Cancer is a major risk disease affecting human survival. The pharmaceutical companies are continuing searching for new drug candidates with promising anticancer activities, and reduced side effects. The current work aimed at synthesized a new tripeptide with potential pharmacological properties. L-Valine methyl ester was used to prepare cyclo (Nα-dinicotinoyl)-bis-[(L-valinyl)-L-lysine methyl ester. The new compound revealed promising in vitro cytotoxic activities against different neuroblastoma, cervical carcinoma, fibrosarcoma as well as hepatocellular carcinomas. Furthermore, we also found that the obtained IC50 of the compound decreased by about 50% during its in vivo anti-prostate cancer evaluation. Furthermore, the mechanism of action studies proposes that the new prepared derivative affects cancer cells trough the inhibition of VEGFR-2 kinase enzyme

Al –Omar, M  A

Amr, A  E

Azab, Mohammad  E.

Elsayed, E  A

Sabry, Nermien  M.

Online Publishing @ NISCAIR

Journal of Scientific & Industrial Research Vol. 79, March 2020, pp. 222-225       Evaluation of in vitro and in vivo Cytotoxic Activities and Kinase Inhibition of Newly Synthesized Cyclo (Nα-Dinicotinoyl)-Bis-[(L-Valinyl)-L-Lysine Methyl Ester] A E Amr1,2, M A Al –Omar2, E A Elsayed3,4*, Mohammad E. Azab5and Nermien M. Sabry2 1 Pharmaceutical Chemistry Department, College of Pharmacy, Drug Exploration & Development Chair (DEDC), King Saud University, Riyadh 11451, Saudi Arabia. 2Applied Organic Chemistry Department, National Research Center, Cairo, Dokki 12622, Egypt. 3Bioproducts Research Chair, Zoology Department, College of Science, King Saud University, Riyadh 11451, Kingdom of Saudi Arabia 4Natural and Microbial Products Department, National Research Centre, Dokki, Cairo 12311, Egypt 5Synthetic Organic Lab, Chemistry Department, Faculty of Science, Ain Shams University, Abbassia, Cairo 11566, Egypt Received 9 November 2019; revised 13 October 2019; accepted 16 January 2020 Cancer is a major risk disease affecting human survival. The pharmaceutical companies are continuing searching for new drug candidates with promising anticancer activities, and reduced side effects. The current work aimed at synthesized a new tripeptide with potential pharmacological properties. L-Valine methyl ester was used to prepare cyclo (Nα-dinicotinoyl)- bis-[(L-valinyl)-L-lysine methyl ester. The new compound revealed promising in vitro cytotoxic activities against different neuroblastoma, cervical carcinoma, fibrosarcoma as well as hepatocellular carcinomas. Furthermore, we also found that the obtained IC50 of the compound decreased by about 50% during its in vivo anti-prostate cancer evaluation. Furthermore, the mechanism of action studies proposes that the new prepared derivative affects cancer cells trough the inhibition of  VEGFR-2 kinase enzyme. Keywords: Macrocyclic tripeptidopyridine ester, in vitro cytotoxicity, in vivo antiprostate, VEGFR-2 kinase activityIntroduction Cancer is one of the most powerful mankind threatening disease, due to its high mortality rate as well as difficult treating procedures and harmful  side effects1,2. The pharmaceutical industry encourages continuous discovery of novel  organic derivatives possessing potential anticancer activities, with the aim to combat cancer3. Heterocyclic ester derivatives have been identified as biologically significant components of medicinal chemistry1, such as: antiviral, anti-inflammatory, antioxidant, anti-HIV, and anticancer agents4,5. On the other hand, peptide candidates have been recognized as interesting exhibiting a broad spectrum of biological and pharmacological activities6-8. In view of these observations and in continuation of our previous work9-17 in heterocyclic and peptide chemistry, herein we reported thein vitro and in vivo cytotoxic activities and kinase inhibition of newly synthesized cyclo (Nα-dinicotinoyl)-bis-[(L-valinyl)-L-lysine methyl ester] derivative. Materials and Methods Synthesis of cyclo (Nα-dinicotinoyl)-bis-[(L-valinyl)-L-lysine methyl ester] (3) Compound 3 was synthesized by reaction of 3, 5-pyridine dicarbonyl dichloride (1) with L-valine methyl ester followed by hydrolysis to corresponding diacid 2, which wascyclized with L-lysine methyl ester by using Mixed anhydride method according to reported procedure. The tested compound 3 was previously characterized by physical and spectroscopic data which were reported by Amr et al.15.  Biological Activities  In vitro cytotoxicity screening Various cancer cell lines (Table 1) were evaluated. Cells were cultivated and propagated using standard medium and conditions using standard cell culture protocols developed by our group18-21. After inoculation, plates were incubated at standard conditions for 24 h. After that, two plates/cell line, were fixed using TCA. Control references were dissolved in DMSO at comparable concentration doses. Plates were then processed as previously mentioned in the protocols. Absorbance was read at 515 nm and the IC50values were calculated from the regression analysis of the data.  ——————— *Author for Correspondence: E-mail: eaelsayed@ksu.edu.sa ELSAYED et al.: EVALUATION OF IN VITRO AND IN VIVO CYTOTOXIC ACTIVITIES   223In vivo cytotoxic activity  During this section, we used male Wistar rats obtained from the Animal House Colony, Research Institute of Ophthalmology, Giza, Egypt. Experiments were performed according to standard protocol22.  VEGFR-2 kinase activity assays by ELISA   This work was carried out according to standard protocol23 using Sorafenibas a positive control. The absorbance was measured at 492 nmand IC50  values were calculated from the inhibition rate percentage equation23.   Results and Discussion Chemistry Macrocyclic tripeptidopyridine candidate 3 was synthesized (Figure 1) and characterized by physical, chemical and spectroscopic evidences in advance according to our previous work19. In the current study, we report the evaluation and activities of these compounds as possible anticancer agents.  Biological Screening Screening of in vitro cytotoxic activities The cytotoxicity of the tested compounds was determined on seventeen different human cancer cell lines (Figure 2). Screening results showed that the prepared compound exhibited variable potentials against difference tested cancer cells. This is in accordance with our previous results obtained showing that different cells respond differently towards investigated drugs15,17-19. This is due to differences between different cell types in terms of membrane structure and cell organization. Furthermore, comparing obtained results (in terms of IC50 values) with those recorded for standard pharmaceutical drugs showed that comparable IC50 Table 1 — Different cell lines and reference drugs used throughout the work Cell line Type Cell line Type Cell line Type KB Cervical carcinoma U937 Leukemia HeLa Cervical carcinoma SKOV-3 Ovarian carcinoma K562 Leukemia MCF-7 Breast carcinoma SF-268 CNS cancer G361 Melanoma HT1080 Fibrosarcoma NCI H460 Non-small lung cancer SK-MEL-28 Melanoma HepG2 Liver carcinoma RKOP27 Colon adenocarcinoma GOTO Neuroblastoma PC-3 Prostate carcinoma HL60 Leukemia NB-1 Neuroblastoma   Reference drugs  Sorafenib Fluorouracil Doxorubicin Cytarabine Gemcitabine Capecitabine Aldesleukin Paclitaxel Epirubicin Imatinib Bicalutamide Flutamide    Fig. 1 — Synthetic route for cyclo (Nα-dinicotinoyl)-bis-[(L-valinyl)-L-lysine methyl ester] 3. J SCI IND RES VOL 79 MARCH 2020   224values were obtained for colon adenocarcinoma  (4.56 and 4.33 nM for compound3 and Capecitabine, respectively), for the neuroblastoma NB-1 (6.0 and 5.15 nM for compound 3 and Doxorubicin, respectively), as well as the prostate cancer PC-3 cell line (9.76 and 8.22 nM for compound 3 and Bicalutamide, respectively). On the other hand, our prepared compound showed higher potential cytotoxic activities, where it showed almost  2-folds activity against the neuroblastoma GOTO cells (IC50: 2.34 and 4.37 nM for compound 3 and Doxorubicin, respectively), 2.6-folds activity against HeLa cells (IC50: 4.55 and 11.8nM for compound 3  and Paclitaxel, respectively), 21-folds activity against the fibrosarcoma HT1080 (IC50: 6.2 and 130.24 nM for compound 3 and Imatinib, respectively) and 5.5-folds activity against the HepG2 cells (IC50: 6.2 and 34.4 nM for compound 3 and Gemcitabine, respectively).  In vivo activity In vivo anti-prostate data showed potential anti-prostate activity for compound 3 in experimental animal model. The obtained significant ED50 value observed for compound (5) (5.26 ± 0.034 μM) was 2.2-folds higher than that obtained for the Flutamide reference drug (11.6 ± 0.09 μM).  VEGFR-2 inhibition Anti-VEGFR-2 results revealed that the synthesized compound (3) exhibited potent VEGFR-2 inhibitory activity, where the obtained IC50 value (1.91nM) showed about 5% activity higher than the Sorafenib reference drug (2 nM).   Conclusion Within the framework of the current investigation, we were able to synthesize a new cyclo  (Nα-dinicotinoyl)-bis-[(L-valinyl)-L-lysine methyl  ester from the reaction of dicarbonyl dichloride and  L-valine methyl ester. The new derivative exhibited potent in vitro cytotoxic activities against neuroblastoma, cervical carcinoma, fibrosarcoma as well as hepatocellular carcinomas in comparison to standard   Fig. 2 — Cytotoxic activities of Cpd. 3 against different evaluated cell lines ELSAYED et al.: EVALUATION OF IN VITRO AND IN VIVO CYTOTOXIC ACTIVITIES   225reference drugs. Furthermore, the new derivative showed 2.2-folds increased in vivo anti-prostate cancer activity, compared to control drug. These results support the potential use of the new derived tripeptide as a promising pharmaceutical molecule with potential in vitro and in vivo anticancer properties.  Abbreviations IC50 Half maximal inhibitory concentration  ED50 Median effective dose  Acknowledgment  Authors are grateful to King Saud University for funding the work through Researchers Supporting Project (Project No. RSP-2019/66)".  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Evaluation of in vitro and in vivo Cytotoxic Activities and Kinase Inhibition of Newly Synthesized Cyclo (Nα-Dinicotinoyl)-Bis-[(L-Valinyl)-L-Lysine Methyl Ester]

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Evaluation of in vitro and in vivo Cytotoxic Activities and Kinase Inhibition of Newly Synthesized Cyclo (Nα-Dinicotinoyl)-Bis-[(L-Valinyl)-L-Lysine Methyl Ester]

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