Synthesis and biological evaluation of 2,6-disubstituted-9H-purine, 2,4-disubstitued-thieno[3,2-d]pyrimidine and -7H-pyrrolo[2,3-d]pyrimidine analogues as novel CHK1 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.03.075

Checkpoint kinase 1 (CHK1) inhibitors can potentiate the effectiveness of deoxyribonucleic acid (DNA) damaging agents in the treatment of cancer. A novel series of 2,6-disubstituted-9H-purine (3a-p, 5a and 5b), 2,4-disubstituted-thieno[3,2-d]pyrimidine (8

Full text of DOI:10.1016/j.ejmech.2018.03.075

Accepted Manuscript
Synthesis and biological evaluation of 2,6-disubstituted-9H-purine, 2,4-disubstitued-
thieno[3,2-d]pyrimidine and -7H-pyrrolo[2,3-d]pyrimidine analogues as novel CHK1
inhibitors
Zifei Han, Chao Tian, Yuanxin Li, Meng Wang, Jiajia Yang, Xiaowei Wang, Zhili
Zhang, Junyi Liu
PII:
S0223-5234(18)30319-2
10.1016/j.ejmech.2018.03.075
EJMECH 10340
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 February 2018
Revised Date: 14 March 2018
Accepted Date: 26 March 2018
Please cite this article as: Z. Han, C. Tian, Y. Li, M. Wang, J. Yang, X. Wang, Z. Zhang, J. Liu, Synthesis
and biological evaluation of 2,6-disubstituted-9H-purine, 2,4-disubstitued-thieno[3,2-d]pyrimidine and
-7H-pyrrolo[2,3-d]pyrimidine analogues as novel CHK1 inhibitors, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.075.
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ACCEPTED MANUSCRIPT
Graphic Abstract
Synthesis and biological evaluation of 2,6-disubstituted-9H-purine,
2,4-disubstitued-thieno[3,2-d]pyrimidine
and
-7H-pyrrolo[2,3-d]pyrimidine
analogues as novel CHK1 inhibitors
Zifei Han, 1,† Chao Tian, 1,* Yuanxin Li, Meng Wang, Jiajia Yang, Xiaowei Wang, Zhili Zhang,
Junyi Liu*
1

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1
2
Synthesis and biological evaluation of 2,6-disubstituted-9H-purine,
2,4-disubstitued-thieno[3,2-d]pyrimidine
and
-7H-pyrrolo[2,3-d]pyrimidine
3
analogues as novel CHK1 inhibitors
4
Zifei Han, 1,† Chao Tian, 1,*,† Yuanxin Li,† Meng Wang,† Jiajia Yang,† Xiaowei Wang,† Zhili Zhang,†
5
Junyi Liu*,†,‡
6
† Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing
100191, China
7
‡
8
State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191,
9
China
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1 Co-first author of this paper. These authors contributed equally.
*Co-corresponding author of this paper. Chao Tian: tianchao@bjmu.edu.cn; Junyi Liu:
jyliu@bjmu.edu.cn.
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2
ABSTRACT
Checkpoint kinase 1 (CHK1) inhibitors can potentiate the effectiveness of deoxyribonucleic acid
3
(DNA) damaging agents in the treatment of cancer. A novel series of 2,6-disubstituted-9H-purine
4
(3a-p,
5a
and
5b),
2,4-disubstituted-thieno[3,2-d]pyrimidine
(8a-c)
and
5
2,4-disbustituted-7H-pyrrolo[2,3-d]pyrimidine (11a-c) analogues were designed and synthesized
as potent CHK1 inhibitors. Compounds (3a, 3d, 3f and 3j-l) with 9H-purine core displayed more
potent inhibition against CHK1. The most potent compound (3l) also exhibited low
anti-proliferative effects towards HT29 and Hek293 cell lines. In addition, 3l showed strong
potentiating effect (7-fold) on the anti-proliferative activity of gemcitabine towards HT29 cells.
The results of cell cycle assay indicated that 3l could strikingly affect the cell cycle distribution of
the gemcitabine-treated HT29 cells and induce a significant S phase accumulation. The kinase
selectivity profile of 3l displayed acceptable selectivity against other kinases. These results
rendered 3l a potent lead compound of CHK1 inhibitor for further investigation.
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10
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19
KEYWORDS
CHK1 inhibitors, 9H-Purine, Potentiating effect, Anti-proliferative effects.
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1. INTRODUCTION
2
3
Cell cycle is well controlled by cell cycle checkpoints, which are indispensable in the growth and
survival of normal cells as well as tumor cells. In response of the DNA damage resulting from
cytotoxic agents or ionizing radiation [1, 2], the cell cycle checkpoints will be activated to induce
the cell cycle arrest at corresponding phase and subsequently recruit the factors involving in the
DNA damage repair mechanism. Checkpoint kinase 1 (CHK1) plays an essential role in the
S-phase and G2/M-phase cell cycle checkpoints and DNA damage response in cells [3]. CHK1 is
a serine-threonine kinase. It is mainly phosphorylated and activated by its upstream kinase ATR
(Ataxia Telangiectasia-Mutated and Rad3-related) in response to single strand breaks in DNA.
After phosphorylated on residues Ser-317 and Ser-345 of CHK1, an auto-phosphorylation takes
place on residue Ser-296 to obtain the activated CHK1 [4, 5]. A number of downstream proteins
including cell division cycle 25 (Cdc25) family are phosphorylated by the activated CHK1 and
finally regulate the cell cycle progression. Furthermore, the repair of damaged DNA was also
promoted by CHK1 through signaling to the repair protein RAD51 [6].
4
5
6
7
8
9
10
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12
13
14
15
16
17
18
19
20
21
22
As we know that nearly half of the tumor cells are p53-defficient ones, which results in an absence
of G1 arrest in cell cycle checkpoints. Based on the crucial role of CHK1 in the cell cycle
checkpoints and DNA damage repair mechanism especially for these p53-mutant tumors, CHK1
has been considered as an excellent target of antitumor agents with potentiating effect. A number
of reports had confirmed that selective inhibition of CHK1 with small molecules, or inactivation
by RNA interference (RNAi) could enhance the effectiveness of chemotherapy or ionizing
radiation therapy [7]. After the discovery of the first CHK1 inhibitor, UCN-01
(7-hydroxystaurosporine) [8, 9], plenty of compounds were reported as potent CHK1 inhibitors
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1
2
3
4
5
[10]. Some of these compounds, including AZD7762 [11], MK-8776 [12], PF477736 [13],
LY2603618 [14], Prexasertib (LY2606368) [15, 16], GDC-0425 [17] and CCT245737 [18, 19],
entered the clinical trials in combination with chemotherapeutic agents. However, only the clinical
trials concerning LY2606368 and CCT245737 are still active on the ClinicalTrials.gov recently.
Most of the others were terminated for toxicity and severe side effects [20-22].
6
7
Figure 1. The representative CHK1 inhibitors and the ATP-binding site of CHK1
As shown in Figure 1, the structures of the CHK1 inhibitors are diverse and can be generally
divided into three fragments (blue, orange and purple) according to the corresponding crystal
structures of the inhibitors bound to the adenosine triphosphate (ATP)-binding site of CHK1. The
“blue fragments” always bound to the “hinge region” of the ATP-binding site of CHK1 which was
generally formed by Glu-85, Tyr-86, Cys-87 and Ser-88 residues. The “orange fragments” always
bound to the “ribose region” which was formed by Gly-90, Glu-91, Glu-134, Asn-135 and
Leu-137 residues. The “purple fragments” usually bound to the “interior pocket” which was
formed by Lys-38, Glu-55, Val-68, Leu-84 Ser-147 and Asp-148 residues. When the inhibitors
bound to the ATP-binding site of CHK1, the “blue fragments” of the inhibitors were in charge of
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1
2
“pushing” the other two fragments into their corresponding binding sites. These three fragments
were indispensable in the scaffold of CHK1 inhibitors.
3
As we know that fused bicyclic pyrimidine derivatives played an important role in the discovery
of kinases inhibitors [23-27]. Replacement of pyrimidine with purine or pyrrolopyrimidine was
also reported to bring many advantages in the biological activities and pharmacokinetic profiles of
kinase inhibitors [28]. In a previous study, 6-morpholino-9H-purine was reported as a CHK1
inhibitor with an IC50 value of micromole [29].The absence of the “interior pocket” binding
4
5
6
7
8
fragment (purple) was considered as a key reason of low inhibitory activity of
9
6-morpholino-9H-purine against CHK1. Based on the reported structure-activity relationships of
CHK1 inhibitors, we thought that introduction of substituted (hetero)aromatic amine group on
position 2 of 6-morpholino-9H-purine was conducive to enhancing its potency in CHK1 inhibition
and potentiating effect. Taking the 6-morpholino-9H-purine and the “interior pocket” binding
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15
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19
20
21
22
fragments of CHK1 inhibitors together, in this work, we designed
a
series of
2,6-disubstituted-9H-purine as novel CHK1 inhibitors (Figure 2). A range of substituted
(hetero)aromatic amines on position 2 were investigated to evaluated the effect of the “interior
pocket”
binding
fragments
on
this
scaffold.
As
a
contrast,
several
2-substituted-N-(tetrahydro-2H-pyran-4-yl)-9H-purin-6-amines,
2,4-disubstituted-thieno[3,2-d]pyrimidines and 2,4-disubstituted-7H-pyrrolo[2,3-d]pyrimidines
were also synthesized to further investigate the effect of the 6-morpholino-9H-purine backbone
structure on the potency of CHK1 inhibition. The inhibition against CHK1 and anti-proliferation
toward HT29 and Hek293 cells of the target compounds were evaluated to discovery potent CHK1
inhibitors with low cytotoxicities. Furthermore, the potentiating effect on gemcitabine-induced
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cell proliferation inhibition, the effect on the cell cycle distribution and kinase selectivity profile of
the most potent compound were further estimated.
1
2
3
4
Figure 2. Design of the novel CHK1 inhibitors
2. RESULTS AND DISCUSSIONS
5
6
2.1. Synthesis of target compounds
7
8
The
target
compounds
were
synthesized
using
2,6-dichloro-9H-purine
(1),
2,4-dichlorothieno[3,2-d]pyrimidine (6) and 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (9) as the
row material. The synthetic route was outlined in Scheme 1. Firstly, morpholine group was
introduced to the 4- or 6-position of the dichloro derivatives (1, 6 and 9) through refluxing in the
9
10
11
12
13
14
15
16
presence
of
trimethylamine
in
ethanol
(EtOH)
for
10
h
to
obtain
2-chloro-6-morpholin-4-yl-9H-purine (2), 2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (7)
and 2-chloro-4-morpholin-4-yl-7H-pyrrolo[2,3-d]pyrimidine (10). A similar reaction was
conducted between 2,6-dichloro-9H-purine and 4-aminotetrahydropyran to achieve
2-chloro-N-(tetrahydro-2H-pyran-4-yl)-9H-purin-6-amine (4). Subsequently, the target compounds
3a-p, 5a, 5b, 8a-c and 11a-c were assembled via a microwave assisted amination of compounds 2,
6

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4, 7 and 10 with different (hetero)aromatic amines (ArNH2) catalyzing by trifluoroacetic acid
(TFA) or palladium(II) acetate (Pd(OAc)2) [30, 31].
1
2
O
N
O
N
N
N
N
ii
N
R
N
H
N
H
N
H
N
Cl
N
Cl
N
2
3a-p
N
N
i
O
O
N
H
Cl
HN
HN
1
N
N
N
N
N
ii
N
R
N
H
N
H
N
N
Cl
N
4
H
5a,5b
O
O
N
Cl
N
N
N
S
ii
S
S
N
i
N
R
N
H
N
Cl
Cl
8a-c
6
7
O
N
O
N
Cl
N
ii
i
N
N
R
N
N
H
N
N
H
N
Cl
Cl
N
H
H
11a-c
9
10
3
4
Scheme 1. The synthesis route of compound 3a-p, 5a, 5b, 8a-c and 11a-c
5
6
7
8
Reagents and conditions: i. Morpholine (or 4-aminotetrahydropyran), trimethylamine, EtOH, reflux; ii. ArNH2,
TFA, 2,2,2-trifluoroethanol (CF3CH2OH), microwave, 140 ºC, 1.5h. or ArNH2, Pd(OAc)2,
4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos), caesium carbonate (Cs2CO3), microwave, 160 ºC,
40 min.
9
10
11
12
13
14
15
2.2. Biological evaluation
2.2.1. CHK1 kinase inhibition
The inhibitory activities of these compounds in the concentration of 10 µM against CHK1 were
evaluated using ADP-Glo kit and listed in Table 1. The thieno[3,2-d]pyrimidine analogues 8a-c
had no inhibition against CHK1. In contrast to 7H-pyrrolo[2,3-d]pyrimidines 11a-c, 9H-purine
analogues 3a-c exhibited moderate to high potency in CHK1 inhibition. From the results of 3a-c,
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1
2
8a-c and 11a-c, a sequence of potencies in CHK1 inhibition could be summarized as follow:
9H-purine analogues > 7H-pyrrolo[2,3-d]pyrimidine analogues >> thieno[3,2-d]pyrimidine
analogues. These results implied that 7-N of 9H-purine might bring some interactions with CHK1
to promote the inhibitory activities. 9H-purine is more suitable to be the backbone structure of
CHK1 inhibitors. Furthermore, 3a displayed 82.4% (10 µM) inhibition against CHK1. However,
the inhibitory activity of its 4-aminotetrahydropyran analogue 5a was only 61.5%. This indicated
that morpholine group had better directly “connect” to the 6-position of 9H-purine. This could
also be demonstrated by the inhibitory activities of 3l and 5b.
3
4
5
6
7
8
9
Our main interests in this work were focusing on the substituent groups on 2-NH of
6-morpholino-9H-purine. From the results of 3a-p, we could summarize some structure-activity
relationships as follow. As the inhibition activity of 3c was lower than 3b, it seemed that
multi-substituted phenyl group on 2-NH would reduce the inhibition against CHK1. From the
inhibitory activities of 3b (p-OCH3), 3d (m-OCH3) and 3e (o-OCH3), we could find that the
substituted position on 2-aminophenyl will significantly affect the inhibition against CHK1. The
sequence of inhibitory activities is m- > p- >> o-. To further investigate the effect of the
substituents, a range of substituents were introduced to m-position of phenyl such as -OCH3, -CH3,
-CF3, -OCF3, -Cl and -F to generate 3d and 3f-k. Among them, m-F substituted analogue (3k) was
the best one, exhibiting 92.0% inhibition against CHK1. Besides, inhibition activities of m-OCH3
and m-CH3 analogues (3d and 3f) were also more than 80%. Beyond our expectation, p-F
substituted analogue 3l also exhibited strong potency in CHK1 inhibition, though a little lower
than that of the m-substituted analogue (3k). The above results indicated that small volume and
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12
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14
15
16
17
18
19
20
21
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1
2
3
4
5
6
7
8
strong electro-withdraw groups on phenyl were conductive to the inhibitory activity. The effect of
the former (volume) was predominant.
Furthermore, three other heteroaromatic rings were introduced to 2-NH of
6-morpholino-9H-purine to generate compounds 3m (3’-pyridinyl), 3o (3’-quinolinyl) and 3p
(2’-pyrazinyl). As the order of inhibitory activities were 3a ≥ 3m > 3p > 3o, phenyl and pyridinyl
ring were undoubtedly the best two aromatic rings on 2-NH of 6-morpholino-9H-purine. Moreover,
compound 3n displayed more than 80% inhibition against CHK1.
Table 1. The inhibitory activities of target compounds against CHK1
9
Inhibition against
CHK1 at 10 µM
(CHK1 IC50 (µM))
Inhibition against
CHK1 at 10 µM
(CHK1 IC50 (µM))
No.
R
No.
R
82.4% (2.57)
51.0%
80.3% (9.87)
38.2%
3a
3b
3c
3d
3e
3n
3o
3p
5a
5b
35.8%
63.1%
84.9% (3.63)
3.8%
61.5%
37.2%
85.2% (1.27)
61.3%
N.D.
N.D.
3f
8a
8b
3g
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31.6%
N.D.
3h
3i
8c
50.0%
44.5%
11a
77.3% (2.83)
92.0% (3.60)
16.7%
26.9%
3j
11b
11c
3k
89.9% (0.684)
82.2% (8.06)
0.006
0.002
3l
AZD7762
3m
CCT245737
1
2
Compounds 3a, 3d, 3f, 3j-l, 3m and 3n, which displayed more than 75% inhibition against CHK1,
were selected to further determine their IC50 values. AZD7762 and CCT245737 were used as the
positive controls. Compounds 3a, 3d, 3f, 3j and 3k with substituted phenyl groups on 2-NH
exhibited moderate inhibitory activities against CHK1, ranging from 1.27 to 3.63 µM. The
pyridinyl analogues 3m and 3n displayed low potencies in CHK1 inhibition (8.06 and 9.87µM).
These results indicated that substituted phenyl groups as substituents on 2-NH of
6-morpholino-9H-purine were better than pyridinyl groups in this scaffold. Despite weaker than
AZD7762 and CCT245737, compound 3l with an IC50 value of 0.684 µM showed the strongest
potency in inhibition of CHK1 among our target compounds.
3
4
5
6
7
8
9
10
11
12
13
14
2.2.2. Anti-proliferative effect toward HT29 cells and Hek293 cells
The anti-proliferative effects of target compounds were assessed on the HT29 cell line
(p53-mutant), which were presented in Table 2. The positive controls, AZD7762 and CCT245737,
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1
2
3
4
5
6
showed moderate to strong potencies in anti-proliferation against HT29 cells, with GI50 values of
0.167 and 9.223 µM, respectively. However, most of our target compounds exhibited low
anti-proliferative potencies on HT29 cells. Among them, the GI50 values of compounds 3a, 3c, 3e,
3h, 3l, 3m and 3o were all above 100 µM, which indicated extremely low anti-proliferative effect
toward HT29 cells.
Table 2. The GI50 values (µM) of target compounds toward HT29 and Hek293 cells
HT29 GI50
(µM)
>100
74.5
Hek293 GI50
(µM)
60.2
HT29 GI50
(µM)
72.2
Hek293 GI50
(µM)
72.2
No.
No.
3a
3b
3c
3d
3e
3f
3n
>100
>100
38.7
>100
25.1
27.2
3o
>100
72.6
22.3
3p
80.3
31.5
5a
>100
67.6
>100
40.5
86.9
>100
>100
>100
>100
64.7
5b
54.5
8a
8b
29.1
31.4
29.7
3g
3h
3i
>100
58.0
>100
73.6
31.6
8c
58.3
11a
34.8
38.4
38.4
63.6
3j
11b
60.8
33.7
50.4
58.7
3k
3l
11c
>100
>100
>100
33.7
0.167
9.223
0.236
1.387
AZD7762
CCT245737
3m
7
8
As low cytotoxicity is necessary for CHK1 inhibitors, all the target compounds were also assessed
their anti-proliferative activities toward Hek293 cell line. As shown in Table 2, AZD7762 and
CCT245737 exhibited strong anti-proliferative effects on Hek293 cells with GI50 values of 0.236
and 1.387 µM, respectively. Contrastingly, our compounds displayed low to moderate potencies in
anti-proliferation on Hek293 cells. To our delight, the most potent 3l showed nearly no
anti-proliferative potency toward Hek293 cells (GI50 > 100 µM). The lower anti-proliferative
activity of 3l implied higher safety profile in drug combination.
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10
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2
3
4
5
6
7
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2.2.3. Colony formation assay
In order to guide the subsequent antitumor potentiation assay, the colony formation assay was
performed to further assess the cytotoxicity of 3l on HT29 cell line. HT29 cells (100 cells per well)
was treated by different concentrations of 3l ranging from 100 nM to 100 µM for 2 weeks. As
shown in Figure 3, 3l did not significantly affect the colony formation of HT29 cells when the
concentration of 3l was no higher than 10 µM. Therefore, in the subsequent potentiation assay, no
more than 10 µM of 3l would be used in combination with gemcitabine in HT29 cells.
9
10
Figure 3. The effect of 3l on the colony formation of HT29 cells.
11
12
2.2.4. Antitumor potentiation assay
13
14
15
16
17
18
To evaluate the antitumor potentiating effect of 3l to DNA-damaging agents, the
concentration-response of HT29 cells to proliferation inhibition by gemcitabine (GEM, ranging
from 1.5 nM to 400 nM) was measured with or without 3l (10 and 1 µM). AZD7762 (50 nM) was
used as the positive control. The ratio of GI50 value of GEM alone over that of GEM in
combination with CHK1 inhibitor was set as potentiation factor (PF) to estimate the anti-tumor
potentiating effect of CHK1 inhibitor [32].
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1
2
3
4
5
6
7
As shown in Figure 4 and Table 3, the GI50 value of GEM was 105.9 nM toward HT29 cells. The
potentiating effect of 3l was dependent on its concentration. Lower concentration of 3l (1 µM) did
not enhance the anti-proliferation of GEM significantly. However, in combination with 10 µM of
3l, the GI50 value of GEM strikingly reduced to 15.2 nM (PF = 7.0), displaying a strong
potentiating effect. Although, the PF value of 3l was less than that of AZD7762 (PF = 15.8), the
potentiation assay demonstrated compound 3l as a good CHK1 inhibitor possessed a strong
potentiating effect to GEM in HT29 cells.
8
9
Figure 4. Anti-proliferation curve of GEM in the presence of 3l and AZD7762. The black curves
in all four plots were anti-proliferative curves of GEM. In contrast, the red curves were the
anti-proliferative curve of GEM in combination with 3l (10 and 1 µM) or AZD7762 (50 nM).
10
11
12
13
Table 3. Potentiation effects of 3l and AZD7762 to GEM in HT29 cells.
GI50 (nM)
105.9
15.2
PFa
GEM
GEM+3l (10 µM)
GEM+3l (1 µM)
7.0
1.9
54.4
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GEM+AZD7762
6.7
15.8
1
2
a. Potentiation factor = ratio of GI50 value of GEM alone over that of GEM in combination with 3l (10 µM and 1
µM) or AZD7762 (50 nM).
3
4
2.2.5. Cell cycle distribution
The cell cycle assay was performed to further study the effect of 3l on the cell cycle distribution.
HT29 cells were treated by 3l alone, GEM alone and GEM in combination with 3l, respectively.
As shown in Figure 5 and 6, in contrast to the untreated control, there was no obvious change in
the cell cycle distribution of HT29 cells after treated by 10 µM of 3l. This was in consistent with
the low anti-proliferation inhibition of 3l. After treated by 25 nM of GEM, the percentage of G2/M
phase cells increased from 7.0% (blank) to 85.2%, indicating a significant G2/M arrest. However,
after treated by GEM in combination with 10 µM of 3l, the population of G2/M phase cells
reduced from 85.2% to 25.2%, meanwhile, the S phase increased from 30.9% to 61.0%. The
above results indicated that compound 3l could induce a significant S phase accumulation in the
GEM treated HT29 cells. This result was in line with the previous report on the cell cycle profiles
of CHK1 inhibitors in combination with GEM in HT29 cells, in which a similar S phase
accumulation was observed in the HT29 cells after treated by GEM plus a CHK1 inhibitor
(CHIR-124) [33].
5
6
7
8
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1
2
Figure 5. Cell cycle distributions of HT29 cells after treated by 3l (10 µM) alone, GEM alone and
GEM plus 3l (10 µM) in relative to untreated control. a. untreated control; b. 3l (10 µM) alone; c.
GEM (25 nM) alone; d. GEM (25 nM) in combination with 3l (10 µM).
3
4
Cell Cycle
GEM + 3l
100
GEM
3l
80
Blank
60
40
20
0
G1
S
G2
Blank
3l
GEM
4.3%
GEM +3l
13.8%
61.0%
25.2%
G1
S
62.1%
30.9%
7.0%
60.0%
29.5%
10.5%
10.5%
85.2%
G2
5
6
7
Figure 6. Histogram of cell cycle distributions of HT29 cells treated by 3l, GEM and GEM in
combination with 3l, respectively.
8
9
2.2.6. Kinase selectivity assay
The kinase inhibitory activities of 3l were also determined against fourteen crucial kinases from
six different kinase families at higher concentration of 10 µM. The inhibition results were
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illustrated in Figure 7. At concentration of 10 µM, 3l exhibited nearly 90% inhibition toward
CHK1. Besides, it also displayed 72% and 73% inhibition against Checkpoint kinase 2 (CHK2)
and Cyclin-dependent kinase 2 (CDK2). Moreover, the inhibitory activities of 3l were all less than
50% against the other 11 kinases. Especially for calcium/calmodulin-dependent protein kinase
type IV (CAMK4), RAC-alpha serine/threonine-protein kinase (AKT1), catalytic subunit α of
protein kinase A (PKAC-α), Anaplastic lymphoma kinase (ALK) and epidermal growth factor
receptor (EGFR) kinases, 3l displayed nearly no inhibitory activities. These results indicated that
3l possessed reasonable kinase selectivity toward CHK1 over the other kinases. Nevertheless,
more work should be done to optimize the structures of our compounds to reduce the inhibitory
activities against CHK2 and CDK2.
3
4
5
6
7
8
9
10
Kinase Selectivity
100
80
60
40
20
0
1
2
4
2
1
α
2
1
β
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4
B
K
K
T
R
F
-
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T
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K
K
A
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K
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K
L
A
C
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S
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A
M
T
H
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H
H
B
G
E
M
A
S
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A
C
C
A
K
P
G
E
K
C
P
M
Inh% a
89.9
Inh% a
73.0
Family
kinase
CHK1
Family
kinase
CDK2
MAPK1
GSK3β
STK3
CMGC
Family
CHK2
72.0
0.0
33.0
47.0
44.0
35.0
0.0
CAMK
Family
CAMK4
MAPKAPK2
AKT1
18.0
0.0
STE Family
TK Family
BTK
AGC Family
TKL Family
PKAC-α
ALK
0.0
EGFR
EPHB4
1.0
23.0
a Inh% is short for Inhibition%
11
12
13
14
Figure 7. Histogram and table of kinase selectivity profile of 3l against representative kinases. 14
kinases from six different kinase families were selected to determine their inhibition by 3l at the
concentration of 10 µM.
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2.3. Molecular modeling study
A molecular modeling study was performed on compound 3l using a reported crystal structure of
CHK1 (PDB: 5F4N) [18]. As shown in Figure 8, the binding mode of 3l indicated that the purine
structure could form two hydrogen bonds with Glu-85 and Cys-87 residues at the hinge area.
However, the hydrogen-bonding interactions in the ribose region and interior pocket were not
obvious. Furthermore, the p-F-phenyl group and purine ring of 3l formed a big planar structure.
The interior pocket was filled with the p-F-phenyl group on 2-NH of 3l. Bulky substituents were
unfavorable to the pocket. This was consistent with our biological assay result that smaller
substituted groups on phenyl ring of 2-NH was conducive to the potency on CHK1 inhibition. The
stretching of morpholine group of 3l into the ribose region was obstructed by Glu-91 residue. The
flexibility of the substituent on position 6 of purine should be reconsidered. This modeling result
revealed that purine was a good backbone structure of CHK1 inhibitor and more appropriate
substituent groups on position 2 and 6 should be further investigated.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Figure 8. Predicting docking mode of 3l with the crystal structure of CHK1 (5F4N). Compound 3l
was displayed in cyan. Hydrogen bonds between 3l and amino acid residue of CHK1 were
indicated with dashed lines in yellow.
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3. CONCLUSION
2
3
A
series of 2,6-disubstituted-9H-purine, 2,4-disubstituted-thieno[3,2-d]pyrimidine and
-7H-pyrrolo[2,3-d]pyrimidine analogues were synthesized as novel CHK1 inhibitors. Among the
three types of compounds, purine analogues possessed the best potency in CHK1 inhibition.
2,6-Disubstituted-9H-purine analogues 3a, 3d, 3f and 3j-n displayed good inhibitory activities
against CHK1. Among them, 3l with an IC50 of 0.684 µM was undoubtedly the best one.
Furthermore, compound 3l exhibited nearly no anti-proliferative activity toward HT29 and
Hek293 cells, which implied a good safety profile. The antitumor potentiating assay showed that
3l could significantly lower the gemcitabine GI50 from 105.9 nM to 15.2 nM. In addition, 3l could
strikingly affect the cell cycle distribution of the gemcitabine treated HT29 cells and induce a
significant S phase accumulation. Kinase selectivity assay revealed that 3l displayed acceptable
selectivity toward 14 kinases. All above results rendered 3l a good lead compound of CHK1
inhibitors for further investigation. A series of 3l analogues varied at 6-position are under
synthesis to discover more potent CHK1 inhibitors.
4
5
6
7
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4. EXPERIMENTAL SECTION
17
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4.1. Chemistry
4.1.1. General information
All the reagents and solvents were purchased from common commercial suppliers without further
purification. Microwave assisted reactions were performed on the Discovery SP (CEM, USA).
Melting points (m.p.) were determined using a SGW X4 apparatus. Mass spectra were recorded on
MDS SCIEX QSTAR system. 1H- and 13C-NMR spectra were recorded with a Varian INOVA-400
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or INOVA-600 with dimethyl sulfoxide-d6 (DMSO-d6) as solvent. Tetramethylsilane (TMS) was
used as an internal standard to express the chemical shift in ppm (parts per million): s, singlet; bs,
broad singlet; d, doublet; t, triplet; q, quartet; quin, quintet m, multiplet; and bs, broad singlet. IR
spectra were recorded on a Nicolet-6700 FT-IR spectrometer (KBr pellets). Thin-layer
chromatography (TLC) with a fluorescent indicator was utilized to monitor the reaction progress,
and the spots were visualized under 254 and 365 nm illumination. All test compounds were
assessed for purity by Agilent Technologies 1260 Infinity HPLC system. HPLC condition:
methanol (CH3OH): H2O = 60:40; flow rate, 1.0 mL/min; UV detection, from 220 to 365 nm;
temperature, ambient; injection volume, 20 µL. The purity of all the test compounds was greater
than 95%.
3
4
5
6
7
8
9
10
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12
13
14
15
16
17
18
19
20
21
22
4.1.2. General procedure for synthesis of compounds 2, 4, 7 and 10
A suspension of 300mg of 2,6-dichloro-9H-purine (1), 2,4-dichlorothieno[3,2-d]pyrimidine (6) or
2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (9) in 10 mL of EtOH was added morpholine or
4-aminotetrahydropyran (1.2 equiv) and 300µL of triethylamine. The mixtrue was refluxed for 10
hours. After the disappearance of compounds 1, 6 or 9 detected by TLC, the solution was
evaporated in vacuum to yield a syrup. Then, it was neutralized by concentrated ammonium
hydroxide. After filtering off, the residue was washed with cold water and cold acetone
successively to remove the triethylammonium hydrochloride.
4.1.2.1. 4-(2-chloro-9H-purin-6-yl)morpholine (2)
Compound 2 was obtained as a white solid (yield 80.8%). m.p. >300 ºC; 1H NMR (400 MHz,
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DMSO-d6) δ: 3.72 (s, 4H, CH2NCH2), 4.18 (bs, 4H, CH2OCH2), 8.16 (s, 1H, CH), 13.23 (s, 1H,
9-NH); 13C NMR (100 MHz, DMSO-d6) δ: 46.0, 66.5, 118.2, 139.4, 152.9, 153.1, 153.8; MS
(ESI): [M+H]+: 240.10. IR (KBr pellet, cm-1): 3437, 3099, 2976, 2832, 1581, 1450, 1359, 1315,
1277, 1262, 1160, 1116, 1032, 959, 940, 833, 636, 524.
3
4
5
6
4.1.2.2. 2-chloro-N-(tetrahydro-2H-pyran-4-yl)-9H-purin-6-amine (4)
7
White solid. m.p. 151-152 ºC; 1H NMR (400 MHz, DMSO-d6) δ: 1.68 (s, 2H, CH2CHCH2) , 1.79
(s, 2H, CH2CHCH2), 3.40 (s, 2H, CH2OCH2), 3.88 (s×2, 2H, CH2OCH2), 4.21 (bs, 1H, NHCH),
8.13 (s, 2H, NH and CH), 13.08 (s, 1H, 9-NH); 13C NMR (100 MHz, DMSO-d6) δ: 19.0, 32.6,
33.6, 46.7, 66.6, 105.0, 118.2, 139.8, 151.0, 153.4, 154.6; MS (ESI): [M+H]+: 254.09. IR (KBr
pellet, cm-1): 3431, 3128, 2971, 2874, 2602, 2519, 2101, 1587, 1520, 1497, 1474, 1450, 1437,
1375, 1299, 1238, 1229, 1206, 1186, 1169, 1080, 1032, 1017, 1001, 988, 952, 905, 869, 856, 767,
655, 636, 556, 484, 418.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
4.1.2.3. 4-(2-chlorothieno[3,2-d]pyrimidin-4-yl)morpholine (7)
Compound 7 was obtained as a white solid (yield 72.3 %). m.p. 193-194 ºC; 1H NMR (400 MHz,
DMSO-d6) δ: 3.71-3.80 (m, 4H, CH2NCH2), 3.87-3.96 (m, 4H, CH2OCH2), 7.41 (d, J = 5.5 Hz,
1H, CH), 8.31 (d, J = 5.5 Hz, 1H, SCH); 13C NMR (100 MHz, DMSO-d6) δ: 46.4, 66.3, 113.0,
124.2, 135.9, 156.4, 158.7, 163.2; MS (ESI): [M+H]+: 256.15. IR (KBr pellet, cm-1): 3096, 3080,
2997, 2967, 2904, 2856, 2773, 1545, 1518, 1488, 1448, 1431, 1367, 1291, 1275, 1242, 1183, 1133,
1114, 1045, 1017, 945, 925, 872, 826, 791, 732, 700, 648, 606, 525.
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4.1.2.4. 4-(2-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)morpholine (10)
Compound 10 was obtained as a white solid (yield 75.4 %). m.p. 259-260 ºC; 1H NMR (400 MHz,
DMSO-d6) δ: 3.72 (s, 4H, CH2NCH2), 3.83 (s, 4H, CH2OCH2), 6.66 (s, 1H, CH), 7.21 (s, 1H,
NHCH), 11.92 (s, 1H, 7-NH); 13C NMR (100 MHz, DMSO-d6) δ: 45.8, 66.4, 101.1, 101.9, 122.5,
152.3, 153.3, 157.4; MS (ESI): [M+H]+: 239.13. IR (KBr pellet, cm-1): 3192, 3114, 2985, 2969,
2895, 2860, 1590, 1569, 1487, 1450, 1439, 1374, 1346, 1322, 1295, 1274, 1253, 1167, 1141, 1120,
1022, 955, 903, 874, 832, 797, 729, 718, 680, 630, 596, 528.
3
4
5
6
7
8
9
4.1.3. General procedure for microwave assisted introduction aromatic amine on position 2 of
compound 2, 4, 7 and 10.
10
11
12
13
14
15
16
17
18
19
20
21
22
General procedure A to obtain compounds 3a-l, 5a, 5b, 8a-c, 11a-c: to a stirred suspension of
50 mg 2-cholro analogues 1, 4, 6 or 9 (1 equiv) and aromatic amine (2 equiv) in 2 ml of 2,2,2-
trifluoroethanol (TFE) was added TFA (40 µl) dropwise. The reagents were placed in a sealed
microwave vial and the reactions were performed under microwave irradiation (140 ºC for 90
min). The solvent was removed in vacuo and the residue was re-dissolved in dichloromethane
(CH2Cl2) (10mL). The solution was washed with saturated sodium bicarbonate solution (3 × 10
mL), and the aqueous extracts were combined and washed with CH2Cl2 (10mL). The combined
organic layers were dried with sodium sulfate anhydrous (Na2SO4). The solvent was removed
under reduced pressure and the crude material was purified by column chromatography on silica
gel.
General procedure B to obtain compounds 3m-p: 4-(2-chloro-9H-purin-6-yl)morpholine
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(50mg, 0.21mmol), heteroaromatic amine (1.2 equiv), 2 mol % Pd(OAc)2, 3 mol % Xantphos,
Cs2CO3 (140mg, 0.43mmol) in dioxane (1.5 mL) was heated to 160 °C in a microwave reactor for
40min. The solvent was removed under reduced pressure and the crude material was purified by
column chromatography on silica gel with CH2Cl2/CH3OH in a 15:1 (v/v) ratio as eluent, to afford
product 3m-p.
3
4
5
6
7
4.1.3.1. 6-morpholino-N-phenyl-9H-purin-2-amine (3a)
8
White solid, yield 81.1%. m.p. 271-272 ºC; 1H NMR (400 MHz, DMSO-d6) δ: 3.68-3.77 (m, 4H,
CH2NCH2), 4.06-4.34 (m, 4H, CH2OCH2), 6.86 (t, J = 7.2 Hz, 1H, Ph-H4’), 7.23 (t, J = 7.8 Hz,
2H, Ph-H3’, Ph-H5’), 7.76 (d, J = 8.0 Hz, 2H, Ph-H2’, Ph-H6’), 7.85 (s, 1H, CH), 8.89 (s, 1H,
2-NH), 12.59 (s, 1H, 9-NH); 13C NMR (100 MHz, DMSO-d6) δ: 45.6, 66.7, 114.7, 118.7, 120.6,
128.8, 136.5, 142.1, 153.5, 153.8, 156.1; HRMS (ESI): calcd for C15H16N6O [M+H]+: 297.1464,
found: 297.1464. IR (KBr pellet, cm-1): 3371, 3105, 2987, 2960, 2868, 1607, 1570, 1531, 1474,
1463, 1453, 1441, 1426, 1393, 1374, 1329, 1290, 1267, 1239, 1154, 1111, 1074, 1018, 937, 827,
786, 756, 731, 692, 638, 518.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
4.1.3.2 N-(4-methoxyphenyl)-6-morpholino-9H-purin-2-amine (3b)
1
White solid, yield 80.3%, m.p. 231-232 ºC; H NMR (400 MHz, DMSO-d6) δ: 3.71 (bs, 7H,
OCH3, CH2NCH2), 4.18 (s, 4H, CH2OCH2), 6.83 (d, J = 8.7 Hz, 2H, Ph-H3’, Ph-H5’), 7.63 (d, J =
8.6 Hz, 2H, Ph-H2’, Ph-H6’), 7.81 (s, 1H, CH), 8.67 (s, 1H, 2-NH), 12.51 (s, 1H, 9-NH); 13C
NMR (100 MHz, DMSO-d6) δ: 45.6, 55.6, 66.7, 114.0, 114.6, 120.5, 135.3, 136.2, 153.6, 153.8,
153.9, 156.4; HRMS (ESI): calcd for C16H18N6O2 [M+H]+: 327.1569, found: 327.1574. IR (KBr
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pellet, cm-1): 3307, 2999, 2963, 2903, 2834, 1600, 1577, 1513, 1476, 1444, 1392, 1317, 1299,
1284, 1268, 1240, 1173, 1154, 1114, 1070, 1034, 937, 884, 831, 811, 785, 685, 626, 556, 525.
3
4
4.1.3.3. 6-morpholino-N-(3,4,5-trimethoxyphenyl)-9H-purin-2-amine (3c)
1
5
White solid, yield 70.3%. m.p. >300 ºC; H NMR (400 MHz, DMSO-d6) δ: 3.60 (s, 3H, OCH3),
6
3.73 (m, 4H, CH2NCH2), 3.75 (s, 6H, OCH3 × 2), 4.20 (s, 4H, CH2OCH2), 7.18 (s, 2H, Ph-H2’,
Ph-H6’), 7.87 (s, 1H, CH), 8.78 (s, 1H, 2-NH), 12.54 (s, 1H, 9-NH); 13C NMR (100 MHz,
DMSO-d6) δ: 49.1, 56.1, 60.6, 66.6, 96.4, 114.6, 131.8, 136.7, 138.3, 153.0, 153.4, 153.8, 156.0;
HRMS (ESI): calcd for C18H22N6O4 [M+H]+: 387.1781, found: 387.1774. IR (KBr pellet, cm-1):
3422, 3280, 2960, 1602, 1584, 1509, 1484, 1453, 1432, 1398, 1354, 1110, 937, 879, 786, 622,
470.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
4.1.3.4. N-(3-methoxyphenyl)-6-morpholino-9H-purin-2-amine (3d)
White solid, yield 76.2%. m.p. 209-210 ºC; 1H NMR (400 MHz, DMSO-d6) δ: 3.73 (s, 7H, OCH3,
CH2NCH2), 4.20 (s, 4H, CH2OCH2), 6.44 (d, J = 7.5 Hz, 1H, Ph-H4’), 7.11 (dd, J = 7.5, 8.0 Hz,
1H, Ph-H2’), 7.26 (d, J = 8.0 Hz, 1H, Ph-H5’), 7.57 (s, 1H, Ph-H6’), 7.86 (s, 1H, CH), 8.89 (s, 1H,
2-NH), 12.60 (s, 1H, 9-NH); 13C NMR (100 MHz, DMSO-d6) δ: 45.6, 55.3, 66.7, 104.4, 106.0,
111.7, 114.7, 129.4, 136.6, 143.3, 153.4, 153.8, 156.0, 160.0; HRMS (ESI): calcd for C16H18N6O2
[M+H]+: 327.1569, found: 327.1566. IR (KBr pellet, cm-1): 3380, 3178, 3003, 2900, 2858, 1605,
1577, 1493, 1439, 1412, 1374, 1306, 1291, 1267, 1215, 1158, 1107, 1025, 939, 870, 833, 785, 773,
729, 695, 632, 515, 459.
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4.1.3.5. N-(2-methoxyphenyl)-6-morpholino-7H-purin-2-amine (3e)
White solid, yield 77.4%. m.p. 220-221 ºC; 1H NMR (400 MHz, DMSO-d6) δ: 3.73 (d, J = 4.1 Hz,
4H, CH2NCH2), 3.88 (s, 3H, OCH3), 4.18 (s, 4H, CH2OCH2), 6.88 - 6.96 (m, 2H, Ph-H3’, Ph-H5’),
7.00 (d, J = 5.6 Hz, 1H, Ph-H4’), 7.41 (s, 1H, Ph-H6’), 7.87 (s, 1H, CH), 8.42 (bs, 1H, 2-NH),
12.64 (s, 1H, 9-NH); 13C NMR (100 MHz, DMSO-d6) δ: 45.7, 56.2, 66.7, 110.8, 115.0, 118.4,
120.8, 121.3, 130.3, 136.7, 148.0, 153.4, 153.9, 155.7; HRMS (ESI): calcd for C16H18N6O2
[M+H]+: 327.1569, found: 327.1573. IR (KBr pellet, cm-1): 3434, 2967, 2856, 1601, 1579, 1537,
1490, 1457, 1435, 1410, 1312, 1288, 1265, 1243, 1217, 1177, 1158, 1113, 1024, 937, 887, 834,
784, 741, 632, 563, 519.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
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22
4.1.3.6. 6-morpholino-N-(m-tolyl)-9H-purin-2-amine (3f)
White solid, yield 83.8%. m.p. 181-182 ºC; 1H NMR (400 MHz, DMSO-d6) δ: 2.27 (s, 3H), 3.73
(s, 4H, CH2NCH2) , 4.20 (s, 4H, CH2OCH2) , 6.68 (d, J = 7.7 Hz, 1H, Ph-H4’) , 7.11 (t, J = 7.6 Hz,
1H, Ph-H5’), 7.53-7.61 (m, 2H, Ph-H2’, Ph-H6’), 7.85 (s, 1H, CH), 8.81 (s, 1H, 2-NH), 12.57 (s,
1H, 9-NH); 13C NMR (100 MHz, DMSO-d6) δ: 21.9, 45.6, 66.7, 114.7, 115.9, 119.2, 121.4, 128.6,
136.5, 137.7, 142.0, 153.5, 153.8, 156.1; HRMS (ESI): calcd for C16H18N6O [M+H]+: 311.1620,
found: 311.1620. IR (KBr pellet, cm-1): 3421, 3330, 2959, 2918, 2859, 1603, 1581, 1530, 1489,
1391, 1315, 1287, 1268, 1220, 1162, 1112, 1071, 1024, 941, 879, 826, 785, 729, 694, 631, 527,
443.
4.1.3.6. 6-morpholino-N-(3-(trifluoromethyl)phenyl)-9H-purin-2-amine (3g)
1
White solid, yield 74.1%. m.p. 209-210 ºC; H NMR (400 MHz, DMSO-d6) δ: 3.74 (s, 4H,
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2
CH2NCH2) , 4.22 (s, 4H, CH2OCH2) , 7.17 (d, J = 7.5 Hz, 1H, Ph-H4’), 7.45 (t, J = 7.8 Hz, 1H,
Ph-H5’), 7.88 (d, J = 8.0 Hz, 1H, Ph-H6’), 7.89 (s, 1H, CH), 8.37 (s, 1H, Ph-H2’), 9.30 (s, 1H,
2-NH), 12.64 (s, 1H, 9-NH); 13C NMR (100 MHz, DMSO-d6) δ: 31.1, 45.7, 66.7, 114.3, 115.1,
116.5, 121.9, 129.8, 136.9, 142.9, 153.3, 153.8, 155.8; HRMS (ESI): calcd for C16H15F3N6O
[M+H]+: 365.1338, found: 365.1341. IR (KBr pellet, cm-1): 3450, 3108, 2995, 2962, 2861, 1585,
1536, 1446, 1399, 1337, 1316, 1281, 1270, 1161, 1111, 1068, 1022, 937, 878, 831, 789, 724, 696,
633, 586, 525, 487.
3
4
5
6
7
8
9
4.1.3.7. 6-morpholino-N-(3-(trifluoromethoxy)phenyl)-9H-purin-2-amine (3h)
1
10
11
12
13
14
15
16
17
18
19
20
21
22
White solid, yield 71.6%. m.p. 201-202 ºC; H NMR (400 MHz, DMSO-d6) δ: 3.73 (s, 4H,
CH2NCH2) , 4.21 (s, 4H, CH2OCH2) , 6.80 (d, J = 7.2 Hz, 1H, Ph-H4’), 7.33 (t, J = 7.9 Hz, 1H,
Ph-H5’), 7.63 (d, J = 7.9 Hz, 1H, Ph-H6’), 7.90 (s, 1H, CH), 8.02 (s, 1H, Ph-H2’), 9.28 (s, 1H,
2-NH), 12.67 (s, 1H, 9-NH); 13C NMR (100 MHz, DMSO-d6) δ: 45.6, 66.7, 110.2, 112.2, 115.0,
117.1, 130.2, 136.9, 143.9, 149.1, 153.2, 153.8, 155.6; HRMS (ESI): calcd for C16H15F3N6O2
[M+H]+: 381.1287, found: 381.1277. IR (KBr pellet, cm-1): 3449, 3100, 2966, 2869, 1585, 1528,
1488, 1444, 1405, 1373, 1313, 1301, 1267, 1208, 1159, 1116, 1070, 1024, 999, 960, 881, 834, 781,
772, 724, 702, 683, 632, 582, 524, 489, 446.
4.1.3.8. 2,2,2-trifluoro-N-(3-((6-morpholino-9H-purin-2-yl)amino)phenyl)acetamide (3i)
1
White solid, yield 66.7%. m.p. 206-207 ºC; H NMR (400 MHz, DMSO-d6) δ: 3.72 (s, 4H,
CH2NCH2), 4.19 (s, 4H, CH2OCH2), 7.07 (d, J = 7.7 Hz, 1H, Ph-H4’), 7.25 (t, J = 8.0 Hz, 1H,
Ph-H5’), 7.55 (d, J = 8.1 Hz, 1H, Ph-H6’), 7.89 (s, 1H, CH), 8.16 (s, 1H, Ph-H2’), 9.14 (s, 1H,
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2
2-NH), 11.19 (s, 1H, NHCOCF3 ), 12.65 (s, 1H, 9-NH); 13C NMR (100 MHz, DMSO-d6) δ: 45.8,
66.7, 112.0, 114.1, 114.6, 116.4, 129.1, 136.6, 136.8, 142.5, 153.2, 153.7, 155.6; HRMS (ESI):
calcd for C17H16F3N7O2 [M+H]+: 408.1396, found: 408.1387. IR (KBr pellet, cm-1): 3286, 2864,
1680, 1606, 1551, 1448, 1407, 1371, 1308, 1285, 1202, 1023, 939, 880, 784, 723, 686, 610, 524.
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4.1.3.9. N-(3-chlorophenyl)-6-morpholino-9H-purin-2-amine (3j)
1
7
White solid, yield 89.5%. m.p. 216-217 ºC; H NMR (400 MHz, DMSO-d6) δ: 3.73 (s, 4H,
8
CH2NCH2) , 4.21 (s, 4H, CH2OCH2) , 6.88 (d, J = 7.7 Hz, 1H, Ph-H4’), 7.24 (t, J = 8.0 Hz, 1H,
Ph-H5’), 7.60 (d, J = 8.2 Hz, 1H, Ph-H6’), 7.89 (s, 1H, CH), 8.06 (s, 1H, Ph-H2’) , 9.17 (s, 1H,
2-NH), 12.69 (s, 1H, 9-NH); 13C NMR (100 MHz, DMSO-d6) δ: 45.7, 66.7, 115.0, 116.9, 117.6,
120.0, 130.3, 133.3, 136.8, 143.7, 153.2, 153.7, 155.6; HRMS (ESI): calcd for C15H15ClN6O
[M+H]+: 331.1074, found: 331.1072. IR (KBr pellet, cm-1): 3404, 3139, 3004, 2859, 1618, 1591,
1520, 1482, 1456, 1438, 1392, 1372, 1313, 1291, 1264, 1218, 1152, 1109, 1072, 1019, 937, 874,
782, 773, 729, 689, 631, 579, 554, 528.
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4.1.3.10. N-(3-fluorophenyl)-6-morpholino-9H-purin-2-amine (3k)
1
White solid, yield 79.0%. m.p. 210-211 ºC; H NMR (400 MHz, DMSO-d6) δ: 3.73 (s, 4H,
CH2NCH2) , 4.21 (s, 4H, CH2OCH2) , 6.64 (t, J = 8.3 Hz, 1H, Ph-H4’), 7.25 (m, 1H, Ph-H5’),
7.44 (d, J = 8.1 Hz, 1H, Ph-H6’), 7.86 (s, 1H, Ph-H2’), 7.89 (s, 1H, CH), 9.18 (s, 1H, 2-NH),
12.67 (s, 1H, 9-NH); 13C NMR (100 MHz, DMSO-d6) δ: 45.6, 66.7, 104.7, 104.9, 106.5, 106.7,
114.3, 114.9, 130.1, 130.2, 136.8, 143.9, 144.0, 153.2, 153.8, 155.7, 161.7, 164.1; HRMS (ESI):
calcd for C15H15FN6O [M+H]+: 315.1370, found: 315.1372. IR (KBr pellet, cm-1): 3289, 3210,
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ACCEPTED MANUSCRIPT
2860, 1598, 1581, 1531, 1490, 1447, 1392, 1315, 1292, 1268, 1140, 1112, 1024, 939, 866, 785,
770, 634, 531.
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4.1.3.11. N-(4-fluorophenyl)-6-morpholino-9H-purin-2-amine (3l)
1
5
White solid, yield 85.2%. m.p. 264-265 ºC; H NMR (400 MHz, DMSO-d6) δ: 3.72 (s, 4H,
6
CH2NCH2), 4.19 (s, 4H, CH2OCH2), 7.08 (t, J = 8.6 Hz, 2H, Ph-H3’, Ph-H5’), 7.71-7.80 (m, 2H,
Ph-H2’, Ph-H6’), 7.85 (s, 1H, CH), 8.94 (s, 1H, 2-NH), 12.59 (s, 1H, 9-NH); 13C NMR (100 MHz,
DMSO-d6) δ: 45.7, 66.7, 114.7, 115.1, 115.3, 120.1, 120.1, 136.5, 138.5, 153.6, 153.8, 155.7,
156.0, 158.1; HRMS (ESI): calcd for C15H15FN6O [M+H]+: 315.1370, found: 315.1370. IR (KBr
pellet, cm-1): 3303, 2857, 1603, 1580, 1529, 1509, 1473, 1442, 1389, 1317, 1287, 1221, 1154,
1115, 1019, 936, 884, 832, 817, 796, 685, 655, 627, 544, 532.
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4.1.3.12. 6-morpholino-N-(pyridin-3-yl)-9H-purin-2-amine (3m)
Yellow solid, yield 38.5 %. m.p. 283-284 ºC; 1H NMR (400 MHz, DMSO-d6) δ: 3.71-3.76 (m, 4H,
CH2NCH2) , 4.10-4.30 (m, 4H, CH2OCH2), 7.27 (dd, J = 8.3, 4.6 Hz, 1H, Ph-H6’), 7.89 (s, 1H,
CH), 8.07 (d, J = 4.6, 2.3 Hz, 1H, Ph-H5’), 8.20 (d, J = 8.4 Hz, 1H, Ph-H4’), 8.91 (d, J = 2.4 Hz,
1H, Ph-H2’), 9.13 (s, 1H, 2-NH), 12.65 (s, 1H, 9-NH); 13C NMR (100 MHz, DMSO-d6) δ: 45.7,
66.7, 115.0, 123.6, 124.9, 136.8, 138.7, 140.7, 141.5, 153.3, 153.8, 155.8; HRMS (ESI): calcd for
C14H15N7O [M+H]+: 298.1416, found: 298.1414. IR (KBr pellet, cm-1): 3371, 2965, 2907, 2865,
2794, 2555, 1593, 1548, 1531, 1503, 1486, 1437, 1423, 1385, 1331, 1282, 1233, 1223, 1192, 1134,
1110, 1081, 1043, 1018, 932, 891, 800, 785, 700, 644, 635, 534, 518.
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ACCEPTED MANUSCRIPT
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4.1.3.13. 5-((6-morpholino-9H-purin-2-yl)amino)picolinamide (3n)
1
White solid, yield 20.8%. m.p. > 300 ºC; H NMR (400 MHz, DMSO-d6) δ: 3.75 (m, 4H,
3
CH2NCH2), 4.22 (s, 4H, CH2OCH2), 7.38 (s, 1H, CONH), 7.89 (s, 1H, CH), 7.94 (bs, 2H, CONH,
Py-H6’), 8.40 (bs, 1H, Py-H5’), 8.93 (bs, 1H, Py-H2’), 9.51 (s, 1H, 2-NH), 12.74 (s, 1H, 9-NH);
13C NMR (100 MHz, DMSO-d6) δ: 45.7, 66.7, 115.2, 122.6, 124.4, 137.1, 138.6, 141.1, 142.1,
153.1, 153.8, 155.3, 166.7; HRMS (ESI-): calcd for C15H16N8O2 [M-H]: 341.1474, found:
341.1473. IR (KBr pellet, cm-1): 3284, 2858, 1721, 1590, 1532, 1488, 1438, 1371, 1282, 1230,
1118, 1011, 890, 857, 789, 703, 658, 567.
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4.1.3.14. 6-morpholino-N-(pyrazin-2-yl)-9H-purin-2-amine (3o)
1
White solid, yield 16.4%.m.p. > 300 ºC; H NMR (400 MHz, DMSO-d6) δ: 3.74 (s, 4H,
CH2NCH2), 4.22 (s, 4H, CH2OCH2), 7.94 (s, 1H, CH), 8.13 (s, 1H), 8.26 (s, 1H), 9.40 (s, 1H),
9.56 (s, 1H, 2-NH), 12.75 (s, 1H, 9-NH); 13C NMR (100 MHz, DMSO-d6) δ: 45.8, 66.7, 110.0,
115.4, 136.0, 136.8, 137.3, 142.6, 151.0, 153.1, 154.3; HRMS (ESI): calcd for C13H14N8O [M+H]+:
299.1369, found: 299.1371. IR (KBr pellet, cm-1): 3422, 3202, 3120, 2921, 2855, 1621, 1585,
1543, 1481, 1420, 1388, 1302, 1283, 1264, 1158, 1145, 1116, 1070, 1025, 1010, 939, 823, 787,
743, 634, 529.
4.1.3.15. N-(6-morpholino-9H-purin-2-yl)quinolin-3-amine (3p)
White solid, yield 49.2%. m.p. 246-247 ºC; 1H NMR (400 MHz, DMSO-d6) δ: 3.73-3.79 (m, 4H,
CH2NCH2), 4.25 (s, 4H, CH2OCH2), 7.50-7.55 (m, 2H), 7.78 (dt, J = 6.1, 2.6 Hz, 1H), 7.88-7.92
(m, 1H), 7.93 (s, 1H, CH), 8.80 (d, J = 2.2 Hz, 1H), 9.07 (d, J = 2.5 Hz, 1H), 9.48 (s, 1H, 2-NH),
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