ACS Medicinal Chemistry Letters
LETTER
Table 1. In Vitro Broad Spectrum Anticancer Activity of Leading Analogues of 1a
μM
compd ID
A-549 (lung)
H-460 (lung)
MCF-7 (breast)
MDA-MB-231 (breast)
OVCAR-3 (ovarian)
PC-3 (prostate)
1
2
2.5 ( 0.06
2.7 ( 0.0
1.7 ( 0.9
1.5 ( 0.4
1.6 ( 0.6
2.8 ( 0.06
2.5 ( 0.5
1.8 ( 0.5
1.6 ( 0.5
1.5 ( 0.4
7.5 ( 0.09
5.7
4.01 ( 0.03
3.3 ( 0.1
2.3 ( 0.2
2.6 ( 0.6
2.5 ( 0.3
14.5 ( 0.0
5.20 ( 0.6
5.00 ( 0.7
5.1 ( 0.6
9.0 ( 0.0
4.8 ( 0.1
4.3 ( 0.1
4.05 ( 0.2
2.5 ( 0.3
2.1 ( 0.1
8
7.0 ( 0.9
6.0 ( 5
4.0 ( 0.9
11
12
a Average IC50 values are shown. Each compound was tested at nine different concentrations, and each drug dilution was repeated four times. Cells
treated with DMSO (equivalent volume) were used as a vehicle control.
through a series of sequential reactions starting from another
intermediate 16 that was also employed in the synthesis of 1.11
Compound 16 was condensed with variously substituted benzyl
isocyanates (17-21) or phenyl isocyanate (22) in acetonitrile at
room temperature to obtain the corresponding ureido inter-
mediates (not shown), which, without isolation, were subjected
to further ring closure, mediated by DBU, to obtain the respec-
tive iminoimidazolone intermediates, 23-28, respectively, in
24-63% yields. The desired final 5:7:5-fused tricyclic hetero-
cycles 8-13 were obtained12 in 53-81% yields by ring closure of
the intermediates 23-28 by heating at reflux with triethyl
orthoformate, catalyzed by concentrated sulfuric acid. Com-
pound 14 was synthesized12 starting from N0-[(Z)-2-amino-
1,2-dicyanovinyl]-N-(2-chlorobenzyl)imidoformamide (29),13
which in turn was prepared by reaction of ethyl (Z)-N-(2-amino-
1,2-dicyanovinyl)formimidate14 with o-chlorobenzyl-amine.13
Ring closure of 29, catalyzed by DBU to obtain 30 (69%), followed
by condensation with p-methoxybenzylisocyanate, gave a mix-
ture (74%) of 32 and its ring-closed product 33. The latter was
also obtained (67%) by treatment of the mixture with catalytic
amount of DBU. Finally, the target 14 was obtained (85%) by
acid-catalyzed ring closure of 33 with triethyl orthoformate. All
new compounds were fully characterized by 1H and 13C NMR,
mass spectral, and elemental microanalytical and/or high-resolution
mass spectral (HRMS) data.
Ph-CH2-) leads to significant loss of activity, (g) movement of
the ortho-methoxy group of 10 to the meta position as in 11 (R3 =
m-OMe-Ph-CH2-) considerably enhances the anticancer activity,
(h) replacement of the para-methoxybenzyl substituent at posi-
tion 3 of 1 with a para-fluorobenzyl group as in 12 (R3 = p-F-
Ph-CH2-) gave closely comparable anticancer activity as that
of 11, (i) replacement of the p-methoxybenzyl group at position
3 of 1 with a directly attached phenyl group with multiple fluoro
substituents at ortho and meta positions as in 13 [R3 = 2,4-(F)2-
Ph] also leads to considerable loss of activity, and finally,
(j) replacement of the para-methoxy substituent at position 7 of
1 with an ortho-chlorobenzyl group as in 14 also leads to con-
siderable loss of activity.
Our preliminary mechanistic studies on anticancer activity
focused on exploring the stage(s) of the cell cycle being affected
by compound 1. To this end, we performed flow cytometry on
MCF-7 cells treated with a 3 μM concentration of 1 for 24 h, and
the results are collected in Figure 1. As shown, the compound
exerted a significant effect on the G1 (Gap1) and S (synthesis)
phases of the cell cycle, while G2 (Gap 2) and M (mitosis) phases
were practically unaffected. The G1 phase of the cell cycle
concerns RNA production and protein synthesis, as well as the
activation of an important cell cycle control mechanism, called
G1 Checkpoint, which ensures that everything is ready for the
subsequent S phase that involves DNA synthesis. Apparently,
MCF-7 cells treated with 1 accumulate in the G1 phase of the cell
cycle, indicating a G1 arrest (79.15% cells in treated vs 65.05% in
untreated control, P = 0.003). Moreover, the treatment with 1
causes a large reduction of cells in the S phase (5.87 vs 18.1%, P =
0.002). On the other hand, the G2/M phase of the cell cycle
exhibited only a minimal effect upon treatment with 1 (14.5%
cells in treated vs 16.15 ( 1.48% in untreated control, P = 0.26)
of the cell cycle. This indicates the G1 cell cycle arrest is one of
the possible mechanisms of action of compound 1. More studies
are currently underway to explore further details on the mecha-
nism of action. One of the prime candidates based on what we have
discovered so far is the effect of 1 on regulation of expression of
DDX3, a member of the DEAD-box RNA helicase family.15,16
Members of this family not only act as molecules that unwind
RNA but also play important roles in virtually all aspects of RNA
synthesis and function as well as in helicase-independent tran-
scriptional regulations.16-20 Not surprisingly, the DDX family of
proteins are important targets for potential anticancer as well
antiviral therapy in many laboratories,21-25 including our own.26
Finally, limited in vivo toxicity studies of 1 were conducted
employing five SCID mice. Following injection of 1 up to 500
μM (≈20 mg/kg), twice a week for 7 weeks, we did not observe
any toxicity in SCID mice. An extensive pathological examination
Compounds 1-14 were screened in vitro against six cancer
cell lines, including A-549 and H-460 (lung cancer), MCF-7 and
MDA-MB-231 (breast cancer), OVCAR-3 (ovarian cancer), and
PC-3 (prostate cancer).12 The compounds that showed the most
promising in vitro broad spectrum anticancer activity are collected
in Table 1.
Some important, albeit limited, SARs emerged from these studies:
(a) replacement of H at the 5-position of 1 (R2 = H) with a phenyl
ring containing an electron-withdrawing group either enhances
or retains activity as in 2 (R2 = p-NO2PhCH2-), whereas (b) an
unsubstituted phenyl ring as in 3 (R2 = Ph) or that containing an
electron-donating group as in 5 (R2 = p-CH3PhCH2-) leads to
loss of activity, (c) multiple substitutions on the aromatic ring as
in 4 [R2 = 2,4,5-(CO2H)3-Ph] also lead to loss of biological activ-
ity despite enhancement in solubility properties, (d) replacement
of H at position 5 with an aliphatic chain as in 6 and 7 [R2 =
-CH2-(CH2)n-CO2H] also leads to loss of biological activity
despite enhancement in solubility properties, (e) replacement of
the p-methoxybenzyl substituent of 1 at position 3 with an unsub-
stituted benzyl group as in 8 (R3 = -CH2Ph) enhanced the anti-
cancer potency in all six cell lines tested, whereas (f) substitution
of the same benzyl group with a single chloro or methoxy group
in ortho position as in 9 or 10 (R3 = o-Cl-PhCH2- or o-OMe-
254
dx.doi.org/10.1021/ml100281b |ACS Med. Chem. Lett. 2011, 2, 252–256