Discovery of a dual tubulin polymerization and cell division cycle 20 homologue inhibitor via structural modification on apcin

Article abstract of DOI:10.1021/acs.jmedchem.9b02097

Apcin is one of the few compounds that have been previously reported as a Cdc20 specific inhibitor, although Cdc20 is a very promising drug target. We reported here the design, synthesis, and biological evaluations of 2,2,2-trichloro-1-aryl carbamate derivatives as Cdc20 inhibitors. Among these derivatives, compound 9f was much more efficient than the positive compound apcin in inhibiting cancer cell growth, but it had approximately the same binding affinity with apcin in SPR assays. It is possible that another mechanism of action might exist. Further evidence demonstrated that compound 9f also inhibited tubulin polymerization, disorganized the microtubule network, and blocked the cell cycle at the M phase with changes in the expression of cyclins. Thus, it induced apoptosis through the activation of caspase-3 and PARP. In addition, compound 9f inhibited cell migration and invasion in a concentration-dependent manner. These results provide guidance for developing the current series as potential new anticancer therapeutics.

Full text of DOI:10.1021/acs.jmedchem.9b02097

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Article
Discovery of a Dual Tubulin Polymerization and Cell Division
Cycle 20 Homologue Inhibitor via Structural Modification on Apcin
Pan Huang, Xiangyang Le, Fei Huang, Jie Yang, Haofeng
Yang, Junlong Ma, Gaoyun Hu, Qianbin Li, and Zhuo Chen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02097 • Publication Date (Web): 15 Apr 2020
Downloaded from pubs.acs.org on April 16, 2020
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Discovery of a Dual Tubulin Polymerization and
Cell Division Cycle 20 Homologue Inhibitor via
Structural Modification on Apcin
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Pan Huang,†,# Xiangyang Le, ‡,# Fei Huang, § Jie Yang,† Haofeng Yang,† Junlong Maǁ, Gaoyun
Hu,† Qianbin Li,† Zhuo Chen*,†
†
Department of Medicinal Chemistry, Xiangya School of Pharmaceutical Sciences, Central
South University, Changsha 410013, Hunan, China
‡
Department of Pharmacy, Yiyang Central Hospital, Yiyang 413000, Hunan, China
§
Center for Medical Experiments, Third Xiangya Hospital of Central South University,
Changsha 410013, Hunan, China
ǁ
Department of Good Clinical Practice, Taihe Hospital, Hubei University of Medicine, Shiyan
442000, Hubei , China
ABSTRACT: Apcin is one of few compounds that have been previously reported as a Cdc20
specific inhibitor, although Cdc20 is a very promising drug target. We reported here the design,
synthesis, and biological evaluations of 2,2,2-trichloro-1-aryl carbamate derivatives as Cdc20
inhibitors. Among these derivatives, compound 9f was much more efficient than the positive
compound apcin in inhibiting cancer cell growth, but it had approximately the same binding
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affinity with apcin in SPR assays. It is possible that another mechanism of action might exist.
Further evidence demonstrated that compound 9f also inhibited tubulin polymerization,
disorganized the microtubule network, and blocked the cell cycle at the M phase, with changed
in the expression of cyclins. Thus, it induced apoptosis through the activation of caspase-3 and
PARP. In addition, compound 9f inhibited cell migration and invasion in a concentration-
dependent manner. These results provide guidance for developing the current series as potential
new anti-cancer therapeutics.
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■ INTRODUCTION
Ubiquitination by the ubiquitin proteasome system (UPS), a post-translational modification,
plays a critical role in regulating a plethora of cellular processes, including cell death, cell cycle
progression, migration, and invasion by governing the degradation of various related proteins.1 It
is a sequential three-enzyme cascade catalyzed by activating (E1), conjugating (E2), and ligating
(E3) enzymes, and notably, E3 ubiquitin ligases provide substrate specificity. Among the E3
ubiquitin ligases, emerging evidence indicates the anaphase-promoting complex/cyclosome
(APC/C, a 1.5MDa ubiquitin ligase comprised of 19 subunits) is the major driving force
controlling the timely cell cycle process, especially in M and G1 phases.2-4 Cell division cycle 20
homologue (Cdc20) and Cdc20 homologue protein 1, Fzr1 (Cdh1), are two related but
functionally distinct activators of APC/C that are also known as APCCdc20 and APCCdh1
,
respectively. These two activator proteins regulate the ubiquitin-mediated degradation of specific
substrates, thus promoting the cell cycle forward in a unidirectional manner.5 Cdc20 activates the
APC/C during early mitosis, while Cdh1 associates with the APC/C during late mitosis and
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extends into the G1 phase. Notably, it has been demonstrated that Cdh1 is a tumor suppressor,
while Cdc20 is an essential developmental gene.6-8
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Cdc20 functions as an oncogenic factor in tumorigenesis, and it is overexpressed in multiple
types of cancer types, including hepatocellular carcinoma, breast cancer, and ovarian cancer.9-10
Its elevated expression correlates with shorter five-year survival, poorer prognosis, and
pathological tumor status in a broad spectrum of human malignancies.11-14 During the cell cycle,
as soon as all sister chromatids are attached to the bipolar spindles, Cdc20 is released from
spindle assembly checkpoint (SAC) and the formation of active APC/Cdc20 is initiated. Cdc20
binds to APC/C, induces the metaphase-anaphase transition, triggers chromosome segregation in
anaphase, and ensures mitotic exit by recruiting downstream substrates for subsequent
proteolysis of 26S proteasomes. Any defect in this process causes abnormal cell division. Since
cell arrest at mitosis is an effective strategy to induce neoplastic cell death, Cdc20 represents a
potential novel target for future cancer treatment.
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Given the vital oncogenic role of Cdc20 in the development and progression of human cancers,
its inhibitors could provide a therapeutic window in multiple human malignancies. Apart from
the majority of small-molecule pan-APC/C inhibitors,15-21 apcin is the only specific
pharmacological inhibitor of Cdc20, which occupies the destruction-box-binding pocket of WD-
40 domain, competitively prevents the recognition of substrates, and disrupts PPIs (protein-
protein interactions) within the Cdc20-substrate complex.22 However, insufficient cytotoxicity
against cancer cells leads to the critical disadvantage of apcin. A recent study reported that the
combination of the APC/C inhibitor with paclitaxel could synergistically inhibit APC/CCdc20 and
microtubule dynamics, and it might represent a new strategy to improve anti-cancer efficacy.23-24
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Microtubules are critical drug targets in anti-cancer chemotherapy. Chemically diverse
compounds interfering with microtubule dynamics are divided into two major classes: the
stabilizing agents, such as taxanes, and the destabilizing agents, such as vinca alkaloid domain
ligands and colchicine site ligands. Similar to APC/C-Cdc20 inhibition, microtubule interfering
agent (MIA) exerts its strikingly anti-tumor effects via blocking the mitotic exit, followed by late
apoptosis.25-27 However, MIAs induce G2/M arrest by SAC activation in response to
perturbations in microtubule dynamics.28 Many cancer cells have a weakened SAC and upon
protracted mitotic arrest, these cells might enter the tetraploid G1 phase state, the phenomenon of
which is referred to as mitotic slippage. Background degradation of cyclin B due to residual
APC/C activity is the most likely cause of mitotic slippage. These tetraploid cells either die from
apoptosis in G1, entering senescence, or continue to cycling, leading to genetic instability and
tumorigenesis, thus fueling tumor resistance against microtubule agents.29 Therefore, inhibiting
Cdc20 is considered to be an effective approach to overcome this side effect. Another
shortcoming of MIAs is their toxicity to normal cells, while studies have reported that cancer
cells are increasingly dependent on APC/C function. In general, the APC/Ccdc20 inhibitor can
enhance the sensitivity of MIAs to tumor cells. Meanwhile, MIA treatment could render tumor
cells more vulnerable to APC/C inhibition. Together these findings suggest that simultaneously
targeting Cdc20 and microtubules, thereby stabilizing cyclin B and preventing mitotic slippage,
may be a promising therapeutic approach in cancer treatment.
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In the present study, a series of Cdc20 inhibitors with the ability to inhibit microtubule
function using a “two-punch strategy” (strong mitotic arrest followed by blocking mitotic exit)
were synthesized.30-31 Their in-vitro effects on HepG2 cells were evaluated and their mechanisms
of action in cancer therapy were elucidated.
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■ RESULTS AND DISSCUSSION
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Chemistry Target compounds 5a-10c were synthesized in a five-step procedure as described in
Scheme 1. Commercially available metronidazole, phenylmethanol, ethanol, 2-pyridylethanol, 4-
morpholineethanol, and (1-methyl-5-nitro-1H-imidazol-2-yl) methanol were reacted with
chloroformate, and the obtained intermediates were subsequently used for the preparation of the
corresponding carbamate derivatives by ammonolysis. The reaction of the amino group of the
carbamate derivatives with chloral hydrate led to the formation of products that were converted
quantitatively into the key intermediates 4a-f by further chlorination. Treatment of 4a-4f with
pyrimidines, fused heteropyrimidines, and benzoylpyridines gave the desired 2,2,2-trichloro-1-
aryl carbamate derivatives 5a-10c (Schemes 2 and 3).
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Scheme 1. Synthesis of Key Intermediates 4a
Cl
Cl
Cl
Cl
Cl
Cl
Cl
O
O
O
NO2
(d)
(a)
(c)
(b)
R1
R1
R1
O
R1 OH
O
NH2
O
N
R1
O
N
OH
H
H
O
O
4
aReagents and conditions: (a) Et3N, 4-nitrophenyl chloroformate, DCM, rt, overnight; (b) NH3•H2O, DCM, 0-10℃, 3h; (c)
chloral hydrate, rt-100℃,18h; (d)Et3N, SOCl2, DCM, 0℃, 15min, reflux, overnight.
Scheme 2. Synthesis of 2,2,2-trichloro-1-aryl Carbamate Derivatives 5a-8ma
2
3
4
2
5a,
5c,
5e,
7a,
7c,
5b,
5d,
R =R =R =H
R =CH3 R3=R4=H
Cl
Cl
R2
N
Cl
Cl
N
NH2
2
2
3
4
R =OCH3 R3=R4=H
R =OH R =R =H
N
N
O
Cl
Cl
Cl
R3
R4
N
N
N
O
N
2
3
4
2
3
4
(e)
(e)
O
N
N
H
5f,
R =NH2 R =R =H
R =Cl R =R =H
H
O2
Cl
N
2
4
3
2
4
3
O
N
N
H
7d
7b,
R =R =Cl R =H
R =R =Cl R =CH3
H
O2N
2
4
3
R =R =Cl R =NH2
5a-f,7a-c
N
N
5
5
5
6a,
6d,
6b,
6c,
R =
R =
R =
Cl
N
Cl
Cl
O
Cl
N
N
Cl
Cl
Cl
N
O
N
N
R5
5
5
N
N
O
N
N
R =
6e,
R =
O
N
H
H
O2N
N
N
H
O2N
6a-e
6
7
6
7
8a,
8c,
8e,
8h,
8k,
8b,
8d,
R =Cl R =H
R =dimethylamino R =H
Cl
R7
4a
Cl
Cl
6
7
6
7
O
O
R =benzamidyl R =H
R =benzamido R =H
N
N
N
6
6
7
6
R =NH2 R7=Cl
R =R =NH2
R =NH2 R7=F
8f,
8g,
N
N
H
O2N
R6
6
7
6
7
6
7
N
8i,
8j,
R =Cl R =NH2
R =Cl R =F
R =OBzl R =NH2
8a-m
6
7
6
7
6
8l,
8m,
R =R =Cl
R =C=O R =formamido
R =NH2 R7=H
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aReagents and conditions: (e) aromatic amine, DCM/MeCN, rt-60℃, overnight.
Scheme 3. Synthesis of 2,2,2-trichloro-1-aryl Carbamate Derivatives 9a-10ca
4
5
6
7
8
9
R7
Cl
Cl
Cl
Cl
Cl
Cl
Cl
O
N
O
6
7
N
(e)
(e)
9a,
R =NH2 R =Cl
O
N
N
O
N
H
H
R6
N
4b
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9a
R7
Cl
6
7
Cl
9b,
9d,
R =NH2 R =Cl
Cl
Cl
Cl
Cl
O
N
O
6
7
N
R =NH2 R =F
N
N
O
N
N
O
N
Cl
H
R6
H
N
N
N
O2N
O2N
9b,9d
4c
4d
6
7
R7
Cl
Cl
Cl
9c,
9e,
R =NH2 R =Cl
Cl
Cl
Cl
Cl
Cl
O
O
N
6
7
R =NH2 R =F
N
O
N
N
O
O
N
H
H
R6
N
9c,9e
6
7
R7
N
R9
N
9f(005),
R =NH2 R =F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
N
Cl
Cl
O
O
6
7
O
O
O
O
O
9g,
N
N
N
R =R =NH2
N
or
or
N
N
8
9
N
10a,
O
N
O
N
N
N
R =NH2 R =H
H
H
H
R6
R8
N
N
4e
4f
9f-g
10a
6
7
9h(705),
R =NH2 R =F
R7
N
R9
N
Cl
Cl
6
7
Cl
Cl
N
Cl
Cl
Cl
Cl
Cl
9i,
R =NH2 R =NH2
O
O
8
9
10b,
10c,
R =NH2 R =H
N
O
N
N
O
N
N
N
N
O
N
H
H
R6
R8
8
9
H
R =OH R =H
N
9h-i
10b-c
aReagents and conditions: (e) aromatic amine, DCM/MeCN, rt-60℃, overnight.
In Vitro Anti-proliferative Activities and SAR Analysis In Table 1, the in vitro anti-
proliferative activities of target compounds against a panel of seven human cancer cell lines:
MCF-7 (breast cancer), A375 (melanoma), A549 (lung cancer), HepG2 (hepatocellular
carcinoma), Hela (cervical cancer), Ovcar-3, and Caov-3 (ovarian cancer) were summarized
using apcin as the reference compound.
Table 1. In Vitro Cell Growth Inhibitory Effects of Compounds
IC50a, μM, ±SD
Cpd
MCF-7
A375
A549
>300
HepG2
>300
Hela
Ovcar-3
>300
Caov-3
>300
5a(Apcin) >300
193.3±13
37.5±5.7
44.1±20.2
>300
220.8±17.9
94.7±6.7
5b
5c
5d
5e
151.5±37.7
186.0±4.4
>300
27.3±3.7
263.8
>300
31.2±8.4
164.5±3.8 154.6±14.4
198.3±6.7 157.4±5.6
79.8±26.8 127.6±6.2
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
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9
5f
>300
20.0±9.1
5.3±5.1
14±3
94.9±10.3
>300
>300
>300
>300
>300
6a
6b
6c
6d
6e
7a
7b
7c
7d
8a
8b
8c
8d
8e
8f
>300
>300
>300
>300
>300
99.6±2.9
141.2±0.2
>300
47.5±25.3
84.3±25.7
>300
22.7±2.7
26.0
9.1±0.7
38.9±0.7
>300
90.1±4.4
72.6±20.3
12.5±0.3
37.3±25.4
>300
109.0±0.1 95.2±2.1
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46.8±5.6
>300
61.4±3.9
>300
81.1±2.1
>300
>300
>300
213.9±15.9
15.6±0.1
27.1±21.0
17.5±1.1
63.2±0.9
>300
32.4±0.8
114.0±5.6
20.6±1.9
159.5±5.0
>300
8.1±0.4
11.9±0.5
14.0±0.2
34.2±1.1
>300
49.5±16.2
23.3±0.1
17.0±0.9
25.0
22.5±3.2
31.2
184.3±30.9 13.6±3.1
31.9±4.2
56.5±2.9
>300
25.8±3.1
25.6±6.1
>300
21.1±2.2
19.2±1.0
109.7
>300
>300
>300
27.3±14.1
>300
191.3±58.2 >300
243.7±30.5 >300
159.8±6.5
>300
>300
264±9.3
>300
>300
225±10.6
243.2
96.0±2.1
>300
>300
148.2±47.7 33.2±16.0 71.8±8.9
>300
245.7±2.7
159.8±14.0 7.3±1.2
218.3±54.3
120.3±1.5
1.9±0.3
130.7±23
>300
143.9±7.8
>300
>300
170.3±15.5 >300
19.7±3.6
0.6
8g
8h
8i
0.8
0.5±0.1
>300
3.6±0.7
1.5±0.1
>300
1.2
>300
>300
>300
57.3±3.9
>300
62.1±16.6
89.1±54.2
218.0±4.2
>300
120.9±1.1
>300
>300
55.1±13.0 >300
>300
>300
8j
146.5±4.7
72.8±4.9
>300
71.1±3.5
54.6±2.6
>300
>300
>300
>300
8k
8l
68.5±27.7
>300
142.3±5.2
>300
162±76.4
>300
224.2±40.1
>300
8m
9a
9b
9c
9d
9e
9f
>300
181.7±38.3 >300
>300
>300
>300
>300
138.1±0.2
53.3±0.4
87.2±34.2
1.5±0.1
0.8
>300
78.5±1.1
61.4±13.7
>300
>300
37.9±2.6
70.4±2.6
65.0±31.7
64.3±11.9
1.2±0.8
43.8±1.4
59.0±11
3.8±0.1
1.4
51.2±0.9
152.0±18.5
0.2
125.3±92.9 59.7±6.0
131.7±1.6 92.9±3.9
2.7±0.5
1.8±0.1
0.7±0.1
0.5±0.1
0.5±0.2
1.5±0.5
0.6±0.4
0.5±0.1
1.7±0.4
0.3±0.1
0.6
0.9
0.2±0.1
0.6
0.3±0.2
1.5±0.1
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9g
125.4±20.8
1.0±0.2
94.7±46.4
0.4±0.1
300
47.2±18.1
3.5±0.6
270.0
8.6±2.1
1.0
47.5±11.9
3.0±0.3
219.7±90.3
<10
11.5±6.7
0.8±0.1
10.5±6.0
1.1±0.1
9h
9i
135.6±112
19.3±10.9
161.6±53.4
>300
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30.9±11.5 29.7±4.0
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>300
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81.3±11.3 109.9±37.5
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aIC50 = compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean ± SD from
the dose−response curves of at least three independent experiments.
Among the (2,2,2-trichloro-1-(pyrimidinylamino)ethyl) carbamate analogues, dichloro-
substituted pyrimidine derivatives 7a-d showed potent anti-proliferative effects against all the
tested tumor cell lines. By contrast, the corresponding non-substituted derivative 6a exhibited an
inhibitory effect identical to that of apcin against most of the cell lines. Intriguingly, the activity
of 6a against the A375 cell line was noticeably improved. The substitution on the pyrimidines
displayed a strong influence on activity, in general of which, electron-withdrawing substituents,
such as carbonyl (5d) and chlorine (5f), showed dramatically reduced activities versus the most
active compound, while the methyl-substituted analogue 5b and methoxy-substituted analogue
5c resulted in considerably better anti-proliferative activity against the seven tumor cell lines. On
the other hand, the amino-substituted analogue 5e led to a decrease of activity, the result was
contrary to our findings. In addition, pyrimidines with bulky substituents suggested improved
potency. For the compounds bearing benzoylpyridines, such as 2-aminoquinoline (6b), 1-
aminoisoquinoline (6c), or 3-aminoiso quinoline (6d), which were larger than the unsubstituted
pyrimidines, increased activities were also observed.
In the case of the fused heteropyrimidine derivatives, a superiority of the purines over the
pyrimidines was observed in the tested cell lines. Among all the derivatives, the greatest activity
occurred when fluorine was located at the purine moiety (8g, 9f and 9h). The insertion of a
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mono-group at the C-6-position of purines generally resulted in a marked loss in anti-
proliferative potency (8a, 8b, 8c, 8d and 8m with IC50 >300 μM). The introduction of additional
substituents into the purines contributed to the improved anti-proliferative potency. Chloro-
substituted analogue 8e, amino-substituted analogue 8f, and fluoro-substituted analogue 8g
showed higher potency than 8m. Likewise, chloro-substituted analogue 8k displayed higher
activity than 8a.
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Finally, we explored the effect by replacing the metronidazole with other groups. However,
compounds with different replacements showed no significant changes in activity (8e vs 9a vs 9b
vs 9c; 8g vs 9d vs 9e vs 9f vs 9h), demonstrating that metronidazole seemed to be an
unnecessary pharmacophore for the anti-proliferative potency of 2,2,2-trichloro-1-aryl carbamate
derivatives. Moreover, the most potent compound identified in this study was the 2-
morpholinoethyl (1-(6-amino-2-fluoro-9H-purin-9-yl)-2,2,2-trichloroethyl)carbamate derivative
9f, which was at least 1000-fold more active than the reference compound apcin among the
seven cancer cell lines. The comparison of pyrazolopyrimidine analogue 10a with 10b clearly
demonstrated, once again, that morpholine was the preferred structural element and contributed
to potency.
In general, fluoro-substituted analogues exhibited the most effectively anti-proliferative
activity. Moreover, dichloro-substituted pyrimidine derivatives showed moderate potency and a
high structural similarity to apcin. The data obtained for seven cell lines were in agreement,
except that HepG2 cells were significantly sensitive to the compounds; thus, HepG2 cells were
selected for further investigation.
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Studies on the Binding of Cdc20 with the Apcin Analogs
To gain insights into the
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interactions between Cdc20 and the selected potent small molecules (7b, 7d, 9f, 8g and 9h), a
SPR based experiment for Cdc20 was performed for subsequent evaluation. Surface plasmon
resonance (SPR) is an optical biosensor detection method that measures the change in the
refractive index at the surface interface that occurs during a binding event. It is used to
investigate the binding efficiency, as it provides information on the affinity and kinetics of
molecular interactions, and the affinity associated with the interaction may be an indicator of the
binding process. The results revealed that some of the selected compounds bound reversibly to
Cdc20 with clear association and dissociation phases. The relatively weak binding affinity of
apcin (KD=123 μM) was consistent with its anti-proliferative potency (Figure 1A). Among all
the compounds, 7d showed a 2- to 3-fold increased binding affinity (KD=49 μM) to Cdc20
compared to apcin (Figure 1B), while 9f suffered a decrease in affinity (KD=119 μM) compared
to 7d (Figure 1C). Moreover, 8g exhibited a similar binding affinity (KD=161 μM) compared to
9f and apcin (Figure S1). The association and dissociation constant of 7b and 9h could not be
determined due to their limited solubility (Figure 1D).
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Figure 1. Sensorgrams for the interaction of (A) apcin, (B) 7d, (C) 9f (005), (D) 7b and 9h (705) with Cdc20. Molecules were
tested in a dilution series starting at 200 μM. The steady state values were calculated and plotted against the concentration. A 1:1
binding model was directly fitted to the sensorgrams and a single binding site model was fitted to the data to calculate KD.
Subsequently, compounds 7d and 9f were performed for a molecular modeling study, a
computational tool which was previously used to determine the apcin binding site of Cdc20 as
well as to elucidate the potential interactions of 7d and 9f with Cdc20. Overall, the overlapped
3D model revealed that 7d and 9f, two close analogues of apcin, were Cdc20 inhibitors, both of
which bound in similar orientations to apcin and were well accommodated within the apcin site.
The hydrophobic trichloromethyl groups of 7d and 9f were found to be buried in the pocket.
However, the metronidazole, morphine, and pyrimidines groups were more peripheral. The
dichloro-substituted pyrimidine of 7d and fluorine-substituted adenine of 9f could successfully
overlap with the binding site of the non-substituted pyrimidine portion of apcin. The
metronidazole moiety of 7d and the morphine of 9f were positioned facing the solvent.
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In brief, both 7d and 9f underwent identical conformational changes. As for predicting the
detailed molecular interactions of 7d and 9f, it was suggested that the phenyl ring of Tyr207
established two CH/π interactions with the linker of both 7d and 9f (Figures 2A and S2). The
amino group of 7d remained to form two hydrogen bonds with backbone atoms from Asp177.
However, the amino group of 9f could only form one hydrogen bond with Asp177 (Figure 2B),
illustrating the higher binding affinity of 7d with Cdc20 compared with 9f.
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The disparate conclusions between SPR studies and anti-proliferative effects prompted us to
perform additional cellular or molecular biological experiments on compounds 7d and 9f using
HepG2 cells.
Figure 2. Predicted docking model for 7d (A) and 9f (B) bound to Cdc20 crystal structure (PDB ID: 4n14). Structures of 7d, 9f
(red skeleton) and apcin (green skeleton with oxygen in red, nitrogen in blue, and proton in gray) were shown as ribbon diagrams
with critical residues labeled in black and carbon atoms in gray.
7d Increased the Protein Level of Substrates Dependent of Cdc20 Rather Than Cdh1 and
9f Improved Cyclin Expression Cdc20 is critical for its ability to trigger the ubiquitination and
degradation of specific substrates during the cell cycle and apoptosis. Cyclins A and B are
critical for mitosis and function as substrates of both Cdc20 and Cdh1, respectively. However,
cyclin B is mainly controlled by Cdc20, while cyclin A is a Cdh1 substrate. Moreover, the pro-
apoptotic molecule Bim is a Cdc20 specific substrate.32 Therefore, the influence of 7d and 9f on
the expression of cyclin B, cyclin A and Bim were examined. The results revealed that treatment
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with compound 7d (30 µM) elevated the Bim level similarly to that in apcin-treated (100 µM)
cells. Cyclins A and B were reported to regulate the mitotic cell cycle, and 7d-treated cells
showed a decrease in Cyclin B degradation (Figure 3A). The expression of Aurora A and Skp2
(Cdh1 specific substrates) was examined to validate the affection of 7d to Cdh1, although there
were no differences in the levels of these two proteins (Figure 3B). The binding affinity between
7d and Cdc20, as well as the increase in substrates caused by 7d, prompted us to elucidate the
exact mechanism behind the delay in mitotic exit. Thus, we followed the degradation of key
Cdc20 proteins, such as cyclin and Bim, in HepG2 cells. As shown in Figure 3C, 7d markedly
increased the stability of Bim and Cyclin B1, which peaked from 2 to 8 h, while the degradation
of Aurora A significantly increased in HepG2 cells. These results reveal that 7d (30 µM)
interfered with the metaphase-to-anaphase transition and led to mitotic delay. However,
treatment with 9f (0.3 µM) seemed to have no effect on the Bim protein level, which might be
due to its low concentration, but a significant increase in cyclins A and B expression was noted,
indicating the efficacy of 9f and 7d in arresting cells in mitosis.
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Figure 3. (A) Cdc20 substrates were analyzed to evaluate cell cycle arrest and apoptosis. Endogenous levels of PHH3, Bim,
Cyclin B1 were determined. (B) Protein levels of Aurora A and SKP2 were measured by Western Blot. The results displayed that
compounds had no effect on levels of Cdh1 substrates. (C) The expression of cyclin B1, Aurora A, Bim and Cleaved PARP were
measured at 1 h, 2 h, 4 h, 8 h and 12 h after the addition of 30 μM 7d.
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7d and 9f Blocked Mitotic Exit in HepG2 Cells The inhibition of substrate degradation did
not strictly correlate with the cell proliferation inhibitory activity of these compounds. Thus,
compounds 7d, 9f, and apcin were evaluated by measuring the mitotic cell cycle marker,
phosphohistone H3 (PHH3) at serine 10 (Figure 4A). Figure 4B demonstrated a dose-dependent
increase of fluorescence intensity of nuclear phosphohistone H3 levels in the compound-treated
cells compared to cells treated with solvent only. The results (Figure 4C) showed that 7d (30
μM) and 9f (100 μM) induced an increase of phosphohistone H3 positive cells compared to
vehicle-treated controls in HepG2 cells for the duration of the treatment (6.9% and 10.0% to
3.8%, respectively). The positive compound, apcin, caused an approximately 2.2-fold increase of
PHH3-positive cells compared with the DMSO control. Likewise, a high protein level of
phopho-histone H3 in treated cells compared to the control was observed (Figure 4D). These
results suggested that 7d and 9f were more potent than apcin in arresting cells at mitotic exit, and
9f was the most potent mitotic blocker.
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Figure 4. 7d, 9f blocked mitotic exit. (A) Cells were treated with DMSO (line 1), 100 μM apcin (line 2), 30 μM 7d (line 3) and
0.3 μM 9f (line 4) for 48 h, p-Histone H3 (Ser 10) were stained for immunofluorometric analysis, representative images were
observed. Cells stained with a specific antibody (red) against phosphohistone H3 (serine 10) and nuclei stained with DAPI (blue)
were visualized. (B) Dose-effect curves of 7d and 9f were constructed. The relative fluorescence values were measured by
comparing to the control group. (C) PHH3-positive cells (red) were counted in randomly selected fields, the histogram showed
the mean number of p-histone H3 (Ser 10)-positive cells in the entire population (%). Cells were counted using Image J software.
Statistical analysis was performed by SPSS, each treatment group was compared to the control group: (***) P < 0.001, (**) P <
0.01, (*) P < 0.05. (D) PHH3 levels were measured by western blot at 24 h after the addition of compounds. The reproducibility
of the results was confirmed by at least two separate experiments.
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Compound 9f Disrupted the Organization of the Cellular Microtubule Network As 9f was
much more effective in blocking the cell cycle than in binding to Cdc20, it was suggested that
another mechanism of action might exist in 9f. Since the disruption of microtubule dynamics
leads to the formation of aberrant mitotic spindles that are unable to align the chromosomes into
the metaphase plate, thereby leading to the accumulation of cells arrested at mitosis, we further
examined the inhibitory effects of 9f on microtubule organization by immunofluorescent staining
in HepG2 cells. As shown in Figure 5, the microtubule networks in vehicle-treated cells
displayed a normal arrangement with fibrous microtubules extending throughout the cell nucleus,
providing cellular structure and shape, and bipolar spindle formation was observed. Meanwhile,
paclitaxel formed multipolar spindles and bundled microtubules that resulted in aggressive
polymerization.33-34 On the contrary, after exposure to 9f at 0.3 μM for 48 h, microtubule
organization in the cytosol was disrupted and showed condensed chromosomes, indicating that
9f induced disruption of the microtubule network. These results clearly demonstrate that 9f
exhibited characteristics of tubulin polymerization inhibitors and had a mechanism of action
different from that of stabilizing agents such as paclitaxel.
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Figure 5. Effects of 7d and 9f on the cellular microtubule network were visualized by immunofluorescence. HepG2 cells were
treated with vehicle control 0.1% DMSO, 30 μM 7d, 0.3 μM 9f and 0.3 μM paclitaxel for 48 h. Then, cells were fixed and stained
with anti-α-tubulin antibody (red), and counterstained with DAPI (blue). Detection of the fixed and stained cells was performed
using a microplate reader.
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To further investigate the effects of 9f on the assembly kinetics of tubulin in vitro, a tubulin
polymerization assay was carried out with the negative control paclitaxel. Accordingly, 9f
inhibited the rate and extent of the assembly of tubulin in a concentration-dependent manner,
indicated that 9f was a potent inhibitor of tubulin assembly (Figure 6). Under similar conditions,
paclitaxel promoted the assembly of tubulin as reported.
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Figure 6. Effect of 9f on in vitro tubulin polymerization was tested. Polymerization of purified tubulin was performed in a cell-
free assay. Tubulin protein was incubated at 37°C in a reaction buffer exposed to vehicle control or test compounds at the
indicated concentrations. Absorbance at 340 nm was monitored at 37 °C every 30 s for 60 min.
Results from the analysis of structure−activity relationships for the derivatives indicated that
incorporating fluorine at the C-2-position in the adenine ring produced highly potent compounds.
To further elucidate how compound 9f interacted with tubulin, the potential binding mode for 9f
was investigated at three different binding sites occupied by the substituted 5- or 6-membered
heterocyclic ring in the tubulin dimer using the molecular operating environment (MOE) 2014
platform.35-36
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The illustration for the close view of the potential binding pose of 9f and 01G (native ligand of
tubulin PDB: 2R75) was shown in Figures 7A and S3. Generally, 9f (blue stick) overlapped
well with 01G (green stick) in the same “L” shape. The 2-fluoroadenine ring of 9f overlapped
well with the adenine ring of 01G and the carbamate overlapped well with the phosphate of 01G.
Similarly, the potential hydrogen bonds were formed between 9f and Phe179, Asn162, Glu135,
Ala67, Thr105, and Gly104. These hydrogen bonds stabilized the interaction of 9f with the
binding pocket. This conclusion was consistent with the X-ray structure of tubulin in complex
with the negative ligand, 8Z8 (PDB: 5NJH) and A9Q (PDB: 5OSK), respectively.37In the
binding mode of Figures 7B and S4, 9f (purple stick) was in close proximity to the native ligand
8Z8 (green stick). The adenine occupied the site where the trifluoro-benzene ring in 8Z8 was
bound, while the trichloromethyl, which was large steric hindrance of 9f crossed through the
adenine ring in 8Z8. Two residues, Tyr 210 and Tyr 224, contributed to the strong binding
affinity of 8Z8 to tubulin. Moreover, arene-arene interaction between the adenine ring of 9f and
Tyr210, as well as arene-H interaction between the carbon with trichloromethyl substitution of 9f
and Tyr224, were also observed. In addition, the binding mode between 9f and tubulin was
consistent with the observation in 5OSK. Furthermore, key amino acids Cys241 and Lys352
formed an arene-H interaction and hydrogen bond with A9Q, respectively. It was noteworthy
that because the linker of 9f projected deeper into the pocket, 9f had an additional H bond with
the surrounding amino acid, Met259 (Figure 7C and S5).
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In conclusion, compound 9f inhibited tubulin polymerization in a concentration-dependent
manner. The attempts to dock the ligands into the binding site of substituted 5- or 6-membered
heterocyclic pointed to the importance of 2-fluoroadenine for the high potency of 9f.38
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Figure 7. Predicted binding mode of 9f (green stick) with three different tubulin complex, (A) PDB code: 2R75, (B) PDB code:
5NJH and (C) PDB code: 5OSK, surrounding amino acid residues were labeled. Hydrogen bonds were shown as dotted orange
lines.
7d and 9f Induced Cell Apoptosis in HepG2 Cells There is evidence to suggest that execution
of cell cycle blockade may induce simultaneous cellular apoptosis. We determined whether 7d
and 9f could trigger cell apoptosis. The Annexin V-FITC/PI assay was conducted in HepG2 cells
which were treated with 100 μM apcin, 30 μM 7d, and 0.3 μM 9f for 48 h. A significant increase
in the number of apoptotic cells was found after treatment with each compound (Figure 8A, B).
As shown in Figure 8C, 7d and 9f induced 34.58 and 42.06% apoptosis at 100 and 0.3 μM,
respectively, and apcin caused 14.7% of cells to become apoptotic at 100 μM. In the untreated
control, only 9.14% of cells underwent apoptosis. These results indicate that 7d and 9f
dramatically stimulated cell apoptosis in HepG2 cells, which was consistent with the inhibition
of cell proliferation. In order to investigate whether mitotic arrest contributed to the regulation of
apoptosis, the levels of the anti-apoptotic BCL-2 family members were examined. The treated
cells prompted elevated levels of cleavage of PARP and caspase-3, respectively, with no
alternation in the BCL-2 level (Figure 8D).
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Figure 8. Effects of 7d and 9f on apoptosis were tested. (A) Cell apoptosis was conducted in HepG2 cells treated with 100 μM
apcin (line 2), 30 μM 7d (line 3), 0.3 μM 9f (line 4) and the solvent control (line 1). (B) Representative immunofluorescence
images of HepG2 cells showed different phenotypes. The reproducibility of the results was confirmed by at least two separate
experiments. (C) Cells were seeded in 6-well plates and treated with compounds for 48 h. Cells were harvested, washed with PBS
and resuspended in binding buffer containing PI and FITC-conjugated anti-Annexin V antibody. Apoptosis was analyzed using a
flow cytometer. (D) Cells were treated for 48 h. Cell lysate was analyzed to investigate apoptotic cell rate. Endogenous protein
levels of caspase3, cleaved caspase3, cleaved PARP and Bcl-2 were determined by Western Blot.
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9f Inhibited Cell Migration and Invasion Small molecules with anti-microtubule potency or
Cdc20 inhibitory effect have been reported to interfere with tumor cell migration and invasion.
To investigate the inhibition of migration by 9f, a scratch assay was used to detect the migratory
activity in HepG2 cells after 9f treatment. Results from wound healing assays suggested that 9f
remarkably suppressed cell migration in a dose-dependent manner (Figures 9A, B and S6). To
further explore whether 9f played a key role in the regulation of hepatoma carcinoma cell
motility, an invasion assay was performed in HepG2 cells treated with 0.3 μM 9f for 48 h. It was
demonstrated that compound 9f significantly retarded the penetration of HepG2 cells through the
Matrigel-coated membrane, suggesting that 9f inhibited cell invasion in HepG2 cells (Figure 9C,
D). Altogether, 9f inhibited cell motility in hepatoma carcinoma cells.
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Figure 9. 9f inhibited cell migration and invasion in HepG2 cells. (A) Cell migration was detected using wound healing assay in
untreated cells (Left panel) and cells treated with 0.3 μM 9f (right panel) during 48 h. (B) Quantitative results were illustrated. (C)
Left panel: inhibitory effect on invasion in untreated HepG2 cells was determined by Transwell invasion assay, right panel: cells
treated with 0.3 μM 9f. (D) Quantitative results were illustrated.
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■ CONCLUSIONS
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Cdc20 has been reported to be a critical factor for tumorigenesis, and depleting endogenous
Cdc20 in various cancer cell lines leads to a mitotic arrest, followed by aberrant proliferation or
apoptotic death.39 Consistently, evidence from several conditional Cdc20 knockout mouse
models suggests that ablation of Cdc20 could completely repress tumors in vivo via apoptosis.40
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By targeting Cdc20, a series of 2,2,2-trichloro-1-aryl carbamate derivatives (5a-10c) were
designed, efficiently synthesized, and evaluated in cellular assays. It was reported that low
concentrations of apcin did not effectively stabilize the APC/C substrates, which might have
been related to the relatively weak interaction between apcin and Cdc20, or the substrates could
be recruited to the APC/C through other mechanisms. In this study, 7d showed higher affinity to
Cdc20 than apcin in the SPR study. Correspondingly, treatment of 7d led to mitotic disturbance
and apoptosis, indicating that it might be a promising lead for discovering more potent Cdc20
inhibitors. However, the most potent compound, 9f, exhibited almost the same binding affinity
with Cdc20 as apcin, and further exploration indicated that the nanomolar potency of 9f against
multiple cancer cell lines was due to its additional inhibition of tubulin polymerization. As two
critical targets acting at different steps of mitosis, microtubules and Cdc20 are closely linked in
function, and the combination of MIAs with APC/C inhibitors seems to be an efficient way to
trigger mitotic cells death. Consistently, the implications of paclitaxel/ProTAME combinatorial
treatment have been reported to reduce chemoresistance, significantly down-regulate
chromosomal instability, and reactivate apoptosis in ovarian cancer and multiple myeloma.
Compound 9f arrested cells in the M phase of the cell cycle, induced HepG2 cell apoptosis, and
disrupted cellular microtubules. Mechanistic considerations suggested that the blockade in M
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phase of cell cycle was associated with alterations in the expression of cyclins, and the induction
of apoptosis was related to activation of caspase-3 and PARP.
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In summary, modification of apcin resulted in the discovery of a novel anti-tumor agent as
tubulin polymerization and Cdc20 inhibitor. The newly developed compound 9f showed marked
biological activity, and it has the potential for further development as an anti-mitotic agent.
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■ EXPERIMENTAL SECTION
General Method Melting points were measured in open capillary tubes on a WRS-2 micro-
computer melting point apparatus. All chemical reagents were purchased and used without
further purification. All reactions were carried out under an atmosphere of dried nitrogen or
argon. Chromatograms were visualized by ultraviolet illumination or by exposure to iodine
vapors. Nuclear magnetic resonance (NMR) spectroscopy was carried out on Bruker
AVANCEШ-400 and AVANCEШ-500 NMR. Chemical shifts were expressed in parts per
million (ppm, δ) downfield from tetramethylsilane (TMS) with coupling constants in hertz (Hz).
Multiplicity was described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and
broad (br). High-resolution mass spectra (HRMS) were recorded using an Advion Expression
CMS mass spectrometer coupled with an Agilent 1260 analytical HPLC. High-performance
liquid chromatography (HPLC) analysis of all final compounds for biological testing were
conducted on an Agilent 1260 Series HPLC with an Agilent Extend-C18 column (150×4.6 mm,
5 μm). All final compounds for biological evaluation were analyzed to achieve a minimum of 95%
purity. Compound names were derived from the structures using ChemDraw Ultra 14.0.
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The readily available substituted ethanol used as the starting material was reacted with 1.2
equivalents of 4-nitrophenyl chloroformate overnight and 1.2 equivalents of anhydrous
triethylamine as a base in anhydrous dichloromethane at room temperature. Product carbamate
was reacted with 28% aqueous ammonia for 3-24 h. Compounds 4a-f were synthesized by the
treatment of chloral hydrate with carbamate at 80-100℃, adding ethyl acetate, and ultrasonically
forming a turbid liquid. The compounds were obtained by suction filtration. The intermediates
were ideally poised to undergo chlorination with chlorosulfoxide by refluxing overnight, and
then removing residual thionyl chloride until the reaction was completed and subjecting it to a
substitution reaction with aromatic amine in the presence of Cs2CO3 in acetone. The turbid
mixture was filtered and concentrated in vacuo, purification by flash column chromatography on
a silica gel to afford the final compounds.
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2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl(2,2,2-trichloro-1-((4-methylpyrimidin-2-yl)
amino)ethyl)carbamate (5b) Following general procedure in Scheme 1 furnished 5b as white
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solid. Yield: 21.42%, mp 209.0~209.9℃, HPLC: 97.19%. H NMR (500 MHz, DMSO-d6):
δ8.27 (d, J = 5.0 Hz, 1H), 8.00 (s, 1H), 7.97 (d, J = 8.8 Hz, 1H), 6.97 (d, J = 9.5 Hz, 1H), 6.73 (d,
J = 5.0 Hz, 1H), 6.60 (t, J = 9.2 Hz, 1H), 4.56-4.46 (m, 3H), 4.35 (dd, J = 10.7, 5.7 Hz, 1H), 2.39
(s, 3H), 2.31 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δ160.69, 155.24, 152.06, 138.88, 133.53,
112.85, 102.88, 70.12, 63.46, 45.90, 24.10, 14.37. HRMS (ESI) m/z calcd for
[C14H16Cl3N7O4+H]+: 452.0329, found: 452.0401.
(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl(2,2,2-trichloro-1-((4-methoxypyrimidin-2-
yl)amino)ethyl)carbamate (5c) Following general procedure in Scheme 1 furnished 5c as
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yellow solid. Yield: 32.66%, mp 81.6~83.3℃, HPLC: 98.63%. 1H NMR (500 MHz, DMSO-d6):
δ8.14 (d, J = 5.7 Hz, 1H), 8.01 (d, J = 16.7 Hz, 2H), 7.05 (s, 1H), 6.57 (d, J = 13.3 Hz, 1H), 6.28
(d, J = 5.7 Hz, 1H), 4.51 (dd, J = 17.4, 4.3 Hz, 3H), 4.38-4.33 (m, 1H), 3.85 (s, 3H), 2.39 (s, 3H).
13C NMR (126 MHz, DMSO-d6): δ170.22, 160.87, 158.59, 155.27, 152.06, 138.88, 133.51,
102.59, 99.44, 70.35, 63.48, 53.74, 45.89, 14.36. HRMS (ESI) m/z calcd for
[C14H16Cl3N7O5+H]+: 468.0278, found: 468.0370.
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2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl(2,2,2-trichloro-1-((6-oxo-1,6-dihydropyrimidin-
2-yl)amino)ethyl)carbamate (5d) Following general procedure in Scheme 1 furnished 5d as
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yellow solid. Yield: 16.24%, mp 192.3~193.2℃, HPLC: 98.39%. H NMR (500 MHz, DMSO-
d6): δ11.14 (s, 1H), 8.71 (d, J = 8.4 Hz, 1H), 8.01 (s, 1H), 7.64 (d, J = 6.1 Hz, 1H), 7.11 (d, J =
9.3 Hz, 1H), 6.49 (t, J = 9.1 Hz, 1H), 5.72 (d, J = 6.1 Hz, 1H), 4.55-4.48 (m, 3H), 4.37 (dd, J =
7.5, 5.0 Hz, 1H), 2.41 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δ162.08, 155.74, 155.43, 153.95,
152.10, 138.89, 133.56, 105.94, 101.66, 69.29, 63.28, 46.00, 14.41. HRMS (ESI) m/z calcd for
[C13H14Cl3N7O5+H]+: 454.0122, found: 454.0200.
2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl(1-((4-aminopyrimidin-2-yl)amino)-2,2,2-
trichloroethyl)carbamate (5e) Following general procedure in Scheme 1 furnished 5e as yellow
1
solid. Yield: 28.69%, mp 104.2~105.0℃, HPLC: 98.55%. H NMR (500 MHz, DMSO-d6):
δ8.02 (s, 1H), 7.95 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 5.3 Hz, 1H), 6.64 (s, 2H), 6.53 (t, J = 9.0 Hz,
1H), 6.15 (s, 1H), 5.87 (d, J = 5.1 Hz, 1H), 4.56-4.46 (m, 3H), 4.32 (d, J = 11.4 Hz, 1H), 2.40 (s,
3H). 13C NMR (126 MHz, DMSO-d6): δ164.54, 160.64, 156.04, 155.13, 152.07, 138.88, 133.52,
103.38, 97.97, 69.98, 63.36, 45.92, 14.37. HRMS (ESI) m/z calcd for [C13H15Cl3N8O4+H]+:
453.0282, found: 453.0360.
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(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl(2,2,2-trichloro-1-((4-chloropyrimidin-2-yl)amino)
ethyl)carbamate (5f) Following general procedure in Scheme 1 furnished 5f as white solid.
Yield: 20.18%, mp: 182.3~183.4℃, HPLC: 98.75%. 1H NMR (500 MHz, DMSO-d6): δ8.40 (d, J
= 4.8 Hz, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.99 (s, 1H), 7.87 (s, 1H), 6.98 (d, J = 5.0 Hz, 1H), 6.55
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(s, 1H), 4.56–4.47 (m, 3H), 4.40-4.35 (m, 1H), 2.41 (s, 3H). C NMR (126 MHz, DMSO-d6):
δ161.18, 155.35, 152.08, 138.90, 133.51, 112.61, 102.13, 70.30, 63.51, 45.88, 14.41. HRMS
(ESI) m/z calcd for [C13H13Cl4N7O4+H]+: 471.9783, found: 471.9864.
(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl(2,2,2-trichloro-1-(pyrimidin-4-ylamino)ethyl)
carbamate (6a) Following general procedure in Scheme 1 furnished 6a as yellow solid. Yield:
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32.24%, mp 179.4~180.2℃, HPLC: 99.79%. H NMR (500 MHz, DMSO-d6): δ8.59-8.48 (m,
2H), 8.21 (d, J = 5.9 Hz, 1H), 8.04-7.94 (m, 2H), 6.88 (d, J = 5.9 Hz, 1H), 6.77 (t, J = 8.6 Hz,
1H), 4.50 (d, J = 11.0 Hz, 3H), 4.35 (dd, J =10.7, 4.5 Hz, 1H), 2.39 (s, 3H). 13C NMR (126 MHz,
DMSO-d6): δ161.10, 158.30, 155.81, 155.59, 152.14, 138.87, 133.56, 107.09, 101.98, 68.80,
63.24, 46.04, 14.41. HRMS (ESI) m/z calcd for [C13H14Cl3N7O4+H]+: 438.0173, found:438.0264.
2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl(2,2,2-trichloro-1-(quinolin-2-ylamino)ethyl)
carbamate (6b) Following general procedure in Scheme 1 furnished 6b as yellow solid. Yield:
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25.62%, mp 119.9~121.0℃, HPLC: 99.75%. H NMR (500 MHz, DMSO-d6): δ8.35 (d, J = 8.8
Hz, 1H), 8.03–7.98 (m, 2H), 7.71 (d, J = 7.9 Hz, 1H), 7.59-7.54 (m, 2H), 7.49 (d, J = 9.2 Hz,
1H), 7.27 (t, J = 7.2 Hz, 1H), 7.13 (d, J = 8.9 Hz, 1H), 6.94 (t, J = 9.0 Hz, 1H), 4.51 (dd, J = 11.7,
4.8 Hz, 3H), 4.35-4.30 (m, 1H), 2.34 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δ155.57, 155.16,
152.16, 147.19, 138.82, 137.80, 133.55, 129.84, 127.96, 126.72, 124.23, 123.01, 113.30, 102.69,
69.54, 63.17, 46.07, 14.38. HRMS (ESI) m/z calcd for [C18H17Cl3N6O4+H]+: 487.0377, found:
487.0463.
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2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl(2,2,2-trichloro-1-(isoquinolin-3-ylamino)ethyl)
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carbamate (6c) Following general procedure in Scheme 1 furnished 6c as yellow solid. Yield:
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28.32%, mp 122.6~124.2℃, HPLC: 99.50%. H NMR (500 MHz, DMSO-d6): δ8.96 (s, 1H),
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8.27 (d, J = 8.7 Hz, 1H), 7.99 (s, 1H), 7.90 (d, J = 8.2 Hz, 1H), 7.64 (d, J = 8.3 Hz, 1H), 7.58-
7.54 (m, 1H), 7.32-7.28 (m, 1H), 7.05 (s, 1H), 6.81-6.72 (m, 2H), 4.49 (d, J = 11.5 Hz, 3H), 4.33
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(d, J = 5.3 Hz, 1H), 2.34 (s, 3H). C NMR (126 MHz, DMSO-d6): δ155.57, 152.99, 152.12,
151.48, 138.86, 138.56, 133.53, 131.03, 128.23, 125.27, 124.18, 123.73, 103.23, 100.17, 70.84,
63.23, 46.02, 14.36. HRMS (ESI) m/z calcd for [C18H17Cl3N6O4+H]+: 487.0377, found:487.0469.
2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl(2,2,2-trichloro-1-(isoquinolin-1-ylamino)ethyl)
carbamate (6d) Following general procedure in Scheme 1 furnished 6d as white solid. Yield:
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27.43%, mp 113.5~114.8℃, HPLC: 97.92%. H NMR (500 MHz, DMSO-d6): δ8.06 (d, J = 8.4
Hz, 1H), 8.02-7.94 (m, 2H), 7.84 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.7 Hz, 1H), 7.73 (t, J = 7.5 Hz,
1H), 7.64 (t, J = 7.6 Hz, 1H), 7.35 (d, J = 8.8 Hz, 1H), 7.18 (d, J = 5.7 Hz, 1H), 7.07 (t, J = 8.7
Hz, 1H), 4.49 (dd, J = 21.6, 11.7 Hz, 3H), 4.37-4.31 (m, 1H), 2.36 (s, 3H). 13C NMR (126 MHz,
DMSO-d6): δ155.11, 152.99, 151.98, 140.88, 138.89, 137.32, 133.50, 130.92, 127.44, 127.06,
122.66, 117.67, 113.11, 103.34, 69.26, 63.53, 45.81, 14.34. HRMS (ESI) m/z calcd for
[C18H17Cl3N6O4+H]+: 487.0377, found: 487.0463.
2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl(1-(1H-benzo[d]imidazol-1-yl)-2,2,2-
trichloroethyl)carbamate (6e) Following general procedure in Scheme 1 furnished 6e as white
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solid. Yield: 22.13%, mp 123.6~124.9℃, HPLC: 97.97%. H NMR (500 MHz, DMSO-d6): δ
9.68 (d, J = 9.8 Hz, 1H), 8.63 (s, 1H), 8.00 (d, J = 6.1 Hz, 1H), 7.96 (t, J = 11.1 Hz, 1H), 7.71 (t,
J = 7.8 Hz, 1H), 7.33 (t, J = 7.4 Hz, 1H), 7.27 (t, J = 7.4 Hz, 1H), 6.88 (t, J = 14.6 Hz, 1H), 4.60-
4.51 (m, 3H), 4.40 (dt, J = 8.5, 3.8 Hz, 1H), 2.38 (s, 3H). 13C NMR (126 MHz, DMSO-d6):
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δ155.67, 152.01, 142.82, 142.73, 138.91, 134.14, 133.57, 123.96, 123.03, 120.18, 112.05, 99.86,
73.06, 63.74, 45.86, 14.34. HRMS (ESI) m/z calcd for [C16H15Cl3N6O4+H]+: 461.0220, found:
461.0127.
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2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl(2,2,2-trichloro-1-((4,6-dichloropyrimidin-2-yl)
amino)ethyl)carbamate (7a) Following general procedure in Scheme 1 furnished 7a as
colorless solid. Yield: 46.23%, mp 175.5~176.3℃, HPLC: 99.73%. 1H NMR (500 MHz, DMSO-
d6): δ8.56 (d, J = 9.2 Hz, 1H), 8.17 (d, J = 9.0 Hz, 1H), 7.98 (s, 1H), 7.21 (s, 1H), 6.45 (t, J = 9.1
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Hz, 1H), 4.55-4.48 (m, 3H), 4.38 (dt, J = 9.5, 4.4 Hz, 1H), 2.42 (s, 3H). C NMR (126 MHz,
DMSO-d6): δ160.73, 155.42, 152.12, 138.90, 133.50, 111.40, 101.52, 70.47, 63.53, 45.90, 14.45.
HRMS (ESI) m/z calcd for [C13H12Cl5N7O4+H]+: 505.9393, found: 505.9472.
2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl(2,2,2-trichloro-1-((4,6-dichloro-5-
methylpyrimidin-2-yl)amino)ethyl)carbamate (7b) Following general procedure in Scheme 1
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furnished 7b as white solid. Yield: 46.66%, mp 140.3~142.1℃, HPLC: 99.82%. H NMR (400
MHz, DMSO-d6): δ8.24 (d, J = 9.3 Hz, 1H), 8.15 (d, J = 9.0 Hz, 1H), 7.99 (s, 1H), 6.40 (t, J =
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9.1 Hz, 1H), 4.56-4.46 (m, 3H), 4.37 (dt, J = 9.3, 3.9 Hz, 1H), 2.42 (s, 3H), 2.26 (s, 3H). C
NMR (101 MHz, DMSO-d6): δ158.23, 155.43, 152.15, 138.90, 133.55, 117.65, 101.74, 70.55,
63.51, 45.92, 15.41, 14.47. HRMS (ESI) m/z calcd for [C14H14Cl5N7O4+H]+: 519.9550, found:
519.9629.
2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl(1-((5-amino-4,6-dichloropyrimidin-2-yl)amino)-
2,2,2-trichloroethyl)carbamate (7c) Following general procedure in Scheme 1 furnished 7c as
yellow solid. Yield: 27.68%, mp 92.9~93.7℃, HPLC: 99.73%. 1H NMR (500 MHz, DMSO-d6):
δ8.00 (d, J = 8.9 Hz, 1H), 7.98 (s, 1H), 7.21 (d, J = 9.5 Hz, 1H), 6.26 (t, J = 9.2 Hz, 1H), 5.16 (s,
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2H), 4.56-4.45 (m, 3H), 4.37 (dt, J = 9.6, 4.6 Hz, 1H), 2.40 (s, 3H). 13C NMR (126 MHz,
DMSO-d6): δ155.34, 152.07, 151.11, 145.55, 138.88, 133.49, 129.80, 102.40, 71.00, 63.41,
45.92, 14.40. HRMS (ESI) m/z calcd for [C13H13Cl5N8O4+H]+: 520.9502, found: 520.9581.
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2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl(1-((2-amino-4,6-dichloropyrimidin-5-yl)amino)-
2,2,2-trichloroethyl) carbamate (7d) Following general procedure in Scheme 1 furnished 7d as
white solid. Yield: 20.18%, mp 125.1~125.7℃, HPLC: 99.14%. 1H NMR (500 MHz, DMSO-d6):
δ8.35 (s, 1H), 8.34 (d, J = 9.5 Hz, 1H), 8.01 (s, 1H), 7.76-7.74 (m, 1H), 7.26 (s, 2H), 5.58 (t, J =
10.0 Hz, 1H), 4.61 (d, J = 10.6 Hz, 1H), 4.47 (d, J = 11.9 Hz, 3H), 4.42–4.35 (m, 1H), 2.40 (s,
3H). 13C NMR (126 MHz, DMSO-d6): δ158.85, 156.40, 155.83, 152.17, 138.83, 133.53, 121.90,
102.23, 75.27, 63.21, 46.09, 14.45. HRMS (ESI) m/z calcd for [C13H13Cl5N8O4+H]+: 520.9502,
found: 520.9574.
2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl(2,2,2-trichloro-1-(6-chloro-9H-purin-9-yl)ethyl)
carbamate (8a) Following general procedure in Scheme 1 furnished 8a as colorless solid. Yield:
21.32%, mp 101.4~101.8℃, HPLC: 96.82%. 1H NMR (500 MHz, DMSO-d6): δ9.76 (d, J = 10.0
Hz, 1H), 8.98 (s, 1H), 8.93 (s, 1H), 7.91 (s, 1H), 7.00 (d, J = 10.0 Hz, 1H), 4.57-4.45 (m, 4H),
2.37 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δ155.56, 153.11, 152.38, 151.86, 150.38, 144.65,
138.98, 133.45, 130.31, 98.77, 71.56, 63.87, 45.75, 14.35. HRMS (ESI) m/z calcd for
[C14H12Cl4N8O4+H]+: 496.9736, found: 496.9782.
2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl(2,2,2-trichloro-1-(6-(dimethylamino)-9H-purin-
9-yl)ethyl)carbamate (8b) Following general procedure in Scheme 1 furnished 8b as white solid.
Yield: 20.23%, mp 113.6~115.2℃, HPLC: 98.22%. 1H NMR (500 MHz, CDCl3): δ8.51 (s, 1H),
8.31 (s, 1H), 7.96 (s, 1H), 7.94 (s, 1H), 6.65 (d, J = 9.7 Hz, 1H), 4.64-4.58 (m, 3H), 4.54-4.49 (m,
ACS Paragon Plus Environment
29