Darpones and water-soluble aminobutoxylated darpone derivatives are distinguished by matrix COMPARE analysis

Article abstract of DOI:10.1016/j.bmcl.2007.01.043

Darpones are a class of compounds with antiproliferative activity for cancer cells in vitro and in mouse models. In order to improve the solubility of the compounds, darpones with aminobutoxy side chains were synthesized. The new derivatives showed retain

Full text of DOI:10.1016/j.bmcl.2007.01.043

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1850–1854
Darpones and water-soluble aminobutoxylated darpone derivatives
are distinguished by matrix COMPARE analysis
Christian Pruhs and Conrad Kunick*
¨
Technische Universita¨t Braunschweig, Institut fu¨r Pharmazeutische Chemie, Beethovenstraße 55, D-38106 Braunschweig, Germany
Received 1 December 2006; revised 12 January 2007; accepted 13 January 2007
Available online 24 January 2007
Abstract—Darpones are a class of compounds with antiproliferative activity for cancer cells in vitro and in mouse models. In order
to improve the solubility of the compounds, darpones with aminobutoxy side chains were synthesized. The new derivatives showed
retained antiproliferative activity for cultured cancer cell lines. However, a change of the selectivity pattern in the in vitro cell line
screening project of the American National Cancer Institute indicates that the solubilized derivatives might act through a different
biological mechanism. A matrix COMPARE analysis of the cancer cell line screening data clearly distinguished darpones with and
without solubilizing aminobutoxy side chains.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The darpones (general formula 1, Fig. 1) constitute a
class of compounds1,2 which have demonstrated anti-
proliferative activity both in the in vitro cell line screen-
ing project (IVCLSP)3–5 of the National Cancer
Institute (NCI) and in vivo in the murine hollow fiber
tumor model.6 Structure–activity relationship (SAR)
studies have shown that for the antiproliferative activity
the secondary lactam structure and both aryl substitu-
ents in 2- and 4-position on the basic heterocyclic ring
system are required.7 The molecular mechanism of the
antiproliferative activity of the title compounds is still
unknown. An isolated remark in the literature reported
the observation that the darpone 682765 (general formu-
la 1, R1, R3 = H, R2 = 40-Cl, X = 0) showed a correla-
tion with the expression of the EGFR ligand TGF-a in
the cancer cell line panel of the IVCLSP.8
Up to date, a straightforward development of the
darpone class is not only hampered by the lack of
knowledge regarding the biological targets but also by
their low water solubility. We therefore designed and
synthesized water-soluble darpone derivatives by attach-
ing aminobutoxy side chains to the darpone basic
structure 2a.
X
O
H
N
H
N
R1
R1
R3
The parent template 2a for structure modification was
chosen to meet three requirements: simple synthesis, sev-
eral options for structure modifications, and a maximum
‘darpone-like’ behavior in the IVCLSP. In the IVCLSP
a test compound is characterized by three parameters
for approximately 60 human cancer cell lines: (1) the
GI50 value (GI50 = molar concentration of the com-
pound that inhibits 50% net cell growth), (2) the TGI
value (TGI, molar concentration of the compound lead-
ing to total inhibition of net cell growth), and (3) the
LC50 value (LC50 = molar concentration of the com-
pound leading to 50% net cell death). Based on these
data mean values are calculated indicating the growth
inhibitory activity averaged over all cell lines. The mean-
graph midpoint log10 GI50 (MG-MID log10 GI50) is the
mean of all 60 log10 GI50 values for the tumor cell lines
R3
N
N
R2
Cl
R2
1
2a-j
Figure 1. General formula 1 of darpones (X = O, S) and modified
darpones 2a–j. For definition of R1–R3, see Table 1.
Keywords: Antiproliferative activity; Darpones; Compare analysis;
Solubility.
*
Corresponding author. Tel.: +49 531 391 2754; fax: +49 531 391
2799; e-mail: c.kunick@tu-braunschweig.de
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.043

C. Pru¨hs, C. Kunick / Bioorg. Med. Chem. Lett. 17 (2007) 1850–1854
1851
from the panel. The inhibition profile (‘fingerprint’)
resulting from the selectivity of a compound over the
60 cell lines is typical for the molecular mechanisms of
the growth inhibition. The analysis of such a pattern is
an established method to identify mechanisms or molec-
ular targets underlying growth inhibitory activity9–11
and might be used to discover prototypes of novel anti-
cancer drugs.12 The characteristic pattern generated by
darpones is clearly different from the patterns of estab-
lished antitumor drugs.2 A frequently used tool for the
comparison of inhibition profiles in the IVCLSP is
COMPARE, a program which generates pairwise corre-
lation coefficients (PCC) between compounds.5,13 In the
‘matrix COMPARE’ mode the tool is suitable to detect
relationships between a multitude of antiproliferative
compounds and to group compounds with similar bio-
logical mechanisms. Employing ‘matrix COMPARE’
for the 44 darpone entities included in the NCI database
of compounds tested in the IVCLSP revealed many high
pairwise intercorrelations within the compound family
(Fig. 2). Successive deletion of darpones with compara-
tively low correlations from the matrix ended with a col-
lection of 15 darpones showing exclusively pairwise
correlations with PCCs between 0.4 and 0.9. Of these,
compound 2a (R1, R2, R3 = H) was selected as parent
structure for aminoalkyl-substituted derivatives because
of the additional two criteria given above. Since the
molecular darpone targets are unknown and side chains
at the wrong position were expected to prevent binding
between the darpone derivative and its target, the solu-
bilizing aminoalkoxy side chains were attached to three
different positions around the darpone molecule (repre-
sented by R1, R2, R3 in formula 2, Fig. 1).
appropriately substituted chalcone 4 in the presence of
lithium hydroxide in THF yielding the Michael adduct
5. The latter is cyclized under oxidative conditions by
heating with ammonium ferric sulfate and ammonium
acetate in glacial acetic acid to furnish the darpone
2d.1 After BBr3-induced ether cleavage14 the resulting
phenol 6 is treated with an excess of 1,4-dibromobutane.
The so obtained bromo derivative 7 is reacted with mor-
pholine or N-methylpiperazine, respectively, to give ter-
tiary amines which are converted to the hydrochlorides
2j and 2m by treatment with hydrochloric acid. The pri-
mary amine 2g is obtained by a classical Gabriel synthe-
sis procedure in which the bromo derivative 7 is reacted
with the potassium salt of phthalimide in DMSO and
subsequent cleavage of the resulting phthalimide deriva-
tive by hydrazinolysis. The other methoxy- and amino-
butoxy-substituted darpones of Table 1 were prepared
following similar procedures starting with educts bear-
ing the methoxy group at appropriate positions.
The determination of solubility in phosphate buffer (pH
4.5) revealed poor solubility (<0.005 gLꢀ1) of the pri-
mary amine hydrochlorides 2e–2g. In contrast, the
hydrochlorides of the morpholino derivative 2j and the
dihydrochlorides of the N-methylpiperazino compounds
2k and 2m showed good solubility (>0.5 gLꢀ1). Obvi-
ously, not only the attachment position of the solubiliz-
ing side chains but also the structure of the amino
moiety has a high impact on solubility. For instance,
while the compound 2g with an aminobutoxy side chain
R3 shows low solubility, the corresponding morpholino-
butoxy analog 2j is much better soluble. The cancer cell
line screening in the IVCLSP revealed that all com-
pounds with aminobutoxy side chains retained antipro-
liferative activity (Table 1). With the exception of
compound 2f these derivatives exhibited an averaged
growth inhibition by single-digit micromolar concentra-
tions. This bioactivity is comparable to that of the par-
ent compound 2a.
The synthesis of the new darpone derivatives is exempli-
fied in Scheme 1 for the preparation of 2g, 2j, and 2m. In
the first step, the cyclic ketone 3 is reacted with an
Of note, the aminobutoxy side chain at R1 enhances the
potency of the compounds. This becomes obvious by the
comparison of the methoxy compound 2b and its
aminobutoxy analogs 2e, 2h, and 2k. The latter are
one order of magnitude more potent as antiproliferative
agents. In contrast, no such enhancement is found for
the two other sets of compounds which bear the solubi-
lizing side chains at R2 or R3, respectively. Another
interesting feature of the test results is that the amino-
butoxy-substituted derivatives seem to show a changed
selectivity pattern in the IVCLSP. This shift is not obvi-
ous on first sight, but is disclosed by a matrix COM-
PARE analysis performed with the compounds 2b–2m
of Table 1 and the 15 most ‘darpone-like’ darpones from
the NCI database (Fig. 3). The matrix COMPARE anal-
ysis assorted the darpones with and without basic side
chains into two groups which are represented by the
orange/yellow squares in the upper left and the lower
right corner of the matrix. While the three chloro-meth-
oxy-substituted darpones 2b–2d (section B) still showed
significant profile correlations with the most characteris-
tic 15 known darpones (section A), no such correlation
is found between darpones with (section C) and without
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
-0.2 0.0
-0.4 -0.2
-0.6 -0.4
Figure 2. Matrix COMPARE analysis of the 44 darpones in the NCI
database of compounds tested in the IVCLSP. Darpones are sorted
from left to right and from top to bottom by increasing ‘darpone-
likeliness’ (the cumulative pairwise correlation coefficient (PCC) with
all other darpones). Individual PCCs are color coded (For PCC limits
refer to color code legend). The arrows indicate the data lines of
compound 2a.

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C. Pru¨hs, C. Kunick / Bioorg. Med. Chem. Lett. 17 (2007) 1850–1854
O
H
N
OCH3
O
H
N
OCH3
i
ii
+
O
O
O
O
Cl
Cl
3
4
5
O
H
N
O
H
N
OCH3
OH
iii
iv
N
N
Cl
2d
O
Cl
6
H
N
O
H
N
O
O
v
N
N
Br
N
X
Cl
Cl
2j (X = O; Hydrochloride)
2m (X = N-CH3; Dihydrochloride)
7
vi
O
H
N
O
H
N
O
O
vii
N
N
O
N
NH2
x HCl
O
Cl
Cl
2g
8
Scheme 1. Synthesis of darpones with aminoalkyl side chains. Reagents and conditions: (i) LiOH, THF, 25 ꢁC, 2 h (65%); (ii) NH4OAc,
NH4Fe(SO4)2, AcOH, D, 2 h (73%); (iii) (1) BBr3, CH2Cl2, 25 ꢁC, 2 h; (2) H2O (61%); (iv) 1,4-dibromobutane, K2CO3, acetone, D, 2 h (58%); (v)
(1) morpholine or N-methylpiperazine, DMF, 80 ꢁC; (2) HCl, EtOH (35% and 28%, respectively); (vi) potassium phthalimide, DMSO, 90 ꢁC, 30 min
(59%); (vii) (1) H2NNH2, EtOH, D, 1 h; (2) NaOH, 1 h; (3) HCl (23%).
(sections A and B) aminobutoxy side chains (blue
areas).Within the group of aminobutoxy-substituted
derivatives clear correlations are found, regardless of
the attachment position for the side chain. Two hypoth-
eses can be deduced from these results: First, the intro-
duction of basic side chains apparently modifies the
mode of antiproliferative activity in the darpone com-
pound class. Second, the attachment point of the basic
side chain is not as relevant as initially considered. For
instance, the N-methylpiperazinobutoxy derivatives 2k,
2l, and 2m all exhibit averaged growth inhibition in
the single-digit micromolar concentration range with
selectivity for distinct cell lines, for example, the colon
carcinoma cell line HCT-15. Consequently, it is ques-
tionable whether the antiproliferative activity of the dar-
pones is resulting from a specific interaction with a
particular target, because in this case it is likely that at
least one subgroup of modified derivatives would have
exhibited diminished growth inhibition. Another expla-
nation for the changed selectivity pattern of the modi-
fied darpones is the possibility that the amino
functions influence cellular uptake or cellular efflux
transport mechanisms.
These results also have consequences for the determina-
tion of molecular drug targets by affinity approaches.
Biologically active drugs decorated by linker chains with
terminal amino groups (e.g., 2e–2g) can easily be bound
to solid matrices. These immobilized molecules then can
be employed for the isolation of proteins that are the po-
tential molecular targets of the basic drug molecule. In a
number of cases, targets of antiproliferative and other
drugs have been successfully identified by this methodol-
ogy.15,16 The findings with the darpones show that care
has to be exercised when interpreting the results of affin-
ity experiments, since the introduction of linker chains
might substantially change the molecular target
repertory.

C. Pru¨hs, C. Kunick / Bioorg. Med. Chem. Lett. 17 (2007) 1850–1854
1853
A
B
C
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
A
-0.2 0.0
-0.4 -0.2
-0.6 -0.4
B
C
Figure 3. Matrix COMPARE analysis of the 15 ‘most characteristic’
darpone derivatives (A); the chloro-methoxy-substituted darpone
derivatives 2b, 2c, 2d (B), and the novel darpones with amine side
chains 2e–2m (C). Individual PCCs are color coded (for PCC limits
refer to color code legend).
Acknowledgments
Funding of this project by the Deutsche Forschungs-
gemeinschaft (to C.P. and C.K., Grant Nos. KU1371/
1-1 and KU1371/1-2) is gratefully acknowledged. We
are grateful to the National Cancer Institute for testing
the compounds in the IVCLSP and especially to Mark
Kunkel and Dan Zaharevitz for introducing us into
the Matrix COMPARE methodology.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.01.043.
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