Synthesis, optical properties and in vitro cell viability of novel spiropyrans and their photostationary states

Article abstract of DOI:10.1016/j.tet.2020.131854

A novel class of spiropyran (SP) was synthesized using a multistep process that involves three key intermediates: (a) diazonium-tetrafluoroborate, (b) hydrazine and (c) indolium iodide. Single crystal X-ray spectroscopy was used to confirm the structure of one of the analogues. The SP analogues were confirmed as being able to isomerize and attain a photostationary state (PSS) upon irradiation with ultraviolet light (UV, 365 nm) in an aqueous environment. UV–visible absorption spectra were recorded to confirm the isomerization properties. The ability of the synthesized compounds to induce growth inhibition of HeLa cervical cancer cells was assessed via the MTT assay after incubation with either the SP or their PSS. The IC₅₀ values of two PSS (PSS-2, 4), were observed to be around 14 ± 4 fold lower (26 ± 3 μM) than their corresponding SPs. The most cytotoxic compounds SP-7 and PSS-7 showed the lowest IC₅₀ values (12 μM). An in vitro tubulin polymerization assay showed that SP-7 and PSS-7 exhibited the greatest difference in tubulin inhibition relative to unirradiated SP-1 and SP-4.

Full text of DOI:10.1016/j.tet.2020.131854

Tetrahedron 80 (2021) 131854
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis, optical properties and in vitro cell viability of novel
spiropyrans and their photostationary states
,
Shiva K. Rastogi c *, Zhenze Zhao c, M. Brenton Gildner c, Ben A. Shoulders c,
Tara L. Velasquez c, Madeleine O. Blumenthal c, Lei Wang a, Xiaopeng Li a,
Todd W. Hudnall c, Tania Betancourt b, Liqin Du c, William J. Brittain c
a Department of Chemistry, University of South Florida, Tampa, FL, 33620, USA
b Material Science, Engineering and Commercialization Program, 601 University Drive, Texas State University, San Marcos, TX, 78666, USA
c Department of Chemistry and Biochemistry, 601 University Drive, Texas State University, San Marcos, TX, 78666, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of spiropyran (SP) was synthesized using a multistep process that involves three key in-
termediates: (a) diazonium-tetrafluoroborate, (b) hydrazine and (c) indolium iodide. Single crystal X-ray
spectroscopy was used to confirm the structure of one of the analogues. The SP analogues were
confirmed as being able to isomerize and attain a photostationary state (PSS) upon irradiation with
ultraviolet light (UV, 365 nm) in an aqueous environment. UVevisible absorption spectra were recorded
to confirm the isomerization properties. The ability of the synthesized compounds to induce growth
inhibition of HeLa cervical cancer cells was assessed via the MTT assay after incubation with either the SP
Received 18 October 2020
Received in revised form
1 December 2020
Accepted 2 December 2020
Available online 27 December 2020
Keywords:
Methoxy-spiropyran
UVeVisible
Photostationary state
Anticancer
Photoactivated
Photopharmacology
or their PSS. The IC50 values of two PSS (PSS-2, 4), were observed to be around 14
(26 M) than their corresponding SPs. The most cytotoxic compounds SP-7 and PSS-7 showed the
lowest IC50 values (12 M). An in vitro tubulin polymerization assay showed that SP-7 and PSS-7
exhibited the greatest difference in tubulin inhibition relative to unirradiated SP-1 and SP-4.
Published by Elsevier Ltd.
4 fold lower
3
m
m
In recent times, stimuli-responsive drug development has
attracted considerable interest as a new paradigm to increase the
specificity of a drug toward the target diseased cells, increasing the
therapeutic effect, and reducing its side-effects. For cancer treat-
ment, in particular, the low specificity of current chemotherapeutic
drugs poses dose limitations and causes severe side-effects [1]. To
overcome these issues, photopharmacology offers tools to design
and develop targeted molecules than can be photoactivated on
demand with both spatial and temporal control [2]. Azobenzene
analogues, azo-combretastatins A-4 (Azo-CA4) are the examples of
photoresponsive anticancer compounds, have been developed
recently [3]. Spiropyran (SP), is another photoresponsive and
photochromic motif that has been utilized in drug delivery systems
for cancer therapy [4], as fluorescence imaging probes of microtu-
bules in living cancer cells [5], and to develop water-soluble pho-
toactivatable cytotoxic moieties [6]. The unique photophysical and
photochromic properties of SP inspired us to explore and tailor its
basic structure to develop novel cytotoxic photo-pharmacophores.
Structurally, SP comprises of an indole ring and a chromene
moiety bound together via a spiro junction (Cspiro-O) and oriented
perpendicular with respect to one another [7]. SP undergoes ther-
mochromism and photochromism in response to photons, reduc-
tion/oxidation, and changes in temperature and pH [8]. In the
photochromism process, near ultraviolet light (UV, ~350 nm) and
blue (~420 nm) light induce photoisomerization in SP between the
colorless SP and colored merocyanine (MC) forms .Scheme- 1b SP
analogues are known as molecular photoswitches, used in a broad
range of research disciplines such as optical data storage [9], smart
materials [10], mechano-chemistry [11], photo induced medicines
[12], DNA detection [13], and photo-controlled drug delivery sys-
tems for cancer therapy [14].
As previously reported, indole [15], chromene [16], and spi-
ropyran [6] have been shown to possess cytotoxic activity. The
unique photo-physical properties of SP1-MC1 isomers and our in-
terest in developing noncytotoxic to toxic photoswitches, promp-
ted us to design and synthesize novel analogues and to investigate
their photophysical and biological properties. Specifically, we have
developed a new class of SP1 analogues by tailoring the SP1
* Corresponding author.
E-mail addresses: skr55@txstate.edu, rastogisk@gmail.com (S.K. Rastogi).
https://doi.org/10.1016/j.tet.2020.131854
0040-4020/Published by Elsevier Ltd.

S.K. Rastogi, Z. Zhao, M.B. Gildner et al.
Tetrahedron 80 (2021) 131854
structure to include the functional groups common to colchicine
and azo-CA4 Fig. 1a on the indole and chromene rings. The PSS of
the corresponding SPs were obtained by irradiation in an aqueous
environment with UV light (365 nm) to produce equilibrium mix-
tures of the SP/MC pairs. We then investigated the ability of both SP
and PSS forms to induce cancer cell growth inhibition. Finally, we
investigated their ability to affect anticancer effects as microtubule
inhibitors through an in vitro tubulin polymerization assay.
The photochromic properties of SP1, SP6, and SP7 in both their
SP and PSS states have been well studied in the literature; however,
their cancer cell growth inhibition activity and interaction with
tubulin has not yet been reported. In this report, the photophysical
properties, anti-cancer effect, and the tubulin polymerization in-
hibition ability of commercially available SP1, previously reported
compounds SP6 [17] and SP7 [18], and four new SPs (SP2 to SP5)
Fig.1b, were investigated in both their SP and photostationary (PSS)
states.
The general route for the synthesis of SP2 to SP5, in five steps, is
shown in scheme ꢀ1. Intermediates 2 and 3 were synthesized as
previously reported in the literature [19]. In the first step, t-butyl
nitrite-mediated nitrogen transfer was carried out on 3,4,5-
trimethoxyaniline 1 to give 2 in 92% yield. This was followed by
the reduction of 2 to 3 using tin (II) chloride. A solution of 3 in dry
dichloromethane (reddish-pink color) was used immediately in the
next step without further purification. A solution if 3 in glacial
acetic acid was refluxed with 3-Methyl-2-butanone to give 4 ESI
(page2-3) and 5 was prepared by the N-methylation of 4 using
iodomethane ESI (page3-4). Finally, the novel SPs (SP2-5) were
synthesized in good yield (65e78%) by condensation of 5 with
~323e351 nm band was attributed to the chromene moiety. Irra-
diation of SP1 with ~365 nm light gives rise to the maximum
isomerization into the open-ring isomer MC1. This photo-
isomerization is a first-order process and showed an appearance of
a new band at ~480e520 nm [4]. Similar patterns were observed in
the UV spectra of SP2 and MC2. However, tailoring of the electron-
donating groups and electron-withdrawing groups in SP3 to SP7
resulted in a loss of the typical absorption peak at 323e352 nm and,
instead the appearance of a band at ~300 nm.
An important feature of the SP-MC system is the equilibrium
constant (Keq ¼ [MC/[SP]). The amount of MC in the PSS depends on
the electron-donating or electron-withdrawing nature of the sub-
stituents on the SP skeleton [20]. As the value of Keq increases, the
MC population the in equilibrium increases. Based on the literature
reports, we compared and arranged the SPs in increasing order of
Keq in non-polar media as KSP7<KSP3<KSP6<KSP5~KSP4<KSP1<KSP2
.
However, the quantification of Keq in an aqueous environment is
important to correlate the amount of SP and MC, and their effect on
biological properties. Hence, we utilized the UVevisible spectra of
SP-PSS, from Table- 1 to determine the value of Keq. The absorption
intensity at 520 nm was used to represent MC and that at 320 nm to
represent SP. The baseline intensity at 630 nm was subtracted from
each SP and MC peak value to calculate the amount of SP and MC
from its spectrum. The increasing order of Keq for SP was observed
as KSP7<KSP6<KSP5~KSP3<KSP2<KSP1<KSP4 and for PSS was observed
as KPSS7~KPSS3<KPSS1<KPSS5<KPSS6<KPSS2<KPSS4. Based on the Keq
order, the concentration of MC was found to be the lowest in the
SP7-PSS7 pair and highest in the SP4-PSS4 pair Table- 2.
The exact percentage of the MC in the equilibrium of PSS was
quantified using NMR. 1H NMR spectra of SP were recorded in a
different aqueous solvent system, such as DMSO‑d6:D2O (1:9 and
1:1; v/v), CD3CN:D2O (3:1; v/v), and CD3CN:1XPBS (9:1; v/v), but
their spectra were unreadable. Only the aqueous mixture of
acetone-d6: D2O (2:1; v/v) produce suitable to produce NMR
spectra for integration quantification. While this solvent mixture is
not comparable to cancer cell growth media, we expected that the
Keq values of SP and PSS would be comparable in both media. Three
to 5 mg of the desired SP compound was dissolved the acetone-d6:
D2O (2:1; v/v) solvent mixture and the 1H NMR spectra of SP were
recorded before and after UV light irradiation. The PSS was pro-
duced by irradiation of 365 nm UV light for 30 min on each cor-
responding SP. The significant differences, in the 1H NMR spectra of
SP1-7 and MC1-7 where the downfield shift of aromatic peaks and
upfield shift of indole alkyl group peaks in the 1H NMR of their
PSS1-7. The integration ratio of the aromatic-hydrogen peaks of
SP1-7 and MC1-7 were used to calculate the percentage of MC in
the PSS. Data are reported in Table- 2 and spectra in the ESI (page
38e44).
various
ortho-hydroxy-benzaldehydes:
2-hydroxy-5-
nitrobenzaldehyde 6a; 2-hydroxybenzaldehyde 6b; 2-hydroxy-3-
methoxybenzaldehyde 6c and 2-hydroxy-3-ethoxybenzaldehyde
6d ESI (page4-6).
The intermediates and final compounds were characterized by
melting point analysis, nuclear magnetic resonance (NMR) spec-
troscopy, infra-red (IR) spectroscopy and high-resolution mass
spectrometry (HRMS); spectra are in the ESI (page9-33). Addi-
tionally, the structure of a representative spiropyran SP3, was
confirmed unambiguously by single crystal x-ray diffraction studies
Fig. 2 and data are listed in the electronic supporting information
(ESI; pages 34e37).
The photoisomerization of SPs and MC, leading to the formation
of an equilibrium state PSS, was characterized using UVevisible
absorption spectrometry Fig. 3. The SP analogues in 5% (v/v)
dimethylsulfoxide (DMSO)/phosphate buffered saline (PBS,
pH ¼ 7.4) were irradiated with UV light (365 nm). The spectrum of
SP1, closed-ring isomer shows typical two localized absorption
bands. The first band at ~272e296 nm corresponded to the pi to pi-
star electronic transition of the indoline moiety, and the
Chemotherapeutic agents are clinically used for the treatment of
Fig. 1. (a) Structures of Colchicine, CA4, Azo-CA4, SP4 and MC4, (b) General structure of the SP-MC equilibrium.
2

S.K. Rastogi, Z. Zhao, M.B. Gildner et al.
Tetrahedron 80 (2021) 131854
Scheme 1. Synthesis of trimethoxy spiropyrans SP2-5.
Further, HeLa cells were viable when incubated with a positive
control (cell culture medium and without any SP or PSS) Figure SI-1
and their growth were inhibited after incubating with colchicine,
shows a sub-micromolar IC50 value Table- 3.
Literature reports show a unique property of SP1 in an aqueous
solution, (i) dramatically higher MC1 populations in its aqueous
PSS1 solution [21], and (ii) a hydrolytic degradation of MC1 to its
precursor compounds of Fisher’s base and the salicaldehyde as
above 45 ꢁC and in strong acidic pH conditions [22]. Further the rate
of SP1-MC1 interconversion is roughly twice that of the hydrolysis
rate of SP1 compound [23]. Literature show these studies were
focused on only 6-nitroSP derivatives, such as SP1 and SP2. As per
our investigation, SP3-7 are predicted to have lower hydrolysis
rates and lower Keq. However, we tested the hydrolytic products
and starting material (1, 2, 3, 4, 5 and ortho-hydroxy aldehydes) of
SP1-7 for the growth inhibition of HeLa Cells. But none of them
showed cell growth activity and all IC50 values were obtained above
Fig. 2. Crystal structure of SP3.
100 mM.
cancer, but their use can be limited due to serious side effects and
toxicity. Hence, researchers are interested in developing effective,
non-destructive, and specific new anticancer agents. Photo-
pharmacology offers an opportunity to create more selective
cancer-targeting compounds, by applying light to selectively acti-
vate photoswitches. Previously we have utilized this elegant
approach to develop novel Azo-CA-4 analogues as tubulin poly-
merization inhibitors [3c].
In the anti-cancer drug discovery research process, colchicine
[24] and combretastatin A-4 (CA-4) [25] have been classified as the
ligands that inhibit the polymerization of the mitotic spindle in
cancer cells. Therefore, we kept the structure of colchicine and azo-
CA-4 in our mind to design new photoresponsive pharmacophores,
we tailor the skeleton of commercial SP1 and MC-1. The pre-
liminary biological evaluation of SP1-7 and PSS1-7 indicated sig-
nificant HeLa cell growth inhibition in the MTT assay. However, to
understand and to investigate the possible biomechanism of the
novel SP-PSS system, we performed an in vitro tubulin polymeri-
zation assay [26] on SP1-PSS1, SP4-PSS4 and SP7-PSS7 as per the
prescribed procedure for the tubulin polymerization assay kit (Cat.
# BK011P) by Cytoskeleton, Inc. ESI (page7). The test results were
compared to three different controls: paclitaxel (known microtu-
bule stabilizer), colchicine (known microtubule destabilizer) and
DMSO (carrier solvent) Fig. 4. Overall, SP1, SP4 and SP7 are weak
tubulin inhibitors with activities between colchicine and paclitaxel.
Little difference was observed between the SP and PSS of SP4 and
SP7 while PSS7 showed the greatest difference in tubulin inhibition
assay. Fig. 4 shows the SP7-PSS7 would be a good hit for further
studies. The four hypothesis (i) no substituent on the SP7 and PSS7
moieties, (ii) the lowest values of Ksp, Kpss, (iii) the smallest ligand
among the SP1-6 and PSS1-6 structures, and (iv) better docking in
the colchicine binding site in tubulin protein (PBD: 1SA0) (data not
reported here) would be making SP7 and PSS7 compound the hit
ligand.
Herein, we have investigated the HeLa cells growth by incu-
bating with SP1-7 (prior to UV exposure) or the corresponding
PSS1-7 (UV exposed SP), where HeLa cells were used as a model for
human cervical adenocarcinoma cells. The stock solutions of SP1-7
were prepared in 100%DMSO, then a serial dilution of each com-
pound was prepared in the cell culture medium (DMEM) to incu-
bate HeLa cells, the procedure is reported in ESI (page7). PSS1-7
were prepared by irradiation with 365 nm light for 30 min in cell
growth medium prior to cell incubation. The activity of SP1-7 and
PSS1-7 on cancer cells was measured with a colorimetric MTT assay
Fig. 3. The intensity of color correlates to the viability of remaining
HeLa cells; IC50 values were determined by curve fitting and sta-
tistical analysis Table- 3. The IC50 values of PSS1-5 are lower than
the corresponding SPs, while no significant change was observed
for those of PSS6 and PSS7. More importantly the IC50 values of
PSS2, 4, 5 were >4e15 fold lower than their related SPs, which
indicates that PSS-1, 2 and PSS-4 have potential effects on inhib-
iting HeLa cell growth. However, SP7 and PSS7 showed the greatest
inhibition activity on HeLa cell growth with the lowest IC50 values.
3

S.K. Rastogi, Z. Zhao, M.B. Gildner et al.
Tetrahedron 80 (2021) 131854
Table 1
UVevisible absorption spectra of SP1-7 recorded at a concentration of 50
m
g/mL (0.18e0.12 mM) in a 5% DMSO/1XPBS buffer (pH ¼ 7.4) (v/v). The solutions were exposed to UV
light (365 nm) at room temperature (23 ꢁC) for 30 min in a quartz cuvette of 1 cm ꢂ 1 cm (width x length); SP ¼ initial spectra before UV irradiation, PSS ¼ spectra after
irradiation with UV light (365 nm) for 30 min.
Table-2
Equilibrium constants (Ksp and Kpss
(0.18e0.12 mM) in a 5% DMSO/1XPBS buffer (pH ¼ 7.4) (v/v), Ksp and Kpss were
calculated based on the UVeVisible absorption intensity Ksp ¼ MC/SP ¼ I520eI630
320eI630), Kpss was calculated from the ratio of the aromatic protons of MC:SP based
on peak integration 1H NMR spectra in acetone-d6/D2O.
) of SP and PSS solutions at 50 mg/mL
/
I
SP or PSS
KSP
KPSS
KPSS/SP
MC:SP Ratio in PSS
1
2
3
4
5
6
7
0.31
0.25
0.23
0.36
0.23
0.21
0.15
0.24
0.45
0.21
0.52
0.35
0.36
0.2
0.75
1.77
0.91
1.5
1.51
1.7
0.25: 1
0.45: 1
0.2:1
0.12:1
0.14:1
0.05:1
0.1:1
Fig. 3. (Top) MTT assay suspension results in HeLa cells in the presence of 5, 25, 125,
and 500 M of SP1 and SP4 in the dark and with UV light. (Bottom) White light
1.3
m
microscopic (phase contrast images using a confluence mask of gold color) images of
HeLa cells incubated for 72 h with SP1 and SP4 in the dark and with UV light.
In conclusion, we have reported the synthesis of four novel
spiropyran analogues (SP2-5) in five steps using t-butyl nitrite
mediated nitrogen-transfer and Fisher-indole condensation-cycli-
zation strategies as key steps. X-ray diffraction study was used to
confirm the structure of representative SP3. Every SP, SP1-7 was
irradiated with UV light (~365 nm) in an aqueous environment to
generate its corresponding SP-MC equilibrium, photostationary
states (PSS). The formation of PSS, quantification of equilibrium
4

S.K. Rastogi, Z. Zhao, M.B. Gildner et al.
Tetrahedron 80 (2021) 131854
Table 3
Science Foundation for NMR instrumentation (CR1IF:MU0946998
and MRI-0821254).
Growth inhibition of HeLa Cells and PSS compositions: [a] IC50 values for samples
without UV treatment (SPs). [b] Standard deviation is based on three separate tests;
student T-test verified that the difference in IC50 is statistically significant for SP1-
PSS1 through SP5-PSS5. [c] IC50 values for UV irradiated samples (PSSs). [d] Cell
culture medium (0.5% DMSO/DMEM).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tet.2020.131854.
Entry
IC50 [a,b] of SP
IC50 [b,c] of PSS
IC50(PSS)/IC50(SP)
1.
2.
3.
4.
5.
6.
7.
>500
m
M
30
32
5
2
m
m
M
M
>17
300 70
>500
300 100
>500
160 60
m
M
10
>4
13
>8
3
5
References
m
M
135
23
61
9
m
m
m
M
M
M
m
M
3
9
m
M
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Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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We thank the National Science Foundation (PREM center for
Interfaces in Materials, DMR-1205670) for financial support of this
research. The authors also acknowledge the support of the National
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