Design and synthesis of Pfmrk inhibitors as potential antimalarial agents.

Article abstract of DOI:10.1016/S0960-894X(01)00578-9

The synthesis and inhibitory activities of 10 potential inhibitors of Pfmrk, a Plasmodium falciparum cyclin-dependent protein kinase, are described. The most potent inhibitor is a 3-phenyl-quinolinone compound with an IC(50) value of 18 microM. It is the first compound reported to inhibit Pfmrk at the micro molar range.

Full text of DOI:10.1016/S0960-894X(01)00578-9

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2875–2878
Design and Synthesis of Pfmrk Inhibitors as Potential
Antimalarial Agents
Zili Xiao,a Norman C. Waters,b Cassandra L. Woodard,b Zhiyu Lib and Pui-Kai Lia,*
aDivision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
bDivision of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA
Received 18May 2001; accepted 14 August 2001
Abstract—The synthesis and inhibitory activities of 10 potential inhibitors of Pfmrk, a Plasmodium falciparum cyclin-dependent
protein kinase, are described. The most potent inhibitor is a 3-phenyl-quinolinone compound with an IC50 value of 18 mM. It is the
first compound reported to inhibit Pfmrk at the micro molar range. # 2001 Elsevier Science Ltd. All rights reserved.
Malaria is one of the most serious tropical diseases and
it affects 400 million people with 1–2 million deaths
every year.1,2 Over the last several decades, drug resis-
tance in malaria parasites has been a major health pro-
blem. There has been a widespread resistance to
chloroquine,3 an antimalarial agent that has been used
for many years. In addition, resistance to suphadoxine/
pyrimethamine, the first line therapy of malaria, is now
emerging in several African countries.4 Therefore,
development of new antimalarial agents has become one
of the highest priorities. Of the four known human
malaria parasites, Plasmodium falciparum is the most
lethal form and the research into the basic biology of
the parasite has begun to search for new molecular tar-
gets for the development of new antimalarial agents.
identified as potential targets for the development of
antimalarial agents.
Although inhibitors of PfPK5 have been identified,7
Pfmrk inhibitors have never been reported. Most of the
CDK inhibitors are designed to compete with the ATP
binding site of the kinases. Known ATP-competitive
human CDK2 inhibitors Olomoucine and Roscovitine
(Fig. 1) failed to inhibit Pfmrk.8 Both inhibitors contain
the purine ring system. Recently, kinase inhibitors con-
taining lactam moiety such as Oxindole and Kenpaul-
lone are reported (Fig. 1).11,12 Kenpaullone is a potent
inhibitor of CDK1, CDK2, and CDK512 while oxindole
is a selective inhibitor of CDK4.11 It appears that the
unsubstituted lactam moiety is critical in forming H-
bonding with the peptide backbone at the ATP binding
site. In Kenpaullone, its CDK1 inhibitory activity
decreases 50-fold when the nitrogen in the lactam moi-
ety is methylated.12
Recently, a family of protein kinases [cyclin-dependent
kinases (CDKs)] that control cell cycle progression has
been the potential targets for drug development.5,6
CDKs are conserved among eukaryotic species and
several CDKs have been isolated from Plasmodium fal-
ciparum. The best characterized Plasmodial CDK is a
homologue of human CDK1 called PfPK5.7 Human
CDK1 inhibitors such as hymenialdisine, indirubin-30-
monooxime and purvalanol show inhibitory activities
against PfPK5.7 Pfmrk is another well characterized
Plasmodial CDK that shows significant homology with
human CDK7.8,9 In humans, CDK7 associates with
TFIIH transcription factor and regulates transcription
and DNA repairs.10 Both PfPK5 and Pfmrk are
In an attempt to identify new lead compound(s) to
inhibit Pfmrk, we design and synthesize 10 compounds
(1–10) containing lactam moiety with five different
structural classes as potential Pfmrk inhibitors (Fig. 2).
The structural classes are: (1) 3-phenyl-quinolinone (1
and 2), (2) dihydro-indolo quinolinone (3), (3) benzo-
furo (3,2-c) quinolinone (4–6), (4) benzopyrano (4,3-c)
quinolinone (7), and (5) benzofuro (2,3-b) quinolinone
(8–10).
The synthesis of compounds 3 was carried out according
to the literature procedure.13 The syntheses of compounds
1 and 2 are shown in Scheme 1. Reacting aldehyde 11
*Corresponding author. Fax: +1-614-688-8556; e-mail: li27@osu.edu
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00578-9

2876
Z. Xiao et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2875–2878
with 3-methoxyphenyl acetic acid 12 yielded the E and
Z (2:1) mixture of o-nitro-a-(3-methoxyphenyl) cin-
namic acid 13. Hydrogenation of 13 followed by intra-
molecular cyclization afforded the dihydroquinolinone
14. Oxidation of 14 with DDQ followed to demethyla-
tion yielded 16. Compound 1 was obtained by reacting
16 with methylbromoacetate followed by ester hydro-
lysis. Reacting 16 with 2-chloroethanol yielded 2.
hydrochloride yielded a mixture of 4 and 8 in a ratio of
2:5.
Compound 7 was synthesized as shown in Scheme 3.
Heating isatin 22, 2-methoxyphenyl acetic acid 23 with
sodium acetate at 200–230 ꢀC for 50 min yielded com-
pound 24. Intramolecular esterification of 24 in anhy-
drous pyridine hydrochloride at reflux afforded 7.
Compounds 4–6 and 8–10 were synthesized from the
same synthetic pathway (Scheme 2). Stirring phenylace-
tate 18 with diethyl oxalate at 60 ꢀC yielded 19, which
underwent thermal decarbonylation at 175 ꢀC to form
20. Refluxing 20 with aniline or p-substituted aniline
afforded 21a–c. Compounds 21a–c are the inter-
mediates for the syntheses of compounds 4–6 and 8–10.
For example, refluxing 21a in anhydrous pyridine
We have developed a microtiter plate drug screen to
identify inhibitors of Pfmrk. Pfmrk activity was assayed
with Pfcyc1, a plasmodial cyclin that stimulates Pfmrk
activity. The carboxy terminal domain of RNA poly-
merase II (CTD) was used as substrate since it has been
shown to be more favorable substrate than Histone H1
(unpublished results). In brief, Pfmrk and Pfcyc1 were
expressed and purified from Escherichia coli as pre-
Figure 1. Known CDK inhibitors.
Figure 2. Proposed Pfmrk inhibitors.
Scheme 1. Synthesis of compounds 1 and 2. Reagents: (a) Ac2O, TEA, reflux 2.5 h; (b) 5% Pd–C/H2, CH3OH, rt, 18h; (c) DDQ, ClCH 2CH2Cl,
reflux 48h; (d) BBr 3, CH2Cl2, rt, 2.5 h; (e) BrCH2CO2CH3, NaH, DMF, rt 6 h; (f) (1) KOH/CH3OH; (2) HCl; (g) ClCH2CH2OH, NaH, DMF, rt,
24 h.

Z. Xiao et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2875–2878
2877
Scheme 2. Synthesis of compounds 4–6 and 8–10. Reagents: (a) diethyl oxalate, EtOK, benzene, 60 ꢀC, 30 min; (b) 175 ꢀC, –CO; (c) p-substituted
aniline, Ph2O, reflux 3.5 h; (d) anhyd pyridine hydrochloride, reflux 1.5 h.
Scheme 3. Synthesis of compound 7. Reagents: (a) AcONa, 200–230 ꢀC, 50 min; (b) Pyr–HCl, reflux 1 h.
viously reported.7 The kinase drug screen was per-
formed in a 96-well filter plate assay. Assay began by
incubating Pfmrk (4 mg) and drug in kinase buffer
(50 mM Tris–HCl pH 7.5, 10 mM MgCl2, 1 mM DTT),
3 mg Pfcyc1 and 10 mg CTD for 5 min at 30 ꢀC to facil-
itate drug binding and followed by the addition of
[g-32P] ATP (5 mCi, 3000 Ci mmol/mL). The mixture was
incubated at 30 ꢀC for 30 min. Following incubation,
plates were washed on a vacuum manifold with six
consecutive washes of 200 mL of 5% phosphoric acid
per well. Following the last wash, scintillation fluid was
added and the plate analyzed in a Topcount microtiter
plate scintillation counter (Packard). Each reaction was
performed in triplicate and controls were included on
each plate in order to subtract background from the
reactions. Counts per min for each set of reactions were
averaged with a variation less than 5%. Each inhibitor
was tested over a range of concentration for determina-
tion of dose-responsiveness and for calculation of IC50
values. The IC50 values of the inhibitors are shown in
Table 1.
due to solubility problems. Compounds containing the
structural classes dihydro-indolo quinolinone (3) and
benzofuro (3,2-c) quinolinone (4–6) are not tolerated by
the enzyme. Both inhibitors 3 and 4 did not exhibit any
inhibitory activities. Substitution of H with OH group
at the 2-position of 4 resulted in inhibitor (5) with very
weak inhibitory activity (539 mM). Benzofuro (2,3-b)
quinolinones (8 and 9) exhibited marginal inhibitory
activities (<371 and 187 mM). Increasing the rotational
degree of freedom on the inhibitors (1 and 2) increase
the inhibitory activities. The most potent inhibitor (2)
has an IC50 value of 18 mM. Inhibitor 2 is the first com-
pound reported to inhibit Pfmrk and at the mM range. It
has been identified as the lead compound for Pfmrk
inhibition and detail structure–activity relationships
studies of the inhibitors are in progress.
References and Notes
1. Organization, W. H. Weekly Epidemiol. Rec. 1997, 72, 269.
2. White, N. J. Ann. Trop. Med. Parasitol. 1998, 92, 449.
3. Zoguereh, D. D.; Delmont, J. Sante 2000, 10, 425.
4. Nzila, A. M.; Mberu, E. K.; Sulo, J.; Dayo, H.; Winstanley,
P. A.; Sibley, C. H.; Watkins, W. M. Antimicrob. Agents Che-
mother. 2000, 44, 991.
The IC50 values range from 18to 539 mM. The IC50
values of inhibitors 6, 7 and 10 could not be determined
5. Toledo, L. M.; Lydon, N. B.; Elbaum, D. Curr. Med.
Chem. 1999, 6, 775.
Table 1. Pfmrk inhibitory activities (IC50 in mM) of compounds 1–10
6. Sielecki, T. M.; Boylan, J. F.; Benfield, P. A.; Trainor, G. L.
J. Med. Chem. 2000, 43, 1.
7. Roch, K. L.; Sestier, D. D.; Dorin, D.; Waters, N.; Kappes,
B.; Chakrabarti, D.; Meijer, L.; Doerig, C. J. Biol. Chem.
2000, 275, 8952.
Compd
IC50 (mM)
Compd
IC50 (mM)
1
2
3
4
5
102
18
No inhibition
No inhibition
539
6
7
8
9
10
Not tested
Not tested
<371
187
Not tested
8. Waters, N. C.; Woodard, C. L.; Prigge, S. T. Mol. Biochem.
Parasitol. 2000, 107, 45.
9. Li, J. L.; Robson, K. J. H.; Chen, J. L.; Targett, G. A. T.;
Baker, D. A. Eur. J. Biochem. 1996, 241, 805.
Purified Pfmrk was assayed with varying concentrations of inhibitors
in the presence of Pfcyc1 and CTD.

2878
Z. Xiao et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2875–2878
10. Fisher, R. P.; Morgan, D. O. Biochim. Biophys. Acta 1996,
1288, 7.
11. Kent, L. L.; Hull-Campbell, N. E.; Lau, T.; Wu, J. C.;
Thompson, S. A.; Nori, M. Biochem. Biophys. Res. Commun.
1999, 260, 768.
12. Schultz, C.; Link, A.; Leost, M.; Zaharevitz, D. W.;
Gussio, R.; Sausville, E. A.; Meijer, L.; Kunick, C. J. Med.
Chem. 1999, 42, 2909.
13. Walser, A.; Silverman, G.; Flynn, T.; Fryer, R. I. J.
Heterocycl. Chem. 1975, 12, 351.