2-Oxotetrahydroquinoline-based antimalarials with high potency and metabolic stability

Article abstract of DOI:10.1021/jm7013138

We report a series of novel inhibitors of protein farnesyl-transferase based on the 2-oxotetrahydroquinoline scaffold. We developed an efficient synthesis of these compounds. These compounds show selective inhibtion of the malaria versus human farnesyltransferase and inhibit the growth of the malaria parasite in the low nanomolar range. Some of the compounds are at least an order of magnitude more stable to metabolic degradation than the corresponding tetrahydroquinolines.

Full text of DOI:10.1021/jm7013138

384
J. Med. Chem. 2008, 51, 384–387
2-Oxotetrahydroquinoline-Based
Antimalarials with High Potency and
Metabolic Stability
Vivek J. Bulbule,† Kasey Rivas,‡
Christophe L. M. J. Verlinde,§ Wesley C. Van Voorhis,‡ and
Michael H. Gelb*,†,§
Departments of Chemistry, Medicine, and Biochemistry, UniVersity
of Washington, Seattle, Washington 98195
ReceiVed October 18, 2007
Abstract: We report a series of novel inhibitors of protein farnesyl-
transferase based on the 2-oxotetrahydroquinoline scaffold. We devel-
oped an efficient synthesis of these compounds. These compounds show
selective inhibtion of the malaria versus human farnesyltransferase and
inhibit the growth of the malaria parasite in the low nanomolar range.
Some of the compounds are at least an order of magnitude more stable
to metabolic degradation than the corresponding tetrahydroquinolines.
Efforts to discover and develop novel antimalarial agents are
being intensified as a result of the widespread development of
resistance to well established antimalarials and the availability
of novel funding mechanisms including the Medicines for
Malaria Venture.1 There are about 300–500 million malaria
cases each year, resulting in 1–3 million deaths, mostly in
Africa.2 In an effort to speed up the development of novel
antimalarial agents, we have explored potential drug targets in
the parasite for which considerable expertise is already available
through efforts in the pharmaceutical industry to develop drugs
for nontropical diseases. We have applied this “piggy-back”
medicinal chemistry toward the development of inhibitors of
the enzyme protein farnesyltransferase as antimalarial agents.3–5
Protein farnesyltransferase from the Plasmodium falciparum
malarial parasite (Pf-PFTa) appears to be an excellent drug
target: (1) Pf-PFT inhibitors are cytotoxic rather than cytostatic
to parasites.6 (2) Inhibitors of human PFT are much less toxic
to mammalian cells; in fact, they are being advanced through
clinical trials for the treatment of leukemia in elderly patients
who do not tolerate other chemotherapeutic regiments very
well.7 (3) Although it has not been possible to obtain recom-
binant Pf-PFT, partially purified enzyme from malaria-infected
red blood cells is available for analysis of thousands of
inhibitors.8 (4) A structural homology model of the active site
of Pf-PFT can be built using numerous X-ray structures of
mammalian PFT with bound inhibitors.9
Figure 1. (Left) Chemical structure of 29, a THQ-based Pf-PFT
inhibitor. (Right) General structure of substituted 2-oxo-THQs. (Below)
X-ray structure of 29 bound to mammalian PFT with the hydrophobic
part of farnesyl diphosphate in cyan, the diphosphate part in red, and
the Zn2+ in green (PDB coordinates available, 2r2l).
an IC50 (concentration that causes 50% enzyme inhibition)
of 0.5 nM and arrests the growth of P. falciparum in human
red cells with an ED50 (concentration that causes 50%
inhibition of growth) of 15 nM.6,12 The X-ray structure of
29 bound to mammalian PFT shows that the nonmethylated
N of the imidazole ring directly binds to the active site Zn2+
.
It also shows that the 6-CN on the tetrahydroquinoline ring,
the group attached to the sulfonamide sulfur, and the group
attached to the sulfonamide N pack into pockets on the
enzyme (Figure 1).9
Values of ED50 in the low nanomolar region are thought to
be adequate for antimalarial drugs. Therefore, compounds such
as 29 have acceptable potency. The problem that we encountered
with THQs in general is their high rate of metabolic degradation
by one or more cytochrome P450s present in liver microsomes.11
This translated to a rapid rate of clearance of THQs from the
serum of mice and rats that were dosed orally.11 Detailed studies
using combined liquid chromatography/mass spectrometry
together with isotopically labeled THQs led to the realization
that the major route of metabolic degradation, at least for some
of the compounds, is loss of the imidazole-containing side chain
attached to N-1 of the THQ ring (Figure 2). This cytochrome
P450-catalyzed reaction can occur by one of two pathways
(Figure 2).
Pathway 1 involves initial electron transfer from N-1 to the
enzyme to give a radical cation, which undergoes further
hydrogen atom transfer to the P450 to give the iminium ion,
which spontaneously breaks down to the amine plus aldehyde.
Pathway 2 involves initial hydrogen atom transfer from the
methylene to the enzyme to form a C-centered radical followed
by hydroxyl rebound to give the carbinolamine, which spon-
taneously decomposes to the observed metabolites, amine plus
aldehyde. We reasoned that placement of a 2-oxo group onto
the THQ ring (2-oxo-THQ, Figure 1) would suppress this
N-dealkylation reaction regardless of which pathway was
In previous work we discovered that tetrahydroquinoline
(THQ)-based PFT inhibitors (Figure 1), first developed at
Bristol-Myers Squibb as inhibitors of human PFT with anti-
cancer potential,10 are potent inhibitors of Pf-PFT and malarial
growth. Our most potent compounds in the series include 29
(Figure 1) that inhibits Pf-PFT enzymatic activity in vitro with
* To whom correspondence should be addressed. Address: Department
of Chemistry, Campus Box 351700, University of Washington, Seattle, WA
98195. Phone: (206) 543-7142. Fax: (206) 685-8665. E-mail: gelb@
chem.washington.edu.
†
Department of Chemistry.
Department of Medicine.
Department of Biochemistry.
‡
§
aAbbreviations: Pf-PFT, protein farnesyltransferase from P. falciparum;
THQ, tetrahydroquinoline.
10.1021/jm7013138 CCC: $40.75
2008 American Chemical Society
Published on Web 01/17/2008

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 385
Table 1. Pf-PFT inhibition and Antimalarial Activity of 2-Oxo-THQsa
a See Figure 1 for the generic structure of 2-oxo-THQs. In the table, “ND” indicates not determined and “(bromo)” indicates that the compound has Br
instead of CN at position 6 of the 2-oxo-THQ ring.
operative. In the context of pathway 1, the addition of a 2-oxo
group leads to a lactam in which the lone pair on N-1 is less
susceptible to oxidation by cytochrome P450 owing to the amide
resonance. If pathway 2 applies, the lone pair on N-1 is less
able to donate into the electron-deficient p-orbital of the
C-centered radical.
A highly efficient route to 2-oxo-THQs, novel structures not
reported previously, is summarized in Scheme 1 (full experi-
mental details given in Supporting Information).
Conversion of the methyl ester to the primary chloride 2 is
carried out by a standard sequence. Malonic ester synthesis gives
lactam 3, which is converted to lactam 4 by acid catalyzed
hydrolysis and decarboxylation. The 3-amino group is sulfonated
and alkylated, and the 6-bromo is converted to 6-CN via Pd-
catalyzed exchange to afford the 2-oxo-THQs. Several 2-oxo-
THQs were made by this route (Supporting Information) and
are shown in Table 1.
We prepared and tested 25 2-oxo-THQs for their ability to
inhibit Pf-PFT in vitro using 3 inhibitor concentrations (0.5, 5,
and 50 nM) (Table 1). We also tested the compounds to
Commercially available aniline 1 undergoes reductive ami-
nation with commercially available 5-formyl-1-methylimidazole.

386 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Letters
Figure 3. In vitro metabolism by mouse liver microsomes of the 2-oxo-
THQ 9 (half-time of 133 min) and the structurally matched THQ lacking
the 2-oxo group (30) (half-time of 16 min).
Figure 2. Two pathways for cytochrome P450-catalyzed loss of the
substituent from N-1 of the THQ. Pathway 1 (top route) involves initial
transfer of a N-1 lone pair electron, whereas pathway 2 (bottom route)
proceeds via a C-centered radical.
Table 2. Half-Times (min) for Metabolic Degradation of 2-Oxo-THQs
and THQs by Mouse Liver Microsomes
Scheme 1a
2-oxo-THQs
half-timea
THQb
half-timea
9
>120, 133
30
31
33
34
16, 10
17
3
11
15
17
19
28
>60, 144
40
21
9
10, 7
31
a Where two numbers are listed, this is the half-time values for two
independent experiments. b Data for THQs is from ref 11. The structure of
the THQ is same as that of 2-oxo-THQ but lacking the 2-oxo group.
Table 3. Inhibition of Rat PFT
rat PFT, % inhibition at indicated concentration
compd
5000 nM
500 nM
50 nM
5 nM
a Reagents and conditions: (a) 5-formyl-1-methylimidazole, CF3CO2H,
EDC, Et3SiH, 50 °C, 48 h (40%); (b) LiAlH4, THF, 0 °C to room temp,
2 h (72%); (c) SOCl2, CHCl3, reflux, 2 h (90%); (d) diethyl acetamido-
malonate, NaH, DMF, 120 °C, 12 h (65%); (e) concentrated HCl, reflux,
12 h, (75%); (f) R1SO2Cl, DIPEA, CH3CN, room temp, 12 h (65%); (g)
R2Br, Cs2CO3, DMF, room temp, 12 h (55%); (h) Zn(CN)2, Pd(PPh3)4,
DMF, microwave 2.5 min (78%).
9
73
56
86
61
92
84
23
0
62
5
75
54
0
0
0
0
23
4
0
0
0
0
0
0
10 (bromo)
11
14
17
19
other than loss of the Zn2+-binding N-methylimidazole arm,
but this was not investigated.
We also tested some of the 2-oxo-THQs for their ability to
inhibit human mammalian (rat) PFT using an in vitro assay
described previously,6 and results are shown in Table 3. It can
be seen that the 2-oxo-THQs are much less potent on rat PFT
compared to Pf-PFT.
In summary, a new series of inhibitors of the malarial protein
farnesyltransferase (Pf-PFT), 2-oxo-THQs, have been designed
on the basis of consideration of the structure of the enzyme–in-
hibitor complex and the knowledge of the pathway of liver
metabolism. Of the 25 compounds prepared, a subset showed
sufficient potency and metabolic stability to warrant further
development of 2-oxo-THQs as novel antimalarial agents. As
this series is expanded, we will focus on continued improvement
in potency against parasite growth while maximizing oral
bioavailability. An efficient route for the synthesis of 2-oxo-
THQs was developed, which is important for reducing the cost
of goods, an important requirement in antimalarial drug
discovery that is often underestimated.
determine the ED50 for the arrest of growth of two strains of P.
falciparum parasites (chloroquine sensitive 3D7 and chloroquine
resistant K1 strains) in human red blood cells (Table 1). In
general there is a good correlation between potency of inhibition
of Pf-PFT in vitro and parasite growth arrest. The importance
of the 6-CN group is apparent from data obtained with two
compounds that have a 6-Br group (5 and 6). Among the R1
groups examined, the N-methylimidazole-4-yl gives the most
potent compounds. The best compound in the series in terms
of antimalarial potency is 17 with an exceptional ED50 of 30
nM. A few other compounds were discovered to have ED50
below 100 nM.
Next we examined the metabolic stability of our most potent
compounds by measuring the half-life for compound loss upon
incubation with mouse liver microsomes in the presence of an
NADPH regeneration system. Results are summarized in Figure
3 and Table 2 along with the half-life for the corresponding
THQs without the 2-oxo group (30-34). Some of the 2-oxo-
THQs show exceptional stability to microsomal degradation;
for example, 9 and 11 are ∼10-fold more stable than the
corresponding THQ analogues lacking the 2-oxo group (Figure
3, Table 2).
Acknowledgment. We are grateful to D. Floyd, L. Lom-
bardo, and D. Williams for helpful discussions.
Despite testing more than 100 THQs for metabolic stability,
we have not been able to find compounds lacking the 2-oxo
groups that displayed half-times greater than 30 min.11 Other
2-oxo-THQs in Table 2 are still destroyed by mouse liver
microsomes within shorter times presumably because of routes
Supporting Information Available: Experimental details in-
cluding the synthesis of all compounds and assay procedures and
HPLC traces of target compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.

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JM7013138