Synthesis and SAR of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as potent and selective CDK4/6 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.08.043

CDK4/6 pathway is an attractive target for development of anti-cancer drugs. Herein, we reported the design and synthesis of a series of 4,5-dihydro-1H-pyrazolo [4,3-h]quinazoline derivatives as selective CDK4/6 inhibitors. Applied with the optimizing strategy to the initial scaffold, it is found that compound 13n is able to selectively inhibit CDK4 and CDK6 with IC₅₀ values 0.01 and 0.026 μM, respectively. The compound showed good anti-proliferative activity when tested in a panel of tumor cell lines with CDK4/6 related mechanism of action, the results clearly suggest that compound 13n works much better than Ly2385219 which is a selective CDK4/6 inhibitor. This compound was also found to have favorable pharmacokinetic parameters. Taken together, compound 13n could be selected for further preclinical evaluation.

Full text of DOI:10.1016/j.ejmech.2018.08.043

Accepted Manuscript
Synthesis and SAR of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as
potent and selective CDK4/6 inhibitors
Hui Zhao, Xiaoxia Hu, Kai Cao, Yue Zhang, Kuantao Zhao, Chunlei Tang, Bainian
Feng
PII:
S0223-5234(18)30712-8
10.1016/j.ejmech.2018.08.043
EJMECH 10654
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 March 2018
Revised Date: 14 August 2018
Accepted Date: 15 August 2018
Please cite this article as: H. Zhao, X. Hu, K. Cao, Y. Zhang, K. Zhao, C. Tang, B. Feng, Synthesis and
SAR of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as potent and selective CDK4/6 inhibitors,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.043.
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Graphical abstract

ACCEPTED MANUSCRIPT
Synthesis and SAR of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as potent and selective
CDK4/6 inhibitors
Hui Zhao, Xiaoxia Hu, Kai Cao, Yue Zhang, Kuantao Zhao, Chunlei Tang*, Bainian Feng**
School of Pharmaceutical Science, Jiangnan University, Wuxi, 214122, China
* Corresponding author. ** Corresponding author.
E-mail address: fengbainian@jiangnan.edu.cn (B. F.); Tel/ Fax: +86 510 68781020
E-mail address: tangcl@jiangnan.edu.cn; Tel/ Fax: +86 510 85197052
Abstract: CDK4/6 pathway is an attractive target for development of anti-cancer drugs. Herein, we reported the
design and synthesis of a series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as selective CDK4/6
inhibitors. Applied with the optimizing strategy to the initial scaffold, it is found that compound 13n is able to
selectively inhibit CDK4 and CDK6 with IC50 values 0.01 and 0.026 µM, respectively. The compound showed
good anti-proliferative activity when tested in a panel of tumor cell lines with CDK4/6 related mechanism of
action, the results clearly suggest that compound 13n works much better than Ly2385219 which is a selective
CDK4/6 inhibitor. This compound was also found to have favorable pharmacokinetic parameters. Taken together,
compound 13n could be selected for further preclinical evaluation.
Keywords: cyclin dependent kinase, anticancer, cell cycle, kinase selectivity
1. Introduction
CDKs (cyclin dependent kinases) which play a critical role in the process of mitosis are frequently over
expressed in human tumors. They are also part of the tumor cell proliferation [1]. There are 21 members in the
CDK family, and many of them are popular targets for drug discovery such as CDK1, CDK2, CDK4/6, CDK5,
CDK7, CDK8, CDK9. Early efforts to block CDKs with nonselective CDK inhibitors led to little efficacy but with
toxicity due to poor selectivity [2]. Result so far indicated that the reason for the clinical failure of pan-CDK
inhibitors is too much toxic side effects and the treatment window is too small. Therefore, finding selective CDK
inhibitors becomes a direction to reduce the toxic side effects of drugs.
CDK4/6 is an attractive target for development of anti-cancer drugs. Several highly selective CDK4/6 inhibitors
such as Ly2385219 [3] and Palbociclib [4] (Fig. 1) have been studied extensively in clinical research. It has been
demonstrated that cell cycle arrest would lead to cell apoptosis. Meanwhile, ongoing clinical trials have produced
promising data on the potential efficacy of CDK4/6 inhibitors, particularly in treating HR advanced breast cancer,
when they improve the response and duration of response to hormonal therapies [5, 6]. It is of great significance to
develop novel, oral CDK4/6 inhibitors with good pharmacokinetic properties.
Ly2835219
Palbociclib
Figure 1. The structure of Ly2835219 and Palbociclib (CDK4/6 inhibitor)
We had discovered 4,4-dimethyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline [7] (Fig. 2) during the literature
investigation. The tricyclic system was previously identified as an inhibitor against many kinases such as Aurora A
(Aur-A) [8], CDK2 [9] and Polo-like kinase 1 (PLK1) [10]. We used computer-aided drug design software to
virtually screen derivatives containing the scaffold. When we introduced 2-aminopyridine fragment contained in
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palbociclib at position 8 of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline (13l), we found that Lowest energy
quantum mechanical model of compound 13l and the crystal structures of Palbociclib were well-overlaid (Fig. 2).
The similar binding patterns prove that they have similar interaction with CDK4/6. During the course of the study,
we used the commercially available Ly2385219 as a control compound to evaluate the anti-tumor effect of our
compounds. CDK4/6 is over-expressed in breast cancer MCF-7 and the growth of MCF-7 cells is dependent on
CDK4/6. Therefore, we chose MCF-7 to evaluate the inhibitory effect of our compounds on the proliferation of
tumor cells. Taken together, We intended to develop a series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline
derivatives as selective CDK4/6 inhibitors and as anti-tumor agent.
A
B
Figure 2. A) The structure of 4,4-dimethyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline scaffold. B) Lowest energy
quantum mechanical model of compound 13l (thick stick) overlaid with X-ray conformations of Palbociclib (thin
stick)
2. Results and Discussion
2.1 Chemistry
According to the related literatures [11-13], the synthetic route to 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline
analogues is shown in Scheme 1. Compounds 1a-1c reacted with 1,1-dimethoxy-N,N-dimethylmethanamine at
60 , ethyl 6-[(dimethylamino)methylene]- 7-oxo-4,5,6,7-tetrahydro-1H-indazole-3-carboxylate derivatives 2a-2c
was obtained. The 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline core 3a-3c was prepared from 2a-2c and
phenylguanidine by cyclization in the presence of dioxane, after hydrolysis and amidation to produce compounds
4a-4f.
Scheme 1. Synthesis of compound 4a-4f. Reagents and conditions: (i) N, N-dimethylformamide dimethyl acetal,
DMF, 60 , 4 h, 90%; (ii) Guanidine hydrochloride, K2CO3, DMF, 100 , 80-90%; (iii) 1.5M KOH in 95% EtOH,
70 ; R3NH2, HOBT, EDC, DMF, rt, 50-80%.
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The synthetic route to different guanidines is shown in Scheme 2. Phenylguanidines 6a-6i were prepared starting
from substituted anilines 5a-5i, which was reacted with cyanamide. The 5-bromine-2-nitropyridine 7 reacted with
4-methylpiperazine to afford compounds 8a-8e. Compounds 8a-8e is reduced to give compounds 9a-9e by iron
powder, and then compounds 9a-9e is substituted and deprotected to give pyridylguanidines compounds 11a-11e.
Compound 2c was coupled with various guanidines 6a-6i, 11a-11e in the presence of DMF to offer compounds
12a-12n in 50-70% yield, then treated with the solution of ammonia to produce the target compounds 13a-13n.
Among the title compounds, fourteen of them 13a-13n have not been reported.
Scheme 2. Synthesis of compounds 13a-13n. Reagents and condition: (i) NCNH2, isopropanol, 80 , 12 h,
60-90%; (ii) DIPEA, acetonitrile, 60 , 4 h, 50-90%; (iii) Fe, AcOH, DCM, 60 , 70-90%; (iv) 1,3-di(tert-butyl
oxycarbonyl)-2-(trifluoromethyl sulfonyl)guanidine, DCM, room temp, 50-80%. (v) DCM, TFA, room temp, 95%;
(vi) dioxane, 90 , 6 h, 50-90%; (vii) NH3, EtOH, 80 , 6 h, 60-90%.
2.2. Biological Activities
Based on the structure of 4,4-dimethyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline scaffold, we modified the
position 3 and 4 of the 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline at first. Biochemical activity against CDK2/A2,
CDK4/D3, CDK6/D3 kinases and in vitro cell proliferation cytotoxicity on MCF-7 cell lines are reported in Table
1. Initial studies showed that both R1 and R2 were substituted with H (compound 3c, 4d-4f) could increase the
inhibitory activity to CDK4/6. When both R1 and R2 were substituted with methyl, compounds 3a, 4a, 4b have
improved inhibitory activity to CDK2 and no inhibitory activity to CDK4/6. Satisfactorily, it is detrimental not
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only for CDK2 inhibition (IC50 = 0.002 µM) but also for the CDK4/6 (IC50 = 0.01/0.06 µM) when R3 is amino (4a,
4d).
With the R3 fixed as NH2, both R1 and R2 fixed as H, compound 4d showing a better activity for CDK4/6 vs
CDK2. Next, attention was turned to the phenylamine at the C-8 position of the
4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline in an attempt to further improve the potency for CDK4/6.
Activity results reported in Table 2 show that most of modifications at R5 (13a-13c) significantly decreased
CDK activity, biochemical activity for the CDK2, 4, 6 kinases and cellular proliferation activity was completely
lost. However, encouraging results were obtained by introducing substituent at the position 4 (13d-13f). These
derivatives (13d-13f) not only maintained acceptable CDK2/A2 activity but they also maintained or even
improved the selectivity toward CDK4/D3 and CDK6/D3. But the simple dimethyl amide derivative 13f showed
4-fold loss of activity in the CDK2, 4 biochemical assay and 3-fold reduction in cell proliferation potency
compared with compound 13g. Analysis of the biochemical data reported in Table 2 shows that in a homogeneous
series, substitution at positions 4 of the benzene ring was inefficient to increase CDK4/6 activity.
Table 1
Results for inhibition of CDK2, 4, 6 and MCF-7 cell lines by compounds 3a, 3b, 4a-4f
a
IC50 (µM)
CDK2/
Cyclin A2
0.62
CDK4/
Cyclin D3
1.0
CDK6/
Cyclin D3
>10
Compd
3a
R1
Me
Me
H
R2
Me
H
R3
MCF-7
6.8
OCH2CH3
OCH2CH3
OCH2CH3
0.83
>10
>10
NA
NA
NA
6.0
3b
H
2.00
3.24
7.36
3c
Me
Me
Me
H
Me
Me
H
NH2
0.004
0.25
0.010
0.450
5.26
2.52
4a
NH CH3
8.56
4b
NH2
0.65
>10
4.3
4c
H
NH2
0.002
0.037
0.12
0.010
0.16
0.058
1.02
5.8
4d
NH CH3
H
H
3.1
4e
H
H
0.45
2.32
NA
4f
a Values are means of two or more experiments. NA means data not available.
The data confirmed 13g and 13i as the most promising compound in terms of cellular activity on MCF-7 cell
line, potency against CDK4/D3 (IC50 = 0.008/0.003 µM) and CDK6/D3 (IC50 = 0.02/0.010 µM). However, there is
no significant improvement of selectivity against CDK4/6.
Analysis of reported SAR around palbociclib [14-15] and X-ray structure of palbociclib bound to CDK6 [16], as
well as molecular modeling studies, suggested that additional selectivity could be obtained by replacing the aniline
with a substituted pyridinylamine. Introduction of the pyridine could enhance selectivity over other kinases via
interactions with the side chain of hinge residue His100. Replacing the benzene ring of compounds 4b-4e with
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pyridine motif gave compounds 13k-13n, which showed increased potency for CDK4/6 as well as improved
selectivity over CDK2. It is indeed demonstrated that the selectivity of compound 13l (selectivity
CDK2/CDK4=28.3) is superior to 13g (selectivity CDK2/CDK4=1.0). The aniline counterpart of 13k-13n are less
selective CDK4/6 inhibitors, as shown with compound 13i. As shown in table 3, introducing the pyridine group led
to the identification of compound 13n (CDK4 IC50=0.010 µM; CDK6 IC50=0.026 µM), which exhibited a 70-fold
selectivity for cdk4 over CDK2. Removal of the piperazine (13f) resulted in reduction in potency, selectivity. The
piperazine moiety proved to be not only a solubilizing group but also an important selectivity determinant due to
deleterious interactions unique to CDK4/6.
Table 2
Results for inhibition of CDK2, 4, 6 and MCF-7 cell lines by compounds 13a-13n
N
O
NH2
HN
N
N
N
X
R5
R4
a
IC50 (µM)
CDK2/
CDK4/
CDK6/
Compd
13a
13b
13c
X
C
C
C
C
C
C
C
C
C
R4
R5
Cyclin A2
1.21
Cyclin D3
1.85
Cyclin D3 MCF-7
H
H
dimethylamino
2.74
7.76
5.79
0.32
0.47
0.045
0.02
0.35
0.010
NA
4.77
NA
piperazin-1-yl
2.41
5.62
H
morpholin-4-yl
3.05
3.65
Cl
H
H
H
H
H
H
0.04
0.06
3.72
NA
13d
13e
Me
0.12
0.25
dimethylamino
piperazin-1-yl
morpholin-4-yl
4-methylpiperazi
n-1-yl
0.03
0.034
0.008
0.28
2.15
0.78
0.94
1.05
13f
0.008
0.33
13g
13h
13i
0.001
0.003
N
N
N
N
N
H
dimethylamino
4.20
1.36
0.85
1.01
0.70
5.70
0.68
0.029
0.03
0.01
>10
0.55
NA
1.81
0.22
0.35
0.19
13j
13k
13l
dimethylamino
piperazin-1-yl
morpholin-4-yl
4-methylpiperazi
n-1-yl
H
H
H
H
0.036
0.04
13m
13n
0.026
0.50
0.008
0.01
0.71
Ly2835219
a Values are means of two or more experiments. NA means data not available.
Derivatives 13n and 13m is proved to be the best compounds of the series, showing anti-proliferative activity in
the low micro molar range in the MCF-7 cell line (IC50 = 0.19 µM), good activity on CDK4/6 (IC50 = 0.010/0.026
µM) and high-level selectivity against CDK2/A3 (IC50 = 0.70 µM). Compounds 13n and 13m were profiled
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against additional cancer cell lines in a 72h proliferation assay (Table 4). The compound resulted active in cells
derived from solid tumors showing a cytotoxicity range from 0.19 µM (HCT116) to 2.30 µM (PANC-1). The
results clearly suggest that compound 13n and 13m works much better than Ly2385219. The compounds were
shown good anti-proliferative activity when tested in two tumor cell lines (MCF-7 and HCT116) with CDK4/6
related mechanism of action. Interestingly compounds 13n and 13m were also shown anti-proliferative activity in
HepG2 and PANC-1 cell lines, which are CDK4/6 unrelated mechanism of action.
Table 3
Results for selective inhibition of CDK2, 4, 6 and MCF-7 cell lines by optimized compounds
IC50a(µM)
CDK4/
Cyclin D3
0.45
Selectivity
IC50a(µM)
Compd
CDK2/
Cyclin A2
0.25
CDK6/
Cyclin D3
8.56
CDK2/CDK4 CDK2/CDK6
MCF-7
0.56
0.20
0.23
1.00
0.33
2.00
28.3
33.3
70.0
0.03
0.03
0.04
0.80
0.10
2.50
23.6
25.2
28.1
6.00
5.80
3.10
0.78
1.05
1.81
0.22
0.35
0.19
4b
4d
0.002
0.037
0.008
0.001
1.36
0.01
0.058
1.02
0.16
4e
0.008
0.003
0.68
0.02
13g
13i
13k
13l
13m
13n
0.010
0.55
0.85
0.029
0.03
0.036
0.04
1.01
0.70
0.01
0.026
a Values are means of two or more experiments.
In view of its better overall profile, compounds 13n and 13m were selected and further analyzed in
pharmacokinetic (PK) experiments. In vivo pharmacokinetic properties were evaluated in mice, following
intravenous (iv) and oral (os). Compound 13n showed pharmacokinetic properties better than 13m with higher
AUC and Cmax (Table 5). Compound 13n showed a good half-life value and oral bioavailability.
2.3. Molecular docking study
Currently, only the crystal complex structure of CDK6 with its inhibitors have been resolved, and the crystal
complex structure of CDK4 with its inhibitors has not been reported [17]. According to related reports [18], CDK6
and CDK4 are high homology, so we used complex structure of CDK6 with Palbociclib (PDB:2EUF) for
molecular docking. We used the CDK4/6 inhibitor Palbociclib for molecular docking with CDK6 and found that
the docking results were the same as those reported in the literature (docking score is 146). So we used the same
method again for our compound 13l (docking score is 135). The two scores are similar, and the similarity of the
binding model prove that they have similar interactions with CDK6.
In the complex structure of CDK6 with 13l (Fig. 3), as expected, 13l binds in the ATP-pocket and most of the
interactions are similar to these found in the past with a close analog. The pyrimidine core of 13l formed a pair of
donor-acceptor-donor hydrogen bonds with the CDK6 hinge region (Val101), The pyridine could enhance
selectivity via interactions with the side chain of hinge residue His100. In addition, the piperazine moiety
contributes to the CDK6 activity since it establishes a polar interaction with the side chain of solvent accessible
region (Asp104) Compound 13l occupies the same binding sites. In addition, the oxygen atom of amide forms a
hydrogen bond to the main-chain of Asp163.
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A
B
Figure 3. A) the features of binding mode of 13l in the ATP pocket of CDK6 (PDB:2EUF). B) Compound 13l
bound to CDK6. Hydrogen bonds are shown as green dashed lines, residues of CDK6 and compound 13l are
shown with carbon, nitrogen and oxygen colored grey, blue and red, respectively.
Table 4
Cell proliferation assay assessing the effects of 13m and 13na on different cell lines
compd
Cell proliferation assay, IC50 /µM
MCF-7
0.35
HCT116
0.27
HepG2
1.58
PANC-1
1.14
13m
13n
0.19
0.13
0.97
2.30
0.71
0.54
NA
5.94
Ly2835219
a Values are means of two or more experiments. NA means data not available.
Table 5
PK parameters for compounds 13m and 13na
compd
In vivo PK (mouse) 10 mg/kg iv
In vivo PK (mouse) 10 mg/kg os
T1/2(h)
0.94
CL(L/h/kg)
4.47
AUC∞(µM h)
5.12
Vss
T1/2(h)
1.56
Cmax
0.32
AUC∞(µM h)
1.14
F(%)
25.0
33.2
3.60
4.31
13m
13n
1.18
4.18
6.36
3.97
0.66
2.30
a n=3 animals per study.
3. Conclusion
A series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamides were designed and evaluated as
CDK4/6 inhibitors. Introduction of a piperazine group on the scaffold through an amide linkage not only improved
the solubility but also significantly strengthened the enzymatic inhibitory potency and the cellular activity against
MCF-7 cell line. It is now demonstrated that the modification of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazolines
including a 2-aminopyridine side chain at the C-8 position could inhibit CDK4/6 with exquisite selectivity. By
further optimization, highly potent and selective CDK4/6 inhibitors 13n was found to have favorable
pharmacokinetic parameters, good potency and selectivity profile. These results support further evaluation and
development of these compounds for the use as selective CDK4/6 inhibitors for cancer therapy. Studies to explore
the mechanism of these compounds are needed and now in progress.
4. Experimental Section
4.1. Chemistry
All chemicals were reagent grade and used as purchased. All reactions were performed under an inert
1
atmosphere of dry argon or nitrogen using distilled dry solvent. H (400 MHz) and 13C (100 MHz) NMR spectra
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were recorded on a Bruker AV 400MHz/100MHz spectrometer. The chemical shift values are reported in parts
per million (ppm) relative to tetramethylsilane as internal standard in DMSO-d6. High-resolution MS data were
obtained on an Agilent TOF G6224 mass spectrometer.
4.1.6. The preparation of compounds 2a-2c
A mixture of compounds 1a-1c (2.0 mmol), dimthyl formide dimethylacetal (4.0 mmol) in DMF (10.0 mL) was
stirred at 60 for 5 h. Progress of reaction was monitored by tlc and after complete conversion of starting material
reaction mixture was cooled to rt. The solvent was evaporated and the residue was purified via column
chromatography to afford compounds 2a-2c.
4.1.6.1ethyl(Z)-6-((dimethylamino)methylene)-1,4,4-trimethyl-7-oxo-4,5,6,7-tetrahydro-1H-indazole-3-carboxylate
1
(2a). yellow solid. Yield: 65%. H-NMR (CDCl3, 400 MHz) δ: 7.59 (s, 1H, -C=CH-), 4.24 (q, J = 6.8 Hz, 2H,
-COOCH2CH3), 4.24 (s, 3H, -NCH3), 3.14 (s, 6H, -N(CH3)2), 2.88 (s, 2H, -CH2C(CH3)2), 1.43 (s, 6H, -C(CH3)2),
1.40 (t, J = 8.0 Hz, 3H, -COOCH2CH3). 13C-NMR (DMSO-d6, 100 MHz): δ 178.7, 162.6, 150.2, 137.8, 129.2,
103.5, 60.7, 43.8, 39.8.6, 24.5, 21.1, 14.1; HRMS (ESI): calcd for C16H23N3O3, [(M+H)+], 306.1818, found
306.1802.
4.1.6.2 ethyl(Z)-6-((dimethylamino)methylene)-1,4-dimethyl-7-oxo-4,5,6,7-tetrahydro-1H-indazole-3-carboxylate
1
(2b). Yellow solid. Yield: 70%; H-NMR(CDCl3, 400 MHz) δ：7.49 (s, 1H, -C=CH-), 4.28 (q, J = 6.8 Hz, 2H,
-COOCH2CH3), 4.24 (s, 3H, -NCH3), 3.14 (s, 6H, -N(CH3)2), 2.96 (t, J = 6.8 Hz, 1H, -CH(CH3)CH2), 2.78 (d, J =
6.8 Hz, 2H, -CH(CH3)CH2), 1.42 (d, J = 8.0 Hz, 3H, -CH2CH(CH3)-), 1.40-1.44 (m, 3H, -COOCH2CH3).
13C-NMR (DMSO-d6, 100 MHz): δ 178.7, 162.6, 150.2, 137.8, 129.2, 103.5, 60.7, 43.8, 39.8.6, 24.5, 21.1, 14.1;
HRMS (ESI): calcd for C15H21N3O3, [(M+H)+], 292.1661, found 292.1632.
4.1.6.3 6-[(dimethylamino)methylene]-1-methyl-7-oxo-4,5,6,7-tetrahydro-1H-pyrazole-3-carboxylate (2c). Yellow
solid. Yield: 68%; 1H-NMR (DMSO-d6, 400 MHz) δ: 7.49 (s, 1H, -C=CH-N-), 4.28 (q, J = 8.0 Hz, 2H,
-COOCH2CH3), 4.31 (s, 3H, -NCH3), 3.12 (s, 6H, -N(CH3)2), 2.90 (t, J = 8.0 Hz, 2H, -C=CCH2CH2-), 2.82 (t, J =
8.0 Hz, 2H, -C=CCH2CH2-), 1.30 (t, J = 8.0 Hz, 3H, -COOCH2CH3). 13C-NMR (DMSO-d6, 100 MHz): δ 178.7,
162.6, 150.2, 137.8, 137.4, 129.2, 103.5, 60.8, 43.8, 39.8, 24.5, 21.0, 14.4; HRMS (ESI): calcd for C14H19N3O3,
[(M+H)+], 278.1505, found 278.1502.
4.1.7. General procedure for the preparation of derivatives 3a-3b.
A solution of compounds 1b-2b (0.24 mmol), phenylguanidine (0.30 mmol) in DMF (2.0 mL) was stirred at
60 ℃for 8 h. Progress of reaction was monitored by tlc and after complete conversion of starting material reaction
mixture was cooled to rt, then poured into H2O (10mL). The aqueous phase was extracted with EtOAc (20.0 mL).
The organic phases were then processed in the usual way and chromatographed (5:1 petroleum ether / EtOAc) to
yield compounds 3a-3b.
4.1.7.1. 1,4,4-trimethyl-8-(phenylamino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylate (3a). Yellow
solid. Yield: 41%. 1H-NMR (DMSO-d6, 400 MHz) δ: 9.58 (s, 1H, Ar-NH-), 8.40 (s, 1H, -N=CH-), 7.72 (d, J = 8.0
Hz, 2H, HAr), 7.30 (t, J = 8.0 Hz, 2H, HAr), 6.97 (t, J = 8.0 Hz, 1H, HAr), 4.35 (s, 3H, -NCH3), 4.30 (q, J = 8.0 Hz,
2H, -COOCH2CH3), 2.71 (s, 2H, -CH2C(CH3)2), 1.32 (t, J =8 Hz, 3H, -COOCH2CH3), 1.30 (s, 6H, -C(CH3)2).
13C-NMR (DMSO-d6, 100 MHz): δ 163.9, 159.2, 158.2, 140.9, 140.7, 130.1, 129.0, 121.9, 119.7, 117.3, 32.0, 25.0,
20.6; HRMS (ESI): calcd for C21H23N5O2, [(M+H)+], 378.1930, found 378.1908.
8

ACCEPTED MANUSCRIPT
4.1.7.2. Ethyl 1,4-dimethyl-8-(phenylamino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylate (3b).
1
Yellow solid. Yield: 55%. H-NMR (CDCl3, 400 MHz) δ: 8.29 (s, 1H, -N=CH-), 7.57 (d, J = 8.0 Hz, 1H, HAr),
7.35 (t, J = 8.0 Hz, 2H, HAr), 7.02 (t, J = 8.0 Hz, 1H, HAr), 4.46 (q, J = 8.0 Hz, 2H, -COOCH2CH3), 4.40 (s, 3H,
-NCH3), 3.26 (m, 1H, -CH2CH(CH3)-), 2.86 (d, J = 8.6 Hz, 2H, -CH2CH(CH3)-), 1.43(t, J = 8.6 Hz, 3H,
-COOCH2CH3), 1.25 (d, J = 8.0 Hz, 3H, -CH(CH3)-). 13C-NMR (DMSO-d6, 100 MHz): δ 163.9, 159.2, 158.2,
140.8, 140.7, 130.1, 128.9, 121.9, 119.7, 117.3, 32.0, 24.9, 20.7; HRMS (ESI): calcd for C20H21N5O2, [(M+H)+],
364.1773, found 364.1759.
4.1.8. General procedure for the preparation of derivative 4a.
A solution of compound 2a (0.73g, 2.4 mmol), phenylguanidine (0.41g, 3.0 mmol) in DMF (12.0 mL) was
stirred at 90 for 8 h. Progress of reaction was monitored by tlc and after complete conversion of starting material
reaction mixture was cooled to rt, then poured into H2O (20mL), filtered. The obtained solid was added to 2M
ethanol solution of ammonia (10.0 mL) and the reaction was stirred at 70
for 8 h. Progress of reaction was
monitored by tlc and after complete conversion of starting material reaction mixture was cooled to rt. The solvent
was evaporated and the residue was purified via column chromatography to afford compound 4a. Yellow solid.
1
Yield: 45%. H-NMR (CDCl3, 400 MHz) δ: 8.21 (s, 1H, -N=CH-), 7.56 (d, J = 7.8 Hz, 2H, HAr), 7.35 (t, J = 7.8
Hz, 2H, HAr), 7.06 (t, J = 7.8 Hz, 1H, HAr), 4.33 (s, 3H, -NCH3), 2.72 (s, 2H, -CH2C(CH3)2), 1.46 (s, 6H,
-CH2C(CH3)2). 13C-NMR (DMSO-d6, 100 MHz): δ 163.9, 159.2, 158.2, 140.9, 140.7, 130.1, 128.9, 121.9, 119.7,
117.3, 32.0, 25.0, 20.7; HRMS (ESI): calcd for C19H20N6O, [(M+H)+], 349.1777, found 349.1770.
4.1.9. General procedure for the preparation of derivative 4b.
A solution of compound 2a (73mg, 0.24 mmol), phenylguanidine (41mg, 0.30 mmol) in DMF (2.0 mL) was
stirred at 90 for 8 h. Progress of reaction was monitored by tlc and after complete conversion of starting material
reaction mixture was cooled to rt, then poured into H2O (10mL), filtered. The obtained solid was added to 2M
ethanol solution of methylamine (3.0 mL) and the reaction was stirred at 70 for 8 h. Progress of reaction was
monitored by tlc and after complete conversion of starting material reaction mixture was cooled to rt. The solvent
was evaporated and the residue was purified via column chromatography (5:1 petroleum ether / EtOAc) to yield
compound 4b. Yellow solid. Yield: 50%. 1H-NMR (CDCl3, 400 MHz) δ: 8.27 (s, 1H, -N=CH-), 7.57 (d, J = 7.8 Hz,
2H, HAr), 7.34 (t, J = 7.8 Hz, 2H, HAr), 7.06 (t, J = 7.8 Hz, 1H, HAr), 6.91(s, 1H, -CONHCH3), 4.31 (s, 3H, -NCH3),
2.98 (s, 3H, -CONHCH3), 2.97(s, 2H, -CH2C(CH3)2), 1.46 (s, 6H, -C(CH3)2). 13C-NMR (DMSO-d6, 100 MHz): δ
163.4, 159.3, 157.5, 152.9, 152.5, 142.3, 142.1, 140.8, 135.0, 131.3, 128.9, 121.9, 119.7, 118.2, 41.2, 32.0, 27.4,
26.3; HRMS (ESI): calcd for C20H22N6O, [(M+H)+], 363.1933, found 363.1917.
4.1.10. General procedure for the preparation of derivative 4c.
A solution of compounds 2b (140mg, 0.24 mmol), phenylguanidine (85mg, 0.30 mmol) in DMF (8.0 mL) was
stirred at 90 for 8 h. Progress of reaction was monitored by tlc and after complete conversion of starting material
reaction mixture was cooled to rt, then poured into H2O (10mL), filtered. The obtained solid was added to 2M
ethanol solution of ammonia (8.0 mL) and the reaction was stirred at 70
for 8 h. Progress of reaction was
monitored by tlc and after complete conversion of starting material reaction mixture was cooled to rt. The solvent
was evaporated and the residue was purified via column chromatography (5:1 petroleum ether / EtOAc) to afford
1
compound 4c. Yellow solid. Yield: 48%. H-NMR (DMSO-d6, 400 MHz) δ: 9.55 (s, 1H, Ar-NH-), 8.86 (s, 1H,
-N=CH-), 8.42 (s, 1H, HAr), 7.84 (d, J = 8.0 Hz, 2H, HAr), 7.73 (t, J = 8.0 Hz, 2H, HAr), 7.30 (t, J = 8.0 Hz, 1H,
HAr), 4.34 (s, 3H, -NCH3), 3.01 (m, 1H, -CH2CH(CH3)-), 2.99 (d, J = 8.6 Hz, 2H, -CH2CH(CH3)-), 1.25 (d, J = 8.6
Hz, 3H, -CH2CH(CH3)-). 13C-NMR (DMSO-d6, 100MHz): δ 163.9, 159.2, 158.2, 140.9, 140.7, 130.1, 128.9, 121.9,
9

ACCEPTED MANUSCRIPT
119.7, 117.3, 37.5, 32.0, 24.9, 20.7; HRMS (ESI): calcd for C18H18N6O, [(M+H)+], 335.1620, found 335.1623.
4.1.11. General procedure for the preparation of derivative 4d.
A solution of compound 2c (71mg, 0.24 mmol), phenylguanidine (42mg, 0.30 mmol) in DMF (2.0 mL) was
stirred at 90 for 8 h. Progress of reaction was monitored by tlc and after complete conversion of starting material
reaction mixture was cooled to rt, then poured into H2O (3 mL), filtered. The obtained solid was added to 2M
ethanol solution of ammonia (3.0 mL) and the reaction was stirred at 70
for 8 h. Progress of reaction was
monitored by tlc and after complete conversion of starting material reaction mixture was cooled to rt. The solvent
was evaporated and the residue was purified via column chromatography (5:1 petroleum ether / EtOAc) to afford
1
compound 4d. Yellow solid. Yield: 60%. H-NMR (DMSO-d6, 400 MHz) δ: 9.57 (s, 1H, Ar-NH-), 8.43 (s, 1H,
-N=CH-), 7.71 (d, J = 8.0 Hz, 2H, HAr), 7.30 (d, J = 8.0 Hz, 2H, HAr), 7.02 (t, J = 8.0 Hz, 1H, HAr), 4.30 (s, 3H,
-NCH3), 2.99 (t, J = 6.8 Hz, 2H, -N=CHCCH2CH2-), 2.89 (t, J = 6.8 Hz, 2H, -N=CHCCH2CH2-). 13C-NMR
(DMSO-d6, 100 MHz): δ 163.4, 159.3, 157.5, 140.8, 134.9, 131.3, 129.0, 121.9, 119.7, 118.2, 32.0, 27.4, 26.3;
HRMS (ESI): calcd for C17H16N6O, [(M+H)+], 321.1464, found 321.1460.
4.1.12. General procedure for the preparation of derivative 4e.
A solution of compound 2c (70mg, 0.24 mmol), phenylguanidine (45mg, 0.30 mmol) in DMF (2.0 mL) was
stirred at 90 for 8 h. Progress of reaction was monitored by tlc and after complete conversion of starting material
reaction mixture was cooled to rt, then poured into H2O (3 mL), filtered. The obtained solid was added to 2M
ethanol solution of methylamine (3.0 mL) and the reaction was stirred at 70 for 8 h. Progress of reaction was
monitored by tlc and after complete conversion of starting material reaction mixture was cooled to rt. The solvent
was evaporated and the residue was purified via column chromatography (5:1 petroleum ether / EtOAc) to afford
1
compound 4e. Yellow solid. Yield: 54%. H-NMR (DMSO-d6, 400 MHz) δ: 9.43 (s, 1H, Ar-NH-), 8.86 (s, 1H,
-N=CH-), 7.74 (d, J = 8.0 Hz, 2H, HAr), 7.30 (t, J = 8.0 Hz, 2H, HAr), 7.02 (t, J = 8.0 Hz, 1H, HAr), 4.30 (s, 3H,
-NCH3), 2.99 (t, J = 6.8 Hz, 2H, -N=CHCCH2CH2-), 2.89 (t, J = 6.8 Hz, 2H, -N=CHCCH2CH2-). 13C-NMR
(DMSO-d6, 100 MHz): δ 166.8, 162.7, 161.0, 159.1, 138.9, 138.6, 129.5, 125.3, 125.1, 122.4, 117.8, 116.2, 37.5,
28.3, 26.3, 18.7; HRMS (ESI): calcd for C18H18N6O, [(M+H)+], 335.1620, found 335.1615.
4.1.13. General procedure for the preparation of derivative 4f.
A solution of compound 2c (0.14g, 0.48 mmol), phenylguanidine (83mg, 0.60 mmol) in DMF (8.0 mL) was
stirred at 60 for 8 h. Progress of reaction was monitored by tlc and after complete conversion of starting material
reaction mixture was cooled to rt, then poured into H2O (20mL), filtered. The obtained solid was added to 2M
ethanol solution of ethylamine (10.0 mL) and the reaction was stirred at 80 for 8 h. Progress of reaction was
monitored by tlc and after complete conversion of starting material reaction mixture was cooled to rt. The solvent
was evaporated and the residue was purified via column chromatography (5:1 petroleum ether / EtOAc) to afford
compound 4f. Yellow solid. Yield: 55%. 1H-NMR (CDCl3, 400 MHz) δ: 8.29 (s, 1H, -N=CH-), 7.57 (d, J = 8.0 Hz,
2H, HAr), 7.35 (t, J = 8.0 Hz, 2H, HAr), 7.02 (t, J = 8.0 Hz, 1H, HAr), 4.46 (q, J = 7.4 Hz, 2H, -CONHCH2CH3),
4.40 (s, 3H, -NCH3), 3.10 (t, J = 8.0 Hz, 2H, -N=CHCCH2CH2-), 2.89 (t, J = 8.0 Hz, 2H, -N=CHCCH2CH2-), 1.43
(t, J = 8.0 Hz, 3H, -CONHCH2CH3). 13C-NMR (DMSO-d6, 100 MHz): δ 166.8, 162.7, 160.7, 159.1, 138.9, 138.6,
129.5, 125.3, 125.1, 122.4, 117.8, 116.2, 37.5, 34.2, 28.3, 18.7, 15.0; HRMS (ESI): calcd for C19H20N6O, [(M+H)+],
349.1777, found 349.1760.
4.1.1. The preparation of compounds 6a-6i
A mixture of compounds 5a-5i (10.0 mmol), concentrated hydrochloric acid (12.2 mmol) and melamine (20.0
10

ACCEPTED MANUSCRIPT
mmol) in isopropyl alcohol (10.0 mL) was refluxed for 8 h. Progress of reaction was monitored by tlc and after
complete conversion of starting material reaction mixture was cooled to rt. The reaction mixture was quenched in
saturated sodium carbonate solution (20.0 mL) and filtered. The solid was washed with water (50.0 mL). The
crude product thus obtained was dried at 50 under reduced pressure to get compounds 6a-6i.
4.1.1.1 1-[3-( dimethylamino) phenyl] guanidine (6a). White solid. Yield: 75%; 1H-NMR (DMSO-d6, 400 MHz) δ:
7.29 (d, J = 7.9 Hz, 1H, HAr),7.22 (s, 1H, HAr), 7.15 (t, J = 8.1 Hz, 1H, HAr), 6.58 (d, J = 7.8 Hz, 1H, HAr), 3.02 (s,
6H, Ar-N(CH3)2). 13C-NMR (DMSO-d6, 100 MHz): δ 157.1, 148.9, 134.6, 130.9, 114.4, 109.7, 42.4; HRMS (ESI):
calcd for C9H14N4, [(M+H)+], 179.1297, found 179.1291.
1
4.1.1.2 1-[3-(4-methylpiperazin-1-yl) phenyl] guanidine (6b). White solid. Yield: 50%; H-NMR (DMSO-d6, 400
MHz) δ: 6.83 (t, J = 7.9 Hz, 1H, HAr), 6.16 – 6.08 (m, 2H, HAr), 6.03 (d, J = 7.4 Hz, 1H, HAr), 4.80 (s, 2H, -NH2),
3.05 – 2.97 (m, 4H, Ar-NCH2CH2N), 2.44 – 2.37 (m, 4H, Ar-NCH2CH2N), 2.20 (s, 3H, -NCH3). 13C-NMR
(DMSO-d6, 100 MHz): δ152.5, 149.7, 129.6, 106.1, 104.6, 101.9, 55.2, 48.7, 46.2; HRMS (ESI): calcd for
C11H17N5, [(M+H)+], 220.1562, found 220.1550.
4.1.1.3 1-(3-morpholinophenyl)guanidine (6c). White solid. Yield: 55%; 1H-NMR (DMSO-d6, 400 MHz) δ: 6.85 (t,
J = 7.9 Hz, 1H, HAr), 6.16 – 6.02 (m, 3H, HAr), 4.84 (s, 2H, -NH2), 3.73 – 3.62 (m, 4H, Ar-NCH2CH2O), 3.03 –
2.92 (m, 4H, Ar-NCH2CH2O). 13C-NMR (DMSO-d6, 100 MHz): δ 152.6, 149.7, 129.7, 106.4, 104.3, 101.6, 66.7,
49.2; HRMS (ESI): calcd for C11H16N4O, [(M+H)+], 221.1402, found 221.1389.
4.1.1.4 1-(4-chlorophenyl)guanidine (6d). White solid. Yield: 80%; 1H-NMR (DMSO-d6, 400 MHz) δ: 7.29 (d, J =
7.9 Hz, 1H, HAr),7.22 (s, 1H, HAr), 7.15 (t, J = 8.1 Hz, 1H, HAr), 6.58 (d, J = 7.8 Hz, 1H, HAr). 13C-NMR
(DMSO-d6, 100 MHz): δ 157.1, 149.1, 145.3, 130.4, 105.8, 104.3, 98.0, 54.0, 41.3; HRMS (ESI): calcd for
C7H8N3Cl, [(M+H)+], 170.0407, found 170.0401.
4.1.1.5 1-(p-tolyl)guanidine (6e). White solid. Yield: 80%; 1H-NMR (DMSO-d6, 400 MHz) δ:
δ 7.07 (d, J = 8.0 Hz,
2H, HAr), 6.81 (d, J = 7.8 Hz, 2H, HAr), 2.25 (s, 3H, Ar-CH3). 13C-NMR (DMSO-d6, 100 MHz): δ 157.1, 149.1,
145.3, 130.4, 105.8, 104.3, 98.0, 54.0, 41.3; HRMS (ESI): calcd for C8H11N3, [(M+H)+], 150.1031, found
150.1025.
4.1.1.6 1-[4-( dimethylamino) phenyl] guanidine (6f). White solid. Yield: 65%; 1H-NMR (DMSO-d6, 400 MHz) δ:
6.56 (d, J = 8.7 Hz, 2H, HAr), 6.49 (d, J = 8.7 Hz, 2H, HAr), 4.38 (s, 2H, -NH2), 2.68 (s, 6H, -N(CH3)2). 13C-NMR
(DMSO-d6, 100 MHz): δ 143.4, 140.7, 115.8, 115.6, 42.3; HRMS (ESI): calcd for C9H14N4, [(M+H)+], 179.2470,
found 179.2465.
4.1.1.7 1-[4-( piperazin-1-yl) phenyl] guanidine (6g). White solid. Yield: 45%; 1H-NMR (DMSO-d6, 400 MHz) δ:
8.91 (s, 1H, Ar-NH-), 7.82(s, 1H, -NH=C-), 6.65(d, J = 8.0 Hz, 2H, HAr), 6.63(s, 2H, -NH=CNH2), 6.49(d, J = 8.0
Hz, 2H, HAr), 3.45 (t, J = 8.0 Hz, 4H, Ar-NCH2CH2NH), 2.35 (t, J = 8.0 Hz, 4H, Ar-NCH2CH2NH). 13C-NMR
(DMSO-d6, 100 MHz): δ 157.2, 139.6, 128.1, 117.3, 113.5, 57.2, 52.0, 46.6; HRMS (ESI): calcd for C11H17N5,
[(M+H)+], 220.1562, found 220.1555.
1
4.1.1.8 1-(4-morpholinophenyl)guanidine (6h). White solid; Yield: 40%; H-NMR (DMSO-d6, 400 MHz) δ: 8.95
(s, 1H, Ar-NH-), 7.83(s, 1H, -NH=C-), 6.65(d, J = 7.8 Hz, 2H, HAr), 6.63(s, 2H, -NH=CNH2), 6.49(d, J = 7.8 Hz,
11

ACCEPTED MANUSCRIPT
2H, HAr), 3.44 (t, J = 8.0 Hz, 4H, Ar-NCH2CH2O), 2.35 (t, J = 8.0 Hz, 4H, Ar-NCH2CH2O). 13C-NMR (DMSO-d6,
100 MHz): δ 157.1, 139.6, 128.0, 117.2, 113.6, 57.2, 52.0, 46.6; HRMS (ESI): calcd for C11H16N4O, [(M+H)+],
221.1402, found 221.1400.
4.1.1.9 1- [4-( 4-methylpiperazin-1-yl) phenyl] guanidine (6i). White solid. Yield: 45%; 1H-NMR (DMSO-d6, 400
MHz) δ: 8.93 (s, 1H, Ar-NH-), 7.84(s, 1H, -NH=C-), 6.65(d, 2H, HAr), 6.63(s, 2H, -NH=CNH2), 6.49(d, 2H, HAr),
3.44 (t, J = 8.0 Hz, 4H, Ar-NCH2CH2N), 2.35 (t, J = 8.0 Hz, 4H, Ar-NCH2CH2O), 2.21 (s, 3H, -NCH2CH2NCH3).
13C-NMR (DMSO-d6, 100 MHz): δ 157.1, 139.6, 128.0, 117.2, 113.6, 57.2, 52.0, 46.6; HRMS (ESI): calcd for
C12H19N5, [(M+H)+], 234.1719, found 234.1712.
4.1.2. The preparation of compounds 8c-8e
A mixture of compound 7 (24.7 mmol), morpholine or substituted piperazine (29.2 mmol) and DIPEA (37.1
mmol) in acetonitrile (100 mL) was refluxed for 8 h. Progress of reaction was monitored by tlc and after complete
conversion of starting material reaction mixture was cooled to rt. The solvent was evaporated and the residue was
purified via column chromatography to afford compounds 8c-8e.
1
4.1.2.1 1-(6-nitropyridin-3-yl)piperazine (8c). Yellow solid. Yield: 82%; H-NMR (DMSO-d6, 400 MHz) δ: 7.96
(d, J = 9.3 Hz, 1H, Pyr-H), 7.88 (d, J = 2.9 Hz, 1H, Pyr-H), 7.26 (dd, J = 9.3, 3.0 Hz, 1H, Pyr-H), 3.64 (t, 4H,
Pyr-NCH2CH2N), 3.32 (t, 4H, Pyr-NCH2CH2N). 13C-NMR (DMSO-d6, 100 MHz): δ 151.7, 146.2, 136.6, 125.9,
118.4, 51.3, 45.8; HRMS (ESI): calcd for C9H12N4O2, [(M+H)+], 209.1039, found 209.1030.
4.1.2.2 1-(6-nitropyridin-3-yl)morpholine (8d). Yellow solid. Yield: 80%; 1H-NMR (DMSO-d6, 400 MHz) δ: 7.95
(d, J = 9.3 Hz, 1H, Pyr-H), 7.88 (d, J = 2.9 Hz, 1H, Pyr-H), 7.25 (dd, J = 9.3, 3.0 Hz, 1H, Pyr-H), 3.60 (t, 4H,
Pyr-NCH2CH2O), 3.32 (t, 4H, Pyr-NCH2CH2O). 13C-NMR (DMSO-d6, 100 MHz): δ 151.7, 146.2, 136.6, 125.9,
118.4, 86.3, 53.3; HRMS (ESI): calcd for C9H11N3O3, [(M+H)+], 210.0879, found 210.0868.
1
4.1.2.3 1-methyl-4-(6-nitropyridin-3-yl)piperazine (8e). Yellow solid. Yield: 85%; H-NMR (CDCl3, 400 MHz) δ:
8.08 (d, J = 12.0, 6.1 Hz, 2H, HAr), 7.13 (dd, J = 9.2, 3.0 Hz, 1H, HAr), 3.46 (t, 4H, Pyr-NCH2CH2N), 2.56 (t, 4H,
Pyr-NCH2CH2N), 2.40 (s, 3H, -NCH3). 13C-NMR (DMSO-d6, 100 MHz): δ 149.9, 133.7, 120.4, 119.7, 52.25, 52.0,
46.7, 11.9; HRMS (ESI): calcd for C10H14N4O2, [(M+H)+], 223.1195, found 223.1189.
4.1.3. The preparation of compounds 9c-9e
A mixture of compounds 8c-8e (26.0 mmol) in acetic acid (10 mL) and methyl alcohol (40 mL) was refluxed,
iron (78.0 mmol) was added. The reaction mixture was heated at 60 for 2 h. The reaction mixture was poured
into saturated aqueous sodium carbonate (100 mL) and extracted with ethyl acetate (3*50 mL), then dried over
sodium sulfate, filtered and concentrated. The crude product was purified via column chromatography to afford
compounds 9c-9e.
1
4.1.3.1 5-(piperazin-1-yl)pyridin-2-amine (9c). White solid. Yield: 68%; H-NMR (DMSO-d6, 400 MHz) δ: 7.62
(d, J = 2.7 Hz, 1H, Pyr-H), 7.17 (dd, J = 8.9, 3.0 Hz, 1H, Pyr-H), 6.40 (d, J = 8.8 Hz, 1H, Pyr-H), 5.44 (s, 2H,
-NH2), 3.43 (t, J = 4.0 Hz, 4H, Pyr-NCH2CH2N), 2.85 (t, J = 4.0 Hz, 4H, Pyr-NCH2CH2N). 13C-NMR (DMSO-d6,
100 MHz): δ 163.0, 162.6, 149.4, 146.9, 133.7, 122.1, 120.3, 43.4, 42.6; HRMS (ESI): calcd for C9H14N4O,
[(M+H)+], 179.1297, found 179.1285.
12

ACCEPTED MANUSCRIPT
4.1.3.2 5-morpholinopyridin-2-amine (9d). White solid. Yield: 55%; 1H-NMR (DMSO-d6, 400 MHz) δ: 7.60 (d, J
= 2.7 Hz, 1H, Pyr-H), 7.15 (dd, J = 8.9, 3.0 Hz, 1H, Pyr-H), 6.37 (d, J = 8.8 Hz, 1H, Pyr-H), 5.42 (s, 2H, -NH2),
3.41 (t, J = 4.0 Hz, 4H, Pyr-NCH2CH2O), 2.83 (t, J = 4.0 Hz, 4H, Pyr-NCH2CH2O). 13C-NMR (DMSO-d6, 100
MHz): δ 163.0, 162.6, 149.4, 146.9, 133.7, 122.1, 120.3, 43.4, 42.6; HRMS (ESI): calcd for C9H13N3O, [(M+H)+],
180.1137, found 180.1130.
1
4.1.3.3 5-(4-methylpiperazin-1-yl)pyridin-2-amine (9e). White solid. Yield: 60%; H-NMR (DMSO-d6, 400 MHz)
δ: 7.60 (d, J = 4.0 Hz, 1H, Pyr-H), 7.16 (dd, J1 = 4.0 Hz, J2 =4.0 Hz, 1H, Pyr-H), 6.40 (d, J = 8.0 Hz, 1H, Pyr-H),
5.40 (s, 2H, -NH2), 2.91 (t, J = 4.0 Hz, 4H, Pyr-NCH2CH2N), 2.42 (t, J = 4.0 Hz, 4H, Pyr-NCH2CH2N), 2.20 (s,
3H, -NCH3). 13C-NMR (DMSO-d6, 100 MHz): δ 153.0, 140.6, 137.0, 128.9, 109.2, 55.1, 50.7, 46.1; HRMS (ESI):
calcd for C10H16N4, [(M+H)+], 193.1426, found 193.1453.
4.1.4. The preparation of compounds 10a-10e
A mixture of compounds 9a-9e (10.0 mmol), TEA (15.6 mmol) and 1,3-di-Boc-2-(trifluoromethyl sulfonyl)
guanidine 10.0 mmol) in DCM (30.0 mL) was stirred at room temperature for 48 h. The solvent was evaporated
and the residue was purified via column chromatography to afford compounds 10a-10e.
4.1.4.1 1,3-di(tert-butyl oxycarbonyl) -2-(pyridine-2-amine)formamidine (10a). White solid. Yield: 55%; 1H-NMR
(CDCl3, 400 MHz) δ: 8.68 (s, 1H, Pyr-NH), 7.86 (s, 1H, Pyr-H), 7.23 (d, J = 7.35 Hz, 1H, Pyr-H), 6.63 (s, 1H,
Pyr-H), 6.53 (s, 1H, Pyr-H), 1.42 (s, 18H, -C=N-Boc + NH-Boc). 13C-NMR (DMSO-d6, 100 MHz): δ 158.5, 158.0,
153.7, 148.1, 138.3, 117.9, 109.7, 27.4; HRMS (ESI): calcd for C16H24N4O4, [(M+H)+], 337.1876, found 337.1868.
4.1.4.2 1,3-di(tert-butyl oxycarbonyl) -2-[5-(dimethylamino)pyridine-2-amine]formamidine (10b). White solid.
Yield: 75%; 1H-NMR (DMSO-d6, 400 MHz) δ: 8.68 (s, 1H, Pyr-NH), 7.95 (s, 1H, Pyr-H), 7.25 (d, J = 7.5 Hz, 1H,
Pyr-H), 6.63 (s, 1H, Pyr-H), 2.92 (s, 6H, -N(CH3)2), 1.48 (s, 18H, -C=N-Boc + -NH-Boc). 13C-NMR (DMSO-d6,
100 MHz): δ 158.9, 157.7, 153.6, 147.7, 134.5, 133.2, 127.7, 110.4, 84.7, 79.8, 41.4, 28.1; HRMS (ESI): calcd for
C18H29N5O4, [(M+H)+], 380.2298, found 380.2290.
4.1.4.3 1,3-di(tert-butyl oxycarbonyl) -2-[5-(piperazin-1-yl)pyridine-2-amine]formamidine (10c). White solid.
Yield: 65%; 1H-NMR (DMSO-d6, 400 MHz) δ: 8.63 (s, 1H, Pyr-NH), 7.95 (s, 1H, Pyr-H), 7.24 (d, J = 7.5 Hz, 1H,
Pyr-H), 6.63 (s, 1H, Pyr-H), 3.62 (t, J = 4.8 Hz, 4H, Pyr-NCH2CH2N), 3.04 (t, J = 7.1 Hz, 4H, Pyr-NCH2CH2N),
1.50 (s, 9H, -C=N-Boc), 1.45 (s, 9H, -NH-Boc). 13C-NMR (DMSO-d6, 100 MHz): δ 158.3, 157.5, 153.6, 147.6,
134.5, 133.2, 127.7, 110.4, 84.7, 79.8, 66.4, 53.1, 28.0; HRMS (ESI): calcd for C20H32N6O4, [(M+H)+], 421.2563,
found 421.2555.
4.1.4.4 1,3-di(tert-butyl oxycarbonyl) -2-[5-(morpholino-1-yl)pyridine-2-amine]formamidine (10d). White solid.
Yield: 78%; 1H-NMR (DMSO-d6, 400 MHz) δ: 8.61 (s, 1H, Pyr-NH), 7.98 (s, 1H, Pyr-H), 7.26 (d, J = 7.5 Hz, 1H,
Pyr-H), 6.65 (s, 1H, Pyr-H), 3.65 (t, J = 4.8 Hz, 4H, Pyr-NCH2CH2O), 3.08 (t, J = 7.1 Hz, 4H, Pyr-NCH2CH2O),
1.53 (s, 9H, -C=N-Boc), 1.45 (s, 9H, -NH-Boc). 13C-NMR (DMSO-d6, 100 MHz): δ 158.5, 158.0, 153.7, 147.8,
134.5, 133.2, 127.7, 110.8, 84.8, 79.8, 66.5, 53.0, 28.4; HRMS (ESI): calcd for C20H31N5O5, [(M+H)+], 422.2403,
found 422.2389.
4.1.4.5 1,3-di(tert-butyl oxycarbonyl) -2-[5-(4-methylpiperazin-1-yl)pyridine-2-amine]formamidine (10e). White
solid. Yield: 75%; 1H-NMR (CDCl3, 400 MHz) δ: 8.21 (s, 1H, Pyr-H), 7.98 (s, 1H, Pyr-H), 7.26 (d, J = 7.5 Hz, 1H,
13

ACCEPTED MANUSCRIPT
Pyr-H), 3.18 (t, J = 4.8 Hz, 4H, Pyr-NCH2CH2N), 2.58 (t, J = 7.1 Hz, 4H, Pyr-NCH2CH2N), 2.36 (s, 3H, -NCH3),
1.53 (s, 9H, -C=N-Boc), 1.51 (s, 9H, -NH-Boc). 13C-NMR (DMSO-d6, 100 MHz): δ 158.5, 158.0, 153.7, 147.7,
135.3, 134.5, 122.7, 110.8, 84.6, 79.7, 57.2, 52.0, 46.6, 28.4; HRMS (ESI): calcd for C21H34N6O4, [(M+H)+],
435.2720, found 435.2712.
4.1.5. The preparation of compounds 11a-11e
A mixture of compounds 10a-10e (2.0 mmol), TFA (3 mL) in DCM (10.0 mL) was stirred for 3 h. The solvent
was evaporated and the reaction mixture was poured into saturated aqueous sodium carbonate (100 mL) and
extracted with ethyl acetate (3*50 mL), then dried over sodium sulfate, filtered and concentrated. The crude
product was purified via column chromatography to afford compounds 11a-11e.
1
4.1.5.1 1-(pyridin-2-yl)guanidine (11a).White solid. Yield: 85%; H-NMR (DMSO-d6, 400 MHz) δ: 8.06 (d, J =
3.7 Hz, 1H, Pyr-H), 7.46 (t, J = 7.7 Hz, 1H, Pyr-H), 6.90 (s, 3H, Pyr-H), 6.66 (dd, J = 14.7, 7.4 Hz, 2H, -NH2).
13C-NMR (DMSO-d6, 100 MHz): δ 144.2, 142.7, 134.5, 129.5, 115.4, 52.8, 46.4, 42.8; HRMS (ESI): calcd for
C11H18N6, [(M+H)+], 137.0827, found 137.0816.
4.1.5.2 1-(5-(dimethylamino)pyridin-2-yl)guanidine (11b). White solid. Yield: 87%; 1H-NMR (DMSO-d6, 400
MHz) δ: 8.01 (d, J = 9.0 Hz, 1H, Pyr-H), 7.92 (d, J = 3.4 Hz, 1H, Pyr-H), 7.30 (d, J = 8.4 Hz, 1H, Pyr-H), 3.12 (s,
6H, -N(CH3)2). 13C-NMR (DMSO-d6, 100 MHz): δ 178.7, 162.6, 150.2, 137.8, 137.4, 129.2, 103.5, 60.8, 43.8,
39.8, 24.5, 21.0, 14.4; HRMS (ESI): calcd for C14H19N3O3, [(M+H)+], 180.1249, found 180.1243.
4.1.5.3 1-(5-(piperazin-1-yl)pyridin-2-yl)guanidine (11c). White solid. Yield: 82%; 1H-NMR (DMSO-d6, 400 MHz)
δ: 7.96 (d, J = 9.3 Hz, 1H, Pyr-H), 7.88 (d, J = 2.9 Hz, 1H, Pyr-H), 7.26 (dd, J = 9.3, 3.0 Hz, 1H, Pyr-H), 3.67 (t, J
= 5.1 Hz, 4H, Pyr-NCH2CH2N), 3.36 (t, J = 5.5 Hz, 4H, Pyr-NCH2CH2N). 13C-NMR (DMSO-d6, 100 MHz): 145.3,
142.8, 135.5, 129.3, 117.8, 114.9, 114.6, 46.4, 42.9; HRMS (ESI): calcd for C10H16N6, [(M+H)+], 221.1515, found
221.1510.
4.1.5.4 1-(5-morpholinopyridin-2-yl)guanidine (11d). White solid. Yield: 89%; 1H-NMR (DMSO-d6, 400 MHz) δ:
7.95 (d, J = 2.6 Hz, 1H, Pyr-H), 7.48 (dd, J = 9.0, 2.6 Hz, 1H, Pyr-H), 6.88 (d, J = 9.0 Hz, 1H, Pyr-H), 3.80 (t, 4H,
Pyr-NCH2CH2O), 3.11 (t, 4H, Pyr-NCH2CH2O). 13C-NMR (DMSO-d6, 100 MHz): δ 178.7, 162.6, 150.2, 137.8,
137.4, 129.2, 103.5, 60.8, 43.8, 39.8, 24.5, 21.0, 14.4; HRMS (ESI): calcd for C10H15N5O, [(M+H)+], 222.1355,
found 222.1302.
4.1.5.5 1-(5-(4-methylpiperazin-1-yl)pyridin-2-yl)guanidine (11e). White solid. Yield: 90%; 1H-NMR (CDCl3, 400
MHz) δ: 7.91 (d, J = 4. 0 Hz, 1H, Pyr-H), 7.84 (dd, J1 = 4.0 Hz, J2 = 4.0 Hz, 1H, Pyr-H), 6.92 (d, J = 8.0 Hz, 1H,
Pyr-H), 3.67 (t, J = 12.0 Hz, 4H, Pyr-NCH2CH2N), 3.14 (t, J = 4.0 Hz, 4H, Pyr-NCH2CH2N), 2.82 (s, 3H, -NCH3).
13C-NMR (DMSO-d6, 100 MHz): δ 144.2, 142.7, 134.5, 129.5, 115.4, 52.8, 46.4, 42.8; HRMS (ESI): calcd for
C11H18N6, [(M+H)+], 235.1671, found 235.1642.
4.1.14. General procedure for the preparation of derivatives 13a-13n.
A solution of compounds 2c (0.24 mmol), substituted phenylguanidines (6a-6i, 11a-11e) (0.30 mmol) in DMF
(2.0 mL) was stirred at 90 for 8 h. Progress of reaction was monitored by tlc and after complete conversion of
starting material reaction mixture was cooled to rt, then poured into H2O (10mL). The aqueous phase was
extracted with EtOAc (20.0 mL). The organic phases were then processed in the usual way and chromatographed
14

ACCEPTED MANUSCRIPT
(5:1 petroleum ether / EtOAc) to yield compounds 12a-12n. Then compounds 12a-12n (0.12 mmol) were added to
2M ethanol solution of ammonia (5.0 mL) and the reaction was stirred at 70 for 8 h. Progress of reaction was
monitored by tlc and after complete conversion of starting material reaction mixture was cooled to rt. The solvent
was evaporated and the residue was purified via column chromatography to afford compounds 13a-13n.
4.1.14.1.8-((3-(dimethylamino)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamid
e (13a). Yellow solid. Yield: 60%. 1H-NMR (DMSO-d6, 400 MHz) δ: 9.19 (s, 1H, Ar-NH-), 8.32 (s, 1H, -N=CH-),
7.45 (d, J = 8.0 Hz, 1H, HAr), 6.73 (d, J = 8.0 Hz, 1H, HAr), 6.60 (d, J = 8.0 Hz, 1H, HAr), 4.30 (s, 3H, -NCH3),
3.02 (s, 6H, Ar-N(CH3)2), 2.97 (t, J = 8.6 Hz, 2H, -N=CHCCH2CH2-), 2.89 (t, J = 8.6 Hz, 2H, -N=CHCCH2CH2-).
13C-NMR (DMSO-d6, 100 MHz): δ 162.2, 159.6, 146.9, 138.0, 130.6, 126.2, 121.7, 117.8, 113.3, 66.8, 60.7, 24.5,
19.8, 14.7; HRMS (ESI): calcd for C19H21N7O, [(M+H)+], 364.1886, found 364.1882.
4.1.14.2.1-methyl-8-((3-(piperazin-1-yl)phenyl)amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide
1
(13b). Yellow solid. Yield: 46%. H-NMR (DMSO-d6, 400 MHz) δ: 9.43 (s, 1H, Ar-NH-), 8.86 (s, 1H, -N=CH-),
8.18 (s, 2H, -CONH2), 7.13 (d, J = 8.0 Hz, 1H, HAr), 6.97 (d, J = 8.0 Hz, 1H, HAr), 6.60 (d, J = 8.0 Hz, 1H, HAr),
6.38 (s, 1H, HAr), 3.95 (s, 3H, -NCH3), 3.46 (t, J = 8.0 Hz, 4H, Ar-NCH2CH2N), 2.99 (t, J = 8.0 Hz, 2H,
-N=CHCCH2CH2-), 2.89 (t, J = 8.0 Hz, 2H, -N=CHCCH2CH2-), 2.78 (t, J = 8.0 Hz, 4H, Ar-NCH2CH2N).
13C-NMR (DMSO-d6, 100 MHz): δ 166.8, 162.7, 161.6, 159.1, 149.1, 143.3, 138.6, 130.4, 125.3, 125.1, 116.2,
107.3, 104.3, 100.7, 54.0, 45.8, 37.5, 28.3, 18.7; HRMS (ESI): calcd for C21H24N8O, [(M+H)+], 405.2151, found
405.2142.
4.1.14.3.1-methyl-8-((3-morpholinophenyl)amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide
(13c). Yellow solid. Yield: 45%. 1H-NMR (CDCl3, 400 MHz) δ: 9.40 (s, 1H, Ar-NH-), 8.41 (s, 1H, -N=CH-), 7.28
(d, J = 7.8 Hz, 2H, HAr), 7.22 (s, 1H, HAr), 7.15 (d, J = 7.8 Hz, 1H, HAr), 6.59 (d, J = 8.0 Hz, 1H, HAr), 4.36 (s, 3H,
-NCH3), 3.75 (t, J = 8.0 Hz, 4H, Ar-NCH2CH2O), 3.08 (t, J = 8.0 Hz, 4H, Ar-NCH2CH2O), 2.99 (t, J = 4.0 Hz, 2H,
-N=CHCCH2CH2-), 2.89 (t, J = 4.0 Hz, 2H, -N=CHCCH2CH2-). 13C-NMR (DMSO-d6, 100 MHz): δ 162.1, 159.2,
157.3, 152.7, 152.0, 141.5, 138.0, 136.5, 129.3, 126.3, 118.8, 111.2, 109.5, 106.8, 66.8, 66.6, 60.6, 49.2; HRMS
(ESI): calcd for C21H23N7O2, [(M+H)+], 406.1992, found 406.1987.
4.1.14.4.8-((4-(dimethylamino)pyridin-2-yl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carbox
amide (13j). Yellow solid. Yield: 58%. 1H-NMR (CDCl3, 400 MHz) δ: 9.51 (s, 1H, Pyr-NH), 8.41 (s, 2H, -N=CH-),
7.92 (s, 1H, Pyr-H), 7.85 (d, J = 6.8 Hz, 1H, Pyr-H), 7.27 (t, J = 7.4 Hz, 1H, Pyr-H), 7.25 (s, 1H, Pyr-H), 4.36 (s,
3H, -NCH3), 3.03 (s, 6H, Pyr-N(CH3)2), 2.99 (t, J = 8.0 Hz, 2H, -N=CHCCH2CH2-), 2.89 (t, J = 8.0 Hz, 2H,
-N=CHCCH2CH2-). 13C-NMR (DMSO-d6, 100 MHz): δ 162.7, 161.6, 159.1, 153.1, 143.3, 138.6, 130.4, 125.3,
125.1, 116.2, 107.3, 104.3, 100.7, 41.3, 37.5, 28.3, 18.7; HRMS (ESI): calcd for C18H20N8O, [(M+H)+], 365.1838,
found 365.1812.
4.1.14.5.1-methyl-8-((4-chlorophenyl)amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (13d).
Yellow solid. Yield: 50%. 1H-NMR (CDCl3, 400 MHz) δ: 9.72 (s, 1H, Ar-NH-), 8.43 (s, 1H, -N=CH-), 7.75 (d, J =
7.80 Hz, 2H, HAr), 7.35 (d, J = 7.8 Hz, 2H, HAr), 4.35 (s, 3H, -NCH3), 2.97 (t, J = 8.0 Hz, 2H, -N=CHCCH2CH2-),
2.85 (t, J = 8.0 Hz, 2H, -N=CHCCH2CH2-). 13C-NMR (DMSO-d6, 100 MHz): δ 162.1, 158.9, 157.4, 152.8, 139.8,
138.0, 136.4, 128.8, 126.5, 125.3, 120.9, 119.3, 60.7, 24.5, 19.6; HRMS (ESI) : calcd for C20H23N9O, [(M+H)+],
406.2104, found 406.2102.
15

ACCEPTED MANUSCRIPT
4.1.14.6.1-methyl-8-(p-tolylamino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (13e). Yellow solid.
1
Yield: 45%. H-NMR (CDCl3, 400 MHz) δ: 8.29 (s, 1H, -N=CH-), 7.65 (d, J = 7.8 Hz, 2H, HAr), 7.35 (d, J =7.8
Hz, 2H, HAr), 4.44 (s, 3H, -NCH3), 3.10 (t, J = 8.0 Hz, 2H, -N=CHCCH2CH2-), 3.00 (s, 3H, Ar-CH3), 2.88 (t, J =
8.0 Hz, 2H, -N=CHCCH2CH2-). 13C-NMR (DMSO-d6, 100 MHz): δ 163.9, 159.2, 158.2, 140.9, 140.7, 130.1,
128.9, 121.9, 119.7, 32.0, 25.0, 20.7; HRMS (ESI): calcd for C20H22N8O2, [(M+H)+], 407.1944, found 407.1939.
4.1.14.7.8-((4-(dimethylamino)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamid
1
e (13f). Yellow solid. Yield: 55%. H-NMR (CDCl3, 400 MHz) δ: 9.19 (s, 1H, Ar-NH-), 8.32 (s, 1H, -N=CH-),
7.45 (d, J = 7.6 Hz, 2H, HAr), 6.72 (d, J = 7.6 Hz, 2H, HAr), 4.32 (s, 3H, -NCH3), 3.02 (s, 6H, -N(CH3)2), 2.95 (t, J
= 8.0 Hz, 2H, -N=CHCCH2CH2-), 2.80 (t, J = 8.0 Hz, 2H, -N=CHCCH2CH2-). 13C-NMR (DMSO-d6, 100 MHz): δ
162.2, 159.6, 146.9, 138.0, 130.6, 126.2, 121.7, 117.8, 113.3, 66.8, 60.7, 24.5; HRMS (ESI): calcd for C19H21N7O,
[(M+H)+], 364.1886, found 364.1878.
4.1.14.8.1-methyl-8-((4-(piperazin-1-yl)phenyl)amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide
(13g). Yellow solid. Yield: 63%. 1H-NMR (CDCl3, 400 MHz) δ: 9.30(s, 1H, Ar-NH-), 8.35 (s, 1H, -N=CH-), 7.55
(d, J = 7.8 Hz, 2H, HAr), 7.00 (d, J = 7.8 Hz, 2H, HAr), 4.32 (s, 3H, -NCH3), 3.00 (t, J = 8.0 Hz, 4H,
Ar-NCH2CH2N), 2.79 (t, J = 8.0 Hz, 2H, -N=CHCCH2CH2-), 2.89 (t, J = 8.0 Hz, 2H, -N=CHCCH2CH2-), 2.78 (t,
J = 8.0 Hz, 4H, Ar-NCH2CH2N). 13C-NMR (DMSO-d6, 100 MHz): δ 162.2, 159.6, 146.9, 138.0, 130.6, 126.2,
121.7, 117.8, 113.3, 66.8, 60.7, 24.5, 19.8; HRMS (ESI): calcd for C21H24N8O, [(M+H)+], 405.2151, found
405.2148.
4.1.14.9.
1-methyl-8-((4-morpholinophenyl)amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide
(13h). Yellow solid. Yield: 43%. 1H-NMR (CDCl3, 400 MHz) δ: 9.29 (s, 1H, Ar-NH-), 8.35 (s, 1H, -N=CH-), 7.53
(d, J = 7.8 Hz, 2H, HAr), 6.90 (d, J = 7.8 Hz, 2H, HAr), 4.32 (s, 3H, -NCH3), 3.00 (t, J = 8.0 Hz, 4H,
Ar-NCH2CH2O), 2.87 (t, J = 8.0 Hz, 4H, Ar-NCH2CH2O), 2.99 (t, J = 8.0 Hz, 2H, -N=CHCCH2CH2-), 2.89 (t, J =
8.0 Hz, 2H, -N=CHCCH2CH2-). 13C-NMR (DMSO-d6, 100 MHz): δ 166.8, 162.7, 161.6, 159.1, 139.6, 138.6,
128.4, 125.3, 125.1, 118.4, 116.2, 113.6, 66.3, 53.3, 37.5, 28.3; HRMS (ESI): calcd for C21H23N7O2, [(M+H)+],
406.1992, found 406.1982.
4.1.14.10 . 8-((5-(dimethylamino)pyridin-2-yl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-
1
carboxamide (13k). Yellow solid. Yield: 56%. H-NMR (CDCl3, 400 MHz) δ: 9.51 (s, 1H, Pyr-NH), 8.41 (s, 1H,
-N=CH-), 7.87 (s, 1H, Pyr-H), 7.28 (d, J = 7.6 Hz, 1H, Pyr-H), 7.25 (d, J = 7.6 Hz, 1H, Pyr-H), 4.36 (s, 3H,
-NCH3), 3.58 (s, 6H, -N(CH3)2), 2.98 (t, J = 8.0 Hz, 2H, -N=CHCCH2CH2-), 2.85 (t, J = 8.0 Hz, 2H,
-N=CHCCH2CH2-). 13C-NMR (DMSO-d6, 100 MHz): δ 162.2, 159.6, 146.9, 138.0, 130.6, 126.2, 121.7, 117.8,
113.3, 66.8, 60.6; HRMS (ESI): calcd for C18H20N8O, [(M+H)+], 365.1838, found 365.1830.
4.1.14.11.1-methyl-8-((5-(piperazin-1-yl)pyridin-2-yl)amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carbo
xamide (13l). Yellow solid. Yield: 59%. 1H-NMR (CDCl3, 400 MHz) δ: 9.62 (s, 1H, Pyr-NH), 8.42 (s, 1H,
-N=CH-), 7.99 (s, 1H, Pyr-H), 7.97 (d, J = 7.6 Hz, 1H, Pyr-H), 7.41 (d, J = 7.6 Hz, 1H, Pyr-H), 4.36 (s, 3H,
-NCH3), 3.02 (t, J = 4.8 Hz, 4H, Pyr-NCH2CH2N), 2.96 (t, J = 8.0 Hz, 2H, -N=CHCCH2CH2-), 2.89 (t, J = 4.8 Hz,
2H, -N=CHCCH2CH2-), 2.84 (t, J = 4.8 Hz, 4H, Pyr-NCH2CH2N). 13C-NMR (DMSO-d6, 100 MHz): δ 157.6,
153.2, 151.8, 148.5, 141.2, 138.3, 133.9, 131.9, 121.9, 121.4, 114.7, 108.2, 56.2, 50.2, 44.9, 41.4, 35.8; HRMS
(ESI): calcd for C19H19N8O2, [(M+H)+], 406.2104, found 406.2100.
16

ACCEPTED MANUSCRIPT
4.1.14.12.1-methyl-8-((5-morpholinopyridin-2-yl)amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxami
1
de (13m). Yellow solid. Yield: 50%. H-NMR (CDCl3, 400 MHz) δ: 9.62 (s, 1H, Pyr-NH), 8.42(s, 1H, -N=CH-),
8.03 (d, J = 8.0 Hz, 1H, Pyr-H), 7.52 (s, 1H, Pyr-H), 7.44 (d, J = 8.0 Hz, 1H, Pyr-H), 4.34 (s, 3H, -NCH3), 3.77 (t,
J = 5.8 Hz, 4H, Pyr-NCH2CH2N), 3.11 (t, J = 4.8 Hz, 4H, Pyr-NCH2CH2N), 2.99 (t, J = 8.0 Hz, 2H,
-N=CHCCH2CH2-), 2.81 (t, J = 8.0 Hz, 2H, -N=CHCCH2CH2-). 13C-NMR (DMSO-d6, 100 MHz): δ 162.1, 158.5,
157.2, 152.8, 145.9, 143.7, 138.0, 136.4, 135.8, 126.3, 125.6, 119.1, 114.0, 100.0, 60.6, 50.3, 46.0; HRMS (ESI):
calcd for C20H22N8O2, [(M+H)+], 407.1944, found 407.1937.
4.1.14.13.1-methyl-8-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline
-3-carboxamide (13n). Yellow solid. Yield: 55%. 1H-NMR (CDCl3, 400 MHz) δ: 9.57 (s, 1H, Pyr-NH), 8.53 (s, 1H,
-N=CH-), 8.40 (s, 2H, -CONH2), 7.98 (s, 1H, Pyr-H), 6.79 (d, J = 8.0 Hz, 1H, Pyr-H), 6.65 (d, J = 8.0 Hz, 1H,
Pyr-H), 3.95 (s, 3H, -NCH3), 3.15 (t, J = 8.0 Hz, 4H, Pyr-NCH2CH2N), 2.99 (t, J = 8.0 Hz, 2H, -N=CHCCH2CH2),
2.89 (t, J = 8.0 Hz, 2H, -N=CHCCH2CH2), 2.35 (t, J = 8.0 Hz, 4H, Pyr-NCH2CH2N), 2.21 (m, 3H,
-NCH2CH2NCH3). 13C-NMR (DMSO-d6, 100 MHz): δ 163.4, 159.3, 157.5, 152.9, 152.5, 142.3, 142.0, 140.8,
134.9, 131.3, 129.0, 122.0, 120.0, 118.2, 41.2, 32.0, 27.4, 26.3; HRMS (ESI): calcd for C21H25N9O, [(M+H)+],
420.2260, found 420.2252.
4.1.14.14.1-methyl-8-((4-(4-methylpiperazin-1-yl)phenyl)amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-ca
1
rboxamide (13i). Yellow solid. Yield: 65%. H-NMR (CDCl3, 400 MHz) δ: 9.29 (s, 1H, Ar-NH-), 8.35 (s, 1H,
-N=CH-), 7.53 (d, J = 8.0 Hz, 2H, HAr), 6.90 (d, J = 8.0 Hz, 2H, HAr), 4.32 (s, 3H, -NCH3), 3.00 (t, J = 8.0 Hz, 4H,
Ar-NCH2CH2N), 2.99 (t, J = 7.8 Hz, 2H, -N=CHCCH2CH2), 2.89 (t, J = 7.8 Hz, 2H, -N=CHCCH2CH2), 2.48 (t, J
= 8.0 Hz, 4H, Ar-NCH2CH2N), 2.23 (s, 3H, -NCH2CH2NCH3). 13C-NMR (DMSO-d6, 100 MHz): δ 162.2, 159.6,
146.8, 137.9, 130.6, 126.2, 121.7, 117.8, 113.3, 66.8, 60.7, 24.5, 19.8, 14.7; HRMS (ESI): calcd for C22H26N8O,
[(M+H)+], 419.2308, found 419.2305.
4.2. Biology
4.2.1. Kinase assay
Kinase assay was performed as previously described [19]. The CDK2/CycA2, CDK4/CycD3 and CDK6/CycD3
were from Carna. The Peptide FAM-P8 and Peptide FAM-P18 were from GL Biochem. The ATP and EDTA were
from Sigma. The kinase reactions were carried out in 30µL volumes in the reaction solution containing the
following: 2µL compound (in 20% DMSO), 18µL kinase in buffer (50mM HEPES, pH 7.5, 5mM MgCl2, 1mM
DTT, 0.0015% Brij-35), 10µL of the mixture of FAM-labeled peptide and ATP. The reaction mixture was
incubated at 30 for 40 min, then add 25µL of stop buffer to stop reaction. Finally, conversion was read from
Caliper, then convert conversion values to inhibition values: percent inhibition = (max-conversion)/(max-min)
*100. “max” stands for DMSO control, “max” stands for low control. IC50 values were fitted by Hill equation with
OriginPro 8.
4.2.2. Cell proliferation assay
MCF-7 cells were collected using 0.25% trypsin-EDTA and seeded in 96-well plates (5*103 cells/well) in
DMEM medium. HCT116, HepG2, and PANC-1 cells were collected using 0.25% trypsin-EDTA and seeded in
96-well plates (5*103 cells/well) in PRMI1640 medium, respectively. After 24 h, cells were incubated for 72 h
with various concentration of the tested compounds. The cells were stained at 37
for 4h with 0.05% MTT
dissolved in PBS. The plates were tested by using CellTiter-Glo assay (Promega) and fluorescence was read at
490nm. Then convert OD values to inhibition values: percent inhibition = (OD1-OD2)/OD1*100%, OD1 stands for
blank control, OD2 stands for drug groups. IC50 of proliferation was fitted by Hill equation with OriginPro 8.
4.2.3. Procedures for mice pharmacokinetic
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In vivo pharmacokinetic properties were evaluated in mice Male ICR mice (20-25 g), following intravenous (iv,
10 mg/kg) and oral (os, 10 mg/kg). All animal experiments were performed in accordance with IACUC protocol or
the regulations effective in China. Blood was collected at multiple time points post dose and transferred to an
EDTA tube. The blood was centrifuged at 8500 rpm for 15 min, and the plasma was transferred to a polypropylene
tube, capped, and stored frozen (-20 ℃) for parent compound analysis. The analysis was conducted by using
HPLC separation coupled with mass spectrometric detection. All pharmacokinetic (PK) parameters were derived
from concentration-time data by non-compartmental analyses. All pharmacokinetic parameters were calculated
with the computer software STATA 12.0.
4.2.4. Molecular modeling
The crystal structure of CDK6 complex with palbociclib (PDB code: 2EUF) was downloaded from Protein Data
Bank (http://www.rcsb.org/) and used for the docking study. The protein was removed from the structure first.
Discovery Studio 3.0 Client was used to analyze the binding sites and force field in ATP package of CDK6. The
structure of the compounds 13l was prepared by using Discovery Studio 3.0 Client.
Acknowledgements
This work was supported in part by the National Natural Science Foundation of China (Grant Nos. 21305051)
and the Jiangsu prospective joint research projiect (BY2015019-25). We thank Ms. Zhou Yin and Ms. Gao
Yueying for useful discussion for technical support.
Supplementary data
Supplementary data (characterization of compounds) associated with this article can be found, in the online
version
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Highlights
Novel 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives were designed
targeting CDK4/6
Introducing a 2-aminopyridine side chain at the C-8 position could inhibit
CDK4/6 with exquisite selectivity over CDK2
Both compounds 13m and 13n displayed potent antiproliferative activities
against MCF-7 cell
The CDK4/6 inhibitor 13n exhibited reasonable pharmacokinetic profiles.
Molecular docking in the active site of CDK6