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11. Spectral data for compounds 2–6, 15, and 16. Compound 2:1H NMR (CD3OD,
400 MHz) d 7.18 (d, J = 8.3 Hz, 1H), 6.70 (s, 1H), 6.60 (s, 1H), 6.46 (dd, J = 8.3,
2.4 Hz, 1H), 6.30 (d, J = 2.4 Hz, 1H), 3.96 (s, 1H), 3.92 (d, J = 11.3 Hz, 1H), 3.68 (d,
J = 11.3 Hz, 1H), 3.01 (d, J = 15.6 Hz, 1H), 2.76 (d, J = 15.6 Hz, 1H); 13C NMR
(CD3OD, 100 MHz) d 157.71, 155.62, 145.55, 145.24, 137.40, 132.22, 131.33,
115.53, 112.89, 112.43, 109.96, 104.24, 78.05, 70.78, 50.95, 42.81. ESI-MS(ꢀ):
Figure 2. The effect of hematoxylin (1) and compound 16 on viral replication.
Terminally differentiated primary macrophages derived from healthy donor C was
infected with HIV-1 and then incubated in serial 10-fold dilutions of compounds
ranging in concentration from
0 to 1 lM. The levels of virus in the culture
285 [MꢀH]; HRMS (ESIꢀ): calcd for
C
16H13O5 (MꢀH+): 285.0763; found:
supernatants were measured at 8 days after inoculation by p24 antigen ELISA. The
data represent the average p24 value from two wells.
285.0755; ½a D25
ꢁ : +84.8 (c = 0.41, CH3OH); (+)ꢀBrazilin (purchased from MP
Biomedicals) ½a 2D5
ꢁ
: +69.5 (c = 0.41, CH3OH). Compound 3: 1H NMR (CD3OD,
400 MHz) d 6.87 (br d, J = 7.6 Hz, 1H), 6.80 (dd, J = 7.6, 7.6 Hz, 1H), 6.74 (s, 1H),
6.67 (d, J = 7.6 Hz, 1H), 6.61 (s, 1H), 4.04 (s, 1H), 4.03 (d, J = 11.2 Hz, 1H), 3.74
(d, J = 11.2 Hz, 1H), 3.03 (d, J = 15.6 Hz, 1H), 2.80 (d, J = 15.6 Hz, 1H); 13C NMR
(CD3OD, 100 MHz) d 146.72, 145.68, 145.25, 142.85, 136.83, 131.33, 125.00,
122.31, 121.87, 114.39, 112.86, 112.51, 78.09, 70.99, 51.51, 42.82; ESI-MS(ꢀ):
285 [MꢀH]ꢀ; HRMS (ESIꢀ): calcd for C16H13O5 (MꢀH+): 285.0763; found:
285.0758. Compound 4: 1H NMR (CD3OD, 400 MHz) d 7.36 (br d, J = 7.8 Hz, 1H),
7.09 (ddd, J = 7.8, 7.5, 1.5 Hz, 1H), 6.95 (ddd, J = 7.5, 7.3, 1.2 Hz, 1H), 6.82 (dd,
J = 8.0, 1.2 Hz, 1H), 6.74 (s, 1H), 6.62 (s, 1H), 4.05 (br s, 1H), 3.96 (dd, J = 11.2,
1.5 Hz, 1H), 3.72 (d, J = 11.2 Hz, 1H), 3.04 (d, J = 15.6 Hz, 1H), 2.79 (d,
showed the comparable high activities in low amounts of virus
infection conditions (donor B and C) to 1, the comparison of the
inhibition of viral p24 antigen production was clearly indicated
that compound 16 is at least twofold more potent anti-viral activ-
ity than 1 (Fig. 2).17
In summary, a novel class of anti-HIV-1 agent based on deoxy-
hematoxylin derivatives was extensively developed. The present
SAR studies suggested that hydroxyl group at C-4-position would
be essential for the high viral replication inhibition activity and
that free hydroxyl groups at C-9- and C-10-positions would in-
crease the cytotoxicity. In addition, catecholic group is not neces-
sary for inhibition of the nuclear import of HIV-1 PICs. The most
potent compound was the C-3-deoxygenated analog 16, which
exhibited most promising biological activities as anti-HIV-1 agent
J = 15.6 Hz, 1H); 13C NMR (CD3OD, 100 MHz)
d 154.93, 145.76, 145.35,
136.87, 131.77, 131.45, 128.49, 124.38, 122.00, 117.92, 112.96, 112.44, 78.00,
70.84, 51.59, 42.90; ESI-MS(ꢀ): 269 [MꢀH]ꢀ; HRMS (ESIꢀ): calcd for C16H13O4
(MꢀH+): 269.0814; found: 269.0809. Compound 5: 1H NMR (CD3CN, 400 MHz)
d 7.43 (dd, J = 8.0, 1.2 Hz, 1H), 7.16–7.12 (m, 2 H), 6.98 (ddd, J = 7.3, 7.3, 1.2 Hz,
1H), 6.93 (br s, 1H), 6.84 (dd, J = 8.8, 1.2 Hz, 1H), 6.65 (br s, 1H), 6.60 (dd, J = 8.3,
2.4 Hz, 1H), 4.08 (s, 1H), 3.95 (dd, J = 11.2, 1.2 Hz, 1H), 3.75 (d, J = 11.2 Hz, 1H),
3.62 (br s, 1H), 3.06 (d, J = 16.3 Hz, 1H), 2.92 (d, J = 16.3 Hz, 1H); 13C NMR
(CD3CN, 100 MHz) d 157.17, 154.37, 142.39, 136.58, 131.64, 128.38, 125.93,
124.31, 121.91, 117.54, 114.43, 112.83, 77.77, 70.45, 50.98, 42.78; ESI-MS(ꢀ):
253 [MꢀH]ꢀ; HRMS (ESIꢀ): calcd for C16H13O3 (MꢀH+): 253.0865; found:
253.0861. Compound 6: 1H NMR (CDCl3, 400 MHz) d 7.45 (br d, J = 7.3 Hz, 1H),
7.34–7.30 (m, 1H), 7.22–7.15 (m, 4 H), 7.05 (dd, J = 7.3, 1.2 Hz, 1H), 6.93 (dd,
J = 8.3, 1.2 Hz, 1H), 4.25 (br s, 1H), 4.05 (dd, J = 11.2, 1.7 Hz, 1H), 3.81 (d,
J = 11.2 Hz, 1H), 3.34 (d, J = 16.1 Hz, 1H), 2.97 (d, J = 16.1 Hz, 1H), 2.49 (s, 1H);
13C NMR (CD3OD, 100 MHz) d 154.98, 145.87, 141.01, 131.91, 128.68, 128.30,
127.88, 126.15, 125.44, 123.94, 122.15, 118.01, 77.88, 70.68, 52.15, 43.18; EI-
MS(ꢀ): m/z 238 [M+], 219; HRMS (EI+): calcd for C16H14O2 (M+): 238.0994;
found: 238.0999. Compound 15: 1H NMR (CDCl3, 400 MHz) d 7.43–7.40 (m, 2
H, aromatic), 7.36–7.32 (m, 2 H, aromatic), 7.29–7.25 (m, 1H, aromatic), 6.95
(dd, J = 7.8, 1.2 Hz, 1H, aromatic), 6.89 (dd, J = 7.8, 7.8 Hz, 1H, aromatic), 6.77
(dd, J = 7.8, 1.2 Hz, 1H, aromatic), 6.70 (s, 1H, aromatic), 6.55 (s, 1H, aromatic),
5.10 (s, 2 H, CH2Ph), 4.11 (d, J = 11 Hz 1H), 4.09 (s, 1H), 3.88 (d, J = 11 Hz 1H),
3.17 (d, J = 16 Hz 1H), 2.83 (d, J = 16 Hz 1H), 2.73 (br s, 1H), 1.64 (s, 3 H), 1.58 (s,
3H). 13C NMR (CDCl3, 100 MHz) d 147.72, 146.72, 146.47, 143.49, 136.98,
136.02, 130.93, 128.45, 127.76, 127.20, 123.34, 122.80, 120.96, 117.74, 112.11,
105.50, 104.94, 70.94, 70.32, 50.70, 41.21, 25.69; EI-MS: m/z 416 [M+], 325;
HRMS (ESI+): calcd for C26H24Na1O5 (M+Na+): 439.1521; found: 439.1538.
Compound 16: 1H NMR (CDCl3, 400 MHz) d 6.95–6.89 (m, 2 H, aromatic), 6.82
(br dd, J = 2.7, 6.8 Hz, 1H, aromatic), 6.71 (s, 1H), 6.56 (s, 1H, aromatic), 4.12–
4.08 (m, 2 H), 3.88 (d, J = 11 Hz, 1H), 3.19 (d, J = 16 Hz, 1H), 2.84 (d, J = 16 Hz,
1H), 2.53 (s, 1H), 1.65 (s, 3 H), 1.59 (s, 3H); 13C NMR (CDCl3, 100 MHz) d 146.87,
146.64, 145.02, 140.48, 135.75, 130.66, 122.52, 121.79, 121.46, 117.90, 113.14,
105.49, 150.02, 77.68, 70.49, 50.56, 41.37, 25.74; ESI-MS(ꢀ): 325 [MꢀH]ꢀ;
HRMS (ESIꢀ): calcd for C19H17O5 (MꢀH+): 325.1076; found: 325.1081.
such as lower cytotoxicity (16:1; >80:40
of nuclear import (0.5:1.3 M), and viral replication in HIV-1-in-
fected TZM-bl cells (24.6:100 M), human peripheral blood mono-
nuclear cells (PMBCs) (30.1 M:toxic). In addition, different spectra
lM), stronger inhibition
l
l
l
of inhibitory activities against infected three healthy humans mac-
rophages with high (donor A) and low (donor B and C) amounts of
virus were also observed. In the case of A, 16 showed 10-times
stronger activity than 1 (16:1; 0.1:<1.0
16 and 1 showed comparable activities in the cases of B and C
(>0.01 and >0.001 M). However, the comparison of the inhibition
lM). On the other hand,
l
of viral p24 antigen production was clearly indicated that com-
pound 16 is at least twofold more potent anti-viral activity than
1. Thus, the present SAR studies could provide valuable informa-
tion for the elaboration of novel class of anti-HIV-1 agents.
Acknowledgments
This study was supported by the program for Promotion of Fun-
damental Studies in Health Sciences of the National Institute of
Biomedical Innovation (NIBIO) of Japan and by a Health Sciences
Research Grant from the Ministry of Health, Labor and Welfare of
Japan (Research on HIV/AIDS).
12. MTT cell viability assay: Molt-4 cells were cultured in RPMI1640
supplemented with 10% FCS, penicillin and streptomycin. Molt-4 cells
(1 ꢂ 105 cells/well) were cultured in 24 well plate at 37 °C for 2 days in the
RPMI1640 containing serially diluted compounds ranging from 0 to 10
lM.
References and notes
MTT analysis was performed using
a standard method as described
previously.18
13. In vitro transport assay: The basic assay for nuclear import assay was
performed as described elsewhere.19 HeLa cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-
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inactivated FCS, penicillin (100 U/ml) and streptomycin (100 lg/ml). Cells
were plated onto glass coverslips 24 h before the assay and grown to 80%
confluence. Cells were washed once in phosphate-buffered saline (PBS) and
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triphosphates. Nuclear import reaction was performed for 15 min at 28 °C.
HeLa Cells were then washed three times with TB and fixed with 4%
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