Discovery of Novel Selective Acetyl-CoA Carboxylase (ACC) 1 Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.7b01547

We initiated our structure-activity relationship (SAR) studies for selective ACC1 inhibitors from 1a as a lead compound. SAR studies of bicyclic scaffolds revealed many potent and selective ACC1 inhibitors represented by 1f; however most of them had physicochemical issues, particularly low aqueous solubility and potent CYP inhibition. To address these two issues and improve the druglikeness of this chemical series, we converted the bicyclic scaffold into a monocyclic framework. Ultimately, this lead us to discover a novel monocyclic derivative 1q as a selective ACC1 inhibitor, which showed highly potent and selective ACC1 inhibition as well as acceptable solubility and CYP inhibition profiles. Since compound 1q displayed favorable bioavailability in mouse cassette dosing testing, we conducted in vivo PD studies of this compound. Oral administration of 1q significantly reduced the concentration of malonyl-CoA in HCT-116 xenograft tumors at doses of more than 30 mg/kg. Accordingly, our novel series of selective ACC1 inhibitors represents a set of useful orally available research tools, as well as potential therapeutic agents for cancer and fatty acid related diseases.

Full text of DOI:10.1021/acs.jmedchem.7b01547

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Article
Discovery of Novel Selective Acetyl-CoA Carboxylase (ACC) 1 Inhibitors
Ryo Mizojiri, Moriteru Asano, Daisuke Tomita, Hiroshi Banno, Noriyuki Nii, Masako Sasaki, Hiroyuki
Sumi, Yoshihiko Satoh, Yukiko Yamamoto, Takeo Moriya, Yoshinori Satomi, and Hironobu Maezaki
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01547 • Publication Date (Web): 12 Dec 2017
Downloaded from http://pubs.acs.org on December 12, 2017
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Discovery of Novel Selective Acetyl-CoA
Carboxylase (ACC) 1 Inhibitors
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Ryo Mizojiri,* Moriteru Asano, Daisuke Tomita, Hiroshi Banno, Noriyuki Nii, Masako Sasaki,
Hiroyuki Sumi, Yoshihiko Satoh, Yukiko Yamamoto, Takeo Moriya, Yoshinori Satomi, and
Hironobu Maezaki
Research, Takeda Pharmaceutical Company Limited, 26ꢀ1,
MuraokaꢀHigashi 2ꢀchome, Fujisawa, Kanagawa 251ꢀ8555, Japan
ABSTRACT
We initiated our structureꢀactivity relationship (SAR) studies for selective ACC1 inhibitors
from 1a as a lead compound. SAR studies of bicyclic scaffolds revealed many potent and
selective ACC1 inhibitors represented by 1f, however most of them had physicochemical issues,
particularly low aqueous solubility and potent CYP inhibition. To address these two issues
and improve the drugꢀlikeness of this chemical series, we converted the bicyclic scaffold into a
monocyclic framework. Ultimately, this lead us to discover a novel monocyclic derivative 1q
as a selective ACC1 inhibitor, which showed highly potent and selective ACC1 inhibition as
well as acceptable solubility and CYP inhibition profiles. Since compound 1q displayed
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favorable bioavailability in mouse cassette dosing testing, we conducted in vivo PD studies of
this compound. Oral administration of 1q significantly reduced the concentration of
malonylꢀCoA in HCTꢀ116 xenograft tumors at doses of more than 30 mg/kg. Accordingly, our
novel series of selective ACC1 inhibitors represents a set of useful orallyꢀavailable research
tools, as well as potential therapeutic agents for cancer and fatty acid related diseases.
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KEYWORDS: AcetylꢀCoA carboxylase (ACC) 1 inhibitor, C acetate uptake inhibition,
solubility, CYP inhibition, MalonylꢀCoA
INTRODUCTION
Since Otto Warburg’s initial observations in the 1920s, it has been known that there are
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metabolic differences between rapidlyꢀproliferating cancer cells and normal cells. Recent
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,3
research has demonstrated that these metabolic differences are actual drivers of tumor growth.
By modulating their metabolic processes, cancer cells are able to divert sugars, fats, and other
energy sources away from energy production to satisfy the ever growing demands of
uncontrolled proliferation. Drugs that intervene with the metabolism of cancer cells,
redirecting them to the normal metabolic course, could present a completely new approach for
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ꢀ7
treating cancer, either as monotherapy or in combination with other therapeutic approaches.
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AcetylꢀCoA carboxylase (ACC) carboxylates acetylꢀCoA to produce malonylꢀCoA,
representing the rate limiting step in fatty acid synthesis. MalonylꢀCoA is an intermediate of
de novo fatty acid synthesis, which acts as a substrate of fatty acid synthase (FAS) for acyl
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chain elongation.
Furthermore, malonylꢀCoA functions as an inhibitor of carnitine
palmitoyltransferase 1 (CPTꢀ1), regulating fatty acid betaꢀoxidation. Therefore, functional
abnormalities of ACC are associated with blocking fatty acid synthesis, disturbing energy
metabolism, and resulting in cell damage.
Two ACC isoforms have been identified in
mammals, ACC1 and ACC2. Recently, it has been reported that ACC1 is overexpressed in
human cancer cells, such as colon, prostate, kidney, spleen, uterine cervix, uterus body, ovary,
and small intestine cancer cells and is likely involved in tumor development and progression.
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Thus, ACC1 is a potential target for developing novel agents as cancer therapeutics.
However, selective ACC1 inhibitors have not been reported even though there are many reports
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for dual ACC1/2 inhibitors
and selective ACC2 inhibitors.
For evaluation of its
potential in cancer therapy, a selective ACC1 inhibitor is required. Therefore, we initiated an
investigation into the generation of potent and selective ACC1 inhibitors. As a starting point
for these studies, we selected a unique 2ꢀazetidylꢀ1,3ꢀbenzoxazole derivative 1a, which had
been found to show moderate ACC1 inhibitory potency with an IC value around 10 ꢀM (48%
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inhibition at 10 ꢀM) from our compound library, as a lead compound (Figure 1). We initiated
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our research program around this compound in the belief that its novel structure could be used
to selectively target ACC1, which has not been possible for known dual ACC1/2 and selective
ACC2 inhibitors.
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Figure 1. Lead compound for selective ACC1 inhibitors
CHEMISTRY
The preparations of 2ꢀazetidylꢀ1,3ꢀbenzoxazole derivatives 1a–d are shown in Scheme 1.
Condensation of 2ꢀchloroꢀ1,3ꢀbenzoxazole 2 with 3ꢀhydroxyazetidine hydrochloride (3)
followed by methanesulfonylation afforded 2ꢀazetidinylꢀ1,3ꢀbenzoxazole derivative 5.
Coupling of mꢀsubstituted phenol 6 with 5 yielded 2ꢀ(3ꢀphenoxyazetidinyl)ꢀ1,3ꢀbenzoxazole
analogue 7. After conversion of the ester group of 7 into a 2ꢀhydroxyethyl group by reduction
of the ester group, Ruꢀcatalyzed oxidation to form the aldehyde, followed by substitution with a
methyl group, enabled synthesis of compounds 1a and 1b by the Ritter reaction using sulfuric
acid in CH CN. Ruꢀcatalyzed oxidation of 8, substitution with a methyl group, and azidation
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with DPPA were carried out to produce the azide 9. The acetamide analogue 11 was prepared
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by Staudinger reaction of 9 and acetylation of the resulting amino group. After removing the
benzyl group of 11 by hydrogenation, compounds 1c and 1d were synthesized by alkylation
with the appropriate alkylbromide.
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Scheme 1
Reagents and conditions: a) 3, DIPEA, rt, overnight, 95%; b) MsCl, Et N, THF, rt, overnight,
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8%; c) 6, Cs CO , DMF, 100 °C, 4 h – overnight, 72–78%; d) i) LiAlH , THF, 0 °C, 30 min;
2 3 4
ii) TPAP, NMO, 4Å molecular sieves, CH CN, rt, 4 h; iii) MeMgBr, THF, 0 °C, 30 min; iv)
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H SO , CH CN, rt, 1 h, 26%; e) LiAlH , THF, 0 °C, 30 min, 25–29%; f) i) TPAP, NMO, 4Å
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molecular sieves, CH CN, rt, 4 h; ii) MeMgBr, THF, 0 °C, 30 min; iii) H SO , CH CN, rt, 1 h,
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1%; g) i) TPAP, NMO, 4Å molecular sieves, CH CN, rt, 4 h; ii) MeMgBr, THF, 0 °C, 30 min;
3
iii) DPPA, DBU, toluene, rt, 2 h, 68%; h) Ph P, water, THF, 60 °C, 2 h, 89%; i) Ac O, pyridine,
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rt, 30 min, 84%; j) H (1 atm), Pd(OH) , MeOH, THF, rt, overnight, 95 %; k) RBr, K CO ,
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DMF, 80 °C, 2 h, 33–70%.
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The preparation of 1e is shown in Scheme 2. 4ꢀPhenoxypiperidine 14 was synthesized from
ꢀhydroxypiperidine 13 by Mitsunobu reaction with phenol 6c. After removal of the benzyl
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group of 14, alkylation with a cyclopropylmethyl group, and removal of the Boc group, the
resulting 17 was coupled with 2ꢀchloroꢀ1,3ꢀbenzoxazole to afford
ꢀpiperidinylꢀ1,3ꢀbenzoxazole derivative 18. Stepwise conversion of the ester group of 18
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into an azido group of 20 was carried out by reduction of the ester using LiAlH , Ruꢀcatalyzed
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oxidation, substitution with MeMgBr, and azidation with DPPA. Finally, compound 1e was
synthesized from azide 20 by hydrogenation and acetylation of the resulting amino group.
Scheme 2
Reagents and conditions: a) 6c, DIAD, Ph P, toluene, rt, overnight, 78%; b) H (1 atm),
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Pd/C, MeOH, rt, 2 h, 45%; c) cPrCH Br, K CO , DMF, 60 °C, overnight, 91%; d) 4 M HCl–
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AcOEt, rt, 4 h, 75%; e) 2, DIPEA, DMF, rt, overnight, 36%; f) i) LiAlH , THF, 0 °C, 30 min; ii)
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TPAP, NMO, 4Å molecular sieves, CH CN, rt, 4 h, 74%; g) i) MeMgBr, THF, 0 °C, 1 h; ii)
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DPPA, DBU, toluene, rt, 4 h, 61%; h) i) H (1 atm), Pd/C, THF, rt, 2 h; ii), Ac O, pyridine, rt, 2
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h, 40%.
The preparation of 1f–h is illustrated in Scheme 3. Coupling reaction of ethyl
ꢀfluorobenzoate (21f) or 5ꢀbromoꢀ2ꢀcyanopyridine (21g) with phenol 22 yielded ethyl
ꢀphenoxybenzoate derivative 23f or 5ꢀphenoxyꢀ2ꢀcyanopyridine derivative 23g, respectively.
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Hydrolysis of the ester or cyano group of 23 under basic conditions gave the corresponding acid
4. Condensation reaction of 24 with aminophenol 25 gave the corresponding amide 26.
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Cyclization of 26 under Mitsunobu reaction conditions afforded 2ꢀphenylꢀ1,3ꢀbenzoxazole
derivative 30f and 2ꢀpyridylꢀ1,3ꢀbenzoxazole derivative 30g. Condensation reaction of 25
with 6ꢀchloronicotinoyl chloride (27) gave the corresponding amide 28.
Mitsunobu
cyclization of 28 gave 2ꢀpyridylꢀ1,3ꢀbenzoxazole analogue 29. Coupling reaction of 29 with
phenol 22 gave the coupling product 30h. Finally, reductive amination of 30f–h followed by
acetylation gave 1f–h.
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Scheme 3
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Reagents and conditions: a) 22, Cs CO , DMF, 100 °C, overnight, 75–98%; b) 2 M NaOH,
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MeOH, THF, rt, 2 h, 97%; c) 2 M NaOH, EtOH, 80 °C, overnight, 80%; d) i) (COCl) , DMF,
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9, 22, Cs CO , DMF, 100 °C, 2 h, 86%; i) NH OAc, NaBH CN, MeOH, 60 °C, overnight,
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7%; j) Ac O, pyridine, rt, 30 min, 51%; k) i) NH OAc, NaBH CN, MeOH, 60 °C, overnight;
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ii) Ac O, pyridine, rt, 30 min, 26–53%.
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The preparation of 1i is outlined in Scheme 4. Nicotinic acid derivative 34 was synthesized
by the coupling of methyl 6ꢀchloronicotinate (32) with phenol 6c followed by hydrolysis.
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Introduction of a thiol group into 4ꢀfluoroꢀ3ꢀnitroacetophenone (35) under basic conditions
gave thiol derivative 36. After reduction of the nitro group into an amino group, condensation
reaction with the acid chloride from 33 afforded benzothiazole precursor 37. Treatment of 37
with NaOMe followed by TFA yielded benzothiazole derivative 38. The synthesis of
acetamide derivative 39 was carried out by reductive amination of the ketone of 38 using
ammonium acetate followed by acetylation. Finally, removal of the benzyl group with TFA in
thioanisole and alkylation with (bromomethyl)cyclopropane afforded 1i.
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Scheme 4
Reagents and conditions: a) 6c, K CO , DMF, 100 °C, 5 h, 71%; b) 2M NaOH, THF, MeOH,
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rt, 2 h, 99%; c) 2ꢀethylhexyl betaꢀmercaptopropionate, K CO , DMF, rt, 2 h, 100%; d) i) Fe,
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NH Cl, EtOH, water, reflux, 30 min; ii) 34, SOCl , THF, 60 °C, 30 min then Et N, THF, rt, 30
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min, 83%; e) NaOMe, THF, rt, 30 min then TFA, THF 60 °C, 20 min, 86%; f) i) NH OAc,
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NaBH CN, THF, MeOH, reflux, 3 h; ii) Ac O, Et N, THF, rt, 1 h, 65%; g) i) TFA, thioanisole,
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5 °C, 30 min; ii) cPrCH Br, K CO , DMF, 50 °C, overnight, 41%.
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The preparation of 1j is illustrated in Scheme 5. Cuꢀcatalyzed coupling reaction of
ꢀbromoiodobenzene (40) with phenol 22 under microwave irradiation afforded
ꢀphenoxybromobenzene 41. After reducing the ketone of 5ꢀacetylbenzothiophene (42), the
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Ritter reaction was carried out to afford acetamide derivative 43. Pdꢀcatalyzed coupling
reaction of 43 with bromobenzene 41 gave the desired benzo[b]thiophene analogue 1j.
Scheme 5
Reagents and conditions: a) 22, CuI, dimethylaminoacetic acid hydrochloride, Cs CO , DME,
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0 °C, overnight, 67%; b) i) NaBH , MeOH, rt, 1 h; ii) H SO , CH CN, rt, 1.5 h, 80%; c) 41,
4 2 4 3
Pd(OAc) , (tertꢀBu) PBF , tertꢀBuOLi, DMA, 120 °C, overnight, 8.6%.
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4
The preparation of 1k is shown in Scheme 6. After reduction of the ester in 23f to produce
benzylalcohol 44, benzylether derivative 46 was prepared by Mitsunobu reaction with
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1
ꢀ(3ꢀhydroxyphenyl)ethanone (45). After reduction of the ketone of 46, compound 1k was
prepared from alcohol 47 by azidation with DPPA, Staudinger reaction and acetylation.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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4
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4
4
5
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0
1
2
3
4
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0
1
2
3
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9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 6
Reagents and conditions: a) LiAlH , THF, 0 °C, 30 min, 98%; b) 45, Ph P, DIAD, THF, rt, 4h,
4
3
9
7%; c) NaBH , EtOH, THF, 0 °C, 30 min, 99 %; d) DPPA, DBU, toluene, rt, 2h, 16%; e) Ph P,
4 3
THF, water, 60 °C, 1h, 81%; f) Ac O, pyridine, rt, 30 min, 90%.
2
The preparation of 1l is displayed in Scheme 7. 2ꢀPhenylꢀ1,3ꢀoxazole derivative 51 was
synthesized from amine 50 and benzoic acid 24f by condensation reaction utilizing
2
ꢀchloroꢀ1ꢀmethylpyridinium iodide, cyclization under Robinson–Gabriel cyclodehydration
conditions using I and Ph P, hydrolysis of the ester, and Weinreb amide formation. Weinreb
2
3
amide 51 was converted to azide 53 in 4 steps by reaction with MeMgBr, reduction of the
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5
5
5
5
5
6
ketone, methanesulfonylation, and substitution with an azido group. Finally, the desired
acetamide derivative 1l was prepared from azide 53 by Staudinger reaction and acetylation.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 7
Reagents and conditions: a) i) 24f, 2ꢀchloroꢀ1ꢀmethylpyridinium iodide, DIPEA, THF, 12 °C,
2
h; ii) I , Ph P, Et N, CH Cl , 12 °C, 1 h; iii) 1M NaOH, THF, MeOH, rt, overnight; iv)
2 3 3 2 2
MeNH(OMe)·HCl, EDC·HCl, HOBt·H O, Et N, DMF, rt, overnight, 50%; b) i) MeMgBr, THF,
2
3
0
8
°C, 1 h; ii) NaBH , EtOH, 0 °C, 30 min, iii) MsCl, Et N, THF, rt, 1 h, 81%; c) NaN , DMF,
4 3 3
0 °C, 2 h, 74 %; d) Ph P, THF, water, 60 °C, 2 h, 82%; e) Ac O, pyridine, rt, 30 min, 53%.
3
2
2
ꢀPhenylꢀ1,3ꢀoxazole derivatives 1m and 1q were prepared as shown in Scheme 8.
Condensation of Nꢀbenzyloxycarbonylglycine (55) and NꢀBocꢀ(S)ꢀalaninol (56) produced the
corresponding ester 57. After deprotection of the benzyloxycarbonyl group by hydrogenation,
condensation with benzoic acid derivative 24f was carried out to give cyclization precursor 58.
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Construction of the oxazole ring by Robinson–Gabriel cyclodehydration yielded 59. Finally,
the desired product 1m was synthesized by removal of the Boc protecting group from 59 and
acetylation. Ureido derivative 1q was synthesized by formation of the 4ꢀnitrophenyl
carbamate followed by treatment with aqueous ammonia solution.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 8
Reagents and conditions: a) 56, EDC·HCl, DMAP, DMF, rt, overnight, 100%; b) i) H (1
2
atm), Pd/C, THF, rt, 4 h; ii) 24f, (COCl) , DMF, THF, rt, 1 h then Et N, THF rt, overnight,
2
3
1
03%; c) I , Ph P, Et N, CH CN, rt, overnight, 74%; d) i) TFA, toluene, rt, 30 min; ii) Ac O,
2 3 3 3 2
pyridine, rt, 30 min, 41%; e) i) formic acid, 40 °C, 30 min, ii) 4ꢀnitrophenyl chloroformate,
Et N, THF, 0 °C, 1 h then 28% aq. NH , rt, 30 min, 41%.
3
3
The preparation of 1o and 1p is outlined in Scheme 9. The 2ꢀpyridylꢀ1,3ꢀoxazole analogue
2 was prepared by condensation with the deprotected amine from 57 followed by Robinson–
6
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2
2
2
2
2
2
2
3
3
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3
3
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3
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3
4
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4
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4
4
4
4
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5
5
5
5
5
5
5
6
Gabriel cyclodehydration. After Cuꢀcatalyzed coupling reaction between 62o and phenol 22,
the desired product 1o was produced by removal of the Boc protecting group and acetylation.
After coupling reaction of 62p with phenol 22 under basic conditions, the desired product 1p
was prepared by removal of the Boc protecting group and acetylation.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 9
Reagents and conditions: a) i) H (1 atm), Pd/C, THF, rt, 2 h; ii) 60, (COCl) , DMF, THF, rt,
2
2
1
9
1
2
h then Et N, THF rt, 1 h, 81%; b) i) H (1 atm), Pd/C, THF, rt, 2 h; ii) 27, Et N, THF rt, 1 h,
3 2 3
8%; c) I , Ph P, Et N, CH CN or CH Cl , rt, overnight, 20–61%; d) 22, 62o, Cs CO , DMF,
2
3
3
3
2
2
2
3
00 °C, 2 h, 21%; e) formic acid, 40 °C, 30 min, 61%; f) Ac O, pyridine, rt, 30 min, 61%; g) i)
2
2, 62p, CuI, picolinic acid, K PO , DMSO, 80–90 °C, 16 h; ii) formic acid 10–15 °C, 5 h; iii)
3
4
Ac O, pyridine, 15–20 °C, 16 h, 10%.
2
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RESULTS AND DISCUSSION
. Investigation of bicyclic derivatives
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
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4
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4
4
4
5
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
Compounds prepared in this study were evaluated for their inhibitory activity against
recombinant human ACC1 and ACC2 expressed in SFꢀ9 cells. To measure the inhibition of de
1
4
novo lipid synthesis in cells of our ACC1 inhibitors, we established a Cꢀacetate uptake assay
in HCTꢀ116 colon cancer cells. In our initial SAR studies of selective ACC1 inhibitors, we
paid particular attention to the novel Nꢀ(1ꢀ(1,3ꢀbenzoxazolꢀ6ꢀyl)ethyl)acetamide moiety of
compound 1a as the core scaffold (Fig. 2).
Figure 2. Synthetic strategies for initial SAR studies
First, we examined the effect of modifications around the tail region. Encouragingly,
investigation of the alkyl chain in the tail region revealed that replacement with nꢀpentyl moiety
could increase ACC1 inhibitory potency and provide good selectivity over ACC2 (compound
1
b: ACC1 IC = 220 nM, ACC2 IC >10000 nM). Furthermore, the improved enzymatic
50 50
potency of compound 1b translated into enhanced cellular potency (acetate uptake IC = 100
5
0
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
5
5
5
6
nM). Replacement of the alkyl tail of compound 1b with an ether chain showed similar
enzymatic activity (compound 1c: ACC1 IC = 210 nM). Since ether 1c displayed higher
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
solubility (8.2 ꢀg/mL at pH 6.8) than alkyl compound 1b (0.44 ꢀg/mL at pH 6.8), we conducted
further modifications of the ether moiety for our SAR campaign in the tail region. Thus,
introduction of a cyclopropylmethoxy group further improved ACC1 inhibitory potency along
with good selectivity over ACC2 (compound 1d: ACC1 IC = 23 nM, ACC2 IC = 5800 nM).
5
0
50
This result indicated that the cyclopropylmethoxy group is important for ACC1 inhibitory
potency. Next, we investigated modifications to the linker section and a series of
cyclopropylmethyl ether derivatives were evaluated. Compound 1e, possessing a 4ꢀphenoxy
piperidine moiety instead of the azetidine linker of 1d, exhibited 10ꢀfold lower ACC1 inhibitory
potency. Notably, 2ꢀ(4ꢀphenoxyphenyl)ꢀ1,3ꢀbenzoxazole derivative 1f showed selective and
highly potent ACC1 inhibition (ACC1 IC = 5.3 nM, ACC2 IC >10000 nM) along with high
5
0
50
cellular potency (acetate uptake IC = 2.2 nM). Thus, the introduction of a phenyl ring at the
5
0
2
ꢀposition of the 1,3ꢀbenzoxazole core was found to significantly improve enzymatic activity as
well as cellular potency, indicating that 2ꢀarylꢀ1,3ꢀbenzoxazle derivatives could be more potent
and selective ACC1 inhibitors. Thus, we conducted further optimization by modification of
the 2ꢀarylbicyclic core.
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a
Table 1. Effects of tail and linker modifications on biological activity
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b
IC (nM)
5
0
c
hACC1
hACC2
>10000
Uptake
>10000
e
1
a
NT
d
d
(
48%)
20
107–441)
10
81.1–532.9)
(22%)
2
100
1
b
>10000
>10000
(
(34.7–309.3)
2
e
1
c
NT
(
2
3
5800
51
(79.0–152.6)
400
1
d
(
14.8–36.6)
30
127.8–396.3)
.3
4.7–5.9)
(3919–8631)
2
1
e
>10000
>10000
(
(186.4–846.4)
2.2
5
1
f
(
(1.6–3.0)
a
b
IC50 values shown are the means of duplicate measurement.
IC50 values and 95%
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2
2
2
2
2
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5
6
confidence limits are calculated from the concentrationꢀresponse curves generated by GraphPad
c
d
e
Prism. Acetate uptake inhibition in HCTꢀ116 cells. % inhibition at 10 ꢀM.
Not tested.
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
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9
0
1
2
3
4
5
6
7
8
9
0
For further SAR studies, we explored other core 2ꢀarylbicyclic scaffolds to generate potent
and selective ACC1 inhibitors. Introduction of a pyridine ring at the 1,3ꢀbenzoxazole
2
ꢀposition produced 2ꢀpyridylꢀ1,3ꢀbenzoxazoles 1g and 1h. These compounds showed potent
and selective inhibition of ACC1 comparable to that of 2ꢀphenylꢀ1,3ꢀbenzoxazole derivative 1f.
Remarkably, compound 1h showed the most potent ACC1 inhibitory activity (IC = 1.8 nM)
5
0
and cellular potency (IC50 = 0.76 nM) of the benzoxazole analogs. Additionally, these
compounds exhibited improved solubility at pH 6.8 (1g: 5.8 ꢀg/mL and 1h: 1.6 ꢀg/mL,
respectively) compared with compound 1f (<0.18 ꢀg/mL) corresponding to their lower
lipophilicity (1g: cLogP 4.44, 1h: cLogP 4.23 and 1f: cLogP 5.62, respectively), brought about
by the introduction of a nitrogen atom. However, ACC1 selectivity over ACC2 for the
2
ꢀpyridyl derivatives decreased to 200ꢀfold (1g) and 75ꢀfold (1h) from that of 1f (>1500ꢀfold).
For further investigation, analogues with other bicyclic core scaffolds were evaluated. They
also displayed potent and selective ACC1 inhibition represented by compound 1i and 1j as
shown in Table 2.
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Table 2. In vitro activity, solubility, and CYP inhibition of inhibitors with modified bicyclic
a
core scaffolds
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b
e
IC (nM)
Solubility
CYP inhibition
5
0
c
d
hACC1
.3
4.7–5.9)
.1
5.4–9.5)
.8
1.5–2.1)
.4
5.2–10.6)
hACC2
>10000
1400
Uptake
2.2
(ꢀg/mL)
2C8 (%)
82.7
2C9 (%)
89.9
5
1
f
<0.18
5.8
(
(
(
(1.6–3.0)
9.9
7
1
g
81.4
81.4
14.4
23.6
90.6
87.0
44.8
33.5
(274.8–7210)
170
(7.3–13.3)
0.76
1
1
h
1.6
(80.6–355)
(0.4–1.5)
14
7
1
i
>10000
>10000
<0.12
<0.090
(
(5.8–33.1)
11
2
6
1
j
(16.4–39.9)
(7.2–17.4)
a
b
IC50 values shown are the means of duplicate measurement.
IC50 values and 95%
confidence limits are calculated from the concentrationꢀresponse curves generated by GraphPad
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
c
d
e
Prism. Acetate uptake inhibition in HCTꢀ116 cells. Solubility in pH6.8.
% inhibition at
1
0 ꢀM.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Thus, by modification of the bicyclic scaffold, multiple potent ACC1ꢀselective inhibitors
were identified. While they generally showed potent and selective ACC1 inhibition as well as
improved cellular potency compared to lead compound 1a, they still bore some liabilities, such
as poor solubility and potent CYP inhibition. Compound 1i and 1j showed lower levels of
CYP inhibition than compound 1f, however, possessed similarly low solubility. Other bicyclic
derivatives we synthesized showed similar imbalances in potency, selectivity and
physicochemical properties. According to these results, we concluded that it would be
difficult to produce more potent and selective ACC1 inhibitors while maintaining favorable
drugꢀlike properties by the modification of these bicyclic derivatives. Therefore, we shifted
our chemistry efforts to identify potent, selective, and more drugꢀlike ACC1 inhibitors suitable
for in vivo use.
2
. Investigation of monocyclic derivatives
It is generally well known that reducing the number of aromatic rings in a compound is
2
7
effective in ameliorating CYP inhibitory potency.
At the same time, increasing
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conformational flexibility enhances the solubility of drugꢀlike compounds.
Taken together,
we thought to replace the bicyclic core structure with a monocycle, which we anticipated would
minimize these issues simultaneously. Based on this consideration, we attempted the two
approaches shown in Figure 3: removal of the oxazole ring to generate monocyclic benzyloxy
derivative 1k (approach A) and removal of a benzene ring to generate monocyclic oxazole
derivative 1l (approach B). Benzyloxy derivative 1k showed moderate ACC1 inhibitory
potency and >70ꢀfold selectivity over ACC2 (ACC1 IC /ACC2 IC = 140 nM/>10000 nM).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
50
As expected, this compound demonstrated reduced CYP inhibitory activity, but with poor
solubility (<0.23 ꢀg/mL at pH 6.8) and weak cellular potency (IC >1000 nM). On the other
5
0
hand, oxazole derivative 1l exhibited slightly improved solubility (2.0 ꢀg/mL at pH 6.8) and an
lower CYP inhibitory activity. Importantly, this compound retained potent ACC1 inhibitory
activity (IC50 = 4.9 nM) and cellular potency (IC50 = 8.6 nM). Accordingly, we chose to
further explore approach B, with compound 1l as our new lead compound for further
optimization to identify a suitable in vivo tool compound with drugꢀlike properties.
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Synthetic strategies to improve CYP inhibition and solubility of ACC1 inhibitors
First, we examined the SAR of the linker between the acetamide moiety and the monocycle.
2
1
Replacement of the alkyl linker of compound 1l with a single enantiomer ether chain
produced highly potent ACC1 inhibitor 1m (ACC1 IC = 0.96 nM). On the other hand, the
5
0
Rꢀisomer 1n showed ca. 300ꢀfold decrease in ACC1 inhibitory activity (ACC1 IC = 240 nM).
5
0
Therefore, we focused on further derivative synthesis maintain the more potent Sꢀisomer
conformation in the linker region. Compound 1m also showed potent cellular activity (ACC1
IC = 3.4 nM) and acceptable solubility (2.7 ꢀg/mL at pH 6.8), along with increased ACC2
5
0
inhibitory potency (ACC2 IC50 = 95 nM). As part of our effort to further improve the
solubility of these monocyclic oxazole derivatives, we investigated the effects of introducing a
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nitrogen atom into compound 1m, a modification that significantly affected the bicyclic
derivatives described above. As a result, replacement of the 2ꢀphenyl group of the 1,3ꢀoxazole
derivatives with a 2ꢀpyridyl group produced improved solubility, as expected (1o: 19 ꢀg/mL
and 1p: 9.4 ꢀg/mL), while maintaining ACC1 inhibitory potency and selectivity over ACC2.
Since increasing the solubility of 1o and 1p relative to 1m would largely depend on lowering
compound lipophilicity, we investigated replacement of the acetamide moiety with a ureido
group. As a result, ureido derivative 1q was prepared and shown to be a highly potent and
selective ACC1 inhibitor (ACC1 IC /ACC2 IC = 0.58 nM/>10000 nM) with potent cellular
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
50
activity (IC = 6.4 nM) and acceptable solubility (1.7 ꢀg/mL at pH 6.8). Accordingly, the
5
0
main effect of introducing the ureido moiety was in reducing ACC2 inhibitory potency. From
our modifications of various monocyclic oxazole derivatives, overall we found compound 1q to
represent the most promising in vivo candidate with high potency and selectivity, as well as
good physicochemical properties.
a
Table 3. In vitro biological activity and solubility of monocyclic oxazole derivatives
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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5
5
5
5
5
5
5
6
O
O
O
O
O
O
O
O
O
O
N
O
O
N
N
H
N
H
N
H
N
1
l
1m
1n
O
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
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5
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7
8
9
0
1
2
3
4
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9
0
1
2
3
4
5
6
7
8
9
0
O
O
N
O
N
N
O
O
N
N
H
N
H
N
NH2
H
N
1o
1p
1q
b
e
IC (nM)
Solubility
CYPinhibition
5
0
c
d
hACC1
.9
3.8–6.2)
.96
0.8–1.1)
40
163–365)
.8
4.3–7.9)
.96
0.9–1.0)
.58
0.5–0.7)
hACC2
860
Uptake
8.6
(ꢀg/mL)
2C8 (%)
41.8
2C9 (%)
4
1
l
2.0
22.4
19.6
(
(
(271.7–2744)
95
(7.1–10.5)
3.4
0
1
m
2.7
13.5
(47.4–191.4)
5700
(2.6–4.4)
710
2
f
f
f
1
n
NT
NT
NT
(
(1068–30304)
930
(580–864)
51
5
1
o
p
q
19
9.4
1.7
4.7
2.4
(
(517.3–1682)
290
(38.1–69.2)
2.8
0
1
23.2
23.5
26.2
14.8
(
(
(139.9–598.4)
(2.2–3.6)
6.4
0
1
>10000
(4.8–8.6)
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a
b
IC50 values shown are the means of duplicate measurement.
IC50 values and 95%
confidence limits are calculated from the concentrationꢀresponse curves generated by GraphPad
c
d
e
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
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2
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5
5
5
6
0
1
2
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5
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0
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0
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0
Prism. Acetate uptake inhibition in HCTꢀ116 cells. Solubility in pH6.8.
% inhibition at
f
1
0 ꢀM. Not tested.
3
. In vivo evaluation of a selective ACC1 inhibitor
Through our SAR studies focused on the bicyclic and monocyclic analogues, highly potent
and selective ACC1 inhibitor 1q was identified. In order to evaluation this compound further,
additional testing of its biological, ADMET, and PK properties was conducted. The overall
profile of this compound is shown in Figure 4. Compound 1q showed good metabolic
stability in mouse liver microsomes, acceptable permeability, and no significant CYP inhibitory
activities in our standard ADME and physicochemical profiling.
Furthermore,
pharmacokinetic (PK) studies in a mouse cassette dosing test (0.1 mg/kg, i.v. and 1 mg/kg, p.o.)
exhibited excellent bioavailability, suggesting it could be used for in vivo evaluation. Based
on the promising in vitro and in vivo profile of 1q, we selected this compound for in vivo
pharmacodynamics (PD) study to evaluate the potential of a selective ACC1 inhibitor in
xenograft tumor models.
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0
1
2
3
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Biological, ADMET, and PK profile of compound 1q
In this preliminary PD study, malonylꢀCoA concentration in tumors was measured as a direct
PD marker. As a result, compound 1q showed potent and sustained malonylꢀCoA suppression
at 16 h after single oral administration in HCTꢀ116 xenograft mice at doses of more than 30
mg/kg.
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9
0
0
.800
.700
#
#
: P<0.05 by Dunnet tꢀtest
0.600
0
0
.500
.400
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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5
5
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0
1
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0
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0
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0
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0
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2
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8
9
0
0.300
#
0
0
.200
.100
#
#
#
#
0.000
Vehicle
30 mg/kg 50 mg/kg 100 mg/kg Vehicle
Compound 1q
30 mg/kg 50 mg/kg 100 mg/kg
Compound 1q
16h
2h
Figure 5. Effects of compound 1q on malonylꢀCoA concentration in HCTꢀ116 xenograft
tumors
CONCLUSIONS
We have described the identification of a novel series of potent and selective ACC1 inhibitors
as a chemical probes for oncology research and potential use as antiꢀcancer agents. Our initial
lead, 2ꢀazetidylꢀ1,3ꢀbenzoxazole derivative 1a, was optimized and converted to
2
ꢀphenylꢀ1,3ꢀbenzoxazole derivative 1f. Compound 1f showed >1800ꢀfold more potent
ACC1ꢀselective inhibition than compound 1a and potent cellular activity, although further
testing suggested the need to improve the physicochemical and ADMET properties of 1f, such
as solubility and CYP inhibition. To address these needs, the bicyclic core was replaced with a
monocyclic scaffold, which ultimately lead us to the discovery of ureido derivative 1q, with an
oxazole core scaffold. Compound 1q showed an improved physicochemical and ADMET
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6
profile, while maintaining potent and subtypeꢀselective ACC1 inhibitory potency. Based on its
promising PK profile and results from initial in vivo PD studies, compound 1q was selected as
an in vivo probe molecule and is undergoing further pharmacological evaluation to determine
the therapeutic potential of selective ACC1 inhibition.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
0
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9
0
1
2
3
4
5
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7
8
9
0
EXPERIMENTAL SECTION
1
General. The proton nuclear magnetic resonance ( H NMR) spectra were measured on a
Bruker Avance 300 (300 MHz), Bruker Avance 400 (400 MHz) or Varian 400 MHz
spectrometer. Chemical shifts are given in δ values (ppm) using tetramethylsilane as the
internal standard. All J values are given in hertz. The following abbreviations are used: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; br s, broad singlet.
Analytical LC was performed on a Shimadzu LCꢀ20AD separations module (Lꢀcolumn2 ODS
(3.0 x50 mm I.D., CERI, Japan); 0.05% TFA in ultrapure water/acetonitrile gradient; UV
detection 220 nm or 254 nm). MS spectra were recorded using a Shimadzu LCMSꢀ2020 with
electrospray ionization. High performance liquid chromatography (HPLC) was performed on
Shimadzu LCꢀ10A series. The purities of all the compounds tested in biological systems were
assessed as being >95% using elemental analysis or analytical HPLC. Purity data were
collected by HPLC with NQAD (nanoquality analyte detector) or Corona CAD (charged aerosol
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detector). The column was an Lꢀcolumn 2 ODS (30 mm × 2.1 mm i.d., CERI) or a Capcell Pak
C18AQ (50 mm × 3.0 mm i.d., Shiseido) with a temperature of 50 °C and a flow rate of 0.5
mL/min. Mobile phases A and B under a neutral conditions were a mixture of 50 mmol/L
AcONH , water, and MeCN (1/8/1, v/v/v) and a mixture of 50 mmol/L AcONH and MeCN (1/9,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
4
v/v), respectively. The ratio of mobile phase B was increased linearly from 5% to 95% over 3
min, 95% over the next 1 min. Melting points (mp) were determined on an OptiMelt MPA100
melting point apparatus and were uncorrected. Elemental analyses (C, H, N) were within ±
0
.4% of theoretical values. For thin layer chromatography (TLC) analysis throughout this work,
Merck precoated TLC plates (silica gel 60 F ) and basic TLC plates (NH silica gel, Fuji Silysia
2
54
Chemical Ltd.) were used. The products were purified on silica gel 60 (0.063ꢀ0.200, E. Merck),
basic silica gel (Chromatorex NH, 100ꢀ200 mesh, Fuji Silysia Chemical Ltd.) or PurifꢀPack (Si or
NH, Shoko Scientific Co., Ltd.). Reagents and solvents were obtained from commercially
sources and used without further purification.
N-(1-(2-(3-(3-methylphenoxy)azetidin-1-yl)-1,3-benzoxazol-6-yl)ethyl)acetamide (1a)
Ethyl 2-(3-hydroxyazetidin-1-yl)-1,3-benzoxazole-6-carboxylate (4): A mixture of ethyl
2
ꢀchloroꢀ1,3ꢀbenzoxazoleꢀ6ꢀcarboxylate (2) (17.5 g, 77.6 mmol), azetidinꢀ3ꢀol hydrochloride (3)
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