Novel 1H-purin-6(9H)-one (D) and 3H-imidazo[4,5-d][1,2,3]trazin-4(7H)-one (E) derivatives were designed, synthesized, and characterized by 1H NMR, 13C NMR and high-resolution mass spectrometry spectra. Their herbicidal activity bioassay showed that compound 7d exhibited relatively good activity with 70.4% inhibition rate against Amaranthus retroflexus in postemergence treatments at 1500 g/ha. Antitumor activity indicated that most of the title compounds displayed potent antitumor activity at 20 μM, among all of the promising compounds possessing lower IC50 values than that of temozolomide, compound 7i demonstrated highest activity inhibiting both HepG-2 and U-118 MG cell lines with IC50 values of 2.0 and 3.8 μM, respectively. The structure-activity relationship analysis revealed that introduction of halogen atoms, a bulky bridging bond between benzene ring and nitrogen atom, longer R2 substituents could contribute to the improvement of antitumor activity. Analysis suggested that compound 7i might have potential as new highly active antitumor agent. Overall, D series had better anticancer activities than E series derivatives.
Novel 1H-purin-6(9H)-one (D) and 3H-imidazo[4,5-d][1,2,3]trazin-4(7H)-one (E) derivatives were designed, synthesized, and characterized by 1H NMR, 13C NMR and high-resolution mass spectrometry spectra. Their herbicidal activity bioassay showed that compound 7d exhibited relatively good activity with 70.4% inhibition rate against Amaranthus retroflexus in postemergence treatments at 1500 g/ha. Antitumor activity indicated that most of the title compounds displayed potent antitumor activity at 20 μM, among all of the promising compounds possessing lower IC50 values than that of temozolomide, compound 7i demonstrated highest activity inhibiting both HepG-2 and U-118 MG cell lines with IC50 values of 2.0 and 3.8 μM, respectively. The structure-activity relationship analysis revealed that introduction of halogen atoms, a bulky bridging bond between benzene ring and nitrogen atom, longer R2 substituents could contribute to the improvement of antitumor activity. Analysis suggested that compound 7i might have potential as new highly active antitumor agent. Overall, D series had better anticancer activities than E series derivatives.
Porphyrin
plays an important role in the biosynthesis of many pigments
crucial for plant survival, during which process protoporphyrinogen
oxidase (PPO) is an essential catalytic enzyme for the conversion
from porphyrin to chlorophyll or heme.[1−3] In recent years, PPO-inhibiting
herbicides have fueled tremendous interest in the field of agrochemical
discovery as novel target-based pesticides.[4] The chemical structure of these herbicides including diphenylethers,
phenylphthalimides, phenylpyrazole, oxazolidinediones, and so on can
be generally summarized as follows:[5] (i)
a heterocycle or heterocyclic ketone with one or more nitrogen atoms;
(ii) a polysubstituted benzene ring linking with the nitrogen atom
of the previous heterocycle, such as azafenidin, butafenacil, and
profluazol (Figure ).
Figure 1
Chemical structures of azafenidin, butafenacil, profluazol, A–F, and temozolimide.
Chemical structures of azafenidin, butafenacil, profluazol, A–F, and temozolimide.In our previous work, pyrazolotetrazinone (A), isoxazoltetrazinone
(B), and pyrazolotriazinone (C) derivatives,
which further demonstrated they acted on PPO enzyme, were designed
and synthesized according to the bioisosteric principle, and some
of these compounds provided good control of weeds.[1,2,3]triazin-4-one Derivatives. J. Agric. Food Chem.. 2008 ">6,7] Nonetheless,
their translation to the application has severely hampered because
of the poor selectivity between crops and grasses. Herein, we report
the design and synthesis of novel 1H-purin-6(9H)-one (D) and 3H-imidazo[4,5-d][1,2,3]triazin-4(7H)-one (E) derivatives, followed by herbicidal activity assay against Amaranthus retroflexus, Bactris campestris, Digitaria sanguinalis, and Echinochloa crusgalli. However, most of the novel
compounds exhibited moderate activity in primary herbicidal bioassay,
but this depressing result stimulated us to explore their bioactivity
in other aspects further. Temozolomide (Figure ), one of azolotetrazinones antitumor agents
highly effective on many kinds of cancer cell lines including brain
tumors, leukemia, melanoma, lymphoma, and solid tumors,[8−10] inspired our interest because of its high similarity to the general
chemical structure of PPO-inhibiting herbicides listed above. Consequently,
initial exploration to their antitumor activity was carried out and
the structure–activity relationships (SAR) were also discussed
using statistical methods based on the analysis of relatively superior
bioactivity results.
Results and Discussion
Synthesis
The synthetic route of 7a–o and 8a–m was shown in Scheme . Intermediate 2 was obtained
by reduction with sodium thiosulfate in an
alkaline solution condition with a pH of 8–9 of water and saturated
sodium bicarbonate. During the process, it was found that temperature
had a great influence on the yield and properties of products. As
the reaction temperature decreased from 35 to 15 °C, the yield
increased and the color of the product became lighter. A possible
mechanism from 2 to 3 was reasonably speculated
(Figure ). The construction
of imidazole ring A had mainly gone through two steps. First, imine
intermediate 3a was synthesized by the nucleophilic substitution
of triethyl orthoate with amine 2 in acetonitrile under
refluxing. Second, the nucleophilic addition reaction at room temperature
of amine R1NH2 with the relatively positively
charged carbon on the cyano group afforded the second imine intermediate 3c, and then the nitrogen atom in R1NH2 attacked the imine first described above to form a five-membered
ring 3d, which ultimately transformed to imidazole 3 after rearrangement and proton transfer. There existed great
differences between different R1 substituents in the aspect
of work-up methods and reaction time.
Scheme 1
Synthetic Route of Title Compounds
Reagents and conditions:
(a)
Na2S2O4, NaHCO3, H2O; (b) (i) RC(OEt)3, MeCN, 80 °C; (ii) R1NH2, MeCN, RT; (c) NaOH, EtOH, reflux; (d) SOCl2, −5 to 0 °C; (e) pyridine, DCM; (f) R2C(OEt)3, Ac2O, reflux; (g) (i) 50% H2SO4, MeOH, RT; (ii) NaNO2, −5 to 0 °C.
Figure 2
Proposed mechanism from compound 2 to 3.
Proposed mechanism from compound 2 to 3.
Synthetic Route of Title Compounds
Reagents and conditions:
(a)
Na2S2O4, NaHCO3, H2O; (b) (i) RC(OEt)3, MeCN, 80 °C; (ii) R1NH2, MeCN, RT; (c) NaOH, EtOH, reflux; (d) SOCl2, −5 to 0 °C; (e) pyridine, DCM; (f) R2C(OEt)3, Ac2O, reflux; (g) (i) 50% H2SO4, MeOH, RT; (ii) NaNO2, −5 to 0 °C.Compound 3 was hydrolyzed and then
converted to its
acid chloride 5, which reacted directly with substituted
aniline 15 in dichloromethane in the presence of pyridine
to furnish amide 6. The intermediates 10–14 were synthesized as the literature described.[11−15] Compound 14 was reduced by stannous
chloride dihydrate in ethanol under refluxing condition to give aniline 15 (Scheme ). Two methods to construct ring B of title compounds were built.
Amide 6 was cyclized with triethyl orthoformate or triethyl
orthoacetate or triethyl orthopropionate in acetic anhydride under
refluxing condition to give purinone derivatives (7a–o, D series). After being activated with 50% sulphuric
acid, amide 6 was reacted with sodium nitrite to give
imidazotriazinone derivatives (8a–m, E series). Table summarized
the newly synthesized compounds D and E.
Scheme 2
Synthesis
Route of Common Intermediate 15
Reagents
and conditions: (a)
NCS, AlCl3,DCM; (b) propyl chlorocarbonate, Et3N, DCM, −5 to 0 C; (c) 98% H2SO4, 65%
HNO3, −5 to 0 C; (d) NaOH, EtOH, RT; (e) K2CO3, 80% propargyl bromide in toluene, DMF; (f) SnCl2, EtOH, reflux.
Table 1
List of Title Compounds
no.
R
R1
R2
no.
R
R1
R2
7a
H
PhCH2
CH3CH2
7o
CH3CH2
PhCH2
CH3
7b
H
PhCH2
H
8a
H
PhCH2
7c
H
PhCH2
CH3
8b
CH3
p-FPhCH2
7d
CH3
PhCH2
CH3CH2
8c
CH3
o-FPhCH2
7e
CH3
PhCH2
CH3
8d
CH3
pyridyl
7f
CH3
Pyridyl
CH3
8e
CH3
PhCH3CH
7g
CH3
PhCH2
H
8f
CH3
Ph
7h
CH3
2,4-difluoroPhCH2
CH3
8g
CH3
m-FPhCH2
7i
CH3
2,4-difluoroPhCH2
CH3CH2
8h
CH3
PhCH2CH2
7j
CH3
pyridyl
CH3CH2
8i
CH3
p-ClPh
7k
CH3
2,4-sifluoroPhCH2
H
8j
CH3
2,4-difluoroPhCH2
7l
CH3
Pyridyl
H
8k
CH3
PhCH2
7m
CH3CH2
PhCH2
CH3CH2
8l
CH3
p-ClPhCH2
7n
CH3CH2
PhCH2
H
8m
CH3CH2
PhCH2
Herbicidal
Activity
Percent inhibition
of title compounds against A. retroflexus, B. campestris, D.
sanguinalis, and E. crusgalli at a concentration of 1500 g/ha was shown in Table . Compounds 7d, 7e, and 7f exhibited relatively better herbicidal activity,
and among them, compound 7d showed 70.4% inhibition activity
against A. retroflexus in postemergence
treatments. Nevertheless, no title compound was found to display 100%
inhibition rate at such relatively high screening concentration. In
a word, the evaluation results of herbicidal activity were less satisfied;
hence, further herbicidal activity tests were not carried out (Scheme ).
Table 2
Herbicidal
Activity of Title Compounds
(Percent Inhibition) (Rate = 1500 g/ha)a
A. retroflexus
B. campestris
D. sanguinalis
E. crusgalli
compd
pre
post
pre
post
pre
post
pre
post
7a
2
20
0
0
0
0
3.4
5.4
7b
0
15
0
4.9
6.2
12.1
8.1
0
7c
0
5
0
5.6
8.4
11.6
11.1
4.2
7d
22.8
70.4
20.1
10.2
2.2
34.5
27
21.5
7e
31
66.4
18.1
16.6
0
12.6
10.9
26.3
7f
11.6
61.5
18.6
16.4
0
15.5
0
21.2
7g
31
48.1
16.6
24.5
0
21.5
22.3
13.5
7h
14.7
20
9
0
0
0
16.7
0
7i
1.6
20
13.8
21.1
0
0
13.4
0
7j
18.9
15.6
20.8
0
0
9.6
18.7
1.1
7k
30
15
5.6
0
30.8
0
14
4.7
7l
9.5
0
23.7
8.3
0
0
13.8
16.3
7m
10.3
34.5
20.5
27.8
0
9.9
20.4
34.7
7n
40.7
19.7
14.4
22.1
17.6
1.7
15.6
40.9
7o
0
18.5
7.5
32.5
3.5
0
12.8
33.2
8a
0
25.9
7.1
0
21.4
8.5
20.4
13.4
8b
20.8
37.4
1.2
0
15.9
14.3
11.6
4.7
8c
0
35.9
0
8.8
0
0
0
0
8d
10.5
33.9
14.9
0
17.1
14.1
14.8
14.9
8e
6.2
32.8
0
0
19
12.6
6.2
3.3
8f
0
28.1
0
0
0
0
9.6
0
8g
0
23.7
0
0
12.2
31.6
3.7
10.2
8h
2.1
20.2
5.7
12.4
25.3
7.8
8.1
31.5
8i
0
9.4
0
0
0
13.8
2.3
25.8
8j
10
5
0
0
0
1.4
16
0
8k
0
0
0
0
0
0
0
0
8l
24.5
0
0
0
0
0
0
0
8m
14.5
29.9
21.6
18.4
0
6.5
8.1
28.7
flumioxazin
100
100
100
100
100
100
100
100
Pre, preemergence; post, postemergence.
Synthesis
Route of Common Intermediate 15
Reagents
and conditions: (a)
NCS, AlCl3,DCM; (b) propyl chlorocarbonate, Et3N, DCM, −5 to 0 C; (c) 98% H2SO4, 65%
HNO3, −5 to 0 C; (d) NaOH, EtOH, RT; (e) K2CO3, 80% propargyl bromide in toluene, DMF; (f) SnCl2, EtOH, reflux.Pre, preemergence; post, postemergence.
Antitumor Activity
The cell lines
used in vitro antitumor activities were liver hepatocellular carcinomaHepG-2 and U-118 MG strocytic glioblastoma, and the results were listed
in Table . In order
to evaluate the tumor control efficacy, the inhibition rates of title
compounds at the concentration of 20 μM were categorized and
sorted into three levels: A (with percentage 70–100), B (with
percentage 40–70), and C (with percentage 0–40). After
calculating statistically, it turned out that percentages of antitumor
activity against HepG-2 sorted into level A, B were 59.3 and 25.9%,
respectively. Similarly, percentages against U-118 MG were 39.1 and
21.7%. This indicated that most of title compounds showed potent inhibitory
activity against HepG-2 and U-118 MG at 20 μM. Among them, compounds 7d, 7h, 7i, and 7k displayed
comparable activity against HepG-2 to positive control temozolomide
with inhibition ratios of 99.0, 99.0, 99.9, and 98.1%, respectively.
It was worth mentioning that some compounds showed better inhibitory
effect on U-118 MG than temozolomide. For instance, the inhibition
rates of 7i, 8i, and 8l were
98.2%, 92.9, and 100%, respectively. IC50 values of compounds
with inhibition ratios classified into level A were further investigated.
Compounds 7d, 7g, 7h, 7i, 7k, 7m, and 7n against
HepG-2 and all of the chosen compounds against U-118 MG (7a, 7c, 7i, 7m, 8e, 8g, 8i, and 8l) exhibited
prominent anticancer activity with their IC50 values lower
than that of temozolomide. Seven compounds (7a, 7c, 7i, 7m, 8e, 8g, and 8l) exhibited a good inhibitory effect
on both HepG-2 and U-118 MG cell lines. Especially, 7i displayed excellent antitumor activities inhibiting HepG-2 and U-118
MG with IC50 values of 2.0 and 3.8 μM, respectively.
Table 3
Antitumor Activity of Title Compounds
against HepG-2 and U-118 MG Cell Lines
20a
IC50 (μM)
log IC50 (μM)
compd
HepG-2
U-118 MG
HepG-2
U-118 MG
HepG-2
U-118 MG
SIe
7a
79.2
79.7
7.3
19.0
0.9
1.3
2.6
7b
50.1
16.6
c
7c
75.7
76.7
4.2
33.8
1.0
1.5
8.1
7d
99.0
68.6
0.5
–0.3
7e
85.9
N
5.0
2.0
7f
20.6
N
7g
77.2
37.1
2.7
0.4
7h
99.0
25.6
1.9
0.3
7i
99.9
98.2
2.0
3.8
0.3
0.6
1.9
7j
65.0
2.3
7k
98.1
46.5
0.4
–0.4
7l
Nb
19.7
7m
86.2
84.4
3.1
12.0
0.5
1.1
3.9
7n
74.2
60.8
3.5
1.9
7o
79.7
47.4
15.1
1.2
8a
48.5
21.7
8b
81.4
88.4
15.0
NDd
1.2
8c
50.0
29.6
8d
63.1
N
8e
76.5
89.4
4.2
13.2
0.6
1.1
3.2
8f
50.2
N
8g
76.0
76.5
31.7
20.2
0.4
1.3
0.6
8h
22.1
15.6
8i
65.7
92.9
2.4
0.4
8j
10.2
N
8k
14.6
27
8l
83.4
100
10.5
6.0
1.0
0.8
0.6
8m
71.6
49.4
31.5
1.5
Controlf
100
62.5
3.9
52.1
0.6
1.7
13.4
Inhibition rate
(%) at 20 μM.
Compound
showing an inhibition rate
<0.
Not tested.
Not determined.
SI = IC50 (U-118 MG)/IC50 (HepG-2).
Temozolomide.
Inhibition rate
(%) at 20 μM.Compound
showing an inhibition rate
<0.Not tested.Not determined.SI = IC50 (U-118 MG)/IC50 (HepG-2).Temozolomide.
Structure–Activity
Relationships
Antitumor Activity against
HepG-2
Percentage (%) of compounds sorted into levels A,
B, and C were counted
and showed in Figure . When R1 was fixed as the benzyl group, the introduction
of fluorine atom in the benzene ring could improve the activity and
the sequence was para > meta > ortho, concretely, shown as 8b > 8c > 8g. The similar
trend for the chlorine
atom in the benzene ring could also be seen from 8i > 8f and 8l > 8k. For the bridging
bond between benzene ring and nitrogen atom on ring A, the corresponding
sequence was PhCH3CH (8e) > Ph (8f) > PhCH2CH2 (8h) > PhCH2 (8k), which indicated that a bulky bridging
bond was
favorable to the improvement of activity. However, there was no definite
rule about the influence of bridging length on the activity, probably
because of the enhancement of activity from the electronic conjugation
effect between benzene and imidazole ring A. The broad bioactivity
of nitrogen-containing heterocycles in drugs and fine chemicals led
to the introduction of the pyridyl group into R1. Keeping
the other chemical structure consistent with each other, compounds
with pyridyl in R1 possessed higher activity regarding D series purinone derivatives and E series imidazotriazinone
derivatives had the opposite trend, which was concluded from biological
activity of some related compounds (7e, 7f, 7d, 7j, 8d, and 8k). As shown in Figure , when R was fixed as CH2CH3, all of compounds
were classified into level A, similarly, 52.6% as CH3,
50% as H, from a statistical point of view, which indicated that the
lengthening of the R carbon chain contributes significantly to the
increase of activity. D series (80%) were sorted into
level A when R2 was fixed as CH3 and CH2CH3, 75% as H. In addition, 20% sorted into level
B as CH2CH3. Therefore, D series
purinone derivatives with a longer R2 group on ring B could
exhibit better inhibition activity, for example, 7f with
CH3 was about 3-fold less potent than 7j with
CH2CH3.
Figure 3
Percentage (%) of compounds sorted into level
A, B, C.
Percentage (%) of compounds sorted into level
A, B, C.
Antitumor
Activity against U-118 MG
As shown in Figure , after statistical analysis, the bioactivity
of title compounds
indicated that shorter R and longer R2 both had higher
probability possessing high activity. For instance, compound 7a (R = H, R2 = CH2CH3) displayed
an inhibition rate of 79.9% at 20 μM and IC50 value
of 19.0 μM, which was a relatively high activity. For R1, it has the same trend regarding the effect of halogen atom
on antitumor activity as discussed above. As shown in Table , compounds 8i, 8l with chlorine atom and 7i with 2,4-difluorobenzyl
showed higher activity than their analogues with IC50 values
of 2.4, 6.0, and 3.8 μM, respectively, which were much lower
than that of temozolomide (IC50 = 52.1 μM). Surprisingly,
compounds 7e, 7g, and 7h, which
had favorable activity against HepG-2 (85.9, 77.2, and 99.9% at 20
μM, respectively) showed low activity against U-118 MG with
inhibition rate <40%. For D series purinone derivatives,
percent inhibition against HepG-2 sorted into level A accounted for
78.6%, and for 30.7% against U-118 MG. The corresponding data of E series imidazotriazinone derivatives were 58.5 and 50%.
In other words, D series showed better activity than E series regarding cancer control, which was consistent to
the hypothesis of higher anticancer activity induced by better biocompatibility
of purinone derivatives. Most of compounds were more sensitive to
HepG-2 cells than U-118 MG cells in vitro, which was summarized by
the analysis mentioned above. Compound 7c showed highest
selectivity to HepG-2 with a selectivity index (SI) of 8.05 (Table ).
Conclusions
In summary, 15 novel 1H-purin-6(9H)-ones (D) and 13 3H-imidazo[4,5-d][1,2,3]triazin-4(7H)-ones (E) were designed and synthesized.
Compound 7d showed
highest herbicidal activity against A. retroflexus in postemergence treatments. Most of the compounds showed good antitumor
activity against HepG-2 and U-118 MG cells. One of the most promising
compounds shows lower IC50 values than that of temozolomide. 7i exhibited excellent activity inhibiting both HepG-2 and
U-118 MG cell lines. The SAR study indicated that introduction of
halogen atoms, a bulky bridging bond between benzene ring and nitrogen
atom on ring A, longer R2 substituents were all favorable
to the improvement of antitumor activity. Given our study and the
good biocompatibility of purinone derivatives, 7i can
serve as a lead compound to search highly active antitumor agents
in our group.
Experimental Section
Synthetic Experiments
General Methods
1H NMR
and 13C NMR spectra were recorded at 400 MHz using a Bruker
AV 400 spectrometer (Bruker CO., Switzerland) in CDCl3 or
DMSO-d6 solution with tetramethylsilane
as the internal standard, and chemical shift values (δ) were
given in ppm. High-resolution mass spectrometry (HRMS) data were obtained
on a Varian QFT-ESI instrument. The melting points were determined
on an X-4 binocular microscope melting point apparatus (Beijing Tech
Instruments Co., Beijing, China) and were uncorrected. Reagents were
all analytically or chemically pure. All solvents and liquid reagents
were dried by standard methods in advance and distilled before use.
Ethyl 2-Amino-2-cyanoacetate (2)
Sodium thiosulfate (62.5 g, 0.352 mol) was added in batches
to a solution of ethyl cyano-(hydroxyimino)acetate (50 g, 0.352 mol)
in saturated NaHCO3 (640 mL) and H2O (150 mL)
and stirred at room temperature for 1 h. More sodium thiosulfate (62.5
g) was added every 2 h until the reaction was completely monitored
by thin-layer chromatography (TLC). The solution was extracted with
DCM and the organic extracts were dried over Na2SO4 and concentrated to give 2 (28.42 g, 63.1%)
as light yellow oil. 1H NMR (400 MHz, CDCl3):
δ 4.46 (s, 1H, CHNH2), 4.34 (q, J = 7.1 Hz, 2H, OCH2CH3), 2.03
(s, 2H, CHNH2), 1.36 (t, J = 7.2 Hz, 3H,
OCH2CH3).
A solution of triethyl orthoacetate
(13.45 g, 81.25 mmol) and ethyl
2-amino-2-cyanoacetate (9.46 g, 78.87 mmol) in 120 mL of acetonitrile
was heated under reflux for
1 h. After cooled down to room temperature, benzyl amine (8.89 g,
0.19 mmol) was added. Stirred at room temperature for overnight, solid
was precipitated out. Filtration and recrystallization with anhydrous
ethanol gave 3–1 as a white solid (8.52 g, 44.5%). 1H NMR (400 MHz, CDCl3): δ 7.42–7.31
(m, 3H, Ar-H), 7.08 (d, J = 6.8 Hz, 2H, Ar-H), 4.99
(s, 2H, NCH2), 4.72 (br s, 1H, NH2), 4.36 (q, J = 7.1 Hz, 2H, CH2CH3), 2.35 (s, 3H, CH3), 1.40 (t, J = 7.1
Hz, 3H, CH2CH3).
A solution of triethyl orthoacetate (26.23 g, 162.23 mmol) and ethyl
2-amino-2-cyanoacetate (18.45 g, 144.07 mmol) in 90 mL of acetonitrile
was heated at reflux for 1 h. After cooled down to room temperature,
aniline (15.10 g, 158.48 mmol) was added. The mixture was stirred
for 18 h at room temperature. The resulting solution was evaporated
under reduced pressure, dissolved with DCM, washed with 10% sodium
hydroxide solution followed by brine. The organic layer was separated,
dried over Na2SO4, and then the solvent was
evaporated under reduced pressure. The residue was purified by flash
column chromatography on silica gel to give 3–3 (5.85 g, 13.5%) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 7.57 (m, 3H, Ar-H), 7.40 (d, J = 7.7 Hz, 2H, Ar-H), 5.66 (s, 2H, NH2), 4.18
(q, J = 7.0 Hz, 2H, CH2CH3), 1.98 (s, 3H, CH3), 1.25 (t, J = 7.0 Hz, 3H, CH2CH3).
A solution of triethyl orthoacetate (33.66 g, 203.58 mmol) and ethyl
2-amino-2-cyanoacetate (23.70 g, 185.07 mmol) in 90 mL of acetonitrile
was heated under reflux for 1 h. After cooled down to room temperature,
1-phenyl ethylamine (25.0 g, 203.58 mmol) was added. The mixture was
stirred for 24 h at room temperature. The resulting solution was evaporated
under reduced pressure, dissolved with DCM, washed with 10% sodium
hydroxide solution followed by brine. The organic layer was separated,
dried over Na2SO4, and then the solvent was
evaporated under reduced pressure. The residue was recrystallized
with anhydrous ethanol to give 3–4 as a white
solid (6.65 g, 14.5%).1H NMR (400 MHz, CDCl3): δ 7.42–7.30 (m, 3H, Ar-H), 7.22 (d, J = 7.8 Hz, 2H, Ar-H), 5.42 (q, J = 7.1 Hz, 1H, CHCH3), 4.48 (br s, 2H, NH2), 4.30
(q, J = 7.1 Hz, 2H, CH2CH3), 2.38 (s, 3H, CH3), 1.86 (d, J = 7.2 Hz, 3H, CHCH3), 1.35 (t, J = 7.1 Hz, 3H, CH2CH3).
A
solution of ethyl 5-amino-1-benzyl-2-methyl-1H-imidazole-4-carboxylate
(4.54 g, 17.52 mmol) in ethanol (180 mL) and 0.5 M NaOH solution (180
mL) was refluxed for 10 h. Volatiles were evaporated, water (5 mL)
was added, and 5% HCl solution was added until pH = 5. The off-white
solid precipitated was filtered and washed with ice water. This material
was dried under vacuum at ambient temperature to give 4–1 (2.63 g, 65.0%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 10.98 (s, 2H, COOH), 7.32 (m, 3H,
Ar-H), 7.08 (d, J = 7.4 Hz, 2H, Ar-H), 6.02 (s, 2H,
NH2), 5.09 (s, 2H, NCH2), 2.07 (s, 3H, CH3).
5-Amino-1-benzyl-2-methyl-1H-imidazole-4-carboxylic
acid (2.63 g, 11.38 mmol) was dissolved in 10 mL of thionyl chloride
at 0 °C and stirred for 3 h. The resulting solution was evaporated
to afford the crude product 5–1 (2.01 g, 70.5%)
as a yellow solid, which was directly used for the next step without
further purification.
5-Amino-1-benzyl-2-methyl-1H-imidazole-4-carbonyl
chloride (2.01 g, 8.02 mmol) was added to a solution of 4-chloro-2-fluoro-5-(prop-2-yn-1-yloxy)aniline
(2.50 g, 11.38 mmol) and pyridine (3 mL) in dichloromethane (35 mL)
at 0 °C. The solution was stirred overnight until TLC showed
the reaction to be complete. The resulting mixture was washed with
1 M HCl, saturated NaHCO3, brine, and dried over Na2SO4. The solvent was evaporated under reduced pressure
and the resulting crude residue was purified by flash column chromatography
on silica gel to give 3–3 (2.31 g, 70%) as a white
solid. 1H NMR (400 MHz, CDCl3): δ 8.82
(s, 1H, CONH), 8.36 (d, J = 6.9 Hz, 1H, Ar-H), 7.36
(m, 3H, Ar-H), 7.16 (d, J = 10.2 Hz, 1H, Ar-H), 7.09
(d, J = 7.0 Hz, 2H, Ar-H), 4.99 (s, 2H, NCH2), 4.78 (d, J = 2.3 Hz, 2H, CH2C≡CH), 4.75 (s, 2H, NH2), 4.12 (t, J = 7.1 Hz, 1H, CH2C≡CH), 2.35 (s, 3H, CH3).
Stannous chloride dihydrate (131.82 g,
0.574 mol) was added to the solution of 1-chloro-5-fluoro-4-nitro-2-(prop-2-yn-1-yloxy)benzene
(26.29 g, 0.114 mol) in ethanol (230 mL). The mixture was heated to
70 °C and refluxed until the reaction was completely monitored
by TLC. The reaction mixture was poured into a large quantity of iced
water and neutralized with 10% NaOH solution to pH = 7–8. The
precipitated white solid was filtered and washed with EtOAc (3 ×
30 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure to afford 15 (22.28 g, 98.2%) as a brown solid. 1H NMR (400 MHz, CDCl3): δ 7.04 (d, J = 10.3 Hz, 1H, Ar-H),
6.56 (d, J = 8.0 Hz, 1H, Ar-H), 4.70 (d, 2H, CH2C≡CH), 3.63 (s, 2H, NH2),
2.56 (t, J = 2.4 Hz, 1H, CH2C≡CH).
1H-Purin-6(9H)-one (D)
Amide 6 (1.17
mmol)
was dissolved in acetic anhydride (6 mL) and triethyl orthoate (6
mL). The reaction solution was heated to 140 °C and refluxed
until the reaction was completely monitored by TCL. The resulting
solution was concentrated under reduced pressure and resulting crude
residue was purified by flash column chromatography on silica gel
to give D as a light yellow solid.
50% Sulphuric acid solution (20 mL) was added to the solution
of amide 6 (1.94 mmol) in methanol (20 mL). The reaction
solution was stirred overnight at room temperature. After being cooled
down below 0 °C, saturated sodium nitrite solution (2 mL) was
added dropwise and stirred for 2.5 h. After the reaction was completely
monitored by TLC, ice water was added, and then extracted with EtOAc.
The organic layer was washed with brine and dried over anhydrous Na2SO4. The solvent was removed under reduced pressure
and purified by flash column chromatography on silica gel to give E as light yellow solids.
Two broadleaf plants and
two grasses including amaranthpigweed (A. retroflexus), rape (B. campestris), crab grass
(D. sanguinalis), and barnyard grass
(E. crusgalli) were used to test the
herbicidal activity of title compounds. Seeds of amaranthpigweed,
barnyard grass, and crab grass were reproduced outdoors and stored
at 4 °C. Seeds of rape were bought from Institute of Crop, Tianjin
Agriculture Science Academy.
Culture
Seeds were planted in 7.0
cm-diameter disposable paper cup (250 mL) containing artificial mixed
soil. Before plant emergence, the cups were covered with plastic film
to keep them moist. Plants were grown in the green house. After 21
days, fresh weight of upground plants was measured after treatment.
Treatment
Dosage (activity ingredient)
for each compound is 1500 g per ha. Purified compounds were dissolved
in 100 μL N,N-dimethylformamide
with the addition of a little Tween 20, and then were sprayed using
a laboratory belt sprayer delivering at 750 L/ha-spray-volume. The
same amount of water was sprayed as control. Preemergency treatment:
compounds were sprayed immediately after seeds planting. Two replicates
each treatment. Postemergency: compounds were sprayed after the first
true leave expanding. The inhibition percent of upground fresh weight is used
to describe the control efficiency of compounds.
Antitumor Activity Assay
The cell
lines used for the antitumor test were liver hepatocellular carcinomaHepG-2 and U-118 MG strocytic glioblastoma. Cells were maintained
in the growth medium Dulbecco’s modified Eagle’s medium
low glucose under a humidified atmosphere of 5% CO2 at
37 °C. The cell line was refreshed according to the routine method
to reach 70–80% cell confluence. Freshly trypsinized cell suspensions
were seeded in 96-well microtiter plates at densities of 3000 cells
per well with compounds. After 72 h in culture with test compounds,
10 μL 5 mg/mL MTT was added per well and OD values of every
well were recorded using microplate reader at 570 nm. Vehicle (dimethyl
sulfoxide) was used as a control. The data were processed by excel
software to calculate the inhibition rate at different concentrations,
and the IC50 values were fitted by SPSS. Inhibiton rate
(%) = {1 – (experimental OD value – blank OD value)/(control
OD value – blank OD value)} × 100%.
Authors: E Lunt; C G Newton; C Smith; G P Stevens; M F Stevens; C G Straw; R J Walsh; P J Warren; C Fizames; F Lavelle Journal: J Med Chem Date: 1987-02 Impact factor: 7.446
Figure 1
Scheme 1
Figure 2
Scheme 2
Figure 3