2-Oxo-tetrahydro-1,8-naphthyridines as selective inhibitors of malarial protein farnesyltransferase and as anti-malarials

Article abstract of DOI:10.1016/j.bmcl.2007.11.104

A new class of 2-oxo-tetrahydro-1,8-naphthyridine-based protein farnesyltransferase inhibitors were synthesized and found to inhibit protein farnesyltransferase from the malaria parasite with potencies in the low nanomolar range. The compounds were much less potent on mammalian protein prenyltransferases. Two of the compounds block the growth of malaria in culture with potencies in the sub-micromolar range. Some of the compounds were found to be much more metabolically stable than previously described tetrahydroquinoline-based protein farnesyltransferase inhibitors.

Full text of DOI:10.1016/j.bmcl.2007.11.104

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 494–497
2-Oxo-tetrahydro-1,8-naphthyridines as selective inhibitors
of malarial protein farnesyltransferase and as anti-malarials
Srinivas Olepu,a Praveen Kumar Suryadevara,a Kasey Rivas,b Kohei Yokoyama,a
Christophe L. M. J. Verlinde,c Debopam Chakrabarti,d
Wesley C. Van Voorhisb and Michael H. Gelba,c,
*
aDepartment of Chemistry, University of Washington, Seattle, WA 98195, USA
bDepartment of Medicine, University of Washington, Seattle, WA 98195, USA
cDepartment of Biochemistry, University of Washington, Seattle, WA 98195, USA
dDepartment of Molecular Biology & Microbiology, University of Central Florida, Orlando, FL 32826, USA
Received 5 October 2007; revised 22 November 2007; accepted 27 November 2007
Available online 3 December 2007
Abstract—A new class of 2-oxo-tetrahydro-1,8-naphthyridine-based protein farnesyltransferase inhibitors were synthesized and
found to inhibit protein farnesyltransferase from the malaria parasite with potencies in the low nanomolar range. The compounds
were much less potent on mammalian protein prenyltransferases. Two of the compounds block the growth of malaria in culture with
potencies in the sub-micromolar range. Some of the compounds were found to be much more metabolically stable than previously
described tetrahydroquinoline-based protein farnesyltransferase inhibitors.
Ó 2007 Elsevier Ltd. All rights reserved.
Malaria remains one of the key parasitic diseases present
today with more than 400 million acute illnesses and at
least 2–3 million deaths annually throughout the tropi-
cal and subtropical regions of the world. The malaria
parasite is able to become resistant to chemotherapeutic
agents. For example, in many places there is a major
problem of resistance to chloroquine, a 4-aminoquino-
line that has been the mainstay of treatment for malaria
for many years. Hence, there is an urgent need for the
development of new drugs against new molecular
targets.1
have been extensively developed as anti-cancer agents
because of their ability to block tumor growth in
experimental animals. We have been pursuing PFTIs
as anti-malarial and anti-trypanosome agents because
these compounds are much more toxic to these parasites
than to mammalian cells, and there are a large number
of lead compounds from which to launch an anti-para-
site drug discovery program (piggy-back medicinal
chemistry).2–4 We have developed tetrahydroquinoline
(THQ)-based PFTIs as anti-malarials with low nanomo-
lar potency on parasite PFT and on parasites cultured
in vitro.3–7 One of our lead compounds, 1 (Fig. 1), kills
Plasmodium falciparum growth in vitro with an ED50 of
16 nM, shows high oral bioavailability, and is able to
cure rats infected with rodent malaria.6
Protein prenylation has been vigorously studied over the
past ꢀ15 years because it is found on several signaling
proteins (including heterotrimeric G proteins) that con-
nect cell surface receptors to intracellular effectors, and
also on Ras proteins, one of the most common oncopro-
teins found in human tumors. Protein farnesyltransfer-
ase (PFT) transfers the farnesyl group from farnesyl
diphosphate to the SH of the cysteine near the C-termi-
nus of proteins such as Ras. PFT inhibitors (PFTIs)
Unfortunately, high doses of 1, 50 mg/kg, were required
for cures in rodents because of rapid compound clear-
ance. In vitro microsome metabolism studies suggest
that the major culprit is cytochrome P450-catalyzed loss
of the imidazole-containing side chain (which binds to
the active site Zn2+ of PFT) leading to 2 (Fig. 1). This
reaction starts either with P450-catalyzed hydrogen
atom abstraction from the CH2 group attached to N1
of the tetrahydroquinoline ring to give a C-centered
radical or enzyme-catalyzed oxidation of N1 to give
Keywords: Malaria; P. falciparum; Anti-malarials; Protein farnesyl-
transferase; Drug discovery; Rational drug design.
*
Corresponding author. E-mail: gelb@chem.washington.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.104

S. Olepu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 494–497
495
O
O
N
N
NH2
O
OEt
O
O
N
N
MeO
MeO
6
a
OEt
OH
N
Cl
5
N
NH
b
N
4
Cl
NC
N
NC
N
N
N
S
N
7
S
N
P450
O
O
O
O
N
H
N
Me
Boc-HN COOCH3
N
2
e
1
CHO
c, d
P(O)(OMe)2
N
N
NH
N
N NH
2
BOCHN
R
N
N
CO2Me
8
10
9
1
N
R
N
NC
S
O
O
O
N
N
Boc-HN COOCH3
Br
Boc-HN COOCH3
Me
N
g
f
3
N
NH
N
NH
N
N
N
N
N
N
12
11
Figure 1. Compound 1 is a tetrahydroquinoline-based PFTI that is
metabolized by cytochrome P450 to give compound 2. Compound 3
shows the general structure of the 2-oxo-tetrahydro-1,8-naphthyridine-
based PFTIs prepared in the current study.
NC
NH2
i
NC
NC
NHBOC
h
N
N
O
N
N
N
O
N
14
13
the N-centered radical cation. Regardless of the mecha-
nism, we envisioned that placement of an oxo group at
the 2-position of the tetrahydroquinoline ring and a N
in place of C-8 would reduce P450-catalyzed radical for-
mation due to a rise in the oxidation potential of the N1
lone pair electrons (due to involvement of the lone pair
in resonance with the carbonyl and the pyridine N).
Thus, we set out to prepare 2-oxo-tetrahydro-1,8-naph-
thyridine-based PFTIs exemplified by 3 (Fig. 1). Consid-
ering the X-ray structure of tetrahydroquinoline PFTIs
bound to mammalian PFT and a homology model of
the active site of malarial PFT,4,6 it appears that addi-
tion of the 2-oxo and 8-aza groups to the tetrahydro-
quinoline scaffold would be tolerated.
N
R2
R2
O
NH
NC
N S R1
k
j
O
O
N
N
O
N
N N
N
15
3
N
N
Scheme 1. Reagents: (a) SOCl2, EtOH, 80%; (b) Et3N, DMF, 60%; (c)
LiAlH4, THF, 70%; (d) MnO2, CH2Cl2, 75%; (e) tetramethylguani-
dine, CH2Cl2, 65%; (f) H2/Pd-C, CH3OH, 50%; (g) Br2, CH3COOH,
65%; (h) Zn(CN)2, Pd(PPh3)4, DMF 35%; (i) 20% CF3COOH,
CH2Cl2, 100%; (j) R2-CHO, NaCNBH3, CH3OH, 55–60%; (k) R1-
SO2Cl, DIPEA, CH2Cl2, 15–20%.
Compounds were prepared following the synthetic se-
quence illustrated in Scheme 1. Ethyl chloronicotinate
5 was prepared from 2-chloro nicotinic acid 4, and
installation of the imidazole was accomplished by nucle-
ophilic substitution conditions to give 7. This was fol-
lowed by reduction of the ester group and subsequent
oxidation to yield 8. The Wittig olefination of com-
pound 8 with Boc protected phosphonoacetate 9 gave
10 followed by catalytic hydrogenation over palladium
in methanol to give 11. Subsequent bromination with
Br2 in acetic acid afforded 6-bromo analogue 12, which
was converted to the corresponding 6-cyano derivative
13 by treatment with zinc cyanide and tetrakis(triphen-
ylphosphine)palladium in dimethylformamide. Removal
of Boc group with trifluoroacetic acid in dichlorometh-
ane afforded the key intermediate 3-amino-6-cyano-2-
oxo-tetrahydro-1,8-naphthyridine 14. Completion of
target molecules was accomplished following a 2-step se-
quence of reductive amination and sulfonamide forma-
tion. If sulfonation was carried out first followed by
alkylation of the sulfonamide N with R2Br, the observed
product was the enamine with a double bond in the 3,4-
position of the lactam ring (due to elimination of the
sulfinate). Full synthetic details are available as Supple-
mentary Data.
Our previous structure–activity data on THQ-based
inhibitors of malarial PFT led to the discovery of com-
pounds with R1 = N-methyl-4-imidazolyl or 2-pyridyl
(i.e., 1) as being potent inhibitors of malarial PFT.7 In
Table 1, we report anti-malarial results obtained with
2-oxo-tetrahydro-1,8-naphthyridine-based PFTIs with
R1 = N-methyl-4-imidazolyl or 2-pyridyl and with varia-
tion of the R2 group. Compounds with R1 = N-methyl-
4-imidazolyl conferred the best in vitro activity against
Plasmodium falciparum PFT (18 and 20 showed 98%
and 95% inhibition at 50 nM, respectively) compared
to compounds with R1 = 2-pyridyl (21 and 19 showed
88% and 48% inhibition at 50 nM, respectively). We also
tested the compounds for their ability to block the
growth of Plasmodium falciparum in human red blood
cell cultures. Values of ED50, the concentration of com-
pound that reduces parasite growth by 50%, are listed in
Table 1. Two malarial strains were studied, 3D7, which
is chloroquine sensitive, and K1, which is chloroquine
resistant. Compounds 18, 20, and 21 showed good
potency, with values of ED50 in the 175–420 nM range
(Table 1). These compounds are also the most potent
in the series studied in inhibiting Plasmodium falciparum
PFT in vitro. Testing on malarial PFT and on parasites
was carried out as described.6

496
S. Olepu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 494–497
Table 1. Structure, PFT inhibition, anti-malarial activity, and microsomal metabolism of 2-oxo-tetrahydro-1,8-naphthyridine-based PFTIs
2
R
O
1
X
N
S R
O
N
N
O
N
N
Compound
X
R1
R2
Malaria PFT enzyme %
ED50 for
Microsome
metabolism
half-timeb (min)
inhibition at the indicated
inhibitor concentration
(nM)
inhibition of
parasite growth
in vitroa (nM)
500
50
5
0.5
3D7
K1
Naph
ND
THQ
ND
N
N
N
16
17
18
Br
H
H
74
28
0
0
>5000
>5000
420
>5000
N
N
N
CN
CN
94
99
66
98
32
80
15
27
>5000
300
ND
52
ND
15.6
O
S
H C
2
O
O
S
19
20
21
CN
CN
CN
91
98
97
48
95
88
3
0
3100
350
>5000
175
15
ND
5.4
3.8
H C
2
N
O
O
N
71
33
16
0
>120
ND
H C
2
N
N
N
N
O
O
O
320
310
H C
2
a ED50 is the concentration of compound that inhibits the growth of parasites by 50% (chloroquine sensitive strain 3D7 or chloroquine resistant strain
K1) in red blood cell cultures (measured according to Ref. 6).
b Given is the half-time for loss of parent compound when incubated with mouse liver microsomes according to the procedure given in Ref. 6.
Compounds tested are: (1) column labeled Naph, the 2-oxo-tetrahydro-1,8-naphthyridines shown in the table; (2) column labeled THQ, the
corresponding tetrahydroquinoline-based PFT (analogs of 1) with the same R1 and R2 groups as the indicated 2-oxo-tetrahydro-1,8-naphthyridine.
ND, not determined.
Table 2. Inhibition of rat PFT and protein geranylgeranyltransferase-I
(PGGT-I) by 2-oxo-tetrahydro-1,8-naphthyridine-based PFTIs
Next, we tested the stability of the 2-oxo-tetrahydro-1,8-
naphthyridine-based inhibitors toward metabolism by
mouse liver microsomes in vitro. For comparison, in Ta-
ble 1 we also include previous data for in vitro metabo-
lism of the corresponding tetrahydroquinolines.
Compound 19 was found to be about 3-folds more sta-
ble than the corresponding tetrahydroquinoline. For
compound 20, the increase in stability is dramatic,
>20-fold. In vitro microsome metabolism studies were
carried out as described.6 All compounds in Table 1
were found to be stable after incubation in aqueous buf-
fer at pH 2 for 24 h.
Compound Rat PFT, % inhibition at the Rat PGGT-I,
indicated inhibitor
% inhibition
at 1 lM inhibitorb
concentrationa (nM)
5000 500 50
5
0.5
16
17
18
20
51
39
47
85
8
8
5
0
0
7
0
0
0
0
16
0
0
1
9
71
0
28
0
5
a PFT assays were carried out as in Ref. 7.
b PGGT-I assays were carried out as for PFT assays except rat PGGT-
I
(5 ng), 5 lM H-Ras-CVLL, and 0.65 lM [3H]geranylgeranyl
pyrophosphate were used.
8
We tested four of the compounds for inhibition of mam-
malian protein prenyltransferases, and results are shown
in Table 2. It can be seen that the compounds are rea-
sonably selective for the malarial versus rat PFT. For
example, compounds 17, 18, and 20 at 5 nM inhibits
malarial PFT by 32%, 80%, and 71%, but inhibit rat
PFT at this concentration only by 0%, 0%, and 7%,
respectively. All four compounds are very poor inhibi-
tors of rat PGGT-I.

S. Olepu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 494–497
497
In conclusion, we have developed a new class of PFT
References and notes
inhibitors based on the 2-oxo-tetrahydro-1,8-naphthyri-
dine scaffold that are more potent on malaria PFT than
on the mammalian enzyme. These compounds are met-
abolically more stable than the tetrahydroquinoline lead
compounds. The route of synthesis of these novel com-
pounds was also developed. The most potent com-
pounds in the series inhibit the malarial PFT with
potencies in the low nanomolar range and kill cultured
parasites in the hundreds of nanomolar range. Future
studies will focus on the oral bioavailability and anti-
malaria efficacy of these compounds in rodents.
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Acknowledgments
This work was supported by funds from the National
Institutes of Health (AI054384) and the Medicines for
Malaria Venture (Geneva).
Supplementary data
Experimental details for the synthesis of representative
compounds are available as Supplementary data. Supple-
mentary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2007.11.104.
8. Yokoyama, K.; Zimmerman, K.; Scholten, J.; Gelb, M. H.
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