Investigations of possible prodrug structures for 2-(2-mercaptophenyl)tetrahydropyrimidines: Reductive conversion from anti-HIV agents with pyrimidobenzothiazine and isothiazolopyrimidine scaffolds

Article abstract of DOI:10.1039/c5ob00301f

3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine (PD 404182) and 3,4-dihydro-2H-benzo[4,5]isothiazolo[2,3-a]pyrimidine are the heterocyclic antiretroviral agents against human immunodeficiency virus type 1 (HIV-1) infection. On the basis of simi

Full text of DOI:10.1039/c5ob00301f

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Biomolecular Chemistry
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Investigations of possible prodrug structures for
2-(2-mercaptophenyl)tetrahydropyrimidines:
reductive conversion from anti-HIV agents with
pyrimidobenzothiazine and isothiazolopyrimidine
scaffolds†
Cite this: Org. Biomol. Chem., 2015,
13, 4706
Shiho Okazaki,a Shinya Oishi,*a Tsukasa Mizuhara,a Kazuya Shimura,b
Hiroto Murayama,b Hiroaki Ohno,a Masao Matsuokab and Nobutaka Fujii*a
3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine (PD 404182) and 3,4-dihydro-2H-benzo-
[4,5]isothiazolo[2,3-a]pyrimidine are the heterocyclic antiretroviral agents against human immunodefi-
ciency virus type 1 (HIV-1) infection. On the basis of similar structure–activity relationships of anti-HIV
activities toward the early-stage of viral infection between these unique scaffolds, the transformations
under the bioassay conditions were investigated. The distinctive S–N bond in the isothiazolopyrimidine
scaffold was immediately cleaved under reductive conditions in the presence of GSH to generate a thio-
phenol derivative. A similar rapid conversion of PD 404182 into the same thiophenol derivative was
observed, suggesting that pyrimidobenzothiazine and isothiazolopyrimidine scaffolds may work as
prodrug forms of the common bioactive thiophenol derivatives.
Received 12th February 2015,
Accepted 9th March 2015
DOI: 10.1039/c5ob00301f
www.rsc.org/obc
revealed that modification of the 9-position of PD 404182 deriva-
tives with hydrophobic aryl groups improved the anti-HIV
Introduction
PD 404182 (1a, 3,4-dihydro-2H,6H-pyrimido[1,2-c][1,3]benzo- activity.7–9 For example, the introduction of an aryl (1b, 1c and
thiazin-6-imine)1–4 is an antiviral agent against human hepa- 1f) or bromo (1d) substituent at the 9-position led to a two- to
titis C virus (HCV),5,6 HIV,6–10 simian immunodeficiency virus three-fold increase in the anti-HIV activity compared with com-
(SIV),6 and herpes simplex virus (HSV) (Fig. 1).10 3,4-Dihydro- pound 1a (Table 1). This improvement was duplicated by the
2H-benzo[4,5]isothiazolo[2,3-a]pyrimidine 2a with a unique modification of benzo[4,5]isothiazolo[2,3-a]pyrimidine deriva-
heterocyclic scaffold also exhibits potent anti-HIV activity tives at the 8-position (2b, 2c, 2d and 2f), which is the equi-
(EC50 = 0.29 0.09 μM) (Fig. 1).11 In our recent studies, we valent position of the 9-position in pyrimido[1,2-c][1,3]-
benzothiazin-6-imine. Similarly, introduction of acetamido
group at the equivalent positions resulted in a complete loss
of the anti-HIV activity of both the PD 404182 derivative (1e)
and isothiazolopyrimidine derivative (2e). Furthermore, a com-
plete loss of anti-HIV activity was observed by the introduction
of the 8-bromo and 7-bromo groups to the parent compounds
(1g and 2g, respectively). These similar structure–activity
relationships implied that the same target molecule(s) or
mechanism(s) of action could possibly be involved in the anti-
Fig. 1 Structures of PD 404182 (1a) and benzo[4,5]isothiazolo[2,3-a]-
HIV activities of PD 404182 and the isothiazolopyrimidine
pyrimidine (2a).
derivatives.
In addition, heterocyclic compounds 1a and 2a both inhib-
aGraduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto
606-8501, Japan. E-mail: soishi@pharm.kyoto-u.ac.jp, nfujii@pharm.kyoto-u.ac.jp;
Fax: +81-75-753-4570; Tel: +81-75-753-4561
bInstitute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan
†Electronic supplementary information (ESI) available. CCDC 1037500. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c5ob00301f
ited early-stage HIV infection including virus attachment and
membrane fusion to host cells as exemplified in comparison
with DS 5000 (adsorption inhibitor)12 and enfuvirtide (fusion
inhibitor).13 Although it was suggested that the antiviral effects
of compound 1a are derived from its virucidal effect against
viral particles,14 its antiviral mechanism of action has not yet
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Table 1 Comparison of the structure–activity relationships of PD 404182 derivatives and benzo[4,5]isothiazolo[2,3-a]pyrimidines
Cmpd
EC50 a,7,8 (μM)
Cmpd
EC50 b,11 (μM)
1a (PD 404182)
R = H
R = Ph
R = m-anisyl
R = Br
R = NHAc
0.44 0.08c
0.24 0.04
0.15 0.05
0.25 0.09c
>10
2a
2b
2c
2d
2e
2f
R = H
R = Ph
R = m-anisyl
R = Br
R = NHAc
0.29 0.09
0.30 0.10
0.10 0.03
0.22 0.07
>10
1b
1c
1d
1e
1f
0.15 0.03
0.16 0.02
1g
>10c
2g
>10d
a EC50 values represent the concentration of the compound required to inhibit the HIV-1 infection by 50%. The data were obtained from three
independent experiments by the MAGI assay. b EC50 values were obtained by the NCK assay. c EC50 values were obtained by the NCK assay,
compound 1a: 0.30 0.06 μM; compound 1d: 0.14 0.02 μM; compound 1g: >10 μM. d Cytotoxicity was observed at 10 μM.
been sufficiently detailed. In the current study, we investigated
the chemical transformations of pyrimido[1,2-c][1,3]benzothia-
zin-6-imine derivatives 1 and benzo[4,5]isothiazolo[2,3-a]pyri-
midine derivatives 2 under the bioassay conditions.
Results and discussion
Synthesis
PD 404182 derivatives 1 and benzo[4,5]isothiazolo[2,3-a]pyri-
midine derivatives
2 were synthesized according to the
protocols in our previous studies (Scheme 1).7,8,11 2-(2-Mercap-
tophenyl)tetrahydropyrimidine derivatives 4 were obtained by
trifluoroacetic acid (TFA)-mediated ethanolysis of the imino
group in pyrimido[1,2-c][1,3]benzothiazin-6-imines 1. The
alternative route via alkaline hydrolysis of pyrimido[1,2-c][1,3]-
benzothiazin-6-thione 3 also generated thiophenol derivatives
4. Some thiophenol derivatives 4 could be isolated as stable
crystals, such as 2-(2-mercaptophenyl)-, 2-(4-bromo-2-mercapto-
phenyl)- or 2-(3-bromo-2-mercaptophenyl)-tetrahydropyrimi-
dine (4a, d, g). Thioanisole derivative 7 was obtained by the
oxidative amidination of the corresponding benzaldehyde
5.15,16 Sulfuric acid-mediated deprotection of N-tosylated tetra-
hydropyrimidine 8, derived from benzaldehyde 6 by the identi-
cal protocol,7 afforded aniline derivative 9.
Scheme 1 Synthesis of benzo[4,5]isothiazolo[2,3-a]pyrimidine and
2-phenyl-1,4,5,6-tetrahydropyrimidine derivatives. Reagents and con-
ditions: (a) TFA, CHCl3–EtOH or EtOH, rt; (b) NaOH, MeOH–H2O, reflux;
(c) PIDA, CHCl3 and/or EtOH, rt; (d) 1,3-propanediamine, I2, K2CO3,
t-BuOH, 70 °C; (e) conc. H2SO4, 100 °C.
Identification of the inactive ingredient by transformation
from PD 404182 in aqueous media
404182 derivatives, we also found that the anti-HIV activity
from the stock solution of 1a in DMSO was often attenuated.
X-ray crystallography of the predominant component from 1a
revealed that the resulting product was 3,4-dihydro-2H,6H-
Recently, Chamoun-Emanuelli et al. reported that the antiviral
activity of PD 404182 (1a) disappeared during storage under
some conditions (for example, in Dulbecco’s phosphate-
buffered saline (pH 7) at 42 °C).10 During our studies on PD
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intracellular environment.17 Interestingly, the imino group of
1a decomposed in 10 mM GSH solution at 37 °C, and was con-
verted into thiophenol 4a within 1 h (Fig. 2 and 3).18 This thio-
phenol derivative 4a exhibited equipotent anti-HIV activity as
the parent compound 1a (EC50 = 0.32 0.06 μM). The mechan-
ism of conversion from 1a into thiophenol 4a could be via the
release of cyanylated glutathione, which was detected by
ESI-MS analysis.19
Our previous structure–activity relationship study of 1a
demonstrated that the 6-imino group and the 7-sulfur atom in
1a were indispensable for the anti-HIV activity (Table 2).7 For
example, substitution of the imino group in compound 1a
with a carbonyl (13) or thiocarbonyl (3a) group resulted in a
significant decrease or loss of activity. Neither pyrimido[1,2-c]-
[1,3]benzoxazines (14a–c) nor pyrimido[1,2-c]quinazolines
(15a–c) exhibited anti-HIV activity (Table 2). In the presence
of GSH, the carbonyl (13) and thiocarbonyl (3a) derivatives
were slowly converted to compound 4a in 26 and 15%
yield, respectively, after 24 h (Table 2, Fig. 3). This is in con-
trast with the rapid and quantitative conversion from 1a
within 1 h, indicating that a more reactive imino group in 1a
was a prerequisite for the efficient generation of 4a with
potent anti-HIV activity. In the absence of GSH, compound 13
was converted into 4a in only 5% yield for 24 h, and 3a did
not produce 4a, indicating that degradation of 13 and 3a
was mainly mediated by GSH.20 Compound 14a was unstable
in the presence or absence of GSH to give a cyclic carbamate
14b and a phenol derivative 16,7,21 which showed no anti-
HIV activity. The carbamate 14b and thiocarbamate 14c
were also degraded into an inactive phenol 16 in 78 and 71%
yield, respectively, in GSH solution over 24 h. The pyrimido-
[1,2-c]quinazolines (15a–c) were stable under identical con-
ditions, and thus, did not produce the possible aniline 9.
In light of these results, the absent or diminished anti-
HIV activity of 3a and 13–15 is likely attributable to decreased
conversion to the potent thiophenol 4a or the poor anti-
HIV activity of the parent compounds and ring-opened
products.
Fig. 2 Transformations of PD 404182 under assay conditions and
under reductive conditions in the presence of GSH at 37 °C. EC50 values
represent the concentration of the compound required to inhibit the
HIV-1 infection by 50%. The data were obtained from three independent
experiments by the NCK assay.
benzo[e]pyrimido[2,1-b][1,3]thiazin-6-one 10 (Fig. 2). An appar-
ent rearrangement of the cyclic amidine substructure in 1a
provided pyrimidothiazinone 10 with no anti-HIV activity. In
contrast, benzo[4,5]isothiazolo[2,3-a]pyrimidine 2a was stable
in acidic and basic solutions, and the assay medium contain-
ing fetal calf serum.
Transformation of PD 404182 and benzo[4,5]isothiazolo[2,3-a]-
pyrimidine derivatives under reductive conditions in the
presence of GSH
We investigated the conversion of PD 404182 (1a) in the pres-
ence of high glutathione (GSH) concentrations that mimic the
Fig. 3 Transformation of compounds 1a (A), 13 (B) and 3a (C) under reductive conditions in the presence of GSH. a The concentrations of com-
pounds were calculated by HPLC analysis using previously determined calibration curves.
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Table 2 Reactions of PD 404182 derivatives under reductive conditions
in the presence of GSH
Fig. 4 Reactions of potent and inactive pyrimido[1,2-c][1,3]benzothia-
zin-6-imine and benzo[4,5]isothiazolo[2,3-a]pyrimidine derivatives
under reductive conditions in the presence of GSH at 37 °C. EC50 values
represent the concentration of the compound required to inhibit the
HIV-1 infection by 50%. The data were obtained from three independent
experiments by the NCK assay.
Cmpd
X
EC50 b,7 (μM)
Conversion (time, yieldc)
PD 404182 (1a)
13
3a
14a
14b
14c
15a
15b
15c
NH
O
S
NH
O
S
NH
O
S
0.44 0.08
8.94 1.07
>10
>10
>10
>10
>10
>10
>10
4a (1 h, quant.)
4a (24 h, 26%)d
4a (24 h, 15%)d
16 + 14be
16 (24 h, 78%)
16 (24 h, 71%)
No reaction
No reaction
No reaction
thiazolopyrimidine derivatives (2a, 2d and 2g) corresponded,
at least in part, to those of the thiophenol derivatives (4a, 4d
and 4g). These results suggest that the tricyclic structures of 1a
and 2a are the prodrug forms, which could be transformed
into 4a under intracellular conditions of high GSH concen-
trations in host cells. Interestingly, inactive analogues with
scaffolds similar to PD 404182 (1a) or compound 2a were not
converted into thiophenol 4a (Fig. 5). For example, compound
10,26 thioanisole derivative 7, benzo[4,5]thiazolo[3,2-a]pyrimi-
dine 11 (the structural isomer of 2a),27 and saccharin-like
derivative 1228 were completely stable under high concen-
trations of GSH for 24 hours. Therefore, the pyrimidoben-
zothiazine scaffold in 1a and the isothiazolopyrimidine
scaffold in 2a structurally satisfied two criteria, which would
make them good prodrugs of 4a.
Taken together, the structure–activity relationships and
antiviral profiles were similar between the PD 404182 deriva-
tives 1 and benzo[4,5]isothiazolo[2,3-a]pyrimidine derivatives
2. Under the reductive conditions corresponding to the intra-
cellular environment, these scaffolds were efficiently converted
to the common ring-opened thiophenol derivatives with
potent anti-HIV activity. In addition, these two scaffolds inhib-
ited not only HIV-1 infection at an early stage, but also
several RNA and DNA viruses as we and others recently
reported.5–8,10,11 In light of these findings, it was suggested
that PD 404182 and benzo-[4,5]isothiazolo[2,3-a]pyrimidine
derivatives may achieve their antiviral activities by acting
a EC50 values represent the concentration of the compound required to
inhibit the HIV-1 infection by 50%. The data were obtained from three
independent experiments by the NCK assay. b EC50 values were
obtained by the MAGI assay. c The product yields were calculated by
HPLC analysis using previously determined calibration curves. d The
reactions were also carried out in the absence of GSH: 5% conversion
from 13 after 24 h; no reaction from 3a. e The accurate product yields
of 14b and 16 by GSH-mediated conversion were not calculated
because of the chemical instability of the substrate 14a.
We next focused on the isothiazolopyrimidine scaffold of 2a
with a characteristic S–N covalent bond, which is contained in
several anti-HIV agents against HIV-1 nucleocapsid protein 7
(NCp7).22–24 We assessed the stability of compound 2a under
identical GSH-containing conditions (Fig. 4). In 10 mM GSH
solution at 37 °C, compound 2a was also immediately con-
verted to a thiophenol derivative 4a with potent anti-HIV
activity (<3 min). The two-fold more potent pyrimidobenzo-
thiazine 1d or isothiazolopyrimidine 2d was also converted
under identical reductive conditions into the corresponding
thiophenol derivative 4d, which has similar anti-HIV activity to
those of 1d and 2d.25 The thiophenol derivative 4g, which was
obtained by conversion from the inactive pyrimidobenzothia-
zine 1g or isothiazolopyrimidine 2g under the GSH-mediated
conditions, exhibited no anti-HIV activity. Thus, the anti-HIV
activities of the PD 404182 derivatives (1a, 1d and 1g) and iso-
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Me4Si as an internal standard. 13C NMR spectra were refer-
enced to the residual solvent signal. Exact mass (HRMS)
spectra were recorded on a JMS-HX/HX 110A mass spectro-
meter or Shimadzu LC-ESI-IT-TOF-MS equipment. For flash
chromatography, Wakogel C-300E (Wako) and CHROMATOREX
NH DM1080 (Fuji Silysia) were employed. For analytical HPLC,
a Cosmosil 5C18-ARII column (4.6 × 250 mm, Nacalai Tesque,
Inc.) was employed with a linear gradient of CH3CN containing
0.1% (v/v) aq. TFA at a flow rate of 1 cm3 min−1, and eluting
products were detected by UV at 254 nm. Preparative HPLC
was performed using a Cosmosil 5C18-ARII preparative column
(20 × 250 mm, Nacalai Tesque, Inc.) with a linear gradient of
CH3CN containing 0.1% (v/v) aq. TFA at a flow rate of 8 cm3
min−1. The purity of the compounds was determined as no
less than 95% by combustion analysis or HPLC analysis. Prepa-
ration of the compounds 1–3, 4d, 6, 8, 13–15 was already
reported from our laboratory.7,8,11,15
2-(2-Mercaptophenyl)-1,4,5,6-tetrahydropyrimidine
(4a).
Method A: TFA (3.70 cm3, 50.0 mmol) was added to a suspen-
sion of PD 404182 (1a) (1.09 g, 5.00 mmol) in CHCl3 (50.0 cm3)
and EtOH (75.0 cm3) dropwise. After being stirred at room
temperature for 1 h, the mixture was quenched with Et3N
(7.20 cm3, 50.0 mmol) at 0 °C and was concentrated. The
crude product was purified by flash chromatography over NH
silica gel with CHCl3–MeOH (10 : 0 to 9 : 1) to give the title
compound 4a as yellow crystals (999 mg, quant.); mp
Fig. 5 Reactions of PD 404182, benzo[4,5]isothiazolo[2,3-a]pyrimidines
and the inactive analogues under reductive conditions in the presence
of GSH at 37 °C. EC50 values represent the concentration of the com-
pound required to inhibit the HIV-1 infection by 50%. The data were
obtained from three independent experiments by the NCK assay.
208–210 °C (from n-hexane–CHCl3–MeOH); IR (neat) νmax
/
through an identical intermediate or via an interaction with a cm−1: 3185–3092 (SH), 1590 (CvN); δH (500 MHz, CD3OD):
similar target molecule(s) or mechanism of action in an intra- 2.05–2.09 (2H, m, CH2), 3.55 (4H, t, J 5.7, CH2), 6.87–6.91 (1H,
cellular compartment. A viral target molecule(s) or mechanism m, Ar), 7.06 (1H, ddd, J1 = J2 8.0, J3 1.7, Ar), 7.28 (1H, dd, J1 7.7,
(s) has not yet been determined,14 but these heterocyclic com- J2 1.4, Ar), 7.56 (1H, dd, J1 8.0, J2 1.1, Ar); δC (125 MHz, DMSO-
pounds could conceivably regulate the defense mechanism(s) d6): 18.1, 38.1 (2C), 118.7, 122.8, 126.7, 128.8, 137.8, 158.9,
of the host cells.
159.8; HRMS (FAB): m/z calcd for C10H13N2S [M + H]+
193.0799; found: 193.0801.
Method B: 3,4-dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothia-
zine-6-thione 3a15 (1.15 g, 4.90 mmol) was suspended in 0.1 N
NaOH in MeOH–H2O (9 : 1, 98.0 cm3). After being stirred
under reflux for 12 h, the mixture was quenched with 1 N HCl
until pH was adjusted to 7. The whole mixture was extracted
with CHCl3–MeOH (95 : 5), and dried over MgSO4. After
concentration, the resulting solid was recrystallized from
Et2O–CHCl3–MeOH to give the title compound 4a as an yellow
solid (655 mg, 70%): spectral data were in good agreement
with those of the compound that was synthesized using
method A.
Conclusions
We have unveiled the chemical transformations of pyrimido-
[1,2-c][1,3]benzothiazin-6-imine and benzo[4,5]isothiazolo-
[2,3-a]pyrimidine scaffolds, which yield compounds with
potent antiviral activities. Under the reductive conditions
meant to mimic intracellular GSH levels, the covalent S–N
bond of the isothiazole substructure in 2a was cleaved to
provide the ring-opened 2-(2-mercaptophenyl)tetrahydropyrimi-
dine 4a with potent anti-HIV activity, which was also generated
from PD 404182 (1a) under identical conditions. The similar
structure–activity relationships and antiviral profiles between
compounds 1a and 2a suggested that these could possibly be
the prodrug forms for the common bioactive substance 4a that
binds with the potential target molecule(s) in host cells.
2-(3-Bromo-2-mercaptophenyl)-1,4,5,6-tetrahydropyrimidine
(4g). By using a procedure similar to that described for the
preparation of the compound 4a from 1a, compound 1g7
(45.0 mg, 0.152 mmol) was converted to the title compound 4g
as a colorless solid (29.6 mg, 72%); mp 260–262 °C (from
n-hexane–CHCl3–MeOH); IR (neat) νmax/cm−1
: 2895–2795
(SH), 1626 (CvN); δH (500 MHz, DMSO-d6): 1.88–1.93 (2H, m,
CH2), 3.44 (4H, t, J 5.7, CH2), 6.58 (1H, dd, J1 = J2 7.7, Ar),
7.29 (1H, d, J 8.0, Ar), 7.53 (1H, d, J 8.0, Ar), 10.85 (2H, br
Experimental section
General
1H NMR spectra were recorded using a JEOL ECA-500 spectro- s); δC (125 MHz, DMSO-d6): 17.8, 38.2 (2C), 117.5,
meter. Chemical shifts are reported in δ (ppm) relative to 126.2, 128.1, 132.8, 133.4, 160.1, 160.7; anal. calcd for
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C10H11BrN2S: C, 44.29; H, 4.09; N, 10.33. Found: C, 44.12; H, (Rint = 0.0220) which were used in all calculations. The final
4.08; N, 10.31.
2-[2-(Methylthio)phenyl]-1,4,5,6-tetrahydropyrimidine
2-(Methylthio)benzaldehyde
wR was 0.0796. The substance was crystallized from toluene.
(7). The CCDC deposition number: 1037500.
5
(90% purity, 0.430 cm3,
3,4-Dihydro-2H-benzo[4,5]thiazolo[3,2-a]pyrimidine
(11).
3.00 mmol) was reacted with propylenediamine (0.300 cm3, Following the reported procedure,27 2-chloro-1,3-benzothiazole
3.60 mmol), K2CO3 (1.24 g, 9.00 mmol) and I2 (952 mg, (0.619 cm3, 5.00 mmol) was converted to the title compound
3.75 mmol). The crude compound was purified by flash 11, as a pale yellow solid (407 mg, 2 steps 73%), by the reaction
chromatography over NH silica with CHCl3–MeOH (10 : 0 to with 3-amino-1-propanol (0.381 cm3, 5.00 mmol). The product
10 : 1) followed by recrystallization from n-hexane–CHCl3 to (80.8 mg) was purified by HPLC to give the compound 11 as a
give the title compound 7 as colorless crystals (294 mg, 48%): colorless solid (72.3 mg, TFA salt): IR (neat) νmax/cm−1
mp 110–112 °C; IR (neat) νmax/cm−1
1621 (CvN); δH 3274–3181 (OH), 1672 (CvO), 1632 (CvN); δH (500 MHz,
:
:
(500 MHz, CDCl3): 1.82–1.87 (2H, m, CH2), 2.43 (3H, s, CH3), CD3OD): 2.31–2.36 (2H, m, CH2), 3.70 (2H, t, J 5.7, CH2), 4.27
3.45 (4H, t, J 5.7, CH2), 4.68 (1H, br s, NH), 7.12 (1H, ddd, J1 = (2H, t, J 6.0, CH2), 7.42–7.45 (1H, m, Ar), 7.57–7.61 (2H, m, Ar),
J2 7.4, J3 1.1, Ar), 7.20 (1H, d, J 6.9, Ar), 7.29 (1H, ddd, J1 = J2 7.84 (1H, d, J 8.0, Ar); δC (125 MHz, CD3OD): 19.2, 41.3, 44.0,
7.6, J3 1.5, Ar), 7.36 (dd, J1 7.4, J2 1.7, 1H, Ar); δC (125 MHz, 113.5, 118.0 (q, J 291.5), 123.1, 124.1, 126.7, 129.2, 140.0, 162.4
CDCl3): 16.0, 20.6, 42.1 (2C), 124.6, 125.5, 128.3, 129.1, 136.7, (q, J 36.0), 166.0; HRMS (FAB): m/z calcd for C10H11N2S
137.0, 154.8; HRMS (ESI): m/z calcd for C11H15N2S [M + H]+ [M + H]+ 191.0643; found: 191.0641.
207.0956; found: 207.0954.
2-(2-Aminophenyl)-1,4,5,6-tetrahydropyrimidine
3,4-Dihydro-2H-benzo[4,5]isothiazolo[2,3-a]pyrimidine 6,6-
(9). A dioxide (12). 50% H2O2 (0.0391 cm3, 0.638 mmol) was added
mixture of 2-[2-N-(p-toluenesulfonylamino)phenyl]-1,4,5,6- to a suspension of compound 2a (48.5 mg, 0.159 mmol, TFA
tetrahydropyrimidine 87 (494 mg, 1.50 mmol) in conc. H2SO4 salt) in TFA (0.610 cm3) dropwise. After being stirred at room
(10.0 cm3) was stirred at 100 °C for 30 min, the mixture temperature for 24 h, the mixture was quenched with Et3N
was cooled to 0 °C, and then pH was adjusted to 12–14 with (1.50 cm3), and the whole mixture was extracted with EtOAc.
2 N NaOH. The whole mixture was extracted with CHCl3, The extract was washed with sat. NaHCO3, brine, and dried
and dried over MgSO4. After concentration, the resulting over MgSO4. After concentration, the residue was purified by
solid was recrystallized from n-hexane–CHCl3 to give the title flash chromatography over aluminum oxide with n-hexane–
compound
9 as colorless crystals (137 mg, 52%): mp EtOAc (3 : 2 to 1 : 1) to give the title compound 12 as colorless
83–85 °C; IR (neat) νmax/cm−1: 2931 (NH2), 2851 (NH2), crystals (25.2 mg, 71%): mp 146–148 °C (from Et2O–MeOH);
1620 (CvN); δH (500 MHz, CDCl3): 1.81–1.86 (2H, m, CH2), IR (neat) νmax/cm−1: 1670 (CvN); δH (500 MHz, CDCl3):
3.48 (4H, t, J 5.7, CH2), 4.71 (1H, br s, NH), 5.72 (2H, br s, NH2), 2.01–2.05 (2H, m, CH2), 3.70 (2H, t, J 5.7, CH2), 3.79 (2H, t,
6.61–6.66 (2H, m, Ar), 7.07–7.10 (1H, m, Ar), 7.23–7.26 (1H, m, J 6.0, CH2), 7.70–7.76 (2H, m, Ar), 7.85–7.88 (1H, m, Ar),
Ar); δC (125 MHz, CDCl3): 20.7, 42.0 (2C), 116.56, 116.61, 119.3, 7.98–8.01 (1H, m, Ar); δC (125 MHz, CDCl3): 19.5, 36.5, 44.2,
126.3, 129.9, 147.1, 155.1; anal. calcd for C10H14N3: C, 68.54; 120.7, 122.8, 129.6, 132.4, 133.6, 134.8, 142.7; anal. calcd for
H, 7.48; N, 23.98. Found: C, 68.28; H, 7.57; N, 23.75.
3,4-Dihydro-2H,6H-benzo[e]pyrimido[2,1-b][1,3]thiazin-6- 4.74; N, 12.36.
one (10). PD 404182 (1a) (20.0 mg, 0.0920 mmol) in DMSO
2-(2-Hydroxyphenyl)-1,4,5,6-tetrahydropyrimidine (16).21
C10H10N2O2S: C, 54.04; H, 4.54; N, 12.60. Found: C, 53.89; H,
A
(0.920 cm3) was dissolved in H2O (92.0 cm3). After being mixture of methyl salicylate (1.29 cm3, 10.0 mmol) and propy-
stirred at 37 °C for 26 h, the mixture was purified by HPLC. lenediamine (2.56 cm3, 30.0 mmol) was refluxed for 16 h. The
The resulting solid was dissolved in EtOAc, and washed with crude product was dissolved in MeOH, and crystallized with
sat. NaHCO3. The extract was dried over MgSO4, and concen- Et2O. The precipitate was filtered, and the unreacted propy-
trated to give the title compound 10 as colorless crystals lenediamine was removed by washing with Et2O. The resulting
(5.2 mg, 27%); mp 132–134 °C (from toluene); IR (neat) νmax
/
solid was purified by flash chromatography over aluminum
cm−1: 1662 (CvO), 1608 (CvN); δH (500 MHz, CDCl3): oxide with CHCl3–MeOH (10 : 0 to 10 : 1) to give the title com-
1.98–2.03 (2H, m, CH2), 3.57 (2H, t, J 5.7, CH2), 4.02 (2H, t, pound 16 as colorless crystals (497 mg, 28%): mp 259–261 °C
J 6.0, CH2), 7.17 (1H, d, J 8.0, Ar), 7.26–7.29 (1H, m, Ar), (from Et2O–MeOH); IR (neat) νmax/cm−1: 3212–3050 (OH), 1620
7.47 (1H, ddd, J1 = J2 7.7, J3 1.6, Ar), 8.24 (1H, dd, J1 8.0, J2 1.1, Ar); (CvN); δH (500 MHz, DMSO-d6): 1.83–1.88 (2H, m, CH2), 3.40
δC (125 MHz, CDCl3): 21.1, 42.3, 46.1, 122.7, 124.0, 126.0, 131.0, (4H, t, J 5.7, CH2), 6.27–6.30 (1H, m, Ar), 6.46 (1H, d, J 8.6, Ar),
133.2, 134.3, 147.0, 161.7; HRMS (FAB): m/z calcd for 7.04–7.08 (1H, m, Ar), 7.45 (1H, dd, J1 8.0, J2 1.7, Ar), 12.10
C11H11N2OS [M + H]+ 219.0592; found: 219.0584. Spectral data (1H, br s, OH); δC (125 MHz, CD3OD): 20.0, 39.3 (2C), 111.2,
were in good agreement with those previously reported.26
114.5, 124.4, 126.3, 135.1, 160.9, 172.5; anal. calcd for
Crystal structure: C11H10N2OS, Mw: 218.27, primitive mono- C10H12N2O: C, 68.16; H, 6.86; N, 15.90. Found: C, 68.13; H,
clinic, a = 10.7456(6), b = 10.9466(6), c = 16.6473(7) Å, β = 7.03; N, 16.09.
97.422(2)°, V = 1941.78(17) Å3, space group P21/n (no. 14), Z =
8. The data were collected with a Rigaku R-AXIS RAPID
Determination of anti-HIV activity
diffractometer using graphite monochromated Mo-Kα radi- The anti-HIV activity of a series of compounds against
ation at −93 K. 18 534 Reflections were measured, 4420 unique HIV-1IIIB was determined by the NCK assay.29 The target
This journal is © The Royal Society of Chemistry 2015
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cells (NCK45-β-Gal; 104 cells per well) were plated in
96-well flat microtiter culture plates. On the following day,
the cells were inoculated with HIV-1 (60 NCK U per well,
giving 60 blue cells after 48 h of incubation) and cultured
in the presence of various concentrations of the test com-
pounds in fresh medium. Forty-eight hours after viral
exposure, all the blue cells stained with X-Gal (5-bromo-
4-chloro-3-indolyl-β-D-galactopyranoside) were counted in each
well. Cytotoxic effects were not observed at 10 μM except for
compound 2g.
3 C. R. Vickery, N. M. Kosa, E. P. Casavant, S. Duan, J. P. Noel
and M. D. Burkart, ACS Chem. Biol., 2014, 9, 1939–1944.
4 Y. T. Ghebremariam, D. A. Erlanson and J. P. Cooke, J. Phar-
macol. Exp. Ther., 2014, 348, 69–76.
5 K. Chockalingam, R. L. Simeon, C. M. Rice and Z. Chen,
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6 A. M. Chamoun, K. Chockalingam, M. Bobardt, R. Simeon,
J. Chang, P. Gallay and Z. Chen, Antimicrob. Agents
Chemother., 2012, 56, 672–681.
7 T. Mizuhara, S. Oishi, H. Ohno, K. Shimura, M. Matsuoka
and N. Fujii, Org. Biomol. Chem., 2012, 10, 6792–6802.
8 T. Mizuhara, S. Oishi, H. Ohno, K. Shimura, M. Matsuoka
and N. Fujii, Bioorg. Med. Chem., 2012, 20, 6434–
6441.
The activity of test compounds was determined as the con-
centration that blocked HIV-1 infection by 50% (50% effective
concentration [EC50]). EC50 was determined by using the fol-
lowing formula:
9 T. Mizuhara, S. Oishi, H. Ohno, K. Shimura, M. Matsuoka
and N. Fujii, Bioorg. Med. Chem., 2013, 21, 2079–2087.
10 A. M. Chamoun-Emanuelli, M. Bobardt, B. Moncla,
M. K. Mankowski, R. G. Ptak, P. Gallay and Z. Chen, Anti-
microb. Agents Chemother., 2014, 58, 687–697.
11 S. Okazaki, T. Mizuhara, K. Shimura, H. Murayama,
H. Ohno, S. Oishi, M. Matsuoka and N. Fujii, Bioorg. Med.
Chem., 2015, 23, 1447–1452.
½logðA=BÞꢀð50ꢁCÞ=ðDꢁCÞþlogðBÞꢂ
EC50 ¼ 10
;
where A, of the two points on the graph that bracket 50% inhi-
bition, the higher concentration of the test compound; B, of
the two points on the graph that bracket 50% inhibition, the
lower concentration of the test compound; C, inhibitory
activity (%) at the concentration B; D, inhibitory activity (%) at
the concentration A.
12 M. Baba, D. Schols, R. Pauwels, H. Nakashima and
E. De Clercq, J. Acquired Immune Defic. Syndr., 1990, 3,
493–499.
13 T. Matthews, M. Salgo, M. Greenberg, J. Chung, R. DeMasi
and D. Bolognesi, Nat. Rev. Drug Discovery, 2004, 3, 215–
225.
14 Of note, it was reported that PD 404182 showed the anti-
viral activities by the virucidal effect via the physical disrup-
tion of virions, see ref. 6 and 10.
15 T. Mizuhara, S. Oishi, N. Fujii and H. Ohno, J. Org. Chem.,
2010, 75, 265–268.
Glutathione-mediated transformation of
pyrimidobenzothiazine and isothiazolopyrimidine derivatives
The compound (1 mM) was incubated in 10 mM GSH (in a
mixture of 50 mM phosphate buffer (pH 7.4) and MeCN
[75 : 25 (v/v) or 1 : 1 (v/v)]) at 37 °C. The sample was analyzed by
HPLC and the peak area was recorded by UV detection at
254 nm. The concentrations of the products were calculated
using previously determined calibration curves. Of note, the
GSH-mediated transformation from 1a was slightly accelerated
by the addition of MeCN in phosphate buffer.
16 M. Ishihara and H. Togo, Tetrahedron, 2007, 63, 1474–
1480.
17 For a review on the function of glutathione, see: R. Franco,
O. J. Schoneveld, A. Pappa and M. I. Panayiotidis, Arch.
Physiol. Biochem., 2007, 113, 234–258 and references cited
therein.
18 Compound 4a was also identified from HTS as an anti-
bacterial agent that inhibits localization of lipoproteins
(Lol) during cell wall biosynthesis, see: H. Ito, A. Ura,
Y. Oyamada, H. Yoshida, J. Yamagishi, S. Narita,
S. Matsuyama and H. Tokuda, Microbiol. Immunol., 2007,
51, 263–270.
Acknowledgements
This work was supported by Grants-in-Aid for Scientific
Research from JSPS, Japan; Platform for Drug Discovery, Infor-
matics, and Structural Life Science from MEXT, Japan; a Grant-
in-Aid for Research on HIV/AIDS from the Ministry of Health
and Welfare of Japan. T.M. is grateful for JSPS Research Fellow-
ships for Young Scientists.
19 During the course of our investigations, an initial report
describing GSH-mediated degradation of PD 404182
appeared, see ref. 4.
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