Novel naphthalimide-benzoic acid conjugates as potential apoptosis-inducing agents: Design, synthesis, and biological activity

Article abstract of DOI:10.1111/j.1747-0285.2011.01232.x

A series of novel naphthalimide derivatives with 4-[4-(3,3-diphenylallyl)piperazin-1-yl]benzoic acid as side chain were designed and synthesized. Their antitumor activities were evaluated against a variety of cancer cell lines in vitro. Preliminary results showed that most of the derivatives had cytotoxic activity comparable with that of amonafide, with IC₅₀ values of 10^-6-10^-5m. Interestingly, compound 12e had the unique antitumor activity against MCF-7 among the cancer cell lines tested. More importantly, flow cytometric analysis indicated that compared with amonafide, the target compounds could effectively induce G₂/M arrest and progress to apoptosis in HL-60 cells after double staining with annexin V-FITC and propidium iodide. The present work provided a novel class of naphthalimide-based derivatives with potential apoptosis-inducing and improved antitumor activity for further optimization.

Full text of DOI:10.1111/j.1747-0285.2011.01232.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01232.x
Chem Biol Drug Des 2011; 78: 941–947
Research Article
Novel Naphthalimide–Benzoic Acid Conjugates
as Potential Apoptosis-Inducing Agents: Design,
Synthesis, and Biological Activity
Aibin Wu1,2,*, Ping Mei1, Yufang Xu2 and
Xuhong Qian2
anticancer drugs have attracted great attention and constituted an
indispensable part in the development of antitumor agents (2),
because of its significantly wide range of applications in photody-
namic therapeutics (3), DNA intercalator (2), inhibitor of topoisomer-
1School of Chemistry and Environmental Engineering, Yangtze
University, Jingzhou 434023, China
ase and telomerase (4), as well as analgesics and fluorescent
markers in medicine and biology (5). Therefore, numerous naphthali-
mide derivatives have been synthesized and evaluated as antitumor
agents (6–8). The continuing success of naphthalimide-based anti-
cancer drugs (9) and recent introduction of its several derivatives
into clinical trials [Amonafide 1 (10), Azonafide 2 (11), and DMP-
840 3 (12), Figure 1] demonstrated that naphthalimide derivatives
represented a group of promising anticancer drugs.
2Shanghai Key Laboratory of Chemical Biology, School of Pharmacy,
East China University of Science and Technology, Shanghai 200237,
China
*Corresponding author: Aibin Wu, abwu@yangtzeu.edu.cn
A series of novel naphthalimide derivatives with
4-[4-(3,3-diphenylallyl)piperazin-1-yl]benzoic acid as
side chainweredesigned andsynthesized. Theiranti-
tumor activities were evaluated against a variety of
cancer cell lines in vitro. Preliminary results showed
that most of the derivatives had cytotoxic activity
comparable with that of amonafide, with IC50 values
of 10)6–10)5 M. Interestingly, compound 12e had the
unique antitumor activity against MCF-7 among the
cancer cell lines tested. More importantly, flow
cytometric analysis indicated that compared with
amonafide, the target compounds could effectively
induce G2 ⁄ M arrest and progress to apoptosis in HL-
60 cells after double staining with annexin V–FITC
and propidium iodide. The present work provided a
novel class of naphthalimide-based derivatives with
However, this conventional cancer chemotherapy was primarily inad-
equate because of the lack of selectivity of the largest majority of
drugs for targeting cancer cells over their non-cancerous counter-
parts (13). Moreover, drug-resistant and multidrug-resistant malig-
nancy could escape treatment (14,15) by a variety of biochemical
mechanisms (16,17). These defects made current anticancer thera-
pies less effective, leading ultimately to the failure (18). Further-
more, there were some problems on the poor solubility and
complicated synthesis of the derivatives. Therefore, it was neces-
sary to attempt neotype structural modification of naphthalimide
with alternative mechanism of action.
potential
apoptosis-inducing
and
improved
In parallel with the growth of knowledge concerning side effects of
this medicine, activation of apoptosis pathways has become an
alternative and promising method of cancer treatment (19,20).
Apoptosis was an evolutionarily conserved and highly regulated pro-
cess, which was used to eliminate defective and unnecessary cells
(21,22). Meanwhile, apoptosis was a cellular process critical to nor-
mal development and homeostasis of multi–cellular organisms (18).
There was a wealth of evidence that disorder of this process was
strongly associated with cancer, and induction of apoptosis was
undoubtedly the most potent defense against cancer (15,19). There-
fore, design and discovery of highly efficient apoptotic inducers,
which could specifically target the abnormal cell death pathway
found in cancer cells, have been paid great attention (15,20,23–26).
The representative reported that apoptotic inducer 4 (23,24), char-
acterized by 4-[4-(3,3-diphenylallyl)piperazin-1-yl] benzoic acid moi-
ety, was illustrated in Figure 1. These inspired us to assume that
naphthalimide conjugated with this specific functional group might
have some improved or different biological activity (27–29).
antitumoractivity forfurther optimization.
Key words: apoptosis, benzoic acid, cell cycle arrest, conjugate,
cytotoxicity, naphthalimide
Abbreviations: A549, human lung cancer cell line; HeLa, human
cervical carcinoma cell line; P388, murine leukemia cell line; HL-60,
human promyelocytic leukemia cell line; MCF-7, human caucasian breast
adenocarcinoma cell line; HCT-8, human ileocecal adenocarcinoma cell
line; A375, human melanoma cell line; MTT, microculture tetrazolium-
formazan; FACS, fluorescence-activated cell sorting; NMR, nuclear mag-
netic resonance; FTIR, Fourier transform infrared spectrum; HRMS (ESI),
high-resolution mass spectrometry (electrospray ionization); DMSO,
dimethyl sulfoxide; TMS tetramethylsilane; CDCl3, deuterated chloro-
form; CD3COCD3, deuterated acetone; m.p., melt point.
Received 15 March 2011, revised 26 March 2011 and accepted for pub-
lication 22 August 2011
The development of highly efficient chemotherapeutic agents
remained an important endeavor (1). Naphthalimide-based
On the basis of above consideration, we focused on modifications
in positions 2 and 6 of naphthalimide (Figure 2). In molecular
941

Wu et al.
Ph
Ph
N
N
NO2
O
O
N
O
N
N
O
N
O
H
N
NH
O
N
O
O
N
O
O2S
N
H
NO2
NH2
O2N
S
NH
Amonafide
1
Azonafide
2
DMP-840
3
4
Figure 1: Structures of some naphthalimide derivatives 1–3 in clinical trials and reported apoptotic inducer 4.
assay. Growth inhibitory effects on the cancer cell lines were mea-
sured by MTT assay (34).
Substituted benzoic acid moiety
Ph
Nitro/hydrogen substitution
R2
O
O
Ph
N
N
R1
Amino substitution
Cell cycle analysis
X
N
HL-60 cells were incubated with different concentration of the com-
pounds. After centrifugation at 1000 rpm for 5 min at room temper-
ature, the supernatant was removed. And then, the cells were
washed twice with PBS solution and fixed with 300 lL of PBS and
700 lL of ice-cold 75% EtOH overnight. Fixed cells were harvested
by centrifugation at 1000 rpm for 10 min at room temperature and
washed twice with PBS. Collected cells were resuspended in 1 mL
of PBS (100 lL ⁄ 1 · 105 cells) and treated with 0.5 lL of RNase A
at 37 ꢀC for 30 min. Propidium iodide was then added to a final
concentration of 50 lg ⁄ mL for DNA staining, and 20 000 fixed cells
were analyzed on a FACScalibur (Becton Dickinson, San Jose, CA,
USA). Cell cycle distribution was analyzed using the Modifit's
program.
Flexible/rigid linker
O
Naphthalimide
R1=-NHCH2CH2SC6H5;
-N(CH2CH2)2S;
R2=-H; -NO2; X: -OCH2CH2-;
-NHSO2C6H4-p;
-NHCH2CH2N(CH3)2;
-NHCH2-2-thiophene;
Figure 2: The design strategy of novel naphthalimide–benzoic
acid conjugates.
design, naphthalimide scaffold was utilized as key prototype struc-
tural unit, and substituted benzoic acid and aliphatic amine func-
tional groups were conjugated to naphthalimide. Amino substituent
introduced to position 6 of naphthalimide was difficult to be acety-
lated (30) and might involve arrest of cell cycle (31). Different types
of linkers, ethanolamine and sulfanilamide (32,33), connected naph-
thalimide with substituted benzoic acid to investigate their effects
on biological activity. As shown in Figure 3, the target compounds
12a–e and 13a–d were prepared. Their in vitro cytotoxicities,
effects on cell cycle, and apoptosis-inducing activities were also
investigated.
Annexin V-FITC staining
Extent of apoptosis was measured through Annexin V–FITC apopto-
sis detection kit (Invitrogen Co., Grand Island, NY, USA) as
described by the manufacture's instruction. Briefly, HL-60 cells were
collected 36 h after the target compounds treatment, washed twice
with PBS, and then resuspended in 400 lL 1· binding buffer
(10 mM HEPES ⁄ NaOH, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). Cells
(100 lL) were transferred to a 5-mL culture tube containing 5 lL of
annexin V–FITC and 10 lL of propidium iodide and then incubated
for 15 min at room temperature in the dark. After 1· binding buffer
was added into each tube, the stained cells were analyzed by flow
cytometry.
Materials and Methods
General
All the reagents were of the commercial quality and were used
without purification. 1H and 13C NMR were obtained with AV-400
spectrometer (Bruker Co., Fꢀllanden, Switzerland) with chemical
shifts reported as ppm (in CDCl3 ⁄ DMSO-d6 ⁄ CD3COCD3, TMS as
internal standard). IR was obtained using 2000 FT-IR spectrometer
(PerkinElmer Co., Boston, MA, USA). HRMS was obtained on HPLC-
Q-Tof MS spectrometer (Micromass Co., Manchester, UK). Melting
points were determined by an X6 micromelting point apparatus,
which were corrected by standard process. Column chromatography
was performed using silica gel 200–300 mesh.
Results and Discussion
Synthesis
The synthetic route of the designed compounds 2-[1,3-dioxo-6-
(substituted amino)-1H-benzo[de]isoquinolin- 2(3H)-yl]ethyl 4-[4-(3,
3-diphenylallyl)piperazin-1-yl]benzoate (12a–e) and N-[4-(1,3-dioxo-
6-[substituted
amino]-1H-benzo[de]isoquinolin-2[3H]-yl)phenylsulfo-
nyl]-4-[4-(3,3-diphenylallyl)piperazin-1-yl]benzamide (13a–d) was
shown in Figure 3. 6-Nitrobenzo[de]isochromene-1,3-dione 5 (35)
was treated with ethanolamine in EtOH for 1 h or treated with
sulfanilamide in glacial acetic acid for 5 h, respectively, affording
intermediates 7 or 8 with satisfactory yield. Nucleophilic substitu-
tion of 7 or 8 with aliphatic amines gave the key intermediates 10
Cytotoxic evaluation in vitro
The target compounds were submitted to the Chinese National Cen-
ter for Drug Screening and School of Pharmacy in East China
University of Science and Technology for in vitro antitumor activity
942
Chem Biol Drug Des 2011; 78: 941–947

Preapoptotic Naphthalimide Conjugates
O
O
O
NO2
b
N
a
SO2NH2
SO2NH2
S
9
OH
OH
c
O
N
O
O
N
O
O
N
O
O
N
O
O
O
O
O
O
d
d
R2
R2
NO2
b
NO2
R1
NO2
R1
Br
7
8
11
10
6
a
f
e
O
O
O2
S
O
O
R2
R1
O
NH
O
N
R2
R1
N
NO2
O
5
O
N
N
12a, 13a : R1 = HNCH2-2-thiophene R2 = H
N
N
R2 = H
12b, 13b : R1 = N(CH2CH2)2S
12c, 13c : R1 = N(CH2CH2)2S
12d, 13d : R1 = HNCH2CH2N(CH3)2
R2 = NO2
R2 = H
R
2 = H
12a–e
12e
:R1 = HNCH2CH2SC6H5
13a–d
Figure 3: Reagents and conditions: (A) ethanolamine, EtOH, reflux 1 h, 95% yield; (B) sulfanilamide, HOAc, reflux 5 h, 75% yield; (C)
thiomorpholine, EtOH, reflux 1 h, 85% yield; (D) corresponding amine, DMF, r.t. 24–36 h, 40–60% yield; (E) 4-[4-(3,3-diphenylallyl)piperazin-
1-yl]benzoic acid (23), CHCl3, EDCI, DMAP, r.t. 24 h, 55–65% yield; (F) 4-[4-(3,3-diphenylallyl)piperazin-1-yl]benzoic acid, DMF, EDCI, DMAP, r.t.
48 h, 50–60% yield.
or 11. 10c and 11c were prepared using 6 (36) as starting mate-
rial. Subsequent condensation of 10 or 11 with 4-[4-(3,3-diphenyl-
allyl)piperazin-1-yl]benzoic acid (23) afforded the target compounds
12a–e or 13a–d with moderate yields of 50–65%, respectively.
The structures of all the newly synthesized compounds were con-
firmed by 1H NMR, 13C NMR, HRMS, and FTIR (see Figures S1–
S27, Supporting Information).
A375 cell lines among each group with IC50 values of 6.70, 10.54,
and 6.63 lM. For A549, HL-60, and MCF-7 cell lines, the highest
cytotoxic compounds were 12d, 12b, and 13a. Among these, IC50
values of compounds 12d, 12b, 13a, and 13d were factors of
10.8, 1.6, 1.2, and 1.1 lower than those of amonafide against A549,
HL-60, MCF-7, and A375 cell lines. In most cases, cytotoxicity
increased in the sequence of 13 and 12, which indicated that the
magnitude and conformation of the linkers had substantial effect on
the cytotoxicity and selectivity of these compounds. For example,
compounds 12a and 13a, respectively, showed the highest cyto-
toxicity against A375 and MCF-7 cell lines. Moreover, the substitu-
ent in position 6 as well as nitro group in position 5 of
naphthalimide also affected their cytotoxicity. We found that com-
pounds 12a–d and 13a–d bearing different substituents showed
a striking contrast to each other. It was interesting that compound
12e with phenylthioethylamino group had exclusive antitumor activ-
ity against MCF-7 (IC50 = 12.21 lM), whereas for the other cancer
cell lines tested, it had no activity (IC50 > 50 lM). This was in
accordance with our previous reported results, indicating that
position 6 of naphthalimide was a crucial active site (37), and
Cytotoxic effects
In vitro antitumor activity of the target compounds was evaluated
by examining their cytotoxic effects using MTT assay (34) against
HeLa, A549, P388, HL-60, MCF-7, HCT-8, and A375 cancer cell lines.
The IC50 represented the drug concentration (lM) required to inhibit
cell growth by 50%. The results were summarized in Table 1.
As shown in Table 1, the target compounds had cytotoxicities com-
parable with those of amonafide against the cancer cell lines
tested except for HCT-8, with IC50 values of 10)6–10)5 M. Com-
pound 13d had the highest cytotoxicity against HeLa, P388, and
Chem Biol Drug Des 2011; 78: 941–947
943

Wu et al.
Table 1: Cytotoxicity of the target compounds against HeLa, A549, P388, HL-60, MCF-7, HCT-8, and A375 cancer cell lines
Cytotoxicity (IC50, lM)a
Compound
HeLa
A549
P388
HL-60
MCF-7
HCT-8
A375
Amonafide
12a
12b
1.40
12.60
7.00
>50
>50
>50
21.00
12.30
7.30
6.70
13.00
>50
2.51
>50
1.22
>50
>50
57.4
>50
>50
4.56
17.96
11.94
11.51
>50
>50
>50
28.11
38.36
12.62
11.92
11.88
11.23
11.92
28.14
32.33
12.21
10.06
24.25
20.37
18.92
9.26
>50
>50
>50
>50
>50
>50
>50
>50
>50
7.51
7.24
7.13
>50
>50
>50
22.04
19.51
6.75
6.63
47.53
30.91
39.42
43.81
>50
39.82
37.87
21.33
10.54
12c
12d
12e
13a
13b
13c
13d
aCytotoxicity values were means of three experiments.
introduction of appropriate substituent to the position could obvi-
ously improve the antitumor selectivity (38). Additionally, it was
worth noting that the target compounds without N,N-dimethylethy-
lenediamine substituent in position 2 of naphthalimide, which was
considered to be important for cytotoxicity (2), still had good activ-
ity. Therefore, manipulation of naphthalimide derivatives to achieve
potent antitumor activity seemed to be closely dependent on struc-
tures and conformation of the substituents and the linkers, which
played important roles in their chemical and biological functions.
sub-G1 portions were increased from 6.44% to 20.68% or from
4.47% to 18.71% versus the untreated control or the control trea-
ted with amonafide, respectively. Meanwhile, the G2 ⁄ M population
increased from 13.29% in the control to 14.85–34.10% in cells trea-
ted with the target compounds. Moreover, on compounds treatment,
HL-60 cells could notably induce morphological changes into round-
ing form (data not listed), which was a primary indication of apop-
tosis and cell death (39). The results in Table 1 and Table 2
indicated that there was no obvious relationship between cytotoxic-
ity and apoptosis-inducing activity. Cell cycle distribution of HL-60
cells in the presence of representative compounds 12b and 13d
was displayed in Figure 4.
Cell cycle profile and apoptosis in HL-60 cells
In the search of a possible mechanism of action responsible for
antitumor activity of the target compounds, we have investigated
their effects on the cell cycle by FACS (26). HL-60 cells were used
in the assay. Cells were treated with the target compounds and
fixed and labeled with propidium iodide. The different phases of
cell cycle were analyzed by flow cytometry. The results were sum-
marized in Table 2.
To further confirm their pro-apoptotic function, FACS analysis was
carried out after double staining cells with propidium iodide and
annexin V–FITC (18,26,39). Early apoptosis corresponded to annexin
V single-positive cells and late apoptosis ⁄ necrosis corresponded to
double-positive cells. As shown in Figure 5, representative com-
pounds 12b and 13d were very effective in induction of apoptosis
in a dose-dependent manner. Treatment of HL-60 cell line by 3.0,
6.0, and 12.0 lM of 12b resulted in 54.38%, 42.30%, and 2.10%
of apoptotic cells as compared to 0.81% of apoptotic cells in an
untreated control. Also, compound 13d had significant effects on
As shown in Table 2, the target compounds could effectively induce
G2 ⁄ M arrest and progress to apoptosis in HL-60 cell line. After
incubation with IC50 concentration of the target compounds, the
Table 2: Cell cycle distribution of HL-60 cell line in the presence of IC50 concentration of the target compounds
Cell cycle distribution (%)a
Compound
Concentration (lM)
G1
S
G2 ⁄ M
sub-G1
Control
Amonafide
12a
0
18
12
12
50
50
50
28
38
13
12
52.86
52.26
62.00
63.47
61.31
68.64
62.02
65.39
71.20
64.95
59.56
33.86
36.68
23.15
21.14
8.25
0.20
3.87
1.93
0.08
13.29
11.06
14.85
15.40
30.45
31.16
34.10
32.68
28.80
15.60
16.32
0.72
2.69
9.32
12b
12c
9.18
10.11
11.74
7.16
21.40
7.20
12d
12e
13a
13b
13c
13d
19.44
24.11
10.43
10.11
aCell cycle distribution values were means of three experiments.
944
Chem Biol Drug Des 2011; 78: 941–947

Preapoptotic Naphthalimide Conjugates
G1 : 63.47%
S : 21.14%
G1 : 59.56%
S : 24.11%
G2 : 16.32%
G1 : 52.86%
S : 33.86%
G
: 15.40%
G
: 13.29%
Apoptosis2: 9.18%
Apoptosis : 10.11%
Apoptosis2: 0.72%
Compound : 12b
Compound : 13d
Control
0
40
80
120
160
200
0
30
60
90
120
0
30
60
90
120
Channels (FL2-A)
Channels (FL2-A)
Channels (FL2-A)
Figure 4: Cell cycle distribution of HL-60 cells in the presence of representative compounds 12b and 13d, respectively. HL-60 cells were
treated with IC50 concentration of the compounds or with 0.1% DMSO. Cells were harvested, fixed, and stained with propidium iodide.
20 000 Stained cells were then subjected to FACScalibur analysis to determine the distribution of cells.
Control
Compound 12b, 3.0 µM
Compound 12b, 6.0 µM
Compound 12b, 12.0 µM
0.00%
1.82%
0.00%
33.80%
0.02%
51.13%
0.00%
91.70%
0.81%
2.10%
42.30%
54.38%
0
1
2
3
4
0
1
2
3
4
0
1
2
3
4
0
1
2
3
4
10
10
10
Annexin-FITC
10
10
10
10
10
10
10
10
10
10
Annexin-FITC
10
10
10
10
10
Annexin-FITC
10
10
Annexin-FITC
Control
Compound 13d, 4.8 µM
Compound 13d, 9.5 µM
Compound 13d, 19.0 µM
0.24%
3.92%
0.00%
9.09%
0.11%
43.04%
1.66%
81.76%
0.51%
3.08%
80.49%
54.41%
0
1
2
3
4
0
1
2
3
4
0
1
2
3
4
0
10
1
2
3
4
10
10
10
10
10
10
10
10
Annexin-FITC
10
10
10
10
10
Annexin-FITC
10
10
10
10
Annexin-FITC
10
10
Annexin-FITC
Figure 5: Compounds 12b and 13d induced apoptosis of HL-60 cells. Cells were treated with corresponding concentration of the com-
pounds or with vehicle solvent (0.1% DMSO), and then stained with annexin V–FITC and propidium iodide. Stained cells were subjected to
FACS analysis to determine the distribution of cells. Early apoptosis corresponded to annexin V single–positive cells and late apoptosis ⁄ necro-
sis corresponded to double–positive cells.
induction of apoptosis. These results collectively suggested that the
target compounds might inhibit the growth of HL-60 cell line by
induction of apoptosis.
with that of amonafide with IC50 values of 10)6–10)5 M. It was inter-
esting that compound 12e exhibited the selective cytotoxicity against
MCF-7 among the cancer cell lines tested. More importantly, flow cy-
tometric analysis indicated that these derivatives could effectively
induce G2 ⁄ M arrest and progress to apoptosis in HL-60 cell line
compared with amonafide. The present work demonstrated that
conjugating the biological active units of naphthalimide and 4-[4-(3,3-
diphenylallyl)piperazin-1-yl]benzoic acid might be able to result in a
novel class of lead compounds with potential apoptosis-inducing and
improved antitumor activity. Further structural optimization and
detailed biological studies on the mechanism of action about the
designed naphthalimide derivatives were under way.
However, the search of the reasonable targets of the synthesized
compounds still remained endeavor. Assays for DNA intercalation
and Top II inhibition of the compounds had been carried out. It was
surprising that the target compounds had less interaction with DNA
and Top II inhibition than amonafide did (data not listed). Maybe,
Bcl-2 ⁄ xL binding or other factors contributed its unique activity?
This section of work was under investigation in our laboratory. We
hope that in near future, we could find the answer.
Conclusions
Acknowledgments
A series of novel naphthalimide–benzoic acid conjugates were
designed and synthesized. Their antitumor activities were evaluated
against a variety of cancer cell lines in vitro. Preliminary results
showed that most of the derivatives had cytotoxic activity comparable
This work was supported by the National Natural Science Founda-
tion of China (20536010) and the Science Research Project of Hubei
Provincial Department of Education (Q20111304) and the Science
Research and Development Foundation of Yangtze University.
Chem Biol Drug Des 2011; 78: 941–947
945

Wu et al.
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Figure S7. 1H NMR spectrum of 12c.
Figure S8. 13C NMR spectrum of 12c.
Figure S9. HRMS spectrum of 12c.
Figure S10. 1H NMR spectrum of 12d.
Figure S11. 13C NMR spectrum of 12d.
Figure S12. HRMS spectrum of 12d.
Figure S13. 1H NMR spectrum of 12e.
Figure S14. 13C NMR spectrum of 12e.
Figure S15. HRMS spectrum of 12e.
Figure S16. 1H NMR spectrum of 13a.
Figure S17. 13C NMR spectrum of 13a.
Figure S18. HRMS spectrum of 13a.
Figure S19. 1H NMR spectrum of 13b.
Figure S20. 13C NMR spectrum of 13b.
Figure S21. HRMS spectrum of 13b.
Figure S22. 1H NMR spectrum of 13c.
Figure S23. 13C NMR spectrum of 13c.
Figure S24. HRMS spectrum of 13c.
Figure S25. 1H NMR spectrum of 13d.
Figure S26. 13C NMR spectrum of 13d.
Figure S27. HRMS spectrum of 13d.
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Supporting Information
Additional Supporting Information may be found in the online ver-
sion of this article:
Figure S1. 1H NMR spectrum of 12a.
Figure S2. 13C NMR spectrum of 12a.
Figure S3. HRMS spectrum of 12a.
Figure S4. 1H NMR spectrum of 12b.
Figure S5. 13C NMR spectrum of 12b.
Please note: Wiley-Blackwell is not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
corresponding author for the article.
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