Biological Evaluation of Newly synthesized Spebrutinibm Analogues: Potential Candidates with Enhanced Activity and Reduced Toxicity Profiles.

Abstract

Background: Undoubtedly, cancer is regarded as a major concern for researchers alongside the whole humanity for its high mortality rates. At this moment, there must be some researchers working hard to design, synthesize, and biologically investigate the effects of some potential candidates to fight back cancer. Materials and methods: In previous unpublished work, the authors successfully designed, synthesized, characterized a potential two spebrutinib analogues. Consequently, these analogues were evaluated with the employment of MCF-7, HCT116, and MDCK cell lines. Results: In respect to the spebrutinib standard, one of these analogues has superior activity against MCF-7 cell line (IC50; 10.744 µg/mL against 13.566 µg/mL for spebrutinib) and an enhanced toxicity profile on MDCK cell line (IC50; 8.653 mg/mL against 4.011 mg/mL for spebrutinib). Conclusion: The two compounds showed good activity against breast and colon cell lines and enhanced toxicity profile against normal kidney cell line in respect to spebrutinib standard.

Full text

ABSTRACT
Background: Undoubtedly, cancer is regarded as a major concern for researchers alongside the whole humanity for its high
mortality rates. At this moment, there must be some researchers working hard to design, synthesize, and biologically investigate
the eects of some potential candidates to ght back cancer.
Materials and methods: In previous unpublished work, the authors successfully designed, synthesized, characterized a
potential two spebrutinib analogs. Consequently, these analogs were evaluated with the employment of MCF-7, HCT116,
and MDCK cell lines.
Results: In respect to the spebrutinib standard, one of these analogs has superior activity against MCF-7 cell line (IC50; 10.744
µg/mL against 13.566 µg/mL for spebrutinib) and an enhanced toxicity prole on madin-darby canine kidney (MDCK) cell
line (IC50; 8.653 mg/mL against 4.011 mg/mL for spebrutinib).
Conclusion: The two compounds showed good activity against breast and colon cell lines and enhanced toxicity prole against
normal kidney cell line in respect to spebrutinib standard.
Keywords: Biological evaluation, Colon cancer, Breast cancer, Spebrutinib analogs, Tyrosine kinase inhibitor, Ic50, Mcf-7,
Hct116, Mdck, Cell Lines
International Journal of Drug Delivery Technology (2019); DOI: 10.25258/ijddt.v9i3.3
How to cite this article: Jaber Al-Obaidi, Z.M., Abdul- Rasheed, O.F., Mahdi, M.F. and Raauf, A.M.R. (0000). Biological
Evaluation of Newly synthesized Spebrutinib Analogues: Potential Candidates with Enhanced Activity and Reduced Toxicity
Proles. International Journal of Drug Delivery Technology, 39(3): 339-346.
Source of support: Nil
Conict of interest: The authors conrm that there is no conict of interest in this research.
Biological Evaluation of Newly synthesized Spebrutinib Analogues:
Potential Candidates with Enhanced Activity and Reduced Toxicity
Proles
Zaid M. Jaber Al-Obaidi,1 Omar F. Abdul- Rasheed,2 Monther F. Mahdi,3 Ayad M.R. Raauf4
1Department of Pharmaceutical Chemistry, College of Pharmacy, University of Kerbala/Iraq
2Department of Chemistry and Biochemistry, College of Medicine, Al- Nahrain University/Iraq
3Department of Pharmaceutical Chemistry, Ashur University College/Iraq
4Department of Pharmaceutical Chemistry, College of Pharmacy, Mustansiriyah University/Iraq
Received: 12th July, 19; Revised: 17th August, 19, Accepted: 06th September, 19; Available Online: 15th September, 2019
INTRODUCTION
Cancer is a major concern of researchers as it is the second
leading cause of deaths worldwide.1 Cancer-related
mortality has increased by almost (40%) in the past 40 years,
although death rates from communicable diseases have
improved worldwide as a result of the medical improvements.
In the next 15 years, a further 60% rise is expected, with about
13 million people estimated to die of cancer in 2030.2
Hence, there is a compelling need to develop new drugs
to treat this life-threatening disease. Cancer treatment using
conventional chemotherapy is associated with several side
eects.3 In recent years, the development of small molecules
such as tyrosine kinase inhibitors (imatinib, spebrutinib,
gefitinib, sunitinib, semaxinib, etc.,) in the treatment of
cancer helped the scientists in the understanding of molecular
RESEARCH ARTICLE
mechanisms of this disease.4 Targeting en zymes involved i n the
signal transduction pathways of protein kinases that regulates
cellular growth and multiplication is one of the approaches in
developing the new anticancer drugs.5,6
Consequently, scientists have recognized tyrosine kinases
as a potential target to suppress or even cure breast cancers.7
Consequently, many tyrosine kinase inhibitors (TKI) have
been developed and tested.8-10 However, the o-target serious
side eects of these TKIs are a major obstacle that has been
encountered.11-14
For some prestigious journal publishers, biological investigation
of newly synthesized potential anticancer candidates is a
must.15 In this work, the authors aimed to biologically evaluate
a previously synthesized novel TKI with superior activity
and reduced toxicity. These compounds were successfully
designed, synthesized, and characterized.
*Author for Correspondence: zaid_alobaidi@hotmail.co.uk

Biological Evaluation of Newly synthesized Spebrutinib Analogues
IJDDT, Volume 9 Issue 3 July 2019 – September 2019 Page 340
MATERIALS AND METHODS
Materials
The materials used in this work are tabulated in Table 1.
Cell lines
The following types of cell lines were used in this study:
MCF-7 Breast cancer
HCT116 colorectal cancer cells
MDCK kidney normal cells.
Cell lines were obtained kindly from the international cell
line collection of Dr. Hamid N. Obied (M.B.CH.B., MSc, PhD
Pharmacology, Lecturer and researcher in anticancer at the
department of clinical pharmacology, College of Medicine,
University of Babylon, and the head of cancer cell research
unit in Al-Fadhil foundation for educational services, training
and development – branch of Babylon).
Instruments
The instruments used in this work are listed in Table 2.
Methods:
Cell lines preparation:
The cell lines were cultured in medium 1640 (RPMI-1640,
Gibco-BRL), with 10% heat-inactivated fetal bovine serum
(FBS) (Gibco). Cell lines were allocated in Celltreat® 96 well
cell culture plates and incubated to grew at 37°C. The time of
cell culture was optimized from 72 hours to be 24 hours and the
steps involved in the cell line part of this work are listed below:
MTT stock solution preparation:
A 25-mg was accurately weighed and transferred into a suitable
ask. Then a 5-mL of DMSO was added and the MTT was
completely dissolved. The 5-mL of 5mg/mL was ltered,
into 12.mL centrifuge tube, with 0.22µm sterile lters and
the tube was foiled with aluminum sheet as the MTT solution
is light sensitive. This solution was kept in the fridge for the
preparation of a working solution.
MTT working solution preparation:
According to the protocols, the working concentrations of MTT
is 0.5mg/ml. This is 10% v/v of the stock solution. For a nal
volume of 12 ml of cell-medium with 10% MTT, the following
dilutions were performed. A 10.600 ml of cell medium was
accurately measured and allocated into a suitable ask. Then
a 2.400 ml of MTT stock solution was added to the medium
and adequately homogenized. The cell-medium with 10%
MTT was ready to be utilized for cell-lines and incubation
of 3hrs period.
Preparation of working concentrations from each test
chemical for the cell-lines:
A suitable amount of each chemical was dissolved in DMSO
to get a stock solution with a concentration of 5mg/mL for
each chemical and standard. After several trials on cell-line,
the concentration was optimized for a 50µg/mL as the higher
concentration from witch a serial dilution was performed.
For each standard and synthesized chemical, a 990-µl of the
medium was accurately measured and a 10-µL from the 5mg/
ml was added and homogenized to get a nal concentration
of 50 µg/ml and a nal concentration of 1% for the DMSO.
Serial dilution was performed for each to get the following
concentrations (50, 25, 12.5, 6.25, 3.125, and 1.5625) µg/mL.
Stock solution preparation:
An accurately-weighed amount of each synthesized chemical
compounds was dissolved in pure DMSO to get a concentration
of 5mg/mL. After complete dissolution, the solutions were
ltered through a 0.2 µm sterile lter. A 10 µL of the above
ltrate was fur ther diluted with 990 µL of RPMI.1640 Mediu m
to get a nal concentration of 50 µg/mL. A serial dilution was
prepared from the above concentration to get (50, 25, 12.5,
6.25, and 3.125) µg/mL.
Application of the chemicals on the cell-lines:
The above serial dilution solutions were added in a 200 µl
portions for each well in triplicates and incubated for 24hours.
After the incubation period, the plates were visualized with
Table 1: Utilized materials with their manufacturers and countries of origin.
# Material Manufacturer Country
1 Spebrutinib AVL-292 (99.48%) BLDpharm CHINA
2 N-(3-((5-uoro-2-((4-(2-methoxyethoxy)phenyl)amino) pyrimidin-4-yl)amino)phenyl)
benzamide (2a) (99.63%)
Medicinal chemistry lab* IRAQ
3 N-(3-((5-uoro-2-((4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)phenyl)
pivalamide (2b) (99.69%)
Medicinal chemistry lab* IRAQ
4 Dimethyl sulphoxide (99%) CDH INDIA
5 Cellulose acetate membrane lter pore size 0.2 µm diameter 25 mm. chm SPAIN
6 MTT (3.[4,5.dimethylthiazol.2.yl] .2,5.diphenyl tetrazolium bromide) Roth GERMANY
7 Celltreat® 96 well cell culture plates CELLTREAT scientic products USA
*These compounds were synthesized and characterized in previous unpublished work.
Table 2: Employed instruments with their manufacturers and countries of
origin.
# Instrument Manufacturer Country
1 4-digit balance Sartorius Lab GERMANY
2 Clean Bench LabTech KOREA
3 Incubator UN 55 Memmert GERMANY
4 Microplate reader 800 TS BioTek USA
5 Inverted Microscope Zeiss GERMANY

Biological Evaluation of Newly synthesized Spebrutinib Analogues
IJDDT, Volume 9 Issue 3 July 2019 – September 2019 Page 341
Table 3: The symbols, IUPAC names, chemical formulas, and the
chemical structures of the spebrutinib and the synthesized analogs.
Chemical
formula (Code)
Structure
Chemical name
C22H22FN5O3
(AVL-292)
Spebrutinib
FO
O
O
N NN
N
N
H
H
H
N-(3-((5-fluoro-2-((4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)phenyl)acrylamide
C26H24FN5O3
(2a)
FO
O
O
N NN
N
N
H
H
H
N-(3-((5-fluoro-2-((4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)phenyl)benzamide
C24H28FN5O3
(2b)
FO
O
O
N NN
N
N
H
H
H
N-(3-((5-fluoro-2-((4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)phenyl)pivalamide
Figure 1: Shows control of colorectal HCT116 cells. An inverted
microscope observation post 24-hours’ incubation.
Figure 2: Shows HCT116 colorectal cell line (subjected to 12.5µg/
ml of spebrutinib standard). An inverted microscope observation post
24-hours’ incubation.
Figure 3: The absorbance of MTT versus concentration of spebrutinib
standard after 24-hours’ incubation of HCT116 colorectal cell line.
Figure 4: The percent viable cells versus concentration of spebrutinib
standard after 24-hours’ incubation of HCT116 colorectal cancer cell line.
Figure 5: Shows HCT116 colorectal cell line (subjected to 12.5µg/mL
of compound 2a). An inverted microscope observation post 24-hours’
incubation.
an inverted microscope and a screenshots was captured for
each well. The media were replaced with 10% MTT media
and incubated for 3 hours. After the 3 hours’ incubation, the
media was removed and the wells were washed with phosphate
buer saline (PBS). Finally, a 200.µL portions of DMSO was
added into each well and left for 30 minutes and were read
with plate reader at 630 nm.
RESULTS
In previous unpublished work, two spebrutinib analogs were
successfully designed, synthesized, and characterized. These
analogs are tabulated in Table 3.
Results of the biological effect of the synthesized
compounds on the cancerous and normal cell-lines:
Eect on HCT116 colorectal cancer cell-line is shown in
Figure 1:
Eect of Spebrutinib is shown in Figures 2, 3, and 4:
Eect of compound 2a is shown in Figures 5, 6, and 7:
Eect of compound 2b is shown in Figure 8, 9, and 10:

Biological Evaluation of Newly synthesized Spebrutinib Analogues
IJDDT, Volume 9 Issue 3 July 2019 – September 2019 Page 342
Figure 7: The percent viable cells versus concentration of compound
2a after 24-hours’ incubation of HCT116 colorectal cancer cell line.
Figure 8: Shows HCT116 colorectal cell line (subjected to 12.5µg/mL
of compound 2b). An inverted microscope observation post 24-hours’
incubation.
Figure 9: The absorbance of MTT versus concentration of compound
2b after 24-hours’ incubation of HCT116 colorectal cancer cell line.
Figure 10: The percent viable cells versus concentration of compound
2a after 24-hours’ incubation of HCT116 colorectal cancer cell line.
Figure 11: shows control of MCF-7 breast cells. An inverted microscope
observation post 24-hours’ incubation.
Figure 12: Shows MCF-7 breast cell line (subjected to 12.5µg/ml
of spebrutinib standard). An inverted microscope observation post
24-hours’ incubation.
Figure 13: the absorbance of MTT versus concentration of spebrutinib
standard after 24-hours’ incubation of MCF-7 breast cancer cell line.
Figure 14: The percent viable cells versus concentration of spebrutinib
standard after 24-hours’ incubation of MCF-7 breast cancer cell line.
Figure 6: the absorbance of MTT versus concentration of compound 2a
after 24-hours’ incubation of HCT116 colorectal cancer cell line.
Eect on MCF-7 breast cancer cell line is shown in
Figures 11, 12, 13, and 14:
Eect of Spebrutinib is shown in Figure 11:
Eect of compound 2a is shown in Figures 15, 16, and 17:
Eect of compound 2b is shown in Figures 18, 19, and 20:
EFFECT ON MDCK KIDNEY NORMAL CELL-
LINE
The ex vivo toxicity of the synthesized spebrutinib analogues

Biological Evaluation of Newly synthesized Spebrutinib Analogues
IJDDT, Volume 9 Issue 3 July 2019 – September 2019 Page 343
were evaluated by applying these synthesized chemicals on
MDCK epithelial cell line (non-cancerous, normal kidney
cells as shown in Figure 21. These cells were derived by S.
H. Madin and N. B. Darby from the kidney tissue of an adult
female cocker spaniel.
Eect of Spebrutinib is shown in Figures 22, 23, and 24:
Eect of compound 2a is shown in Figure 25, 26, and 27:
Figure 15: Shows MCF-7 breast cell line (subjected to 12.5µg/ml of
compound 2a). An inverted microscope observation post 24-hours’
incubation.
Figure 16: The absorbance of MTT versus concentration of compound
2a after 24-hours’ incubation of MCF-7 breast cancer cell line.
Figure 17: The percent viable cells versus concentration of compound
2a after 24-hours’ incubation of MCF-7 breast cancer cell line.
Figure 18: Shows MCF-7 breast cell line (subjected to 12.5µg/ml of
compound 2b). An inverted microscope observation post 24-hours’
incubation.
Figure 20: The percent viable cells versus concentration of compound
2b after 24-hours’ incubation of MCF-7 breast cancer cell line.
Figure 19: The absorbance of MTT versus concentration of compound
2b after 24-hours’ incubation of MCF-7 breast cancer cell line.
Figure 21: Shows control MDCK kidney normal cell line. An inverted
microscope observation post 24-hours’ incubation.
Figure 22: Shows MDCK kidney normal cell line (subjected to 12.5µg/
ml of spebrutinib standard). An inverted microscope observation post
24-hours’ incubation.
Figure 23: the absorbance of MTT versus concentration of spebrutinib
standard after 24-hours’ incubation of MDCK kidney normal cell line.
Figure 24: the percent viable cells versus concentration of spebrutinib
standard after 24-hours’ incubation of MDCK kidney normal cell line.

Biological Evaluation of Newly synthesized Spebrutinib Analogues
IJDDT, Volume 9 Issue 3 July 2019 – September 2019 Page 344
Eect of compound 2b is shown in Figure 28, 29, and 30:
The IC50 for the synthesized compounds and the
spebrutinib standard for the HCT116, MCF-7, and the MDCK
cell lines are calculated in Table 4.
DISCUSSION
Results of the biological eect of the synthesized compounds
on the cancerous and normal cell-lines:
The time of incubation was optimized to 48 hours instead
of 72 hours and eventually 24 hours’ incubation period was
selected. The concentrations of the synthesized chemicals that
were applied to the cells were optim ized though. Starting with
the highest concentration of 1 mg/L and reaching the optimized
high concentration of 50 µg/mL.
Thereafter, the serial dilution performed from the 50 µg/mL was
mathematically accepted to give good representative curves.
The sketched curves possess good correlation coecients.
Furthermore, the half-maximal inhibitory concentration (IC50)
values were mostly in the middle of the curves to exclude any
proposed drift in the curves if any. The IC50 is a measure of
the eectiveness of the synthesized chemical compound in
inhibiting cell growth.
The cell lines were meaningfully chosen and accurately
selected. For colorectal cell lines, excluding skin cancers,
colorectal cancer is classied as third cancer in occurrence in
US for both sexes. In the same context, in the United States,
the American Cancer Society has proposed the number of the
cases for colorectal cancer for 2019 as (44,180) new cases of
rectal cancer and (101,420) new cases of colon cancer [16].
Regarding breast cancer cell line, excluding skin cancers,
breast cancer is classied as rst cancer in occurrence in the
US for American women. In terms of numbers, in the United
States, the American Cancer Society has estimated the number
of the cases for the breast cancer for 2019 as (268,600) new
cases of invasive breast cancer and (62,930) new cases of non-
invasive breast cancer.17
Notably, to evaluate the selectivity of the newly synthesized
compounds toward the cancer cells, it is essential to test their
toxicity on normal (non-cancerous) cells.18-20 A dramatic
number of authors utilized the MDCK cell line for cell viability
studies.21-29 According to this fact besides their availability,
the MDCK cell lines were selected.
Figure 27: The percent viable cells versus concentration of compound
2a after 24-hours’ incubation of MDCK kidney normal cell line.
Figure 26: The absorbance of MTT versus concentration of compound
2a after 24-hours’ incubation of MDCK kidney normal cell line.
Figure 28: Shows MDCK normal kidney cell line (subjected to 12.5µg/ml of
compound 2b). An inverted microscope observation post 24-hours’ incubation.
Figure 29: The absorbance of MTT versus concentration of compound
2b after 24-hours’ incubation of MDCK kidney normal cell line.
Figure 30: The percent viable cells versus concentration of compound
2b after 24-hours’ incubation of MDCK kidney normal cell line.
Table 4: A summary for IC50 for the cell lines and chemical compounds
specied.
Cell line IC50 AVL-292 IC50 2a IC50 2b
HCT116 11.73 µg/mL 34.05 µg/mL 25.53 µg/mL
MCF-7 13.566 µg/mL 10.744 µg/mL 19.23 µg/mL
MDCK 4.011 mg/mL 8.653 mg/mL 1.705 mg/mL
Figure 25: Shows MDCK normal kidney cell line (subjected to
12.5µg/ml of compound 2a). An inverted microscope observation post
24-hours’ incubation.

Biological Evaluation of Newly synthesized Spebrutinib Analogues
IJDDT, Volume 9 Issue 3 July 2019 – September 2019 Page 345
Table 4 shows the net results of this work as it reveals the
biological eects of the chemically synthesized compounds on
the cancerous and normal cells. This brief comparison gives
valuable information. To critically analyze the observed data,
it will be categorized for each compound and then for each
cell as below:
• For the spebrutinib standard, it shows close values of IC50
for both HCT116 and MCF-7 cell lines with priority for
the HCT116 cells. In the same context of spebrutinib, the
IC50 for MDCK cells shows approximately 300-fold the
concentration required for the activity against both HCT116
and MCF-7 cell lines. This reveals an excellent selectivity
toward the cancerous cells rather than the normal non-
cancerous cells.
• For compound 2a, the IC50 value for the HCT116 cells is
3-fold its value for MCF-7 cells. In other words, compound
2a has superior activity on MCF-7 cells rather than for
the HCT116 cells. Moreover, the IC50 of compound 2a
for MDCK shows approximately 250-fold and 800-fold
of the concentration required for the activity against the
HCT116 and MCF-7 cell lines, respectively. This indicates
an excellent selectivity toward the cancerous cells rather
than the normal non-cancerous cells with better selectivity
toward the MCF-7 cell line.
• For compound 2b, the IC50 value for the HCT116 cells is
1.3-fold its value for MCF-7 cells. Accordingly, compound
2b has better activity on MCF-7 cells rather than for the
HCT116 cells. Moreover, the IC50 of compound 2b for
MDCK shows approximately 67-fold and 89-fold of the
concentration requi red for the ac tivity agains t the HCT116
and MCF-7 cell lines, respectively. This indicates an
acceptable selectivity margin toward the cancerous cells
rather than the normal non-cancerous cells with better
selectivity toward the MCF-7 cell line.
According to the European Medicines Agency (EMA),
with respect to the “Nonclinical Evaluation for Anticancer
Pharmaceuticals” The EMA concludes the following
statement “A common approach for many small molecules
is to set a start dose at 1/10 the Severely Toxic Dose in 10%
of the animals (STD 10) in rodents.30 If the non-rodent is
the most appropriate species, then 1/6 the Highest Non-
Severely Toxic Dose (HNSTD) is considered an appropriate
starting dose. The HNSTD is dened as the highest dose
level that does not produce evidence of lethality, life-
threatening toxicities or irreversible ndings”.30 In all the
previously discussed ndings the proposed therapeutic
doses are much lower than the “one-tenth” portion of
the STD 10. Accordingly, the therapeutic doses can be
reduced to be equal or below the micromolar concentrations
recommended by many types of research.31-33
• For HCT116 cell line, the IC50 for the spebrutinib standard
has the lowest value, followed by compound 2b and lastly
compound 2a. Where as, compound 2b has twice the IC50
value in respect to spebrutinib standard, compound 2a has
trice the IC50 value for the standard.
• For the MCF-7 cell line, it has the most interesting ndings.
Compound 2a has the lowest IC50 value, which indicates
better cytotoxic activity than the spebrutinib standard.
Whereas, spebrutinib has the middle value for IC50 (1.25
times of that of compound 2a). Finally, compound 2b has
the highest IC50 value.
• For the MDCK cell line, the IC50 values have a piece
of appreciated information. For example, compound 2b
has the lowest IC50 value which means the most toxic in
respect to compounds 2a and standard. On the other hand,
the IC50 for compound 2a has approximately 2-times the
IC50 value of that of spebrutinib standard. To sum up,
compound 2a has better activity and lower toxicity than
the spebrutinib standard.
CONCLUSIONS
In this stu dy, two of the sy nthesized compounds were found to
have biological activity. The biological eects are concluded
for compound 2a which has approximately 2-times the IC50
value of that of spebrutinib, hence compound 2a has the lowest
IC50 value, which indicates better cytotoxic activity than the
spebrutinib standard. Whereas, spebrutinib has the middle
value for IC50 (1.25 times of that of compound 2a).
SUPPLEMENTARY MATERIALS
Graphical abstract will be available in the supplementary
material’s pattern when published.
DATA AVAILABILITY
“The data used to support the findings of this study are
available from the corresponding author upon request”.
ACKNOWLEDGMENT
The authors would like to acknowledge the support supplied
by the College of Pharm acy, Un iversity of Kerbala, the College
of Pharmacy, Mustansiriyah University, and the College of
Medicine, University of Babylon for their laboratories and
assistance.
ABBREVIATIONS
HCT116, Human Colorectal Carcinoma; MCF-7, Michigan
Cancer Foundation -7; MDCK, Madin-Darby Canine Kidney;
TK, Tyrosine Kinase; TKI, Tyrosine Kinase Inhibitor.
REFERENCES
1. Papac RJ. (2001). Origins of cancer therapy. The Yale journal
of biology and medicine. 74 (6):3916.
2. Kuipers EJ, Rösch T & Bretthauer M. (2013). Colorectal cancer
screening — optimizing current strategies and new directions.
Nat. Rev. Clin. Oncol. 10, 130 –142.
3. Verhoef MJ, Rose MS, White M and Balneaves LG.
(2008). Declining conventional cancer treatment and using
complementary and alternative medicine: a problem or a
challenge? Current Oncology. 15 (Suppl 2):s101.

Biological Evaluation of Newly synthesized Spebrutinib Analogues
IJDDT, Volume 9 Issue 3 July 2019 – September 2019 Page 346
4. Haddad JJ. (2012). The immunopharmacologic potential of
Semaxanib and new generation directed therapeutic drugs:
Receptor tyrosine kinase regulation with anti-tumorigenensis/
angiogenesis properties. Saudi Pharmaceutical Journal. 20
(2):103-23.
5. Broekman F, Giovannetti E and Peters GJ. (2011). Tyrosine
kinase inhibitors: Multi-targeted or single-targeted? World J
Clin Oncol. 2 (2):80-93.
6. Wolle, P., et al. (2019). “Targeting the MKK7–JNK (Mitogen-
Activated Protein Kinase Kinase 7–c-Jun N-Terminal Kinase)
Pathway with Covalent Inhibitors.” Journal of Medicinal
Chem istr y.
7. Burstein, H. J., et al. (2008). “Phase II study of sunitinib malate,
an oral multitargeted tyrosine kinase inhibitor, in patients with
metastatic breast cancer previously treated with an anthracy-cline
and a taxane.” Journal of Clinical Oncology 26(11): 1810-1816.
8. Bensinger, D., et al. (2019). “Virtual Screening Identies
Irreversible FMS-like Tyrosine Kinase 3 Inhibitors with
Activity Toward Resistance-Conferring Mutations.” Journal
of Medicinal Chemistry.
9. Popow, J., et al. (2019). “Highly Selective PTK2 Proteolysis
Targeting Chimeras to Probe Focal Adhesion Kinase
Scaolding Functions.” Journal of Medicinal Chemistry.
10. Simpson, G. L., et al. (2019). “Identication and Optimization
of Novel Small c-Abl Kinase Activators Using Fragment and
HTS Methodologies.” Journal of Medicinal Chemistry 62(4):
2154 -2171.
11. Steegmann, J. L., et al. (2012). “O-target eects of BCR–ABL1
inhibitors and the ir pot enti al long-term implicat io ns in pa tients
with chronic myeloid leukemia.” Leukemia & lymphoma
53(12): 2351-2361.
12. Yang, B. and T. Papoian (2012). “Tyrosine kinase inhib-itor
(TKI)‐induced cardiotoxicity: approaches to narrow the gaps
between preclinical safety evaluation and clinical outcome.”
Journal of Applied Toxicology 32(12): 945-951.
13. Force, T. and R. Kerkelä (2008). “Cardiotoxicity of the new
cancer therapeutics–mechanisms of, and approaches to, the
problem.” Drug discovery today 13(17-18): 778-784.
14. Kerkela, R., et al. (2009). “Sunitinib‐induced cardio-toxicity
is mediated by o‐target inhibition of AMP‐activated protein
kinase.” Clinical and translational science 2(1): 15-25.
15. American Chemical Society, “Author Guide”, 2018, https://
pubs.acs.org/paragonplus/submission/orlef7/orlef7_authguide.
pdf; Accessed 28.02.2019.
16. The American Cancer Society, “Key Statistics for Colorectal
Cancer”, 2019, https://www.cancer.org/cancer/colon-rectal-
cancer/about/key-statistics.html, Accessed 12.02.2019.
17. The American Cancer Society, “Current year estimates for
breast cancer”, 2019, https://www.cancer.org/cancer/breast-
cancer/about/how-common-is-breast-cancer.html, Accessed
12.02.2019.
18. Jie Liu, Xiao-Hua Zou, Qian-Ling Zhang, Wen-Jie Mei, Jian-
Zhong Liu, and Liang-Nian Ji, “Synthesis, Characterization
and Antitumor Ac tivi ty of a Series of Polypyr id yl Complexes,”
Metal-Based Drugs, vol. 7, no. 6, pp. 343-348, 2000.
19. Hanelt, M., Gareis, M., & Kollarczik, B. (1994). Cytotoxicity
of mycotoxins evaluated by the MTT-cell culture assay.
Mycopathologia, 128(3), 167–174.
20. Kovačević, S. Z., Karadžić, M. Ž., Vukić, D. V., Vukić,
V. R., Podunavac-Kuzmanović, S. O., Jevrić, L. R., &
Ajduković, J. J. (2018). Toward steroidal anticancer drugs:
Non-parametric and 3D-QSAR modeling of 17-picolyl and
17-picolinylidene androstanes with antiproliferative activity on
br ea st a de noca rcin oma cells. Jour nal of Molecular Graph ics and
Modelling.
21. Mazzawi, N., Kimmel, E., & Tsarfaty, I. (2019). The eect of
low-intensity ultrasound and met signaling on cellular motility
and morphology.
22 . Appl ied Ac Tsai, C.-P., & Tsai, H.-J. (2018). In uenz a B Viruses
in Pigs, Taiwan. Inuenza and Other Respiratory oustics, 143,
1–6.
23. Alam, J., Jeon, S., & Choi, Y. (2019). Determination of
Anti-aquaporin 5 Autoantibodies by Immunouorescence
Cytochemistry. Methods in Molecular Biology, 79–87.
24. Singh, G., Arora, A., Kalra, P., Maurya, I. K., Ruizc, C. E.,
Estebanc, M. A., … Sehgal, R. (2019). A strategic approach
to the synthesis of ferrocene appended chalcone linked
triazole allied organosilatranes: Antibacterial, Antifungal,
Antiparasitic and Antioxidant studies. Bioorganic & Medicinal
Chem istr y.
25. Jia, S.-F., Hao, X.-L., Wen, Y.-Z., & Zhang, A. Y. (2019).
Synthesis, cytotoxicity, apoptosis and cell cycle arrest of a
monoruthenium(II)-substituted Dawson polyoxotungstate.
Journal of Coordination Chemistry, 1–9.
26. Xiong, W., Zhao, G. D., Yin, X., Linghu, K. G., Chu, J. M.
T., Wong, G. T. C., Wang, Y. T. (2019). Brij-grafted-chitosan
copolymers with function of P-glycoprotein modulation:
Synthesis, characterization and in vitro investigations.
Carbohydrate Polymers.
27. Yu, Y., Tazeem, T., Xu, Z., Du, L., Jin, M., Dong, C. Wu, S.
W. (2018). Design and Synthesis of Heteroaromatic Based
Be nzenesulfo na mide Der ivatives as Potent Inh ibitors of H5N1
Inuenza A Virus. MedChemComm.
28. Dar’in, D., Zarubaev, V., Galochkina, A., Gureev, M., &
Krasavin, M. (2019). Non-chelating p-phenylidene-linked bis-
imidazoline analogs of known inuenza virus endonuclease
inhibitors: Synthesis and anti-inuenza activity. European
Journal of Medicinal Chemistry, 161, 526–532.
29. Li, H., Li, M., Xu, R., Wang, S., Zhang, Y., Zhang, L., Xiao, S.
(2018). Synthesis, structure activity relationship and in vitro
anti-inuenza virus activity of novel polyphenol-pentacyclic
triterpene conjugates. European Journal of Medicinal
Chem istr y.
30. The European Medicines Agency, “ICH S9 Nonclinical
Evaluation for Anticancer Pharmaceuticals”, 2019, https://
www.ema.europa.eu/documents/scientific-guideline/ich-s-
9-nonclinical-evaluation-anticancer-pharmaceuticals-note-
guidance-nonclinical-evaluation_en.pdf, Accessed 13.02.2019.
31. A. H. Sheikh, A. Khalid, F. Khan, A. Begum, ChemistrySelect
2019, 4, 228.
32. Jiao , Y. H., et al. (2019) . “S ynthe si s of a nove l p-hydroxyci nna mic
amid e with a nt icancer capabilit y a nd its interaction with human
serum albumin.” Exp Ther Med 17(2): 1321-1329.
33. Serhii Holota, Anna Kryshchyshyn, Halyna Derkach, Yaroslava
Trun, Inna Demchuk, Andrzej Gzella, Philippe Grellier,
Roman Lesyk, Synthesis of 5-enamine-4-thiazolidinone
derivatives with trypanocidal and anticancer activity,
Bioorganic Chemistry, Volume 86, 2019, Pages 126-136, ISSN
0045-2068.