Design, synthesis and bioevaluation of novel maleamic amino acid ester conjugates of 3,5-bisarylmethylene-4-piperidones as cytostatic agents

Article abstract of DOI:10.1016/j.bmcl.2009.09.069

A novel series of maleamic amino acid ester conjugates of 3,5-bisarylmethylene-4-piperidones were prepared to investigate the efficacy of micronutrient conjugation in enhancing cytotoxic potency by improving selectivity and delivery. These compounds, prep

Full text of DOI:10.1016/j.bmcl.2009.09.069

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6364–6367
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and bioevaluation of novel maleamic amino acid ester
conjugates of 3,5-bisarylmethylene-4-piperidones as cytostatic agents
Dani Youssef a,b, Elizabeth Potter b, Mamta Jha b, Erik De Clercq c, Jan Balzarini c,
James P. Stables d, Amitabh Jha b,
*
a Département des Sciences, Université Sainte-Anne, Pointe-de-l’Église, NÉ, Canada BOW 1MO
b Department of Chemistry, Acadia University, Wolfvile, NS, Canada B4P 2R6
c Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
d National Institute of Neurological Disorders and Stroke, Rockville, MD 20852, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of maleamic amino acid ester conjugates of 3,5-bisarylmethylene-4-piperidones were pre-
pared to investigate the efficacy of micronutrient conjugation in enhancing cytotoxic potency by improv-
ing selectivity and delivery. These compounds, prepared as anticancer agents, were expected to
Received 25 August 2009
Revised 15 September 2009
Accepted 17 September 2009
Available online 22 September 2009
demonstrate enhanced selectivity towards malignant cells through the inhibition of topoisomerase II
a
via protein thiolation. The cytostatic effects of these compounds were evaluated against three cell lines,
namely murine L1210 leukemia cells, human Molt 4/C8 and CEM T-lymphocyte cells. All compounds
were found to have greater potency than the reference drug melphalan. Several compounds were found
Keywords:
3,5-Bisarylmethylene-4-piperidones
Cytostatic activity
Anticancer
to potently inhibit topoisomerase II
a
and displayed cytostatic activity in the nanomolar range.
Ó 2009 Elsevier Ltd. All rights reserved.
Topoisomerase inhibitions
QSAR
Since the discovery of chemotherapeutic agents capable of
treating cancer, chemists have sought to develop novel compounds
with the ability to selectively induce apoptosis in cancerous tis-
sues, while leaving healthy normal tissues unharmed. A number
of 3,5-bisarylmethylene-4-piperidone derivatives have been inves-
tigated over recent years, which have demonstrated promising
poisoning occurs, the double stranded nicks created by the enzyme
cannot religate, resulting in potentially lethal lesions in the DNA
frame work, which may then lead to apoptosis.17,18 In rapidly prolif-
erating cancerous cells, topoisomerase IIa expression is enhanced,
thus thiol-alkylators targeting this enzyme offer a means of selectiv-
ity targeting these malignant cells to inhibit tumour growth.
The interest in 3,5-bisarylmethylene-4-piperidones stems from
the high antiproliferative activity which was shown by the parent
molecule 1a (Fig. 1) towards leukemia and colon cancer cell lines.7,14
Not only was compound 1a highly selective, but the hydrochloride
salt was found to be well-tolerated in mice, as no mortalities were
noted after five consecutive daily doses of 240 mg/kg.1,14 Various
substitutions in the aryl rings led to the synthesis of 1b–f. A National
Cancer Institute (NCI) screen of 1a–c and 1f against 54 human
tumour cell lines from eight types of cancers, including leukemia,
melanoma, colon, non-small-cell lung, small-cell lung, central ner-
vous system, ovarian and renal cancers, indicated both selectivity
and potency were affected by the nature of the aryl substituent. In
general, members of series 1, were found to have greater or similar
potency to melphalan (AlkeranÒ), an established alkylating agent
for cancer treatment.1,7
cytotoxic and anticancer properties.1–16 As
a,b-unsaturated ke-
tones, these compounds are capable of undergoing Michael addi-
tion, resulting in alkylation of cellular nucleophiles. These
compounds possess a greater affinity towards cellular thiols than
towards amino and hydroxyl groups, thus compounds of this nat-
ure may be devoid of the genotoxic side effects associated with
many alkylating agents.2,4
Topoisomerase IIa, an enzyme rich in cysteine residues and
essential for cell proliferation,17–19 has been identified through
preliminary work in our laboratory as a potential target of 3,5-bisa-
rylmethylene-4-piperidone derivatives.1,20,21 This enzyme creates
transient double stranded nicks in the DNA helix, releasing torsional
strain and facilitating separation of the DNA template, allowing rep-
lication and transcription processes to occur.17,18,22–24 Interruption
of these processes via catalytic inhibition causes replication and
transcription to stall, impeding cell growth. If topoisomerase
The conversion of series 1 to N-acryloyl amides (series 2, Fig. 1)
provided an additional site for thiolation. In general, these
compounds were found to be more potent than 1, and on average,
a 26-fold increase was observed in activity against P388 screens,
* Corresponding author.
E-mail address: ajha@acadiau.ca (A. Jha).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.069

D. Youssef et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6364–6367
6365
involved which are capable of differentiating between the
L
- and
D
-isomers of the amino acid.25b The bioavailability of the
L-valyl es-
ter of acyclovir was found to be 3–4 times greater than the parent
molecule.25b Tamoxifen, an anticancer agent, was also found to
have increased water solubility when conjugated to amino acid
esters.26 Doxorubicin derivatives incorporating an amino acid
functionality have also been found to express enhanced bioavail-
ability, as well as reduced toxicity.27 The
L-leucine conjugate of
doxorubicin resulted in a fourfold decrease in toxicity, while main-
taining equal potency against L1210 murine leukemia.26,27 It was
also found to be active in three of nine doxorubicin-resistant
malignancies.27
L-Leucine-doxorubicin is slowly hydrolyzed to
the parent molecule in vivo, and is found to remain in the tumour
for a longer period of time.27
The 3,5-bisarylmethylene-4-piperidone derivatives reported in
this investigation have been designed to improve drug delivery
via conjugation of amino acid derivatives which may engage amino
acid transporters, facilitating entry to the cell. The amino acid con-
jugates are expected to display enhanced selectivity towards rap-
idly dividing cells, as these are essential micronutrients for
biosynthesis.
In the present investigation, we decided to develop series 8 and
9, in which the side chain aryl moiety of 5 and 6, respectively, has
been replaced with an amino acid ethyl ester. It was hypothesized
that this exercise would lead to molecules with improved bioavail-
ability and site-specific delivery to cancer cells resulting in en-
hanced efficacy. While such conjugations may be deemed as
prodrug formation, it should be noted that the molecules incorpo-
rate the 3 enone pharmacophore and therefore may act as drugs on
their own right. Compounds 1a and 1e were chosen as precursors
as their N-acryloyl derivatives, viz. 2a and 2e, were found to be
highly potent cytotoxics.15
Figure 1. Structures of series 1 and 2. The substituents in the aryl rings were as
follows: a, R = H; b, R = 4-Cl; c, R = 3,4-Cl; d, 4-F; e, 4-NO2; f, 4-OCH3.
when compared to the parent molecule.1,2,5 Placement of electron-
withdrawing substituents on the molecule improved potency, due
to an increase in the electrophilicity of the olefinic bond where thi-
olation is believed to occur;7 compound 2e was found to have the
greatest potency.1,15
The promising activity displayed by N-acryloyl derivatives of
3,5-bisarylmethylene-4-piperidones made the development of ser-
ies 3–6 (Fig. 2) a logical progression in the investigation of the
activity of these molecules. Replacement of a terminal acryloyl
hydrogen with an aryl ring or an arylcarbamoyl moiety enabled
the electrophilicity of the acryloyl moiety to be investigated, as it
relates to thiol alkylation and cytotoxicity, by modification of the
aryl substituents.1,2,4–6 In general, compounds of series 3 were less
potent than their precursor 2a and were found to display greater
selectivity than melphalan, the reference drug.3,4
The target molecules 8a–c and 9a–h were prepared as shown in
Scheme 1. The 3,5-bisarylmethylene-4-piperidones, 1a,e, were pre-
pared via condensation of 4-piperidone monohydrate hydrochlo-
ride with the appropriate aromatic aldehyde, as previously
The arylcarbamoyl containing Z congeners, 4, were generally
found to have greater potency than the parent molecule, 1a and
were significantly more potent than melphalan.2 Both 5 and 6 were
found to have comparable potencies to melphalan.5
described.7 Ethyl esters of eight different natural
L-amino acids re-
acted with maleic anhydride, following a procedure reported in the
literature,28 to yield the corresponding maleamic acids, 7a–h. The
final step of the synthesis, condensing 7a–h to the appropriate
3,5-bisarylmethylene-4-piperidone, was performed following
chemistry reported previously by our group.6 All reaction products
were purified by column chromatography and fully characterized
based on their spectroscopic data.29
To evaluate the cytotoxicity of compounds 8a–c and 9a–h
against malignant cell lines, the compounds were evaluated for
cytostatic activity against murine L1210 leukemia cells, as well
as human Molt4/C8 and CEM T-lymphocyte cells, following litera-
ture procedure.30 The results are presented in Table 1.
The high cytotoxicity of all of the compounds tested provides
sufficient evidence that these molecules are indeed potential po-
tent antitumour agents as well as promising lead molecules for fur-
ther development. Compared to melphalan,4 an established
anticancer agent, the compounds generally display significantly
greater potency, with the exception of 8a–c which show lower
cytotoxicity than melphalan in the L1210 screen.
Malignant tumours, which are marked by rapid cell division,
have a constant requirement for amino acids for protein biosynthe-
sis. Thus, drugs conjugated with amino acids have the potential to
improve both site specificity and absorption, as the drug is deliv-
ered to areas where the amino acid requirement is high. It is com-
monly known that polar amino acids and their corresponding
esters are moieties capable of enhancing aqueous solubility25a
facilitating bioavailability.25b Conjugation of acyclovir, an antiviral
indicated against infections caused by the herpes virus, with amino
acid esters was found to significantly enhance bioavailability.25b
The chirality of the amino acid moiety was found to influence
activity, as the
corresponding
L
-isomers were found to be more active than the
D
-isomers.25b This indicates transporters must be
Previous literature indicates the placement of a nitro group at
the para position of the arylidene ring results in enhanced cytotox-
icity due to the electronic effects of the substituent.1,5,11,16 In accor-
dance with this observation, 9a–h were all found to have greater
potency than compounds 8a–c and melphalan against all cell lines,
displaying activity at nanomolar concentrations.
The cytostatic activity of compounds 8a–c and 9a–h were all
found to have greater potency than the parent pharmacophores
1a and 1e, respectively (Table 1). Thus, the antiproliferative activ-
ity of these compounds is likely due to the intact molecule rather
Figure 2. Investigated analogs of series 2.

6366
D. Youssef et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6364–6367
Scheme 1. Synthesis of compounds 7a–c and 8a–h. The substituents were as follows: a, R = H; b, R = CH2CH(CH3)2; c, R = CH2(4-OH-C6H4), d, R = CH3; e, R = CH(CH3)2; f,
R = CH2CH2SCH3; g, R = CH2Ph;h, R = CH2(3-indolyl).
Table 1
are presented in Table 2. A representative gel picture is presented
in Figure 3.
Evaluation of compounds 8a–c and 9a–h against murine L1210 leukemic cells and
human Molt 4/C8 and CEM T-lymphocyte cells
Compounds 8a–c were found to be inactive at 50
lM towards
Compound
IC50 (lM)
TOPO II . As these molecules were shown to be cytotoxic agents,
a
their interaction with topoisomerase is likely not their primary
mode of action. In contrast, compounds 9a–h were all found to
L1210
Molt 4C/8
CEM
1.70 0.02
4.47 2.28
1.48 0.32
0.26 0.02
1.3 0.1
1aa
7.96 0.11
32.09 4.2
8.69 0.73
0.42 0.07
6.1 1.2
1.67 0.15
8.28 0.75
1.42 0.27
0.15 0.06
1.4 0.1
1ea
be catalytic inhibitors active towards TOPO II
several derivatives, namely 9a,f–h retaining their inhibitory activ-
ity at 10 M. Further study of these compounds at concentrations
below 10 M may be valuable to determine the limit of their activ-
ity. Comparison of activity towards TOPO II and the potency of
these molecules yielded no significant trends.
a at 100 lM, with
2ab
2eb
l
l
8a
8b
8c
9a
9b
9c
9d
9e
4.7 1.1
4.6 1.3
1.1 0.2
1.2 0.0
a
0.57 0.11
0.23 0.01
0.21 0.02
0.28 0.06
0.24 0.02
0.22 0.01
0.25 0.01
0.24 0.02
0.27 0.08
3.24 0.79
0.35 0.02
0.11 0.05
0.17 0.03
0.17 0.05
0.083 0.015
0.17 0.06
0.19 0.07
0.13 0.06
0.19 0.06
2.47 0.03
0.84 0.07
0.24 0.08
0.60 0.09
0.58 0.20
0.44 0.07
0.58 0.08
0.20 0.03
0.62 0.31
2.13 0.03
Two representative compounds 9a and 9b were evaluated for
murine toxicity in vivo following a literature procedure.33 The
compounds were injected intraperitoneally into mice at doses of
30, 100 and 300 mg/kg. The animals were then monitored for 0.5
and 4 h. The compounds where found to be well tolerated, with
9b showing no neurotoxicity. Neurological deficit was displayed
at a dose of 100 mg/kg after 0.5 h in one out of eight animals stud-
ied in the case of compound 9a (1/8), no neurotoxicity was ob-
served at 300 mg/kg for the same duration, or after 4 h at all
concentrations studied. These results indicate that compounds of
series 9 are well tolerated and are not general biocidal agents.
Future studies on this novel series of potential anticancer mol-
ecules will be carried out to establish the effectiveness of micronu-
trient conjugation in improving selectivity and delivery in the
appropriate models.
In conclusion, the present investigation reveals amino acid con-
jugates of 3,5-bisarylmethylene-4-piperidones to be promising
leads for the development of novel cytostatic agents. Series 9
was found to be significantly more potent than the reference drug
melphalan against L1210, Molt C4/8 and CEM cell proliferation.
QSAR results indicate conjugation with more complex and increas-
ingly lipophilic amino acids may further accentuate bioactivity. As
these compounds have been shown to inhibit topoisomerase II, and
as they are structurally divergent from known topoisomerase II
inhibitors, they may be of value in treating drug-resistant tumours.
9f
9g
9h
Melphalanc
a
b
c
The data for 1a,e are reproduced from Ref. 14.
The data for 2a,e are reproduced Ref. 15.
The data for melphalan are reproduced from Eur. J. Med. Chem. 2001, 35, 970.
than the products of hydrolysis, which would yield the parent
pharmacophore and the corresponding maleamic acid.
In an attempt to comprehend the effect of structural and elec-
tronic parameters on the cytostatic activity of compounds 9a–h,
a quantitative structure–activity relationship (QSAR) study was
undertaken using certain physicochemical parameters as descrip-
tors of bioactivity. The extent to which the lipophilicity of the ami-
no acid side chain affected the bioactivity was evaluated by
comparing log P of the side chain (calculated using commercial
software)31 with the IC50 values of the three cell lines. Likewise,
molar refractivity (MR) values of the amino acid side chain were
also obtained using commercial software31 and utilized as a means
of accessing the effect of the steric properties of the side chain with
respect to bioactivity.
A positive correlation (p <0.05) was observed between log P and
antiproliferative activity against L1210 cells. This indicates that the
exploration of derivatives with increasingly lipophilic substituents
is of value to fine tune the anticancer potential of these molecules.
While no significant correlations (p >0.05) were observed between
MR and activity, a positive correlation approaching significance
was observed against Molt4/C8 and CEM cell lines (p = 0.094 and
0.056, respectively). This would indicate that the inclusion of more
complex and bulky amino acid side chains (or possibly conjugation
with di/tri-peptides) would enhance the inhibitory ability of these
agents against human cell lines.
Table 2
Topoisomerase II
+ = active; À = inactive
a inhibitory activity displayed by compounds 4a–c and 5a–f;
Compound
Concentration employed in assay (lM)
100
50
25
10
8a
8b
8c
9a
9b
9c
9d
9e
9f
À
À
+
+
+
+
+
+
+
+
+
À
À
À
+
À
À
À
+
À
À
À
+
À
À
À
À
+
À
À
+
+
As compounds of this nature have been proposed as TOPO II
a
+
+
+
+
À
+
+
+
+
inhibitors,1,21 appropriate assays were undertaken to identify if
these derivatives did indeed result in inhibition of this vital en-
9g
9h
+
+
zyme. TOPO II
human DNA TOPO II
a
inhibition assays were performed using purified
+
a
and supercoiled pRYG DNA.32 The results

D. Youssef et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6364–6367
6367
10. Pati, H. N.; Das, U.; Das, S.; Bandy, B.; De Clercq, E.; Balzarini, J.; Kawase, M.;
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Inhib. Med. Chem. 2007, 22, 451.
13. Das, U.; Das, S.; Bandy, B.; Stables, J. P.; Dimmock, J. R. Bioorg. Med. Chem. 2008,
16, 3602.
14. Dimmock, J. R.; Arora, V. K.; Wonko, S. L.; Hamon, N. W.; Quail, J. W.; Jia, Z.;
Warrington, R. C.; Fang, W. D.; Lee, J. S. Drug Des. Deliv. 1990, 6, 183.
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Motaganahalli, N. L.; Zello, G. A.; Quail, J. W.; Oloo, E. O.; Kraatz, H. B.;
Prisciak, J. S.; Allen, T. M.; Santos, C. L.; Balzarini, J.; De Clercq, E.; Manavathu, E.
K. J. Med. Chem. 2001, 44, 586.
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T. V.; Odinets, I. L. Eur. J. Med. Chem. 2009, 44, 2135.
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Figure 3. TOPO II
Lane 2, DNA with TOPO II; Lane 3, DNA with TOPO II in presence of DMSO control;
Lane 4, DNA with TOPO II in presence of 100 M VP-16, an established TOPO II
poison; Lanes 5–8; DNA with TOPO II in presence of 9h at 10, 25, 50, 100 lM,
respectively.
a inhibition by compound 9h. Lane 1, supercoiled pRYG DNA;
l
21. Duffield, K., Honours Thesis, Acadia University, 2004.
22. Wang, J. Nat. Rev. Mol. Cell Biol. 2002, 3, 430.
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In vivo neurotoxicity experiments reveal the compounds to be
well-tolerated by mice at concentrations up to 300 mg/kg body
weight. These results indicate that the investigation of 3,5-bisa-
rylmethylene derivatives conjugated to amino acids is a promising
area of study to fine tune the anticancer potential of these
molecules.
28. Liwschitz, Y.; Zilkha, A. J. Am. Chem. Soc. 1955, 77, 1265.
Acknowledgements
29. General procedure for the synthesis of compounds 8a–c and 9a–h: To dry THF
under anhydrous conditions, the appropriate maleamic amino acid ester (7a-f)
was added. The reaction flask was cooled on ice and TEA (1.1 equiv) was added
dropwise over 10 min, while maintaining the reaction mixture on ice. Ethyl
chloroformate (1.1 equiv) was then added dropwise over 10 min. The reaction
mixture was stirred for 30 min. The appropriate 3,5-bisarylmethylene-4-
piperidone was added and the reaction was allowed to stir at room
temperature for 24 h. Solvent was removed by rotary evaporation and the
product purified in 0–5% DCM/methanol. Analytical data for 9f: Yellow
powder; yield: 38%; mp 97–100 °C. 1H NMR (300 MHz; CDCl3): d 1.31 (t,
J = 6.9 Hz, 3H, CH3), 2.00–2.182 (m, 5H, SCH3, CH2), 2.50 (t, J = 7.3 Hz, 2H, SCH2),
4.24 (q, J = 6.9 Hz, 2H, OCH2), 4.56–4.74 (m, 3H, COCH, 2 Â NCH), 4.89–5.11 (AB
quartet, J = 16.7 Hz, 2H, 2 Â NCH), 5.97 and 6.25 (d, J = 11.9 Hz, 1H each, 2 Â Z-
vinylic-H), 6.97 (br s, 1H, NH), 7.55 (d, J = 8.1 Hz, 2H, ArH), 7.67 (d, J = 8.4 Hz,
2H, ArH), 7.84 and 7.88 (s, 1H each, 2 Â vinylic-H), 8.30–8.34 (m, 4H, ArH). 13C
NMR (75 MHz; CDCl3): d 14.18, 15.48, 29.94. 31.32, 42.66, 47.08, 51.85, 61.88,
123.21, 124.09, 127.79, 130.78, 130.86, 131.08, 132.29, 133.65, 133.81, 135.14,
The authors thank the following agencies for financial support,
the Nova Scotia Health Research Foundation (A.J., E.P.) and the
Concerted Research Actions (GOA 05/19) (J.B.). Appreciation is ex-
tended to Mrs. Lisette van Berckelaer for conducting the Molt 4/C8,
CEM, and L1210 assays, and the National Institute of Neurological
Disorders and Stroke, USA, for undertaking the short term toxicity
studies in mice. Dr. Sherri McFarland (Acadia University) is
thanked for demonstrating the topoisomerase assay protocol.
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136.13, 140.52, 140.63, 147.89, 163.47, 166.67, 171.50, 185.29. IR (KBr; mmax):
1734, 1623, 1517, 1437, 1384, 1273, 1173, 1108, 1004, 984, 854, 805,
681 cmÀ1. UV (CHCl3; kmax): 217, 329 nm. ESI-MS (amu): 645 [M+Na]+.
30. Balzarini, J.; De Clerq, E.; Mertes, M.; Shugar, D.; Torrence, P. F. Biochem.
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31. ChemBioDraw, 11.0.1; CambridgeSoft: 2007.
32. Assays investigating the inhibition of TOPO II
protocol supplied by TopoGEN Inc., the source for purified human DNA TOPO
II (p170 form) and pRYG DNA. The enzyme was incubated with the
a were carried out as per the
a
compound for 30 min at 37 °C. Subsequently SDS and proteinase K were
added. The reaction was stopped with the appropriate TopoGEN stop buffer
after 15 min of incubation. The assay and all appropriate controls were loaded
on a 1% agarose gel in 1Â TAE and run at 60 V for 2 h. The gel was then
visualized using ethidium bromide staining. Compounds were assayed at
100 lM, decreasing the concentrations to 50 lM, 25 lM and 10 lM if
compounds still exhibited inhibitory activity. All assays were completed in
duplicate.
33. Molecular and Cellular Targets for Antiepileptic Drugs; Stables, J. P., Kupferberg,
H. J., Eds.; John Libbey: London, 1997.