Derivatives of aryl amines containing the cytotoxic 1,4-dioxo-2-butenyl pharmacophore

Article abstract of DOI:10.1016/j.bmcl.2010.01.098

Several series of compounds containing the 1,4-dioxo-2-butenyl moiety have been prepared as candidate cytotoxins, including the methyl N-arylmaleamates, methyl N-arylfumaramates, and N-arylmaleimides. In addition, the N-arylisomaleimides were synthesized which are the structural isomers of N-arylmaleimides. These compounds were evaluated against human Molt 4/C8 and CEM T-lymphocytes as well as murine L1210 cells. Methyl N-arylfumaramates showed the highest cytotoxic potencies and, in particular, methyl N-(3,4-dichlorophenyl)fumaramate is six times more potent than melphalan towards L1210 cells and is equipotent with this drug in the Molt 4/C8 assay. Electrophilicity of compounds under investigation was demonstrated by carrying out thiolation using model benzyl mercaptan on representative compounds. Methyl N-(3,4-dichlorophenyl)fumaramate and methyl N-(4-chlorophenyl)maleamate inhibited human N-myristoyltransferase, a possible molecular target, in high micromolar range. QSAR and molecular modeling revealed some correlations between different structural features of a number of the molecules and cytotoxic potencies. Methyl N-arylfumaramates were well tolerated in mice in comparison to the analogs in other series of compounds tested. The data obtained in this investigation affords guidelines for preparing new series of molecules with greater potencies.

Full text of DOI:10.1016/j.bmcl.2010.01.098

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1510–1515
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Derivatives of aryl amines containing the cytotoxic 1,4-dioxo-2-butenyl
pharmacophore
Amitabh Jha a, , Chandrani Mukherjee a,b, Ashok K. Prasad b, Virinder S. Parmar b, Manjula Vadaparti a,
*
Umashankar Das c, Erik De Clercq d, Jan Balzarini d, James P. Stables e, Anuraag Shrivastav f,
Rajendra K. Sharma f, Jonathan R. Dimmock c
a Department of Chemistry, Acadia University, Wolfville, NS, Canada B4P 2R6
b Department of Chemistry, University of Delhi, Delhi 110 007, India
c College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK, Canada S7N 5C9
d Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
e National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD 20852, USA
f Department of Pathology, College of Medicine, University of Saskatchewan, and Cancer Research Unit, Saskatoon, SK, Canada S7N 4H4
a r t i c l e i n f o
a b s t r a c t
Article history:
Several series of compounds containing the 1,4-dioxo-2-butenyl moiety have been prepared as candidate
cytotoxins, including the methyl N-arylmaleamates, methyl N-arylfumaramates, and N-arylmaleimides.
In addition, the N-arylisomaleimides were synthesized which are the structural isomers of N-arylmalei-
mides. These compounds were evaluated against human Molt 4/C8 and CEM T-lymphocytes as well as
murine L1210 cells. Methyl N-arylfumaramates showed the highest cytotoxic potencies and, in particular,
methyl N-(3,4-dichlorophenyl)fumaramate is six times more potent than melphalan towards L1210 cells
and is equipotent with this drug in the Molt 4/C8 assay. Electrophilicity of compounds under investiga-
tion was demonstrated by carrying out thiolation using model benzyl mercaptan on representative com-
pounds. Methyl N-(3,4-dichlorophenyl)fumaramate and methyl N-(4-chlorophenyl)maleamate inhibited
human N-myristoyltransferase, a possible molecular target, in high micromolar range. QSAR and molec-
ular modeling revealed some correlations between different structural features of a number of the mol-
ecules and cytotoxic potencies. Methyl N-arylfumaramates were well tolerated in mice in comparison to
the analogs in other series of compounds tested. The data obtained in this investigation affords guidelines
for preparing new series of molecules with greater potencies.
Received 5 December 2009
Accepted 19 January 2010
Available online 25 January 2010
Keywords:
1,4-Dioxo-2-butenyl
Cytotoxicity
N-Arylmaleamates
N-Arylfumaramates
N-Arylmaleimides
N-Arylisomaleimides
Structure–activity relationships
Molecular modeling
Ó 2010 Elsevier Ltd. All rights reserved.
A major interest of these laboratories is the design, syntheses,
and bioevaluations of
,b-unsaturated ketones.1–7 These com-
tigation since it has not only the 1,4-dioxo-2-butenyl group
(hereafter referred to as the enedione moiety) but also an aryl ring
into which can be placed different substituents to modulate po-
tency. These compounds were evaluated against human Molt
4/C8 and CEM T-lymphocytes as well as murine L1210 cells and
a
pounds have an affinity for thiols but are either unreactive or less
reactive towards amino and hydroxy groups.8,9 Since these latter
two functionalities are present in nucleic acids, administration of
conjugated enones may not cause the genotoxic effects which are
displayed in a number of contemporary anticancer drugs.10 Thiols
react at the b carbon atom of conjugated enones. Hence the place-
ment of an additional carbonyl group on the b carbon atom should
deplete the electron density on the olefinic carbon atoms and in-
crease the rates of thiol alkylation in the cell compared to the en-
one moiety. One of the objectives of the present investigation was
to ascertain whether the 1,4-dioxo-2-butenyl group is a cytotoxic
pharmacophore when incorporated into a variety of ligands.
Several years ago, the N-arylmaleamic acids 1 (Fig. 1) were pre-
pared and this series was chosen as the starting point in this inves-
were either weakly active (IC50 values of 43–290
lM) or had IC50
figures in excess of 500
l
M.11 They were well tolerated in mice
whereby doses up to and including 300 mg/kg did not cause any
mortalities.
The reasons for the design of analogs of series 1 are as follows.
The low potencies of 1a–i may have been due to the polar carboxy
group which impedes passage via the cell membrane of cancer
cells. Hence the formation of the corresponding esters 2 was sug-
gested. The hypothesis was formulated that the cytotoxic poten-
cies of the compounds related to the N-arylmaleamic acids is
controlled principally by the olefinic and aryl groups and also the
topographical relationship between these two structural moieties.
Such considerations led to the proposal to synthesize series 3–6.
The olefinic double bond in series 1 and 2 adopts the Z configura-
* Corresponding author. Tel.: +1 902 585 1515; fax: +1 902 585 1114.
E-mail address: ajha@acadiau.ca (A. Jha).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.098

A. Jha et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1510–1515
1511
Figure 1. Molecular structures of compounds studied.
tion and a comparison with the potencies of the E-isomers 3 and 4
was planned. In order to examine the effect of compressing the
enedione group into a ring, series 5 was suggested. The decision
was made to prepare the N-arylmaleisoimides 6 in which the ole-
finic group is adjacent to both carbonyl and imino moieties. As a
null hypothesis, the reduction of the olefinic double bond of 1a
(or 3a), 2a (or 4a), and 5a leading to 7–9 (Fig. 1) was considered
in order to confirm that the olefinic group is important in confer-
ring cytotoxic properties. Confirmation of the thiol-alkylating
properties of these compounds was proposed, whereby represen-
tative compounds in each of the series 1–6 would react with a
model thiol, namely benzyl mercaptan. In addition, such experi-
ment with benzyl mercaptan would indicate at which olefinic car-
bon atoms thiolation takes place.
corresponding acid 7 from which the ester 8 and dehydrated prod-
uct 9 were obtained.
All compounds of series 2–6 and compounds 6–9 were evalu-
ated against human Molt 4/C8, and CEM T-lymphocytes as well
as murine leukemic L1210 cells. The results are given in Table 1.
Representative compounds, namely 2c,d,4b,c,e and 3b–e,g were
evaluated as inhibitors of human N-myristoyltransferase (hNMT).
Murine toxicity of 2b–d,g,h, 3a–i, 4a–c,e–g,i, 5a–h, 6b,c,g,h, and
8 was assessed by injecting doses of 30, 100, and 300 mg/kg into
mice and the animals were examined at the end of 0.5–4 h for
mortalities.
The cytotoxic evaluation of 2a–e,g–i, 3a–i, 4a–h, 5a–h, and 6–8,
9a–i was undertaken using Molt 4/C8 and CEM T-lymphocytes in
order to assess whether the compounds demonstrate antineoplas-
tic properties towards human transformed cells. The murine L1210
bioassay was used as a number of anticancer drugs inhibit the
growth of this cell line15 and hence this screen may reveal lead
compounds. The reference anticancer drug used in this study was
the alkylating agent melphalan. Of particular note is the observa-
tion that 3c is six times more potent than melphalan towards
L1210 cells and equipotency with melphalan was achieved by
3c,e in the Molt 4/C8 assay and by 3e in the L1210 test.
The choice of aryl substituents in series 1–6 was made on the
basis of their differing electronic, hydrophobic and steric proper-
ties. Thus groups with positive (+) or negative (À) Hammett
descriptor of electronic substituent effect on aromatic rings) and
Hansch (a descriptor of hydrophobic/hydrophilic character) val-
ues were utilized viz + /+ /+ (d/e), + /À (g,h), and
(b,c), À
(f) while the molar refractivity (MR; a descriptor of steric
r (a
p
r
p
r
p
r p
Àr/Àp
bulk) figures of the substituents b–i ranged from 5.65 (d) to
11.18 (h).
Correlations between the structures of the compounds in series
2–5 and cytotoxic potencies were sought in order to provide guide-
lines for expansion of this study. Three approaches were employed
in the analysis of the biodata. First, qualitative and quantitative
comparisons of the IC50 values between different series of com-
pounds were undertaken. Second, the importance of various phys-
icochemical constants of the aryl substituents in determining
cytotoxic potencies was assessed. Third, molecular modeling was
used to determine if the relative positions of certain functional
groups influenced the potencies of the compounds.
Comparisons of the relative cytotoxic potencies between clus-
ters of molecules enabled the following conclusions to be drawn.
(1) The biodata presented in Table 1 reveal that the esters and
amides which contain the enedione moiety, namely series 2–4, in-
hibit the growth of all three cell lines with the exception of 2a.
Hence the hypothesis that the enedione is a cytotoxic pharmaco-
phore appears to be valid. The conclusion was strengthened by
the fact that when the olefinic double bond in 2a, 3a and 4a was
reduced, leading to 7 and 8, cytotoxic properties were abolished.
(2) The conversion of the N-arylmaleamic and N-fumaramic acids
to the corresponding methyl esters led to increases in cytotoxicity
in most cases. Thus a comparison between the literature IC50 val-
ues of series 111 with corresponding methyl esters 2 revealed
greater cytotoxic potencies in series 2. Similarly all of the com-
pounds in series 4 had greater cytotoxic potencies than the precur-
sor N-arylfumaramic acids 3. (3) In order to compare the potencies
of the compounds in series 2–4, the following procedures were
adopted. The IC50 values of the compounds containing the same
aryl substituent were compared and a score of 3 (greatest potency),
In summary, the objectives of the present study were as follows.
First, an evaluation would be made to determine whether the
enedione group is a cytotoxic pharmacophore which could be used
as a building block in developing more complex ligands. In this re-
gard, the recent recommendations of starting projects with lead
molecules having low molecular weights of 300–400 and moderate
potencies in the micromolar range is of relevance.12 Second, an
estimate would be made of the influence of the location of this
putative pharmacophore in different series of compounds in regard
to cytotoxic potencies. Third, the spatial relationship between the
aryl ring and the enedione group may influence the IC50 values.
Fourth, the possibility exists that the nature of the aryl substitu-
ents exerts a statistically significant effect on the magnitude of
the biological responses. The comparisons of the potencies of the
compounds was planned to be achieved using the Molt 4/C8,
CEM, and L1210 screens.
The synthetic chemical routes followed in producing the com-
pounds in series 1–9 are outlined in Scheme 1. The reaction of an
aryl amine with maleic anhydride led to the synthesis of the
N-arylmaleamic acids 1 which were converted into the corre-
sponding methyl esters 2. While 2f was formed, it was very unsta-
ble reverting to 1f and hence it was not evaluated for bioactivity.
The dehydration of 1a–h led to 5a–h under thermodynamically
controlled conditions and 6a–h when the reaction was controlled
kinetically.13,14 Reaction of various aryl amines with fumaryl chlo-
ride led to the isolation of the corresponding N-arylfumaramic
acids 3a–i which were esterified with methanol leading to 4a–i.
Condensation of aniline with succinic anhydride produced the

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A. Jha et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1510–1515
Scheme 1. Reagents and conditions: (i) CH3OH/H+; (ii) (CH3CO)2O, CH3COOÀNa+; (iii) (CF3CO)2/dioxane; (iv) (a) NaOH; (b) HCl.
Table 1
addition, the average IC50 values of 3a–e,g,h are lower than the
analogs in series 2 and 4. The perceptible lower IC50 figures of
the compounds in series 4 than 2 suggests that the shape of 4 is
preferred, such as its reduced flexibility and the relative positions
of various atoms and groups being in different locations (vide in-
fra). (4) In general, the maleimides 4a–h have greater cytotoxic
potencies towards Molt 4/C8, CEM and L1210 cells than the isoma-
leimides 5. Thus when comparisons were made between com-
pounds possessing the same aryl substituents, greater potency
was displayed by series 4 and 5 in 63% and 12% of the cases, respec-
tively, while equipotency was observed in 25% of the comparisons
made. One may note that in both series 4 and 5 the murine L1210
cells are more sensitive to these compounds than the Molt 4/C8
and CEM T-lymphocytes. Also, 3,4-dichloro substitution (c) led to
the most potent compounds in each of the series 2–5 in general.
Second, an investigation was conducted in order to gain insight
into the contributions that the aryl substituents in series 2b–e,g–i,
4a–i, and 5a–h made to cytotoxic potencies. Accordingly linear
(lin) and semilogarithmic (sl) plots were made between the IC50 val-
Evaluation of 2a–e,g–i, 3a–i, 4a–i, 5a–h, 6a–h, and 7–9 against Molt 4/C8, CEM and
L1210 cells
Compounda,b
IC50 (lM)
Molt 4/C8
CEM
L1210
2b
2c
2d
2h
4a
4b
4c
4d
4e
4f
4g
4h
4i
5c
5d
5e
41
41
37
50
42
1
7
5
9
0
95 62
55 25
78
39
47 15
184 22
40
7.5 2.4
0.37 0.29
9.3 0.4
2.2 0.3
18 10
3
6
36
44
40
4
3
1
4
8.2 0.1
2.4 0.3
9.6 0.1
4.8 0.1
21
4.6 2.3
10
7.3 0.7
4
15
1
7.6 0.00
0
37
11
32
31
44
42
35
3
1
2
5
4
1
7
6.2 0.8
35
26
43
43
29
2
2
1
1
3
17
34
37
7.8 0.4
8.1 0.1
5
7
9
Melphalan
3.2 0.8
2.5 0.0
2.1
0
ues obtained for each series of compounds with the Hammett
r and
a
Compounds 2a,e,g,i, 3a–i, 5a,b,f, 6a,d,f,h, 7, 8, and 9 were inactive against the
three cell lines tested (IC50 >150 M).
Compounds 5g,h, 6b,c,e,g were moderately active with IC50 values in the range
50–250 M against the three cell lines tested with at least one value <100 M (see
Supplementary data).
*
Taft
r values, then the Hansch p figures and finally the molecular
l
b
refractivity (MR) constants in the three bioassays. In addition, since
l
l
all the MR values (but not the or figures) are positive, logarith-
r
p
mic/semilogarithmic (lsl) and logarithmic (l) plots were constructed
between the IC50 values in series 2, 4, and 5 and the MR constants. A
negative correlation was noted between the IC50 figures of 5a–h in
2 and 1 (lowest potency) were made. Standard deviations were ta-
ken into account and six points were invariably awarded, for exam-
ple, if two compounds had greater potencies than a third molecule,
then the scoring would be 2½, 2½ and 1, respectively. A compari-
son of the IC50 values of 2a–e,g,h, 3a–e,g,h, and 4a–e,g,h were
made and the results are summarized in Table 2. The results indi-
cate that in terms of the potency scores, the N-arylfumaramate es-
ters 3, having the E conformation of the olefinic double bond, are
the most potent series of compounds. The N-arylmaleamates 2
and the N-arylmaleimides 4 in which the olefinic group adopts
the Z conformation have substantially lower potency scores. In
the CEM screen and the
p values (lin, sl plots, p <0.05). In addition,
a trend towards significance (sl plots, p <0.1) was observed between
the IC50 data of 4a–i in the CEM test and 5a–h in the Molt 4/C8 bio-
assay with the
p constants. These observations indicate that in the
future the placement of highly lipophilic substituents in the aryl ring
of the compounds in series 4 and 5 may lead to analogs with in-
creased potencies. No other correlations (p <0.05) nor trends to
significance (p <0.1) were observed.
Third, molecular modeling with representative molecules was
undertaken in order to find if the relative locations of portions of

A. Jha et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1510–1515
1513
Table 2
Comparison of the relative potencies of 2a–e,g,h, 4a–e,g,h, and 5a–e,g,h towards Molt 4/C8, CEM and L1210 cells
Compounds
Molt 4/C8
CEM
L1210
OVERALL
Potency scorea Average IC50
(l
M) Potency scorea Average IC50
(l
M) Potency scorea Average IC50
(lM) Potency score Average IC50 (lM)
2a–e,g,h
4a–e,g,h
5a–e,g,h
10.5
20
11.5
>152
17.2
120
11
21
10
>164
17.1
141
8.5
19.5
14
>180
11.8
70.0
30
60.5
35.5
>165
15.4
110
a
The calculation of the potency scores is explained in the text.
the enediones 2, 4, and 5 influence cytotoxic potencies. Models were
built of 2a, 4a, and 5a since they differ in potencies, that is, Table 1
revealed that 4a > 5a > 2a which reflects the relative potencies in
general of series 2, 4, and 5 as indicated in Table 2. The relative loca-
tions of the C1, C2, O1, and O2 atoms of the enedione moiety of 2a, 4a,
and 5a are likely important determinants of cytotoxic potencies.
These positions are referenced to the aryl ring which could also con-
tribute to bioactivity by van der Waals bonding at a complementary
binding site. An axis was constructed through carbon atoms 2 and 5
of the aryl ring as indicated in Figure 2 and the relative positions of
captan, under simulated physiological conditions These experi-
ments were undertaken in order to evaluate whether the
compounds in these series are in fact thiol alkylators and, if so,
to determine the locus of thiolation. The results are summarized
in Scheme 2. In the case of 1d, 2d, and 4d, thiolation occurs at the
olefinic carbon atom adjacent to the amidic group leading to 10
(from 1d) and 11 (from 2d and 4d). On the other hand, thiolation
of the E-isomer 3d gave rise to 12 as the major product along
with 10. The regiochemistry of thiol addition leading to com-
pounds 10, 11, and 12 were confirmed by 1D NOESY experiments.
One would expect thiolation to take place on the unsaturated car-
bon beta to amide carbonyl in case of Z and E acids (1d and 3d)
and on the unsaturated carbon beta to ester carbonyl in case of Z
and E esters (2d and 4d) on account of relatively higher electro-
philicity of these carbonyl carbons. The anomalous thiol addition
in case of 1d is presumably due to the increase in positive char-
acter of the carboxylate carbonyl which engages in intramolecular
H-bonding with the amide proton. Geometry of 3d does not per-
mit the formation of an intramolecular H-bond. While the reac-
tion of benzyl mercaptan with 5d led to the expected product
13, the isomaleimide 6d reacted with 2 M equiv of benzyl mer-
captan to yield 14. This product presumably arose from an initial
attack on the carbonyl carbon atom leading to ring opening and
acylation of benzyl mercaptan followed by thiol addition at the
olefinic bond. The conclusions drawn from these thiolation reac-
tions are as follows. First, the compounds in series 1–6 alkylate
thiols which is presumably one general way whereby cytotoxicity
is mediated. Secondly, the differential reactivity leading to regio-
selective thiolation of N-tolylmaleamic acid (1d) and N-tolylfuma-
ramic acid (3d) was found to be intriguing.
the C1, C2, O1, and O2 atoms determined from the d1–d4 and w1
–w4
measurements. These data are presented in Table 3.
The following observations were made. (1) The locations of the
O1 and C1 atoms in 2a, 4a, and 5a are similar in relation to the aryl
ring. Thus the
less than 8%. On the other hand the d3, d4,
D
% figures for d1, d2,
w
1, and w2 determinations are
3, and w4 measure-
w
ments varied considerably. Thus the reason for the potency differ-
ences between the compounds in series 2, 4, and 5 is controlled to
a large extent by the relative positions of the C2 and O2 atoms. (2)
The d3 and d4 spans are considerably higher in the most potent
compound 4a than either 2a or 5a. In addition, the lower w3 and
w4 values of 4a indicate a different positioning of the terminal
group (@C–C(O)OCH3). (3) The higher potency of 5a than 2a is
likely associated with differences in the locations of the C2 and
O2 atoms is evidenced by shorter d3 and d4 spans and larger w3
and w4 angles. Amplification of this project should bear in mind
these results. For example, rigid analogs should ensure that the
d3 and d4 distances are similar or larger than the figures for 4a
and that the w3 and w4 angles do not exceed 50°.
The compounds under investigation (series 1–6), all being Mi-
chael acceptors, were expected to cause protein thiolation. A
number of studies report that protein thiolation plays an impor-
tant role in biochemical processes. Some representative examples
of this phenomenon include inhibition of human cytomegalovirus
protease,16 inhibition of Ref-1, a therapeutic target for asthma,17
inactivation of cysteine proteases,18 blockade of platelet-derived
growth factor BB-stimulated Akt phosphorylation,19 catalytic
inhibition of human topoisomerase-2 alpha, an established target
for the development of anticancer agents,20,21 etc. This encour-
aged us the study the reactivity of representative compounds in
each of the series 1–5 and 9 towards a model thiol, benzyl mer-
The confirmation of the thiol-alkylating properties of the com-
pounds in series 1–6 suggests that interactions with important cel-
lular constituents containing
a mercapto group may be a
significant contributor to the bioactivity observed. The enzyme
hNMT catalyzes the transfer of a myristoyl group to the amino
moiety of the terminal glycine residue of various proteins and
the active site is believed to contain the mercapto group of the
169-cysteine amino acid.22 In addition, a previous study revealed
that the IC50 figures of 1a,d,g,i towards hNMT are 203, 53, 246,
and 434
lM, respectively, while a concentration of 242 lM of 1c
stimulated the enzymatic activity by 50%.11
Figure 2. (A) The distances d1–d4 are the spans between the center of the aryl ring and the O1, C1, C2 and O2 atoms, respectively, as illustrated by 3a. (B) The bond angle w1
between axis 1 and the O1 atom is indicated. The w2 4 angles created between axis 1 and the C1, C2, and O2 atoms, respectively, are not shown for reasons of clarity.
3, and
,
w
w

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A. Jha et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1510–1515
Table 3
Some interatomic distances and bond angles of 2a, 3a, and 4a
Compound
Interatomic distances (Å)
Bond angles (°)
d1
d2
d3
5.98
6.36
5.16
5.83
d4
5.06
8.77
4.06
5.96
w1
w2
w3
w4
2a
3a
4a
3.88
4.05
4.06
4.00
3.00
5.24
5.23
5.16
5.21
0.96
25.61
28.29
25.27
26.39
7.20
50.17
54.47
52.75
52.46
4.37
61.56
47.39
67.65
58.87
19.50
85.37
48.15
95.14
76.22
36.83
Average figure
a
D
%
11.5
47.2
a
The
D% figures are the quotients of the maximum deviation from the average figure and the average figure expressed as a percentage.
Scheme 2. Reaction of 1d, 2d, 3d, 4d, 5d and 6d with benzylmercaptan (BnSH). Reagents and conditions: (i) BnSH/MOPS buffer (pH 7.4): DMSO (1:1), 37 °C.
In order to assess the ability of the compounds under investigation
to inhibit hNMT, the IC50 values of the following compounds against
hNMT were obtained, namely 2c: 431.6 6.92 M; 2d: >10 mM;
3b: 1.60 0.35 mM; 3c: 4.38 1.59 mM; 3d: 8.35 0.21 mM; 3e:
3a–i, like the N-arylmaleamic acids (1),11 did not cause any deaths in
this bioassay. Detailed results on compounds showing mortality are
as follows: Compound ID (dose in mg/kg, number of animals dead/
number of animals treated) after 0.5 h, namely 2d (300, 1/4) and
5a (300, 2/4) while mortalities were noted at the end of the 4 h per-
iod after administration of 2c (300, 2/2), 4d (100, 4/4; 300,1/2), 2g
(300, 2/2), 2h (300, 2/2), 5a (30, 2/2; 100, 4/4; 300, 2/2), 5b (100, 4/
4; 300, 2/2), 5c (300, 2/2), 5d (100, 4/4; 300, 2/2), 5e (100, 3/4), 5f
(100, 4/4; 300, 2/2), 5g (100, 2/4; 300, 2/2), 5h (100, 4/4; 300, 2/2),
6b (300, 1/2), 6c (30, 1/2; 100, 3/4; 300, 2/2), 6g (100, 1/4; 300, 2/
2), and 6h (300,1/2). These observations reveal a clear-cut advantage
for pursuing the N-arylfumaramic acid esters 4 in contrast to the
analogs in series 2, 5, and 6.
In conclusion, this study has revealed that the enedione is a poten-
tial cytotoxic pharmacophore. In particular, the N-arylfumaramic
esters 4 are promising leads due to the cytotoxic potencies displayed
andtheirtolerancebymice. Henceanalog developmentofthesenovel
cytotoxins 4 should be actively pursued in different ways. For exam-
ple, the N-arylfumaramoyl group could be attached by ester or amidic
linkages to other cytotoxic molecules in order to produce a bifurcate
toxicity to neoplasms. Quantitative and qualitative structure–activity
relationships revealed various correlations between physicochemical
properties andthe IC50 figures generated. The reactionsofrepresenta-
tive compounds with a model thiol, namely benzyl mercaptan, con-
firmed that the enediones are thiol alkylators.
l
7.55 2.2 mM; 3g: 2.56 0.97 mM; 4b: 400.0 1.5
lM; 4c: >8.00
mM, and 4e: 2.33 0.81 mM.
The IC50 values of 2c and 4b are 432 and 400
lM, respectively.
However the inhibition of the growth of Molt 4/C8, CEM and L1210
cells by these two compounds is achieved by substantially lower
concentrations as the data in Table 1 indicates. Furthermore the
IC50 values of 2d, 3c,e, and 3b–e,g towards hNMT are all in excess
of 500 lM. Hence inhibition of hNMT contributes only a very mar-
ginal effect in some cases towards the cytotoxicity displayed by the
compounds prepared in this study whose principal modes of action
are by other mechanisms.
Finally in considering one or more of the series 2–6 as lead mole-
cules for further development, the tolerance of representative com-
pounds in mice was undertaken. Doses of 30, 100, and 300 mg/kg of
2b–d,g,h, 3a–i, 4a–c,e–g,i, 5a–h, 6b,c,g,h, and 9 were administered
to mice and the animals examined at the end of 0.5 and 4 h. No mor-
talities were noted in the case of 2b, 3a–i, 4a–c,e–g,i (i.e., all mem-
bers of series 3 which were examined were tolerated by the mice)
and 9. On the other hand, the remaining compounds viz 2c,d,g,h,
all members of series 5 and 6 which were evaluated, namely 5a–h
and 6b,c,g,h, led to deaths of theanimals. The N-arylfumaramic acids

A. Jha et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1510–1515
1515
4. Jha, A.; Mukherjee, C.; Rolle, A. J.; De Clercq, E.; Balzarini, J.; Stables, J. P. Bioorg.
Med. Chem. Lett. 2007, 17, 4545.
Acknowledgments
5. Jha, A.; Zhao, J.; Cameron, T. S.; De Clercq, E.; Balzarini, J.; Manavathu, E. K.;
Stables, J. P. Lett. Drug Des. Discovery 2006, 3, 304.
6. Dimmock, J. R.; Jha, A.; Zello, G. A.; Quail, J. W.; Oloo, E. O.; Nienaber, K. H.;
Kowalczyk, E. S.; Allen, T. M.; Santos, C. L.; De Clercq, E.; Balzarini, J.;
Manavathu, E. K.; Stables, J. P. Eur. J. Med. Chem. 2002, 37, 961.
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The authors thank the following agencies for financial support,
namely the Nova Scotia Health Research Foundation (A.J.),
Canadian Institutes of Health Research (A.J., J.R.D., R.K.S), Fonds
voor Wetenschappelijk Onderzoek-Vlaanderen (J.B., E.D.C.), and
the Department of Biotechnology, DBT, New Delhi (A.K.P.). Appre-
ciation is extended to Mrs. Lizette van Berckelaer for conducting
the Molt 4/C8, CEM and L1210 assays, and the National Institute
of Neurological Disorders and Stroke, MD, U.S.A. for undertaking
the short-term toxicity studies in mice. Dr Mike Lumsden of Atlan-
tic Region Magnetic Resonance Centre, Dalhousie University is
thanked for his help in recording 1D NOESY experiments on certain
samples.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.01.098.
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