Stereoselective Assembly of Multifunctional Spirocyclohexene Pyrazolones That Induce Autophagy-Dependent Apoptosis in Colorectal Cancer Cells

Article abstract of DOI:10.1021/acs.joc.9b01098

Enantio- and diastereoselective synthesis of multifunctional spiropyrazolone scaffolds has been achieved using secondary amine-catalyzed [4 + 2] annulations of α,β,γ,δ-unsaturated pyrazolones with aldehydes. The pyrazolone substrates serve as C4 synthons to produce 6-membered, carbocycle-based, chiral spiropyrazolone derivatives. The synthesized chiral compounds showed potent toxicity against a panel of cancer cell lines. The most potent compound 3h-induced cell cycle arrest and macroautophagy in HCT116 colorectal cancer cells, triggering autophagy-dependent apoptotic cell death.

Full text of DOI:10.1021/acs.joc.9b01098

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Article
Stereoselective assembly of multifunctional spirocyclohexene pyrazolones
that induce autophagy-dependent apoptosis in colorectal cancer cells
Xiang Li, Fei-Yu Chen, Jing-Wen Kang, Jin Zhou, Cheng
Peng, Wei Huang, Mu-Ke Zhou, Gu He, and Bo Han
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b01098 • Publication Date (Web): 25 Jun 2019
Downloaded from http://pubs.acs.org on June 26, 2019
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Stereoselective assembly of multifunctional spirocyclohexene
pyrazolones that induce autophagy-dependent apoptosis in
colorectal cancer cells
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Xiang Li, † Fei-Yu Chen, † Jing-Wen Kang, † Jin Zhou, † Cheng Peng,*† Wei Huang, † Mu-Ke Zhou, ‡
Gu He*‡ and Bo Han*†
†
State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional
Chinese Medicine, Chengdu 611137, China. E-mail: pengcheng@cdutcm.edu.cn or hanbo@cdutcm.edu.cn
‡ State Key Laboratory of Biotherapy and Department of Dermatology, West China Hospital, Sichuan University and Collaborative
Innovation Center for Biotherapy, Chengdu, 610041, China. E-mail: hegu@scu.edu.cn
R1
R3
R4
O
OH
R2
amine
catalysis
O
N
N
EWG
+
EWG
N
[4+2]
N
R4
O
R1
R3
spiropyrazolone framwork
R2
C4 synthon
HOMO activation
S
G1
G2
Cell Death
M
cell cycle arrest apoptosis
autophagy
Abstract: Enantio- and diastereoselective synthesis of multifunctional spiropyrazolone
scaffolds has been achieved using secondary amine-catalyzed [4+2] annulations of α,β,γ,δ-
unsaturated pyrazolones with aldehydes. The pyrazolone substrates serve as C4 synthons to
produce six-membered, carbocycle-based, chiral spiropyrazolone derivatives. The synthesized
chiral compounds showed potent toxicity against a panel of cancer cell lines. The most potent
compound 3h induced cell cycle arrest and macroautophagy in HCT116 colorectal cancer cells,
triggering autophagy-dependent apoptotic cell death.
Introduction
The unique combination of a lactam functionality and two adjacent nitrogen atoms makes the
pyrazolone framework a privileged structure in medicinal chemistry and drug discovery.1 Of
particular interest are spirocyclohexane(ene) pyrazolones, which contain a six-membered chiral
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carbocyclic ring and show attractive biological activities.2 Cyclohexane(ene)-fused
spiropyrazolones with multiple functional groups show insecticidal, antibacterial, analgesic,
anti-inflammatory and antitumor properties (Figure. 1). Since the stereochemistry of spirocyclic
pyrazolones strongly affects their antitumor activity,3 convenient and efficient methods to
asymmetrically synthesize spirocyclohexane(ene) pyrazolones are highly desirable.4
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OH
O
Ph
Cl
O
O2N
Tol
MeOOC
N
Ph
Ph
O
O
O
OH
O
N N
N N
N N
Ph
Ph
Ph
III analgesia
I antitumor
II anti-inflammation
Ph
O
NO2
O
O
O
N
N
N
N
S
N
H
Fe
CF3
OH
IV RalA inhibitor
V acetyl-CoA carboxylase inhibitor
Figure 1. Selected bioactive compounds containing a cyclohexane(ene)-fused spiropyrazolone
skeleton. RalA, Ras-like proto-oncogene A.
Pyrazolin-5-one substrates have shown great potential in asymmetric organocatalytic
reactions to construct six-membered carbocycle-based chiral spiropyrazolone scaffolds
(Scheme 1a).5-7 The simple pyrazolin-5-one (edaravone) serves as a nucleophilic C1 synthon in
domino [1+2+3] or [1+5] cyclizations.5 The α-arylidene pyrazolinones serve as versatile
electrophilic C2 synthons in various asymmetric [2+2+2] or [2+4] annulations,6 while α,β-
unsaturated pyrazolones bearing a γ-hydrogen have been exploited in [3+3] cyclizations
involving vinylogous γ-additions.7 However, few reports exist of catalytic asymmetric [4+2]
cyclizations using pyrazolone substrates as four-carbon building blocks. This led us to search
for suitable C4 surrogates and to develop novel, alternative strategies to synthesize
pharmacologically interesting spiropyrazolone derivatives, as part of our continuing interest in
combining asymmetric synthesis and medicinal chemistry.8
We designed an amine-catalyzed asymmetric [4+2] annulation of α,β,γ,δ-unsaturated
pyrazolone 1 with aldehyde 2 to produce the target framework (Scheme 1b). In this reaction,
the α,β,γ,δ-unsaturated pyrazolone substrate 1 serves as the electron-deficient C4 building block
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(a) Asymmetric synthesis of spirocyclohexane(ene)-pyrazolone hybrids
electrophilic site;
nucleophilic site; FG: functional group
6
7
8
9
R'
[1+5]
ref. 5
[2+4]
N
N
ref. 6
N
N
O
O
O
R
R
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FG
R
N
C1 synthon
C2 synthon
R'
R'
N
FG'
EWG
ref. 7
[3+3]
N
N
N
N
O
[4+2]
underdeveloped
O
R
R
C3 synthon
C4 synthon
(b) This work
R1
R3
R4
O
OH
R2
amine
catalysis
O
N
N
EWG
+
EWG
N
[4+2]
N
O
R4
R1
R3
R2
electron-deficient
branched diene
HOMO
activation
spirocyclohexene-fused
pyrazolone framwork
Scheme 1. Strategies to construct six-membered carbocycle-based chiral spiropyrazolone
scaffolds.
as well as branched diene for spiro-construction,9 while the aldehyde 2 is activated via a
HOMO-raising strategy.10 The resulting spirocyclohexene-pyrazolone hybrids can provide a
chiral spirocycle library for bioactive screening as part of the combinatorial pharmacophore
strategy, which can be effective for lead discovery.
RESULTS AND DISCUSSION
Initially, the reaction of α,β,γ,δ-unsaturated pyrazolone 1a with n-propanal 2a was employed
as a model reaction to probe the validity of the proposed organocatalytic annulation reaction.
In the presence of Hayashi-Jørgensen catalyst Cat. I in toluene at 25 oC, the desired spirocyclic
product was isolated with good yield and good ee value albeit in moderate dr value (Table 1,
entry 1). In efforts to improve stereoselectivity as well as yield, other catalysts were evaluated;
however, only a slightly increasing ee and dr value was observed (entry 2 and 3). Then we
attempted to investigate other various reaction parameters such as solvent, additive, and
reaction temperature. We found that the properties of different solvents showed significantly
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Table 1. Optimization of the model reactiona
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8
Ph
5
O
OR
O
Ph
Ar
I:
PG = TMS, Ar = Ph
PG = TES, Ar = Ph
N
N
COOEt
Cat.
, Additive
Ar
OPG
Cat.
COOEt
N
II:
+
N
Solvent, Temp
N
H
III:
PG = TES,
O
Ph
3a
4a
Ph
9
Ar = 3,5-(CF3)2C6H3
R
R
5 = H
5 = TMS
1a
2a
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TMSCl, TEA
Yield
(%)b
Entry
Cat.
Solvente
Additive
Temp (oC)
drc
ee (%)d
1
I
Tol
BzOH
25
25
25
25
25
25
25
25
25
25
25
0
51
52
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62
60
76
76
74
78
74
63
76
75
70
67
62:38
62:38
65:35
70:30
68:32
73:27
75:25
75:25
72:28
73:27
70:30
80:20
84:16
84:16
84:16
43
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53
66
68
76
80
78
76
78
68
90
97
96
94
2
II
Tol
BzOH
3
III
III
III
III
III
III
III
III
III
III
III
III
III
Tol
BzOH
4
CH2Cl2
CHCl3
MeCN
THF
THF
THF
THF
THF
THF
THF
THF
THF
BzOH
5
BzOH
6
BzOH
7
BzOH
8
2-FBzOH
3-NO2BzOH
AcOH
9
10
11
12
13
14
－
BzOH
BzOH
BzOH
BzOH
-20
-30
-20
15f
a
Reactions were performed with 1a (0.15 mmol), 2a (0.30 mmol), catalyst (20 mol%) and acidic additive (30
mol%) in 1.5 mL of solvent at 25 oC. b Yield of isolated major isomer 4a over two steps. c Calculated based on
1H NMR analysis of the crude reaction mixture. Determined by chiral HPLC analysis of the major
d
diastereoisomer. e The ratio of solvent/H2O was 20:1 in v/v. f 10 mol% catalyst was used.
impact on stereoselectivity as well as yield (entry 4-7) and using tetrahydrofuran delivered the
desirable product with 80% ee from 53% ee and 75:25 dr from 65:35 dr (entry 7). Various acid
additives were also investigated, and benzoic acids with an electron-withdrawing group on the
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aromatic ring or acetic acid slightly affected stereoselectivity of the product while retaining
good yield (entry 8-10). Besides, inferior stereoselectivity of the reaction was observed without
the promotion of acid additive (entry 11). After lowering the reaction temperature in order to
enhance diastereoselectivity and enantioselectivity, an excellent 97% ee and 84:16 dr was
provided when the reaction was conducted at -20 oC (entry 12-14). Reducing the load of catalyst
decreased the yield of the reaction with maintaining in high stereoselectivity (entry 15).
These optimized conditions were used to investigate substrate scope with various kinds of
α,β,γ,δ-unsaturated pyrazolones 1 (Scheme 2). The reaction tolerated a broad range of
substrates with diverse electronic and steric properties, affording the corresponding products 4
in satisfying yields (69-84%) with good to high diastereoselectivities (up to 86:14 d.r.) and
excellent enantioselectivites (up to 99% ee). For example, the reaction tolerated electron-
deficient and -rich substituents on the aryl ring at position R1 at ortho-, meta-, and para-
positions of 1, giving the spirocyclohexene-pyrazolones 4a-h in yields of 71-84% with up to
98% ee and up to 85:15 dr. Also suitable as substrates were α,β,γ,δ-unsaturated pyrazolones
containing thienyl or 2-naphthyl groups, which provided the desired products 4i and 4j in
reasonable yield, although the 2-naphthyl moiety led to low diastereoselectivity. Changing the
electronic properties of the functional groups at position R2 slightly affected the
enantioselectivity of the reaction in a few cases (4k-m), although it did reduce dr values. The
absolute configuration of 4m was assigned based on single-crystal X-ray diffraction analysis
(CCDC 1886929), and the configurations of products 3 and 4 were assigned by analogy.11
Impressively, the reaction performed quite well in the presence of a bulky phenyl group at
position R3, generating 4n. Switching the aldehyde 2 from propanal to n-butanal or 4-methyl-
2-pentenal led to good yields and diastereoselectivities for products 4o and 4p, although
enantioselectivity was only moderate for the latter product. Treating 3a with PCC generated the
oxidation product 5a in excellent yield. This product can diversify the framework of bioactive
spirocyclohexene-pyrazolones. Further modifying ester group of 1 with phenyl group failed to
deliver the target products due to the inert reactivity of substrates. Moreover, we successfully
obtained some TMS-protected diastereoisomers of 4’ with moderate dr values. The relative
configuration of minor diastereoisomer 4k’ was assigned based on single-crystal X-ray
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diffraction analysis (CCDC 1910043), and the configurations of products 4’ were assigned by
5
6
analogy (see details in ESI).12
7
8
9
R1
5
R3
O
OR
R1
R4
COOEt
Cat. III
BzOH (30 mol% )
R2
(20 mol%)
O
N
N
COOEt
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+
N
N
R2
THF/H2O, -20 o
C
R4
O
R3
3
R
5 = H
1
2
TMSCl, TEA, DCM, 0 o
C
4
R
5 = TMS
5
5
O
O
OR
OR
Ph
Ph
5
O
5
OR
O
N
N
N
OR
Ph
COOEt
COOEt
Ph
N
N
N
N
COOEt
COOEt
N
F
Br
Br
3a
4a
, 85%; , 75%
97% ee, 84:16 dr
3b
4b
, 88%; , 82%
93% ee, 85:15 dr
3c
4c
, 90%; , 84%
3d
4d
, 91%; , 82%
97% ee, 82:18 dr
97% ee, 82:18 dr
5
5
O
O
OR
OR
Ph
Ph
5
O
5
OR
N
N
O
N
N
OR
Ph
COOEt
COOEt
Ph
N
N
N
N
COOEt
COOEt
Me
Cl
Cl
4e
OMe
4g
, 80%; , 71%
OMe
4h
, 80%; , 73%
98% ee, 73:27 dr
3e
3f
4f
, 81%; , 74%
90 % ee, 78:22 dr
3g
3h
, 80%; , 72%
93% ee, 80:20 dr
90% ee, 70:30 dr
Cl
5
O
OR
Ph
5
O
OR
N
N
F
N
N
N
N
Ph
COOEt
N
N
O
O
COOEt
Ph
OR5
Ph
OR5
S
COOEt
COOEt
3i
4i
, 87%; , 78%
93% ee, 83:17 dr
3j
4j
, 78%; , 69%
99% ee, 68:32 dr
3k
4k
3l
4l
, 82%; , 74%
, 80%; , 72%
95% ee, 62:38 dr
94% ee, 60:40 dr
Me
Ph
Ph
N
N
N
N
N
N
O
O
O
OR5
Ph
OR5
Ph
OR5
Ph
COOEt
COOEt
4m
COOEt
3m
3n
4n
3o
4o
, 81%;
, 72%
, 85%; , 77%
, 88%; , 80%
95% ee, 86:14 dr
4m
CCDC 1886929 (
)
99% ee, 65:35 dr
96% ee, 85:15 dr
Ph
Ph
N
N
N
N
O
O
O
Ph
OR5
Ph
COOEt
COOEt
, 78%
94% ee, 83:17 dr
3p
4p
5a
, 78%; , 70%
66% ee, 80:20 dr
Scheme 2. Substrate scope of the annulation reaction. Yield was calculated from the isolated
diastereomer of 3 or 4; dr values, from 1H NMR analysis of the crude mixture of 3 and 4; and ee values,
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from chiral HPLC analysis of major isomer 4. Compound 3a was oxidized with PCC in CH2Cl2 at 40 oC
for 6 h to yield 5a.
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Figure 2. (a) Heat map of mean half maximal inhibitory concentrations (IC50) of compounds 3a–3p, 4a
and 5a against a panel of normal or cancer cell lines. Mean values were determined from triplicate
experiments. (b) Cell cycle analysis of HCT116 cells incubated with 0.5 or 2.0 μM 3h for 24 h. (c)
Apoptosis levels in HCT116 cells incubated with 0.5 or 2.0 μM 3h for 24 h, based on dual staining with
annexin-V/propidium iodide followed by flow cytometry. (d) Nuclear morphology in HCT116 cells
incubated with 0.5 or 2.0 μM 3h for 24 h, followed by Hoechst 33258 staining. Scale bar: 6 μm.
Next we wanted to screen the cyclohexene-fused spiropyrazolone products of our reaction
for potential bioactivity.13 According to Lipinski’s rules, 3 showed a more favorable lipo-hydro
partition coefficient and number of hydrogen bond donors and acceptors than 4.14 Therefore we
mainly focused our bioactivity screening efforts on 3. Compounds 3a-3p, 4a and 5a showed
various degrees of toxicity against a panel of cancer cell lines without remarkable effects on
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BEAS-2B human bronchial epithelial cells (Figure 2a, Table S1). Several modifications
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substantially reduced this cytotoxicity: protecting the OH group of 3a with TMS (4a),
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Figure 3. (a) Analysis of autophagic vacuoles in HCT116 cells after incubation with 0.5 μM 3h for 24
h. Cells were analyzed using transmission electron microscopy. Scale bar: 1 μm. (b) HCT116 cells were
transfected with GFP-LC3 plasmid, then incubated with 0.5 μM 3h for 24 h. Nuclei were visualized using
propidium iodide staining, while autophagosomes were imaged as GFP-LC3 puncta under a fluorescence
microscope. Scale bar: 6 μm. (c) HCT116 cells were treated with 2.0 μM 3h with or without 1 mM 3-
MA, then cell viability was assayed using the MTT method. ** p < 0.01. (d) HCT116 cells were treated
with 2.0 μM 3h with or without 1 mM 3-MA, then apoptotic cells were quantified based on dual staining
with annexin V and propidium iodide. (e) HCT116 cells were incubated with 2.0 μM 3h for 24 h with or
without 1 mM 3-MA, then levels of apoptosis- and autophagy-related proteins were assessed using
Western blotting.
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introducing a 2-naphthyl at R1 (3j), introducing a phenyl group at R3 (3n) or adding a long-
chain alkyl group at R4 (3o, 3p). In contrast, oxidizing the hydroxyl group to a ketone (5a) only
slightly reduced toxicity. Substituting an N-phenyl group at R2 on the pyrazolone moiety (3k-
3m) did not improve cytotoxicity. In contrast, adding an m- or p-methoxyl-substituted phenyl
group at R1 (3g and 3h) did improve toxicity.
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In order to begin to identify potential mechanisms of 3h cytotoxicity, we focused on the
compound's effects on cell cycle progression and apoptosis in HCT116 colorectal cancer cells.
After 24 h incubation, 3h induced cell cycle arrest at G0/G1 phase in a dose-dependent manner
(Figure 2b). The compound also induced apoptotic cell death, based on staining with annexin-
V/propidium iodide (Figure 2c) and altered nuclear morphology as revealed by Hoechst 33258
staining (Figure 2d).
In addition, 24 h incubation with 0.5 μM 3h potently induced macroautophagic vacuole
formation in HCT116 cells expressing GFP-LC3. These vacuoles were visualized using
transmission electron microscopy (Figure 3a) and fluorescence microscopy, which detected
GFP-LC3 puncta in cytoplasm (Figure 3b).15 To examine whether this macroautophagy may
help explain the observed cytotoxicity of 3h, we incubated HCT116 or SW480 cells with 3h
alone or in combination with the autophagy inhibitor 3-methyladenine (3-MA). This inhibitor
reversed the ability of 3h to induce cytotoxicity and apoptosis, to up-regulate expression of
proapoptotic PARP, Caspase-3, Bax and cytochrome c, and to down-regulate anti-apoptotic
Bcl-2. Similarly, 3-MA reversed 3h-induced changes in LC3 cleavage, SQSTM1 degradation
and ULK1 phosphorylation at Ser555 (Figure 3c-3e).16 These results suggest that 3h induces
autophagy-dependent apoptotic cell death in HCT116 colorectal cancer cells.17
CONCLUSIONS
Here we report a highly enantioselective organocatalyzed [4+2] cyclization of α,β,γ,δ-
unsaturated pyrazolones and aldehydes that facilely delivers a variety of drug-like
spirocyclohexene pyrazolones bearing contiguous stereogenic centers and multiple functional
groups. A small preliminary library of these pyrazolones was found to contain compounds that
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suppressed proliferation in a panel of cancer cell lines. The most potent compound, 3h, was
found to arrest HCT116 cells in G0/G1 phase and induce macroautophagy and apoptosis in a
dose-dependent manner. These anticancer effects of 3h were reversed by the autophagy
inhibitor 3-MA, suggesting that 3h exerts its effects by stimulating autophagy-dependent
apoptosis. These results demonstrate the potential of our cyclization procedure for generating
novel spiropyrazolone derivatives that can kill cancer cells by inducing programmed cell death.
Further studies to explore novel chiral small molecules against colorectal cancer are ongoing
in our laboratory.
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EXPERIMENTAL SECTION
General information
NMR data were obtained for 1H at 400 MHz and for 13C at 100 MHz, or for 1H at 600 MHz and
for 13C at 150 MHz. Chemical shifts were reported in parts per million (ppm) using tetramethyl
silane as internal standard with solvent resonance in CDCl3 or DMSO-d6. Enantiomeric ratios
were determined by comparing HPLC analyses of products on chiral columns with results
obtained using authentic racemates. UV detection was performed at 254 nm. ESI-HRMS
spectra were measured with a Q-TOF instrument. Column chromatography was performed on
a silica gel (200-300 mesh) using an eluent of ethyl acetate and petroleum ether. TLC was
performed on glass-backed silica plates; products were visualized using UV light. Melting
points were determined on a Mel-Temp apparatus.
Cell culture and cellular proliferation assay
The human lung cancer cell line A549, human breast cancer cell line MCF-7, human pancreatic
cancer cell line PANC-1, human renal cell carcinoma cell line 786O and human colorectal cell
line HCT116 were incubated under sterile conditions at 37 ℃ and were maintained in a
humidified atmosphere 5% (vol/vol) CO2 with RPMI-1640 or DMEM medium containing 10%
fetal bovine serum (GIBCO, Waltham, MA, USA). MTT assay was performed to evaluate the
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cellular proliferation inhibitory activities of tested compounds by a panel of cancer cell lines.
In general, cells were seeded into 96-well plates and treated with a series of concentration of
test drugs for 24 h. The MTT reagent (5 mg/ml) was added per well for 3 h at 37 oC. After that,
the MTT was removed and 150 μL DMSO was added to dissolve the formazan crystals. Then,
optical density (OD) was measured at 570 nm of the solution. The control group consisted of
untreated cells. The percentage of cell viability averaged from three individual experiments.
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Cell cycle, apoptosis assay by Flow Cytometry (FCM) and fluorescent microscopy
HCT116 cells were seeded in 6-well plates for 12 h, and then treated with compound 3h (0.5
or 2 μM, respectively) or vehicle (DMSO) for 16 h. Cells were collected, then fixed with 75%
ice-cold ethanol and stored at -20 °C for 1 h. After centrifugation, the cells were washed with
ice-cold PBS twice, then stained with PI at 4 °C for 20 min in the dark. Cell cycle analysis was
performed in an FACS Calibur flow cytometer. Apoptosis induction assay processed by FACS
in HCT116 cells treated with compound 3h. HCT116 cells treated with compound 3h or saline
(control) were gently trypsinized without EDTA and centrifuged at 2000 rpm for 5 minutes.
Then, the harvested cells were washed with 1.0 ml ice cold PBS and re-suspended in 500 μL
binding buffer, and incubated with 5 µL of Annexin V-FITC and 5 µL of propidium iodide (PI)
for 15 min at room temperature. Followed by FCM (BD FACS Calibur, BD, USA) using the
FL1 channel for Annexin V-FITC and the FL2 channel of PI. Both early apoptotic (Annexin
V+/PI-) and late apoptotic (Annexin V+/PI+) cells are included in the cell assay of apoptosis.
In separate experiments, the extent of apoptosis was assessed based on alteration of nuclear
morphology detected using the nucleus-specific dye Hoechst 33342 (Keygen, Nanjing, China).
Cells were seeded into 6-well plates containing coverslips (4×105 cells per well), cultured for
24 h, then treated with compound 3h (0.5 or 2 μM, respectively) or vehicle (DMSO) for 24 h.
Cells were then washed twice with PBS, fixed with 4% paraformaldehyde for 20 min at room
temperature, stained with Hoechst 33342 (5 µg/ml) in the dark at 37 °C for 30 min and analyzed
using inverted fluorescence microscopy (TE-2000, Nikon, Japan). Cells containing uniformly
stained nuclei were scored as healthy, while cells containing condensed or fragmented nuclei
were scored as apoptotic.
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Autophagy assays
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HCT116 cells were transfected with a plasmid encoding a fusion of GFP with the C-terminal
domain of LC3 protein (also known as microtubule-associated protein 1A/1B light chain 3B).
Transfected cultures were treated with compound 3h (0.5 μM) and observed using fluorescence
microscopy (Axio Observer A1, Zeiss, Germany). Autophagy-positive cells were defined as
those containing GFP-LC3 puncta. The HCT116 cells were treated with compound 3h (0.5 μM)
for 24 h, collected and washed with ice-cold PBS, then fixed with 4% glutaraldehyde in 0.1 M
sodium cacodylate for 2 h. Cells were then post-fixed with 1% OsO4 for 1.5 h, washed,
dehydrated and embedded in Epon-Araldite resin. Ultrathin sections (80 nm) were prepared,
stained with 3% aqueous uranyl acetate for 1 h, and counterstained with 0.3% lead citrate.
Sections were analyzed using transmission electron microscopy (HT7700, Hitachi, Japan).
Western blot analysis
The compound 3h treated HCT116 cells with or without combination of autophagy inhibitor 3-
MA were harvested and washed with cold 1×PBS. Total cell lysates were prepared in lysis
RIPA buffer (Invitrogen, CA, USA) on ice for 30 min, followed by centrifugation at 13000 rpm
o
for 30 min at 4 C. After collecting supernatant, protein concentration was determined by a
bicinchoninic acid protein assay kit (Thermo, USA). The protein was resolved on a 10-15%
SDS-polyacrylamide gel, electro blotted onto nitrocellulose membranes, and then incubated
with proper primary antibodies which were purchased from Cell Signaling Technology or Santa
Cruz Biotechnology and secondary antibodies before visualization by chemiluminescence Kit
(Millipore, USA).
Preparation of α,β,γ,δ-unsaturated pyrazolones 1
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The solution of phenylhydrazine (324.4 mg, 3.0 mmol) with ethyl acetoacetate (468.5 mg, 3.6
mmol, 1.2 equiv) in acetic acid (2.0 mL) was refluxed for 8 h in oil bath, and left to cool. The
pure product could be obtained by recrystallization by using acetic acid as a yellow solid, which
could be used directly in next step without further purification. To a solution of the above
product (348.4 mg, 2.0 mmol) in THF (5.0 mL) under argon atmosphere was added ethyl (E)-
4-oxo-4-phenylbut-2-enoate (408.5 mg, 2.0 mmol, 1.0 equiv) and pyridine (474.6 mg, 6.0 mmol,
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3.0 equiv). The mixture was stirred at -10 C followed by addition of Titanium isopropoxide
(1.42 g, 5.0 mmol, 2.5 equiv). The solution was stirred at room temperature until the reaction
had stopped progressing as observed by TLC analysis, then diluted with EtOAc and washed
with 1N aqueous HCl, Na2CO3, and brine. The organic layer was dried over Na2SO4, removed
under reduced pressure, and purified by column chromatography (n-hexane/ethyl acetate = 20:1
to 15:1) to provide 1a. Other α,β,γ,δ-unsaturated pyrazolones 1 were prepared according to the
same procedure.
Ethyl-(2E,4E)-4-(3-methyl-5-oxo-1-phenyl-1,5-dihydro-4H-pyrazol-4-ylidene)-4-
phenylbut-2-enoate (1a): red solid (n-hexane/ethyl acetate = 20:1 for purification), 583.8 mg,
54% yield, E/Z >20:1, m.p. 118–119 oC; 1H NMR (600 MHz, CDCl3) δ (ppm) 9.39 (d, J = 1
Hz, 1H), 7.92 (d, J = 9.0 Hz, 2H), 7.53 – 7.47 (m, 3H), 7.43 – 7.40 (m, 2H), 7.23 – 7.19 (m,
3H), 5.90 (d, J = 15.6 Hz, 1H), 4.25 (q, J = 7.2 Hz, 2H), 1.49 (s, 3H), 1.33 – 1.29 (t, J = 7.2 Hz,
3H); 13C{1H} NMR (150 MHz, CDCl3) δ (ppm) 165.8, 162.7, 154.7, 148.9, 140.0, 138.0, 134.8,
134.6, 129.8, 128.9, 128.9, 128.7, 127.6, 125.2, 119.1, 61.3, 17.0, 14.3; HRMS (ESI-TOF) m/z:
[M + Na]+ Calcd for C22H20N2O3Na 383.1372; found 383.1370.
Ethyl-(2E,4E)-4-(4-fluorophenyl)-4-(3-methyl-5-oxo-1-phenyl-1,5-dihydro-4H-pyrazol-4-
ylidene)but-2-enoate (1b): red solid (n-hexane/ethyl acetate = 18:1 for purification), 578.9
mg, 51% yield, E/Z >20:1, m.p. 153–154 oC; 1H NMR (600 MHz, CDCl3) δ (ppm) 9.36 (d, J =
15.8 Hz, 1H), 7.93 – 7.90 (m, 2H), 7.43 – 7.40 (m, 2H), 7.23 – 7.21 (m, 3H), 7.21 – 7.19 (m,
2H), 5.87 (d, J = 15.6 Hz, 1H), 4.25 (q, J = 7.2 Hz, 2H), 1.53 (s, 3H), 1.31 (t, J = 7.2 Hz, 3H);
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13C{1H} NMR (150 MHz, CDCl3) δ (ppm) 165.6, 163.5 (d, JCF = 249.3 Hz), 162.5, 153.3,
148.4, 140.0, 137.8, 134.4, 130.91 (d, JCF = 8.6 Hz), 130.67 (d, JCF = 3.5 Hz), 128.9, 127.9,
125.2, 119.0, 116.1, 115.9, 61.2, 17.1, 14.2; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for calcd
for C22H19FN2O3Na 401.1277; found 401.1275.
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Ethyl-(2E,4E)-4-(3-bromophenyl)-4-(3-methyl-5-oxo-1-phenyl-1,5-dihydro-4H-pyrazol-
4-ylidene)but-2-enoate (1c): red solid (n-hexane/ethyl acetate = 18:1 for purification), 777.6
mg, 59% yield, E/Z >20:1, m.p. 132–133 oC; 1H NMR (600 MHz, CDCl3) δ (ppm) 9.36 (d, J =
15.6 Hz, 1H), 7.92 – 7.90 (m, 2H), 7.66 (ddd, J = 8.4, 2.4, 1.2 Hz, 1H), 7.43 – 7.40 (m, 3H),
7.38 (t, J = 7.8 Hz, 1H), 7.22 – 7.19 (m, 1H), 7.19 – 7.17 (m, 1H), 5.88 (d, J = 15.6 Hz, 1H),
4.26 (q, J = 7.2 Hz, 2H), 1.54 (s, 3H), 1.32 (t, J = 7.2 Hz, 3H); 13C{1H} NMR (150 MHz,
CDCl3) δ (ppm) 165.5, 162.4, 152.2, 148.3, 139.4, 137.8, 136.6, 134.5, 132.8, 131.6, 130.3,
128.9, 127.8, 127.5, 125.2, 122.8, 119.0, 61.3, 17.1, 14.2; HRMS (ESI-TOF) m/z: [M + Na]+
Calcd for C22H19BrN2O3Na 461.0477; found 461.0475.
Ethyl-(2E,4E)-4-(4-bromophenyl)-4-(3-methyl-5-oxo-1-phenyl-1,5-dihydro-4H-pyrazol-
4-ylidene)but-2-enoate (1d): red solid (n-hexane/ethyl acetate = 18:1 for purification), 764.4
mg, 58% yield, E/Z >20:1, m.p. 137–138 oC, n-hexane/ethyl acetate = 18:1; 1H NMR (600 MHz,
CDCl3) δ (ppm) 9.35 (d, J = 15.6 Hz, 1H), 7.91 (dd, J = 9.0, 1.2 Hz, 2H), 7.66 – 7.64 (m, 2H),
7.41 (dd, J = 8.4, 7.8 Hz, 2H), 7.20 (tt, J = 8.0, 1.2 Hz, 1H), 7.13 – 7.11 (m, 2H), 5.86 (d, J =
15.6 Hz, 1H), 4.25 (q, J = 7.2 Hz, 2H), 1.54 (s, 3H), 1.31 (t, J = 7.2 Hz, 3H); 13C{1H} NMR
(150 MHz, CDCl3) δ (ppm) 165.5, 162.4, 152.9, 148.3, 139.6, 137.8, 134.4, 133.6, 132.0, 130.5,
128.99, 127.7, 125.2, 124.2, 119.0, 61.3, 17.2, 14.2; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd
for C22H19BrN2O3Na 461.0477; found 461.0480.
Ethyl-(2E,4E)-4-(3,4-dichlorophenyl)-4-(3-methyl-5-oxo-1-phenyl-1,5-dihydro-4H-
pyrazol-4-ylidene)but-2-enoate (1e): red solid (n-hexane/ethyl acetate = 18:1 for purification),
618.2 mg, 48% yield, E/Z >20:1, m.p. 170–171 oC; 1H NMR (600 MHz, CDCl3) δ (ppm) 9.33
(d, J = 15.6 Hz, 1H), 7.90 (dd, J = 8.4, 0.6 Hz, 2H), 7.60 (d, J = 7.8 Hz, 1H), 7.41 (t, J = 7.8
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Hz, 2H), 7.36 (d, J = 1.8 Hz, 1H), 7.22 – 7.19 (m, 1H), 7.10 (dd, J = 7.8, 1.8 Hz, 1H), 5.86 (d,
J = 16.2 Hz, 1H), 4.25 (q, J = 7.2 Hz, 2H), 1.57 (s, 3H), 1.31 (t, J = 7.2 Hz, 3H); 13C{1H} NMR
(150 MHz, CDCl3) δ (ppm) 165.3, 162.2, 151.0, 147.9, 139.2, 137.7, 134.4, 134.4, 133.4, 130.9,
130.7, 128.9, 128.2, 127.9, 125.3, 118.9, 61.3, 17.3, 14.2; HRMS (ESI-TOF) m/z: [M + Na]+
Calcd for C22H18Cl2N2O3Na 451.0592; found 451.0595.
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Ethyl-(2E,4E)-4-(3-methyl-5-oxo-1-phenyl-1,5-dihydro-4H-pyrazol-4-ylidene)-4-(o-
tolyl)but-2-enoate (1f): red solid (n-hexane/ethyl acetate = 18:1 for purification), 730.2 mg,
65% yield, E/Z >20:1, m.p. 110–111 oC; 1H NMR (600 MHz, CDCl3) δ (ppm) 9.44 (d, J = 15.6
Hz, 1H), 7.93 (dd, J = 8.4, 1.2 Hz, 2H), 7.43 – 7.37 (m, 3H), 7.31 (d, J = 7.8 Hz, 2H), 7.22 –
7.18 (m, 1H), 7.06 (d, J = 7.8 Hz, 1H), 5.85 (d, J = 15.6 Hz, 1H), 4.25 (q, J = 7.2 Hz, 2H), 2.17
(s, 3H), 1.43 (s, 3H), 1.31 (t, J = 7.2 Hz, 3H); 13C{1H} NMR (150 MHz, CDCl3) δ (ppm) 165.7,
162.6, 154.4, 148.9, 138.9, 137.9, 135.3, 134.2, 133.6, 130.6, 129.6, 128.8, 128.5, 127.7, 126.1,
125.1, 118.9, 61.2, 19.5, 16.1, 14.2; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C23H22N2O3Na
397.1528; found 397.1525.
Ethyl-(2E,4E)-4-(3-methoxyphenyl)-4-(3-methyl-5-oxo-1-phenyl-1,5-dihydro-4H-
pyrazol-4-ylidene)but-2-enoate (1g): red solid (n-hexane/ethyl acetate = 15:1 for purification),
702.8 mg, 60% yield, E/Z >20:1, m.p. 125–126 oC; 1H NMR (600 MHz, CDCl3) δ (ppm) 9.37
(d, J = 15.6 Hz, 1H), 7.93 – 7.92 (m, 2H), 7.42 – 7.38 (m, 3H), 7.21 – 7.18 (m, 1H), 7.03 (dd,
J = 8.4, 2.4 Hz, 1H), 6.80 (d, J = 7.2 Hz, 1H), 6.75 (d, J = 1.8 Hz, 1H), 5.95 (d, J = 15.6 Hz,
1H), 4.25 (q, J = 7.2 Hz, 2H), 3.85 (s, 3H), 1.56 (s, 3H), 1.31 (t, J = 7.2 Hz, 3H); 13C{1H} NMR
(150 MHz, CDCl3) δ (ppm) 165.7, 162.7, 159.6, 154.3, 148.8, 139.7, 137.9, 135.9, 134.4, 129.8,
128.8, 127.4, 125.1, 121.1, 118.9, 114.9, 114.5, 61.2, 55.4, 16.7, 14.2; HRMS (ESI-TOF) m/z:
[M + Na]+ Calcd for C23H22N2O4Na 413.1477; found 413.1476.
Ethyl-(2E,4E)-4-(4-methoxyphenyl)-4-(3-methyl-5-oxo-1-phenyl-1,5-dihydro-4H-
pyrazol-4-ylidene)but-2-enoate (1h): red solid (n-hexane/ethyl acetate = 15:1 for purification),
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726.2 mg, 62% yield, E/Z >20:1, m.p. 126–127 oC; 1H NMR (600 MHz, CDCl3) δ (ppm) 9.29
(d, J = 15.6 Hz, 1H), 7.93 (dd, J = 8.4, 0.6 Hz, 2H), 7.41 (dd, J = 8.4, 7.2 Hz, 2H), 7.19 (t, J =
7.2 Hz, 1H), 7.15 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H), 5.95 (d, J = 15.6 Hz, 1H), 4.25
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127.5, 127.0, 125.0, 119.0, 114.1, 61.1, 55.4, 17.1, 14.2; HRMS (ESI-TOF) m/z: [M + Na]+
Calcd for C23H22N2O4Na 413.1477, found 413.1474.
Ethyl-(2E,4E)-4-(3-methyl-5-oxo-1-phenyl-1,5-dihydro-4H-pyrazol-4-ylidene)-4-
(thiophen-2-yl)but-2-enoate (1i): red solid (n-hexane/ethyl acetate = 15:1 for purification),
714.5 mg, 65% yield, E/Z = 15:1, m.p. 120–121 oC; 1H NMR (600 MHz, CDCl3) δ (ppm) 9.23
(d, J = 15.6 Hz, 1H), 7.92 (dd, J = 8.4, 1.2 Hz, 2H), 7.59 (dd, J = 4.8, 1.2 Hz, 1H), 7.41 (dd, J
= 8.4, 7.8 Hz, 2H), 7.20 (t, J = 7.2 Hz, 1H), 7.17 (dd, J = 5.4, 4.8 Hz, 1H), 7.10 (dd, J = 3.0,
1.2 Hz, 1H), 6.15 (d, J = 15.6 Hz, 1H), 4.26 (t, J = 7.2 Hz, 2H), 1.72 (s, 3H), 1.32 (t, J = 7.2
Hz, 3H); 13C{1H} NMR (150 MHz, CDCl3) δ (ppm) 165.6, 162.2, 148.5, 147.2, 140.6, 137.8,
134.4, 134.2, 130.7, 129.3, 129.1, 128.8, 127.5, 125.1, 119.0, 61.2, 16.2, 14.2; HRMS (ESI-
TOF) m/z: [M + Na]+ Calcd for C20H18SN2O3Na 389.0936; found 389.0939.
Ethyl-(2E,4E)-4-(3-methyl-5-oxo-1-phenyl-1,5-dihydro-4H-pyrazol-4-ylidene)-4-
(naphthalen-2-yl)but-2-enoate (1j): red solid (n-hexane/ethyl acetate = 20:1 for purification),
615.7 mg, 50% yield, E/Z = 13:1, m.p. 156–157 oC; 1H NMR (600 MHz, CDCl3) δ (ppm) 9.44
(d, J = 15.6 Hz, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.95 – 7.93 (m, 3H), 7.90 – 7.88 (m, 1H), 7.72
(s, 1H), 7.62 – 7.60 (m, 2H), 7.42 (dd, J = 8.4, 7.8 Hz, 2H), 7.32 (dd, J = 8.4, 1.8 Hz, 1H), 7.20
(t, J = 7.2 Hz, 1H), 5.90 (d, J = 15.6 Hz, 1H), 4.23 (q, J = 7.2 Hz, 2H), 1.45 (s, 3H), 1.29 (t, J
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= 7.2 Hz, 3H); C{1H} NMR (150 MHz, CDCl3) δ (ppm) 165.6, 162.6, 154.5, 148.8, 140.0,
137.9, 134.6, 133.4, 132.5, 132.1, 128.8, 128.6, 128.5, 128.2, 127.9, 127.8, 127.4, 127.2, 126.0,
125.0, 118.9, 61.1, 17.1, 14.2; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C26H22N2O3Na
433.1528; found 433.1530.
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Ethyl-(2E,4E)-4-(1-(2-fluorophenyl)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazol-4-ylidene)-
4-phenylbut-2-enoate (1k): red solid (n-hexane/ethyl acetate = 18:1 for purification), 647.1
mg, 57% yield, E/Z = 18:1, m.p. 104–105 oC; 1H NMR (600 MHz, CDCl3) δ (ppm) 9.36 (d, J
= 15.6 Hz, 1H), 7.52 – 7.49 (m, 3H), 7.47 (dd, J = 7.8, 1.8 Hz, 1H), 7.36 – 7.32 (m, 1H), 7.25
– 7.21 (m, 4H), 5.90 (d, J = 15.6 Hz, 1H), 4.23 (q, J = 7.2 Hz, 2H), 1.48 (s, 3H), 1.29 (t, J = 7.2
Hz, 3H); 13C{1H} NMR (150 MHz, CDCl3) δ (ppm) 165.7, 163.0, 156.70 (d, JCF = 251.3 Hz),
155.1, 149.3, 139.8, 134.7, 134.6, 129.7, 129.31 (d, JCF = 7.8 Hz), 128.7, 128.6, 127.2, 126.4,
124.74 (d, JCF = 11.9 Hz), 124.41 (d, JCF = 3.8 Hz), 116.9, 116.8, 61.2, 16.9, 14.2; HRMS (ESI-
TOF) m/z: [M + Na]+ Calcd for C22H19FN2O3Na 401.1277; found 401.1275.
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Ethyl-(2E,4E)-4-(1-(3-chlorophenyl)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazol-4-ylidene)-
4-phenylbut-2-enoate (1l): red solid (n-hexane/ethyl acetate = 18:1 for purification), 651.5 mg,
55% yield, E/Z = 15:1, m.p. 105–106 oC; 1H NMR (600 MHz, CDCl3) δ (ppm) 9.35 (d, J = 15.6
Hz, 1H), 8.01 (t, J = 2.4 Hz, 1H), 7.90 (ddd, J = 8.4, 1.8, 0.6 Hz, 1H), 7.53 – 7.51 (m, 2H), 7.50
– 7.48 (m, 1H), 7.33 (t, J = 8.4 Hz, 1H), 7.22 (dd, J = 7.2, 1.2 Hz, 2H), 7.16 (ddd, J = 7.8, 1.8,
0.8 Hz, 1H), 5.91 (d, J = 15.6 Hz, 1H), 4.26 (q, J = 7.2 Hz, 2H), 1.49 (s, 3H), 1.31 (t, J = 7.2
Hz, 3H); 13C{1H} NMR (150 MHz, CDCl3) δ (ppm) 165.6, 162.7, 155.1, 149.3, 139.8, 138.9,
134.8, 134.6, 129.9, 129.8, 128.8, 128.7, 127.2, 124.9, 118.6, 116.5, 61.2, 16.9, 14.2; HRMS
(ESI-TOF) m/z: [M + Na]+ Calcd for C22H19ClN2O3Na 417.0982; found 417.0980.
Ethyl-(2E,4E)-4-(3-methyl-5-oxo-1-(p-tolyl)-1,5-dihydro-4H-pyrazol-4-ylidene)-4-
phenylbut-2-enoate (1m): red solid (n-hexane/ethyl acetate = 18:1 for purification), 685.2 mg,
61% yield, E/Z = 18:1, m.p. 107–108 oC; 1H NMR (600 MHz, CDCl3) δ (ppm) 9.39 (d, J = 15.6
Hz, 1H), 7.79 (d, J = 8.4 Hz, 2H), 7.51 – 7.47 (m, 3H), 7.22 – 7.20 (m, 4H), 5.89 (d, J = 15.6
Hz, 1H), 4.25 (q, J = 7.2 Hz, 2H), 2.35 (s, 3H), 1.48 (s, 3H), 1.31 (t, J = 7.2 Hz, 3H); 13C{1H}
NMR (150 MHz, CDCl3) δ (ppm) 165.7, 162.5, 154.4, 148.6, 140.0, 135.5, 134.8, 134.4, 129.6,
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Calcd for C23H22N2O3Na 397.1528; found 397.1525
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Ethyl-(2E,4E)-4-(5-oxo-1,3-diphenyl-1,5-dihydro-4H-pyrazol-4-ylidene)-4-phenylbut-2-
enoate (1n): red solid (n-hexane/ethyl acetate = 18:1 for purification), 646.4 mg, 51% yield,
E/Z = 15:1, m.p. 166–167 oC; 1H NMR (600 MHz, CDCl3) δ (ppm) 9.41 (d, J = 15.6 Hz, 1H),
8.02 (dd, J = 8.4, 1.2 Hz, 2H), 7.43 (dd, J = 8.4, 7.2 Hz, 2H), 7.24 – 7.21 (m, 1H), 7.14 – 7.11
(m, 1H), 7.08 – 7.05 (m, 1H), 7.02 – 7.00 (m, 2H), 6.98 – 6.96 (m, 4H), 6.95 – 6.93 (m, 2H),
6.03 (d, J = 15.6 Hz, 1H), 4.27 (q, J = 7.2 Hz, 2H), 1.32 (t, J = 7.2 Hz, 3H); 13C{1H} NMR (150
MHz, CDCl3) δ (ppm) 165.7, 163.1, 156.0, 151.3, 140.7, 138.0, 134.8, 134.2, 132.2, 130.7,
129.9, 128.8, 128.0, 127.9, 127.7, 127.6, 125.5, 125.3, 119.3, 61.2, 14.2; HRMS (ESI-TOF)
m/z: [M + Na]+ Calcd for C27H22N2O3Na 445.1528; found 445.1525.
(E)-4-((E)-1,3-diphenylallylidene)-5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (1o):
red solid (n-hexane/ethyl acetate = 18:1 for purification), 688.7 mg, 63% yield, E/Z = 18:1, m.p.
160–161 oC; 1H NMR (600 MHz, CDCl3) δ (ppm) 9.32 (d, J = 15.6 Hz, 1H), 7.97 (dd, J = 8.4,
1.2 Hz, 2H), 7.58 – 7.56 (m, 2H), 7.53 – 7.49 (m, 3H), 7.41 (dd, J = 8.4, 7.2 Hz, 2H), 7.35 –
7.33 (m, 3H), 7.29 – 7.28 (m, 2H), 7.20 – 7.17 (m, 1H), 6.70 (d, J = 15.6 Hz, 1H), 1.47 (s, 3H);
13C{1H} NMR (150 MHz, CDCl3) δ (ppm) 163.7, 158.7, 149.2, 147.9, 138.3, 136.0, 135.9,
130.4, 129.2, 129.0, 128.9, 128.8, 128.6, 128.4, 126.7, 124.8, 123.3, 119.1, 16.8; HRMS (ESI-
TOF) m/z: [M + Na]+ Calcd for C25H20N2ONa 387.1473; found 387.1470.
Procedure for the asymmetric synthesis of 3 and 4 (including some
diastereoisomers of 4’)
To α,β,γ,δ-unsaturated pyrazolone 1 (0.15 mmol), amine catalyst Cat. III (20 mol%), benzoic
acid (30 mol%), and THF/H2O (20:1 in v/v, 1.5 mL) in a standard glass vial with stir bar was
added aldehyde 2 (0.30 mmol). The reaction mixture was stirred at reaction temperature of -20
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oC until the reaction completed (monitored by TLC). The reaction mixture was concentrated
and the residue was purified by flash chromatography on silica gel (n-hexane/ethyl acetate =
4:1) to give the spirocyclohexene-pyrazolone derivate 3. To a solution of spirocyclohexene-
pyrazolone derivate 3 in CH2Cl2 (1.0 mL) was added TEA (0.3 mmol in 0.5 mL CH2Cl2) at ice
bath, after which TMSCl (0.2 mmol in 0.5 mL CH2Cl2 ) was added. The reaction mixture was
stirred until the reaction completed (monitored by TLC). Then the reaction was quenched with
aqueous NaHCO3, extracted with CH2Cl2. The organic layer was dried over Na2SO4 and
concentrated. The residue was purified by chromatography on silica gel (n-hexane/ethyl acetate
= 20:1) to give the TMS-protected spirocyclohexene-pyrazolone derivate 4 which was dried
under vacuum and further analysed by 1H-NMR, 13C{1H}-NMR, HRMS, chiral HPLC analysis,
etc. The diastereoisomers of 4’ were synthesized and analysed according to the same procedure.
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Ethyl-(5S,8R,9R,10S)-10-hydroxy-1,9-dimethyl-4-oxo-3,6-diphenyl-2,3-diazaspiro[4.5]-
deca-1,6-diene-8-carboxylate (3a): white solid, 53.4 mg, 85% yield, dr 84:16, m.p. 189-190
oC; [α]D20 = +26.9 (c = 0.21 in CH2Cl2); 1H NMR (600 MHz, DMSO-d6) δ (ppm) 7.82 (d, J =
7.2 Hz, 2H), 7.44 (t, J = 7.8 Hz, 2H), 7.26 – 7.25 (m, 3H), 7.20 (t, J = 7.2 Hz, 1H), 6.98 (dd, J
= 6.6, 3.0 Hz, 2H), 6.12 (d, J = 4.8 Hz, 1H), 5.66 (d, J = 6.6 Hz, 1H), 4.30 (dd, J = 10.8, 6.0
Hz, 1H), 4.21 – 4.14 (m, 2H), 3.57 (t, J = 6.0 Hz, 1H), 2.90 – 2.83 (m, 1H), 1.80 (s, 3H), 1.26
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(t, J = 7.2 Hz, 3H), 1.00 (d, J = 7.2 Hz, 3H); C{1H} NMR (150 MHz, DMSO-d6) δ (ppm)
172.9, 172.1, 162.1, 139.5, 138.5, 136.2, 130.0, 129.5, 129.1, 128.6, 126.6, 125.2, 118.7, 69.8,
66.0, 61.1, 48.2, 29.5, 14.7, 14.6, 14.3; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for
C25H26N2O4Na 441.1790; found 441.1792.
Ethyl-(5S,8R,9R,10S)-1,9-dimethyl-4-oxo-3,6-diphenyl-10-((trimethylsilyl)oxy)-2,3-di-
azaspiro[4.5]deca-1,6-diene-8-carboxylate (4a): oil, 55.2 mg, 75% yield, dr 84:16, ee 97%,
[α]D20 = +16.2 (c = 0.12 in CH2Cl2); 1H NMR (400 MHz, CDCl3) δ (ppm) 7.94 (d, J = 7.6 Hz,
2H), 7.41 (t, J = 8.0 Hz, 2H), 7.23 – 7.18 (m, 4H), 7.04 (dd, J = 6.8, 2.4 Hz, 2H), 6.15 (d, J =
2.4 Hz, 1H), 4.27 – 4.17 (m, 2H), 3.85 (d, J = 10.4 Hz, 1H), 3.28 – 3.21 (m, 1H), 3.05 (dd, J =
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10.4, 2.4 Hz, 1H), 1.78 (s, 3H), 1.29 (t, J = 7.2 Hz, 3H), 1.09 (d, J = 6.4 Hz, 3H), 0.05 (s, 9H);
13C{1H} NMR (100 MHz, CDCl3) δ (ppm) 172.1, 171.9, 160.3, 139.0, 138.3, 134.8, 130.0,
128.8, 128.7, 128.1, 126.3, 124.8, 118.9, 65.5, 61.2, 50.7, 30.5, 29.7, 16.9, 14.7, 14.2, 0.8;
HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C28H34N2O4SiNa 513.2186; found 513.2189.
Ethyl-(5S,8R,9R,10S)-6-(4-fluorophenyl)-10-hydroxy-1,9-dimethyl-4-oxo-3-phenyl-2,3-
diazaspiro[4.5]deca-1,6-diene-8-carboxylate (3b): white solid, 57.6 mg, 88% yield, dr 85:15,
m.p. 167-168 oC; [α]D20 = +18.2 (c = 0.11 in CH2Cl2); 1H NMR (600 MHz, DMSO-d6) δ (ppm)
7.81 (d, J = 8.4 Hz, 2H), 7.44 (t, J = 7.8 Hz, 2H), 7.21 (t, J = 7.8 Hz, 1H), 7.11 (t, J = 9.0 Hz,
2H), 7.01 (dd, J = 8.4, 5.4 Hz, 2H), 6.12 (d, J = 4.8 Hz, 1H), 5.67 (d, J = 6.0 Hz, 1H), 4.29 (dd,
J = 11.4, 6.6 Hz, 1H), 4.21 – 4.14 (m, 2H), 3.57 (t, J = 5.4 Hz, 1H), 2.87 – 2.81 (m, 1H), 1.82
(s, 3H), 1.26 (t, J = 7.2 Hz, 3H), 0.99 (d, J = 7.2 Hz, 3H); 13C{1H} NMR (150 MHz, DMSO-
d6) δ (ppm) 172.8, 172.0, 162.2 (d, JCF = 243.6 Hz), 162.0, 138.4, 135.8 (d, JCF = 3.5 Hz), 135.1,
130.4, 129.5, 128.7 (d, JCF = 8.6 Hz), 125.3, 118.8, 116.1 (d, JCF = 21.5 Hz), 69.8, 66.1, 61.1,
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48.1, 29.6, 14.7, 14.6, 14.3; F NMR (565 MHz, DMSO-d6) δ (ppm) -113.82; HRMS (ESI-
TOF) m/z: [M + Na]+ Calcd for C25H25FN2O4Na 459.1696; found 459.1693.
Ethyl-(5S,8R,9R,10S)-6-(4-fluorophenyl)-1,9-dimethyl-4-oxo-3-phenyl-10-((trimethylsilyl)
oxy)-2,3-diazaspiro[4.5]deca-1,6-diene-8-carboxylate (4b): oil, 62.6 mg, 82% yield, dr 85:15,
ee 93%, [α]D20 = +10.2 (c = 0.09 in CH2Cl2); 1H NMR (400 MHz, CDCl3) δ (ppm) 7.90 – 7.88
(m, 2H), 7.43 – 7.39 (m, 2H), 7.20 (t, J = 7.6 Hz, 1H), 7.04 (dd, J = 8.8, 5.2 Hz, 2H), 6.89 (t, J
= 8.8 Hz, 2H), 6.11 (d, J = 5.6 Hz, 1H), 4.64 (d, J = 11.2 Hz, 1H), 4.27 – 4.20 (m, 2H), 3.56 (t,
J = 5.6 Hz, 1H), 3.10 – 3.01 (m, 1H), 1.89 (s, 3H), 1.35 (t, J = 7.2 Hz, 3H), 1.06 (d, J = 6.8 Hz,
3H), 0.03 (s, 9H); 13C{1H} NMR (100 MHz, CDCl3) δ (ppm) 172.1, 171.8, 162.1 (d, JCF = 246.1
Hz), 160.4, 137.6, 135.2, 134.6 (d, JCF = 3.4 Hz), 129.2, 128.4, 127.9 (d, JCF = 8.1 Hz), 124.6,
118.6, 115.1 (d, JCF = 21.4 Hz), 72.2, 65.8, 60.6, 47.9, 29.8, 24.9, 14.6, 14.0, 13.9, 0.3; 19F NMR
(565 MHz, DMSO-d6) δ (ppm) -113.58; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for
C28H33FN2O4SiNa 531.2091; found 531.2093.
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Ethyl-(5S,8R,9R,10S)-6-(3-bromophenyl)-10-hydroxy-1,9-dimethyl-4-oxo-3-phenyl-2,3-
diazaspiro[4.5]deca-1,6-diene-8-carboxylate (3c): white solid, 67.2 mg, 90% yield, dr 82:18,
m.p. 156-157 oC; [α]D20 = +20.6 (c = 0.16 in CH2Cl2); 1H NMR (600 MHz, DMSO-d6) δ (ppm)
7.78 (d, J = 7.8 Hz, 2H), 7.47 – 7.43 (m, 3H), 7.25 – 7.20 (m, 2H), 7.13 (s, 1H), 6.99 (d, J = 7.8
Hz, 1H), 6.22 (d, J = 5.4 Hz, 1H), 5.64 (d, J = 5.4 Hz, 1H), 4.29 (dd, J = 11.4, 6.6 Hz, 1H), 4.21
– 4.14 (m, 2H), 3.58 (t, J = 6.0 Hz, 1H), 2.87 – 2.83 (m, 1H), 1.83 (s, 3H), 1.26 (t, J = 7.2 Hz,
3H), 1.00 (d, J = 6.6 Hz, 3H); 13C{1H} NMR (150 MHz, DMSO-d6) δ (ppm) 172.7, 171.8,
161.8, 141.6, 138.3, 134.7, 131.4, 131.3, 129.5, 129.3, 125.8, 125.5, 122.3, 119.1, 69.8, 65.9,
61.1, 48.2, 29.6, 14.7, 14.6, 14.3; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C25H25BrN2O4Na
519.0895; found 519.0897.
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Ethyl-(5S,8R,9R,10S)-6-(3-bromophenyl)-1,9-dimethyl-4-oxo-3-phenyl-10-((trimethyl-
silyl)oxy)-2,3-diazaspiro[4.5]deca-1,6-diene-8-carboxylate (4c): oil, 71.8 mg, 84% yield, dr
82:18, ee 97%, [α]D20 = +9.6 (c = 0.13 in CH2Cl2); 1H NMR (600 MHz, CDCl3) δ (ppm) 7.89
(d, J = 7.8 Hz, 2H), 7.41 (t, J = 7.8 Hz, 2H), 7 .21 – 7.14 (m, 2H), 6.95 – 6.91 (m, 1H), 6.83 –
6.80 (m, 2H), 6.16 (d, J = 5.4 Hz, 1H), 4.64 (d, J = 10.8 Hz, 1H), 4.27 – 4.21 (m, 2H), 3.57 (t,
J = 5.4 Hz, 1H), 3.10 – 3.04 (m, 1H), 1.89 (s, 3H), 1.35 (t, J = 7.2 Hz, 3H), 1.06 (d, J = 6.6 Hz,
3H), 0.03 (s, 9H); 13C{1H} NMR (150 MHz, CDCl3) δ (ppm) 172.5, 172.2, 160.7, 141.0, 138.0,
135.4, 131.3, 130.5, 130.3, 130.1, 128.9, 125.3, 124.9, 122.7, 119.4, 72.7, 66.0, 61.2, 48.4, 30.3,
15.1, 14.6, 14.5, 0.8, 0.6; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C28H33BrN2O4Si+Na
591.1291; found 591.1294.
Ethyl-(5S,8R,9R,10S)-6-(4-bromophenyl)-10-hydroxy-1,9-dimethyl-4-oxo-3-phenyl-2,3-
diazaspiro[4.5]deca-1,6-diene-8-carboxylate (3d): white solid, 67.9 mg, 91% yield, dr 82:18,
m.p. 162-163 oC; [α]D20 = +19.8 (c = 0.15 in CH2Cl2); 1H NMR (600 MHz, DMSO-d6) δ (ppm)
7.81 – 7.79 (m, 2H), 7.49 – 7.47 (m, 2H), 7.45 – 7.43 (m, 2H), 7.21 (t, J = 7.8 Hz, 1H), 6.92 (d,
J = 8.4 Hz, 2H), 6.16 (d, J = 4.8 Hz, 1H), 5.67 (d, J = 6.0 Hz, 1H), 4.28 (dd, J = 11.4, 6.0 Hz,
1H), 4.21 – 4.14 (m, 2H), 3.57 (t, J = 5.4 Hz, 1H), 2.87 – 2.80 (m, 1H), 1.82 (s, 3H), 1.26 (t, J
= 7.2 Hz, 3H), 0.99 (d, J = 6.6 Hz, 3H); 13C{1H} NMR (150 MHz, DMSO-d6) δ (ppm) 172.7,
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171.9, 161.9, 138.6, 138.4, 135.0, 132.1, 130.8, 129.5, 128.8, 125.3, 121.9, 118.9, 69.8, 65.9,
61.1, 48.1, 29.5, 14.7, 14.6, 14.3; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C25H25BrN2O4Na
519.0895; found 519.0898.
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Ethyl-(5S,8R,9R,10S)-6-(4-bromophenyl)-1,9-dimethyl-4-oxo-3-phenyl-10-((trimethyl-
silyl)oxy)-2,3-diazaspiro[4.5]deca-1,6-diene-8-carboxylate (4d): oil, 70.1 mg, 82% yield, dr
82:18, ee 97%, [α]D20 = +53.4 (c = 0.18 in CH2Cl2); 1H NMR (600 MHz, CDCl3) δ (ppm) 7.89
(d, J = 7.8 Hz, 2H), 7.41 (t, J = 8.4 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 7.20 (t, J = 7.2 Hz, 1H),
6.93 (d, J = 9.0 Hz, 2H), 6.13 (d, J = 5.4 Hz, 1H), 4.63 (d, J = 10.8 Hz, 1H), 4.27 – 4.20 (m,
2H), 3.56 (t, J = 5.4 Hz, 1H), 3.10 – 3.03 (m, 1H), 1.88 (s, 3H), 1.35 (t, J = 7.2 Hz, 3H), 1.06
(d, J = 6.6 Hz, 3H), 0.03 (s, 9H); 13C{1H} NMR (150 MHz, CDCl3) δ (ppm) 172.4, 172.1,
160.6, 137.9, 137.8, 135.5, 131.7, 129.9, 128.8, 128.2, 125.0, 122.3, 118.9, 72.6, 66.0, 61.0,
48.3, 30.2, 15.0, 14.4, 14.3, 0.6; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for
C28H33BrN2O4SiNa 591.1291; found 591.1290.
Ethyl-(5S,8R,9R,10S)-6-(3,4-dichlorophenyl)-10-hydroxy-1,9-dimethyl-4-oxo-3-phenyl-
2,3-diazaspiro[4.5]deca-1,6-diene-8-carboxylate (3e): white solid, 58.5 mg, 80% yield, dr
80:20, m.p. 166-167 oC; [α]D20 = +6.3 (c = 0.22 in CH2Cl2); 1H NMR (600 MHz, DMSO-d6) δ
7.79 (dd, J = 9.0, 1.2 Hz, 2H), 7.58 (d, J = 8.4 Hz, 1H), 7.47 – 7.44 (m, 2H), 7.22 (dt, J = 7.2,
1.2 Hz, 1H), 7.16 (d, J = 1.8 Hz, 1H), 6.96 (dd, J = 8.4, 1.2 Hz, 1H), 6.30 (d, J = 5.4 Hz, 1H),
5.75 (d, J = 6.6 Hz, 1H), 4.28 (dd, J = 11.4, 6.6 Hz, 1H), 4.19 – 4.14 (m, 2H), 3.61 – 3.59 (m,
1H), 2.84 – 2.80 (m, 1H), 1.84 (s, 3H), 1.26 (t, J = 7.2 Hz, 3H), 0.99 (d, J = 7.2 Hz, 3H);
13C{1H} NMR (150 MHz, DMSO-d6) δ (ppm) 172.6, 171.7, 161.9, 139.8, 138.2, 133.6, 132.0,
131.8, 131.5, 131.4, 131.2, 129.5, 128.4, 126.9, 125.5, 119.0, 69.7, 65.7, 61.2, 48.1, 29.5, 14.7,
14.6, 14.3; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C25H24Cl2N2O4Na 509.1011; found
509.1014.
Ethyl-(5S,8R,9R,10S)-6-(3,4-dichlorophenyl)-1,9-dimethyl-4-oxo-3-phenyl-10-((trimethyl
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silyl)oxy)-2,3-diazaspiro[4.5]deca-1,6-diene-8-carboxylate (4e): oil, 60.4 mg, 72% yield, dr
80:20, ee 93%, [α]D20 = -35.6 (c = 0.10 in CH2Cl2); 1H NMR (600 MHz, CDCl3) δ (ppm) 7.88
(d, J = 8.4 Hz, 2H), 7.44 (t, J = 7.8 Hz, 2H), 7.29 – 7.27 (m, 1H), 7.24 – 7.22 (m, 2H), 6.89 (dd,
J = 8.4, 1.8 Hz, 1H), 6.18 (d, J = 5.4 Hz, 1H), 4.65 (d, J = 11.4 Hz, 1H), 4.30 – 4.23 (m, 2H),
3.59 (t, J = 5.4 Hz, 1H), 3.12 – 3.04 (m, 1H), 1.93 (s, 3H), 1.37 (t, J = 7.2 Hz, 3H), 1.08 (d, J =
7.2 Hz, 3H), 0.05 (s, 9H);13C{1H} NMR (150 MHz, CDCl3) δ (ppm) 172.2, 172.0, 160.4, 138.8,
137.8, 134.5, 132.7, 132.3, 130.8, 130.7, 130.6, 128.8, 125.5, 125.2, 119.2, 72.6, 65.9, 61.2,
48.3, 30.2, 15.0, 14.5, 14.4, 0.7; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for
C28H32Cl2N2O4SiNa 581.1406; found 581.1408.
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Ethyl-(5S,8R,9R,10S)-10-hydroxy-1,9-dimethyl-4-oxo-3-phenyl-6-(o-tolyl)-2,3-diaza-
spiro[4.5]deca-1,6-diene-8-carboxylate (3f): white solid, 52.6 mg, 81% yield, dr 78:22, m.p.
173-174 oC; [α]D20 = -17.3 (c = 1.05 in CH2Cl2); 1H NMR (600 MHz, DMSO-d6) δ (ppm) 7.48
(dd, J = 9.0, 1.2 Hz, 2H), 7.30 (dd, J = 8.4, 7.8 Hz, 2H), 7.14 (d, J = 7.8 Hz, 1H), 7.11 – 7.08
(m, 1H), 7.06 (td, J = 7.2, 1.2 Hz, 1H), 6.98 (t, J = 7.2 Hz, 1H), 6.80 (d, J = 7.8 Hz, 1H), 5.77
(d, J = 6.6 Hz, 1H), 5.75 (d, J = 3.0 Hz, 1H), 4.17 – 4.13 (m, 2H), 3.92 (dd, J = 11.4, 6.6 Hz,
1H), 3.30 (dd, J = 10.2, 3.0 Hz, 1H), 2.33 (s, 3H), 2.29 (s, 3H), 1.21 (t, J = 7.2 Hz, 2H), 1.10
(d, J = 6.6 Hz, 3H); 13C{1H} NMR (150 MHz, DMSO-d6) δ (ppm) 174.2, 172.8, 159.7, 138.0,
137.6, 136.7, 132.8, 130.9, 130.7, 129.3, 128.1, 127.4, 125.4, 125.2, 118.7, 74.8, 67.7, 61.3,
50.2, 33.7, 20.6, 17.4, 17.0, 14.6; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C26H28N2O4Na
455.1947; found 455.1945.
Ethyl-(5S,8R,9R,10S)-1,9-dimethyl-4-oxo-3-phenyl-6-(o-tolyl)-10-((trimethylsilyl)oxy)-
2,3-diazaspiro[4.5]deca-1,6-diene-8-carboxylate (4f): oil, 56.0 mg, 74% yield, dr 78:22, ee
90%, [α]D20 = -20.7 (c = 0.09 in CH2Cl2); 1H NMR (600 MHz, CDCl3) δ (ppm) 7.40 – 7.39 (m,
2H), 7.20 – 7.27 (m, 2H), 7.07 (d, J = 7.8 Hz, 1H), 7.00 – 6.98 (m, 2H), 6.89 (t, J = 7.8 Hz,
1H), 6.82 (d, J = 7.8 Hz, 1H), 5.71 (d, J = 2.4 Hz, 1H), 4.18 – 4.13 (m, 3H), 3.15 (dd, J = 10.2,
2.4 Hz, 1H), 2.54 – 2.50 (m, 1H), 2.37 (s, 3H), 2.30 (s, 3H), 1.23 (t, J = 7.2 Hz, 3H), 1.08 (d, J
= 6.6 Hz, 3H), -0.08 (s, 9H); 13C{1H} NMR (150 MHz, CDCl3) δ (ppm) 173.7, 172.7, 159.6,
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137.6, 136.7, 136.6, 133.6, 130.6, 129.9, 128.6, 127.9, 127.6, 125.0, 124.9, 119.3, 76.5, 67.9,
61.3, 50.4, 33.9, 20.4, 17.4, 17.3, 14.3, 0.5; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for
C29H36N2O4SiNa 527.2342; found 527.2344.
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Ethyl-(5S,8R,9R,10S)-10-hydroxy-6-(3-methoxyphenyl)-1,9-dimethyl-4-oxo-3-phenyl-
2,3-diazaspiro[4.5]deca-1,6-diene-8-carboxylate (3g): white solid, 53.8 mg, 80% yield, dr
70:30, m.p. 76-77 oC; [α]D20 = +20.3 (c = 0.07 in CH2Cl2); 1H NMR (600 MHz, DMSO-d6) δ
7.84 (dd, J = 8.4, 1.2 Hz, 2H), 7.44 (dd, J = 8.4, 7.2 Hz, 2H), 7.22 – 7.17 (m, 2H), 6.83 – 6.82
(m, 1H), 6.60 (dd, J = 6.6, 0.6 Hz, 1H), 6.46 (t, J = 1.8 Hz, 1H), 6.15 (d, J = 5.4 Hz, 1H), 5.69
(d, J = 6.6 Hz, 1H), 4.29 (dd, J = 10.8, 6.6 Hz, 1H), 4.21 – 4.13 (m, 2H), 3.57 (dd, J = 5.4, 2.4
Hz, 1H), 3.52 (s, 3H), 2.88 – 2.82 (m, 1H), 1.82 (s, 3H), 1.26 (t, J = 7.2 Hz, 3H), 0.99 (d, J =
7.2 Hz, 3H); 13C{1H} NMR (150 MHz, DMSO-d6) δ 173.0, 172.1, 162.3, 159.5, 140.8, 138.5,
135.9, 130.3, 130.1, 129.5, 125.2, 119.1, 118.5, 114.2, 111.6, 69.8, 66.0, 61.1, 55.2, 48.1, 29.5,
14.7, 14.6, 14.3; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C26H28N2O5Na 471.1896; found
471.1894.
Ethyl-(5S,8R,9R,10S)-6-(3-methoxyphenyl)-1,9-dimethyl-4-oxo-3-phenyl-10-((trimethyl-
silyl)oxy)-2,3-diazaspiro[4.5]deca-1,6-diene-8-carboxylate (4g): oil, 55.4 mg, 71% yield, dr
70:30, ee 90%, [α]D20 = -22.1 (c = 0.25 in CH2Cl2); 1H NMR (600 MHz, CDCl3) δ (ppm) 7.92
(dd, J = 9.0, 1.2 Hz, 2H), 7.41 – 7.38 (m, 2H), 7.18 (tt, J = 7.8, 1.2 Hz, 1H), 7.13 – 7.10 (m,
1H), 6.76 (ddd, J = 8.2, 2.4, 0.6 Hz, 1H), 6.67 – 6.66 (m, 1H), 6.60 – 6.58 (m, 1H), 6.16 (d, J
= 5.4 Hz, 1H), 4.65 (d, J = 10.8 Hz, 1H), 4.26 – 4.22 (m, 2H), 3.56 (t, J = 5.4 Hz, 1H), 3.55 (s,
3H), 3.11 – 3.06 (m, 1H), 1.89 (s, 3H), 1.35 (t, J = 7.2 Hz, 3H), 1.06 (d, J = 6.6 Hz, 3H), 0.04
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(s, 9H); C{1H} NMR (150 MHz, CDCl3) δ (ppm) 172.9, 172.4, 161.2, 159.6, 140.4, 138.3,
136.5, 129.7, 129.4, 128.9, 125.0, 119.2, 118.9, 114.4, 111.4, 72.8, 66.3, 61.1, 55.1, 48.5, 30.3,
15.1, 14.6, 14.5, 0.8; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C29H36N2O5SiNa 543.2291;
found 543.2288.
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Ethyl-6-(3-methoxyphenyl)-1,9-dimethyl-4-oxo-3-phenyl-10-((trimethylsilyl)oxy)-2,3-
diazaspiro[4.5]deca-1,6-diene-8-carboxylate (4g’): oil, 14.1 mg, 18% yield, dr 30:70, ee 39%,
[α]D20 = -24.1 (c = 0.10 in CH2Cl2); 1H NMR (600 MHz, CDCl3) δ (ppm) 7.96 (dd, J = 9.0, 1.2
Hz, 2H), 7.40 (dd, J = 8.4, 7.2 Hz, 2H), 7.18 (tt, J = 7.2, 1.2 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H),
6.76 (ddd, J = 8.4, 2.4, 0.6 Hz, 1H), 6.66 – 6.64 (m, 1H), 6.57 (t, J = 2.4 Hz, 1H), 6.17 (d, J =
3.0 Hz, 1H), 4.26 – 4.18 (m, 2H), 3.85 (d, J = 10.8 Hz, 1H), 3.55 (s, 3H), 3.29 – 3.15 (m, 1H),
3.05 (dd, J = 10.2, 2.4 Hz, 1H), 1.81 (s, 3H), 1.29 (t, J = 7.2 Hz, 3H), 1.08 (d, J = 6.6 Hz, 3H),
0.05 (s, 9H); 13C{1H} NMR (150 MHz, CDCl3) δ (ppm) 172.2, 171.9, 160.5, 159.5, 140.3,
138.2, 134.7, 129.9, 129.7, 128.8, 124.8, 118.8, 118.7, 114.4, 110.9, 76.7, 65.5, 61.3, 55.0, 50.6,
30.5, 16.9, 14.7, 14.2, 0.8; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C29H36N2O5SiNa
543.2291; found 543.2294.
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Ethyl-(5S,8R,9R,10S)-10-hydroxy-6-(4-methoxyphenyl)-1,9-dimethyl-4-oxo-3-phenyl-
2,3-diazaspiro[4.5]deca-1,6-diene-8-carboxylate (3h): white solid, 53.8 mg, 80% yield, dr
73:27, m.p. 83-84 oC; [α]D20 = +30.3 (c = 0.22 in CH2Cl2); 1H NMR (600 MHz, DMSO-d6) δ
(ppm) 7.82 (d, J = 7.2 Hz, 2H), 7.44 (t, J = 7.2 Hz, 2H), 7.20 (t, J = 7.2 Hz, 1H), 6.90 (d, J =
7.8 Hz, 2H), 6.81 (d, J = 9.0 Hz, 2H), 6.05 (d, J = 4.8 Hz, 1H), 5.62 (d, J = 5.4 Hz, 1H), 4.28
(dd, J = 10.8, 6.0 Hz, 1H), 4.19 – 4.14 (m, 2H), 3.68 (s, 3H), 3.56 – 3.53 (m, 1H), 2.85 – 2.81
(m, 1H), 1.80 (s, 3H), 1.26 (td, J = 7.2, 1.8 Hz, 3H), 0.98 (d, J = 6.0 Hz, 3H); 13C{1H} NMR
(150 MHz, DMSO-d6) δ (ppm) 173.0, 172.2, 162.3, 159.5, 138.6, 135.7, 131.8, 129.5, 128.9,
127.7, 125.2, 118.7, 114.5, 69.8, 66.1, 61.0, 55.6, 48.1, 29.6, 14.7, 14.7, 14.3; HRMS (ESI-
TOF) m/z: [M + Na]+ Calcd for C26H28N2O5Na 471.1896; found 471.1893.
Ethyl-(5S,8R,9R,10S)-6-(4-methoxyphenyl)-1,9-dimethyl-4-oxo-3-phenyl-10-((trimethyl-
silyl)oxy)-2,3-diazaspiro[4.5]deca-1,6-diene-8-carboxylate (4h): oil, 57.0 mg, 73% yield, dr
73:27, ee 98%, [α]D20 = +4.3 (c = 0.13 in CH2Cl2); 1H NMR (600 MHz, CDCl3) δ (ppm) 7.92
(d, J = 7.8 Hz, 2H), 7.41 (t, J = 8.4 Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 6.98 (d, J = 8.4 Hz, 2H),
6.72 (d, J = 9.0 Hz, 2H), 6.08 (d, J = 5.4 Hz, 1H), 4.64 (d, J = 11.4 Hz, 1H), 4.26 – 4.20 (m,
2H), 3.73 (s, 3H), 3.55 (t, J = 5.4 Hz, 1H), 3.08 – 3.04 (m, 1H), 1.87 (s, 3H), 1.34 (t, J = 7.2
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Hz, 3H), 1.05 (d, J = 6.6 Hz, 3H), 0.03 (s, 9H); 13C{1H} NMR (150 MHz, CDCl3) δ (ppm)
172.7, 172.4, 161.2, 159.3, 138.1, 136.0, 131.4, 128.7, 128.6, 128.4, 127.7, 124.8, 118.9, 113.8,
72.7, 66.2, 60.9, 55.1, 48.3, 30.2, 15.1, 14.4, 14.3, 0.7; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd
for C29H36N2O5SiNa 543.2291; found 543.2290.
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Ethyl-6-(4-methoxyphenyl)-1,9-dimethyl-4-oxo-3-phenyl-10-((trimethylsilyl)oxy)-2,3-
diazaspiro[4.5]deca-1,6-diene-8-carboxylate (4h’): oil, 13.3 mg, 17% yield, dr 27:73, ee 12%,
[α]D20 = -26.5 (c = 0.11 in CH2Cl2); 1H NMR (600 MHz, CDCl3) δ (ppm) 7.95 (dd, J = 9.0, 1.2
Hz, 2H), 7.41 (dd, J = 8.4, 7.2 Hz, 2H), 7.19 (td, J = 7.8, 1.2 Hz, 1H), 6.97 (d, J = 9.0 Hz, 2H),
6.73 (d, J = 9.0 Hz, 2H), 6.09 (d, J = 3.0 Hz, 1H), 4.27 – 4.16 (m, 2H), 3.84 (d, J = 10.8 Hz,
1H), 3.74 (s, 3H), 3.25 – 3.18 (m, 1H), 3.03 (dd, J = 10.2, 2.4 Hz, 1H), 1.79 (s, 3H), 1.29 (t, J
= 7.2 Hz, 3H), 1.08 (d, J = 6.6 Hz, 3H), 0.05 (s, 9H); 13C{1H} NMR (150 MHz, CDCl3) δ (ppm)
172.2, 172.0, 164.7, 159.4, 138.3, 134.2, 131.4, 128.9, 128.8, 127.5, 124.8, 118.9, 113.9, 99.9,
76.7, 65.6, 61.2, 55.2, 50.7, 30.6, 16.8, 14.6, 14.2, 0.77; HRMS (ESI-TOF) m/z: [M + Na]+
Calcd for C29H36N2O5SiNa 543.2291; found 543.2289.
Ethyl-(5S,8R,9R,10S)-10-hydroxy-1,9-dimethyl-4-oxo-3-phenyl-6-(thiophen-2-yl)-2,3-
diazaspiro[4.5]deca-1,6-diene-8-carboxylate (3i): white solid, 55.4 mg, 87% yield, dr 83:17,
m.p. 158-159 oC; [α]D20 = +25.8 (c = 0.30 in CH2Cl2); 1H NMR (600 MHz, DMSO-d6) δ (ppm)
7.83 (d, J = 7.8 Hz, 2H), 7.44 – 7.39 (m, 3H), 7.20 (t, J = 7.2 Hz, 1H), 6.92 (d, J = 3.0 Hz, 1H),
6.69 (s, 1H), 6.36 (d, J = 5.4 Hz, 1H), 5.70 (d, J = 8.4 Hz, 1H), 4.30 (dd, J = 10.8, 6.0 Hz, 1H),
4.19 – 4.14 (m, 2H), 3.59 (t, J = 5.4 Hz, 1H), 2.78 – 2.70 (m, 1H), 1.95 (s, 3H), 1.25 (t, J = 6.6
Hz, 3H), 0.98 (d, J = 6.6 Hz, 3H); 13C{1H} NMR (150 MHz, DMSO-d6) δ (ppm) 172.4, 171.8,
162.2, 141.2, 138.6, 129.6, 129.4, 128.2, 126.3, 125.2, 123.9, 118.8, 69.8, 66.0, 61.1, 48.1, 29.9,
14.7, 14.6, 14.2; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C23H24SN2O4Na 447.1354; found
447.1354.
Ethyl-(5S,8R,9R,10S)-1,9-dimethyl-4-oxo-3-phenyl-6-(thiophen-2-yl)-10-((trimethyl-
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silyl)oxy)-2,3-diazaspiro[4.5]deca-1,6-diene-8-carboxylate (4i): oil, 58.1 mg, 78% yield, dr
83:17, ee 93%, [α]D20 = +48.3 (c = 0.26 in CH2Cl2); 1H NMR (600 MHz, CDCl3) δ (ppm) 7.92
– 7.90 (m, 2H), 7.38 (t, J = 7.8 Hz, 2H), 7.18 – 7.15 (m, 1H), 7.07 – 7.06 (m, 1H), 6.80 (dd, J
= 4.8, 3.6 Hz, 1H), 6.67 (dd, J = 3.6, 1.2 Hz, 1H), 6.35 (d, J = 6.0 Hz, 1H), 4.66 (d, J = 11.4
Hz, 1H), 4.23 – 4.17 (m, 2H), 3.52 (t, J = 6.0 Hz, 1H), 2.93 (ddd, J = 11.4, 6.6, 6.0 Hz, 1H),
1.99 (s, 3H), 1.31 (t, J = 7.2 Hz, 3H), 1.02 (d, J = 7.2 Hz, 3H), 0.02 (s, 9H); 13C{1H} NMR (150
MHz, CDCl3) δ (ppm) 172.4, 172.0, 161.5, 140.7, 138.2, 129.7, 128.9, 128.9, 127.8, 125.0,
124.9, 123.8, 118.9, 72.8, 66.0, 61.2, 48.4, 30.6, 15.2, 14.5, 14.5, 0.8; HRMS (ESI-TOF) m/z:
[M + Na]+ Calcd for C26H32SN2O4SiNa 519.1750; found 519.1748.
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Ethyl-(5S,8R,9R,10S)-10-hydroxy-1,9-dimethyl-6-(naphthalen-2-yl)-4-oxo-3-phenyl-2,3-
diazaspiro[4.5]deca-1,6-diene-8-carboxylate (3j): white solid, 54.8 mg, 78% yield, dr 68:32,
m.p. 79-80 oC; [α]D20 = +15.2 (c = 0.13 in CH2Cl2); 1H NMR (600 MHz, DMSO-d6) δ (ppm)
7.82 (dd, J = 9.0, 1.2 Hz, 2H), 7.81 – 7.80 (m, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.62 – 7.61 (m,
1H), 7.48 (d, J = 1.2 Hz, 1H), 7.45 – 7.40 (m, 4H), 7.22 – 7.19 (m, 1H), 7.08 (dd, J = 8.4, 1.8
Hz, 1H), 6.20 (d, J = 2.4 Hz, 1H), 5.78 (d, J = 6.6 Hz, 1H), 4.14 – 4.10 (m, 2H), 3.80 (dd, J =
11.4, 6.6 Hz, 1H), 3.23 (dd, J = 10.2, 2.4 Hz, 1H), 2.92 – 2.88 (m, 1H), 1.71 (s, 3H), 1.18 (t, J
= 7.2 Hz, 3H), 1.02 (d, J = 6.6 Hz, 3H); 13C{1H} NMR (150 MHz, DMSO-d6) δ (ppm) 172.7,
172.4, 162.4, 138.6, 136.9, 134.4, 133.1, 132.8, 131.0, 129.6, 128.8, 128.3, 128.0, 127.2, 126.9,
125.3, 125.2, 124.4, 118.8, 73.3, 65.5, 61.2, 50.4, 30.2, 16.3, 14.7, 14.5; HRMS (ESI-TOF) m/z:
[M + Na]+ Calcd for C29H28N2O4Na 491.1947; found 491.1949.
Ethyl-(5S,8R,9R,10S)-1,9-dimethyl-6-(naphthalen-2-yl)-4-oxo-3-phenyl-10-((trimethyl-
silyl)oxy)-2,3-diazaspiro[4.5]deca-1,6-diene-8-carboxylate (4j): oil, 55.9 mg, 69% yield, dr
68:32, ee 99%, [α]D20 = +16.6 (c = 0.17 in CH2Cl2); 1H NMR (600 MHz, CDCl3) δ (ppm) 7.92
(d, J = 7.8 Hz, 2H), 7.74 (d, J = 7.2 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.65 – 7.61 (m, 2H), 7.52
(s, 1H), 7.45 – 7.43 (m, 3H), 7.23 – 7.19 (m, 2H), 6.27 (d, J = 4.8 Hz, 1H), 4.71 (d, J = 11.4
Hz, 1H), 4.30 – 4.24 (m, 2H), 3.63 (t, J = 5.4 Hz, 1H), 3.18 – 3.14 (m, 1H), 1.87 (s, 3H), 1.37
(t, J = 7.2 Hz, 3H), 1.10 (d, J = 6.6 Hz, 3H), 0.05 (s, 9H); 13C{1H} NMR (150 MHz, CDCl3) δ
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(ppm) 172.8, 172.4, 161.0, 138.1, 136.5, 136.4, 133.1, 132.8, 129.8, 128.8, 128.3, 128.1, 127.5,
126.3, 126.2, 125.5, 125.0, 124.6, 119.2, 72.8, 66.2, 61.0, 48.4, 30.2, 15.1, 14.5, 14.4, 0.7;
HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C32H36N2O4SiNa 563.2342; found 563.2339.
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Ethyl-1,9-dimethyl-6-(naphthalen-2-yl)-4-oxo-3-phenyl-10-((trimethylsilyl)oxy)-2,3-
diazaspiro[4.5]deca-1,6-diene-8-carboxylate (4j’): oil, 15.4 mg, 19% yield, dr 32:68, ee 7%,
1H NMR (600 MHz, CDCl3) δ (ppm) 7.96 (dd, J = 9.0, 1.2 Hz, 2H), 7.76 – 7.74 (m, 1H), 7.69
(d, J = 8.4 Hz, 1H), 7.63 – 7.61 (m, 1H), 7.50 (d, J = 1.8 Hz, 1H), 7.46 – 7.43 (m, 2H), 7.43 –
7.41 (m, 2H), 7.23 (tt, J = 7.8, 1.2 Hz, 1H), 7.18 (dd, J = 8.4, 1.8 Hz, 1H), 6.29 (d, J = 2.4 Hz,
1H), 4.26 – 4.18 (m, 2H), 3.91 (d, J = 10.8 Hz, 1H), 3.32 – 3.26 (m, 1H), 3.11 (dd, J = 10.8,
2.4 Hz, 1H), 1.77 (s, 3H), 1.60 (s, 3H), 1.29 (t, J = 7.2 Hz, 3H), 1.12 (d, J = 6.6 Hz, 3H), 0.06
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(s, 9H); C{1H} NMR (150 MHz, CDCl3) δ (ppm) 172.3, 171.9, 160.4, 138.2, 136.3, 134.8,
133.1, 132.8, 130.4, 128.9, 128.4, 128.1, 127.5, 126.4, 126.2, 125.3, 125.0, 124.3, 119.1, 76.7,
65.5, 61.3, 50.8, 30.6, 16.9, 14.7, 14.2, 0.8; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for
C32H36N2O4SiNa 563.2342; found 563.2345.
Ethyl-(5S,8R,9R,10S)-3-(2-fluorophenyl)-10-hydroxy-1,9-dimethyl-4-oxo-6-phenyl-2,3-
diazaspiro[4.5]deca-1,6-diene-8-carboxylate (3k): white solid, 53.7 mg, 82% yield, dr 62:38,
m.p. 177-178 oC; [α]D20 = +32.6 (c = 0.21 in CH2Cl2); 1H NMR (600 MHz, DMSO-d6) δ (ppm)
7.42 – 7.41 (m, 2H), 7.31 (dd, J = 5.4, 1.8 Hz, 3H), 7.29 (dd, J = 4.8, 1.8 Hz, 2H), 7.14 (dd, J
= 6.0, 2.4 Hz, 2H), 6.09 (d, J = 2.4 Hz, 1H), 5.79 (d, J = 6.0 Hz, 1H), 4.16 – 4.12 (m, 2H), 3.80
(dd, J = 10.8, 6.0 Hz, 1H), 3.21 (dd, J = 10.2, 2.4 Hz, 1H), 2.88 – 2.82 (m, 1H), 1.71 (s, 3H),
1.21 (t, J = 7.2 Hz, 3H), 1.04 (d, J = 6.6 Hz, 3H); 13C{1H} NMR (150 MHz, DMSO-d6) δ (ppm)
172.2, 171.7, 161.5, 156.1 (d, JCF = 250.5 Hz), 138.8, 133.9, 129.8, 129.2, 128.5, 127.2 (d, JCF
= 0.8 Hz), 126.7, 125.8, 124.7 (d, JCF = 3.5 Hz), 116.6 (d, JCF = 19.7 Hz), 72.5, 63.1, 60.5, 49.6,
29.4, 15.7, 14.0, 13.8; 19F NMR (565 MHz, DMSO-d6) δ (ppm) -118.87; HRMS (ESI-TOF)
m/z: [M + Na]+ Calcd for C25H25FN2O4Na 459.1696; found 459.1693.
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Ethyl-(5S,8R,9R,10S)-3-(2-fluorophenyl)-1,9-dimethyl-4-oxo-6-phenyl-10-((trimethyl
silyl)oxy)-2,3-diazaspiro[4.5]deca-1,6-diene-8-carboxylate (4k): oil, 56.5 mg, 74% yield, dr
62:38, ee 95%, [α]D20 = +17.3 (c = 0.30 in CH2Cl2); 1H NMR (600 MHz, CDCl3) δ (ppm) 7.46
(td, J = 7.8, 1.8 Hz, 1H), 7.35 – 7.31 (m, 1H), 7.27 – 7.26 (m, 2H), 7.23 – 7.20 (m, 2H), 7.17 –
7.15 (m, 2H), 6.15 (d, J = 2.4 Hz, 1H), 4.23 – 4.15 (m, 2H), 3.89 (d, J = 10.8 Hz, 1H), 3.30 –
3.23 (m, 1H), 3.06 (dd, J = 10.8, 2.4 Hz, 1H), 1.78 (s, 3H), 1.26 (d, J = 7.2 Hz, 3H), 1.11 (d, J
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= 6.6 Hz, 3H), 0.13 (s, 9H); C{1H} NMR (150 MHz, CDCl3) δ (ppm) 172.5, 171.9, 160.7,
157.0 (d, JCF = 252.0 Hz), 139.0, 134.8, 130.1, 129.3 (d, JCF = 8.0 Hz), 128.7, 128.2, 127.1 (d,
JCF = 1.4 Hz), 126.5, 125.1 (d, JCF = 12.2 Hz), 124.4 (d, JCF = 4.1 Hz), 117.0 (d, JCF = 19.5 Hz),
64.1, 61.3, 50.9, 30.6, 16.9, 14.7, 14.3, 0.9; 19F NMR (565 MHz, DMSO-d6) δ (ppm) -118.40;
HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C28H33FN2O4SiNa 531.2091; found 531.2090.
Ethyl-3-(2-fluorophenyl)-1,9-dimethyl-4-oxo-6-phenyl-10-((trimethylsilyl)oxy)-2,3-
diazaspiro[4.5]deca-1,6-diene-8-carboxylate (4k’): white solid, 8.4 mg, 11% yield, dr 38:62,
ee 82%, m.p. 153-154 oC; [α]D20 = -49.8 (c = 0.11 in CH2Cl2); 1H NMR (600 MHz, CDCl3) δ
(ppm) 7.46 (td, J = 7.2, 1.2 Hz, 1H), 7.35 – 7.31 (m, 1H), 7.27 – 7.26 (m, 2H), 7.24 – 7.22 (m,
2H), 7.21 – 7.19 (m, 1H), 7.17 – 7.15 (m, 2H), 6.15 (d, J = 2.4 Hz, 1H), 4.23 – 4.15 (m, 2H),
3.89 (d, J = 10.8 Hz, 1H), 3.30 – 3.23 (m, 1H), 3.06 (dd, J = 10.8, 3.0 Hz, 1H), 1.78 (s, 3H),
1.26 (t, J = 7.2 Hz, 3H), 1.10 (d, J = 6.6 Hz, 3H), 0.13 (s, 9H); 13C{1H} NMR (150 MHz,
CDCl3) δ (ppm) 172.4, 171.8, 160.6, 156.89 (d, JCF = 251.7 Hz), 138.9, 134.7, 130.0, 129.2 (d,
JCF = 7.8 Hz), 128.6, 128.3, 128.1, 127.1 (d, JCF = 1.1 Hz), 126.4, 124.3 (d, JCF = 4.1 Hz), 116.9
(d, JCF = 19.5 Hz), 76.5, 63.9, 61.2, 50.8, 30.5, 16.8, 14.7, 14.2, 0.8; 19F NMR (565 MHz, CDCl3)
δ (ppm) -118.03; HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C28H33FN2O4SiNa 531.2091;
found 531.2094.
Ethyl-(5S,8R,9R,10S)-3-(3-chlorophenyl)-10-hydroxy-1,9-dimethyl-4-oxo-6-phenyl-2,3-
diazaspiro[4.5]deca-1,6-diene-8-carboxylate (3l): white solid, 54.4 mg, 80% yield, dr 60:40,
m.p. 169-170 oC; [α]D20 = 25.4 (c = 0.16 in CH2Cl2); 1H NMR (600 MHz, DMSO-d6) δ (ppm)
7.80 (d, J = 8.4 Hz, 2H), 7.35 – 7.32 (m, 2H), 7.31 – 7.29 (m, 1H), 7.14 – 7.11 (m, 2H), 7.05
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