Enantioselective synthesis of derivatives and structure-activity relationship study in the development of NA255 as a novel host-targeting anti-HCV agent

Article abstract of DOI:10.1016/j.bmcl.2012.10.083

Hepatitis C virus (HCV) infection represents a serious health-care problem. Previously we reported the identification of NA255 from our natural products library using a HCV sub-genomic replicon cell culture system. Herein, we report how the absolute stereochemistry of NA255 was determined and an enantioselective synthetic method for NA255 derivatives was developed. The structure-activity relationship of the NA255 derivatives and rat pharmacokinetic profiles of the representative compounds are disclosed.

Full text of DOI:10.1016/j.bmcl.2012.10.083

Bioorganic & Medicinal Chemistry Letters 23 (2013) 336–339
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Enantioselective synthesis of derivatives and structure–activity relationship
study in the development of NA255 as a novel host-targeting anti-HCV agent
⇑
Ken-ichi Kawasaki , Miyako Masubuchi, Tadakatsu Hayase, Susumu Komiyama, Fumio Watanabe,
Hiroshi Fukuda, Takeshi Murata, Yasuaki Matsubara, Kouhei Koyama, Hidetoshi Shindoh,
Hiroshi Sakamoto, Kohichi Okamato, Atsunori Ohta, Asao Katsume, Masahiro Aoki, Yuko Aoki,
Nobuo Shimma, Masayuki Sudoh, Takuo Tsukuda
Research Division, Chugai Pharmaceutical Co., Ltd, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 July 2012
Revised 15 October 2012
Accepted 17 October 2012
Available online 30 October 2012
Hepatitis C virus (HCV) infection represents a serious health-care problem. Previously we reported the
identification of NA255 from our natural products library using a HCV sub-genomic replicon cell culture
system. Herein, we report how the absolute stereochemistry of NA255 was determined and an enantio-
selective synthetic method for NA255 derivatives was developed. The structure–activity relationship of
the NA255 derivatives and rat pharmacokinetic profiles of the representative compounds are disclosed.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
HCV
Host-targeting anti-HCV agent
Serine palmytoyltransferase
NA255
Natural product
Total synthesis
HCV is a worldwide infectious pathogen that causes chronic li-
ver diseases, including hepatic fibrosis, hepatic cirrhosis and hepa-
We set out to identify a novel HCV replication inhibitor that
would manipulate host cell factors to avoid the resistance issue.
Using a HCV replicon cell culture system,9 we identified a lead
compound, NA255, from natural screening sources and found that
its target was a host protein, serine palmytoyltransferase.10,11
tocellular carcinoma.1 Pegylated
a-interferon and ribavirin
combination therapy is the standard-of-care (SOC), but the therapy
usually involves adverse side effects and yields a limited sustained
virological response of approximately 50% for the patients with the
predominant genotype 1-infection.2,3 Therefore, HCV represents a
huge unmet medical need that has triggered intensive research ef-
forts to develop more effective drugs. In the past decade, the viral
NS3/4A serine protease and NS5B RNA-dependent RNA polymerase
have been the most intensively pursued anti-HCV targets for treat-
ing HCV infection. These efforts resulted in the recent regulatory
approval of boceprevir and telaprevir, NS3 protease inhibitors that
improved cure rates when added to the SOC.4,5 In addition, several
other NS3 protease inhibitors, as well as NS5B nucleoside and non-
nucleoside inhibitors and NS5A inhibitors, are in various stages of
clinical trials.6 However, the high mutation rate of HCV and its ra-
pid replication inevitably lead to drug resistance7,8 so it is extre-
mely desirable to further develop new, safer and even more
effective agents against HCV infection and acquired resistance.
HO
HO
O
O
O
5
HO
3
13
20
2
O
O
NH
R
HO
(3S,4S,2'S)
O
R=
NA255
Viridiofungin A
*
R=H
NA255 has strong potency in a HCV replicon assay; however, isolat-
ing NA255 from culture extracts requires multiple consecutive
chromatographic manipulations, in addition to having a low fer-
mentation titer and thus providing only limited quantities of mate-
rial. The difficulties associated with fermentation-based approaches
spurred our efforts toward achieving the total synthesis of NA255
⇑
Corresponding author.
E-mail address: kawasakikni@chugai-pharm.co.jp (K. Kawasaki).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.083

K. Kawasaki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 336–339
337
O
CO2H
CO2H
CO2Me
HO
HO2C
HO
HO2C
O
HO
MeO2C
(
)
( )
5
n-C7H15
O
n-C7H15
O
(
)
5
n-C7H15
OH
5
(a),(b)
(c)
NA255
O
O
NH
NH
O
NH
HO
HO
O
O
O
O
1 = viridiofungin A trimethylester
2
3
(d) - (f)
Scheme 1. Reagents and conditions for transforming NA255 to viridiofungin A trimethyl ester and compound 2 and 3: (a) (trimethylsilyl)diazomethane, DCM/MeOH, rt, 43%;
(b) 6 N-HCl, methanol, rt, 7 h, 65%; (c) H2-Pd/C, methanol, rt, 34%; (d) 4-methylbenzenesulfonohydrazide, MeOH, reflux, 1.5 h; (e) catecholborane, chloroform, rt, 3 h, then
MeOH, DMSO, sodium acetate, heat, 54%, 2 steps; (f) LiOH, EtOH, H2O, rt, 35%.
derivatives in hopes of developing the SAR on this new chemical
class and finding a time-saving way to supply adequate derivatives
for further biological evaluation.
C5/C6 double bond with a modest E/Z ratio. Based on their prec-
edent we developed an efficient 18-step enantioselective total
synthesis of NA255 derivatives, with around 3% of total yield
in the longest linear steps for the representative compound, such
as in 39, as shown in Scheme 3. The major differences from the
previous synthesis are, firstly, the direct introduction of an alke-
nyl side chain with high E-configuration instead of using alkene
cross-metathesis and, secondly, simultaneous transformation of
two primary alcohols to tert-butyl esters, which helped reduce
the number of steps.
Herein, we report an enantioselective synthetic route for NA255
derivatives, the SAR obtained on their HCV replication inhibitory
activity, and the PK properties of the promising compounds.
NA255 has the same skeleton as a natural product, viridiofun-
gin A, which was reported to be an antifungal agent.12 Because
NA255 transformed into viridiofungin A trimethyl ester via two
steps, its absolute stereochemistry was determined as (3S, 4S,
20S) so esterification with TMS diazomethane provided the
corresponding trimethyl ester of NA255, and subsequent acid
Total synthesis of NA255 derivatives:
Structures of compound 15–43.
CO2H
O
HO
CO2H
O
HO
HO2C
HO2C
R1
O
( )n
(
)
n-C7H15
5
O
NH
O
NH
R2
HO
O
R1 =
n-C9H19
O
O
n =
3
S
*
*
*
*
*
15
16
17
18
19
20
21
22
23
24
25
HO2C
HO2C
HO2C
HO2C
HO2C
26
27
28
n-C5H11
7
5
5
5
5
*
*
*
*
29
30
31
*
*
*
c-hexyl
Ph
HO2C
HO2C
HO2C
32
33
5
34: R3= Me
38: R3= phenyl
*
O
N
R3
35: R3= n-propyl 39: R3= but-2-yn-1-yl
*
n-C3H7
5
5
5
5
36: R3= n-butyl
37: R3= benzyl
40: R3= 3-aminopropyl
n-C6H13
n-C7H15
n-C8H17
Ar
41: Ar= phenyl
42: Ar= 3-methoxyphenyl
43: Ar= 3-pyridyl
(=2)
*
HO2C
The synthesis was initiated from 8, which was transformed to allyl
alcohol 9 according to the reported method with some minor
modifications. Katsuki–Sharpless AE provided the epoxide 10 in an
excellent enantiomeric excess and isolated yield. Then we examined
introducing the alkenyl side chain using alkenyl Grignard reagent,
which was prepared in situ by reacting the terminal acetylene 7
with Schwartz salt to form alkenyzirconium, followed by
treatment removed the 3-methylbut-2-en-1-yl group, as shown
in Scheme 1. Comparison of 1H and 13C NMR spectra and [
a]
D
was consistent with the reported analytical data.11,13 The first
enantioselective total synthesis of viridiofungin A was reported
in 2005 by Hatakeyama and co-workers,14 who employed
Katsuki–Sharpless asymmetric epoxidation (AE) to set absolute
stereochemistry and alkene cross-metathesis to construct the

338
K. Kawasaki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 336–339
O
O
O
(c)
(a)
O
( )
O
(b)
O
R1
R1
(
)
( )
n
(
)
n
OH
N
n
n
4
5
6
7
Scheme 2. Reagents and conditions for synthesizing terminal alkyne: (a) N,O-dimethylhydroxyamine hydrochloride, EDC, HOBT, DMF, rt, 94% yield⁄; (b) RMgX, THF, ꢀ10 °C,
93% yield⁄; (c) ethyleneglycol, cat. p-TsOH, toluene, reflux, 98% yield⁄. ⁄Indicated yields are for synthesis of 2-heptyl-2-(oct-7-yn-1-yl)-1,3-dioxolane (n = 5 and R1 = n-C7H15).
OTPS
TPSO
TPSO
HO
O
HO
O
)
O
R
(a)-(g)
(i)
(h)
1
(
TPSO
O
n
TPSO
OH
TPSO
OH
HO
8
9
10
11
OH
CO2tBu
O
)
O
)
O
(o), (p)
(l), (m), (n)
(j), (k)
t-BuO2C
1
1
(
R
(
n
R
HO
n
O
O
O
CO2H
O
HO
HO2C
O
12
13
(
)
5
n-C7H15
O
O
HO
NH
O
HO CO2H
HO2C
CO2tBu
O
HO
t-BuO2C
(q), (r)
(s)
1
1
(
)
n
R
(
)
n
R
O
24a
O
NH
R
O
OH
2
CO2H
OH
HO
HO2C
15 ~ 43
14
(
)
5
n-C7H15
(t)
O
O
NH
HO
O
24b
Scheme 3. Reagents and conditions: (a) TBDPSCl, imidazole, DMF, rt; (b) n-BuLi, (CH2O)n, THF; (c) Red-Al, 0 °C, then I2, THF ꢀ40 °C; (d) DHP, PPTS, DCM, rt; (e) n-BuLi,
(CH2O)n, THF, ꢀ78 °C to 0 °C; (f) TBDPSCl, imidazole, DMF, rt; (g) PPTS, EtOH. 28.6% over 7 steps; (h)
L-(+)-DET, Ti(Oi-Pr)4, TBHP, DCM, 97%, >95% ee; (i) Terminal alkyne 7 in
Scheme 2, Cp2ZrClH, MeMgCl, CuI, THF, ꢀ20 °C, 91% yield⁄; (j) 2,2-dimethoxypropane, PPTS, DCM, 85% yield⁄; (k) TBAF, AcOH, THF, 89% yield⁄; (l) oxalyl chloride, DMSO,
triethylamine, DCM, ꢀ78 °C; (m) NaClO2, NaH2PO4, 2-methyl-2-butene, t-BuOH-H2O; (n) N,N-dimethylformamide di-tert-butyl acetal, 58% yield in 3 steps⁄; (o) 80% AcOH,
THF, rt, 90% yield⁄; (p) Jones reagent, aqueous acetone, ꢀ10 °C, 80% yield⁄; (q) the corresponding amine, HATU, Hunig base, 85% yield⁄; (r) TFA, anisole, DCM, 90% yield⁄; (s)
H2-Pd/C, EtOH, 80%; (t) NaBH4, THF, MeOH, 93% yield. ⁄yields when n = 5 and R1 = n-C7H15
.
transmetallation with methylmagnesium chloride.15 The acetylene
7 was synthesized in a concise manner as shown in Scheme 2. The
copper-catalyzed ring-opening reaction proceeded smoothly, pro-
viding the desired coupling product 11 with high regioselectivity
and an exclusive E configuration. Then, protection of hydroxyl
groups in 11 as acetonide, followed by deprotection of TPS groups,
afforded diol 12. Successive Swern oxidation, sodium chlorite oxida-
tion and esterification by N,N-dimethylformamide di-tert-butyl ace-
tal allowed simultaneous conversion of two hydroxymethyl groups
into the corresponding tert-butyl esters, providing 13. Then, aceto-
nide and ketal were deprotected with acetic acid, and the resulting
primary alcohol was oxidized to carboxylic acid 14. The coupling
reaction of 14 with a variety of primary amines generally worked
well. The amidation of 14 with secondary amines was not successful
and resulted in lactone formation. Finally, treatment of the amide
with trifluoroacetic acid furnished carboxylic acid derivatives 15–
43.16 Several analogues were further synthesized from 24, thus
treatment with sodium borohydride gave 24b as a diastereomer
mixture. Hydrogenation of C5/C6 double bond provided analogue
24a.
substituent of the 3-methylbut-2-en-1-yl group. To our delight,
compound 24 (bearing isopentyl group on phenolic oxygen of tyro-
sine moiety which is isosteric to the 3-methylbut-2-en-1-yl group)
showed similar potency to the lead compound so we then explored
the SAR of the right-hand side lipophilic side chain, keeping the
isopentyl group. The results are shown in Table 1. First, compound
24a, which resulted from hydrogenation of the double bond of
compound 24, showed significant loss of inhibitory activity, sug-
gesting an important role of the E geometry between C5 and C6
in restricting the exiting vector of the side chain. In addition,
reduction of the ketone in this side chain to an alcohol 24b or to
a methylene group 3 renders molecules less active.
Table 1
Anti-replicon activity and cell viability of NA255 and its derivatives: modification of
side chain
a
b
a
b
Compd Replicon IC50 CellTox CC50 Compd Replicon IC50 CellTox CC50
M) M) M) M)
(
l
(l
(l
(l
NA255 0.0020
>5
>5
>50
>5
>1
>50
>50
>1
22
15
16
17
18
21
20
19
0.80
>5
>1
>1
>1
>1
>1
>1
>1
2
0.0019
0.0014
0.15
0.025
0.155
0.0069
0.019
0.0051
0.0014
0.0061
0.0057
>1
24
24a
24b
3
Confirmation of the correct stereochemistry of this synthesis
was provided by converting NA255 to compound 2, as shown in
Scheme 1, for comparison with analytical data of 24 obtained by
this total synthesis.11
NA255 was found to be unstable under acidic condition as men-
tioned above. First of all, because it is incompatible with the final
step in the synthetic scheme, we decided to find an alternative
25
23
0.29
0.032
a
Values are the mean of two experiments.
Concentration at which cell viability was reduced by 50%.
b

K. Kawasaki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 336–339
339
Table 2
although incorporating a 3-pyridyl group (43) was not beneficial.
Of this set of derivatives, benzyl ether analogue 37 provided the
best potency in vitro in this study.
Following this SAR analysis, compound 37, 39 and 42 were se-
lected for further evaluation of their pharmacokinetic properties
in rats. The compounds were dosed subcutaneously in male Spra-
gue–Dawley rats at 10 mg/kg and the data are reported in
Table 3.18
These data demonstrate that all compounds showed relatively
short half-life in plasma but had relatively high liver exposure.
Since HCV replicates in hepatocytes, this feature could be a virtue
for an anti-HCV drug and may provide a wide therapeutic window
by avoiding systemic distribution. This liver-targeted tissue distri-
bution profile could be the result of substrate recognition by organ-
ic anion transporter polypeptides, which are highly expressed in
hepatocytes.17 In addition, the liver exposures of 37 and 42 were
remarkably increased compared to 39 by having significantly long-
er half-lives in the liver. These compounds were selected for fur-
ther development and an efficacy study on these molecules in an
animal model will be reported in due course.19
Anti-replicon activity and cell viability of NA255 derivatives: modification of amino
acid moiety
a
b
a
b
Compd Replicon IC50 CellTox CC50 Compd Replicon IC50 CellTox CC50
M) M) M) M)
(
l
(
l
(
l
(l
26
29
32
27
28
30
31
33
34
>5
1.0
0.012
0.017
0.49
>5
>1
0.014
0.0064
>5
>5
>1
13.9
>1
>5
>1
>5
>5
35
36
39
40c
38
37
41
42
43
0.0026
0.0012
0.0012
0.038c
0.0019
0.00032
0.0016
0.0026
0.010
>5
>5
>5
>1
>5
>5
>5
>1
>1
a
b
c
Values are means of two experiments.
Concentration at which cell viability was reduced by 50%.
TFA salt was used.
Table 3
Rat PK parameters for selected compounds
Parameter
Compd 37a
Compd 39a
Compd 42a
(CH4630806)
(CH4630808)
(CH4861321)
Acknowledgments
Plasma
Liver
Plasma Liver
Plasma
Liver
5.92c
82.9
21.3
16.1
c
Cmax
AUC (
T1/2 (h)
MRT (h)
(
l
l
g/mL)
0.209b
2.69
9.81
6.59c 0.604b
100
27.3
17.0
7.10
49.6
4.75
7.30
0.351b
2.59
We would like to thank Yoshiko Itezono for NMR analysis, and
Isamu Kusanagi for biological testing. We also thank Shige
Nagahashi for helpful discussion, and Sally Matsuura for editorial
assistance.
g h/mL)
3.05
3.99
5.48
14.8
13.6
12.9
Compounds were dosed subcutaneously at 10 mg/kg (n = 2).
Administered using 10%PEG300–10%HPCD in water as a vehicle.
a
References and notes
Tri-sodium salt was used.
Concentration at 0.5 h postdosing.
b
c
Concentration at 2 h postdosing.
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chain and the data obtained are shown in Table 1. Even though
compound 25, in which the side chain was elongated by adding
one carbon atom at the terminal, did not provide an additional
benefit, shortening the length of the side chain led to largely de-
creased potency, depending on its length (22, 23). In addition,
incorporating a cyclic substituent was explored, and little tolerance
was observed for an aromatic ring, such as 20, but it is interesting
to note that cyclohexyl analogue 19 showed 10 times better inhib-
itory activity. As expected, small modifications were tolerated,
such as a branched side chain at the terminal of 18 and terminal
olefin in 17. As mentioned above, ketone at the position of C13
may have a role as an HB acceptor, but shifting it to various differ-
ent positions was found to be well tolerated (15, 16).
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results are shown in Table 2. Removing carboxylic acid (compound
29) or incorporating (R)-amino acid (compound 26) resulted in a
huge drop in inhibitory activity. Compounds 32 and 27 showed
similar potency, 10-fold less active than 24, suggesting that ether
linkage plays a role in high potency. In addition, replacing tyrosine
with several unnatural amino acids was explored, and b-naphtyl
analogue 33 showed retained potency. Next, we explored SAR
around the ether linkage by replacing the isopentyl group of 24
with groups of varied length, such as methyl, n-propyl and n-butyl
groups. Alkylation with longer groups provided an increase in po-
tency, in the order of compound 34 < 35 < 36, suggesting the
hydrophobic nature of its surroundings. Compound 39, bearing
but-2-en-1-yl group, was also well tolerated, which may reflect
the flexibility of the pocket. When we tried to test this assumption
by incorporating a benzene ring, both phenyl ether analogue 38
and biphenyl analogue 41 showed strong inhibitory activity,
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cartridge.
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18. The animals used in this experiment were treated in accordance with Chugai
Pharmaceutical’s ethical guidelines of animal care, handling, and termination.
These guidelines meet the generally accepted international criteria of human
treatment, sparing the animals needless pain and suffering and that the
experiments conducted are of actual scientific benefit to mankind.
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Sudoh, M.; Kohara, M. in publication.