INTRODUCTION:

This work presents the data from a study of cytotoxic activity of 2-(morpholin-4-yl)-4,5-bis(2’’,2’’,2’’-trinitroethoxy)-1,3,5-triazine which was previously synthesized in our institution.¹ The interest to this substance as a potential anti-tumour agent is supported by the fact that a number of nitroaromatic compounds, including pyrazoline², quinolone³, aminoquinoline⁴, pyrimidine⁵, oxadiazole^6,7, indole⁸, hydantoin⁹, coumarin¹⁰, triazole¹¹ and triazine derivatives¹² have already been tested and used as anti-tumour therapies. Hexamethylmelamine (Altretamine, Figure 1A) which is used in ovarian cancer chemotherapy regimen is an example of such compounds.

Another 1,3,5-triazine derivative, trimethyloltrimethylmelamine (trimelamol, Figure 1B), was used in clinical trials but its high toxicity, poor stability and solubility made it a poor drug candidate, despite its good efficacy for a number of malignancies. A number of substituted triazines were screened for cytotoxicity against cell lines derived from leukemia and solid tumors and showed a promising effect on malignant cells vs. normal ones¹³. A review¹⁴summarizes the data on anti-tumor activity of triazine derivatives collected worldwide to date demonstrating that such compounds may act as cell signaling modulators and have potent antitumor effects against broad spectrum of the malignancies including colon cancer, breast cancer, melanoma, glioblastoma, leukemias, etc.

Therefore, triazine derivatives in many cases possess antitumor activities and, as such, can be articles of interest as the potential pharmacological agents. In our previous study^1, we synthesized 2-(morpholin-4-yl)-4,5-bis(2’’,2’’,2’’-trinitroethoxy)-1,3,5-triazine (Figure 1C). Its chemical structure is similar to many antitumor triazine-based agents, including altretamine, with replacement of amines on the triazine core for morpholino which is thought to be beneficial in the terms of antitumor activity¹⁴, and for trinitroethoxy groups which could improve NO donor activity. Note that triazine derivatives with polynitroethoxy moieties have not previously been tested as the potential antitumor agents.

A B C

Figure 1. Structures of 1,3,5-triazine derivatives having an anti-tumor activity. A, altretamine; B, trimelamol; C, 2-(morpholin-4-yl)-4,5-bis(2’’,2’’,2’’-trinitroethoxy)-1,3,5-triazine.

The aim of this work is to study an in vitro cytotoxic effect of 2-(morpholin-4-yl)-4,5-bis(2’’,2’’,2’’-trinitroethoxy)-1,3,5-triazine on human fibroblasts, peripheral blood mononuclear cells and breast tumour cell cultures.

MATERIALS AND METHODS:

Test compound:

2-(Morpholin-4-yl)-4,5-bis(2’’,2’’,2’’-trinitroethoxy)-1,3,5-triazine was used in the experiments as a test compound. This compound was tested for physicochemical properties, potential biological effects and therapeutic targets using PASS Online (http://www.way2drug.com/passonline/index.php¹⁵) web resource. A significant effect was assumed to meet activity level (Р_а) ≥ 0.7 for separate enzymes and metabolic pathways and ≥ 0.6 for cytotoxicity towards tumour cell lines.

The compound was dissolved in dimethyl sulfoxide (Panreac Quimica SAU, Spain) to the final concentration of 100µM and used ex tempora in appropriate concentrations. Doxorubicin (LENS Farm LLC, Russia), a cytostatic agent commonly used in chemotherapy regimen for many cancers, was used as a reference drug within its concentration range of 0.05-50 nM which is shown to include LD₅₀ for BT474, the tumour cells tested in the study¹⁶. All experiments were performed at least in triplicate.

Cell lines:

Human primary dermal fibroblast culture isolated in Cell Culture Laboratory, Samara State Medical University, Russia, PHA activated peripheral blood mononuclear cells (PBMC), and BT474 HER2-positive breast cancer cell line obtained from the Bioresource Collection of Vertebrate Cells Cultures, Institute of Cytology, Russian Academy of Sciences, all were used to assess the compound cytotoxic activity. Such an approach would ensure comparison of the test compound effect on the malignant target cells, connective tissue cells presenting in most organs and body parts, and blood cells inherently contacting any substance being administered by parenteral mode.

BT474 cells stored in liquid nitrogen were thawed at 37°С using a water bath, centrifuged for 10 min at 200 g, and resuspended in RPMI-1640 medium. Viable cells were counted using microscopy with Trypan Blue, and grown in RPMI-1640 supplemented with 10% foetal calf serum, 2mM L-glutamine, and 1 µg/mL gentamycin (PanEco Biolot, Russia) in МСО-17АI СО₂ incubator (Sanyo, Japan) at 37°С, 5% СО₂ and constant humidity until a confluent monolayer was obtained. The confluent adhesive cells were washed out with 0.25% trypsin/Versene solution and placed into a 96-well plate with the cultural media for subsequent incubation with the test compound and cytotoxicity tests.

Fibroblasts were grown using a primary explant technique¹⁷ with complete cell culture medium (199 medium supplemented with 10% foetal calf serum and 40 µg/mL gentamycin, PanEco Biolot, Russia) in 96-well plates inoculated with 2×10⁴ cells/cm² at 37°С, 5% СО₂ and constant humidity. Before the experiments, the culture was identified and characterised using morphological and genetical methods. This examination confirmed that the cells were unipotential fibroblastic lineage cells. PCR showed no culture contamination, including by mycoplasmas and cytomegalovirus. Upon obtaining a confluent monolayer, the appropriate wells were incubated with the test compound and used in the cytotoxicity tests.

PBMCs were isolated from the heparinized venous blood of adult healthy volunteers by gradient centrifugation with 1.077g/cm³ Ficoll solution (PanEco, Russia). Cells were counted and assessed for viability in a counting chamber using 0.1% Trypan Blue, considering >90% viability satisfactory. Then PBMCs were incubated with 5µg/mL phytohemagglutinin A (PHA, Sigma-Aldrich, USA) and the test compound for 5 days in the complete RPMI-1640 medium supplemented with L-glutamine, streptomycin, 20 mg/mL HEPES, 10% foetal calf serum (PanEco Biolot, Russia) at 37°С, 5% СО₂and constant humidity. PBMC count was 800,000 cells/400 µL. After 5 days, the plates were removed from an incubator and used for cytotoxicity testing.

Cell viability/cytotoxicity tests:

After cultivation and counting, the cells in the 96-well plates with RPMI-1640 were added with the test compounds in various concentrations, incubated for 5 days at 37°С, 5% СО₂ and constant humidity and subjected to cytotoxicity testing. The wells with cells and no test compounds were used as control samples, and the wells without both cells and test compound were blanks. Cytotoxicity tests used in the study included MTT and LDH activity tests. MTT test was done in 96-well plate (200µL well volume) on Tecan Infinite М200 plate reader (Tecan Instruments, Austria). LDH activity was measured in 1-mL cells on SF-56 spectrophotometer (LOMO, Russia). The procedures were performed as per Assay Guidance Manual.¹⁸

Statistical processing:

The data were statistically processed in Microsoft Office Excel 2016. The results considered significant at p < 0.05.

RESULT AND DISCUSSION:

Predicted properties:

Estimated solubility of the test compound in aqueous media is ~10^-4 М (ALOGPS 2.1, http://www.vcclab.org/lab/alogps/). The predicted bioactivity and cytotoxicity values are listed in Table 1.

Table 1 shows that 2-(morpholin-4’-yl)-4,6-bis(2’’,2’’,2’’-trinitroethoxy)-1,3,5-triazine potentially has anti-inflammatory, anti-anginal, anti-ischemic activities and can cause cytotoxic effect on childhood acute myeloid leukemia, prostate carcinoma and breast cancer cells. To confirm the information on the predicted cytotoxic activity of the test compound, in vitro studies were performed yielding the following results.

Toxicity assessment:

Figure 2 shows MTT test results which illustrate that 2-(morpholin-4’-yl)-4,6-bis(2’’,2’’,2’’-trinitroethoxy)-1,3,5-triazine below 100 nM did not affect fibroblasts, PHA-activated mononuclear cells and cancer cells. With higher concentrations, the test compound produced much more significant effect on BT474 breast cancer cells vs. fibroblasts and PBMCs. The test compound significantly affected the tumor cells at concentrations higher than 600 nM (p < 0.05) whereas such cut-offs were approximately 1.5 µM for fibroblasts and 2.0 µM for PBMCs. At the same time, LD₅₀ for BT474 cells was 1.4±0.3 µM, and at higher concentrations of the test compound (2.5 - 5.0 µM), BT474 viability was twice lower vs. normal cells.

Table 1. Predicted bioactivities and cytotoxicity of 2-(morpholin-4’-yl)-4,6-bis(2’’,2’’,2’’-trinitroethoxy)-1,3,5-triazine

Pa	Pi	Activity	Cell line	Tumour
0.948	0.001	Increase in lipid catabolism	-	-
0.836	0.002	Interleukin agonist	-	-
0.799	0.001	Interleukin 12 agonist	-	-
0.778	0.004	Restenosis therapy	-	-
0.747	0.005	Inflammatory bowel disease treatment	-	-
0.691	0.030	Anti-ischemic effect	-	-
0.701	0.076	Phobic disorders treatment	-	-
0.624	0.018	Antianginal effect	-	-
0.611	0.082	Membrane permeability inhibitor	-	-
0.578	0.013	Cytotoxicity	Kasumi 1	Childhood acute myeloid leukaemia
0.578	0.017	Cytotoxicity	PC-3	Prostate carcinoma
0.554	0.002	Cytotoxicity	MDA-MB-361	Breast adenocarcinoma

Figure 2. MTT test results. Cell viability against test compound concentration.

Taken together, these results suggest that 2-(morpholin-4’-yl)-4,6-bis(2’’,2’’,2’’-trinitroethoxy)-1,3,5-triazine could be considered as an anti-tumor drug candidate for HER2-positive breast cancer treatment and recommended for further pre-clinical studies.

Upon that, the suggested therapeutic concentration range for this compound is 0.6-2.0 µM (as indicated with a green area in the Figure 2) as these concentrations significantly reduce cancer cells viability with no changes in the viability of normal cells vs. control values.

Because 2-(morpholin-4’-yl)-4,6-bis(2’’,2’’,2’’-trinitroethoxy)-1,3,5-triazine had a similar concentration-dependent effect on fibroblasts and PBMC viability (correlation coefficient is 0.93, p < 0.01), LDH test was only performed for fibroblasts and BT474 cells.

The test results were essentially the same as for MTT test (Figure 3), except for the cut-off concentration value for fibroblasts which was of 3.6µM, with LD₅₀ for BT474 cells of 2.5±0.4µM.

Figure 3. LDH activity test results. Cell viability against test compound concentration.

Correlation coefficients for MTT vs. LDH tests were 0.78 and 0.96 (p < 0.05) for fibroblasts and BT474 cells, respectively. This means that both LDH and MTT tests yielded a similar estimation of the test agent cytotoxicity for cancer cells, with only an insignificant difference in the sensitivity for fibroblasts.

However, for the safety reasons we recommend to use the therapeutic range of 0.6 - 2.0µM as determined by MTT test.

Comparison to doxorubicin:

We also used MTT test to compare cytopathic effects of the test compound and a widely used anti-tumor drug, doxorubicin. This study has shown that doxorubicin in the concentration range less than 5nM had a mild, statistically insignificant stimulatory effect on BT474 cells proliferation which disappeared with higher concentrations (5-30nM), where the number of survived cells was actually the same as in the control (Figure 4). Significant cytotoxicity was only revealed using LDH test for doxorubicin concentrations of 30-50nM, but MTT test did not suggest any effect of these concentrations on the tumor cells.

Figure 4. BT474 cell viability vs. doxorubicin concentration.

At the same time, there are reports that, with 7-day exposure, LD₅₀ of doxorubicin for BT474 cell line was about 10nM, and the concentrations of about 100nM caused almost 100% cell death¹⁶. This discrepancy seems to be associated with shorter incubation time (5 days) in our experiments. Note that other malignant cell lines are characterized by different effective doxorubicin concentrations vs. the above ones, e.g., LNCaP and HepG2 cells (prostate and liver cancer, respectively) are only susceptible to doxorubicin in micromolar range,^19,20 and doxorubicin therapeutic index (a ratio of 75% inhibitory to 10% lethal dose) in mice is about 0.3²¹.

Thus, 2-(morpholin-4’-yl)-4,6-bis(2’’,2’’,2’’-trinitroethoxy)-1,3,5-triazine in the concentrations larger than 0.6 µM had a significant cytotoxic effect for a breast cancer cell line as demonstrated by both tests. This suggests that the test compound has two order higher cytotoxic concentrations vs. doxorubicin; however, clear therapeutic range makes it a suitable candidate for further studies.

CONCLUSION:

1. 2-(Morpholin-4-yl)-4,5-bis(2’’,2’’,2’’-trinitroethoxy)-1,3,5-triazine possesses a cytotoxic activity for breast cancer cells with a smaller effect on fibroblasts and blood mononuclear cells.

2. A preliminary therapeutic range of this compound is 0.6 - 2.0µM.

3. 2-(Morpholin-4-yl)-4,5-bis(2’’,2’’,2’’-trinitroethoxy)-1,3,5-triazine is a promising candidate for further anti-tumor efficacy studies.

The data obtained makes a basis allowing to conduct further studies of 2-(morpholin-4-yl)-4,5-bis(2’’,2’’,2’’-trinitroethoxy)-1,3,5-triazine which, in a case of their success, could become an active substance of a novel chemotherapy for breast cancer treatment.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

This work was funded within the framework of Prioritet-2030 Programme of Ministry of Education and Science of the Russian Federation.

REFERENCES:

1. Gidaspov AA, Zalomlenkov VA, Bakharev VV, Parfenov VE, Yurtaev EV, Struchkova MI, Palysaeva NV, Suponitsky KY, Lempert DB, Sheremetev AB. Novel trinitroethanol derivatives: high energetic 2-(2,2,2-trinitroethoxy)-1,3,5-triazines. RSC Advances. 2016; 6(41): 34921-34, 10.1039/c6ra05826d.

2. Murugan V, Revathi S, Sumathi K, Geetha KM, Divekar K. Synthesis of Some 1-[Bis-N,N-(2-Chloroethyl)Aminoacetyl]-3,5-Disubstituted-1,2-Pyrazolines as Possible Alkylating Anticancer Agents. Asian J Research Chem. 2010; 3(2): 496-9.

3. Fernandes J, Kumar A, Kumar P. Synthesis and Biological Activity of Some Novel Quinolinyl Chalcones Derived from N-Substituted 2-Quinolones. Research J Pharm and Tech. 2013; 6(12): 1336-9.

4. Battin SN, Manikshete AH, Sarasamkar SK, Asabe MR, Sathe DJ. Synthesis, Spectral, Antibacterial, Antifungal and Anticancer activity Studies of Schiff bases Derived from O-Vanillin and Aminoquinolines. Asian J Research Chem. 2017; 10(5): 660-8, 10.5958/0974-4150.2017.00112.2.

5. Kumar MS, Aanandhi MV. An Insight into the Therapeutic Potential of Pyridopyrimidines as Anticancer Agents. Research J Pharm and Tech. 2018; 11(3): 1259-69. 10.5958/0974-360X.2018.00235.4.

6. Modi V, Shah RS. Synthesis, Characterization and Biological Activities of Some Novel Oxadiazole Derivatives. Asian J Research Chem. 2013; 6(7): 671-5.

7. Kalkotwar RS, Saudagar RB. Synthesis and QSAR Studies of Some 2,5-Diaryl Substituted-1,3,4-Oxadiazole Derivatives. Asian J Research Chem. 2013; 6(11): 985-91.

8. Radhika C, Venkatesham, A, Venkateshwar, RJ, Sarangapani M. Synthesis and Cytotoxic Activity of New Indole Derivatives. Asian J Research Chem. 2010; 3(4): 965-8.

9. Ganatra SH, Patle MR, Bhagat GK. Studies of Quantitative Structure-Activity Relationship (QSAR) of Hydantoin Based Active Anti-Cancer Drugs. Asian J Research Chem. 2011; 4(10): 1643-8.

10. Prasad RK, Loksh KR. Anticancer Potential of Coumarin derivatives: A Review. Asian Journal of Pharmacy and Technology. 2022; 12(4): 391-400.

11. Kubba AARM, Shihab WA, Al-Shawi N, N,. Insilico and in vitro Approach for Design, Synthesis, and Anti-proliferative Activity of Novel Derivatives of 5-(4-Aminophenyl)-4-Substituted Phenyl-2, 4-Dihydro-3H-1, 2, 4-Triazole-3-Thione. Research J Pharm and Tech. 2020; 13(7): 3329-39, 10.5958/0974-360X.2020.00591.0.

12. Kumar R, Kumar N, Roy RK, Singh A. Triazines – A comprehensive review of their synthesis and diverse biological importance. Current Medical and Drug Research. 2017; 1(1): 01-12.

13. Arya K, Dandia A. Synthesis and cytotoxic activity of trisubstituted-1,3,5-triazines. Bioorg Med Chem Lett. 2007; 17(12): 3298-304, 10.1016/j.bmcl.2007.04.007.

14. Dai Q, Sun Q, Ouyang X, Liu J, Jin L, Liu A, He B, Fan T, Jiang Y. Antitumor Activity of s-Triazine Derivatives: A Systematic Review. Molecules. 2023; 28(11). 10.3390/molecules28114278.

15. Filimonov DA, Lagunin AA, Gloriozova TA, Rudik AV, Druzhilovskii DS, Pogodin PV, Poroikiv VV. Prediction of the Biological Activity Spectra of Organic Compounds Using the Pass Online Web Resource. Chemistry of Heterocyclic Compounds. 2014; 50: 444-457,

16. Harris LN, Yang L, Liotcheva V, Pauli S, Iglehart JD, Colvin OM, Hsieh TS. Induction of topoisomerase II activity after ErbB2 activation is associated with a differential response to breast cancer chemotherapy. Clin Cancer Res. 2001; 7(6): 1497-504,

17. Grinberg KM, Kukharenko VI, Lyashko VN, Terekhov SM, Pichugina EM, Freydin MI, Chernikov VG. [Human fibroblast culture for hereditary disease diagnostics]. In [Cell culture methods]. Edited by Pinaev GP. Leningrad: Nauka; 1988. pp. 250-257.

18. Markossian S, Grossman A, Brimacombe K, Arkin M, Auld D, Austin C, Baell J, Chung TDY, Coussens NP, Dahlin JL, Devanarayan V, Foley TL, Glicksman M, Gorshkov K, Haas JV, Hall MD, Hoare S, Inglese J, Iversen PW, Kales SC, Lal-Nag M, Li Z, McGee J, McManus O, Riss T, Saradjian P, Sittampalam GS, Tarselli M, Trask OJ, Wang Y, Weidner JR, Wildey MJ, Wilson K, Xia M, Xu X, editors. Assay Guidance Manual. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004.

19. Bowyer C, Lewis AL, Lloyd AW, Phillips GJ, Macfarlane WM. Hypoxia as a target for drug combination therapy of liver cancer. Anticancer Drugs. 2017; 28(7): 771-80, 10.1097/CAD.0000000000000516.

20. Kim E, Jung Y, Choi H, Yang J, Suh JS, Huh YM, Kim K, Haam S. Prostate cancer cell death produced by the co-delivery of Bcl-xL shRNA and doxorubicin using an aptamer-conjugated polyplex. Biomaterials. 2010; 31(16): 4592-9, 10.1016/j.biomaterials.2010.02.030.

21. Arakawa H, Morita M, Kodera T, Okura A, Ohkubo M, Morishima H, Nishimura S. In vivo anti-tumor activity of a novel indolocarbazole compound, J-107088, on murine and human tumors transplanted into mice. Jpn J Cancer Res. 1999; 90(10): 1163-70, 10.1111/j.1349-7006.1999.tb00691.x.

Received on 02.11.2023 Revised on 28.10.2024

Accepted on 30.05.2025 Published on 08.11.2025

Available online from November 13, 2025

Research J. Pharmacy and Technology. 2025;18(11):5142-5146.

DOI: 10.52711/0974-360X.2025.00742

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.