Novel N-(4-piperidinyl)benzamide antimalarials with mammalian protein farnesyltransferase inhibitory activity

Article abstract of DOI:10.1248/cpb.53.1324

Protein farnesyltransferase of Plasmodium falciparum is a potential target in the treatment of malaria for which increased drug resistance is observed. The design, synthesis and evaluation of a series of N-(4-piperidinyl)benzamides is reported. The most potent compounds showed in vitro activity against the parasite at submicromolar concentrations.

Full text of DOI:10.1248/cpb.53.1324

1324
Notes
Chem. Pharm. Bull. 53(10) 1324—1326 (2005)
Vol. 53, No. 10
Novel N-(4-Piperidinyl)benzamide Antimalarials with Mammalian Protein
Farnesyltransferase Inhibitory Activity
Adina RYCKEBUSCH,a Pauline GILLERON,a Régis MILLET,a Raymond HOUSSIN,a Amélie LEMOINE,a
Nicole POMMERY,a Philippe GRELLIER,b Christian SERGHERAERT,c and Jean-Pierre HÉNICHART*,a
a Institut de Chimie Pharmaceutique Albert Lespagnol, EA 2692, Université de Lille 2; 3 rue du Professeur Laguesse, B.P.
83, 59006 Lille, France: b Museum National d’Histoire Naturelle, USM 0504; 61 rue Buffon, 75005 Paris, France: and
c Institut de Biologie et Institut Pasteur de Lille, UMR CNRS 8525, Université de Lille 2; 1 rue du Professeur Calmette, B.P.
447, 59021 Lille, France. Received February 7, 2005; accepted June 12, 2005
Protein farnesyltransferase of Plasmodium falciparum is a potential target in the treatment of malaria for
which increased drug resistance is observed. The design, synthesis and evaluation of a series of N-(4-
piperidinyl)benzamides is reported. The most potent compounds showed in vitro activity against the parasite at
submicromolar concentrations.
Key words farnesyltransferase; malaria; Plasmodium falciparum; N-(4-piperidinyl)benzamide derivative; inhibitor
Antimalarial drugs saved millions of lives during the 20th cations: (i) replacement of methioninate by phenylalaninate
century. However, owing to the onset of drug resistance, or isoleucinate (R1) with the aim of increasing selectivity
there is now an urgent need to identify new drug targets in versus geranylgeranyl transferase, based on our previous re-
Plasmodium falciparum and to develop effective new anti- sults14) (ii) replacement of the cysteinyl moiety by a known
malarial agents.1,2) Prenylated proteins have been shown to metal chelator, i.e. (1-benzylimidazol-5-yl)methyl substituted
function in important cellular processes including signal in para position (R2) of benzyl in order to increase cellular
transductions. Among them, protein farnesyl transferase uptake (iii) reduction of the benzoyl group into benzyl (R3)
(PFT) has been a major target in the conception of new anti- in order to introduce flexibility into this region of the mole-
cancer drugs.3)
cule. Target compounds 15a—c, 16c, 17c, 18 were obtained
In an effort to identify a new and more effective drug tar- by reductive amination between adequate imidazolylcar-
get, Chakrabarti4,5) found that the peptidomimetic L-745,631 baldehydes 8a—c and 4-aminopiperidines 12—14. The syn-
(Chart 1) was the best inhibitor of P. falciparum PFT and thesis of 5-formylimidazoles 8a—c was completed (Chart 3)
also a good inhibitor of parasite growth.
in 5 steps according to a strategy16,17) described for the prepa-
This finding suggests the real potential of designing or ration of regiochemically substituted imidazoleacetic esters:
identifying inhibitors of P. falciparum prenyl transferase as tritylation of N1-imidazole (4), protection of primary alcohol
an approach to malaria therapy. In addition, several works as ester (5), benzylation of N3 and detritylation of N1 (6a—
have reported mammalian PFT inhibitors displaying potent c). The aldehyde function was finally created by chemical ox-
antimalarial activities in vitro and more recently in vivo.6) idation of the hydroxymethyl group (7a—c) resulting from
Small inhibitors of mammalian FTase developed as anti- hydrolysis of the ester (6a—c). Piperidines 9—11 were also
cancer drugs like BMS-2146627—11) are also effective in- prepared (Chart 4) from reductive amination of N-Boc-
hibitors of P. falciparum growth.12,13)
piperidone with adequate a-amino esters. Benzoylation and
deprotection of piperidine (compounds 12—14, 18), possibly
followed by reduction of benzoyl into benzyl for 12, was the
Results and Discussion
Our work has focused on peptidomimetic inhibitors based last step before aminative reduction which yielded final com-
on the CA1A2X tetrapeptide, known to be responsible for pounds 15—17 and 19.
interaction with the mammalian FTase, where the A1A2
Antimalarial activities (Table 1) of piperidines 1, 15a—c,
peptide is replaced by the structurally restricted N-(4- 16c, 17c, and 19 range from 4.4 to 0.85 mM with submicro-
piperidinyl)benzamide scaffold. Initial studies14) led us to molar activity for the most potent compound, 15b. Introduc-
compound 1 which possessed an IC50 (isolated enzyme tion of a trifluoromethyl substituent into the para of the ben-
FTase) as low as 22.8 nM, but did not inhibit the proliferation zyl group increases potency about 3-fold when compared to
of tumor cells in culture (Chart 2). More recently,15) we syn- the non substituted compound 15a. The nature of the benzyl
thesized a series of derivatives of compound 1, of general
structure 2 which was the outcome of three structural modifi-
Chart 1
Chart 2
∗ To whom correspondence should be addressed. e-mail: jean-pierre.henichart@univ-lille2.fr
© 2005 Pharmaceutical Society of Japan

October 2005
1325
substituent is important, as replacement of CF3 by CN lowers
potency about 3-fold (compound 15c). This result suggests
that lipophilicity at this region of the molecule could have an
important role in antimalarial activity. Replacement of the
benzyl-4H-imidazol-4-ylmethyl moiety of compound 15a by
a cysteinyl residue (compound 1) at the piperidinyl nitrogen
increased antimalarial activity, which could also be attributed
to stronger lipophilicity of the imidazolyl fragment compared
to that of the cysteinyl one. Replacement of methioninate in
compound 15c by other amino acid residues such as pheny-
lalaninate (16c) or isoleucinate (17c) led to an increase in an-
timalarial activity, as high as 2-fold in the case of 17c. Aro-
maticity and greater lipophilicity in this region of the mole-
cule seem to be favourable to antimalarial potency. Reduc-
tion of benzamide into benzylamine decreases antimalarial
activity (15c vs. 19). As previously seen for other series of
FTase mammalian inhibitors with potent antimalarial activi-
ties,4,18,19) no correlation can be observed between the two bi-
ological activities. The most potent inhibitor of FTase in the
series (19) displays the lowest antimalarial activity. On the
other hand, the most potent antimalarial derivative, 15b, also
displays good inhibition activity on FTase whereas com-
pound 15a, possessing the lowest FTase inhibition, also dis-
plays low antimalarial activity. Taken together, these results
suggest that in the set of compounds reported here, inhibitory
activity of mammalian FTase is not sufficient to explain the
antimalarial activities observed. Several works16,18,19) suggest
that the active-site recognition properties of malarial FTase
should be different from those of the mammalian enzyme.
This difference could partly account for the lack of correla-
tion noted between the two activities. However, a more recent
work25) suggests that the active-site residues of the farnesyl-
transferase of P. falciparum and rat are nearly identical de-
spite low overall sequence identity due to several additional
external loops of the P. falciparum enzyme. Except for struc-
tural microscopic differences between the two enzymes, there
are other factors which could account for the differences
noted. Pharmacological properties such as lipophilicity or
weak basic character could be involved in cellular uptake in-
side the parasite. Additional mechanisms cannot be excluded
such as complexation of imidazole moiety with ferriproto-
Reagents and conditions: (a) (C6H5)3CCl, NEt3, DMF, rt, 72 h, 71%; (b) (CH3CO)2O,
pyridine, rt, 18 h, 84%; (c) i) (p)R2-C6H4CH2Br, EtOAc, 55 °C, 24 h; ii) MeOH, reflux,
18 h, 69—77% for 6a, b or TFA, rt, 1 h, 57% for 6c; (d) 2 N NaOH, MeOH, rt, 0.5—2 h,
36—99%; (e) MnO2, dioxane, 30—40 °C, 3 h, 70—97%.
Chart 3
Reagents and conditions: (a) Boc2O, DIEA, dioxane/H2O (4 : 1), rt, 24 h, 75%; (b) H-
(Met, Phe or Ile)-OCH3, NEt3, NaBH3CN, MeOH, 50 °C, 48 h, 40—68%; (c) Benzoyl
chloride, NEt3, CH2Cl2, 0 °C, 24 h, 30—55%; (d) HCl/MeOH, rt, 18 h, 95%; (e) i)
BH3.THF, THF, 0 °C, 18 h; ii) 6 N HCl, H2O, reflux, 10 min, 30%; (f) i) 8a—c, NEt3,
MeOH; ii) NaBH3CN, MeOH, 50 °C, 18 h, 42—52%; (g) i) 8c, NEt3, MeOH; ii)
NaBH3CN, MeOH, 50 °C, 18 h, 47%.
Chart 4
Table 1. Biological Evaluation of Compounds 1, 15a—c, 16c, 17c, and 19
IC50a,b) (nM)
IC5022) (mM)
P. falciparum
Compd.
R1
R2
R3
FTasec)
GGTase-1c)
CQd)
1
—
—
—
—
H
CF3
CN
CN
CN
CN
—
—
Nde)
22.8ꢀ2.1
397ꢀ84
43.9ꢀ4.2
4.60ꢀ2.36
22.0ꢀ6.6
32.7ꢀ3.8
2.35ꢀ0.48
Nd
ꢁ100
Nd
0.13ꢀ0.03
1.6ꢀ0.3
2.9ꢀ0.9
0.85ꢀ0.14
3.0ꢀ0.9
7.0ꢀ0.1
1.4ꢀ0.4
4.4ꢀ0.1
15a
15b
15c
16c
17c
19
(CH2)2SCH3
(CH2)2SCH3
(CH2)2SCH3
CH2C6H5
CH(CH3)CH2CH3
(CH2)2SCH3
COC6H5
COC6H5
COC6H5
COC6H5
COC6H5
CH2C6H5
Nd
ꢁ2000
ꢁ2000
ꢁ2000
ꢁ2000
a) Values are means of three determinations. b) See refs. 15, 20, 21. c) Mammalian enzyme. d) Chloroquine. e) Not determined.

1326
Vol. 53, No. 10
Table 2. Antiproliferative Effects of 4-Aminopiperidine FTIs on L-1210
and DLD-1 Cell Lines
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IC50a) (mM)
Compd.
L-1210
DLD-1
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1
ꢁ10 (ꢁ6.2)b)
ꢁ10 (3.5)
ꢁ10 (ꢁ12)
0.020ꢀ0.006 (0.007)
4.95ꢀ0.29 (0.7)
6.62ꢀ0.32 (4.7)
3.95ꢀ0.22 (0.9)
18.1ꢀ2.3 (11)
27.1ꢀ6.6 (9.3)
63.1ꢀ18.6 (74)
9.20ꢀ2.47 (3.0)
7.74ꢀ3.62 (1.6)
26.5ꢀ6.3 (19.0)
12.9ꢀ3.8 (2.9)
15a
15b
15c
16c
17c
19
a) See ref. 27. b) The ratio IC50 compound/IC50 P. falciparum is given in parentheses.
porphyrin IX (FP) reported for potent in vitro imidazole anti-
malarials,26) which could also help to explain the differences
noted.
Cytotoxicities on mammalian cells (Table 2) were evalu-
ated on two selected cell lines (murine leukemia cell line L-
1210 and human colon cell line DLD-1 which expresses K-
Ras isoform in wild type) in order to determine the ratio be-
tween antiproliferative effects of compounds and in vitro ac-
tivity against P. falciparum. The compounds have IC50 values
against L-1210 and DLD-1 in the micromolecular range, ex-
cept for 15c (on L-1210). As seen with the direct comparison
of the IC50 values, there is no significant relationship. For ex-
ample, 15b has better activity for P. falciparum than for L-
1210 and DLD-1 cell lines, which was not observed for 15c.
This shows that it is possible to develop antimalarial drugs
without cytotoxic side effects.
In conclusion, new N-(4-piperidinyl)benzamide derivatives
have been designed as FTase mammalian inhibitors display-
ing significant antimalarial micromolar and submicromolar
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22) In vitro P. falciparum culture and drug assay. P. falciparum strains were
piperidinyl)benzamide core are not exactly similar, suggest-
ing that selective inhibition of parasitic growth over mam-
malian FTase inhibition should be possible. This study en-
ables us to highlight a new antimalarial lead compound, 15b.
Further modulations to this compound are ongoing in order
to optimize its antiparasitic activity. The first goal consists in
combining the presence of diverse hydrophobic and bulky R1
substituents with the replacement of methionine by
isoleucine or other lipophilic groups. This work, together
with other previous studies, shows that despite the absence of
recombinant P. falciparum enzyme, the screening of FTase
mammalian inhibitors on parasite growth can provide a num-
ber of new antimalarial leads.
maintained continuously in culture on human erythrocytes.23) In vitro
antiplasmodial activity was determined using a modification of the
semi-automated micro-dilution technique.24) Plasmodium falciparum
chloroquine-resistant (FcB1R/Colombia) strains were used in sensitiv-
ity testing. Stock solutions of chloroquine diphosphate and test com-
pounds were prepared in sterile distilled water and DMSO, respec-
tively. Drug solutions were serially diluted with culture medium and
introduced to asynchronous parasite cultures (0.5% parasitemia and
1% final hematocrite) on plates comprising 96-well plates for 24 h at
37 °C prior to the addition of 0.5 mCi of [3H]hypoxanthine (1 to
5 Ci/mmol; Amersham, Les Ulis, France) per well, for 24 h. The
growth inhibition of each drug concentration was determined by com-
parison of the radioactivity incorporated into the treated culture with
that in the control culture (without drug) maintained on the same plate.
IC50 was obtained from the drug concentration–response curve and the
results were expressed as meanꢀstandard deviations, determined from
several independent experiments. The DMSO concentration never ex-
ceeded 0.1% and did not inhibit parasite growth.
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