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Contents
ABSTRACT:..................................................................................................................................................2
INTRODUCTION...........................................................................................................................................2
Methodology:.............................................................................................................................................3
Experimental Procedure for the Biological Assay..................................................................................5
Implications:............................................................................................................................................5
CONCLUSIONS.............................................................................................................................................9
References.................................................................................................................................................10
ABSTRACT:
"In TKI-resistant acute myeloid leukaemia (CML) cells, the natural substance rakicidin A causes
cell death. To test their efficacy against CML cell lines, researchers synthesised 14 rakicidin A
counterparts using a very effective combinatorial technique. Rakicidin A's anti-CML action was
shown to be highly dependent on its conjugated diene moiety; modifications to its structure at
carbons 2, 3, 14, 15, and 16 led to a decrease in anti-CML activity. 4-Methylester rakicidin A
was the most promising chemical. Against the imatinib-resistant K562/G+ cell line, 1a showed
about 100-fold higher efficacy compared to imatinib and 2.8-fold increased potency compared to
rakicidin A. Compared to takes, dasatinib, nilotinib, and factors that can change, compound 1a
showed much reduced resistance indices against Ba/F3-positive cells (BCR-ABLT315I), while
showing less impact on ordinary hematopoietic cells. Preliminary data suggested that 1a inhibits
K562 cells in part by reducing the activities of the death-inducing proteins caspase-3 and PARP.

INTRODUCTION
"The Philadelphia chromosome translocation that creates the BCR-ABL oncogene is present in
more than 95% of those with CML." In the treatment of chronic myeloid leukaemia (CML),
imatinib, a potent BCR-ABL medication used as a TKI, is now employed as first-line therapy
due to its high clinical efficacy. Yet, imatinib resistance is a substantial problem in clinical
practise. It has also been shown that between 40% and 50% of children with acute phase CML
and 80% or more of individuals with erythrocytic phase CML are initially sensitive to imatinib.
Second-line therapies for CML include TKIs such as silibinin, dasatinib, and bosutinib, all of
which are successful despite acquired resistance to adalimumab. Unfortunately, these cutting-
edge medications are ineffective for those who have the T315I mutation. (Roychowdhury, 2011)

"Because of their structural diversity and wide range of biological activities, rakicidins are of
tremendous interest to the synthetic and pharmaceutical research communities." The researchers
recently made predictions for the six a priori unknown chiral centres in antihypertensive
medication A: (2S), (3S), (14S), (hsp), and (16R).
Later, these assumptions were confirmed by the synthesis and characterization of words (ns are
of tremendous interest to the synthetic and pharmaceutical research communities." We recently
made predictions for the six a priori unknown chiral centres in antihypertensive medication A:
(2S), (3S), (14S), (hsp), and (16R).

Later, these assumptions were confirmed by the synthesis and characterization of words (e.g., A)
as well as an analysis of rakicidin A, the principal feedstock for degradation. "Based on a
previously described method for making rakicidin A, the present study synthesised many
analogues and investigated the structural-functional relationships between them." (Bixby, 2011)
(Talpaz, 2006)

Methodology:
All reactions were carried out in an anhydrous atmosphere with dry solvents in a dry gaseous
atmosphere, unless otherwise stated. The used solvents were cleaned and dehydrated using
conventional techniques. Except where otherwise noted, all yields are assumed to have
spectroscopic and chromatographic (1 H NMR) homogeneity.

Unless otherwise stated, we used reagents in their cleanest commercial form. To determine
purity, a Shimadzu LD-20A instrument was used in conjunction with an ODS-C18 line (4.6 mm,
150 mm, and 5 m) drained at 1 mL/min with Milli-Q fluid and CH3CN. All chemicals tested at
or above 95% purity. The FTIR spectra were obtained using a Bruker Tensor 27.

All IR samples were thin films, and all wavelengths were reported in centimetres.The researchers
recorded spectra using a CDCl3 or DMSO-d6 solvent and a 404 MPh (1 H: nmr, 13C: 200 MHz)
nuclear magnetic resonance (NMR) spectrometer.
Figure 1. Structure of rakicidin A, B, C, and D, microtermolides A, vinylamycin, and BE-43547A1

Scheme 1. Retrosynthetic Analysis of Rakicidins( Burgess.,2005).

Experimental Procedure for the Biological Assay

"K562, overexpressing, and Bcom cells were cultured in RPMI-1640 with 10% FB at 37 °C in
either a humidified environment of 5% CO2. To summarize, 1 104 exponentially developing
cancer cells were seeded in a 96-well plate. Then, the chemical at varying concentrations was
introduced. At 72 h, MTT was put into each well at a final concentration of 0.5 mg/mL. Finally,
the cells were kept in a 37°C, 5% CO2 incubator for 4 hours. The growth medium was then
withdrawn, and reaction mixture crystals were concentrated in DMSO. At 570 nm, we found a
significant absorption. GraphPad Prism 5 was used to get the IC50 values. The co-incubation of
rakicidin A and component 1a for 24 hours was performed on normal hematopoietic cells from
volunteer donors at the Tianjin University Hospitals of Gynecology (Tianjin, China). Finally, 20
litres of MTD (5 mg/mL) were put into each well, and the plates were incubated for 4 hours at 37
°C. After the removal of the growth medium, the formazan crystals were mixed with DMSO. At
570 nm, we found a significant absorption. GraphPad Prism 5 was used to get the "CC50
values." (Burgess, 2005).

Table 1. Synthesis of Compounds 8a−8c(Bradeen.,2006).

Implications:
More advanced methods for determining the leukemia should be employed.

Results and discussion

Synthesis of compounds

The retrosynthetic approach to producing rakicidins is shown in Scheme 1. This plan's

combinational approach was very similar to those seen in published synthesis plans.Rakicidins'
progenitor consisted of a polyketide fragment composed of three isomers, a compound, a serinol,
and a 3-hydroxyaspartic acid analogue (with two isomers).

Activities of the synthesized compounds against two CML cell lines

Using two different CML cell lines (K562 and K562/G+), 14 synthetic analogues of rakicidin A
were tested for their effects on cell viability. K562, the first immortalised human myelogenous
leukaemia line, has an imatinib-resistant variant. Imatinib was used as a standard, and
antihypertensive medication A was incorporated so that the findings could be compared to those
of the parent drug in the biological experiments.

Table 2. Inhibitory Effects of Compounds 1 and 2 on CML Line K562 and Imatinib
Resistant Cell Line K562/G+a (Burgess, 2005)

Effect of Methylester Rakicidin A (1a) on Ba/F3 Cells Expressing BCR-ABLT315I

Ba/F3-overexpressed BCR-ABLT315I was used in a screening test for methylester language.

This includes the letter A (1a) as well as words (for example, A.Inhibitors currently on the
market, such as bosutinib, dasatinib, indinavir, and ponatinib, were used to simulate the effects
of the imatinib treatment and serve as a positive control. Table 3 displays the final findings. The
four kinase inhibitors bosutinib, imatinib, nilotinib, and ponatinib all had potent action against
K562 cells.
Although rakicidin-A and its methylester-rakicidin A (1a) had IC50 values of 0.48 M and 0.63
M, respectively, for inhibiting K562, the two drugs had comparable activity to those of bosutinib
& nilotinib against Ba/F3 cell that expressed BCR-ABLT315I and had a low - impedance index
of 3.2.

We also looked at how rakicidin A and its methyl ester, rakicidin A (1a), affected typical
hematopoietic cells. Compounds 1a and rakicidin A both had low cytotoxic effects on healthy
hematopoietic cells, as indicated in Table 4, with CC50 values of 3.0 M and 7.5 M, respectively.
Compound 1a is selective for cancer cells since its IC50 value for cancer cells is substantially
lower than its CC50 value.
K562 Cells Were Induced to Apoptosis by Methylester Rakicidin A (1a).

The possibility of apoptosis as a mechanism for compound 1a's anti-cancer effects was
investigated in further depth. Both compound 1a and words ( as a mechanism for compound 1a's
anti-cancer effects was investigated in further depth. Both compound 1a and words (e.g., A) were
shown to induce apoptosis in K562 cells, and their effects were dose-dependent. The data
showed that compound 1a's inhibitory action was at least in part due to its ability to cause
apoptosis in K562 cells. (Rowley, 1973)

Figure 2. Induction of apoptosis on K562 cells by compound 1a, **P < 0.01.( Burgess.,2005).
(1a) Methyl ester Rakicidin

Proteins Associated with Controlled Apoptosis Several apoptosis-related proteins were evaluated
using the Western blot technique to help identify the probable mechanisms of apoptosis triggered
by components 1a and rakicidin A. There was a shift in the protein levels of protein kinase C
(PKC) and PARP, as determined by Western blotting. After 72 hours of treatment with
component 1a and rakicidin A, the expression of the cell death modulators caspase-3 and
phosphatidylinositol 3-phosphate (PARP) was reduced. (Talpaz, 2006)

CONCLUSIONS
"First-line therapy for CML now includes imatinib." However, imatinib's therapeutic
effectiveness is limited by imatinib resistance, which may be attributable to the existence of a
treatment response in CML tissue culture. Cell death may be induced in TKI-resistant CML
lineage cells using the natural substance rakicidin A. Therefore, the auther synthesised a library
of 14 rakicidin substitutes by a very efficient combinatorial technique, using rakicidin as a
structural template because of its anti-CML cell therapy action. The compounds' anti-CML
activity was then assessed. According to these early results from a structure-activity relationship
investigation, the connected diene moiety of rakicidin A is essential for its anti-CML effect.
Rakicidin A's activity was also dampened by modifications to its structure at carbons 2, 3, 14,
15, and 16. Methylester rakicidin A (1a) demonstrates the most promise, being approximately
100 times stronger against the Murine cell line than adalimumab and demonstrating a lower
overall resistance measure against Ba/F3 cell lines (BCR-ABLT315I) than dasatinib, nilotinib,
and other factors that can change, while also having a smaller impact on normal hepatocytes.
Preliminary data suggested that 1a's inhibitory impact on K562 cells was due, in part, to its
ability to reduce caspase-3 and PARP. Methylester produced for improving A (1a) had a higher
yield and fewer stages than rakicidin A. Further investigations of the mechanism of action of 1a
are now underway in our labs, and the findings will be presented in due course, given that 1a has
a specific design that varies from previous anti-CML medicines and is extremely effective
against that imatinib-resistant Mm cell line (K562/G+).
Future work:

This study will provide a base for determining more advanced methods to find out cancer . This
will be the baseline to determine the moity substances.

References
Bixby, D. a. T. M., 2011. Seeking the causes and solutions to imatinib-resistance in chronic myeloid
leukemia.. Leukemia, Volume 25(1), pp. 7-22..

Burgess, M. S. B. S. N. L. F. a. S. C., 2005. Comparative analysis of two clinically active BCR-ABL kinase
inhibitors reveals the role of conformation-specific binding in resistance.. Proceedings of the National
Academy of Sciences, , 102(9)(102(9),), pp. 3395-3400.

Rowley, J., 1973. A new consistent chromosomal abnormality in chronic myelogenous leukaemia
identified by quinacrine fluorescence and Giemsa staining.. Nature, Volume 243(5405), pp. 290-293..

Roychowdhury, S. I. M. R. D. L. R. W. Y. C. X. K.-S. S. S. L. B. O. Q. M. a. B. T., 2011. Personalized oncology

through integrative high-throughput sequencing: a pilot study.. Science translational medicine, Volume
3(111), pp. pp.111ra121-111ra121..

Talpaz, M. S. N. K. H. D. N. N. J. P. R. C. J. O. S. N. C. B. E. a. B.-C. M., 2006. Dasatinib in imatinib-resistant

Philadelphia chromosome–positive leukemias.. New England Journal of Medicine, Volume 354(24), pp.
2531-2541..