e. Thus the attachment of the 3-(2-hydroxyethylsulfanyl)-
propanoyl group onto the 3,5-bis(benzylidene)-4-piperidone
scaffold affords a cluster of novel lead molecules 4a-e. In series
4, compounds 4a-f are potent MDR-revertants while 4g-k are
inactive. The replacement of a hydroxy substituent in the side
chain of 4c by a mercapto group retained MDR-reversal
properties while a sulfonic acid group (SO3H) (4i) led to almost a
complete loss of this property. The three most potent compounds
(4b-d) have 9-13 times the potency of verapamil hydrochloride
and one may note that even the least potent compound among 4a-
f, namely 4a, has approximately the same FAR value as the
reference drug. In regard to 4a-e, the two most potent
compounds are 4b and 4d which have the highest Hansch π
values of the aryl substituents of 0.56 and 0.71, respectively.20 It
is conceivable therefore that analogs with increased lipophilicity
may have greater MDR-revertant potencies. The FAR value of 4f
is approximately half that of the oxo isostere 4c. The molecular
refractivity values of aliphatic hydroxy and mercapto groups are
2.55 and 8.76, respectively21 and thus the smaller size of the
hydroxy substituent in 4c may contribute to its greater potency
than 4f. Clearly the replacement of the hydroxy and mercapto
group of the N-acyl side chain by a sulfonic acid group leading to
4g-k virtually abolishes MDR-revertant properties. This effect
could be due to the sulfonic group causing a misalignment of 4g-
k with P-gp and/or the highly polar nature of the sulfonic group
substituent which inhibits penetration of the molecule via cell
membranes.
A review of the biodata in Table 1 reveals that 4a-f and 4k have
average CC50 values below 10 µM. All of these compounds are
substantially more potent than melphalan, e.g., 4e has 36 times the
potency of this reference drug. The SI values of 4d and 4e are greater
than 10 and in particular 4e has a similar selectivity as melphalan.
Thus both 4d and 4e achieve PL10 status. In order to quantify
potency and selectivity, the product of the average CC50 figures and
the SI values were obtained and the potency-selectivity expression
(PSE) data are given in Table 1. The PSE values reveal that the N-[3-
(2-hydroxyethylsulfanyl)propanoyl] analogs 4a-e are important lead
molecules, especially 4d and 4e. Both of these compounds contain
strongly electron-withdrawing substituents in the aryl ring (the
Hammett σp values of the chloro and nitro group are 0.23 and 0.78,
respectively20), and thus the creation of analogs with such aryl
substituents as 4-cyano (Hammett σp=0.66) and 4-trifluoromethyl
(Hammett σp=0.54)20 should be considered. The retention of one or
more nitro groups in the aryl rings requires careful consideration. On
the one hand, under the hypoxic conditions of certain solid tumours,
the rate of reduction of the nitro group to a variety of cytotoxins is
likely to be faster than in the corresponding normal cells. Thus
selective toxicity for tumours may result. On the other hand,
reduction products such as nitroxyl and nitro radicals as well as
nitroso groups can lead to a variety of pathological conditions25 and
unacceptable side effects. Thus to avoid these issues associated with a
nitro group, an isosteric replacement of 4-nitrophenyl group in 4e by
a 4-pyridyl group should be carried out in future. Among the sulfonic
acid derivatives 4g-k, only 4k had a greater PSE figure than
melphalan.
A ten-fold increase in concentration, i.e., 40 µg/mL was also
employed with a view to identifying MDR revertants which are
either inactive or display weak potencies at the lower
concentration. The quotients of the FAR values at 40 µg /mL and
4 µg /mL are presented in Table 1. A large increase in the FAR
figures for 4a,f was observed at the higher concentration.
However in most cases there are little differences in the FAR
values at 4 µg /mL and 40 µg /mL while for some compounds the
FAR values diminished at the higher concentration, e.g., 4b.This
phenomenon suggests that in these instances, viz similar or lower
FAR values at 40 µg /mL than 4 µg /mL, other biochemical
processes are activated which diminish the MDR-revertant
potencies. This phenomenon has been noted previously.15,22 With
the exception of the low potency of 4i, raising the concentration
of 4g-k to 40 µg/mL did not reveal any compounds with
noteworthy MDR-revertant properties.
In conclusion, the noteworthy MDR-revertants among series 4 are
4b-d. The introduction of a 3-(2-hydroxyethylsulfanyl)- propanoyl
scaffold onto 3,5-bis(benzylidene)-4-piperidones in series 1 led to a
novel group of potent P-gp modulators. In terms of cytotoxicity, SI
values and PSE figures, 4d and 4e emerge as the lead molecules.
Hence the optimal dual agent having MDR reversal properties and
selective-cytotoxicity is 4d which should be pursued in a variety of
directions. First, investigation of molecular mechanisms by which 4d
binds to P-gp which will give an insight for further designing of
potent P-gp modulators. Second, evaluation of 4d against other efflux
transporters such as MRPs and BCRPs. Third, the isosteric
replacement of 4-chloro or 4-chloroaryl groups with the groups
possessing similar electronic, hydrophobic and steric properties
should be undertaken and the compounds evaluated for MDR-
revertant properties and selective toxicity for neoplasms.
The next phase of the investigation was to identify the optimal
dual agents, i.e., those compounds which display both MDR-
revertant properties and greater toxicity to neoplasms than non-
malignant cells. The cytotoxicity of the compounds in series 4
have been assessed against human HSC-2 and HSC-4 squamous
cell carcinomas as well as human HL-60 promyelocytic leukemia
cells16,19 using a reported methodology23 and the average CC50
values are given in Table 1. In addition, 4a-k were evaluated
against human HGF gingival fibroblasts, HPC pulp cells and
HPLF peridontal ligament fibroblasts which are non-malignant
cells.16,19 From these data, selectivity index (SI) figures were
generated (average CC50 values towards normal cells/average
CC50 figures versus neoplasms) which are displayed in Table 1.
Of particular interest is the identification of those compounds
achieving PL10 status, i.e., a Promising Lead compound having
an average CC50 value of < 10 µM towards cancer cells and a SI
value of >10.11,24
Acknowledgments
The authors thank the Canadian Institutes of Health Research for
financial support.
References and notes
1.
2.
Pati, H. N.; Das, U.; Sharma, R. K.; Dimmock, J. R. Mini-Rev. Med.
Chem. 2007, 7, 131-139.
Dimmock, J. R.; Elias, D. W.; Beazely, M. A.; Kandepu, N. M. Curr.
Med. Chem. 1999, 6, 1125-1149.
3.
4.
5.
Dimmock, J. R.; Wong, M. C. L. Can. J. Pharm.Sci. 1976, 11, 35-53.
Baluja, G.; Municio, A. M.; Vega, S. Chem. Ind. 1964, 2053-2054.
Mutus, B.; Wagner, J. D.; Talpas, C. J.; Dimmock, J. R.; Phillips, O.
A.; Reid, R. S. Anal. Biochem. 1989, 177, 237-243.
Okey, A. B.; Harper, P. A. In Principles of Medical Pharmacology;
Kalant, H.; Grant, D. M.; Mitchell, J., Eds., 7th ed.; Elsevier: Toronto,
Canada, 2007; p. 902.
6.
Das, Umashankar
