Synthesis of novel strobilurin-pyrimidine derivatives and their antiproliferative activity against human cancer cell lines

Article abstract of DOI:10.1016/j.bmcl.2013.04.045

A series of new strobilurin-pyrimidine analogs were designed and synthesized based on the structures of our previously discovered antiproliferative compounds I and II. Biological evaluation with two human cancer cell lines (A549 and HL60) showed that most of these compounds possessed moderate to potent antiproliferative activity. Two potent candidates (8f, IC₅₀ = 2.2 nM and 11d, IC₅₀ = 3.4 nM) were identified with nanomolar activity against leukemia cancer cell line HL60 for further development. This activity represents a 1000- to 2500-fold improvement compared to the parent compounds I and II and is 20- to 30-fold better than the chemotherapy drug, doxorubicin. The present work provides strong incentive for further development of these strobilurin-pyrimidine analogs as potential antitumor agents for the treatment of leukemia.

Full text of DOI:10.1016/j.bmcl.2013.04.045

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3505–3510
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel strobilurin–pyrimidine derivatives
and their antiproliferative activity against human cancer cell lines
Baoshan Chai a,b, Shuyang Wang a, Wenquan Yu a, Huichao Li b, Chuanjun Song a, Ying Xu b,
Changling Liu b, , Junbiao Chang a,
⇑
⇑
a College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou, Henan Province 450001, PR China
b State Key Laboratory of the Discovery and Development of Novel Pesticide, Shenyang Research Institute of Chemical Industry Co. Ltd, Shenyang, Liaoning Province 110021, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new strobilurin–pyrimidine analogs were designed and synthesized based on the structures of
our previously discovered antiproliferative compounds I and II. Biological evaluation with two human
cancer cell lines (A549 and HL60) showed that most of these compounds possessed moderate to potent
antiproliferative activity. Two potent candidates (8f, IC50 = 2.2 nM and 11d, IC50 = 3.4 nM) were identified
with nanomolar activity against leukemia cancer cell line HL60 for further development. This activity
represents a 1000- to 2500-fold improvement compared to the parent compounds I and II and is
20- to 30-fold better than the chemotherapy drug, doxorubicin. The present work provides strong incen-
tive for further development of these strobilurin–pyrimidine analogs as potential antitumor agents for
the treatment of leukemia.
Received 8 November 2012
Revised 10 April 2013
Accepted 18 April 2013
Available online 26 April 2013
Keywords:
Strobilurin–pyrimidine
Antiproliferative activity
Lung cancer
Ó 2013 Elsevier Ltd. All rights reserved.
Leukemia
Structure–activity relationship
Strobilurins, isolated from specific fungi, constitute a large
family of compounds that possess a broad spectrum of fungicidal
activity with low toxicity towards mammalian cells and environ-
mentally benign characteristics.1,2 Strobilurin derivatives are also
used as agrochemical,1–6 antiviral,7 anti-malarial,8 and anti-micro-
bial agents.9 In particular, the strobilurin–pyrimidine moiety has
been extensively utilized as a drug scaffold in medicinal chemistry,
and at present, three commercialized fungicide products, including
azoxystrobin, fluoxastrobin, and fluacrypyrim, incorporate this
substructure (Fig. 1).1,2,5,6 In addition, it has been found that stro-
bilurin–pyrimidine analogs have antitumor activity through inhi-
bition of STAT3 activation.10 For example, fluacrypyrim ( Fig. 1)
inhibited leukemia cancer cell growth by predominantly G1 arrest
with significant decreases of cyclin D1 protein and mRNA levels.10
Our research group has been investigating the potential of stro-
bilurin–pyrimidine derivatives for antitumor applications. Previ-
ously, we discovered two analogs I and II (Fig. 2), which possess
Our structural exploration strategy for the hit compounds (I and
II) is illustrated in Figure 3. For compound I, first, the 5-n-butyl
group was fused with the 6-methyl group on the pyrimidine sub-
structure to form a 5- or six-member carbocycle (8a, 8e) to restrict
the possible conformations. Second, a series of halogens and/or al-
kyl groups (8b–d, 8f–g) was introduced to the 2-phenylamino moi-
ety to study substituent effects on SAR. Similarly, for compound II,
the phenylamino group was replaced with substituted anilines
(9a–f) or aliphatic amines (10a–g). Next, incorporation of a methyl
group at the 5-position of the pyrimidine gave the analog 11 series.
Furthermore, replacement of the toxophore (b-methoxyacrylate,
Q1) moiety with groups Q2–Q5 afforded derivatives 12–15.
Key building blocks for the construction of strobilurin–pyrimi-
dine analogs 8–15 were the 2-aminopyrimidin-4-ols 4–7, which
were generally synthesized via condensation of guanidines with
the corresponding b-keto esters (Scheme 1). Guanidines 1 and 2
were readily prepared according to our previously reported meth-
ods.14,15 Most of the b-keto esters were purchased from commercial
sources, and intermediate 3 was prepared by methylation of ethyl
4,4,4-trifluoro-3-oxobutanoate with methyl bromide.16 Benzyl ha-
lides (III–V, Schemes 2 and 3) were prepared according to literature
procedures.17–20 Nucleophilic substitution of the benzyl halides
with phenols 4–7 in the presence of potassium carbonate as base
afforded the target molecules 8–13 in 65–85% yields (Schemes 2
and 3). The structure of compound 9b was further confirmed by
X-ray crystallography (see ‘Supplementary data’). Treatment of
good antiproliferative activity against lung cancer (I, IC50 = 3.4
II, IC50 = 3.0 M, A549) and leukemia (I, IC50 = 5.5 M, II,
IC50 = 3.3
lM,
l
l
l
M, HL60) cell lines.11 As a continuation of our previous
work,11–15 herein we report a follow-up lead optimization to im-
prove the potency, and also structure–activity relationship (SAR)
investigation results.
⇑
Corresponding authors.
E-mail addresses: liuchangling@sinochem.com (C. Liu), changjunbiao@zzu.e-
du.cn (J. Chang).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.045

3506
B. Chai et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3505–3510
Figure 1. Structure of representative strobilurin–pyrimidine scaffolds.
50% inhibition of tumor growth) of tested compounds are summa-
rized in Tables 1 and 2.
Fusion of the 5-n-butyl and 6-methyl groups on the pyrimidine
substructure as a 6-member ring (8a) did not improve the antipro-
liferative activity. However, elimination of one ring carbon gave
the cyclopenta[d]pyrimidine analog (8e), which showed equally
good or better activity against both cancer cell lines compared to
compound I. Thus, building on the structure of 8e, introduction
of halogens (8f–g) to the aniline moiety was probed to improve po-
tency. For example, the 2,4-dichloro-3-methyl analog (8g,
IC50 = 0.2
l
M) led to a 17-fold improvement of activity against
M). A break-
the human lung cancer cell line A549 (vs I, IC50 = 3.4
l
Figure 2. Structures of antiproliferative strobilurin–pyrimidine derivatives I and II.
through was the 2,3,4-trifluoro analog (8f), which possessed an
compounds 12 and 13 with methylamine gave derivatives 14 and
15, respectively, in good yields (Scheme 3).
IC50 of 2.2 nM against leukemia cancer cell line HL60. This activity
represents an increased potency of 2500-fold better than
I
The in vitro antiproliferative activity of strobilurin–pyrimidine
analogs against two human cancer cell lines (A549 and HL60)
was evaluated using MTT or SRB assays according to Mosmann’s
(IC50 = 5.5
(IC50 = 0.082
l
M)
and
a
37-fold
greater
than
doxorubicin
lM),
chemotherapy drug with broad-spectrum
antitumor activity. Overall, in this series, substituent effects for
the 5- and 6-positions of the pyrimidine ring could be ranked as
methods.21 IC50
(l
M) values (concentration required to achieve
NH
O
NH
O
2
N
N
O
O
O
1
cyclization
R1
N
N
O
O
3
NH
O
O
6
O
4
5
N
N
O
O
8a
O
I
n
n=1,2
8
NH
O
N
N
O
NH
O
O
N
N
O
O
8e
F3C
O
II
Q1
R1
R1
R1
NH
O
NH
NH
O
N
N
O
O
N
N
Q
N
N
O
O
F3C
O
F3C
O
F3C
O
9
CH3
CH3
11
12-15
R2
R3
N
O
O
O
O
Q=
N
Q2=
N
O
Q4=
N
N
O
O
O
N
O
O
H
F3C
O
O
10
O
Q3=
Q5=
N
N
N
O
H
Figure 3. Structure exploration strategy based on compounds I and II.

B. Chai et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3505–3510
3507
O
O
O
n=1,2
n=1,2
b
N
R1
N
H
4
N
OH
H
N
NH2
NH
NH2
a
R1
R1
CF3
N
1/2 H2CO3
1
b
N
R1
N
OH
O
O
H
5
F3C
O
R2
R3
NH2
NH
S
R2
c
N
NH
R3
CF3
N
H2N
NH
+
d
N
1/2 H2SO4
1/2 H2SO4
R2
2
N
R3
OH
O
O
6
F3C
O
H
N
NH2
NH
R1
CF3
1/2 H2CO3
b
N
1
R1
N
H
N
OH
O
O
O
O
O
e
CH3Br
+
F3C
O
7
F3C
3
Scheme 1. Synthesis of 2-aminopyrimidin-4-ols 4–7. Reagents and conditions: (a) NH2CN, concd. HCl, Na2CO3; (b) toluene, reflux; (c) H2O; (d) NaOCH3, CH3OH; (e) NaH,
toluene.
OH
n
N
R1
R1
R1
N
H
4
N
n=1,2
NH
NH
O
O
N
N
O
O
O
N
N
O
O
OH
O
O
O
F3C
O
N
R2
n
Cl
N
R3
N
CF3
9
8
n=1,2
III
5
a
OH
R2
N
R3
R1
N
O
N
R1
NH
O
N
O
O
N
H
N
CF3
N
N
O
O
F3C
O
6
F3C
O
11
OH
10
N
R1
N
N
CF3
H
7
Scheme 2. Synthesis of strobilurin–pyrimidines 8–11. Reagents and conditions: (a) K2CO3, DMF, 80 °C.

3508
B. Chai et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3505–3510
O
R1
O
N
R1
O
NH
O
NH
O
Br
N
N
b
N
N
O
O
N
N
O
N
H
IV
F3C
O
F3C
O
a
CF3
N
12
14
O
N
R1
O
N
O
V
R1
N
H
OH
R1
NH
O
NH
Br
O
7
O
O
b
N
N
N
O
N
N
N
N
H
a
F3C
O
F3C
O
13
Scheme 3. Synthesis of strobilurin–pyrimidines 12–15. Reagents and conditions: (a) K2CO3, DMF, 80 °C; (b) NH2CH3, CH3OH.
15
Table 1
Structure–activity relationship study of compounds 8–11 in A549 and HL60 cell lines
R1
R1
R1
R2
N
R3
N
NH
NH
N
NH
O
O
O
O
N
N
N
N
O
O
O
O
N
N
O
O
O
O
O
F3C
O
F3C
O
F3C
O
n
11
IC50
10
9
8
Compounds
R1
n
R2
R3
(lM)
A549a
HL60b
I
8a
8b
8c
8d
8e
8f
8g
—
H
—
2
2
2
2
1
1
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
3.4
9.5
16.4
1.1
25.6
3.1
1.0
5.5
11.8
1.1
8.2
2.3
1.9
0.0022
7.7
3.3
2,3-Di-Cl
2,4-Di-F
2,3,4-Tri-F
H
2,3,4-Tri-F
2,4-Di-Cl-3-Me
0.2
3.0
II
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
9a
9b
9c
9d
9e
9f
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
4-Cl
2,4-Di-Cl
2-F, 4-Cl
2,4-Di-F
2,6-Di-F
2,3,4-Tri-F
—
—
—
—
—
—
—
H
4-Cl
—
—
—
—
—
—
Me
Et
Me
Et
iPr
—
—
—
—
—
—
H
1.3
>100
1.3
1.0
41.4
1.0
93.7
3.2
6.0
17.9
9.8
10
19.0
>100
2.7
4.8
12.1
13.6
76.7
0.3
11.1
15.0
3.4
6.5
51.6
0.7
0.7
2.9
H
Me
Et
H
Cyclopropyl
–(CH2)5–
H
4.9
7.4
5.5
0.4
3.1
5.0
9.8
0.5
—
—
—
—
—
—
—
—
—
—
—
—
—
—
2,4-Di-Cl
2,4-Di-F
2,3,4-Tri-F
11d
11e
Fluacrypyrim
Doxorubicin
0.0034
0.8
6.9
0.082
a
IC50s were measured using SRB assay.
IC50s were measured using MTT assay.
b
follows: 5-cyclic fused pyrimidine (8e–g) >5-n-butyl-6-methyl
pyrimidine (I) >6-cyclic fused pyrimidine (8a–d).
According to the encouraging SAR results from the derivative I
series, halogens were introduced into the aniline substructure of
compound II to give analogs 9. Most of these derivatives (9a, 9c–
d and 9f) exhibited slightly improved activity against the human
lung cancer cell line A549 compared to compound II. It is worth
noting that introduction of chlorine (9b) to the 2-position instead

B. Chai et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3505–3510
3509
Table 2
Structure–activity relationship study of compounds 12–15 in A549 and HL60 cell lines
R1
R1
R1
R1
NH
O
NH
NH
O
NH
O
O
N
O
N
O
N
N
O
O
N
N
N
N
N
O
N
N
O
N
H
N
N
H
F3C
O
F3C
O
F3C
O
F3C
O
15
M)
14
12
13
Compounds
R1
IC50 (l
A549a
HL60b
II
—
3.0
83.5
82.1
>100
26.9
>100
>100
>100
>100
3.3
48.1
50.5
68.1
66.4
25.3
>100
>100
>100
12a
12b
13a
13b
14a
14b
15a
15b
2,4-Di-F
2,3,4-Tri-F
2,4-Di-F
2,3,4-Tri-F
2,4-Di-F
2,3,4-Tri-F
2,4-Di-F
2,3,4-Tri-F
a
IC50s were measured using SRB assay.
IC50s were measured using MTT assay.
b
SMMC7721 and breast cancer cell line MCF-7. As shown in
Table 3, analog 8f with high potency against leukemia cancer cell
line HL60 displayed good selectivity over the other four cell lines
(A549, EC9706, SMMC7721 and MCF-7). Analog 11c exhibited both
good activity and selectivity against lung (A549) and esophagus
(EC9706) cancer cell lines over cell lines HL60, SMMC7721 and
MCF-7.
In this Letter, in order to develop effective chemotherapeutic
anticancer drug candidates, we designed and synthesized a series
of new strobilurin–pyrimidine derivatives (8–15) based on our
previously discovered antiproliferative compounds I and II. Their
biological profiles were evaluated with two human tumor cell lines
(A549 and HL60), using the SRB and MTT assays, respectively, and
most of the analogs possessed moderate to potent antiproliferative
activity. The SAR investigation indicated that compounds contain-
ing a 5-cyclic fused pyrimidine, fluorinated aniline, and/or 5-
methyl-6-trifluoromethyl-pyrimidine substructures possessed
good activity. Meanwhile, two potent candidates (8f, IC50 = 2.2 nM
and 11d, IC50 = 3.4 nM) were identified with nanomolar antiprolif-
erative activity against leukemia cancer cell line HL60, which rep-
resents a 1000- to 2500-fold improvement in activity compared to
Table 3
The antiproliferative activity of compounds 8f and 11c against various human cancer
cell lines
Compounds
IC50 (lM)
EC9706b
A549a
HL60b
SMMC7721b
MCF-7b
8f
11c
1.0
0.4
0.0022
2.9
8.4
0.44
1.9
4.6
24.2
14.2
a
IC50s were measured using SRB assay.
IC50s were measured using MTT assay.
b
of hydrogen (9a) or fluorine (9c), completely eliminated antiprolif-
erative activity (IC50 >100 M, A549; IC50 >100 M, HL60), which
indicated that this position would not tolerate large substituents.
Next, the entire aniline moiety was replaced with alkylamino
groups (10). In this series, only the ethylamino analog 10b
l
l
(IC50 = 0.3
l
M) showed 11-fold improvement in activity against
the leukemia cancer cell line HL60 (vs II, IC50 = 3.3
lM). Further-
more, 5-methylation of the pyrimidine ring (11) significantly
improved antiproliferative activity (vs 9 series). In particular, the
IC50 of compound 11d (3.4 nM) was a 1000- to 1400-fold improve-
ment compared to the parent analogs (II, IC50 = 3.3
IC50 = 4.8 M) and was 24-fold better than doxorubicin
(IC50 = 0.082 M) against the leukemia cancer cell line HL60. Even
the parent compounds I (IC50 = 5.5
20- to 30-fold greater potency than the chemotherapy drug doxo-
rubicin (IC50 = 0.082 M). Further evaluation indicated that ana-
lM) and II (IC50 = 3.3 lM), and
l
lM and 9d,
logs 8f and 11c possess good selectivity against HL60 and A549/
EC9706 cell lines, respectively. This study provides strong impetus
to further develop these strobilurin–pyrimidine analogs as poten-
tial antitumor agents for the treatment of leukemia.
l
l
for the inactive compound 9b, 5-methylation (11c) also greatly
improved the antiproliferative activity against both cell lines
(A549, IC50 = 0.4 lM; HL60, IC50 = 2.9 lM). Thus, in the II series, a
Acknowledgments
5-methyl substitution of the pyrimidine ring was optimal.
Generally, most of the compounds with a b-methoxyacrylate
(Q1) group (9–11) showed moderate to potent antiproliferative
activity. However, further replacement of this toxophoric moiety
with Q2–Q5 groups (12–15) greatly reduced compound potency
against both cancer cell lines. This result demonstrated that the
b-methoxyacrylate (Q1) group is both necessary and an optimal
substructure for maintaining compounds’ activity.
To further investigate the antiproliferative activity, compounds
8f and 11c were selected as representatives of two structural clas-
ses (I and II) and tested in more human cancer cells commonly
used: esophagus cancer cell line EC9706, liver cancer cell line
We are grateful to the Outstanding Young Scholarship from the
National Natural Science Foundation of China (NSFC, J.C.
30825043), NSFC (J.C. 21172202), and the Outstanding Scholar
Foundation of Henan Province (#094100510019) for financial
support. This project was also supported by the National Basic Re-
search Program of China (973 Program: 2010CB126105, 2010CB7
35601 and 2012CB724501) and the National Key Technologies
R&D Program (Nos. 2011BAE06B00, 2011BAE06B01, 2011BAE06
B02, 2011BAE06B03 and 2011BAE06B05). We thank Dr. John E.
Reiner for critical reading of the manuscript.

3510
B. Chai et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3505–3510
8. Alzeer, J.; Chollet, J.; Heinze-Krauss, I.; Hubschwerlen, C.; Matile, H.; Ridley, R.
Supplementary data
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CCDC 893933 contains the supplementary crystallographic data
of compound 9b. These data can be obtained free of charge at
www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge
Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge
CB2 1EZ, UK (Fax: +44 1223 336033; email: deposit@ccdc.cam.a-
c.uk or http://www.ccdc.cam.ac.uk).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.04. 045.
14. Chai, B. S.; Liu, C. L.; Li, H. C.; He, X. M.; Luo, Y. M.; Huang, G.; Zhang, H.; Chang,
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