Preparation of Rhodium(III) complexes with 2(1H)-quinolinone derivatives and evaluation of their in vitro and in vivo antitumor activity

Article abstract of DOI:10.1016/j.ejmech.2018.03.074

A series of 2(1H)-quinolinone derivatives and their rhodium (III) complexes were designed and synthesized. All the rhodium (III) complexes exhibited higher in vitro cytotoxicity for Hep G2, HeLa 229, MGC80-3, and NCI-H460 human tumor cell lines than their ligands and cisplatin, and among them complex 9 was found to be selectively cytotoxic to tumor cells. Further investigation revealed that complex 9 caused cell cycle arrest at the G2/M phase and induced apoptosis, and inhibited the proliferation of Hep G2 cells by impeding the phosphorylation of epidermal growth factor receptor (EGFR) and its downstream enzymes. Complex 9 also up-regulated the proapoptotic proteins Bak, Bax, and Bim, which altogether activated caspase-3/9 to initiate cell apoptosis. Notably, complex 9 effectively inhibited tumor growth in the NCI-H460 xenograft mouse model with less adverse effect than cisplatin.

Full text of DOI:10.1016/j.ejmech.2018.03.074

Accepted Manuscript
Preparation of Rhodium(III) complexes with 2(1H)-quinolinone derivatives and
evaluation of their in vitro and in vivo antitumor activity
Xing Lu, Yi-Ming Wu, Jing-Mei Yang, Feng-E. Ma, Liang-Ping Li, Sheng Chen, Ye
Zhang, Qing-Ling Ni, Ying-Ming Pan, Xue Hong, Yan Peng
PII:
S0223-5234(18)30317-9
10.1016/j.ejmech.2018.03.074
EJMECH 10339
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 February 2018
Revised Date: 23 March 2018
Accepted Date: 25 March 2018
Please cite this article as: X. Lu, Y.-M. Wu, J.-M. Yang, F.-E. Ma, L.-P. Li, S. Chen, Y. Zhang, Q.-L. Ni,
Y.-M. Pan, X. Hong, Y. Peng, Preparation of Rhodium(III) complexes with 2(1H)-quinolinone derivatives
and evaluation of their in vitro and in vivo antitumor activity, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.03.074.
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ACCEPTED MANUSCRIPT

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Preparation of Rhodium(III) Complexes with 2(1H)-Quinolinone Derivatives
and Evaluation of Their in Vitro and in Vivo Antitumor Activity
a
a
a
a
a
a
Xing Lu , Yi-Ming Wu , Jing-Mei Yang , Feng-E Ma , Liang-Ping Li , Sheng Chen ,
a
a
a
b,*
a,*
Ye Zhang , Qing-Ling Ni , Ying-Ming Pan , Xue Hong , Yan Peng .
a
State Key Laboratory for Chemistry and Molecular Engineering of Medicinal
Resources, School of Chemistry and Pharmacy, Guangxi Normal University, 15 Yucai
Road , Guilin 541004, P. R. China
b
State Key Laboratory of Organ Failure Research, Guangdong Provincial Institute of
Nephrology Division of Nephrology, Nanfang Hospital, Southern Medical University,
1
838 Guangzhou North Street, Guangzhou 510515, P. R. China
*
Corresponding Authors
Xue Hong: E-mail: hongxue0203@163.com. Phone: 086-020-62786324. Fax:
0
86-020-62786324.
Yan Peng: E-mail: pengyan630@aliyun.com. Phone: 086-773-2120958. Fax:
86-773-21209958.
Running title: Antitumor of Rh complexes with 2(1H)-Quinolinone derivatives
0

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ABSTRACT
A series of 2(1H)-quinolinone derivatives and their rhodium(III) complexes were
designed and synthesized. All the rhodium(III) complexes exhibited higher in vitro
cytotoxicity for Hep G2, HeLa 229, MGC80-3, and NCI-H460 human tumor cell lines
than their ligands and cisplatin, and among them complex 9 was found to be
selectively cytotoxic to tumor cells. Further investigation revealed that complex 9
caused cell cycle arrest at the G2/M phase and induced apoptosis, and inhibited the
proliferation of Hep G2 cells by impeding the phosphorylation of epidermal growth
factor receptor (EGFR) and its downstream enzymes. Complex 9 also up-regulated
the proapoptotic proteins Bak, Bax, and Bim, which altogether activated caspase-3/9
to initiate cell apoptosis. Notably, complex 9 effectively inhibited tumor growth in the
NCI-H460 xenograft mouse model with less adverse effect than cisplatin.
KEYWORDS: 2(1H)-quinolinone; rhodium complex; antitumor activity; apoptosis

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1
. Introduction
Platinum compounds have been widely used as antitumor drugs since the
discovery of cisplatin’s antitumor activity by Rosenberg et al. [1]. However, clinically
they have also shown various drawbacks, including nephrotoxicity, neurotoxicity,
acquired resistance, etc., and their applicability is at times limited [2–4]. As a result,
new metal-based agents with fewer side effects are desired [5,6]. One of the relevant
research directions focused on developing antitumor complexes of non-platinum
metals [7–11]. For example, rhodium(III) complexes have recently attracted much
interest due to their various pharmacology properties. Zhong et al. identified a
rhodium(III) complex as a potent and selective NAE inhibitor that showed in vivo
anti-inflammatory activity [12]. In addition, Yang et al. also reported a rhodium(III)
complex that could target LSD1 and hence possessed anticancer activity [13].
Quinolines and their oxo-derivatives are known to exert biological actions such as
antioxidation, anti-osteoporosis, anti-influenza, and anticancer activities [14–17].
Dovitinib, which contains the 3-substituted 2(1H)-quinolinone skeleton, has been
developed as a multi-target anticancer agent and is currently under clinical
investigation [18–20]. In addition, some naphthoquinone and anthraquinone
derivatives were reported as multi-target antitumor agents. For example, Federica
Prati et al. reported that the 2-phenoxy-1,4-naphthoquinones as dual molecules are
able to inhibit glycolysis and mitochondrial respiration simultaneously [21], Bahia et
al. reported that some quinonoid compounds act as antitumor agents [22], and Belluti
et al. reported a series of quinine-coumarin hybrids as dual-targeted
glyceraldehyde-3-phosphate dehydrogenase/trypanothione reductase inhibitors [23].
However,
transition
metal
complexes
derived
from
3
-benzimidazole-2(1H)-quinolinone have not been explored as potential antitumor

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agents. In this work, as part of our continuing quest for novel metal-based anticancer
agents, we designed, prepared, and characterized a series of 2(1H)-quinolinone
derivatives and their rhodium(III) complexes. Among them, complex 9 showed the
best in vitro and in vivo antitumor activity. The antitumor mechanism of complex 9
was carefully investigated.
2
. Results and Discussion
2
.1. Synthesis and Characterization
The design of the rhodium(III) complexes was based on the following
consideration: 1. Both 2(1H)-quinolinone derivatives and benzimidazol derivatives
are potential antitumor agents [24]. The combination of 2(1H)-quinolinone and
benzimidazol may afford improved antitumor activity. 2. The combination of
2
(1H)-quinolinone and benzimidazol forms bidentate ligands, which could contribute
to the formation of stable metal complexes. 3. Rhodium complexes are known as
potential antitumor agents. The unique geometries of rhodium complexes may lead to
improved enzyme inhibition and selectivity [25].
Five 2(1H)-quinolinone derivatives bearing different substituents were
synthesized. Scheme 1 shows the synthetic route for compounds 4–8. These
compounds were prepared by treating 3 with different o-phenylenediamine
derivatives in methanol. Compound 3 was synthesized according to our reported
method [14]. The structures of compounds 4–8 were characterized by NMR
spectrometry and ESI-MS (Supporting Information, Figures S1−S14). Compound 4
was additionally characterized by single-crystal X-ray diffraction analysis (Figure 1).
The selected bond lengths (Å) and bond angles (deg) of 4 are listed in Table S1
(Supporting Information), and the crystal data and structure refinement parameters of

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4
are given in Table S2.
2
.2. Crystal Structures of Rhodium Complexes
Solvothermal method is often used for the synthesis of metal complexes, in
which the ligand and metal are reacted in the presence of a solvent in a sealed reactor
under heating. Five rhodium complexes (complex 9–13) were obtained by
solvothermal synthesis from RhCl ·3H O and compounds 4–8 in methanol and
3
2
chloroform (Scheme 2), respectively, and their structure was characterized by NMR
spectrometry, ESI-MS and X-ray crystallography (Figure 1, Figures S15−S29, and
Table S3–S8). All these rhodium complexes were isomorphous (Figure 1). Among the
five isostructural complexes, complex 9 was selected for detailed crystallographic
analysis. Complex 9 had a mononuclear structure and the coordination of Rh(III) had
a distorted octahedral geometry. The Rh(III) atom was chelated by a ligand and
associated with three chlorine atoms and one methanol molecule. The N(3), O(1),
O(2), and Cl(2) atoms formed the equatorial plane, and the two Cl(1), Cl(3) atoms
were located at the apical positions. The ligand and the Rh(III) atom formed a
six-membered ring by chelation. Selected bond lengths (Å) and bond angles (deg) of
9
–13 are reported in Table S8, and the crystal data and structure refinement
parameters of 4 are given in Table S3–S7.
2
.3. Stability of Ligands and Rhodium Complexes in Solution
The stability of compounds 4−13 was tested under physiological conditions (PBS
buffer solution containing 1% DMSO, pH = 7.3) via UV-Vis spectroscopy. All
compounds were found to be stable in PBS for 24 h at room temperature based on the
observations from the UV-Vis spectra (Figure S30). The stability of 4–13 was further
tested by HPLC, and all compounds were found to be stable for 24 h in DMSO stock
solution (Figure S31).

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2
.4. In vitro Cytotoxicity
Compounds 4–13 were tested against four human tumor cell lines (Hep G2,
HeLa 229, MGC80-3, NCI-H460) and the normal human liver HL-7702 cells by MTT
assay to evaluate their in vitro cytotoxicity. For comparison, the cytotoxicity of
RhCl ·3H O and cisplatin were also tested and used as controls. After the cells were
3
2
incubated with the test compounds (2.5, 5, 10, 20, 40 µM) for 24 h, the IC values
5
0
were calculated. Table 1 shows that the in vitro antitumor activity of the tested
compound generally fell in the following order: Rh(III) complex > cisplatin >
ligand > RhCl ·3H O. The Rh(III) complexes 9–13 appeared to be far more cytotoxic
3
2
than RhCl ·3H O for all tested cell lines, and they also showed enhanced potency in
3
2
comparison with their corresponding ligand 4–8. That is, a synergy that enhanced the
antitumor activity took effect when the Rh(III) ion was combined with the ligands. In
particular, complex 9 was more potent than cisplatin for all tested cancer cell lines but
was less cytotoxic to the normal human liver HL-7702 cell line (IC = 22.8 ± 1.4 µM)
5
0
than cisplatin (IC = 12.8 ± 0.9 µM). Therefore, complex 9 was selected for further
5
0
detailed studies.
2
.5. EGFR Inhibition
The epidermal growth factor receptor (EGFR) plays a pivotal role in cellular
signaling related to cell growth, proliferation, survival, and migration. Aberrant EGFR
activity is a key enabler of the development and growth of tumor cells and is
associated with the onset and progression of cancer [26–31]. Therefore, we tested the
inhibitory potency of 4–13 against wild type EGFR. The phosphorylation state of an
artificial substrate, which was measured by the homogeneous time-resolved
fluorescence resonance energy transfer (HTRF) method, was used as the indicator of
the compounds' activity. Figure 2 shows that at the same concentration (1 µM), the

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Rh(III) complexes 9–13 exhibited stronger inhibitory activity than their corresponding
ligands 4–8. Complex 9 showed an inhibition rate of 100%, the highest EGFR
inhibitory activity of all tested compounds. Hence, complex 9 might be an interesting
drug candidate for EGFR-driven cancer.
2
.6. Western Blot Analysis of Anti-proliferative Activity against Hep G2 Cell Line
We examined the potential of complex 9 to inhibit the phosphorylation of EGFR
and its downstream enzymes (Akt and Erk1/2) by Western blot analysis. Figure 3
shows that the Hep G2 cells experienced a significant reduction in EGFR
phosphorylation after they were treated with complex 9 (10 µM). This result suggests
that complex 9 inhibited the phosphorylation of EGFR in the Hep G2 cells.
Additionally, since the phosphorylation of Akt and ERK1/2 is required for
EGFR-mediated cell proliferation, we examined the effects of complex 9 on the
activation of Akt and ERK1/2. Again, it was found that the phosphorylation of Akt
and ERK1/2 was inhibited by complex 9 (10 µM). Hence, complex 9 could inhibit the
proliferation of Hep G2 cells by impeding the phosphorylation of EGFR and its
downstream enzymes.
2
.7. Gene Expression Profile
The gene expression profile is a useful for discovering cancer biomarkers and
therapeutic targets [32,33]. To enhance mechanistic understandings, Hep G2 cells
treated with complex 9 were examined on an Illumina Hiseq 2500 sequencing
platform to measure the gene expression profile and evaluate the global changes in
transcription levels. It was found that complex 9 changed the expression of 498 genes,
among which 141 genes (e.g., F8, ITGB3, HIST1H2BD, E2F3) were up-regulated
and 357 genes (e.g., E2F1, CCNE1, CCNB1, CCNA2) were down-regulated. Figure
4
A shows the relative expression profiles of 76 genes that experienced significant

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change (P < 0.5, fold change). The GO and KEGG analyses were carried out to find
out relevant pathways through which target genes were significantly enriched (Figure
4
B). The results suggested that complex 9 could change the expression of genes
involved in cell cycle signaling in Hep G2 cells.
2
.8. Cell Cycle Arrest
The Hep G2 cells were treated with complex 9 for 24 h and the cell cycle
progression was measured. Figure 5 shows that compared with control group (G1:
4.61%, G2/M: 6.23%, S: 29.16%), the treated cells exhibited an accumulation in the
G2/M phase, and the level of accumulation was dependent on the dose of complex 9
1 µM, 15.19%; 2 µM, 17.54%; 5 µM, 18.46%). The results indicated G2/M phase
arrest of the Hep G2 cells.
6
(
2
.9. Induction of Apoptosis
The ability of complex 9 to induce apoptosis in Hep G2 cells was measured by
Annexin V-PI staining and flow cytometry analyses. Figure 6 shows that after the Hep
G2 cells were treated with complex 9 at 2, 5, 10 µM for 24 h, the population of early
apoptotic cells increased from 5.4% in the control group to as much as 40.9%.
Therefore, complex 9 is a potent apoptosis inducer.
2
.10. Assessment of Caspase-3/9 Activation
Caspase-3 and caspase-9 are members of the cysteine protease family, and the
sequential activation of caspases is essential in cell apoptosis. After the proapoptotic
molecules activate the initiator caspase-9, caspase-9 will cleave and activate the
executioner caspases-3, which results in the degradation of cellular components
[
34,35]. We explored whether complex 9 could activate caspase-3 and caspase-9 and
consequently induce apoptosis in Hep G2 cells by treating the cells with complex 9 (5,
0, 15, 20 µM). Spectrophotometry results (Figure 7) showed that compared with the
1

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control group, the absorbance ratio of the treated cells increased with rising dose of
complex 9. Hence, complex 9 induced cell apoptosis by activating caspase-3/9 in Hep
G2 cells.
2
.11. Detection of Bcl-2 protein family by Western Blot Assay
The Bcl-2 protein family consists of members that either inhibit apoptosis (e.g.,
Bcl-2, Bcl-xl) or promote apoptosis (e.g., Bax, Bak, Bim) [36]. Therefore, it is of
interest to determine which of the Bcl-2 family proteins participated in the death
signaling after cells were treated with complex 9. The changes in the expression of
candidate proteins were examined by Western blot assay. Figure 8 shows that complex
9
up-regulated the expression of Bak, Bax, and Bim in Hep G2 cells, whereas other
proteins (Bcl-2, Bcl-xl) experienced no changes that were dependent on the dose of
complex 9.
2
.12. Toxicological Evaluation in Mice
The toxicity of complex 9 in the mouse was evaluated extensively. Twelve male
and twelve female four-week-old Kunming mice were divided randomly into four
groups (n = 6). The control group received intraperitoneal saline injection once a day.
The two test groups received 12.5 and 25 mg/kg body weight of intraperitoneal
injection of complex 9 once a day, and the last group was treated once every two days
with cisplatin at 2 mg/kg body weight. The mice were treated and monitored for the
two weeks. During this time, mice treated with complex 9 showed no adverse reaction
or significant loss of body weight, whereas the body weight of the mice treated with
cisplatin was 13.8% less than the body weight of mice in the control group (Figure
9
A). The mice were sacrificed after two weeks, and the heart, liver, and kidney were
collected and weighed. Figure 9B shows that no significant change was observed in
the tissues of mice treated with complex 9. The decreased liver and kidney weights of

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the mice in the cisplatin group could be attributed to their decreased body weight. The
histological morphology of tissue sections was examined by haematoxylin and eosin
(H&E) staining to reveal drug-related pathological changes of major organs. No
significant pathological change in the heart, liver, and kidney was observed in the
mice treated with complex 9. In contrast, mice treated with cisplatin showed renal
tubular injury (Figure 9C). In conclusion, complex 9 showed an acceptable safety
profile since the mice tolerated treatment with it at 25 mg/kg body weight.
2
.13. Growth Inhibition of NCI-H460 and Hep G2 Xenograft in Vivo
To further examine the therapeutic potential of complex 9, we compared the
antitumor efficacy of 9 and cisplatin in NCI-H460 and Hep G2 subcutaneous
xenograft mice models. Tumor-bearing mice were randomly assigned and received
intraperitoneal injection of complex 9, vehicle control, or cisplatin.
Figure 10 shows that continuous treatment with complex 9 for two weeks
significantly slowed the tumor growth in the NCI-H460 xenograft model. The
inhibition of tumor growth was dependent on the dose of complex 9. The relative
tumor increment rate (T/C) was of 47.1% (P < 0.01) and 56.1% (P < 0.05) when the
dosage was 25 and 12.5 mg/kg body weight, respectively. After the mice were
sacrificed on day 14, end point tumor weight was recorded and the inhibitory rate was
calculated. Complex 9 exhibited significant antitumor activity in the NCI-H460 model
with an inhibitory rate of 49.8% (P < 0.01), lower than that of cisplatin (63.2%, P <
0
.01).
The tumor tissues of the NCI-H460 xenograft model after treatment with complex
were excised for further pathological studies. The H&E-stained tissue sections of
9
the mice treated with complex 9 and the mice in the control group showed notable
differences in their tumor tissue morphology. Figure 10E shows increased necrosis in

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the tumor tissues from the mice treated with complex 9 or cisplatin compared with
those from the control group, and cisplatin was less effective than complex 9 to
promote tumor tissues necrosis.
The Hep G2 xenograft model was also tested similarly for two weeks, and Figure
S32 shows that complex 9 (25 mg/kg body weight) had a low activity on the in vivo
growth of Hep G2 tumor (inhibitory rate: 24.0%).
In summary, the results indicated that compared with cisplatin, complex 9 was
effective in inhibiting tumor growth in the NCI-H460 xenograft mouse model and
incurred less adverse effect.
3
. Conclusions
A series of 2(1H)-quinolinone derivatives and their rhodium(III) complexes were
synthesized and characterized, and their in vitro antitumor activities were tested. The
Rh(III) complexes showed higher cytotoxicity than their ligands and cisplatin. In
particular, compared with cisplatin, complex 9 was more potent for all tested cancer
cell lines and less cytotoxic to the normal human liver HL-7702 cell line. It was found
that complex 9 caused cell cycle arrest at the G2/M phase and induced apoptosis, and
it inhibited the proliferation of Hep G2 cells by impeding the phosphorylation of
EGFR and its downstream enzymes. Complex 9 up-regulated the proapoptotic
proteins Bak, Bax, and Bim, all of which helped to activate caspase-3/9 and initiate
cell apoptosis. The in vivo tests showed that complex 9 could effectively inhibit tumor
growth in the NCI-H460 xenograft mouse model, and the side effects of complex 9
were less severe than those of cisplatin. Therefore, complex 9 has the potential for
further development into an efficient anticancer agent with low toxicity.

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4
. Experimental
4
.1. Materials
All reagents and solvents were purchased from Alfa Aesar and Xilong Chemical
Co., Ltd. and used directly without further purification. All compounds used in
pharmacological studies had >95% purity. The HTRF kinEASE TK kit was purchased
from Cisbio. The apoptosis detection kit was purchased from BD Biosciences. The
caspase-3/9 assay kit was obtained from Biovision. All antibodies were purchased
from Abcam. All cell lines were purchased from Shanghai Institute for Biological
Science.
4
.2. Instruments
Electrospray ionization mass spectrometry (ESI-MS) tests were carried out on a
Bruker HCT mass spectrometer. NMR spectra were recorded on a Bruker AV-500
NMR spectrometer. HPLC analyses were carried out on an Elite P230II instrument
(Dalian, China). MTT assays were performed with an M1000 microplate reader. Cell
cycle analyses were performed with an FACS Aria II flow cytometer. Western blot
assays were run on an ECL Western blot system. Gene expression profiles were
analyzed using the Illumina Hiseq 2500 sequencing platform.
4
.3. Synthesis of Ligands
A solution of 3 (1.87 g, 10 mmol) and o-phenylenediamine derivatives (10 mmol)
in methanol (80 mL) was stirred at 65 °C for 4 h, then cooled to room temperature
and filtered to give a yellow powder.
Compound 4 (yield 90%). Anal. Calc. for C H N O: C 74.17; H 4.76; N 15.26;
1
7
13
3
1
O 5.81%, Found: C 74.13; H 4.82; N 15.22; O 5.83%. H NMR (500 MHz,
DMSO-d6) δ 12.65 (s, 1H), 12.40 (s, 1H), 9.00 (d, J = 1.7 Hz, 1H), 7.74 – 7.70 (m,
1
H), 7.69 (s, 1H), 7.68 – 7.63 (m, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.33 (d, J = 8.4 Hz,

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13
1
H), 7.22 – 7.17 (m, 2H), 2.36 (s, 3H). C NMR (125 MHz, DMSO-d6) δ 160.79,
1
47.96, 142.83, 138.90, 136.85, 134.49, 133.15, 131.89, 128.38, 122.41, 122.05,
+
1
19.93, 119.19, 118.38, 115.29, 112.88, 20.56. ESI-MS: m/z 276.11 [M+ H] .
Compound 5 (yield 88%). Anal. Calc. for C H N O: C 75.23; H 5.65; N 13.85;
1
9
17
3
1
O 5.27%, Found: C 75.21; H 5.66; N 13.83; O 5.3%. H NMR (500 MHz, DMSO-d6)
δ 12.41 (s, 1H), 12.35 (s, 1H), 8.97 (s, 1H), 7.72 (s, 1H), 7.45 (s, 2H), 7.43 (d, J = 1.8
Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 2.39 (s, 3H), 2.33 (s, 6H). ESI-MS: m/z 304.14
+
[
M+ H] .
Compound 6 (yield 91%). Anal. Calc. for C H F N O: C 65.59; H 3.56; N
1
7
11
2
3
1
1
3.50; O 5.14%, Found: C 65.61; H 3.55; N 13.53; O 5.16%. H NMR (500 MHz,
DMSO-d6) δ 12.82 (s, 1H), 12.44 (s, 1H), 8.97 (s, 1H), 7.69 (s, 1H), 7.66 (d, J = 8.5
13
Hz, 2H), 7.44 (dd, J = 8.4, 1.6 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 2.37 (s, 3H). C
NMR (125 MHz, DMSO-d6) δ 160.53, 149.77, 147.79, 145.92, 138.94, 138.26,
1
2
36.83, 133.22, 131.80, 129.90, 128.35, 119.29, 118.99, 115.23, 105.46, 100.55,
+
0.45. ESI-MS: m/z 312.09 [M+ H] .
Compound 7 (yield 85%). Anal. Calc. for C H Cl N O: C 59.32; H 3.22; N
1
7
11
2
3
1
1
2.21; O 4.65%, Found: C 59.30; H 3.23; N 12.24; O 4.67%. H NMR (500 MHz,
DMSO-d6) δ 12.89 (s, 1H), 12.46 (s, 1H), 9.04 (s, 1H), 7.94 (s, 1H), 7.91 (s, 1H),
13
7
.75 (s, 1H), 7.48 (dd, J = 8.4, 1.6 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 2.39 (s, 3H). C
NMR (125 MHz, DMSO-d6) δ 174.75, 160.98, 150.88, 142.88, 140.27, 137.51,
1
34.46, 133.99, 132.35, 130.10, 128.95, 124.91, 119.78, 115.74, 114.56, 99.99, 20.92.
-
ESI-MS: m/z 342.02 [M-H] .
Compound 8 (yield 84%). Anal. Calc. for C H Br N O: C 47.14; H 2.56; N
1
7
11
2
3
1
9
.70; O 3.69%, Found: C 47.16; H 2.57; N 9.68; O 3.71%. H NMR (500 MHz,
DMSO-d6) δ 12.87 (s, 1H), 12.47 (s, 1H), 9.03 (s, 1H), 8.10 (s, 1H), 8.05 (s, 1H),

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1
3
7
.74 (s, 1H), 7.47 (d, J = 9.7 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 2.39 (s, 3H). C NMR
(125 MHz, DMSO-d6) δ 160.95, 150.63, 143.82, 140.32, 137.49, 135.32, 133.97,
1
32.31, 130.08, 128.94, 122.92, 119.42, 117.68, 116.61, 116.55, 115.72, 20.93.
-
ESI-MS: m/z 431.91 [M-H] .
4
.4. Synthesis of Rh(III) Complexes
A mixture of RhCl ·3H O (0.05 mmol, 0.010 g), ligand 4–8 (0.025 mmol),
3
2
methanol (0.45 mL), and chloroform (0.15 mL) was placed in a thick Pyrex tube (ca.
5 cm long) and frozen by liquid N . The tube was vacuumed, sealed, and then heated
2
2
at 80 °C for 3 days to give red block crystals suitable for X-ray diffraction analysis.
Complex 9 (yield 70%) Anal. Calc. for C H Cl N O Rh: C 41.59; H 3.86; N
1
9
21
3
3
3
1
7
.66; O 8.75%, Found: C 41.53; H 3.82; N 7.69; O 8.71%. H NMR (500 MHz,
DMSO-d6) δ 14.24 (s, 1H), 14.02 (s, 1H), 9.06 (s, 1H), 8.69 (d, J = 8.4 Hz, 1H), 7.82
(s, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.70 (d, J = 9.5 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H),
13
7
.44 (t, J = 7.6 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 2.49 (s, 3H). C NMR (125 MHz,
DMSO-d6) δ 164.60, 145.69, 142.05, 140.90, 136.40, 135.81, 135.50, 134.42, 128.74,
1
25.22, 122.94, 122.65, 120.92, 119.37, 117.16, 112.41, 21.06. ESI-MS: m/z 603.97
+
[
M+2DMSO-MeOH-Cl] .
Complex 10 (yield 72%) Anal. Calc. for C H Cl N O Rh : C 43.57; H 4.59; N
4
3
54
6
6
7
2
1
7
.09; O 9.45%, Found: C 43.55; H4.53; N 7.14; O 9.41%. H NMR (500 MHz,
DMSO-d6) δ 13.97 (s, 2H), 9.00 (s, 1H), 8.45 (s, 1H), 7.81 (s, 1H), 7.69 (dd, J = 8.6,
1
3
1
.6 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.49 (s, 1H), 2.49 (s, 3H), 2.41 (s, 3H), 2.32 (s,
1
3
H). C NMR (125 MHz, DMSO-d6) δ 164.57, 144.58, 140.72, 140.25, 136.21,
35.54, 135.39, 134.50, 133.01, 131.60, 128.63, 122.19, 120.94, 119.57, 117.10,
+
1
12.00, 21.07, 20.86, 20.50. ESI-MS: m/z 632.01 [M+2DMSO-MeOH-Cl] .
Complex 11 (yield 68%) Anal. Calc. for C H Cl F N O Rh : C 38.99; H 3.52;
39
42
6
4
6
7
2

ACCEPTED MANUSCRIPT
1
N 7.00; O 9.32%, Found: C 38.93; H3.57; N 6.92; O 9.36%. H NMR (500 MHz,
DMSO-d6) δ 14.56 (s, 1H), 14.06 (s, 1H), 9.05 (s, 1H), 8.64 (dd, J = 11.9, 7.8 Hz,
1
H), 7.88 (dd, J = 9.9, 7.3 Hz, 1H), 7.81 (s, 1H), 7.71 (dd, J = 8.6, 1.7 Hz, 1H), 7.65
13
(
d, J = 8.5 Hz, 1H), 2.48 (s, 3H). C NMR (125 MHz, DMSO-d6) δ 163.99, 149.21,
1
47.22, 145.39, 140.86, 137.04, 135.98, 135.53, 135.11, 129.84, 128.30, 120.36,
118.43,
116.70,
109.27,
100.44,
20.53.
ESI-MS:
m/z
639.96
+
[
M+2DMSO-MeOH-Cl] .
Complex 12 (yield 70%) Anal. Calc. for C H Cl N O Rh ：C 36.53; H 3.22; N
3
9
41 10
6
8
2
1
6
.55; O 9.98%, Found: C 36.55; H3.20; N 6.58; O 9.92%. H NMR (500 MHz,
DMSO-d6) δ 14.64 (s, 1H), 14.08 (s, 1H), 9.09 (s, 1H), 8.93 (s, 1H), 8.06 (s, 1H),
1
3
7
.83 (s, 1H), 7.78 – 7.70 (m, 1H), 7.66 (d, J = 8.5 Hz, 1H), 2.49 (s, 3H). C NMR
(125 MHz, DMSO-d6) δ 164.52, 148.19, 141.96, 141.19, 136.60, 136.23, 135.65,
1
34.13, 128.87, 127.78, 125.65, 123.22, 120.83, 118.74, 117.22, 114.17, 21.05.
-
ESI-MS: m/z 629.87 [M+ DMSO-H] .
Complex 13 (yield 65%) Anal. Calc. for C H Br Cl N O Rh ：C 32.42; H 2.93;
3
9
42
4
6
6
7
2
1
N 5.82; O 7.75%, Found: C 32.47; H 2.89; N 5.58; O 7.68%. H NMR (500 MHz,
DMSO-d6) δ 14.58 (s, 1H), 14.07 (s, 1H), 9.09 (s, 1H), 9.09 (s, 1H), 8.16 (s, 1H),
1
3
7
.82 (s, 1H), 7.73 (d, J = 8.7 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 2.49 (s, 3H). C NMR
(125 MHz, DMSO-d6) δ 164.52, 147.86, 141.99, 136.62, 136.25, 135.64, 134.88,
1
7
4
31.04, 128.86, 126.42, 120.83, 119.71, 118.69, 117.62, 117.22, 117.12. ESI-MS: m/z
-
19.74 [M+ DMSO-H] .
.5. X-ray Crystallography
The X-ray crystallography data of 4 and complexes 9–13 were collected on a
Bruker SMART Apex II CCD diffractometer equipped with graphite monochromated
Mo-Kα radiation (λ =0.710 73 Å) at room temperature. All crystal structures were

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solved with direct methods and refined using SHELX-97 programs [37]. Tables
S2–S7 list the crystallographic data of compound 4 and complexes 9–13, and Tables
S1 and S8 show selected bond lengths and bond angles.
4
.6. Cell Culture and other Experimental Methods
The Hep G2, HeLa 229, MGC80-3, NCI-H460 and HL-7702 cells were obtained
from Shanghai Institute for Biological Science. The cell lines were cultured in the
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine
serum (FBS). The procedures of the cytotoxicity assay, cell cycle analysis, apoptosis
analysis, Western blot and in vivo xenograft model assay were similar to those
reported by Zhang et al. [38]. The assessment of caspase-3/9 activation was
performed as reported by Duan et al. [39]. The EGFR inhibition tests were performed
as reported by Engel et al. [29]. The haematoxylin and eosin (H&E) staining tests
were performed as reported by Guo et al.[40]. The NCI-H460 xenograft mouse
models were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China;
st
approval No. SCXK 2014-004). The animal procedures were approved by the 181
Hospital of Chinese People’s Liberation Army (Guilin, China; approval No. SYXK
2
013-0004). All animal experiments were performed in accordance with the NIH
guidelines for the care and use of laboratory animals. Statistical analyses were also
carried out [38].
4
.7. Acute Toxicity Studies
Four-week old Kunming (KM) mice (18−22 g body weight, twelve female and
twelve male) were randomly divided into four groups (n = 6). Complex 9 was
administered into two groups of mice at a dose of 25 and 12.5 mg/kg body weight,
respectively. The mice in the control group received saline injection. The body weight
of the mice was recorded daily. After treatment for two weeks, the major organs (heart,

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liver, and kidney) were collected and weighed.

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ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China
Grants No. 81673473, 21501032). Natural Science Foundation of Guangxi Province
Grants No. 2014GXNSFAA118165, 2015GXNSFAA139010), the Innovative Team
(
(
&
Outstanding Talent Program of Colleges and Universities in Guangxi(2017-38) and
Guangxi Natural Science Foundation of China (No. 2016GXNSFAA380300 and
014GXNSFBA118050), State Key Laboratory for Chemistry and Molecular
Engineering of Medicinal Resources (Guangxi Normal University)
CMEMR2015-A09, CMEMR2015-B12, ACMEMR2015-B10, and
CMEMR2016-A09), and Guilin Scientific Research and Technology Development
020 Project (Grant No. 20160210). Innovation Project of Guangxi Graduate
2
(
1
Education: YCBZ2017029. Gene expression profile experiments were performed by
Sheng Ting Bio-tech, Zhejiang, China.
CONFLICTS OF INTEREST
The authors declare no conflict of interest.
Supporting Information
Additional figures illustrating X-ray crystallography data, ESI-MS, NMR spectra,
crystal data (CCDC No:1818157-1818162), UV-vis absorption of spectra, HPLC
spectra and growth inhibition of Hep G2 xenograft in vivo.

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Figure legends
Figure 1. Crystal structures of 4, 9–13 with atom labeling. The free methanol in 9–13
is omitted for clarity.
Figure 2. Inhibition rates of 4−13 (1 µM) for EGFR. Afatinib (1 µM) was used as
positive control.
Figure 3. (A) Complex 9 inhibits the phosphorylation of the EGFR and its
downstream enzymes (Akt and Erk1/2) in Hep G2 cells. (B) Western blot bands
quantified with ImageJ (from three independent measurements).
Figure 4. (A) Relative expression profiles of 76 cancer-related genes in Hep G2 cells
after treatment with complex 9 (10 µM) for 24 h. (B) Gene ontology terms associated
with the altered genes that show strong enrichment.
Figure 5. Cell cycle distribution of Hep G2 cells treated with complex 9 at 1, 2, 5 µM
for 24 h. (A) Cell cycle arrest analysis by flow cytometry. (B) Histogram of the
percentage of cells in G1, S, and G2/M phases.
Figure 6. Induction of apoptosis by complex 9 in Hep G2 cells. (A) Annexin V-PI
staining and flow cytometry analysis. (B) Histogram of the percentage of early
apoptotic cells.
Figure 7. Activation of (A) caspase-3 and (B) caspase-9 in Hep G2 cells treated with
complex 9 (5, 10, 15, and 20 µM) for 24 h.
Figure 8. (A) Expression levels of Bcl-2 family proteins in Hep G2 cells treated with
complex 9. (B) Western blot bands quantified with ImageJ (from three independent
measurements).
Figure 9. (A) Body weight of Kunming mice (n = 6) treated with complex 9 (12.5 and
2
5 mg/kg body weight, intraperitoneal injection once daily) or cisplatin (2 mg/kg
body weight, intraperitoneal injection once every two days) for two weeks. (B) The

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weight of harvested organs (heart, liver, kidney) after treatment for two weeks. (C)
Histological morphology of H&E-stained tissue sections of mice in different groups.
Figure 10. In vivo anticancer activity of complex 9 in NCI-H460 xenograft model. (A)
Body weight of nude mice treated with complex 9 (12.5 and 25 mg/kg body weight,
intraperitoneal injection once daily), cisplatin (2 mg/kg body weight, intraperitoneal
injection once every two days), or vehicle control (5% DMSO in saline, v/v) for 2
weeks. (B) Effect of complex 9, cisplatin, or vehicle control on the growth of tumor
3
xenograft. The mean tumor volume (mm ) ± SD (n = 6) was used to indicate tumor
growth and calculate the relative tumor increment rate (T/C, %). (C) Tumor weight
distribution at day 14 of NCI-H460 xenograft tumors. (D) Photographs of tumor from
mice in different groups. (E) Histological morphology of H&E-stained tumor tissue
sections of representative nude mice in different groups.
Scheme 1. Synthesis of 4–8. (a) acetic anhydride, hydrochloric acid, r.t. (b) POCl3,
DMF, 90 °C. (c) 70% acetic acid aqueous solution, 95 °C. (d) o-phenylenediamine
derivatives, MeOH, 65 °C.
Scheme 2. Synthesis of the rhodium complexes. (a) RhCl ·3H O, MeOH/CHCl , 80
3
2
3
°
C.

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a
Table 1. IC50 (µM) values of compounds 4−13 towards normal liver cell HL-7702 and four tumor cell lines.
SI
SI
HeLa229
0.7
SI
SI
Compound
Hep G2
HeLa229
MGC80-3
NCI-H460
HL-7702
Hep G2
0.9
1.1
−
MGC80-3 NCI-H460
4
5
6
7
8
9
26.6 ± 0.4
25.3 ± 0.6
>40
33.5 ± 1.4
30.1 ± 0.7
>40
22.8 ± 0.7
29.7 ± 0.9
37.5 ± 1.0
30.1 ± 0.5
24.7 ± 0.5
10.2 ± 0.6
9.4 ± 0.7
6.1 ± 0.5
10.6 ± 0.6
6.3 ± 0.2
>40
35.7 ± 0.7
33.7 ± 1.2
>40
22.8 ± 1.4
28.5 ± 0.9
>40
1.0
1.0
−
0.6
0.8
−
0.9
−
22.4 ± 1.6
20.3 ± 0.5
6.1 ± 0.2
8.8 ± 0.6
7.9 ± 0.8
9.6 ± 0.7
9.6 ± 0.7
>40
26.5 ± 1.2
>40
>40
29.1 ± 0.7
30.4 ± 0.9
22.0 ± 0.7
13.6 ± 1.7
18.4 ± 1.3
12.5 ± 1.5
9.5 ± 1.2
>40
1.3
1.5
3.6
1.5
2.3
1.3
1.0
−
1.1
1.0
1.2
2.2
1.4
3.0
1.2
1.5
−
−
22.8 ± 0.6
6.3 ± 0.3
8.2 ± 1.1
9.6 ± 0.7
10.3 ± 0.8
10.2 ± 0.5
>40
−
1.3
3.5
1.7
1.9
1.2
0.9
−
8.4 ± 0.8
10.4 ± 1.2
11.2 ± 0.9
7.3 ± 0.5
11.5 ± 1.0
>40
2.6
1.3
1.6
1.7
0.8
−
10
11
1
1
2
3
RhCl ·3H O
3
2

ACCEPTED MANUSCRIPT
b
Cisplatin
8.3 ± 0.6
14.5 ± 0.7
11.1 ± 0.5
17.5 ± 0.9
12.8 ± 0.9
1.5
0.9
1.2
0.7
a
b
IC values are presented as the mean ± SD (standard error of the mean) from five independent experiments. Cisplatin was dissolved in saline.
5
0
SI (Selectivity Index)=(IC for HL-7702)/ (IC for the respective cancer cell line)
50
50