Imidazopyridine-based fatty acid synthase inhibitors that show anti-HCV activity and in vivo target modulation

Article abstract of DOI:10.1021/ml300335r

Potent imidazopyridine-based inhibitors of fatty acid synthase (FASN) are described. The compounds are shown to have antiviral (HCV replicon) activities that track with their biochemical activities. The most potent analogue (compound 19) also inhibits rat FASN and inhibits de novo palmitate synthesis in vitro (cell-based) as well as in vivo.

Full text of DOI:10.1021/ml300335r

Letter
pubs.acs.org/acsmedchemlett
Imidazopyridine-Based Fatty Acid Synthase Inhibitors That Show
Anti-HCV Activity and in Vivo Target Modulation
Johan D. Oslob,* Russell J. Johnson, Haiying Cai, Shirley Q. Feng, Lily Hu, Yuko Kosaka, Julie Lai,
Mohanram Sivaraja, Samnang Tep, Hanbiao Yang, Cristiana A. Zaharia, Marc J. Evanchik,
and Robert S. McDowell
3-V Biosciences, Inc., 1050 Hamilton Court, Menlo Park, California 94025, United States
S
* Supporting Information
ABSTRACT: Potent imidazopyridine-based inhibitors of fatty
acid synthase (FASN) are described. The compounds are
shown to have antiviral (HCV replicon) activities that track
with their biochemical activities. The most potent analogue
(compound 19) also inhibits rat FASN and inhibits de novo
palmitate synthesis in vitro (cell-based) as well as in vivo.
KEYWORDS: FASN, inhibitor, antiviral, HCV, structure−activity relationship, in vivo, target modulation, reversible
the current work was two-fold. First, we wished to identify
uman fatty acid synthase (hFASN) is a key enzyme in the
Hde novo synthesis of fatty acids (FAs) including potent, small-molecule inhibitors of FASN with a reversible
palmitate.1,2 While the putative involvement of FASN in cancer
mode of inhibition. Second, to enable studies in cell-based
has been well documented,3−5 other potential therapeutic uses
assays, good cell permeability was essential. Compounds with
these characteristics were expected to be useful tools for
evaluating the potential of FASN as a small-molecule drug
target for the treatment of HCV.17 Furthermore, to pave the
way for subsequent drug discovery efforts, the compounds
needed to have good activity against both human and rodent
FASN, along with reasonable rodent pharmacokinetic (PK)
profiles, enabling pharmacodynamic (PD) studies.18
In spite of having very limited existing structure−activity
relationships (SAR) or published data, compound 3 (Chart 1)
and its close analogue 4 were among the more promising
starting points at the inception of our explorations.14,19
However, while these molecules were potent biochemical
inhibitors of human FASN (hFASN, Scheme 1), members of
this class were found to undergo cleavage of the secondary
amide moiety in rodent plasma resulting in the formation of a
potentially toxic primary aniline. Furthermore, the weak
activities of 3 and 4 against rat FASN (rFASN) precluded
their use in preclinical in vivo studies. A search for modified
structures, devoid of these issues, was therefore initiated
(Scheme 1).
of FASN inhibitors are beginning to emerge including
cardiovascular disease and diabetes.6−8 Furthermore, it has
recently been shown that FASN is up-regulated during hepatitis
C virus (HCV) infection and that reduction of FASN levels
using siRNA leads to diminished replication of the virus.9
Infection by HCV has become a serious health issue with a
significant portion (∼3%) of the world's population
affected.10,11 While the majority of the newer HCV treatments
are direct-acting antivirals (DAAs) that target viral proteins,10 it
has been proposed that blocking the infectious cycle by
modulating cellular factors such as FASN may result in a higher
barrier to the emergence of resistance.12,13
Although a number of inhibitors of FASN have been
reported, the majority of them have had limited utility due to
modest inhibitory activity or low cell permeability or were
irreversible binders; the latter include the widely used tool
compounds C75 and Cerulenin (Chart 1).5,14−16 The goal of
Chart 1. Inhibitors of FASN
In an attempt to prevent amide cleavage, the parity of the
amide was initially reversed, resulting in an equally active
hFASN compound, 5 (Scheme 1), with good plasma stability
(t1/2 > 10 h). However, the activity against rFASN was poor,
and initial attempts to optimize this new series met with limited
success. Hence, additional scaffold modifications were explored
(Scheme 1). Cyclizing the reversed amide (5) yielded the
Received: October 10, 2012
Accepted: December 2, 2012
Published: December 2, 2012
© 2012 American Chemical Society
113
dx.doi.org/10.1021/ml300335r | ACS Med. Chem. Lett. 2013, 4, 113−117

ACS Medicinal Chemistry Letters
Letter
yielded a compound (10) devoid of activity against hFASN and
rFASN. Overall, as shown previously, the antiviral activities
track with biochemical inhibition of hFASN.17,20
Scheme 1. Compound Progression Leading to
Imidazopyridine Motif
A preliminary survey of the imidazopyridine portion was
undertaken next (Table 2). While replacing the pyrrolidine
moiety in 6 with a hydrogen resulted in modest loss in potency
against hFASN, a more significant decrease in activity was
observed against the rat enzyme (compound 6 vs 11, Table 2).
The corresponding unsubstituted benzimidazole analogue 12
showed further loss of activity.
Replacing the pyrrolidine moiety in 6 with polar substituents
(compounds 13 and 14) rendered more soluble analogues but
with a concomitant loss of rodent activity. In an attempt to
regain some activity against rFASN (see above, compound 9,
Table 1), the R3-methyl was introduced to yield piperazine
derivative 15, which indeed diplayed improved activity against
the rodent enzyme as compared to its R3 = hydrogen
counterpart, compound 14. Therefore, subsequent analogues
were prepared with the R3 = Me substitution pattern. While the
corresponding unsubstituted piperazine analogue 16 did not
show any improvement in activity, the noncharged morpholine
and methyl ether derivatives (17 and 18) displayed good
activities across the assays, including inhibition of rFASN and
antiviral activity. Combining the original pyrrolidine moiety
with the R3 = Me substitution pattern (compound 19) resulted
in potent inhibition of hFASN and HCV replication. The
activity against the rodent enzyme was also improved. While
the corresponding azetidine derivative (20) showed a modest
loss of activity, introduction of a methyl-sulfone in place of the
pyrrolidne led to a more significant decrease, in particular
against rFASN. Consistent with the SAR shown above,
changing the position of the nitrogen in the imidazopyridine
moiety resulted in minor changes in activities (compounds 20
vs 23 and 21 vs 22), whereas the effect of moving the X
substituent is more pronounced (compound 20 vs 24). As
noted above and previously, the cell-based antiviral activities are
found to track well with the human biochemical activities.20
Further characterization using dialysis and washout studies
indicated that compounds from this series are reversible
inhibitors of FASN, both biochemically as well as in cell-
based assays.17,18
corresponding imidazopyridine derivative 6. The new deriva-
tive, devoid of the secondary amide motif, had good plasma
stability (t1/2 > 10 h). It also displayed promising biochemical
activities against both hFASN and rFASN. The scaffold was
therefore selected for further profiling and optimization.
Compound 6 had good antiviral activity in a cell-based HCV
replicon assay with a minor shift from the biochemical assay
and an acceptable selectivity index (SI = EC50/CC50, where
CC50 is cell viability EC50, Table 1). These observations are
consistent with good cell permeability as confirmed in an
Madin−Darby canine kidney (MDCK) permeability assay (Papp
= 8.0 × 10−6 cm/s, efflux ratio = 2.5). Next, a methyl-walk
around the central ring was conducted (Table 1). Replacing the
R1-methyl with a hydrogen resulted in modest loss of human
and rodent activities (compound 6 vs 7), whereas a methyl at
R2 (compound 8) completely abolished biochemical and
antiviral activities. On the other hand, replacing the R3-
hydrogen with a methyl yielded an analogue (9) that had good
activity against hFASN and also improved activity against the
rodent enzyme. Finally, introducing a methyl at R4 again
The analogues described above were prepared using a
general route exemplified by the preparation of compound 19
in Scheme 2. Treatment of 2,4-dimethylbenzoic acid (25) with
Table 1. Preliminary SAR around Compound 6: Methyl-Walk, Antiviral, and FASN Inhibitory Activities
a
b
c
d
e
compd
R1
R2
R3
R4
hFASN (IC50, μM)
rFASN (IC50, μM)
HCV (EC50, μM)
CC50 (IC50, μM)
SI
6
7
Me
H
H
H
H
0.107
0.130
>50
1.72
2.95
0.175
0.550
>10
>20
>3.0
>10
8.6
>114
>5.0
N/A
57
H
H
H
8
H
Me
H
H
H
>50
9
H
Me
H
H
0.060
>50
0.635
>50
0.150
>10
10
H
H
Me
1.9
<0.19
a
b
c
Biochemical inhibition of human FASN. Biochemical inhibition of rat FASN. Inhibition of HCV RNA in the replicon system. Telaprevir was used
d
e
as a positive control. Inhibition of cell viability. SI = EC50/CC50; n ≥ 2, and CV ≤ 50%.
114
dx.doi.org/10.1021/ml300335r | ACS Med. Chem. Lett. 2013, 4, 113−117

ACS Medicinal Chemistry Letters
Letter
Table 2. Imidazopyridine SAR, Antiviral, and FASN Inhibitory Activities
a
b
c
Biochemical inhibition of human FASN. Biochemical inhibition of rat FASN. Inhibition of HCV RNA in the replicon system, typically
determined only for more active compounds. See the Supporting Information for correlations across a larger number of analogues. Telaprevir was
d
e
used as a positive control. Inhibition of cell viability. SI = EC50/CC50; n ≥ 2, and CV ≤ 50%.
a
28 with diamino-pyridine derivative 29 under oxidative
Scheme 2. Preparation of Compound 19
conditions to give target compound 19 in 73% yield.
In the proton NMR spectrum of 19, the signal for the R3-
methyl was split (Δδ ≈ 40 Hz, d6-DMSO), suggesting a
possible hindered rotation around the phenyl-carbonyl bond.
Preliminary estimates of rotational barriers in compound 19
were determined using VT-NMR.22,23 At a moderately elevated
temperature (Tc < 323 K), the R3-methyl peaks coalesce,
suggesting a fast interconversion between rotamers (<0.01 s at
37 °C).22−27 In a similar analysis of the rotation around the
carbonyl-nitrogen bond, the coalescence temperature of
piperidine protons (Tc ≈ 357 K, Δδ ≈ 65 Hz, d6-DMSO)
implies a rotamer half-life of ∼0.1 s at 37 °C. Taken together,
these preliminary observations suggest that 19 does not
represent a mixture of discrete atropisomers.
a
Reagents and conditions: (a) NaIO4, I2, H2SO4, AcOH. (b) n-BuLi,
THF, −78 °C, then DMF. (c) 4-(Piperidine-4-yl)benzonitrile, HBTU,
DIEA, DMF. (d) Pyrrolidine, K2CO3, MeCN, 70 °C (91%). (e) H2,
Pd/C, MeOH (94%). (f) Compound 28, Na2S2O5, DMF, 100 °C.
Having identified potent inhibitors of human and rat FASN,
we next studied the relationship between exposure and target
modulation in vivo using 19 as a sentinel compound. De novo
synthesis of palmitate was selected as a PD marker since
palmitate is directly produced by FASN.1,18 As expected, 19
was a potent inhibitor of palmitate synthesis in both rat and
human cells (Table 3). While compound 19 had moderate iv
clearance and a short half-life in the rat (Table 4), its oral
bioavailability and exposure over time were considered
acceptable for an initial assessment of in vivo target modulation.
iodine and sodium periodate in a mixture of sulfuric acid and
acetic acid furnished the iodinated aryl compound 26 in 82%
yield. Lithium-halogen exchange followed by addition of N,N-
dimethylformamide (DMF) gave the corresponding aldehyde
derivative (27) in good yield (74%). Amide coupling with 4-
(piperidin-4-yl)benzonitrile21 was then achieved using HBTU
in DMF to afford intermediate 28 (84%). Subsequent
imidazole formation was accomplished by reacting aldehyde
115
dx.doi.org/10.1021/ml300335r | ACS Med. Chem. Lett. 2013, 4, 113−117

ACS Medicinal Chemistry Letters
Letter
Notes
Table 3. In Vitro and in Vivo Inhibition of de Novo
Palmitate Synthesis by Compound 19
The authors declare the following competing financial
interest(s): All authors are current or former employees of 3-
V Biosciences, Inc..
human IC50 (HeLa, μM)
rat IC50 (NMU, μM)
in vitro
0.012
0.062
ACKNOWLEDGMENTS
b
c
■
rat no.
[19]liver (μM)
% inhibition
Excellent productivity and chemistry support from Dr.
Tongqian Chen and Xiaojuan Hu and their team at Pharmaron
is gratefully acknowledged.
a
in vivo: 15 mg/kg
1
2
3
4
5
1.7
33
2
0.16
0.13
9
ABBREVIATIONS
2.9
35
70
■
8.1
BuLi, butyl lithium; DIEA, N,N-diisopropylethylamine; DMF,
N,N-dimethylformamide; FASN, fatty acid synthase; HCV,
hepatitis C virus; MDCK, Madin−Darby canine kidney; PD,
pharmacodynamic; PK, pharmacokinetic; SAR, structure−
activity relationships; SI, selectivity index
a
in vivo: 50 mg/kg
1
2
3
4
5
23.6
14.4
12.6
9.8
91
96
52
100
40
3.4
REFERENCES
■
a
b
c
Oral dose. Liver level of 19 at 8 h. % inhibition of de novo
palmitate synthesis at 8 h as compared to vehicle group.
(1) Chirala, S. S.; Wakil, S. J. Structure and function of animal fatty
acid synthase. Lipids 2004, 39 (11), 1045−1053.
(2) Liu, H.; Liu, J. Y.; Wu, X.; Zhang, J. T. Biochemistry, molecular
biology, and pharmacology of fatty acid synthase, an emerging
therapeutic target and diagnosis/prognosis marker. Int. J. Biochem. Mol.
Biol. 2010, 1 (1), 69−89.
Table 4. Rat PK Profile of Compound 19
c
d
e
f
g
route Cl (mL/min/kg)
V (L/kg) t1/2 (h) AUC (h ng/mL) % F
a
iv
36
2.0
0.7
3.2
460
(3) Kuhajda, F. P. Fatty-acid synthase and human cancer: new
perspectives on its role in tumor biology. Nutrition 2000, 16 (3), 202−
208.
b
po
18200
88
a
b
c
d
e
1 mg/kg. 50 mg/kg. Clearance. Volume of distribution. Half-life.
f
g
(4) Menendez, J. A.; Lupu, R. Fatty acid synthase and the lipogenic
phenotype in cancer pathogenesis. Nat. Rev. Cancer 2007, 7 (10),
763−777.
(5) Abramson, H. N. The lipogenesis pathway as a cancer target. J.
Med. Chem. 2011, 54 (16), 5615−5638.
(6) Menendez, J. A.; Vazquez-Martin, A.; Ortega, F. J.; Fernandez-
Real, J. M. Fatty acid synthase: Association with insulin resistance, type
2 diabetes, and cancer. Clin. Chem. 2009, 55 (3), 425−38.
(7) Wakil, S. J.; Abu-Elheiga, L. A. Fatty acid metabolism: target for
metabolic syndrome. J. Lipid Res. 2009, 50 (Suppl.), S138−S143.
(8) Wu, M.; Singh, S. B.; Wang, J.; Chung, C. C.; Salituro, G.;
Karanam, B. V.; Lee, S. H.; Powles, M.; Ellsworth, K. P.; Lassman, M.
E.; Miller, C.; Myers, R. W.; Tota, M. R.; Zhang, B. B.; Li, C.
Antidiabetic and antisteatotic effects of the selective fatty acid synthase
(FAS) inhibitor platensimycin in mouse models of diabetes. Proc. Natl.
Acad. Sci. U.S.A. 2011, 108 (13), 5378−5383.
Area under the curve. Oral bioavailability.
The in vivo PD response was assessed in the liver since
hepatocytes are the site of HCV infection.11 Livers were
harvested 8 h after treating animals with oral doses of
compound 19 delivered as a methylcellulose suspension (15
mg/kg, 50 mg/kg, or vehicle only), and the levels of compound
and newly synthesized palmitate were determined. As shown in
Table 3, the extent of palmitate synthesis inhibition 8 h after
dosing is dose- and exposure-dependent. Strong inhibition of
hepatic palmitate synthesis is observed at the higher dose (50
mg/kg), and a weaker response occurs at 15 mg/kg. The data
in Table 3 indicate that the degree of suppression is correlated
to the level of compound in the liver.
In summary, the present study outlines the discovery of a
potent FASN inhibitor that blocks palmitate synthesis in the
liver. While compound 19 has desirable attributes including
good cell permeability (MDCK Papp = 8.3 × 10−6 cm/s) and
notable antiviral activity, its highly lipophilic nature (clogP =
4.99, cLipE = 2.75)28−30 and relatively high molecular weight
(Mw = 504.6 g/mol, LE = 15.21)31,32 pose challenges to further
development.33 Subsequent manuscripts will present our efforts
to improve these properties while maintaining desired bio-
logical activities using the results and SAR presented in the
current study as a foundation.
(9) Yang, W.; Hood, B. L.; Chadwick, S. L.; Liu, S.; Watkins, S. C.;
Luo, G.; Conrads, T. P.; Wang, T. Fatty acid synthase is up-regulated
during hepatitis C virus infection and regulates hepatitis C virus entry
and production. Hepatology 2008, 48 (5), 1396−1403.
(10) Melnikova, I. Hepatitis CPipeline update. Nat. Rev. Drug
Discovery 2011, 10 (2), 93−94.
(11) Lemon, S. M.; Walker, M. C.; Alter, M. J.; Yi, M. Hepatitis C
Virus. In Fields Virology; Knipe, D. M., Howley, P. M., Griffin, D. E.,
Lamb, R. A., Martin, M. A., Roizman, B., Straus, S. E., Eds.; Lippincott
Williams & Wilkins: Philadelphia, 2006; Vol. 1, pp 1253−1304.
(12) Prussia, A.; Thepchatri, P.; Snyder, J. P.; Plemper, R. K.
Systematic Approaches towards the Development of Host-Directed
Antiviral Therapeutics. Int. J. Mol. Sci. 2011, 12 (6), 4027−52.
(13) Schwegmann, A.; Brombacher, F. Host-directed drug targeting
of factors hijacked by pathogens. Sci. Signal 2008, 1 (29), re8.
(14) Flavin, R.; Peluso, S.; Nguyen, P. L.; Loda, M. Fatty acid
synthase as a potential therapeutic target in cancer. Future Oncol. 2010,
6 (4), 551−562.
ASSOCIATED CONTENT
* Supporting Information
Biological assays and experimental procedures. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
(15) When tested in our biochemical assays, C75 and Cerulenin had
weak activities (>5 μM) against human and rat fatty acid synthase.
(16) For a recent example of a novel inhibitor scaffold, see Abdel-
Magid, A. F. Fatty Acid Synthase Inhibitors as Possible Treatment for
Cancer. ACS Med. Chem. Lett. 2012, 3 (8), 612−613.
AUTHOR INFORMATION
Corresponding Author
*E-mail: johan.oslob@3vbio.com.
■
116
dx.doi.org/10.1021/ml300335r | ACS Med. Chem. Lett. 2013, 4, 113−117

ACS Medicinal Chemistry Letters
Letter
(17) Kemble, G.; McDowell, R.; Moese, S.; Oslob, J. D.; Patick, A. K.;
Yendluri, S.; Parsey, M. Potent Hepatitis C Antiviral Activity by
Inhibiting Fatty Acid Synthase. The 63rd Annual Meeting of the
American Association for the Study of Liver Diseases, Boston, MA, 2012.
(18) Evanchik, M.; Cai, H.; Feng, Q. S.; Hu, L.; Johnson, R.; Kemble,
G.; Kosaka, Y.; Lai, J.; Oslob, J. D.; Sivaraja, M.; Tep, S.; Yang, H.;
Zaharia, C. A.; McDowell, R. TVB-2640, a Novel Anti-HCV Agent,
Safely Causes Sustained Host-Target Inhibition In Vivo. The 63rd
Annual Meeting of the American Association for the Study of Liver
Diseases, Boston, MA, 2012.
(19) Wallenius, L.; Butlin, R.; Kjellstedt, A.; Lofgren, L.; Oakes, N. A
Novel Fatty Acid Synthase Inhibitor Suppresses De Novo Lipogenesis
but induces Hepatic Steatosis, Dermatitis and does not enhance
Insulin Sensitivity in Obese Zucker Rats. American Diabetes Association
68th Scientific Sessions, San Francisco, CA, 2008.
(20) See Graph S1 in the Supporting Information.
(21) Pieper, H.; Linz, G.; Himmelsbach, F.; Austel, V.; Muller, T.;
Weisenberger, J.; Guth, B. Carboxylic acid derivatives, pharmaceutical
compositions containing these compounds and processes for preparing
them. U.S. 5442064, 1995.
(22) Sandstrom, J. Dynamic NMR Spectroscopy; Academic Press:
̈
London, New York, 1982.
(23) Friebolin, H., Basic One- and Two-Dimensional NMR Spectros-
copy; Wiley-VCH: Weinheim, New York, 1998.
(24) Clayden, J.; Moran, W. J.; Edwards, P. J.; LaPlante, S. R. The
challenge of atropisomerism in drug discovery. Angew. Chem., Int. Ed.
Engl. 2009, 48 (35), 6398−6401.
(25) Guile, S. D.; Bantick, J. R.; Cooper, M. E.; Donald, D. K.;
Eyssade, C.; Ingall, A. H.; Lewis, R. J.; Martin, B. P.; Mohammed, R.
T.; Potter, T. J.; Reynolds, R. H.; St-Gallay, S. A.; Wright, A. D.
Optimization of monocarboxylate transporter 1 blockers through
analysis and modulation of atropisomer interconversion properties. J.
Med. Chem. 2007, 50 (2), 254−263.
(26) LaPlante, S. R.; Edwards, P. J.; Fader, L. D.; Jakalian, A.; Hucke,
O. Revealing atropisomer axial chirality in drug discovery.
ChemMedChem 2011, 6 (3), 505−513.
(27) Laplante, S. R.; Fader, L. D.; Fandrick, K. R.; Fandrick, D. R.;
Hucke, O.; Kemper, R.; Miller, S. P.; Edwards, P. J. Assessing
atropisomer axial chirality in drug discovery and development. J. Med.
Chem. 2011, 54 (20), 7005−7022.
(28) Leeson, P. D.; Springthorpe, B. The influence of drug-like
concepts on decision-making in medicinal chemistry. Nat. Rev. Drug
Discovery 2007, 6 (11), 881−90.
(29) Ryckmans, T.; Edwards, M. P.; Horne, V. A.; Correia, A. M.;
Owen, D. R.; Thompson, L. R.; Tran, I.; Tutt, M. F.; Young, T. Rapid
assessment of a novel series of selective CB(2) agonists using parallel
synthesis protocols: A Lipophilic Efficiency (LipE) analysis. Bioorg.
Med. Chem. Lett. 2009, 19 (15), 4406−4409.
(30) Here, cLipE = p(HCV EC50) − clogP.
(31) Perola, E. An analysis of the binding efficiencies of drugs and
their leads in successful drug discovery programs. J. Med. Chem. 2010,
53 (7), 2986−2997.
(32) Here, LE = p(hFAS IC50)/Mw × 1000.
(33) Edwards, M. P.; Price, D. A. Role of Physicochemical Properties
and Ligand Lipophilicity Efficiency in Addressing Drug Safety Risks. In
Annual Reports in Medicinal Chemistry; Macor, J. E., Ed.; Academic
Press: Amsterdam, Boston, 2010; Vol. 45, Chapter 23, pp 380−391.
117
dx.doi.org/10.1021/ml300335r | ACS Med. Chem. Lett. 2013, 4, 113−117