Synthesis and Antitumor Activity of Novel Pyridoxine-Based Bioisosteric Analogs of trans-Stilbenes

A series of trans-6-phenylethenyl substituted pyridoxine derivatives, novel bioisosteric analogs of drugs based on trans-stilbene scaffold, were synthesized using the Wittig reaction of a bis-triphenylphosphonium pyridoxine derivative with various aromatic aldehydes. Two compounds demonstrated high activity against the estrogen-dependent MCF-7 (breast cancer) cell line with IC50 in the range of 1.9–7.9 μM and very good selectivity for other studied normal and tumor cells, including the estrogen receptor negative MDA-MB-231 breast cancer cells. The active compounds possessed an intense blue fluorescence, and this feature allowed us to effectively visualize them in cytoplasm and in nucleus. The obtained results make the described chemotype a promising starting point for the development of new anticancer agents for the therapy of estrogen-dependent malignancies.

In this work, we studied a series of trans-6-phenylethenyl substituted pyridoxine derivatives for their cytotoxic activity against a panel of normal and cancer cell lines.The underlying idea was to design a novel chemotype of bioisosteric analogs of the well-known estrogen receptor targeting drugs such as diethylstilbestrol, tamoxifen, and raloxifene (Figure 1).All these drugs are bioisosteric mimetics of the natural estrogen receptor (ER) agonists such as estradiol (I).Their characteristic feature is the presence in their molecular structures of a trans-diarylethylene fragment (structures II-IV).These agents also typically contain the hydroxy or alkoxy groups at the remote axial positions of the molecule (e.g., at parapositions of phenyl rings).In some cases, for example, in tamoxifen, the hydroxyl groups appear as a result of metabolic transformations in human body.Such molecular features provide electronic and spatial compatibility to the ER active site.The mentioned drugs are clinically important ER modulators, widely used in the therapy of the estrogendependent malignancies.For example, diethylstilbestrol is an inexpensive and effective option for prostate cancer treatment before chemotherapy [13].Persistently increased blood levels of estrogens are associated with an increased risk of breast cancer.Selective estrogen receptor modulators (SERMs), such as tamoxifen and raloxifene, are a class of compounds actively used for breast cancer prevention [14].Compounds of general formula V (Figure 1) studied in this work represent the substituted azaheterocyclic analogs of trans-stilbene.The presence in their structures of the characteristic trans-diarylethylene fragment makes them novel bioisosteric analogs of the natural and synthetic drugs based on trans-stilbene scaffold, and this feature prompted us to investigate their potential as selective estrogen-dependent cytotoxic agents.The synthetic route to compounds V has been reported in our recent work [8].Herein, we report synthesis of one novel compound from this structural series and also describe biological and physicochemical properties of this interesting chemotype.In accordance with the underlying hypothesis, we have found that compounds of general formula V, which contain the hydroxy or alkoxy groups at the remote axial positions of the molecule, possess high activity against an estrogen-dependent breast cancer cell line and very good selectivity for other studied normal and tumor cells.The obtained compounds also possess an intense blue fluorescence that makes it possible to visualize them in an intracellular environment.The obtained results suggest promising potential of the active compounds as new anticancer agents potentially useful in the therapy of the estrogendependent malignancies such as breast cancer or as novel fluorescent probes for molecular biological applications.

Materials and Methods
2.1.Synthetic Procedures.Chromatographic purification of compounds was carried out using column chromatography on Acros silica gel (60-200 mesh).Reaction progress and purity of compounds were monitored by TLC on Sorbfil PTLC-AF-A-UF plates.Melting points of the products were determined using a Stanford Research Systems MPA-100 OptiMelt appliance. 1H and 13 C NMR spectra were recorded on a Bruker Avance 400 spectrometer (400.17 and 100.62 MHz).Signals of chloroform (H 7.26, C 77.16) were used as references in the 1 H and 13 C NMR spectra.Coupling constants (J) are reported in Hz.HPLC/MS-experiment was carried out using a Triple TOF 5600, AB Sciex superhigh resolution mass spectrometer from the solution in methanol by turboionic spray (TIS) ionization method with the collision energy with nitrogen molecules of 10 eV.The excitation and emission spectra have been recorded using TECAN M200 Pro plate reader.
Synthesis and analytical characteristics of compounds a, b, b, and a-c were reported in our recent papers [7,8].
The effect of synthesized compounds on the proliferation of the cells was measured by MTT-assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Promega, USA).The cells were seeded in a 96-well plate at a concentration of 2000 cells per well and were cultured in supplemented -MeM (180 L) according to the standard culturing conditions for 1 day.The cell viability was measured at 72 h after treatment with the test agent (20 L).Then, the medium was exchanged for the supplemented medium (80 L).MTT solution (5 mg mL −1 ) was added to a volume of 20 L in each well and incubated for 3.5 h.Then the solution was removed and DMSO (100 L) was added to solubilize the formazan crystals.After 10 min, absorbance was measured at 555 nm (the reference wavelength 650 nm) by a TECAN plate reader (Switzerland).The results were presented as the percent ratio to the control sample treated with PBS.For each compound tested, the IC 50 values ( < 0.05) were generated from the dose-response curves for each cell line.Then the cover slides with the cells were washed again and transferred to microscope slides with the use of a mounting medium Shandon Consul-Mount (Thermo Scientific).The received slides were analyzed on Zeiss LSM 800 confocal laser scanning microscope with ×100 immersion objective and ×20 air objective.The samples with compound b were excited using 405 nm laser and detected in the range of 410-476 nm.

Results and Discussion
The studied compounds were obtained from pyridoxine hydrochloride according to our previously reported approach [8] (Scheme 1).The Wittig reactions of monohydrate with various aromatic aldehydes a-c were performed in CH 2 Cl 2 in the presence of NaH (6 equiv.) at reflux for 30 hours.The target 5-methyl-6-alkenyl substituted derivatives a-c with a trans-configuration of the alkene fragment were the major products of this reaction.The reaction typically led to a number of additional products such as cisalkenes, 5,6-bis-alkenyl substituted compounds, and some minor hyperconjugated structures.Compounds a-c and a cis-alkene compound b could be separated from other products by silica gel column chromatography and were obtained in low to moderate isolated yields (9-30%) as white and yellow crystalline substances.Characteristic features of compounds a-c were the signals of trans-alkene protons at 6.50-7.70ppm (AB-pattern, 3  HH = 15.6 Hz).Synthesis and analytical characteristics of compounds a and b and b have been reported in our recent work [8], while compound c is described in this paper for the first time.
b, 9% [8] (2) RCHO 3a-c Scheme 1: Synthesis of compounds studied in this work.The obtained compounds were screened for cytotoxicity against two cell lines, the estrogen-dependent MCF-7 (breast cancer) cells and the human embryonic kidney 293 (HEK-293) cells (Table 1).To explore SAR dependencies, we also tested the three structurally related trans-5-phenylethenyl substituted pyridoxine derivatives a-c (Figure 2) described in our recent work [7].The widely used anticancer anthracycline antibiotic doxorubicin was used as a broad-spectrum reference drug.In this screening experiment, compounds b and c demonstrated high cytotoxic activity against the MCF-7 cells with IC 50 in the range of 1.9-7.9M, which is comparable to cytotoxic activity of the reference drug (IC 50 1.2 M).At the same time, the cytotoxic activity of b and c against the normal HEK-293 cells (IC 50 22.8 and Figure 2: 5-Phenylethenyl substituted pyridoxine derivatives described in our recent work [5] and studied in the cytotoxicity screening experiment. >293 M, respectively) was significantly lower than that of doxorubicin (IC 50 1.2 M).As a result, compounds b and c have significantly better selectivity index (SI 12 and 37, respectively) as compared to doxorubicin (SI 1.0).
Of note, the active compounds contain in their structures the hydroxy or methoxy groups in para-position of the phenyl ring, which is consistent with the general aforementioned SAR requirements for this class of ER modulators [16].
Compounds b and c were then tested against an extended panel of normal and cancer cell lines (Table 2).In agreement with our underlying theoretical hypothesis, a very good selectivity for the estrogen-dependent MCF-7 cell line was observed for the active compounds as compared to other   An interesting and practically significant property of the studied compounds is their intense blue fluorescence with the maximum at 416 nm (Figure 3).The fluorescence appears as a result of -conjugation in the trans-diarylethylene scaffold.
This feature allowed us to assess their cellular permeability using the visible light microscopy.Figure 4 demonstrates that one of the leading compounds b effectively penetrates through cellular membrane.It can be effectively visualized in cytoplasm and, moreover, is accumulated in nucleus, the cellular compartment where the ERs are localized.
The reasons for the observed effective penetration of the studied compounds through the cell membrane are probably related to their relatively high lipophilicity (log P in the range of 4.3-4.5)and the presence of a basic aromatic nitrogen atom, which are optimal for effective passive diffusion through the lipid bilayers.On the other hand, the presence of pyridoxine moiety can enhance transmembrane transport of the obtained derivatives via active mechanisms.First of all, many cells have specific pyridoxine transporters in their membranes (e.g., [17]).Pyridoxine is a cofactor for more than 140 essential enzymatic reactions, and pyridoxine-modified molecules are often recognized by many pathogenic cells and microorganisms as endogenous.As a result, they have increased cellular permeability and increased bioactivity.For example, the development of pyridoxine-modified nanoparticles for efficient intracellular delivery of doxorubicin (DOX-B6-SA-NP) was reported [18].It was demonstrated that the treatment with DOX-B6-SA-NP kept higher doxorubicin accumulation inside the cells than conventional lipid nanoparticles.

Conclusion
Herein, we have described a novel pyridoxine-based chemotype of bioisosteric analogs of the natural and synthetic drugs based on trans-stilbene scaffold.Despite inherent complications, the synthetic route to these compounds is rather straightforward and can be performed in an industrial scale.The active compounds b and c demonstrate high activity against the estrogen-dependent MCF-7 (breast cancer) cell line with IC 50 in the range of 1.9-7.9M and very good selectivity for other studied tumor and normal cells.The leading compound b also demonstrated clear selectivity for estrogen-dependent breast cancer MCF-7 cells as compared to ER negative MDA-MB-231 breast cancer cells (selectivity index 10), suggesting a potential role of estrogen receptors as targets for the active compounds.The active compounds possessed an intense blue fluorescence with the maximum at 410-420 nm, and this feature allowed us to assess their cellular permeability using the confocal microscopy.Thus, compound b could be effectively visualized in cytoplasm and in nucleus, where the ERs are localized.
The obtained results make the described chemotype a promising starting point for the development of new anticancer agents potentially useful in the therapy of the estrogen-dependent malignancies such as breast cancer or as a novel fluorescent probe for molecular biological applications.The active compounds are currently under active investigation within our group, and their in vitro and in vivo properties will be reported and discussed in a complementary paper.

2 VFigure 1 :
Figure 1: The natural ER agonist estradiol (I), examples of ER targeting drugs (II-IV), and compounds studied in this work (V); highlighted is the trans-diarylethylene motif characteristic of the bioisosteric analogs of the natural agonist.

2. 3 .
Visual Microscopy.Intracellular accumulation and distribution of compound b was assessed by confocal microscopy.Breast cancer cells MCF-7 were seeded into glass slides on a 6-well cultural plate in number of 200000 cells per well.Next day, compound b was added to each well in cultural media from the stock solution in DMSO to reach final concentration of 28 M and maintained 20 minutes at 37 ∘ C. Then the cells were washed by Hanks's solution and fixed for 10 min at 4 ∘ C in 4% paraformaldehyde.

Table 1 :
Results of the cytotoxicity screening experiment against two cell lines.

Table 2 :
Cytotoxicity of compounds b