Nagham Mahmood Aljamali¹*and Imad Kareem Alwan Alsabri²

¹Synthetic Chemistry Field, Department of Chemistry, Iraq.

²Master in Oncology, College of Medicine, Kufa University, Iraq.

Corresponding Author E-mail : dr.nagham_mj@yahoo.com

DOI : https://dx.doi.org/10.13005/bpj/1925

Abstract

Cancer tumors cause not few deaths among the total deaths in the world, despite the great progress in the treatment of cancer, so it has become necessary to understand the molecular explanations that contribute to the development and progress of cancer and to search for more effective and less toxic treatments to treat this disease.The work included the innovation and development of the drug trimethoprim by linking it with the innovative compound sulfazane and  creation of several derivatives of sulfazane –trimethoprim for the first time then studying the behavior and impact of innovative compounds on vital activities as anti-cancer tumors by following an innovative  procedures of preparation for sulfazane compounds ,then linking them with several chemical reactions with the drug trimethoprim to develop its effectiveness and efficiency. Several spectral techniques were used to diagnose and demonstrate the formation of innovative derivatives (sulfazane- trimethoprem) thereof (FT.IR - Spectra ,H.NMR – Spectra , HMBC- Spectrum ,Mass Spectra), chemical properties, Flowing  through  TLC ,in addition to  laboratory study of cancerous tumors. The results indicated  to formation of these drug derivatives by appearance of new bands and disappearance of bands in starting compounds., besides to conclusions from our paper that gave good data for inhibition efficiency for these drug derivatives  against cancer cells.

Keywords

Azo; Coupling; Cancer; Innovation; Invented Compounds; New Reaction of  Diazonium; Sulfide-Azo; Sulfazane; Tumor; Toxicity; Trimethoprim; (-S-N=N-)

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Copy the following to cite this article: Aljamali N. M, Alsabri I. K. A. Development of Trimethoprim Drug and Innovation of Sulfazane-Trimethoprim Derivatives as Anticancer Agents. Biomed Pharmacol J 2020;13(2).

Copy the following to cite this URL: Aljamali N. M, Alsabri I. K. A. Development of Trimethoprim Drug and Innovation of Sulfazane-Trimethoprim Derivatives as Anticancer Agents. Biomed Pharmacol J 2020;13(2). Available from: https://bit.ly/2UJpCEB

Introduction

The newly  innovative  sulfazane  compounds  that  were  invented  and  prepared  for the first  time  by the researcher (Dr. Nagham Aljamali in year 2019  in the first research work⁽¹⁾) The researcher laid the first foundations for the innovative sulfazane compounds in terms of (methods of preparation , their properties , chemical and physical characteristics, their colors, stability, some of their vital applications and behavior towards some types of bacteria and fungi).^(1,2) Now in this research work continued to prepare other drug derivatives by linking sulfazane to the drug trimethoprim via (-S-N=N-) sulfazane  group to study its effect on cancerous tumors.Trimethoprim contains the active ingredient trimethoprim, an antibiotic used to treat infections with bacteria.Bacterial cells in order to grow and multiply need a genetic material (DNA) to produce DNA and need folic acid (folate).^(3-10) Methoprim works by preventing bacteria from producing folic acid and without it bacteria cannot produce DNA, and thus become unable to reproduce^(11-22) and increase numbers, so methoprim stops the spread of infection, and kills the remaining bacteria by the immune system.^(23-34)

Trimethoprim (C14H18N4O3) : 5-(3,4,5-Trimethoxybenzyl)pyrimidine-2,4-diamine., its  names (Proloprim, Monotrim, Triprim, others) ,M.Wt : 290.32 g/mol .,It is used to treat bacterial infections in the urinary tract and also to prevent frequent bacterial infections in the urinary tract,^(35-43) as well as to treat bacterial infections in the lungs and bronchi (respiratory system), such as acute bronchitis, chronic bronchitis or pneumonia.^(44-56) The cancer  is a type of non-infectious disease that is characterized by uncontrolled growth of cells, there are more than (100) different types of cancer, which were classified according to the type of cell that was affected at the beginning, and cancer causes a death rate of more than 20% of the total deaths in the world on according to the World Health Organization, where lung cancer is the most prevalent,^(57-59) followed by breast, colon and prostate cancer, despite significant progress in cancer treatment, the death rate has not decreased, so it has become necessary to understand the molecular explanations that contribute to the development and progress of cancer and search for more treatments. Efficacy and least toxic for a processor with this disease,^(60-63) there are many methods for treating this disease, including tumor lift, multiple chemical treatments, and radiological treatments

Experimental  Part

All chemicals compounds supplied from Sigma –chemicals company and fluka –chemicals company.

An innovative method^{(1, 2)} was used to develop the trimethoprim drug by linking it to an innovative sulfazane compounds through several steps, chemical reactions and reaction conditions that differ for each of the innovative derivatives prepared in this research. The chemical composition of the innovative derivatives prepared in this research was proven by several  investigation techniques (FT-IR spectra (FT-IR 8300 Shimadzu) with the range (400-4000)cm^-1  using discs of  KBr., 1H.NMR–Spectra in  solvent (d-DMSO) Fourier transformation broker spectrometer ,operating at (400MHz)., HMBC- Spectrum ,Mass spectra for some of them) in Kashan university, and the preparation was followed by (TLC), then a laboratory study of cancer cells line to know the effectiveness and efficiency of  the innovative compounds.

Paths  of  Synthesis^{(1, 2)}

Innovation and Creation of Sulfazane- Trimethoprim {1}

P-thiol aniline (0.01 mole) with (0.01 mole) ammonium thiocyanate in bromine with glacial acetic acid ,after rotation for (2 hrs) at (10 C°) via three reactions ,then precipitation filtered, washed, dried, purification by recrystallization, then (0.01 mole) from precipitation dissolved in basic alcoholic solution, and added  to  acidic diazo- trimethoprim salt in three steps according to invented  procedure in papers^{(1, 2)} ,after (3 days), filtered ,washed ,dried ,recrystallized to yield  sulfazane- trimethoprim {1}.

Scheme 1: Creation of Sulfazane- Trimethoprim {1} Click here to View Scheme

 

Innovation and Creation of Sulfazane- Trimethoprim {2}

Cystein(0.01 mole) in basic alcoholic solution by two steps, then added  to acidic diazo- trimethoprim salt in three steps according to invented  procedure in papers^{(1, 2)} ,after (3 days), filtered ,washed ,dried ,recrystallized to yield  sulfazane- trimethoprim {2}.

Scheme 2: Creation of Sulfazane- Trimethoprim {2} Click here to View Scheme

 

Innovation and Creation of Sulfazane- Trimethoprim {3}

Thiol benzoic acid (0.01 mole) in basic alcoholic solution by two steps, then added  to acidic diazo- trimethoprim salt in three steps according to invented  procedure in papers^{(1, 2)} , after (3 days), filtered ,washed ,dried ,recrystallized to yield  sulfazane- trimethoprim {3}.

Scheme 3: Creation of Sulfazane-Trimethoprim {3} Click here to View Scheme

 

Innovation and Creation of Sulfazane- Trimethoprim {4}

2-Thiol imidazole in previously steps was prepared ,then (0.01 mole) dissolved in basic alcoholic solution by two steps, then added to acidic diazo-trimethoprim salt in three steps according to invented procedure in papers^{(1, 2)} , after (3 days), filtered ,washed ,dried ,recrystallized to yield  sulfazane- trimethoprim {4}.

Scheme 4: Creation of Sulfazane-Trimethoprim {4} Click here to View Scheme

 

Results

Our research described  invented  procedure^{(1, 2)}  via  original  process to  creation and preparation of  Sulfazane  compounds linked with trimethoprim drug via designation of reaction conditions, type of  used catalyst, mechanism  of this  reaction, we called it (Name–Sulfazane) as a first and Original preparation to this  novel type of trimethoprim- derivatives., then all these  drug derivatives  investigated via numerous spectral techniques and other  chemical with physical studies :

Spectral   Evidences

FT.IR- Spectra

The infrared spectrum is the first technique that has demonstrated, with certain evidence, the creation of innovative, advanced derivatives in this study through the emergence of effective and functional grouping frequencies in derivatives indicating the reason for the accuracy of their preparation and the correctness of the innovative procedure to prepare them :

Sulfazane- Trimethoprim {1}

Bands at (-OCH₃) methoxy group: 1183 ,(-N=N-S-) Azo-Sulfide: (1398, 1489, 1500) , (S-CH-) Sulfide : 1226 ,(C-S) endocyle of benzothiazole : 756 ,(C=N) endocycle of pyrimidine: 1652 ,(-NH₂) amine group: (3358 ,3409).

Sulfazane- Trimethoprim {2}

Bands at (-OCH₃) methoxy group: 1118 ,(-N=N-S-) Azo-Sulfide: (1363, 1454, 1496) , (S-CH₂-) Sulfide : 1224 , (C=N) endocycle of pyrimidine: 1664 ,(-NH₂) amine group: (3314 ,3379), (CO-O-)carbonyl of carboxyl : 1729 .

Sulfazane- Trimethoprim {3}

Bands at (-OCH₃) methoxy group: 1134 ,(-N=N-S-) Azo-Sulfide: (1377, 1462, 1499) , (S-CH-) Sulfide : 1231 , (C=N) endocycle of pyrimidine: 1651 ,(CO-O-)carbonyl of carboxyl : 1721 .

Sulfazane- Trimethoprim {4}

Bands at (-OCH₃) methoxy group: 1130 ,(-N=N-S-) Azo-Sulfide: (1382, 1460, 1502) , , (C=N) endocycle of pyrimidine: 1657 ,(NH) in  imidazole : 3210 ,(C=N) endocycle of imidazole: 1632. Other active groups  are shown in some selected  spectra (1 , 2) .

Figure 1: I.R Spectrum of Invented Sulfazane-Trimethoprim{1} Click here to View Figure

 

Figure 2: I.R Spectrum of  Invented Sulfazane-Trimethoprim {2} Click here to View Figure

 

¹H.NMR- Spectra

The nuclear resonance spectrum is the  second technique that has demonstrated, with certain evidence, the creation of innovative, advanced derivatives in this study through the emergence of effective and functional grouping frequencies in derivatives indicating the reason for the accuracy of their preparation and the correctness of the innovative procedure to prepare them  by disappearance of  amine signal of (-NH₂) as a result of  formation of (sulfazane)- group^{(1, 2)} in  derivatives ,all spectra  showed signals at (2. 50) for  solvent (d-DMSO) ,besides to other  signals like:

Sulfazane- Trimethoprem{1}

It  gave signals  at (5. 40) due to proton  of amine group (NH₂) .,(6. 78–7. 74) to protons of aromatic ring ,(2. 98) for (O-CH₃) protons of  methoxy  groups.

Sulfazane- Trimethoprem{2}

It  gave signals  at (5. 11) due to proton  of amine group (NH₂) .,(6. 36–7. 42) to protons of aromatic ring ,(2. 94) for (O-CH₃) protons of  methoxy  groups ,(COOH) proton of carboxyl group : (13. 26) , (S-CH₂-CH-N-): (3. 56 , 3. 76 ).

Sulfazane- Trimethoprem{3}

It  gave signals  at (6. 77–7. 57) to protons of aromatic ring ,(2. 86) for (O-CH₃) protons of  methoxy  groups ,(COOH) proton of carboxyl group : (13. 08) .

Sulfazane- Trimethoprem{4}

It  gave signals  at (6. 69–7. 52) to protons of aromatic ring ,(2. 92) for (O-CH₃) protons of  methoxy  groups ,(NH) proton of amine in  imidazole ring: (8. 14) . Other  protons  of  functional groups  appeared  in some spectra (3 , 4) .

Figure 3: H.NMR-Spectrum of Invented Sulfazane-Trimethoprim {1} Click here to View Figure

 

Figure 4: H.NMR-Spectrum of Invented Sulfazane-Trimethoprim { 2} Click here to View Figure

 

HMBC- Spectrum

The  HMBC- spectrum is the  third  technique that has demonstrated, with certain evidence, the creation of innovative, advanced derivatives in this study through the emergence of effective and functional grouping frequencies in derivatives indicating the reason for the accuracy of their preparation and the correctness of the innovative procedure to prepare them via appearance of  functional  groups for  invented derivative, Figures (5).

Figure 5: HMBC -Spectrum of Invented Sulfazane-Trimethoprim { 2} Click here to View Figure

 

Mass  Spectra of  Sulfazane –Trimethoprim Derivatives

The  mass spectrum is the  fourth  technique that has demonstrated, with certain evidence, the creation of innovative, advanced derivatives in this study through the emergence of effective and functional grouping frequencies in derivatives indicating the reason for the accuracy of their preparation and the correctness of the innovative procedure to prepare them via appearance of fragments of  some of  invented derivatives, Figures (6, 7):

Figure 6: Mass Spectrum of Invented Sulfazane -Trimethoprim {1} Click here to View Figure

 

Figure 7: Mass Spectrum of Invented Sulfazane -Trimethoprim {2} Click here to View Figure

 

All chemical with physical  properties  and information  about  TLC for  invented derivatives in Table(1)

Table 1: All  chemical with physical  properties  and information  of  TLC 

Invented Derivatives	Product  %	Color	M .P (C °)	Rf	Solvents (TLC)
Sulfazane-Trimethoprim {1}	74	Deep Yellow	202	0.68	Ethanol : Benzene
Sulfazane-Trimethoprim {2}	80	Yellowish Orange	168	0.60	Ethanol : Benzene
Sulfazane-Trimethoprim {3}	78	Yellowish Orange	182	0.62	Ethanol : Benzene
Sulfazane-Trimethoprim {4}	82	Pale  Orange	194	0.70	Ethanol : Benzene

 

Discussion Innovative Derivatives Test  Against Breast CancerInitialization of  Cancer Cell Line⁽¹³⁾

Line processing and implantation of breast cancer cells and  live cell line were carried out at Biotechnology Center – the Nahrain (MCF-7 cell line) and (WRL cell line grew in  95% of RPMI–1640) supplemented with (10% FBS), cell suspension and incubation^{(13, 53)} at (37 °C) in incubator {(CO₂) % 5}. The suspended cells were centrifuged at (250 g) for (10 minutes) and the supernatant was removed, the cells were re-suspended in a freezing medium, then placed at (-70 °C) in  beaker for (1-3) days, the beaker was transferred from the standard freezer boxes to the liquid (N₂) container.

Processing  Method

MTT was used to determine cell viability by chromatic examination^(64-70) of two (MCF-7 and WRL cell lines):

Cell suspension (100 µL) was added to the wells of a small flat plate bottom.

The solution was prepared by dissolving the crystals of 5 mg MTT in 1 ml of PBS solution (phosphate buffer solution).

The concentrations of each innovative derivative of the prepared derivatives were used in this research (500, 250, 125, 62.5 , 31.5 , 15.6 (µg/ml of methanol, which were added to each well (three replicates per concentration).

A 10 ml MTT solution was added to each well of a plate containing 96 wells and then incubated for 4 hours with a test sample at 37 °C (the solution became yellow).

DMSO was added (200 µL (to each hole and stirred for 5 minutes (to become a purple DMSO solution).

After the complete dissolution of the dye, the absorption of the colored solution from the living cells was read at (575 nm) using the ELISA reader.7- The mean absorption was calculated for each group of iterations and the validity ratio of the cells exposed to different treatments was obtained as follows⁽¹³⁾:

Cell Vitality% = [(Absorption from the treated sample /Absorption from the untreated sample) X 100

Table 2: Mean Percentage (%) for each cell line (Respond to Treatment) for  Derivative{1}

Sulfazane- Trimethoprim{1}	IC50    (μg/ml)  : ( 190. 1654 )
	Mean Percentage  (%)  for each cell line (Respond to treatment )
	Conc (μg/ml)	Killing and inhibition of Carcinoma Cells %	Toxic Effect  on  Normal Cell Line
	500	66 %	30 %
	250	58 %	25 %
	125	50 %	25 %
	62.5	46%	20 %
	31.5	46 %	20 %
	15.6	44 %	20 %

 

Table 3: Mean Percentage (%) for each cell line (Respond to Treatment) for  Derivative{2}

Sulfazane- Trimethoprim{2}	IC50    (μg/ml)  : (228. 1930)
	Mean Percentage  (%)  for each cell line (Respond to treatment )
	Conc (μg/ml)	Killing and inhibition of Carcinoma Cells %	Toxic Effect  on  Normal Cell Line
	500	56 %	20 %
	250	52 %	14 %
	125	46 %	14  %
	62.5	40 %	14 %
	31.5	32 %	10 %
	15.6	30 %	10 %

 

Table 4: Mean Percentage (%) for each cell line (Respond to Treatment) for  Derivative{3}

Sulfazane- Trimethoprim{3}	IC50    (μg/ml)  : (258. 5202 )
	Mean Percentage  (%)  for each cell line (Respond to treatment )
	Conc (μg/ml)	Killing and inhibition of Carcinoma Cells %	Toxic Effect  on  Normal Cell Line
	500	50 %	12 %
	250	38 %	10 %
	125	36 %	10 %
	62.5	34 %	8 %
	31.5	30 %	8 %
	15.6	30 %	4 %

 

Table 5: Mean Percentage (%) for each cell line (Respond to Treatment) for  Derivative{4}

Sulfazane- Trimethoprim{4}	IC50    (μg/ml)  : (210. 3871 )
	Mean Percentage  (%)  for each cell line (Respond to treatment )
	Conc (μg/ml)	Killing and inhibition of Carcinoma Cells %	Toxic Effect  on  Normal Cell Line
	500	60 %	22 %
	250	50 %	18 %
	125	44 %	18 %
	62.5	40 %	16 %
	31.5	30 %	16 %
	15.6	30 %	16 %

 

Conclusions

The results indicated  to formation of these drug derivatives by appearance of new bands and disappearance of bands in starting compounds., besides to conclusions from our paper that gave good data for inhibition efficiency for these drug derivatives  against cancer cells.  Our  results appeared   good  inhibition for carcinoma  cells line for all  invented  derivatives , and  gave high  response for  derivatives {1  and  4} more  than other  invented  derivatives ,(it was  = 66 %  response percentage  of  derivative {1} for  inhibition and  killing  of cancer cells due  to sulfazane^{(1, 2)} group(-S-N=N- Drug ) that linked with  drug which gave  it  more response  against cancer cells also due to  thiazole core  in this derivative{1}. 

Acknowledgments

We would like to express our heartfelt thanks to  Biotechnology Center-the Nahrain for providing assistance  samples of cells and  bioinformatics analysis.

Conflict of Interest

The authors declare that there is no conflict of interest.   

Funding Source

None

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44. Wu Y-J, Yang BV (2009) Chapter 5.5: Five-membered ring systems: with N and S(Se). In: Gribble GW, Joule JA (eds) Progress in heterocyclic chemistry, vol 21. Elsevier, OxfordGoogle Scholar.
45. Nawfel M B ,Hayder H K, Noor H D, Nawfel Muhammed Baqer ,Nagham Mahmood Aljamali., “Preparation of Chemical Inhibitors to Treat the Corrosion and Erosion of Machines”, International Journal of Engineering, Applied and Management Sciences Paradigms., 2019, 54, 3,89-93p.
46. Wu Y-J, Yang BV (2011) Chapter 5.5: Five-membered ring systems: with N and S(Se). In: Gribble GW, Joule JA (eds) Progress in heterocyclic chemistry, vol 23. Elsevier, OxfordGoogle Scholar.
47. Farmer LJ, Bemis G, Britt SD, Cochran J, Connors M, Harrington EM, Hoock T, Markland W, Nanthakumar S, Taslimi P, Ter Haar E, Wang J, Zhaveri D, Salituro FG (2008) Bioorg Med Chem Lett 18:6231–6235CrossRefGoogle Scholar.
48. Nagham Mahmood Aljamali. “Synthesis and Biological Study of Hetero (Atoms and Cycles) Compounds”, Der Pharma Chemica, 2016, 8,6, 40-48.
49. Zhou C, Garcia-Calvo M, Pinto S, Lombardo M, Feng Z, Bender K, Pryor KD, Bhatt UR, Chabin RM, Geissler WM, Shen Z, Tong X, Zhang Z, Wong KK, Roy RS, Chapman KT, Yang L, Xiong Y (2010) J Med Chem 53:7251–7263CrossRefGoogle Scholar.
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51. Eicher, T.; Hauptmann, S. (2003). The Chemistry of Heterocycles: Structure, Reactions, Syntheses, and Applications. ISBN 978-3-527-30720-3.
52. Mak, Jeffrey Y.W.; Xu, Weijun; Fairlie, David P. (2015). Peptidomimetics I Topics in Heterocyclic Chemistry. 48. Springer Berlin Heidelberg. pp. 235–266. doi:10.1007/7081_2015_176. ISBN 978-3-319-49117-2.
53. Imad Kareem Alwan Alsabri, Hasaneen Kudhair Abdullabass ,Nagham Mahmood Aljamali, Imad K A A, Hasaneen K A., Invention of (Gluta.Sulfazane-Cefixime) Compounds as Inhibitors of Cancerous Tumors., Journal of Cardiovascular Disease Research, 2020,11, 2.
54. Kriek, M.; Martins, F.; Leonardi, R.; Fairhurst, S. A.; Lowe, D. J.; Roach, P. L. (2007). “Thiazole Synthase from Escherichia coli: An Investigation of the Substrates and Purified Proteins Required for Activity in vitro” (PDF). J. Biol. Chem. 282 (24): 17413–17423 . doi:10.1074/jbc.M700782200 .  PMID 17403671.
55. Dondoni, A.; Merino, P. (1995). “Diastereoselective Homologation of D-(R)-Glyceraldehyde Acetonide using 2-(Trimethylsilyl)thiazole”. Organic Syntheses. 72: 21.; Collective Volume, 9, p. 952
56. Nagham Mahmood Aljamali.; Intisar Obaid Alfatlawi., “Synthesis of Sulfur Heterocyclic Ligands and Study of Expected Biological Activity”,Research J. Pharm. and Tech., 2015, 8,9 ,1225-1242 , DOI: 10.5958/0974-360X.2015.00224.3.
57. Gitai, Z . “The new bacterial cell biology: moving parts and sub cellular architecture”. Cell, 2005 , 120 ,5, 577–86.
58. Vanita S .; Supriya, M. “Development of furfuraldehyde formazans as potential antitubercular agents “., Der Pharma Chemica, 2016,8,18, 144-148.
59. Park, S.; Park, B.; Yun, S.;  Kang, H.  and Yun,  “Antimicrobial activities of honey bee venom against pathogens isolated from clinical bovine mastitis in Korea” , Planta Med., 2013, 79, , PL16.
60. Habibe,T .;Mehmet, L. A. “Electrochemical properties of 1-(o−, m−, pnitrophenyl)-3-(m−nitrophenyl)-5-phenylformazans and their nickel complexes”.,Turk J Chem ,2010,34, 465 – 479.
61. Angela, K.; Lucica, V.;  Nicoleta,  C . “Synthesis and structural studies of complexes of Cu, Co, Ni and Zn with isonicotinic acid hydrazide and isonicotinic acid (1-naphthyl methylene) hydrazide”, J. Serb. Chem. Soc, 2010, 75 ,2, 229-242.
62. Wright, G.D. ” The antibiotic resistome: the nexus of chemical and genetic diversity”., Nature Reviews Microbiology, 2007, 5, 175-186.
63. Nagham Mahmood Aljamali., “(Synthesis, Investigation, Chromatography, Thermal)-Behavior of (Five, Seven)- Membered Ring with Azo and Anil Compounds”, Pak. J. Biotechnol. 2018; 15(1): 219-239.
64. Hegazi, A.G.; Abdou, A.M. and Abd, A. F. “Evaluation of the antibacterial activity of bee venom from   different sources”, World Applied Sciences Journal, 2014, 30 ,3, 266-270.
65. Aseel Mahmood Jawad, Nagham Mahmood Aljamali, Saher Mahmood Jwad, Aseel M J, Saher M J., Development and Preparation of ciprofloxacin Drug Derivatives for Treatment of Microbial Contamination in Hospitals and Environment, Indian Journal of Forensic Medicine & Toxicology, 2020,14, 2, p:1115-1122.
66. Intisar Obaid Alfatlawi ,Nuha Salman S, Zainab Mahmood Jawad , Nagham Mahmood Aljamali., Synthesis of New Organic Ligands Via Three Components Reaction with Studying of (Identification ,Thermal Behavior, Bioactivity  on Bacteria  of Teeth)., Journal of Global Pharma Technology. 2017; 11, 9 ,157-164.
67. Mieaad Mohamd, Nagham Mahmood Aljamali ,Wassan Ala Shubber ,Sabreen Ali Abdalrahman .,”New Azomethine- Azo Heterocyclic Ligands Via Cyclization of Ester”., Research J. Pharm. and Tech. 11, 6 , 2018 .
68. Al-Shammari, A.M.; Salman, M.I.; Saihood, Y.D.; Yaseen, N.Y.; Raed, K.; Shaker, H.K.; Ahmed, A.; Khalid, A.; Duiach, A. (2016) In vitro synergistic enhancement of Newcastle Disease Virus to 5-fluorouracil cytotoxicity against tumor cells. Biomedicines, 4(3): 1-10.
69. Nagham Mahmood Aljamali., “(Synthesis, Investigation, Chromatography, Thermal)- Behavior of (Five, Seven)- Membered Ring with Azo and Anil Compounds”, Pak. J. Biotechnol. ,2018; 15(1): 219-239.
70. Kaur, K.; Kumar, V.; Sharma, A.K.; Gupta, G.K. (2014) Isoxazoline containing natural products as anticancer agents: a review. European journal of medicinal chemistry, 77(2014): 121-133.