Design, synthesis and biological evaluation of methylenehydrazine-1-carboxamide derivatives with (5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-indole scaffold: Novel potential CDK9 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104064

In continuation of our previous work on the investigation of CDK9 inhibitors bearing indole moiety for the discovery of novel anticancer agents, novel methylenehydrazine-1-carboxamide derivatives with (5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-indole scaffold were designed, synthesized, and evaluated for the CDK9 inhibitory activity and anticancer activity. Biological activity results demonstrated that most of these derivatives possessed good inhibitory on the kinase activity of CDK9 such as blocking its phosphorylation function and inhibiting HIV-1 transcription. Compound 12i was found to be the most potent CDK9 inhibitor and exhibited excellent anticancer activity against HepG2, A375, MCF-7, and A549, but low toxic on normal cells including HaCaT and MCF-10A. Further studies revealed that as a result of CDK9 inhibition and subsequent inhibition of phosphorylation at Serine 2 of the RNAPII CTD, the representative compound 12i dose-dependently increased cleaved PARP level, exerting its antiproliferative effect through induction of apoptosis in cancer cells. Finally, the molecular docking analysis implied that 12i had a good binding affinity with CDK9. In summary, 12i is a potent CDK9 inhibitor and can be considered as a good lead-candidate for developing potential anticancer drugs.

Full text of DOI:10.1016/j.bioorg.2020.104064

Journal Pre-proofs
Design, synthesis and biological evaluation of methylenehydrazine-1-carbox-
amide derivatives with (5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-indole
scaffold: novel potential CDK9 inhibitors
Hongyu Hu, Jun Wu, Mingtao Ao, Xiaoping Zhou, Boqun Li, Zhenzhen Cui,
Tong Wu, Juanli Wang, Yuhua Xue, Zhen Wu, Meijuan Fang
PII:
S0045-2068(20)31361-4
DOI:
https://doi.org/10.1016/j.bioorg.2020.104064
YBIOO 104064
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
19 March 2020
25 June 2020
29 June 2020
Please cite this article as: H. Hu, J. Wu, M. Ao, X. Zhou, B. Li, Z. Cui, T. Wu, J. Wang, Y. Xue, Z. Wu, M. Fang,
Design, synthesis and biological evaluation of methylenehydrazine-1-carboxamide derivatives with (5-((4-
(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-indole scaffold: novel potential CDK9 inhibitors, Bioorganic Chemistry
(2020), doi: https://doi.org/10.1016/j.bioorg.2020.104064
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Design, synthesis and biological evaluation of
methylenehydrazine-1-carboxamide derivatives with (5-((4-
(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-indole scaffold: novel
potential CDK9 inhibitors
Hongyu Hua,b#, Jun Wua,#, Mingtao Aoa,#, Xiaoping Zhoua, Boqun Lia, Zhenzhen Cuia,
Tong Wua, Juanli Wanga, Yuhua Xuea,*, Zhen Wua,*, Meijuan Fanga,*
a
School of Pharmaceutical Sciences and the Key Laboratory for Chemical Biology of Fujian
Province, Xiamen University, South Xiang-An Road, Xiamen, 361102, China.
b Xingzhi College, Zhejiang Normal University, LanXi, 321004, China
# These authors contributed equally to this paper
* Corresponding authors. Tel.: +86-592-2189868; Fax: +86-592-2189868.
E-mail address: fangmj@xmu.edu.cn (Meijuan Fang); wuzhen@xmu.edu.cn (Zhen Wu) and
xueyuhua@xmu.edu.cn (Yuhua Xue)

Abstract:
In continuation of our previous work on the investigation of CDK9 inhibitors bearing
indole moiety for the discovery of novel anticancer agents, novel methylenehydrazine-
1-carboxamide derivatives with (5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-indole
scaffold were designed, synthesized, and evaluated for the CDK9 inhibitory activity
and anticancer activity. Biological activity results demonstrated that most of these
derivatives possessed good inhibitory on the kinase activity of CDK9 such as blocking
its phosphorylation function and inhibiting HIV-1 transcription. Compound 12i was
found to be the most potent CDK9 inhibitor and exhibited excellent anticancer activity
against HepG2, A375, MCF-7, and A549, but low toxic on normal cells including
HaCaT and MCF-10A. Further studies revealed that as a result of CDK9 inhibition and
subsequent inhibition of phosphorylation at Serine 2 of the RNAPII CTD, the
representative compound 12i dose-dependently increased cleaved PARP level, exerting
its antiproliferative effect through induction of apoptosis in cancer cells. Finally, the
molecular docking analysis implied that 12i had a good binding affinity with CDK9. In
summary, 12i is a potent CDK9 inhibitor and can be considered as a good lead-
candidate for developing potential anticancer drugs.
Keywords: 4-(pyridin-3-yl)pyrimidine; indole; CDK9; anticancer; apoptosis.
1. Introduction

Cancer is one of the most serious diseases threatening the health of humans worldwide.
The activation of oncogenes and the dysfunction of tumor suppressors are essential for
tumorigenesis and induce the cancer cells to proliferate uncontrollably.[1-4] Most of the
oncogenes and cancer suppressors are tightly regulated by transcription. For instance,
the activity of c-Myc and p53 (two most frequently mutated genes) could be influenced
by the cyclin-dependent protein kinase 9 (CDK9) which plays a key role in transcription
regulation, but in external roles regarding cell cycle regulation.[5-7] Besides, the
overexpression of CDK9 has been demonstrated in many tumors including leukemia
and malignant melanoma.[8-11] CDK9 and its partner T (T1, T2a, and T2b) establish the
main core of the positive transcription elongation factor b (P-TEFb) complex. P-TEFb
is essential for the transcription regulations, especially in the transcription elongation
of human immunodeficiency virus 1 (HIV-1) and the transcription regulation of short-
lived anti-apoptotic proteins that are important for the survival of many tumor cells.[12-
14]
It has been reported that CDK9 inhibitors can bind to CDK9, and block its
phosphorylation and kinase activity, thereby preventing PTEFb-mediated activation of
RNA Pol II and leading to the inhibition of gene transcription of various anti-apoptotic
proteins.[15] For example, CDK9 inhibitors such as Flavopiridol, SNS-032, and
LDC000067 can decrease the mRNA level of c-Myc and increase the transcription level
of p53, thus induce the death of cancer cells and repress tumor growth.[1]
In recent years, increasing CDK9 inhibitors of various structure classes (Figure 1) have
been reported, which can effectively suppress tumor growth.[16-21] However, despite
mass data indicated that CDK9 is a promising target for HIV-1 and cancer, but the
CDK9 related therapies have not been wildly employed due to unacceptable toxicity or
undesirable side effects. For example, Flavopiridol has many adverse reactions that
occurred in phase Ⅰ and Ⅱ clinical trials, such as thrombocytopenia, thrombosis,
neutropenia, and fatigue. So, the current challenge is to develop a variety of novel
CDK9 inhibitors with more potent and low cytotoxicity to normal cell lines.
Insert Figure 1 here.

Figure 1. Structures of representative CDK9 inhibitors.
In our previous work,[22] two 2,5-disubstituted indole derivatives 2c and 3b (Figure 2)
were found to exhibit good CDK9 inhibitory and anticancer activity. Herein, to
optimize/improve the biological activities, two series of target compounds were
designed based on the structures of lead compounds 2c and 3b by using scaffold
hopping, hybridization, and substitution decorating strategies (Figure 2). For hit
compound 2c, we changed the indole scaffold to a phenyl ring with an amino group,
replaced flexible linker and phenyl group with the 2-(2-azanyl)-4-(pyridin-3-
yl)pyrimidine moiety, and hybridized with the formohydrazide structure of 3b,
constructing the structure of target compounds 4a-4n (series 1). Meanwhile, for hit
compound 3b, we retained the indole-2carboxamide scaffold, similarly changed the
formamide substitution at the 5-position of the indole ring to 2-(2-azanyl)-4-(pyridin-
3-yl)pyrimidine moiety, and last introduced different substitutions at the position of the
methylene group, building the structure of the target compounds 12a-12l (series 2).
Insert Figure 2 here.
Figure 2. Design strategy and structure of novel CDK9 inhibitors as anticancer agents.
As a continuation of our previous research, we synthesized the new two series of target
compounds and studied their CDK9 inhibitory and anticancer activities. The inhibitory
activity on CDK9 was determined by using a luciferase transcription assay for HIV-1
transcription efficiency and the western blotting analysis for the phosphorylated Ser2
level of RNAPII CTD. Three compounds (12d, 12f, and 12i) identified as good CDK9
inhibitors were then evaluated for the anti-cancer activity against a panel of cancer cell
lines and the toxic effect on normal cells. Furthermore, we performed cell apoptosis
analysis and docking studies for the hit compound 12i obtained from the study. The
results showed that compound 12i was the most potent CDK9 inhibitor with excellent
anticancer activity and low toxic activity on normal cells.

2. Results & discussion
2.1 Chemistry
The
synthesis
of
novel
N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)
amino)phenyl)-2-methylenehydrazine-1-carboxamide derivatives 4a-4n is depicted in
Scheme1. The key intermediate 3 was obtained by a two-step synthetic rout as
24]
described previously.[23,
Briefly, treatment of 6-methyl-N1- (4-(pyridin-3-yl)
pyrimidin-2-yl)benzene-1,3-diamine (1) with phenyl carbonochloridate in the presence
of trimethylamine resulted in phenyl (4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-
yl)amino)phenylcarbamate (2), followed by the reaction with hydrazine hydrate to get
the
corresponding
4-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-
ylamino)phenyl)semicarbazide (3). Finally, the intermediate 3 was condensed with
different aldehyde under the refluxing condition to yield the targeted of Series 1 (4a-
4n).
Insert Scheme 1 here.
Scheme 1. Synthetic Route of N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)- 2-
methylenehydrazine-1-carboxamide derivatives 4a-4n (target compounds of Series 1).
N'-methylene-5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-indole-2-carbohydrazide
derivatives 12a-12l were prepared as described in Scheme 2. The key intermediate 8
was synthesized from the commercially available (4-nitrophenyl)hydrazine (5) using a
procedure reported in our previous work.[22] Then, ethyl 5-(4-(pyridin-3-yl)pyrimidin-
2-ylamino)-1H-indole-2-carboxylate (10) was made from the intermediate 8 using the
method reported previously,[25] followed by refluxing with Hydrazine hydrate for 6
hours to produce the 5-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-1H-indole-2-
carbohydrazide (11). Finally, the intermediate 11 refluxed with different aldehydes in

EtOH for 8 hours, converting to the corresponding final compounds 12a-12l (Series 2).
Insert Scheme 2 here.
Scheme 2. N'-methylene-5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-indole-2-carbohydrazide
derivatives 12a-12l
1
The chemical structures of target compounds were characterized by HRMS, H
13
NMR, and C NMR. These acylhydrazone compounds were further identified as a
single E-isomer according to our previous work[26] and other similar studies[27-30]
.
Moreover, the interpretations of the spectral data for two representative examples, 4l
and 12i, have been added to the supplementary materials (Table S1 and Figure S1-
S6).
2.2. Identification of CDK9 inhibitors from target compounds
2.2.1. The inhibition of HIV-1 transcription provides a rapid readout of CDK9-
inhibiting activity and SAR analysis.
CDK9 is an attractive target for the treatment of cancer and we’d like to discover new
potential CDK9 inhibitors as anticancer agents. CDK9 is a key component of the P-
TEFb complex which plays an important role in HIV-1 transcription. CDK9 inhibitors
reported can effectively inhibit HIV-1 transcription.[31] Herein, the effect of all target
compounds (4a-4n and 12a-12l) on the HIV-1 transcription was evaluated using a
luciferase transcription assay in NH2 cells (HeLa cells with an integrated HIV-1 LTR-
luciferase reporter gene and stably expressing PCDNA3 Tat-flag)[32] By comparing to
DMSO-treated NH2 cells, the HIV-1 transcription rate of NH2 cells treated with
synthesized compounds at three different concentrations were presented in Table 1 and
Table 2. The lower rate of HIV-1 transcription reveals a higher potential inhibitory of
the tested compound on CDK9 activity. The results showed that, among these two series

of target compounds, 9 compounds performed higher inhibitory activities with the
transcription rate of lower than 0.50 (the HIV-1 transcription inhibition rate > 50%) at
2.0 µM, implying that they had better inhibitory on HIV-1 transcription than our
previous reported compounds 2c and 3b.[22] Especially, compound 12i exhibited similar
inhibitory activity compared with the positive compound FLP, revealing that 12i might
be a great potential CDK inhibitor. Attempts were made to establish relationships
between the inhibitory activity of HIV-1 transcription and the structure of the target
compounds. In combination with our obtained data, several points for SARs analysis
were discussed as follows.
The inhibitory effect of N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)
phenyl)-2-methylenehydrazine-1-carboxamide derivatives (4a-4n). By analysis of
the data from Table 1, we found that all compounds except 4a showed moderate
inhibitory activity against HIV-1 transcription with the HIV transcription rate of less
than 0.60 (the HIV-1 transcription inhibition rate > 40%) at 20 µM. Among those
compounds containing unsubstituted aryl groups, aromatic heterocyclic substitutions
have more potential to inhibit HIV-1 transcription than benzene and naphthalene
substitutions, the inhibitory potency at 20 µM was decreased in the order 2-furyl (4k) >
2-thienyl (4l) > 3-pyridyl (4m) > 1-naphthyl (4n) > phenyl (4b). To evaluate the
importance of the substituted position and the nature of the substituted group at the
phenyl ring, compounds 4c-4j were subsequently synthesized. We found that the
substitution at the phenyl ring had an obvious influence on the inhibitory activity.
Compared with compound 4b (phenyl), compounds 4c (4-Cl-phenyl), 4d (2-Cl-phenyl),
4h (3,4-OCH3-phenyl), and 4i (3,4-Cl-phenyl) showed enhanced the inhibitory activity.
In particular, compound 4c with 4-Cl-phenyl substitution suppressed HIV-1
transcription to 0.41±0.02 at 2.0 µM and 0.57±0.09 at 0.2 µM, displaying better
inhibitory activity than previously reported compounds 2c (0.56±0.02 at 2.0 µM and
0.86±0.02 at 0.2 µM) and 3b (0.64±0.05 at 2.0 µM and 0.76±0.06 at 0.2 µM).
Insert Table 1 here.

Table 1. The inhibitory activity of target compounds 4a-4n.
The inhibitory effect of N'-methylene-5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-
1H-indole-2-carbohydrazide derivatives (12a-12l). The results in Table 2, indicated
that most target compounds of series II at both 20 µM and 2.0 µM displayed good
inhibitory activities with the HIV transcription rate of less than 0.50. By comparing the
inhibitory activity of N-(4-methyl-3-((4-substitute phenyl) hydrazine-1-carboxamide
derivatives with that of the corresponding 5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-
1H-indole-2-carbohydrazide derivatives, we could conclude that the replacing phenyl
moiety with indole as the skeletal structure had an obvious improvement in the
inhibition activity of HIV-1 transcription. For example, with the same 2-Cl substitution
on the terminal phenyl, compound 12d (with the HIV transcription rate of 0.20±0.05 at
2.0 µM) displayed a more potent inhibitory effect on HIV-1 transcription than
compound 4d (with the HIV transcription rate of 0.56±0.02 at 2.0 µM). Similar results
can be observed between compound 12f and 4h (both with 3,4-OCH3-phenyl
substitution) and their HIV-1 transcription rates were 0.30±0.02 and 0.57±0.04 at 2.0
µM, respectively. To further evaluate the inhibition effect of different substituted
phenyl rings on HIV-1 transcription, compounds 12a (3-OCH3), 12b (4-SCH3), 12c (4-
COOH), and 12e (2,6-OCH3) were subsequently synthesized. The 50% inhibiting
concentration value of compound 12c was over 20.0 µM, suggesting that the
introduction of a strong electron-withdrawing group to the terminal phenyl might be
unfavorable for the inhibition of HIV-1 transcription. Among compounds 12g-12l,
compound 12i with 3-indolyl substitution had the HIV-1 transcription rate of the HIV-
1 transcription of 0.20±0.10 at 0.2 µM, displaying the highest inhibition of HIV-1
transcription. Collectively, the SAR studies led to the discovery of a novel 2,5-
disubstituted indole derivative 12i which exhibited a good potency in inhibiting HIV-1
transcription (IC50 < 0.2 µM) and might be an excellent CDK9 inhibitor. Compound
12i was then assessed in a dose-dependent reporter gene assay. As shown in Figure
3A, compound 12i showed a dose-dependent effect on the inhibition of the HIV-1
transactivation, and 0.5 μM 12i displayed a similar inhibitory effect when compared

with 0.1 μM FLP as a reference.
Insert Table 2 here.
Table 2. The inhibitory activity of target compounds 12a-12l.
2.2.2. The inhibitory effect of 12i, 12d, and 12f on the phosphorylation at Ser2 of
RNAPII CTD
Based on the above experiments’ data, compounds 12i, 12d, and 12f were found to be
the most three effective inhibitors of HIV-1 transcription in NH2 cells. It is well known
that CDK9 plays a key role in the phosphorylation at Ser2 of RNAPII CTD.[33] To
confirm these three compounds as CDK9 inhibitors, we then checked the
phosphorylated Ser2 (p-Ser2) level of RNAPII CTD using western blot analysis.
Figure 3(B) showed the p-Ser2 level of RNAPII CTD slightly decreased when HeLa
cells treated with 12d and 12f at 2.0 µM for 3 hours, but the changes were obvious
when treated with 12i at 2.0 µM for 3 hours. It indicated that all these three compounds
could block the phosphorylation function of CDK9 and compound 12i was the most
potential CDK9 inhibitor. To further test the inhibitory effect of compound 12i on
CDK9 kinase activity, HeLa cells were treated with various concentrations of 12i for
3h and the p-Ser2 level of RNAPII CTD was then checked. The results in Figure 3(C)
showed that compound 12i decreased the p-Ser2 level in a dose-dependent manner.
Taken together, these data indicate that compound 12i and other derivatives could act
as CDK9 inhibitors to block phosphorylation and repress HIV-1 transcription.
Insert Figure 3 here.
Figure 3. (A) The HIV-1 transcription inhibition of compound 12i at different concentrations in
NH2 cells; (B) Western blotting analysis of the phosphorylated Ser2 (p-Ser2) level of RNAPII CTD
in Hela cells with the treatment of compounds 12i, 12d, and 12f; (C) Western blotting analysis of
the phosphorylated Ser2 (p-Ser2) level of RNAPII CTD in HeLa cells treated with 12i at different

concentrations.
2.3. In vitro antitumor activity and safety of compounds 12d, 12f, and 12i acting as
CDK9 inhibitors.
To check whether this kind of indole derivatives have the antitumor activity,
compounds 12d, 12f, and 12i with significant inhibition on the kinase activity of CDK9
were evaluated the cytotoxicity on four cancer cell lines including A375 (skin cancer),
A549 (lung cancer), HepG2 (liver cancer), and MCF-7 (breast cancer) using an MTT
assay with FLP as a reference. As shown in Table 3, all three compounds had moderate
anticancer activity and compound 12i was more potent than compounds 12d and 12f
against the four tested cancer cell lines, which is consistent with the luciferase assay
and western blot analysis. Compound 12i displayed the strongest cytotoxicity on the
tested four cell lines with the IC50 values ranging from 0.07 μM to 0.53 μM. It implied
that compound 12i acted as a good CDK9 inhibitor to result in excellent antitumor
activity.
To verify the safety of these synthesized compounds, compounds 12d, 12f, and 12i
were then selected to test the cytotoxicities in two normal cell lines including MCF-
10A and HaCaT cell lines. As demonstrated in Table 3, the results showed that though
these three compounds effectively repress the growth of cancer cells, they have low
cytotoxicity on the two normal cells (IC50 >50 μM).
Insert Table 3 here.
Table 3. The in vitro cytotoxicity of compounds 12d, 12f, and 12i on cancer and normal
cells.
2.4. Effects of Compound 12i on cell apoptosis
The induction of apoptosis is a common way for cancer therapy, and numerous studies

have demonstrated that the CDK9 inhibitors could effectively induce cell apoptosis of
cancer.[34] Herein, we further evaluated compound 12i for its ability effect on the
induction of apoptosis by checking the expression level of the specific apoptosis-
associated protein (cleaved PARP) and detecting apoptosis cells in 12i-treated cancer
cells.
2.4.1. Induction of PARP cleavage by compound 12i.
We checked the effect of 12i on the cleaved PARP levels in four types of cancer cell
lines (HepG2 cells, A375 cells, MCF-7 cells, and A549 cells) by using western blot
analysis. The assays were performed at six different doses (0, 0.2, 0.5, 1.0, 2.0, and 5.0
M) for 24 hours, the results of which are summarized in Figure 4. The western blot
results showed that the cleaved PARP increased obviously in A375 cells and MCF-7
cells after treated with 0.5 μM 12i for 24 hours. However, in HepG2 cells and A549
cells, the PARP cleavage could only be detected after treated with1.0 μM 12i for 24
hours. These results implied that compound 12i induced cell apoptosis in these four
tested cancer cell lines.
Insert Figure 4 here.
Figure 4. Induction of PARP cleavage by compound 12i. Different cancer cell lines were treated
with compound 12i at indicated concentrations for 24 h. Cleaved PARP was tested by western
blotting.
2.4.2. Cell apoptosis analysis in 12i-treated HepG2 cells.
Compound 12i has the best antitumor activity against HepG2 cells with the IC50 value
of 0.07 μM, and the PARP cleavage was obvious in 12i-treated HepG2 cells. To further
elucidate the proliferation inhibition on HepG2 cells, we analyzed HepG2 cells treated
with different concentrations of compound 12i by flow cytometry after Annexin V-
FITC/PI double staining. As observed in Figure 5, after 24 h of treatment, 12i displayed
an obvious apoptotic induction on HepG2 cells. The treatment of HepG2 with 12i at

1.0 and 5.0 μM concentrations resulted in 24.2% and 36.3% apoptotic cells,
respectively.
These results imply that compound 12i has a severe effect on cellular apoptosis in
cancer cells, indicating that cell apoptosis induced by 12i contributes to its antitumor
effect.
Insert Figure 5 here.
Figure 5. Apoptosis assay of compound 12i on HepG2 cells. HepG2 cells were treated with
compound 12i at indicated concentrations for 24 h, then subjected to double stating and detected
through FACS.
2.5. Molecular docking
To characterize the binding nature of 12i to CDK9, we performed a molecular docking
study for 12i and CDK9. The docking study revealed that compound 12i exhibited a
strong binding affinity with CDK9, with a docking score of -12.822 kcal/mol for the
first top-scored docking mode shown in Figure 6. The docking study showed that 12i
was well accommodated into the LBP of CDK9 (Figure 6(A)). The specific non-
bonded interactions between 12i and the residues of CDK9 were shown in Figure 6
(B)&(C). We observed three important interactions between 12i and CDK9, including
hydrogen bonds, hydrophobic interactions, and the π-π stacking interaction. The moiety
of 4-(pyridin-3-yl) pyrimidine inserted deeply into the hydrophobic pocket formed by
PHE105, PHE103, VAL79, LEU70, IEL25, and GLY26. Moreover, the N-1 atom of
pyrimidine and near amino nitrogen formed a hydrogen bond with the backbone's
amino-group and hydroxyl of amino acid residue CYS106, respectively. Besides, the
right indole group extended out of the pocket and interacted with ASP167 and LYS151
via forming a hydrogen bond and the π-π stack, respectively. These molecular docking
results further supported the above biological assay data and suggested that 12i may be
a potential CDK9 inhibitor.

Insert Figure 6 here.
Figure 6. Molecular docking study of 12i and CDK9.
3. Conclusion
In summary, 14 new N-(4-methyl-3-((4-substituted phenyl) hydrazine-1-carboxamide
derivatives (4a-4n) and 12 new 5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-indole-
2-carbohydrazide derivatives (12a-12l) were designed, synthesized, and evaluated for
the inhibitory activity on CDK9 and the anticancer activity. Compounds 12d, 12f, and
12i showed higher inhibitory activity on HIV-1 transcription mediated by CDK9 than
other synthesized compounds, and they also effectively inhibited the phosphorylation
of Ser2 residues in the CTD of RNAPII (the substrate of CDK9). Among them,
compound 12i was found to be the most potent CDK9 inhibitor which suppressed both
HIV-1 transcription and the phosphorylation at Serine 2 of the RNAPII CTD in a dose-
dependent manner. Moreover, compound 12i showed excellent cytotoxicity against all
four tested cancer cell lines, and it was also found to exert its antiproliferative effect
through the induction of apoptosis in HepG2 cells. Finally, the molecular docking
analysis indicated compound 12i as a good CDK9 inhibitor and predicted their binding
modes. In future work, we will use compound 12i as a good lead-candidate for the
optimization of new potent anticancer agents with the indole scaffold and the inhibitory
activity on CDK9.
4. Experimental section
4.1 Chemistry
All reagents were purchased and used without further purification unless otherwise
indicated. Reactions were magnetically stirred and monitored by thin-layer
chromatography (TLC) on Merck silica gel 60F-254 by fluorescence. All of the final
compounds were purified by column chromatography. 1H-NMR and 13C-NMR spectra

were obtained using a Bruker AV2 600 Ultra shield spectrometer at 600 and 150 MHz,
Chemical shifts were given in parts per million (ppm) relative to tetramethylsilane
(TMS) as an internal standard. Multiplicities were abbreviated as follows: single (s),
doublet (d), doublet-doublet (dd), doublet-triplet (dt), triplet (t), triplet-triplet (tt),
triplet-doublet (td), quartet (q), quartet-doublet (qd), multiplet (m), and broad signal
(brs). High-resolution mass spectral (HRMS) data were acquired on a Q Exactive.
Melting points were measured on a SGW X-4 micro-melting point spectrometer and
were uncorrected. The purity of 12i was assessed by high performance liquid
chromatography (HPLC) with a COSMOSIL C18-MS-II column (250 mm * 4.6 mm
i.d., 5 m). The HPLC analysis was performed on an Agilent Technologies 1100 Series
HPLC system. The mobile phase was acetonitrile (A) and water (B), eluted as the
following gradient program: A from 80% to 100% and B from 20% to 0% during 0-20
min. The flow rate was 1.0 mL/min and the detection wavelengths were 254 nm and
280 nm.
4.1.1. phenyl (4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)carbamate (2)
6-methyl-N1-(4-(pyridin-3-yl)pyrimidin-2-yl)benzene-1,3-diamine (0.277 g, 1.0 mmol) was
dissolve in dichloromethane (10 mL). The obtained solution was cooled down on an ice-water bath.
Then phenyl carbonochloridate (0.15 mL, 1.2 mmol), triethylamine (0.17 mL, 1.2 mmol) were
added in order. The reaction mixture was stirred for 5 h when TLC indicated that the reaction had
gone to completion, the mixture was concentrated and purified by column chromatography using
1
appropriate mixtures of EtOAc and PE. White solid (62%), m.p. 176.3~176.6
℃
. H-NMR (600
MHz, CDCl3): δ 9.25(s, 1H), 8.71 (d, J = 3.8 Hz, 1H), 8.53 (d, J = 5.0 Hz, 2H), 8.51 (brs, 1H), 7.42
(t, J = 8.0 Hz, 2H), 7.36 (dd, J = 5.0, 7.5 Hz, 1H), 7.28 (s, 1H), 7.27 (d, J = 7.5 Hz, 1H), 7.24 (s,
1H), 7.23 (s, 1H), 7.21-7.18 (m, 2H), 7.13 (d, J = 8.0 Hz, 1H), 7.10 (s, 1H), 2.36 (s, 3H). 13C-NMR
(150 MHz, CDCl3): δ 162.7, 160.5, 159.0, 151.7, 151.3, 150.7, 148.4, 137.9, 136.1, 135.1, 132.7,
130.9, 129.4, 125.6, 123.8, 123.0, 121.7, 113.7, 111.4, 108.4, 17.6; ESI-HRMS (+): m/z calcd for
C23H20N5O2 [M+H]+ 398.1612, found 398.1609; calcd for C23H19N5O2Na+ [M+Na]+ 420.1431,
found 420.1426.
4.1.2. N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)hydrazinecarboxamide (3)

To a solution of phenyl (4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl) carbamate
(0.397 g 1.0 mmol) in 95% ethyl alcohol (10 mL) hydrazine hydrate (2 mL) was added and the
reaction mixture was heated under reflux for 8 h. The hydrazide was obtained in quantitative yields
directly after evaporation of the reaction solution without any additional purification step. White
solid (86%), m.p.175.5~176.3
℃
. 1H-NMR (600 MHz, DMSO-d6): δ 9.26 (s, 1H), 8.91 (s, 1H), 8.69
(d, J = 4.0 Hz, 1H), 8.55 (brs, 1H), 8.50 (d, J = 5.0 Hz, 1H), 8.46 (d, J = 8.0 Hz, 1H), 7.80 (brs, 1H),
7.54 (dd, J = 5.0, 7.6 Hz, 1H), 7.41 (d, J = 5.0 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H), 7.09 (d, J = 8.1
Hz, 1H), 2.16 (s, 3H); 13C-NMR (150MHz, DMSO-d6): δ 161.6, 161.2, 159.5, 157.5, 151.4, 148.2,
137.9, 137.8, 134.5, 132.3, 130.1, 125.3, 123.9, 115.1, 114.7, 107.5, 107.4, 17.5. ESI-HRMS(+):
m/z calcd for C17H18N7O+ [M+H]+ 336.1567, found 336.1570; calcd for C17H17N7ONa+ [M+Na]+
358.1387, found 358.1391.
4.1.3. Genera method for the synthesis of compounds (E)-2-substitute-N-(4-methyl-3-((4-(pyridine
-3-yl)pyrimidin-2-yl)amino)phenyl)hydrazine-1-carboxamide (4a-4n)
To a solution of N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl) hydrazinecar -
boxamide (0.335 g, 1.0 mmol) in abs ethyl alcohol (5 mL) aliphatic, aromatic or heteroaromatic
aldehyde (1.2 mmol) was added, and the reaction mixture was heated at reflux for 8 h, and monitored
by TLC analysis. After completion of the reaction, the mixture was concentrated and purified by
column chromatography using appropriate mixtures of CH2Cl2/MeOH to produce (E)-2-substitute-
N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)hydrazine-1-carboxamide (4a-4n).
Compound 4a: Yellow solid; Yield 88%; m.p. 125.2~127.3 ℃. 1H-NMR (600 MHz, DMSO-d6):
δ 10.27 (s, 1H), 9.29 (d, J = 1.5 Hz, 1H), 8.97 (s, 1H), 8.72 (dd, J = 1.5, 4.8 Hz, 1H), 8.54-8.49 (m,
3H), 7.90 (s, 1H), 7.58 (dd, J = 4.8, 7.8 Hz, 1H), 7.43 (d, J = 1.5 Hz, 1H), 7.29-7.24 (m, 2H), 7.14-
7.10 (m, 1H), 2.21 (dd, J = 1.5, 7.8 Hz, 2H), 2.18 (s, 3H), 1.50 (m, 2H), 0.91 (t, J = 7.4Hz, 3H); 13C-
NMR (150 MHz, DMSO-d6): δ 161.6, 159.9, 153.5, 153.4, 151.2, 148.1, 145.2, 138.1, 137.6, 135.5,
133.0, 130.4, 126.6, 124.5, 118.9, 116.3, 107.9, 34.2, 20.1, 18.0, 14.1. ESI-HRMS (+): m/z calcd
for C21H24N7O+ [M+H]+ 390.2037, found 390.2032; calcd for C21H23N7ONa+ [M+Na]+ 412.1856,
found 412.1854.
Compound 4b: Yellow solid; Yield 85%; m.p. 188.1~189.2 ℃. 1H-NMR (600 MHz, DMSO-d6):
δ 10.76 (s, 1H), 9.29 (d, J = 1.8 Hz, 1H), 8.98 (s, 1H), 8.88 (s, 1H), 8.69 (dd, J = 1.8, 4.8 Hz, 1H),

8.51 (d, J = 6.5 Hz, 1H), 8.46 (td, J = 1.8, 8.0 Hz, 1H), 8.00-7.96 (m, 2H), 7.89-7.81 (m, 2H), 7.51
(dd, J = 4.8, 8.0 Hz, 1H), 7.44-7.36 (m, 5H), 7.17 (d, J = 6.5 Hz, 1H), 2.22 (s, 3H); 13C-NMR (150
MHz, DMSO-d6): δ 162.1, 161.7, 159.9, 153.5, 151.8, 148.6, 141.1, 138.2, 137.5, 134.9, 134.8,
132.7, 130.4, 129.8, 129.0, 127.5, 127.1, 124.2, 117.4, 117.0, 107.9, 18.0. ESI-HRMS (+): m/z calcd
for C24H22N7O+ [M+H]+ 424.1880, found 424.1873; calcd for C24H21N7ONa+ [M+Na]+ 446.1700,
found 446.1705.
Compound 4c: Yellow solid; Yield 83%; m.p. 221.1~221.6 ℃. 1H NMR (600 MHz, DMSO-d6):
δ 10.82 (brs, 1H), 9.28 (s, 1H), 8.98 (s, 1H), 8.92 (brs, 1H), 8.68 (dd, J = 1.8, 5.0 Hz, 1H), 8.51 (d,
J = 5.0 Hz, 1H), 8.46 (d, J = 8.1 Hz, 1H), 7.97-7.92(m, 2H), 7.89 (d, J = 8.1 Hz, 2H), 7.51 (dd, J =
5.0, 8.1 Hz, 1H), 7.46 (d, J = 8.1 Hz, 2H), 7.43-7.40 (m, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.17 (d, J =
8.1 Hz, 1H), 2.21 (s, 3H); 13C NMR (150 MHz, DMSO-d6): δ 162.1, 161.7, 159.9, 153.5, 151.8,
148.6, 139.7, 138.2, 137.5, 134.8, 134.2, 133.9, 132.7, 130.4, 129.2, 129.1, 127.2, 124.3, 117.5,
117.1, 107.9, 18.0. ESI-HRMS(+): m/z calcd for C24H21ClN7O+ [M+H]+ 458.1491, found 458.1480;
calcd for C24H20ClN7ONa+ [M+Na]+ 480.1310, found 480.1316.
Compound 4d: Yellow solid; Yield 85%; m.p. 204.4~204.7 ℃. 1H NMR (600 MHz, DMSO-d6):
δ 10.99 (s, 1H), 9.28 (d, J = 1.8 Hz, 1H), 8.98 (s, 1H), 8.95 (s, 1H), 8.68 (dd, J = 1.8, 5.0 Hz, 1H),
8.51 (d, J = 5.0 Hz, 1H), 8.46 (td, J = 2.0, 8.0 Hz, 1H), 8.38 (dd, J = 2.0, 8.0 Hz, 1H), 8.35 (s, 1H),
7.95 (d, J = 1.8 Hz, 1H), 7.56-7.46 (m, 2H), 7.42 (d, J = 5.0 Hz, 1H), 7.41-7.34 (m, 3H), 7.17 (d, J
= 8.0Hz, 1H), 2.21 (s, 3H); 13C NMR (150 MHz, DMSO-d6): δ 162.1, 161.7, 159.9, 153.4, 151.8,
148.6, 138.2, 137.4, 136.9, 134.8, 133.0, 132.7, 132.1, 131.2, 130.4, 130.1, 128.0, 127.8, 127.2,
124.3, 117.6, 117.1, 107.9, 18.0. ESI-HRMS(+): m/z calcd for C24H21ClN7O+ [M+H]+ 458.1491,
found 458.1487; calcd for C24H20ClN7ONa+ [M+Na]+ 480.1310, found 480.1296.
Compound 4e: Yellow solid; Yield 82%; m.p. 190.4~191.6 ℃. 1H NMR (600 MHz, DMSO-d6):
δ 10.60 (s, 1H), 9.28 (d, J = 1.8 Hz, 1H), 8.97 (s, 1H), 8.80 (s, 1H), 8.69 (dd, J = 1.8, 5.0 Hz, 1H),
8.51 (d, J = 5.0 Hz, 1H), 8.47 (td, J = 1.8, 8.0 Hz, 1H), 7.94 (d, J = 1.8 Hz, 1H), 7.89 (s, 1H), 7.78
(d, J = 8.0 Hz, 2H), 7.52 (dd, J = 5.0, 8.0 Hz, 1H), 7.42 (d, J = 5. 0 Hz, 1H), 7.37 (dd, J = 1.8, 8.0
Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H), 6.96 (d, J = 8. 0 Hz, 2H), 3.79 (s, 3H), 2.20 (s, 3H); 13C NMR
(150 MHz, DMSO-d6): δ 162.1, 161.7, 160.8, 159.9, 153.6, 151.8, 148.6, 141.0, 138.2, 137.6, 134.8,
132.7, 130.4, 129.0, 127.5, 127.0, 124.3, 117.3, 116.8, 114.5, 107.9, 55.7, 18.0. ESI-HRMS (+): m/z
+
calcd for C25H24N7O2 [M+H]+ 454.1986, found 454.1986; calcd for C25H23N7O2Na+ [M+Na]+

476.1805, found 476.1815.
Compound 4f: Yellow solid; Yield 83%; m.p. 222.4~224.7 ℃. 1H-NMR (600MHz, DMSO-d6): δ
11.10 (s, 1H), 9.32 (d, J = 1.8 Hz, 1H), 9.04 (d, J = 7.8 Hz, 2H), 8.73 (dd, J = 1.8, 5.0 Hz, 1H), 8.60-
8.56 (m, 1H), 8.54 (d, J = 5.0 Hz, 1H), 8.27-8.21 (m, 2H), 8.17-8.11(m, 2H), 8.05 (s, 1H), 7.95 (d,
J = 1.8 Hz, 1H), 7.62 (dd, J = 5.0, 8.1 Hz, 1H), 7.45 (d, J = 5.0 Hz, 1H), 7.38 (dd, J = 1.8, 8.1 Hz,
1H), 7.19 (d, J = 8.1 Hz, 1H), 2.22 (s, 3H); 13C-NMR (150 MHz, DMSO-d6): δ 161.6, 161.0, 160.0,
153.3, 150.6, 147.8, 147.5, 141.4, 138.6, 138.1, 137.3, 136.2, 133.2, 130.4, 128.3, 127.3, 124.8,
+
124.2, 117.7, 117.3, 108.0, 18.0. ESI-HRMS (+): m/z calcd for C24H21N8O3 [M+H]+ 469.1731,
found 469.1732; calcd for C24H20N8O3Na+ [M+Na]+ 491.1551, found 491.1559.
Compound 4g: Yellow solid; Yield 81%; m.p. 220.4~221.7 ℃. 1H NMR (600 MHz, DMSO-d6):
δ 10.83 (s, 1H), 9.28 (d, J = 1.8 Hz, 1H), 8.98 (s, 1H), 8.91 (s, 1H), 8.68 (dd, J = 1.8, 5.1 Hz, 1H),
8.51 (d, J = 5.1 Hz, 1H), 8.46 (td, J = 1.8, 8.0 Hz, 1H), 7.94 (s, 1H), 7.92 (s, 1H), 7.82 (d, J = 8..0
Hz, 2H), 7.59 (d, J = 8.0 Hz, 2H), 7.52 (dd, J =5.0, 8.0Hz, 1H), 7.42 (d, J = 5.0 Hz, 1H), 7.38 (dd,
J = 1.8, 8.0 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 2.21 (s, 3H); 13C NMR (150 MHz, DMSO-d6): δ
162.1, 161.7, 159.9, 153.5, 151.8, 148.6, 139.8, 138.2, 137.4, 134.8, 134.2, 132.7, 132.0, 130.4,
129.4, 127.2, 124.3, 123.0, 117.5, 117.1, 107.9, 18.0. ESI-HRMS (+): m/z calcd for C24H21BrN7O+
[M+H]+ 502.0985, found 502.0982; calcd for C24H20BrN7ONa+ [M+Na]+ 524.0805, found 524.0895.
Compound 4h: Yellow solid; Yield 80%; m.p. 168.4~169.6 ℃. 1H NMR (600MHz, DMSO-d6): δ
10.60 (s, 1H), 9.32 (s, 1H), 9.03 (s, 1H), 8.85 (s, 1H), 8.75 (d, J = 3.8 Hz, 1H), 8.61 (d, J = 8.1 Hz,
1H), 8.54 (d, J = 5.0 Hz, 1H), 7.94 (d, J = 1.9 Hz, 1H), 7.88 (s, 1H), 7.66 (dd, J = 5.0, 8.1 Hz, 1H),
7.52 (d, J = 1.9 Hz, 1H), 7.46 (d, J = 5.0 Hz, 1H), 7.36 (dd, J = 1.9, 8.2 Hz, 1H), 7.22 (dd, J = 1.9,
8.1 Hz, 1H), 7.17 (d, J = 8.1 Hz, 1H), 6.97 (d, J = 8.1 Hz, 1H), 3.82 (s, 3H), 3.79 (s, 3H), 2.20 (s,
3H);13C NMR (150MHz, DMSO-d6): δ 161.5, 161.2, 160.0, 159.0, 158.7, 153.6, 150.6, 149.5, 146.5,
141.4, 138.0, 137.6, 133.7, 130.4, 127.7, 127.1, 125.3, 121.7, 117.4, 117.2, 111.8, 109.5, 108.0, 56.1,
+
56.0, 18.0. ESI-HRMS(+): m/z calcd for C26H26N7O3 [M+H]+ 484.2092, found 484.2081, calcd for
C26H25N7O3Na+ [M+Na]+ 524.0805, found 524.0895.
Compound 4i: Yellow solid; Yield 89%; m.p. 225.8~226.3 ℃. 1H NMR (600 MHz, DMSO-d6): δ
10.92 (s, 1H), 9.28 (d, J = 1.9Hz, 1H), 9.00 (d, J = 8.1 Hz, 2H), 8.68 (dd, J = 1.8, 5.0 Hz, 1H), 8.51
(d, J = 5.0 Hz, 1H), 8.46 (td, J = 1.9, 8.1 Hz, 1H), 8.24 (d, J = 1.9 Hz, 1H), 7.94 (d, J = 1.3 Hz, 1H),
7.91 (s, 1H), 7.79 (dd, J = 2.0, 8.1Hz, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.51 (dd, J = 5.0, 8.1 Hz, 1H),

7.42 (d, J = 5.0 Hz, 1H), 7.38 (dd, J = 1.8, 8.1 Hz, 1H), 7.18 (d, J = 8. 1 Hz, 1H), 2.21 (s, 3H); 13C-
NMR (150 MHz, DMSO-d6): δ 162.1, 161.7, 159.9, 153.4, 151.8, 148.6, 138.4, 138.2, 137.4, 135.8,
134.8, 132.7, 132.1, 131.8, 131.1, 130.3, 128.6, 127.8, 127.3, 124.2, 117.9, 117.4, 107.9, 18.0. ESI-
HRMS (+): m/z calcd for C24H20Cl2N7O+ [M+H]+ 492.1101, found 492.1094; calcd for
C24H19Cl2N7ONa+ [M+Na]+ 514.0920, found 514.0914.
Compound 4j: Yellow solid; Yield 87%; m.p. 239.3~240.3 ℃. 1H NMR (600 MHz, DMSO-d6):
δ 10.99 (s, 1H), 9.28 (d, J = 1.8 Hz, 1H), 8.97 (d, J = 8.8 Hz, 2H), 8.68 (dd, J = 1.8, 5.0 Hz, 1H),
8.51 (d, J = 5.0 Hz, 1H), 8.48-8.41 (m, 2H), 8.28 (s, 1H), 7.96-7.93 (m, 1H), 7.63 (dd, J = 2.4, 8.0
Hz, 1H), 7.51 (dd, J = 5.0, 8.0 Hz, 1H), 7.41 (d, J = 5.0 Hz, 1H), 7.37 (dd, J = 2.4, 8.0 Hz, 1H), 7.31
(dt, J = 2.4, 8.0 Hz, 1H), 7.17 (d, J = 8.0 Hz, 1H), 2.21 (s, 3H); 13C NMR (150 MHz, DMSO-d6): δ
163.5, 162.1, 161.8, 161.2(d, J = 277.5 MHz), 153.4, 151.8, 148.7, 138.3, 138.2, 137.4,134.9, 132.7,
130.4, 130.0, 129.1, 128.2, 127.3, 124.3(d, J = 9.6 MHz), 123.6(d, J = 8.9 MHz), 120.9, 117.4(d, J
= 73.5 MHz), 115.9, (d, J = 15 MHz), 107.9, 18.0. ESI-HRMS (+): m/z calcd for C24H20BrFN7O+
[M+H]+ 520.0891, found 520.0887; calcd for C24H19BrFN7ONa+ [M+Na]+ 542.0711, found
542.0705.
Compound 4k: Yellow solid; Yield 86%; m.p. 198.8~199.9 ℃. 1H NMR (600 MHz, DMSO-d6):
δ 10.74 (s, 1H), 9.29 (d, J = 1.8 Hz, 1H), 8.97 (s, 1H), 8.69 (dd, J = 1.8, 4.8 Hz, 1H), 8.67 (s, 1H),
8.51 (d, J = 5.0 Hz, 1H), 8.47 (td, J = 1.8, 8.0 Hz, 1H), 7.96 (s, 1H), 7.89 (s, 1H), 7.78 (d, J = 1.8
Hz, 1H), 7.52 (dd, J = 4.8, 8.0 Hz, 1H), 7.41 (d, J = 5.0 Hz, 1H), 7.32 (dd, J = 1.8, 8.0 Hz, 1H), 7.16
(d, J = 8.0 Hz, 1H), 6.95 (d, J = 3.0 Hz, 1H), 6.61 (dd, J = 1.8, 3.0 Hz, 1H), 2.21 (s, 3H); 13C NMR
(150 MHz, DMSO-d6): δ 162.0, 161.7, 159.9, 153.3, 151.7, 150.1, 148.6, 144.8, 138.3, 137.4, 134.9,
132.8, 131.5, 130.5, 126.9, 124.3, 116.9, 116.5, 112.5, 112.1, 107.9, 18.0. ESI-HRMS (+): m/z calcd
+
for C22H20N7O2 [M+H]+ 414.1673, found 414.1674; calcd for C22H19N7O2Na+ [M+Na]+ 436.1492,
found 436.1501.
Compound 4l: Yellow solid; Yield 84%; m.p. 155.1~157.8 ℃. 1H NMR (600 MHz, DMSO-d6): δ
10.73 (s, 1H), 9.34 (d, J = 1.8 Hz, 1H), 9.05 (s, 1H), 8.78 (dd, J = 1.8, 5.0 Hz, 1H), 8.65 (td, J = 1.8,
8.0 Hz, 1H), 8.63 (s, 1H), 8.55 (d, J = 5.0 Hz, 1H), 8.16 (s, 1H), 7.91 (s, 1H), 7.69 (dd, J = 5.0, 8.0
Hz, 1H), 7.61 (d, J = 5.0 Hz, 1H), 7.47 (d, J = 5.0 Hz, 1H), 7.43 (d, J = 3.6 Hz, 1H), 7.30 (dd, J =
2.3, 8.0 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H), 7.11 (dd, J = 3.6, 5.0 Hz, 1H), 2.20 (s, 3H); 13C NMR
(150MHz, DMSO-d6): δ 161.5, 161.4, 160.0, 153.2, 150.0, 147.0, 139.5, 138.1, 137.4, 136.5, 133.5,

130.5, 129.9, 128.4, 128.2, 127.0, 125.1, 118.9, 116.9, 116.6, 108.0, 18.0. ESI-HRMS (+): m/z calcd
for C22H20N7OS+ [M+H]+ 430.1445, found 430.1432; calcd for C22H19N7OSNa+ [M+Na]+ 452.1264,
found 452.1258.
1
Compound 4m: Yellow solid; Yield: 83%; 231.2~232.4 ℃. H NMR (600 MHz, DMSO-d6): δ
10.91 (s, 1H), 9.28 (d, J = 1.8 Hz, 1H), 9.02-8.92 (m, 3H), 8.68 (dd, J = 1.8, 5.0 Hz, 1H), 8.56 (dd,
J = 1.8, 5.0 Hz, 1H), 8.51 (d, J = 5.0 Hz, 1H), 8.46 (td, J = 1.8, 8.0 Hz, 1H), 8.31 (td, J = 1.8, 8.0
Hz, 1H), 7.97 (s, 1H), 7.94 (d, J = 1.8 Hz, 1H), 7.52 (dd, J = 5.0, 8.0 Hz, 1H), 7.45-7.41 (m, 2H),
7.38 (dd, J = 1.8, 8.0 Hz, 1H), 7.17 (d, J = 8.0 Hz, 1H), 2.21 (s, 3H);13C NMR (150 MHz, DMSO-d6):
δ 162.1, 161.7, 159.9, 153.4, 151.8, 150.4, 149.0, 148.6, 138.2, 138.1, 137.4, 134.8, 134.1, 132.7,
130.8, 130.3, 127.2, 124.3, 124.1, 117.6, 117.1, 107.9, 18.0. ESI-HRMS (+): m/z calcd for
C23H21N8O+ [M+H]+ 425.1833, found 425.1837; calcd for C23H20N8ONa+ [M+Na]+ 447.1652,
found 447.1668.
Compound 4n: Yellow solid; Yield 82%; m.p. 215.8~218.3 ℃. 1H NMR (600 MHz, DMSO-d6):
δ 10.81 (s, 1H), 9.29 (d, J = 1.8 Hz, 1H), 8.99 (s, 1H), 8.92 (s, 1H), 8.80 (s, 1H), 8.68 (dd, J = 1.8,
5.0 Hz, 1H), 8.52 (d, J = 5.0 Hz, 1H), 8.47 (td, J = 1.8, 8.0 Hz, 1H), 8.36 (d, J = 8.0 Hz, 1H), 8.31
(d, J =8.0 Hz, 1H), 8.03-7.95(m, 3H), 7.68-7.61 (m, 1H), 7.61-7.55 (m, 2H), 7.52 (dd, J = 5.0, 8.0
Hz, 1H), 7.43 (d, J = 5.0 Hz, 1H), 7.39 (dd, J = 1.8 8.0 Hz, 1H), 7.18 (d, J = 8.0 Hz, 1H), 2.22 (s,
3H); 13C NMR (150 MHz, DMSO-d6):δ 162.1, 161.7, 159.9, 153.5, 151.8, 148.6, 139.3, 138.2,
137.5, 134.8, 133.9, 132.7, 130.8, 130.4, 130.1, 130.1, 129.3, 127.5, 127.0, 126.5, 126.0, 125.7,
124.3, 123.2, 117.3, 116.9, 107.9, 18.0. ESI-HRMS (+): m/z calcd for C28H24N7O+ [M+H]+
474.2037, found 474.2039; calcd for C28H23N7ONa+ [M+Na]+ 496.1856, found 496.1882.
4.1.4. (E)-ethyl 2-(2-(4-nitrophenyl)hydrazono)propanoate (6)
A solution of 1-(4-nitrophenyl) hydrazine (10.0 g, 65.0 mmol,) in EtOH (100 mL) was treated with
ethyl 2-oxopropanoate (8.4 g, 72.0 mmol,), The mixture has been stirred at 78℃ for 2 h, when TLC
indicated that the reaction had gone to completion. On cooling, Precipitated complex was filtered,
washed with EtOH, and dried to afford (E)-ethyl 2-(2-(4-nitrophenyl)hydrazono)propan-oate (6) as
a yellow solid. Yield: 14.5 g (89%). 1H-NMR (600 MHz, CDCl3): δ 8.21 (d, J = 9.0 Hz, 2H), 8.09
(s, 1H), 7.28 (d, J = 9.0 Hz, 2H), 4.39-4.32 (m, 2H), 2.17 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H).
4.1.5. Ethyl 5-nitro-1H-indole-2-carboxylate (7)

The solution of (E)-ethyl 2-(2-(4-nitrophenyl)hydrazono)propanoate 6 (10 g, 42.0 mmol) in
polyphosphoric acids was stirred at 100 for 2 h, and monitored by TLC analysis. After
℃
completion of the reaction, the mixture was diluted with a lot of water. The precipitated complex
was filtered, washed with water, and dried to afford ethyl 5-nitro-1H-indole-2-carboxylate (3) as a
green solid. Yield: 8.1 g (82%). 1H-NMR (600 MHz, DMSO-d6): δ 12.58 (brs, 1H), 8.71 (d, J = 2.1
Hz, 1H), 8.12 (dd, J = 2.1, 9.2 Hz, 1H), 7.61 (d, J = 9.2 Hz, 1H), 7.42 (d, J =1.0 Hz, 1H), 4.37 (q, J
= 7.1 Hz, 2H), 1.35 (t, J = 7.1 Hz, 3H).
4.1.6. Ethyl 5-amino-1H-indole-2-carboxylate (8)
Compound 7 (10 mmol, 2.34 g) was dissolved in ethanol (150 mL), 10% Pd/C was added (300 mg),
and the mixture was subjected to H2 (38 psi) using a Parr hydrogenator for 3.5 h. The mixture was
filtered over Celite, which was washed with methanol. After concentration and drying under vacuum,
1.86 g of a brown powder (91%) was isolated. 1H-NMR (600 MHz, DMSO-d6): δ 11.38 (brs, 1H),
7.19-7.16 (m, 1H), 6.84 (m, 1H), 6.73-6.69 (m, 2H), 4.63 (brs, 2H), 4.29 (q, J = 7.1 Hz, 2H), 1.31
(t, J = 7.1 Hz, 3H);13C-NMR (150 MHz, DMSO-d6): δ 161.9, 142.5, 132.0, 128.2, 127.1, 117.2,
+
113.2, 106.6, 103.6, 60.6, 14.8. ESI-HRMS (+): m/z calcd for C11H13N2O2 [M+H]+ 205.0972, found
205.0972; calcd for C11H12N2O2Na+ [M+Na]+ 227.0791, found 227.0793.
4.1.7. ethyl 5-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-1H-indole-2-carboxylate (10)
In 100 mL round bottom flask, ethyl 5-amino-1H-indole-2-carboxylate (4) (3.37 g, 16.6 mmol), and
cyanamide (1.59 g, 38.1 mmol) were added to ethanol (20 mL). While being stirred, concentrated
HCl (2.1 mL, 24.8 mmol) was dropped slowly into the mixture. The mixture was heated to reflux
for 15 h. Ethanol was removed by reduced pressure distillation, and water (20 mL) was added into
the residue. Under 0℃, NH4NO3 (2.64 g, 33.1 mmol) solution was dropped for 30 min. Then the
mixture was stirred for 30 min, and filtered, giving the crude product. The crude product needs to
be purified by being washed with ether, and finally a white solid 9 (4.01 g) was maintained.
Compound 9 was used directly in the next to reaction without purification.
In a round-bottom flask, compound 9 (3.09 g, 10 mmol), 3-dimethylamino-1-(3-pyridyl)-2-propen-
1-one (1.76 g, 10.0 mmol), sodium hydroxide (0.48 g, 12.0 mmol) was dissolved in alcohol (20
mL). The mixture was stirred and heated to reflux for 24 h. Then alcohol was removed completely

by reduced pressure distillation. EtOAc (50 mL) and water (30 mL) were added into the residue,
and the mixture was stirred for 20 min. The organic phase was separated from the aqueous phase,
and the aqueous phase was extracted by EtOAc (15 mL). The combined organic phase was washed
by water (15 mL), saturated sodium chloride (15 mL). The organic layer was dried over Na2SO4,
filtered, and distilled under vacuum to give the crude product. The crude product was purified by
recrystallization from ether and EtOAc, giving a yellow solid (2.67 g, 45%). 1H-NMR (600 MHz,
DMSO-d6): δ11.79 (brs, 1H), 9.63 (s, 1H), 9.35 (s, 1H), 8.73 (d, J = 5.0 Hz, 1H), 8.58 (d, J = 5.0
Hz, 1H), 8.51 (d, J = 7.9 Hz, 1H), 8.18 (s, 1H), 7.62-7.57 (m, 2H), 7.46-7.41 (m, 2H), 7.13 (s, 1H),
4.35 (q, J = 7.1 Hz, 2H), 1.36 (t, J = 7.1 Hz, 3H); 13C-NMR (150 MHz, DMSO-d6): δ 162.0, 161.8,
161.1, 159.8, 151.9, 148.6, 134.8, 134.3, 133.8, 132.8, 128.1, 127.2, 124.4, 120.5, 112.8, 111.9,
108.0, 60.8, 14.8. ESI-HRMS (+): m/z calcd for C20H18N5O2+ [M+H]+ 360.1455, found 360.1460;
calcd for C20H17N5O2Na+ [M+Na]+ 382.1274, found 382.1283.
4.1.8. 5-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-1H-indole-2-carbohydrazide (11)
To a solution of ethyl 5-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-1H-indole-2-carboxylate (1.73 g,
5.0 mmol) in 95% ethyl alcohol (10 mL) hydrazine hydrate (10 mL) was added and the reaction
mixture was heated under reflux for 8 h. The hydrazide was obtained in quantitative yields directly
after evaporation of the reaction solution without any additional purification step. White solid; Yield:
1
89 %; m.p. >320℃. H-NMR (600 MHz, DMSO-d6): δ 11.50 (brs, 1H),9.73(brs, 1H), 9.57 (brs,
1H), 9.35 (brs, 1H), 8.73 (brs, 1H), 8.57 (brs, 1H), 8.49 (d, J = 7.0 Hz, 1H), 8.09 (brs, 1H), 7.62-
7.55 (m, 1H), 7.51 (d, J = 7.0 Hz, 1H), 7.42 (brs, 1H), 7.38 (d, J = 7.0 Hz, 1H), 7.05 (brs, 1H), 4.50
(brs, 2H); 13C-NMR (150 MHz, DMSO-d6): δ 162.0, 161.7, 161.1, 159.8, 151.8, 148.6, 134.8, 133.5,
133.2, 132.9, 131.3, 127.5, 124.3, 118.8, 112.5, 111.6, 107.8, 102.2. ESI-HRMS (+): m/z calcd for
C18H16N7O+ [M+H]+ 346.1411, found 346.1417; calcd for C18H15N7ONa+ [M+Na]+ 368.1230,
found 368.1237.
4.1.9. General method for the synthesis of compounds 12a-12l
To a solution of 5-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-1H-indole-2-carbohydrazide 11 (0.345
g, 1 mmol) in ethyl alcohol (5 mL) aliphatic, aromatic or heteroaromatic aldehyde (1.2 mmol) was
added, and the reaction mixture was heated at reflux for 8 h, and monitored by TLC analysis. After
completion of the reaction, the mixture was concentrated and purified by column chromatography

using appropriate mixtures of CH2Cl2/MeOH to Produce 12a-12l.
Compound 12a: Yellow solid; Yield 82%; m.p. 231.2~232.4 ℃. 1H NMR (600 MHz, DMSO-
d6): δ 11.96 (brs, 1H), 11.78 (brs, 1H), 9.65 (s, 1H), 9.38 (brs, 1H), 8.73 (d, J = 4.6 Hz, 1H), 8.57
(d, J = 4. 6 Hz, 1H), 8.51-8.44 (m, 2H), 8.26 (brs, 1H), 7.57 (dd, J = 4.6, 8.0 Hz, 2H), 7.47 (d, J =
8.0 Hz, 1H), 7.41 (d, J = 4.6 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.35-7.29 (m, 2H), 7.01 (d, J = 8.0
Hz, 1H), 3.81 (s, 3H); 13C NMR (150 MHz, DMSO-d6): δ 161.9, 161.1, 160.0, 159.8, 158.2, 151.9,
148.6, 147.4, 136.3, 134.8, 133.8, 132.9, 130.8, 130.4, 127.5, 124.3, 120.5, 119.7, 116.6, 112.7,
+
111.7, 107.9, 104.1, 55.6. ESI-HRMS (+): m/z calcd for C26H22N7O2 [M+H]+, 464.1829, found
464.1831; calcd for C26H21N7O2Na+ [M+Na]+ 486.1649, found 486.1651
Compound 12b: Yellow solid; Yield: 84%; m.p. 286.7~288.9 ℃. 1H NMR (600 MHz, DMSO-
d6): δ 11.86 (brs, 1H), 11.76 (brs, 1H), 9.70 (s, 1H), 9.43 (s, 1H), 8.84 (d, J = 4.9 Hz, 1H), 8.70 (d,
J = 8.0 Hz, 1H), 8.62 (d, J = 4.9 Hz, 1H), 8.44 (brs, 1H), 8.19 (brs, 1H), 7.77 (dd, J =4.9, 8.0 Hz,
1H), 7.70 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 4.9 Hz, 1H), 7.45 (d, J = 8.0 Hz,
1H), 7.35 (d, J = 8.0 Hz, 2H), 7.31 (brs, 1H), 2.54 (s, 3H);13C NMR (150 MHz, DMSO-d6): δ 161.9,
161.1, 159.8, 158.3, 158.0, 151.9, 148.6, 147.1, 141.3, 134.8, 133.7, 132.9, 131.3, 130.9, 127.9,
127.5, 126.1, 124.4, 119.6, 112.7, 111.6, 107.9, 104.1, 67.8. ESI-HRMS (+): m/z calcd for
C26H22N7OS+ [M+H]+, 480.1601, found 480.1605; calcd for C26H21N7OSNa+ [M+Na]+ 502.1421
found 502.1425.
Compound 12c: Yellow solid; Yield: 85%，m.p. >320 ℃. 1H-NMR (600 MHz, DMSO-d6): δ
12.06 (brs, 1H), 11.80 (brs, 1H), 9.65 (s, 1H), 9.39 (brs, 1H), 8.76 (d, J=5.0 Hz, 1H), 8.60 (d, J=5.0
Hz, 1H), 8.53 (d, J=8.0 Hz, 2H), 8.26 (brs, 1H), 8.06 (d, J=5.0 Hz, 2H), 7.90 (d, J=8.0 Hz, 2H),
7.61 (dd, J=5.0, 8.0 Hz, 1H), 7.55 (d, J=8.0 Hz, 1H), 7.45 (d, J=5.0 Hz, 2H), 7.35 (brs, 1H);13C-
NMR (150MHz, DMSO-d6): δ 167.4, 161.9, 161.1, 159.9, 158.2, 151.7, 148.5, 146.2, 138.9, 135.0,
133.8, 133.8, 132.9, 132.0, 130.6, 130.3, 127.5, 124.4, 119.8, 112.7, 111.7, 108.0, 104.3. ESI-
-
HRMS (-) m/z: calcd for C26H18N7O3 [M-H]- 476.1472, found 476.1467.
Compound 12d: Yellow solid; Yield: 82%; m.p. 248.2~250.9 ℃. 1H NMR (600 MHz, DMSO-
d6): δ 12.20 (brs, 1H), 11.85 (brs, 1H), 9.63 (s, 1H), 9.37 (brs, 1H), 8.87 (brs, 1H), 8.74 (brs, 1H),
8.58 (d, J =5.0 Hz, 1H), 8.50 (d, J = 8.0 Hz, 1H), 8.25 (brs, 1H), 8.06 (brs, 1H), 7.59 (dd, J = 5.0,
8.0 Hz, 1H), 7.54 (d, J = 8.0 Hz, 2H), 7.49-7.41(m, 4H), 7.33 (brs, 1H); 13C NMR (150 MHz,
DMSO-d6): δ 161.9, 161.1, 159.8, 158.2, 151.8, 148.5, 143.2, 134.9, 133.8, 133.6, 132.9, 132.2,

131.8, 130.6, 130.4, 128.1, 127.5, 127.2, 124.4, 119.8, 112.7, 111.7, 107.9, 104.0. ESI-HRMS (+):
m/z calcd for C25H19ClN7O+ [M+H]+, 468.1334, found 468.1328, calcd for C25H18ClN7ONa+
[M+Na]+ 490.1154, found 490.1146.
Compound 12e: Yellow solid; Yield: 81%; m.p. 305.3~307.0 ℃. 1H NMR (600 MHz, DMSO-
d6): δ 11.93 (brs, 1H), 11.74 (brs, 1H), 9.63 (s, 1H), 9.38 (d, J = 1.6 Hz, 1H), 8.79-8.72 (m, 2H),
8.59 (d, J = 5.0Hz, 1H), 8.51 (d, J =8.0Hz, 1H), 8.25 (brs, 1H), 7.60 (dd, J = 5.0, 8.0 Hz, 1H), 7.57-
7.51 (m, 2H), 7.47-7.41 (m, 2H), 7.33 (brs, 1H), 7.21-7.09 (m, 2H), 3.86 (s, 3H), 3.84 (s, 3H); 13C
NMR (150 MHz, DMSO-d6): δ 161.9, 161.1, 159.9, 158.0, 153.2, 151.9, 148.6, 148.4, 142.9, 134.8,
133.7, 132.9, 130.8, 128.3, 127.5, 124.9, 124.4, 119.6, 117.5, 114.6, 112.7, 111.6, 107.9, 103.9, 61.7,
+
56.2. ESI-HRMS (+): m/z calcd for C27H24N7O3 [M+H]+ 494.1935, found 494.1937; calcd for
C27H23N7O3Na+ [M+Na]+ 516.1755, found 516.1754.
Compound 12f: Yellow solid; Yield: 93%; m.p. 255.7~258.3 ℃. 1H-NMR (600MHz, DMSO-
d6): δ 11.75 (brs, 1H), 11.72-11.63 (m, 1H), 9.61 (brs, 1H), 9.37 (brs, 1H), 8.74 (brs, 1H), 8.58 (brs,
1H), 8.50 (d, J = 7.9 Hz, 1H), 8.40 (brs, 1H), 8.27-8.16 (m, 1H), 7.59 (brs, 1H), 7.53 (d, J = 7.9 Hz,
1H), 7.44 (brs, 2H), 7.37 (brs, 1H), 7.28 (brs, 1H), 7.23 (brs, 1H), 7.05 (brs, 1H), 3.84 (s, 3H), 3.82
(s, 3H); 13C-NMR (150MHz, DMSO-d6): δ 161.9, 161.1, 159.8, 157.9, 151.8, 151.2, 149.6, 148.5,
147.8, 134.9, 133.7, 132.9, 131.0, 127.6, 124.4, 122.3, 119.5, 112.7, 112.0, 111.6, 108.7, 107.9,
103.8, 56.1, 55.9. ESI-HRMS (+): m/z calcd for C27H24N7O3+ [M+H]+ 494.1935, found 494.1935;
calcd for C27H23N7O3Na+ [M+Na]+ 516.1755, found 516.1754.
Compound 12g: Yellow solid; Yield: 88%; m.p. 231.7~233.9 ℃. 1H NMR (600 MHz, DMSO-
d6): δ 12.40 (brs, 1H), 11.87 (brs, 1H), 9.72 (s, 1H), 9.43 (s, 1H), 8.89 (brs, 1H), 8.84 (d, J = 5.0 Hz,
1H), 8.70 (d, J = 8.0 Hz, 1H), 8.63 (d, J = 5.0 Hz, 1H), 8.50 (d, J = 8.0 Hz, 1H), 8.24 (brs, 1H), 8.20
(brs, 1H), 8.12 (s, 1H), 7.78 (dd, J = 5.0, 8.0 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.50 (d, J = 5.0 Hz,
1H), 7.47 (d, J = 8.0 Hz, 1H), 7.37 (brs, 1H); 13C-NMR (150 MHz, DMSO-d6): δ 161.2, 160.9,
159.9, 159.0, 158.7, 149.9, 146.8, 137.0, 136.9, 133.7, 130.2, 129.9, 128.6, 127.4, 125.3, 124.8,
123.6, 122.9, 122.9, 120.2, 112.8, 112.0, 108.1. ESI-HRMS (+): m/z calcd for: C24H19N8O+ [M+H]+
435.1676, found 435.1680; calcd for C24H18N8ONa+ [M+Na]+ 457.1496, found 457.1499.
Compound 12h: Yellow solid; Yield: 88%; m.p. 224.4~225.9 ℃; 1H-NMR (600 MHz, DMSO-d6):
δ 12.09 (brs, 1H), 11.80 (brs, 1H), 9.64 (s, 1H), 9.37 (brs, 1H), 8.74 (d, J=5.0 Hz, 1H), 8.64 (d,
J=5.0 Hz, 1H), 8.58 (d, J=5.0 Hz, 1H), 8.51 (d, J=7.0 Hz, 2H), 8.26 (brs, 1H), 8.02 (d, J=5.0 Hz,

1H), 7.90 (brs, 1H), 7.60 (dd, J=5.0, 7.0 Hz, 1H), 7.55 (d, J=7.0 Hz, 1H), 7.47-7.40 (m,3H), 7.34
(brs, 1H);13C-NMR (150MHz, DMSO-d6): δ 161.8, 161.0, 159.8, 158.3, 153.7, 151.7, 149.9, 148.4,
147.5, 137.5, 135.0, 133.8, 133.8, 133.0, 130.5, 127.5, 124.8, 124.5, 120.5, 119.9, 112.7, 111.5,
108.0, 104.4. ESI-HRMS (+): m/z calcd for C24H19N8O+ [M+H]+ 435.1676, found 435.1671; calcd
for C24H18N8ONa+ [M+Na]+ 457.1496, found 457.1490.
Compound 12i: Yellow solid; Yield: 86%; m.p. 218.7~220.9 ℃. 1H-NMR(600 MHz, DMSO-d6) :
δ 11.68 (brs, 1H), 11.65 (brs, 1H), 11.58 (s, 1H), 9.75 (s, 1H), 9.45 (s, 1H), 8.88 (d, J = 5.0 Hz, 1H),
8.79 (d, J = 8.0Hz, 1H), 8.66 (s, 1H), 8.64 (d, J = 5.0 Hz, 1H), 8.33 (d, J = 8.0 Hz, 1H), 8.15 (s, 1H),
7.89-7.83 (m, 2H), 7.55 (d, J = 8.0Hz, 1H), 7.51 (d, J = 5.0 Hz, 1H), 7.48 (d, J = 5.0, 1H), 7.46(d,
J = 8.0, 1H), 7.29 (s, 1H), 7.25-7.21 (m, 1H), 7.20-7.16 (m, 1H); 13C-NMR (150 MHz, DMSO-d6):
δ 160.9, 159.0, 158.8, 157.5, 148.9, 145.9, 144.9, 137.5, 134.1, 133.7, 133.3, 131.6, 130.8, 127.6,
125.7, 124.8, 123.1, 122.4, 120.9, 119.4, 116.9, 115.0, 112.7, 112.3, 112.2, 108.0, 103.3. ESI-
HRMS (+): m/z calcd for C27H21N8O+ [M+H]+ 473.1833, found 473.1832; calcd for C27H20N8ONa+
[M+Na]+ 495.1652, found 495.1651. Purity: 94.3% (by HPLC).
Compound 12j: Yellow solid; Yield: 87%; m.p. 232.0~234.9 ℃. 1H-NMR (600MHz, DMSO-d6): δ
11.67 (s, 1H), 11.54 (s, 1H), 11.50 (s, 1H), 9.73 (s, 1H), 9.44 (d, J=3.0 Hz, 1H), 8.86 (d, J=3.0 Hz,
1H), 8.75 (d, J=8.0 Hz, 1H), 8.73 (s, 1H), 8.63 (d, J=5.0 Hz, 1H), 8.26 (d, J=7.1 Hz, 1H), 8.17 (s,
1H), 7.82 (dd, J=5.0, 8.0 Hz, 1H), 7.54 (dd, J=5.0, 8.0 Hz, 1H), 7.50 (d, J=5.0 Hz, 1H), 7.47 (d,
J=8.0 Hz, 1H), 7.36 (d, J=7.1 Hz, 1H), 7.28 (s, 1H), 7.17-7.11 (m, 2H), 2.57 (s, 3H);13C-NMR (150
MHz, DMSO-d6) :δ 161.0, 160.9, 159.9, 159.0, 158.8, 157.4, 149.3, 146.2, 144.5, 140.6, 137.7,
136.2, 133.9, 133.7, 133.3, 131.7, 127.6, 125.8, 125.5, 122.3, 121.7, 120.7, 119.3, 117.0, 115.1,
112.7, 112.0, 111.3, 108.0, 108.0, 103.1, 12.0. ESI-HRMS (+): m/z calcd for C28H23N8O+ [M+H]+
487.1989, found 487.1988; calcd for C28H22N8ONa+ [M+Na]+ 509.1809, found 509.1808.
Compound 12k: Yellow solid; Yield: 88%，m.p. 277.7~278.9 ℃. 1H NMR (600 MHz, DMSO-d6):
δ 11.98 (brs, 1H), 11.71 (brs, 1H), 9.67 (brs, 1H), 9.44 (d, J = 8.0 Hz, 1H), 9.41 (brs, 1H), 9.20 (brs,
1H), 8.78 (d, J = 5 Hz, 1H), 8.61 (d, J = 5 Hz, 1H), 8.57 (d, J = 8.0 Hz, 1H), 8.26 (brs, 1H), 8.07 (d,
J =8.0 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.64 (dd, J = 5, 8 Hz, 2H), 7.60-7.53 (m, 2H), 7.51-7.43
(m, 3H), 7.41 (brs, 1H), 4.05 (s, 3H);13C-NMR (150 MHz, DMSO-d6): δ 161.7, 161.1, 159.9, 158.2,
158.0, 151.3, 148.1, 145.1, 135.4, 133.8, 133.7, 133.1, 131.2, 131.0, 129.3, 129.0, 128.5, 127.6,
126.4, 124.6, 124.5, 119.6, 114.9, 113.9, 112.7, 111.8, 108.0, 104.0, 57.2. ESI-HRMS (+): m/z calcd

+
for C30H24N7O2 [M+H]+ 514.1986, found 514.1981; calcd for C30H23N7O2Na+ [M+Na]+ 536.1805,
found 536.1801.
Compound 12l: Yellow solid; Yield: 82%; m.p. 303.7~306.1 ℃. 1H NMR (600 MHz, DMSO-d6)
: δ 12.33 (brs, 1H), 11.86 (brs, 1H), 9.72 (s, 1H), 9.53-9.31 (m, 1H), 9.01 (s, 1H), 8.95 (brs, 1H),
8.84 (d, J =5.0 Hz, 1H), 8.71 (d, J = 8.0 Hz, 1H), 8.63 (d, J = 5.0 Hz, 1H), 8.29-8.21 (m, 2H), 8.00
(d, J = 8.0 Hz, 1H), 7.88 (t, J = 8.0Hz, 1H), 7.78 (dd, J = 5.0, 8.0 Hz, 1H), 7.71 (t, J =8.0 Hz, 1H),
7.58 (dd, J =5.0, 8.0 Hz, 1H), 7.50 (d, J = 5.0 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.39 (brs, 1H); 13C
NMR (150MHz, DMSO-d6) : δ 161.1, 160.9, 159.9, 159.0, 158.7, 158.2, 149.6, 149.0, 147.6, 146.5,
137.3, 136.1, 134.0, 132.2, 130.5, 129.5, 128.3, 128.1, 127.5, 127.4, 126.7, 120.0, 117.0, 115.1,
112.8, 112.0, 108.1, 104.5. ESI-HRMS (+): m/z calcd for C28H20ClN8O+ [M+H]+ 519.1443, found
519.1456; calcd for C28H19ClN8ONa+ [M+Na]+ 541.1263, found 541.1274.
4.2 Biological activity
4.2.1. Cell lines and reagents
All the cell lines used in this research were from our lab storage. The normal breast
MCF-10A cells were cultured in MEBMTM Basal Medium (Lonza, CC-3151) with
MEGMTM SingleQuotsTM Supplements (Lonza, CC-4136) and 10% FBS (GIMINI,
100-500). Except for MCF-10A, other cell lines were cultured inDulbecco's Modified
Eagle Medium (DMEM, ThermoFisher scientific, 11965092) with 10% FBS. The
primary antibodies, anti-RNA Poll CTD p-Ser2 (A300-654A); anti--Tubulin (T5168)
and anti-PARP1 (9542) were obtained from Bethyl, sigma, and CST, respectively. Both
anti-mouse and anti-rabbit IgG were purchased from Pierce.
4.2.2. Luciferase assay
To measure the inhibition effect of target compounds on HIV-1 transcription, luciferase
assay was performed in NH2 cells as previously described with minor modifications.[35]
Briefly, NH2 cells in the 6-well plate were treated with different compounds or
flavopiridol (positive control) for 8 h, the whole-cell extracts (WCE) were normalized
and objected to luciferase activity measure using luciferase kit (Promega, E1500).
4.2.3. MTT assay

MTT assay was performed according to previously described.[22] All types of cancer
cells used for MTT assay were cultured in DMEM medium (ThermoFisher scientific)
with 10% FBS. Cells were cultured in the 96-well plate with 100 uL DMEM, and the
total amount of cells in each well was about 1x104 when compounds added. Six
concentration gradients of each compound and six repeats of each treatment were made.
Meanwhile, dimethyl sulfoxide (DMSO) was used as a negative control. After 48 h
treatment, 10 uL MTT solution (5 mg/mL, M2128, sigma) was added to each well, and
cells were cultured for another 4 h in cell culture incubator. Then the medium in each
well was discarded and 100 uL DMSO was added for dissolution. After a short time
sharking, the light intensity was detected at the wavelength of 490 nm, and the IC50
values were calculated with GraphPad prism5 software (GraphPad Software, San Diego,
CA, USA)
4.2.4. Western blot analysis
After compounds treatment, cells in the plate were washed with 1xPBS, and added
1xSDS loading buffer for cell lysing. Then the samples were boiled (100℃, 10min) and
subjected to western blot analysis. The primary antibodies including RNA Poll CTD p-
Ser2 (A300-654A, Bethyl); α-Tubulin (T5168, sigma); PARP1 ((9542, CST, Shanghai,
China). Both anti-mouse and anti-rabbit IgG were purchased from Pierce.
4.2.5. Annexin-V-FITC apoptosis assay
Cells were cultured in 6-well plate (about 1x105 cells in each well) and treated with
varying concentrations of 12i for 24h. Then the cells were harvested and objected to
apoptosis analysis following the protocols (BD Bioscience, Franklin Lakes, NJ, USA).
After stating with FITC annexin V/PI, the apoptosis status of cells was analyzed using
Attune NxT flow cytometer (Thermo, USA) and the data were analyzed with FlowJo7.6
software.
4.3 Molecular docking
Schrodinger suite (Schrödinger, LLC, New York, NY, 2016) was employed in our
structure-based virtual screening. The 2D structures of the compounds were

transformed into 3D by the LigPrep module in Maestro 10.5. The crystal structure of
the CDK9 complex was retrieved from the Protein Data Bank (www.rcsb.org) (PDB
ID: 3BLR) and was prepared by the Protein Preparation Wizard module in Maestro
10.5. Molecular docking was performed by the Glide module in Maestro 10.5. XP (extra
precision) scoring was used. Molecular graphics were prepared with PyMOL
(http://www.pymol.org/).
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (No.
81773600 and 81672955), the Natural Science Foundation of Fujian Province of China
(No. 2018J01132), the Fundamental Research Funds for the Central Universities (No.
20720180051), and the Health Commission of Zhejiang Province (2020KY628).
Appendix A. Supplementary material
Supplementary data to this article can be found online at …….
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