Design, synthesis, and X-ray structure of potent memapsin 2 (β-secretase) inhibitors with isophthalamide derivatives as the P 2 P3-ligands

Article abstract of DOI:10.1021/jm061338s

Structure-based design and synthesis of a number of potent and selective memapsin 2 inhibitors are described. These inhibitors were designed based upon the X-ray structure of memapsin 2-bound inhibitor 3 that incorporates methylsulfonyl alanine as the P

Full text of DOI:10.1021/jm061338s

J. Med. Chem. 2007, 50, 2399-2407
2399
Design, Synthesis, and X-ray Structure of Potent Memapsin 2 (â-Secretase) Inhibitors with
Isophthalamide Derivatives as the P2-P3-Ligands
Arun K. Ghosh,*,† Nagaswamy Kumaragurubaran,† Lin Hong,‡,§ Sarang S. Kulkarni,† Xiaoming Xu,† Wanpin Chang,§
Vajira Weerasena,‡ Robert Turner,‡ Gerald Koelsch,‡,§ Geoffrey Bilcer,‡ and Jordan Tang§,
Departments of Chemistry and Medicinal Chemistry, Purdue UniVersity, West Lafayette, Indiana 47907, Athenagen Inc, Oklahoma City,
Oklahoma 73104, Protein Studies Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, and Department of
Biochemistry and Molecular Biology, UniVersity of Oklahoma Health Science Center, Oklahoma City, Oklahoma 73104
ReceiVed NoVember 17, 2006
Structure-based design and synthesis of a number of potent and selective memapsin 2 inhibitors are described.
These inhibitors were designed based upon the X-ray structure of memapsin 2-bound inhibitor 3 that
incorporates methylsulfonyl alanine as the P2-ligand and a substituted pyrazole as the P3-ligand. Of particular
importance, we examined the ability of the substituted isophthalic acid amide derivative to mimic the key
interactions in the S2-S3 regions of the enzyme active sites of 3-bound memapsin 2. We investigated various
substituted phenylethyl, R-methylbenzyl, and oxazolylmethyl groups as the P3-ligands. A number of inhibitors
exhibited very potent inhibitory activity against mempasin 2 and good selectivity against memapsin 1. Inhibitor
5d has shown low nanomolar enzyme inhibitory potency (Ki ) 1.1 nM) and very good cellular inhibitory
activity (IC50 ) 39 nM). Furthermore, in a preliminary study, inhibitor 5d has shown 30% reduction of
Aâ40 production in transgenic mice after a single intraperitoneal administration (8 mg/kg). A protein-
ligand X-ray crystal structure of 5d-bound memapsin 2 provided vital molecular insight that can serve as
an important guide to further design of novel inhibitors.
Introduction
cellular protein catabolism.12 Therefore, development of selec-
tivity against these other aspartyl proteases may be important
due to the potential toxicity. We have recently designed and
developed very potent and highly selective memapsin 2 inhibi-
tors that incorporate methylsulfonyl alanine as the P2-ligand
along with pyrazole- and oxazole-derived heterocycles as the
P3-ligands. These inhibitors (3 and 4) exhibited enhanced
potency against memapsin 2 and excellent selectivity over
memapsin 1 and cathepsin D.10 The protein-ligand X-ray
structure of the pyrazole-bearing inhibitor 3 provided important
molecular insight into the specific cooperative ligand-binding
site interactions for selectivity design.10 The inhibitor 3-bound
memapsin 2 X-ray structure indicated a number of important
interactions in the S2 and S3 sites. The P2-sulfone oxygens
appear to interact with a tightly bound water molecule as well
as involve in hydrogen bond with Arg-235 in the S2 site. Also,
one of the P3-pyrazole nitrogens is within hydrogen-bonding
distance to Thr-232, and the dimethyl groups on the pyrazole
ring appear to effectively fill the S3 pocket. In our continuing
efforts toward development of novel memapsin 2 inhibitors, we
now have investigated the suitability of substituted isophthala-
mide-based ligands to interact with various residues at the S2
and S3 regions of the enzyme active site. A number of reports
incorporating isopthalamide-based P2-ligand in memapsin 2
inhibitor design have appeared in literature.13 Recently, Coburn
and co-workers incorporated an N-sulfonyl derivative at the
3-position of the isopthalamide ring to interact with residues in
the S2 pocket.14a Herein, we report the results of our preliminary
studies regarding the design and synthesis of a series of potent
memapsin 2 inhibitors that incorporate substituted isopthala-
mides as P2-P3 ligands in combination with the Leu-Ala
hydroxyethylene dipeptide isostere. A number of inhibitors have
shown excellent memapsin 2 inhibitory potency. One of these
inhibitors 5d has shown good selectivity over memapsin 1 and
cathepsin D as well as good cellular inhibitory activity of
memapsin 2 in Chinese hamster ovary cells. Furthermore, the
The deposition of amyloid â-peptide (Aâ) in the brain is one
of the major events in the pathogenesis of Alzheimer’s disease
(AD).1,2 Aâ is formed by an initial cleavage of the membrane-
anchored â-amyloid precursor protein (APP) by two proteases
known as â- and γ-secretases. â-Secretase (BACE-1, memapsin
2) cleaves APP first in its luminal domain to form a membrane-
bound C-terminal fragment of APP. This cleavage followed by
hydrolysis by γ-secretase thus generates the 40-42 residue Aâ
peptides.3 Since â-secretase catalyzes the first step in Aâ
production, it has become an attractive therapeutic target for
Alzheimer’s disease.4
On the basis of preliminary substrate specificity and kinetic
information, we designed a number of potent peptidomemetic
inhibitors incorporating a nonhydrolyzable Leu-Ala-based hy-
droxyethylene dipeptide isostere.5,6 One of these inhibitors,
OM99-2 (1), exhibited a Ki value of 1.6 nM against memapsin
2.7 The X-ray crystal structure of 1-bound memapsin 2 provided
important molecular insight into the ligand binding interactions
in the active site.8 Our structure-based modifications led to the
discovery of potent memapsin 2 inhibitors with reduced
molecular weight.9 However, these inhibitors lacked important
selectivity against other aspartyl proteases. For example, inhibi-
tor 2 showed Ki values of 1.6 nM and 1.2 nM against human
memapsin 2 and memapsin 1, respectively.10 The selectivity of
memapsin 2 inhibitors over other human aspartic proteases is
expected to be important, particularly against memapsin 1 and
cathepsin D. Memapsin 1 has specificity similarity to memapsin
2,11 and it is presumed to have important physiological functions.
Cathepsin D is abundant in cells and plays an important role in
* To whom correspondence should be addressed. Phone: (765)-494-
5323; fax: (765)-496-1612; e-mail:akghosh@purdue.edu.
† Purdue University.
‡ Athenagen Inc.
§ Oklahoma Medical Research Foundation.
University of Oklahoma Health Science Center.
10.1021/jm061338s CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/14/2007

2400 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Ghosh et al.
Scheme 2. Synthesis of Isophthalamide Ligands (6a-g)
Scheme 3. Synthesis of Oxazolylamines 13 and 18
Figure 1. Structure of inhibitors 1-4.
Scheme 1. General Synthetic Route to Inhibitors
9. The requisite synthesis of isophthalamide derivatives from
commercially available dimethyl 5-aminoisophthalate 10 is
shown in Scheme 2. Mesylation of amine 10 with mesyl chloride
in pyridine followed by alkylation of the resulting mesylate using
sodium hydride and methyl iodide, as reported by Stachel and
co-workers,14b provided the corresponding N-methylsulfonamide
derivative. Selective hydrolysis of the bis-ester with aqueous
sodium hydroxide afforded mono-carboxylic acid 11. Coupling
of acid 11 with various amines (12-18) using EDC/HOBT in
the presence of i-Pr2NEt afforded the respective amide deriva-
tives in moderate to good yield (40-65%). Saponification of
the resulting ester provided the required isophthalic acid
derivatives 6a-g in good yields.
Amines 12 and 14-17 were obtained from commercial
sources and amines 13 and 18 were prepared as shown in
Scheme 3. Reduction of the known15 oxazole ester 19 with
excess of Dibal-H in CH2Cl2 at -78 °C afforded the corre-
sponding alcohol. Azidation of the resulting alcohol with
diphenylphosphoryl azide in the presence of DBU provided the
corresponding azide. Reduction of azide with triphenyl phos-
phine in THF afforded amine 13. The synthesis of racemic
amine 18 was also carried out from the same oxazole ester 19.
As shown, selectivereduction of ester 19 with 1.2 equiv of
protein-ligand X-ray structure of this inhibitor provided
important molecular insight into ligand-binding site interactions
for further design.
Chemistry. Our general strategy for the synthesis of inhibitors
containing the Leu-Ala hydroxyethylene isostere and isoph-
thalamide derivatives is outlined in Scheme 1. Various inhibitors
can be synthesized by coupling isophthalic acid derivatives 6
and the amine derived from BOC-derivative 7. Amine 7 is
obtained by coupling of Leu-Ala acid 8 and valine derivative

Memapsin 2 (â-Secretase) Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2401
Table 1. Structure and Inhibitory Potency of Inhibitors
Scheme 4. Synthesis of Inhibitors 5a-g
Scheme 5. Synthesis of Inhibitors 22 and 24
Dibal-H afforded the corresponding aldehyde. Reaction of the
resulting aldehyde with methyl magnesium bromide in THF
furnished racemic alcohol 20. This alcohol was then converted
to amine 18 by azidation followed by reduction as described
for compound 13.
examined the ability of an unsubstituted isophthalimide deriva-
tive to function as the P2-ligand with a Leu-Ala isostere. As
shown, incorporation of propyl isophthalamide as the P2- and
P3-ligands in place of P2-methylsulfonyl alanine and P3-
oxazolylmethyl ligands provided inhibitor 22 with a Ki value
of 788 nM. This inhibitor is considerably less potent than
inhibitors with amino acid-derived ligands described previously.9a
In an effort to interact with the residues in the S2-site, we
examined the potential of the N-methylsulfonamide functionality
on the P2-isophthalamide ligand. The corresponding inhibitor
5a exhibited a significant improvement in memapsin 2 inhibitory
potency compared to 22. This is consistent with our energy-
minimized model structure of inhibitor 4 and a prototype
inhibitor 5b where P2-methylsulfonyl alanine is replaced with
a N-methylsulfonyl-substituted isophthalimide P2-ligand, and P3-
oxazolyl urethane is replaced with an oxazolylmethyl amide as
shown in Figure 2. The model of inhibitor 4 was created based
upon the crystal structure of inhibitor 3-bound memapsin 2
reported by us recently.10 Compound 5b exhibited a Ki value
of 1.8 nM, a 28-fold potency enhancement over propylamide
The general synthesis of inhibitors containing the Leu-Ala
hydroxyethylene isostere and isophthalamide derivatives is
shown in Scheme 4. Coupling of previously described7,9 Leu-
Ala isostere 8 with valine derivative 9 using EDC and HOBt in
the presence of i-Pr2NEt afforded amide derivative 7. Removal
of the BOC group by exposure of 7 to trifluoroacetic acid in
CH2Cl2 at room temperature provided the corresponding amine
derivative. Reaction of the resulting amine with various acids
6a-g provided the respective coupling product. Removal of
the TBS-group with tetrabutylammonium fluoride in THF
furnished inhibitors 5a-g in good yields. Inhibitor 22 was
prepared by analogous procedure using 7-derived amine and
the propyl amide of isophthalic acid 21 (Scheme 5). The known
â-secretase inhibitor 24 was prepared from commercially
available BOC-oxirane 23 as described in the literature.14b
Results and Discussion
Structure and inhibitory potency of various inhibitors against
recombinant memapsin 2 are shown in Table 1. We initially

2402 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Ghosh et al.
Figure 2. Inhibitors 4 (red) and 5b (green) modeled into the active site of memapsin 2. Inhibitor model 4 was created based upon X-ray structure
of 3-bound memapsin 2.
derivative 5a. The molecular model in Figure 2 suggests that
the N-methylsulfonamide derivative in 5b may interact with
active site residues similar to the P2-sulfone of inhibitor 4. In
addition, the model suggests an additional interaction which may
contribute to the increased potency of compound 5b. The P3-
dimethyloxazole in 5b appears to effectively fill in the hydro-
phobic pocket in the S3-subsite. On the basis of this speculation,
we explored the suitability of other hydrophobic groups as P3-
ligands. As can be seen, inhibitor 5c containing a P3-benzyl
amide is significantly less potent than 5b. Incorporation of an
R-methylbenzyl amide (R-configuration) resulted in a very
potent memapsin 2 inhibitor 5d. This inhibitor exhibited a
memapsin 2 Ki of 1.1 nM, a 120-fold improvement over the
benzylamide derivative 5c. The inhibitor with S-configuration
(5e), however, proved to be significantly less potent, thus
indicating a strong stereochemical preference at this position.
This is consistent with the observation reported by Stachel and
co-workers in their studies with hydroxyethylamine isostere.14b
Also, the polar R-hydroxymethyl substituent with S-configura-
tion (inhibitor 5f) was not very effective. Since the R-methyl
group of the P3-ligand in inhibitor 5d is preferred by the S3-
subsite of memapsin 2, we decided to incorporate an R-methyl
group in the P3-oxazolylmethyl derivative of inhibitor 5b. For
preliminary evaluation, we prepared inhibitor 5g as a diaster-
eomeric mixture (1:1) at the P3-stereocenter, and we have
determined the inhibitory potency with this mixture of diaster-
eomers. While still a potent inhibitor (Ki ) 27 nM), this
compound displayed reduced potency compared to unsubstituted
inhibitor 5b. However, actual contribution of individual dia-
stereomers has not been determined.
In a preliminary investigation, we have also examined the in
ViVo inhibitory effect on brain Aâ-production in transgenic mice
by inhibitor 5d. Intraperitoneal administration of inhibitor 5d
to Tg2576 mice18 effected a 30% reduction of Aâ40 production
at 4 h after a single administration at 8 mg/kg (Figure 3). The
inhibition of memapsin 2 activity in ViVo was significant relative
to the vehicle-treated control animals (p ) 0.001) and relative
to the baseline Aâ40 levels for the treated group (p ) 0.01).
The exhibition of Aâ40 inhibition at 4 h after a single injection
of 5d follows the established Aâ40 inhibition profile by the
prototypic conjugated pseudopeptide inhibitor OM003-DR9
designed in our laboratories.16 Since Aâ production in this
mouse model is brain specific at the age used in the study18
and the efflux of Aâ from brain to plasma occurs rapidly,16
there is a good possibility that the observed plasma Aâ40
reduction is mainly due to an inhibition of Aâ40 production in
the brain. However, further studies will be needed to verify the
pharmacokinetic aspects of 5d including the brain penetration.
In order to obtain molecular insight into the enzyme-inhibitor
interactions of inhibitor 5d, we determined the crystal structure
of inhibitor-bound memapsin 2 at 2.5 Å resolution. A stereoview
of 5d-bound memapsin 2 structure is shown in Figure 4. The
transition-state hydroxyl group of the Leu-Ala isostere forms
two tight hydrogen bonds (2.4 and 2.7 Å bond distances) with
both active site aspartic acid residues (Asp32 and Asp228).7
The P3-phenyl ring occupies a unique position which spans S3
and S4 subsites and causes a significant positional shift of a
protein loop containing residues from 8 to 13 (the 10’s loop)19
located in the S3/S4 pocket. This conformational change to
accommodate the bound inhibitor identifies a flexible part of
the active site cleft which can be further exploited for ligand
design. This binding pocket consists of residues 11-14, 229-
232, 307, and 335 which make direct contact with the P3-phenyl
ring group.
The P2-sulfonamide functionality fits well into the S2 pocket
and makes extensive interactions with memapsin 2. One of the
sulfone oxygen atoms is within hydrogen-bonding distance with
the Asn233 amide nitrogen and the Ser325 hydroxyl oxygen
(both at 3.0 Å bond distance). The same sulfone oxygen also
makes ionic interactions with the guanidine side chain of
Arg235. This side chain appeared to be quite flexible and
adopted a different conformation than our previously reported
structures.7, 20 The other sulfonamide oxygen makes hydrogen
bonds to Thr232 and Asn233 main chain nitrogen atoms (3.2
and 2.8 Å bond distances, respectively). Significant Van der
Waal interactions with the nonpolar atoms of Thr231, 232,
Asn233, and Arg235 are also evident. The P3′-isopropyl group
makes Van der Waal contacts with the flap residues Pro70,
We then evaluated the potencies of selected inhibitors against
recombinant memapsin 1 and human cathepsin D. Furthermore,
we determined the cellular memapsin 2 inhibitory potency of
these compounds in Chinese hamster ovary cells.16 The results
are shown in Table 2. Inhibitor 5a showed a modest reduction
of potency against memapsin 1 and cathepsin D. Compound
5b, however, is a very potent and selective memapsin 2 inhibitor.
It is over 308-fold selective against memapsin 1 and over 136-
fold selective against cathepsin D. Its cellular IC50 value is
similar to inhibitor 4.10 Inhibitor 5d is the most potent memapsin
2 inhibitor in this series. It exhibited a modest selectivity against
memapsin 1 (28-fold) and cathepsin D (37-fold). Of particular
note, 5d displayed excellent cellular inhibition of memapsin 2
(39 nM). In comparison, an in-house prepared inhibitor 2414b
with a hydroxyethylamine isostere showed little to no selectivity
against memapsin 1 and cathespin D.17 This inhibitor is also
less potent in our cellular assay.16

Memapsin 2 (â-Secretase) Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2403
Table 2. Inhibitory and Cellular Potency of Inhibitors
and benzophenone; dichloromethane, pyridine, triethylamine, and
diisopropylethylamine, distilled from CaH2. All other solvents were
HPLC grade. Column chromatography was performed with What-
man 240-400 mesh silica gel under low pressure of 5-10 psi.
TLC was carried out with E. Merck silica gel 60-F-254 plates. 1H
and 13C NMR spectra were recorded on Varian Mercury 300 and
Bruker Avance 400 and 500 spectrometers. Infrared spectra were
recorded on a Mattson Genesis II FTIR instrument. Optical rotations
were measured using a Perkin-Elmer 341 polarimeter.
compd M2a (nM) M1b (nM) CDc (nM) M1/M2 CD/M2 IC50 (µM)
5a
5b
5c
5d
5f
52.5 ( 7.4 340 ( 37 483 ( 96
1.8 ( 1.1 556 ( 16 245 ( 17
6.5
308
3.8
28
1
9.2
136
-
3.6 ( 3.2
1.8 ( 0.34
5.6 ( 1.85
136 ( 8.5 523 ( 302
nd
1.1 ( 0.37 31 ( 10.7 41 ( 12.4
438 ( 52 397 ( 123 633 ( 116
27 ( 1.8 408 ( 97 490 ( 103
233 ( 23 240 ( 47 448 ( 368
37 0.039 ( 0.013
1.4
18
2
190 ( 309
10.3 ( 5.7
0.078 ( 0.009
5g
24
15
1
Leu-Ala Derivative 7. The dipeptide isostere 87 (209 mg, 0.5
mmol) and the amine 910 (82 mg, 0.52 mmol) were dissolved in
CH2Cl2 (5 mL). To this solution EDC (115 mg, 0.6 mmol) and
HOBT (81 mg, 0.6 mmol) followed by diisopropylethylamine (0.5
mL) were successively added and stirred overnight at room
temperature. After this period, water was added and extracted with
ethyl acetate. The combined organic layers were washed with
aqueous NaHCO3 and brine and dried over anhydrous Na2SO4. The
solvents were evaporated under reduced pressure and purified by
silica gel flash column chromatography (30% EtOAc in hexanes)
to provide 7 in 94% yield (0.26 g) as a white solid. 1H NMR (300
MHz, CDCl3): δ 0.04 (3H, s), 0.05 (3H, s), 0.86-0.96 (21H, m),
1.07-1.22 (10H, m), 1.31-1.50 (11H, m), 1.54-1.62 (1H, m),
1.71-1.80 (1H, m), 1.99-2.06 (1H, m), 2.47-2.54 (1H, m), 3.60-
3.72 (2H, m), 3.99-4.09 (2H, m), 4.49 (1H, br d J ) 9.9 Hz),
6.17 (1H, br d J ) 7.5 Hz), 6.33 (1H, br d J ) 8.9 Hz). 13C NMR
(75 MHz, CDCl3): δ 176.4, 170.2, 156.2, 79.2, 72.0, 59.0, 50.1,
42.0, 41.2, 38.3, 37.24, 30.7, 28.5, 25.9, 24.9, 23.1, 22.7, 22.5, 22.2,
19.3, 18.7, 18.1, 16.6, -4.0, -4.4.
a M2: Memapsin-2/BACE-1. b M1: Memapsin-1/BACE-2. c CD: Cathe-
psin D.
Figure 3. Inhibitor 5d reduces Aâ40 production following a single
intraperitoneal administration at 8 mg/kg. Plasma Aâ40 assessed at 4 h
postinjection was expressed relative to baseline levels of individual
mice and was significantly lower than the vehicle-treated control group
(p ) 0.001).
3-Methoxycarbonyl-5-(N-methylmethan-5-ylsulfonamido)-
benzoic Acid 11. To a stirred solution of dimethyl 5-aminoisoph-
thalate (2g, 10 mmol) in CH2Cl2 (30 mL) at 0 °C was added
pyridine (2.4 mL, 30 mmol) followed by methanesulfonyl chloride
(0.9 mL, 11 mmol). The resulting solution was allowed to warm
to 23 °C and stirred for 14 h. The solvent was removed under
reduced pressure. Ethyl acetate was added to the residue which
resulted in the formation of a precipitate. The precipitate was filtered
and washed with hexane to give the corresponding sulfonamide as
an amorphous solid (2.7 g, 95%).
To a stirred suspension of NaH (0.4 g, 10 mmol, 60% oil
dispersion) in DMF (10 mL) at 23 °C, the above sulfonamide (1.4
g, 5 mmol) followed by methyl iodide (0.6 mL, 10 mmol) was
added. The reaction mixture was stirred at 23 °C for 5 h. After this
period, the reaction was carefully quenched with water, and the
aqueous layer was extracted with ethyl acetate (3×). The combined
organic layer was washed with brine, dried over anhydrous Na2-
SO4, and concentrated under reduced pressure to provide the
corresponding methyl sulfonamide (1.4 g, 91%).
Thr72, and Arg128. It appears that the isopropyl/flap interactions
facilitate two directional hydrogen bonds with the P2′-carbonyl
to Tyr198 hydroxyl group and the P3′-NH to carbonyl of Pro70.
The protein/ligand interactions at P1, P1′, and P2′ are very similar
to protein-ligand structures reported by us previously.7,20,21
Conclusion
In conclusion, our structure-based design strategies led to
design of novel memapsin 2 inhibitors incorporating various
substituted isophthalamides as P2-P3 ligands in inhibitors with
a hydroxyethylene dipeptide isostere. The inhibitors were
designed to interact with various residues at the S2- and S3-
sites of the memapsin 2 active site. Of particular interest, we
examined the structural requirements for selectivity over other
aspartyl proteases and improvement of cellular inhibitory
potency. Inhibitor 5b exhibited excellent potency against
memapsin 2 and selectivity over memapsin 1 and Cathepsin D.
However, its cellular memapsin 2 inhibitory potency was not
improved. Inhibitor 5d, on the other hand showed very good
enzyme inhibitory activity as well as cellular inhibitory potency
in Chinese hamster ovary cells (46-fold improvement over 5b).
Intraperitoneal administration of inhibitor 5d to Tg2576 mice
resulted in a 30% reduction of Aâ40 production at 4 h after a
single dose of 8 mg/kg. An inhibitor-bound X-ray crystal
structure of 5d-bound memapsin 2 (2.5 Å) indicated extensive
critical interactions in the enzyme active site. Particularly notable
are the interactions with the P2-sulfonamide functionality at the
S2-region of memapsin 2 and an interesting conformational
change in the 10’s loop upon inhibitor binding to accommodate
the P3-phenyl ring. This molecular insight will be utilized to
design more potent and selective inhibitors. Further design and
synthesis of inhibitors based upon the current molecular insight
are in progress.
To a stirred solution of above diester (1.4 g, 4.5 mmol) in a
mixture (1:1:1) of THF, MeOH, and water (15 mL) at 0 °C, solid
NaOH (180 mg, 4.5 mmol) was added and the mixture was stirred
for 14 h at 23 °C. After this period, the solvent was evaporated
and saturated NaHCO3 (10 mL) was added. The aqueous layer was
extracted with toluene (2×) to remove unreacted diester. Aqueous
layer was then acidified with 1 N HCl to pH 3 and extracted with
EtOAc (3×). The combined organic layer was dried over anhydrous
Na2SO4 and concentrated under reduced pressure to provide mono
1
acid 11 as an amorphous solid (1.1 g, 82%). H NMR (500 MHz,
CD3OD): δ 2.97 (s, 3H), 3.42 (s, 3H), 3.99 (s, 3H), 8.29-8.31
(m, 2H), 8.57-8.59 (m, 1H).
2,5-Dimethyloxazol-4-ylmethanamine 13. To a stirred solution
of (2,5-dimethyloxazol-4-yl)methanol (127 mg, 1.0 mmol) in
toluene (5 mL) were added DPPA (216 µL, 1.0 mmol) and DBU
(180 µL, 1.2 mmol) at 0 °C. The reaction mixture was warmed to
room temperature and stirred. After 17 h, the reaction mixture was
quenched with 10% aqueous dil HCl and extracted with EtOAc.
The combined layers were washed with brine, dried over anhydrous
Na2SO4, and concentrated under reduced pressure. The crude
product thus obtained was purified by silica gel flash column
chromatography (3% MeOH/CHCl3) to give the corresponding azide
Experimental Section
General. All moisture sensitive reactions were carried out under
nitrogen or argon atmosphere. Anhydrous solvents were obtained
as follows: THF, diethyl ether, and benzene, distilled from sodium
1
in 77% yield (117.5 mg). H NMR (300 MHz, CDCl3): δ 2.25
(3H, s), 2.37 (3H, s), 4.12 (2H, s).

2404 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Ghosh et al.
Figure 4. Stereoview of inhibitor 5d-bound memapsin 2 X-ray structure. The residues contacting the inhibitor (green) are shown in yellow, and
the protein/ligand hydrogen bonds are shown in white dotted lines.
The above azide (117 mg, 0.7 mmol) was taken up in THF, and
PPh3 (183.6 mg, 0.7 mmol) was added. The reaction mixture was
stirred at room temperature for 7 h. Then, a few drops of water
were added and refluxed at 55 °C for overnight. Evaporation of
solvent followed by flash column chromatography over silica gel
(10-15% MeOH/CHCl3 and 2% Et3N) afforded amine 13 as a
to remove organic impurities. The aqueous layer was cooled to 0
°C, acidified with 1 N HCl, extracted with EtOAc, and dried over
anhydrous Na2SO4. The solvent was evaporated and dried under
reduced pressure to give pure acid 21 as a white solid in 69% yield
1
(105 mg). H NMR (CD3OD, 300 MHz): δ 0.9 (t, J ) 6.5 Hz,
3H), 1.58 (q, J ) 6.5 Hz, 2H), 3.3 (m, 2H), 7.5 (t, J ) 8.4 Hz,
1H), 7.93 (d, J ) 8 Hz, 1H), 8.1 (d, J ) 8 Hz, 1H), 8.4 (s, 1H), 8.6
(bs, 1H). MS-CI (m/z): [M + H]+ 208.15.
1
yellow liquid (52 mg, 41%) after two steps. H NMR (300 MHz,
CDCl3): δ 2.13 (3H, s), 2.27 (3H, s), 2.54 (2H, br), 3.54 (2H, s).
13C NMR (75 MHz, CDCl3): δ 159.5, 143.2, 134.6, 36.9, 13.9,
10.0.
3-(N-Methylmethan-5-ylsulfonamido)-5-(propylcarbomoyl)-
benzoic Acid 6a. To a solution of the mono acid 11 (143.5 mg,
0.5 mmol) in DMF/CH2Cl2 (1:3 mL), EDC (114.6 mg, 0.6 mmol)
and HOBT (81 mg, 0.6 mmol) were added followed by n-
propylamine (12) (82 µL, 1 mmol) and diisopropylethylamine (0.5
mL). The reaction mixture was stirred at room temperature for 13
h. After this time, water was poured into reaction mixture, and the
mixture was extracted with EtOAc and dried over anhydrous Na2-
SO4. The solvent was evaporated under reduced pressure, and
purification by silica gel flash column chromatography yielded the
n-propylamide derivative which further underwent hydrolysis with
1 N NaOH in THF/MeOH to provide the acid 6a in 71% yield as
a white solid. 1H NMR (300 MHz, CDCl3 + CD3OD): 0.88 (3H,
t, J ) 7.5 Hz), 1.55 (2H, m), 2.83 (3H, s), 3.27 (3H, s), 3.30 (2H,
m), 7.82 (1H, s), 7.91 (1H, s), 8.05 (1H, m), 8.27 (1H, s). 13C NMR
(75 MHz, CDCl3 + CD3OD): δ 166.9, 141.3, 135.5, 129.8, 128.1,
127.0, 41.8, 37.7, 35.2, 22.4, 11.2.
3-{[(2,5-Dimethyloxazol-4-yl)methyl]carbamoyl}-5-(N-meth-
ylmethan-5-ylsulfonamido)benzoic Acid 6b. To a stirred solution
of the mono acid 11 (172 mg, 0.6 mmol) in DMF/CH2Cl2 (1:5
mL) were added EDC (143.2 mg, 0.75 mmol) and HOBT (101.2
mg, 0.75 mmol) followed by (2,5-dimethyloxazol-4-yl)methanamine
(13) (76 mg, 0.6 mmol) and diisopropylethylamine (0.5 mL). The
reaction mixture was stirred at room temperature for 13 h. Then
workup similar to that for 6a followed by silica gel flash column
chromatography gave the corresponding amide derivative which
was further hydrolyzed in presence of 1 N NaOH in THF/MeOH
to give the benzoic acid 6b in 33% yield as a white solid. 1H NMR
(300 MHz, CDCl3 + CD3OD): δ 2.37 (3H, s), 2.39 (3H, s), 2.84
(3H, s), 3.32 (3H, s), 4.39 (2H, s), 8.13 (1H, s), 8.18 (1H, m), 8.73
(1H, m). 13C NMR (75 MHz, CDCl3 + CD3OD): δ 166.6, 166.0,
159.7, 145.45, 141.70, 135.18, 132.0, 129.9, 128.7, 126.6, 37.1,
34.7, 34.3, 12.5, 9.0. MS-ESI (m/z): [M + Na]+ 403.96.
2,5-Dimethyloxazol-4-yl-1-ethanamide 18. To a stirred solution
of ethyl 2,5-dimethyloxazole-4-carboxylate (350 mg, 2 mmol) in
CH2Cl2 (5 mL) at -78 °C was was added DIBAL-H (3 mL, 4.5
mmol, 1.5 M soln in toluene). The mixture was stirred for 5 min
at -78 °C, and the reaction was quenched with saturated aqueous
potassium sodium tartrate solution. The solution was diluted with
CH2Cl2 and vigorously stirred at 23 °C for 30 min. The aqueous
layer was extracted with CH2Cl2 (2×). The combined layers were
dried on anhydrous Na2SO4. Evaporation of solvent under reduced
pressure provided the corresponding aldehyde as a colorless liquid
(200 mg). This aldehyde was used for the next step without further
purification.
To a stirred solution of the above aldehyde in THF (5 mL) at
-78 °C was added methylmagnesium bromide (640 µL, 1.9 mmol,
3 M soln in ether). The reaction mixture was warmed to 23 °C
over a period of 3 h. The reaction was quenched with saturated
aqueous NH4Cl and was extracted with EtOAc (3×). The combined
organic layers were dried on anhydrous Na2SO4. Evaporation of
solvent under reduced pressure gave secondary alcohol 20 as a
colorless liquid (143 mg).
To a stirred solution of above alcohol and diphenyl phosphoryl
azide (260 µL, 1.2 mmol) in toluene (3 mL) at 0 °C was added
DBU (180 µL, 1.2 mmol). The reaction was warmed to 23 °C and
stirred for 17 h. After this time, the reaction mixture was quenched
with 10% aqueous HCl and extracted with EtOAc (3×). The
combined organic layer was washed with brine, dried over
anhydrous Na2SO4, and concentrated under reduced pressure. The
crude product thus obtained was purified by silica gel column
chromatography (3% MeOH/CHCl3) to provide the corresponding
azide (159 mg).
The above azide was dissolved in THF (5 mL) at 23 °C, and
PPh3 (262 mg, 1 mmol) was added. The reaction mixture was stirred
at 23 °C for 7 h. After this period, a few drops of water were added,
and the reaction mixture was heated at 55 °C for 12 h. After this
period, the mixture was concentrated and the residue was purified
by silica gel chromatography (10-25% MeOH/CHCl3 and 2%
Et3N) to provide amine 18 as a yellow liquid (57.5 mg, 20%, three
3-(Benzylcarbamoyl)-5-(N-methylmethan-5-ylsulfonamido)-
benzoic Acid 6c. The acid 6c was prepared by following a
procedure similar to that described above. To the mono acid 11
(144.8 mg, 0.4 mmol), EDC (96 mg, 0.5 mmol), and HOBT (67.5
mg, 0.5 mmol) in DMF/CH2Cl2 (1:4 mL) at room temperature was
added benzylamine (14) (57 µL, 0.5 mmol) followed by diisopro-
pylethylamine (0.5 mL). The reaction mixture was stirred at room
temperature for 20 h. Then workup similar to that for 6a, followed
by silica gel flash column chromatography, provided the corre-
sponding amide which was further hydrolyzed by using 1 N NaOH
in presence of THF/MeOH to give the benzoic acid 6c in 61%
1
steps). H NMR (500 MHz, CDCl3): δ 1.44 (d, J ) 6.5 Hz, 3H),
2.20 (s, 3H), 2.32 (s, 3H), 4.13 (q, J ) 6.5 Hz, 2H), 5.69 (br, 2H).
3-Propylcarbamoylbenzoic Acid 21. To a stirring solution of
methyl 3-(propylcarbamoyl)benzoate (167 mg, 0.75 mmol) in THF:
H2O mixture (1 mL:1 mL) at 0 °C was added solid LiOH (47 mg,
1.1 mmol), and the resulting solution was stirred at 23 °C for 4 h.
After this period, the reaction mixture was extracted with toluene
1
yield. H NMR (300 MHz, CDCl3 + CD3OD): δ 2.84 (3H, s),

Memapsin 2 (â-Secretase) Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2405
3.08 (3H, s), 4.56 (2H, d, J ) 5.4 Hz), 7.28 (5H, m), 7.86 (1H,
bs), 7.97 (1H, s), 8.09 (1H, m), 8.32 (1H, m).
DMF/dichloromethane (1:2 mL) and stirred at room temperature
for 44 h. Then water (5 mL) was added to the reaction mixture
and extracted with ethyl acetate (2 × 10 mL). The organic layers
were dried over Na2SO4 and concentrated under reduced pressure.
The TBS-protected inhibitor thus obtained was used for the next
step.
(R)-3-(N-Methylmethan-5-ylsulfonamido)-5-((1-phenylethyl)-
carbomoyl)benzoic Acid 6d. Acid 6d was prepared by following
a similar procedure to that described for acid 6b. To mono acid 11
(215 mg, 0.75 mmol), EDC (172 mg, 0.9 mmol), and HOBT (122
mg, 0.5 mmol) in DMF/CH2Cl2 (1:5 mL) at room temperature was
added (R)-R-methylbezylamine (15) (100 µL, 0.75 mmol) followed
by diisopropylethylamine (0.5 mL). The reaction mixture was stirred
at room temperature for 20 h. Then workup similar to 6a, followed
by silica gel flash column chromatography, provided the corre-
sponding amide which was further hydrolyzed by using 1 N NaOH
in presence of THF/MeOH to give the benzoic acid 6d (198 mg,
60%) as a white solid. 1H NMR (500 MHz, CDCl3 + CD3OD): δ
1.56 (3H, d, J ) 7.5 Hz), 2.87 (3H, s), 3.32 (3H, s), 5.24 (1H, m),
To the stirred solution of TBS-protected inhibitor in THF (3 mL)
was added TBAF (1 M THF, 0.23 mL) at room temperature, and
the resulting mixture was stirred overnight. The mixture was
concentrated under reduced pressure and purified by silica gel flash
column chromatography (4% methanol in chloroform) to give the
1
inhibitor 5a as a white solid in 61% yield (17.5 mg). H NMR
(500 MHz, CDCl3 + CD3OD): δ 0.75 (3H, d, J ) 6.5 Hz), 0.79
(3H, d, J ) 7.0 Hz), 0.90 (6H, d, J ) 6.5 Hz), 0.94 (3H, t, J ) 7.0
Hz), 1.09-1.11 (9H, m), 1.38 (1H, m), 1.51 (1H, m), 1.59-1.70
(5H, m), 1.79-1.85 (1H, m), 2.68 (1H, m), 2.88 (3H, s), 3.33-
3.39 (5H, m), 3.62 (1H, m), 3.90 (1H, d, J ) 8.5 Hz), 3.97 (1H,
m), 4.17 (1H, m), 7.96 (2H, s), 8.29 (1H, s). MS-ESI (m/z): [M +
Na]+ 662.17. HRMS [M + Na]+ calcd for C31H53N5NaO7S
662.3558, found 662.3560.
Inhibitor 5b. To a stirred solution of Boc-protected hydroxyethyl
dipeptide isostere (7) (41.8 mg, 0.075 mmol) in dichloromethane
(1.5 mL) was added TFA (0.5 mL), and the mixture was stirred at
room temperature. The reaction mixture was stirred for 30 min and
then concentrated under reduced pressure to provide the corre-
sponding amine.
The amine was dissolved in dichloromethane (1 mL), and
diisopropylethyl amine (0.2 mL) was added. The combined mixture
was added to a stirred solution of acid 6b (27.2 mg, 0.075 mmol),
HOBT (13.5 mg, 0.1 mmol), and EDC (19.2 mg, 0.1 mmol) in
DMF/dichloromethane (1:2 mL) and stirred at room temperature
for 28 h. Then water (5 mL) was added to the reaction mixture
and extracted with ethyl acetate (2 × 10 mL). The organic layers
were dried over Na2SO4 and concentrated under reduced pressure.
The crude TBS-protected inhibitor thus obtained was used as such
for the next step.
7.19-7.36 (5H, m), 7.97 (1H, m), 8.11 (1H, m), 8.35 (1H, m). 13
C
NMR (75 MHz, CDCl3 + CD3OD): δ 168.6, 166.0, 143.0, 141.3,
135.3, 134.9, 129.3, 128.3, 127.9, 126.61, 125.64, 49.1, 37.2, 34.6,
20.8.
(S)-3-(N-Methylmethylsulfonamido)-5-(1-phenylethylcarbam-
oyl)benzoic Acid 6e. Acid 6e was prepared by following a
procedure similar to that described for acid 6b. To mono acid 11
(100 mg, 0.35 mmol), EDC (77 mg, 0.4 mmol), and HOBT (54
mg, 0.5 mmol) in DMF/CH2Cl2 (1:3 mL) at room temperature was
added (S)-R-methylbezylamine (16) (49 µL, 0.4 mmol) followed
by diisopropylethylamine (0.5 mL). The reaction mixture was stirred
at room temperature for 20 h. Then workup similar to that for 6a,
followed by silica gel flash column chromatography, provided the
corresponding amide which was further hydrolyzed by using 1 N
NaOH in presence of THF/MeOH to give the benzoic acid 6e as a
1
white solid in 45% yield (60 mg). H NMR (500 MHz, CD3OD):
1.61 (d, J ) 7 Hz, 3H), 2.98 (s, 3H), 3.40 (s, 3H), 5.28 (m, 1H),
7.27-7.45 (m, 5H), 8.11 (s, 1H), 8.11 (s, 1H), 8.11 (s, 1H). MS-
ESI (m/z): [M + Na]+ 398.90.
(R)-3-{[2-Hydroxy-1-phenylethyl]carbamoyl}-5-(N-methyl-
methan-5-ylsulfonamido)benzoic Acid (6f). Acid 6f was prepared
by following a procedure similar to that described for acid 6b. To
mono acid 11 (115 mg, 0.4 mmol), EDC (96 mg, 0.5 mmol), and
HOBT (66 mg, 0.5 mmol) in DMF/CH2Cl2 (1:4 mL) at room
temperature was added (R)-(2)-phenylglycinol (17) (69 mg, 0.4
mmol) followed by diisopropylethylamine (0.5 mL). The reaction
mixture was stirred at room temperature for 20 h. Then workup
similar to that for 6a, followed by silica gel flash column
chromatography, provided the corresponding amide which was
further hydrolyzed by using 1 N NaOH in presence of THF/MeOH
to give the benzoic acid 6f.
The stirred solution of TBS-protected inhibitor in THF (3 mL),
TBAF (1 M THF, 0.38 mL) was added at room temperature, and
the resulting mixture was stirred overnight. The mixture was
concentrated under reduced pressure and purified by column
chromatography (2% methanol in chloroform) to give the inhibitor
1
5b as a white solid in 28% yield (15 mg). H NMR (500 MHz,
CDCl3): δ 0.83 (3H, d, J ) 6.5 Hz), 0.87 (3H, d, J ) 6.5 Hz),
0.93 (3H, d, J ) 6.5 Hz), 0.95 (3H, d, J ) 6.5 Hz), 1.08 (3H, d,
J ) 6.5 Hz), 1.13 (3H, d, J ) 6.5 Hz), 1.19 (3H, d, J ) 7.0 Hz),
1.43-1.47 (1H, m), 1.62-1.72 (3 H, m), 1.82-1.84 (1H, m), 1.96-
2.02 (1H, m), 2.40 (3H, s), 2,45 (3H, s), 2.71-2.75 (1H, m), 2.86
(3H, s), 3.33 (3H, s), 3.76 (1H, m), 3.98-4.02 (1H, m), 4.10-
4.19 (2H, m), 4.33 (1H, dd, J ) 5.0, 15.0 Hz), 4.44 (1H, dd, J )
5.5, 15.0 Hz), 6.60 (1H, b), 6.81 (1H, b), 6.96 (1H, b), 8.02 (2H,
d, J ) 5.5 Hz), 8.09 (1H, b), 8.33 (1H, s). MS-ESI (m/z): [M +
Na]+ 729.15. HRMS [M + Na]+ calcd for C34H54N6NaO8S
729.3616, found 729.3646.
Inhibitor 5c. The procedure described for synthesis of inhibitor
5a was used for the synthesis of 5c. Accordingly, Boc-protected
hydroxyethyl dipeptide isostere 7 (42 mg, 0.075 mmol), HOBT
(14 mg, 0.1 mmol), EDC (20 mg, 0.1 mmol), and acid 6c (27 mg,
0.075) gave the TBS-protected inhibitor. Exposure to TBAF
followed by column chromatography (5% methanol in chloroform)
yielded inhibitor 5c as a white solid in 64% yield (33 mg). 1H NMR
(500 MHz, CDCl3 + CD3OD): δ 0.75 (3H, d, J ) 6.5 Hz), 0.78
(3H, d, J ) 7.0 Hz), 0.91 (6H, d, J ) 6.5 Hz), 1.08 (6H, t, J ) 6.5
Hz), 1.11 (3H, d, J ) 7.0 Hz), 1.36-1.41 (1H, m), 1.51-1.57 (1H,
m), 1.60-1.72 (3H, m), 1.80-1.85 (1H, m), 2.72 (1H, m), 2.88
(3H, s), 3.34 (3H, s), 3.66 (1H, m), 3.93 (2H, m), 4.19 (1H, m),
4.59 (2H, ABq, υ ) 17.5 Hz, J ) 15 Hz, 20 Hz,), 7.22-7.33 (5H,
m), 7.98 (1H, d, J ) 1.5 Hz), 8.03 (1H, t, J ) 1.5 Hz), 8.41 (1H,
s). MS-ESI (m/z): [M + Na]+ ) 710.17. HRMS [M + Na]+ calcd
for C35H53N5NaO7S 710.3558, found 710.3557.
3-{[1-(2,5-Dimethyloxazol-4-yl)ethyl]carbamoyl}-5-(N-meth-
ylmethan-5-ylsulfonamido)benzoic Acid 6g. Acid 6g was prepared
by following a procedure similar to that described for acid 6b. To
mono acid 11 (115 mg, 0.4 mmol), EDC (96 mg, 0.5 mmol), and
HOBT (66 mg, 0.5 mmol) in DMF/CH2Cl2 (1:4 mL) at room
temperature was added (2,5-dimethyloxazol-4yl)ethanamine (18)
(56 mg, 0.4 mmol) followed by diisopropylethylamine (0.5 mL).
The reaction mixture was stirred at room temperature for 20 h.
Then workup similar to that for 6a, followed by silica gel flash
column chromatography, provided the corresponding amide which
was further hydrolyzed by using 1 N NaOH in presence of THF/
MeOH to give the benzoic acid 6g as a white solid in 40% (63
1
mg) yield. H NMR (300 MHz, CDCl3): δ 1.61 (3H, d, J ) 7.5
Hz), 2.43 (3H, s), 2.46 (3H, s), 2.86 (3H, s), 3.37 (3H, s), 5.45
(1H, m), 8.27 (1H, s), 8.32 (1H, m), 9.10 (1H, m), 9.33 (1H, br).
MS-ESI (m/z): [M + Na]+ 418.04.
Inhibitor 5a. To a stirred solution of Boc-protected hydroxyethyl
dipeptide isostere (7) (25 mg, 0.045 mmol) in dichloromethane (1.5
mL) was added TFA (0.5 mL). The reaction mixture was stirred at
room temperature for 30 min and then concentrated under reduced
pressure to give the corresponding amine.
The amine was dissolved in dichloromethane (1 mL), and
diisopropylethylamine (0.2 mL) was added. The combined mixture
was added to a stirred solution of acid 6a (16 mg, 0.05 mmol),
HOBT (8 mg, 0.054 mmol) and EDC (10.3 mg, 0.054 mmol) in
Inhibitor 5d. The procedure described for synthesis of inhibitor
5a was used for the synthesis of 5d. Accordingly, Boc-protected

2406 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Ghosh et al.
hydroxyethyl dipeptide isostere 7 (62 mg, 0.11 mmol), HOBT (17
mg, 0.125 mmol), EDC (24 mg, 0.125 mmol) and acid 6d (41 mg,
0.11) gave the TBS-protected inhibitor. Exposure to TBAF followed
by column chromatography (5% methanol in chloroform) yielded
inhibitor 5d as a white solid in 26% yield (20 mg). 1H NMR (500
MHz, CDCl3): δ 0.80 (3H, d, J ) 6.5 Hz), 0.85 (3H, d, J ) 6.5
Hz), 0.90 (3H, d, J ) 6.5 Hz), 0.92 (3H, d, J ) 6.5 Hz), 1.10 (6H,
d, J ) 6.5 Hz), 1.13 (3H, d, J ) 7.0 Hz), 1.39-1.43 (1H, m), 1.51
(3H, d, J ) 7.0 Hz), 1.59-1.64 (2H, m), 1.66-1.72 (1 H, m), 1.73-
1.79 (1H, m), 1.87-1.93 (1H, m), 2.77 (1H, m), 2.84 (3H, s), 3.32
(3H, s), 3.72 (1H, m), 3.98 (1H, m), 4.10 (1H, t, J ) 9.0 Hz), 4.25
(2H, m), 5.26 (1H, m), 6.38 (1H, d, J ) 7.0 Hz), 6.99 (1H, d, J )
9.5 Hz), 7.17 (1H, d, J ) 9.0 Hz), 7.23 (1H, t, J ) 7.0 Hz), 7.30
(2H, t, J ) 7.0 Hz), 7.36 (2H, d, J ) 7.5 Hz), 7.50 (1H, d, J ) 7.5
Hz), 8.02 (2H, d, J ) 1.5 Hz), 8.42 (1H, s). MS-ESI (m/z): [M +
Na]+ ) 724.18. HRMS [M + Na]+ calcd for C36H55N5NaO7S
724.3714, found 724.3715.
protected hydroxyethyl dipeptide isostere 7 (25 mg, 0.045 mmol),
HOBT (8 mg, 0.054 mmol), EDC (10 mg, 0.054 mmol), and acid
21 (23 mg, 0.05 mmol) gave the TBS-protected inhibitor. Exposure
to TBAF followed by column chromatography (5% methanol in
chloroform) yielded inhibitor 22 as a white solid in 41% yield (11
1
mg). H NMR (500 MHz, CDCl3 + CD3OD): δ 0.76 (d, J ) 7
Hz, 3H), 0.81 (d, J ) 7 Hz, 3H), 0.91-0.97 (m, 9H), 1.11 (d, J )
7 Hz, 12H), 1.36-1.41 (m, 1H), 1.49-1.56 (m, 1H), 1.59-1.73
(m, 5H), 1.83-1.87 (m, 1H), 2.68 (q, J ) 7 Hz, 1H), 3.32-3.40
(m, 4H), 3.63-3.66(m, 1H), 3.91 (d, J ) 8 Hz, 1H), 3.94-4.00
(m, 1H), 4.16-4.18 (m, 1H), 7.50 (t, J ) 8 Hz, 1H), 7.92 (d, J )
8 Hz, 1H), 8.01 (d, J ) 8 Hz, 1H), 8.28 (s, 1H). MS-ESI (m/z):
[M + Na]+ 555.20. HRMS [M + Na]+ calcd for C29H48N4NaO5
555.3522, found 555.3539.
Preliminary in Vivo Inhibition of Aâ Production by Inhibitor
5d. Inhibitor 5d was dissolved in DMSO to 4 mg/mL and diluted
into an equal volume of Solutol HS-15/propylene glycol (1:2). The
resulting solution was diluted further with an equal volume of H2O,
to a final concentration of 1 mg/mL 5d. Tg257617 female mice age
3 months were injected intraperitoneally with 8 mL/kg of 5d
formulated as above (8 mg/kg/bw dose). Animals were housed at
the Oklahoma Medical Research Foundation, and procedures
adhered to IACUC guidelines to minimize stress to experimental
subjects. Blood was sampled from the saphenous vein prior to
injection in lithium-heparin Microvette tubes (Sarstedt). Mice in
two groups of three each were sampled at 4 h postinjection. Three
control mice, receiving vehicle alone (8 mL/kg), were likewise
injected and their blood sampled. Plasma was separated by
centrifugation at 1000g and stored at -70 °C. Aâ40 was analyzed
by sandwich ELISA (Invitrogen). Aâ40 was expressed relative to
baseline level for each individual animal to allow utilization of
smaller cohorts of animals. Data is reported as average ( SEM
(standard error of the mean) and analyzed by Student’s t-test using
a critical value of 0.05.
Inhibitor 5e. The procedure described for synthesis of inhibitor
5a was used for the synthesis of 5e. Accordingly, Boc-protected
hydroxyethyl dipeptide isostere 7 (62 mg, 0.11 mmol), HOBT (17
mg, 0.125 mmol), EDC (24 mg, 0.125 mmol), and acid 6e (42 mg,
0.11) gave the TBS-protected inhibitor. Exposure to TBAF followed
by column chromatography (5% methanol in chloroform) yielded
1
inhibitor 5e as a white solid in 44% yield (34 mg). H NMR (500
MHz, CDCl3 + CD3OD): δ 0.83 (d, J ) 7 Hz, 3H), 0.86 (d, J )
7 Hz, 3H), 1.13-1.19 (m, 9H), 1.42-1.46 (m, 1H), 1.63 (d, J ) 7
Hz, 6H), 1.65-1.81 (m, 5H), 1.92-1.95 (m, 1H), 2.00-2.10 (m,
1H), 1.83-1.87 (m, 1H), 2.76 (q, J ) 7 Hz, 1H), 2.88 (s, 3H),
3.38 (s, 3H), 3.78 (t, J ) 6 Hz, 1H), 4.02-4.10 (m, 2H), 4.26-
4.27 (m, 1H), 5.32 (t, J ) 7 Hz, 1H), 6.18 (bs, 1H), 6.93 (d, J )
9 Hz, 1H), 9.97 (d, J ) 8 Hz, 1H), 7.33-7.39 (m, 5H) 8.05 (s,
1H), 8.34 (s, 1H). MS-ESI (m/z): [M + Na]+ ) 724.16. HRMS
[M + H]+ calcd for C36H56N5O7S 702.3900, found 702.3907.
Inhibitor 5f. The procedure described for synthesis of inhibitor
5a was used for the synthesis of 5f. Accordingly, Boc-protected
hydroxyethyl dipeptide isostere 7 (32 mg, 0.057 mmol), HOBT
(10 mg, 0.07 mmol), EDC (14 mg, 0.07 mmol), and acid 6f (22
mg, 0.057) gave the TBS-protected inhibitor. Exposure to TBAF
followed by column chromatography (3-5% methanol in chloro-
form) yielded inhibitor 5f as a white solid in 48% yield (20 mg).
1H NMR (500 MHz, CDCl3): δ 0.76 (3H, d, J ) 7.0 Hz), 0.82
(3H, d, J ) 7.0 Hz), 0.89-0.93 (6H, m), 1.11 (6H, d, J ) 6.5 Hz),
1.16 (3H, d, J ) 7.0 Hz), 1.33-1.45 (2H, m), 1.57-1.69 (2 H, m),
1.78-1.93 (2H, m), 2.75 (1H, m), 2.85 (3H, s), 2.95 (1H, m), 3.31
(3H, s), 3.71 (1H, m), 3.85-3.92 (2H, m), 3.97-4.02 (1H, m),
4.10 (1H, m), 4.25 (1H, m), 5.20 (1H, m), 6.63 (1H, d, J ) 8.5
Hz), 7.00 (1H, d, J ) 8.5 Hz), 7.12 (1H, d, J ) 8.5 Hz), 7.24-
7.35 (5H, m), 8.00 (1H, s), 8.05 (1H, s), 8.21 (1H, d, J ) 7.0 Hz),
8.38 (1H, s). MS-ESI (m/z): [M + Na]+ 740.26. HRMS [M +
Na]+ calcd for C36H55N5NaO8S 740.3669, found 740.3672.
Inhibitor 5g. The procedure described for synthesis of inhibitor
5a was used for the synthesis of 5g. Accordingly, Boc-protected
hydroxyethyl dipeptide isostere 7 (14 mg, 0.025 mmol), HOBT (4
mg, 0.03 mmol), EDC (6 mg, 0.03 mmol), and acid 6g (10 mg,
0.025) gave the TBS-protected inhibitor. Exposure to TBAF
followed by column chromatography (10% methanol in chloroform)
Acknowledgment. Financial support by the National Insti-
tutes of Health (AG 18933) is gratefully acknowledged.
Supporting Information Available: HPLC and HRMS data
for compounds 5a-g and Figure 5. This material is available free
of charge via the Internet at http://pubs.acs.org.
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1
yielded inhibitor 5g as a white solid in 36% yield (6 mg). The H
1
NMR showed a mixture of diastereomers. H NMR (500 MHz,
CDCl3): δ 0.78-0.88 (6H, m), 0.91-0.98 (6H, m1.07-1.09 (3H,
m), 1.13-1.16 (3H, m), 1.18 (3H, d, J ) 7.0 Hz), 1.48-1.52 (3H,
m), 1.54-1.65 (2H, m), 1.76 (2H, m), 1.83-1.99 (2H, m), 2.32
and 2.38 (6H, 2s, diastereomers), 2.69 (1H, m), 2.86 (3H, s), 2.98
(1H, m), 3.33-3.54 (3H, m), 3.68-3.83 (1H, m), 3.96-4.06 (1H,
m), 4.22-4.25 (2H, m), 5.12-5.18 (1H, m), 6.19 and 6.58 (1H, d,
J ) 8.0 Hz, diastereomers), 6.75 (1H, t, J ) 8.0 Hz), 6.85 and
6.94 (1H, d, J ) 8.0 Hz, diasteromers), 7.98-8.06 (2H, m), 8.22
and 8.29 (1H, 2s, diastereomers). MS-ESI (m/z): [M + Na]+
743.30. HRMS [M + Na]+ calcd for C35H56N6NaO8S 743.3778,
found 743.3785.
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JM061338S