The first total synthesis of novel human chymase inhibitor SPF32629A

Article abstract of DOI:10.1016/j.tetlet.2008.08.048

The first total synthesis of SPF32629A, a novel human chymase inhibitor, has been accomplished from the readily available precursor 4-nitropyridine N-oxide, following an efficient strategy with a sequence of eight straightforward steps.

Full text of DOI:10.1016/j.tetlet.2008.08.048

Tetrahedron Letters 49 (2008) 6297–6299
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Tetrahedron Letters
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The first total synthesis of novel human chymase inhibitor SPF32629A
a
b
Srinivasa Rao Vegi a,b, Shanthaveerappa K. Boovanahalli a, , Arun Prakash Sharma , K. Mukkanti
*
a Medicinal Chemistry Laboratory, GVK Biosciences Pvt Ltd, Plot No. 28, IDA, Nacharam, Hyderabad, 500 076 AP, India
b Chemistry Division, Institute of Science and Technology, JNT University, Kukatpally, Hyderabad, 500 072 AP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The first total synthesis of SPF32629A, a novel human chymase inhibitor, has been accomplished from the
readily available precursor 4-nitropyridine N-oxide, following an efficient strategy with a sequence of
eight straightforward steps.
Received 8 May 2008
Revised 8 August 2008
Accepted 13 August 2008
Available online 19 August 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Human chymase (EC 3.4.21.39) is a chymotrypsin-like serine
protease localized in the secretory granules of mast cells. Several
reports demonstrated the involvement of chymase in the progres-
sion of dermatitis,1 chronic inflammation following cardiac2,3 and
pulmonary fibrosis.4 Therefore, chymase inhibitors are expected
to be potential therapeutic agents for the treatment of cardiovas-
cular diseases, allergic inflammation, and fibrotic disorders.5
In 2006, Shimatani and coworkers6a reported the isolation of
two new human chymase inhibitors, SPF-32629A and B, (Fig. 1)
from the cultured broth of Penicilliumsp., SPF-32629. The structural
elucidation of these chymase inhibitors was accomplished on the
basis of complementary evidence garnered from FAB-MS, HRFAB-
MS, IR, and 1H (COSY, HMBC) spectral data. Analysis for optical
purity was carried out by four types of chiral HPLC columns; how-
ever the absolute configurations of these molecules remain to be
established. The specific inhibition of human chymase among four
serine proteases—chymase, chymotrypsin, cathepsinG, and Elas-
tase by 1 and 2 constitutes an important lead toward developing
new chymase inhibitors. But so far, there is no report of the total
synthesis of these molecules. Considering the important and signifi-
cant biological activities6a,b as well as their interesting structures,
we initiated a program to develop a flexible strategy for the total
synthesis of these molecules. In this preliminary communication,
we report the first total synthesis of SPF32629A.
OH
R
O
N
H
O
O
1: R = H, SPF-32629A
2: R = COOH, SPF-32629B
Figure 1. Human chymase inhibitors SPF-32629 A and B.
2-position of 2 by reacting it with 1.18 equiv of trimethylsilyl
cyanide following a modified Reissert–Henze protocol.8 This trans-
formation, pyridine N-oxide into the corresponding pyridinecarbo-
nitrile 3, was done in dichloromethane using 1.2 equiv of diethyl or
dimethylcarbamyl chloride as the activating electrophile in 84%
yield.
Grignard reaction of nitrile 3 with 1.5 equiv of phenylmagne-
sium bromide in ether at À30 °C to rt furnished ketone 4 in 76%
yield. The pyridine ring in compound 4 was transformed into its
corresponding 2-pyridone derivative 6 by employing N-oxidation,
followed by reaction with acetic anhydride and hydrolysis proto-
col. Thus, mCPBA oxidation of compound 4 yielded its N-oxide
derivative 5 in DCM at rt in 91% yield, which smoothly rearranged
to the corresponding 2-pyridone derivative 6 in 45% yield under
the influence of boiling acetic anhydride followed by hydrolysis
with aqueous ethanol. Reduction of the keto group in 6 using so-
dium borohydride in methanolic THF furnished alcohol 7 in 84%
yield. Further, coupling of alcohol 7 with isovaleric acid in the pres-
ence of EDCÁHCl and DMAP in DCM at 10 °C to rt furnished the cor-
responding isovaleryl ester 9 in 86% yield along with bis-isovaleryl
derivative 8 as a by-product in 5–10% yield. Interestingly, when
compound 8 was subjected to basic hydrolysis using LiOHÁH2O,
selective de-protection of isovaleric ester occurred leading to the
formation of compound 9 in 88% yield. Finally, debenzylation
Our synthesis commenced from the commercially available
starting material, 4-nitropyridine N-oxide (Scheme 1). Firstly, we
wanted to introduce a protected hydroxyl group at the 4-position
of the pyridine moiety, and it is well established that the nitro
group at the 4-position of pyridine N-oxide can be easily displaced
by nucleophilic reagents. Accordingly, displacement of the nitro
group with benzyl alcohol in the presence of metallic sodium at
10 °C to rt afforded the corresponding benzyloxy derivative 2 in
high yield (81%).7 Then, the cyano group was introduced at the
* Corresponding author. Tel.: +91 40 6451 3333x1163; fax: +91 40 2715 2999.
E-mail address: sveerappa.boovanahal@gvkbio.com (S. K. Boovanahalli).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.08.048

6298
S. R. Vegi et al. / Tetrahedron Letters 49 (2008) 6297–6299
NO2
N
OBn
OBn
OBn
N
a
c
b
N
N
CN
O
1
O
2
O
4
3
d
OBn
OBn
OBn
f
e
O
N
H
O
N
H
N
O
OH
7
O
6
O
5
g
OBn
N
OBn
OH
O
i
+
O
O
N
H
O
N
H
O
O
O
O
O
O
8
9
SPF-32629A
h
Scheme 1. Total synthesis of SPF-32629A (1). Reagents and conditions: (a) BnOH, Na, 10 °C to rt, 12 h, 81%; (b) TMSCN, N,N-diethylcarbamoyl chloride, DCM, 5 °C to rt, 12 h,
84%; (c) PhMgBr, THF, À30 °C to rt, 5 h, 77%; (d) m-CPBA, DCM, rt, 12 h, 91%; (e) (Ac)2O, 145 °C, 4 h, then EtOH, H2O, 100 °C, 4 h, 40%; (f) NaBH4, THF/MeOH (8:2), rt, 6 h, 85%;
(g) isovaleric acid, EDCÁHCl, DMAP, DCM, 10 °C to rt, 6 h, 87%; (h) LiOHÁH2O, THF, H2O, rt, 3 h, 88%; (i) Pd/C (10%), THF/MeOH (8:2), H2, 50 psi, rt, 30 min (90%).
was accomplished under catalytic hydrogenation using Pd/C at 50
psi in a mixture of methanolic THF to obtain racemic SPF-32629A
in 90% yield, which exhibited 1H and 13C NMR spectral data closely
comparable to those reported in the literature.6a,9 Further confir-
mation of the structure of synthetic SPF-32629A was accomplished
on the basis of 13C DEPT, 1H–1H COSY, and gHMBC (Gradient heter-
onuclear multiple bond coherence) spectral data. The key features
obtained from COSY and HMBC are depicted in Figure 2.
As compound 7 consists of amide and alcohol functionalities, a
significant amount of bis-isovaleryl derivative 8 was also observed
as a by-product during the coupling reaction of 7 with isovaleric
acid, which was again converted to the desired compound 9 under
basic conditions. To overcome this difficulty and to further confirm
the position of isovaleryl ester at carbon-7, we protected the amide
group in compound 6 with Cbz, and followed a similar sequence of
reactions as in Scheme 1 to obtain SPF-32629A in satisfactory
yields (Scheme 2).9 Thus, reaction of compound 6 with CbzCl using
TEA as base in DCM furnished Cbz protected compound 10 in 91%
yield. Compound 10 upon reduction with NaBH4 followed by ester-
OH
12
4
3
5
11
10
13
8
O
N
H
6
7
2
9
15
18
O
14
16
O
17
Figure 2. 1H–1H COSY (bold lines) and HMBC (arrows) correlations observed for
SPF-32629A.
OBn
OBn
OBn
k
j
O
N
O
N
O
N
H
Cbz OH
Cbz
O
10
O
6
11
l
OBn
m
SPF-32629A
O
N
Cbz
O
O
12
Scheme 2. Reagents and conditions: (j) CbzCl, TEA, DCM, 0 °C to rt, 4 h, 91%; (k) NaBH4, THF/MeOH (8:2), rt, 2 h, 92%; (l) isovaleric acid, EDC.HCl, DMAP (cat), DCM, 0 °C to rt,
6 h, 95%; (m) Pd/C (10%), EtOAc/MeOH (8:2), H2, 50 psi, rt, 30 min, 89%.

S. R. Vegi et al. / Tetrahedron Letters 49 (2008) 6297–6299
6299
(C), 132.7 (CH), 130.8 (2CH), 128.6 (2CH), 128.3 (CH), 127.9 (2CH), 127.4 (2CH),
113.1 (CH), 110.6 (CH), 69.9 (CH2); MS (APCI) m/z 290.3 (M+H)+; LCMS (ES) m/z
calcd for C19H16NO2, [M+H]+: 290.12, found: 290.30. Compound 5: Pale yellow
ification using isovaleric acid afforded compound 12 in good yield.
Finally, de-protection of both Cbz and benzyl groups was achieved
in a single step using Pd/C to obtain racemic SPF-32629A in 89%
yield, the 1H and 13C NMR spectral data are in agreement with
the data obtained from Scheme 1.
In conclusion, we have accomplished the first total synthesis of
natural human chymase inhibitor SPF-32629A starting from the
readily available 4-nitropyridine N-oxide employing an efficient
strategy with a sequence of eight straightforward reactions. Syn-
thetic studies toward another chymase inhibitor SPF-32629B and
asymmetric induction during the carbonyl reduction using chiral
reducing agents are currently underway in our laboratory, and will
be reported in due course.
solid; mp 138–140 °C; IR (KBr pellet): m ;
max 1673, 1217 cmÀ1 1H NMR (400 MHz,
CDCl3): d 8.12 (1H, dd, J 5.6 and 2 Hz), 7.85 (1H, s), 7.83 (1H, d, J 1.2 Hz), 7.59
(1H, t, J 7.6 Hz), 7.48–7.36 (7H, m), 6.99 (2H, dd, J 6.4 and 2.8 Hz), 5.11 (2H, s);
13C NMR (100 MHz, CDCl3): d 188.6 (C), 156.5 (C), 147.3 (C), 140.5 (CH), 134.6
(C), 134.4 (C), 134.0 (CH), 129.2 (2CH), 128.66 (2CH), 128.6 (2CH) 128.5 (CH),
127.4 (2CH), 113.9 (CH), 111.0 (CH), 71.0 (CH2); MS (ESI) m/z 306 (M+H)+; LCMS
(ES) m/z calcd for C19H16NO3, [M+H]+: 306.11, found: 306.28. Compound 6:
Cream colored solid; mp 158–160 °C; IR (KBr pellet): mmax 1644, 1620,
1600 cmÀ1 1H NMR (400 MHz, CDCl3): d 9.4 (1H, br s, D2O exchangeable),
;
7.77 (2H, d, J 7.6 Hz), 7.64 (1H, t, J 7.4 Hz), 7.5 (2H, t, J 8 Hz), 7.42–7.36 (5H, m),
6.50 (1H, d, J 2 Hz), 6.18 (1H, d, J 2 Hz), 5.02 (2H, s); 13C NMR (100 MHz, CDCl3): d
188.1 (C), 166.6 (C), 164.3 (C), 139.0 (C), 135 (C), 134.5 (CH), 133.1 (CH), 129.3
(2CH), 128.69 (2CH), 128.61 (2CH), 128.5 (C), 127.7 (2CH), 108.8 (CH), 102.9
(CH), 70.7 (CH2); MS (APCI) m/z 306.05 (M+H)+; LCMS (ESI) m/z calcd for
C
19H16NO3, [M+H]+: 306.11, found: 306.28. Compound 7: Colorless solid; mp
350 °C (decomposed); IR (KBr pellet): mmax 3429, 3268, 1644, 1628 cmÀ1 1H
;
Acknowledgments
NMR (400 MHz, CD3OD): d 7.44–7.31 (10H, m), 6.39 (1H, s), 6.07 (1H, s), 5.66
(1H, s), 5.17 (2H, s); 13C NMR (100 MHz, CDCl3): d 171.9 (C), 166.3 (C), 154.1 (C),
142.1 (C), 136.5 (C), 129.77 (2CH), 129.73 (2CH), 129.53 (2CH), 129.51 (CH),
128.87 (2CH), 127.8 (CH), 101.8 (CH), 96.5 (CH), 73.0 (CH), 72.0 (CH2); MS (ESI)
m/z 308.08 (M+H)+; LCMS (ES) m/z calcd for C19H18NO3, [M+H]+: 308.13, found:
308.31. Compound 8: Colorless syrup; IR (CHCl3 film): mmax 1764, 1741,
We thank G.V.K. Biosciences Pvt Ltd for the financial support
and encouragement. Help from the analytical department for the
analytical data is appreciated. We thank Dr. Balaram Patro, Dr.
Milind Sindkhedkar and Dr. Lakshmana Rao Vadali for their helpful
suggestions.
1602 cmÀ1 1H NMR (400 MHz, CDCl3): d 7.38 (6H, m), 7.33–7.25 (4H, m), 6.92
;
(1H, d, J 2 Hz), 6.75 (1H, s), 6.56 (1H, d, J 1.6 Hz), 5.07 (2H, s), 2.44 (2H, d, J
7.2 Hz), 2.30 (2H, d, J 7.2 Hz), 2.22 (1H, m), 2.14 (1H, m), 1.04 (3H, s), 1.02 (3H, s),
0.95 (3H, s), 0.93 (3H, s); 13C NMR (100 MHz, CDCl3): d 171.6 (C), 170.8 (C), 167.8
(C), 159.5 (C), 158.8 (C), 138.6 (C), 135.1 (C), 128.76 (2CH), 128.54 (CH), 128.45
(2CH), 128.15 (CH), 127.7 (2CH), 127.3 (2CH), 106.9 (CH), 101.0 (CH), 76.9 (CH),
70.4 (CH2), 43.4 (CH2), 43.3 (CH2), 25.7 (CH), 25.5 (CH), 22.4 (2CH3), 22.3 (2CH3);
MS (ESI) m/z 476.3 (M+H)+; LCMS (ES) m/z calcd for C29H34NO5, [M+H]+: 476.24,
found: 476.49. Compound 9: Colorless solid; mp 132–134 °C; IR (KBr pellet):
References and notes
1. (a) Tomimori, Y.; Muto, T.; Fukami, H.; Saito, K.; Horikawa, C.; Tsuruoka, N.;
Saito, M.; Sugiura, N.; Yamashiro, K.; Sumida, M.; Kakutani, S.; Fukuda, Y. Lab.
Invest. 2002, 82, 789–794; (b) Watanabe, N.; Tomimori, Y.; Saito, K.; Miura, K.;
Wada, A.; Tsudzuki, M.; Fukuda, Y. Int. Arch. Allergy Immunol. 2002, 128, 229–
234.
2. (a) Kokkonen, J. O.; Lindstedt, K. A.; Kovanen, P. T. Circulation 2003, 107, 2522–
2524; (b) Matsumoto, T.; Wada, A.; Tsutamoto, T.; Ohnishi, M.; Isono, T.;
Kinoshita, M. Circulation 2003, 107, 2555–2558.
3. Kanemitsu, H.; Takai, S.; Tsuneyoshi, H.; Nishina, T.; Yoshikawa, K.; Miyazaki,
M.; Ikeda, T.; Komeda, M. Hypertens. Res. 2006, 29, 57–64.
4. (a) Tomimori, Y.; Muto, T.; Saito, K.; Tanaka, T.; Maruoka, H.; Sumida, M.;
Fukami, H.; Fukuda, Y. Eur. J. Pharmacol. 2003, 478, 179–185; (b) Sakaguchi, M.;
Takai, S.; Jin, D.; Okamoto, Y.; Muramatsu, M.; Kim, S.; Miyazaki, M. Eur. J.
Pharmacol. 2004, 493, 173–176.
mmax 1744, 1646 cmÀ1 1H NMR (400 MHz, CDCl3):
; d 9.22 (1H, br s, D2O
exchangeable CONH), 7.39–7.26 (10H, m), 6.59 (1H, s), 5.89 (1H, d, J 1.6 Hz), 5.85
(1H, d, J 2.4 Hz), 4.96 (2H, s), 2.32 (2H, d, J 7.2 Hz), 2.12 (1H, m), 0.94 (3H, s), 0.92
(3H, s); 13C NMR (100 MHz, CDCl3): d 171.5 (C), 168.2 (C), 166 (C), 146.4 (C),
136.4 (C), 135.1 (C), 128.9 (CH), 128.8 (2CH), 128.7 (2CH), 128.5 (CH), 127.8
(2CH), 127.2 (2CH), 99.4 (CH), 97 (CH), 72.9 (CH), 70.3 (CH2), 43.1 (CH2), 25.6
(CH), 22.36 (CH3), 22.33 (CH3); MS (ESI) m/z 392.2 (M+H)+; LCMS (ES) m/z calcd
for C24H26NO4, [M+H]+: 392.19, found: 392.42. Compound 10: Colorless solid; 1
H
NMR (400 MHz, CDCl3): d 8.09 (2H, d, J 1.6 Hz), 7.59 (2H, m), 7.56–7.33 (12H, m),
6.87 (1H, d, J 2.4 Hz), 5.29 (2H, s), 5.27 (2H, s); MS (ESI) m/z 440.1 (M+H)+, 462.1
(M+Na)+. Compound 11: Colorless solid; mp 142–145 °C; IR (KBr pellet): mmax
3435, 1758, 1649 cmÀ1 1H NMR (400 MHz, CDCl3): d 9.65 (1H, br s, D2O
;
exchangeable), 7.42–7.32 (15H, m), 6.42 (1H, s), 5.91 (1H, d, J 2.4 Hz), 5.84 (1H,
5. (a) Takai, S.; Jin, D.; Muramatsu, M.; Okamoto, Y.; Miyazaki, M. Eur. J. Pharmacol.
2004, 501, 1–8; (b) Muto, T.; Fukami, H. IDrugs 2002, 5, 1141–1150.
6. (a) Shimatani, T.; Hosotani, N.; Ohnishi, M.; Kumagai, K.; Saji, I. J. Antibiot. 2006,
591, 29–34; (b) Shimatani, T.; Hosoya, Y. Japanese Patent 2004067584, 2004.
Chem. Abstr. 2004, 140, 216278.
d, J 2.0 Hz), 5.17 (2H, psq, J 12.4 Hz), 4.94 (2H, s); MS (ESI) m/z 442.2 (M+H)+,
464.2 (M+Na)+. Compound 12: Colorless liquid; IR (CHCl3 film):
m
max 1753, 1643,
1602 cmÀ1
;
1H NMR (400 MHz, CDCl3): d 7.42–7.28 (15H, m), 6.98 (1H, d, J
2.0 Hz), 6.61 (1H, s), 6.57 (1H, d, J 1.6 Hz), 5.16 (2H, d, J 1.6 Hz), 5.05 (2H, s), 2.44
(2H, d, J 7.2 Hz), 2.22 (1H, m), 1.04 (3H, s), 1.02 (3H, s); MS (ESI) m/z 526.3
(M+H)+, 548.2 (M+Na)+. SPF32629A: Pale yellow solid; mp 98–102 °C; IR (KBr
7. Ochiai, E. J. Org. Chem. 1953, 18, 534–551.
8. Fife, W. K. J. Org. Chem. 1983, 48, 1375–1377.
pellet): mmax 3435 (br), 2960, 1746, 1643, 1617, 1454 cmÀ1 1H NMR (400 MHz,
;
9. Yields refer to the isolated pure compounds and the products from all reactions
were purified either by flash column chromatography using silica gel 60 (230–
400 mesh Kieselgel 60) or by crystallization unless otherwise indicated. All new
compounds were fully characterized on the basis of IR, 1H NMR, 13C NMR, and
LCMS spectral data and are consistent with their structures. Spectral data for all
the synthesized compounds are as follows: Compound 2: Pale yellow solid; mp
CDCl3): d 9.2 (2H, br s, D2O exchangeable CONH and OH), 7.35–7.30 (5H, m),
6.62 (1H, s), 5.97 (1H, d, J 1.6 Hz), 5.87 (1H, d, J 2 Hz, D2O exchangeable pyridone
3-CH), 2.31 (2H, d, J 7.2 Hz), 2.11 (1H, m), 0.92 (3H, d, J 0.8 Hz), 0.90 (3H, d, J
0.4 Hz); 1H NMR (400 MHz, DMSO-d6): d 11.22 (1H, br s, D2O exchangeable
CONH), 10.52 (1H, br s, D2O exchangeable OH), 7.44–7.33 (5H, m), 6.43 (1H, s),
5.75 (1H, br s), 5.41 (1H, br s, D2O exchangeable pyridone 3-CH), 2.32 (2H, d, J
6.8 Hz), 2.04–1.99 (1H, m), 0.89 (3H, s), 0.88 (3H, s); 13C NMR (100 MHz, CDCl3):
d 171.8 (C), 170.0 (C), 165.7 (C), 146.1 (C), 135.9 (C), 129.1 (CH), 128.9 (2CH),
127.18 (2CH), 101.8 (CH), 98.4 (CH), 72.7 (CH), 43.1 (CH2), 25.6 (CH), 22.29
(CH3), 22.27 (CH3); MS (ESI) m/z 302.24 (M+H)+; LCMS (ES) m/z calcd for
179–181 °C (lit. 175–177 °C); IR (KBr pellet): mmax 1622, 1225 cmÀ1 1H NMR
;
(400 MHz, CDCl3): d 8.13 (2H, dd, J 5.6 and 2 Hz), 7.43–7.38 (5H, m), 6.87 (2H,
dd, J 5.2 and 2 Hz), 5.11 (2H, s); 13C NMR (100 MHz, CDCl3): d 156.4 (C), 139.6
(2CH), 134.5 (C), 128.4 (2CH), 128.2 (CH), 127.1 (2CH), 112.2 (2CH), 70.5 (CH2);
MS (APCI) m/z 202.02 (M+H)+. Compound 3: Colorless solid; mp 90–91 °C; IR
17H20NO4, [M+H]+: 302.14, found: 302.3; column used: Develosil ODS MG-3
mobile phase: A: 5 mM ammonium bicarbonate, B:
(KBr pellet): mmax 2237, 1590 cmÀ1 1H NMR (400 MHz, CDCl3): d 8.5 (1H, d, J
;
C
(4.6 Â 50 mm), 2.5
l,
6 Hz), 7.44–7.37 (5H, m), 7.27 (1H, d, J 2.4 Hz), 7.06 (1H, dd, J 5.8 and 2.2 Hz),
5.15 (2H, s); 13C NMR (100 MHz, CDCl3): d 164.8 (C), 152.1 (CH), 134.8 (C), 134.3
(C), 128.7 (2CH), 128.6 (CH), 127.4 (2CH), 117.0 (CH), 116.0 (CN), 113.3 (CH),
70.4 (CH2); MS (ESI) m/z 211.27 (M+H)+; LCMS (ES) m/z calcd for C13H11N2O,
[M+H]+: 211.09, found: 211.27. Compound 4: Colorless crystalline solid; mp 79–
acetonitrile, T/%B: 0/10, 4/50, 6/90, 10/90, 10.1/10; flow rate: 1 mL/min,
diluent: acetonitrile; UV: 215 nm, rt = 3.60, purity = 97.08%; Chiral HPLC:
Method-1: racemic mixture; column used: Chiral PAK-AD-H (4.6 Â 250 mm),
5 l, mobile phase: A: hexane/IPA (90:10) Isocratic, flow rate: 1 mL/min, diluent:
80 °C; IR (KBr pellet): mmax 1661, 1582 cmÀ1 1H NMR (400 MHz, CDCl3): d 8.52
;
mobile phase, run time: 25 min, UV: 286 nm, rt = 5.72 and 7.01. Chiral HPLC:
Method-2: racemic mixture; column used: Chiral PAK-IA (4.6 Â 250 mm), 5
l,
(1H, d, J 6 Hz), 8.07 (1H, s), 8.053 (1H, m), 7.64 (1H, d, J 2.8 Hz), 7.59–7.55 (1H,
m), 7.49–7.36 (7H, m), 7.04 (1H, dd, J 6 and 2.8 Hz), 5.19 (2H, s); 13C NMR
(100 MHz, CDCl3): d 193.4 (C), 165.4 (C), 156.6 (C), 149.7 (CH), 136.1 (C), 135.1
mobile phase: A: hexane/ethanol (70:30) isocratic, flow rate: 0.8 mL/min,
diluent: mobile phase, run time: 25 min, UV: 286 nm, rt = 5.37 and 6.76.