Synthesis and SAR of α-acylaminoketone ligands for control of gene expression

Article abstract of DOI:10.1016/S0960-894X(02)00980-0

A lead discovery library and a follow-up focused library of α-acylaminoketones were designed based on known dibenzoylhydrazine ecdysone agonists, including GS-E. The compounds were assayed in mammalian cells expressing the ecdysone receptor from Bombyx mori for their ability to cause expression of a reporter gene downstream of an ecdysone response element. The most potent α-acylaminoketones were comparable to GS-E in this assay.

Full text of DOI:10.1016/S0960-894X(02)00980-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 475–478
Synthesis and SAR of ꢀ-Acylaminoketone Ligands for Control
of Gene Expression
Colin M. Tice,a,* Robert E. Hormann,a Christine S. Thompson,a Jennifer L. Friz,a
Caitlin K. Cavanaugh,a Enrique L. Michelotti,b,y Javier Garcia,b,c
Ernesto Nicolasc and Fernando Albericioc
aRHeoGene, PO Box 949, 727 Norristown Road, Spring House, PA 19477-0949, USA
bRohm and Haas Company, PO Box 904, 727 Norristown Road, Spring House, PA 19477-0904, USA
cDepartment of Organic Chemistry, University of Barcelona, 08028-Barcelona, Spain
Received 8 August 2002; accepted 14 October 2002
Abstract—A lead discovery library and a follow-up focused library of a-acylaminoketones were designed based on known diben-
zoylhydrazine ecdysone agonists, including GS –E. The compounds were assayed in mammalian cells expressing the ecdysone
TM
receptor from Bombyx mori for their ability to cause expression of a reporter gene downstream of an ecdysone response element.
TM
The most potent a-acylaminoketones were comparable to GS –E in this assay.
# 2002 Elsevier Science Ltd. All rights reserved.
The ability to control the level and timing of gene
expression is a powerful tool in biological systems and
has potential applications in human gene therapy and
the production of therapeutic proteins.1À3 A number of
such ‘gene-switch’ systems have been described, among
them some based on the insect ecdysone receptor
(EcR).4À8 These have the advantage that they are
intrinsically orthogonal to mammalian steroid hormone
receptors and can be activated by certain ecdysteroids
and by non-steroidal ecdysone agonist ligands.9,10 As
part of our effort to develop the RheoPlexTM system of
orthogonal ‘gene switches’ based on natural and muta-
ted ecdysone receptors, we sought novel chemotypes
with ecdysone agonist activity.11
ketones 2 in which X was varied, R1 and R2 were either
both methyl or were joined to form a cyclopentane ring,
and Y was fixed as hydrogen. The geminal dimethyl
compounds were prepared from Boc-Aib–OH (3) as
shown in Scheme 1.12 Thus, 3 was coupled with N,O-
dimethylhydroxylamine to afford Weinreb amide 4.
Reaction of 4 with PhMgBr gave aminoketone 5a
directly in modest yield, rather than the expected Boc
derivative. Coupling of 5a with benzoic acids using
standard protocols afforded the desired a-acylaminoke-
tones 2aa–2af (Table 1).13 Similarly, acylation of 1-
amino-1-benzoylcyclopentane (5b), which is available
by amination of cyclopentyl phenyl ketone,14 afforded
compounds 2ba–2bf.
Dibenzoyl derivatives of t-butylhydrazine e.g., RH-5849
(1a) and GS –E (1d) are well known as ecdysone ago-
The compounds were assayed at a single dose in a cell
line engineered to express Bombyx mori EcR and to
contain a b-galactosidase gene under the control of an
ecdysone response element.5,15 The assay results are
presented as fold induction relative to a DMSO control
(Table 1). No increase in the expression of b-galactosi-
dase above background levels was observed with the
geminal dimethyl compounds 2aa–2ac or 2ae; however,
a 2-fold increase in expression was observed with 2ad and
2af. These compounds bear the X=4-ethyl and 2-
methyl-3-methoxy substitution patterns seen in the com-
mercial ecdysone agonist insecticides tebufenozide (1b)
and methoxyfenozide (1c). Similarly, in the cyclopentane
TM
nists (Fig. 1).10 Examination of structure 1 suggested
that replacement of the N-t-butyl moiety with an
appropriately chosen dialkylmethylene group (CR1R2)
to afford a-acylaminoketone 2 might lead to a new class
of ecdysone agonists. To test the validity of this concept,
we prepared a lead discovery library of a-acylamino-
*Corresponding author. Fax: +1-215-619-1665; e-mail: ctice@
rheogene.com
yPresent address: Locus Discovery Inc., Four Valley Square, 512
Township Line Road, Blue Bell, PA 19422, USA.
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00980-0

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C. M. Tice et al. / Bioorg. Med. Chem. Lett. 13 (2003) 475–478
series, 2bd and 2bf stood out, causing substantial
increases in gene expression. These results suggested
that, for 2 to effectively promote gene expression,
greater bulk was necessary at R1 and R2 than was pro-
vided by the geminal dimethyl moiety and, possibly,
that R1 and R2 should be constrained in a ring.
To further optimize potency, a focused library of a-
acylaminoketones 2 in which X was fixed as 2-Me-3–
MeO was designed. The substitution pattern X=2-Me-
3–MeO, rather than X=4-Et, was used in the focused
library to render the resulting target compounds 2
somewhat less lipophilic. R1 and R2 were selected to be
–(CH2)4-; Et, Et; or Me, i-Pr. Finally Y was chosen to
include methyl and methoxy at the 2-, 3- and 4-positions
as well as the 3,5-dimethyl group that is present in the
commercial ecdysone agonist insecticides 1b and 1c, and
Figure 1. Dibenzoylhydrazine ecdysone agonists 1a–d and isosteric
a-acylaminoketones 2.
TM
in GS –E (1d).
Initially Weinreb amides 8 (Scheme 2) were anticipated
to be pivotal intermediates in this synthesis. Thus,
treatment of the commercially available a,a-dis-
ubstituted amino acids 6b–d with 2.5 equiv of 2-methyl-
3-methoxybenzoyl chloride in pyridine afforded azlac-
tones 7b–d. These were ring opened by treatment with
N,O-dimethylhydroxylamine in the presence of pyridine
to afford 8b–d.16 Treatment of 8b with 3-methoxy-
phenylmagnesium bromide in THF at room tempera-
ture afforded the desired a-acylaminoketone 2bj in 63%
yield; however, when the more sterically congested 8d
was treated under similar conditions the major product
was N-(hydroxymethyl)-N-methylamide 9d; 13C NMR
indicated that little or none of the desired ketone 2dj
was present.17 Given this result the reaction of Weinreb
amides 8 with Grignard reagents was deemed insuffi-
ciently general for library production.
Scheme 1. Synthesis of lead discovery library of a-acylaminoketones.
(a) MeNHOMe, EDC, i-Pr2NEt, CH2Cl2, rt, 24 h; (b) R4MgBr
(5 equiv), THF, rt, 5 h; (c) PS–HOBt–O2CPhX, i-Pr2NEt, CH2Cl2,
12 h, rt.
Table 1. Single dose transactivation assay resultsa
Fold Inductionb,c (33 mM)
2a
2b
X
Y
Me, Me
–(CH2)4–
a
b
c
d
e
f
H
2-Me
3-MeO
H
H
H
H
H
H
1
1
1
2
1
2
2
2
2
133
1
71
4-Et
3,4-OCH2O
2-Me–3-MeO
As an alternative, we undertook the longer sequence
depicted in Scheme 3. The previously prepared azlac-
tones 7b–d were reduced with sodium borohydride in
THF to provide the primary alcohols 10b–d.18 These
aSee ref 15 for assay protocol.
bRatio of light measured in treated cells versus a DMSO control.
cAverage of two replicates.
Scheme 3. Amidoaldehyde route to a-acylaminoketones. (a) NaBH4,
THF, rt: (b) Dess–Martin periodinane, CH2Cl2, rt; (c) Y–PhMgBr
Scheme 2. Weinreb amide route to a-acylaminoketones. (a) 2-Me–3-
MeO–PhCOCl (2.5 equiv), pyridine, rt; (b) MeNHOMe.HCl, pyridine,
CH2Cl2, rt; (c) 3-MeOPhMgBr (4 equiv), THF, rt, 5 h.
(4 equiv), THF, À70 ꢀC
!
rt; (d) Dess–Martin periodinane,
CH2Cl2, rt.

C. M. Tice et al. / Bioorg. Med. Chem. Lett. 13 (2003) 475–478
477
Table 2. Single dose transactivation assay resultsa
Fold Inductionb,c (33 mM)
The initial screening results of the focused library of a-
acylaminoketones are shown in Table 2. Library mem-
bers which caused >50-fold induction of b-galactosi-
dase expression at 33 mM were screened in a dose
response assay in the same cell line. The results are
reported in Table 3 in terms of LC50 and maximum fold
2b
–(CH2)4–
2c
Et,Et
2d
Me, i-Pr
X
Y
f
2-Me–3-MeO
2-Me–3-MeO
2-Me–3-MeO
2-Me–3-MeO
2-Me–3-MeO
2-Me–3-MeO
2-Me–3-MeO
2-Me–3-MeO
2-Me–3-MeO
2-Me–3-MeO
H
2-Me
2-MeO
3-Me
3-MeO
4-Me
4-MeO
4-F
71
30
1196
291
127
21
11
5
384
525
256
8
4
60
223
1005
71
452
599
370
35
TM
induction compared to GS –E (1d). An effective ligand
g
h
i
j
k
l
m
n
o
must combine a low LC50 value with a high maximum
fold induction. By these criteria, a-acylaminoketones
2bo and 2do, which can be considered as isosteres of the
commercial insecticide methoxyfenozide (1c), were the
best compounds in the library.
31
d
5
d
218
487
660
3-Me-4-F
3,5-diMe
265
751
In conclusion, we have described the discovery of a new
class of ecdysone agonists useful for the control of gene
expression in ecdysone responsive systems. Levels of
reporter gene expression induced by the most potent
compounds 2bo and 2do approached those seen with the
aSee ref 15 for assay protocol.
bRatio of light measured in treated cells versus a DMSO control.
cAverage of two replicates.
dCompound not made.
TM
GS –E (1d).
were cleanly oxidized to the corresponding aldehydes
11b–d using the Dess–Martin periodinane. The overall
yields of aldehydes 11b–d from the corresponding ami-
noacids 6b–d were 62, 47 and 88%, respectively. Alde-
Acknowledgements
hydes 11b–d were reacted with
a
variety of
This work was supported in part by NIST Advanced
Technology Project Grant 70NANB0H3012.
phenylmagnesium bromides in THF at low temperature
to afford the secondary alcohols 12, which were once
again oxidized with the Dess–Martin periodinane to
afford the desired a-acylaminoketones 2 (Table 2).19
The last two steps of the sequence proved to be amen-
able to manual parallel synthesis. Aqueous workups
were performed using Varian Chem-Elut cartridges20
and polymer supported tosyl hydrazide was used to
scavenge unreacted aldehyde from ketone product.
Fractionation of the final products on silica gel
SPE cartridges routinely afforded material of
>85% purity. Yields for conversion of aldehydes
References and Notes
1. DeMayo, F. J.; Tsai, S. Y. Trends in Endocrinology &
Metabolism 2001, 12, 348.
2. Clackson, T. Gene Therapy 2000, 7, 120.
3. Zuo, J.; Chua, N.-H. Curr. Opin. Biotechnology 2000, 11,
146.
4. Christopherson, K. S.; Mark, M. R.; Bajaj, V.; Godowski,
P. J. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 6314.
5. Suhr, S. T.; Gil, E. B.; Senut, M.-C.; Gage, F. H. Proc.
Natl. Acad. Sci. U.S.A. 1998, 95, 7999.
6. Martinez, A.; Sparks, C.; Hart, C. A.; Thompson, J.; Jep-
son, I. The Plant Journal 1999, 19, 97.
7. Hoppe, U. C.; Marban, E.; Johns, D. C. Molecular Therapy
2000, 1, 159.
8. Kumar, M. B.; Fujimoto, T.; Potter, D. W.; Deng, Q.;
Palli, S. R. Proc. Natl. Acad. Sci. U.S.A., in press.
9. Wing, K. D.; Slawecki, R. A.; Carlson, G. R. Science 1988,
241, 470.
10. Carlson, G. R.; Cress, D. E.; Dhadialla, T. S.; Hormann,
R. E.; Le, D. P. U.S. Patent 6,258,603, 2001; Chem. Abstr.
2001, 135, 72148.
11 to a-acylaminoketones
2 were not optimized
and ranged from 9 to 60%. The lowest yields
were encountered with products derived from hin-
dered aldehyde 11d. All compounds were characterized
by 1H and 13C NMR and selected compounds were
more fully characterized.21
Table 3. Dose response transactivation assay resultsa
LC50 (mM)b/Rel Max FIc
2b
–(CH2)4–
2c
Et,Et
2d
Me, i-Pr
X
Y
11. For other orthogonal systems see: (a) Moradpour, D.;
Englert, C.; Blum, H. E. Biol. Chem. 1998, 379, 1189. (b) Shi,
Y.; Koh, J. T. Chemistry & Biology 2001, 8, 501. (c) Tedesco,
R.; Thomas, J. A.; Katzenellenbogen, B. S.; Katzenellenbo-
gen, J. A. Chemistry & Biology 2001, 8, 277. (d) Doyle, D. F.;
Braasch, D. A.; Jackson, L. K.; Weiss, H. E.; Boehm, M. F.;
Mangelsdorf, D. J.; Corey, D. R. J. Am. Chem. Soc. 2001, 123,
11367.
d
d
f
2-Me–3-MeO
2-Me–3-MeO
2-Me–3-MeO
2-Me–3-MeO
2-Me–3-MeO
2-Me–3-MeO
2-Me–3-MeO
2-Me–3-MeO
2-Me–3-MeO
2-Me–3-MeO
H
2-Me
2-MeO
3-Me
3-MeO
4-Me
4-MeO
4-F
2.7/0.14
d
33.3/0.04
18.0/0.26
15.0/0.20
8.6/0.47
g
h
i
j
k
l
m
n
o
5.2/0.70
3.4/0.46
8.8/0.12
6.2/0.29
6.6/0.25
33.3/0.01
25.5/0.12
20.0/0.05
d
d
d
d
e
e
15.0/0.14
3.5/0.43
1.9/0.85
13.7/0.04
10.4/0.07
4.5/0.60
12. Garcia, J.; Nicolas, E.; Albericio, F.; Michelotti, E. L.;
Tice, C. M. Tetrahedron Lett. 2002, 43, 7495.
3-Me-4-F
3,5-diMe
41.8/0.24
2.1/0.75
13. Compound 2aa has been reported previously: Hassner, A.;
Burke, S. S.; Jesse, C. I. J. Am. Chem. Soc. 1975, 97, 4692.
14. Farnum, D. G.; Carlson, G. R. Synthesis 1972, 191.
15. A bulk transformed HEK-293 cell line, created as descri-
bed in ref 5, was provided by Gage and Suhr. Dilution cloning
was used to isolate individual clones. Clone Z3 was selected
using 450 mg mLÀ1 G418 and 100 ng mLÀ1 puromycin. Cells
aSee ref 15 for assay protocol.
bDose affording 50% of maximum transactivation.
cRatio of maximum level of gene expression of compound to max-
imum level of gene expression with 1d.
dCompound not tested.
eCompound not made.

478
C. M. Tice et al. / Bioorg. Med. Chem. Lett. 13 (2003) 475–478
were trypsinized and diluted to a concentration of 2.5 ꢁ
104 cells mLÀ1. One hundred microliters of cell suspension was
placed in each well of a 96 well plate (Dynex, 14-245-182) and
incubated at 37 ꢀC under 5% CO2 for 24 h. Ligand stock
solutions were prepared (10 mM in DMSO) and diluted 100-
fold in media. 50 mL of diluted ligand solution was added to
each well. The final concentration of DMSO was maintained
at 0.03% in both controls and treatments. b-Galactosidase
reporter gene expression was measured 48 h after treatment of
the cells using Gal ScreenTM bioluminescent reporter gene
assay system from Tropix (GSY1000). Luminescence was
detected at room temperature using a Dynex MLX microtiter
plate luminometer. Fold inductions were calculated by divid-
ing relative light units (RLU) in ligand treated cells with RLU
in DMSO treated cells.
(50 mL). Solid Na2S2O3 (2.13 g, 8.6 mmol) was added and the
mixture was stirred for 0.5 h. The mixture was extracted with
ether (150 mL). The ether extract was washed with satd aq
NaHCO3 (50 mL), dried and evaporated under reduced pres-
sure to afford 11d (293 mg, 103%) as an oil. 1H NMR
(300 MHz, CDCl3) d 0.98 (d, J=6.6 Hz, 3H), 1.04 (d,
J=6.6 Hz, 3H), 1.51 (s, 3H), 2.27 (s, 3H), 2.29 (m, 1H), 3.84 (s,
3H), 6.30 (br s, 1H), 6.91 (m, 1H), 6.96 (m, 1H), 7.18 (m, 1H),
9.60 (s, 1H). (4) Preparation of 2di: An oven dried vial equip-
ped with a stir bar was flushed with N2, charged with a stock
solution of 11d in dry THF (1 mL of 0.5 M, 0.5 mmol) and
cooled in dry ice acetone. 3-Methyl-phenylmagnesium bro-
mide in THF (1.0 M, 2 mL, 2.0 mmol) was added and the
mixture was stirred at rt for 2 h. The reaction was quenched by
addition of satd aq NaHCO3 (5 mL), applied to a 10-mL
ChemElut cartridge, allowed to stand for 5 min and eluted
with CH2Cl2 (25 mL). The eluate was evaporated to leave
crude carbinol 12di (180 mg). To a stirred solution of crude
12di in CH2Cl2 (2 mL) was added Dess–Martin periodinane
(1.4 mL, 15 wt.% in CH2Cl2, ꢂ0.65 mmol). The mixture was
stirred at rt for 6 h, diluted with satd aq NaHCO3 (5 mL) and
treated with solid Na2S2O3 (ꢂ1 g, 6.3 mmol). The mixture was
stirred for 0.5 h, applied to a 10-mL ChemElut cartridge,
allowed to stand for 5 min and eluted with CH2Cl2 (20 mL).
The eluate was evaporated to leave crude ketone 2di (95 mg).
The crude ketone was taken up in CH2Cl2 (4 mL) treated with
16. Kemp, A.; Ner, S. K.; Rees, L.; Suckling, C. J.; Tedford,
M. C.; Bell, A. R.; Wrigglesworth, R. J. Chem. Soc., Perkin
Trans. 2 1993, 741.
17. Graham, S. L.; Scholz, T. H. Tetrahedron Lett. 1990, 31,
6269.
18. Ruble, J. C.; Fu, G. C. J. Am. Chem. Soc. 1998, 120,
11532.
19. The following procedure for the preparation of 2di is
typical. (1) Preparation of 7d: To a stirred suspension of
a-methylvaline (2.62 g, 20 mmol) in pyridine (40 mL) cooled to
ꢂ5 ꢀC was added solid 3-methoxy-2-benzoyl chloride (8.31 g,
45 mmol). The mixture was allowed to warm to rt, stirred for 1
week, evaporated under reduced pressure to remove pyridine
and taken up in ether (150 mL) and water (50 mL). The
organic layer was separated, washed with 5% aq HCl (50 mL)
and satd aq NaHCO3 (50 mL), and dried over MgSO4.
Removal of the solvent left 7d (6.41 g, 122%) as an oil. 1H
NMR (300 MHz, CDCl3) d 0.97 (d, J=6.6 Hz, 3H), 1.09 (d,
J=6.6 Hz, 3H), 1.51 (s, 3H), 2.12 (m, 1H), 2.50 (s, 3H), 3.85 (s,
3H), 7.02 (m, 1H), 7.24 (m, 1H), 7.40 (m, 1H). (2) Preparation
of 10d: To a stirred solution of 7d (1.76 g, 6.7 mmol) in THF
(30 mL) at rt was added solid sodium borohydride (0.15 g,
4.0 mmol). The mixture was stirred for 16 h. Removal of the
solvent under reduced pressure left a white glassy solid which
was taken up in CH2Cl2 (150 mL), washed with 1% aq HCl
(50 mL) and satd aq NaHCO3 (50 mL) and dried over MgSO4.
Removal of the solvent left 10d (1.25 g, 69%) as a white solid,
mp 142–145 ꢀC. 1H NMR (300 MHz, CDCl3) d 0.97 (d,
J=6.6 Hz, 3H), 0.98 (d, J=6.6 Hz, 3H), 1.11 (s, 3H), 2.26 (s,
3H), 2.50 (m, 1H), 3.75 (m, 2H), 3.84 (s, 3H), 5.39 (br s, 1H),
5.83 (br s, 1H), 6.90 (m, 2H), 7.18 (m, 1H); 13C NMR
(75 MHz, CDCl3) d 12.4, 16.8, 17.0, 18.3, 31.1, 55.5, 62.4, 68.1,
111.2, 118.1, 124.0, 126.6, 138.2, 157.9, 171.3. (3) Preparation
of 11d: To a stirred solution of 10d (285 mg, 1.1 mmol) in
CH2Cl2 (10 mL) at rt was added Dess–Martin periodinane
solution (15% by weight, 2.4 mL, ꢂ1.1 mmol). The mixture
was stirred at rt for 4 h and poured into satd aq NaHCO3
Argonaut PS-TsNHNH2 resin (0.20 g, 2.9 mmol gÀ1
,
0.58 mmol) and allowed to stand for 6 h. The mixture was fil-
tered and washed with CH2Cl2 and ether. The filtrate was
evaporated to leave a solid which was fractionated on a 2 g
silica cartridge eluted sequentially with 0, 25, 50, 75 and 100%
ether in hexanes (10 mL of each). The 4th fraction (75% ether
in hexanes) contained ketone 2di (27 mg, 15%) as an off-white
solid. 1H NMR (CDCl3) d 0.94 (d, J=6.8 Hz, 3H), 1.09 (d,
J=6.7 Hz, 3H), 1.67 (s, 3H), 2.01 (s, 3H), 2.37 (s, 3H), 2.50 (m,
1H), 6.36 (br s, 1H), 6.76 (d, J=7.6 Hz, 1H), 6.84 (d, J=8.1 Hz,
1H), 7.12 (m, 1H), 7.40 (m, 2H), 7.78 (m, 2H). 13C NMR
(75 MHz, CDCl3) d 12.1, 17.1, 17.4, 17.5, 21.5, 34.3, 55.6, 67.5,
111.3, 118.3, 125.0, 125.1, 126.5, 127.7, 129.2, 132.5, 136.7,
137.6, 137.9, 158.0, 168.9, 201.0. The 3rd fraction (50% ether in
hexanes) contained additional 2di (34.5 mg, 20%) of lower purity.
20. Breitenbucher, J. G.; Arienti, K. L.; McClure, K. J. J.
Comb. Chem. 2001, 3, 528.
21. Characterization of 2bo: mp 130–132 ꢀC. 1H NMR
(CDCl3) d 1.80 (m, 4H), 1.85 (s, 3H), 2.06 (m, 2H), 2.31 (s,
6H), 2.58 (m, 2H), 3.77 (s, 3H), 6.46 (br s, 1H), 6.52 (d,
J=7.6 Hz, 1H), 6.79 (d, J=8.1 Hz, 1H), 7.04 (t, J=7.9 Hz,
1H), 7.10 (s, 1H), 7.46 (s, 2H)). 13C NMR (75 MHz, CDCl3) d
11.7, 21.3, 24.9, 37.8, 55.6, 70.9, 111.2, 118.2, 124.8, 126.1,
126.3, 133.2, 136.7, 137.3, 137.4, 157.9, 169.0, 201.7. IR
(CDCl3) 3431, 1684, 1664 cmÀ1. MS (ESI, +ve ion) m/z 366
(M+1).