Design and synthesis of novel fluoro amino acids: synthons for potent macrocyclic HCV NS3 protease inhibitors

Article abstract of DOI:10.1016/j.tetlet.2010.04.010

The Hepatitis C Virus (HCV) is a major health hazard and its infection is a leading cause of chronic liver disease world wide. In our efforts toward the discovery of a back up to our first clinical candidate, Boceprevir (SCH 503034), we approached the depeptidization of the molecule through macrocyclization. Herein we report the design and synthesis of fluoro amino acids with desired stereochemistry required for the synthesis of macrocyclic inhibitors with fluorine at various positions of the aliphatic chain. Biological activities of representative examples are also reported.

Full text of DOI:10.1016/j.tetlet.2010.04.010

Tetrahedron Letters 51 (2010) 3057–3061
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Design and synthesis of novel fluoro amino acids: synthons for potent
macrocyclic HCV NS3 protease inhibitors
*
Latha G. Nair , Stephane Bogen, Frank Bennett, Kevin Chen, Bancha Vibulbhan, Yuhua Huang, Weing Yang,
Ronald J. Doll, N.-Y. Shih, F. George Njoroge
Merck-Research Laboratories, Kenilworth, NJ 07033, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 December 2009
Revised 2 April 2010
Accepted 5 April 2010
Available online 9 April 2010
The Hepatitis C Virus (HCV) is a major health hazard and its infection is a leading cause of chronic liver
disease world wide. In our efforts toward the discovery of a back up to our first clinical candidate, Boce-
previr (SCH 503034), we approached the depeptidization of the molecule through macrocyclization.
Herein we report the design and synthesis of fluoro amino acids with desired stereochemistry required
for the synthesis of macrocyclic inhibitors with fluorine at various positions of the aliphatic chain. Biolog-
ical activities of representative examples are also reported.
Ó 2010 Elsevier Ltd. All rights reserved.
p2
Around 3% of the world population is affected by Hepatitis C
Virus and its infection is a leading cause of chronic liver des-
ease.1a–d The current standard of care treatment is a combination
p1'
NH2
cap
N
O
of subcutaneous pegylated interferon-a with oral nucleoside drug
ribavirin.2 Due to the urgent need for a more tolerable and effica-
cious regimen, the last decade has seen an emergence of therapies
targeting the key enzymes involved in the replication and matura-
tion of the virus.3 Extensive efforts have been focused on NS3-4A
protease research4 recently and a number of novel inhibitors
including SCH 503034 (Fig. 1) Boceprevir5 are reported.
N
N
O
O
N
O
O
p1
p3
Modeling studies and the X-ray crystal structure on enzyme
surface for Boceprevir, the clinical candidate, revealed the proxim-
ity of P2 and P4 residues as well as the P1 and P3 residues. We
envisioned the macrocyclization of these two units6 as an effort
to depeptidize the HCV protease inhibitor. Macrocyclization has
been an approach widely used for depeptidization and there are re-
ports which show that this type of modification could improve the
physicochemical properties with improved Pharmacokinetics
(PK).7 Encouraged by the results from the fluorinated congener of
Boceprevir8 we decided to block the metabolic site of the aliphatic
chain of the P1 or P3 with fluorine. Previous results from our
group6 showed that a 16-membered macrocycle would be optimal
for P1–P3 ring. Herein we report the design and synthesis of the
desired fluoro amino acids as synthons for macrocyclic inhibitors.
Retrosynthetic analysis of the 16-membered macrocycle 2 is de-
picted in Figure 2. Tripeptide 3 was obtained by the sequential cou-
pling of the proline derivative 5 with the fluoro amino acid 4,
hydrolysis of the corresponding methyl ester followed by the cou-
Ki* =14 nM
EC90= 350 nM
Figure 1. Boceprevir (SCH 503034).
pling of the allyl glycine 6. Ring-closing metathesis (RCM)9 was
used as the key step for the construction of macrocycle 2.
The synthesis of the fluoro amino acid 4 started with the 6-oxo
piperidine carboxylic acid derivative 7 (Scheme 1). Grignard addi-
tion of the butenyl magnesium bromide 8 to the piperidinone 7 re-
sulted in the keto amino acid 9 with the desired stereochemistry.
The Grignard reagent was synthesized in situ with the alkenyl bro-
mide and magnesium turnings in THF. Keto amino acid 9 was con-
verted to the corresponding difluoro derivative 10 with DAST. We
were interested in investigating the effect of fluorine at both P1
and P3 units of the macrocycle. Difluoroamino acid 10 was used
as the synthon for both 10,10-difluoromacrocycle and 12,12-diflu-
oromacrocycle at P3 and P1, respectively (color coded blue in
Fig. 3). Boc deprotection of the amino acid 10 afforded the amine
salt 11 that will be coupled with the proline derivative 5 to get
the fluoro side chain at the P3 position. Ester hydrolysis of the
* Corresponding author. Tel.: +1 908 740 4692; fax: +1 908 740 7305.
E-mail address: latha.nair@spcorp.com (L.G. Nair).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.04.010

3058
L. G. Nair et al. / Tetrahedron Letters 51 (2010) 3057–3061
1.
MgBr
8
NHBoc
CO2Et
N
CO2Me
N
CO2Me
O
N
N
CO2Et
N
F
F
DAST
2.
H
N
H
N
Boc
O
O
Boc
O
Boc
O
3
15
13
16
Scheme 2. Synthesis of fluoro amino acid.
F
F
F
F
2
ester could also be used at both P1 and P3 areas to construct mac-
rocycles with fluorine at 13,13 and 9,9 positions, respectively.
A unique synthesis (Scheme 3) was designed for the symmetri-
cal fluoro analog (11,11-difluoro derivative, green colored in Fig. 3).
5-Hydroxy pentanal 16 was treated with allyl magnesium bromide
to the corresponding allyl alcohol. Protection of the primary alco-
hol with the benzoyl group followed by oxidation of the secondary
alcohol with Dess–Martin Periodinane resulted in the allyl ketone
17. Difluorination of 17 was achieved with DAST in 61% isolated
yield. Basic hydrolysis attempts of the benzyl ester under aqueous
conditions were unsuccessful. Thus, the difluorocompound was
further treated with ethyl magnesium bromide and the alcohol
was oxidized with Dess–Martin Periodinane to get the aldehyde
18. Aldehyde 18 was treated with the phosphonate 19 to get the
unsaturated amino acid 20. Enantioselective reduction of enamide
20 was accomplished with 8% w/w of chiral rhodium catalyst and
the resulting acetamide was exchanged with Boc-protecting group.
The Boc methyl ester was then hydrolyzed to the chiral amino acid
21 with LiOH in 85% combined yield in three steps.
O
O
N
HCl
H2N
CO2Me
OH
O
O
+
+
N
H
O
6
5
4
F
F
OEt
O
N
Boc
O
7
Figure 2. Retrosynthetic analysis of 10,10-difluoromacrocycle 2.
MgBr, Et2O
82%
8
O
NHBoc
CO2Et
7
9
Incorporation of fluorine in the macrocycle was outlined with a
representative example shown in Scheme 4. Dipeptide 22 was syn-
thesized via a coupling of the difluoroamino acid 4 and the P2 pro-
line methyl ester 5. The synthesis of the P2 proline derivative 5 was
reported in earlier publications.12 The dipeptide 22 was then
hydrolyzed under basic conditions to the acid which was then cou-
pled to the allyl glycine 6 to obtain the tripeptide 3. Although the
initial attempts for the Ring-closing Metathesis with Grubbs first
generation catalyst were not successful, we were able to convert
the tripeptide 3 to the corresponding 16-membered macrocycle
in good yield using Grubbs second generation catalyst. The result-
ing isomeric mixture of olefins was hydrogenated using Pd/C at
1 atm. pressure with an excellent yield to afford the methyl ester
2. Reduction of the methyl ester 2 to the corresponding alcohol
with lithium borohydride followed by oxidation with Dess–Martin
periodinane to the aldehyde and the Passerini reaction with cyclo-
propyl isocyanide resulted in the acetyl amide 23.6c Deprotection
of the acetyl group followed by the oxidation of the hydroxyl group
resulted in the ketoamide 24.
Improvement in the yield of the fluorination was achieved with
Deoxofluor as the fluorinating agent. One representative example
is outlined in Table 1. Change of fluorinating agent from DAST to
deoxofluor for ketone 14 improved the yield almost twofold. The
yield for the coupling of amino acid of ester 14 with proline 5
was low (30%). Twofold betterment in the yield in two steps was
achieved by reversing the sequence of coupling and fluorination.
Thus, coupling of the amino acid of keto ester 14 with the proline
5 (98% isolated yield) followed by reaction of the resulting dipep-
tide 26 with deoxofluor afforded the corresponding difluoro dipep-
tide in 70% yield.
DAST, 50%
NHBoc
F
F
CO2Et
1
10
M
A
q
1
.
0
0
%
LiOH
HCl/Dioxane
M
100%
4
NHBoc
CO2H
NH2HCl
CO2Et
F
F
F
F
11
4
Scheme 1. Synthesis of fluoro amino acid.
O
N
OMe
N
O
BocHN
O
12
13
11
9
10
12
Figure 3. Macrocycle with color code for fluorine substitution.
amino acid 10 with LiOH resulted in the Boc-protected amino acid
4. Incorporation of the fluorine at the desired position in the P1
unit was achieved via the coupling of the amino acid 4 with the
dipeptide 25 under standard HATU conditions (Scheme 4).
In order to achieve the synthesis of macrocycles with fluorine at
9,9 and 13,13 positions (colored red in Fig. 3) we designed amino
acid 1510 (scheme 2). Pyroglutamate derivative 13 was opened
up with butenyl Grignard reagent11 8 as described before and the
resulting keto amino acid 14 (Table 1) was fluorinated with DAST
to afford 15. Boc-protected amino acid of ester 15 or its free amino
Biological activities of selected fluoromacrocycles are outlined
in Table 2. All inhibitors were tested in the HCV continuous enzy-
matic assay13 usÃing the NS4A-tethered single chain NS3 serine
protease.14 The Ki values reflected the equilibrium constant deter-
mined by the reversible covalent bond formed between the ketone
and serine and other interactions between the inhibitors and the
enzyme.15 The concentration required for the inhibition of 90% of
virus replication, EC90, was obtained as a measure of replicon

L. G. Nair et al. / Tetrahedron Letters 51 (2010) 3057–3061
3059
O
O
OH
1) AllylMgBr , Et O, -78ºC
2
1) DAST, DCM (61%)
2) EtMgBr , Et O
2
2) PhCOCl , Et N, DMAP
3
16
17
3) Dess-Martin Oxidation
O
3) Dess-Martin oxidation
60%
O
O
H
O
N
N
O
O
O
1) H , EtOH
2
O
O
P
O
O
O
19
3% w/w Rh* (S,S) duphos
O
F
2) (Boc) O, DMAP, THF, 60ºC
2
F
DBU, DCM
3) LiOH , MeOH, 0ºC
85%
F
F
54% (3 steps)
18
20
Used as crude
DCM solution
O
O
N
OH
F
F
21
Scheme 3. Synthesis of fluoromacrocyclic precursor for the symmetrical (11,11-difluoro) analog.
OH
HATU,NMM, 6
N
95%
O
O
NHBoc
5
25
HATU,NMM,
OMe
4, 35%
N
O
O
F
F
NHBoc
22
O
1.aq.LiOH/THF, 100%
1.RCM,80%
N
22
OMe
N
2.HATU, 6
2.H2, Pd/C,99%
O
BocHN
68%
O
3
F
F
1.LiBH4 2.Dess-Martin
85% combined
O
OAc
O
N
N
N
N
N
OMe
3.
NC, HAC
90%
O
O
O
N
BocHN
O
O
O
2
23
F
F
F
F
1. K2CO3, quantitative
O
N
3. Dess-Martin oxidation
92%
N
N
O
O
BocHN
O
24
F
F
Scheme 4. Synthesis of fluoromacrocycles.

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L. G. Nair et al. / Tetrahedron Letters 51 (2010) 3057–3061
Table 1
Fluorination of amino acids
In conclusion, we have designed and accomplished the synthe-
ses of new fluoroamino acids in such a way as to have the difluoro-
substitution at various desired positions of the macrocycles, at
both the P3 and the P1 positions. Macrocyclization of the fluoro
olefins were achieved via Grubbs Ring-closing metathesis using
second generation catalyst. These macrocycles are biologically ac-
tive against the HCV NS3 serine protease.
Reagent
Ketoamino acid
Yield (%)
40
NHBoc
CO2Et
DAST
O
14
NHBoc
CO2Et
Acknowledgments
Deoxo-fluor
75
70
O
L.G.N. would like to thank Structural Chemistry and Virology
groups of legacy Schering-Plough for spectral analysis and
in vitro assays, respectively.
14
OMe
N
Supplementary data
O
Deoxo-fluor
BocHN
O
26
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.04.010.
O
References and notes
O
N
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OMe
N
Deoxo-fluor
50
O
BocHN
O
27
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Biological activity of fluorinated macrocycles
O
N
N
N
O
16
O
O
N
O
O
Linker
KiÃ
(lM)
Compound
Linker
EC90
0.30
(lM)
28
0.096
0.42
0.6
F
F
F
F
29
30
0.80
NA
F
F
24
0.4
0.900
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F
F
cellular potency.16 Inhibitors were tested for the activity against
one of the most structurally closely related serine protease, human
neutrophil elastase (HNE) to determine the selectivity between
HCV and HNE.

L. G. Nair et al. / Tetrahedron Letters 51 (2010) 3057–3061
3061
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