Design, synthesis and "in vitro" anti-leukemic evaluation of ferulic acid analogues as BCR-Abl inhibitors

Article abstract of DOI:10.1039/c6ra10106b

Semi-synthetic modification of ferulic acid isolated from Salicornia brachiata was performed and the derivatives showed gratifying in silico binding scores with the BCR-Abl protein, comparable with imatinib. Anti-proliferative activity against K562, U937 and Hep G2 cancer cell lines and the BCR-Abl kinase inhibitory activity using an ADP-Glo assay were investigated. Compounds 2i and 3j were potent BCR-Abl inhibitors and were also active against K562 cells.

Full text of DOI:10.1039/c6ra10106b

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Subramaniam, M. C. Raja, H. M. Venkatasubba Rao, S. R, V. Ulaganathan, B. Pemiah, S. Kar Mahapatra and
A. Sivasubramanian, RSC Adv., 2016, DOI: 10.1039/C6RA10106B.
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In the present analysis, all the backbone atoms in ligands are acetamide derivatives. Then to 3, the prepared N-Phenyl acetamide
interacting with the ATP binding site residues of BCR-Abl (GLU286 & was added and refluxed with K2CO3 and DMF (Fig. 2). Compounds
GLU282) by forming hydrogen bonds. In addition, hydrophobic from 3m-3w have been prepared by O- Alkylation of alkyl bromides
residues such as Val, Ile, Leu and Phe and charged residues are with 3. Reaction between compound 2 with isocyanate, gave the
forming the classical cationic-hydrophobic residue dyads. Among all compound 3x. In this case, DABCO has been used as catalyst and
the 39 compounds, (E)-4-(3-((4-fluorophenyl) thio)-3-oxoprop-1-en- THF, as the solvent. A type of Friedel-Crafts reaction of ferulic acid
1-yl)-2-methoxyphenyl acetate (2i), Methyl (E)-3-(3-methoxy-4-(2- with 2-naphthol and resorcinol by using lewis acid AlCl3, yielded 1,
oxo-2-thiomorpholinoethoxy) phenyl) acrylate (3j) shows good glide 2-dihydro-3H-benzo[f]chromen-3-one
derivatives.
Figure
3
score with BCR-Abl proteins (Table S1†). Figure 1 clearly expresses represents the scheme for the synthesis of 1, 2-dihydro-3H-
the binding sites of imatinib, ferulic acid and two ferulic acid benzo[f]chromen-3-one compounds (4a to 4c). The structure of the
analogues 2i and 3j, on BCR-Abl protein.
compound and the formation of lactone ring have been confirmed
with proton and carbon NMR and unequivocally confirmed with
single crystal XRD (Fig S4) †.
To further the study, the semi-synthetic modifications on FA was
performed. Figure 2 represents the schemes used for the synthesis
of compounds 2a-2k. Compounds 2a-2g have been prepared by
acid-amine coupling reactions of (E)-3-(4-acetoxy-3-methoxyphenyl)
acrylic acid chloride with aliphatic and heterocyclic amines. Then
the compounds (E)-2-methoxy-4-(3-oxo-3-(phenylthio)prop-1-en-1-
yl)phenyl acetate (2h), (E)-4-(3-((4-fluorophenyl)thio)-3-oxoprop-1-
en-1-yl)-2-methoxyphenyl acetate (2i), (E)-2-methoxy-4-(3-oxo-3-(p-
tolylthio)prop-1-en-1-yl)phenyl acetate (2j), phenyl (E)-3-(4-
acetoxy-3-methoxyphenyl)acrylate (2k) have been prepared by the
addition of 4-fluro-thiophenol, thio-cresol, thio-phenol and phenol
with FA-acid chloride respectively. The above three kind of
reactions were done with same reaction condition, of using
triethylamine catalyst at the temperature of 10°C for 3 h. The
structures of the all compounds have been confirmed with proton
and carbon NMR studies†.
Fig. 3 Schemes for the synthesis of compounds 3a-3x and 4a-4c.
All the synthesized FA analogues were screened for anti-
proliferative activity against K562 (human chronic myeloid
leukemia), U937 (human acute monocytic myeloid leukemia), Hep
G2 (Human liver cancer) cancer cell lines†. Cell viability was checked
in a dose dependent manner of different concentrations for FA
analogues (0.1 to 100 µg/mL). Also the effect of FA analogues on
normal PBM (peripheral blood mononuclear) cells were analysed
and they were found to be safe (Table S1)†. Among all the tested
compounds on K562 cells, 2i and 3j significantly (p<0.05) decreased
the cell viability. 2i and 3j had the IC50 values of 6.3µg/mL and
4µg/mL respectively. 2h and 2j also presented the IC50 values of
35µg/mL and 60 µg/mL respectively. Rest of the compounds shows
minor activity against K562 cells (with more than 100 µg/mL of IC50
value) (Table S1). The purity of the key compounds are depicted by
HPLC-MS (Fig S5-S8). As the FA analogues were active against K562
cells, the synthesized FA derivatives were then subjected to BCR-Abl
kinase inhibitory activity with the established ADP-Glo assay7. 16
compounds exhibited Bcr-Abl antiproliferative activity, comparable
to imatinib (Table 1).
Fig. 2 Schemes for the synthesis of compounds 2a-2k.
Figure 3 represents that the schematic diagram of reactions
involved in the synthesis of compounds from 3a-3x and 4a-4c.
Compound 3, methyl ferulate was prepared by the esterification of
ferulic acid with methanol and sulfuric acid at 80°C for 3 h. The
formation of 3 was confirmed with proton NMR†. Compound 3 was
taken for the synthesis of derivatives from 3a-3x. Synthesis of
To find the mechanism of apoptosis, ROS generation studies8,
biochemical parameters and DNA fragmentation studies, were done
on 2i and 3j. Further, annexin V-FITC/PI dual staining followed by
FACS analysis was carried out to evaluate the extent of apoptosis.
compounds 3a to 3l has done in two steps.
In the first step the
respective amine was converted into halogenated N-Phenyl
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Malondialdehyde (MDA) is used for the estimation of damage by
reactive oxygen species. MDA is a major reactive aldehyde resulting
from the peroxidation of biological membranes11. MDA level was
significantly (p < 0.01) increased in K562 cells treated with FA, 2i
and 3j by 100%, 156% and 176% respectively, as compared to their
control group. Glutathione is an important antioxidant in cellular
system. So, to understand glutathione level, we have measured
both reduced and oxidized form of glutathione. The reduced
glutathione (GSH) level was decreased (26.67%) significantly (p <
0.05) in FA treated K562 cells and more significantly (p < 0.01) in the
cells treated with 2i (73%) and 3j (60%), as compared with control
group. The oxidized glutathione (GSSG) level was increased (150%)
significantly (p < 0.05) in FA treated cells and more significantly (p <
0.01) in the cells treated with 2i (270%) and 3j (230%), as compared
with control group. The redox ratio was decreased significantly (p <
0.05) K562 cells treated with 2i (12 folds) and 3j (7.8 folds), as
compared with control group. (Fig. 5).
Docking score
against
Bcr-abl protein
-6.29
-8.17
-6.21
-6.08
-6.38
-6.98
-7.02
NB
Bcr-Abl Kinase
inhibition
(IC50 , µM)a,b
0.024
0.255
0.012
2.25
Compound
code
S.No
1
2
3
4
5
6
7
8
1
2
2a
2b
2c
2d
2e
2f
2h
2i
0.019
0.44
>500
1.69
0.012
0.019
0.44
>500
1.69
0.44
0.32
>500
1.69
0.024
0.89
0.017
58
0.024
16.2
0.012
0.024
2.65
2.58
0.015
1.69
9
-7.63
-8.86
-7.03
-6.66
-6.48
-7.89
-6.81
-7.16
NB
-7.52
-7.38
-7.65
-7.64
-8.26
-7.79
-7.12
-7.72
-5.98
-6.54
-5.8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
2j
2k
3
3a
3b
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
Induction of apoptosis is the most important key event and also
most studied for anticancer strategy. In the present study, induction
of apoptosis in leukemic cells by Ferulic acid derivatives was
monitored by DNA fragmentation12 and Et–Br/AO staining13 and
Annexin V-FITC/propidium iodide stating14
.
-6.52
-6.15
-5.6
-6.42
-6.81
-5.98
-7.52
-7.2
1.44
3s
3t
>500
0.019
0.032
0.15
0.24
0.18
3u
3v
3w
3x
4a
4b
4c
-8.25
-8.22
-6.8
0.24
0.022
0.015
0.074
Imatinib
-7.78
Table 1. In silico docking and Bcr-abl kinase inhibitory studies of
ferulic acid derivatives. a- Mean value; b- Boldface: IC50 ≤ imatinib
Reactive oxygen species (ROS) are formed as a natural by-product
of normal metabolism and play crucial roles in cell signalling and
homeostasis9. However, an increase in the intracellular ROS may
result in significant damage to cell morphology, which ultimately
leads to cell death10. DCFH2-DA is widely used as an oxidant-
sensitizer, is non-fluorescent, and switches to a highly fluorescent
DCF when oxidized by ROS and other peroxides. The addition of
Fig. 4 FA, 2i and 3j induced ROS generation by estimation of DCF
Fluorescence by (A) spectrofluorometry, (B) FACS analysis and (C)
DCFH2-DA to the cells treated with FA, 2i and 3j showed strong fluorescence microscopy. *p<0.05, **p<0.01 indicate the significant
fluorescence at 520 nm, when compared with control untreated
cells, indicating the drug induced ROS generation in cells. There was
a 2fold, 5 fold and 4 fold increase in the ROS generation observed in
the cells treated with FA, 2i and 3j respectively, when compared
with control untreated cells (Fig. 4a). Further FA, 2i and 3j induced
ROS generation in K562 cells was confirmed by analysing the cells in
fluorescence activated cell shorter (FACS, BD) (Fig. 4b) and
observing the cells under fluorescence microscope (Fig. 4c).
differences compared with untreated cells.
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its mRNA expression16, a study of the BCR-Abl kinase domain
expression by conventional RT-PCR and real time PCR in K562 cells
after treatment with FA, 2i and 3j, in comparison with imatinib, was
carried out (Fig. 7). It was found that, BCR-Abl kinase domain was
significantly (p<0.01) decreased upon treatment with 2i and 3j, and
was comparable with the known BCR-Abl inhibitor, imatinib.
Plausibly, the decrease in BCR-Abl kinase domain can be linked and
correlated with the activity of 2i and 3j, on K562 cells.
Fig. 5 Biochemical estimation of (A) GSH, (B) GSSG, (C) GSH/GSSG
ratio, and (D) lipid peroxidation in K562 cells after tretatment with
FA, 2i, 3j and imatinib. *p<0.05, **p<0.01 indicate the significant
differences compared with untreated cells.
Fig. 7 FA, 2i, 3j and Imatinib decreased the Bcr-Abl kinase domain
mRNA expression by (A) conventional RT-PCR and (B) quantitative
real-time PCR. **p<0.01 indicates the significant differences
compared with untreated cells.
Fig. 6 Flow cytometric detection of apoptosis by Annexin V/PI
staining of K562 cells with FA, 2i, 3j and Imatinib.
Significant (p < 0.01) DNA fragmentation was observed in the cells
treated with 2i and 3j, as compared with control group (Fig S9). Et–
Br/AO staining images showed that, AO was taken up by both viable
and nonviable cells and emits green fluorescence, whereas Et–Br
was taken up only by non-viable cells and emits orange-red
fluorescence because of the intercalation into DNA. It is clear that
K562 cells treated with FA, 2i and 3j, showed orange-red
fluorescence, suggesting the presence of non-viable cells 15(Fig S9
C).
Fig. 8 Proposed mechanisms of 2i and 3j on Bcr-Abl protein
Based on the anti-proliferative studies of Ferulic acid derivatives,
the plausible anti-leukemic pathway of 2i and 3j is represented in
Fig. 8. When the compounds bind to the receptor site of BCR-Abl
protein, it inhibits the ATP Signalling, followed by inhibition of
cancer cell survival and proliferation signals. This leads to apoptosis
and the proliferations of CML cell proliferation will be controlled.
To evaluate the extent the apoptosis, the annexin V-FITC/PI dual
staining followed by FACS analysis was carried out (Fig. 6). Results
showed that 4.23 % untreated K562 cells and 12.54% FA treated
K562 cells undergo apoptosis and, 48.56% -2i treated and 42.49% -
3j K562 cells experience apoptosis, respectively. Results of annexin
V-FITC and PI-staining on K562 cells, indicates late stage apoptosis.
Thus, this result confirms the pivotal role of 2i and 3j in inducing
apoptosis in K562 cells. As BCR-Abl kinase activity is depended on
Conclusions
In conclusion, a series of ferulic acid derivatives were synthesized
and evaluated for anti-proliferative activity against K562, U937, Hep
G2 cells. The compounds were further tested for BCR-Abl kinase
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inhibition. In silico studies on the Bcr-Abl protein with these ferulic
acid analogues demonstrated good docking scores, comparable
with the drug imatinib. Among the tested compounds for in-vitro
anti-leukemic activity against K562 cells, compounds 2i and 3j
showed excellent activity, deserving further development of anti-
leukemic drugs from the derivatives of ferulic acid.
Acknowledgements
The authors would like to thank the management, SASTRA
UNIVERISTY, for providing the necessary facilities and the TRR
Funding. The financial support of SERB-DST, Government of India,
under the EMR scheme (EMR/2016/000326) is earnestly
acknowledged.
References
1. J. Seralathan, P. Stevenson, S. Subramaniam, R. Raghavan,
B. Pemaiah, A. Sivasubramanian and A. Veerappan,
Spectrochem Acta Mol Biomol Spectrosc., 2014, 118, 349-
355.
2. (a) N. Kumar and V. Pruthi, Biotech Reports, 2014, 4, 86-
91; (b) C. Mancuso and R. Santangelo, Food Chem Toxicol.,
2014, 65, 185-195; (c) A. Sgarbossa, D. Giacomazza and M.
Carlo, Nutrients, 2015, 7, 5764-5782.
3. (a) N. Kumar, S. Kumar, S. Abbat, K. Nikhil, S.M. Sondhi,
P.V. Bharatam, P. Roy and V. Pruthi, Med Chem Res.,
2016, 1-18; (b) L.C. Lopes, B. Roman, M.A. Medeiros, A.
Mukhopadhyay, P. Utrilla, J. Galvez, S.G. Maurino, V.
Moltiva, A. Lourenco and A.S. Feliciano, Tetrahedron let.,
2011, 52, 6392-6295; (c) W. Li, N. Li, Y. Tang, B. Li, L. Liu,
X. Zhang, H. Fu and J. Duan, Bioorg Med Chem Lett., 2012,
22, 6085-6088 ; (d) S. Oliver, O. Vittorio, G. Cirillo and C.
Boyer, Polym Chem., 2016,7, 1529-1544; (e) A. Piazzon, U.
Vrhovsek, D. Masuero, F. Mattivi, F. Mandoj and M.
Nardini, J Agric Food Chem., 2012, 60, 12312−12323.
4. M. Deininger, J.M. Goldman and J.V. Melo, Blood, 96,
3343-3358.
5. (a) R. Ren, Reviews, 2005, 5, 172-183; (b) S. Salesse and C.
M. Verfaillie, Oncogene, 2002, 21, 8547-8559.
6. M. Deininger, E. Buchdunger and B.J. Druker, Blood, 2005,
105, 2640-2650.
7. X. Pan, J. Dong, R. Shao, P. Su, Y. Shi, J. Wang, L. He,
Bioorg. Med. Chem. Lett., 2015, 25, 4164–4168.
8. D. Wu and P. Yotnda, J Vis Exp, 2011, 57, 1-4.
9. A. Bhattacharyya, R. Chattopadhyay, S. Mitra, S.E. Crowe,
Physiol Rev., 2014, 94, 329–354.
10. S. Fulda, A.M. Gorman, O. Hori, A. Samali, Int J Cell Biol.,
2010, Article ID 214074.
11. D.R. Janero, Free Radic Biol Med., 1990, 9,515-40.
12. K.A.E. Diab, A.I. Elmakwy, O.M. Elmoneim and H.A. Sharaf,
J Cytol Histol., 2012, 1, 3-5.
13. M.G. Curcic, M.S. Stankovic, E.M. Mrkalic, Z.D. Matovic,
D.D. Bankovic, D.M. Cvetkovic, D.S. Dacic and S.D.
Markovic, Int. J. Mol. Sci., 2012, 13, 2521-2534.
14. K.B.A. Ahmed, S. K. Mahapatra, M.R.C. Raja, S.
Subramanian, M. Sengan, N. Rajendran, S.K. Das, K.
Haldar, S. Roy, A. Sivasubramanian and V. Anbazhagan,
RSC Adv, 2016, 6, 18980-18989.
15. S.K. Mahapatra, S.P. Chakraborty, S. Das and S. Roy, Oxid
Med Cell Longev., 2009, 2, 222-230.
16. Y. Dai, M. Rahmani, S.J. Corey, P. Dent, S. Grant S, J Biol
Chem., 2004, 279, 34227–34239.
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