Practical and efficient method for amino acid derivatives containing β-quaternary center: application toward synthesis of hepatitis C virus NS3 serine protease inhibitors

Article abstract of DOI:10.1016/j.tetlet.2007.07.002

A practical and efficient route toward synthesis of amino acid derivatives containing β-quaternary center has been developed using diastereoselective Strecker reaction. The method was employed for preparation of >100 g of β-methylcyclohexyl glycine deriva

Full text of DOI:10.1016/j.tetlet.2007.07.002

Tetrahedron Letters 48 (2007) 6343–6347
Practical and efficient method for amino acid derivatives containing
b-quaternary center: application toward synthesis of hepatitis
C virus NS3 serine protease inhibitors
Ashok Arasappan,* Srikanth Venkatraman, Angela I. Padilla, Wanli Wu, Tao Meng,
Yan Jin, Jesse Wong, Andrew Prongay, Viyyoor Girijavallabhan and F. George Njoroge
Schering Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, United States
Received 28 February 2007; revised 2 July 2007; accepted 3 July 2007
Available online 7 July 2007
Abstract—A practical and efficient route toward synthesis of amino acid derivatives containing b-quaternary center has been devel-
oped using diastereoselective Strecker reaction. The method was employed for preparation of >100 g of b-methylcyclohexyl glycine
derivative, 21. Incorporation of some of the hindered amino acid derivatives at the P3 position resulted in potent HCV NS3 serine
protease inhibitors.
Ó 2007 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) infection is a global health cri-
sis leading to liver cirrhosis, hepatocellular carcinoma
and liver failure in humans.1 SCH 503034, a HCV
NS3 serine protease inhibitor, discovered in our labs is
currently undergoing clinical studies in humans.2 The
structure of 503034 is shown in Figure 1. The ketoamide
moiety acts as the electrophile, trapping the active site
serine. X-ray structure of the inhibitor bound to the pro-
tease revealed P1, P2, P3, and P3 cap moieties occupied
the corresponding pockets on the enzyme, which
resulted in the observed potency.
P2
O
H
N
NH2
N
H
H
O
O
N
N
O
O
P3 cap
P1
P3
Figure 1. Structure of SCH 503034.
During the course of our studies leading to potent HCV
NS3 serine protease inhibitors, we identified L-tert-leu-
cine and L-cyclohexylglycine as suitable P3 residues. In
an effort to further explore the P3 area, we decided to
introduce b-methyl cyclohexyl (or b-methyl cycloalkyl)
glycine, combination of the aforementioned amino acid
derivatives containing b-quaternary center, at that posi-
tion. Hence we turned our attention toward the synthe-
sis of these hindered amino acids.
synthesis. However, a-amino acids containing b-quater-
nary center are not trivial to synthesize.4 We decided to
employ the Strecker reaction for preparation of these
nonproteinogenic hindered amino acid derivatives. Only
few reports have appeared in the literature detailing use
of Strecker protocol for construction of amino acids
containing b-quaternary center.5 Herein we describe a
practical and efficient method for the diastereoselective
synthesis of b-methylcycloalkyl glycine derivatives. Fur-
thermore, P3 exploration with these nonnatural amino
acids resulted in potent HCV NS3 serine protease
inhibitors.
b-Quaternary centered amino acids are useful class of
nonnatural amino acids that occur as components in
pharmaceuticals and natural products.3 Numerous
methods are available for stereoselective a-amino acid
Aldehyde precursors for the diastereoselective Strecker
reaction were prepared as shown in Scheme 1.
*
Corresponding author. E-mail: ashok.arasappan@spcorp.com
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.07.002

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A. Arasappan et al. / Tetrahedron Letters 48 (2007) 6343–6347
Table 1.
CO2H
CHO
R1 R2
5-8
a
b
SM
Amino nitrile
Ratio a:ba
Yieldb (%)
CO2Me
a
b
1
2, 3
1
2
4
3
9a
9b
8:1
9:1
7:1
6:1
85
26
37
32
10a
11a
12a
10b
11b
12b
c
R1 R2
R1 R2
a Ratio determined from 1H NMR of crude material after Strecker
reaction or after hydrolysis to the amino methylester.
b Yield reported for spectroscopically pure product from correspond-
ing SM.
OH
5
8
4
6
7
Scheme 1. Reagents and conditions: (a) (i) MeOH, concd HCl (cat.),
reflux, 16 h; (ii) DIBAL or LAH, CH2Cl2, À78 °C to rt, 16 h; (iii) PCC
or DM periodinane, CH2Cl2, 0 °C to rt, 16 h; (b) (i) LDA or KHMDS,
MeI, THF, 0–10 °C, 2 h; then repeat step (a) (ii) and (iii); (c) repeat
step (a) (iii).
While the above described reaction sequence (Scheme 3)
worked well on a small scale, conversion of the amino
nitrile 9, 10a/b to amino ester 13, 14a/b were generally
low on a larger scale (>20 mmol).7 Careful analysis of
the reaction mixture showed the presence of amino
amide 13, 14c, resulting from partial hydrolysis of nitrile
functionality. It was interesting to note that the amino
amides 13, 14c were isolated (via crystallization or chro-
matography) as single diastereomer with S-stereochem-
istry at the a-center. These amino amides were also
Commercially available acid 1 was converted to the
corresponding methyl ester that was subsequently
processed to aldehyde 5 via a two-step reduction/oxida-
tion process. Aldehydes 6 and 7 were prepared from the
respective methyl esters 2 and 3 by alkylation followed
by the reduction/oxidation method. Aldehyde 8 was
synthesized by oxidation of the commercial alcohol 4
using PCC.
H
N
H
N
MeO2C
R1
Ph
MeO2C
R1
Ph
The aforementioned aldehydes were then subjected to
stereoselective Strecker reaction (Scheme 2). Thus, treat-
ment of various aldehydes with R-2-phenyl glycinol in
anhydrous chloroform at room temperature resulted in
in situ formation of the corresponding imines. Addition
of TMSCN to the cooled solution of the imines followed
by slow warming to room temperature afforded the de-
sired amino nitriles 9–12 with high diastereoselectivity.
The required S-amino nitrile was obtained as the major
product in all cases. Following completion of the reac-
tion, part of the product mixture remained protected
as the TMS–ether. The free alcohol could be easily
regenerated by treatment with aqueous 3 N HCl. The
diastereoselectivity and yield for the Strecker reaction
with various aldehydes are listed in Table 1.6
a
+
9-12a/b
13-16a
OH
OH
R2
R2
13-16a
13-16b
MeO2C
R1
NHBoc
HO2C
NHBoc
b
c
R1
R2
17-20
R2
21-24
=
R1
R2
9, 13
10, 14
18, 22
11, 15
19, 23
12, 16
20, 24
17, 21
Scheme 3. Reagents and conditions: (a) 6 M HCl/MeOH, 0 °C to rt,
16 h, add another portion of 6 M HCl/MeOH reflux, 16 h (43–45%);
(b) H2, 10% Pd/C or Pd(OH)2, Boc2O, EtOAc or MeOH, rt, 16 h (88–
100%); (c) aq 1 M LiOH, THF, MeOH, rt, 4–48 h.
The mixture of amino nitrile products described above
were hydrolyzed to the respective methyl esters 13–
16a/b using anhydrous HCl/MeOH conditions. It
should be noted that amino esters 13–16a/b were easier
to separate on silica gel column compared to their amino
nitrile counterpart 9–12a/b. Thus, the required major S-
isomers 13–16a was separated and the chiral auxiliary
was removed under hydrogenation conditions, with con-
comitant protection of the liberated amino residue to
give N-t-Boc intermediates 17–20. Hydrolysis of the
methyl ester using LiOH provided the expected N-pro-
tected amino acid derivatives 21–24.
H
H2NOC
N
Ph
a
+
13, 14a/b
9, 10a/b
13, 14c
OH
n = 0,1
13, 14c
H
N
HO2C
Ph
HO2C
NHBoc
b
c
OH
H
N
H
N
NC
R1
Ph
NC
R1
Ph
a
CHO
R1 R2
5-8
n = 0,1
n = 1, 25; n = 0, 26
n = 0,1
+
21, 22
OH
OH
R2
R2
Scheme 4. Reagents and conditions: (a) Condition (a) as in Scheme 3
(13c—40%, 14c—32%); (b) aq 6 M HCl, reflux, 16 h (91–100%); (c) (i)
H2, 10% Pd/C MeOH, rt, 16 h; (ii) Boc2O, NaOH, dioxane/water, rt,
16 h.
9-12a
9-12b
Scheme 2. Reagents and conditions: (a) R-2-Phenylglycinol (1 equiv),
CHCl3, rt, 2 h. Then add TMSCN (2 equiv), 0 °C to rt, 16 h.

A. Arasappan et al. / Tetrahedron Letters 48 (2007) 6343–6347
6345
was refluxed with aqueous 6 N HCl when the acid 25
precipitated out (on cooling the reaction mixture). Re-
moval of the chiral auxiliary using catalytic hydrogena-
tion condition followed by protection of the amino
residue resulted in the desired compound 21 as a solid
in 53% yield (for three steps).8
H
N
H
N
NC
Ph
HO2C
Ph
a
OH
OH
25
HO2C
9a
HO2C
NH2
NHBoc
Having obtained the hindered amino acid derivatives,
we then set out to incorporate those at the P3 position
of NS3 serine protease inhibitors. Synthesis of inhibitors
derived from 21 is shown in Scheme 6. Modified proline
derivative 289 was coupled to 21 under standard
coupling conditions (HATU, DIPEA). Basic hydrolysis
resulted in acid 32, which was subsequently coupled with
previously described intermediate 29.2 Oxidation under
modified Moffatt protocol afforded the desired inhibitor
34. Urea capped inhibitor 35 was obtained from 30 as
follows: removal of t-Boc functionality followed by
treatment with t-butylisocyanate provided dipeptide
31. Further processing to inhibitor 35 followed proce-
dures described in Scheme 6. Inhibitors 36–38 described
in Table 2 were also prepared from the corresponding
P3 amino acid derivatives, 22 and 23, in a similar
manner.
b
c
27
21
Scheme 5. Reagents and conditions: (a) (i) 6 M HCl/MeOH, 0 °C to
rt, 3 d; (ii) aq 6 M HCl, reflux, 2 d; (b) H2, 10% Pd/C MeOH, rt, 16 h;
(c) Boc2O, NaOH, dioxane/water, rt, 16 h (53% for three steps).
progressed further to the required N-protected amino
acid derivatives 21, 22 as shown in Scheme 4.
Although we were able to identify all the products of
amino nitrile hydrolysis and maximize the yield of the
required amino acid derivatives 21 and 22, this method
was not very practical on a large scale. Since we needed
modified cyclohexylglycine derivative 21 in larger quan-
tities, a practical and efficient route to this hindered
amino acid was then investigated. Thus, our optimized
conditions are shown in Scheme 5. After the diastereo-
selective Strecker reaction, the major S-amino nitrile
9a was obtained via silica gel column chromatography.
Amino nitrile 9a was converted to acid 25 in a one-pot
two-step process. Initial treatment with 6 M HCl in
MeOH resulted in a mixture of amino ester and amino
amide. The solvent was then removed and the residue
Targets 34–38 were then tested for HCV NS3 serine pro-
tease inhibition10 using the continuous spectrophoto-
metric assay described earlier11 and the results are
summarized in Table 2. Cyclization of two of the methyl
groups of L-tert-leucine (see Fig. 1) to a cyclopropyl ring
resulted in a drastic loss in potency as evidenced by the
KÃi value of inhibitors 37 and 38, 700 nm and 280 nm,
respectively. Increasing the ring size resulted in inhibitor
36 containing b-methyl cyclopentylglycine moiety at
P3, with improved potency (KÃi = 23 nm) compared to
OMe
N
c
a
H
OMe
O
X
N
N
O
HCl.
H
O
O
28
30, X = O
b
31, X = NH
OH
HCl.H2N
NH2
O
H
N
OH
O
NH2
N
H
N
N
29
O
H
X
O
O
O
X
N
O
O
d
O
34, X = O
32, X = O
33, X = NH
35, X = NH
Scheme 6. Reagents and conditions: (a) 21, HATU (1.3 equiv), DIPEA (3 equiv), CH2Cl2/DMF, À20–0 °C, 16 h; (b) (i) 4 M HCl/dioxane, rt, 3 h; (ii)
tert-butylisocyanate (1.3 equiv), DIPEA (2.5 equiv), CH2Cl2, 0 °C, 16 h (55%); (c) aq 1 M LiOH, THF, MeOH, rt, 4 h; (d) (i) 29, HATU (1.3 equiv),
DIPEA (3 equiv), CH2Cl2/DMF, À20–0 °C, 2 d; (ii) EDCI (10 equiv), dichloroacetic acid (5 equiv), DMSO/toluene, 0 °C to rt, 5 h (for 34—46%
after four steps, for 35—59% after two steps).

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A. Arasappan et al. / Tetrahedron Letters 48 (2007) 6343–6347
Table 2.
at P3 was in hydrophobic contact with the enzyme,
while the cyclohexyl residue was solvent exposed.
O
H
N
In summary we have developed a practical and efficient
diastereoselective route toward amino acids containing
b-quaternary center using Strecker chemistry. Some of
the hindered amino acids prepared were evaluated as
P3 surrogates resulting in potent HCV NS3 serine prote-
ase inhibitors. From SAR studies, b-methyl cycloalkyl-
glycine residue containing inhibitors exhibited P3 cap/
P3 synergy similar to earlier P3 tert-leucine containing
inhibitors. Inhibitor 35, with b-methyl cyclohexylglycine
moiety at P3 and urea cap afforded the best binding and
cellular replicon potency, similar to SCH 503034 that is
currently undergoing human clinical studies.
NH2
N
H
N
O
O
X
O
O
n
a
Compd #
X
n
KÃi (nm)
EC90 (nM)
37
38
36
34
35
O
1
1
3
4
4
700
280
23
120
13
nt
nt
500
nt
400
NH
NH
O
NH
a nt = not tested.
Acknowledgements
b-methyl cyclopropyl P3 compound 38. Incorporation
of the six-membered ring, b-methyl cyclohexylglycine,
at P3 position afforded inhibitors 34 and 35. While the
tert-butyl carbamate capped inhibitor 34 was less po-
tent, the tert-butyl urea capped compound 35 exhibited
the best binding (KÃi = 13 nm) and cellular potency (rep-
licon EC90 = 400 nM).12 Previously, we discovered
interesting synergy between the P3 cap and P3 moiety.2
Thus, for P3 L-cyclohexylglycine containing inhibitors,
tert-butyl carbamate cap provided better potency. On
the other hand, for P3 L-tert-leucine containing inhibi-
tors, tert-butyl urea cap was optimal, which resulted in
discovery of SCH 503034. In our present study, from
the potency data (Table 2) it was clear that inhibitors
with b-methyl cycloalklglycine P3 moiety exhibited syn-
ergy similar to those containing tert-leucine P3. Thus,
urea capped targets were more potent than their respec-
tive carbamate capped compounds.
We thank the Structural Chemistry group for providing
NMR and Mass Spectral assistance. We thank the
Virology group (Dr. B. Malcolm, Dr. R. Liu, N. But-
kiewicz, J. Pichardo) for providing KÃi and replicon
EC90 data.
References and notes
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Med. Chem. 2006, 49, 6074.
X-ray crystal structure of the inhibitor 35 bound to the
protease is shown in Figure 2. The core interactions of
35 with the protease resembled those of our previous
inhibitors.2 Interestingly, the quaternary methyl group
3. (a) Magnin, D. R.; Robl, J. A.; Sulsky, R. B.; Augeri, D.
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Discafani, C.; Beyer, C.; Greenberger, L. M.; Loganzo, F.;
Ayral-Kaloustian, S. J. Med. Chem. 2004, 47, 4774.
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Hasegawa, M.; Taniyama, D.; Tomioka, K. Tetrahedron
2000, 56, 10153; (c) Chowdari, N. S.; Suri, J. T.; Barbas,
C. F. Org. Lett. 2004, 6, 2507.
5. (a) Chakraborty, T. K.; Hussain, K. A.; Reddy, G. V.
Tetrahedron 1995, 51, 9179; (b) Pellicciari, R.; Costantino,
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Jaquith, J. B.; Neelagiri, V. R.; Saleh-Hanna, S.; Durst, T.
Figure 2. X-ray structure of 35 bound to the protease.

A. Arasappan et al. / Tetrahedron Letters 48 (2007) 6343–6347
6347
Org. Lett. 2002, 4, 695; (d) Noe, M. C.; Natarajan, V.;
Snow, S. L.; Mitchell, P. G.; Lopresti-Morrow, L.; Reeves,
L. M.; Yocum, S. A.; Carty, T. J.; Barberia, J. A.; Sweeny,
F. J.; Liras, J. L.; Vaughn, M.; Hardink, J. R.; Hawkins, J.
M.; Tokar, C. Bioorg. Med. Chem. Lett. 2005, 15, 2808.
6. Selected spectroscopic data: For minor isomer 9b—1H
NMR (CDCl3, 300 MHz) d 0.93 (s, 3H), 1.15–1.52 (m,
10H), 3.06 (s, 1H), 3.30 (s, 3H), 3.57–3.67 (m, 3 H), 7.21–
7.35 (m, 5H); for major isomer 9a—1H NMR (CDCl3,
300 MHz) d 0.93 (s, 3H), 1.11–1.40 (m, 10H), 2.95 (s, 1H),
3.51–3.60 (m, 2H), 3.64–3.70 (m, 1H), 3.70 (s, 3H), 7.27–
7.33 (m, 5H).
8. The optimized procedure was employed for large scale
(ꢀ100 g) synthesis of 21 starting from >100 g of aldehyde
5.
9. Mamai, A.; Zhang, R.; Natarajan, A.; Madalengoitia, J.
S. J. Org. Chem. 2001, 66, 455.
10. The HCV NS3 Serine protease inhibitory value is provided
as KÃi . For a definition of KÃi and discussion, see Morrison,
J. F., Walsh, C. T., in Adv. Enzymol. Relat. Areas Mol.
Biol., Meister, A., Ed.; 1988, Vol. 61, pp 201–301.
11. Zhang, R.; Beyer, B. M.; Durkin, J.; Ingram, R.; Njoroge,
F. G.; Windsor, W. T.; Malcolm, B. A. Anal. Biochem.
1999, 270, 268. For the present study, the substrate
Ac-DTEDVVP(Nva)-O-PAP was employed.
7. Only 9, 10a/b were subjected to larger scale (>20 mmol)
hydrolysis since we required those amino acid derivatives
for SAR studies.
12. For the replicon assay, see: Lohmann, V.; Korner, F.;
Koch, J.-O.; Herian, U.; Theilmann, L.; Bartenschlager,
R. Science 1999, 285, 110.