryštof and coworkers reported that
thiomorpholine-containing pyrimidine compound 3 exhibited potential antitumor activities against a
panel of cancer cell lines [17]. More interestingly, indole-pyrimidine hybrid 4 with a morpholine
group was identified as a potent PI3K inhibitor with a low nanomolar IC50 value and used in the
treatment of PI3K-mediated disorders such as inflammation and cancer [18].
<Insert fig. 1 here>
As the heterocyclic compounds consisting of multiple cores receive much attention in recent
years [19-23], and in view of the above mentioned prominence, we have focused on the design and
efficient synthesis of novel indole-pyrimidine systems bearing a morpholine or thiomorpholine
moiety. In the present study, morpholine and thiomorpholine moieties replaced the piperazine group
of the C-2 position of the pyrimidine moiety of compound 1 (Fig. 2) and a series of novel hybrids of
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indole-pyrimidine containing a morpholine or thiomorpholine ring 11–32 were synthesized via an
efficient one-pot multistep synthetic methodology. Furthermore, these compounds were evaluated
for the antiproliferative and tubulin polymerization inhibitory activities.
65
<Insert fig. 2 here>
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2. Chemistry
The synthesis of the target compounds 11–32 was illustrated in Scheme 1. Substituted ethyl
(2-cyanophenyl)carbamates 6 were obtained by condensation of the appropriately substituted
2-aminobenzonitriles 5 and ethyl chloroformate under reflux. The subsequent Thorpe–Ziegler
cyclization with various α-bromoketones using K2CO3 as a base in dimethylformamide provided the
common intermediates N-1-ethoxycarbonyl-2-substituted-3-aminoindoles 7. These intermediates
were subsequently N-deprotected by alkaline hydrolysis using NaOH in aqueous ethanol to generate
the corresponding 3-amino-1H-indoles 8, as illustrated in detail in our previously reported approach
[24].
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The expected indole-pyrimidine derivatives 11–32, containing a morpholine or thiomorpholine
ring, were generally obtained via a one-pot, multistep synthetic operation of equimolar amounts of
substituted 3-amino-indoles 7 or 8, 2,4-dichloro-5-nitropyrimidne 9 and morpholine or
thiomorpholine in dry acetone (Scheme 1). The structures of target compounds 11–32 were
1
characterized with spectroscopic techniques including H NMR, 13C NMR and HRMS, and the
spectral data agree with the proposed structures.
<Insert scheme 1 here>
3. Results and Discussion
3.1 In vitro antiproliferative activity
The synthesized compounds 11–32 were evaluated for their in vitro antiproliferative activities
against four human cancer cell lines including HeLa, MDA-MB-231, MCF-7, and HCT116 using
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the MTT assay [25]. The assay results expressed as IC50 (µM) were summarized in Table 1 and
compared with the inhibitory activities of two reference compounds, CA-4, a potent natural
tubulin-binding anticancer agent, and our previously reported compound 1. Here, the IC50 value
represents the concentration of a compound resulting in 50% inhibition of cell growth after 48 h
incubation, and is the average of three independent experiments.
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<Insert table 1 here>
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As shown in Table 1, it is clear that the first series of indole-pyrimidine hybrids (11–29)
bearing a morpholine ring at the C-2 position of the pyrimidine, exhibit generally higher potency
than the corresponding hybrids (30–32) with a thiomorpholine ring, indicating that the morpholine
substitution on the pyrimidine ring might play a crucial role in modulating the antitumor activity.
Among the morpholine series (11–29), most compounds showed moderate to excellent
antiproliferative activities against the four tested cancer cell lines. The IC50 values of the most
promising compound 15 were 0.29, 4.04, and 9.48 µM against MCF-7, HeLa, and HCT116 cell
lines, respectively, which indicated that this compound was 48.0, 4.9, and 1.8 folds more active than
the lead compound 1. More interestingly, compound 17 showed potent in vitro antiproliferative
activities against all the tested cancer cell lines with the IC50 values ranged from 2.13 to 5.52 µM.
Notably, compound 17 was two folds more active than positive control CA-4 in inhibiting HCT116
cell proliferation with IC50 value of 2.99 µM. It was worth noting that, the compound did not affect
the normal human embryonic kidney cells, HEK-293.
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Further analysis clearly revealed that different antiproliferative activities were observed when
various substituents R1, R2, R3 were introduced into the indole ring. Compounds 11–17, without any
substituent at 6-position (R1) of the indole ring, displayed strong antiproliferative activity against
the four tested cancer cell lines. The introduction of an electron-withdrawing or electron-donating
group at the position led to an obvious decline in antitumour activities, which could be further
confirmed by compounds 18–23, and 24–29. For the substituent R2, compounds with a bulky
4-phenylphenyl substituent, exhibit much higher antiproliferative activities against HeLa and
MCF-7 cell lines: 15, 23, 29 versus 11–13, 18–21, 24–28, respectively. However, replacement of
the phenyl ring (R2) with a cyclopropyl group resulted in a decrease in the antiproliferative
4

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activities (16 versus 11–15). It should be noted that introduction of ethoxycarbonyl group (R3)
enhanced the inhibitory activity against MDA-MB-231, MCF-7 and HCT116 (17 versus 13, 18,
22).
125
To study the effect of the synthesized compounds on the cell cycle progression,
fluorescence-activated cell sorting analysis (FACS) was performed. Compound 14 was selected and
tested against HeLa cell lines at given concentrations (1.25, 2.5, 5 µM). As shown in Fig. 3 and
Table 2, the G2/M peak significantly increased from 9.12% to 12.63% (1.25 µM), 58.34% (2.5 µM),
and 88.58% (5 µM) after 12 h of incubation with the compound. These cell cycle analysis results
revealed that the compound arrested the cell cycle at G2/M phase in a concentration-dependent
manner when compared to untreated control cells.
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<Insert fig. 3 here>
<Insert table 2 here>
3.2. Inhibition of in vitro tubulin polymerization
Based on the obtained IC50 values of target hybrids against the four cancer cell lines employed
in this study, nine representative hybrids were selected for the evaluation of their inhibitory
activities against in vitro tubulin polymerization at 10 µM and CA-4 was also used as a reference.
The results were summarized in Table 3. Notably, there is a clear correlation between anti-tubulin
activity and antiproliferative activity of the tested compounds. For example, compounds 15 and 17
exhibited approximately 40% inhibition of the tubulin polymerization at 10 µM and
antiproliferative activity in low micromolar concentrations against all four cell lines tested.
Meanwhile, thiomorpholine-linked indole-pyrimidine hybrid 30 lost its inhibitory potential against
tubulin polymerization as well as anticancer activities against all four tested cancer cell lines.
Additionally, compound 15 displayed an anti-tubulin activity with an IC50 value of 19.3 µM, which
was comparable with that of compound 1 (IC50 = 11.2 µM).
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<Insert table 3 here>
3.3. Molecular docking study
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Understanding of the binding mode of a lead compound in a molecular target provides an
opportunity to rationally design inhibitors of the target more potent than the lead compound in a
drug discovery project. We, therefore, performed docking simulations and the relative binding free
energy calculation of the most active compound 15 and its relative binding free energy calculation
in order to investigate its potential binding mode (PDB ID: 1SA0) [26]. Compound 15 was docked
in the colchicine binding pocket in the beta chain of tubulin using Glide and the pose with the best
emodel score(docking score -6.8, glide emodel -103.6 kcal/mol) was used for MM-GBSA relative
binding free energy calculation using Prime (Glide, version 6.9, Schrodinger, LLC, New York, NY,
2015 and Prime, version 4.2, Schrodinger, LLC, New York, NY, 2015.). As shown in Fig. 4,
docking studies revealed various hydrogen bonding and hydrophobic interactions that appear to
play a role in the binding mode. 1-NH of the indole moiety of compound 15 forms a hydrogen
bonding interaction with Thr353. Meanwhile, the indole moiety forms hydrophobic interaction with
Leu248. The biaryl moiety of compound 15 occupies the hydrophobic pocket formed by Tyr202,
Val238, Cys241, Leu242, Ala250, Leu252, Leu255, Ala316, Ala317 and Ile378. The morpholine
moiety is located at close proximity to Asn101, Ser178, Thr179, and Tyr224 of the alpha chain of
tubulin.
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<Insert fig. 4 here>
4. Conclusion
In the present study, the piperazine ring of the lead compound 1 was replaced by a morpholine
or thiomorpholine group to generate a series of novel indole-pyrimidine hybrids. The
antiproliferative activities of the synthesized compounds were evaluated in vitro against HeLa,
MDA-MB-231, MCF-7, and HCT116 cell lines. Most compounds demonstrated significant
antiproliferative activities against four human cancer cell lines employed in this study. The IC50
values of the most promising compound 15 were 0.29, 4.04, and 9.48 µM against MCF-7, HeLa,
and HCT116 cell lines, respectively, which was 48.0, 4.9, and 1.8 folds more active than the lead
compound 1. Moreover, the result from FACS analysis revealed that compound 14, showing the
highest activity against HeLa cell lines (IC50 = 2.51 µM) among the synthesized compounds,
displayed a significant effect on G2/M cell-cycle arrest in a concentration-dependent manner in
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HeLa cells. Notably, the compound 15 also exhibited the most potent anti-tubulin activity showing
42% inhibition at 10 µM. These preliminary results encourage further investigation on
indole-pyrimidine hybrids aiming to the development of new potential tubulin polymerization
inhibitors and anticancer agents.
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5. Experimental protocols
5.1 Chemistry
13
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200
1H NMR and C NMR spectra were recorded on a Mercury-Plus 400 spectrometer in CDCl3
or DMSO-d6 solution and chemical shifts were recorded in parts per million (ppm) with TMS as the
internal reference. MS spectra were obtained using a Micromass ZQ 4000 mass spectrometer, and
signals were given in m/z. Elemental analyses were performed on a Vario EL III elemental analysis
instrument. Melting points (mp) were taken on a Buchi B-545 melting point apparatus and are
uncorrected. Unless otherwise noted, reagents were purchased from commercial suppliers and used
without further purification while all solvents were redistilled before use.
General
procedure
for
the
one-pot
reactions
of
substituted
3-aminoindoles,
2,4-dichloro-5-nitro-pyrimidine, and morpholine or thiomorpholine.
2,4-Dichloro-5-nitro-pyrimidine 9 (1.0 mmol) was added to a stirred solution of substituted
3-aminoindoles 7 or 8 (1.0 mmol) in 5.0 mL of anhydrous acetone, and the mixture were heated at
40°C for 2–3h. Morpholine or thiomorpholine 10 (1.1 mmol) was then added and stirred for about
30 min. After the reaction was complete according to the TLC detection, solvent was removed
under reduced pressure and the residue was purified by column chromatography using a mixture of
petroleum ether and acetone as an eluent to give the target compounds in yields of 56.4–78.5%.
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5.1.1 (3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indol-2-yl)(phenyl)methanone (11). Yield,
70.3%; mp 178.0–179.0°C; 1H NMR (400 MHz, CDCl3) δ: 3.50 (d, J = 4.4 Hz, 2H, CH2), 3.58 (d, J
= 4.4 Hz, 2H, CH2), 3.74 (d, J = 4.4 Hz, 2H, CH2), 3.93 (d, J = 4.4 Hz, 2H, CH2), 7.19 (t, J = 7.4 Hz,
1H, ArH), 7.39–7.45 (m, 3H, ArH), 7.47–7.54 (m, 2H, ArH), 7.66 (d, J = 8.0 Hz, 1H, ArH), 7.75 (t,
13
J = 4.2 Hz, 2H, ArH), 8.92 (s, 1H, NH), 8.93 (s, 1H, pyrimidine-H), 10.28 (s, 1H, NH). C NMR
(100 MHz, DMSO-d6) δ: 187.9, 159.9, 157.9, 154.2, 138.2, 136.3, 132.6, 129.1, 128.6, 126.8, 126.2,
122.9, 122.7, 120.4, 120.2, 119.5, 113.4, 66.2, 44.7. HRMS (ESI) m/z: calcd for C23H20N6O4
[M+H]+ 445.1546, found 445.1630.
7

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5.1.2 (4-bromophenyl)(3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indol-2-yl)methanone (12).
1
Yield, 57.5%; mp 235.1–236.0°C; H NMR (400 MHz, CDCl3) δ: 3.50 (s, 2H, CH2), 3.58 (s, 2H,
215
CH2), 3.73 (s, 2H, CH2), 3.95 (s, 2H, CH2), 7.19 (t, J = 7.4 Hz, 1H, ArH), 7.42–7.49 (m, 2H, ArH),
7.55 (d, J = 8.4 Hz, 2H, ArH), 7.66 (t, J = 8.6 Hz, 3H, ArH), 8.89 (s, 1H, NH), 8.99 (s, 1H,
pyrimidine-H), 10.39 (s, 1H, NH). 13C NMR (100 MHz, CDCl3) δ: 186.5, 159.5, 157.3, 154.1,
136.4, 135.7, 131.7, 130.0, 127.6, 127.1, 126.0, 123.1, 122.5, 120.8, 120.5, 119.7, 112.5, 66.7, 44.7.
HRMS (ESI) m/z: calcd for C23H19BrN6O4 [M+H]+ 525.0631, found 525.0718.
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5.1.3 (4-methoxyphenyl)(3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indol-2-yl)methanone
1
(13). Yield, 78.3%; mp 164.7–165.5°C; H NMR (400 MHz, CDCl3) δ: 3.50 (d, J = 3.6 Hz, 2H,
CH2), 3.58 (s, 2H, CH2), 3.73 (s, 2H, CH2), 3.85 (s, 3H, CH3), 3.92 (d, J = 7.6 Hz, 2H, CH2), 6.88
(d, J = 8.8 Hz, 2H, ArH), 7.18 (t, J = 7.4 Hz, 1H, ArH), 7.41 (t, J = 7.6 Hz, 1H, ArH), 7.48 (d, J =
8.4 Hz, 1H, ArH), 7.66 (d, J = 8.4 Hz, 1H, ArH), 7.78 (d, J = 8.8 Hz, 2H, ArH), 8.95 (s, 1H, NH),
13
8.99 (s, 1H, pyrimidine-H), 10.37 (s, 1H, NH). C NMR (100 MHz, DMSO-d6) δ: 186.6, 163.1,
159.9, 158.0, 154.1, 136.1, 131.6, 130.6, 126.8, 125.9, 122.8, 122.7, 120.5, 120.0, 119.1, 114.0,
113.4, 66.2, 56.0, 44.7. HRMS (ESI) m/z: calcd for C24H22N6O5 [M+H]+ 475.1652, found 475.1740.
5.1.4 (3-bromophenyl)(3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indol-2-yl)methanone (14).
Yield, 59.2%; mp 233.0–234.7°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.49 (s, 2H, CH2), 3.63 (s, 2H,
CH2), 3.84 (s, 2H, CH2), 7.11 (t, J = 7.6 Hz, 1H, ArH), 7.36 (dd, J = 12.0 and 7.6 Hz, 2H, ArH),
7.50 (d, J = 8.4 Hz, 1H, ArH), 7.59 (d, J = 8.0 Hz, 1H, ArH), 7.72 (dd, J1 = 8.0 Hz, J2 = 12.4 Hz, 2H,
230
235
13
ArH), 7.79 (s, 1H, ArH), 8.90 (s, 1H, NH), 10.38 (s, 1H, pyrimidine-H), 11.90 (s, 1H, NH). C
NMR (100 MHz, DMSO-d6) δ: 186.3, 159.9, 158.0, 154.3, 140.3, 136.5, 135.0, 131.6, 130.7, 127.9,
126.8, 126.5, 123.2, 122.5, 121.6, 120.4, 120.3, 119.7, 113.4, 66.2, 44.7. HRMS (ESI) m/z: calcd
for C23H19BrN6O4 [M+H]+ 525.0631, found 525.0720.
5.1.5 biphenyl-4-yl(3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indol-2-yl)methanone (15).
Yield, 61.7%; mp 116.3–117.9°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.39 (s, 2H, CH2), 3.52 (s, 2H,
CH2), 3.64 (s, 2H, CH2), 3.84 (s, 2H, CH2), 7.12 (t, J = 7.4 Hz, 1H, ArH), 7.36 (t, J = 7.6 Hz, 1H,
ArH), 7.44 (t, J = 7.2 Hz, 1H, ArH), 7.52 ((t, J = 7.2 Hz, 3H, ArH), 7.64–7.70 (m, 5H, ArH), 7.85 (d,
J = 8.0 Hz, 2H, ArH), 8.85 (s, 1H, NH), 10.64 (s, 1H, pyrimidine-H), 11.88 (s, 1H, NH). 13C NMR
(100 MHz, CDCl3) δ: 187.3, 160.0, 157.6, 154.4, 145.6, 139.7, 136.4, 135.6, 129.0, 128.9, 128.2,
127.3, 127.0, 126.8, 126.5, 123.3, 122.2, 120.7, 120.5, 119.3, 112.4, 66.7, 44.6. HRMS (ESI) m/z:
calcd for C29H24N6O4 [M+H]+ 521.1859, found 521.1960.
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5.1.6 cyclopropyl(3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indol-2-yl)methanone (16).
Yield, 74.6%; mp 234.8–236.9°C; 1H NMR (400 MHz, CDCl3) δ: 1.03–1.07 (m, 2H,
cyclopropane-H), 1.28–1.33 (m, 2H, cyclopropane-H), 2.58–2.62 (m, 1H, cyclopropane-H), 3.43 (d,
J = 4.0 Hz, 2H, CH2), 3.50 (d, J = 4.0 Hz, 2H, CH2), 3.72 (s, 2H, CH2), 3.96 (s, 2H, CH2), 7.15 (t, J
8

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= 7.4 Hz, 1H, ArH), 7.39 (t, J = 7.6 Hz, 1H, ArH), 7.44 (d, J = 8.4 Hz, 1H, ArH), 7.57 (d, J = 8.0 Hz,
13
1H, ArH), 9.08 (s, 1H, NH), 9.14 (s, 1H, pyrimidine-H), 10.52 (s, 1H, NH). C NMR (100 MHz,
250
DMSO-d6) δ: 193.4, 160.0, 158.3, 153.6, 135.9, 126.6, 126.2, 124.2, 121.9, 120.8, 119.7, 119.3,
113.3, 66.1, 44.8, 18.5, 11.5, 9.5. HRMS (ESI) m/z: calcd for C20H20N6O4 [M+H]+ 409.1546, found
409.1636.
5.1.7 ethyl 2-(4-methoxybenzoyl)-3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indole-1-
carboxylate (17). Yield, 60.5%; mp 183.5–184.9°C; 1H NMR (400 MHz, CDCl3) δ: 1.10 (t, J = 7.0
Hz, 3H, CH3), 3.43 (s, 2H, CH2), 3.54 (s, 2H, CH2), 3.71 (s, 2H, CH2), 3.88 (s, 3H, CH3), 3.93 (s,
2H, CH2), 4.16 (dd, J = 14.4 and 7.2 Hz, 2H, CH2), 6.94 (d, J = 8.8 Hz, 2H, ArH), 7.32 (d, J = 7.6
Hz, 1H, ArH), 7.51 (t, J = 7.8 Hz, 1H, ArH), 7.57 (d, J = 8.0 Hz, 1H, ArH), 7.85 (d, J = 8.8 Hz, 2H,
ArH), 8.29 (d, J = 8.4 Hz, 1H, ArH), 9.02 (s, 1H, pyrimidine-H), 10.37 (s, 1H, NH). 13C NMR (100
MHz, DMSO-d6) δ: 185.4, 163.9, 159.8, 158.1, 154.7, 150.5, 135.0, 131.3, 130.4, 129.0, 127.7,
125.2, 123.6, 123.5, 122.7, 120.6, 115.1, 114.5, 66.3, 56.0, 49.0, 44.7, 13.8. HRMS (ESI) m/z: calcd
for C27H26N6O7 [M+H]+ 547.1863, found 547.1951.
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260
5.1.8
(6-chloro-3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indol-2-yl)(phenyl)methanone
1
(18). Yield, 56.6%; mp 210.2–211.5°C; H NMR (400 MHz, CDCl3) δ: 3.50 (d, J = 4.4 Hz, 2H,
CH2), 3.60 (d, J = 4.0 Hz, 2H, CH2), 3.74 (d, J = 4.0 Hz, 2H, CH2), 3.94 (d, J = 4.0 Hz, 2H, CH2),
7.15 (d, J = 8.4 Hz, 1H, ArH), 7.41 (t, J = 7.8 Hz, 2H, ArH), 7.52 (dd, J = 16.2 and 9.0 Hz, 2H,
ArH), 7.59 (d, J = 8.8 Hz, 1H, ArH), 7.74 (d, J = 7.6 Hz, 2H, ArH), 8.94 (s, 1H, NH), 9.08 (s, 1H,
265
13
pyrimidine-H), 10.26 (s, 1H, NH). C NMR (100 MHz, DMSO-d6) δ: 187.8, 159.8, 157.9, 154.2,
137.9, 136.4, 132.8, 130.7, 129.1, 128.6, 127.6, 124.4, 121.8, 120.8, 120.4, 119.6, 112.7, 66.4, 44.7.
HRMS (ESI) m/z: calcd for C23H19ClN6O4 [M+H]+ 479.1156, found 479.1245.
270
275
280
5.1.9 (6-chloro-3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indol-2-yl)(4-chlorophenyl)
methanone (19). Yield, 53.3%; mp 247.8–248.5°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.36 (s, 2H,
CH2), 3.51 (s, 2H, CH2), 3.63 (s, 2H, CH2), 3.84 (s, 2H, CH2), 7.13 (dd, J = 8.8 and 1.6 Hz, 1H,
ArH), 7.49 (dd, J = 9.8 and 5.0 Hz, 3H, ArH), 7.67 (d, J = 8.4 Hz, 1H, ArH), 7.76 (d, J = 8.4 Hz, 2H,
ArH), 8.91 (s, 1H, NH), 10.54 (s, 1H, pyrimidine-H), 12.00 (s, 1H, NH). 13C NMR (100 MHz,
DMSO-d6) δ: 186.6, 159.8, 157.9, 154.2, 137.7, 136.6, 136.5, 130.9, 128.7, 127.4, 124.5, 121.8,
120.9, 120.4, 119.8, 112.7, 66.2, 46.1. HRMS (ESI) m/z: calcd for C23H18Cl2N6O4[M+H]+ 513.0767
found 513.0856.
5.1.10 (6-chloro-3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indol-2-yl)(4-methoxyphenyl)
methanone (20). Yield, 58.4%; mp 232.6–234.1°C; 1H NMR (400 MHz, CDCl3) δ: 3.51 (d, J = 4.0
Hz, 2H, CH2), 3.60 (s, 2H, CH2), 3.73 (s, 2H, CH2), 3.86 (s, 3H, CH3), 3.94 (s, 2H, CH2), 6.89 (d, J
= 8.8 Hz, 2H, ArH), 7.15 (dd, J = 8.8 and 1.6 Hz, 1H, ArH), 7.48 (d, J = 1.2 Hz, 1H, ArH), 7.59 (d,
J = 8.8 Hz, 1H, ArH), 7.77 (d, J = 8.8 Hz, 2H, ArH), 8.92 (s, 1H, NH), 8.96 (s, 1H, pyrimidine-H),
9

ACCEPTED MANUSCRIPT
10.34 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 186.4, 163.3, 159.9, 158.0, 154.1, 136.2,
131.6, 130.4, 127.7, 124.4, 121.7, 120.6, 119.1, 114.0, 112.6, 66.2, 56.0, 44.7. HRMS (ESI) m/z:
calcd for C24H21ClN6O5 [M+H]+ 509.1262, found 509.1369.
285
290
295
300
5.1.11 (6-chloro-3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indol-2-yl)(thiophen-2-yl)
methanone (21). Yield, 52.8%; mp 268.4–269.7°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.40 (s, 2H,
CH2), 3.50 (s, 2H, CH2), 3.62 (s, 2H, CH2), 3.84 (s, 2H, CH2), 7.14 (dd, J = 8.4 and 1.6 Hz, 1H,
ArH), 7.25 (t, J = 4.2 Hz, 1H, thiophene-H), 7.53 (d, J = 1.6 Hz, 1H, ArH), 7.69 (d, J = 8.8 Hz, 1H,
ArH), 7.98 (d, J = 3.6 Hz, 1H, thiophene-H), 8.07 (d, J = 4.8 Hz, 1H, thiophene-H), 8.98 (s, 1H,
13
NH), 11.01 (s, 1H, pyrimidine-H), 12.02 (s, 1H, NH). C NMR (100 MHz, DMSO-d6) δ: 178.9,
159.9, 158.2, 153.8, 142.9, 136.5, 135.7, 134.6, 130.7, 129.0, 126.7, 124.9, 121.3, 120.7, 120.6,
119.8, 112.8, 66.4, 44.8. HRMS (ESI) m/z: calcd for C21H17ClN6O4S [M+H]+ 485.0721, found
485.0815.
5.1.12 ethyl 2-benzoyl-6-chloro-3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indole-1-
carboxylate (22). Yield, 54.5%; mp 193.6–195.8°C; 1H NMR (400 MHz, DMSO-d6) δ: 1.00 (t, J =
14.0 Hz, 3H, CH3), 3.47 (s, 2H, CH2), 3.60 (s, 2H, CH2), 3.66 (s, 2H, CH2), 3.82 (s, 2H, CH2), 4.14
(dd, J1 = 7.2 Hz, J2 = 14.4 Hz, 2H, CH2), 7.42 (dd, J = 8.6 and 1.6 Hz, 1H, ArH), 7.49 (t, J = 7.6 Hz,
2H, ArH), 7.62 (d, J = 7.6 Hz, 1H, ArH), 7.70 (d, J = 8.4 Hz, 1H, ArH), 7.77 (d, J = 8.0 Hz, 2H,
ArH), 8.15 (d, J = 1.6 Hz, 1H, ArH), 8.93 (s, 1H, pyrimidine-H), 10.28 (s, 1H, NH). 13C NMR (100
MHz, DMSO-d6) δ: 186.5, 159.8, 158.1, 154.6, 150.1, 137.5, 135.5, 134.0, 132.5, 129.2, 128.8,
124.5, 124.1, 124.0, 120.7, 115.0, 66.2, 64.8, 44.7, 13.7. HRMS (ESI) m/z: calcd for C26H23ClN6O6
[M+H]+ 551.1368, found 551.1458.
5.1.13 biphenyl-4-yl(6-chloro-3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indol-2-yl)
methanone (23). Yield, 75.0%; mp 276.5–277.7°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.39 (s, 2H,
CH2), 3.53 (s, 2H, CH2), 3.64 (s, 2H, CH2), 3.84 (s, 2H, CH2), 7.14 (dd, J = 8.6 and 1.6 Hz, 1H,
ArH), 7.44 (t, J = 7.2 Hz, 1H, ArH), 7.52 (dd, J = 8.4 and 6.4 Hz, 3H, ArH), 7.65–7.70 (m, 5H,
ArH), 7.84 (d, J = 8.0 Hz, 2H, ArH), 8.85 (s, 1H, NH), 10.63 (s, 1H, pyrimidine-H), 12.03 (s, 1H,
305
310
13
NH). C NMR (100 MHz, DMSO-d6) δ: 187.3, 159.9, 157.8, 154.3, 144.5, 139.5, 136.7, 136.5,
130.8, 129.8, 129.5, 128.7, 127.6, 127.4, 126.8, 124.5, 121.8, 120.8, 120.5, 119.7, 112.7, 66.2, 44.7.
HRMS (ESI) m/z: calcd for C29H23ClN6O4 [M+H]+ 555.1467, found 555.1558.
5.1.14 (6-methyl-3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indol-2-yl)(phenyl)methanone
1
(24). Yield, 63.7%; mp 210.1–210.8°C; H NMR (400 MHz, DMSO-d6) δ: 2.44 (s, 3H, CH3), 3.40
(s, 2H, CH2), 3.52 (s, 2H, CH2), 3.63 (s, 2H, CH2), 3.84 (s, 2H, CH2), 6.94 (d, J = 8.4 Hz, 1H, ArH),
7.28 (s, 1H, ArH), 7.42 (t, J = 7.6 Hz, 2H, ArH), 7.52 (dd, J = 7.8 and 5.0 Hz, 2H, ArH), 7.75 (d, J =
7.2 Hz, 2H, ArH), 8.87 (s, 1H, NH), 10.62 (s, 1H, pyrimidine-H), 11.69 (s, 1H, NH). 13C NMR (100
315
10

ACCEPTED MANUSCRIPT
MHz, DMSO-d6) δ: 187.7, 159.9, 157.9, 154.1, 138.3, 136.9, 136.0, 132.5, 129.0, 128.5, 126.2,
122.6, 122.3, 120.9, 120.4, 119.9, 112.7, 66.4, 44.7, 22.1. HRMS (ESI) m/z: calcd for C24H22N6O4
[M+H]+459.1703, found 459.1793.
320
325
5.1.15 ((4-bromophenyl)(6-methyl-3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indol-2-yl)
methanone (25). Yield, 65.2%; mp 258.6–259.9°C; 1H NMR (400 MHz, DMSO-d6) δ: 2.44 (s, 3H,
CH3), 3.39 (s, 2H, CH2), 3.51 (s, 2H, CH2), 3.64 (s, 2H, CH2), 3.84 (s, 2H, CH2), 6.95 (d, J = 8.4 Hz,
1H, ArH), 7.26 (s, 1H, ArH), 7.52 (d, J = 8.4 Hz, 1H, ArH), 7.62 (d, J = 8.4 Hz, 2H, ArH), 7.68 (d, J
13
= 8.4 Hz, 2H, ArH), 8.91 (s, 1H, NH), 10.62 (s, 1H, pyrimidine-H), 11.70 (s, 1H, NH). C NMR
(400 MHz, DMSO-d6) δ: 186.6, 159.8, 157.9, 154.1, 137.4, 137.0, 136.3, 131.5, 131.0, 126.3, 125.9,
122.6, 122.5, 120.9, 120.4, 120.2, 112.7, 66.4, 44.7, 22.1. HRMS (ESI) m/z: calcd for
C24H21BrN6O4 [M+H]+ 538.0787, found 539.0872.
5.1.16 (4-methoxyphenyl)(6-methyl-3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indol-2-yl)
1
methanone (26). Yield, 62.6%; mp 231.5–232.5°C; H NMR (400 MHz, DMSO-d6) δ: 2.44 (d, J =
330
335
2.4 Hz, 3H, CH3), 3.42 (s, 2H, CH2), 3.52 (s, 2H, CH2), 3.64 (s, 2H, CH2), 3.81 (s, 2H, CH2), 3.82
(s, 3H, CH3), 6.93–6.99 (m, 3H, ArH), 7.28 (s, 1H, ArH), 7.53 (t, J = 4.0 Hz, 1H, ArH), 7.79 (dd, J
= 8.6 and 3.0 Hz, 2H, ArH), 8.91 (d, J = 3.2 Hz, 1H, NH), 10.75 (d, J = 2.4 Hz, 1H, pyrimidine-H),
13
11.64 (s, 1H, NH). C NMR (100 MHz, DMSO-d6) δ: 186.4, 163.0, 159.9, 158.0, 154.0, 136.7,
135.6, 131.6, 130.8, 126.3, 122.6, 122.2, 120.8, 120.5, 119.5, 114.0, 112.7, 66.3, 56.0, 44.7, 22.0.
HRMS (ESI) m/z: calcd for C25H24N6O5 [M+H]+ 489.1808, found 489.1891.
5.1.17 (4-chlorophenyl)(6-methyl-3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indol-2-yl)
methanone (27). Yield, 71.4%; mp 248.9–249.8°C; 1H NMR (400 MHz, DMSO-d6) δ: 2.44 (s, 3H,
CH3), 3.40 (s, 2H, CH2), 3.51 (s, 2H, CH2), 3.64 (s, 2H, CH2), 3.84 (s, 2H, CH2), 6.95 (d, J = 8.4 Hz,
1H, ArH), 7.27 (s, 1H, ArH), 7.50 (dd, J = 17.2 and 8.4 Hz, 3H, ArH), 7.76 (d, J = 8.0 Hz, 2H, ArH),
8.91 (s, 1H, NH), 10.62 (s, 1H, pyrimidine-H), 11.71 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6)
δ: 186.5, 159.8, 157.9, 154.0, 137.4, 137.0, 136.3, 130.9, 128.6, 126.0, 122.7, 122.4, 120.9, 120.4,
120.2, 112.7, 66.4, 44.7, 22.1. HRMS (ESI) m/z: calcd for C24H21ClN6O4 [M+H]+ 493.1313, found
493.1285.
340
5.1.18 (6-methyl-3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indol-2-yl)(thiophen-2-yl)
methanone (28). Yield, 72.3%; mp 281.2–282.6°C; 1H NMR (400 MHz, DMSO-d6) δ: 2.46 (s, 3H,
CH3), 3.44 (s, 2H, CH2), 3.52 (s, 2H, CH2), 3.64 (s, 2H, CH2), 3.87 (s, 2H, CH2), 6.97 (d, J = 8.0 Hz,
1H, ArH), 7.29 (d, J = 16.0 Hz, 2H, ArH, thiophene-H), 7.57 (d, J = 8.4 Hz, 1H, ArH), 8.04 (t, J =
345
350
13
13.4 Hz, 2H, thiophene-H), 9.01 (s, 1H, NH), 11.12 (s, 1H, pyrimidine-H), 11.72 (s, 1H, NH). C
NMR (100 MHz, DMSO-d6) δ: 178.8, 159.9, 158.2, 153.6, 143.3, 137.1, 136.0, 135.2, 134.3, 128.9,
125.2, 123.2, 122.3, 120.6, 120.4, 120.3, 112.9, 66.3, 44.8, 31.1, 22.1. HRMS (ESI) m/z: calcd for
C22H20N6O4S [M+H]+ 465.1267, found 465.1214.
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5.1.19 biphenyl-4-yl(6-methyl-3-(2-morpholino-5-nitropyrimidin-4-ylamino)-1H-indol-2-yl)
methanone (29). Yield, 58.7%; mp 256.7–258.2°C; 1H NMR (400 MHz, DMSO-d6) δ: 2.44 (s, 3H,
CH3), 3.42 (s, 2H, CH2), 3.53 (s, 2H, CH2), 3.64 (s, 2H, CH2), 3.84 (s, 2H, CH2), 6.95 (d, J = 8.4 Hz,
1H, ArH), 7.29 (s, 1H, ArH), 7.44 (t, J = 7.4 Hz, 1H, ArH), 7.50–7.55 (m, 3H, ArH), 7.65–7.70 (m,
4H, ArH), 7.84 (d, J = 8.0 Hz, 2H, ArH), 8.85 (s, 1H, NH), 10.68 (s, 1H, pyrimidine-H), 11.73 (s,
1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 187.2, 159.9, 157.8, 154.2, 144.2, 139.6, 137.2, 137.0,
136.1, 129.8, 129.5, 128.6, 127.3, 126.8, 126.2, 122.6, 122.4, 120.9, 120.5, 120.0, 112.8, 66.2, 44.7,
22.1. HRMS (ESI) m/z: calcd for C30H26N6O4 [M+H]+ 535.2016, found 535.2036.
355
360
365
5.1.20 (3-(5-nitro-2-thiomorpholinopyrimidin-4-ylamino)-1H-indol-2-yl)(phenyl)methanone (30).
1
Yield, 56.7%; mp 172.6–173.8°C; H NMR (400 MHz, CDCl3) δ: 2.49 (t, J = 4.6 Hz, 2H, CH2),
2.68 (t, J = 4.6 Hz, 2H, CH2), 3.76 (t, J = 4.6 Hz, 2H, CH2), 4.21 (t, J = 4.6 Hz, 2H, CH2), 7.19 (t, J
= 7.6 Hz, 1H, ArH), 7.41 (dd, J = 16.0 and 8.0 Hz, 3H, ArH), 7.47–7.54 (m, 2H, ArH), 7.65 (d, J =
8.0 Hz, 1H, ArH), 7.75 (d, J = 7.6 Hz, 2H, ArH), 8.94 (s, 1H, NH), 9.07 (s, 1H, pyrimidine-H),
10.24 (s, 1H, NH). 13C NMR (100 MHz, CDCl3) δ: 187.7, 159.9, 157.7, 154.4, 137.8, 135.6, 132.5,
128.4, 126.8, 126.4, 123.4, 122.5, 120.6, 120.4, 119.6, 112.4, 47.0, 46.9, 27.6. HRMS (ESI) m/z:
calcd for C23H20N6O3S [M+H]+ 461.1318, found 461.1401.
5.1.21 (4-bromophenyl)(3-(5-nitro-2-thiomorpholinopyrimidin-4-ylamino)-1H-indol-2-yl)
1
methanone (31). Yield, 59.0%; mp 265.3–266.6°C; H NMR (400 MHz, CDCl3) δ: 2.51 (s, 2H,
370
CH2), 2.71 (s, 2H, CH2), 3.77 (s, 2H, CH2), 4.25 (s, 2H, CH2), 7.21 (s, 1H, ArH), 7.47 (dd, J = 16.4
and 6.8 Hz, 2H, ArH), 7.57 (d, J = 7.6 Hz, 2H, ArH), 7.65 (d, J = 7.6 Hz, 3H, ArH), 8.96 (s, 1H,
NH), 9.02 (s, 1H, pyrimidine-H), 10.45 (s, 1H, NH). 13C NMR (100 MHz, CDCl3) δ: 186.4, 174.1,
159.3, 157.2, 154.2, 136.4, 135.7, 131.8, 130.0, 127.6, 127.1, 126.0, 123.2, 122.7, 120.9, 119.9,
112.5, 47.2, 27.7. HRMS (ESI) m/z: calcd for C23H19BrN6O3S [M+H]+ 540.0402, found 541.0483.
375
380
5.1.22 (4-methoxyphenyl)(3-(5-nitro-2-thiomorpholinopyrimidin-4-ylamino)-1H-indol-2-yl)
1
methanone (32). Yield, 63.5%; mp 254.8–255.9°C; H NMR (400 MHz, CDCl3) δ: 2.50 (s, 2H,
CH2), 2.70 (s, 2H, CH2), 3.77 (s, 2H, CH2), 3.85 (d, J = 3.6 Hz, 3H, CH3), 4.24 (s, 2H, CH2), 6.88
(d, J = 8.8 Hz, 2H, ArH), 7.19 (t, J = 7.4 Hz, 1H, ArH), 7.41 (t, J = 7.6 Hz, 1H, ArH), 7.50 (d, J =
8.4 Hz, 1H, ArH), 7.64 (d, J = 8.0 Hz, 1H, ArH), 7.79 (d, J = 8.4 Hz, 2H, ArH), 8.97 (s, 1H, NH),
9.14 (s, 1H, pyrimidine-H), 10.44 (s, 1H, NH). 13C NMR (100 MHz, CDCl3) δ: 186.4, 163.4, 159.0,
156.7, 154.2, 135.4, 131.0, 130.1, 126.7, 126.5, 123.3, 122.3, 120.6, 118.7, 113.7, 112.5, 55.5, 47.2,
27.6. HRMS (ESI) m/z: calcd for C24H22N6O4S [M+H]+ 491.1423, found 491.1509.
5.2 Biological evaluation
5.2.1 Antitumor activity
385
The antitumor activities of compounds 11–32 were evaluated with HeLa, MDA-MB-231,
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MCF-7, and HCT116 cell lines with the standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay in vitro. The cancer cell lines were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were
splitted at 70-80% confluence, about twice a week by trypsinization.
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395
400
Exponentially growing cells were plated in 96-well plates (1×104 cells/well) and incubated at
37 °C for 24 h for attachment. Test compounds were prepared by dissolving in dimethyl sulfoxide
(DMSO) at 10 mM and diluted with the medium into a series of concentrations. The culture
medium was then changed, and cells grew in medium with the test compounds. DMSO (0.1%) was
used as negative control. Cells were incubated at 37 °C for 48 h with or without compounds. Then
the medium was replaced with MTT solution (5 mg/mL, 100 µL) followed by incubation for
another 4 h. The medium was then aspirated and formazan crystals were dissolved in DMSO (100
µL) for about 10 min. The absorbance at 570 nm (Abs) of the suspension was measured with a
spectrophotometer. The inhibition percentage was calculated using the following formula: %
inhibition = (Abscontrol－Abscompound)/Abscontrol×100%. The IC50 values of the test compounds and
CA-4 were measured by treating cells with various concentrations of compounds, and analyzed by
use of the prism statistical package (GraphPad Software, San Diego, CA, U.S.A.).
5.2.2 In vitro tubulin polymerization assay
Pig brain microtubule protein was isolated by employing three cycles of temperature-dependent
assembly/disassembly according to method described by Shelanski, et al [27]. Homogeneous
tubulin was prepared from microtubule protein by phosphocellulose (P11) chromatography as has
been described previously [28]. The purified proteins were stored in aliquots at -70 °C.
Microtubule polymerization of tubulin protein, in solutions containing different concentrations
of compounds in PEM buffer (100 mM PIPES, 1 mM MgCl2, and 1 mM EGTA), 1 mM GTP, and
5% glycerol, was monitored at 37 °C by using light scattering at 340 nm with a SPECTRA MAX
190 (MD) spectrophotometer. Plateau absorbance values were used for calculations. CA-4 was used
as standard inhibitor of tubulin polymerisation, while DMSO was used as negative control. The
percent inhibition values for selected compounds were compared to the value of CA-4 and
measured the same day under the same conditions.
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5.2.3 Molecular docking study
A tubulin pdb structure (PDB ID: 1SA0) was imported from the PDB data bank
(www.rcsb.org). It was prepared for computational simulations using Protein Preparation Wizard
that performed assignment of bond orders, reassignment of H atoms, generation of zero-order bonds
to metals, removal of waters, generation of metal binding states, reassignment of H-bond networks
and restrained minimization of H atoms [29]. A grid of the complex of tubulin alpha and beta was
generated using Glide: van der Waals radius scaling factor 1.0, partial charge cutoff 0.25, ligand in
the workspace as the grid centroid, and the grid box dimension of 10Å×10Å×10Å. LigPrep was
used to prepare ligands for docking simulations (LigPrep, version 3.6, Schrodinger, LLC, New
York, NY, 2015). Docking simulations were performed in the SP and XP modes using Glide
MM-GBSA relative binding free energy calculation of the ligand was performed with Prime.
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Acknowledgments
We should thank Dr. Hong-Bing Zhao (Shanghai TANG Bioscience Co., Ltd.) for the test of
tubulin polymerisation activity. The present work was supported by National Natural Science
Foundation of China (21372113 and 21102069), and the Special Funds for the Cultivation of
Guangdong College Students' Scientific and Technological Innovation. (“Climbing Program”
Special Funds.) (No. pdjh2016b0108)
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References
[1] F. Mora-Bermúdez, W.B. Huttner. Novel insights into mammalian embryonic neural stem cell
division: focus onmicrotubules, Mol Biol Cell. 26 (2015) 4302–4306.
[2] R. Kaur, G. Kaur, R.K. Gill, R. Soni, J. Bariwal. Recent developments in tubulin
polymerization inhibitors: an overview, Eur. J. Med. Chem. 87 (2014) 89–124.
[3] M. Driowya M, J. Leclercq, V. Verones, A. Barczyk, M. Lecoeur, N. Renault, N. Flouquet, A.
Ghinet, P. Berthelot, N. Lebegue. Synthesis of triazoloquinazolinone based compounds as
tubulin polymerizationinhibitors and vascular disrupting agents. Eur. J. Med. Chem. 115 (2016)
393–405.
[4] Z. Gan-Or, G.A. Rouleau, E.E. Benarroch. Dynamics of microtubules and their associated
proteins: Recent insights and clinical implications, Neurology. 87 (2016) 2173.
[5] A.S. Kumar, M.A. Reddy, N. Jain, C. Kishor, T.R. Murthy, D. Ramesh, B. Supriya, A.
Addlagatta, S.V. Kalivendi, B. Sreedhar. Design and synthesis of biaryl aryl stilbenes
/ethylenes as antimicrotubule agents, Eur. J. Med. Chem. 60 (2013) 305–324.
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455
[6] P. Strzyz. Cytoskeleton: Microtubules set the beat, Nat Rev Mol Cell Biol. 17 (2016) 333.
[7] C. Dumontet, M.A. Jordan, Microtubule-binding agents: a dynamic field of cancer therapeutics,
Nat. Rev. Drug Discov. 9 (2010) 790-803.
[8] M.J. Hu, B. Zhang, H.K. Yang, Y. Liu, Y.R. Chen, T.Z. Ma, L. Lu, W.W. You, P.L. Zhao, Design,
synthesis and molecular docking studies of novel indole-pyrimidine hybrids as tubulin
polymerization inhibitors, Chem. Biol. Drug Des. 86 (2015) 1491–1500.
[9] B. Pasquier, Y. El-Ahmad, B. Filoche-Rommé, C. Dureuil, F. Fassy, P.Y. Abecassis, M.
Mathieu, T. Bertrand, T. Benard, C. Barrière, S. El Batti, J.P. Letallec, V. Sonnefraud, M.
Brollo, L. Delbarre, V. Loyau, F. Pilorge, L. Bertin, P. Richepin, J. Arigon, J.R. Labrosse, J.
Clément, F. Durand, R. Combet, P. Perraut, V. Leroy, F. Gay, D. Lefrançois, F. Bretin, J.P.
Marquette, N. Michot, A. Caron, C. Castell, L. Schio, G. McCort, H. Goulaouic, C. Garcia-
Echeverria, B. Ronan. Discovery of (2S)-8-[(3R)-3-methylmorpholin-4-yl]-1-(3-methyl-2-
oxobutyl)-2-(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one: a novel potent
andselective inhibitor of Vps34 for the treatment of solid tumors. J Med Chem. 58 (2015)
376–400.
460
465
470
475
480
[10] W. Peng, Z.C. Tu, Z.J. Long, Q.t. Liu, G. Lu. Discovery of 2-(2-aminopyrimidin-5-yl)-4-
morpholino-N-(pyridin-3-yl)quinazolin-7-amines as novel PI3K/mTOR inhibitors and anticancer
agents, Eur. J. Med. Chem. 108 (2016) 644–654.
[11] D.P. Sutherlin, L. Bao, M. Berry, G. Castanedo, I. Chuckowree, J. Dotson, A. Folks, L.
Friedman, R. Goldsmith, J. Gunzner, T. Heffron, J. Lesnick, C. Lewis, S. Mathieu, J. Murray, J.
Nonomiya, J. Pang, N. Pegg, W.W. Prior, L. Rouge, L. Salphati, D. Sampath, Q. Tian, V. Tsui,
N.C. Wan, S. Wang, B. Wei, C. Wiesmann, P. Wu, B.Y. Zhu, A. Olivero, Discovery of a potent,
selective, and orally available Class I phosphatidylinositol 3-kinase (PI3K)/mammalian target
of rapamycin (mTOR) kinase inhibitor (GDC-0980) for the treatment of cancer, J. Med. Chem.
54 (2011) 7579–7587.
[12] J.J. Wallin, K.A. Edgar, J.E. Guan, M. Berry, W.W. Prior, L. Lee, J.D. Lesnick, C. Lewis, J.
Nonomiya, J.D. Pang, L. Salphati, A.G. Olivero, D.P. Sutherlin, C. O'Brien, J.M. Spoerke, S.
Patel, L. Lensun, R. Kassees, L. Ross, M.R. Lackner, D. Sampath, M. Belvin, L.S. Friedman,
GDC-0980 is a novel class I PI3K/mTOR kinase inhibitor with robust activity in cancer models
driven by the PI3K pathway, Mol. Cancer Ther. 10 (2011) 2426–2436.
[13] F.I. Raynaud, S. Eccles, P.A. Clarke, A. Hayes, B. Nutley, S. Alix, A. Henley, F. Di-Stefano, Z.
Ahmad, S. Guillard, L.M. Bjerke, L. Kelland, M. Valenti, L. Patterson, S. Gowan, A. de Haven
Brandon, M. Hayakawa, H. Kaizawa, T. Koizumi, T. Ohishi, S. Patel, N. Saghir, P. Parker, M.
Waterfield, P. Workman, Pharmacologic characterization of a potent inhibitor of class I
15

ACCEPTED MANUSCRIPT
phosphatidylinositide 3-kinases, Cancer Res. 67 (2007) 5840–5850.
485
490
495
500
505
510
515
[14] S. Park, N. Chapuis, V. Bardet, J. Tamburini, N. Gallay, L. Willems, Z.A. Knight, K.M. Shokat,
N. Azar, F. Viguie, N. Ifrah, F. Dreyfus, P. Mayeux, C. Lacombe, D. Bouscary, PI-103, a dual
inhibitor of Class IA phosphatidylinositide 3-kinase and mTOR, has antileukemic activity in
AML, Leukemia 22 (2008) 1698–1706.
[15] M.T. Burger, S. Pecchi, A. Wagman, Z.J. Ni, M. Knapp, T. Hendrickson, G. Atallah, K. Pfister,
Y. Zhang, S. Bartulis, K. Frazier, S. Ng, A. Smith, J. Verhagen, J. Haznedar, K. Huh, E.
Iwanowicz, X. Xin, D. Menezes, H. Merritt, I. Lee, M. Wiesmann, S. Kaufman, K. Crawford,
M. Chin, D. Bussiere, K. Shoemaker, I. Zaror, S.M. Maira, C.F. Voliva, Identification of
NVP-BKM120 as a potent, selective, orally bioavailable class I PI3 kinase inhibitor for treating
cancer, ACS Med. Chem. Lett. 2 (2011) 774–779.
[16] J. Rodon, I. Brana, L.L. Siu, M.J. De Jonge, N. Homji, D. Mills, E. Di Tomaso, C. Sarr, L.
Trandafir, C. Massacesi, F. Eskens, J.C. Bendell, Phase I doseescalation and -expansion study
of buparlisib (BKM120), an oral pan-Class I PI3K inhibitor, in patients with advanced solid
tumors, Invest. New. Drugs 32 (2014) 670–681.
[17] L. Vymětalová, L. Havlíček, A. Šturc, Z. Skrášková, R. Jorda, T. Pospíšil, M. Strnad, V. Kryštof.
5-Substituted 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)Hpyrazolo[4,3-d]pyrimidines with
anti-proliferative activity as potent and selective inhibitors of cyclin-dependent kinases, Eur. J.
Med. Chem. 110 (2016) 291–301.
[18] G. Castanedo, D.M. Goldstein, R. K. Kondru, M.C. Lucas, W.S. Palmer, S. Price, B. Safina,
P.P.A. Savy, E.M. Seward, D.P. Sutherlin. et al. Preparation of bicyclic indolylpyrimidine
derivatives for use as PI3K-p110d inhibitors and useful in treatment of PI3 kinase mediated
disorders, WO 2010136491.
[19] R.A. Rane, P. Bangalore, S.D. Borhade, P.K. Khandare, Synthesis and evaluation of novel
4-nitropyrrole-based 1,3,4-oxadiazole derivatives as antimicrobial and anti-tubercular agents.
Eur. J. Med. Chem. 70 (2013) 49–58.
[20] Gregorić, M. Sedić, P.A. Grbčić, K.P.S. Tomljenović, M. Cetina, R. Vianello, S. Raić-Malić.
Novel pyrimidine-2,4-dione-1,2,3-triazole and furo[2,3-d]pyrimidine-2-one-1,2,3-triazole
hybrids as potential anti-cancer agents: Synthesis, computational and X-ray analysis and
biological evaluation. Eur. J. Med. Chem. 125 (2016) 1247–1267.
[21] L.Y. Ma, Y.C. Zheng, S.Q. Wang, B. Wang, Z.R. Wang, L.P. Pang, M. Zhang, J.W. Wang, L.
Ding, J. Li, C. Wang, B. Hu, Y. Liu, X.D. Zhang, J.J. Wang, Z.J. Wang, W. Zhao, H.M. Liu.
Design, synthesis, and structure-activity relationship of novel LSD1 inhibitors based on
pyrimidine-thiourea hybrids as potent, orally active antitumor agents. J Med Chem. 58 (2015)
1705–1716.
[22] R. Sribalan, G. Banuppriya, M. Kirubavathi, A. Jayachitra, V. Padmini, Multiple biological
16

ACCEPTED MANUSCRIPT
520
525
530
535
activities
and
molecular
docking
studies
of
newly
synthesized
3-(pyridin-4-yl)-1H-pyrazole-5-carboxamide chalcone hybrids. Bioorg Med Chem Lett. 26
(2016) 5624–5630.
[23] S. Bhakta, N. Scalacci, A. Maitra, A.K. Brown, S. Dasugari, D. Evangelopoulos, T.D. McHugh,
P.N. Mortazavi, A. Twist, E. Petricci, F. Manetti, D. Castagnolo. Design and Synthesis of
1-((1,5-Bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl)methyl)-4-methylpiperazine
and N-Adamantan-2-yl-N'-((E)-3,7-dimethylocta-2,6-dienyl)ethane-1,2-diamine
(BM212)
(SQ109)
Pyrrole Hybrid Derivatives: Discovery of Potent Antitubercular Agents Effective against
Multidrug-Resistant Mycobacteria, J Med Chem. 59 (2016) 2780–2793.
[24] H.K. Yang, W.W. You, G.H. Yan, Z.H. Jiang, P.L. Zhao, Z.Z. Zhou. Efficient one-pot synthesis
of 3-amino-7-azaindoles under microwave irradiation. Synthetic Commun. 44 (2014):
1165–1171.
[25] P.L. Zhao, W.F. Ma, A.N. Duan, M. Zou, Y.C. Yan, W.W. You, S.G. Wu. One-pot synthesis of
novel isoindoline-1,3-dione derivatives bearing 1,2,4-triazole moiety and their preliminary
biological evaluation, Eur. J. Med. Chem. 54 (2012) 813–822.
[26] R.B. Ravelli, B. Gigant, P.A. Curmi, I. Jourdain, S. Lachkar, A. Sobel, M. Knossow. Insight
into tubulin regulation from a complex with colchicine and a stathmin-like domain, Nature 428
(2004): 198–202.ꢀ
[27] M.L. Shelanski, F. Gaskin, C.R. Cantor, Microtubule assembly in the absence of added
nucleotides, Proc. Natl. Acad. Sci. U.S.A. 70 (1973)765–768.
540
545
[28] D.M. Barron, S.K. Chatterjee, R. Ravindra, R. Roof, E. Baloglu, D.G.I. Kingston, S. Bane, A
fluorescence-based high-throughput assay for antimicrotubule drugs. Anal. Biochem. 315
(2003) 49–56.
[29] M.H.M. Olsson, C.R. Søndergard, M. Rostkowski, J.H. Jensen. PROPKA3: consistent
treatment of internal and surface residues in empirical pKa predictions. J. Chem. Theor.
Comput. 7 (2011), 525–537.
550
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Fig. captions
Fig. 1. Our previously reported the indole-pyrimidine hybrid 1 and selected morpholine or thiomorpholine-linked
pyrimidine and indole derivatives with antitumor activity.
Fig. 2. Design strategy of the title compounds.
Fig. 3. Effect of compound 14 on cell cycle and apoptosis in HeLa cells. Flow cytometry analysis of HeLa cells
treated with 14 for 48 h. (A) Control; (B) 14, 1.25 µM; (C) 14, 2.5 µM; (D) 14, 5 µM.
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Fig. 4. Docking pose of compound 15 in tubulin with (a) and without (b) the surface of the pocket (PDP ID:
1SA0). Amino acid residues of tubulin beta chain are labeled in yellow and those in tubulin alpha in orange.
MM-GBSA ΔG is -83.1 kcal/mol.
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Fig. 1. Our previously reported the indole-pyrimidine hybrid 1 and selected morpholine or thiomorpholine-linked
pyrimidine and indole derivatives with antitumor activity.
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Fig. 2. Design strategy of the title compounds.
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615
A
B
C
D
Fig. 3. Effect of compound 14 on cell cycle and apoptosis in HeLa cells. Flow cytometry analysis of HeLa cells
treated with 14 for 48 h. (A) Control; (B) 14, 1.25 µM; (C) 14, 2.5 µM; (D) 14, 5 µM.
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A
B
Fig. 4. Docking pose of compound 15 in tubulin with (A) and without (B) the surface of the pocket (PDP ID:
1SA0). Amino acid residues of tubulin beta chain are labeled in yellow and those in tubulin alpha in orange.
MM-GBSA ΔG is -83.1 kcal/mol.
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650
Scheme 1 One-pot synthesis of compounds 11–32
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660
Table 1 Antiproliferative activities of compounds 11–32 against a panel of human cancer and
normal cell lines
In vitro antiproliferative IC50(µM) a
Comp.
X
R1
R2
R3
HeLa
82.55
54.30±3.69
MDA-MB-231
92.63 ±0.96
34.12±0.78
MCF-7
89.2±1.26
85.39±5.26 33.28±2.22
HCT116
55.77±2.13
HEK293 b
NT c
11
12
O
O
H
H
C6H5
4-BrC6H4
H
H
NT
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
O
O
O
O
O
O
O
H
H
H
H
H
4-MeOC6H4
3-BrC6H4
4-PhC6H4
H
H
H
H
4.32±0.29
2.51±0.18
4.04±0.37
49.65±2.56
8.69±0.33
9.42±0.25
10.35±0.23
89.36±2.34
3.01±0.18
>100
57.12±2.36
1.42±0.09
0.29±0.02
89.36±2.39 73.49±2.13
2.13±0.14
>100
6.40±0.14 14.77±0.15
11.42±0.23 10.06±0.53
9.48±0.11 78.63±1.25
NT
4-MeOC6H4 CO2Et 5.52±0.26
2.99±0.13
>100
>100
Cl
Cl
C6H5
4-ClC6H4
H
H
>100
>100
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
>100
NT
>100
83.65±3.25
>100
O
O
O
O
O
O
O
O
O
O
S
Cl 4-MeOC6H4
Cl
Cl
Cl
CH3
CH3
H
H
CO2Et
H
H
H
H
H
H
H
H
H
H
47.05±3.65
94.65±0.99
>100±0.
16.57±2.61
47.09±3.56
>100
90.53±2.15
>100
>100
7.46±0.21
>100
>100
>100
35.03±1.53 24.35±2.36
69.36±1.03 >100
78.39±5.39 80.56±5.36
42.69±1.23 >100
56.81±1.03 80.63±0.26
>100
>100
98.68±4.26
>100
C6H5
4-PhC6H4
C6H5
35.79±0.15
58.69±1.29
76.32±3.25
>100
89.36±0.15
>100
4-BrC6H4
>100
43.95±4.42
>100
39.11±3.26
>100
CH3 4-MeOC6H4
CH3
CH3
CH3
H
H
H
4-ClC6H4
93.65±0.32
>100
4-PhC6H4
C6H5
4-BrC6H4
4-MeOC6H4
1
60.36±3.68
>100
>100
>100
>100
>100
>100
>100
S
>100
>100
19.91±1.23
0.23±0.018
>100
>100
S
5.01±0.15
270.0±1.65
13.93±0.29 17.43±1.02
0.041±0.013 6.10±0.14
CA-4
a
50% inhibitory concentration and mean
±
SD of three independent experiments performed in duplicate. b Normal human embryonic
kidney (HEK-293) cell lines. c NT: not tested.
665
Table 2. Effect of compound 14 on cell cycle distribution in HeLa cells
Concentration G0/G1(%) a S(%) b G2/M(%) c
0µM
1.25µM
2.5µM
5µM
61.74
53.32
15.31
5.02
29.14
34.05
26.35
6.40
9.12
12.63
58.34
88.58
a G0/G1: to prepare the cell for DNA synthesis. b S: DNA is manufactured during the phase.
c G2/M: is the phase in which DNA replication completed, start to mitosis.
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Table 3 Tubulin polymerization inhibitory activities of selected representative compounds
Tubulin polymerization
Comp.
X
R1
R2
R3
% inhibition a
IC50(µM)
b
11
13
14
15
17
18
20
21
30
O
O
O
O
O
O
O
O
S
H
H
H
H
H
Cl
C6H5
H
H
H
H
23
14
36
42
19
9
14
19
2
-
4-MeOC6H4
3-BrC6H4
4-PhC6H4
-
-
19.3±1.18
4-MeOC6H4 CO2Et
-
-
-
-
C6H5
H
H
H
H
Cl 4-MeOC6H4
Cl
H
C6H5
1
-
47
11.2±1.13
CA-4
80
4.22±0.22
a Compounds were tested at a final concentration of 10µM. b -: not tested.
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Highlights
• 22 novel indole-pyrimidine hybrids were designed and synthesized. •
Antiproliferative activities of these compounds were evaluated. • Compound 14
arrested HeLa cells in G2/M phase of cell cycle. • Hybrid 15 exhibited powerful
tubulin inhibitory activity. • Molecular modeling suggested that 15 binds well in the
colchicine binding site of α,β-tubulin.