Design and synthesis of highly potent benzodiazepine γ-secretase inhibitors: Preparation of (2S,3R)-3-(3,4- difluorophenyl)-2-(4-fluorophenyl)-4-hydroxy-N-((3S)-1-methyl-2-oxo-5- phenyl-2,3-dihydro-1H-benzo[e][1,4]-diazepin-3-yl)butyramide by use of an as

Article abstract of DOI:10.1021/jm034058a

Novel benzodiazepine-containing γ-secretase inhibitors for potential use in Alzheimer's disease have been designed that incorporate a substituted hydrocinnamide C-3 side chain. A syn combination of α-alkyl or aryl and β-hydroxy or hydroxymethyl substituen

Full text of DOI:10.1021/jm034058a

J . Med. Chem. 2003, 46, 2275-2278
2275
the proteases R-secretase and γ-secretase, leading to
non-amyloidogenic fragments. Alternative processing by
stepwise cleavage mediated by â-secretase and γ-secre-
tase leads to the production of Aâ, and it is inhibitors
of the latter enzyme that were targeted in the current
work.9-19
A whole-cell γ-secretase inhibition assay using
SH-SY5Y neuroblastoma cells in which human γ-secre-
tase catalyzes the breakdown of the overexpressed
exogenous substrate A4CTF has been developed.20
We have previously disclosed21,22 benzodiazepines 1 and
2, which have demonstrated in vitro inhibition of
Design a n d Syn th esis of High ly P oten t
Ben zod ia zep in e γ-Secr eta se In h ibitor s:
P r ep a r a tion of (2S,3R)-3-(3,4-
Diflu or op h en yl)-2-(4-flu or op h en yl)-4-
h yd r oxy-N-((3S)-1-m eth yl-2-oxo-5-
p h en yl-2,3-d ih yd r o-1H-ben zo[e][1,4]-
d ia zep in -3-yl)bu tyr a m id e by Use of a n
Asym m etr ic Ir ela n d -Cla isen
Rea r r a n gem en t
Ian Churcher,*,† Susie Williams,† Sonia Kerrad,†
Timothy Harrison,† J ose´ L. Castro,†
Mark S. Shearman,‡ Huw D. Lewis,‡ Earl E. Clarke,‡
J onathan D. J . Wrigley,‡ Dirk Beher,‡
Yui S. Tang,§ and Wensheng Liu§
Department of Medicinal Chemistry and Department of
Biochemistry & Molecular Biology, The Neuroscience
Research Centre, Merck Sharp & Dohme Research
Laboratories, Terlings Park, Eastwick Road, Harlow, Essex,
CM20 2QR, U.K., and Department of Drug Metabolism,
Merck Research Laboratories, 126 East Lincoln Avenue,
Rahway, New J ersey 07065
Aâ(1-40) secretion in this assay. Here, we describe the
subsequent optimization of these leads resulting in the
identification of novel, hydroxy-functionalized deriva-
tives possessing picomolar levels of potency for the in
vitro inhibition of Aâ peptide synthesis.
Ch em istr y. Initially the introduction of a hydroxyl
group into the â-position of the hydrocinnamide side
chain was investigated. In the case where the R-sub-
stituent was methyl, this was carried out using the
Evans oxazolidinone chiral auxiliary methodology23 to
yield the syn- or anti-aldol adduct with either absolute
stereochemistry. Coupling to the known, homochiral
benzodiazepine24 7 yielded the desired amides 3-6 as
shown in Scheme 1.
Functionalization of syn-aldol 3 was possible to yield
adducts 8-11; alternatively, oxidation of 3 gave the
â-keto amide 12 (exclusively in the keto form in CDCl3
solution) as a mixture of diastereomers. Treatment with
the appropriate hydroxylamine yielded the oximes 13
and 14, again as mixtures of isomers.
When the above route was applied to the preparation
of R-(4-fluorophenyl)-substituted aldol analogues, sig-
nificant retro-aldol reaction was observed at the LiOH/
H2O2-mediated auxiliary cleavage step, necessitating
the use of a silyl protecting group. This modification
allowed the preparation of the desired syn isomers
15-18 as outlined in Scheme 2.
The synthesis of â-hydroxymethyl analogue 22 pro-
ceeded via carbene addition to methyl methacrylate,
yielding after saponification the dibromocyclopropane
1925 (Scheme 3). Subsequent silver-mediated ring ex-
pansion26 gave the 4-bromofuran-2-one 20, which was
subjected to Suzuki coupling27 and hydrogenation to
yield the syn-lactone 21. When this racemic lactone was
opened with benzodiazepine 7 under aluminum cataly-
sis,28 a kinetic resolution occurred to yield adduct 22
as a single diastereomer together with recovered lac-
tone. Prolonged reaction times failed to give conversion
of the slower-reacting enantiomer of the lactone. At-
tempts to determine the relative stereochemistry of
adduct 22 were unsuccessful.
Received March 6, 2003
Abstr a ct: Novel benzodiazepine-containing γ-secretase in-
hibitors for potential use in Alzheimer’s disease have been
designed that incorporate a substituted hydrocinnamide C-3
side chain. A syn combination of R-alkyl or aryl and â-hydroxy
or hydroxymethyl substituents was shown to give highly
potent compounds. In particular, (2S,3R)-3-(3,4-difluorophe-
nyl)-2-(4-fluorophenyl)-4-hydroxy-N-((3S)-2-oxo-5-phenyl-2,3-
dihydro-1H-benzo[e][1,4]diazepin-3-yl)butyramide (34) dem-
onstrated excellent in vitro potency (IC50 ) 0.06nM). 34 could
also be selectively methylated to give [3H]-28, which is of use
in radioligand binding assays.
In tr od u ction . The 40-42 amino acid amyloid-â (Aâ)
peptide is the major component of the extracellular
proteinaceous plaques seen in Alzheimer’s disease (AD),
and much evidence suggests a pivotal role for Aâ in the
disease process.1,2 In particular, individuals possessing
autosomal dominant mutations in the genes encoding
for amyloid-â precursor protein (âAPP) or the membrane-
bound protein homologues presenilin 1 and 2 have
elevated Aâ levels and suffer from aggressive forms of
early onset AD.3-6 These observations have led to the
hypothesis that Aâ, in its soluble form or when ag-
gregated into oligomers, fibrils, and subsequently plaques,
is responsible for neuronal toxicity and cell death.7
Inhibition of Aâ synthesis is thus an attractive target
for therapeutic intervention in AD.
Αâ is derived by processing of the 695-770 residue,
type I transmembrane protein âAPP.8 The major meta-
bolic pathway of âAPP involves sequential cleavage by
* To whom correspondence should be addressed. Telephone: +44
1279 440000. Fax: +44 1279440390. E-mail: ian_churcher@merck.com.
† Department of Medicinal Chemistry, Merck Sharp & Dohme
Research Laboratories, Terlings Park.
‡ Department of Biochemistry & Molecular Biology, Merck Sharp
& Dohme Research Laboratories, Terlings Park.
The lactone-opening route used above was deemed
unsuitable for the preparation of R-(4-fluorophenyl)-
§ Department of Drug Metabolism, Merck Research Laboratories,
Rahway.
10.1021/jm034058a CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/13/2003

2276 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12
Sch em e 1. Synthesis of Aldol Isomers and Derivativesa
Letters
Sch em e 3. Synthesis of â-Hydroxymethyl Analogue 22a
a
Reagents (a) CHBr3, NaOH, BnEt3N+Cl-, H2O; (b) LiOH, THF/
H2O; (c) AgO2CCF3, CF3CH2OH, reflux; (d) 3,4-difluorophenyl-
boronic acid, Pd(PPh3)4, Na2CO3, DME/H2O, 90 °C; (e) 40 psi of
H2, Pd/C, EtOAc; (f) 7, AlMe3, CH2Cl2, reflux.
Sch em e 4. Preparation of Alternative â-Substituted
Analogues by Use of an Asymmetric Ireland-Claisen
Rearrangementa
a
Reagents: (a) 3,4-dichlorobenzaldehyde, Bu2BOTf, Et3N,
CH2Cl2, -78 °C; (b) H2O2, LiOH, THF/H2O; (c) 7, HBTU, CH3CN;
(d) 3,4-dichlorobenzaldehyde, 2 equiv of Bu2BOTf, iPr2NEt, Et2O,
-78 °C; (e) Me2SO4, NaH, DMF/THF; (f) ClSO2NCO, THF then
Na2S2O5; (g) ClSO2NH2, pyridine, CH2Cl2; (h) ICH2CONH2, NaH,
DMF/THF; (i) Dess-Martin periodinane, CH2Cl2; (j) H2NOR.HCl,
pyridine, EtOH.
Sch em e 2. Preparation of syn-R-(4-Fluorophenyl)aldol
Analoguesa
a
Reagents: (a) MeI, K2CO3, DMF; (b) DIBAL-H, THF, -10
°C; (c) 4-fluorophenylacetyl chloride, Et3N, CH2Cl2, 0 °C; (d) 24,
BBr3, Et3N, PhMe (see text for details); (e) 40 psi of H2, Pd/C,
EtOH; (f) 7, EDC, HOBt, CH2Cl2; (g) O3, MeOH, CH2Cl2, -78 °C,
then NaBH4, -78 °C to room temp; (h) PPh3, CBr4, CH2Cl2; (i)
Bu3SnH, AIBN, PhH, reflux.
a
Reagents: (a) 4-fluorophenylacetyl chloride, nBuLi, THF, -78
°C; (b) 3,4-dichlorobenzaldehyde or 3,4-difluorobenzaldehyde,
Bu2BOTf, Et3N, CH2Cl2, -78 °C; (c) TBSOTf, 2,6-lutidine, CH2Cl2,
0 °C; (d) H2O2, LiOH, THF/H2O; (e) TBAF, THF; (f) 7, EDC, HOBt,
CH2Cl2.
benzodiazepine 7 to give complete conversion to a single
diastereomer by NMR and HPLC analysis.
An improvement in the yield and ease of reaction was
observed by modifying the reaction conditions (for the
full procedure, see Supporting Information). Addition
of the ester to the bromoborane catalyst derived from
bis-sulfonamide 2429 was carried out at -78 °C, and
stirring was maintained at this temperature for 1 h.
Subsequent slow warming to ambient temperature over
16 h afforded, after purification, a 79% yield of the
desired isomer with the ee undiminished. This reaction
protocol was amenable to scale (>5 g) with the chiral
ligand routinely reisolated and reused after recrystal-
lization (dichloromethane/isohexane).
substituted adducts analogous to 22 because of the
likelihood of epimerization of the syn-3,4-bis-aryllactone
analogous to 21 and its potentially low reactivity toward
aminolysis. This, coupled with the desire for a flexible,
enantioselective route prompted us to investigate the
use of an asymmetric Ireland-Claisen reaction.29,30 The
appropriate substrate 23 for this rearrangement was
easily accessible from 3,4-difluorocinnamic acid as
shown in Scheme 4. Use of the standard Corey protocol29
(rearrangement at -78 °C/24h then +4 °C/48h) gave the
desired syn pentenoic acid derivative 25 in good (63%)
yield. The enantiomeric excess (ee) was determined to
be >99% by coupling to excess, homochiral (>99% ee)
The substituted pentenoic acid 25 was hydrogenated
and coupled to benzodiazepine 7 to yield the â-ethyl
compound 26. Alternatively, direct coupling to give 27,

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12 2277
Ta ble 1. Inhibitory Activities of Aldol Adducts and Derivatives
1-6 and 8-18
Sch em e 5. Preparation of Alternative Hydroxylated
Derivatives and Tritiated 28a
a
a
compd
IC50 ( SD (nM)
compd
IC50 ( SD (nM)
1
2
3
4
5
6
8
9
10
320 ( 200
15 ( 8
11
12
13
14
15
16
17
18
4750 ( 2700
930 ( 700
35 ( 19
19 ( 6
1340 ( 540
880 ( 310
380 ( 190
59 ( 25
2450 ( 55
450 ( 290
>10000
1.2 ( 0.9
0.8 ( 0.7
67 ( 50
9.6 ( 6
a
IC50 values were determined over the range of 0.3 nM to 10
µM and are the mean of at least three determinations.
Ta ble 2. Inhibitory Activities of Further â-Functionalized
Analogues
a
a
compd
IC50 ( SD (nM)
compd
IC50 ( SD (nM)
22
26
27
28
29
6.9 ( 4
30
32
34
36
1.5 ( 0.9
212 ( 160
100 ( 70
0.07 ( 0.04
6.7 ( 4
35 ( 23
0.06 ( 0.03
0.07 ( 0.03
a
IC50 values were determined over the range of 0.3 nM to 10
µM or 0.003-100 nM as appropriate and are the mean of at least
three determinations.
a
Reagents: (a) BnBr, K2CO3, DMF; (b) BH3‚THF, THF, then
H2O2; (c) TBSOTf, 2,6-lutidine, CH2Cl2, -78 °C; (d) 40 psi of H2,
Pd/C, MeOH; (e) 33, EDC, HOBt, CH2Cl2; (f) TBAF, AcOH, THF,
40 °C; (g) 25, EDC, HOBt, CH2Cl2; (h) O3, MeOH, CH2Cl2, -78
°C, then NaBH4, -78 °C to room temp; (i) CT3I, NaH, THF, 40
°C, 2 h.
Oxidation to the â-keto amide 12 (927 nM) was
attended by a large reduction in potency relative to 3,
although formation of the oxime 13 (35 nM as a
diastereomeric mixture) allowed activity to be regained.
The inactivity of methyl oxime 14 may suggest an
important role for the hydroxyl function in the binding
of these side chains.
followed by ozonolysis with in situ reduction yielded
alcohol 28. This was deoxygenated by conversion to the
bromide 29 followed by radical debromination (Bu3SnH)
to afford the â-methyl analogue 30.
To investigate the effect of increasing further the
length of the linking chain to the hydroxyl group, the
â-hydroxyethyl compound 32 was prepared as shown
in Scheme 5. Ireland-Claisen product 25 was benzyl-
protected and hydroborated to give alcohol 31. Subse-
quent manipulation of protecting groups and coupling
to benzodiazepine 3324 yielded the desired analogue
32.
The â-hydroxymethyl side chain was also coupled to
the N(1)-H benzodiazepine 33 and racemic 1,5-benzo-
diazepine 3531 to yield the analogues 34 and 36 (after
isomer separation), respectively. Selective N-methyla-
tion of 34 was also possible, and by use of CT3I, this
procedure proved to be amenable to the preparation of
the radioligand (CT3)-28.
Resu lts a n d Discu ssion . Previous work within our
group had identified 1-methyl-2-oxo-5-phenyl-2,3-dihy-
dro-1H-benzo[e][1,4]diazepines bearing an N-hydrocin-
namide side chain at the 3-position to be a novel class
of γ-secretase inhibitors.21 Introduction of a substituent
at the R-position of the side chain as in 1 (320 nM) or 2
(15 nM) was found to greatly increase potency.
Further development focused on introduction of a
â-substituent, and the inhibitory activities of a range
of such compounds are summarized in Table 1. Prepa-
ration of the four aldol stereoisomers (3-6) demon-
strated an increase in potency with the (2R,3R) isomer
only (3, 19 nM), the other isomers being at best
equipotent to analogue 1. The preference for the (2R)
configuration is opposite that seen with non-â-substi-
tuted derivatives.8 Hydroxyl derivatization was gener-
ally poorly tolerated (8-11) with only the â-methoxy
analogue 8 (59 nM) retaining moderate potency.
Previous studies had shown that use of an R-(4-
fluorophenyl) substituent allowed preparation of more
potent analogues (cf. 1 vs 2), and this was also the case
in the current â-hydroxy series of compounds. Thus, 15
(1.2 nM) showed an increase in potency relative to
methyl-substituted 3 with the (2R,3R) stereochemistry
again preferred (2S,3S isomer 17, 67 nM). Exchange of
the chlorine substituents for fluorine, a modification
that had previously been shown to improve potency,21
gave 16 (IC50 ) 0.8 nM).
Although the initial investigation of substituents at
the â-position had suggested a limited degree of steric
tolerance (cf. 8-11), the increase in potency observed
on going from ketone 12 to oxime 13 suggested that
introduction of a homologated alcohol moiety may be
advantageous. Thus, we next prepared compounds
bearing a â-hydroxymethyl function, and activities of
representative compounds are summarized in Table 2.
Initial synthesis of the chemically more tractable
R-methyl analogue 22 (6.9 nM) as a single, undefined
syn isomer demonstrated an increase in potency relative
to the corresponding â-hydroxy compound 3.
By use of a modified asymmetric Ireland-Claisen
rearrangement, the corresponding R-(4-fluorophenyl)-
â-hydroxymethyl analogue was next prepared with the
syn (now 2S,3R) stereochemistry (corresponding to 16)
targeted. Use of the Lewis acid catalyst derived from
(S,S)-bis-sulfonamide 24 in the key rearrangement
yielded desired 28, which demonstrated greatly en-
hanced potency (IC50 ) 0.07 nM). Analogues bearing a
lipophilic â-substituent (26, 27, 29, 30) all showed
reduced levels of potency with only the â-methyl ana-
logue 30 retaining moderate activity, suggesting an

2278 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12
Letters
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Harden, P.; Scardina, J . M.; Mavunkel, B.; Luedtke, G. R.;
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interaction of the hydroxyl group to be important in the
binding of these analogues.
Further hydroxyl homologation to the â-hydroxylethyl
analogue 32 led to a large reduction in potency. The
optimal C-3 amide side chain could also be introduced
onto other benzodiazepine cores to yield, for example,
34 or 36 with maintenance of excellent levels of potency.
Selective N-methylation of 34 was also possible, and this
transformation using CT3I was utilized in the synthesis
of radiolabel (CT3)-28, which has found use in in vitro
binding studies.32
Con clu sion s. A series of benzodiazepine γ-secretase
inhibitors bearing a C-3 hydrocinnamide side chain has
been developed and yielded highly potent compounds
(e.g., 34, 0.06 nM) resulting from systematic optimiza-
tion of hydroxylated derivatives. This compound was
prepared using a modified Corey asymmetric Ireland-
Claisen rearrangement. The N(1)-H analogue 34 could
also be radiolabeled by N-methylation to yield the useful
radioligand (CT3)-28.
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Ack n ow led gm en t. The authors thank Beth Oxley,
Robert Newman, and Ian Gowers for assistance in
screening and the Terlings Park Physical Methods
group for analytical support.
Su p p or tin g In for m a tion Ava ila ble: Spectral data for all
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