Design and synthesis of a series of bioavailable fatty acid synthase (FASN) KR domain inhibitors for cancer therapy

Article abstract of DOI:10.1016/j.bmcl.2018.05.014

We designed and synthesized a new series of fatty acid synthase (FASN) inhibitors with potential utility for the treatment of cancer. Extensive SAR studies led to highly active FASN inhibitors with good cellular activity and oral bioavailability, exemplified by compound 34. Compound 34 is a potent inhibitor of human FASN (IC₅₀ = 28 nM) that effectively inhibits proliferation of A2780 ovarian cells (IC₅₀ = 13 nM) in lipid-reduced serum (LRS). This cellular activity can be rescued by addition of palmitate, consistent with an on-target effect. Compound 34 is also active in many other cell types, including PC3M (IC₅₀ = 25 nM) and LnCaP-Vancouver prostate cells (IC₅₀ = 66 nM), and is highly bioavailable (F 61%) with good exposure after oral administration. In a pharmacodynamics study in H460 lung xenograft-bearing mice, oral treatment with compound 34 results in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate, fully consistent with the desired target engagement.

Full text of DOI:10.1016/j.bmcl.2018.05.014

Accepted Manuscript
Design and synthesis of a series of bioavailable fatty acid synthase (FASN) KR
domain inhibitors for cancer therapy
Tianbao Lu, Carsten Schubert, Maxwell D. Cummings, Gilles Bignan, Peter J.
Connolly, Karine Smans, Donald Ludovici, Michael H. Parker, Christophe
Meyer, Christian Rocaboy, Richard Alexander, Bruce Grasberger, Sabine De
Breucker, Norbert Esser, Erwin Fraiponts, Ron Gilissen, Boudewijn Janssens,
Danielle Peeters, Luc Van Nuffel, Peter Vermeulen, James Bischoff, Lieven
Meerpoel
PII:
S0960-894X(18)30403-7
https://doi.org/10.1016/j.bmcl.2018.05.014
BMCL 25831
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 April 2018
4 May 2018
7 May 2018
Please cite this article as: Lu, T., Schubert, C., Cummings, M.D., Bignan, G., Connolly, P.J., Smans, K., Ludovici,
D., Parker, M.H., Meyer, C., Rocaboy, C., Alexander, R., Grasberger, B., De Breucker, S., Esser, N., Fraiponts, E.,
Gilissen, R., Janssens, B., Peeters, D., Nuffel, L.V., Vermeulen, P., Bischoff, J., Meerpoel, L., Design and synthesis
of a series of bioavailable fatty acid synthase (FASN) KR domain inhibitors for cancer therapy, Bioorganic &
Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.05.014
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Bioorganic & Medicinal Chemistry Letters
Design and synthesis of a series of bioavailable fatty acid synthase (FASN) KR
domain inhibitors for cancer therapy
Tianbao Lu  a, Carsten Schuberta, Maxwell D. Cummingsa, Gilles Bignana, Peter J. Connollya, Karine
Smansb, Donald Ludovicia, Michael H. Parkera, Christophe Meyerc, Christian Rocaboyd, Richard
Alexandera, Bruce Grasbergera, Sabine De Breuckerb, Norbert Esserb, Erwin Fraipontsb, Ron Gilissenb,
Boudewijn Janssensb, Danielle Peetersb, Luc Van Nuffelb, Peter Vermeulenb, James Bischoffa, and Lieven
Meerpoelb
aJanssen Research & Development, LLC, 1400 McKean Roads, Spring House, PA 19477, United States of America
bJanssen Research & Development, Turnhoutseweg 30, B-2340, Beerse, Belgium
cJanssen Research & Development, CS10615, Campus de Maigremont 27106 Val de Reuil, France
dVillapharma Research, Parque Tecnologico de Fuente Alamo, Carretera El Estrecho-Lobosillo, 30320 Murcia, Spain
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
We designed and synthesized a new series of fatty acid synthase (FASN) inhibitors with
potential utility for the treatment of cancer. Extensive SAR studies led to highly active FASN
inhibitors with good cellular activity and oral bioavailability, exemplified by compound 34.
Compound 34 is a potent inhibitor of human FASN (IC50 = 28 nM) that effectively inhibits
proliferation of A2780 ovarian cells (IC50 = 13 nM) in lipid-reduced serum (LRS). This cellular
activity can be rescued by addition of palmitate, consistent with an on-target effect. Compound
34 is also active in many other cell types, including PC3M (IC50 = 25 nM) and LnCaP-
Vancouver prostate cells (IC50 = 66 nM), and is highly bioavailable (F 61%) with good exposure
after oral administration. In a pharmacodynamics study in H460 lung xenograft-bearing mice,
oral treatment with compound 34 results in elevated tumor levels of malonyl-CoA and decreased
tumor levels of palmitate, fully consistent with the desired target engagement.
Keywords:
Fatty acid synthase; Enzyme inhibitor;
x-Ray crystal structure; cell proliferation;
SAR; Cancer
2009 Elsevier Ltd. All rights reserved.
———
 Corresponding author. Tel.: +1-215-628-7090; fax: +1-215-540-4612; e-mail: tlu3@its.jnj.com

Human Fatty Acid Synthase (FASN) is a complex homodimeric enzyme (552-kDa) that carries out de novo fatty acid (palmitate)
synthesis from acetate and malonate in mammalian cells in the presence of NADPH.1,2,3 Studies have recently associated FASN
with a variety of human diseases and adverse health conditions including obesity, inflammation, cardiovascular disease, and
especially cancer. 4,5,6,7,8 FASN is up-regulated in cancer cells, providing fatty acid building blocks for rapid cell growth and cell
division. Increased FASN expression is correlated with disease progression and poor prognosis in many malignancies including
prostate, breast, ovary, colon, and lung cancers.9,10,11,12 FASN has been demonstrated to play an important role in carcinogenesis by
protecting cells from apoptosis, and inhibition with the non-specific FASN inhibitors orlistat or cerulenin prevents tumor growth in
various mouse xenograft models.13,14 Thus, FASN is an attractive emerging target for cancer drug discovery.
Recent publications disclose several FASN inhibitor chemotypes that share a common pharmacophore involving an aromatic group
and an acylated cyclic amine attached to a central scaffold (e.g. 1H-imidazo[4,5-c]pyridine).15,16,17 We postulated that a 5,5-
disubstituted-spirocyclic imidazolinone core would be an acceptable and drug-like scaffold, inspired by the precedent of irbesartan,
an approved antihypertensive drug in which a spirocyclopentyl-imidazolinone core replaces the substituted imidazole ring of
losartan, an older approved agent from the same drug class (Figure 1). Working from this hypothesis we have discovered a new
class of spirocyclic imidazolinone based FASN inhibitors.
General synthesis of the new series of imidazolinone FASN inhibitors is outlined in Scheme 1 starting from, as an example, 1-
amino-cyclopropane-1 carboxamide (I). First, the aminoamide was coupled with a substituted bromophenylcarboxylic acid (II)
under standard peptide coupling conditions. The resulting product III was then cyclized under basic conditions (NaOH in methanol)
to give the imidazolinone (IV), which in turn was allowed to react with an appropriate alkylating reagent such as (R)-tert-butyl 3-
(bromomethyl)pyrrolidine-1-carboxylate (V) to produce product VI. Boc de-protection, acylation, and final Suzuki coupling gave
the desired products VIII. Alternatively, the Suzuki coupling was performed first, followed by Boc de-protection and acylation, to
prepare the final products VIII.
Figure 1. Design principle for new FASN inhibitors

Scheme 1. General synthetic procedure
Using the spiro-cyclopropane-imidazolinone core substituted with (4-(1-methyl-1H-indazol-5-yl)phenyl) at the 2-position as a
starting point, we began structure-activity relationship (SAR) studies of the linker between the imidazolinone and the terminal acyl
group (Table 1, see supplementary data for assay conditions). The most potent linker is (R)-pyrrolidine (1), with an IC50 of 30 nM.
(S)-pyrrolidine (2) is 8-fold less potent, whereas azetidine (3) is only 2-fold less potent. 4-Piperidine (4) and (S)-3-piperidine (5) are
45-fold and 280-fold less potent than 1, respectively, and (R)-3-piperidine (6) is completely inactive at >10 M.
Table 1. SAR (full length FASN enzyme) of the linker between the imidazolinone and acyl group
Compound #
Linker
FASN IC50 (nM)
1
2
3
4
30
250
69
1,380

5
6
8,510
>10,000
We also explored the SAR at the 5-position of the imidazolinone (Table 2). Compared to the benchmark spiro-cyclopropyl analogue
1, spiro-cyclopentyl (7), 5,5-dimethyl (8), and 5-phenyl-5-methyl (10) are less potent and 5-methyl-5-methoxymethyl (9) is much
less potent. Incorporation of either an oxygen or nitrogen atom into the spirocycle (11-15) reduces potency by at least 6-fold. The
weaker potency of N-i-propylpiperidine (12) compared to cyclopropanecarbonylpiperidine (13) suggests that the 5-substituent
interacts with a region of FASN that does not tolerate positive charge. This observation will be discussed further below in the
context of an x-ray crystal structure of compound 1.
Table 2. SAR (full length FASN enzyme) at the 5- position of the imidazolinone
Compound #
1
X-Y
FASN IC50 (nM)
30
7
8
83
140
9
490
160
10
11
12
300
1,100
13
200
14
15
550
460
Table 3. SAR (full length FASN enzyme) for central aryls

Compound #
1
Linker
FASN IC50 (nM)
30
16
17
22
51
18
19
130
300
20
21
22
790
460
8,710
23
24
33,100
8,910
Investigation of the 2-substituent on the core was conducted in two parts. First, SAR for the central aryl ring between the
imidazolinone and terminal aryl was explored (Table 3). Comparing again to benchmark compound 1 having a central phenyl ring
(IC50 = 30 nM), fluorophenyl (16) and methylphenyl (17) with substitution ortho to the imidazolinone have similar potencies (IC50
= 22 and 51 nM, respectively), while fluorophenyl (18) and methylphenyl (19) with substitution ortho to the terminal aryl group
show decreased potency (IC50 = 130 and 300 nM respectively). Pyridinyl replacements (20 and 21) of phenyl are at least 15-fold
less potent, and pyrimidinyl (22 and 23) or thiophenyl (24) analogues are dramatically less potent.
Table 4. SAR (full length FASN enzyme) for terminal aryl groups
Compound #
1
R
FASN IC50 (nM)
30
25
26
32
33

27
28
29
30
31
32
42
20
20
25
120
160
33
34
23
28
35
36
50
41
37
38
360
590
The right-hand terminus of the 5-subustutent (Table 4) tolerates a variety of aryl and heteroaryl rings. Bicyclic heteroaryls including
indole (36), benzothioazole (27), indazole (25, 26), naphthalene (30), quinolone (29) and isoquinoline (28) all show good activity,
with potency comparable to or slightly better than 1. Biaryls such as 1-methyl-1H-pyrazol-4-ylphenyl (34) and 4-pyridinylphenyl
(33) are also potent (IC50 = 28 and 23 nM respectively). 6-Isoquinoline (28) and 7-quinoline (29) are the most potent (IC50 = 20 nM)
and 6-quinoline (32) is the least potent (IC50 = 160 nM) among all quinolines and isoquinolines (31). 3-Indazole (37) is
approximately 10-fold less potent than 4-or 5-indazole (26, 25). 3-Chlorophenyl (35) and 5-benzo[d]thiazole (27) both maintain
good potency (50 and 42 nM), but 2-benzo[d]imidazole (38) is much less potent (590 nM).
Table 5. SAR (full length FASN enzyme) for acyl groups
Compound #
R
FASN IC50 (nM)
39
40
41
42
46
36
28
590

43
44
170
79
45
46
300
450
In the last part of our SAR effort, the amide substituent on the pyrrolidine ring was investigated. Cyclopropyl (39) with hydrogen or
small substitutions such OH (40) or methyl (41) are allowed (IC50 = 46, 36, and 28 nM, respectively) but CN (42) reduces potency
more than 10-fold compared to compound 39. Replacement of cyclopropyl with trifluoroethyl (43) also reduces potency. (S)-
Tetrahydrofuran (44) is 4-fold more potent than (R)-tetrahydrofuran (45), and 1-methylpyrazol-3-yl (46) is 10-fold less potent than
cyclopropyl (39).
In order to further understand the SAR and mechanism of inhibition for this novel chemotype we determined the X-ray crystal
structure of a partial FASN construct comprising the functionally relevant ketoreductase (KR) and the enzymatically inactive
pseudo-methyltransferase and pseudo-KR (ψME-ψKR) domains, complexed with compound 1 and NADPH (Figure 2, see
Supplementary Data for details).
The bound inhibitor adopts an overall “V” shape, with the imidazolinone serving as a central core or scaffold from which the
substituted acylpyrrolidine projects toward the catalytic center and the bound NADPH co-factor. The biaryl moiety projects into a
large hydrophobic pocket formed at the interface of the KR and ψME-ψKR domains, whereas the cyclopropyl substituent of the
imidazolinone core projects away from the binding site and into solvent. Interestingly, analysis of the apo structure of pig FASN
inhibitor complexes18,19,20 had previously led us to select for small-molecules that could position a carbonyl moiety similarly to what
is observed for bound compound 1.
Seven co-planar heavy atoms of the terminal cyclopropylamide group stack in an offset face-to-face manner against the
nicotinamide moiety of NADPH, with the amide carbonyl forming intermolecular hydrogen bonds with the sidechains of the
catalytically important residues Ser2021 and Tyr2034. These residues in turn contribute to a more extensive hydrogen bonding
network involving additional residues (Figure 2 and Figure S2). The positioning of the amide carbonyl and the size and nature of the
terminal substituent are critical for the inhibitory potency of this inhibitor series, with clear preferences seen for ring size, ring
stereochemistry, and substituent size that are fully consistent with the structure of the compound 1 complex (Tables 1, Figure 2).
The terminal cyclopropyl group fits snugly in this region of the FASN active site, consistent with our observation of limited
tolerance for alternatives at this position. The complicated hydrogen bonding contacts of the amide carbonyl make the positioning
of this key carbonyl moiety exquisitely sensitive to changes in both the amide ring and the terminal substituent.
The central imidazolinone group makes extensive contact with the enzyme, sandwiched between the sidechains of Asn2028 and
Val2080 and hydrogen bonding with the hydroxyl of Ser2081. The amide carbonyl and phenyl substituents adjacent to the
exocyclic methylene favor the observed ca. 90° N-to-C torsion between the imidazolinone and the methylene, and the methylene is
an axial substituent on the pyrrolidine ring (Figure 2). These two structural features result in a specific conformational relationship
between the imidazolinone and pyrrolidine rings, with the planes of the two rings projecting in parallel, offset by one C-C bond,
allowing for the precise positioning of the very specific amide carbonyl (see discussion above and Figure 2). Both the structure and
the sensitivity to cycloamide ring size suggest that this conformational feature is a key contributor to inhibitor potency. The
selectivity seen at the spirocyclopropyl position of the imidazolinone is somewhat surprising given the degree of exposure and lack
of binding contacts for this part of the inhibitor (Table 2, Figure 2). However, since the enzymatic data is acquired in the context of
full-length FASN rather than the truncated version used here for structural studies, we cannot exclude the possibility that the
cyclopropyl ring interacts with other components of the full-length enzyme in a manner that is not captured in this artificial system.
The central aryl ring of the imidazolinone 2-substituent occupies a tight pocket or tunnel that imposes strict conformational and
chemical constraints (Figure 2). The imidazolinone-phenyl torsion is ca. 80° with a bulky substituent at one of the adjacent
imidazolinone positions, whereas the phenyl-indazole torsion is 35° with all four positions ortho to this latter torsion
unsubstituted.21 Additionally, each of the four unsubstituted positions of the central phenyl ring make reasonably close contact(s)
with the binding site, with each having at least one protein atom within 4.1 Å; one of the positions ortho to the imidazolinone-
phenyl torsion is marginally less crowded than the others. These structural features of the complex are reflected in the observed
SAR data (Table 3), satisfying both inhibitor conformation and binding site complementarity considerations. Small substituents are
acceptable on the imidazolinone side of the phenyl ring but detrimental on the indazole side. Aromatic nitrogens appear to be
unacceptable at all four phenyl positions in question (Table 3), fully consistent with poor binding site complementarity and an
increased energetic cost of desolvation for such groups.

The SAR for the terminal aryl group is relatively flat (Table 4), as might be expected for this part of the inhibitor which projects
into a large inter-domain cavity. The tolerance for bulk at positions adjacent to the torsion (to the central phenyl/aryl ring) is
somewhat surprising given the observed torsion angle and the snugness of the binding site at the proximal end of this group in the
bound structure. In related complex structures rings in this region are typically oriented similarly to that of the present complex,
although some adjustment of the inter-domain interface at this region of the binding site has been observed to accommodate
deviation from this plane in some cases (unpublished results).
Figure 2. Binding mode of compound 1 in the active site of the FASN-KR domain (PDB code: 5C37). Upper Schematic summary
of inhibitor binding mode, shown graphically in lower panel. Lower The acylpyrrolidine amide carbonyl group of the inhibitor
(lime carbons) hydrogen-bonds with the catalytic residues S2021 and Y2034 (white ball & stick) and forms a stacking interaction
with NADPH (teal carbons). The terminal aryl group projects into a large hydrophobic cavity at the interface of the KR (wheat) and
ψME-ψKR (grey) domains. Contact distances are indicated in Å. Residues 2063-2083 of the KR domain are omitted from the
surface representation and depicted in loop representation, for clarity.
Table 6. Inhibition of cell proliferation in multiple cell lines measured by ATP-GLO after 4 days incubation with compound 34

LRS
EC50 (nM)
LRS+25 µM PA
EC50 (nM)
Cell Line
A2780
13
>10,000
PC3M
LNCaP
K562
25
66
NT
NT
110
55
9,330
6,760
6,310
OCI-LY1
HL-60
120
U2932
190
3,020
MM1R
U266
170
10,000
3,720
59
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
NT
H929
MV4-11
H460
36
BT20
10,000
7,940
20
HT29
A549
>10,000
9,330
LL
590
MDA-MB-468
LP1
26
>10,000
>10,000
10,000
Based on its FASN potency and attractive properties, compound 34 was chosen for additional in vitro and in vivo profiling.
Compound 34 shows comparable enzyme inhibition on full length FASN and the FASN KR domain (IC50 = 28 and 54 nM,
respectively), further confirming inhibition is through binding to the KR domain. Compound 34 also strongly inhibits cell
proliferation in LRS in several cancer cell lines including A2780 (ovarian), PC3M (prostate), LNCaP (prostate), OCI-LY1
(lymphoma), MV4-11 (leukemia/lymphoma/myeloma), H460 (lung), A549 (lung), and MDA-MB-468 (breast) (Table 6). The
inhibition can be rescued by addition of 25 µM palmitic acid (PA), confirming FASN target engagement for the series.
Compound 34 possesses favorable in vitro ADME proprieties. IC50 values against most major human P450 isozymes (CPY1A2,
CPY2C8, CPY2C19, CPY2D6, CPY3A4) were determined to be > 10 µM, while CPY2C9 was inhibited at 7.9 µM. The compound
is stable in mouse microsomes, highly permeable (CaCo-2 Papp = 7.1 x 106 cm/s), and gives no indication of being a substrate of
efflux transporters. Compound 34 is stable in mouse plasma; the human and mouse plasma protein binding is 94.1% and 96.6%,
respectively.
In vivo pharmacokinetic properties were also evaluated following intravenous and oral delivery in mice. Compound 34 showed
promising pharmacokinetic behavior following a 10 mg/kg oral dose, with Tmax = 2.0 h, T1/2 = 2.5 h, Cmax = 2,030 ng/mL, AUC =
11,100 ng.h/mL, and F = 60.6 %. In a pharmacodynamics study in NCI-H460 lung xenograft-bearing mice, oral treatment with
compound 34 at 3, 10, and 30 mg/kg dose-dependently increased malonyl-CoA (MaCoA) levels at 2 hours post dose (see Figure 3),
while MaCoA levels reached a plateau after 4 to 12 hours. The increase in MaCoA was concomitant with a decrease in palmitate
levels (data not shown).

Figure 3. In vivo PD modulation for compound 34 in a NCI-H460 xenograft model. Mice bearing tumors of 300-400 mm3 were
treated for 2 days BID at the indicated doses. Tumors were collected at 2, 4, and 12 hours post last dose and MaCoA levels
determined by Mass Spectrometry.
In conclusion, we designed a new series of potent imidazolinone FASN KR domain inhibitors using a structure-based approach
guided by x-ray crystallography. Thorough exploration of the SAR for this inhibitor chemotype led to the discovery of compound
34, a potent FASN inhibitor with favorable ADME properties and significant oral exposure in mice. These features make
compound 34 a useful in vivo tool compound that will facilitate a better understanding of FASN as a therapeutic target for cancer,
obesity, and other human diseases.
Acknowledgments
Use of the IMCA-CAT beamline 17-ID (or 17-BM) at the Advanced Photon Source is supported by the companies of the Industrial
Macromolecular Crystallography Association through a contract with Hauptman-Woodward Medical Research Institute.
ABBREVIATIONS
FASN, Fatty acid synthase; LRS, lipid-reduced serum; NADPH, nicotinamide adenine dinucleotide phosphate. KR, ketoreductase
domain; ER, enoylreductase domain, ψME, pseudo-methyltransferase domain; ψKR, pseudo-ketoreductase domain.
A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/
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21. In the two monomers present in the crystallographic asymmetric unit the observed values of the imidazolinone-phenyl torsion are 74.6°
and 84.2°, and those of the phenyl-indazole torsion are 34.7° and 35.6°.

This is optional but I can’t complete the online submission without it
Highlights
 A new series of imidazolinone fatty acid synthase inhibitors.
 Crystal structure shows binding to the fatty acid synthase ketoreductase domain.
 Strong antiproliferative activity in several cancer cell lines.
 Favorable ADME properties and significant oral exposure in mice.

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Design and synthesis of a series of bioavailable fatty acid synthase (FASN)
KR domain inhibitors for cancer therapy
Tianbao Lu*, a, Carsten Schuberta, Maxwell D. Cummingsa, Gilles Bignana, Peter J. Connollya, Karine
Smansb, Donald Ludovicia, Michael H. Parkera, Christophe Meyerc, Christian Rocaboyd, Richard
Alexandera, Bruce Grasbergera, Sabine De Breuckerb, Norbert Esserb, Erwin Fraipontsb, Ron Gilissenb,
Boudewijn Janssensb, Danielle Peetersb, Luc Van Nuffelb, Peter Vermeulenb, James Bischoffa, Lieven
Meerpoelb