Discovery of novel polycyclic spiro-fused carbocyclicoxindole-based anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2016.12.021

A series of novel polycyclic spiro-fused carbocyclicoxindoles were synthesized and investigated for their in?vitro antiproliferative activities against nine human cancer cell lines. Five compounds (10i, 10l, 10n, 10p, and 10r) demonstrated anticancer activities against A2780s cells with IC₅₀values of less than 30?μM. In particular, compound 10i showed anticancer activities against seven cancer cell lines and stronger activities than cisplatin in A2780s, A2780T, CT26, and HCT116?cells. Further studies illustrated that compound 10i arrested cell cycle in G1 phase and induced apoptosis of HCT116?cells. This compound also effectively increased the protein levels of cleaved caspase-3, p53, and MDM2. Molecular docking results revealed that compound 10i could bind well to the p53-binding site on MDM2, indicating that it might work by blocking the MDM2-p53 interactions.

Full text of DOI:10.1016/j.ejmech.2016.12.021

Accepted Manuscript
Discovery of novel polycyclic spiro-fused carbocyclicoxindole-based anticancer
agents
Lidan Zhang, Wen Ren, Xiaoyan Wang, Jiaying Zhang, Jie Liu, Lifeng Zhao, Xia
Zhang
PII:
S0223-5234(16)31027-3
10.1016/j.ejmech.2016.12.021
EJMECH 9112
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 October 2016
Revised Date: 30 November 2016
Accepted Date: 9 December 2016
Please cite this article as: L. Zhang, W. Ren, X. Wang, J. Zhang, J. Liu, L. Zhao, X. Zhang, Discovery
of novel polycyclic spiro-fused carbocyclicoxindole-based anticancer agents, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2016.12.021.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of novel polycyclic spiro-fused carbocyclicoxindole-based
anticancer agents
Lidan Zhang a, †, Wen Ren a, †, Xiaoyan Wang c, Jiaying Zhang d, Jie Liu a, ∗, Lifeng Zhao b ∗, and
Xia Zhang a,
∗
a State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan
University and Collaborative Innovation Center for Biotherapy, Chengdu 610041, China
b Chengdu University, Sichuan Industrial Institute of Antibiotics, Chengdu 610052, China
c Analytical & Testing Center, Sichuan University, Chengdu 610064, China
d Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu 610041,
China
∗ Corresponding authors. E-mail addresses: liujie2011@scu.edu.cn (J. L.), lifengzhao@scu.edu.cn
(L. Z.), zhang-xia@scu.edu.cn (X. Z.). Tel./fax: +86 28 85503817;
† These authors contributed equally.
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Abstract
A series of novel polycyclic spiro-fused carbocyclicoxindoles were synthesized
and investigated for their in vitro antiproliferative activities against nine human cancer
cell lines. Five compounds (10i, 10l, 10n, 10p, and 10r) demonstrated anticancer
activities against A2780s cells with IC50 values of less than 30 ꢀM. In particular,
compound 10i showed anticancer activities against seven cancer cell lines and stronger
activities than cisplatin in A2780s, A2780T, CT26, and HCT116 cells. Further studies
illustrated that compound 10i arrested cell cycle in G1 phase and induced apoptosis of
HCT116 cells. This compound also effectively increased the protein levels of cleaved
caspase-3, p53, and MDM2. Molecular docking results revealed that compound 10i could
bind well to the p53-binding site on MDM2, indicating that it might work by blocking the
MDM2-p53 interactions.
Keywords: polycyclic spiro-fused carbocyclicoxindoles; antiproliferative activity; p53
inducer; MDM2
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1. Introduction
The p53 protein is a well-known tumor suppressor, which has a pivotal role in
defending against tumor-associated DNA damage and regulating cell division.[1, 2]
Under non-stressed conditions, p53 is maintained at low levels mainly by its negative
regulator MDM2 protein through an auto-regulatory feedback loop.[3, 4] In response to
cellular stresses including DNA damage and hyperproliferation, the protein levels of p53
are elevated, leading to cell cycle arrest, apoptosis, or senescence. The outcome of these
events blocks cell proliferation or eliminates the cancer cells, and thus suppresses the
development of cancer.[5]
Dysregulation of the p53 pathway, through mutations in TP53 (the human gene
that encodes p53) or inactivation of the components of the p53 pathway, is a common
feature in human cancers.[6, 7] In about 50% of tumors, p53 is nonfunctional as a result
of mutations in TP53.[7] The mutations in TP53 inactivated p53 by altering the DNA-
binding ability of p53 or changing the interactions of p53 with other proteins.[8] In
cancers in which TP53 is not mutated or deleted, the mdm2 gene is usually found
amplified or overexpressed to inhibit the function of p53.[9] As the primary negative
regulator of p53, MDM2 deregulates the protein levels and the activity of p53 by
promoting p53 degradation and blocking the binding of p53 to its targeted DNA.[10, 11]
Thus, blockade of MDM2-p53 interaction may provide a novel method for the treatment
of a broad spectrum of cancers.
The X-ray crystal structure of MDM2 with a transactivation domain peptide of
p53 revealed that the p53 peptide binds to a hydrophobic cleft on MDM2.[12] Three p53
residues Phe19, Trp23, and Leu26, which are also involved in the transactivation, insert
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deeply into the hydrophobic cavity on MDM2. Although targeting protein-protein
interactions is usually viewed as a challenging task due to the typical flat binding sites,
the existence of the well-defined p53-binding cavity on MDM2 suggests the possibility of
the design of non-peptide inhibitors that bind to MDM2 and disrupt the MDM2-p53
interactions.[13]
In the past years, intensive efforts have been devoted to the search for efficient
small-molecule inhibitors of the MDM2-p53 interactions.[1, 14, 15] A breakthrough in
this field came from the discovery of the first series of small-molecule inhibitors Nutlins.
The crystal structure of MDM2/Nutlin-2 complex illustrated that the inhibitor mimicked
the structure of p53 peptide where the imidazoline scaffold of Nutlin-2 replaced the p53
peptide backbone and three substituents of Nutlin-2 positioned into the MDM2 pockets
which were normally occupied by residues Phe19, Trp23, and Leu26 of p53. To date, ten
small-molecule inhibitors have advanced into clinical trials as anticancer agents.[16-22]
The representative structures of these inhibitors are shown in Figure 1. Among them, the
spirooxindole-based inhibitor SAR405838 (MI-77301, compound 3, Figure 1)
demonstrated a high binding affinity to MDM2 with a Ki value of 0.88nM.[20]
Researchers found that the spirooxindole core structure of SAR405838 was essential for
its binding affinity. The spirooxindole structure filled the Trp23 (p53) pocket on MDM2
and positioned other substituents to occupy the Phe19 (p53) and Leu26 (p53) pockets on
MDM2.
To us, spirooxindole-based inhibitors such as SAR405838 represented a good
opportunity for further investigations due to its high potency.[20, 23, 24] Based on the
strategy of diversity-oriented synthesis, we developed a methodology for the efficient
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construction of functionalized polycyclic spiro-fused carbocyclicoxindoles via an
asymmetric organocatalytic quadruple-cascade reaction.[25] We postulated that by
keeping the spirooxindole scaffold, the compounds produced by our methodology should
retain the ability of mimicking the Trp23 residue of p53 and positioning other
substituents to fill the pockets on MDM2. In addition, by using fused ring systems, we
imposed structural constraints onto the ligands, which may reduce the entropic cost
associated with the binding event. Herein, we report the anticancer activity of these
polycyclic spiro-fused carbocyclicoxindoles and the preliminary mechanism studies of
their anticancer effects.
2. Results and Discussion
2.1. Chemistry
The general synthetic route for the targeted compounds 10a-s is outlined in
Scheme 1. The easily accessible oxindole 6 was treated with salicylaldehyde 7 to obtain
the key intermediate (E)-3-(2-hydroxybenzylidene)oxindole (8). The commercially
available starting material crotonaldehyde (9) then reacted with 8 in the presence of α,α-
L-diphenylprolinol trimethylsilyl ether and 2-(trifluoromethyl)benzoic acid in a
quadruple-cascade manner to yield the polycyclic spiro-fused carbocyclicoxindole 10.
The reactions provided the targeted compounds in moderate to high yields (up to 90%).
All the products were characterized by 1H NMR, 13C NMR, and ESI-MS analysis. The
absolute configuration of compound 10i was determined by X-ray crystallography. [26]
Details of the discussions of this methodology was under separate cover.[25]
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2.2. Biological evaluation
2.2.1. In vitro antiproliferative activity
The in vitro antiproliferative activities of the compounds 10a-s were evaluated
against multiple cell lines. The IC50 (the concentration causing 50% inhibition of the
tumor cell proliferation) values or percent inhibition (if the IC50 of the corresponding
compound was greater than 30 ꢀM) of the tested compounds against A2870s and
A2870T cells were listed in Table 1. The IC50 values of the tested compounds against
H1299, BGC823, CT26, HCT116, A549, MCF7, and H1975 cells were listed in Table 2.
Several compounds (compounds 10i, 10l, 10n, 10p, and 10r) demonstrated moderate to
favorable anti-proliferative activities against the A2780s cell line. Steric hindrance of the
substituents on the carbocyclic group of the polycyclic portion decreased the activities of
these compounds (compounds 10p vs. 10a, 10q-r). The chlorine atom on the oxindole
ring enhanced the compound potency (compounds 10l vs. 10a) in A2780s cells.
Substituents on the distal phenyl ring of the polycyclic moiety did not affect the activity
of the compounds dramatically in most cases (compounds 10b-h vs. 10a), with the
bromide group enhancing the potency the most (10f vs. 10a) in A2780s cells. The percent
inhibition of compound 10o (47.2%) against A2780s was much larger than that of
compound 10a (23.5%), indicating that the dihydro-pyridine substructure worked better
than dihydro-pyran in A2730s cells. In addition, three compounds 10i, 10n, and 10p
showed activities of less than 30 ꢀM in multiple cell lines (Figure 2). Particularly,
compound 10i exhibited IC50 values ranging from 7.9 ꢀM to 23.4 ꢀM against a broad
spectrum of cancer cell lines including A2780s, A2780T, CT26, HCT116, A549, MCF7,
and H1975. It also showed stronger activity than the positive control cisplatin in A2780s,
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A2780T, CT26, and HCT116 cells. However, it did not inhibit the p53-deficient cell line
H1299 with an IC50 value of less than 30 ꢀM, probably because compound 10i worked by
blocking the interactions between MDM2 and p53 and did not exert inhibitory effects on
the p53-deficient cell lines. Interestingly, the compound 10n showed antiproliferative
activities against the the p53-deficient H1299 cell line, which indicated it might act
through additional non-p53-related inhibitory mechanisms. Because of the strong
activities of compound 10i in multiple cell lines, the mechanisms of its antiproliferative
effects were further investigated.
2.2.2. Cell cycle analysis
To elucidate the molecular mechanism by which compound 10i suppressed
proliferation of HCT116 cells, the effects of compound 10i on cell cycle distribution were
examined using flow cytometry. As shown in Figure 3, after exposure to compound 10i at
7.5 ꢀM and 10 ꢀM for 24 h, significant accumulation of HCT116 cells in G1 phase,
accompanied by a decrease of cells in S and G2 phases, was observed. The percentage of
cells in G1 phase increased from 43.79% in the vehicle group to 46.10% and 75.18% in
the groups treated with compound 10i at concentrations of 7.5 ꢀM and 10 ꢀM,
respectively. When treated with compound 10i for 48 h, the HCT116 cells in G1 phase
increased more dramatically. 82.32% of cells with treatment of compound 10i at 10 ꢀM
for 48 h accumulated in G1 phase. These results suggested that compound 10i inhibited
proliferation of HCT116 cells through inducing G1 phase arrest in a concentration- and
time-dependent manner. Nutlin-3a, a selective inhibitor of MDM2-p53 interactions also
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effectively arrested cell-cycle progression in HCT116 cells, but it induced both G1 and
G2 arrest.[27]
2.2.3. Induced apoptosis by compound 10i
The ability of compound 10i to provoke apoptosis in HCT116 cells was then
evaluated. After 24 h of treatment with compound 10i, HCT116 cells displayed the
morphological features of apoptotic cells including cell shrinkage (Figure 4A). The
fluorescence microscopic examination of Hoechst 33342 staining cells further confirmed
the apoptosis-inducing effects of compound 10i. As shown in Figure 4B, nuclei
condensation, cell-volume reduction, and nuclear fragmentation were observed in
HCT116 cells after 24 h of treatment with compound 10i. The flow cytometric analysis
with PI-staining was then used to quantitatively assess the apoptotic effects of compound
10i. As illustrated in Figure 5, the percentage of sub-G1 HCT116 cells in the 10i-treated
group increased in a concentration- and time-dependent manner. The apoptosis rate
increased from 1.73% (control) to 3.18%, 15.92%, and 37.03% when cells were treated
with compound 10i at 7.5, 10 and 12.5 ꢀM for 24 h, respectively. The apoptosis
augmented more dramatically with a rate up to 64.26% after 48 h of treatment with 10i at
12.5 ꢀM. In addition, the flow cytometric analysis with Annexin V-FITC/PI fluorescence
staining in HCT116 cells was used to further examine the apoptosis-inducing ability of
compound 10i. The experimental results indicated that the treatment with 10i
significantly induced concentration-dependent apoptosis in HCT116 cells (Figure 6). The
percentage of apoptotic HCT116 cells treated with 10i for 48 h increased from 6.27% to
66.06% when the concentration of 10i increased from 7.5 ꢀM to 12.5 ꢀM. Similar results
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were observed in HCT116 cells with treatment of 10i for 24 h. These data confirmed that
10i induced apoptosis of HCT116 cells in a concentration- and time-dependent manner.
2.2.4. Upregulation of caspase-3, p53, and MDM2 by 10i
The protein levels of procaspase-3, caspase-3, p53, and MDM2 in HCT116 cells
treated with 10i was monitored through western blot analysis. It is well known that
caspase-3 plays an essential role in apoptosis.[28] The common events in apoptosis
including cell shrinkage, chromatin condensation, and DNA fragmentation all require
caspase-3. The results of the western blotting demonstrated that the treatment with 10i for
24 h resulted in significant increase of the cleaved caspase-3 in a concentration-
dependent manner (Figure 7). The activation of caspase-3 suggested that 10i induced
apoptosis in HCT116 cells through a cascade-dependent pathway. It was also observed
that the treatment with 10i enhanced the protein levels of p53 and MDM2. Notably,
small-molecule inhibitors of MDM2-p53 interactions was expected to activate p53 and to
increase the levels of p53 in cells with wild-type p53. Moreover, the activation of p53 by
these inhibitors led to the induction of mdm2, a p53targeted gene, and as a result the
protein levels of MDM2 were increased.[24] Thus, the phenomenon of enhanced levels
of p53 and MDM2 by 10i was consistent with that of the reported MDM2-p53 interaction
inhibitors, suggesting that 10i may work by blocking the MDM2-mediated p53
degradation and consequently lead to accumulation of p53 and transcriptional activation
of the mdm2 gene.[24, 29]
2.2.5. Molecular docking analysis
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The docking study of 10i into the p53-binding site on MDM2 was then performed
using GOLD software (version 5.0)[30]. The predicted binding mode of 10i was
illustrated in Figure 8. Similar to the reported spirooxindole-based MDM2-p53
interaction inhibitors, the NH on the oxindole group of 10i exerted hydrogen-bonding
interactions with the Leu54 backbone carbonyl group on MDM2. The oxindole group of
10i extended into the Trp23 (p53) pocket on MDM2 and interacted with the hydrophobic
sidechains of Leu54 and Ile99. In addition, the polycyclic portion of 10i overlaid the
Phe19 residue of p53 and formed intensive hydrophobic interactions with Ile61, Met62,
Try67, Val75, Phe91, and Val93. Importantly, the distal phenyl group of the naphthalenyl
moiety filled the Phe19 (p53) pocket. The other compounds without the naphthalene
group presumably lacked the favorable hydrophobic interactions and shape
complementarity, which may explain why those compounds have lower anticancer
activities compared with 10i.
3. Experimental Section
3.1. Synthesis
3.1.1. Materials and methods
1H NMR and 13C NMR spectra were recorded on a Bruker 400 MHz NMR
spectrometer (Bruker BioSpin AG, Fällanden, Switzerland) at 400 and 100 MHz,
respectively. Chemical shifts (δ) were reported in ppm with tetramethylsilane as an
internal standard. ESI-HRMS spectra were recorded on a Waters SYNAPT G2.
Methanol was used to dissolve the sample. Melting points were recorded at SGW X-4
Melting point instrument (Shanghai precision & scientific instrument Co., Ltd, Shanghai,
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China). All reagents and solvents were obtained from Alfa, Aldrich, and Energy
Chemical and used without further purification. Column chromatography was performed
on silica gel (300–400 mesh, Qingdao Marine Chemical Ltd, Qingdao, China).
3.1.2. General procedures for the preparation of substituted oxindole
Substituted isatin (10 mmol) was dissolved in hydrazine hydrate (98%, 10 mL,
32.5 mmol) and refluxed for 15-30 min (130 °C). The reaction mixture was then poured
into cold water, extracted with ethyl acetate. The organic layer was then dried over
sodium sulfate. Evaporation of the solvent and recrystallization from hexane/ethyl acetate
provided the substituted oxindole.
3.1.3. General procedures for the preparation of substituted (E)-3-(2-
hydroxybenzylidene)oxindole
The reaction mixture of the substituted oxindole (1 equiv), the substituted
salicylaldehyde (1.2 equiv) and piperidine (0.1 equiv) in ethanol (1-2 mL/1 mmol) was
stirred at 90 °C for 3-5 h. After the reaction mixture cooled down, the precipitate was
filtered and washed with cold ethanol and diethyl ether, successively. Allowed to dry, the
product was used in subsequent reaction without further purification.
3.1.4. General procedures for the preparation of polycyclic spiro-fused
carbocyclicoxindoles
The crotonaldehyde (0.244 mmol) was added to a dry tube containing a
suspension of (E)-3-(2-hydroxybenzylidene)oxindole (0.1 mmol), α,α-L-diphenylprolinol
trimethylsilyl ether (0.02 mmol), 2-(trifluoromethyl)benzoic acid (0.06 mmol) and dry
solvent (1 mL). The reaction mixture was stirred at 40 °C for 24 h and then heated up to
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60 °C for 12 h. The reaction mixture was subjected to silica gel column chromatography
to yield the corresponding product (petroleum ether/ ethyl acetate, 2/1, V/V).
3.1.5. Analytical and Spectral Characterization Data
6,9-dimethyl-2'-oxo-6,6a,9,10a-tetrahydrospiro[benzo[c]chromene-10,3'-indoline]-8-
carbaldehyde (10a)
Light yellow solid; 80% yield; >99% ee; 8：1 dr; m.p. 125 °C -127 °C; [α]D22 = -49.8 (c
= 1.53 in CHCl3); HPLC (DAICEL CORPORATION AS-H), 2-propanol/n-hexane =
10/90, flow rate = 1.0 mL/min, λ = 254 nm, retention time: 13.69 min (major) and 17.80
min (minor); 1H NMR (400 MHz, CDCl3) δ = 9.53 (s, 1H), 8.86 (s, 1H), 7.28 (s, 1H),
7.13 (d, J = 7.2 Hz, 1H), 6.98 (t, J = 7.2 Hz, 2H), 6.90 (d, J = 7.6 Hz, 1H), 6.86-6.73 (m,
2H), 6.41 (t, J = 7.6 Hz, 1H), 5.99 (d, J = 7.6 Hz, 1H), 4.40 (dq, J = 11.6, 6.0 Hz, 1H),
3.67 (d, J = 10.4 Hz, 1H), 3.21 (t, J = 10.4 Hz, 1H), 2.81 (q, J = 6.4 Hz, 1H), 1.60 (d, J =
6.0 Hz, 3H), 1.19 (d, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ = 193.0, 179.1,
154.6, 147.0, 143.7, 140.0, 131.0, 128.6, 127.9, 126.0, 125.8, 124.2, 122.1, 120.4, 117.2,
110.2, 77.7, 50.6, 44.0, 36.5, 34.8, 20.8, 18.9. HRMS (ESI-TOF) calcd for C23H21NO3
[M+Na]+ = 382.1414, Found 382.1415.
4-fluoro-6,9-dimethyl-2'-oxo-6,6a,9,10a-tetrahydrospiro[benzo[c]chromene-10,3'-
indoline]-8-carbaldehyde (10b)
White solid; 75% yield; >99% ee; 6:1 dr; m.p. 59 °C-61 °C; [α]D22 = -50.0 (c = 0.074 in
CHCl3); HPLC (DAICEL CORPORATION AS-H), 2-propanol/n-hexane = 15/85, flow
rate = 1.0 mL/min, λ = 254 nm, retention time: 11.99 min (major) and 14.07 min (minor);
1H NMR (400 MHz, CDCl3) δ = 9.54 (s, 1H), 7.95 (s, 1H), 7.32 (t, J = 7.6 Hz, 1H), 7.17
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(d, J = 7.6 Hz, 1H), 7.07-6.94 (m, 2H), 6.88-6.77 (m, 2H), 6.37 (td, J = 8.0, 5.2 Hz, 1H),
5.79 (d, J = 8.0 Hz, 1H), 4.48 (dq, J = 12.0, 6.0 Hz, 1H), 3.69 (d, J = 10.4 Hz, 1H), 3.24
(t, J = 10.8 Hz, 1H), 2.84 (q, J = 6.8 Hz, 1H), 1.68 (d, J = 6.0 Hz, 3H), 1.21 (d, J = 7.2
Hz, 3H). 13C NMR (100 MHz, CDCl3) δ = 192.9, 179.3, 146.3, 143.8, 142.7, 140.0,
130.8, 128.7, 128.3, 125.9, 122.2, 119.9, 119.3, 114.8, 114.6, 110.4, 78.3, 50.7, 43.9,
36.5, 34.8, 20.7, 18.9. HRMS (ESI-TOF) calcd for C23H20FNO3 [M+Na]+ = 400.1319,
Found 400.1325.
3-methoxy-6,9-dimethyl-2'-oxo-6,6a,9,10a-tetrahydrospiro[benzo[c]chromene-10,3'-
indoline]-8-carbaldehyde (10c)
Light yellow solid; 65% yield; >99% ee; 5:1 dr; m.p.70 °C-72 °C; [α]D22 = -42.3 (c =
0.40 in CHCl3); HPLC (DAICEL CORPORATION AS-H), 2-propanol/n-hexane =
10/90, flow rate = 1.0 mL/min, λ= 254 nm, retention time: 17.63 min (major) and 21.38
min (minor); 1H NMR (400 MHz, CDCl3) δ = 9.53 (s, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.19
(d, J = 7.6 Hz, 1H), 7.03 (t, J = 7.6 Hz, 1H), 6.96 (d, J = 7.6 Hz, 1H), 6.83 (d, J = 2.4 Hz,
1H), 6.38 (d, J = 2.4 Hz, 1H), 6.00 (dd, J = 8.8, 2.4 Hz, 1H), 5.88 (d, J = 8.8 Hz, 1H),
4.38 (dq, J = 12.2, 6.0 Hz, 1H), 3.65 (s, 4H), 3.23 (t, J = 10.4 Hz, 1H), 2.81 (q, J = 6.8
Hz, 1H), 1.62 (d, J = 6.0 Hz, 3H), 1.19 (d, J = 4.8 Hz, 3H). 13C NMR (100 MHz, CDCl3)
δ = 193.0, 179.4, 159.5, 155.7, 146.9, 143.9, 140.1, 131.1, 128.6, 126.0, 124.9, 122.1,
117.5, 110.3, 106.0, 102.8, 77.6, 55.2, 50.9, 44.0, 36.1, 34.7, 20.7, 19.0. HRMS (ESI-
TOF) calcd for C24H23NO4 [M+Na]+ = 412.1519, Found 412.1533.
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2-fluoro-6,9-dimethyl-2'-oxo-6,6a,9,10a-tetrahydrospiro[benzo[c]chromene-10,3'-
indoline]-8-carbaldehyde (10d)
White solid; 72% yield; 98% ee; 7:1 dr; m.p. 61 °C-63 °C; [α]D22 = -24.5 (c = 0.33 in
CHCl3); HPLC (DAICEL CORPORATION AS-H), 2-propanol/n-hexane = 10/90, flow
rate = 1.0 mL/min, λ = 254 nm, retention time: 15.51 min (major) and 18.96 min (minor);
1H NMR (400 MHz, CDCl3) δ = 9.53 (s, 1H), 8.00 (s, 1H), 7.34 (t, J = 7.6 Hz, 1H), 7.18
(d, J = 7.6 Hz, 1H), 7.08-6.97 (m, 2H), 6.81 (d, J = 2.4 Hz, 1H), 6.76-6.66 (m, 2H), 5.73
(dd, J = 10.0, 2.4 Hz, 1H), 4.37 (dq, J = 12.0, 6.0 Hz, 1H), 3.64 (d, J = 10.4 Hz, 1H), 3.18
(t, J = 10.8 Hz, 1H), 2.83 (q, J = 6.8 Hz, 1H), 1.59 (d, J = 6.0 Hz, 3H), 1.21 (d, J = 6.8
Hz, 3H). 13C NMR (100 MHz, CDCl3) δ = 192.9, 179.2, 150.6, 146.6, 143.7, 140.1,
130.4, 129.0, 127.1, 127.1, 126.0, 122.3, 117.9, 117.8, 114.3, 114.1, 111.5, 111.3, 110.6,
77.5, 50.6, 43.8, 36.5, 34.8, 20.7, 18.8. HRMS (ESI-TOF) calcd for C23H20FNO3
[M+Na]+ = 400.1319, Found 400.1329.
2-chloro-6,9-dimethyl-2'-oxo-6,6a,9,10a-tetrahydrospiro[benzo[c]chromene-10,3'-
indoline]-8-carbaldehyde (10e)
White solid; 65% yield; 98% ee; 6:1 dr; m.p. 54 °C-56 °C; [α]D22 = -61.3 (c = 0.266 in
CHCl3); HPLC (DAICEL CORPORATION AS-H), 2-propanol/n-hexane = 5/95, flow
rate = 1.0 mL/min, λ = 254 nm, retention time: 31.17 min (major) and 39.05 min (minor);
1H NMR (400 MHz, CDCl3) δ = 9.53 (s, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.18 (d, J = 7.6
Hz, 1H), 7.10-6.99 (m, 2H), 6.95 (dd, J = 8.4, 2.0 Hz, 1H), 6.82 (d, J = 2.4 Hz, 1H), 6.72
(d, J = 8.4 Hz, 1H), 5.94 (d, J = 1.2 Hz, 1H), 4.38 (dq, J = 12.0, 6.0 Hz, 1H), 3.63 (d, J =
10.4 Hz, 1H), 3.21 (t, J = 10.4 Hz, 1H), 2.84 (q, J = 6.8 Hz, 1H), 1.60 (d, J = 6.0 Hz, 3H),
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ACCEPTED MANUSCRIPT
1.21 (d, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 192.9, 178.8, 153.3, 146.3,
143.8, 140.1, 130.4, 129.0, 127.8, 126.9, 126.0, 125.0, 124.7, 122.3, 118.3, 110.4, 77.7,
50.5, 43.5, 36.5, 34.7, 20.6, 18.9. HRMS (ESI-TOF) calcd for C23H20ClNO3 [M+Na]+ =
416.1024, Found 416.1033.
2-bromo-6,9-dimethyl-2'-oxo-6,6a,9,10a-tetrahydrospiro[benzo[c]chromene-10,3'-
indoline]-8-carbaldehyde (10f)
White solid; 70% yield; 98% ee; 7:1 dr; m.p. 94 °C-96 °C; [α]D22 = -72.6 (c = 1.15 in
CHCl3); HPLC (DAICEL CORPORATION AS-H), 2-propanol/n-hexane = 5/95, flow
rate = 1.0 mL/min, λ= 254 nm, retention time: 32.81 min (major) and 37.87 min (minor);
1H NMR (400 MHz, CDCl3) δ = 9.54 (s, 1H), 7.70 (s, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.19
(d, J = 7.6 Hz, 1H), 7.12-7.00 (m, 3H), 6.82 (d, J = 2.4 Hz, 1H), 6.67 (d, J = 8.8 Hz, 1H),
6.08 (d, J = 1.2 Hz, 1H), 4.38 (dq, J = 12.0, 6.0 Hz, 1H), 3.64 (d, J = 10.4 Hz, 1H), 3.21
(t, J = 11.2 Hz, 1H), 2.85 (q, J = 6.8 Hz, 1H), 1.61 (d, J = 6.0 Hz, 3H), 1.22 (d, J = 7.2
Hz, 3H). 13C NMR (100 MHz, CDCl3) δ = 192.9, 178.9, 153.8, 146.3, 143.8, 140.0,
130.7, 130.4, 129.0, 127.7, 127.3, 126.0, 122.4, 118.7, 112.4, 110.4, 77.7, 50.6, 43.4,
36.5, 34.6, 20.6, 18.9. HRMS (ESI-TOF) calcd for C23H20BrNO3 [M+Na]+ = 460.0519,
Found 460.0525.
2-nitro-6,9-dimethyl-2'-oxo-6,6a,9,10a-tetrahydrospiro[benzo[c]chromene-10,3'-
indoline]-8-carbaldehyde (10g)
White solid; 35% yield; 98% ee; 4:1 dr; m.p. 104 °C-106 °C; [α]D22 = -87.1 (c = 0.31 in
CHCl3); HPLC (DAICEL CORPORATION AD-H), 2-propanol/n-hexane = 20/80, flow
15

ACCEPTED MANUSCRIPT
rate = 1.0 mL/min, λ = 254 nm, retention time: 11.05 min (major) and 17.27 min (minor);
1H NMR (400 MHz, CDCl3) δ = 9.58 (s, 1H), 7.93 (dd, J = 9.2, 2.4 Hz, 1H), 7.78 (s, 1H),
7.43 (t, J = 7.6 Hz, 1H), 7.22 (d, J = 7.6 Hz, 1H), 7.17-7.07 (m, 2H), 7.04 (d, J = 1.6 Hz,
1H), 6.86 (d, J = 8.8 Hz, 2H), 4.53 (dq, J = 12.4, 6.0 Hz, 1H), 3.70 (d, J = 10.4 Hz, 1H),
3.31 (t, J = 10.8 Hz, 1H), 2.92 (q, J = 6.8 Hz, 1H), 1.71 (d, J = 6.0 Hz, 3H), 1.26 (d, J =
7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ = 192.7, 178.7, 160.4, 145.0, 144.3, 140.9,
140.3, 129.9, 129.4, 125.8, 125.0, 124.3, 122.5, 121.7, 117.3, 111.1, 79.0, 50.5, 42.7,
36.6, 34.4, 20.4, 19.1. HRMS (ESI-TOF) calcd for C23H20N2O5 [M+Na]+ = 427.1264,
Found 427.1277.
2,6,9-trimethyl-2'-oxo-6,6a,9,10a-tetrahydrospiro[benzo[c]chromene-10,3'-indoline]-
8-carbaldehyde (10h)
White solid; 75% yield; >99% ee; 5:1 dr; m.p. 75 °C-77 °C; [α]D22 = -44.3 (c = 0.33 in
CHCl3); HPLC (DAICEL CORPORATION AS-H), 2-propanol/n-hexane = 10/90, flow
rate = 1.0 mL/min, λ= 254 nm, retention time: 10.61 min (major) and 12.35 min (minor);
1H NMR (400 MHz, CDCl3) δ = 9.54 (s, 1H), 7.71 (s, 1H), 7.32 (t, J = 7.6 Hz, 1H), 7.18
(d, J = 7.6 Hz, 1H), 7.07-6.94 (m, 2H), 6.82 (dd, J = 14.8, 5.2 Hz, 2H), 6.69 (d, J = 8.0
Hz, 1H), 5.78 (s, 1H), 4.37 (dq, J = 12.0, 6.0 Hz, 1H), 3.66 (d, J = 10.4 Hz, 1H), 3.20 (t, J
= 10.4 Hz, 1H), 2.83 (q, J = 7.2 Hz, 1H), 1.84 (s, 3H), 1.60 (d, J = 6.0 Hz, 3H), 1.22 (d, J
= 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ = 193.1, 179.3, 152.3, 147.2, 143.7, 140.2,
131.1, 129.3, 128.6, 128.3, 126.2, 125.3, 125.1, 122.1, 116.8, 110.0, 50.7, 44.1, 36.5,
34.7, 20.7, 20.7, 18.9. HRMS (ESI-TOF) calcd for C24H23NO3 [M+Na]+ = 396.1570,
Found 396.1575.
16

ACCEPTED MANUSCRIPT
2,5-dimethyl-2'-oxo-2,4a,5,12c-tetrahydrospiro[dibenzo[c,f]chromene-1,3'-indoline]-
3-carbaldehyde (10i)
White solid; 65% yield; >99% ee; 1:4 dr; m.p. 60 °C-62 °C; [α]D22 = 55.6 (c = 0.16 in
CHCl3); HPLC (DAICEL CORPORATION AD-H, 2-propanol/n-hexane = 20/80, flow
rate = 1.0 mL/min, λ = 254 nm, retention time: 8.20 min (major) and 9.28 min (minor);
1H NMR (400 MHz, CDCl3) δ = 9.71 (s, 1H), 7.60-7.50 (m, 2H), 7.33 (t, J = 7.2 Hz, 1H),
7.15 (t, J = 7.6 Hz, 1H), 7.05 (dd, J = 16.0, 8.4 Hz, 4H), 6.72 (d, J = 7.8 Hz, 1H), 6.62 (q,
J = 8.4 Hz, 2H), 4.92 (dd, J = 6.4, 4.0 Hz, 1H), 3.66 (dt, J = 12.0, 4.4 Hz, 1H), 3.50-3.44
(m, 1H), 3.17 (d, J = 6.8 Hz, 1H), 1.33 (d, J = 6.8 Hz, 4H), 0.98 (d, J = 7.2 Hz, 3H). 13C
NMR (100 MHz, CDCl3) δ = 191.1, 178.6, 152.3, 151.9, 142.2, 141.8, 134.8, 133.0,
129.3, 129.2, 128.5, 128.0, 124.3, 123.8, 123.2, 122.8, 122.5, 120.1, 114.5, 109.9, 70.7,
56.4, 43.3, 42.5, 41.4, 22.7, 14.1. HRMS (ESI-TOF) calcd for C27H23NO3 [M+Na]+ =
432.1570, Found 432.1585.
5',6,9-trimethyl-2'-oxo-6,6a,9,10a-tetrahydrospiro[benzo[c]chromene-10,3'-
indoline]-8-carbaldehyde (10j)
White solid; 67% yield; 98% ee; 6:1 dr; m.p. 122 °C-124 °C; [α]D22 = -56.4 (c = 0.69 in
CHCl3); HPLC (DAICEL CORPORATION AS-H), 2-propanol/n-hexane = 5/95, flow
rate = 1.0 mL/min, λ = 254 nm, retention time: 20.85 min (major) and 26.50 min (minor);
1H NMR (400 MHz, CDCl3) δ = 9.53 (s, 1H), 8.34 (s, 1H), 7.08 (d, J = 8.0 Hz, 1H), 7.03-
6.95 (m, 2H), 6.82 (t, J = 8.0 Hz, 3H), 6.45 (t, J = 7.6 Hz, 1H), 6.03 (d, J = 7.6 Hz, 1H),
4.40 (dq, J = 12.0, 6.0 Hz, 1H), 3.65 (d, J = 10.4 Hz, 1H), 3.20 (t, J = 10.4 Hz, 1H), 2.80
17

ACCEPTED MANUSCRIPT
(q, J = 6.8 Hz, 1H), 2.28 (s, 3H), 1.61 (d, J = 6.0 Hz, 3H), 1.20 (d, J = 7.2 Hz, 3H). 13C
NMR (100 MHz, CDCl3) δ = 193.1, 179.9, 154.6, 147.0, 143.9, 137.8, 131.4, 131.0,
128.9, 127.8, 126.6, 126.0, 124.5, 120.5, 117.1, 110.2, 77.7, 50.8, 44.1, 36.5, 34.8, 21.3,
20.8, 19.0. HRMS (ESI-TOF) calcd for C24H23NO3 [M+Na]+ = 396.1570, Found
396.1581.
5'-fluoro-6,9-dimethyl-2'-oxo-6,6a,9,10a-tetrahydrospiro[benzo[c]chromene-10,3'-
indoline]-8-carbaldehyde (10k)
White solid; 80% yield; 96% ee; 6:1 dr; m.p. 109 °C-111 °C; [α]D22 = -43.7 (c = 0.35 in
CHCl3); HPLC (DAICEL CORPORATION AS-H), 2-propanol/n-hexane = 10/90, flow
rate = 1.0 mL/min, λ= 254 nm, retention time: 13.96 min (major) and 20.39 min (minor);
1H NMR (400 MHz, CDCl3) δ = 9.54 (s, 1H), 7.96 (s, 1H), 7.02 (dt, J = 8.8, 4.4 Hz, 2H),
6.96-6.87 (m, 2H), 6.88-6.76 (m, 2H), 6.48 (t, J = 7.6 Hz, 1H), 6.00 (d, J = 7.6 Hz, 1H),
4.40 (dq, J = 12.0, 6.0 Hz, 1H), 3.63 (d, J = 10.4 Hz, 1H), 3.19 (t, J = 10.8 Hz, 1H), 2.84
(q, J = 6.8 Hz, 1H), 1.61 (d, J = 6.0 Hz, 3H), 1.21 (d, J = 6.4 Hz, 3H). 13C NMR (100
MHz, CDCl3) δ = 192.8, 179.2, 159.8, 154.6, 146.8, 143.4, 136.1, 132.7, 128.0, 125.5,
124.0, 120.5, 117.3, 115.0, 113.8, 110.8, 92.8, 77.6, 51.2, 44.1, 36.6, 34.8, 20.8, 18.8.
HRMS (ESI-TOF) calcd for C23H20FNO3 [M+Na]+ = 400.1319, Found 400.1320.
6'-chloro-6,9-dimethyl-2'-oxo-6,6a,9,10a-tetrahydrospiro[benzo[c]chromene-10,3'-
indoline]-8-carbaldehyde (10l)
Light yellow solid; 90% yield; >99% ee; 7:1 dr; m.p. 139 °C-141 °C; [α]D22 = -29.0 (c =
1.12 in CHCl3); HPLC (DAICEL CORPORATION AS-H), 2-propanol/n-hexane =
18

ACCEPTED MANUSCRIPT
10/90, flow rate = 1.0 mL/min, λ = 254 nm, retention time: 13.96 min (major) and 19.58
min (minor); 1H NMR (400 MHz, CDCl3) δ = 9.53 (s, 1H), 8.99 (s, 1H), 7.09-6.92 (m,
4H), 6.84 (dd, J = 12.4, 5.2 Hz, 2H), 6.48 (t, J = 7.6 Hz, 1H), 5.99 (d, J = 7.6 Hz, 1H),
4.40 (dq, J = 12.0, 6.0 Hz, 1H), 3.65 (d, J = 10.4 Hz, 1H), 3.18 (t, J = 10.4 Hz, 1H), 2.80
(q, J = 6.8 Hz, 1H), 1.63 (d, J = 6.0 Hz, 3H), 1.17 (d, J = 6.8 Hz, 3H). 13C NMR (100
MHz, CDCl3) δ = 193.0, 180.1, 154.6, 147.0, 143.5, 141.5, 134.3, 129.4, 128.1, 126.6,
125.6, 124.0, 122.1, 120.6, 117.3, 111.3, 77.7, 50.7, 44.1, 36.4, 34.8, 20.8, 18.9. HRMS
(ESI-TOF) calcd for C23H20ClNO3 [M+Na]+ = 416.1024, Found 416.1023.
1',6,9-trimethyl-2'-oxo-6,6a,9,10a-tetrahydrospiro[benzo[c]chromene-10,3'-
indoline]-8-carbaldehyde (10m)
White solid; 31% yield; 94% ee; 10:1 dr; m.p. 62 °C-64 °C; [α]D22 = -52.0 (c = 0.10 in
CHCl3); HPLC (DAICEL CORPORATION AS-H), 2-propanol/n-hexane = 10/90, flow
rate = 1.0 mL/min, λ= 254 nm, retention time: 17.80 min (major) and 22.07 min (minor);
1H NMR (400 MHz, CDCl3) δ = 9.53 (s, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.28 (s, 1H), 7.08
(t, J = 7.6 Hz, 1H), 6.99 (dd, J = 7.2, 4.0 Hz, 2H), 6.79 (d, J = 8.8 Hz, 2H), 6.41 (t, J =
7.6 Hz, 1H), 5.86 (d, J = 7.6 Hz, 1H), 5.02-4.87 (m, 1H), 3.79-3.69 (m, 2H), 3.19 (s, 3H),
2.69 (dd, J = 14.0, 6.8 Hz, 1H), 1.42 (d, J = 6.4 Hz, 3H), 1.24 (d, J = 7.2 Hz, 3H). 13C
NMR (100 MHz, CDCl3) δ = 192.8, 177.5, 154.9, 146.9, 143.4, 143.2, 130.7, 128.7,
127.9, 125.6, 125.6, 124.2, 122.1, 120.1, 117.2, 108.8, 77.3, 77.2, 77.0, 76.7, 73.5, 58.5,
50.4, 41.4, 34.7, 19.1, 18.4, 17.8. HRMS (ESI-TOF) calcd for C24H23NO3 [M+Na]+ =
396.1570, Found 396.1573.
19

ACCEPTED MANUSCRIPT
6,9-dimethyl-2'-oxo-1'-phenyl-6,6a,9,10a-tetrahydrospiro[benzo[c]chromene-10,3'-
indoline]-8-carbaldehyde (10n)
White solid; 45% yield; 92% ee; 12:1 dr; m.p. 72 °C-74 °C; [α]D22 = -25.6 (c = 0.50 in
CHCl3); HPLC (DAICEL CORPORATION AS-H, 2-propanol/n-hexane = 3/97, flow
rate = 1.0 mL/min, λ = 254 nm, retention time: 32.66 min (major) and 37.55 min (minor);
1H NMR (400 MHz, CDCl3) δ = 7.49 (t, J = 7.6 Hz, 2H), 7.39 (d, J = 7.6 Hz, 1H), 7.34
(t, J = 6.8 Hz, 4H), 7.10 (t, J = 7.2 Hz, 1H), 7.03 (t, J = 7.2 Hz, 1H), 6.95 (d, J = 7.6 Hz,
1H), 6.83 (d, J = 7.6 Hz, 1H), 6.79 (d, J = 2.0 Hz, 1H), 6.49 (t, J = 7.2 Hz, 1H), 6.14 (d, J
= 7.6 Hz, 1H), 5.05-4.87 (m, 1H), 3.86-3.74 (m, 2H), 2.91 (q, J = 6.8 Hz, 1H), 1.44 (d, J
= 6.4 Hz, 3H), 1.31 (d, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ = 192.8, 177.0,
155.1, 146.8, 143.3, 134.2, 130.4, 129.6, 128.6, 128.2, 128.0, 126.7, 125.9, 125.8, 124.2,
122.6, 120.1, 117.3, 110.1, 73.6, 50.4, 41.5, 34.9, 31.1, 19.0, 17.8. HRMS (ESI-TOF)
calcd for C29H25NO3 [M+Na]+ = 458.1727, Found 458.1728.
6',9'-dimethyl-2-oxo-6',6a',9',10a'-tetrahydro-5'H-spiro[indoline-3,10'-
phenanthridine]-8'-carbaldehyde (10o)
Light orange solid; 83% yield; >99% ee; 2.5:1 dr; m.p. 121 °C-123 °C; [α]D22 = -88.8 (c =
0.55 in CHCl3); HPLC (DAICEL CORPORATION AD-H), 2-propanol/n-hexane =
20/80, flow rate = 1.0 mL/min, λ= 254 nm, retention time: 19.96 min (major) and 17.34
min (minor); 1H NMR (400 MHz, CDCl3) δ = 9.54 (s, 1H), 8.37 (s, 1H), 7.24 (d, J = 7.6
Hz, 1H), 7.12 (d, J = 7.2 Hz, 1H), 7.00-6.89 (m, 3H), 6.86 (t, J = 7.6 Hz, 1H), 6.48 (d, J =
7.6 Hz, 1H), 6.21 (t, J = 7.6 Hz, 1H), 5.94 (d, J = 7.6 Hz, 1H), 3.65-3.46 (m, 2H), 2.92 (t,
J = 10.0 Hz, 1H), 2.80 (q, J = 6.8 Hz, 1H), 1.47 (d, J = 6.0 Hz, 3H), 1.18 (d, J = 6.8 Hz,
20

ACCEPTED MANUSCRIPT
3H). 13C NMR (100 MHz, CDCl3) δ = 193.4, 180.2, 149.0, 144.5, 143.3, 140.1, 131.5,
128.3, 127.2, 125.8, 124.1, 123.0, 121.9, 117.0, 113.5, 110.3, 53.8, 51.0, 43.3, 37.8, 35.0,
22.4, 18.9. HRMS (ESI-TOF) calcd for C23H22N2O2 [M+Na]+ = 381.1573, Found
381.1584.
2'-oxo-6,6a,9,10a-tetrahydrospiro[benzo[c]chromene-10,3'-indoline]-8-carbaldehyde
(10p)
Light yellow solid; 55% yield; 64% ee; 1:2.5 dr; m.p. 98 °C-100 °C; [α]D22 = 14.6 (c =
0.35 in CHCl3); HPLC (DAICEL CORPORATION AS-H), 2-propanol/n-hexane =
20/80, flow rate = 1.0 mL/min, λ = 254 nm, retention time: 16.65 min (major) and 23.05
min (minor); 1H NMR (400 MHz, CDCl3) δ = 9.57 (s, 1H), 8.28 (s, 1H), 7.32 (t, J = 7.6
Hz, 1H), 7.22 (d, J = 7.6 Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H), 7.06-6.93 (m, 2H), 6.82 (s,
1H), 6.78 (d, J = 8.0 Hz, 1H), 6.44 (t, J = 7.2 Hz, 1H), 6.18 (d, J = 8.0 Hz, 1H), 4.68 (dd,
J = 10.0, 4.0 Hz, 1H), 4.18-4.07 (m, 1H), 3.95 (t, J = 11.2 Hz, 1H), 3.51 (d, J = 10.4 Hz,
1H), 2.78 (d, J = 18.8 Hz, 1H), 2.65 (d, J = 18.8 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ
= 192.7, 179.7, 154.8, 146.3, 139.9, 138.3, 134.5, 128.7, 128.4, 125.4, 123.6, 122.9,
122.6, 120.4, 117.1, 110.4, 70.2, 47.5, 41.4, 35.6, 34.4. HRMS (ESI-TOF) calcd for
C21H17NO3 [M+Na]+ = 354.1101, Found 354.1103.
6,9-diethyl-2'-oxo-6,6a,9,10a-tetrahydrospiro[benzo[c]chromene-10,3'-indoline]-8-
carbaldehyde (10q)
Yellow solid; 60% yield; >99% ee; 3:1 dr; m.p. 53 °C-55 °C; [α]D22 = -86.3 (c = 0.23 in
CHCl3); HPLC (DAICEL CORPORATION AD-H, 2-propanol/n-hexane = 5/95, flow
21

ACCEPTED MANUSCRIPT
rate = 1.0 mL/min, λ= 220 nm, retention time: 22.05 min (major) and 30.51 min (minor);
1H NMR (400 MHz, CDCl3) δ = 9.58 (s, 1H), 8.03 (s, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.21
(d, J = 7.6 Hz, 1H), 7.00 (dd, J = 16.4, 7.6 Hz, 3H), 6.85 (dd, J = 13.6, 5.2 Hz, 2H), 6.46
(t, J = 7.6 Hz, 1H), 5.99 (d, J = 7.6 Hz, 1H), 4.33-4.17 (m, 1H), 3.70 (d, J = 10.4 Hz, 1H),
3.19 (t, J = 10.0 Hz, 1H), 2.74 (t, J = 5.8 Hz, 1H), 2.12-2.03 (m, 1H), 1.93-1.84 (m, 1H),
1.78 (dt, J = 14.4, 7.2 Hz, 1H), 1.54-1.45 (m, 1H), 1.17 (t, J = 7.2 Hz, 3H), 0.73 (t, J =
7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ = 193.1, 179.5, 154.9, 147.6, 142.6, 140.1,
131.0, 128.6, 127.8, 126.7, 125.7, 123.9, 122.3, 120.5, 117.2, 110.3, 82.1, 51.2, 42.2,
40.8, 37.6, 27.3, 26.4, 13.1, 9.2. HRMS (ESI-TOF) calcd for C25H25NO3 [M+Na]+ =
410.1727, Found 410.1725.
2'-oxo-6,9-dipropyl-6,6a,9,10a-tetrahydrospiro[benzo[c]chromene-10,3'-indoline]-8-
carbaldehyde (10r)
Yellow solid; 78% yield; >99% ee; 4:1 dr; m.p. 129 °C-131 °C; [α]D22 = -84.7 (c = 0.66
in CHCl3); HPLC (DAICEL CORPORATION AD-H), 2-propanol/n-hexane = 7/93, flow
rate = 1.0 mL/min, λ = 220 nm, retention time: 13.68 min (major) and 21.51 min (minor);
1H NMR (400 MHz, CDCl3) δ = 9.57 (s, 1H), 8.60 (s, 1H), 7.30 (td, J = 7.6, 0.8 Hz, 1H),
7.19 (d, J = 7.2 Hz, 1H), 7.06-6.93 (m, 3H), 6.83 (t, J = 5.6 Hz, 2H), 6.46 (t, J = 7.6 Hz,
1H), 5.98 (d, J = 7.6 Hz, 1H), 4.39- 4.19 (m, 1H), 3.69 (d, J = 10.4 Hz, 1H), 3.16 (td, J =
10.8, 1.6 Hz, 1H), 2.76 (t, J = 5.6 Hz, 1H), 1.99-1.88 (m, 1H), 1.79-1.72 (m, 3H), 1.66-
1.55 (m, 1H), 1.46-1.34 (m, 1H), 1.23-1.15 (m, 1H), 1.05 (t, J = 7.2 Hz, 4H), 0.96-0.79
(m, 3H), 0.75 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ = 193.1, 180.3, 154.8,
147.3, 143.1, 140.2, 131.0, 128.6, 127.7, 126.8, 125.4, 124.0, 122.3, 120.6, 117.3, 110.6,
22

ACCEPTED MANUSCRIPT
80.9, 51.2, 42.7, 39.9, 37.6, 36.6, 36.0, 22.1, 18.2, 14.2, 14.1. HRMS (ESI-TOF) calcd for
C27H29NO3 [M+Na]+ = 438.2040, Found 438.2042.
6,6,9-trimethyl-2'-oxo-6,6a,9,10a-tetrahydrospiro[benzo[c]chromene-10,3'-indoline]-
8-carbaldehyde (10s)
White solid; 50% yield; 97% ee; 5:1 dr; m.p. 65 °C-67 °C; [α]D22 = -59.2 (c = 0.31 in
CHCl3); HPLC (DAICEL CORPORATION AS-H, 2-propanol/n-hexane = 10/90, flow
rate = 1.0 mL/min, λ = 254 nm, retention time: 12.13 min (major) and 14.02 min (minor);
1H NMR (400 MHz, CDCl3) δ = 9.54 (s, 1H), 8.19 (s, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.19
(d, J = 7.2 Hz, 1H), 7.06-6.91 (m, 3H), 6.81 (dd, J = 14.4, 5.2 Hz, 2H), 6.49 (t, J = 7.6
Hz, 1H), 6.06 (d, J = 7.6 Hz, 1H), 3.65 (d, J = 10.8 Hz, 1H), 3.25 (d, J = 10.8 Hz, 1H),
2.81 (q, J = 6.8 Hz, 1H), 1.61 (s, 3H), 1.43 (s, 3H), 1.23 (d, J = 7.2 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ = 192.9, 179.7, 154.5, 147.7, 143.3, 140.1, 131.1, 128.5, 128.0,
127.7, 125.8, 123.8, 122.1, 120.8, 117.7, 110.3, 79.6, 50.7, 48.2, 34.5, 33.0, 28.8, 24.5,
18.8. HRMS (ESI-TOF) calcd for C24H23NO3 [M+Na]+ = 396.1570, Found 396.1576.
3.2. Biological evaluation
3.2.1. Antiproliferative activity assay
The MTT method was used to evaluate the antiproliferative activities of the
synthesized polycyclic spiro-fused carbocyclicoxindole derivatives. Targeted cancer cell
lines (3×103/well) were seeded in 96-well plates and cultured for 24 h, followed by the
treatment with compounds 10a-s (final concentrations of 30, 15, 7.5, 3.8, 1.9, and 0.9
ꢀM) for 48 h. A solution of 20 ꢀL 5 mg/mL MTT was added per well and incubated for
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another 2-4 h at 37 °C, and then the supernatant fluid was removed and 150 ꢀL DMSO
were added per well for 15-20 min. The absorptions (OD) were measured at 570 nm
using Spectra MAX M5 microplate spectrophotometer (Molecular Devices, LLC,
Sunnyvale, CA, USA). The effect of compounds on cancer cell viability was expressed
by IC50 of each cell line. Each assay was carried out at least three times.
3.2.2. Cell-cycle distribution and apoptosis detection analysis by flow cytometry
Cell cycle distributions in cells were determined through PI staining. HCT116
Cells were seeded in 6-well plates at the density of 1×105 cells/well and cultured for 24 h,
followed by the treatment of compound 10i with concentration of 0, 7.5, 10, and 12.5 ꢀM
for another 24 h and 48 h. Next, cells were harvested. Sediments were re-suspended in 1
mL hypotonic fluorochrome solution (50 ꢀg/mL PI in 0.1% sodium citrate plus 0.1%
Triton X-100), and then analyzed by flow cytometer (Novo Cyte, ACEA bioscience,
USA). Cell cycle distribution was then estimated with software NovoExpress 1.1.2.
The extent of apoptosis induced by compound 10i was quantitatively measured
using PI staining and Annexin V-FITC/PI staining assays. Following the instructions of
the PI and Annexin V-FITC/PI detection kits, HCT116 cells (1×106 cells/mL) were
seeded in 6-well plates for 24 h and then were treated with compound 10i (0, 7.5, 10, and
12.5 ꢀM) for another 24 h and 48 h. Cells were then collected and washed twice with
PBS and stained with 5 ꢀL PI solution or 5 ꢀL of Annexin V-FITC and 1 ꢀL of PI (100
ꢀg/mL) in 1×binding buffer (10 mM HEPES, pH 7.4, 140 mM NaOH, 2.5 mM CaCl2) at
room temperature for 30 minutes in the dark. Apoptotic cells were quantified using flow
cytometry. The percentages of early apoptotic (Annexin V-FITC positive, PI negative)
and late apoptotic (double positive of Annexin V-FITC and PI) cells were determined
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using software NovoExpress 1.1.2.
3.2.3. Hoechst staining
HCT116 cells (1×105/well) were seeded in 6-well plates and cultured for 24 h,
followed by the treatment of 10i with different final concentrations (0, 7.5, 10, and 12.5
ꢀM) for another 24 h. After rinsing with phosphate buffered saline (PBS), the cells were
fixed using 75% of ethanol. The morphological change of cells was examined by inverted
microscope. After that, the cells were stained with Hoechst 33342 (2 ꢀg/mL, in PBS) and
analysed under fluorescence microscope (Zeiss, Axiovert 200, Germany) to identify the
apoptotic cells.
3.2.4 Western blot analysis
After treatment with 10i, HCT116 cells were lysed in radio-immunoprecipitation
assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Nantong, Jiangsu
Province, China) with 1% cocktail (Sigma-Aldrich, St. Louis, MO, USA) on ice. Next,
lysates were centrifuged at 13000 g for 20 min at 4 °C. The supernatant was harvested
and the protein concentration was measured by the BCA method. Equal amounts of total
proteins were subjected to SDS-PAGE and transferred onto polyvinylidene fluoride
(PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were incubated
with each antibody and detected by immunoblot analysis. All antibodies were purchased
from Cell Signaling Technology, Inc. (Boston, MA, USA) and diluted in accordance with
the manufacturer’s instruction. After incubation with the specific primary and secondary
antibodies, the protein bands were visualized using an enhanced chemiluminescent
substrate to horseradish peroxidase (Amersham, Piscataway, NJ).
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3.3. Molecular docking analysis
Predicted bound configurations of compound 10i were obtained using GOLD
software (version 5.0)[30] with 4OAS.pdb[21] representing the human MDM2 structure.
The preparation of the protein structure including adding hydrogen atoms and removing
water molecules was conducted using the docking wizard in GOLD. The three-
dimensional structure of compound 10i was constructed and minimized using
ChemBio3D ultra (version 12.0, CambridgeSoft Corporation, USA). CHEMPLP was
selected as the scoring function for pose prediction. The docking pose of compound 10i
was visualized using PyMOL (version 0.99rc6, Schrödinger Inc., New York, NY, USA).
4. Concluding Remarks
In conclusion, we synthesized a novel series of polycyclic spiro-fused
carbocyclicoxindoles and evaluated their anticancer activities against multiple cancer cell
lines. Five compounds (10i, 10l, 10n, 10p, and 10r) exhibited IC50 values of less than 30
ꢀM in A2780s cells. The most potent compound 10i in A2780s cells (IC50 = 7.9 ꢀM) also
displayed inhibitory activities against a panel of other cancer cell lines including
A2870T, CT26, HCT116, A549, MCF7, and H1975. Notably, 10i outperformed cisplatin
in A2870s, A2870T, CT26, and HCT116 cells. The Hoechst 33342 staining assays and
the flow cytometric analyses with PI staining and Annexin V-FITC/PI staining indicated
that 10i induced apoptosis. The western blotting results illustrated that the treatment of
10i led to upregulation of cleaved caspase-3 in HCT116 cells and suggested that 10i
induce apoptosis through a cascade-dependent pathway. 10i also enhanced the protein
levels of p53 and MDM2, indicating it blocked MDM2-mediated p53 degradation. The
molecular docking study of 10i into the p53-binding site on MDM2 further supported that
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10i might work as an MDM2-p53 interaction inhibitor. However, the exact molecular
mechanism of the upregulation of p53 and MDM2 requires additional investigation.
Acknowledgement
We appreciate the funding support from the National Natural Science Foundation
of China (Nos. 81573290 and 81202403). We also thank Sichuan University Analytical
& Testing Center for NMR analysis.
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