Design and synthesis of biotin- or alkyne-conjugated photoaffinity probes for studying the target molecules of PD 404182

Article abstract of DOI:10.1016/j.bmc.2013.01.016

To investigate the mechanism of action of the potent antiviral compound PD 404182, three novel photoaffinity probes equipped with a biotin or alkyne indicator were designed and synthesized based on previous structure-activity relationship studies. These probes retained the potent anti-HIV activity of the original pyrimidobenzothiazine derivatives. In photoaffinity labeling studies using HIV-1-infected H9 cells (H9IIIB), eight potential proteins were observed to bind PD 404182.

Full text of DOI:10.1016/j.bmc.2013.01.016

Bioorganic & Medicinal Chemistry 21 (2013) 2079–2087
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Bioorganic & Medicinal Chemistry
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Design and synthesis of biotin- or alkyne-conjugated photoaffinity
probes for studying the target molecules of PD 404182
a
b
b
Tsukasa Mizuhara a, Shinya Oishi a, , Hiroaki Ohno , Kazuya Shimura , Masao Matsuoka ,
⇑
Nobutaka Fujii a,
⇑
a Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
b Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
To investigate the mechanism of action of the potent antiviral compound PD 404182, three novel photo-
affinity probes equipped with a biotin or alkyne indicator were designed and synthesized based on
previous structure–activity relationship studies. These probes retained the potent anti-HIV activity of
the original pyrimidobenzothiazine derivatives. In photoaffinity labeling studies using HIV-1-infected
H9 cells (H9IIIB), eight potential proteins were observed to bind PD 404182.
Received 12 December 2012
Revised 5 January 2013
Accepted 5 January 2013
Available online 16 January 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Anti-HIV agents
PD 404182
Photoaffinity labeling
Pyrimidobenzothiazine
1. Introduction
Photoaffinity labeling is an efficient approach to identify the
target protein(s) of biologically active molecules.14 In modern drug
3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine
(PD 404182) (1)1–3 is a potent antiviral agent against the human
immunodeficiency virus (HIV) and the hepatitis C virus (HCV)
(Fig. 1).4,5 In structure–activity relationship (SAR) studies5,6 of
compound 1 using a series of facile synthetic procedures,7,8 we
identified several derivatives 2–4 that exhibited two- or three-fold
more potent anti-HIV activity than compound 1. The comparative
time of drug addition study using standard anti-HIV agents dem-
onstrated that compound 1 showed a similar antiviral profile
against HIV-1IIIB infection with that of DS 5000 (adsorption inhib-
itor)9 and enfuvirtide (fusion inhibitor),10 indicating that com-
pound 1 impaired virus replication at the early-stage of HIV
infection.5 Additionally, the antiviral activities of compound 1
against multiple HIV clades suggest that the target molecule of
compound 1 is not chemokine receptors (CC chemokine receptor
type 511 or CXC chemokine receptor type 412).5 Recently, the viru-
cidal effects of compound 1 against HCV, HIV and the simian
immunodeficiency virus have also been reported.13 However, the
mode of action and mechanism of antiviral activity of compound
1 has not yet been fully elucidated.
discovery, there have been a number of successful examples that
have determined the target molecules and identified the binding
site through the formation of a covalent bond between the ligand
and the specific protein.15 In general, photoaffinity probes contain
three functional groups: a bioactive scaffold, a photoreactive group
and an indicator group. A biotin-tag is widely employed as an indi-
cator because biotinylated proteins can be detected and isolated by
several immunological methods or through a biotin-avidin interac-
tion.16 A terminal alkyne is an alternative indicator for Huisgen
cycloaddition-mediated conjugation with various azide-modified
reporters, such as fluorescent-azide and biotin-azide after the
crosslinking reaction onto the target protein(s).17
In this article, the design and synthesis of biotin- or alkyne-con-
jugated photoaffinity probes based on previous SAR studies, and its
application for photoaffinity labeling studies are described.
2. Results and discussion
2.1. Design of biotin- or alkyne-conjugated photoaffinity probes
from PD 404182
Trifunctional probes for the target protein(s) of compound 1
and the derivatives were designed on the basis of our previous
SAR investigations.5,6 In our previous study, the introduction of a
hydrophobic group on the benzene ring and the cyclic amidine
Abbreviations: MAGI, multinuclear activation of a galactosidase indicator.
Corresponding authors. Tel.: +81 75 753 4551; fax: +81 75 753 4570.
E-mail addresses: soishi@pharm.kyoto-u.ac.jp (S. Oishi), nfujii@pharm.kyoto-u.
⇑
ac.jp (N. Fujii).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.016

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T. Mizuhara et al. / Bioorg. Med. Chem. 21 (2013) 2079–2087
benzophenone group on the pyrimidobenzothiazine scaffold
N
S
PD 404182 (1)
EC50 = 0.44 0.08 µM
CC50 > 100 µM
would be tolerated. Additionally, the N-alkoxycarbonyl piperidine
group onto the amidine substructure of 1 reproduced potent
anti-HIV activity (compound 5), indicating that this part could be
used as a linkage position for the addition of functional groups.
With this in mind, we designed three photoaffinity probes.
Compound 6 was modified with indicator biotin via a photoreac-
tive benzophenone group onto the benzene ring substructure
(Fig. 2). Compound 7 equips the biotin and benzophenone groups
on the right-part amidine moiety. The biotin moiety is conjugated
with benzophenone via a polyethylene glycol (PEG) linker as the
spacer. Compound 8 is an alkyne-containing derivative.
N
NH
N
S
N
10
R
N
N
9
R
NH
S
NH
2a (R = Br, EC50 = 0.25 0.09 µM) 3a (R = Br, EC50 = 0.24 0.05 µM)
2b (R= Ph, EC50 = 0.24 0.04 µM) 3b (R= Ph, EC50 = 0.24 0.05 µM)
2.2. Synthesis of biotin-conjugated probe 6
Synthesis of the probe 6 started with the preparation of benzo-
phenone boronic acid pinacol ester 11 (Scheme 1). Condensation of
p-(hydroxymethyl)benzoic acid 9 and N,O-dimethylhydroxylamine
followed by TBDPS protection of a primary hydroxy group gave an
amide 10. Subsequent nucleophilic addition of an in situ-generated
organolithium compound easily provided the desired boronate 11.18
We next assembled the components to synthesize the biotin-
conjugated probe 6 (Scheme 1). Alkylation of compound 2a with
p-methoxybenzyl (PMB) bromide followed by Suzuki–Miyaura
cross coupling with compound 11 afforded a benzophenone-conju-
gated pyrimidobenzothiazine 13. Desilylation of 13 and the subse-
quent reaction with p-nitrophenyl chloroformate afforded the
carbonate 16. The biotin moiety was incorporated by reaction of
16 with biotin-PEG-NH2 (15), which was prepared by catalytic
hydrogenation of azide 14.19 TFA-mediated deprotection of the
PMB group in compound 17 provided the desired probe 6.
N
N
R1
S
NH
R2
2c (R1 = OMe, R2 = H, EC50 = 0.15 0.05 µM)
2d (R1, R2 = -OCH2O-, EC50 = 0.15 0.03 µM)
N-CO2Me
N
N
S
N
N
S
NH
Br
NH
4
5
EC50 = 0.24 0.04 µM
EC50 = 0.44 0.02 µM
2.3. Synthesis of biotin-conjugated probe 7
Figure 1. Structures and anti-HIV activity of PD 404182 and the derivatives 2–5.
Synthesis of the biotin-conjugated probe 7 is outlined in
Scheme 2. PMB protection of compound 186 followed by selective
removal of the PMB group on the piperidine ring provided
compound 20. Separately, the synthesis of biotin-benzophenone
adduct 23 started from 4-(tert-butyldiphenylsilyloxy)methyl-40-
(hydroxymethyl)benzophenone 21.20 The treatment of 21 with
substructures effectively improved antiviral activity (compounds
2–4, Fig. 1). We expected that these moieties would potentially
take part in a favorable interaction(s) with the target molecule(s),
and the incorporation of
a hydrophobic and photoreactive
N
N
O
O
O
NH
H
O
HN
(
)
3
N
H
N
H
O
S
NH
S
H
O
O
6
O
O
O
O
HN
H
(
)
3
NH
N
O
O
N
H
N
H
N
S
H
N
O
7
Br
S
NH
N
N
O
S
NH
O
8
Figure 2. Structures of photoaffinity probes 6–8.

T. Mizuhara et al. / Bioorg. Med. Chem. 21 (2013) 2079–2087
2081
O
O
O
a,b
c
OH
NMe(OMe)
TBDPSO
HO
TBDPSO
Bpin
9
10
11
N
N
N
e
N
TBDPSO
S
NPMB
Br
S
NR
2a (R = H)
12 (R = PMB)
O
d
13
N
f,g
O
O
N
NH
H
O
(
HN
)
3
N
H
R ( -NO )C H OCO
2
S
NPMB
p
2
6
4
S
H
14 (R = N3)
15 (R = NH2)
O
16
i
h
N
S
O
O
O
N
NH
H
O
HN
(
)
j
N
H
N
O
NR
3
H
S
H
O
17 (R = PMB)
6 (R = H)
Scheme 1. Synthesis of biotin-conjugated probe 6. Reagents and conditions: (a) HNMe(OMe)ꢀHCl, EDCꢀHCl, HOBtꢀH2O, Et3N, DMF, rt; (b) TBDPSCl, Et3N, DMAP, CH2Cl2, rt, 49%
[2 steps (a,b)]; (c) 2-(4-bromophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, t-BuLi, THF, pentane, ꢁ78 to rt, 83%; (d) t-BuOK, DMF, 0 °C, then PMBBr, rt, 98%; (e) 11,
Pd(PPh3)4, PdCl2(dppf)ꢀCH2Cl2, K2CO3, toluene, EtOH, H2O, reflux, 96%; (f) TBAF, THF, rt; (g) p-nitrophenyl chloroformate, pyridine, CH2Cl2, reflux; (h) Et3N, DMF, rt to 40 °C,
46% [3 steps (f–h)]; (i) H2, 10% Pd-C, MeOH, rt; (j) MS4Å, TFA, CHCl3, rt, 35%.
chloroformate furnished a carbonate 22. Biotin-PEG-NH2 15 was
successfully conjugated onto 22 to give the biotin-benzophenone
adduct 23. Desilylation of 23, treatment with p-nitrophenyl chloro-
formate and coupling with 20 provided biotin/benzophenone-con-
jugated 26. PMB deprotection of 26 afforded the desired probe 7.
(EC50 = 0.64 lM). These probes 6–8 represent promising tools for
the identification of the target molecule(s) of compound 1 and
the derivatives.
2.6. Photoaffinity labeling experiment using biotin-conjugated
probes for HIV-1-infected H9 cells
2.4. Synthesis of alkyne-containing probe 8
Probes 6 and 7 were applied to the experiment for target iden-
tification of compound 1 and the derivatives. After HIV-1-infected
H9 cells (H9IIIB) were incubated with a probe (6 or 7) for 1 h, the
cells were exposed to UV–vis light (>300 nm) for 1 min. After cell
lysis, the biotinylated proteins were captured with NeutrAvidin
agarose beads. The whole was subjected to separation by SDS–
PAGE followed by Western blot analysis.
We next investigated the synthesis of alkyne-containing probe
8 (Scheme 3). Suzuki–Miyaura cross coupling of compound 275
with boronate 11 gave compound 28. Subsequent modifications
including desilylation, propargylation, and removal of the tert-
butyl group provided the expected alkyne-conjugated probe 8.
2.5. Anti-HIV activity of biotin- or alkyne-conjugated probes
Eight bands of 95, 80, 75, 70, 60, 55, 48 and 40 kDa proteins
were observed from the cell samples incubated with probe 6 (Lane
A, Fig. 3). These bands were competed by unlabeled compound 3a,
suggesting that the labeling was PD 404182-specific (Lane C). In
contrast, these bands, with the exception of the 70 and 40 kDa
bands, were not detected in the cells incubated with probe 7 (Lane
B). This observation indicated that the potential target proteins did
not fully interact with the benzophenone group on the right-part
amidine moiety in the pyrimidobenzothiazine scaffold of 7.
This preliminary experiment demonstrated that the synthe-
sized probe 6 could be useful for the identification of the target
protein(s) of compound 1. Efforts of the crosslinking experiments
using alkyne-conjugated probe 8 are also currently in progress.
The antiviral activities of probes 6–8 against HIV-1IIIB were
measured by multinuclear activation of a galactosidase indicator
(MAGI) assay. In this assay, the inhibitory activity against HIV
infection at the early stage, including virus attachment and
membrane fusion to host cells, can be evaluated.21 Both biotin-
conjugated probes 6 and 7 showed potent anti-HIV activity with
EC50 values of 6.87 and 5.11 lM, respectively (Table 1). These
activities were slightly lower than that of compound 1; however,
the incorporation of large functional groups including benzophe-
none, the PEG linker and the biotinyl reporter was largely toler-
ated. Alkyne-conjugated probe 8 potently inhibited HIV infection

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T. Mizuhara et al. / Bioorg. Med. Chem. 21 (2013) 2079–2087
N-PMB
NH
N
S
N
b
N
N
Br
NR
Br
NPMB
S
20
18 (R = H)
19 (R = PMB)
a
NO2
O
TBDPSO
OH
c
TBDPSO
O
O
O
O
22
21
d
O
O
O
HN
H
O
(
)
NH
RO
O
N
H
N
H
3
S
H
23 (R = TBDPS)
24 (R = H)
25 [R = CO2C6H4(p-NO2)]
O
e
f
g
O
O
O
O
O
HN
H
O
(
)
3
NH
N
O
N
H
N
H
N
S
S
H
N
O
26 (R = PMB)
7 (R = H)
Br
NR
h
Scheme 2. Synthesis of biotin-conjugated probe 7. Reagents and conditions: (a) t-BuOK, DMF, 0 °C, then PMBBr, rt, 81%; (b) 1-chloroethyl chloroformate, Et3N, CH2Cl2, 0 °C,
then MeOH, reflux; (c) 4-nitrophenyl chloroformate, pyridine, CH2Cl2, reflux; (d) 15, Et3N, DMF, rt, quant. [2 steps (c,d)]; (e) HF-pyridine, THF, 0 °C to rt, 73%; (f) 4-nitrophenyl
chloroformate, pyridine, CH2Cl2, reflux, 80%; (g) 20, Et3N, DMF, rt; (h) MS4Å, TFA, CHCl3, rt, 36% [2 steps (g,h)].
Wakogel C-300E (Wako) or aluminum oxide 90 standardized
(Merck) were employed. For preparative TLC, TLC silica gel 60
F254 (Merck) or TLC aluminum oxide 60 F254 basic (Merck) were
employed. For analytical HPLC, a Cosmosil 5C18-ARII column
(4.6 ꢂ 250 mm, Nacalai Tesque, Inc., Kyoto, Japan) was employed
with a linear gradient of CH3CN containing 0.1% (v/v) NH3 at a flow
rate of 1 mL/min on a Shimadzu LC-10ADvp (Shimadzu Corp., Ltd,
Kyoto, Japan), and eluting products were detected by UV at
254 nm. Preparative HPLC was performed using a COSMOSIL
5C18-ARII column (20 ꢂ 250 mm, Nacalai Tesque Inc.) with a lin-
ear gradient of MeCN containing 0.1% (v/v) NH3 at a flow rate of
8 mL/min on Shimadzu LC-6AD (Shimadzu corporation, Ltd). The
purity of the compounds 6–8 was determined by HPLC analysis
as >95%.
3. Conclusions
In conclusion, we have designed and synthesized novel
photoaffinity probes of antiviral PD 404182 with photoreactive
benzophenone, and biotin or alkyne indicators. The probes exhib-
ited equipotent or slightly less potent anti-HIV activities when
compared with the activity of the parent compound 1. Preliminary
photoaffinity labeling experiments suggest that these probes could
be useful in the identification of a potential target protein(s), the
binding site on the target protein(s) and the mechanism(s) of
action of PD 404182 derivatives.
4. Experimental
4.1. Synthesis
4.1.2. 4-[(tert-Butyldiphenylsilyloxy)methyl]-N-methoxy-N-
methylbenzamide (10)
4.1.1. General methods
1H NMR spectra were recorded using a JEOL AL-400 or a JEOL
ECA-500 spectrometer. Chemical shifts are reported in d (ppm) rel-
ative to Me4Si (CDCl3) or DMSO (DMSO-d6) as internal standards.
13C NMR spectra were referenced to the residual solvent signal. Ex-
act mass (HRMS) spectra were recorded on a JMS-HX/HX 110A
mass spectrometer. Melting points were measured by a hot stage
melting point apparatus (uncorrected). For flash chromatography,
To
a mixture of 4-(hydroxymethyl)benzoic acid 9 (4.6 g,
30.0 mmol), N,O-dimethylhydroxylamine hydrochloride (14.6 g,
150.0 mmol), Et3N (21.7 mL, 150.0 mmol) in DMF (300 mL) were
added EDCꢀHCl (11.5 g, 60.0 mmol) and HOBtꢀH2O (9.2 g,
60.0 mmol). After being stirred at rt overnight, solvent was evapo-
rated. The residue was dissolved in EtOAc, and washed with 1 N
HCl, satd NaHCO3, brine, and dried over MgSO4. The filtrate was

T. Mizuhara et al. / Bioorg. Med. Chem. 21 (2013) 2079–2087
2083
N
a
N
Br
S
Nt-Bu
27
N
S
N
b,c
TBDPSO
N -Bu
t
O
28
N
N
O
S
NR
O
29 (R = t-Bu)
8 (R = H)
d
Scheme 3. Synthesis of alkyne-conjugated probe 8. Reagents and conditions: (a)
11, Pd(PPh3)4, PdCl2(dppf)ꢀCH2Cl2, K2CO3, toluene, EtOH, H2O, reflux, 71%; (b) TBAF,
THF, rt; (c) NaH, THF, propargyl bromide, 0 °C to rt, 60% [2 steps (b,c)]; (d) MS4Å,
TFA, CHCl3, reflux, 92%.
Figure 3. Western blot analysis of the photolabeled proteins with biotin-conju-
gated probes 6 and 7. H9IIIB cells were incubated with (A) 20
lM probe 6, (B)
Table 1
20 M probe 7, and (C) 20 M probe 6 and 40 M compound 3a. The cells were
l
l
l
Anti-HIV activities of the probes 6–8
exposed to UV light for 1 min and were lysed. The resulting photolabeled proteins
were captured onto NeutrAvidin-agarose and the whole was subjected to SDS–
PAGE. The resulting gel was analyzed by Western blotting with streptavidin–HRP.
a
Compound
EC50
(
l
M)
PD 4041825
6
7
8
0.44 0.08
6.87 2.22
5.11 1.31
0.64 0.06
was added dropwise over 3 min. After being stirred at the same
temperature for additional 20 min, compound 10 (3.25 g,
7.5 mmol) was added. The reaction mixture was warmed to rt over
1 h and quenched with satd NH4Cl. The whole was extracted with
EtOAc and the extract was dried over MgSO4. After concentration,
the residue was purified by silica gel chromatography with n-hex-
ane/EtOAc (9:1) to give the title compound 11 as yellow oil (3.60 g,
83%): IR (neat) cmꢁ1: 1659 (C@O); 1H NMR (400 MHz, CDCl3) d:
1.11 (s, 9H, 3 ꢂ CH3), 1.37 (s, 12H, 4 ꢂ CH3), 4.85 (s, 2H, CH2),
7.37–7.46 (m, 8H, Ar), 7.69 (d, J = 6.6 Hz, 4H, Ar), 7.75–7.80 (m,
4H, Ar), 7.92 (d, J = 8.0 Hz, 2H, Ar); 13C NMR (100 MHz, CDCl3) d:
19.3, 24.8 (4C), 26.8 (3C), 65.2, 84.2 (2C), 125.6 (2C), 127.8 (4C),
128.9 (2C), 129.8 (2C), 130.2 (2C), 133.2 (2C), 134.5 (2C), 134.8,
135.5 (4C), 136.2, 140.0, 146.0, 196.6; HRMS (FAB): m/z calcd for
a
EC50 values represent the concentration of com-
pound required to inhibit the HIV-1 infection by 50%,
and were obtained from three independent
experiments.
concentrated to give crude Weinreb amide (4.05 g, ca. 20.7 mmol).
To the mixture of the Weinreb amide, a solution of Et3N (8.98 mL,
62.1 mmol) and DMAP (252.9 mg, 2.1 mmol) in CH2Cl2 (138 mL)
was slowly added TBDPSCl (5.83 mL, 22.8 mmol). After being stirred
at rt for 3 h, the reaction mixture was quenched with water. After
concentration, the residue was dissolved in EtOAc. The mixture
was washed with satd NaHCO3, brine, and dried over MgSO4. After
concentration, the residue was purified by flash column chromatog-
raphy over silica gel with n-hexane/EtOAc (3:1) to give the title
compound 10 as colorless oil (6.98 g, 49%): IR (neat) cmꢁ1: 1644
(C@O); 1H NMR (400 MHz, CDCl3) d: 1.10 (s, 9H, 3 ꢂ CH3), 3.36 (s,
3H, CH3), 3.57 (s, 3H, CH3), 4.80 (s, 2H, CH2), 7.36–7.43 (m, 8H,
Ar), 7.65–7.70 (m, 6H, Ar); 13C NMR (100 MHz, CDCl3) d: 19.3, 26.8
(3C), 33.8, 61.0, 65.2, 125.4 (2C), 127.7 (4C), 128.2 (2C), 129.8 (2C),
132.6, 133.3 (2C), 135.5 (4C), 143.8, 169.9; HRMS (FAB): m/z calcd
for C26H32NO3Si [M+H]+ 434.2152; found: 434.2160.
C
36H42BO4Si [M+H]+ 577.2945; found: 577.2949.
4.1.4. 9-Bromo-3,4-dihydro-N-(p-methoxybenzyl)-2H,6H-
pyrimido[1,2-c][1,3]benzothiazin-6-imine (12)
To the flask containing 2a (740.4 mg, 2.50 mmol) and t-BuOK
(561.1 mg, 5.00 mmol) was added DMF (10.0 mL) at 0 °C under
an Ar atmosphere. After being stirred at the same temperature
for 30 min, PMB-Br (729.0 lL, 5.00 mmol) was added. After being
stirred at rt for 1 h, the reaction mixture was quenched with
H2O. The whole was extracted with EtOAc, and washed with satd
NaHCO3, brine, and dried over MgSO4. After concentration, the res-
idue was purified by flash column chromatography over aluminum
oxide with n-hexane/EtOAc (3:1) to give the title compound 12 as
pale yellow amorphous (1.02 g, 98%): IR (neat) cmꢁ1: 1661 (C@N),
1510 (C@N); 1H NMR (400 MHz, CDCl3) d: 1.97-2.03 (m, 2H), 3.64
(t, J = 5.7 Hz, 2H, CH2), 3.80-3.84 (m, 5H, OCH3, CH2), 4.14 (s, 2H,
CH2), 6.86 (d, J = 8.5 Hz, 2H, Ar), 7.21-7.27 (m, 3H, Ar), 7.38 (dd,
4.1.3. 4-[(tert-Butyldiphenylsilyloxy)methyl]-40-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzophenone (11)
To a solution of 1,4-dibromobenzene (3.13 g, 13.3 mmol) and 2-
isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (2.80 mL,
13.8 mmol) in anhydrous THF (60 mL) was added t-BuLi
(19.4 mL, 1.55 M in pentane, 30.0 mmol) dropwise over 3 min at
ꢁ78 °C under an Ar atmosphere. After being stirred at ꢁ78 °C for
30 min, additional t-BuLi (19.4 mL, 1.55 M in pentane, 30.0 mmol)

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T. Mizuhara et al. / Bioorg. Med. Chem. 21 (2013) 2079–2087
J = 8.2, 1.8 Hz, 1H, Ar), 7.43 (d, J = 1.8 Hz, 1H, Ar); 13C NMR
(100 MHz, CDCl3) d: 19.8, 38.7, 44.3, 47.7, 55.3, 111.9, 114.1 (2C),
124.8, 127.9, 129.5, 130.2, 130.3 (2C), 132.6, 133.4, 138.7, 147.6,
159.1; HRMS (FAB): m/z calcd for C19H19N3OS [M+H]+ 416.0432;
found: 416.0431.
1H NMR (500 MHz, CDCl3) d: 1.39–1.45 (m, 2H, CH2), 1.57–1.74
(m, 4H, 2 ꢂ CH2), 2.03–2.08 (m, 2H, CH2), 2.20 (t, J = 6.9 Hz, 2H,
CH2), 2.70 (d, J = 12.6 Hz, 1H, CH), 2.87 (dd, J = 12.6, 4.6 Hz, 1H,
CH), 3.12 (d, J = 11.7, 4.6 Hz, 1H, CH), 3.40–3.43 (m, 4H, 2 ꢂ CH2),
3.54–3.71 (m, 14H, 7 ꢂ CH2), 3.77 (s, 3H, CH3), 3.88 (t, J = 6.0 Hz,
2H, CH2), 4.19 (s, 2H, CH2), 4.26–4.29 (m, 1H, CH), 4.45–4.47 (m,
1H, CH), 5.17 (s, 1H, NH), 5.20 (s, 2H, CH2), 5.65 (s, 1H, NH), 6.07
(s, 1H, NH), 6.48 (s, 1H, NH), 6.84 (d, J = 8.0 Hz, 2H, Ar), 7.26–7.28
(m, 2H, Ar), 7.44–7.62 (m, 7H, Ar), 7.81 (d, J = 8.0 Hz, 2H, Ar),
7.85 (d, J = 8.0 Hz, 2H, Ar); 13C NMR (125 MHz, CDCl3) d: 19.8,
25.5, 28.0, 28.1, 35.9, 39.0, 39.1, 40.4, 40.9, 44.3, 47.7, 55.2, 55.5,
60.1, 61.7, 65.8, 69.9, 69.9, 70.0, 70.2, 70.3 (2C), 112.2, 114.0 (2C),
125.7, 127.1 (2C), 127.4 (2C), 127.6, 128.6, 129.5, 130.2 (2C),
130.3 (2C), 130.6 (2C), 135.0, 136.5, 136.7, 137.1, 141.4, 142.0,
143.7, 148.2, 156.3, 158.9, 163.9, 173.2, 195.7; HRMS (FAB): m/z
calcd for C52H62N7O9S2 [M+H]+ 992.4050; found: 992.4050.
4.1.5. 9-{4-[4-(tert-Butyldiphenylsilyloxy)methyl]-
benzoylphenyl}-3,4-dihydro-N-(p-methoxybenzyl)-2H,6H-
pyrimido[1,2-c][1,3]benzothiazin-6-imine (13)
Pd(PPh3)4 (32.8 mg, 4 mol %) and PdCl2(dppf)ꢀCH2Cl2 (17.4 mg,
3 mol %) were added to a solution of 12 (296.2 mg, 0.71 mmol)
and 11 (409.4 mg, 0.71 mmol) in toluene (7.1 mL)-EtOH (4.3 mL)-
1 M aq K2CO3 (7.1 mL). After being stirred at reflux for 1 h, the mix-
ture was extracted with CHCl3. The extract was dried over MgSO4
and concentrated. The residue was purified by flash chromatogra-
phy over aluminum oxide with n-hexane/EtOAc (1:0 to 9:1) to give
the title compound 13 as pale yellow amorphous (536.2 mg, 96%):
IR (neat) cmꢁ1: 1658 (C@O), 1607 (C@N), 1511 (C@N); 1H NMR
(400 MHz, CDCl3) d: 1.12 (s, 9H, 3 ꢂ CH3), 2.03–2.08 (m, 2H), 3.70
(t, J = 5.5 Hz, 2H, CH2), 3.77 (s, 3H, CH3), 3.88 (t, J = 5.9 Hz, 2H,
CH2), 4.19 (s, 2H, CH2), 4.86 (s, 2H, CH2), 6.84 (d, J = 8.5 Hz, 2H,
Ar), 7.28 (m, 1H, Ar), 7.38–7.56 (m, 14H, Ar), 7.71 (dd, J = 7.6,
1.2 Hz, 4H, Ar), 7.81 (d, J = 8.0 Hz, 2H, Ar), 7.86 (d, J = 8.0 Hz, 2H,
Ar); 13C NMR (125 MHz, CDCl3) d: 19.3, 19.8, 26.8 (3C), 39.0,
44.3, 47.7, 55.2, 65.1, 112.2, 113.9 (2C), 125.6 (2C), 125.7, 127.0
(2C), 127.7 (4C), 128.7, 129.5, 129.8 (2C), 130.1 (2C), 130.2, 130.3
(2C), 130.5 (2C), 133.1 (2C), 135.0, 135.5 (4C), 136.2, 136.4,
137.0, 142.1, 143.5, 146.0, 148.2, 158.9, 195.8; HRMS (FAB): m/z
calcd for C49H48N3O3SSi [M+H]+ 786.3186; found: 786.3178.
4.1.8. 4-[4-(6-Imino-2,3,4,6-tetrahydrobenzo[e]pyrimido[1,2-
c][1,3]thiazin-9-yl)benzoyl]benzyl {13-oxo-17-[(3aS,4S,6aR)-2-
oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]-3,6,9-trioxa-12-
azaheptadecyl}carbamate (6)
TFA (2.0 mL) was added to
a mixture of 17 (62.9 mg,
0.063 mmol) in small amount of CHCl3 (1 or 2 drops) and molecu-
lar sieves 4 Å (300 mg, powder, activated by heating). After being
stirred at rt for 4 h, Et3N was added dropwise to the stirring mix-
ture at 0 °C to adjust pH to 8–9. The whole was extracted with
CHCl3, and washed with satd NaHCO3, brine, and dried over MgSO4.
After concentration, the residue was purified by flash chromatogra-
phy over aluminum oxide with CHCl3/MeOH (1:0–95:5) followed
by preparative HPLC to give the title compound 6 as colorless solid
(19.3 mg, 35%): IR (neat) cmꢁ1: 1699 (C@O), 1654 (C@O), 1621
(C@O), 1601 (C@N), 1574 (C@N); 1H NMR (500 MHz, CDCl3) d:
1.39–1.44 (m, 2H, CH2), 1.60–1.76 (m, 4H, 2 ꢂ CH2), 1.99–2.04
(m, 2H, CH2), 2.20 (t, J = 7.4 Hz, 2H, CH2), 2.71 (d, J = 12.6 Hz, 1H,
CH), 2.88 (dd, J = 12.6, 5.0 Hz, 1H, CH), 3.11 (d, J = 11.7, 5.0 Hz,
1H, CH), 3.40–3.43 (m, 4H, 2 ꢂ CH2), 3.54–3.63 (m, 12H,
6 ꢂ CH2), 3.73 (t, J = 5.4 Hz, 2H, CH2), 4.06 (t, J = 6.0 Hz, 2H, CH2),
4.28 (t, J = 6.0 Hz, 1H, CH), 4.47 (t, J = 6.0 Hz, 1H, CH), 5.20 (s, 2H,
CH2), 5.44 (s, 1H, NH), 5.73 (s, 1H, NH), 6.37 (s, 1H, NH), 6.66 (s,
1H, NH), 7.32 (s, 1H, Ar), 7.48 (d, J = 8.0 Hz, 2H, Ar), 7.52 (d,
J = 8.6 Hz, 1H, Ar), 7.69 (d, J = 8.0 Hz, 2H, Ar), 7.81 (d, J = 8.0 Hz,
2H, Ar), 7.88 (d, J = 8.0 Hz, 2H, Ar), 8.36 (d, J = 8.6 Hz, 1H, Ar); 13C
NMR (125 MHz, CDCl3) d: 20.8, 25.6, 28.0, 28.2, 35.9, 39.0, 40.4,
40.9, 43.9, 44.7, 51.2, 55.6, 60.1, 61.7, 65.7, 69.9, 70.0, 70.1, 70.3
(2C), 122.0, 125.2, 125.8, 126.9 (2C), 127.4 (2C), 129.6, 129.7,
130.2 (2C), 130.7 (2C), 137.0, 141.5, 142.2, 142.9, 144.8, 146.6,
152.9, 156.3, 164.1, 173.3, 195.6; HRMS (FAB): m/z calcd for
4.1.6. N-(2-[2-{2-(2-Aminoethoxy)ethoxy}ethoxy]ethyl)-5-
[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-
yl]pentanamide (15)
To the solution of 14 (116.0 mg, 0.26 mmol) in MeOH (2.0 mL)
was added 10% Pd-C (wetted with ca. 55% water, 160.0 mg). After
being stirred at rt overnight under H2 atmosphere, the mixture
was filtered through a celite pad and concentrated. The crude prod-
uct was used for the next step without further purification.
4.1.7. 4-(4-{6-[(4-Methoxybenzyl)imino]-2,3,4,6-tetra-
hydrobenzo[e]pyrimido[1,2-c][1,3]thiazin-9-yl}benzoyl)benzyl
{13-oxo-17-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-
d]imidazol-4-yl]-3,6,9-trioxa-12-azaheptadecyl}carbamate (17)
To a solution of 13 (157.2 mg, 0.20 mmol) in THF (2.0 mL) was
added TBAF in THF (1 M, 0.50 mL, 0.50 mmol). After being stirred
at rt overnight, the reaction mixture was quenched with satd
NH4Cl. The whole was extracted with CHCl3 and dried over MgSO4.
After concentration, the residue was subjected to flash column
chromatography over aluminum oxide with n-hexane/EtOAc
(5:1–0:1) to give the desilylated compound. To a solution of the
resulting compound in CH2Cl2 (6.0 mL) were added p-nitrophenyl
C
44H54N7O8S2 [M+H]+ 872.3475; found: 872.3481.
4.1.9. N-[9-Bromo-10-(4-methoxybenzyl)-2H-spiro(benzo[e]-
pyrimido[1,2-c][1,3]thiazine-3,40-piperidin)-6(4H)-ylidene]-1-
(4-methoxyphenyl)methanamine (19)
chloroformate (60.5 mg, 0.30 mmol) and pyridine (64.6 lL,
By a procedure identical with that described for synthesis of 12
from 2a, the imine 18 (274.3 mg, 0.57 mmol) was converted into
19 as colorless amorphous (275.1 mg, 81%): IR (neat) cmꢁ1: 1668
(C@N), 1510 (C@N); 1H NMR (400 MHz, CDCl3) d: 1.61–1.64 (m,
4H, 2 ꢂ CH2), 2.36–2.42 (m, 2H, CH2), 2.45–2.51 (m, 2H, CH2),
3.45 (s, 2H, CH2), 3.47 (s, 2H, CH2), 3.55 (s, 2H, CH2), 3.80 (s, 3H,
CH3), 3.81 (s, 3H, CH3), 4.12 (s, 2H, CH2), 6.82-6.87 (m, 4H, Ar),
7.19–7.23 (m, 5H, Ar), 7.38 (dd, J = 8.2, 1.8 Hz, 1H, Ar), 7.44 (d,
J = 2.0 Hz, 1H, Ar); 13C NMR (100 MHz, CDCl3) d: 28.2, 32.4 (2C),
39.1, 48.7 (2C), 54.6, 55.2, 55.3, 55.4, 62.6, 111.9, 113.7 (2C),
113.9, 114.1 (2C), 124.8, 128.0, 129.7, 130.0, 130.2 (4C), 133.4,
133.4, 138.6, 147.1, 158.8, 159.1; HRMS (FAB): m/z calcd for
0.8 mmol). After being stirred under reflux for 1 h, additional p-
nitrophenyl chloroformate (12.0 mg, 0.06 mmol) was added. After
being stirred under reflux for additional 30 min, the reaction mix-
ture was washed with brine, and dried over MgSO4. After concen-
tration, the solution of resulting residue (crude 16) in DMF (2.0 mL)
was added to the solution of 15 (ca. 0.26 mmol) and Et3N (86.7 lL)
in DMF (3.0 mL). After being stirred at rt for 8 h, the reaction mix-
ture was stirred at 40 °C overnight. After concentration, the residue
was purified by flash column chromatography over aluminum
oxide with CHCl3/MeOH (1:0–95:5) followed by flash column chro-
matography over silica gel with CHCl3/MeOH (1:0–9:1) to give the
title compound 17 as pale yellow amorphous (90.6 mg, 46%): IR
(neat) cmꢁ1: 1699 (C@O), 1656 (C@O), 1607 (C@N), 1511 (C@N);
C
31H34BrN4O2S [M+H]+ 605.1586; found: 605.1585.

T. Mizuhara et al. / Bioorg. Med. Chem. 21 (2013) 2079–2087
2085
4.1.10. N-[9-Bromo-2H-spiro(benzo[e]pyrimido[1,2-c][1,3]-
thiazine-3,4’-piperidin)-6(4H)-ylidene]-1-(4-methoxyphenyl)-
methanamine (20)
64.2, 65.7, 69.9, 69.9 (2C), 70.1, 70.3 (2C), 126.4 (2C), 127.3 (2C),
130.2 (2C), 130.2 (2C), 136.2, 137.1, 141.3, 146.4, 156.4, 164.1,
173.5, 196.0; HRMS (FAB): m/z calcd for C34H47N4O9S [M+H]+
687.3064; found: 687.3058.
To a solution of 19 (60.6 mg, 0.10 mmol) in CH2Cl2 (0.5 mL)
were added Et3N (28.9
lL, 0.20 mmol) and 1-chloroethyl chlorofor-
mate (21.8 L, 0.20 mmol) at 0 °C under an Ar atmosphere. After
l
4.1.13. 4-(4-{[(4-Nitrophenoxy)carbonyloxy]methyl}benzoyl)-
benzyl 13-oxo-17-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-
d]imidazol-4-yl]-3,6,9-trioxa-12-azaheptadecylcarbamate (25)
To a solution of 24 (28.2 mg, 0.04 mmol) in CH2Cl2 (1.2 mL)
were added p-nitrophenyl chloroformate (24.8 mg, 0.12 mmol)
being stirred at the same temperature for 30 min, the reaction mix-
ture was concentrated. The residue was dissolved in MeOH
(2.0 mL). After being stirred under reflux for 10 min, the reaction
mixture was concentrated. The residue was dissolved in CHCl3,
and was washed with satd NaHCO3, brine, and dried over MgSO4.
After concentration, the crude product was used for the next step
without further purification.
and pyridine (13.2 lL, 0.16 mmol). After being stirred under re-
flux for 1 h, the reaction mixture was washed with brine, and
dried over MgSO4. After concentration, the residue was purified
by preparative TLC over aluminum oxide with CHCl3/MeOH
(9:1) to give the title compound 25 as colorless amorphous
(27.9 mg, 80%): IR (neat) cmꢁ1: 1768 (C@O), 1698 (C@O), 1656
(C@O), 1612 (C@O); 1H NMR (400 MHz, CDCl3) d: 1.38–1.45 (m,
2H, CH2), 1.59–1.76 (m, 4H, 2 ꢂ CH2), 2.20 (t, J = 7.4 Hz, 2H,
CH2), 2.72 (d, J = 12.7 Hz, 1H, CH), 2.88 (dd, J = 12.7, 4.9 Hz, 1H,
CH), 3.12 (dd, J = 11.8, 7.4 Hz, 1H, CH), 3.38–3.44 (m, 4H,
2 ꢂ CH2), 3.55–3.63 (m, 12H, 6 ꢂ CH2), 4.28 (t, J = 6.0 Hz, 1H,
CH), 4.47 (t, J = 6.0 Hz, 1H, CH), 5.19 (s, 2H, CH2), 5.38 (s, 2H,
CH2), 5.52 (br s, 1H, NH), 5.69 (br s, 1H, NH), 6.44 (br s, 1H,
NH), 6.66 (br s, 1H, NH), 7.41 (d, J = 9.3 Hz, 2H, Ar), 7.47 (d,
J = 8.0 Hz, 2H, Ar), 7.56 (d, J = 8.0 Hz, 2H, Ar), 7.79 (d, J = 8.0 Hz,
2H, Ar), 7.84 (d, J = 8.0 Hz, 2H, Ar), 8.29 (d, J = 9.3 Hz, 2H, Ar);
13C NMR (CDCl3, 100 MHz) d: 25.5, 28.1, 28.2, 35.9, 39.1, 40.5,
40.9, 55.5, 60.2, 61.8, 65.8, 69.9, 70.0, 70.0 (2C), 70.2, 70.4 (2C),
121.7 (2C), 125.3 (2C), 127.5 (2C), 128.1 (2C), 130.2 (2C), 130.4
(2C), 136.8, 137.9, 138.6, 141.7, 145.5, 152.4, 155.4, 156.3,
4.1.11. 4-[4-(tert-Butyldiphenylsilyloxymethyl)benzoyl]benzyl
{13-oxo-17-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-
d]imidazol-4-yl]-3,6,9-trioxa-12-azaheptadecyl}carbamate (23)
To
(15.0 mL) were added p-nitrophenyl chloroformate (151.2 mg,
0.75 mmol) and pyridine (161.4 L, 2.00 mmol). After being stir-
a
solution of 2120 (240.3 mg, 0.50 mmol) in CH2Cl2
l
red under reflux for 1 h, the reaction mixture was washed with
brine, and dried over MgSO4. After concentration, the solution
of the resulting residue in DMF (7.5 mL) was added to a mixture
of 15 (ca. 0.20 mmol) and Et3N (216.8 lL) in DMF (5.0 mL). After
being stirred at rt overnight, the mixture was concentrated. The
residue was purified by flash column chromatography over silica
gel with CHCl3/MeOH (1:0–95:5) to give the title compound 23 as
colorless amorphous (471.5 mg, quant.): IR (neat) cmꢁ1: 1700
(C@O), 1656 (C@O), 1609 (C@O); 1H NMR (400 MHz, CDCl3) d:
1.12 (s, 9H, 3 ꢂ CH3), 1.39–1.46 (m, 2H, CH2), 1.61–1.76 (m, 4H,
2 ꢂ CH2), 2.19–2.23 (m, 2H, CH2), 2.69–2.76 (m, 1H, CH), 2.85–
2.90 (m, 1H, CH), 3.09–3.15 (m, 1H, CH), 3.39–3.43 (m, 4H,
2 ꢂ CH2), 3.54–3.66 (m, 12H, 6 ꢂ CH2), 4.26–4.33 (m, 1H, CH),
4.45–4.51 (m, 1H, CH), 4.85 (s, 2H, CH2), 5.19 (s, 2H, CH2), 5.54
(br s, 1H, NH), 5.68 (br s, 1H, NH), 6.55 (br s, 1H, NH), 6.72 (br
s, 1H, NH), 7.36–7.48 (m, 10H, Ar), 7.69 (d, J = 7.6 Hz, 2H, Ar),
7.70 (d, J = 7.6 Hz, 2H, Ar), 7.77 (d, J = 5.5 Hz, 2H, Ar), 7.79 (d,
J = 5.5 Hz, 2H, Ar); 13C NMR (100 MHz, CDCl3) d: 19.3, 25.5, 26.8
(3C), 28.1, 28.2, 35.9, 39.1, 40.5, 40.9, 55.5, 60.1, 61.7, 65.1,
65.8, 69.9, 70.0, 70.0, 70.2, 70.4 (2C), 125.6 (2C), 127.4 (2C),
127.8 (4C), 129.8 (2C), 130.1 (2C), 130.2 (2C), 133.2 (2C), 135.5
(4C), 136.1, 137.4, 141.2, 146.0, 156.3 163.9, 173.2, 196.0; HRMS
(FAB): m/z calcd for C50H65N4O9SSi [M+H]+ 925.4242; found:
925.4246.
163.9, 173.3, 195.5; HRMS (FAB): m/z calcd for C41H50N5O13
S
[M+H]+ 852.3126; found: 852.3127.
4.1.14. 4-(4-{3,17-Dioxo-21-[(3aS,4S,6aR)-2-oxohexahydro-1H-
thieno[3,4-d]imidazol-4-yl]-2,7,10,13-tetraoxa-4,16-
diazahenicosyl}benzoyl)benzyl 9-bromo-6-imino-4,6-dihydro-
2H-spiro(benzo[e]pyrimido[1,2-c][1,3]thiazine-3,4’-piperidine)-
1’-carboxylate (7)
To a solution of 20 (ca. 0.027 mmol) in DMF (0.4 mL) were
added Et3N (11.7 lL, 0.081 mmol) and the solution of 25
(23.3 mg, 0.027 mmol) in DMF (0.4 mL) at rt. After being stirred
at the same temperature for 1 h, the reaction mixture was con-
centrated. The residue was subjected to preparative TLC over sil-
ica gel with CHCl3/MeOH (9:1) to give crude imine 26. By a
procedure identical with that described for synthesis of 6 from
17, the crude 26 was converted into 7 as a colorless amorphous
(10.4 mg, 36%): IR (neat) cmꢁ1: 1699 (C@O), 1655 (C@O), 1612
(C@O), 1573 (C@N); 1H NMR (400 MHz, CDCl3) d: 1.39–1.46 (m,
2H, CH2), 1.53 (d, J = 5.6 Hz, 4H, 2 ꢂ CH2), 1.61–1.72 (m, 4H,
2 ꢂ CH2), 2.20 (t, J = 7.3 Hz, 2H, CH2), 2.71 (d, J = 12.7 Hz, 1H,
CH), 2.89 (dd, J = 12.7, 4.9 Hz, 1H, CH), 3.12 (d, J = 12.1, 7.3 Hz,
1H, CH), 3.39–3.44 (m, 4H, 2 ꢂ CH2), 3.53–3.63 (m, 18H,
9 ꢂ CH2), 3.93 (s, 2H, CH2), 4.28 (t, J = 5.7 Hz, 1H, CH), 4.47 (t,
J = 6.5 Hz, 1H, CH), 5.14 (s, 1H, NH), 5.19 (s, 2H, CH2), 5.22 (s,
2H, CH2), 5.68 (s, 1H, NH), 6.01 (s, 1H, NH), 6.52 (s, 1H, NH),
7.22 (d, J = 2.0 Hz, 1H, Ar), 7.34 (dd, J = 8.8, 2.0 Hz, 1H, Ar), 7.45
(d, J = 8.0 Hz, 2H, Ar), 7.46 (d, J = 8.0 Hz, 2H, Ar), 7.79 (m, 4H,
Ar), 8.10 (d, J = 8.8 Hz, 1H, Ar); 13C NMR (100 MHz, CDCl3) d:
25.5, 28.1, 28.1, 29.6, 32.2 (2C), 35.8, 39.1, 39.9 (2C), 40.5, 40.9,
49.9, 54.6, 55.4, 60.1, 61.8, 65.8, 66.4, 69.9, 70.0 (2C), 70.2,
70.4 (2C), 125.0, 125.3, 126.0, 127.3 (2C), 127.4 (2C), 129.6,
130.2 (2C), 130.3 (2C), 130.4, 130.6, 137.0, 137.1, 141.4, 141.5,
145.1, 152.6, 155.0, 156.3, 163.8, 173.3, 195.7; HRMS (FAB): m/
4.1.12. 4-[4-(Hydroxymethyl)benzoyl]benzyl {13-oxo-17-
[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]-
3,6,9-trioxa-12-azaheptadecyl}carbamate (24)
To a solution of 23 (383.0 mg, 0.41 mmol) in THF (8.2 mL) was
added HF-pyridine (617.7 lL) at 0 °C. After being stirred at rt over-
night, the reaction was quenched with satd NaHCO3. The whole
was extracted with CHCl3, and washed with water and brine, and
dried over MgSO4. After concentration, the residue was purified
by preparative TLC over silica gel with CHCl3/MeOH (85:15) to give
the title compound 24 as colorless oil (204.2 mg, 73%): IR (neat)
cmꢁ1: 1696 (C@O), 1650 (C@O), 1609 (C@O); 1H NMR (400 MHz,
CDCl3) d: 1.34–1.41 (m, 2H, CH2), 1.55–1.73 (m, 4H, 2 ꢂ CH2),
2.07 (br s, 1H, OH), 2.16 (t, J = 7.4 Hz, 2H, CH2), 2.68 (d,
J = 12.9 Hz, 1H, CH), 2.85 (dd, J = 12.9, 4.9 Hz, 1H, CH), 3.08 (dd,
J = 11.8, 7.4 Hz 1H, CH), 3.37–3.42 (m, 4H, 2 ꢂ CH2), 3.51–3.64
(m, 12H, 6 ꢂ CH2), 4.23 (t, J = 6.2 Hz, 1H, CH), 4.43 (t, J = 6.2 Hz,
1H, CH), 4.78 (s, 2H, CH2), 5.18 (s, 2H, CH2), 5.51 (br s, 1H, NH),
5.82 (br s, 1H, NH), 6.34 (br s, 1H, NH), 6.75 (br s, 1H, NH), 7.45
(d, J = 8.3 Hz, 2H, Ar), 7.48 (d, J = 8.3 Hz, 2H, Ar), 7.76 (d,
J = 8.0 Hz, 2H, Ar), 7.77 (d, J = 8.0 Hz, 2H, Ar); 13C NMR (125 MHz,
CDCl3) d: 25.5, 28.0, 28.2, 35.8, 39.1, 40.4, 40.9, 55.6, 60.2, 61.8,
z
calcd for
C
50H62BrN8O10S2 [M+H]+ 1077.3214; found:
1077.3213.

2086
T. Mizuhara et al. / Bioorg. Med. Chem. 21 (2013) 2079–2087
4.1.15. N-(tert-Butyl)-9-{4-[4-(tert-butyldiphenylsilyloxy)-
methyl]benzoylphenyl}-3,4-dihydro-2H,6H-pyrimido[1,2-
c][1,3]benzothiazin-6-imine (28)
195.7; HRMS (FAB): m/z calcd for C28H24N3O2S [M+H]+ 466.1589;
found: 466.1589.
Compound 27 (2.17 g, 6.17 mmol) was subjected to the general
cross-coupling procedure as described for the synthesis of 13 to
give the title compound 28 as colorless solid (3.16 g, 71%): mp
152–153 °C (from CHCl3/n-hexane): IR (neat) cmꢁ1: 1656 (C@O),
1623 (C@N), 1593 (C@N); 1H NMR (400 MHz, CDCl3) d: 1.12 (s,
9H, 3 ꢂ CH3), 1.41 (s, 9H, 3 ꢂ CH3), 1.91-1.97 (m, 2H), 3.65 (t,
J = 5.4 Hz, 2H, CH2), 3.90 (t, J = 6.2 Hz, 2H, CH2), 4.86 (s, 2H, CH2),
7.37-7.48 (m, 10H, Ar), 7.69-7.71 (m, 6H, Ar), 7.81 (d, J = 8.3 Hz,
2H, Ar), 7.88 (d, J = 8.3 Hz, 2H, Ar), 8.30 (d, J = 8.5 Hz, 1H, Ar); 13C
NMR (100 MHz, CDCl3) d: 19.3, 21.9, 26.8 (3C), 30.0 (3C), 45.2,
45.5, 54.2, 65.2, 123.0, 124.9, 125.7 (2C), 126.9 (2C), 127.4, 127.8
(4C), 129.1, 129.8 (2C), 129.9, 130.2 (2C), 130.7 (2C), 133.2 (2C),
135.5 (4C), 136.2, 137.2, 138.0, 141.7, 143.2, 146.1, 147.6, 195.9;
HRMS (FAB): m/z calcd for C45H48N3O2SSi [M+H]+ 722.3237; found:
722.3244.
4.2. Determination of anti-HIV activity
The sensitivity of HIV-1IIIB strain was determined by the MAGI
assay. The target cells (HeLa-CD4/CCR5-LTR/b-gal; 104 cells/well)
were plated in 96-well flat microtiter culture plates. On the follow-
ing day, the cells were inoculated with the HIV-1 (60 MAGI U/well,
giving 60 blue cells after 48 h of incubation) and cultured in the
presence of various concentrations of the test compounds in fresh
medium. Forty-eight hours after viral exposure, all the blue cells
stained with X-Gal (5-bromo-4-chloro-3-indolyl-b-D-galactopy-
ranoside) were counted in each well. The activity of test com-
pounds was determined as the concentration that blocked HIV-1
infection by 50% (50% effective concentration [EC50]). EC50 was
determined by using the following formula:
EC50 ¼ 10^½logðA=BÞ ꢂ ð50 ꢁ CÞ=ðD ꢁ CÞ þ logðBÞꢃ
4.1.16. N-(tert-Butyl)-3,4-dihydro-9-[4-(4-propargyloxymethyl)-
benzoylphenyl]-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-
imine (29)
wherein
A: of the two points on the graph which bracket 50% inhibition,
the higher concentration of the test compound,
B: of the two points on the graph which bracket 50% inhibition,
the lower concentration of the test compound,
C: inhibitory activity (%) at the concentration B,
D: inhibitory activity (%) at the concentration A.
To a solution of 28 (200.0 mg, 0.28 mmol) in THF (2.8 mL) was
added TBAF in THF (1 M, 0.55 mL, 0.55 mmol). After being stirred
at rt for 2 h, the reaction mixture was quenched with satd NH4Cl.
The whole was extracted with EtOAc, and washed with brine,
and dried over MgSO4. The filtrate was concentrated. To the solu-
tion of the resulting residue in THF (2.8 mL) was added NaH
(22.8 mg, 0.55 mmol, 60% oil suspension) at 0 °C. After being stir-
red at the same temperature for 30 min, propargyl bromide
4.3. Photoaffinity labeling experiments using HIV-1-infected H9
cells (H9IIIB)
(31.5 lL, 0.42 mmol) was added dropwise. After being stirred at
1 lL of probe 6 or 7 (10 mM solution in DMSO) was added to H9
rt overnight, the reaction was quenched with water. The whole
was extracted with EtOAc, and washed with brine, and dried over
MgSO4. After concentration, the residue was purified by flash col-
umn chromatography over aluminum oxide with n-hexane/EtOAc
(5:1) to give the title compound 29 as colorless solid (87.2 mg,
cells chronically infected with HIV-1 (H9IIIB) in D-MEM with 10%
fetal bovine serum (500
evaluation (Fig. 3, lane C), 2
l
L, 0.5 ꢂ 106 cells). For the competitive
l
L of compound 3a (10 mM solution
in DMSO) was also added. The cells were incubated at 37 °C for
1 h. Then the cells were photolabeled by irradiation by UV
(MUV-202U, Moritex Co., Japan) at room temperature for 1 min
at a distance of 3 cm through a longpass filter (LU0300, Asahi spec-
tra Co.). The mixture was centrifuged at 200 ꢂ g for 5 min and the
supernatant was removed. The cells were washed with PBS once
and were lysed in RIPA buffer containing 1% protease inhibitor
cocktail (Nacalai Tesque, Inc., Japan) at 4 °C for 30 min. After cen-
trifugation at 16500 ꢂ g for 15 min, the supernatant was used for
the next experiment.
60%): mp 133–135 °C (from CHCl3/n-hexane): IR (neat) cmꢁ1
:
1656 (C@O), 1620 (C@N), 1593 (C@N); 1H NMR (400 MHz, CDCl3)
d: 1.41 (s, 9H, 3 ꢂ CH3), 1.91–1.97 (m, 2H), 2.50 (t, J = 2.3 Hz, 1H,
CH), 3.65 (t, J = 5.5 Hz, 2H, CH2), 3.90 (t, J = 6.1 Hz, 2H, CH2), 4.25
(d, J = 2.3 Hz, 2H, CH2), 4.71 (s, 2H, CH2), 7.39 (d, J = 1.7 Hz, 1H,
Ar), 7.46–7.50 (m, 3H, Ar), 7.70 (d, J = 8.0 Hz, 2H, Ar), 7.82 (d,
J = 8.0 Hz, 2H, Ar), 7.87 (d, J = 8.0 Hz, 2H, Ar), 8.30 (d, J = 8.3 Hz,
1H, Ar); 13C NMR (100 MHz, CDCl3) d: 21.9, 30.0 (3C), 45.2, 45.4,
54.2, 57.6, 70.9, 75.0, 79.3, 123.0, 124.8, 126.9 (2C), 127.3, 127.5
(2C), 129.1, 129.9, 130.2 (2C), 130.7 (2C), 136.9, 137.0, 137.9,
141.6, 142.2, 143.4, 147.5, 195.7; HRMS (FAB): m/z calcd for
NeutrAvidin-agarose beads (50
ibrated with RIPA buffer, were treated with the supernatant con-
taining 180 g of proteins and were incubated at 4 °C for 1 h. The
lL, Thermo), which were equil-
l
C
32H32N3O2S [M+H]+ 522.2215; found: 522.2207.
beads were then centrifuged at 9100 ꢂ g for 30 sec and washed
with RIPA buffer (repeated three times). After heating the beads
at 95 °C for 5 min in sample buffer [50 mM Tris–HCl (pH 8.0), 2%
SDS, 0.1% BPB, 10% glycerol, 2% b-ME], the supernatants were sub-
jected to SDS–PAGE electrophoresis (SuperSep™Ace, 5–20%, Wako)
and the separated proteins were transferred onto a PVDF mem-
brane. The membrane was blocked with Blocking One (Nacalai Tes-
que, Inc.) at room temperature for 1 h, and was then incubated
with a streptavidin–HRP conjugate (Invitrogen; 1:5000 in PBS with
0.1% Tween) at 4 °C overnight. The membrane was treated with
Chemi-Lumi One L (Nacalai Tesque, Inc.). Biotinylated proteins
were detected by Image Quant LAS 4000mini (GE Healthcare).
4.1.17. 3,4-Dihydro-9-[4-(4-propargyloxymethyl)-
benzoylphenyl]-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-
imine (8)
Using a procedure identical with that described for synthesis of
6 from 17, the imine 29 (42.8 mg, 0.08 mmol) was allowed to react
under reflux for 1 h with TFA (2.0 mL) and MS4Å (300 mg). Purifi-
cation by flash chromatography over aluminum oxide with n-hex-
ane/EtOAc (9:1 to 1:1) gave the title compound 8 as colorless solid
(35.4 mg, 92%): mp 159–160 °C (from CHCl3/n-hexane): IR (neat)
cmꢁ1: 1654 (C@O), 1619 (C@N), 1573 (C@N); 1H NMR (400 MHz,
CDCl3) d: 1.96–2.04 (m, 2H), 2.50 (t, J = 2.4 Hz, 1H, CH), 3.72 (t,
J = 5.6 Hz, 2H, CH2), 4.05 (t, J = 6.1 Hz, 2H, CH2), 4.25 (d, J = 2.4 Hz,
2H, CH2), 4.71 (s, 2H, CH2), 7.26–7.31 (m, 2H, Ar, NH), 7.48–7.51
(m, 3H, Ar), 7.67–7.89 (m, 6H, Ar), 8.33 (d, J = 8.5 Hz, 1H, Ar); 13C
NMR (100 MHz, CDCl3) d: 21.0, 43.8, 45.0, 57.6, 70.9, 75.0, 79.3,
122.0, 125.1, 126.3, 126.9 (2C), 127.5 (2C), 129.6, 129.7, 130.2
(2C), 130.7 (2C), 137.0, 137.1, 142.2, 142.3, 143.0, 146.2, 153.0,
Acknowledgments
This work was supported by Grants-in-Aid for Scientific Re-
search and Platform for Drug Discovery, Informatics, and Structural
Life Science from MEXT; and Health and Labor Science Research

T. Mizuhara et al. / Bioorg. Med. Chem. 21 (2013) 2079–2087
2087
Nelson, K.; Bodart, V.; Wong, R.; Fricker, S.; Huskens, D.; Schols, D. Bioorg. Med.
Chem. Lett. 2011, 21, 6950.
Grants (Research on HIV/AIDS, Japan). T.M. is grateful for JSPS Re-
search Fellowships for Young Scientists.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.016.
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