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

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

Photoinduced Isomerization and Hepatoxicities of

Semaxanib, Sunitinib and Related 3-Substituted Indolin-2-ones

Mun Hong Ngai,[a] Choon Leng So,[a] Michael B Sullivan,[b] Han Kiat Ho,[a] and

Christina L L Chai*[a]

Introduction

The 3-substituted indolin-2-ones (oxindoles) are an important

class of compounds that have been well-explored for their

bio-logical 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

com-pounds 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

and negative results in phase II/III studies.[5–7]Subsequent stud-ies 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 ad-vanced renal cell cancer and imatinib-resistant or imatinib-in-tolerant 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 phar-maceutical implications of this isomerization process, for exam-ple, the stabilities of the administered drug, have been recog-nized for both semaxanib and sunitinib, leading to the devel-opment of analytical methods for detection of the iso-mers.[11–14] However, not much has been reported with regard

to the photoisomerization process of semaxanib,[11–13] suniti-nib,[15]and related 3-pyrrolylmethylidene oxindoles, or whether the stereoisomers display differential biological activities In the latter context, we were specifically interested in determin-ing whether the E and Z isomers display different toxicities that may lead to undesired side effects later in the drug devel-opment process This is especially relevant in view of the toxic-ity 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

3-Substituted indolin-2-ones are an important class of

com-pounds that display a wide range of biological activities

Suniti-nib 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

re-lated 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

1H NMR spectroscopy The rate constants for

were tested for cytotoxicity in the TAMH liver cell line E/Z mix-tures of four compounds were also assessed for toxicity in the TAMH and HepG2 cell lines In some cases, the

stereochemical-ly pure drug was more toxic than the E/Z mixtures, but a

gener-al statement cannot be made Our studies show that each ste-reoisomer could contribute differently to toxicity, suggesting that stereochemical purity issues that could arise from isomeri-zation cannot be ignored

[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: phacllc@nus.edu.sg

[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

to recapitulate in vivo bioactivation events, and in another

liver cell line, HepG2, to validate our findings

Results and Discussion

Synthesis

The 3-substituted indolin-2-ones were prepared following

re-ported procedures using a Knoevenagel condensation

be-tween substituted indoline-2-ones and aldehydes in the

pres-ence of piperidine in ethanol (Scheme 1).[4]Oxindole, 5-fluoro-,

and 5-nitrooxindoles were commercially available

5-Acetylox-indole was prepared via Friedel–Crafts acetylation of

oxin-dole.[16]Oxidation of oxindole with

phenyliodine(III)-bis(trifluor-oacetate) (PIFA) gave 5-hydroxyoxindole, which was

subse-quently methylated to give 5-methoxyoxindole.[17]

5-Acetami-doxindole was synthesized from 5-nitrooxindole in two steps,

according to a literature procedure.[18]For all of the

3-substitut-ed indoline-2-ones with R1=H (1, 5a–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(5h–i) were

iso-lated exclusively as the E isomer (i.e., 5h) or as mixtures of

both the Z and E isomers (i.e., 5i; Table 1) The 5-amino

indoli-none 5g was synthesized by reduction of 5-nitro indoliindoli-none

5c (Scheme 2).[19]

N1-substituted compounds 6a–c were synthesized by

acyla-tion or alkylaacyla-tion of semaxanib Acylaacyla-tion and alkylaacyla-tion of

semaxanib occurred exclusively at the nitrogen atom of the

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

ratios of the stereoisomers for 7a–c were determined by

1H 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 condens-ing indoline-2-one with pyrrole-2-carboxaldehyde under stan-dard Knoevenagel conditions.[4]

Scheme 1 Synthesis of 3-substituted indolin-2-ones Reagents and

condi-tions: a) piperidine, EtOH, 758C

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

Scheme 2 Synthesis of 5-amino indolinone 5g Reagents and conditions:

a) Pd/C (10 %), H2, EtOH, RT, 16 h, 34%

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

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

spectrosco-py, 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 their1H 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

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-hy-droxy compound 5 f and 5-amino compound 5g, 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-2-ones were investigated Figure 1 shows the formation of the

E isomer of semaxanib in solution after exposure to light at

dif-ferent time intervals Prior to light exposure, only

(Z)-semaxa-nib 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

isomeri-zation of (Z)-semaxanib to (E)-semaxanib was determined to

be 0.048 h¢1 (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

room temperature, and1H NMR spectra were recorded at dif-ferent 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 h¢1

In a similar manner, the rate constants of Z!E or E!Z pho-toisomerization and reversion of semaxanib and related 3-sub-stituted 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 5g) to 0.09 h¢1 With the exception of 5h, the rate constants measure Z!E photoisomerization For those with measurable reversion, the rate constants ranged from 0.001 to 0.061 h¢1 Thus there

is little discernible correlation between the rate constants for isomerization and reversion with the nature of the substitu-ents

Theoretical studies

We attempted to rationalize the observed ease of photoisome-rization by assessing the HOMO and LUMO energies of com-pounds 5a–h and 6a 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

Table 2 Indolinone Z/E isomer ratios before (t0) and after 24 h light

expo-sure (t24), and chemical shifts (d) of the vinyl protons

t0 t24 Z isomer E isomer

Figure 1 Kinetics of Z!E photoisomerization of semaxanib (1) in [D6]DMSO Data were determined at various time points by1H NMR spectroscopy

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

Compd R1 R2 R3 Rate constant [h¢1][a]

Isomerization Reversion

[a] Concentration of the samples was 10 mm in [D6]DMSO [b] Isomeriza-tion from E to Z

An examination of the oscillator strengths (f) of each pair of

isomers in the UV region of ~400 nm shows that compounds

1, 5a–c, 5e, 5h, and 6a have high f values, in the range of

~415 nm to 430 nm, as compared with compounds 5d, 5 f,

and 5g With the exception of 5d and 5h, 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 5h, E!Z isomerization occurs readily, as is

reflect-ed by the high f value for the E isomer Basreflect-ed solely on our

re-sults, the isomerization of 5d does not fit the trend of the

others The UV/Vis results for 5d 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, 5a–h, and 6a may provide

fur-ther 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=C¢C¢N 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 5h, where both the E and Z isomers have a

dihe-dral angle of ~308, which is the consequence of N-methylation

at the pyrrole ring, which in turn, does not allow for

intramo-lecular hydrogen bonding with the carbonyl group on the

in-dolinone Consistent with experimental observations, calcula-tions show that the Z isomer is the more stable isomer for compounds 1, 5a–g, and 6a, while the E isomer is the more stable isomer for 5h

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 re-produce the toxicity observed with classical hepatotoxicants, such as acetaminophen.[22] The 50% inhibitory concentration value (IC50) was estimated using GraphPad Prism 6 (Table 6)

When comparing the compounds with a hydrogen atom on the N1’ of the pyrrole ring (i.e., 1, 2, 5a, 6a–c, 8), (Z)-8 was found to be the least toxic to TAMH cells (IC50= 21.53 mm) The

IC50value of (Z)-2 was 11.28 mm, which is higher than for indoli-nones 1, 5a, and 6a–c Compounds substituted at the N1

posi-Table 4 Calculated electronic spectra for selected indolinones

Compd l [nm] Oscillator

strength Compd l [nm] Oscillatorstrength

361.1

305.3

0.679 0.230 0.025

358.1 295.6

0.604 0.182 0.026 (Z)-5a 423.9

367.2

307.6

0.625 0.309 0.026

(E)-5a 421.3

365.0 298.5

0.543 0.241 0.028 (Z)-5b 421.6

375.2

348.2

0.749 0.009 0.309

(E)-5b 415.4

376.4 348.5

0.645 0.010 0.159 (Z)-5c 525.1

415.5

356.3

0.022 0.864 0.353

(E)-5c 526.5

412.5 359.8

0.030 0.667 0.237 (Z)-5d 439.0

395.7

304.6

0.232 0.699 0.025

(E)-5d 437.8

385.6 295.0

0.228 0.542 0.026 (Z)-5e 424.9

373.9

307.4

0.606 0.343 0.023

(E)-5e 420.9

369.8 300.9

0.510 0.263 0.052 (Z)-5 f 439.6

390.4

305.2

0.297 0.623 0.025

(E)-5 f 438.3

382.2 295.6

0.285 0.486 0.027 (Z)-5g 476.4

401.0

303.8

0.087 0.825 0.021

(E)-5g 473.8

390.2 297.7

0.122 0.634 0.061 (Z)-5h 427.5

365.0

325.3

0.613 0.191 0.028

(E)-5h 419.3

362.1 317.6

0.570 0.085 0.026 (Z)-6a 430.2

346.4

315.1

0.717 0.086 0.109

(E)-6a 419.2

346.8 324.0

0.704 0.105 0.039

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

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

[a] Values are the meanœSD of three independent experiments

tion of the oxindole ring, such as (Z)-6b (R3= CH3) and (Z)-6c

(R3=Boc), were more toxic (IC50values of 3.76 and 0.69 mm,

re-spectively) (Z)-6a (R3= Ac) was less toxic (IC50= 9.77 mm) than

the unsubstituted (Z)-1 (R3=H; IC50= 6.28 mm) Fluorine

in-creased toxicity relative to compound (Z)-1 In contrast,

com-pounds with substitution at the nitrogen atom of the pyrrole

ring were generally less toxic to the TAMH cell line With the

exception of 7c (IC50= 11.94 mm), all compounds in this series

showed IC50values greater than 50 mm

Overall, the toxicities of the compounds varied Boc

substitu-tion at the N1 posisubstitu-tion of the indolin-2-ones resulted in 6c

and 7c (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

pyrrol-2-yl)methylidenyl]indolin-2-one series

To determine if the E isomers were indeed less toxic, the E/Z

mixtures of 1, 2, 5a, and 8 were also tested against the TAMH

and HepG2 cell lines 8 is the parent compound, while

(Z)-1 (semaxanib) and (Z)-2 (sunitinib) are potent anticancer

agents 5a 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

in-dependently 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 5a, for

which the percentage of (E)-5a was significantly lower

(Table 7)

For 1 and 5a, the E/Z mixtures were more toxic than

(Z)-1 and (Z)-5a, while the reverse was true for 2 and 8 in the

TAMH cell line The differences between the IC50 values

be-tween the pure Z isomers and the E/Z mixtures were not large

for 5a 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 IC50value 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, 5a, 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

mix-ture 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 depen-dent 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 sem-axanib (1) and sunitinib (2) were synthesized in yields of 15%

to 90 % For 5a–g, 6a–c, and 8, only the Z isomers were ob-tained In contrast, only the E isomer of 5h was obtained, while 5i and 7a–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 isomeri-zation 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 via-bility to a greater extent The E/Z mixtures of the four selected compounds that were of clinical importance (1, 2, 5a, 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 IC50values between the pure Z isomers and the E/Z mix-tures for 5a and 2 were not large For the HepG2 cell line,

Z isomers of 1, 5a, and 2 were more toxic than the E/Z mix-tures The differences in the observed trends between the two liver cells lines are indicative of the sensitivities of the

respons-es of the cell linrespons-es 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 find-ings could prompt the appropriate handling of clinically avail-able 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 activi-ties and toxiciactivi-ties Removing such unsaturation and/or

design-Table 7 Z/E isomer ratios of the indolinones and their corresponding IC50values against TAMH and HepG2 cell lines

[a] Values are the meanœSD of three independent experiments

ing stereochemically stable compounds may be feasible,

pro-vided that the activities are not compromised This would

avoid the issue of E/Z isomerization

Experimental Section

General methods: (Z)-2 (Sunitinib malate) was used as purchased

from LC laboratories All other reagents and solvents were

pur-chased 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

chromatogra-phy (TLC) was performed on Merck precoated silica gel plates

Vis-ualization was accomplished with UV light or by staining with

chromatogra-phy on columns using Merck silica gel 60 (230–400 mesh) unless

otherwise specified The purity of the compounds were

deter-mined using analytical HPLC with a Phenomenex Kinetex 2.6 mm

C18 100 æ (150Õ 4.60 mm) column at 254 nm All compounds were

>95% pure Mass spectra were recorded on an Applied

Biosys-tems MDS SCIEX API 2000 mass spectrometer High-resolution

mass spectra (HRMS) were recorded on an Agilent mass

spectrom-eter using electrospray ionization–time of flight (ESI-TOF)

Analyti-cal LC–MS was performed on a Phenomenex Luna 3 mm C18 100 æ

(50Õ3.0 mm) column Melting points (mp) were recorded on a

Gal-lenkamp melting point apparatus and are uncorrected NMR

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

was used as reference in13C NMR (CDCl3: d=77.0; [D6]DMSO: d=

39.5) The following abbreviations were used to describe the

sig-nals: s=singlets, d=doublet, t=triplet, m=multiplet, br=broad

signal

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 758C for 4 h The

reac-tion mixture was then cooled to room temperature, and the

result-ing precipitate was filtered and washed with cold EtOH to give the

5-substituted-2-oxindole compound

(3Z)-3-[(3,5-Dimethyl-1H-pyrrol-2-yl)methylidene]-1,3-dihydro-2H-indol-2-one ((Z)-1): Following the general procedure, a solution

of 3,5-dimethyl-2-carboxaldehyde (4a; 111 mg, 0.90 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)-1 as an orange solid

in 62 % yield (111 mg): mp: 232–2348C (lit.[23]220–2228C);1H NMR

(400 MHz, [D6]DMSO): d=13.35 (brs, 1H), 10.77 (brs, 1H), 7.71 (d,

J=7.2 Hz, 1H), 7.55 (s, 1H), 7.09 (m, 1H), 6.97 (m, 1H), 6.87 (d, J=

7.6 Hz, 1H), 6.00 (d, J=2.4 Hz, 1H), 2.32 (s, 3H), 2.30 ppm (s, 3H);

13C 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)-3-((3,5-Dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoroindolin-2-one ((Z)-5a): Following the general procedure, a solution of

3,5-di-methyl-2-carboxaldehyde (4a; 50 mg, 0.41 mmol),

5-fluoroindolin-2-one (61 mg, 0.41 mmol), and piperidine (3 drops) in EtOH (3 mL)

was stirred at 758C for 12 h Purification by silica gel column

chro-matography (CH2Cl2) afforded (Z)-5a as an orange solid in 58%

(400 MHz, CDCl3): d=13.14 (brs, 1H), 7.65 (brs, 1H), 7.33 (s, 1H), 7.17 (m, 1H), 6.81 (m, 2H), 6.00 (s, 1H), 2.38 (s, 3H), 2.33 ppm (s,

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 (Z)-5-Acetyl-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)indolin-2-one (5b): Following the general procedure, a solution of 5-acetyl-2-oxindole (100 mg, 0.57 mmol) and 3,5-dimethyl-2-carboxalde-hyde (4a; 84 mg, 0.68 mmol), and piperidine (3 drops) in EtOH (7 mL) was stirred at 758C for 4 h to give 5-acetyl derivative 5b as

a brown solid (84.8 mg, 53%): mp: 297–2988C (decomposed);

8.35 (d, J=1.6 Hz, 1H), 7.75–7.77 (m, 2H), 6.96 (d, J=8.4 Hz, 1H), 6.05 (d, J=2 Hz, 1H), 2.59 (s, 3H), 2.36 (s, 3H), 2.34 ppm (s, 3H);

13C 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-2-one (5c): Following the general procedure, a solution of 5-nitro-2-oxindole (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 758C for 4 h The resulting precipitate was filtered and washed with cold EtOH to give 5-nitro 5c as a brown solid (114.2 mg, 75 %): mp: >3008C (lit.[25]>2808C); 1H NMR (400 MHz, [D6]DMSO): d=13.35 (s, 1H), 11.43 (s, 1H), 8.78 (d, J=2.4 Hz, 1H), 8.03 (dd, J=8.4, 2.4 Hz, 1H), 7.95 (s, 1H), 7.04 (d, J=8.4 Hz, 1H),

(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

(Z)-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-methoxyindolin-2-one (5d): Following the general procedure, a solution of 5-me-thoxy-2-oxindole (75 mg, 0.46 mmol) and 3,5-dimethyl-2-carboxal-dehyde (4 a; 68 mg, 0.55 mmol), and piperidine (3 drops) in EtOH (7 mL) was stirred at 758C for 6 h The residue was purified by

derivative 5d as a brown solid (90.1 mg, 73%): mp: 256–2578C

10.56 (s, 1H), 7.57 (s, 1H), 7.39 (d, J=2.4 Hz, 1H), 6.75 (d, J= 8.4 Hz, 1H), 6.67 (dd, J=8.4, 2.4 Hz, 1H), 6.00 (d, J=2.0 Hz, 1H), 3.77 ppm (s, 3H); 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

(Z)-N-(3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-2-oxoindolin-5-yl)acetamide (5e): Following the general procedure, a solution of 5-acetamide-2-oxindole (64 mg, 0.34 mmol) and 3,5-dimethyl-2-car-boxaldehyde (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

5-acet-amide derivative 5 e as a yellow solid (70.8 mg, 71 %): mp: >3508C

10.70 (s, 1H), 9.75 (s, 1H), 7.77 (d, J=2.0 Hz, 1H), 7.36 (s, 1H), 7.22 (dd, J=8.4, 2.0 Hz, 1H), 6.79 (d, J=8.4 Hz, 1H), 6.01 (d, J=2.0 Hz, 1H), 2.32 (s, 3H), 2.28 (s, 3H), 2.02 ppm (s, 3H);13C NMR (100 MHz,

[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-((3,5-Dimethyl-1H-pyrrol-2-yl)methylene)-5-hydroxyindolin-2-one (5 f): Following the general procedure, a solution of

5-hy-droxy-2-oxindole (34 mg, 0.23 mmol) and

3,5-dimethyl-2-carboxal-dehyde (4 a; 33 mg, 0.27 mmol), and piperidine (3 drops) in EtOH

(5 mL) was stirred at 758C for 16 h The residue was purified by

column chromatography (CH2Cl2/MeOH, 97:3 to 95:5) to give

(400 MHz, [D6]DMSO) d13.41 (s, 1H), 10.46 (s, 1H), 7.40 (s, 1H), 7.09

(d, J=2.4 Hz, 1H), 6.65 (d, J=8.4 Hz, 1H), 6.53 (dd, J=8.4, 2.4 Hz,

1H), 5.98 (d, J=2.4 Hz, 1H), 2.30 (s, 3H), 2.28 ppm (s, 3H);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

(Z)-5-Amino-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)indolin-2-one (5g): 10 % Pd/C (18 mg, 0.017 mmol Pd) was added to a

sus-pension of 5-nitro compound 5 c (60 mg, 0.21 mmol) in EtOH

(2 mL) The reaction mixture was stirred under hydrogen

atmos-phere overnight The reaction mixture was then filtered through

a pad of Celite The residue was purified by column

chromatogra-phy (CH2Cl2/MeOH, 1:0 to 99:1) to give 5-amino derivative 5g as

an orange solid (17.8 mg, 34%): mp: 249–2518C (decomposed);

1H NMR (400 MHz, [D6]DMSO): d=13.39 (s, 1H), 10.34 (s, 1H), 7.29

(s, 1H), 6.89 (s, 1H), 6.56 (d, J=8.0 Hz, 1H), 6.38 (dd, J=8.0, 1.6 Hz,

1H), 5.96 (s, 1H), 4.59 (brs, 2H), 2.30 (s, 3H), 2.26 ppm (s, 3H);

13C 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

(E)-3-((1,3,5-Trimethyl-1H-pyrrol-2-yl)methylene)indolin-2-one

((E)-5h): Following the general procedure, a solution of 4b

(200 mg, 1.46 mmol), indolin-2-one (162 mg, 1.21 mmol), and

pi-peridine (3 drops) in EtOH (5 mL) was stirred at 758C for 24 h

Pu-rification by silica gel column chromatography (petroleum ether/

EtOAc, 4:1!100% EtOAc) afforded (E)-5 h as an orange solid in

d=7.67 (s, 1H), 7.64 (s, 1H), 7.17 (m, 2H), 6.95 (dt, J=4.0, 8.0 Hz,

1H), 6.87 (d, J=8.0 Hz, 1H), 5.94 (s, 1H), 3.48 (s, 3H), 2.29 (s, 3H),

1.97 ppm (s, 3H);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

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

(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 758C 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)-5i=97:3)

E isomer (400 MHz, CDCl3): d=8.52 (s, 1H), 7.68 (s, 1H), 6.86 (m,

3H), 5.97 (s, 1H), 3.49 (s, 3H), 2.29 (s, 3H), 1.98 ppm (s, 3H);

13C 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

(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 piperi-dine (3 drops) in EtOH (2 mL) was stirred at 908C for 3 h The re-sulting precipitate was filtered, washed with cold EtOH, and dried

to give (Z)-8 as an orange solid in 66 % yield (104 mg): mp: 234– 2368C (lit.[26] 210–2138C); 1H NMR (400 MHz, [D6]DMSO): d=13.34 (brs, 1H), 10.88 (brs, 1H), 7.74 (s, 1H), 7.63 (d, J=7.2 Hz, 1H), 7.35 (brs, 1H), 7.14 (td, J=7.6, 1.2 Hz, 1H), 7.00 (m, 1H), 6.88 (d, J=

(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 General procedure for the acylation reaction for the synthesis of 6a, 7a, 6 c, and 7c: 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 CH2Cl2was stirred under nitrogen at room tempera-ture The reaction was monitored using TLC until no (Z)-1 or (E)-5h 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

1-Acetyl-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)indolin-2-one (6a): Following the general procedure, a mixture of

(Z)-1 (200 mg, 0.84 mmol), DMAP ((Z)-15 mg, 0.(Z)-13 mmol), triethylamine

(6 mL) was stirred under nitrogen at room temperature Purifica-tion by silica gel column chromatography (petroleum ether/EtOAc, 4:1) afforded (Z)-6a as an orange solid in 78% yield (184 mg): mp: 196–1988C;1H NMR (400 MHz, CDCl3): d=12.61 (brs, 1H), 8.25 (m, 1H), 7.49 (m, 1H), 7.40 (s, 1H), 7.20 (m, 2H), 6.04 (s, 1H), 2.80 (s, 3H), 2.42 (s, 3H), 2.35 ppm (s, 3H);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 (Z)-tert-Butyl 3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-2-oxoin-doline-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)-6c as

an orange solid in 90% yield (51.8 mg):1H NMR (400 MHz, CDCl3): d=12.82 (brs, 1H), 7.73 (m, 1H), 7.49 (m, 1H), 7.39 (s, 1H), 7.17 (m, 2H), 6.02 (s, 1H), 2.37 (s, 3H), 2.34 (s, 3H), 1.70 ppm (s, 9H);

13C 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

1-Acetyl-3-((1,3,5-trimethyl-1H-pyrrol-2-yl)methylene)indolin-2-one (7a): Following the general procedure, a mixture of (E)-5h (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) af-forded 7a as an E/Z mixture ((E)-7 a:(Z)-7a=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, 1H), 7.65 (s, 1H), 7.31 (m, 1H), 7.17 (m, 2H), 6.00 (s, 1H), 3.48 (s, 3H), 2.66 (s, 3H), 2.28 (s, 3H),

170.5, 167.7, 138.5, 137.1, 128.1, 126.4, 126.2, 125.7, 124.3, 122.9,

121.3, 118.8, 115.4, 111.5, 31.6, 26.4, 13.9, 12.3 ppm; HRMS

295.1439

2-oxo-3-((1,3,5-trimethyl-1H-pyrrol-2-yl)methyle-ne)indoline-1-carboxylate ((E)-7c): Following the general

proce-dure, a mixture of (E)-4a (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

7c as an E/Z mixture ((E)-7 c:(Z)-7c=86:14) as an orange oil in

90% (63.4 mg) combined yield:1H NMR E isomer (400 MHz, CDCl3):

d=7.89 (m, 1H), 7.68 (s, 1H), 7.24 (m, 2H), 7.06 (m, 1H, H-7), 5.97

(s, 1H), 3.46 (s, 3H), 2.28 (s, 3H), 1.94 (s, 3H), 1.67 ppm (s, 9H);

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

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 6b;

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 (3Õ10 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

oth-erwise 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

(Z)-3-((3,5-Dimethyl-1H-pyrrol-2-yl)methylene)-1-methylindolin-2-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

ex-tracted Purification by silica gel column chromatography

(petrole-um ether/EtOAc, 4:1) afforded (Z)-6b as an orange solid in 53 %

13.25 (brs, 1H), 7.51 (d, J=8.0 Hz, 1H), 7.40 (s, 1H), 7.19 (dt, J=

1.2, 8.0 Hz, 1H), 7.08 (dt, J=1.2, 8.0 Hz, 1H), 6.88 (d, J=8.0H, 1H),

5.97 (s, 1H), 3.38 (s, 3H), 2.38 (s, 3H), 2.33 ppm (s, 3H); 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

(E)-1-methyl-3-((1,3,5-trimethyl-1H-pyrrol-2-yl)methylene)indo-lin-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

ex-tracted Purification by silica gel column chromatography

(petrole-um ether/EtOAc, 4:1) afforded 7b as an E/Z mixture

((E)-7b:(Z)-7b=89:11) as an orange oil in 85% (47.5 mg) combined yield:

1H NMR (E)-isomer (400 MHz, CDCl3): d=7.65 (s, 1H), 7.21 (m, 2H),

6.97 (m, 1H), 6.83 (d, J=8.0 Hz, 1H), 5.93 (s, 1H), 3.47 (s, 3H), 3.31

(s, 3H), 2.28 (s, 3H), 1.96 ppm (s, 3H);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

(ESI-TOF): (m/z) calcd for C17H18N2O [M+H]+267.1497, found 267.1496

Isomerization study: Photoisomerization experiments were per-formed with 10 mm [D6]DMSO solutions of 3-substituted indoline-2-one derivatives in NMR tubes The NMR tubes were then ex-posed to fluorescent light (Philips Tornado 5W ES 6500 K cool day-light, 285 lumens) at ambient temperature (22–248C), with a light intensity of ~1700 lux (measured with a Gossen Luna-Pro F light meter) The samples were analyzed by1H NMR spectroscopy at var-ious 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 vari-ous time points (0.5, 1, 2, 3, 4, 5, 6, 12, 24, 48, 72 h), samples were analyzed by1H NMR spectroscopy

Isomerization of 100mm compound stocks: The 100 mm drug stocks of (Z)-1, (Z)-5a, (Z)-2, and (Z)-10 were exposed to fluores-cent 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

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 ¢20 8C TAMH cells were maintained in

a T75 flask with Dulbecco’s modified Eagle’s medium

insulin, 5 mgmL¢1transferrin, 5 ngmL¢1selenium), 10 mm

HepG2 was maintained in minimal essential medium in the pres-ence of 10% fetal bovine serum Both lines were incubated at 378C in 95% air and 5% CO2

MTT cell proliferation assay: The TAMH cells were trypsinized to pro-duce a single-cell suspension and were resuspended using 0.5 mgmL¢1 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 15000 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 mgmL¢1 in each well, and the plates were incubated at 378C 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

deter-mined using GraphPad Prism 6 Software

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

Computational Resource Centre (for M.B.S.) for the use of its

high-performance computing facilities

Keywords: indolin-2-ones · computational chemistry ·

cytotoxicity · kinetics · photoisomerization

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