SYNTHESIS AND PHOTOCHROMIC PROPERTIES OF A NOVEL CHROMENE DERIVATIVE

A convenient synthetic approach to a novel 2,2-diphenyl-2H - chromene derivative is developed starting from vanillin or acetovanillone. The photochromic properties of the resulting chromene are studied. The kinetic characteristics of this compound show its potential for the design of new efficient DNA intercalators.


Introduction
Small molecules that are able to recognize a DNA sequence are promising therapeutic agents and molecular tools for studying and regulating specific gene expression [1,2].Of particular interest is the investigation of DNA binding with organic molecules that can intercalate between two complementary base pairs, thereby changing its functioning.This process is of high importance for the search for new drugs for the treatment of various diseases, including different types of cancer.
In developing selective drugs for chemotherapy, special attention is given, in particular, to the photoactive derivatives of chromene [3].Owing to the structural features, a chromene molecule in a closed form does not interact with DNA; however, upon irradiation, it undergoes a photochromic transformation into an open, planar merocyanine form that can coordinate with DNA, for example, of cancer cells [4].
It should be noted that there is only a limited number of reports devoted to the application of chromene derivatives as photoswitchable ligands for DNA.Therefore, the goal of this work was to synthesize a novel chromene derivative and to study its photochromic characteristics.A relatively stable open form of the resulting derivative opens the way to the investigation of its interaction with DNA, which will become a subject for further research.

Syntheses
Chromene derivative 1 was chosen as a target compound.The introduction of electron-donating substituents at positions 6 and 7 of a benzopyran moiety leads to a bathochromic shift of an absorption band relative to that of the unsubstituted chromene, which can provide a greater color contrast between closed and open forms [5].The presence of a bromine atom in the oxyalkyl substituent ensures a possibility of further synthesis of chromene conjugates with other molecules, for example, styryl dyes that exhibit affinity to DNA.The length of this spacer can affect the simultaneous binding of both of the conjugate moieties with a DNA molecule.
As is known, one of the methods for synthesizing this type of chromenes is the interaction of phenols with diphenylpropargyl alcohol 2 in the presence of acids [6][7][8][9][10].Compound 1 was obtained by reacting phenol 3 with 2 in the presence of p-toluenesulfonic acid upon heating in toluene (Scheme 1).

Scheme 1. Synthesis of chromene derivative 1.
The formation of a linear chromene instead of an angular one was confirmed by the presence of two singlets in the 1 H NMR spectrum of chromene 1, which correspond to the protons at positions 5 and 8 of the benzopyran moiety.
Initial phenol 3 can be obtained in three stages starting from commercially available vanillin (4а, R = H) or acetovanillone (4b, R = Me) (Scheme 2).At the first step, substituted phenols 4a,b were subjected to the O-alkylation upon prolonged refluxing with a double excess of 1,5-dibromopentane in the presence of potassium carbonate and potassium iodide in acetonitrile [11].The next step was the Baeyer-Villiger oxidation (rearrangement).The interaction of 5a,b with m-chloroperoxybenzoic acid in dichloromethane under an argon atmosphere was accomplished according to the published procedure [12].The hydrolysis of esters 6a,b was carried out by heating with 7% aq.hydrochloric acid.It should be noted that the hydrolysis of acetate ester 6b required a much higher temperature.At the same time, the hydrolysis of formic acid ester 6а afforded target phenol 3 in a higher yield.

Photochromic properties
It is well known that chromenes undergo structural transformations under the action of UV radiation, which afford two, the most stable isomers, the so-called transoid-cis (TC) and transoid-trans (TT) forms (Scheme 3) [13,14].These isomers feature high closure stability, which defines the total lifetime of the open form.The phototransformation can be monitored by UV-Vis spectroscopy.The open forms exhibit absorption bands in the visible region, which gradually disappear after the cessation of irradiation.Thus, the irradiation of a solution of chromene 1 leads to the growth of two bands in the spectrum with λmax = 380 and 470 nm (Fig. 1).The stability of the photoinduced forms can be defined from kinetic characteristics of the thermal relaxation: the reaction rate constant (k) and half-decoloration time (τ½).The kinetics of the thermal relaxation of chromene 1 was studied at four temperatures: 294, 298, 303, and 313 К (Fig. 2).
As can be seen from Fig. 2a, the absorption spectrum at T = 294 K did not conform to the initial form (Fig. 1, A), which may evidence the formation of a stable photoinduced form (TT).A temperature increase was accompanied by a decrease in the residual optical density in the visible region (Fig. 2b,c): the values of optical density at λ = 470 nm in 3000 s after cessation of irradiation were 0.21 (294 K), 0.19 (298 K), 0.17 The processes that take place after cessation of irradiation can be characterized by the following scheme of transformations: where TT and TC are the open forms of the chromene, CF is the closed (initial) form of the chromene.A total expression for the observed changes in the intensities of absorption bands of the open forms in time has a biexponential character and can be described by the following equation: where  TT and  TC are the rate constants of sequential steps of the decoloration process,  0 TC and  0 TT are the initial absorbances of each form (at the beginning of the reverse reaction).The resulting kinetic characteristics for each temperature are summarized in Table 1.The temperature dependence of the rate constant of the thermal relaxation corresponds to the Arrhenius equation: where   is the reaction activation energy,  is the preexponential factor,  is the universal gas constant, and T is the temperature.
The results of the investigation of the kinetics of thermal relaxation for both of the reactions are compared in Fig. 3 in the form of the Arrhenius plot.The dash lines in Fig. 3 represent the linear approximation of the data, and the line slopes were used to calculate the activation energies.The resulting thermodynamic characteristics (Fig. 3) of the thermal relaxation of the colored forms are in good agreement with the literature data [15].
The closure of the open TC and TT forms can be accelerated not only by a temperature rise.While conducting the photochemical relaxation through irradiation with λ = 405 nm, the absorption spectrum of the open form also returns to its initial form (Fig. 4).

Experimental General remarks
All the reagents were purchased from commercial sources and used without purification.The 1 Н and 13 C NMR spectra were registered on a Bruker Avance TM 400 spectrometer at the operating frequencies of 400 and 100 MHz using deuterated solvents (СDCl3, CD3COCD3, CD3CN).The residual solvent signals were used as internal standards.The UV-Vis spectra were measured on an AvaSpec-2048 spectrophotometer.The irradiation was performed with a light diode (405 nm), a FIL-105 photographic flash (flash guide 34 at ISO 100, discharge energy 68 J), and a high-pressure mercury lamp (DRSh 120 W).The certain spectrum lines of the Hg lamp were separated using glass filters from a standard set of samples of color optical glasses (313 nm).The UV-Vis spectra of the colored form were registered during continuous irradiation of the samples with the filtered radiation of the mercury lamp to obtain the maximal concentration of the open forms.The dark relaxation was carried out in total darkness; the scanning light required for the registration of the UV-vis spectrum was turned off between measurements, its exposure time to the sample P. S. Perevozchikova et al., INEOS OPEN, 2021, 4 (1), 29-34 during spectrum registration composed less than 1 s per 1 spectrum (point on the kinetic curve).The elemental analyses were performed at the Laboratory of Microanalyses of INEOS RAS.The EI mass spectra were registered on a Finnigan Polaris Q instrument (ion trap).The energy of ionizing electrons was 70 eV.The melting points were measured on a PTP(M) apparatus.

Conclusions
Two schemes for the synthesis of the new chromene derivative were developed that consist of four steps and utilize vanillin and acetovanillone as key precursors.Taking into account the higher yields at each step and availability of vanillin, the approach based on it seems to be optimal.
The photochromic properties of the resulting chromene derivative were explored at different temperatures.The kinetic studies revealed the high closure stability of its open form, which can be used for further investigation of the interaction with DNA.
(303 K), and 0.08 (313 K).It is obvious that a temperature rise leads to the acceleration of both reverse processes.Owing to this, the stable open ТТ form converts to the less stable TC form, which finally leads to the complete closure of a pyran ring.During dark relaxation at T = 313 K (Fig. 2d), the long-wave absorption P. S. Perevozchikova et al., INEOS OPEN, 2021, 4 (1), 29-34 band disappears.At the same time, the absorption spectrum of the light-exposed solution kept in the dark became almost indistinguishable from that of the solution before irradiation.

Figure 3 .
Figure 3. Plot of the natural logarithm of the rate constants of thermal relaxation ( TCtriangles,  TTsquares) as a function of the inverse temperature (Arrhenius plot).The values of the activation energies are indicated next to the plots.

Figure 4 .
Figure 4. Relaxation of a solution of chromene 1 (4×10 -5 M, MeCN) during irradiation with λirr = 405 nm; an inset shows changes in the absorbance at λ = 470 nm over time.The application of NMR spectroscopy for the investigation of photochromic transformations allows one to unambiguously define the structures of the photoinduced forms and their stabilities.The irradiation of a solution of chromene 1 led to the appearance of signals of the open forms in the 1 Н NMR spectrum, which changed their intensity depending on the irradiation duration.A characteristic signal of the TC form is a peak of a proton at position 3 observed at 8.57 ppm.This shift is caused by a deshielding effect of the closely located carbonyl group (Fig.5b).When irradiation was stopped, the reverse process was observed: the signals of the closed form increased and those of the open ones reduced (Fig.5c).To fully escape the signals of the open form (TT), a solution after thermal relaxation was irradiated with λirr = 405 nm (Fig.5d).

Figure 5 .
Figure 5. Observation of the photochromism of chromene 1 by NMR spectroscopy: closed form (a), irradiation with 313 nm (b), thermal relaxation after irradiation for 1 h (c), relaxation after irradiation with 405 nm (d).

Table 1 .
Kinetic characteristics of the thermal relaxation