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