Photoinduced isomerization and hepatoxicities semaxanib, sunitinib and related 3 substituted indolin 2 ones

DOI: 10.1002/cmdc.201500475

Full Papers

Photoinduced Isomerization and Hepatoxicities of
Semaxanib, Sunitinib and Related 3-Substituted Indolin-2ones
Mun Hong Ngai,[a] Choon Leng So,[a] Michael B. Sullivan,[b] Han Kiat Ho,[a] and
Christina L. L. Chai*[a]
tion ranged from 0.009 to 0.048 hÀ1. Selected compounds
were tested for cytotoxicity in the TAMH liver cell line. E/Z mixtures of four compounds were also assessed for toxicity in the
TAMH and HepG2 cell lines. In some cases, the stereochemically pure drug was more toxic than the E/Z mixtures, but a general statement cannot be made. Our studies show that each stereoisomer could contribute differently to toxicity, suggesting
that stereochemical purity issues that could arise from isomerization cannot be ignored.

3-Substituted indolin-2-ones are an important class of compounds that display a wide range of biological activities. Sunitinib is an orally available multiple tyrosine kinase inhibitor that
has been approved by the US Food and Drug Administration
(FDA) for the treatment of renal cell cancer. Sunitinib and a related compound, semaxanib, exist as thermodynamically stable
Z isomers, which photoisomerize to E isomers in solution. In
this study, 17 3-substituted indolin-2-ones were synthesized,
and the kinetics of their photoisomerization were studied by
1
H NMR spectroscopy. The rate constants for photoisomeriza-

Introduction
and negative results in phase II/III studies.[5–7] Subsequent studies focused on structural modifications of semaxanib, leading
to the discovery of sunitinib (2), a new multitargeted receptor
tyrosine kinase inhibitor.[8] Sunitinib has been approved by the
US Food and Drug Administration (FDA) for treatment of advanced renal cell cancer and imatinib-resistant or imatinib-intolerant gastrointestinal stromal tumors (GISTs).[9, 10]
Both semaxanib and sunitinib have Z stereochemistry at the
double bond, and both can undergo photoisomerization to
the E isomer. The Z isomer is the thermodynamically stable
form, due to the presence of intramolecular hydrogen bonding

between the C2 carbonyl group of the oxindole and the NH
group of the pyrrole ring.[4] Thermal reversion of the less stable
E isomer to the Z isomer is also reported to occur. Some pharmaceutical implications of this isomerization process, for example, the stabilities of the administered drug, have been recognized for both semaxanib and sunitinib, leading to the development of analytical methods for detection of the isomers.[11–14] However, not much has been reported with regard
to the photoisomerization process of semaxanib,[11–13] sunitinib,[15] and related 3-pyrrolylmethylidene oxindoles, or whether
the stereoisomers display differential biological activities. In
the latter context, we were specifically interested in determining whether the E and Z isomers display different toxicities
that may lead to undesired side effects later in the drug development process. This is especially relevant in view of the toxicity effects observed with semaxanib in clinical trials.
In this study, we report the photoisomerization studies of
semaxanib and 3-substituted indolin-2-one analogues and
report the toxicities of the compounds and their isomers, first
against the TAMH cell line as a metabolically competent model

The 3-substituted indolin-2-ones (oxindoles) are an important
class of compounds that have been well-explored for their biological activities and continue to attract much interest due to
their promise in drug development.[1] Of these oxindoles, the
3-pyrrolylmethylidene oxindoles are particularly exciting, as
they have been shown to inhibit receptor tyrosine kinases
(RTKs). The selectivities of these oxindoles against particular
RTKs are dependent on the substituents, especially at the C3
position. Semaxanib (1) is an example of this class of compounds and shows potent activity against vascular endothelial
growth factor (VEGF).[2–4] Semaxanib proceeded to clinical
trials, but its development was halted due to toxicity issues

[a] Dr. M. H. Ngai,+ C. L. So,+ Prof. Dr. H. K. Ho, Prof. Dr. C. L. L. Chai
Department of Pharmacy, National University of Singapore
18 Science Drive 4, Singapore 117543 (Singapore)
E-mail:

[b] Dr. M. B. Sullivan
Institute of High-Performance Computing

Agency for Science Technology and Research, Singapore
1 Fusionopolis Way, #16-16 Connexis, Singapore 138632 (Singapore)
[+] These authors contributed equally to this work.

ChemMedChem 2016, 11, 72 – 80

72

 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


Full Papers
to recapitulate in vivo bioactivation events, and in another
liver cell line, HepG2, to validate our findings.

Table 1. Yield and Z/E isomer ratios of 3-substituted indolin-2-ones.

Results and Discussion
Synthesis
The 3-substituted indolin-2-ones were prepared following reported procedures using a Knoevenagel condensation between substituted indoline-2-ones and aldehydes in the presence of piperidine in ethanol (Scheme 1).[4] Oxindole, 5-fluoro-,

Compd

R1

R2

R3

1

5a
5b
5c
5d
5e
5f
5g
5h
5i
6a
6b
6c
7a
7b
7c
8

H
H
H
H
H
H
H
H
CH3
CH3
H
H
H

CH3
CH3
CH3
–

H
F
Ac
NO2
OMe
NHAc
OH
NH2
H
F
H
H
H
H
H
H
–

H
H
H
H
H
H
H

H
H
H
Ac
CH3
Boc
Ac
CH3
Boc
–

Yield [%]

Z/E isomer ratio

62
58
53
74
73
71
57
34
46
15
78
53
90
58
85

90
66

100:0
100:0
100:0
100:0
100:0
100:0
100:0
100:0
0:100
3:97
100:0
100:0
100:0
16:84
11:89
14:86
100:0

doline-2-one ring. The same acylation and alkylation method
was applied to the synthesis of N-methylpyrrole compounds
7 a–c, and a mixture of E and Z isomers were obtained, despite
starting with the pure E isomer of 5 h (Scheme 3). The relative

Scheme 1. Synthesis of 3-substituted indolin-2-ones. Reagents and conditions: a) piperidine, EtOH, 75 8C.

and 5-nitrooxindoles were commercially available. 5-Acetyloxindole was prepared via Friedel–Crafts acetylation of oxindole.[16] Oxidation of oxindole with phenyliodine(III)-bis(trifluoroacetate) (PIFA) gave 5-hydroxyoxindole, which was subsequently methylated to give 5-methoxyoxindole.[17] 5-Acetamidoxindole was synthesized from 5-nitrooxindole in two steps,
according to a literature procedure.[18] For all of the 3-substituted indoline-2-ones with R1 = H (1, 5 a–f), only the Z isomer was

obtained. This was verified with NOE experiments in which an
NOE effect was observed between the vinylic proton and the
C4 aromatic proton. Compounds with R1 = CH3 (5 h–i) were isolated exclusively as the E isomer (i.e., 5 h) or as mixtures of
both the Z and E isomers (i.e., 5 i; Table 1). The 5-amino indolinone 5 g was synthesized by reduction of 5-nitro indolinone
5 c (Scheme 2).[19]
N1-substituted compounds 6 a–c were synthesized by acylation or alkylation of semaxanib. Acylation and alkylation of
semaxanib occurred exclusively at the nitrogen atom of the in-

Scheme 3. Synthesis of N1-substituted indolin-2-one derivatives 6 a–c and
N1-substituted N1’-methylindolin-2-one derivatives 7 a–c. Reagents and conditions: a) R3 = Ac: Ac2O, Et3N, DMAP, CH2Cl2, RT; R3 = CH3 : NaH, MeI, DMF
(6 b) or THF (7 b), RT; R3 = Boc: (Boc)2O, DMAP, CH2Cl2, RT.

ratios of the stereoisomers for 7 a–c were determined by
H NMR spectroscopy (Table 1). It was observed that the vinyl
hydrogen for the Z isomer resonated downfield to the E isomer
by ~ 0.2–0.3 ppm. Compound 8 was synthesized by condensing indoline-2-one with pyrrole-2-carboxaldehyde under standard Knoevenagel conditions.[4]
1

Scheme 2. Synthesis of 5-amino indolinone 5 g. Reagents and conditions:
a) Pd/C (10 %), H2, EtOH, RT, 16 h, 34 %.

ChemMedChem 2016, 11, 72 – 80

www.chemmedchem.org

73

 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
Photoinduced isomerization studies
In order to assess the effect of substituents on the ease of the
photoisomerization process, isomerization studies were carried
out. The isomerization was monitored by 1H NMR spectroscopy, as this method can be carried out in real-time, is rapid and
quantitative, and can be carried out in a neutral solvent
([D6]DMSO). In a typical experiment, the indolin-2-ones in
[D6]DMSO were exposed to fluorescent light, and their 1H NMR
spectra were acquired at various time intervals (0.25, 0.5, 1, 2,
3, 4, 5, 6, 12, and 24 h). Table 2 shows the ratio of Z/E isomers

Figure 1. Kinetics of Z!E photoisomerization of semaxanib (1) in [D6]DMSO.
Data were determined at various time points by 1H NMR spectroscopy.

Table 2. Indolinone Z/E isomer ratios before (t0) and after 24 h light exposure (t24), and chemical shifts (d) of the vinyl protons.

Compd

R1

R2

R3

1
5a
5b
5c
5d
5e

5f
5g
5h
5i
6a
6b
6c
7a
7b
7c
2
8

H
H
H
H
H
H
H
H
CH3
CH3
H
H
H
CH3
CH3
CH3
–

-

H
F
Ac
NO2
OMe
NHAc
OH
NH2
H
F
H
H
H
H
H
H
–
–

H
H
H
H
H
H
H
H
H

H
Ac
CH3
Boc
Ac
CH3
Boc
–
–

t0

Z/E ratio
t24

100:0
100:0
100:0
100:0
100:0
100:0
100:0
100:0
0:100
3:97
100:0
100:0
100:0
16:84
11:89

14:86
100:0
100:0

64:36
70:30
89:11
94:6
69:31
88:12
100:0
100:0
18:82
28:72
74:26
83:17
72:28
23:77
20:80
25:75
65:35
55:45

Table 3. Rate constants of Z!E isomerization in light and reversion in
dark of semaxanib and 3-substituted indolin-2-one analogues in
[D6]DMSO.

d [ppm]
Z isomer
E isomer

7.55
7.63
7.77
7.95
7.57
7.77
7.40
7.29
7.59
7.67
7.66
7.60
7.63
7.81
7.66
7.75
7.73
7.74

7.33
7.38
7.41
7.48
7.32
7.49
NA
NA
7.42
7.50
7.51

7.41
7.46
7.65
7.51
7.58
7.40
7.51

www.chemmedchem.org

R1

R2

R3

1
5a
5b
5c
5d
5e
5f
5g
5h
6a

H
H
H

H
H
H
H
H
CH3
H

H
F
Ac
NO2
OMe
NHAc
OH
NH2
H
H

H
H
H
H
H
H
H
H
H
Ac

Rate constant [hÀ1][a]
Isomerization
Reversion
0.048
0.048
0.043
0.009
0.019
0.021
None
None
0.090[b]
0.041

0.002
0.003
0.018
0.006
0.061
0.001
–
–
None
0.033

[a] Concentration of the samples was 10 mm in [D6]DMSO. [b] Isomerization from E to Z.

room temperature, and 1H NMR spectra were recorded at different time intervals to study the E-to-Z isomer conversion. The
E isomer of semaxanib reverted back to the Z isomer in the
dark with a slower observed rate constant of 0.002 hÀ1.

In a similar manner, the rate constants of Z!E or E!Z photoisomerization and reversion of semaxanib and related 3-substituted indolin-2-one derivatives were measured as shown in
Table 3. In general, the rate constants for isomerization ranged
from 0 (no isomerization for compounds 5 f and 5 g) to
0.09 hÀ1. With the exception of 5 h, the rate constants measure
Z!E photoisomerization. For those with measurable reversion,
the rate constants ranged from 0.001 to 0.061 hÀ1. Thus there
is little discernible correlation between the rate constants for
isomerization and reversion with the nature of the substituents.

of the indolin-2-one before exposure to light and the Z/E ratio
after exposure to light for 24 h. With the exception of 5-hydroxy compound 5 f and 5-amino compound 5 g, the Z/E ratio
of all other indolinones changed upon exposure to fluorescent
light, in the range of ~ 6–36 %.
The kinetics of the photoisomerization of selected indolin-2ones were investigated. Figure 1 shows the formation of the
E isomer of semaxanib in solution after exposure to light at different time intervals. Prior to light exposure, only (Z)-semaxanib was present, and the formation of the E isomer reached
equilibrium at 36 % after 24 h. The formation of the E isomer
followed first-order kinetics, and the rate constant for isomerization of (Z)-semaxanib to (E)-semaxanib was determined to
be 0.048 hÀ1 (Table 3). The E isomer of semaxanib was not
stable in solution and reverted back to the Z isomer when left
in the dark. To determine the reversion kinetics of semaxanib
in the dark, the compound was exposed to fluorescent light
for 24 h, following which, the sample was kept in the dark at
ChemMedChem 2016, 11, 72 – 80

Compd

Theoretical studies
We attempted to rationalize the observed ease of photoisomerization by assessing the HOMO and LUMO energies of compounds 5 a–h and 6 a. We used a similar approach to that
taken by Tomasi et al.,[20] using B3LYP/6-311 + + G(d,p)//B3LYP/
6-311G(d,p) with IEF-PCM solvation in DMSO, as implemented

in Gaussian 09.[21] A summary of the calculated UV/Vis results is
shown in Table 4.
74

 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


Full Papers
Table 4. Calculated electronic spectra for selected indolinones.

Table 5. HOMO and LUMO energies of selected (Z)-indolinones.

Compd

l [nm]

Oscillator
strength

Compd

l [nm]

Oscillator
strength

Compd

(Z)-1

422.3
361.1
305.3
423.9
367.2
307.6
421.6
375.2
348.2
525.1
415.5
356.3
439.0
395.7
304.6
424.9
373.9
307.4
439.6
390.4
305.2
476.4
401.0
303.8
427.5
365.0
325.3
430.2
346.4
315.1

0.679
0.230
0.025
0.625
0.309
0.026
0.749
0.009
0.309
0.022
0.864
0.353
0.232
0.699
0.025
0.606
0.343
0.023
0.297
0.623
0.025
0.087
0.825
0.021
0.613
0.191
0.028
0.717
0.086

0.109

(E)-1

415.1
358.1
295.6
421.3
365.0
298.5
415.4
376.4
348.5
526.5
412.5
359.8
437.8
385.6
295.0
420.9
369.8
300.9
438.3
382.2
295.6
473.8
390.2
297.7
419.3
362.1

317.6
419.2
346.8
324.0

0.604
0.182
0.026
0.543
0.241
0.028
0.645
0.010
0.159
0.030
0.667
0.237
0.228
0.542
0.026
0.510
0.263
0.052
0.285
0.486
0.027
0.122
0.634
0.061
0.570

0.085
0.026
0.704
0.105
0.039

1
5a
5b
5c
5d
5e
5f
5g
5h
6a

(Z)-5 a

(Z)-5 b

(Z)-5 c

(Z)-5 d

(Z)-5 e

(Z)-5 f

(Z)-5 g

(Z)-5 h

(Z)-6 a

(E)-5 a

(E)-5 b

(E)-5 c

(E)-5 d

(E)-5 e

(E)-5 f

(E)-5 g

(E)-5 h

(E)-6 a

www.chemmedchem.org

Z isomer
HOMO

À2.27
À2.34

À2.39
À2.89
À2.25
À2.33
À2.27
À2.22
À2.22
À2.48

À5.47
À5.55
À5.57
À5.70
À5.46
À5.53
À5.45
À5.31
À5.45
À5.59

HOMO-1
À6.33
À6.31
À6.54
À6.82
À5.86
À6.23
À5.93
À5.66
À6.27

À6.69

dolinone. Consistent with experimental observations, calculations show that the Z isomer is the more stable isomer for
compounds 1, 5 a–g, and 6 a, while the E isomer is the more
stable isomer for 5 h.
Cytotoxicity studies
The cytotoxicities of the compounds were first determined
using the MTT cell proliferation assay against the TAMH cell
line, selected for its ability to metabolize xenobiotics and to reproduce the toxicity observed with classical hepatotoxicants,
such as acetaminophen.[22] The 50 % inhibitory concentration
value (IC50) was estimated using GraphPad Prism 6 (Table 6).

Table 6. Cytotoxicity and Z/E isomer ratios of 3-substituted indolin-2ones.

An examination of the oscillator strengths (f) of each pair of
isomers in the UV region of ~ 400 nm shows that compounds
1, 5 a–c, 5 e, 5 h, and 6 a have high f values, in the range of
~ 415 nm to 430 nm, as compared with compounds 5 d, 5 f,
and 5 g. With the exception of 5 d and 5 h, these are reflective
of the ease of Z!E isomerization, such that those with large
f values for the Z isomers are easier to isomerize and vice
versa. With 5 h, E!Z isomerization occurs readily, as is reflected by the high f value for the E isomer. Based solely on our results, the isomerization of 5 d does not fit the trend of the
others. The UV/Vis results for 5 d and 5 f are very similar, but
the rates of their photoisomerization are different.
If one considers that photoisomerization is a consequence
of S0 to S1 transitions, then the calculated HOMO and LUMO
energies of the Z isomers of 1, 5 a–h, and 6 a may provide further insight into their ease of isomerization. As shown in
Table 5, the FMO energies are broadly similar, and the energy
difference between the HOMO and LUMO does not explain the
observed isomerization rates.

From these calculations, the dihedral angle of C=CÀCÀN for
the Z isomers is roughly zero, indicating planarity, while the
same dihedral angle is ~ 108 out of plane for the E isomers. The
exception is 5 h, where both the E and Z isomers have a dihedral angle of ~ 308, which is the consequence of N-methylation
at the pyrrole ring, which in turn, does not allow for intramolecular hydrogen bonding with the carbonyl group on the inChemMedChem 2016, 11, 72 – 80

LUMO

Compd

R1

R2

R3

1
5a
5h
5i
6a
6b
6c
7a
7b
7c
2
8

H

H
CH3
CH3
H
H
H
CH3
CH3
CH3
–
–

H
F
H
F
H
H
H
H
H
H
–
–

H
H
H
H
Ac

CH3
Boc
Ac
CH3
Boc
–
–

Z/E ratio

IC50 [mm][a]

100:0
100:0
0:100
3:97
100:0
100:0
100:0
16:84
11:89
14:86
100:0
100:0

6.28 Ỉ 1.24
2.17 Ỉ 0.47
> 50
> 50
9.77 Ỉ 0.67

3.76 Ỉ 0.61
0.69 Ỉ 0.12
> 50
> 50
11.94 Ỉ 0.08
11.28 Ỉ 0.65
21.53 Ỉ 2.40

[a] Values are the mean Ỉ SD of three independent experiments.

When comparing the compounds with a hydrogen atom on
the N1’ of the pyrrole ring (i.e., 1, 2, 5 a, 6 a–c, 8), (Z)-8 was
found to be the least toxic to TAMH cells (IC50 = 21.53 mm). The
IC50 value of (Z)-2 was 11.28 mm, which is higher than for indolinones 1, 5 a, and 6 a–c. Compounds substituted at the N1 posi75

 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


Full Papers
tion of the oxindole ring, such as (Z)-6 b (R3 = CH3) and (Z)-6 c
(R3 = Boc), were more toxic (IC50 values of 3.76 and 0.69 mm, respectively). (Z)-6 a (R3 = Ac) was less toxic (IC50 = 9.77 mm) than
the unsubstituted (Z)-1 (R3 = H; IC50 = 6.28 mm). Fluorine substitution on the oxindole core (compound 5 a; R2 = F) had increased toxicity relative to compound (Z)-1. In contrast, compounds with substitution at the nitrogen atom of the pyrrole
ring were generally less toxic to the TAMH cell line. With the
exception of 7 c (IC50 = 11.94 mm), all compounds in this series
showed IC50 values greater than 50 mm.
Overall, the toxicities of the compounds varied. Boc substitution at the N1 position of the indolin-2-ones resulted in 6 c
and 7 c (R3 = Boc), and these were the most toxic compounds
within their series (R1 = H or Me). Moreover, it could be inferred
that methylation at the N1’ position and/or the E isomeric
forms could decrease the toxicity of the 3-[(substituted pyrrol2-yl)methylidenyl]indolin-2-one series.

To determine if the E isomers were indeed less toxic, the E/Z
mixtures of 1, 2, 5 a, and 8 were also tested against the TAMH
and HepG2 cell lines. (Z)-8 is the parent compound, while (Z)1 (semaxanib) and (Z)-2 (sunitinib) are potent anticancer
agents. (Z)-5 a was also selected, because it is similar to (Z)1 but has an isosteric replacement (R2 = F). The stability of
100 mm drug stocks of these compounds in glass vials was independently determined. They were exposed to fluorescent
lighting, and after 24 h, the E/Z ratios were determined using
NMR spectroscopy by diluting 50 mL of the stocks solution to
500 mL in [D6]DMSO. In all of the cases above, isomerization
was observed. The observed E/Z ratios were only slightly lower
than those listed in Table 2, with the exception of 5 a, for
which the percentage of (E)-5 a was significantly lower
(Table 7).
For 1 and 5 a, the E/Z mixtures were more toxic than (Z)1 and (Z)-5 a, while the reverse was true for 2 and 8 in the
TAMH cell line. The differences between the IC50 values between the pure Z isomers and the E/Z mixtures were not large
for 5 a and 2 (2.17 mm vs. 1.59 mm and 11.28 mm vs. 16.66 mm,
respectively). The IC50 value was 6.28 mm for (Z)-1 but was
2.99 mm for the E/Z mixture, which contained 31 % E isomer
and 69 % Z isomer. For (Z)-8, the IC50 value was 21.53 mm, while
its E/Z mixture was less toxic to the TAMH cell line (IC50 >
50 mm). For HepG2 cell line, Z isomers of 1, 5 a, and 2 were
more toxic than their respective E/Z mixtures, while the E/Z
mixture of 8 was not significantly more toxic than (Z)-8 (2.38
and 2.17 mm, respectively). It was observed that the E/Z mixture of 8 was the most toxic compound in the HepG2 cell line.

Thus, E/Z isomerism can affect the toxicity of the compounds.
Whether the E/Z mixtures or the isomerically pure samples
were more toxic could not be generalized, as this was dependent on the structures of the compounds and the differential
handling of the compounds by the host cell line (Table 7).

Conclusions

A total of 16 compounds that were structurally related to semaxanib (1) and sunitinib (2) were synthesized in yields of 15 %
to 90 %. For 5 a–g, 6 a–c, and 8, only the Z isomers were obtained. In contrast, only the E isomer of 5 h was obtained,
while 5 i and 7 a–c were obtained as E/Z mixtures but with the
E form predominating. Overall, a majority of the compounds
tested were able to undergo isomerization. The rate of isomerization and the amount of the less stable isomer varied. The
stabilities of the E/Z mixtures were also examined, and it was
found that without continual exposure to the light source,
some mixtures converted back to the more stable form, with
variable rates of reversion.
All compounds were tested in the TAMH cell line. Generally,
compounds with N1’ substitutions that had the E isomers as
the predominant form were less toxic to the TAMH cell line,
while compounds that were in the Z forms decreased cell viability to a greater extent. The E/Z mixtures of the four selected
compounds that were of clinical importance (1, 2, 5 a, and 8)
were tested for toxicity in the TAMH and HepG2 cell lines. With
respect to TAMH cell line toxicity, the E/Z mixture of 1 was
more toxic to the cells than (Z)-1, while the reverse was true
for 2 and 8. However, it should be noted that the differences
in the IC50 values between the pure Z isomers and the E/Z mixtures for 5 a and 2 were not large. For the HepG2 cell line,
Z isomers of 1, 5 a, and 2 were more toxic than the E/Z mixtures. The differences in the observed trends between the two
liver cells lines are indicative of the sensitivities of the responses of the cell lines to the stereoisomers. We note that HepG2 is
a liver cancer cell line, and the effects of these RTK inhibitors
on HepG2 cannot be dissociated from the toxicity.
The significance of our study should be realized. Such findings could prompt the appropriate handling of clinically available drugs (e.g., sunitinib) and demonstrate that protection of
the drugs from light could be vital. For future lead compounds
that contain exocyclic double bonds, it may be advantageous
to separately investigate the contributions of their more stable
isomers, less stable isomers, and/or their E/Z mixtures to activities and toxicities. Removing such unsaturation and/or design-

Table 7. Z/E isomer ratios of the indolinones and their corresponding IC50 values against TAMH and HepG2 cell lines.

Compd
1
2
5a
8

Z/E ratio
100:0
100:0
100:0
100:0

TAMH

IC50 [mm][a]

6.28 Ỉ 1.24
11.28 Ỉ 0.65
2.17 Æ 0.47
21.53 Æ 2.40

Z/E ratio

HepG2

8.17 Æ 0.39
5.85 Æ 1.20
12.11 Æ 0.26
2.38 Ỉ 1.67

69:31
69:31
83:17
56:44

TAMH

IC50 [mm][a]

2.99 Ỉ 0.36
16.66 Ỉ 1.52
1.59 Ỉ 0.29
> 50

HepG2

37.55 Ỉ 10.03
10.36 Ỉ 4.43
21.57 Ỉ 2.74
2.17 Ỉ 1.13

[a] Values are the mean Ỉ SD of three independent experiments.

ChemMedChem 2016, 11, 72 – 80

www.chemmedchem.org

76

 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


Full Papers
ing stereochemically stable compounds may be feasible, provided that the activities are not compromised. This would
avoid the issue of E/Z isomerization.

matography (CH2Cl2) afforded (Z)-5 a as an orange solid in 58 %
yield (60.9 mg): mp: 275–277 8C (lit.[24] 271–273 8C); 1H NMR
(400 MHz, CDCl3): d = 13.14 (br s, 1 H), 7.65 (br s, 1 H), 7.33 (s, 1 H),
7.17 (m, 1 H), 6.81 (m, 2 H), 6.00 (s, 1 H), 2.38 (s, 3 H), 2.33 ppm (s,
3 H); 13C NMR (100 MHz, CDCl3): d = 169.9, 168.9, 160.2, 157.9,
137.9, 133.7, 132.7, 128.1, 128.0, 127.2, 124.4, 113.1, 111.8, 111.6,
109.6, 109.5, 104.7, 104.4, 14.0, 11.7 ppm (number of carbon signals
was greater than expected due to F coupling); HRMS (ESI-TOF): (m/
z) calcd for C15H13FN2O [M + H] + 257.1090, found 257.1090.

Experimental Section
General methods: (Z)-2 (Sunitinib malate) was used as purchased
from LC laboratories. All other reagents and solvents were purchased from Sigma–Aldrich or Alfa Aesar and were used without
further purification unless otherwise specified. Reactions involving
air- or moisture-sensitive reagents were performed with dried
glassware under a nitrogen atmosphere. Thin layer chromatography (TLC) was performed on Merck precoated silica gel plates. Visualization was accomplished with UV light or by staining with
KMnO4 solution. Compounds were purified by flash chromatography on columns using Merck silica gel 60 (230–400 mesh) unless
otherwise specified. The purity of the compounds were determined using analytical HPLC with a Phenomenex Kinetex 2.6 mm
C18 100 Š (150 ” 4.60 mm) column at 254 nm. All compounds were
> 95 % pure. Mass spectra were recorded on an Applied Biosystems MDS SCIEX API 2000 mass spectrometer. High-resolution
mass spectra (HRMS) were recorded on an Agilent mass spectrometer using electrospray ionization–time of flight (ESI-TOF). Analytical LC–MS was performed on a Phenomenex Luna 3 mm C18 100 Š
(50 ” 3.0 mm) column. Melting points (mp) were recorded on a Gallenkamp melting point apparatus and are uncorrected. NMR spectra were recorded at 400 MHz for 1H and at 100 MHz for 13C on
a Bruker spectrometer with CDCl3 or [D6]DMSO as solvent. The

chemical shifts are given in ppm, using the proton solvent residue
signal (CDCl3 : d = 7.26; [D6]DMSO: d = 2.50) as a reference in the
1
H NMR spectrum. The deuterium coupled signal of the solvent
was used as reference in 13C NMR (CDCl3 : d = 77.0; [D6]DMSO: d =
39.5). The following abbreviations were used to describe the signals: s = singlets, d = doublet, t = triplet, m = multiplet, br = broad
signal.

(Z)-5-Acetyl-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)indolin-2one (5 b): Following the general procedure, a solution of 5-acetyl2-oxindole (100 mg, 0.57 mmol) and 3,5-dimethyl-2-carboxaldehyde (4 a; 84 mg, 0.68 mmol), and piperidine (3 drops) in EtOH
(7 mL) was stirred at 75 8C for 4 h to give 5-acetyl derivative 5 b as
a brown solid (84.8 mg, 53 %): mp: 297–298 8C (decomposed);
1
H NMR (400 MHz, [D6]DMSO): d = 13.33 (s, 1 H), 11.14 (br s, 1 H),
8.35 (d, J = 1.6 Hz, 1 H), 7.75–7.77 (m, 2 H), 6.96 (d, J = 8.4 Hz, 1 H),
6.05 (d, J = 2 Hz, 1 H), 2.59 (s, 3 H), 2.36 (s, 3 H), 2.34 ppm (s, 3 H);
13
C NMR (100 MHz, [D6]DMSO): d = 196.9, 169.8, 141.8, 136.7, 133.0,
130.5, 126.9, 126.6, 125.9, 124.7, 118.5, 113.0, 111.3, 108.8, 26.6,
13.5, 11.4 ppm; HRMS (ESI-TOF): (m/z) calcd for C17H16N2O2 [M + H] +
281.1290, found 281.1287.
(Z)-3-((3,5-Dimethyl-1H-pyrrol-2-yl)methylene)-5-nitroindolin-2one (5 c): Following the general procedure, a solution of 5-nitro-2oxindole (96 mg, 0.54 mmol) and 3,5-dimethyl-2-carboxaldehyde
(4 a; 80 mg, 0.65 mmol), and piperidine (3 drops) in EtOH (8 mL)
was stirred at 75 8C for 4 h. The resulting precipitate was filtered
and washed with cold EtOH to give 5-nitro 5 c as a brown solid
(114.2 mg, 75 %): mp: > 300 8C (lit.[25] > 280 8C); 1H NMR (400 MHz,
[D6]DMSO): d = 13.35 (s, 1 H), 11.43 (s, 1 H), 8.78 (d, J = 2.4 Hz, 1 H),
8.03 (dd, J = 8.4, 2.4 Hz, 1 H), 7.95 (s, 1 H), 7.04 (d, J = 8.4 Hz, 1 H),
6.10 (d, J = 2.0 Hz, 1 H), 2.38 (s, 3 H), 2.36 ppm (s, 3 H); 13C NMR
(100 MHz, [D6]DMSO): d = 169.8, 142.9, 142.0, 138.3, 134.9, 127.2,
126.8, 126.4, 121.5, 113.8, 113.6, 109.8, 108.9, 13.6, 11.4 ppm; HRMS

(ESI-TOF): (m/z) calcd for C15H13N3O3 [M + H] + 284.1035, found
284.1039.

General procedure for Knoevenagel condensation: Piperidine
(3 drops) was added to a solution of 5-substitiuted-2-oxindole
(1.0 equiv) and 3,5-dimethyl-2-carboxaldehyde (1.2 equiv) in EtOH
(8 mL). The reaction mixture was heated at 75 8C for 4 h. The reaction mixture was then cooled to room temperature, and the resulting precipitate was filtered and washed with cold EtOH to give the
5-substituted-2-oxindole compound.

(Z)-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-methoxyindolin2-one (5 d): Following the general procedure, a solution of 5-methoxy-2-oxindole (75 mg, 0.46 mmol) and 3,5-dimethyl-2-carboxaldehyde (4 a; 68 mg, 0.55 mmol), and piperidine (3 drops) in EtOH
(7 mL) was stirred at 75 8C for 6 h. The residue was purified by
column chromatography (CH2Cl2/MeOH, 99:1) to give 5-methoxy
derivative 5 d as a brown solid (90.1 mg, 73 %): mp: 256–257 8C
(decomposed); 1H NMR (400 MHz, [D6]DMSO): d = 13.44 (s, 1 H),
10.56 (s, 1 H), 7.57 (s, 1 H), 7.39 (d, J = 2.4 Hz, 1 H), 6.75 (d, J =
8.4 Hz, 1 H), 6.67 (dd, J = 8.4, 2.4 Hz, 1 H), 6.00 (d, J = 2.0 Hz, 1 H),
3.77 ppm (s, 3 H); 13C NMR (100 MHz, [D6]DMSO): d = 169.5, 154.7,
135.5, 132.0, 131.6, 126.8, 126.6, 123.6, 113.2, 112.4, 111.9, 109.6,
104.1, 55.6, 13.5, 11.3 ppm. HRMS (ESI-TOF): (m/z) calcd for
C16H16N2O2 [M + H] + 269.1290, found 269.1286.

(3Z)-3-[(3,5-Dimethyl-1H-pyrrol-2-yl)methylidene]-1,3-dihydro2H-indol-2-one ((Z)-1): Following the general procedure, a solution
of 3,5-dimethyl-2-carboxaldehyde (4 a; 111 mg, 0.90 mmol), indolin2-one (100 mg, 0.75 mmol), and piperidine (3 drops) in EtOH (2 mL)
was stirred at 90 8C for 3 h. The resulting precipitate was filtered,
washed with cold EtOH, and dried to give (Z)-1 as an orange solid
in 62 % yield (111 mg): mp: 232–234 8C (lit.[23] 220–222 8C); 1H NMR
(400 MHz, [D6]DMSO): d = 13.35 (br s, 1 H), 10.77 (br s, 1 H), 7.71 (d,
J = 7.2 Hz, 1 H), 7.55 (s, 1 H), 7.09 (m, 1 H), 6.97 (m, 1 H), 6.87 (d, J =
7.6 Hz, 1 H), 6.00 (d, J = 2.4 Hz, 1 H), 2.32 (s, 3 H), 2.30 ppm (s, 3 H);
13

C NMR (100 MHz, [D6]DMSO): d = 169.3, 138.0, 135.5, 131.4, 126.5,
125.7, 125.6, 123.3, 120.7, 117.9, 112.6, 112.4, 109.1, 13.4, 11.2 ppm;
HRMS (ESI-TOF): (m/z) calcd for C15H14N2O [M + H] + 239.1184, found
239.1182.

(Z)-N-(3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-2-oxoindolin-5yl)acetamide (5 e): Following the general procedure, a solution of
5-acetamide-2-oxindole (64 mg, 0.34 mmol) and 3,5-dimethyl-2-carboxaldehyde (4 a; 50 mg, 0.41 mmol), and piperidine (3 drops) in
EtOH (5 mL) was stirred at 75 8C for 4 h. The residue was purified
by column chromatography (CH2Cl2/MeOH, 99:1) to give 5-acetamide derivative 5 e as a yellow solid (70.8 mg, 71 %): mp: > 350 8C
(decomposed); 1H NMR (400 MHz, [D6]DMSO): d = 13.36 (s, 1 H),
10.70 (s, 1 H), 9.75 (s, 1 H), 7.77 (d, J = 2.0 Hz, 1 H), 7.36 (s, 1 H), 7.22
(dd, J = 8.4, 2.0 Hz, 1 H), 6.79 (d, J = 8.4 Hz, 1 H), 6.01 (d, J = 2.0 Hz,
1 H), 2.32 (s, 3 H), 2.28 (s, 3 H), 2.02 ppm (s, 3 H); 13C NMR (100 MHz,

(Z)-3-((3,5-Dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoroindolin-2one ((Z)-5 a): Following the general procedure, a solution of 3,5-dimethyl-2-carboxaldehyde (4 a; 50 mg, 0.41 mmol), 5-fluoroindolin2-one (61 mg, 0.41 mmol), and piperidine (3 drops) in EtOH (3 mL)
was stirred at 75 8C for 12 h. Purification by silica gel column chroChemMedChem 2016, 11, 72 – 80

www.chemmedchem.org

77

 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


Full Papers
[D6]DMSO): d = 169.5, 167.7, 135.7, 134.2, 133.1, 131.5, 126.4, 125.7,
122.8, 118.0, 112.8, 112.5, 110.1, 109.1, 23.7, 13.5, 11.2 ppm; HRMS
(ESI-TOF): (m/z) calcd for C17H17N3O2 [M + H] + 296.1399, found
296.1404.

(Z)-3-((1H-pyrrol-2-yl)methylene)indolin-2-one ((Z)-8): Following
the general procedure, a solution of 1H-pyrrole-2-carbaldehyde
(86 mg, 0.9 mmol), indolin-2-one (100 mg, 0.75 mmol), and piperidine (3 drops) in EtOH (2 mL) was stirred at 90 8C for 3 h. The resulting precipitate was filtered, washed with cold EtOH, and dried
to give (Z)-8 as an orange solid in 66 % yield (104 mg): mp: 234–
236 8C (lit.[26] 210–213 8C) ; 1H NMR (400 MHz, [D6]DMSO): d = 13.34
(br s, 1 H), 10.88 (br s, 1 H), 7.74 (s, 1 H), 7.63 (d, J = 7.2 Hz, 1 H), 7.35
(br s, 1 H), 7.14 (td, J = 7.6, 1.2 Hz, 1 H), 7.00 (m, 1 H), 6.88 (d, J =
7.6 Hz, 1 H), 6.83 (m, 1 H), 6.36–6.34 ppm (m, 1 H); 13C NMR
(100 MHz, [D6]DMSO): d = 169.1, 138.9, 129.5, 126.8, 126.2, 125.5,
125.1, 121.1, 120.1, 118.4, 116.7, 111.3, 109.4 ppm; HRMS (ESI-TOF):
(m/z) calcd for C13H10N2O [M + H] + 211.0871, found 211.0873.

(Z)-3-((3,5-Dimethyl-1H-pyrrol-2-yl)methylene)-5-hydroxyindolin2-one (5 f): Following the general procedure, a solution of 5-hydroxy-2-oxindole (34 mg, 0.23 mmol) and 3,5-dimethyl-2-carboxaldehyde (4 a; 33 mg, 0.27 mmol), and piperidine (3 drops) in EtOH
(5 mL) was stirred at 75 8C for 16 h. The residue was purified by
column chromatography (CH2Cl2/MeOH, 97:3 to 95:5) to give 5-hydroxy derivative 5 f as an orange solid (33.2 mg, 57 %): 1H NMR
(400 MHz, [D6]DMSO) d13.41 (s, 1 H), 10.46 (s, 1 H), 7.40 (s, 1 H), 7.09
(d, J = 2.4 Hz, 1 H), 6.65 (d, J = 8.4 Hz, 1 H), 6.53 (dd, J = 8.4, 2.4 Hz,
1 H), 5.98 (d, J = 2.4 Hz, 1 H), 2.30 (s, 3 H), 2.28 ppm (s, 3 H); 13C NMR
(100 MHz, [D6]DMSO): d = 169.4, 152.2, 135.2, 131.1, 130.9, 126.8,
126.4, 122.9, 113.5, 112.7, 112.3, 109.6, 105.3, 13.5, 11.2 ppm; HRMS
(ESI-TOF): (m/z) calcd for C15H14N2O2 [M + H] + 255.1134, found
255.1132.

General procedure for the acylation reaction for the synthesis of
6 a, 7 a, 6 c, and 7 c: A mixture of (Z)-1 or (E)-5 h (1.0 equiv), DMAP
(0.15 equiv), and (Boc)2O or Ac2O (1.2 equiv) with triethylamine
(1.2 equiv) in CH2Cl2 was stirred under nitrogen at room temperature. The reaction was monitored using TLC until no (Z)-1 or (E)-5 h
could be detected. The solvent was evaporated, and the mixture
was purified using column chromatography (petroleum ether/
EtOAc, 4:1), unless otherwise specified. If a mixture of E and

Z isomers was obtained, the analytical data reported correspond to
the major isomer. The minor isomer is not reported.

(Z)-5-Amino-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)indolin-2one (5 g): 10 % Pd/C (18 mg, 0.017 mmol Pd) was added to a suspension of 5-nitro compound 5 c (60 mg, 0.21 mmol) in EtOH
(2 mL). The reaction mixture was stirred under hydrogen atmosphere overnight. The reaction mixture was then filtered through
a pad of Celite. The residue was purified by column chromatography (CH2Cl2/MeOH, 1:0 to 99:1) to give 5-amino derivative 5 g as
an orange solid (17.8 mg, 34 %): mp: 249–251 8C (decomposed);
1
H NMR (400 MHz, [D6]DMSO): d = 13.39 (s, 1 H), 10.34 (s, 1 H), 7.29
(s, 1 H), 6.89 (s, 1 H), 6.56 (d, J = 8.0 Hz, 1 H), 6.38 (dd, J = 8.0, 1.6 Hz,
1 H), 5.96 (s, 1 H), 4.59 (br s, 2 H), 2.30 (s, 3 H), 2.26 ppm (s, 3 H);
13
C NMR (100 MHz, [D6]DMSO): d = 169.2, 143.1, 134.7, 130.3, 129.4,
126.3, 122.0, 114.1, 112.3, 112.1, 109.6, 104.3, 13.4, 11.2 ppm; HRMS
(ESI-TOF): (m/z) calcd for C15H15N3O [M + H] + 254.1293, found
254.1292.

(Z)-1-Acetyl-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)indolin-2one ((Z)-6 a): Following the general procedure, a mixture of (Z)1 (200 mg, 0.84 mmol), DMAP (15 mg, 0.13 mmol), triethylamine
(102 mg, 1.01 mmol), and Ac2O (121 mg, 1.01 mmol) in CH2Cl2
(6 mL) was stirred under nitrogen at room temperature. Purification by silica gel column chromatography (petroleum ether/EtOAc,
4:1) afforded (Z)-6 a as an orange solid in 78 % yield (184 mg): mp:
196–198 8C ;1H NMR (400 MHz, CDCl3): d = 12.61 (br s, 1 H), 8.25 (m,
1 H), 7.49 (m, 1 H), 7.40 (s, 1 H), 7.20 (m, 2 H), 6.04 (s, 1 H), 2.80 (s,
3 H), 2.42 (s, 3 H), 2.35 ppm (s, 3 H); 13C NMR (100 MHz, CDCl3): d =
171.4, 168.8, 138.1, 136.2, 134.5, 127.3, 126.4, 126.1, 124.4, 124.0,
116.3, 116.3, 113.5, 110.1, 27.1, 14.1, 11.7 ppm; HRMS (ESI-TOF): (m/
z) calcd for C17H16N2O2 [M + H] + 281.1290, found 281.1293.

(E)-3-((1,3,5-Trimethyl-1H-pyrrol-2-yl)methylene)indolin-2-one
((E)-5 h): Following the general procedure, a solution of 4 b

(200 mg, 1.46 mmol), indolin-2-one (162 mg, 1.21 mmol), and piperidine (3 drops) in EtOH (5 mL) was stirred at 75 8C for 24 h. Purification by silica gel column chromatography (petroleum ether/
EtOAc, 4:1!100% EtOAc) afforded (E)-5 h as an orange solid in
46 % yield (140 mg): mp: 170–172 8C; 1H NMR (400 MHz, CDCl3):
d = 7.67 (s, 1 H), 7.64 (s, 1 H), 7.17 (m, 2 H), 6.95 (dt, J = 4.0, 8.0 Hz,
1 H), 6.87 (d, J = 8.0 Hz, 1 H), 5.94 (s, 1 H), 3.48 (s, 3 H), 2.29 (s, 3 H),
1.97 ppm (s, 3 H); 13C NMR (100 MHz, CDCl3): d = 170.1, 140.2, 135.3,
128.1, 126.8, 125.5, 125.3, 124.7, 123.0, 122.4, 121.7, 111.0, 109.3,
31.8, 13.8, 12.6 ppm; HRMS (ESI-TOF): (m/z) calcd for C16H16N2O
[M + H] + 253.1341, found 253.1342.

(Z)-tert-Butyl 3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-2-oxoindoline-1 carboxylate ((Z)-6c): Following the general procedure,
a mixture of (Z)-1 (50 mg, 0.21 mmol), DMAP (4 mg, 0.03 mmol),
and (Boc)2O (37 mg, 0.17 mmol) in CH2Cl2 (4 mL) was stirred under
nitrogen at room temperature. Purification by silica gel column
chromatography (petroleum ether/EtOAc, 4:1) afforded (Z)-6 c as
an orange solid in 90 % yield (51.8 mg): 1H NMR (400 MHz, CDCl3):
d = 12.82 (br s, 1 H), 7.73 (m, 1 H), 7.49 (m, 1 H), 7.39 (s, 1 H), 7.17 (m,
2 H), 6.02 (s, 1 H), 2.37 (s, 3 H), 2.34 (s, 3 H), 1.70 ppm (s, 9 H);
13
C NMR (100 MHz, CDCl3): d = 167.9, 149.4, 138.1, 135.6, 134.1,
127.3, 126.0, 125.7, 123.7, 123.6, 116.6, 114.7, 113.3, 110.0, 84.2,
28.2, 14.0, 11.7 ppm; HRMS (ESI-TOF): (m/z) calcd for C20H22N2O3
[M + Na] + 361.1528, found 361.1523.

(E)-5-Fluoro-3-((1,3,5-trimethyl-1H-pyrrol-2-yl)methylene)indolin2-one ((E)-5i): Following the general procedure, a solution of 4 b
(157 mg, 1.14 mmol), 5-fluoroindolin-2-one (208 mg, 1.37 mmol),
and piperidine (3 drops) in EtOH (5 mL) was stirred at 75 8C for
12 h. Purification by silica gel column chromatography (petroleum
ether/EtOAc, 4:1) afforded 5 i as an E/Z mixture ((E)-5 i:(Z)-5 i = 97:3)
as an orange solid in 15 % (46.2 mg) combined yield: 1H NMR

E isomer (400 MHz, CDCl3): d = 8.52 (s, 1 H), 7.68 (s, 1 H), 6.86 (m,
3 H), 5.97 (s, 1 H), 3.49 (s, 3 H), 2.29 (s, 3 H), 1.98 ppm (s, 3 H);
13
C NMR E isomer (100 MHz, CDCl3): d = 170.2, 159.9, 157.6, 136.2,
136.1, 126.7, 126.7, 126.2, 126.2, 114.3, 114.0, 110.2, 109.9, 109.5,
109.4, 31.8, 13.9, 12.7 ppm (number of carbon signals was greater
than expected due to F coupling); HRMS (ESI-TOF): (m/z) calcd for
C16H15FN2O [M + H] + 271.1247, found 271.1239.
ChemMedChem 2016, 11, 72 – 80

www.chemmedchem.org

(E)-1-Acetyl-3-((1,3,5-trimethyl-1H-pyrrol-2-yl)methylene)indolin2-one ((E)-7 a): Following the general procedure, a mixture of (E)5 h (100 mg, 0.40 mmol), DMAP (7 mg, 0.06 mmol), triethylamine
(61 mg, 0.60 mmol), and Ac2O (72 mg, 0.60 mmol) in CH2Cl2 (6 mL)
was stirred under nitrogen at room temperature. Purification by
silica gel column chromatography (petroleum ether/EtOAc, 4:1) afforded 7 a as an E/Z mixture ((E)-7 a:(Z)-7 a = 84:16) as an orange
solid in 58 % (68.2 mg) combined yield: 1H NMR E isomer (400 MHz,
[D6]DMSO): d = 8.18 (d, J = 8 Hz, 1 H), 7.65 (s, 1 H), 7.31 (m, 1 H), 7.17
(m, 2 H), 6.00 (s, 1 H), 3.48 (s, 3 H), 2.66 (s, 3 H), 2.28 (s, 3 H),
1.87 ppm (s, 3 H); 13C NMR E isomer (100 MHz, [D6]DMSO): d =
170.5, 167.7, 138.5, 137.1, 128.1, 126.4, 126.2, 125.7, 124.3, 122.9,

78

 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


Full Papers
121.3, 118.8, 115.4, 111.5, 31.6, 26.4, 13.9, 12.3 ppm; HRMS (ESITOF): (m/z) calcd for C18H18N2O2 [M + H] + 295.1447, found
295.1439.

Isomerization study: Photoisomerization experiments were performed with 10 mm [D6]DMSO solutions of 3-substituted indoline2-one derivatives in NMR tubes. The NMR tubes were then exposed to fluorescent light (Philips Tornado 5W ES 6500 K cool daylight, 285 lumens) at ambient temperature (22–24 8C), with a light
intensity of ~ 1700 lux (measured with a Gossen Luna-Pro F light
meter). The samples were analyzed by 1H NMR spectroscopy at various time points (0.25, 0.5, 1, 2, 3, 4, 5, 6, 12, 24 h). For the analysis
of isomerization in the dark, after the samples were exposed to
light for 24 h, the samples were then protected from light. At various time points (0.5, 1, 2, 3, 4, 5, 6, 12, 24, 48, 72 h), samples were
analyzed by 1H NMR spectroscopy.

(E)-tert-Butyl
2-oxo-3-((1,3,5-trimethyl-1H-pyrrol-2-yl)methylene)indoline-1-carboxylate ((E)-7 c): Following the general procedure, a mixture of (E)-4 a (50 mg, 0.20 mmol), DMAP (4 mg,
0.03 mmol), and (Boc)2O (52 mg, 0.24 mmol) in CH2Cl2 (5 mL) was
stirred under nitrogen at room temperature. Purification by silica
gel column chromatography (petroleum ether/EtOAc, 4:1) afforded
7 c as an E/Z mixture ((E)-7 c:(Z)-7 c = 86:14) as an orange oil in
90 % (63.4 mg) combined yield: 1H NMR E isomer (400 MHz, CDCl3):
d = 7.89 (m, 1 H), 7.68 (s, 1 H), 7.24 (m, 2 H), 7.06 (m, 1 H, H-7), 5.97
(s, 1 H), 3.46 (s, 3 H), 2.28 (s, 3 H), 1.94 (s, 3 H), 1.67 ppm (s, 9 H);
13
C NMR E isomer (100 MHz, [D6]DMSO): d = 165.5, 148.8, 138.0,
136.6, 128.2, 126.3, 125.8, 125.3, 123.6, 122.3, 121.5, 119.0, 114.0,
111.3, 83.3, 31.5, 27.7, 13.9, 12.3 ppm; HRMS (ESI-TOF): (m/z) calcd
for C21H24N2O3 [M + Na] + 375.1685, found 375.1678.

Isomerization of 100 mm compound stocks: The 100 mm drug
stocks of (Z)-1, (Z)-5 a, (Z)-2, and (Z)-10 were exposed to fluorescent lighting. At the end of 24 h, the E/Z ratios were determined
using NMR spectroscopy by diluting 50 mL of the stock solutions to
500 mL using [D6]DMSO.

General procedure for the alkylation reaction for the synthesis
of 6 b and 7 b: (Z)-1 (1.0 equiv) in DMF or THF (2 mL) was added to

a stirring suspension of NaH (1.0 equiv for the synthesis of 6 b;
2.2 equiv for 7 b) under nitrogen. After 1 h, iodomethane (1.0 equiv
for the synthesis of 6 b; 2.2 equiv for 7 b) was added. The reaction
was monitored using TLC until no (Z)-1 could be detected. Upon
completion, 0.5 mL of saturated ammonium chloride was added.
The mixture was extracted with EtOAc (3 ” 10 mL). The combined
organic layers were washed with brine and dried with Na2SO4. The
solvent was evaporated, and the mixture was purified using
column chromatography (petroleum ether/EtOAc, 4:1), unless otherwise specified. If a mixture of E and Z isomers was obtained, the
analytical data correspond to the major isomer. The minor isomer
is not reported.

Biology
Reagents and general procedures: Gentamicin and the soybean
trypsin inhibitor were from Invitrogen. Phosphate-buffered saline
(PBS) was purchased from Bio-Rad, and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazoliumbromide (MTT) dye was purchased from
Duchefa. Drug stocks (10 mm and 100 mm) were prepared in
DMSO and were stored at À20 8C. TAMH cells were maintained in
a T75 flask with Dulbecco’s modified Eagle’s medium (DMEM):Nutrient Mixture F-12 (DMEM/F-12) supplemented with ITS (5 mg mLÀ1
insulin, 5 mg mLÀ1 transferrin, 5 ng mLÀ1 selenium), 10 mm nicotinamide, 100 nm dexamethasone, and 10 mg mLÀ1 gentamicin, while
HepG2 was maintained in minimal essential medium in the presence of 10 % fetal bovine serum. Both lines were incubated at
37 8C in 95 % air and 5 % CO2.

(Z)-3-((3,5-Dimethyl-1H-pyrrol-2-yl)methylene)-1-methylindolin2-one ((Z)-6b): Following the general procedure, (Z)-1 (20 mg,
0.08 mmol) in DMF (2 mL) was added to a stirring suspension of
NaH (3.5 mg, 0.09 mmol) under nitrogen. After 1 h, iodomethane
(13 mg, 0.09 mmol) was added. The mixture was quenched and extracted. Purification by silica gel column chromatography (petroleum ether/EtOAc, 4:1) afforded (Z)-6 b as an orange solid in 53 %
yield (10.7 mg): mp: 162–164 8C; 1H NMR (400 MHz, CDCl3): d =
13.25 (br s, 1 H), 7.51 (d, J = 8.0 Hz, 1 H), 7.40 (s, 1 H), 7.19 (dt, J =
1.2, 8.0 Hz, 1 H), 7.08 (dt, J = 1.2, 8.0 Hz, 1 H), 6.88 (d, J = 8.0 H, 1 H),

5.97 (s, 1 H), 3.38 (s, 3 H), 2.38 (s, 3 H), 2.33 ppm (s, 3 H); 13C NMR
(100 MHz, CDCl3): d = 168.4, 139.7, 136.5, 132.1, 127.0, 125.6, 125.5,
123.1, 121.6, 117.0, 112.5, 111.9, 107.8, 26.1, 13.9, 11.6 ppm; HRMS
(ESI-TOF): (m/z) calcd for C16H16N2O [M + H] + 253.1341, found
253.1343.

MTT cell proliferation assay: The TAMH cells were trypsinized to produce a single-cell suspension and were resuspended using
0.5 mg mLÀ1 of soybean trypsin inhibitor. After centrifugation, the
cell pellet was resuspended using the DMEM/F-12 media. After
counting the cells using a hemocytometer, the cell suspension was
diluted to provide the desired density of 15 000 cells per well and
then seeded into 96-well plates, where the cells were allowed to
attach for 24 h. The spent media was removed. Drug stocks were
diluted appropriately using DMEM/F-12 medium immediately
before each assay. Stocks (200 mL) were added to each well and
were incubated for 24 h. Cell viability was determined by reduction
in MTT by viable cell dehydrogenases. MTT was added to give
a final concentration of 400 mg mLÀ1 in each well, and the plates
were incubated at 37 8C for 3 h before aspirating the supernatant
and solubilizing the insoluble formazan product using 100 mL
DMSO. Absorbance at 570 nm was measured using an Infinite 200
microplate reader (Tecan). Cell viability in percentage was plotted
against the concentrations of the drug. IC50 values were determined using GraphPad Prism 6 Software.

(E)-1-methyl-3-((1,3,5-trimethyl-1H-pyrrol-2-yl)methylene)indolin-2-one ((E)-7b): Following the general procedure, (Z)-1 (50 mg,
0.21 mmol) in THF (2 mL) was added to a stirring suspension of
NaH (20 mg, 0.48 mmol) under nitrogen. After 1 h, iodomethane
(69 mg, 0.48 mmol) was added. The mixture was quenched and extracted. Purification by silica gel column chromatography (petroleum ether/EtOAc, 4:1) afforded 7 b as an E/Z mixture ((E)-7 b:(Z)7 b = 89:11) as an orange oil in 85 % (47.5 mg) combined yield:
1
H NMR (E)-isomer (400 MHz, CDCl3): d = 7.65 (s, 1 H), 7.21 (m, 2 H),

6.97 (m, 1 H), 6.83 (d, J = 8.0 Hz, 1 H), 5.93 (s, 1 H), 3.47 (s, 3 H), 3.31
(s, 3 H), 2.28 (s, 3 H), 1.96 ppm (s, 3 H); 13C NMR (E)-isomer (100 MHz,
CDCl3): d = 168.9, 143.2, 134.9, 128.1, 126.8, 125.2, 124.8, 122.7,
122.2, 121.6, 110.8, 107.5, 31.7, 26.1, 13.8, 12.6 ppm; HRMS (ESITOF): (m/z) calcd for C17H18N2O [M + H] + 267.1497, found 267.1496.
ChemMedChem 2016, 11, 72 – 80

www.chemmedchem.org

Acknowledgements
The authors thank Ms. David P. Sheela (National University of
Singapore) for her assistance with some of the biological assays.
This work was supported by a National University of Singapore
(NUS) start-up grant to C.L.L.C. (R148000146133) and the A*STAR
79

 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


Full Papers
Computational Resource Centre (for M.B.S.) for the use of its
high-performance computing facilities.
Keywords: indolin-2-ones · computational
cytotoxicity · kinetics · photoisomerization

chemistry

[16] T. C. Crawford (Pfizer Inc., New York), US Pat. No. US4652658, 1987.
[17] J. Caballero, C. Munoz, J. H. Alzate-Morales, S. Cunha, L. Gano, R. Bergmann, J. Steinbach, T. Kniess, Eur. J. Med. Chem. 2012, 58, 272 – 280.
[18] M. H. Kim, A. L. Tsuhako, E. W. Co, D. T. Aftab, F. Bentzien, J. Chen, W.
Cheng, S. Engst, L. Goon, R. R. Klein, D. T. Le, M. Mac, J. J. Parks, F. Qian,

M. Rodriquez, T. J. Stout, J. H. Till, K. A. Won, X. Wu, F. M. Yakes, P. W. Yu,
W. T. Zhang, Y. P. Zhao, P. Lamb, J. M. Nuss, W. Xu, Bioorg. Med. Chem.
Lett. 2012, 22, 4979 – 4985.
[19] J. Spencer (University of Greenwich), Int. PCT Pub. No.
WO 2012025726 A1, 2012.
[20] J. Tomasi, B. Mennucci, R. Cammi, Chem. Rev. 2005, 105, 2999 – 3093.
[21] Gaussian 09, Rev. D.01: M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci,
G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K.
Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O.
Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro,
M. J. Bearpark, J. Heyd, E. N. Brothers, K. N. Kudin, V. N. Staroverov, R.
Kobayashi, J. Normand, K. Raghavachari, A. P. Rendell, J. C. Burant, S. S.
Iyengar, J. Tomasi, M. Cossi, N. Rega, N. J. Millam, M. Klene, J. E. Knox,
J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L.
Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, Ö. Farkas, J. B. Foresman, J. V. Ortiz,
J. Cioslowski, D. J. Fox, Gaussian Inc., Wallingford, CT (USA), 2009.
[22] a) J. C. Wu, G. Merlino, K. Cveklova, B. Mosinger, Jr., N. Fausto, Cancer
Res. 1994, 54, 5964 – 5973; b) R. H. Pierce, C. C. Franklin, J. S. Campbell,
R. P. Tonge, W. Chen, N. Fausto, S. D. Nelson, S. A. Bruschi, Biochem.
Pharmacol. 2002, 64, 413 – 424.
[23] J. Lubkoll, A. Millemaggi, A. Perry, R. J. K. Taylor, Tetrahedron 2010, 66,
6606 – 6612.
[24] T. Kniess, R. Bergmann, M. Kuchar, J. Steinbach, F. Wuest, Bioorg. Med.
Chem. 2009, 17, 7732 – 7742.
[25] T. Kniess, M. Kuchar, F. Wuest, Synth. Commun. 2008, 38, 3017 – 3022.
[26] J. Spencer, B. Z. Chowdhry, S. Hamid, A. P. Mendham, L. Male, S. J. Coles,
M. B. Hursthouse, Acta Crystallogr. Sect. C 2010, 66, o71 – o78.

·

[1] A. Millemaggi, R. J. K. Taylor, Eur. J. Org. Chem. 2010, 4527 – 4547.
[2] T. A. T. Fong, L. K. Shawver, L. Sun, C. Tang, H. App, T. J. Powell, Y. H. Kim,
R. Schreck, X. Y. Wang, W. Risau, A. Ullrich, K. P. Hirth, G. McMahon,
Cancer Res. 1999, 59, 99 – 106.
[3] R. M. Shaheen, D. W. Davis, W. Liu, B. K. Zebrowski, M. R. Wilson, C. D.
Bucana, D. J. McConkey, G. McMahon, L. M. Ellis, Cancer Res. 1999, 59,
5412 – 5416.
[4] L. Sun, N. Tran, F. Tang, H. App, P. Hirth, G. McMahon, C. Tang, J. Med.
Chem. 1998, 41, 2588 – 2603.
[5] R. Longo, R. Sarmiento, M. Fanelli, B. Capaccetti, D. Gattuso, G. Gasparini, Angiogenesis 2002, 5, 237 – 256.
[6] B. C. Kuenen, L. Rosen, E. F. Smit, M. R. N. Parson, M. Levi, R. Ruijter, H.
Huisman, M. A. Kedde, P. Noordhuis, W. J. F. van der Vijgh, G. J. Peters,
G. F. Cropp, P. Scigalla, K. Hoekman, H. M. Pinedo, G. Giaccone, J. Clin.
Oncol. 2002, 20, 1657 – 1667.
[7] K. Lv, L. L. Wang, M. L. Liu, X. B. Zhou, S. Y. Fan, H. Y. Liu, Z. B. Zheng, S.
Li, Bioorg. Med. Chem. Lett. 2011, 21, 3062 – 3065.
[8] L. Sun, C. Liang, S. Shirazian, Y. Zhou, T. Miller, J. Cui, J. Y. Fukuda, J.-Y.
Chu, A. Nematalla, X. Wang, H. Chen, A. Sistla, T. C. Luu, F. Tang, J. Wei,
C. Tang, J. Med. Chem. 2003, 46, 1116 – 1119.
[9] M. Atkins, C. A. Jones, P. Kirkpatrick, Nat. Rev. Drug Discovery 2006, 5,
279 – 280.
[10] S. Faivre, G. Demetri, W. Sargent, E. Raymond, Nat. Rev. Drug Discovery
2007, 6, 734 – 745.
[11] A. Sistla, N. Shenoy, Drug Dev. Ind. Pharm. 2005, 31, 1001 – 1007.
[12] a) Y. Zhao, J. Sukbuntherng, J. Pharm. Biomed. Anal. 2005, 38, 479 – 486;
b) Y. Zhao, J. Sukbuntherng, L. Antonian, J. Pharm. Biomed. Anal. 2004,
35, 513 – 522.
[13] A. Sistla, W. L. Yang, N. Shenoy, J. Chromatogr. A 2006, 1110, 73 – 80.
[14] M. C. Etienne-Grimaldi, N. Renee, H. Izzedine, G. Milano, J. Chromatogr.
B 2009, 877, 3757 – 3761.

[15] a) M. Maafi, L. Y. Lee, J. Pharm. Biomed. Anal. 2015, 110, 34 – 41; b) S. K.
Dewan, S. Sangwan, R. Thapar, M. Prasad, J. Appl. Chem. 2014, 3, 1055 –
1058.

ChemMedChem 2016, 11, 72 – 80

www.chemmedchem.org

Received: October 14, 2015
Published online on November 23, 2015

80

 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim