Structure-based design of highly selective β-secretase inhibitors: Synthesis, biological evaluation, and protein-ligand x-ray crystal structure

Article abstract of DOI:10.1021/jm3008823

The structure-based design, synthesis, and X-ray structure of protein-ligand complexes of exceptionally potent and selective β-secretase inhibitors are described. The inhibitors are designed specifically to interact with S₁′ active site residues to provide selectivity over memapsin 1 and cathepsin D. Inhibitor 5 has exhibited exceedingly potent inhibitory activity (K_i = 17 pM) and high selectivity over BACE 2 (>7000-fold) and cathepsin D (>250000-fold). A protein-ligand crystal structure revealed important molecular insight into these selectivities. These interactions may serve as an important guide to design selectivity over the physiologically important aspartic acid proteases.

Full text of DOI:10.1021/jm3008823

Article
pubs.acs.org/jmc
Structure-Based Design of Highly Selective β‑Secretase Inhibitors:
Synthesis, Biological Evaluation, and Protein−Ligand X‑ray Crystal
Structure
Arun K. Ghosh,*,† Kalapala Venkateswara Rao,† Navnath D. Yadav,† David D. Anderson,†
Navnath Gavande,† Xiangping Huang,‡ Simon Terzyan,‡ and Jordan Tang‡
†Department of Chemistry and Department of Medicinal Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana
47907, United States
‡Protein Studies Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Science Center, 825 NE 13th
Street, MS 28, Oklahoma City, Oklahoma 73104, United States
S
* Supporting Information
ABSTRACT: The structure-based design, synthesis, and X-ray structure of
protein−ligand complexes of exceptionally potent and selective β-secretase
inhibitors are described. The inhibitors are designed specifically to interact with
S1′ active site residues to provide selectivity over memapsin 1 and cathepsin D.
Inhibitor 5 has exhibited exceedingly potent inhibitory activity (Ki = 17 pM) and
high selectivity over BACE 2 (>7000-fold) and cathepsin D (>250000-fold). A
protein−ligand crystal structure revealed important molecular insight into these
selectivities. These interactions may serve as an important guide to design selectivity over the physiologically important aspartic
acid proteases.
INTRODUCTION
■
Alzheimer's disease (AD) is a progressive, degenerative brain
disorder with no effective treatment to date, and the
development of new drugs is an urgent priority in medicine.1
The hallmark of AD is the formation of neutritic plaques
containing 40/42 residue amyloid-β (Aβ) peptides and
neurofibrillary tangles in the brain.2 β-Secretase [memapsin 2,
β-site APP cleaving enzyme 1 (BACE 1)] is one of two
proteases that cleaves β-amyloid precursor protein (APP) and
generates Aβ and its aggregation product.3 There is
considerable evidence that excess Aβ leads to brain
inflammation, neuronal death, and AD.4 Consequently, β-
secretase has become a major therapeutic target for drug
development.5,6 Since our design of initial transition-state
inhibitor (1, Figure 1) and subsequent determination of
inhibitor-bound memapsin 2 X-ray structure, nearly a decade
ago, steady progress has been made toward the evolution of
potent small molecule and brain-penetrable inhibitor drugs.7,8
Recently, we have shown that administration of β-secretase
inhibitor 2 rescued cognitive decline in transgenic AD mice,
validating β-secretase as an important drug design target.9,10
However, the development of clinical β-secretase inhibitor drug
is faced with numerous formidable challenges, including lack of
Figure 1. Structures of β-secretase inhibitors 1−3.
selectivity against other physiologically important aspartic acid
proteases and issues of poor pharmacological profiles including
blood−brain penetration.7,8 In our continuing work toward the
design of potent small molecule and selective inhibitors, we
Special Issue: Alzheimer's Disease
have been particularly interested in developing tools for
selectivity against relevant physiologically important aspartic
Received: June 22, 2012
acid proteases, especially cathepsin D (CD) and β-site APP
© XXXX American Chemical Society
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cleaving enzyme 2 (BACE 2). BACE 2 has specificity similarity
to BACE 1, and this is known to have important physiological
functions.11 CD plays a key role in important biological
functions like protein catabolism.12 The abundance of CD in
various cells, especially in central nervous system tissue cells, is
very high. Furthermore, CD gene knockout studies in mice
showed marked phenotypic response including high mortality
rate.13 Therefore, the selective inhibition of β-secretase over
CD and BACE 2 is very critical to reduce toxicity and other side
effects of β-secretase inhibitor drugs.
of stereochemistry on the potency and selectivity. We have also
synthesized and evaluated inhibitors 7−9 to examine the
importance of various functional groups at the P1′ and P2′ sites
on the potency.
CHEMISTRY
■
The synthesis of inhibitors 4−6 is shown in Scheme 1. Amino
acids 10a−c were coupled with isobutyl amine in the presence
Scheme 1. Synthesis of Inhibitors 4−6
As described by us previously, the X-ray crystal structure of
inhibitor 1-bound β-secretase showed an interesting hydrogen
bonding between the P2′-carbonyl and the hydroxyl of Tyr-198,
forming a rare kink at the P2′ site.8 We have exploited this
interaction in the design and synthesis of very potent and
highly selective β-secretase inhibitors such as 3 by incorporating
hydroxyethylene isosteres.14 However, the cellular β-secretase
inhibitory activity of this class of inhibitors was only in the
micromolar range. In an attempt to design small molecule
inhibitors with improved selectivity and cellular activity
exploiting this unique interaction, we have further explored β-
secretase inhibitors with a reduced amide isostere and
incorporated functionality to improve potency and selectivity.
The basic amine functionality in the reduced amide isostere
may also improve cell permeability.15 Herein, we report our
structure-based design and synthesis of very potent and
exceptionally selective inhibitors with excellent cellular
inhibitory properties. A protein−ligand X-ray structure
provided important molecular insight into the specific
cooperative ligand-binding site interactions for selectivity.
The inhibitors containing reduced amide isostere have been
reported; however, they exhibited only marginal selectivity
against memapsin 1 (BACE 2).6,16 A reduced amide β-secretase
inhibitor 4 was synthesized by us, and this compound has
exhibited a BACE 1 Ki of 27 nM and marginal selectivity
against BACE 2 and CD in our in-house enzyme inhibitory
assays. An energy-minimized model of 4 was created based
upon the protein−ligand X-ray structure of 2-bound β-
secretase.9 Our preliminary model suggested that an
introduction of a hydroxyl group with S-configuration on the
ethyl group of homoalanine moiety would make enhanced
interactions in the active site and possibly enhance selectivity.
On the basis of this molecular insight, we have designed,
synthesized, and evaluated inhibitors 5 and 6 (Figure 2) to
investigate the influence of the hydroxyl group and also the role
i
of EDC, HOBt, and Pr2NEt to afford the corresponding
amides in 71−91% yield. Removal of the Boc group with
trifluoroacetic acid followed by reductive amination of the
resulting amine with aldehyde 1117 provided compounds 12a−
c in 49−76% yields. Treatment of 12a−c with trifluoroacetic
acid followed by coupling with acid 1318 provided the inhibitors
4−6 in 68−75% yields.
The synthesis of inhibitors 7−9 is shown in Schemes 2 and 3.
Coupling of Boc-protected allothreonine derivative 10b with N-
Scheme 2. Synthesis of Inhibitors 7 and 8
i
isobutyl-N-methyl amine using EDC, HOBt, and Pr2NEt
followed by Boc removal using trifluoroacetic acid afforded
(2S,3S)-2-amino-3-hydroxy-N-isobutyl-N-methyl-butanamide
in 49% yield over two steps. Reductive amination of this amine
with known aldehyde 11 provided 12d in 30% yield. Inhibitor 7
was synthesized in 61% yield from 12d by Boc removal using
trifluoroacetic acid followed by coupling of the resulting amine
with the known acid 13 (Scheme 2). As shown in Scheme 2,
inhibitor 8 was prepared from 15 following the similar reaction
sequence utilized for the synthesis of inhibitors 4−7 (Schemes
1 and 2). Compound 15, in turn, has been synthesized by O-
Figure 2. Structure of isophthalamide-derived β-secretase inhibitors
4−9.
B
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Scheme 3. Synthesis of Inhibitor 9
Scheme 4. Synthesis of Carboxylic Acid 25 and Inhibitors 18
and 21
methylation of 14 using MeI and Ag2O19 followed by
hydrolysis using aqueous LiOH.
The synthesis of inhibitor 9 has been carried out as shown in
Scheme 3. Treatment of 14 with TBSOTf in the presence of
Et3N followed by reaction with DiBALH provided the
corresponding aldehyde. Reductive amination of this crude
aldehyde with isobutyl amine afforded 16 in 66% yield over
three steps. Removal of the Boc group using trifluoroacetic acid
followed by reductive amination of the resulting amine with the
aldehyde 11 afforded compound 17.
The synthesis of tricyclic indole derivative in inhibitors 21
and 22 has been carried out from butyl 3-chloropropanesul-
fonate 26 as shown in Scheme 5. In a one-pot two-step
Scheme 5. Synthesis of Carboxylic Acid 30
Reaction of compound 17 with trifluoroacetic acid followed
i
by coupling with acid 13 using EDC, HOBt, and Pr2NEt
provided TBS-protected inhibitor, which upon treatment with
TBAF gave the inhibitor 9 in 46% yield over three steps. On
the basis of the results obtained from these inhibitors, we have
directed our attention to reduce the peptidic nature of 5 by
keeping all of the key hydrogen-bonding interactions of the
prime region intact. In this direction, we have designed and
synthesized the inhibitors 18−22 (Figure 3) containing 7,6,5-
tricyclic indole moieties as P2 ligands.
reaction, 26 was treated first with BuLi followed by treatment
again with BuLi, and BOMCl provided the corresponding butyl
1-(benzyloxymethyl)cyclopropanesulfonate in 80% yield. Hy-
drolysis of this sulfonate with KSCN followed by refluxing of
the resulting potassium sulfonate with SOCl2 afforded sulfonyl
chloride 27 in 91% yield over two steps. Reaction of indole
derivative 2822 with 27 in the presence of pyridine and a
catalytic amount of DMAP furnished sulfonamide 29 in 74%
yield. Hydrogenolysis of 29, followed by mesylation of the
resulting alcohol using mesyl chloride and Et3N, afforded the
corresponding mesylate. This mesylate was subjected to a one-
pot cyclization and N-methylation using NaH and MeI
followed by hydrolysis in the presence of aqueous NaOH to
obtain the desired carboxylic acid 30 in 65% yield over two
steps.
The synthesis of (2S,3S)-2-amino-3-hydroxy-N-isobutyl (or
isopropyl)hexanamide moiety present in the prime region of the
inhibitors 19, 20, and 22 has been carried out as shown in
Scheme 6. Asymmetric dihydroxylation of 31 using AD-mix α
followed by reaction of the resulting dihydroxy compound with
p-nitrobenzenesulfonyl chloride in the presence of Et3N
afforded nosylate 32 in 32% yield over two steps.23 Treatment
Figure 3. Structure of indole-derived β-secretase inhibitors 18−22.
For the synthesis of inhibitor 18, known indolecarboxylic
acid 23 was prepared as described in the literature.20 The
synthesis of α,α-dimethyl tricyclic indole derivative 25, present
in inhibitor 20, was synthesized as shown in Scheme 4.
Reaction of the known tricyclic indole derivative 2421 with MeI
in the presence of NaH provided the corresponding α,α-
dimethyl derivative. Saponification of the resulting ester with
aqueous NaOH afforded α,α-dimethylindole carboxylic acid 25
(37% yield over two steps). Inhibitor 18 was synthesized by
treatment of Boc derivative 12b with trifluoroacetic acid
followed by coupling of the resulting amine with acid 2320
(68% yield). Similarly, coupling of the above amine with
cyclopropyl indole derivative 30 provided inhibitor 21.
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To gain further molecular insight, we have determined the X-
ray structure of 5-bond to β-secretase at a 2.2 Å resolution. As
shown in Figure 4, the amine functionality of the reduced
amide isostere forms two tight hydrogen bonds (2.4 and 2.7 Å
bond distances) with active site aspartic acid Asp228.7
Interestingly, the other active site Asp32 is not directly
interacting with the reduced amide isostere. Asp32 is
extensively hydrogen bonded to three groups: the amide
nitrogen of Gly34 (2.8 Å), the hydroxyl group of Ser35 (2.5−3
Å with rotations of the involved groups), and the amide
nitrogen of Gly230 (3.3 Å). These interactions appear to lock
Asp32 in a rigid conformation; thus, its hydrogen bonding to
Asp228 produced a network of hydrogen bond interactions to
include the interaction of the inhibitor and the protease. A
similar inhibitor−enzyme interacting pattern has been reported
for the crystal structure of reduced amide isosteres.16b The fact
that inhibitors 2 and 5 are highly potent suggests that the
interaction of both active site carboxyls with the transition-state
isostere is not a necessary feature for the design of potent
inhibitors. The P3-phenyl ring occupies a unique position that
spans S3 and S4 subsites and causes a significant positional shift
of a protein loop containing residues from 8 to 13 (the 10s
loop)26 located in the S3/S4 pocket similar to inhibitor 2. This
flexible part of the active site cleft can be further exploited for
ligand design. The P2′-carbonyl as well as P2′-NH are within
proximity to form hydrogen bonds with Thr72 and Gly34,
respectively. Most significantly, the allothreonine hydroxyl
group is oriented toward the Tyr-198 hydroxyl group. This
interaction is presumably absent in inhibitor 4. Also, the P1′-
hydroxyl group stereochemistry is optimal for critical hydrogen
bonding with Tyr-198. The combinations of active site
interactions are responsible for the potency and selectivity of
inhibitor 5.
The X-ray crystal structure of 5-bound memapsin 2
demonstrates the importance of the allothreonine moiety as
it forms key hydrogen-bonding interactions with the prime
region of memapsin 2. Therefore, inhibitors 18−22, with a
7,6,5-tricyclicindole moiety as the P2 ligand, were designed with
a view to reduce labile amide bonds in isophthalic acid amide-
derived ligand. Synthetic inhibitors 18−22 were evaluated
against recombinant BACE 1, and the results are summarized in
Table 2. Inhibitor 18, containing a known 7,6,5-tricyclic moiety
as the P2 ligand, showed a low nanomolar activity toward BACE
1 (Ki = 7.3 nM). This inhibitor is substantially less potent than
inhibitor 5; however, the ratio of cell inhibitory to enzyme
inhibitory efficacy was improved significantly (3 vs >58),
indicating better cell permeability for compound 18. Inhibitor
19 with a sterically more demanding propyl group in the P1′
region has shown around 18-fold improvement in the potency
(entries 1 and 2). Inhibitor 20 with a dimethyl-substituted
indole derivative as the P2 ligand resulted in >10-fold potency
enhancement over unsubstituted inhibitor 19. This inhibitor
exhibited a cellular IC50 value of 15 nM. The ligand was also
designed especially to halt the possibility of retro-Michael
reaction of the P2-α,α-unsubstituted sultam functionality in
inhibitors 18 and 19.27 Inhibitors 21 and 22, designed by
replacing the two methyl groups of P2 ligand in 20 with
cyclopropyl group, also exhibited impressive potency.
Scheme 6. Synthesis of Inhibitors 19, 20, and 22
of 32 with NaN3 followed by hydrogenation of the
corresponding azide using 10% Pd−C in the presence of
(Boc)2O afforded 33 in 80% yield. Hydrolysis using aqueous
LiOH followed by coupling with isobutyl amine and isopropyl
i
amine using EDC, HOBt, and Pr2NEt afforded 34a and 34b,
respectively. Boc removal using trifluoroacetic acid followed by
reductive amination of the resulting amine with aldehyde 11
gave 35a and 35b in 49 and 74% yields, respectively. Inhibitors
19, 20, and 22 were synthesized by coupling of the amine,
obtained by Boc removal of 35a and 35b using trifluoroacetic
acid, with tricyclic indole derivatives 23 or 25 or 30.
RESULTS AND DISCUSSION
■
The BACE 1 inhibitory activity of synthetic inhibitors 4−9 was
determined against recombinant β-secretase using our
previously reported assay protocols.24 The results are shown
in Table 1. As can be seen, inhibitor 4 with a homoalanine P1′
side chain has shown a Ki of 27 nM (entry 1). Inhibitor 5 with
an allothreonine P1′ side chain has exhibited remarkable BACE
1 inhibitory activity with a Ki of 17 pM (entry 2). Inhibitor 6
with a threonine P1′ side chain has shown significant reduction
of BACE 1 activity over the deoxyderivative 4 or the inhibitor 5
with an allothreonine P1′ side chain (entries 1−3). We have
also evaluated the cellular inhibition of β-secretase in
neuroblastoma cells.25 Consistent with potent BACE 1
inhibitory activity, inhibitor 5 exhibited an average cellular
IC50 value of 1 nM. The corresponding inhibitor 6 with a
threonine P1′ side chain has shown a cellular IC50 value of >1
μM in the same assay. Inhibitor 7 with a N-methyl amide
abolished all BACE 1 inhibitory activity. Inhibitor 8 with an
“OMe” group in the P1′ region also showed substantial
reduction in inhibitory potency (entry 5) as compared to
inhibitor 5 with hydroxyl group. Inhibitor 9 with a reduced
amide in the P2′ region resulted in a total loss in potency. These
results clearly demonstrate the significance of hydrogen-
bonding interactions of inhibitor 5 with the prime region of
the BACE 1 active site.
We then evaluated the potencies of selected inhibitors
against recombinant BACE 2 and human CD, and the results
are shown in Table 3. Interestingly, inhibitor 5 displayed very
impressive selectivity against BACE 2 (Ki = 120 nM, selectivity
>7000-fold) and CD (Ki = 4.3 μM, selectivity >250000-fold) as
D
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Table 1. BACE 1 Inhibitory and Cellular Activity of Inhibitors 4−9
a
The IC50 was determined in neuroblastoma cells. GRL-8234 exhibited Ki = 1.8 nM and IC50 = 2.5 nM in this assay.
well. In comparison, deoxyinhibitor 4 has shown a BACE 2 Ki
of 1450 nM (selectivity >55-fold) and CD Ki of 8264 nM
(selectivity >300-fold). This result suggested that the
allothreonine hydroxyl group on the P1′ side chain is critical
to the selectivity and potency of inhibitor 5. Inhibitor 18 has
also exhibited good selectivity (over 970-fold selective) toward
BACE 1 over CD (entry 3). Inhibitor 19 with a propyl side
chain has shown improvement in CD selectivity (entry 4).
Inhibitor 20 displayed good selectivity against BACE 2 and
excellent selectivity against CD (entry 5). Inhibitor 22 has also
shown a >4200-fold selectivity over CD (entry 6).
CONCLUSION
■
In conclusion, we have designed, synthesized, and examined the
biological activity of isophthalamide-based BACE 1 inhibitors
containing various functional groups in the prime region. The
selectivity of inhibitors 4 and 5 against BACE 2 and CD was
also examined. Inhibitor 5 with an allothreonine moiety
E
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Table 3. Selectivity Studies of BACE 1 Inhibitors against
BACE 2 and CD
Ki (nM)
selectivity
BACE 2/
BACE 1
CD/
BACE 1
entry inhibitor BACE 1 BACE 2
CD
1
2
3
4
5
6
4
5
27.12
0.017
7.3
1450
120
8264
4300
7100
690
>55
>300
>250000
>970
>7000
18
19
20
22
0.4
>1700
0.036
0.47
11
530
>300
>14700
>4200
2000
exhibited superior potency and exceptionally high selectivity
when compared to inhibitors with a threonine moiety or an
ethylglycine (homoalanine) moiety. Inhibitors with N-isobutyl-
N-methylamide and P2′ reduced amide groups on the prime
side lost their efficacy. These results clearly demonstrate the
significance of prime region of the inhibitors on the potency
and selectivity. The X-ray structure of 5-bond β-secretase also
showed the presence of an effective hydrogen bond between
the prime side of the inhibitor and Thr72, Gly34, and Tyr 198
residues of BACE 1. On the basis of this molecular insight, we
have further designed and synthesized inhibitors by replacing
the isophthalamide moiety with conformationally constrained
7,6,5-tricyclicindole moieties and by keeping the allothreonine
moiety intact. These inhibitors have also shown very good
potency and selectivity against CD. These results further
support the significance of hydrogen-bonding interactions in
the prime region for the potency and selectivity. The
combination of active site interactions along with the Tyr-198
may be responsible for the observed selectivity. This molecular
insight may aid further design of selectivity against other
aspartic acid proteases. Further investigations into the origin of
selectivity are in progress.
Figure 4. X-ray structure of 5 (green)-bound−β-secretase complex.
Hydrogen bonds are shown in dotted lines (PDB ID: 4GID).
Table 2. BACE 1 Inhibitory and Cellular Activity of
Inhibitors 18−22
EXPERIMENTAL SECTION
■
General. All anhydrous solvents were obtained according to the
following procedures: diethyl ether and tetrahydrofuran (THF) were
distilled from sodium/benzophenone under argon; dichloromethane
was from calcium hydride. Other solvents were used without
purification. All moisture-sensitive reactions were carried out in
flame-dried flasks under an argon atmosphere. Reactions were
monitored by thin-layer chromatography (TLC) using Silicycle 60A-
F254 silica gel precoated plates. Flash column chromatography was
performed using Silicycle 230−400 mesh silica gel. Yields refer to
chromatographically and spectroscopically pure compounds. 1H NMR
and 13C NMR spectra were recorded on a Varian Inova-300 (300 and
75 MHz, respectively), Bruker Avance ARX-400 (400 and 100 MHz),
and Bruker Avance DRX-500 (500 and 125 MHz). High- and low-
resolution mass spectra were carried out by the Mass Spectroscopy
Center at Purdue University. The purity of all test compounds was
determined by HRMS and HPLC analysis. All test compounds showed
≥95% purity.
Synthesis of Compound 12a. To a mixture of (S)-2-[(tert-
butoxycarbonyl)amino]butanoic acid 10a (2.3 mmol, 0.47 g) and
iPr2NEt (2.76 mmol, 0.48 mL) in CH2Cl2 (12 mL), HOBt·H2O (2.76
mmol, 0.37 g), isobutyl amine (2.76 mmol, 0.27 mL), and EDC.HCl
(2.76 mmol, 0.53 g) were added simultaneously at 23 °C, and the
resulting mixture was stirred for 17 h at 23 °C. The reaction mixture
was quenched with saturated aqueous NaHCO3 solution and extracted
with CH2Cl2. The combined extracts were dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The
resulting residue was purified by silica gel column chromatography
(1−3% MeOH/CH2Cl2) to furnish (S)-tert-butyl [1-(isobutylamino)-
1-oxobutan-2-yl]carbamate in 86% yield (0.51 g). 1H NMR (400
a
The IC50 was determined in neuroblastoma cells. GRL-8234
exhibited Ki = 1.8 nM and IC50 = 2.5 nM in this assay.
F
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MHz, CDCl3): δ 6.24 (brs, 1H), 5.08 (brs, 1H), 4.07−3.89 (m, 1H),
3.18−2.96 (m, 2H), 1.92−1.70 (m, 2H), 1.62 (hept, J = 7.3 Hz, 1H),
1.43 (s, 9H), 0.93 (t, J = 7.4 Hz, 3H), 0.89 (d, J = 6.8 Hz, 6H).
To a solution of (S)-tert-butyl [1-(isobutylamino)-1-oxobutan-2-
yl]carbamate (1.97 mmol, 0.51 g) in CH2Cl2 (9 mL), trifluoroacetic
acid (3 mL) was added at 0 °C, and the resulting mixture was stirred
for 1.5 h at 23 °C. Excess trifluoroacetic acid and CH2Cl2 were
removed under reduced pressure, and the resulting residue was
purified by silica gel column chromatography [2−5% (5%NH3/
MeOH)/CH2Cl2] to furnish (S)-2-amino-N-isobutylbutanamide in
96% yield (0.298 g).
carbamate following the procedure described for the synthesis of
compound 12a. H NMR (400 MHz, CDCl3): δ 7.35−7.27 (m, 3H),
7.26−7.19 (m, 1H), 7.17 (d, J = 7.4 Hz, 2H), 4.53 (d, J = 8.9 Hz, 1H),
4.05−3.86 (m, 2H), 3.39 (brs, 1H), 3.13−3.00 (m, 2H), 2.99 (d, J =
5.7 Hz, 1H), 2.87−2.71 (m, 2H), 2.67 (dd, J = 12.1, 4.4 Hz, 1H), 2.58
(dd, J = 12.1, 7.6 Hz, 1H), 1.73 (hept, J = 6.7 Hz, 1H), 1.41 (s, 9H),
1.18 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 7.1 Hz, 6H).
1
Synthesis of Inhibitor 5. Inhibitor 5 has been prepared (yield 71%,
over two steps) from 12b by Boc deprotection followed by coupling
with (R)-3-(N-methylmethylsulfonamido)-5-[(1-phenylethyl)-
carbamoyl]benzoic acid 13 following a similar reaction procedure
described for the inhibitor 4. 1H NMR (400 MHz, CDCl3): δ 8.13 (s,
1H), 7.93 (s, 1H), 7.88 (s, 1H), 7.39−7.27 (m, 6H), 7.26−7.14 (m,
6H), 5.27 (p, J = 7.3 Hz, 1H), 4.48−4.30 (m, 1H), 4.04−3.94 (m,
1H), 3.24 (s, 3H), 3.09 (d, J = 4.3 Hz, 1H), 3.02−2.88 (m, 3H), 2.88−
2.77 (m, 1H), 2.80 (s, 3H), 2.73 (dd, J = 12.4, 3.7 Hz, 1H) 2.64 (dd, J
= 12.3, 7.7 Hz, 1H), 1.64 (hept, J = 6.7 Hz 1H), 1.56 (d, J = 6.9 Hz,
3H), 1.04 (d, J = 6.2 Hz, 3H), 0.80 (d, J = 6.8 Hz, 3H), 0.80 (d, J = 6.8
Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 172.37, 166.01, 164.75,
142.91, 142.03, 137.54, 135.90, 135.77, 129.16, 128.65, 128.59, 127.89,
127.62, 127.43, 126.65, 126.23, 123.87, 68.21, 68.06, 52.16, 51.60,
49.66, 46.40, 39.00, 37.83, 35.56, 28.34, 21.65, 20.06, 18.21. HRMS-
ESI (m/z): [M + H]+ calcd for C35H48N5O6S, 666.3325; found,
666.3321.
Synthesis of Compound 12c. tert-Butyl [(2S,3R)-3-hydroxy-1-
(isobutylamino)-1-oxobutan-2-yl]carbamate has been synthesized
(yield 91%) by coupling of (2S,3R)-2-[(tert-butoxycarbonyl)amino]-
3-hydroxybutanoic acid 10c with isobutyl amine in the presence of
iPr2NEt, HOBt, and EDC as described for (S)-tert-butyl [1-
(isobutylamino)-1-oxobutan-2-yl]carbamate. 1H NMR (400 MHz,
CDCl3): δ 6.71 (brs, 1H), 5.52 (d, J = 7.6 Hz, 1H), 4.45−4.30 (m,
1H), 3.97 (dd, J = 8.2, 1.9 Hz, 1H), 3.22−3.08 (m, 1H), 3.07−2.92
(m, 1H), 1.77 (hept, J = 6.7 Hz, 1H), 1.45 (s, 9H), 1.18 (d, J = 6.4 Hz,
3H), 0.90 (d, J = 6.9 Hz, 6H).
Compound 12c was prepared from tert-butyl [(2S,3R)-3-hydroxy-1-
(isobutylamino)-1-oxobutan-2-yl]carbamate following the procedure
described for the synthesis of compound 12a (yield 49% over two
steps). 1H NMR (400 MHz, CDCl3): δ 7.37−7.26 (m, 2H), 7.25−7.13
(m, 4H), 4.59 (d, J = 8.5 Hz, 1H), 4.04−3.75 (m, 2H), 3.15−2.96 (m,
2H), 2.94 (d, J = 5.2 Hz, 1H), 2.80 (d, J = 6.2 Hz, 2H), 2.70−2.55 (m,
2H), 1.92 (brs, 1H), 1.74 (hept, J = 6.7 Hz, 1H), 1.40 (s, 9H), 1.18 (d,
J = 6.3 Hz, 3H), 0.87 (d, J = 6.4 Hz, 6H).
Synthesis of Inhibitor 6. Inhibitor 6 has been prepared (yield 75%,
over two steps) from 12c by Boc deprotection followed by coupling
with (R)-3-(N-methylmethylsulfonamido)-5-[(1-phenylethyl)-
carbamoyl]benzoic acid 13 following a similar reaction procedure
described for the inhibitor 4. 1H NMR (400 MHz, CDCl3): δ 8.15 (s,
1H), 7.92 (s, 1H), 7.87 (s, 1H), 7.48 (d, J = 7.7 Hz, 1H), 7.38−7.27
(m, 5H), 7.26−7.15 (m, 5H), 7.08 (t, J = 5.9 Hz, 1H), 5.27 (p, J = 7.1
Hz, 1H), 4.42−4.27 (m, 1H), 3.79 (p, J = 6.3 Hz, 1H), 3.22 (s, 3H),
3.01−2.87 (m, 4H), 2.84 (dd, J = 13.7, 7.7 Hz, 1H), 2.78 (s, 3H),
2.71−2.59 (m, 2H), 1.69−1.59 (m, 1H), 1.56 (d, J = 7.0 Hz, 3H), 1.14
(d, J = 6.2 Hz, 3H), 0.79 (d, J = 6.4 Hz, 3H), 0.78 (d, J = 6.6 Hz, 3H).
13C NMR (100 MHz, CDCl3): δ 172.78, 165.82, 164.81, 143.03,
142.05, 137.58, 135.88, 135.77, 129.14, 128.61, 127.78, 127.64, 127.39,
126.64, 126.23, 123.80, 69.13, 68.38, 52.04, 50.90, 49.68, 46.54, 38.59,
37.79, 35.56, 28.33, 21.68, 20.03, 19.68. HRMS-ESI (m/z): [M + H]+
calcd for C35H48N5O6S, 666.3325; found, 666.3335.
To a solution of the above (S)-2-amino-N-isobutylbutanamide (1.87
mmol, 0.296 g) and (S)-tert-butyl (1-oxo-3-phenylpropan-2-yl)-
carbamate 11 [prepared from the corresponding Weinreb amide (2
mmol) following a similar literature procedure]7 in CH2Cl2 (20 mL),
Na(OAc)3BH (2.62 mmol, 0.55 g) was added at 0 °C. The resulting
mixture was stirred for 1 h at 0 °C and 15 h at 23 °C. The reaction
mixture was quenched with saturated aqueous NaHCO3 solution and
extracted with CH2Cl2. The combined extracts were dried over
anhydrous Na2SO4, filtered, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography (1−3%
MeOH/CH2Cl2) to furnish the corresponding adduct 12a in 79%
1
yield (0.58 g). H NMR (400 MHz, CDCl3): δ 7.34−7.26 (m, 2H),
7.25−7.11 (m, 4H), 4.50 (brs, 1H), 3.95 (brs, 1H), 3.10−2.96 (m,
2H), 2.92 (dd, J = 7.3, 5.0 Hz, 1H), 2.86−2.69 (m, 2H), 2.62 (dd, J =
11.9, 4.3 Hz, 1H), 2.52 (dd, J = 12.0, 7.9 Hz, 1H), 1.80−1.63 (m, 2H),
1.63−1.50 (m, 1H), 1.41 (s, 9H), 0.92 (t, J = 7.5 Hz, 3H), 0.85 (d, J =
6.5 Hz, 6H).
Synthesis of Inhibitor 4. To a solution of 12a (0.72 mmol, 0.282 g)
in CH2Cl2 (9 mL), trifluoroacetic acid (3 mL) was added at 0 °C.
After the reaction mixture was stirred for 1 h at 23 °C, CH2Cl2 and
trifluoroacetic acid were removed under reduced pressure. The
resulting residue was purified by silica gel column chromatography
[2−6% (5%NH3/MeOH)/CH2Cl2] to furnish the corresponding
amine in 93% yield (0.195 g).
To a solution of the above amine (0.125 mmol, 36.4 mg) in CH2Cl2
(10 mL), iPr2NEt (0.03 mL), HOBt·H2O (0.16 mmol, 21.6 mg), (R)-
3-(N-methylmethylsulfonamido)-5-[(1-phenylethyl)-carbamoyl]-
benzoic acid 13 (0.16 mmol, 60.2 mg), and EDC·HCl (0.16 mmol,
30.7 mg) were added simultaneously at 23 °C, and the resulting
mixture was stirred for 14 h at the same temperature. The reaction
mixture was quenched with saturated aqueous NaHCO3 solution and
extracted with CH2Cl2. The combined extracts were dried over
anhydrous Na2SO4, filtered, and concentrated under reduced pressure.
The resulting residue was purified by silica gel column chromatog-
raphy (1−3% MeOH/CH2Cl2) to furnish the inhibitor 4 in 73% yield
(59.6 mg). 1H NMR (400 MHz, CDCl3): δ 8.26 (s, 1H), 8.03 (s, 1H),
7.98 (s, 1H), 7.48 (d, J = 7.8 Hz, 1H), 7.39−7.34 (m, 2H), 7.33−7.24
(m, 5H), 7.24−7.18 (m, 3H), 6.71 (t, J = 6.2 Hz, 1H), 5.31 (p, J = 7.5
Hz, 1H), 4.40−4.26 (m, 1H), 3.29 (s, 3H), 3.04−2.91 (m, 3H), 2.88−
2.83 (m, 1H), 2.81 (s, 3H), 2.81−2.73 (m, 2H), 2.55 (dd, J = 12.3, 4.1
Hz, 1H), 1.75−1.58 (m, 2H), 1.57 (d, J = 7.0 Hz, 3H), 1.55−1.48 (m,
1H), 0.89 (t, J = 7.4 Hz, 3H), 0.77 (d, J = 6.7 Hz, 3H), 0.76 (d, J = 6.6
Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 174.15, 165.23, 164.53,
143.06, 142.23, 137.64, 135.65, 129.1, 128.62, 128.58, 127.93, 127.87,
127.33, 126.64, 126.23, 123.39, 65.20, 51.68, 50.24, 49.56, 46.41,
38.55, 37.84, 35.52, 28.36, 26.96, 21.60, 19.98, 19.95, 10.33. HRMS-
ESI (m/z): [M + H]+ calcd for C35H48N5O5S, 650.3376; found,
650.3370.
Synthesis of Compound 12d. To a mixture of (2S,3S)-2-[(tert-
Synthesis of Compound 12b. tert-Butyl [(2S,3S)-3-hydroxy-1-
(isobutylamino)-1-oxobutan-2-yl]carbamate was synthesized in 71%
yield by coupling of (2S,3S)-2-[(tert-butoxycarbonyl)amino]-3-hydrox-
ybutanoic acid 10b with isobutyl amine in the presence of EDC,
butoxycarbonyl)amino]-3-hydroxybutanoic acid (10b) (1 mmol, 0.22
g) and Pr2NEt (1.2 mmol, 0.21 mL) in CH2Cl2 (5 mL) were added
i
HOBt.H2O (1.2 mmol, 0.162 g), N-isobutyl-N-methylamine (1.2
mmol, 0.14 mL), and EDC·HCl (1.2 mmol, 0.23 g) at 23 °C, and the
resulting reaction mixture was stirred for 15 h at 23 °C. The reaction
mixture was quenched with saturated aqueous NaHCO3 solution and
extracted with CH2Cl2. The combined extracts were dried over
anhydrous Na2SO4, filtered, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography (1−3%
MeOH/CH2Cl2) to obtain tert-butyl {(2S,3S)-3-hydroxy-1-[isobutyl-
(methyl)amino]-1-oxobutan-2-yl}carbamate.
i
HOBt, and Pr2NEt as described for (S)-tert-butyl [1-(isobutylamino)-
1-oxobutan-2-yl]carbamate. 1H NMR (400 MHz, CDCl3): δ 6.47 (brs,
1H), 5.53 (brs, 1H), 4.10−3.75 (m, 3H), 3.23−3.09 (m, 1H), 3.06−
2.92 (m, 1H), 1.78 (hept, J = 6.7 Hz, 1H), 1.45 (s, 9H), 1.29 (d, J =
6.1 Hz, 3H), 0.91 (d, J = 6.5 Hz, 6H).
Compound 12b was synthesized in 52% yield (for two steps) from
tert-butyl [(2S,3S)-3-hydroxy-1-(isobutylamino)-1-oxobutan-2-yl]-
G
dx.doi.org/10.1021/jm3008823 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
To a solution of the above tert-butyl {(2S,3S)-3-hydroxy-1-
[isobutyl(methyl)amino]-1-oxobutan-2-yl}carbamate in CH2Cl2 (6
mL) was added trifluoroacetic acid (2 mL) at 0 °C. The resulting
reaction mixture was warmed to 23 °C and stirred for 1.5 h at 23 °C.
The reaction mixture was concentrated under reduced pressure, and
the resulting residue was purified by silica gel column chromatography
[2−5% (5%NH3/MeOH)/CH2Cl2] to furnish (2S,3S)-2-amino-3-
hydroxy-N-isobutyl-N-methyl-butanamide in 49% (93.2 mg) yield over
3.09−2.98 (m, 1H), 1.77 (hept, J = 6.7 Hz, 1H), 1.44 (s, 9H), 1.18 (d,
J = 6.3 Hz, 3H), 0.91 (d, J = 6.8 Hz, 6H).
To a solution of tert-butyl [(2S,3S)-1-(isobutylamino)-3-methoxy-1-
oxobutan-2-yl]-carbamate (0.624 mmol, 0.18 g) in CH2Cl2 (6 mL)
was added trifluoroacetic acid (2 mL) at 0 °C, and the resulting
mixture was stirred for 1.5 h at 23 °C. Trifluoroacetic acid and CH2Cl2
were removed under reduced pressure, and the resulting residue was
diluted with EtOAc and basified with saturated aqueous NaHCO3
solution. The organic layer was separated, and the aqueous layer was
extracted several times with EtOAc. The combined organic layers were
dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure to obtain crude (2S,3S)-2-amino-N-isobutyl-3-
methoxybutanamide in 88% yield.
To a solution of the above crude amine (0.55 mmol, 0.104 g) and
(S)-tert-butyl (1-oxo-3-phenylpropan-2-yl)carbamate 11 [prepared
from the corresponding Weinreb amide (0.55 mmol) following a
similar literature procedure]7 in CH2Cl2 (6 mL), Na(OAc)3BH (0.825
mmol, 0.175 g) was added at 0 °C. The resulting mixture was stirred
for 1 h at 0 °C and 15 h at 23 °C. The reaction mixture was quenched
with saturated aqueous NaHCO3 solution and extracted with CH2Cl2.
The combined extracts were dried over anhydrous Na2SO4, filtered,
and concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (20−45% EtOAc/hexanes) to
furnish the corresponding adduct 12e in 67% yield (0.155 g). 1H
NMR (400 MHz, CDCl3): δ 7.35 (brs, 1H), 7.29−7.21 (m, 2H),
7.21−7.10 (m, 3H), 4.64 (d, J = 8.2 Hz, 1H), 3.94 (brs, 1H), 3.77−
3.68 (m, 1H), 3.29 (s, 3H), 3.27 (d, J = 4.0 Hz, 1H), 3.00 (t, J = 6.4
Hz, 2H), 2.82 (dd, J = 12.9, 5.7 Hz, 1H), 2.69 (dd, J = 13.6, 7.7 Hz,
1H), 2.58−2.46 (m, 2H), 1.68 (hept, J = 6.7 Hz, 1H), 1.39 (s, 9H),
1.00 (d, J = 6.4 Hz, 3H), 0.83 (d, J = 6.6 Hz, 6H).
1
two steps. H NMR (400 MHz, CDCl3): δ 3.89−3.76 (m, 1H), 3.67
and 3.64 (two doublets, J = 4.7 Hz, 4.6 Hz, 1H), 3.36 (dd, J = 13.2, 8.0
Hz, 0.5H), 3.19 (dd, J = 14.4, 8.3 Hz, 0.5H), 3.10 (dd, J = 14.3, 7.0 Hz,
0.5H), 3.05 (s, 1.7H), 3.03−2.98 (m, 0.5H), 2.91 (s, 1.3H), 2.51 (brs,
3H), 2.00−1.87 (m, 1H), 1.10 (two doublets, J = 6.3, 6.3 Hz, 3H),
0.93−0.81 (m, 6H).
Compound 12d was synthesized from (2S,3S)-2-amino-3-hydroxy-
N-isobutyl-N-methyl-butanamide by reductive amination with alde-
hyde 11 following a similar procedure described for the synthesis of
1
compound 12a (yield 30%). H NMR (300 MHz, CDCl3): δ 7.33−
7.10 (m, 5H), 4.80−4.64 (m, 1H), 3.95−3.78 (m, 2H), 3.43 (dd, J =
11.8, 4.4 Hz, 1H), 3.35−3.09 (m, 2H), 3.06−2.96 (m, 2.5H), 2.91 (s,
1.5H), 2.84 (brs, 2H), 2.72−2.59 (m, 1H), 2.48−2.33 (m, 1H), 2.01−
1.87 (m, 1H), 1.39 (s, 9H), 1.10 and 1.06 (two doublets, J = 6.4, 6.4
Hz, 3H), 0.90 and 0.86 (two doublets, J = 6.7, 6.3 Hz, 6H).
Synthesis of Inhibitor 7. Inhibitor 7 was synthesized from
compound 12d by Boc removal using trifluoroacetic acid followed
by coupling of the resulting amine with the known acid 13 using EDC,
i
HOBt, and Pr2NEt following a similar procedure described for the
1
synthesis of inhibitor 4 (yield 61%, over two steps). H NMR (400
MHz, CDCl3): δ 8.21 and 8.17 (two singlets, 1H), 8.07−7.92 (m, 2H),
7.45−7.30 (m, 4H), 7.30−7.15 (m, 6H), 7.07 (t, J = 7.8 Hz, 1H),
5.40−5.26 (m, 1H), 4.34−4.19 (m, 1H), 3.97−3.82 (m, 1H), 3.55 and
3.53 (two doublets, J = 4.2, 4.2 Hz, 1H), 3.33 (s, 3H), 3.23−3.03 (m,
2.5H), 3.01 (s, 2H), 2.95−2.87 (m, 1H), 2.85 and 2.84 (two singlets,
3H), 2.77 (s, 1H), 2.73−2.57 (m, 2.5H), 1.96−1.78 (m, 1H), 1.60 (d, J
= 6.9 Hz, 3H), 1.13 and 1.10 (two doublets, J = 6.4, 6.5 Hz, 3H), 0.89
and 0.84 (two doublets, J = 6.6, 6.5 Hz, 2.7H), 0.79 and 0.75 (two
doublets, J = 6.7, 6.7 Hz, 3.3H). HRMS-ESI (m/z): [M + H]+ calcd
for C36H50N5O6S, 680.3482; found, 680.3478.
Synthesis of Inhibitor 8. To a solution of 12e (0.073 mmol, 30.8
mg) in CH2Cl2 (3 mL) was added trifluoroacetic acid (1 mL) at 0 °C.
The resulting reaction mixture was warmed to 23 °C and stirred for
1.5 h at 23 °C. The reaction mixture was concentrated under reduced
pressure, and the resulting residue was used in the next step without
any further purification.
To a solution of the above residue in CH2Cl2 (5 mL) were added
iPr2NEt (0.1 mL), HOBt·H2O (0.14 mmol, 18.9 mg), acid 13 (0.07
mmol, 26.3 mg), and EDC·HCl (0.14 mmol, 26.8 mg) simultaneously
at 23 °C. The resulting reaction mixture was stirred for 17 h at 23 °C.
The reaction mixture was quenched with saturated aqueous NaHCO3
solution and extracted with CH2Cl2. The combined extracts were dried
over anhydrous Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy (2%MeOH/CH2Cl2) to obtain the inhibitor 8 in 67% yield
(32.1 mg). 1H NMR (400 MHz, CDCl3): δ 8.19 (s, 1H), 8.02 (s, 1H),
7.96 (s, 1H), 7.42−7.29 (m, 5H), 7.28−7.25 (m, 2H), 7.22 (d, J = 7.4
Hz, 3H), 7.14−7.02 (m, 3H), 5.32 (p, J = 7.2 Hz, 1H), 4.48−4.36 (m,
1H), 3.73−3.61 (m, 1H), 3.33 (s, 3H), 3.30 (s, 3H), 3.27 (d, J = 4.2
Hz, 1H), 3.04−2.94 (m, 3H), 2.90−2.80 (m, 4H), 2.76−2.65 (m, 2H),
1.69−1.61 (m, 1H), 1.61 (d, J = 6.9 Hz, 3H), 1.04 (d, J = 6.4 Hz, 3H),
0.80 (d, J = 6.4 Hz, 6H). 13C NMR (125 MHz, CDCl3): δ 171.84,
165.41, 164.41, 142.87, 142.20, 137.42, 135.67, 135.64, 129.11, 128.60,
128.57, 127.80, 127.73, 127.38, 126.63, 126.18, 123.41, 65.95, 56.52,
51.99, 51.19, 49.53, 46.33, 38.73, 37.83, 35.43, 28.32, 21.63, 20.00,
14.62. HRMS-ESI (m/z): [M + H]+ calcd for C36H50N5O6S,
680.3482; found, 680.3488.
Synthesis of tert-Butyl {(2R,3S)-3-[(tert-Butyldimethylsilyl)oxy]-1-
(isobutylamino)butan-2-yl}carbamate (16). To a solution of
(2S,3S)-methyl 2-[(tert-butoxycarbonyl)amino]-3-hydroxybutanoate
(14) (2.2 mmol, 0.513 g) in CH2Cl2 (11 mL) were added Et3N
(3.3 mmol, 0.46 mL) and TBSOTf (2.86 mmol, 0.66 mL) at 0 °C. The
resulting reaction mixture was warmed to 23 °C and stirred for 2 h at
the same temperature. The reaction mixture was diluted with H2O and
CH2Cl2. The organic layer was separated, and the aqueous layer was
extracted with CH2Cl2. The combined extracts were dried over
anhydrous Na2SO4, filtered, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography (10%
EtOAc/hexanes) to obtain (2S,3S)-methyl 2-[(tert-butoxycarbonyl)-
amino]-3-[(tert-butyldimethylsilyl)oxy]butanoate in 88% yield (0.67
Synthesis of (2S,3S)-2-[(tert-Butoxycarbonyl)amino]-3-methoxy-
butanoic Acid (15). To a solution of (2S,3S)-methyl 2-[(tert-
butoxycarbonyl)amino]-3-hydroxybutanoate (14) (2.14 mmol, 0.5 g)
in CH3CN (21 mL) were added Ag2O (10.7 mmol, 2.48 g) and MeI
(21.4 mmol, 1.3 mL) at 23 °C, and the resulting reaction mixture was
stirred for 8 days at 23 °C. The reaction mixture was filtered and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (20−25% EtOAc/hexanes) to
furnish (2S,3S)-methyl 2-[(tert-butoxycarbonyl)amino]-3-methoxybu-
tanoate in 55% yield (0.29 g). 1H NMR (400 MHz, CDCl3): δ 5.27 (d,
J = 9.3 Hz, 1H), 4.41 (dd, J = 8.6, 3.6 Hz, 1H), 3.74 (s, 3H), 3.66−
3.55 (m, 1H), 3.34 (s, 3H), 1.43 (s, 9H), 1.18 (d, J = 6.4 Hz, 3H).
To a solution of (2S,3S)-methyl 2-[(tert-butoxycarbonyl)amino]-3-
methoxybutanoate (0.86 mmol, 0.213 g) in THF (4 mL) and H2O (2
mL) was added LiOH.H2O (5.16 mmol, 0.216 g) at 23 °C. The
resulting reaction mixture was stirred for 5 h at 23 °C. The reaction
mixture was diluted with H2O and diethyl ether. The organic layer was
separated, and the aqueous layer was carefully acidified with 2 N HCl
and extracted with ethyl acetate. The combined extracts were dried
over anhydrous Na2SO4, filtered, and concentrated under reduced
pressure to provide crude (2S,3S)-2-[(tert-butoxycarbonyl)amino]-3-
methoxybutanoic acid (15). This crude acid was used in the coupling
reaction without any further purification.
Synthesis of Compound 12e. The synthesis of tert-butyl [(2S,3S)-
1-(isobutylamino)-3-methoxy-1-oxobutan-2-yl]carbamate has been
carried out by coupling of the above crude acid 15 with isobutyl
i
amine using EDC, HOBt, and Pr2NEt following the procedure
described for the synthesis of (S)-tert-butyl [1-(isobutylamino)-1-
1
oxobutan-2-yl]carbamate (yield 74%, over two steps). H NMR (300
MHz, CDCl3): δ 6.22 (brs, 1H), 5.15 (brs, 1H), 4.10 (dd, J = 7.7, 6.8
Hz, 1H), 3.59 (p, J = 6.4 Hz, 1H), 3.34 (s, 3H), 3.21−3.10 (m, 1H),
H
dx.doi.org/10.1021/jm3008823 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
g). 1H NMR (400 MHz, CDCl3): δ 5.25 (d, J = 8.4 Hz, 1H), 4.16 (dd,
J = 8.3, 3.7 Hz, 1H), 4.05−3.96 (m, 1H), 3.66 (s, 3H), 1.36 (s, 9H),
1.16 (d, J = 6.3 Hz, 3H), 0.79 (s, 9H), −0.03 (s, 6H).
Synthesis of Inhibitor 18. Inhibitor 18 was synthesized from
compound 12b by Boc removal using trifluoroacetic acid followed by
coupling of the resulting amine with known acid 23 using EDC,
i
To a solution of (2S,3S)-methyl 2-[(tert-butoxycarbonyl)amino]-3-
[(tert-butyldimethyl-silyl)oxy]butanoate (0.288 mmol, 0.1 g) in Et2O
(3 mL) was added DiBAL-H (0.63 mmol, 0.63 mL, 1 M solution in
CH2Cl2) at −78 °C, and the resulting mixture was stirred for 1.5 h at
−78 °C. The reaction mixture was quenched with 0.5 mL of EtOAc
and diluted with Et2O and saturated aqueous sodium potassium
tartrate. After it was stirred for 0.5 h, the organic layer was separated,
and the aqueous layer was extracted with Et2O. The combine extracts
were washed with brine, dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure. The resulting residue was used
in the next step without any further purification.
To a solution of the above aldehyde in CH2Cl2 (6 mL) were added
isobutyl amine (0.864 mmol, 0.086 mL) and Na(OAc)3BH (0.576
mmol, 0.122 g) at 0 °C, and the resulting reaction mixture was stirred
for 1.5 h at 0 °C and 15 h at 23 °C. The reaction mixture was
quenched with saturated aqueous NaHCO3 solution and extracted
with CH2Cl2. The combined extracts were dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The
resulting residue was purified by silica gel column chromatography
[3% (5%NH3/MeOH)/CH2Cl2] to obtain (tert-butyl {(2R,3S)-3-
[(tert-butyldimethylsilyl)oxy]-1-(isobutylamino)butan-2-yl}-carbamate
(16) in 75% yield (80.7 mg). 1H NMR (400 MHz, CDCl3): δ 5.36 (d,
J = 7.9 Hz, 1H), 4.11−3.98 (m, 1H), 3.57−3.42 (m, 1H), 2.90 (dd, J =
12.2, 5.3 Hz, 1H), 2.62 (dd, J = 12.2, 4.3 Hz, 1H), 2.44−2.30 (m, 2H),
1.72 (hept, J = 6.7 Hz, 1H), 1.43 (s, 9H), 1.14 (d, J = 6.4 Hz, 3H),
0.92−0.84 (m, 15H), 0.04 (s, 3H), 0.03 (s, 3H).
HOBt, and Pr2NEt following a similar procedure described for the
1
synthesis of inhibitor 4 (yield 68%, over two steps). H NMR (400
MHz, CDCl3): δ 7.85 (s, 1H), 7.47 (s, 1H), 7.38−7.28 (m, 3H), 7.24−
7.19 (m, 2H), 6.84 (s, 1H), 6.47 (d, J = 8.0 Hz, 1H), 4.62−4.36 (m,
3H), 4.02 (p, J = 6.0 Hz, 1H), 3.90−3.81 (m, 2H), 3.47 (s, 3H), 3.12
(d, J = 5.0 Hz, 1H), 3.10−2.93 (m, 4H), 2.84 (dd, J = 12.2, 4.7 Hz,
1H), 2.77 (dd, J = 11.1, 6.8 Hz, 1H), 2.72 (q, J = 7.5 Hz, 2H), 1.76−
1.65 (m, 1H), 1.30 (t, J = 7.5 Hz, 3H), 1.15 (d, J = 6.4 Hz, 3H), 0.85
(d, J = 6.7 Hz, 3H), 0.84 (d, J = 6.6 Hz, 3H). 13C NMR (100 MHz,
CDCl3): δ 172.75, 167.90, 137.52, 133.84, 130.72, 129.28, 128.65,
127.72, 127.23, 126.75, 125.78, 120.26, 118.03, 117.60, 68.32, 67.48,
56.77, 52.30, 51.39, 46.41, 43.60, 39.66, 39.03, 28.42, 20.11, 18.95,
17.93, 14.25. HRMS-ESI (m/z): [M + H]+ calcd for C31H44N5O5S,
598.3063; found, 598.3060.
Synthesis of Compound 25. To a solution of 24 (0.43 mmol, 0.145
g) in DMF (4 mL) was added NaH (1.72 mmol, 68.8 mg, 60% NaH in
mineral oil) at 23 °C, and the resulting mixture was stirred for 15 min
at the same temperature. Iodomethane (1.72 mmol, 0.11 mL) was
added to the reaction mixture, and stirring was continued for further
2.5 h at 23 °C. The reaction mixture was quenched with methanol and
then diluted with EtOAc. The resulting solution was washed with H2O
and brine, dried over anhydrous Na2SO4, filtered, and concentrated
under reduced pressure. The resulting residue was purified by silica gel
column chromatography (30% EtOAc/hexanes) to obtain the
1
corresponding α,α-dimethylated adduct in 51% (76.8 mg) yield. H
NMR (400 MHz, CDCl3): δ 8.18 (d, J = 1.0 Hz, 1H), 7.74 (s, 1H),
6.81 (s, 1H), 4.14 (s, 2H), 3.93 (s, 3H), 3.55 (s, 3H), 2.76 (q, J = 7.5
Hz, 2H), 1.57 (s, 6H), 1.32 (t, J = 7.5 Hz, 3H).
Synthesis of Compound 17. Compound 17 was synthesized by
Boc removal of tert-butyl {(2R,3S)-3-[(tert-butyldimethylsilyl)oxy]-1-
(isobutylamino)butan-2-yl}carbamate (16) using trifluoroacetic acid
followed by reductive amination with known aldehyde 11 following
the procedure described for the synthesis of 12e. 1H NMR (400 MHz,
CDCl3): δ 7.31−7.23 (m, 2H), 7.23−7.15 (m, 3H), 5.20 (brs, 1H),
3.94−3.75 (m, 2H), 2.95−2.81 (m, 1H), 2.79−2.67 (m, 2H), 2.58 (dd,
J = 12.4, 5.0 Hz, 2H), 2.50−2.43 (m, 2H), 2.38 (qd, J = 11.5, 6.9 Hz,
2H), 1.74 (hept, J = 6.7 Hz, 1H), 1.40 (s, 9H), 1.10 (d, J = 6.3 Hz,
3H), 0.90 (d, J = 6.6 Hz, 6H), 0.87 (s, 9H), 0.04 (s, 3H), 0.02 (s, 3H).
Synthesis of Inhibitor 9. Boc removal of compound 17 using
trifluoroacetic acid followed by coupling of the resulting amine with
known acid 13 using EDC, HOBt, and iPr2NEt following the
procedure described for the synthesis of inhibitor 8 afforded the
A mixture of the above ester (0.219 mmol, 76.7 mg) and NaOH (20
mmol, 10 mL, 2 N NaOH) in EtOH (10 mL) and THF (10 mL) was
stirred for 2.5 days at 23 °C. The solvent was removed under reduced
pressure, and the resulting mixture was diluted with H2O and diethyl
ether. The organic layer was separated, and the aqueous layer was
acidified with aqueous 1 N HCl and extracted with ethyl acetate. The
combined extracts were dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure to furnish the corresponding
crude acid (25) in 72% yield (53.2 mg), which was used directly in the
coupling reaction without any further purification. LRMS-ESI (m/z):
[M + Na]+, 359.19
Synthesis of 1-(Benzyloxymethyl)cyclopropanesulfonyl Chloride
(27). Solutions of BuLi [17.2 mmol, 6.9 mL (2.5 M in hexanes) in 15
mL of THF] and butyl 3-chloro-1-propanesulfonate (26) (16.3 mmol,
3.5 g in 15 mL of THF) were added at the same time via cannula to an
oven-dried flask containing THF (100 mL) at −78 °C, and the
resulting mixture was stirred for 5−10 min at −78 °C and 30 min at 0
°C. The reaction mixture was cooled back to −78 °C, and a solution of
BuLi [19.6 mmol, 7.8 mL (2.5 M in hexanes)] was added to this
mixture. After it was stirred for 15 min at −78 °C, BOMCl (19.6
mmol, 2.7 mL) was added to the reaction flask, and stirring was
continued for further 2 h at −78 °C and 3 h at 23 °C. The reaction
mixture was quenched with H2O, and THF was removed under
reduced pressure. The resulting mixture was diluted with CH2Cl2 and
H2O. The organic layer was separated, and the aqueous layer was
extracted with CH2Cl2. The combined extracts were dried over
anhydrous Na2SO4, filtered, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography (8−12%
EtOAc/hexanes) to furnish butyl 1-(benzyloxymethyl)-
cyclopropanesulfonate in 80% yield (3.9 g). 1H NMR (300 MHz,
CDCl3): δ 7.40−7.25 (m, 5H), 4.55 (s, 2H), 4.23 (t, J = 6.6 Hz, 2H),
3.79 (s, 2H), 1.73−1.58 (m, 2H), 1.48 (ABq, J = 6.9, 5.0 Hz, 2H),
1.44−1.30 (m, 2H), 1.11 (ABq, J = 7.0, 5.1 Hz, 2H), 0.90 (t, J = 7.4
Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 137.41, 128.33, 127.78,
127.61, 73.09, 70.66, 69.84, 37.81, 31.04, 18.55, 13.41, 10.72.
To a mixture of butyl 1-(benzyloxymethyl)cyclopropanesulfonate
(13 mmol, 3.88 g) in DME (40 mL) and H2O (40 mL), KSCN (13.65
mmol, 1.33 g) was added at 23 °C, and the resulting reaction mixture
was refluxed for 15 h. The reaction mixture was cooled to 23 °C and
1
corresponding amide in 70% yield. H NMR (400 MHz, CDCl3): δ
8.20 (s, 1H), 8.00 (s, 1H), 7.94 (d, J = 7.8 Hz, 1H), 7.92 (s, 1H), 7.66
(d, J = 7.6 Hz, 1H), 7.40 (d, J = 7.5 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H),
7.29−7.19 (m, 5H), 7.20−7.12 (m, 1H), 5.29 (p, J = 7.1 Hz, 1H),
4.20−4.06 (m, 1H), 4.06−3.93 (m, 1H), 3.28 (s, 3H), 3.20 (dd, J =
12.4, 8.2 Hz, 1H), 3.10−2.98 (m, 2H), 2.86 (dd, J = 13.6, 6.4 Hz, 1H),
2.82−2.76 (m, 4H), 2.76−2.67 (m, 3H), 2.60 (dd, J = 12.4, 7.1 Hz,
1H), 1.81 (hept, J = 6.8 Hz, 1H), 1.59 (d, J = 7.0 Hz, 3H), 1.11 (d, J =
6.4 Hz, 3H), 0.86 (s, 9H), 0.75 (d, J = 6.7 Hz, 3H), 0.75 (d, J = 6.7 Hz,
3H), 0.07 (s, 3H), 0.05 (s, 3H).
To a solution of the above amide (0.104 mmol, 79.7 mg) in THF
(10 mL) was added TBAF (1.25 mmol, 1.25 mL, 1 M solution in
THF) at 0 °C, and the resulting reaction mixture was stirred for 18 h
at 23 °C. The solvent was removed under reduced pressure, and the
resulting residue was purified by silica gel column chromatography
[3−5% (5%NH3/MeOH)/CH2Cl2] to obtain inhibitor 9 in 65% (43.9
1
mg) yield. H NMR (400 MHz, CDCl3): δ 8.24 (s, 1H), 8.01−7.93
(m, 2H), 7.40 (d, J = 7.3 Hz, 2H), 7.37−7.24 (m, 7H), 7.24−7.16 (m,
3H), 5.31 (p, J = 7.3 Hz, 1H), 4.37−4.25 (m, 1H), 3.93−3.83 (m,
1H), 3.31 (s, 3H), 3.04 (dd, J = 13.5, 5.8 Hz, 1H), 2.81 (s, 3H), 2.80−
2.67 (m, 5H), 2.44−2.27 (m, 3H), 1.70−1.54 (m, 4H), 1.13 (d, J = 6.8
Hz, 3H), 0.81 (d, J = 6.5 Hz, 6H). 13C NMR (100 MHz, CDCl3): δ
165.78, 164.64, 143.13, 142.13, 137.87, 135.80, 129.24, 128.63, 128.53,
128.16, 127.46, 127.37, 126.53, 126.34, 123.78, 68.48, 60.61, 57.62,
51.94, 49.74, 49.48, 49.30, 38.67, 37.88, 35.46, 27.73, 21.78, 20.41,
20.38, 20.34. HRMS-ESI (m/z): [M + H]+ calcd for C35H50N5O5S,
652.3533; found, 652.3543.
I
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Journal of Medicinal Chemistry
Article
diluted with H2O and ethyl acetate. The organic layer was separated,
and the aqueous layer was concentrated under reduced pressure to
provide the crude potassium 1-(benzyloxymethyl)-
cyclopropanesulfonate, which was used directly in the next step
without additional purification.
3H), 3.48 (s, 3H), 2.77 (q, J = 7.5 Hz, 2H), 1.53 (t, J = 6.6 Hz, 2H),
1.31 (t, J = 7.5 Hz, 3H), 1.03−0.95 (m, 2H).
A mixture of the above 7,6,5-tricyclic indole derivative (0.2 mmol,
69.7 mg) and NaOH (20 mmol, 0.8 g in 10 mL of H2O) in EtOH (5
mL) and THF (10 mL) was stirred for 4 days at 23 °C. The solvent
was removed under reduced pressure, and the resulting mixture was
diluted with H2O and diethyl ether. The organic layer was separated,
and the aqueous layer was acidified with aqueous 1 N HCl and
extracted with ethyl acetate. The combined extracts were dried over
anhydrous Na2SO4, filtered, and concentrated under reduced pressure
to furnish the corresponding crude acid (30) in 75% yield (50 mg),
which was used directly in the coupling reaction without any further
purification. LRMS-ESI (m/z): [M + Na]+, 357.26
A mixture of potassium 1-(benzyloxymethyl)cyclopropanesulfonate
in SOCl2 (35 mL) and DMF (3.5 mL) was refluxed for 1.5 h, and
excess SOCl2 was removed under reduced pressure. Water was added
carefully to the resulting mixture and extracted with ethyl acetate. The
combined extracts were dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (5−10% EtOAc/hexanes) to obtain
1-(benzyloxymethyl)cyclopropanesulfonyl chloride 27 in 91% yield
1
Synthesis of (2R,3S)-Ethyl 3-Hydroxy-2-{[(4-nitrophenyl)sulfonyl]-
oxy}hexanoate (32). To a solution of (E)-ethyl hex-2-enoate (31)
(12.6 mmol, 1.79 g) in tBuOH (30 mL) and H2O (30 mL) were added
AD-mix α (17.6 g) and MeSO2NH2 (15.1 mmol, 1.44 g) at −1 °C,
and the resulting reaction mixture was stirred for 7 days at −1 °C. The
reaction mixture was quenched with saturated aqueous Na2S2O3
solution and extracted with ethyl acetate. The combined extracts
were dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (20−35% EtOAc/hexanes) to furnish (2R,3S)-ethyl
(3.1 g) (over two steps). H NMR (300 MHz, CDCl3): δ 7.46−7.28
(m, 5H), 4.63 (s, 2H), 4.01 (s, 2H), 1.85−1.77 (m, 2H), 1.46−1.37
(m, 2H). 13C NMR (75 MHz, CDCl3): δ 136.99, 128.41, 127.89,
127.61, 73.29, 68.38, 52.39, 14.03.
Synthesis of Sulfonamide 29. To a mixture of amine 28 (3.66
mmol, 0.8 g), pyridine (11 mmol, 0.89 mL), and DMAP (0.73 mmol,
89.2 mg) in CH2Cl2 at 0 °C was added a solution of 1-
(benzyloxymethyl)-cyclopropanesulfonyl chloride (27) (3.84 mmol,
1 g in 5 mL of CH2Cl2), and the resulting mixture was stirred for 44 h
at 23 °C. The reaction mixture was diluted with CH2Cl2, washed with
aqueous 1 N HCl and brine, and dried over anhydrous Na2SO4.
Dichloromethane solution was filtered and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (20−30% EtOAc/hexanes) to afford the correspond-
ing sulfonamide 29 in 74% yield (1.2 g). 1H NMR (400 MHz,
CDCl3): δ 9.38 (s, 1H), 8.25 (s, 1H), 7.87 (s, 1H), 7.49 (d, J = 7.1 Hz,
2H), 7.42 (t, J = 7.4 Hz, 2H), 7.39−7.33 (m, 1H), 6.79 (s, 1H), 6.69
(s, 1H), 4.73 (s, 2H), 3.91 (s, 3H), 3.90 (s, 2H), 2.75 (q, J = 7.5 Hz,
2H), 1.30 (t, J = 7.5 Hz, 3H), 1.11−0.99 (m, 2H), 0.77−0.65 (m, 2H).
13C NMR (100 MHz, CDCl3): δ 167.62, 136.35, 135.01, 128.91,
128.63, 128.44, 122.14, 121.20, 120.85, 120.76, 120.61, 120.36, 74.15,
73.10, 51.84, 38.91, 18.09, 14.25, 10.25.
Synthesis of 7,6,5-Tricyclic Indole Derivative 30. To a solution of
sulfonamide 29 (2.7 mmol, 1.19 g) in MeOH (75 mL) and AcOH (25
mL), 10% Pd/C (0.2 g) was added under argon. The argon balloon
was now replaced with a H2 balloon, and the resulting mixture was
stirred for 16 h at 23 °C. The reaction mixture was filtered through
Celite and washed with MeOH. The solvent was removed under
reduced pressure, and the resulting residue was diluted with toluene
and concentrated under reduced pressure to furnish the corresponding
alcohol in 95% yield.
To a mixture of the above alcohol (0.6 mmol, 0.21 g) and Et3N (0.9
mmol, 0.12 mL) in CH2Cl2 (25 mL) at 0 °C was added
methanesulfonyl chloride (0.63 mmol, 0.049 mL), and the resulting
mixture was stirred for 2 h at 23 °C. The reaction mixture was diluted
with CH2Cl2, washed with aqueous 1 N HCl and brine, and dried over
anhydrous Na2SO4. Dichloromethane solution was filtered and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (5−15% diethyl ether/CH2Cl2) to
afford the corresponding mesylate in 46% (70% BRSM) yield (0.12 g,
69 mg of alcohol was recovered). 1H NMR (400 MHz, CDCl3): δ 9.24
(s, 1H), 8.26 (s, 1H), 7.87 (d, J = 1.0 Hz, 1H), 7.41 (s, 1H), 7.07 (s,
1H), 4.58 (s, 2H), 3.93 (s, 3H), 3.16 (s, 3H), 2.79 (q, J = 7.5 Hz, 2H),
1.38−1.28 (m, 5H), 1.06−0.97 (m, 2H).
1
2,3-dihydroxyhexanoate in 60% yield (1.34 g). H NMR (300 MHz,
CDCl3): δ 4.29 (q, J = 7.2 Hz, 2H), 4.10−4.04 (m, 1H), 3.96−3.84
(m, 1H), 3.07 (d, J = 5.2 Hz, 1H), 1.90 (d, J = 7.7 Hz, 1H), 1.68−1.38
(m, 4H), 1.32 (t, J = 7.1 Hz, 3H), 0.96 (t, J = 7.2 Hz, 3H).
To a solution of (2R,3S)-ethyl 2,3-dihydroxyhexanoate (6.7 mmol,
1.18 g) in CH2Cl2 (30 mL) were added Et3N (10 mmol, 1.39 mL) and
4-nitrobenzenesulfonyl chloride (6.7 mmol, 1.48 g) at 0 °C. The
resulting reaction mixture was stirred for 24 h at 23 °C. The reaction
mixture was diluted with H2O and CH2Cl2. The organic layer was
separated, and the aqueous layer was extracted with CH2Cl2. The
combined extracts were dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (10−25% EtOAc/hexanes) to
obtain (2R,3S)-ethyl 3-hydroxy-2-{[(4-nitrophenyl)sulfonyl]oxy}-
1
hexanoate (32) in 54% yield (1.3 g). H NMR (400 MHz, CDCl3):
δ 8.38 (d, J = 8.8 Hz, 2H), 8.16 (d, J = 8.9 Hz, 2H), 4.96 (d, J = 2.9
Hz, 1H), 4.15 (q, J = 7.1 Hz, 2H), 4.12−4.04 (m, 1H), 2.07 (brs, 1H),
1.62−1.44 (m, 3H), 1.43−1.30 (m, 1H), 1.21 (t, J = 7.1 Hz, 3H), 0.92
(t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 166.74, 150.75,
141.85, 129.45, 124.18, 80.97, 71.23, 62.30, 34.98, 18.47, 13.89, 13.63.
Synthesis of (2S,3S)-Ethyl 2-[(tert-Butoxycarbonyl)amino]-3-
hydroxyhexanoate (33). To a solution of (2R,3S)-ethyl 3-hydroxy-
2-{[(4-nitrophenyl)sulfonyl]oxy}hexanoate (32) (3.6 mmol, 1.3 g) in
DMF (10 mL), NaN3 (5.76 mmol, 0.374 g) was added at 23 °C, and
the resulting mixture was heated at 55 °C for 15 h. The reaction
mixture was cooled to 23 °C and diluted with ethyl acetate, and the
resulting solution was washed with H2O, dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (15%
EtOAc/hexanes) to obtain (2S,3S)-ethyl 2-azido-3-hydroxyhexanoate
in 83% yield (0.6 g). 1H NMR (400 MHz, CDCl3): δ 4.29 (qd, J = 7.2,
1.3 Hz, 2H), 3.94 (s, 2H), 2.32 (brs, 1H), 1.59−1.45 (m, 3H), 1.44−
1.37 (m, 1H), 1.33 (t, J = 7.2 Hz, 3H), 0.94 (t, J = 6.9 Hz, 3H). 13C
NMR (100 MHz, CDCl3): δ 168.92, 71.56, 66.15, 61.99, 35.03, 18.52,
14.06, 13.74.
To a solution of the above mesylate (0.69 mmol, 0.297 g) in DMF
(30 mL) was added NaH (2.76 mmol, 0.11 g, 60% dispersion in
mineral oil) at 23 °C. After the mixture was stirred for 3 h at 23 °C,
iodomethane (3.5 mmol, 0.22 mL) was added to the flask, and stirring
was continued for further 1 h at 23 °C. The reaction mixture was
carefully quenched with MeOH, diluted with ethyl acetate, and washed
with aqueous 1 N HCl. The ethyl acetate solution was filtered and
concentrated under reduced pressure. The resulting residue was
purified by silica gel column chromatography (35−40% EtOAc/
hexanes) to afford the corresponding 7,6,5-tricyclic indole derivative in
To a mixture of (2S,3S)-ethyl 2-azido-3-hydroxyhexanoate (2.98
mmol, 0.6 g) and (Boc)2O (4.47 mmol, 0.975 g) in EtOH (15 mL)
was added 10% Pd−C (0.15 g) at 23 °C under argon. The argon
balloon was replaced with a H2 balloon, and the reaction mixture was
stirred for 15 h under H2 atmosphere. The reaction mixture was
filtered through Celite, washed with ethyl acetate, and concentrated
under reduced pressure. The residue was purified by silica gel column
chromatography (10−25% EtOAc/hexanes) to obtain (2S,3S)-ethyl 2-
[(tert-butoxycarbonyl)amino]-3-hydroxyhexanoate (33) in 96% yield
1
(0.79 g). H NMR (300 MHz, CDCl3): δ 5.52 (d, J = 7.2 Hz, 1H),
4.42−4.28 (m, 1H), 4.27−4.13 (m, 2H), 3.94−3.80 (m, 1H), 3.03
(brs, 1H), 1.59−1.30 (m, 13H), 1.26 (t, J = 7.1 Hz, 3H), 0.89 (t, J =
1
87% yield (0.21 g). H NMR (400 MHz, CDCl3): δ 8.26 (d, J = 1.1
Hz, 1H), 7.83 (d, J = 1.0 Hz, 1H), 6.78 (s, 1H), 4.32 (s, 2H), 3.93 (s,
J
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6.9 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 170.68, 155.98, 80.20,
72.67, 61.48, 58.34, 35.29, 28.18, 18.86, 14.06, 13.82. LRMS-ESI (m/
z): [M + Na]+, 298.26.
with 7,6,5-tricyclic indole derivative 30 as described for the inhibitor 4.
1H NMR (400 MHz, CDCl3): δ 7.88 (s, 1H), 7.47 (s, 1H), 7.36 (t, J =
6.0 Hz, 1H), 7.33−7.27 (m, 2H), 7.26−7.17 (m, 3H), 6.77 (s, 1H),
6.55 (d, J = 8.2 Hz, 1H), 4.56−4.43 (m, 1H), 4.27 (s, 2H), 4.03 (p, J =
6.2 Hz, 1H), 3.41 (s, 3H), 3.12 (d, J = 5.1 Hz, 1H), 3.08−2.99 (m,
2H), 2.95 (dd, J = 6.5, 4.4 Hz, 2H), 2.82 (dd, J = 12.2, 4.7 Hz, 1H),
2.78−2.65 (m, 3H), 1.69 (hept, J = 6.6 Hz, 1H), 1.52 (t, J = 6.3 Hz,
2H), 1.28 (t, J = 7.5 Hz, 3H), 1.13 (d, J = 6.4 Hz, 3H), 1.00 (t, J = 6.4
Hz, 2H), 0.83 (d, J = 6.7 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H). 13C NMR
(100 MHz, CDCl3): δ 172.75, 167.89, 137.56, 134.44, 130.80, 129.26,
128.62, 128.14, 127.09, 126.71, 126.13, 120.33, 118.15, 117.90, 68.26,
67.53, 52.30, 52.23, 51.39, 46.39, 43.05, 39.77, 39.02, 28.40, 20.09,
18.86, 17.94, 14.19, 12.67. HRMS-ESI (m/z): [M + H]+ calcd for
C33H46N5O5S, 624.3220; found, 624.3223.
Synthesis of tert-Butyl [(2S,3S)-3-Hydroxy-1-(isobutylamino)-1-
oxohexan-2-yl]carbamate (34a). To a solution of (2S,3S)-ethyl 2-
[(tert-butoxycarbonyl)amino]-3-hydroxyhexanoate (33) (0.67 mmol,
0.18 g) in THF (6 mL) and H2O (3 mL) was added LiOH·H2O (3.45
mmol, 0.145 g). The resulting reaction mixture was stirred for 12 h at
23 °C. The reaction mixture was diluted with H2O and ethyl acetate.
The organic layer was separated, and the aqueous layer was acidified
with 1 N HCl and extracted with ethyl acetate. The combined extracts
were dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure. The residue was used in the coupling reaction
without any further purification.
The synthesis of tert-butyl [(2S,3S)-3-hydroxy-1-(isobutylamino)-1-
oxohexan-2-yl]carbamate (34a) has been carried out by coupling of
(2S,3S)-2-[(tert-butoxycarbonyl)amino]-3-hydroxyhexanoic acid with
Synthesis of Compound 35b. (2S,3S)-2-Amino-3-hydroxy-N-
isopropylhexanamide was synthesized from (2S, 3S)-2-[(tert-
butoxycarbonyl)amino]-3-hydroxyhexanoic acid by coupling with
i
i
isobutyl amine using EDC, HOBt, and Pr2NEt following a similar
isopropyl amine using EDC, HOBt, and Pr2NEt followed by Boc
procedure described for the synthesis of (S)-tert-butyl [1-(isobutyla-
mino)-1-oxobutan-2-yl]carbamate (yield 60%, over two steps). 1H
NMR (400 MHz, CDCl3): δ 6.51 (brs, 1H), 5.62 (d, J = 7.2 Hz, 1H),
3.98−3.82 (m, 2H), 3.69 (brs, 1H), 3.21−3.08 (m, 1H), 3.06−2.92
(m, 1H), 1.77 (hept, J = 6.7 Hz, 1H), 1.63−1.46 (m, 3H), 1.44 (s,
9H), 1.41−1.32 (m, 1H), 0.97−0.83 (m, 9H).
removal using trifluoroacetic acid following the procedure described
for the synthesis of (2S,3S)-2-amino-3-hydroxy-N-isobutyl-N-methyl-
butanamide (yield 79% over two steps). 1H NMR (400 MHz, CDCl3):
δ 7.22 (brs, 1H), 4.14−3.92 (m, 1H), 3.81−3.67 (m, 1H), 3.22 (d, J =
6.2 Hz, 1H), 2.40 (brs, 2H), 1.62−1.24 (m, 4H), 1.18−1.08 (m, 6H),
0.91 (t, J = 6.9 Hz, 3H).
Synthesis of Compound 35a. Compound 35a was synthesized
from tert-butyl [(2S,3S)-3-hydroxy-1-(isobutylamino)-1-oxohexan-2-
yl]carbamate (34a) by Boc removal using trifluoroacetic acid followed
by reductive amination of the resulting amine with aldehyde 11
following the procedure described for the synthesis of compound 12a
(yield 49%, over two steps). 1H NMR (400 MHz, CDCl3): δ 7.28 (t, J
= 7.2 Hz, 2H), 7.22 (d, J = 7.0 Hz, 1H), 7.19−7.13 (m, 2H), 4.63 (d, J
= 7.4 Hz, 1H), 3.92 (brs, 1H), 3.84−3.74 (m, 1H), 3.12−2.94 (m,
3H), 2.86−2.71 (m, 2H), 2.64 (dd, J = 12.1, 4.6 Hz, 1H), 2.55 (dd, J =
12.0, 7.4 Hz, 1H), 1.71 (hept, J = 6.7 Hz, 1H), 1.58−1.21 (m, 13H),
0.90 (t, J = 7.0 Hz, 3H), 0.86 (d, J = 6.6 Hz, 6H).
Compound 35b was prepared from (2S,3S)-2-amino-3-hydroxy-N-
isopropylhexanamide by reductive amination with aldehyde 11
following the procedure described for the synthesis of compound
1
12a (yield 81%). H NMR (400 MHz, CDCl3): δ 7.28 (t, J = 7.4 Hz,
2H), 7.22 (d, J = 7.3 Hz, 1H), 7.20−7.13 (m, 2H), 7.00 (br, 1H), 4.62
(d, J = 8.1 Hz, 1H), 4.08−3.85 (m, 2H), 3.83−3.72 (m, 1H), 2.98 (d, J
= 5.3 Hz, 1H), 2.82 (dd, J = 13.7, 6.5 Hz, 1H), 2.75 (dd, J = 13.3, 7.4
Hz, 1H), 2.60 (dd, J = 12.2, 4.7 Hz, 1H), 2.54 (dd, J = 12.1, 7.3 Hz,
1H), 1.59−1.20 (m, 13H), 1.11 (d, J = 6.5 Hz, 3H), 1.06 (d, J = 6.6
Hz, 3H), 0.90 (t, J = 7.0 Hz, 3H).
Synthesis of Inhibitor 22. Inhibitor 22 was synthesized by Boc
Synthesis of Inhibitor 19. Inhibitor 19 has been synthesized from
compound 35a by Boc removal using trifluoroacetic acid followed by
coupling of the resulting amine with known acid 23 using EDC,
removal of 35b using trifluoroacetic acid followed by coupling of the
i
resulting amine with the acid 30 using EDC, HOBt, and Pr2NEt
following a similar procedure described for the synthesis of inhibitor 4
(yield 65% over two steps). 1H NMR (400 MHz, CDCl3): δ 7.88 (d, J
= 1.1 Hz, 1H), 7.46 (d, J = 1.0 Hz, 1H), 7.36−7.28 (m, 2H), 7.28−
7.20 (m, 3H), 6.97 (d, J = 8.2 Hz, 1H), 6.78 (s, 1H), 6.38 (d, J = 8.2
Hz, 1H), 4.54−4.42 (m, 1H), 4.28 (ABq, J = 20.6, 14.9 Hz, 2H),
4.08−3.95 (m, 1H), 3.84−3.75 (m, 1H), 3.44 (s, 3H), 3.09 (d, J = 4.9
Hz, 1H), 2.98 (d, J = 6.8 Hz, 2H), 2.81 (dd, J = 12.3, 4.9 Hz, 1H),
2.77−2.69 (m, 3H), 1.56−1.33 (m, 5H), 1.33−1.24 (m, 4H), 1.07 (t, J
= 6.9 Hz, 6H), 1.01−0.96 (m, 2H), 0.87 (t, J = 7.1 Hz, 3H). 13C NMR
(125 MHz, CDCl3): δ 171.67, 167.77, 137.47, 134.36, 130.68, 129.20,
128.58, 128.06, 126.97, 126.69, 126.05, 120.23, 118.05, 117.83, 71.81,
66.05, 52.15, 51.63, 50.99, 43.01, 40.92, 39.60, 38.81, 35.05, 22.46,
18.90, 17.89, 14.15, 13.86, 12.64. HRMS-ESI (m/z): [M + H]+ calcd
for C34H48N5O5S, 638.3376; found, 638.3371.
Determination of X-ray Structure of β-Secretase−Inhibitor 5
Complex. Expression and purification of recombinant human β-
secretase, crystal growing, inhibitor soaking of the crystal, and
diffraction data collection were performed as previously described.8
The structure was determined by molecular replacement implemented
with the program AMoRe using the C molecule of previously
determined memapsin 2 structure (PDB ID: 1FKN) as a search
model8 with removed inhibitor and water molecules. Rotation and
translation functions followed by the rigid-body refinement with data
from 15 to 3.5 Å resolution in space group P21 gave unambiguous
solutions for the four memapsin 2 molecules in the asymmetric unit. A
random selection of 7% of reflections (9028 reflections) was set aside
as the test set for cross-validation during the refinement. The refined
model had well-defined electron density for the inhibitor, and its
corresponding structure was built into the active site. The four
molecules in the crystallographic asymmetric unit have essentially
identical structures. The crystal form was determined to be monoclinic
with a resolution of 2.0 Å. The unit cell parameters are a = 86.4 Å, b =
130.3 Å, c = 88.4 Å, and β = 97.5. The coordinates and structure
i
HOBt, and Pr2NEt following a similar procedure described for the
1
synthesis of inhibitor 8 (yield 50%). H NMR (400 MHz, CDCl3): δ
7.85 (d, J = 1.0 Hz, 1H), 7.46 (s, 1H), 7.35−7.28 (m, 2H), 7.26−7.19
(m, 3H), 6.84 (s, 1H), 6.41 (d, J = 8.2 Hz, 1H), 4.54−4.44 (m, 3H),
3.89−3.76 (m, 3H), 3.47 (s, 3H), 3.13 (d, J = 4.9 Hz, 1H), 3.11−2.99
(m, 2H), 2.97 (d, J = 6.8 Hz, 2H), 2.83 (dd, J = 12.3, 4.8 Hz, 1H),
2.79−2.68 (m, 3H), 1.71 (hept, J = 6.7 Hz, 1H), 1.54−1.33 (m, 4H),
1.30 (t, J = 7.5 Hz, 3H), 0.91−0.80 (m, 9H). 13C NMR (100 MHz,
CDCl3): δ 172.89, 167.85, 137.51, 133.83, 130.72, 129.30, 128.67,
127.72, 127.26, 126.78, 125.83, 120.25, 117.93, 117.56, 72.00, 66.43,
56.81, 51.98, 51.20, 46.44, 43.62, 39.61, 38.94, 35.26, 28.42, 20.10,
18.98, 17.94, 14.26, 13.94. HRMS-ESI (m/z): [M + H]+ calcd for
C33H48N5O5S, 626.3376; found, 626.3369.
Synthesis of Inhibitor 20. Inhibitor 20 has been synthesized from
compound 35a by Boc removal using trifluoroacetic acid followed by
coupling of the resulting amine with acid 25 using EDC, HOBt, and
iPr2NEt following the similar procedure described for the synthesis of
1
inhibitor 8 (yield 52%). H NMR (400 MHz, CDCl3): δ 7.71 (d, J =
0.8 Hz, 1H), 7.40 (s, 1H), 7.34−7.28 (m, 2H), 7.26−7.18 (m, 3H),
6.80 (s, 1H), 6.39 (d, J = 8.2 Hz, 1H), 4.54−4.40 (m, 1H), 4.11 (s,
2H), 3.80 (p, J = 4.2 Hz, 1H), 3.50 (s, 3H), 3.13 (d, J = 4.8 Hz, 1H),
3.11−2.99 (m, 2H), 2.97 (d, J = 6.8 Hz, 2H), 2.82 (dd, J = 12.3, 5.0
Hz, 1H), 2.79−2.64 (m, 3H), 1.70 (hept, J = 6.7 Hz, 1H), 1.56 (s,
6H), 1.53−1.33 (m, 4H), 1.30 (t, J = 7.5 Hz, 3H), 0.93−0.79 (m, 9H).
13C NMR (125 MHz, CDCl3): δ 172.59, 167.99, 137.38, 134.28,
130.53, 129.22, 128.81, 128.59, 127.37, 126.83, 126.71, 120.29, 116.36,
114.86, 71.83, 66.29, 65.68, 56.50, 51.89, 51.03, 46.38, 39.40, 38.81,
35.15, 28.33, 22.49, 22.46, 20.03, 19.71, 18.90, 17.88, 14.02, 13.87.
HRMS-ESI (m/z): [M + H]+ calcd for C35H52N5O5S, 654.3689;
found, 654.3696.
Synthesis of Inhibitor 21. Inhibitor 21 was synthesized (yield 65%
over two steps) from 12b by Boc deprotection followed by coupling
K
dx.doi.org/10.1021/jm3008823 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
factors of the β-secretase and 5 complex have been deposited in
8234 Rescues Age-related Cognitive Decline in APP Transgenic Mice.
FASEB J. 2011, 25, 775−784.
Protein Data Bank28 with PDB ID: 4GID.
(11) Turner, R. T.; Loy, J. A.; Nguyen, C.; Devasamudram, T.;
Ghosh, A. K.; Koelsch, G.; Tang, J. Specificity of Memapsin 1 and Its
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Selectivity. Biochemistry 2002, 41, 8742−8746.
ASSOCIATED CONTENT
* Supporting Information
HPLC and HRMS data of inhibitors 4−9 and 18−22;
crystallographic data collection and refinement statistics for
inhibitor 5. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
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Accession Codes
The PDB accession code for 5-bound β-secretase X-ray
structure is 4GID.
AUTHOR INFORMATION
Corresponding Author
*Tel: 765-494-5323. Fax: 765-496-1612. E-mail: akghosh@
purdue.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support by the National Institutes of Health (AG
18933) is gratefully acknowledged. We thank Dr. Cuthbert D.
Martyr (Purdue University) for his help with the MS data.
ABBREVIATIONS
■
AD, Alzheimer's disease; BACE 1, β-site APP cleaving enzyme
1; BACE 2, β-site APP cleaving enzyme 2; APP, β-amyloid
precursor protein; CD, cathepsin D
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