Cytotoxicity of aporphines in human colon cancer cell lines HCT-116 and Caco-2: An SAR study

Article abstract of DOI:10.1016/j.bmcl.2011.06.005

A series of synthetic aporphine derivatives structurally related to domesticine and nantenine (ring A, N6 and ring C truncated analogs), was evaluated in MTS cytotoxicity assays against the human colon cancer cell lines, HCT-116 and Caco-2. In general, the C1 position of ring A is tolerant of alkoxy substituents as well as a benzoyl ester functionality. Other modifications evaluated resulted in a decrease in cytotoxic activity. The most potent compounds identified had IC₅₀ values in the range 23-38 μM, comparable to the known cytotoxic agent, etoposide.

Full text of DOI:10.1016/j.bmcl.2011.06.005

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4462–4464
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Cytotoxicity of aporphines in human colon cancer cell lines HCT-116 and Caco-2:
An SAR study
Shashikanth Ponnala a, Sandeep Chaudhary a, Antonio González-Sarrias b, Navindra P. Seeram b,
Wayne W. Harding a,
⇑
a Chemistry Department, Hunter College, CUNY, 695 Park Avenue, New York, NY 10065, USA
b Bioactive Botanical Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, 41 Lower College Road, University of Rhode Island,
Kingston, RI 02881, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of synthetic aporphine derivatives structurally related to domesticine and nantenine (ring A, N6
and ring C truncated analogs), was evaluated in MTS cytotoxicity assays against the human colon cancer
cell lines, HCT-116 and Caco-2. In general, the C1 position of ring A is tolerant of alkoxy substituents as
well as a benzoyl ester functionality. Other modifications evaluated resulted in a decrease in cytotoxic
Received 27 April 2011
Revised 31 May 2011
Accepted 2 June 2011
Available online 14 June 2011
activity. The most potent compounds identified had IC50 values in the range 23–38
the known cytotoxic agent, etoposide.
lM, comparable to
Keywords:
Aporphine
Nantenine
Cytotoxic
HCT-116
Caco-2
Ó 2011 Elsevier Ltd. All rights reserved.
Cancer is the second leading cause of death in developed coun-
tries and colon cancer is the third most common cancer in the
world, with prevalence mainly in Western countries. Despite the
fact that many new chemotherapeutic drugs have been developed
and many aggressive treatments are available, cancer death rates
continue to rise.1 Many chemotherapeutic agents act through cyto-
toxicity which leads to inhibition of carcinogenesis. Development
of resistance to approved chemotherapy medications as well as a
myriad of side-effects of these compounds in cancer patients, has
contributed to the continued challenge in treating various forms
of cancer. Thus, there is a constant search for new cytotoxic agents
that can serve as leads for the development of chemotherapeutics.
Furthermore, the discovery of new cytotoxic agents may afford the
opportunity to obtain a more detailed understanding of the mech-
anistic underpinnings of this deadly scourge.
Aporphine alkaloids are endowed with a range of biologicalactiv-
ities and may well be considered to be privileged drug discovery
scaffolds. For example, naturally occurring and synthetic aporphines
have been investigated as acetylcholinesterase inhibitors,2–4 CNS
receptor ligands,5–8 and as antimicrobial,9 antimalarial10,11 and anti-
viral12,13 agents. In addition, there are a number of reports on the
cytotoxic activity of some members of this alkaloid class.14–16 Prior
studies on aporphines as cytotoxic agents have focused exclusively
on naturally-occurring aporphines. The biological targets involved
in the cytotoxic activity of aporphines are yet to be fully elucidated
although DNA-mediated and topoisomerase-related mechanisms
appear to play a role in some instances.17–20 Interpretation of cur-
rently available information with regards to the SAR of these mole-
cules as cytotoxic agents is unreliable because of the diversity of
assay systems and cell lines employed by various investigators. To
date, no systematic study of the structure–activity relationships
(SARs) of aporphines as cytotoxic agents has been conducted. An
understanding of structure–activity effects is a necessary aspect of
any future undertaking to further understand the mechanism of ac-
tion of these molecules as well as to capitalize on their therapeutic
potential.
Herein, we present results from an SAR evaluation of the cyto-
toxic activity of a set of synthetic aporphine derivatives (some of
which we have acquired in the process of other investigations) in
the human colon cancer cell lines HCT-116 and Caco-2.
Based on the structural similarity of domesticine (1) and nant-
enine (2) (Table 1) to other known cytotoxic aporphines, we ini-
tially screened these compounds in HCT-116 and Caco-2 colon
cancer cell lines. In these assays, 1 had moderate activity, (approx-
imately three- and four-fold less potent, respectively, than the
benchmark etoposide). However, we were enthused to find that
2 had potency comparable to that of etoposide in both cell lines.
This suggested that a C1 phenolic group on the aporphine scaffold
⇑
Corresponding author.
E-mail address: whardi@hunter.cuny.edu (W.W. Harding).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.005

S. Ponnala et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4462–4464
4463
Table 1
group removed by treatment with ZnBr2, and the amine alkylated
via reductive amination to give the bromo-nantenine analog, com-
pound 6. (We also attempted to synthesize 6 via direct NBS bro-
mination of 2 but found that this method gave poly-halogenation
of the aromatic rings.)
Compounds 1–14 were evaluated in MTS assays as described by
us23 with modifications.24 Table 2 summarizes the data from this
evaluation. Replacement of the C1 hydroxyl group of domesticine
(1) with alkyloxy groups (i.e., compounds 2–4) resulted in im-
proved cytotoxicity versus the cell lines tested. The branched alkyl
analog 3 was the most potent against the cell lines evaluated and
was approximately two-fold selective for colon cancer cells versus
normal colon fibroblasts (CCD-18Co).
Ring A, N6 and seco-ring C analogs evaluated.
R2
3
O
O
O
O
O
2
A
B
NR3
N
N
R1O
1
6
C
D
10
9
O
O
O
O
O
O
14
13
1-12
The n-pentyl analog 4 was slightly less potent than nantenine
(2) in the HCT-116 cell line but was equipotent in the Caco-2 cell
line.
Together, these results suggest that C1 alkyl branching results
in higher cytotoxicity but also indicates that increasing n-alkyl
chain length is detrimental to activity versus HCT-116 cells. The
benzoyl ester 5 had slightly improved activity as compared to 2
in HCT-116 and Caco-2 cells and was moderately more selective
with regards to toxicity in non-tumorigenic human colon CCD-
18Co cells. Addition of a bromine atom to position 3 of nantenine
(i.e., compound 6) decreased cytotoxic activity in all cell lines.
Replacement of the N-methyl group of nantenine (2) with acetyl,
ethyl carbamate or methanesulfonyl groups was detrimental to
cytotoxic activity (compounds 7–9 respectively). However, com-
parison of domesticine (1) to its N-ethyl carbamate analog 10 re-
vealed no significant difference in cytotoxicity. Although C1
alkylation gave improved activity in the N-methyl series, this
structural change was not associated with improved activity in
the N-ethyl carbamate series of analogs, as comparison of 10 with
11 and 12 reveals. Cumulatively, the evaluation of 1–12 is indica-
tive of a greater preference for the N-methyl group of the apor-
phine skeleton as compared to other N-protected functionalities
in relation to cytotoxic activity. Additionally, the presence of an
N-ethyl carbamate functionality appears to be better tolerated
with a C1 hydroxy group than with a C1 methoxy group on the
aporphine skeleton. The ring truncated analogs 13 and 14 had sig-
nificantly lower activity as compared to 2. This points to a require-
ment for ring C of the aporphine nucleus to be intact for cytotoxic
activity.
In conclusion, our study has uncovered potent cytotoxic activity
of the aporphine alkaloid nantenine (2), the C1 alkoxy derivative
(3) and the C1 benzoate derivative (5). Of these, 3 and 5 exhibit
marginally higher selective cytotoxicity versus normal cells. These
compounds have potencies comparable to that of the clinically
available anticancer therapeutic drug, etoposide in vitro. This is
the first account of the cytotoxic activity of aporphines in HCT-
116 and Caco-2 human colon cancer cell lines. Our SAR evaluation
indicates that the N-methyl group and the intact aporphine nu-
cleus of 2 are important for its cytotoxic activity. Interestingly, this
study also indicates that 2 may be manipulated via substitution of
Compound #
R1
R2
R3
1
2
3
4
5
6
7
8
H
CH3
H
H
H
H
H
Br
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3CO
CH3CH2OCO
CH3SO2
(CH3CH2)2CHCH2
CH3(CH2)3CH2
PhCO
CH3
CH3
CH3
CH3
H
CH3CH2
PhCH2O
9
10
11
12
CH3CH2OCO
CH3CH2OCO
CH3CH2OCO
was detrimental to activity and we therefore decided to investigate
the effect of replacement of this phenolic group with other moie-
ties. Thus compounds 3–5 were targeted for cytotoxicity studies
to aid in understanding the SAR at this position. In addition, to
probe the effects of other structural changes in ring A and N6 of
the aporphine nucleus, compounds 6–12 were evaluated. An
assessment of the requirement for an intact aporphine skeleton
was obtained via evaluation of the seco-ring C analogs 13 and 14.
Compounds 1, 2, 4 and 7–13 were prepared as described by us
previously.2,7,8,21,22 Compound 3 was prepared in three steps by
alkylation of readily available phenol 108 followed by treatment
of the carbamate product with lithium aluminium hydride
(Scheme 1) and alkylation of the secondary amine product thus
formed (due to cleavage of the N-carbamate group). Compound 5
(Scheme 1) was synthesized via lithium aluminium hydride reduc-
tion of 10. This reduction gave predominantly the secondary
amine; the crude mixture was subsequently subjected to reductive
amination to yield domesticine (1). Acylation of 1 with benzoic
anhydride afforded 5.
Phenol 15, was prepared in a manner analogous to the synthesis
of 10,8 and was brominated with NBS to give 16 (Scheme 2). In a
sequential manner, compound 16 was O-methylated, the boc
1. LiAlH4, THF
1. 1-Bromo-2-ethylbutane
O
O
2. HCHO, NABH(OAc)
DCM
K2CO3, acetone
reflux
O
O
3 O
O
N
3. benzoic anhydride,
Et3N, DMAP
2. LiAlH4, THF
3. HCHO, NaBH(OAc)3
DCM
N
NCO2Et
HO
O
O
O
O
O
O
5
3
10
Scheme 1. Synthesis of compounds 3 and 5.

4464
S. Ponnala et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4462–4464
Br
1. CH3I, K2CO3
acetone, reflux
2. ZnBr2, DCM
3, HCHO, NaBH(OAc)3,
DCM
Br
HO
O
HO
O
O
O
NBoc
NBoc
N
NBS, DCM
O
O
O
O
15
O
16
O
6
Scheme 2. Synthesis of compound 6.
5. Cabedo, N.; Berenguer, I.; Figadere, B.; Cortes, D. Curr. Med. Chem. 2009, 16,
2441.
6. Zhang, A.; Zhang, Y.; Branfman, A. R.; Baldessarini, R. J.; Neumeyer, J. L. J. Med.
Chem. 2007, 50, 171.
Table 2
Cytotoxicity of compounds 1–14 against human colon cancer (HCT-116, Caco-2) and
normal colon (CCD-18Co) cell lines
Compound #
IC50 (
Caco-2
l
M)a
7. Pecic, S.; Makkar, P.; Chaudhary, S.; Reddy, B. V.; Navarro, H. A.; Harding, W. W.
Bioorg. Med. Chem. 2010, 18, 5562.
8. Chaudhary, S.; Pecic, S.; Legendre, O.; Navarro, H. A.; Harding, W. W. Bioorg.
Med. Chem. Lett. 2009, 19, 2530.
9. Chen, J. H.; Du, Z. Z.; Shen, Y. M.; Yang, Y. P. Arch. Pharm. Res. 2009, 32, 3.
10. Ayers, S.; Zink, D. L.; Mohn, K.; Powell, J. S.; Brown, C. M.; Murphy, T.; Brand, R.;
Pretorius, S.; Stevenson, D.; Thompson, D.; Singh, S. B. Planta Med. 2007, 73,
296.
11. Waechter, A. I.; Cave, A.; Hocquemiller, R.; Bories, C.; Munoz, V.; Fournet, A.
Phytother. Res. 1999, 13, 175.
HCT-116
CCD-18Co
1
2
3
4
5
6
7
8
73.3 4.6
38.3 1.6
26.6 1.1
46.3 2.6
25.2 2.2
100.3 2.1
185.7 3.4
81.4 1.2
196.1 3.4
72.7 2.4
73.3 4.6
93.3 4.7
190.9 4.0
195.4 5.7
25.5 2.1
73.5 3.2
36.2 2.4
22.8 3.5
35.3 2.4
25.9 1.8
100.7 1.7
159.6 6.4
83.2 3.8
195.8 4.3
71.7 2.8
73.5 3.2
99.8 2.3
191.3 2.5
189.8 5.7
18.6 1.8
133.5 3.1
56.5 1.9
50.7 2.0
73.4 2.1
45.1 0.8
180.6 3.5
188.5 2.2
111.6 1.6
n.d.
127.9 5.4
133.5 3.1
109.6 0.6
n.d.
12. Boustie, J.; Stigliani, J. L.; Montanha, J.; Amoros, M.; Payard, M.; Girre, L. J. Nat.
Prod. 1998, 61, 480.
13. Montanha, J. A.; Amoros, M.; Boustie, J.; Girre, L. Planta Med. 1995, 61, 419.
14. Makarasen, A.; Sirithana, W.; Mogkhuntod, S.; Khunnawutmanotham, N.;
Chimnoi, N.; Techasakul, S. Planta Med. 2011. doi:10.1055/s-0030-1270743.
15. Mohamed, S. M.; Hassan, E. M.; Ibrahim, N. A. Nat. Prod. Res. 2010, 24, 1395.
16. Stevigny, C.; Block, S.; De Pauw-Gillet, M. C.; de Hoffmann, E.; Llabres, G.;
Adjakidje, V.; Quetin-Leclercq, J. Planta Med. 2002, 68, 1042.
17. Hoet, S.; Stevigny, C.; Block, S.; Opperdoes, F.; Colson, P.; Baldeyrou, B.;
Lansiaux, A.; Bailly, C.; Quetin-Leclercq, J. Planta Med. 2004, 70, 407.
18. Goren, A. C.; Zhou, B. N.; Kingston, D. G. Planta Med. 2003, 69, 867.
19. Woo, S. H.; Reynolds, M. C.; Sun, N. J.; Cassady, J. M.; Snapka, R. M. Biochem.
Pharmacol. 1997, 54, 467.
9
10
11
12
13
14
Etoposide
n.d.
44.4 1.8
n.d. = not determined.
a
Determined at 72 h.
20. Stevigny, C.; Bailly, C.; Quetin-Leclercq, J. Curr. Med. Chem. Anticancer Agents
2005, 5, 173.
21. Chaudhary, S.; Pecic, S.; Legendre, O.; Harding, W. W. Tetrahedron Lett. 2009, 50,
2437.
22. Legendre, O.; Pecic, S.; Chaudhary, S.; Zimmerman, S. M.; Fantegrossi, W. E.;
Harding, W. W. Bioorg. Med. Chem. Lett. 2010, 20, 628.
23. Li, L.; Henry, G. E.; Seeram, N. P. J. Agric. Food Chem. 2009, 57, 7282.
24. Cell lines and culture conditions: Human colon cancer cell lines Caco-2
(adenocarcinoma) and HCT-116 (carcinoma) were obtained from American
Type Culture Collection (Rockville, USA). Caco-2 cells were grown in EMEM
medium supplemented with 10% v/v fetal bovine serum, 1% v/v nonessential
the C1 methyl group with other alkyl groups without severely
compromising cytotoxicity. The findings herein are relevant to
the study of aporphines as potential anticancer drugs and are also
significant in other therapeutic areas (where cytotoxicity of the
molecules described may not be desirable).
The importance of substituents on other portions of the nante-
nine aporphine core (e.g., ring D) remains to be determined.
Furthermore, the selectivity versus other cancer cell lines and
mechanism(s) involved in the cytotoxic actions of 2, 3 and 5 are
unknown at present. These are dimensions that we intend to
explore in future.
amino acids, 1% v/v L-glutamine and 1% v/v antibiotic solution (Sigma). HCT-
116 cells were grown in McCoy’s 5a medium supplemented with 10% v/v fetal
bovine serum, 1% v/v nonessential amino acids, 2% v/v HEPES and 1% v/v
antibiotic solution. Cells were maintained at 37 °C in an incubator under a 5%
CO2/95% air atmosphere at constant humidity. Cells were counted using a
hemacytometer and were plated at 5000–3000 cells per well, in a 96-well
format for 24 or 48 h prior to MS extract or pure compounds addition
depending on the cell line. All of the test samples were solubilized in DMSO
Acknowledgments
(<0.5% in the culture medium) and were filter sterilised (0.2 lm) prior to
addition to the culture media. Control cells were also run in parallel and
subjected to the same changes in medium with a 0.5% DMSO. In addition, cells
were treated as indicated above for 24, 48 or 72 h to unravel the potential
cytotoxicity of compounds against all cell lines.
Cell cytotoxicty (MTS assay): At the end of 2 or 3 days of treatment with serially
diluted test samples, 20 lL of the MTS reagent, in combination with the
electron coupling agent, phenazine methosulfate, was added to the wells and
cells were incubated at 37 °C in a humidified incubator for 3 h. Absorbance at
490 nm (OD490) was monitored with a spectrophotometer (SpectraMax M2,
Molecular Devices Corp., operated by SoftmaxPro v.4.6 software, Sunnyvale,
This publication was made possible by Grant Number RR03037
from the National Center for Research Resources (NCRR) of the
National Institutes of Health (NIH). Its contents are solely the
responsibility of the authors and do not necessarily represent the
official views of NCRR or NIH.
References and notes
CA, USA), to obtain the number of cells relative to control populations. 20 lL of
1. Jemal, A.; Siegel, R.; Xu, J.; Ward, E. C. A. Cancer J. Clin. 2010, 60, 277.
2. Pecic, S.; McAnuff, M. A.; Harding, W. W. J. Enzyme Inhib. Med. Chem. 2011, 26,
46.
3. Tang, H.; Wei, Y. B.; Zhang, C.; Ning, F. X.; Qiao, W.; Huang, S. L.; Ma, L.; Huang,
Z. S.; Gu, L. Q. Eur. J. Med. Chem. 2009, 44, 2523.
etoposide 4 mg/mL (Sigma) was assayed as a negative control of proliferation.
The results are expressed as the concentration that inhibit growth of cell by
50% versus control cells (control medium used as negative control), IC50. Data
are presented as the mean S.D. of three separated experiments on each cell
line. Etoposide provided consistent IC50 values of 15–25
and Caco-2 cells.
lM for the HCT-116
4. Adsersen, A.; Kjolbye, A.; Dall, O.; Jager, A. K. J. Ethnopharmacol. 2007, 113, 179.