Synthesis of New 1 , 2 , 3-Triazolo-naphthalimide / phthalimide Conjugates via ‘ Click ’ Reaction : DNA Intercalation and Cytotoxic Studies

All reagents and solvents were obtained from commercial suppliers and were used without further purification. Analytical thin layer chromatography (TLC) was performed on Merck precoated silica gel 60-F254 (0.5 mm) aluminum plates. Visualization of the spots on TLC plates was achieved by UV light. 1 H and 13 C NMR spectra were recorded on Varian Gemini 200 or Varian Unity 400 or Varian Inova 500 or Bruker Avance 300 MHz, making a solution of samples in CDCl3 solvent using tetramethylsilane (TMS) as the internal standard. Chemical shifts for 1 H and 13 C are reported in parts per million (ppm) downfield from tetramethylsilane. Spin multiplicities are described as s (singlet), bs (broad singlet), d (doublet), dd (double doublet), t (triplet), q (quartet), and m (multiplet). Coupling constant (J) values are reported in hertz (Hz). HRMS were determined with Agilent QTOF mass spectrometer 6540 series instrument. Wherever required, column chromatography was performed using silica gel (60-120 or 100-200) or neutral alumina. The anhydrous reactions are carried under nitrogen positive pressure using freshly distilled solvents. All evaporation of solvents was carried out under reduced pressure on Heidolph rotary evaporator below 45 °C.


Introduction
Cancer is considered as one of the lethal diseases worldwide and the incidence of cancer has been rising in major regions of the world with predicted substantive increase to 19.3 million by the year 2025. 1 Although the scientific advances focused on finding exact pathophysiology of the disease and tremendous efforts have been made on early detection of cancer, the overall mortality rate has not declined. 2In this connection, there is an immense need to design and synthesize effective new chemotherapeutic agents to combat cancer.Due to complexity with redundant and robust biological networks, cancer cells exhibits uncontrolled cell proliferation, metastasis and also become resistant to apoptotic signals. 3As cancer cells are highly proliferative tissues, the DNA becomes one of the most promising biological targets to develop antitumor agents. 4DNA replication has an invaluable role in cancer cell division, hence polycyclic planar molecules such as doxorubicin, acridines, anthraquinones, distamycins, naphthalimides and phenanthrene derivatives are well recognized as DNA targeting antitumor agents. 5aphthalimides are aromatic heterocycles with profound biological significance and they serve as core scaffold for many anti-tumor, anti-inflammatory, antidepressant, antiprotozoal and antiviral compounds. 6Planarity is the most important prerequisite for DNA intercalation and also facilitates embedding into DNA base pairs. 7Owing to their tricyclic planar structure, naphthalimide is primarily responsible for its intercalation with DNA to perturb the cellular events, thereby prevents cancer cell divison. 8epresentative examples of naphthalimide based molecules such as mononaphthalimide (i.e., amonafide, Figure 1) and bisnaphthalimides (i.e., elinafide) have reached the clinical trials, but due to central neurotoxicity and limited efficacy in solid tumors, the clinical development was regrettably terminated. 9However, their structural modification lead to potent antitumor agents with improved efficacy and toxicological profile. 10In 2005, Cholody et al. 11 investigated asymmetrical bi-functional antitumor agents by conjugating imidazoacridone moiety to the naphthalimide core.This imidazoacridone linked nitro naphthalimide conjugate efficiently interacted with DNA and induction of apoptosis.Moreover, naphthalimides conjugated with pyrrolo[2,1-c] [1,4]benzodiazepines through a pyrazine moiety with an alkane spacer has also been proved to increase the DNA binding affinity and anticancer activity. 12n the dimeric form of intercalators such as bisphenanthridines, bisnaphthalimides and bisphenazine carboxamides have also displayed potent antitumor potential.The promising in vivo activities of drugs such as bisnafide (bisnaphthalimide) 13 and WP631 (bisanthracyclines) 14 have stimulated to search for novel bifunctional DNA ligands.The basic principle behind the design of dimers is to enhance sequence recognition and DNA binding affinity.However, in some instances, the second chromophore does not play the role of intercalator, instead it can serve as a hook to trap DNA binding proteins and cofactors.
On the other hand, 1,2,3-triazoles are considered as privileged building blocks in the context of bioconjugates.They are highly stable under basic and acid hydrolysis including oxidative and reductive conditions.Moreover, this heterocycle is the bioisostere of amide and is capable of interacting with biomolecular targets through hydrogen-bonding. 15This scaffold is well recognized for their multitude of biological properties such as anti-malarial, anti-leishmanial, anti-trypanosomal, anti-allergic, antifungal, antibacterial, antitubercular, anti-HIV, anticancer, antimalarial and antiviral properties. 16ascinatingly, it can interact with DNA and also acts as a supporting motif for DNA targeting drugs. 17,2,3-Triazoles tethered to various bioactive molecules such as β-carbolines and phenanthrenes also resulted in enhanced antitumor potential through effective DNA intercalation. 18Quian and co-workers 19 have reported that 1,8-naphthalimide substituted triazole derivatives could enhance the cytotoxicity and improves the affinity towards calf thymus (CT)-DNA.The enhanced cytotoxicity has been attributed to the presence of triazole nucleus.Prompted by the above reports, in the present study we have designed and synthesized a library of 1,2,3-triazolo based naphthalimide/phthalimide conjugates and evaluated their in vitro cytotoxicity and DNA intercalating capabilities.
The Cu I -catalyzed 1,3-dipolar cycloaddition of azide and alkynes (CuAAC) or 'click chemistry' can rapidly yield bioactive molecules linked through 1,2,3-triazole. 20Cu I catalyst allows the cycloaddition to provide 1,4-regioselectivity in 1,2,3-triazole ring formation.Currently, the most widely used reaction conditions for the 1,4-regiospecific method is the use of an organic solvent, such as t-BuOH or CH 2 Cl 2 , water, CuSO 4 .5H 2 O and sodium ascorbate.Despite the numerous advantages of the CuAAC reactions, the organic azides employed therein are hazardous and their handling is very risky. 21n this context, efficient protocols that eliminate the handling of organic azides are highly preferable. 22In continuation of our earlier efforts dedicated to triazole chemistry towards the synthesis of diverse bioactive molecules, 23 recently, we have also reported a novel, simple and efficient bis[(tetrabutylammonium)di-µ-iododiiododicuprate(I)] catalyzed one-pot, three component 1,3-dipolar cycloaddition of alkyl/benzyl bromides with sodium azide and alkynes for the regioselective synthesis of 1,4-disubstituted 1,2,3-triazoles in water. 24Herein we have applied this efficient protocol for the synthesis of a variety of diversely substituted 1,2,3-triazolo-naphthalimide/ phthalimide conjugates.The advantages of this onepot reaction are the use of water as solvent and in situ generation of azides from alkyl halides and thus handling of hazardous azides is avoided.The newly synthesized compounds were evaluated for their in vitro cytotoxicity and DNA binding affinity.

Pharmacology Cytotoxic activity
The newly synthesized 1,2,3-triazolo-naphthalimide/ phthalimide conjugates were evaluated for their in vitro cytotoxicity against different cancer cell lines such as lung (A549), prostate (PC-3), breast (MCF-7), cervical cancer (HeLa) and a normal cell line (RPE1) by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. 25Cell lines were treated with the compounds at 50 µM concentration for 48 h and growth inhibition percentage was noted.Among them, some of the representative compounds 6b, 6c, 6d, 6j, 6k, 6p, 6u, 6v and 7a displayed cytotoxicity above 50% at 50 µM and were selected to screen for the dose dependent responses and IC 50 (concentration required to inhibit 50% of the tumor cells) values for these active compounds were reported.The IC 50 (µM) values of the active compounds and the reference drug doxorubicin are listed in Table 1.
It is observed from the cytotoxicity results that derivatives containing planar phthalimide or naphthalimide nucleus in their structures showed moderate cytotoxicity and compound 6c, possessing naphthalimide and triazole cores linked through a 4 carbon linker, displayed good cytotoxic activity on A549 lung cancer cells with an IC 50 value of 7.6 ± 0.78 µM (Table 1).Some of the tested compounds 6d, 6k, 6u, and 7a displayed moderate cytotoxicity against the cervical and prostate cancer cells.From careful analysis of IC 50 values, it was observed that most of the dimers containing phthalimide or naphthalimide scaffolds linked through various triazole linkers were adequately active against the cancer cell lines.Further, cytotoxicity of the most active compounds was also tested on normal cell line (RPE1).Interestingly, most of the compounds were nontoxic to the normal cells and the most active compound 6c showed selectivity index (ratio of cytotoxicity of normal cell line to that of cancer cells) of 6 folds approximately.Based on the observed cytotoxicity results, one of the active compounds 6c was taken-up for examining the mechanism of cancer cell growth inhibition through cell staining techniques.Additionally, to prove the DNA interaction of these planar molecules, we have also investigated the relative viscosity alterations of these new molecules 6c, 6d, 6p and performed molecular docking studies to explain the possible mode of interaction with DNA.

Acridine orange/ethidium bromide (AO/EB) staining
Acridine orange/ethidium bromide (AO/EB) fluorescent staining assay was performed to differentiate the live, apoptotic, and necrotic cells. 26AO penetrates the healthy cells and stains the nuclei green, whereas EB enters only into membrane disintegrated cells and stain the nucleus red.
It can be observed from Figure 2 that the green color of the control cells is due to their normal morphology.From the Figure, it was clear that the cell viability is decreased in concentration dependent manner.Fluorescence microscopic images of the cells treated with 20 µM of compound 6c clearly showed the altered morphological characteristics, suggesting that the compound 6c induced cell death in A549 cells.DAPI staining DAPI (4',6-diamidino-2-phenylindole) is a fluorescent stain that effectively binds to A-T rich regions in DNA and reveals the nuclear damage.DAPI permeates the live cell membrane with less efficiency.Therefore, the intensity of staining in live cells is lesser, whereas apoptotic cells are stained bright due to the presence of condensed nucleus.Therefore, we have examined the effect of compound 6c on A549 cells by using DAPI staining. 27This technique differentiates dead and live cells based on nuclear morphology.DAPI stains the nucleus bright blue by forming a fluorescent complex with chromatin.As observed from Figure 3, the nuclear structure of control cells was intact whereas compound 6c treated A549 cells exhibited condensed nuclei, indicating the characteristic features of cancer cell death.

Relative viscosity study
Relative viscosity studies are helpful to predict the interactions of tested compounds with DNA.These studies were performed for the active compounds 6c, 6d, and 6p along with ethidium bromide, Hoechst 33258, and doxorubicin to study their interactions with DNA by measuring the changes in relative viscosity.Intercalation of small molecules like ethidium bromide between the base pairs of DNA leads to the substantial increase in viscosity; whereas a minimal change in viscosity is observed with groove binders like Hoechst 33258.
Doxorubicin is a well-known intercalator, which shows a rapid increase in viscosity down the concentration.The addition of these compounds 6c, 6d, and 6p to CT-DNA results in a gradual increase of viscosity in concentration  depending manner.This infers that the naphthalimidetriazole congeners bind to DNA by intercalation.The Figure 4 shows a graph plotted between concentration (compound/CT-DNA) and viscosity ((η/η 0 ) 1/3 ) on x and y-axis, respectively, with ethidium bromide, Hoechst 33258 and doxorubicin as standards.

Molecular docking
Molecular docking studies were performed for the most active compounds 6c, 6d and 6p using duplex DNA (PDB: 209D) 28 by XP Glide 7.4 (Schrödinger 2017-1) 29 with default settings (Figure 5).The perfect planar insertion of the naphthalimide ring between G-C base pairs in the site of intercalation was observed.Further, the triazole and alkyl chain acts as the linker and orients the complementary substitution (naphthalimide in 6c and 6p and phthalimide moiety in 6p) into the minor groove.Moreover, the docked poses were stabilized by π-π stacking interactions of naphthalimide ring and hydrogen-bonding interactions between the carbonyl oxygens of naphthalimide towards base pairs of DNA in the minor groove.Thereby, dual mode of binding with DNA (DNA intercalation and minor groove binding) was observed with these compounds supporting their efficacy as DNA interacting agents.These results were compatible with in vitro cytotoxicity and relative viscosity studies.

Conclusions
In the present work, a new series of 1,2,3-triazolonaphthalimide/phthalimide conjugates were synthesized by employing Cu I complex.This protocol involved  three component 1,3-dipolar cycloaddition of alkyl/ benzyl bromides, sodium azide and alkynes.The synthesized compounds were evaluated for their in vitro cytotoxic potential against lung (A549), prostate (PC-3), breast (MCF-7) and cervical cancer (HeLa) cell lines.Compound 6c displayed IC 50 of 7.6 ± 0.78 µM against A549 lung cancer cells.Compound 6c also caused the death of cancer cells and induced apoptosis in concentration dependent manner as observed from DAPI and AO/EB staining.Further, relative viscosity studies indicated that increase in concentration of compounds 6c, 6d and 6p proportionally enhanced viscosity suggesting that these compounds binds to DNA by intercalation.Molecular docking studies also supported the DNA interaction of these compounds.Overall, the current study established that these derivatives have the potential to be advanced as DNA interactive agents for the treatment of lung cancer.

Figure 2 .
Figure 2. AO/EB staining in A549 cells treated with various concentrations of compound 6c.Apoptotic features and dead cells were observed.

Figure 3 .
Figure 3. DAPI staining: A549 cells treated with compound 6c and stained with DAPI.The images were captured with fluorescence microscope with a DAPI filter.

Figure 4 .
Figure 4. Graphical representation of relative viscosity studies using compounds 6c, 6d and 6p with doxorubicin, Hoechst-33258, and ethidium bromide as reference standards.

Figure 5 .
Figure 5. Side view (a) and top view (b) of 6c; side view (c) and top view (d) of 6d; side view (e) and top view (f) of 6p, interaction with duplex DNA.DNA is represented as ribbon in blue color and compounds 6c, 6d and 6p are represented in yellow, green and orange color, respectively.