First synthesis of novel 3,3′-bipyridazine derivatives as new potent antihepatocellular carcinoma agents

Article abstract of DOI:10.1016/j.tet.2015.07.063

Hepatocellular carcinoma is one of the most common kind of cancers in clinical, and its clinical treatment is quite difficult. The latest research suggests that pyridazinone with a novel molecular skeleton shows excellent activity against hepatocellular c

Full text of DOI:10.1016/j.tet.2015.07.063

Tetrahedron 71 (2015) 7670e7675
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First synthesis of novel 3,30-bipyridazine derivatives as new potent
antihepatocellular carcinoma agents
,
a,
Qiming Yue a, Yi Zhao a, Li Hai a, Tao Zhang b, Li Guo a , Yong Wu
*
*
a Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Chengdu 610041, PR China
b ChengDu Military General Hospital, Chengdu 610083, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 June 2015
Received in revised form 20 July 2015
Accepted 25 July 2015
Available online 30 July 2015
Hepatocellular carcinoma is one of the most common kind of cancers in clinical, and its clinical treatment
is quite difficult. The latest research suggests that pyridazinone with a novel molecular skeleton shows
excellent activity against hepatocellular carcinoma in vitro and in vivo. Considering YHHU-759 as the
leading compound for reasonable structure modification, a series of 3,30-bipyridazine derivatives in-
cluding 8 novel compounds in this study were designed, synthesized and screened for their anti-hep-
atoma activities against liver cancer cell lines SMMC-7721, BEL-7402 and QGY-7703 in vitro. The result
shows that all of the 3,30-bipyridazine derivatives have good anti-hepatoma activities.
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
3,30-Bipyridazine derivatives
anti-Hepatoma
Synthesis
Activity
1. Introduction
of 3,30-bipyridazine derivatives including eight new compounds in
this study (Fig.1) were designed, synthesized and screened for their
Human hepatocellular carcinoma (HCC) is one of the most
common kind of cancers in clinical, and its clinical treatment is
quite difficult.1,2 In recent years, liver cancer incidence and mor-
tality rates are on the rise worldwide, ranked fifth of the most
common human cancer in solid tumors.3e6 One reason for the high
mortality rate is that effective drugs are limited for the treatment of
unresectable HCC.7,8 Given that there is no effective chemotherapy
for HCC, the development of new liver-cancer-specific drugs for the
treatment of HCC will be of great significance.
anti-hepatoma activities against liver cancer cell lines SMMC-7721,
BEL-7402 and QGY-7703 in vitro. We hope that this sort of 3,30-
bipyridazine derivatives can satisfy anti-hepatoma drugs.
2. Results and discussion
2.1. Chemistry
To the best of our knowledge, only few papers have reported the
synthesis of 3,30-bipyridazine derivatives.11e13 But the anti-
hepatocellular carcinoma activities of them have not been reported
by any literature up to the date. In this study, a new approach to
In recent years, Hu et al.9,10 have reported a range of novel
pyridazinone derivatives with excellent anti-hepatoma activity,
among which compound YHHU-759 (Fig. 1) has been proved with
superior anti-hepatoma activity to that of multikinase inhibitor
Sorafenib which is approved by the FDA and the EU for hepato-
cellular carcinoma treatment. But the metabolism time of YHHU-
759 is relatively short, which will greatly reduce the druggability.
And the structural modification in previous studies by Hu is limited
to the side chain modification, which may lead to the loss of po-
tentially better compounds.
Therefore, we considered YHHU-759 as the lead compound for
our subsequent structure modifications, in hopes of finding a po-
tential drug candidate with better anti-hepatoma activity. A series
* Corresponding authors. Tel.: þ86 028 85503777 (L.G.); tel.: þ86 028 85503235
(Y.W.); e-mail addresses: guoli@scu.edu.cn (L. Guo), wyong@scu.edu.cn (Y. Wu).
Fig. 1. Compound YHHU-759 and 3,30-bipyridazine derivatives.
http://dx.doi.org/10.1016/j.tet.2015.07.063
0040-4020/Ó 2015 Elsevier Ltd. All rights reserved.

Q. Yue et al. / Tetrahedron 71 (2015) 7670e7675
7671
synthesize derivative 11a was designed and presented (Scheme 1).
The approach began with changing the commercially available 3,6-
dichloropyridazine 1 into 3,6-diiodopyridazine 2, using NaI and HI
under 80 ꢀC.14 The intermediate compound 3 was resulted from
a mono nucleophilic substitution of one iodine atom of compound
2 with sodium methylate.15 Starting from compound 3 and using
Sonogashira coupling reaction, the alkynylpyridazine 4 was ob-
tained.16 In this case, the deprotection of the trimethylsilyl group
could be easily carried out with TBAF in THF, leading to ethy-
nylpyridazine 5, which was then treated with H2SO4 and HgSO4 at
80 ꢀC for 2 h to give 6 (50%) as a pale yellow solid.17 Compound 7
could be obtained by treating 6 with ethyl trifluoropyruvate under
Compound 10a was obtained by treating 9 with morpholine in
dioxane. Because of the electron withdrawing effect tri-
fluoromethyl, it tended to undergo a mono nucleophilic sub-
stitution at the ortho-position of trifluoromethyl rather than the
other. It is worth noting that, at the beginning of this study, com-
pound 9 was directly reacted with morpholine in DMF at 80 ꢀC
(Table 1, entry 1).23 However, it was found the main product was
compound 12 (95% yield) rather than 10a. Lowering the reaction
temperature increased the yield of the desired product 10a to 73%
(Table 1, entry 4). Among various solvents examined, dioxane
turned out to be the best choice, while others such as DMF, EtOH,
THF, acetone, CH2Cl2, ethyl acetate, CH3CN, DMSO and toluene were
less effective (Table 1, entries 6e14). In terms of the molar pro-
portion of the reactants, a 1:5 M ratio of 10a and morpholine was
found to be adequate, as neither bigger nor smaller proportions
showed better yields (Table 1, entries 14e19). In summary, as ob-
served in this study, the optimized reaction conditions tend to be:
compound 10a (2.0 mmol), morpholine (10 mmol) in dioxane at
15 ꢀC. In the last step, the chlorine atom of 10a was substituted with
different catalysts, such as L
-pro,18,19 ZnCl2 and other Lewis acids.20
Following previous studies,18e20 using ZnCl2 as the catalyst, com-
pound 6 was smoothly transformed into compound 7 with a high
yield. It then subsequently underwent the ring closing reaction21
using hydrazine hydrate to afford the desired compound 8 with
acceptable to good yield. Then, by treating compound 8 with
POCl3,22 dichlorobipyridazine 9 was obtained.
Scheme 1. Preparation of derivative 11a. Reagents and Conditions: (a) NaI, HI, acetone, 80 ꢀC, 1 h. Yield: 99%. (b) CH3ONa, CH3OH, H2O, 80 ꢀC, 1 h. Yield: 97%. (c) Pd(PPh3)2Cl2,
(CH3)3SiCCH, CuI, Et3N, THF, 0 ꢀC-rt, 2 h. Yield: 98%. (d) TBAF, THF, rt, 0.5 h. Yield: 80%. (e) H2SO4, HgSO4, 80 ꢀC, 2 h. Yield: 50%. (f) ethyl trifluoropyruvate, ZnCl2, DMF,110 ꢀC, 25 h. Yield:
76%. (g) hydrazine hydrate, acetic acid, 100 ꢀC, 7 h. Yield: 53%. (h) POCl3, 80 ꢀC, 3 h. Yield: 84%. (i) morpholine, dioxane, 15 ꢀC, 10 h. Yield: 89%. (j) acetic acid, 100 ꢀC, 2 h. Yield: 91%.
Table 1
Screening of the reaction conditionsa
Entry
9: Morpholine
Solvent
Temp (ꢀC)
Time (h)
Yieldb (%) 10a
Yieldb (%) 12
1
2
3
4
5
6
7
8
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:1
1:2
1:4
1:7.5
1:10
DMF
DMF
DMF
DMF
DMF
EtOH
THF
Acetone
CH2Cl2
Ethyl acetate
CH3CN
DMSO
Toluene
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
80
50
30
15
0
0.5
3
8
0
15
34
73
59
75
61
69
58
56
63
71
49
89
64
70
79
73
65
95
79
51
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6
10
24
10
10
10
10
10
10
10
10
10
10
10
10
10
10
15
15
15
15
15
15
15
15
15
15
15
15
15
15
9
10
11
12
13
14
15
16
17
18
19
18
a
Unless noted otherwise, reactions were performed with 9 (2.0 mmol) and morpholine in solvent (10 mL).
Isolated yield. Compounds 10a and 12 had been separated by chromatography.
b

7672
Q. Yue et al. / Tetrahedron 71 (2015) 7670e7675
hydroxy in acetic acid at 100 ꢀC to give 11a with a high yield
(91%).24e26
On the basis of Scheme 2, 8 novel 3,30-bipyridazine derivatives
were synthesized. And the structures of these new compounds
were characterized by MS and NMR.
Fig. 2. Pharmacophore models of compounds YHHU-759 and 11a (Pharmacophore
features were color-coded with red spheres for hydrogen-bond acceptor (HBA), blue
spheres for hydrogen-bond donor (HBD), yellow spheres for repellence and brown
rings for aromatic ring center).
Scheme 2. Preparation of the 3,30-bipyridazine derivatives. Reagents and conditions:
(a) RH, dioxane, 15 ꢀC, 10 h. (b) acetic acid, 100 ꢀC, 2 h.
2.2. anti-Hepatoma activity
The anticancer activities of these 3,30-bipyridazine derivatives
were also examined. Three liver cancer cell lines, SMMC-7721, BEL-
7402 and QGY-7703 were used. Data suggests that these derivatives
have good anticancer activities. The results were summarized in
Table 2.
contrast, the inactive compound 12 might be a result of not having
the feature of hydrogen-bond donor. This in turn suggested that the
hydrogen-bond donor feature might be critical for the biological
activity.
3. Conclusion
Table 2
The GI50 of 3,30-bipyridazine derivativesa
In this study, we have designed and prepared a range of new
3,30-bipyridazine derivatives 11aeh as the new anti-hepatoma
drugs. These compounds were subsequently evaluated for their
anti-hepatoma activities against liver cancer cell lines SMMC-7721,
BEL-7402 and QGY-7703. Our preliminary data showed that all of
the 3,30-bipyridazine derivatives have good anti-hepatoma activi-
ties. The results are very promising and more work is in progress,
including the development of better animal models for compre-
hensive in vivo evaluations.
Compound
GI50 (nM)b
SMMC-7721
BEL-7402
QGY-7703
12
11a
11b
11c
11d
11e
11f
11g
N/Ac
123.2
59.2
252.6
3648
22.9
1,538,000
285.1
101.5
825.7
1319
N/A
177.9
98.6
516.8
1352
42.0
56.7
88.1
166.0
487.5
516.3
469.1
1346
83.3
178.7
195.4
28.7
128.1
347.4
30.2
11h
YHHU-759
Sorafenib
4. Experimental section
4.1. General
6512
8451
a
SMMC-7721, BEL-7402 and QGY-7703 cells were incubated at 37 ꢀC/5% CO2 for
72 h with test compounds.
b
Growth inhibition rate (GI)¼(1ꢁOD sample/OD negative)ꢂ100%, where OD
sample
All reactions were carried out under an argon atmosphere. Most
chemicals and solvents were analytical grade and used without
further purification. TLC was performed using precoated silica gel
GF254 (0.2 mm), while column chromatography was performed
using silica gel (100e200 mesh). The melting point was measured
on an YRT-3 melting point apparatus (Shantou Keyi Instrument &
Equipment Co. Ltd, Shantou, China). NMR spectra were taken on
a Varian INOVA 400 or 600 MHz (Varian, Palo Alto, CA, USA) using
CDCl3 or DMSO-d6 as solvent. Chemical shifts are expressed in
and OD negative were the optical density values of the wells (with the derivatives or
the positive drug) and negative wells (DMSO dilutions without cells), respectively.
GI50 was calculated by Graphpad Prism 5.0.
c
This means that the compounds of the cells’ the maximum inhibition rate did
not reach 50%, or no inhibition within the detection range.
In Table 2, the GI50 data of the new compounds, YHHU-759 and
Sorafenib against human hepatoma cell lines (SMMC-7721, BEL-
7402, QGY-7703) are presented. Compound 12 barely has antican-
cer activity. It may be because the dimorpholine derivative did not
have the enol structure, which may be an essential functional group
for the anticancer activity. All derivatives 11aeh showed much
better anti-hepatoma activities than Sorafenib in these cancer cell
lines. More interestingly, improved potencies in anti-cancer activity
were found in compounds 11b and 11e against BEL-7402 cell line
than the lead compound YHHU-759. Most of other derivatives
showed the comparable activities against the three cell lines with
YHHU-759.
In order to better understand the structure-activity-relationship
of the derivatives, pharmacophore models were constructed for
YHHU-759 and 11a (Fig. 2). The model suggested that the two
compounds shared the same pharmacophore features, thus
explained the comparable activities of the derivatives 11aeh. In
d
(ppm), with tetramethylsilane (TMS) functioning as the internal
reference, and coupling constants (J) were expressed in Hertz (Hz).
High resolution mass spectroscopy data of the product were col-
lected on a Waters Micromass GCT or a Bruker Apex IV FTMS
instrument.
4.2. Synthesis
4.2.1. Synthesis of compound 2.14 To a solution of compound 1
(2.00 g, 13.4 mmol) in acetone (50 mL) were added NaI (10.0 g,
67.1 mmol) and cat. HI. The mixture was heated at 80 ꢀC for 1 h,
followed by being poured into water (20 mL). The resulting pre-
cipitate was collected by filtration, dried and purified by silica gel
column chromatography to give compound 2 (4.44 g, 99%) as a light

Q. Yue et al. / Tetrahedron 71 (2015) 7670e7675
7673
pink solid, mp 156e158 ꢀC. 1H NMR (CDCl3, 400 MHz)
J¼9.2 Hz).
d: 7.69 (d, 2H,
calcd for C12H13F3N2O5þNaþ 345.0674, found 345.0676. Elemental
Analysis: C, 44.73; H, 4.07; F, 17.69; N, 8.69; found C, 44.82; H, 4.23;
F, 17.51; N, 8.73.
4.2.2. Synthesis of compound 3.14 To a solution of compound 2
(1.40 g, 4.22 mmol) in methanol (20 mL) was added CH3ONa
(0.34 g, 6.33 mmol), and the mixture was stirred at 80 ꢀC for 1 h.
The reaction was quenched by addition of water (20 mL), then
extracted with CH2Cl2 (10 mL ꢂ3). The combined organic layer was
dried over sodium sulfate, filtered, concentrated under reduced
pressure and purified by silica gel column chromatography to give
compound 3 (0.97 g, 97%) as a colored solid, mp 96e98 ꢀC. 1H NMR
4.2.7. Synthesis of compound 8. To a solution of compound 7
(0.77 g, 2.39 mmol) in acetic acid (15 mL) was added hydrazine
hydrate (0.72 mL, 7.17 mmol). After stirring for 7 h at 100 ꢀC, the
reaction was quenched by addition of saturated aqueous sodium
carbonate, then extracted with ethyl acetate (30 mL ꢂ3). The
combined organic layer was dried over sodium sulfate, filtered,
concentrated under reduced pressure and purified by silica gel
column chromatography to give compound 8 (0.35 g, 53%) as
(CDCl3, 400 MHz)
(d, 1H, J¼9.2 Hz).
d
: 4.09 (s, 3H, OCH3), 6.69 (d, 1H, J¼9.2 Hz), 7.64
a brown oil. 1H NMR (DMSO-d6, 400 MHz)
(d, 1H, J¼9.2 Hz), 8.18 (d, 1H, J¼9.2 Hz), 8.65 (s, 1H), 14.07 (br, 1H,
OH). 13C NMR (CDCl3, 100 MHz)
177.21(1C), 161.87(1C),
d: 4.09 (s, 3H, OCH3), 7.38
4.2.3. Synthesis of compound 4.16 To a solution of compound 3
(2.08 g, 8.81 mmol) in anhydrous THF (25 mL) and Et3N (10 mL)
were added copper(I) iodide (0.17 g, 0.88 mmol), bis(-
triphenylphosphine)palladium(II) dichloride (0.31 g, 0.44 mmol)
followed by trimethylsilylacetylene (1.04 g, 10.6 mmol) dropwise at
d:
152.36(1C), 147.17(1C), 142.61(1C), 131.55(2C), 130.73(2C),
58.65(1C). ESI-HRMS: calcd for C10H7F3N4O2þNaþ 295.0419, found
295.0418. Elemental Analysis: C, 44.13; H, 2.59; F, 20.94; N, 20.58,
found C, 44.25; H, 2.47; F, 21.13; N, 20.46.
0
ꢀC. After stirring for 2 h at room temperature, the reaction was
quenched by addition of water (15 mL), then extracted with ethyl
acetate (10 mL ꢂ3). The combined organic layer was dried over
sodium sulfate, filtered, concentrated under reduced pressure and
purified by silica gel column chromatography to give compound 4
(1.78 g, 98%) as a brown solid, mp 70e72 ꢀC. 1H NMR (CDCl3,
400 MHz) d: 0.28 (s, 9H, Si(CH3)3), 4.15 (s, 3H, OCH3), 6.90 (d, 1H,
J¼9.2 Hz), 7.44 (d, 1H, J¼9.2 Hz).
4.2.8. Synthesis of compound 9. To a solution of POCl3 (2 mL,
7.40 mmol) at 0 ꢀC, compound 8 (0.10 g, 0.37 mmol) was added in
portions. The mixture was heated at 80 ꢀC for 3 h, followed by being
poured into water (10 mL). The pH was adjusted to 7 with saturated
aqueous sodium bicarbonate, then extracted with ethyl acetate
(30 mL ꢂ3). The combined organic layer was dried over sodium
sulfate, filtered, concentrated under reduced pressure and purified
by silica gel column chromatography to give compound 9 (92 mg,
4.2.4. Synthesis of compound 5.16 To a stirred solution of compound
4 (1.70 g, 8.24 mmol) in tetrahydrofuran (30 mL) was added TBAF
(1.30 g, 4.12 mmol) at room temperature. After stirring for 30 min,
the reaction was quenched with saturated aqueous NH4Cl, and the
reaction mixture was extracted with ethyl acetate (10 mL ꢂ3). The
combined organic layer was dried over sodium sulfate, filtered,
concentrated under reduced pressure and purified by silica gel
column chromatography to give compound 5 (0.88 g, 80%) as
84%) as a brown oil. 1H NMR (CDCl3, 400 MHz)
d: 7.78 (d, 1H,
J¼9.2 Hz), 8.79 (d, 1H, J¼9.2 Hz), 9.09 (s, 1H). 13C NMR (CDCl3,
100 MHz)
d: 160.77(1C), 152.17(1C), 146.68(1C), 142.33(1C),
130.75(2C), 130.42(2C), 120.25(1C). ESI-HRMS: calcd for
C9H3Cl2F3N4þNaþ 316.9585, found 316.9588. Elemental Analysis: C,
36.64; H,1.02; Cl, 24.03; F,19.32; N,18.99, found C, 36.61; H,1.08; Cl,
24.11; F, 19.37; N, 19.16.
a colorless oil. 1H NMR (CDCl3, 400 MHz)
d
: 3.31 (s, 1H, CH), 4.16 (s,
4.2.9. Synthesis of compound 10aeh. To a solution of 9 (2.0 mmol)
in dioxane (10 mL) was added amine (10 mmol). The resulting
mixture was stirred at 15 ꢀC for 10 h, and ethyl acetate (10 mL) and
water (10 mL) were added. After stirring for 10 min, the organic
solution was separated and the aqueous solution was extracted
with ethyl acetate (10 mL ꢂ3). The combined organic layer was
washed with saturated aqueous sodium chloride (10 mL ꢂ3), dried
over sodium sulfate, filtered and concentrated in vacuo. The resi-
due was purified by silica gel column chromatography to give
10aeh.
3H, OCH3), 6.94 (d, 1H, J¼9.2 Hz), 7.47 (d, 1H, J¼9.2 Hz).
4.2.5. Synthesis of compound 6.26 To a solution of compound 5
(1.80 g, 13.4 mmol) in l4% H2SO4 (25 mL) was added HgSO4 (0.40 g,
1.34 mmol). After stirring for 2 h at 80 ꢀC, the reaction was
quenched by addition of saturated aqueous sodium carbonate,
then extracted with ethyl acetate (30 mL ꢂ3). The combined or-
ganic layer was dried over sodium sulfate, filtered, concentrated
under reduced pressure and purified by silica gel column chro-
matography to give compound 6 (1.02 g, 50%) as a pale yellow
solid, mp 125e127 ꢀC. 1H NMR (CDCl3, 400 MHz)
d: 2.79 (s, 3H,
4.2.9.1. Compound 10a. Yellow solid. Yield 89%, mp 156e158 ꢀC.
COCH3), 4.21 (s, 3H, OCH3), 7.04 (d, 1H, J¼9.2 Hz), 8.02 (d, 1H,
1H NMR (CDCl3, 400 MHz)
d
: 3.69 (t, 4H, J¼4.8 Hz, OeCH2 ꢂ2), 3.90
J¼9.2 Hz).
(t, 4H, J¼4.8 Hz, NeCH2 ꢂ2), 7.69 (d, 1H, J¼9.2 Hz), 8.69 (d, 1H,
J¼9.2 Hz), 8.85 (s, 1H). 13C NMR (CDCl3, 100 MHz)
d: 166.22(1C),
4.2.6. Synthesis of compound 7. To a solution of compound 6
(0.58 g, 3.81 mmol) in DMF (45 mL) were added ethyl 3,3,3-
trifluoro-2-oxopropanoate (2.59 g, 15.3 mmol) and ZnCl2 (0.78 g,
5.72 mmol) at ꢁ20 ꢀC. After completion of the addition, the mixture
was moved to 110 ꢀC and stirred for 25 h. The reaction was
quenched by addition of saturated aqueous ammonium chloride,
then extracted with ethyl acetate (30 mL ꢂ3). The combined or-
ganic layer was dried over sodium sulfate, filtered, concentrated
under reduced pressure and purified by silica gel column chro-
matography to give compound 7 (0.93 g, 76%) as a brown oil. 1H
157.80(1C), 140.30(1C), 145.40(1C), 132.44(2C), 131.01(2C),
120.54(1C), 66.54(2C), 49.66(2C). ESI-HRMS: calcd for
C
13H11ClF3N5OþNaþ 368.0502, found 368.0501. Elemental Analy-
sis: C, 45.17; H, 3.21; Cl, 10.26; F, 16.49; N, 20.26, found C, 45.08; H,
3.29; Cl, 10.15; F, 16.40; N, 20.34.
4.2.9.2. Compound 10b. Yellow solid. Yield 91%, mp 140e142 ꢀC.
1H NMR (CDCl3, 400 MHz)
d
: 1.75e1.77 (m, 6H, CH2 ꢂ3), 3.62e3.65
(m, 4H, NeCH2 ꢂ2), 7.66 (d,1H, J¼8.8 Hz), 8.68 (d,1H, J¼8.8 Hz), 8.77
(s, 1H). 13C NMR (CDCl3, 100 MHz)
: 166.01(1C), 157.95(1C),
d
NMR (CDCl3, 600 MHz)
d
: 1.30 (t, 3H, J¼7.2 Hz, CH3), 4.25 (s, 3H,
150.24(1C),145.33(1C),135.39(2C),131.20(2C),116.05(1C), 49.14(2C),
OCH3), 4.39 (q, 2H, J¼7.2 Hz, CH2), 7.09 (d, 1H, J¼9.6 Hz), 8.01 (d, 1H,
25.69(2C), 22.71(1C). ESI-HRMS: calcd for
C
14H13ClF3N5þNaþ
J¼9.6 Hz). 13C NMR (CDCl3, 100 MHz)
d
: 205.09(1C), 173.95(1C),
366.0709, found 366.0711. Elemental Analysis: C, 48.92; H, 3.81; Cl,
10.31; F, 16.58; N, 20.37, found C, 48.85; H, 3.89; Cl, 10.22; F, 16.43; N,
20.51.
171.68(1C), 139.18(1C), 135.39(1C), 125.90(1C), 121.10(1C),
89.07(1C), 61.53(1C), 56.06(1C), 23.89(1C), 12.25(1C). ESI-HRMS:

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Q. Yue et al. / Tetrahedron 71 (2015) 7670e7675
4.2.9.3. Compound 10c. White solid. Yield 93%, mp 161e162 ꢀC.
1H NMR (CDCl3, 400 MHz)
: 1.23e1.39 (m, 3H), 1.46e1.55 (m, 2H),
solution was extracted with ethyl acetate (10 mL ꢂ3). The com-
d
bined organic layer was washed with saturated aqueous sodium
chloride (10 mL ꢂ3), dried over sodium sulfate, filtered and con-
centrated in vacuo. The residue was purified by silica gel column
chromatography to give 12. (0.74 g, 95%) as a white solid, mp
1.68e1.71 (m, 1H), 1.77e1.81 (m, 2H), 2.18e2.21 (m, 2H), 4.43 (s, 1H,
CH), 5.09 (br, 1H, NH), 7.65 (d, 1H, J¼8.8 Hz), 8.65 (d, 1H, J¼8.8 Hz),
8.72 (s, 1H). 13C NMR (CDCl3, 100 MHz)
d: 163.11(1C), 156.55(1C),
146.29(1C), 142.20(1C), 130.85(2C), 130.36(2C), 123.22(1C),
49.14(1C), 32.95(2C), 25.69(1C), 23.89(2C). ESI-HRMS: calcd for
209e211 ꢀC. 1H NMR (CDCl3, 400 MHz)
d
: 3.57 (t, 4H, J¼4.8 Hz,
OeCH2 ꢂ2), 3.75 (t, 4H, J¼4.8 Hz, OeCH2 ꢂ2), 3.87e3.90 (m, 8H,
NeCH2 ꢂ2), 7.07 (d, 1H, J¼10.0 Hz), 8.49 (d, 1H, J¼10.0 Hz), 8.83 (s,
C
15H15ClF3N5þNaþ 380.0866, found 380.0865. Elemental Analysis:
C, 50.36; H, 4.23; Cl, 9.91; F, 15.93; N, 19.58, found C, 50.24; H, 4.15;
Cl, 10.03; F, 15.87; N, 19.65.
1H). 13C NMR (CDCl3, 100 MHz)
d: 162.57(1C), 158.20(1C),
149.47(1C), 142.10(1C), 130.93(2C), 130.48(2C), 121.83(1C),
66.38(4C),
C
49.84(2C),
49.01(2C).
ESI-HRMS:
calcd
for
4.2.9.4. Compound 10d. Yellow solid. Yield 70%, mp 213e215 ꢀC.
17H19F3N6O2þNaþ 419.1419, found 419.1417. Elemental Analysis: C,
1H NMR (CDCl3, 400 MHz)
d
: 2.41 (s, 3H, CH3), 2.66 (s, 4H, CH2 ꢂ2),
3.75 (s, 4H, NeCH2 ꢂ2), 7.68 (d, 1H, J¼8.8 Hz), 8.69 (d, 1H, J¼8.8 Hz),
8.82 (s, 1H). 13C NMR (CDCl3, 100 MHz)
: 165.42(1C), 155.35(1C),
148.69(1C), 143.16(1C), 132.61(2C), 132.01(2C), 120.00(1C),
51.51; H, 4.83; F, 14.38; N, 21.20, found C, 51.55; H, 4.80; F, 14.34; N,
21.26.
d
4.2.11. Synthesis of compound 11aeh. To a solution of acetic acid
(10 mL) was added compound 10aeh (2.0 mmol). After stirring for
2 h at 100 ꢀC, the mixture was concentrated under reduced pres-
sure, and the residue was purified by silica gel column chroma-
tography to give 11aeh.
55.04(2C),
49.51(2C),
30.33(1C).
ESI-HRMS:
calcd
for
C
14H14ClF3N6þNaþ 381.0818, found 381.0815. Elemental Analysis:
C, 46.87; H, 3.93; Cl, 9.88; F, 15.89; N, 23.43, found C, 46.95; H, 3.86;
Cl, 9.91; F, 15.94; N, 23.37.
4.2.9.5. Compound 10e. Yellow solid. Yield 78%, mp 149e151 ꢀC.
4.2.11.1. Compound 11a. Yellow solid. Yield 91%, mp
1H NMR (CDCl3, 400 MHz)
d
: 2.06e2.09 (m, 4H, CH2 ꢂ2), 3.84 (s, 4H,
NeCH2 ꢂ2), 7.63 (d, 1H, J¼9.2 Hz), 8.66 (d, 1H, J¼9.2 Hz), 8.78 (s,
1H). 13C NMR (CDCl3, 100 MHz)
160.87(1C), 152.61(1C),
242e244 ꢀC. 1H NMR (CDCl3, 400 MHz)
d
: 3.63 (t, 4H, J¼4.2 Hz,
OeCH2 ꢂ2), 3.89 (t, 4H, J¼4.2 Hz, NeCH2 ꢂ2), 7.17 (d, 1H, J¼9.6 Hz),
d
:
8.40 (s, 1H), 8.58 (d, 1H, J¼9.6 Hz), 12.71 (br, 1H, OH). 13C NMR
146.67(1C), 143.36(1C), 129.73(2C), 129.17(2C), 122.65(1C),
(CDCl3, 100 MHz) d: 162.37(1C), 157.26(1C), 149.19(1C), 142.16(1C),
49.31(2C), 25.65(2C). ESI-HRMS: calcd for
C
13H11ClF3N5þNaþ
130.89(2C), 130.36(2C), 124.51(1C), 66.59(2C), 50.60(2C). ESI-
HRMS: calcd for C13H12F3N5O2þNaþ 350.0841, found 350.0840.
Elemental Analysis: C, 47.71; H, 3.70; F, 17.42; N, 21.40, found C,
47.66; H, 3.78; F, 17.35; N, 21.44.
352.0553, found 352.0556. Elemental Analysis: C, 47.36; H, 3.36; Cl,
10.75; F, 17.29; N, 21.24, found C, 47.41; H, 3.39; Cl, 10.68; F, 17.34; N,
21.29.
4.2.9.6. Compound 10f. White solid. Yield 88%, mp 168e170 ꢀC.
4.2.11.2. Compound 11b. Yellow solid. Yield 90%, mp
1H NMR (CDCl3, 400 MHz)
d
: 1.02 (d, 3H, J¼6.4 Hz, CH3), 1.36e1.45
238e240 ꢀC. 1H NMR (CDCl3, 600 MHz)
d: 1.72e1.75 (m, 6H, CH2
(m, 3H, CH2þCH), 1.81 (d, 1H, J¼13.2 Hz, CH2), 3.17 (t, 2H,
J¼12.0 Hz), 4.13 (d, 2H, J¼13.2 Hz, NeCH2), 7.66 (d, 1H, J¼9.2 Hz),
ꢂ3), 3.54e3.57 (m, 4H, NeCH2 ꢂ2), 7.13 (d, 1H, J¼10.0 Hz), 8.31 (s,
1H), 8.57 (d, 1H, J¼10.0 Hz), 12.41 (s, 1H). 13C NMR (CDCl3, 150 MHz)
8.68 (d, 1H, J¼9.2 Hz), 8.78 (s, 1H). 13C NMR (CDCl3, 100 MHz)
d:
d: 162.37(1C), 157.95(1C), 148.31(1C), 142.50(1C), 130.84(2C),
160.30(1C), 153.73(1C), 147.26(1C), 141.52(1C), 128.83(2C),
128.08(2C), 121.67(1C), 49.98(2C), 33.55(2C), 23.27(1C), 19.92(1C).
ESI-HRMS: calcd for C15H15ClF3N5þNaþ 380.0866, found 380.0869.
Elemental Analysis: C, 50.36; H, 4.23; Cl, 9.91; F, 15.93; N, 19.58,
found C, 50.42; H, 4.18; Cl, 9.82; F, 16.02; N, 19.63.
130.33(2C), 123.99(1C), 51.31(2C), 25.72(2C), 24.12(1C). ESI-HRMS:
calcd for C14H14F3N5OþNaþ 348.1048, found 348.1046. Elemental
Analysis: C, 51.69; H, 4.34; F, 17.52; N, 21.53, found C, 51.52; H, 4.28;
F, 17.63; N, 21.59.
4.2.11.3. Compound 11c. Yellow solid. Yield 90%, mp
4.2.9.7. Compound 10g. Yellow solid. Yield 74%, mp 129e131 ꢀC.
263e264 ꢀC. 1H NMR (CDCl3, 400 MHz)
d: 1.24e1.34 (m, 3H),
1H NMR (CDCl3, 400 MHz)
d
: 3.35 (s, 6H, NeCH3 ꢂ2), 7.66 (d, 1H,
1.43e1.52 (m, 2H), 1.66e1.69 (m, 1H), 1.76e1.79 (m, 2H), 2.15e2.18
(m, 2H), 4.36 (d, 1H, J¼6.8 Hz, NeCH), 4.95 (d, 1H, J¼6.8 Hz, NH),
7.11 (d, 1H, J¼9.6 Hz), 8.21 (s, 1H), 8.54 (d, 1H, J¼9.6 Hz), 12.41 (s, 1H,
J¼9.2 Hz), 8.67 (d, 1H, J¼9.2 Hz), 8.80 (s, 1H). 13C NMR (CDCl3,
100 MHz)
d: 162.35(1C), 157.25(1C), 149.18(1C), 142.15(1C),
130.88(2C), 130.35(2C), 124.50(1C), 42.23(2C). ESI-HRMS: calcd for
OH). 13C NMR (CDCl3, 100 MHz)
d: 162.74(1C), 157.63(1C),
C
11H9ClF3N5þNaþ 326.0396, found 326.0397. Elemental Analysis: C,
146.24(1C), 142.75(1C), 130.75(2C), 130.17(2C), 122.03(1C),
50.55(1C), 32.69(2C), 25.53(1C), 24.63(2C). ESI-HRMS: calcd for
43.51; H, 2.99; Cl, 11.67; F, 18.77; N, 23.06, found C, 43.46; H, 2.87;
Cl, 11.77; F, 18.86; N, 22.99.
C
15H16F3N5OþNaþ 362.1205, found 362.1209. Elemental Analysis:
C, 53.10; H, 4.75; F, 16.80; N, 20.64, found C, 53.04; H, 4.71; F, 16.83;
N, 20.69.
4.2.9.8. Compound 10h. Yellow solid. Yield 67%, mp 136e138 ꢀC.
1H NMR (CDCl3, 400 MHz)
d
: 1.04 (t, 3H, J¼7.2 Hz, CH3), 1.74e1.83
(m, 2H, CH2), 3.75 (q, 2H, J¼6.0 Hz, NeCH2), 5.26 (br, 1H, NH), 7.64
4.2.11.4. Compound 11d. Yellow solid. Yield 93%, mp
(d,1H, J¼8.8 Hz), 8.65 (d,1H, J¼8.8 Hz), 8.71 (s,1H). 13C NMR (CDCl3,
290e292 ꢀC. 1H NMR (DMSO-d6, 400 MHz)
d: 2.61 (s, 3H, CH3), 3.04
100 MHz)
d:
161.54(1C), 152.21(1C), 147.04(1C), 142.41(1C),
(s, 4H, CH2 ꢂ2), 3.58 (s, 4H, CH2 ꢂ2), 7.11 (d, 1H, J¼8.8 Hz), 8.25 (s,
131.32(2C), 130.80(2C), 122.35(1C), 44.05(1C), 22.71(1C), 13.01(1C).
ESI-HRMS: calcd for C12H11ClF3N5þNaþ 340.0553, found 340.0551.
Elemental Analysis: C, 45.37; H, 3.49; Cl, 11.16; F, 17.94; N, 22.04,
found C, 45.31; H, 3.53; Cl, 11.22; F, 17.87; N, 22.11.
1H), 8.37 (d, 1H, J¼8.8 Hz), 13.54 (br, 1H, OH). 13C NMR (CDCl3,
150 MHz)
d: 162.37(1C), 157.25(1C), 149.19(1C), 142.15(1C),
130.88(2C), 130.37(2C), 124.50(1C), 57.20(2C), 50.67(2C), 29.66(1C).
ESI-HRMS: calcd for C14H15F3N6OþNaþ 363.1157, found 363.1156.
Elemental Analysis: C, 49.41; H, 4.44; F, 16.75; N, 24.70, found C,
49.46; H, 4.38; F, 16.81; N, 24.74.
4.2.10. Synthesis of compound 12. To a solution of 9 (0.58 g,
2.0 mmol) in DMF (10 mL) was added morpholine (0.86 g,
10 mmol). The resulting mixture was stirred at 80 ꢀC for 0.5 h, and
ethyl acetate (10 mL) and water (10 mL) were added. After stirring
for 10 min, the organic solution was separated and the aqueous
4.2.11.5. Compound 11e. Yellow solid. Yield 91%, mp
260e262 ꢀC. 1H NMR (DMSO-d6, 400 MHz)
3.67 (s, 4H, Ne CH2 ꢂ2), 7.07 (d, 1H, J¼9.2 Hz), 8.10 (s, 1H), 8.35 (d,
d
: 1.98 (s, 4H, CH2 ꢂ2),

Q. Yue et al. / Tetrahedron 71 (2015) 7670e7675
7675
1H, J¼9.2 Hz), 13.35 (s, 1H, OH). 13C NMR (CDCl3, 150 MHz)
d
:
derivatives) and control wells (DMSO dilutions without cells), re-
spectively. For each sample, the final optical density was the aver-
age value measured from six wells in parallel. Data analysis and
GI50 values were calculated on the four-parameter logic fitting
module of the software Graphpad Prism 5.0. The result was pre-
sented in Table 2.
162.57(1C), 153.73(1C), 145.09(1C), 142.98(1C), 130.76(2C),
130.19(2C), 123.00(1C), 50.06(2C), 25.52(2C). ESI-HRMS: calcd for
C
13H12F3N5OþNaþ 334.0892, found 334.0894. Elemental Analysis:
C, 50.16; H, 3.89; F, 18.31; N, 22.50, found C, 50.11; H, 3.94; F, 18.26;
N, 22.57.
4.2.11.6. Compound 11f. Yellow solid. Yield 89%, mp
Acknowledgements
258e260 ꢀC. 1H NMR (CDCl3, 400 MHz)
d: 1.00 (d, 3H, J¼6.4 Hz,
CH3), 1.34e1.44 (m, 2H, CH2), 1.67e1.69 (m, 1H, CH), 1.77e1.80 (m,
2H, CH2), 3.13 (t, 2H, J¼12.8 Hz), 4.03 (d, 2H, J¼13.2 Hz), 7.12 (d, 1H,
J¼10.0 Hz), 8.29 (s, 1H), 8.56 (d, 1H, J¼10.0 Hz), 11.88 (s, 1H). 13C
This work was supported by the National Natural Science
Foundation of China (No. 21472130 & No. 81373259).
NMR (CDCl3, 100 MHz)
d: 162.74(1C), 152.62(1C), 146.25(1C),
142.75(1C), 130.76(2C), 130.18(2C), 122.03(1C), 50.65(2C),
Supplementary data
32.68(2C),
24.64(1C),
20.12(1C).
ESI-HRMS:
calcd
for
C
15H16F3N5OþNaþ 362.1205, found 362.1208. Elemental Analysis:
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2015.07.063.
C, 53.10; H, 4.75; F, 16.80; N, 20.64, found C, 53.06; H, 4.69; F, 16.84;
N, 20.69.
References and notes
4.2.11.7. Compound 11g. Yellow solid. Yield 90%, mp
230e232 ꢀC. 1H NMR (DMSO-d6, 400 MHz)
d
: 3.17 (s, 6H, NeCH3
ꢂ2), 7.08 (d, 1H, J¼10.0 Hz), 8.12 (s, 1H), 8.35 (d, 1H, J¼10.0 Hz),
13.39 (s, 1H, OH). 13C NMR (CDCl3, 150 MHz)
: 162.36(1C),
1. Jemal, A.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman, D. CAeCancer J.
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d
2. Liese, J.; Abhari, B. A.; Fulda, S. Cancer Lett. 2015, 365, 47e56.
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157.30(1C), 149.24(1C), 142.25(1C), 130.57(2C), 130.88(2C),
124.37(1C), 41.88(2C). ESI-HRMS: calcd for
308.0735, found 308.0732. Elemental Analysis: C, 46.32; H, 3.53; F,
19.98; N, 24.55, found C, 46.28; H, 3.57; F, 20.01; N, 24.46.
C
11H10F3N5OþNaþ
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4.2.11.8. Compound 11h. White solid. Yield 91%, mp
223e224 ꢀC. 1H NMR (CDCl3, 400 MHz)
d
: 1.04 (t, 3H, J¼7.2 Hz, CH3),
1.73e1.82 (m, 2H, CH2), 3.70e3.75 (m, 2H, CH2), 5.18 (br, 1H, NH),
7.11 (d, 1H, J¼10.0 Hz), 8.21 (s, 1H), 8.53 (d, 1H, J¼10.0 Hz), 11.54 (s,
1H, OH). 13C NMR (CDCl3, 150 MHz)
d: 162.33(1C), 153.50(1C),
146.48(1C), 142.85(1C), 130.75(2C), 130.28(2C), 121.57(1C),
43.90(1C),
22.27(1C),
11.30(1C).
ESI-HRMS:
calcd
for
C
12H12F3N5OþNaþ 322.0892, found 322.0893. Elemental Analysis:
C, 48.16; H, 4.04; F, 19.05; N, 23.40, found C, 48.11; H, 4.09; F, 18.97;
N, 23.44.
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cultured in Dulbecco’s modified Eagle’s medium (DMEM), supple-
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microplates with 50
After incubation for 6 h, each well was added with 50
(20 M, 2 M, 0.2 M, 0.02 M, 0.002 M, 0.0002 M). After 72 h
incubation, 20 L MTS reagent (Invitrogen, USA) was added to each
mL of culture media (about 4000 cells/well).
mL derivatives
m
m
m
m
m
m
m
well and incubated for another 2 h. The optical density value (OD
value) of each well was measured with a microplate reader (Flex-
station 3, Molecular Devices Co., USA) at 490 nm. The cell viability
(%) relative to control cells cultured in media without derivatives
was calculated from OD sample/OD negativeꢂ100%, where OD
sample
and OD negative were the optical density values of the wells (with the