Preparation and anti-HIV activities of retrojusticidin B analogs and azalignans

Article abstract of DOI:10.1016/j.bmc.2004.05.036

Ten lignans (2-11) and a series of azalignans including 1-aryl-pyrronaphthalenes 20-24 and 3-N-alkylaminomethyl-1-arylnaphthalenes 25-28, structurally related to two HIV reverse transcriptase inhibitors, retrojusticidin B and phyllamyricin A, were prepare

Full text of DOI:10.1016/j.bmc.2004.05.036

Bioorganic & Medicinal Chemistry 12 (2004) 4045–4054
Preparation and anti-HIV activities of retrojusticidin B analogs
and azalignans
Kadali S. Sagar,a Chia-Chuan Chang,a Wei-Kung Wang,b Jung-Yaw Linc and
Shoei-Sheng Leea,*
aSchool of Pharmacy, College of Medicine, National Taiwan University, Taipei 100, Taiwan, ROC
bInstitute of Microbiology, College of Medicine, National Taiwan University, Taipei 100, Taiwan, ROC
cInstitute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei 100, Taiwan, ROC
Received 30 April 2004; revised 27 May 2004; accepted 27 May 2004
Available online 19 June 2004
Abstract—Ten lignans (2–11) and a series of azalignans including 1-aryl-pyrronaphthalenes 20–24 and 3-N-alkylaminomethyl-1-
arylnaphthalenes 25–28, structurally related to two HIV reverse transcriptase inhibitors, retrojusticidin B and phyllamyricin A, were
prepared from phyllanthin (1) for evaluation of anti-HIV activities. Anti-HIV activity of these compounds on a R5 pseudotype
virus, ConB/pNL43E-L+, in the U87-CD4-CCR5 cells has been measured. Compounds 5, 22, 23, and 28 showed good anti-HIV
activity with IC50 value of 0.25, 1.07, 0.01, 0.32 lg/mL, respectively.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
preparation of anti-HIV drugs.7 Many synthetic entries
to the arylnaphthalene type of lignan have been devel-
oped.8–10 Of these, oxidative aryl–benzyl coupling reac-
tion of the diarylbutane lignan, phyllanthin (1), using
DDQ under ultrasonication, has been particularly useful
and leads to an arylnaphthalene skeleton in one step.11
Since our prior study had revealed 1 to be the most
abundant constituent (6.63%) present in the alcoholic
extract of Phyllanthus urinaria,12 we utilized it as starting
material to construct the arylnaphthalene skeleton. By
using appropriate chemical reactions, a series of retro-
justicidin B and phyllamiricin B related compounds and
azalignans were prepared. The anti-HIV activity of the
prepared compounds was evaluated. The following de-
scribe the outcome of this effort.
Reverse transcriptase (RT) is essential for the replication
of human immunodeficiency virus (HIV), thus many
attempts have been made to develop RT inhibitors for
the treatment of AIDS.1 Lignans, found in the roots,
stems, bark, fruit, and seeds of many plant species, have
been demonstrated to possess activities against cancers,
viruses, and bio-oxidation.2;3 Of these, lignans of aryl-
tetrahydronaphthalene4 type exhibit antiviral activity
against HIV by inhibiting RT; those of the dibenzyl
butanolide type inhibit both HIV protein production
and reverse transcriptase.3 In addition, two aryl naph-
thalene lignans, phyllamiricin B and retrojusticidin B
isolated from Phyllanthus myrtifolius (Euphorbiaceae),
possess strong inhibition on HIV-1 reverse transcriptase
(HIV-1 RT) (IC50 3.5 and 5.5 lM, respectively) and are
relatively inactive toward human DNA polymerase-a
(hDNAP) (IC50 289 and 989 lM, respectively).5–7 This
potent and selective property and a computer-aided
molecular modeling study of these two molecules sug-
gested that they would be good lead compounds for the
2. Results and discussions
Phyllanthin (1) was oxidized by DDQ under ultrasonic
condition11a to give the aryl naphthaldehyde 2 (92%),
dC-9 192.4 (d) and dH-9 10.38 (s) whose structure has been
described previously in the literature.11 Oxidation of the
aldehyde 2 by sodium chlorite in the presence of sodium
phosphate and 2-methyl-butene13 produced the acid 3
(85%), dC-9 170.9 (s). O-Demethylation of the 90-meth-
oxy group by treating with trifluoroacetic acid at
room temperature yielded the primary alcohol, which
Keywords: Retrojusticidin B analogs; Azalignans; Semisynthesis; Anti-
human immunodeficiency virus.
* Corresponding author. Tel./fax: +886-2-2321-1127; e-mail: shoeilee@
ha.mc.ntu.edu.tw
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.05.036

4046
K. S. Sagar et al. / Bioorg. Med. Chem. 12 (2004) 4045–4054
spontaneously lactonized to give the lactone 4 (98%),14
(2 · aryl-CH3) 2.43 (s) and 2.10 (s) and d (2 · aryl-CH3)
20.9 (q) and 17.3 (q). O-Perdemethylation of 7 under
similar conditions to those used for 4 yielded the catechol
0
dH-9 5.26 and 5.17 (each doublet, J ¼ 15:0 Hz), and dC-9
0
171.6 (s), dC-9 69.6 (t) whose structure has been previ-
ously reported.10;15;19 The lactone moiety of 4, similar to
that found in retrojusticidin B was thus accomplished in
two steps. O-Perdemethylation of 4 with melted pyrid-
inium hydrochloride (190 ꢁC)16 afforded the catechol 5
(79%),17 which had been reported as an intermediate but
none of its physical data had been listed. To compensate
this, the physical data were measured and listed in the
Experimental section. Treating 4 with the common
O-demethylating agent BCl3,16 however, gave 5 in a
poor yield. O,O-Methylenation of the catechol groups in
8 (57%), whose H NMR spectrum (methanol-d4) re-
1
vealed the absence of the four methoxy singlets. O,O-
Methylenation of 8 by dichloromethane in DMF pro-
duced the known compound 9 (55%),22 d (2 · –OCH2O–)
6.02 and 5.93, and d(2 · –OCH2O–) 101.0 (t) and 100.8 (t).
For comparison of biologic activity, another two com-
pounds, 10 and 11, were prepared (Scheme 1). Reduc-
tion of the aldehydic group in
2 with sodium
borohydride produced the alcohol 10 (75% yield), dH-9
4.79 and 4.74, each doublet (J ¼ 12:2 Hz); dC-9 70.7 (t).
Treatment of 1 with POCl3 in dry benzene and trifluo-
roacetic acid under reflux yielded the tetrahydrofuran 11
5 to give the known compound justicidin E (6),18–20
d
(–OCH2O–) 6.07 (2H, an AB system) and 6.06 (2H, an
AB system), d (–OCH2O–) 101.8 (t) and 101.4 (t), was
achieved by treating with dichloromethane in DMF
(67%) (Scheme 1).
(45%),11a dH-9=H-9 3.89 (2H, dd, J ¼ 8:6, 6.7 Hz) and 3.51
0
0
(2H, dd, J ¼ 8:6, 6.0 Hz); dC-9=C-9 73.3 (t).
In order to confirm that the c-lactone moiety on com-
pound 4 was an essential pharmacophore, the corre-
sponding compounds 7–9, devoid of this moiety, were
prepared (Scheme 1). Catalytic hydrogenation of 2 yiel-
ded the dimethyl naphthalene 7 (92%), which has been
Bioassay of these prepared lignans 2–11 against HIV-1
RT was performed and it was found that 4,5,30,40-tet-
rahydroxy-2,70-cycloligna-7,70-dien-9,90-olide (5) showed
significant activity with an IC50 of 35 lM. When the
catechol functions were protected, lower anti-HIV RT
activity was observed [4 (tetra-O-methylated), IC50
reported many times previously in the literature,21
d
O
MeO
MeO
CO2H
MeO
MeO
MeO
MeO
O
O
c
CH2OMe
OMe
OMe
OMe
OMe
OMe
OMe
4
d
3
11
g
b
O
O
9
7
MeO
MeO
5
4
1
CHO
HO
HO
MeO
MeO
CH2OMe
CH2OMe
8
CH2OMe
7'
a
9'
3'
OMe
OMe
OH
4'
OMe
OMe
OH
1
2
5
h
10 8-CH2OH
f
e
O
MeO
MeO
Me
Me
R1O
R2O
Me
Me
9
O
O
O
d
9'
OMe
OR3
OR4
O
OMe
O
8 R1~R4= H
9 R1+R2=R3+R4= CH2
6
7
e
Scheme 1. Preparation of compounds 2–11 from phyllanthin (1). Reagents and conditions: (a) DDQ, HOAc, ultrasonic wave, 50 ꢁC, 40 min (92%);
(b) 2-methyl-butene, t-BuOH, NaClO2, NaH2PO4, rt, 18 h (85%); (c) TFA, MeCN, rt, 24 h (98%); (d) pyridinium HCl, N2, 190 ꢁC, 30 min (5: 79%; 8:
57%); (e) CH2Cl2, DMF, 110 ꢁC, 5 h (6: 67%; 9: 55%); (f) H2, 10% Pd–C, HOAc, rt, 18 h (92%); (g) POCl3, C6H6, TFA, 0 ꢁC, 6 h (45%); (h) NaBH4,
CH2Cl2, MeOH, rt, 10 h (75%).

K. S. Sagar et al. / Bioorg. Med. Chem. 12 (2004) 4045–4054
4047
226 lM and 6 (dimethylenated), IC50 609 lM]. The
remaining compounds in the series were almost inactive
(IC50 >1000 lM). These data suggest that the 9,90-c-
lactone in this series is essential for anti-HIV activity,
consistent with conclusions from molecular modeling
studies.7 Since the c-lactone moiety would be hydro-
lyzed easily in vivo and the catechol functionality is not
stable, modification of these moieties is essential from
the viewpoint of drug development. Hence a series of
azalignans possessing a pyrrole moiety, corresponding
to the c-lactone, and a series of 9-N-alkylamino deriv-
atives of 2, were prepared (Scheme 2) in order to avoid
the pharmacokinetic issue and to study the structure–
activity relationship.
H
H
7.30 8.27
10.20
NH-CH2-CH2-CH3
3a
4
5
MeO
4.04
N+-CH2-CH2-CH3
Br-
2
2a
3.78
MeO
4.56d
8
4.45d (18.0)
H
6.96
N+Pr
6.82
1'
NHPr
H
H
Jo 8.2
Jm 1.8
4.03m
6.90
4.02 m
1.78m
3'
2.00 m
7.03
1.03t (6.6)
H
1.19 t (6.7)
OMe
3.85
4'
3.95
OMe
Figure 1. 1H NMR data, NOESY (solid curves) and major COSY-45
(dash lines) of 16 (CDCl3).
Treatment of 2 with dry HBr in CH2Cl2 yielded the 90-
0
bromo derivative 12 (89%), dH-9 4.97 (1H, d) and 4.73
(1H, d), and dH-7 8.17 (s). Treatment of 2 with dry HBr
and bromine in CH2Cl2 yielded the 6,90-dibromolignan
13 (46%), dH-7 8.68 (s), and the 60,90-dibromolignan 14
coupling between the C-3a singlet (d 160.0) and the H-4
singlet (HMBC) (Fig. 2), confirming the assigned
structure for 16. With the assistance of these 2D NMR
data, the complete assignment of 1H and 13C NMR data
of 16 was made unambiguously and they are given in
Figure 1 and listed in the Experimental section, respec-
tively. The HR-EI-MS spectra of 15–19 gave the ex-
pected molecular ions as listed in the Experimental
Section, and support the structural assignments.
0
0
(7.6%), dH-2 6.92 (s) and dH-5 7.28 (s), in addition to 12
(8.9%). Reaction of 12 with alkylamines, including
methylamine, propylamine, isopropylamine, and isobu-
tylamine, yielded N-alkyl 3a-alkylamino-5H-pyrro[4,3-
b]-naphthalenes 15 (51%), 16 (41%), 17 (46%), and 18
(58%). The same reaction of 13 and methylamine yielded
19 (65%). These assigned structures were supported by
the following common spectral evidences: IR absorption
The formation of these diamino adducts could be
attributed to the mechanism shown in Scheme 3. The
secondary amine formed by the reaction between the
amine and the bromomethyl group, could react with
the aldehyde to form an aminol that is air-oxidized to
the lactam. Tautomerization of the lactam, followed by
nucleophilic attack of alkylamine and subsequent
dehydration would yield the products 15–19.
1
near 1649 cmꢀ1 typical for C@N stretching, H NMR
signal for the 3a-NH near 10 ppm (br s) and 13C NMR
signal for a quaternary carbon near 160 ppm (C-3a). The
2D NMR spectra of 16 showed the vicinal coupling
relationship of the protons in 3a-NHnPr (COSY-45), an
NOE relationship between H-4 (d 8.27) and 3a-
NHCH2– (d 4.02) (NOESY) (Fig. 1), and the three-bond
R1
9
9
6
MeO
MeO
CHO
Br
MeO
MeO
CHO
MeO
MeO
5
CHO
1
a
OMe
Br
+
3
9'
R2
6'
OMe
OMe
OMe
NHR
OMe
OMe
OMe
12
b
2
13 R1= Br, R2= H
14 R1= H, R2= Br
d
9
5
4
3a
MeO
MeO
4a
3
2
MeO
MeO
MeO
6
7
NHR
OMe
N+R
Br-
NR
c
8a
2a
MeO
1
8
1'
3'
OMe
OMe
OMe
4'
OMe
OMe
OMe
20 R= CH3 ( 74%)
21 R= nPr ( 66%)
22 R= iPr ( 74%)
23 R= iBu ( 72%)
15 R= CH3, ( 51%)
16 R= nPr ( 41%)
17 R= iPr ( 46%)
18 R= iBu ( 58%)
25 R= CH3 ( 99%)
26 R= nPr ( 98%)
27 R= iPr ( 98%)
28 R= iBu ( 98%)
24 R= CH3, 2- Br ( 75%) (from 19)
19 R= CH3, 2-Br ( 65%) (from 13)
Scheme 2. Preparation of azalignans 20–28 from the arylnaphthaldehyde 2. Reagents and conditions: (a) HBrg (12) or HBrg/Br2 (12–14); (b) RNH2,
CH2Cl2; (c) NaBH4, MeOH; (d) RNH2, NaBH4, MeOH.

4048
K. S. Sagar et al. / Bioorg. Med. Chem. 12 (2004) 4045–4054
Compounds 2–11 and 20–28 were tested for anti-HIV
H
H
H
activity on a R5 pseudotype virus, ConB/pNL43E-L+,
in the U87-CD4-CCR5 cells. The IC50 and IC90 values
were determined by using a virus infectivity assay and
the CC50 and CC90 of these compounds were determined
for comparison. The results (Table 1) indicate that
compounds 5, 22, 23, and 28 have good activity against
HIV. It was noted that in the 9,90-c-lactone series (4–6),
the dicatechol 5 is much more active than the tetra-
methoxy (4) and the dimethylenedioxy (6) derivatives,
and the N-isobutyl azalignans 23 and 28 were the most
active among the series of compounds 20–24, 25–28.
Most strikingly the N-isobutyl-pyrro[4,3-b]naphthalene
23 possessed both best potency (IC50 0.01 lg/mL) and
therapeutic index (CC50/IC50 ¼ 60,000). It could serve as
a lead for the development of anti-HIV drug.
NH-CH2-CH2-CH3
N+-CH2-CH2-CH3
MeO
MeO
H
H
H
OMe
OMe
Figure 2. HMBC correlation in compound 16 (CDCl3).
Sodium borohydride reduction of 15–19, respectively,
yielded N-alkyl-4-aryl-2,5-H-pyrro[4,3-b]naphthalenes
20 (74%), 21 (66%), 22 (74%), 23 (72%), and 24 (75%)
(Scheme 2). The structures of compounds 21–24 were
1
verified by H NMR data, for instance in 20 dH-3a 3.84
3. Experimental
(s) and dH-2a 4.05 (1H) and 4.08 (1H), an AB system,
J ¼ 12:5 Hz, and 13C NMR data dC-2a 61.07 (t) and dC-3a
60.02 (t). Reductive N-alkylation of 2 with methylamine,
propylamine, isopropylamine, and isobutylamine by
sodium borohydride yielded 9-N-alkylaminolignans 25–
28 (Scheme 2) quantitatively. Compounds 25–28 showed
positive color reaction to the Dragendorff’s reagent and
their structures were verified by HR-EI-MS and 1H
3.1. General experimental conditions
The physical data of the prepared compounds were
obtained from the following instruments: Melting
points: Fisher–Johns melting apparatus and not cor-
rected; IR (KBr): JASCO FT/IR-410 spectrometer; UV
(MeOH): Hitachi U-2000 spectrophotometer; NMR:
Bruker Avance 400 MHz in deuterated solvent, using the
residual solvent peak as internal standard (CDCl3: dH
7.24 and dC 77.0; methanol-d4: dH 3.30, dC 49.0; acetone-
d6: dH 2.04 and dC 29.8). Mass: Finnigan MAT 95S (EI,
0
NMR data, for instance in 25 dH-9 4.03 (s), dH-9 4.29
(1H) and 4.34 (1H), an AB system, J ¼ 10:2 Hz, and
dNHMe 2.25 (s), and 13C NMR data dC-9 58.3 (t), dC-9 70.3
0
(t), and dNHMe 34.5 (q).
Scheme 3. Proposed mechanism for the formation of compounds 15–19 from 12 reacting with amines.
Table 1. Anti-HIV activity (IC)a and cytotoxicity (CC) of tested compounds (lg/mL)a
Compound
IC50 (lg/mL)
CC50 (lg/mL)
CC50/IC50
IC90 (lg/mL)
CC90 (lg/mL)
CC90/IC90
5
0.25
1.07
0.01
0.32
74.8
63.5
60.0
12.8
299.2
59.3
17.6
15.43
2.76
4.38
134.5
114.3
106.4
79.9
7.6
7.4
22
23
28
6000
40.0
38.6
18.2
a AZT, the positive control, showed IC100 0.2 lg/mL.

K. S. Sagar et al. / Bioorg. Med. Chem. 12 (2004) 4045–4054
4049
70 eV). Chromatographic system: 230–400 mesh silica
gel for column; silica gel TLC: solvent A EtOAc–
petroleum ether (30–70 ꢁC) (6:4), solvent B EtOAc–
petroleum ether (4:6).
3.5. 4,5,30,40-Tetrahydroxy-2,70-cycloligna-7,70-dien-9,90-
olide (5)
A mixture of 4 (10 mg, 0.03 mmol) and pyridinium HCl
(300 mg, 2.6 mmol) was stirred under N2 at 190 ꢁC for
30 min and cooled. The mixture was partitioned between
satd NaHCO3 (10 mL) and EtOAc (10 mL · 2). The
combined organic layers were washed with brine, dried
by Na2SO4, and concentrated to give 5 (6.7 mg, 79%):
white amorphous solid; Rf 0.40 [MeOH–CHCl3 (2:8)];
UV kmax (log e) 204 (4.40), 223 (4.33), 257 (4.45), 322
3.2. 4,5,30,40,90-Pentamethoxy-9-oxo-2,70-cycloligna-7,70-
diene (2)
A mixture of phyllanthin (1, 2.9 g, 6.9 mmol), DDQ
(4.92 g, 21.7 mmol), and acetic acid (glacial, 300 mL) was
placed in an ultrasonic bath (37 3 kHz) at 50 ꢁC for
40 min and filtered. The filtrate was evaporated under
vacuum and the residue (3.4 g) was recrystallized from
hexane–EtOAc (9:1) to give 2 as colorless needles (2.6 g,
92%). The spectral and other properties were identical to
those reported previously.11
1
(3.86) nm; H NMR (acetone-d6) d 8.14 (1H, s, H-7),
7.48 (1H, s, H-6), 7.22 (1H, s, H-3), 7.00 (1H, d,
J ¼ 8:0 Hz, H-50), 6.90 (1H, d, J ¼ 2:0 Hz, H-20), 6.76
(1H, dd, J ¼ 8:0, 2.0 Hz, H-60), 5.23 (1H, d, J ¼ 8:1 Hz,
H-90), 5.20 (1H, d, J ¼ 8:0 Hz, H-90); 13C NMR (ace-
tone-d6) d 171.9 (s, C-9), 150.0 (s, C-4), 147.9 (s, C-30),
146.2 (s, C-5), 145.9 (s, C-40), 137.6 (s, C-70), 132.7 (s, C-
8), 132.5 (s, C-10), 130.8 (s, C-80), 128.7 (s, C-1), 123.6 (d,
C-7), 121.6 (d, C-60), 121.4 (s, C-2), 117.1 (d, C-20), 116.4
(d, C-50), 111.9 (d, C-3), 108.5 (d, C-6), 69.9 (t, C-90).
3.3. 4,5,30,40,90-Pentamethoxy-2,70-cycloligna-7,70-dien-9-
oic acid (3)
To a mixture of 2 (400.1 mg, 1.0 mmol) and 2-methyl-
butene (0.78 mL) in t-BuOH (16 mL) was added aqueous
solution (7.9 mL) of NaClO2 (788.0 mg, 8.7 mmol) and
NaH2PO4 (995.8 mg, 8.3 mmol). The resultant suspen-
sion was stirred overnight at rt and then concentrated.
The residue was suspended in H2O (20 mL) and the
suspension was adjusted to pH 1 by adding concd
H2SO4 dropwise, and was extracted with CHCl3
(20 mL · 2). The CHCl3 layers were concentrated to give
a residue, which was passed through a silica gel column
(230–400 mesh, 2 g, solvent A) to give 3 (354.0 mg, 85%):
white amorphous solid; Rf 0.12 (solvent A); UV kmax
3.6. 4,5:30,40-Bis(methylenedioxy)-2,70-cycloligna-7,70-
dien-9,90-olide (6) (justicidin E)
A mixture of 5 (10 mg, 0.03 mmol), CH2Cl2 (0.2 mL,
3.12 mmol), DMF (0.2 mL), and K2CO3 (28 mg) was
stirred in a sealed tube at 110 ꢁC for 5 h, cooled, and
concentrated. The residue was, partitioned between H2O
(5 mL) and CHCl3 (5 mL · 3). The combined CHCl3
layers were washed by brine, dried by Na2SO4, and
concentrated to give 6 (7.2 mg, 67%) as white amor-
phous solid. The spectral and other properties were
identical to those reported previously.10;19
1
(log e) 204 (4.50), 250 (4.67), 286 (3.97) nm; H NMR
(CDCl3) d 8.39 (1H, s, H-7), 7.21 (1H, s, H-6), 7.01 (1H,
d, J ¼ 7:8 Hz, H-50), 6.86 (1H, br d, J ¼ 7:8 Hz, H-60),
6.84 (1H, br s, H-20), 6.76 (1H, s, H-3), 4.52 (1H, d,
J ¼ 10:6 Hz, H-90), 4.45 (1H, d, J ¼ 10:6 Hz, H-90), 4.00
(3H, s, 5-OMe), 3.99 (3H, s, 40-OMe), 3.85 (3H, s, 30-
OMe), 3.72 (3H, s, 4-OMe), 3.33 (3H, s, 90-OMe); 13C
NMR (CDCl3) d 170.9 (s, C-9), 151.2 (s, C-4), 150.2 (s,
C-5), 148.7 (s, C-40), 148.5 (s, C-30), 140.0 (s, C-70), 131.0
(s, C-8), 130.6 (d, C-7), 130.6 (s, C-10), 128.3 (s, C-1),
128.3 (s, C-80), 127.6 (s, C-2), 122.6 (d, C-60), 113.4 (d, C-
20), 110.9 (d, C-50), 107.1 (d, C-3), 105.9 (d, C-6), 70.2 (t,
C-90), 58.1 (q, 90-OMe), 56.0 (q, 5-OMe), 56.0 (q, 30-
OMe), 55.7 (q, 4-OMe), 55.7 (q, 40-OMe); EIMS m=z
[M]þ 412 (53), 380 (76), 379 (100), 349 (17), 335 (7), 305
(10), 181 (9); HREIMS m=z 412.1534 (calcd for
C23H24O7, 412.1517).
3.7. 4,5,30,40-Tetramethoxy-2,70-cycloligna-7,70-diene (7)
A mixture of 2 (1.0 g, 2.52 mmol), EtOAc (200 mL), 10%
Pd–C (697 mg), and acetic acid (20 mL) was stirred un-
der H2 at rt for 18 h, filtered through a layer of Celite
pad, and concentrated. The residue was suspended in
H2O and the suspension was adjusted to pH 7 by adding
NH4OHaq dropwise, and was extracted with CHCl3
(30 mL). The CHCl3 layer was washed with brine, dried
over Na2SO4, and concentrated to give 7 (826.5 mg,
92%) as an off-white amorphous solid. The spectral and
other properties were identical to those reported previ-
ously.21
3.8. 4,5,30,40-Tetrahydroxy-2,70-cycloligna-7,70-diene (8)
3.4. 4,5,30,40-Tetramethoxy-2,70-cycloligna-7,70-dien-9,90-
olide (4)
Under similar reaction conditions and workup proce-
dure for the preparation of 5, 8 (9.6 mg, 57%) was ob-
tained from 7 (20 mg, 0.06 mmol) and pyridinium HCl
(350 mg, 3.0 mmol). Compound 8: white amorphous
solid; mp 244–245 ꢁC; Rf 0.24 (solvent A); 1H NMR
(methanol-d4) d 7.26 (1H, s, H-7), 6.92 (1H, s, H-6), 6.83
(1H, d, J ¼ 8:0 Hz, H-50), 6.61 (1H, s, H-3), 6.55 (1H, d,
J ¼ 1:8 Hz, H-20), 6.43 (1H, dd, J ¼ 8:0, 1.8 Hz, H-60),
2.32 (3H, s, H-9), 2.01 (3H, s, H-90); 13C NMR
To a mixture of 3 (148.7 mg, 0.36 mmol) and MeCN
(3 mL) was added TFA (130 lL) drop by drop. The
reaction mixture was stirred at rt for 24 h, concentrated,
and passed through a silica gel column (230–400 mesh,
solvent A) to give 4 (135.0 mg, 98%) as white amorphous
solid. The spectral and other properties were identical to
those reported previously.10;19

4050
K. S. Sagar et al. / Bioorg. Med. Chem. 12 (2004) 4045–4054
(methanol-d4) d 146.4 (s, C-4), 146.4 (s, C-5), 146.3 (s, C-
40), 145.1 (s, C-30), 138.2 (s, C-70), 134.2 (s, C-8), 133.4 (s,
C-10), 131.1 (s, C-80), 129.3 (s, C-1), 129.3 (s, C-2), 126.1
(d, C-7), 122.7 (d, C-60), 118.5 (d, C-20), 116.3 (d, C-50),
110.1 (d, C-3), 109.9 (d, C-6), 21.1 (q, C-9), 17.5 (q, C-
90); EIMS m=z [M]þ 296 (13), 196 (8), 153 (14), 125 (16),
111 (32), 99 (28), 85 (62), 71 (100), 57 (68); HREIMS
m=z [M]þÅ 296.3317 (calcd for C18H16O4, 296.1049).
3.12. Bromo-4,5,30,40-tetramethoxy-9-oxo-2,70-cyclo-
ligna-7,70-dienes (12–14: 12, 90-Br; 13, 6-Br and 90-Br; 14,
60-Br and 90-Br)
HBr gas, prepared by adding Br2 to tetralin, was bub-
bled through a solution of 2 (500 mg) in anhyd CH2Cl2
(10 mL), via a CaCl2 drying tube, for 30 min. The reac-
tion mixture was capped and allowed to stand at room
temperature for 3 h. Removal of the solvent yielded a
residue, which was treated with H2O (5 mL), acetone
(10 mL), and BaCO3 (500 mg), heated under reflux for
1 h. After cooling, the reaction mixture was extracted
with CHCl3 (10 mL · 3). The combined organic layers
were dried over anhyd Na2SO4 and evaporated to give a
residue (580 mg), which was crystallized from hexane–
EtOAc (19:1) to furnish the light yellowish needles of 12
(501 mg, 89%).
3.9. 4,5:30,40-Bis(methylenedioxy)-2,70-cycloligna-7,70-
diene (9)
Under similar reaction conditions and workup proce-
dure for the preparation of 6, 9 (1.9 mg, 55%) was ob-
tained from 8 (3.2 mg, 0.01 mmol), CH2Cl2 (0.3 mL,
4.68 mmol), DMF (0.1 mL), and K2CO3 (16 mg). The
spectral and other properties were identical to those
reported previously.22
During the process for preparation of 12 as described
above a few drops of bromine was added after HBr
bubbling and workup as that for 12 yielded a residue
(125 mg) from 2 (100 mg, 0.28 mmol). This residue was
chromatographed on silica gel (4 g) eluted with 10%
EtOAc–hexane to give 12 (10 mg, 8.9%), (13) (61 mg,
46%), and 14 (10 mg, 7.6%).
3.10. 4,5,30,40,90-Pentamethoxy-9-hydroxy-2,70-cyclo-
ligna-7,70-diene (10)
To 2 (34.6 mg, 0.09 mmol) dissolved in CH2Cl2 (3 mL)
and MeOH (2 mL) at rt was added NaBH4 (60 mg,
0.63 mmol) portionwise and the mixture was stirred for
10 h. The mixture was concentrated to give a residue,
which was partitioned between H2O (60 mL) and CHCl3
(60 mL). The organic layer was washed with brine, dried
over anhyd Na2SO4, and concentrated to give 10 (26 mg,
Compound 12: mp 174–75 ꢁC; Rf 0.44 (solvent B); IR
1
m
max: 2958, 1689, 1617, 1506, 1255, 1026, 958 cmꢀ1; H
NMR (CDCl3) d 10.26 (1H, s, H-9), 8.17 (1H, s, H-7),
7.22 (1H, s, H-6), 7.00 (1H, d, J ¼ 8:4 Hz, H-50), 6.92
(1H, d, J ¼ 1:8 Hz, H-20), 6.86 (1H, dd, J ¼ 8:4, 1.8 Hz,
H-60), 6.69 (1H, s, 1H, H-3), 4.97 (1H) and 4.73 (1H)
(each d, J ¼ 9:2 Hz, H-2a), 4.00 (3H, s, 5-OCH3), 3.94
(3H, s, 30-OCH3), 3.85 (3H, s, 40-OCH3), 3.69 (3H, s, 4-
OCH3); 13C NMR (CDCl3) d 192.1 (s, C-9), 152.1 (s, C-
4), 150.5 (s, C-5), 148.8 (s, C-30), 148.6 (s, C-40), 140.2 (s,
C-80), 139.1 (s, C-70), 135.4 (d, C-7) 130.6 (s, C-8), 129.6
(s, C-2), 129.2 (s, C-1), 128.2 (s, C-10), 122.0 (d, C-60),
112.9 (d, C-50), 111.1 (d, C-20), 107.5 (d, C-6), 106.8 (d,
C-3), 56.1 (q, 5-OMe), 56.0 (q, 30-OMe), 55.9 (q, 40-
OMe), 55.8 (q, 4-OMe), 29.4 (t, C-90); EIMS m=z (rel.
int.) 444 (8, Mþ), 442 (90), 364 (80), 349 (49) 333 (19),
189 (38); HREIMS m=z [M]þ 444.0575 (calcd for
C22H21O5Br79, 444.0572), 446.0565 (calcd for
C22H21O5Br81, 446.0553).
1
75%): white amorphous solid; Rf 0.17 (solvent A); H
NMR (CDCl3) d 7.74 (1H, s, H-7), 7.13 (1H, s, H-6),
6.99 (1H, d, J ¼ 8:4 Hz, H-50), 6.86 (1H, br d,
J ¼ 8:4 Hz, H-60), 6.85 (1H, br s, H-20), 6.76 (1H, s, H-
3), 4.79 (1H, d, J ¼ 12:2 Hz, H-9a), 4.74 (1H, d,
J ¼ 12:2 Hz, H-9b), 4.43 (1H, d, J ¼ 10:4 Hz, H-90), 4.38
(1H, d, J ¼ 10:4 Hz, H-90), 3.99 (3H, s, 5-OMe), 3.98
(3H, s, 40-OMe), 3.84 (3H, s, 30-OMe), 3.70 (3H, s, 4-
OMe), 3.29 (3H, s, 90-OMe); 13C NMR (CDCl3) d 149.8
(s, C-4), 149.5 (s, C-5), 148.6 (s, C-40), 148.2 (s, C-30),
139.6 (s, C-70), 136.7 (s, C-8), 131.5 (s, C-10), 129.9 (s, C-
80), 129.2 (s, C-1), 128.2 (s, C-2), 127.2 (d, C-7), 122.6 (d,
C-60), 113.5 (d, C-20), 110.8 (d, C-50), 106.3 (d, C-3),
106.0 (d, C-6), 70.7 (t, C-9), 65.0 (t, C-90), 58.1 (q, 90-
OMe), 55.9 (q, 5-OMe), 55.9 (q, 30-OMe), 55.9 (q, 40-
OMe), 55.7 (q, 4-OMe); EIMS m=z 398 (71, [M]þ), 367
(24), 366 (100), 335 (18), 305 (16), 275 (19); HREIMS
m=z [M]þ: 398.1723 (calcd for C23H26O6, 398.1729).
1
Compound 13: Rf 0.46 (solvent B); H NMR (CDCl3)
d 10.33 (1H, s, H-9), 8.68 (1H, s, H-7), 7.00 (1H, d,
J ¼ 8:2 Hz, H-50), 6.91 (1H, d, J ¼ 1:8 Hz, H-20), 6.86
(1H, dd, J ¼ 8:2, 1.6 Hz, H-60), 6.74 (1H, s, H-3), 5.00
(1H) and 4.74 (1H) (each d, J ¼ 9:2 Hz, H-90), 3.97
(3H, s, 5-OCH3), 3.91 (3H, s, 30-OCH3), 3.87 (3H, s,
40-OCH3), 3.70 (3H, s, 4-OMe); 13C NMR (CDCl3) d
192.1 (d, C-9), 154.1 (s, C-5), 148.9 (s, C-4), 148.8 (s, C-
30), 148.2 (s, C-40), 140.5 (s, C-80), 139.1 (s, C-70),
135.4(d, C-7), 130.1 (s, C-8), 129.6 (s, C-2), 129.2 (s,
C-1), 128.8 (s, C-10), 122.1 (d, C-60), 117.5 (d, C-6),
112.9 (d, C-50), 111.1 (d, C-20), 106.75 (d, C-3), 60.8
(q, 5-OCH3), 56.0 (q, 30-OCH3), 55.9 (q, 40-OCH3), 55.8
(q, 4-OCH3), 28.8 (t, C-90); EIMS m=z (rel. int. %) 526
(2, [M+4]þ), 524 (4, [M+2]þ), 522 (2, [M]þ), 446 (81),
444 (100), 385 (9), 383 (8), 355 (10), 353 (9), 306 (34),
189 (12).
3.11. 3,4,30,40-Tetramethoxy-9,90-epoxylignan (11)
To a mixture of 1 (20 mg, 0.05 mmol), dry benzene
(0.5 mL), and trifluoroacetic anhydride (0.1 mL) was
added POCl3 (247 mg, 1.6 mmol) at 0 ꢁC. The reaction
solution was refluxed for 6 h and concentrated to give a
residue, which was partitioned between 5% aqueous
K2CO3 (10 mL), and EtOAc (10 mL). The organic layer
was washed by brine, dried over anhyd Na2SO4, and
concentrated to give 11 (8.0 mg, 45%). The spectral and
other properties were identical to those reported previ-
ously.11a

K. S. Sagar et al. / Bioorg. Med. Chem. 12 (2004) 4045–4054
4051
Compound 14: Rf 0.45 (solvent B); 1H NMR (CDCl3) d
10.32 (1H, s, H-9), 8.12 (1H, s, H-7), 7.28 (1H, s, H-50),
7.22 (1H, s, H-6), 6.92 (1H, s, H-20), 6.56 (1H, s, H-3),
5.35 (1H) and 4.31 (1H) (each d, J ¼ 9:2 Hz, H-90), 4.00
(3H, s, 5-OCH3), 3.98 (3H, s, 30-OCH3), 3.87 (3H, s, 40-
OCH3), 3.75 (3H, s, 4-OMe); EIMS m=z (rel. int. %) 526
(1, [M+4]þ), 524 (2, [M+2]þ), 522 (1, [M]þ), 446 (88),
444 (100), 364 (52), 306 (100), 189 (18).
3a-NHC2H4CH3), 11.0 (q, NþC2H4CH3), and for the rest
of the data see above; NOESY data, see Figure 1; HMBC
data, see Figure 2; FABMS m=z (rel. int. %) [M+H]þ 463
(100), 154 (24), 136 (22); HREIMS m=z [M]þ 462.2500
(calcd for C28H34N2O4, 462.2518); EIMS m=z (rel. int. %)
462 (100, [M]þ), 433 (75), 420 (56), 378 (60).
1
Compound 17: Rf 0.43 (8% MeOH–CHCl3); H NMR
(CDCl3) d 9.98 (1H, br s, 3a-NHC3H7), 8.31 (1H, s, H-
4), 4.44 (1H) and 4.36 (1H) (each d, J ¼ 18:4 Hz, H-2a),
5.61 (1H, m, NþCHMe2), 4.84 (1H, m, 3a-NHCHMe2),
1.35 (6H, d, J ¼ 6:4 Hz, 3a-NHCHMe2), 1.28 (6H, d,
J ¼ 6:5 Hz, NþCHMe2), and for the rest of the data see
Figure 1; 13C NMR (CDCl3) d 55.8 (t, C-2a), 158.7 (s, C-
3a), 49.4 (d, NþCHMe2), 48.2 (d, 3a-NHCHMe2), 23.3
(q) and 23.2 (q) (3a-NHCHMe2), 20.4 (2C, q,
NþCHMe2), and the rest data see above; FABMS m=z
(rel. int. %) 495 (50, [M+Na]þ), 463 (100, [M+H]þ), 448
(16), 154 (60), 136 (55); HRMS m=z [M]þ 462.2496 calcd
for C28H34N2O4, 462.2518); EIMS m=z (rel. int. %) 462
(100, [M]þ), 447 (75), 432 (10), 419 (56), 405 (38).
3.13. 1-(30,40-Dimethoxyphenyl)-6,7-dimethoxy-[2-alkyl-
amino-N-alkyl]-5H-pyrro[4,3-b]-naphthalenes (16: al-
kyl ¼ nPr; 17: alkyl ¼ iPr; 18: alkyl ¼ iBu; 19: 5-Br
and alkyl ¼ Me)
The mixture of 12 (459 mg, 1.03 mmol), CH2Cl2 (15 mL),
and methylamine (197 lL) was stirring for 3 h and was
evaporated to get a residue (660 mg). This residue was
chromatographed on silica gel (15 g) eluted with 2%
MeOH–CHCl3 to give compound 15 (258 mg, 51%).
Using similar reaction conditions and workup to the
preparation of 15, compounds 16 (303 mg, 41%), 17
(336 mg, 46%), 18 (420 mg, 58%), and 19 (70 mg, 65%)
were produced from 12 (600 mg, 1.35 mmol for 16 and 17;
560 mg, 1.26 mmol for 18), and 13 (100 mg, 0.19 mmol for
19) by reacting with propylamine (279 lL, 3.37 mmol,
2.5 equiv for 16), isopropylamine (270 lL, 3.37 mmol,
2.5 equiv for 17), isobutylamine (237 lL, 3.15 mmol,
2.5 equiv for 18), and methylamine (43 lL for 19).
1
Compound 18: Rf 0.42 (7% MeOH–CHCl3); H NMR
(CDCl3) d 10.38 (1H, br s, 3a-NHC3H7), 8.25 (1H, s, H-
4), 4.53 (1H) and 4.45 (1H) (each d, J ¼ 18:4 Hz, H-2a),
4.04 (2H, m, NþCH2CHMe2), 3.86 (2H, m, 3a-
NHCH2CHMe2), 2.36 (1H, m, 3a-NHCH2CHMe2),
2.21 (1H, m, NþCH2CHMe2), 1.13 (6H, d, J ¼ 6:4 Hz,
3a-NCH2CHMe2), 1.03 (6H, d, J ¼ 6:4 Hz, NþCH2
CHMe2), and for the rest of the data see Figure 1; 13C
NMR (CDCl3) d 55.8 (t, C-2a), 160.4 (s, C-3a), 53.9 (t,
NþCH2CHMe2), 50.8 (t, 3a-NHCH2CHMe2), 29.1 (d,
3a-NHCH2CHMe2), 27.5 (d, NþCH2CHMe2), 20.1 (2C,
q, 3a-NCH2CHMe2), 19.6 (2C, q, NþCH2CHMe2), and
for the rest of the data see above; FABMS m=z (rel. int.
%) 491 (62, [M+H]þ), 391 (18), 307 (22), 154 (100), 136
(65); HRMS m=z [M]þ 490.2833 (calcd for C30H38N2O4,
490.2831); EIMS m=z (rel. int. %) 490 (80, [M]þ), 475 (8),
447 (100), 434 (2), 392 (22), 378 (95).
1
Common H NMR data for 15–18: see Figure 1 for 16.
Common 13C NMR data for 15–18: d (CDCl3) 133.8–
134.1 (s, C-2), 133.5–133.7 (s, C-1), 129.3–129.5 (s, C-4a),
124.3–124.6 (d, C-4), 123.4–123.6 (s, C-3), 107.8–108.0
(d, C-5), 150.1–150.6 (s, C-6), 152.5 (s, C-7), 103.9–104.0
(d, C-8), 130.7 (s, C-8a), 128.3–128.5 (s, C-10), 111.9–
120.0 (d, C-20), 149.4–149.5 (s, C-30), 149.2 (s, C-40),
111.7–111.8 (d, C-50), 121.4 (d, C-60), 56.13–56.20 (q, 6-
OMe), 55.92–56.0 (q, 7-OMe), 55.88–55.90 (q, 30-OMe),
Compound 19: semi-solid, Rf 0.42 (8% MeOH–CHCl3);
1H NMR (CDCl3) d 10.83 (1H, s, H-3a), 8.92 (1H, s, H-
4), 7.04 (1H, d, J ¼ 8:2 Hz, H-50), 7.00 (1H, s, 8-H), 6.88
(1H, dd, J ¼ 1:8 and 8.2 Hz, H-60), 6.80 (1H, d,
J ¼ 1:8 Hz, H-20), 4.60 (1H, d, J ¼ 18:8 Hz, H-2a), 4.54
(1H, d, J ¼ 18:8 Hz, H-2a), 3.98 (3H, s, 6-OCH3), 3.97
(3H, s, 30-OCH3), 3.88 (3H, s, 40-OCH3), 3.79 (3H, s, 7-
OCH3), 3.71 (3H, s, NþMe), 3.68 (3H, s, NHMe); 13C
NMR: dC 161.1 (s, C-3a), 155.3 (s, C-7), 149.6 (s, C-30),
149.5 (s, C-40), 148.5 (s, C-6), 135.6 (s, C-4a), 134.2 (s, C-
2), 133.2 (s, C-1), 130.7 (s, C-8a), 128.9 (s, C-10), 125.5
(d,C-4), 124.8 (s, C-3), 121.4 (d, C-60), 118.3 (d, C-5),
111.9 (d, C-20 and C-50), 104.7 (d, C-8), 60.9 (q, 6-OMe),
56.2 (q, 30-OMe), 56.0 (q, 40- and 7-OMe), 35.2 (q,
NþCH3), 31.4 (q, 3a-NHCH3); EIMS m=z (rel. int. %)
483 (100, [M)H]þ), 469 (20), 456 (13), 262 (12).
56.1 (q, 40-OMe), (d6-OMe > d4 -OMe > d7-OMe > d3 -OMe).
0
0
Compound 15: Rf 0.41 (8% MeOH–CHCl3); IR mmax 3417
(NH), 2964, 1649, 1619, 1506, 1257, 1119, 1026, 947 cmꢀ1
;
1H NMR (CDCl3) d 10.19 (1H, br s, 3a-NHMe), 8.45 (1H,
s, H-4), 4.50 (1H) and 4.57 (1H) (each d, J ¼ 18:4 Hz, H-
2a), 3.64 (3H, s, NþMe), 3.60 (3H, s, 3a-NHMe), and for
the rest of the data see Figure 1; 13C NMR (CDCl3) d 57.3
(t, C-2a), 161.2 (s, C-3a), 35.3 (q, s, NþMe), 31.3 (q, 3a-
NHMe), and for the rest of the data see above; FABMS
m=z (rel. int. %) [M+H]þ 407 (55), 307 (13), 289 (12), 154
(100), 136 (83); HREIMS m=z [M]þ 406.1840 (calcd for
C24H26N2O4, 406.1892), [M)Me]þ 391.1643 (calcd for
C23H23N2O4, 391.1657); EIMS m=z (rel. int. %) 406 (100,
[M]þ), 405 (100, [M)H]þ), 389 (18), 149 (34).
Compound 16: Rf 0.42 (8% MeOH–CHCl3); IR (KBr)
3.14. 1-(30,40-Dimethoxyphenyl)-6,7-dimethoxy-N-alkyl-
200,500H-pyrro[4,3-b]naphthalenes (20: N-Me; 21: N-nPr;
22: N-iPr; 23: N-iBu; 24: 5-Br and N-Me)
m
max: 3416 (NH), 2964, 1649, 1619, 1506, 1257, 1119,
1
1026, 947 cmꢀ1; H NMR (CDCl3), see Figure 1; 13C
NMR (CDCl3) d 57.1 (t, C-2a), 160.0 (s, C-3a), 45.4 (t,
3a-NHCH2C2H5), 48.8 (t, NþCH2C2H5), 23.4 (t, 3a-
NHCH2CH2CH3), 21.0 (t, NþCH2CH2CH3), 11.3 (q,
To the solution of 15 (81 mg, 0.17 mmol) in methanol
(5 mL) was added sodium borohydride (31 mg,

4052
K. S. Sagar et al. / Bioorg. Med. Chem. 12 (2004) 4045–4054
0.83 mmol, 5 equiv) portionwise. After stirring 30 min at
rt, the suspension was evaporated under vacuum and the
residue was partitioned between H2O (5 mL) and CHCl3
(5 mL · 3). The combined organic layers were dried over
anhyd Na2SO4 and evaporated to give a residue
(105 mg), which was chromatographed on silica gel (3 g),
eluted with 1% MeOH–CHCl3, to give compound 20
(47 mg, 74%).
H-2a), 3.86 (2H, s, H-3a), 2.75 (1H, m, NCHMe2), 1.16
(6H, d, J ¼ 6:2 Hz, NCHMe2), and for the rest of the
data see above; 13C NMR (CDCl3) d 135.3 (s, C-1),
136.2 (s, C-2), 57.2 (t, C-2a), 56.6 (t, C-3a), 54.9 (d,
NCHMe2), 21.5 (2C, q, NCHMe2), and for the rest of
the data see above; HRMS m=z [M]þ 407.1809 (calcd for
C25H29NO4, 407.2096), [M)2H]þ 405.1934 (calcd for
C25H27NO4, 405.1940); EIMS m=z (rel. int. %) 407 (50,
Mþ), 406 (100), 392 (64), 349 (24), 196 (10).
Under similar reaction conditions and workup to the
preparation of 20, compounds 21 (41 mg, 66%), 22
(46 mg, 74%), 23 (38 mg, 72%), and 24 (30 mg, 75%) were
produced from 16 (82 mg, 0.15 mmol) for 21, 17 (82 mg,
0.15 mmol) for 22, 18 (72 mg, 0.13 mmol) for 23, and 19
(50 mg, 0.09 mmol) for 24 by reduction with sodium
borohydride (28 mg, 0.75 mmol, 5 equiv for 21 and 22;
23 mg, 0.63mmol for 23; 17 mg, 0.44 mmol for 24).
1
Compound 23: Rf 0.42 (5% MeOH–CHCl3); H NMR
(CDCl3) d 4.07 (1H) and 4.01 (1H) (each d, J ¼ 12:5 Hz,
H-2a), 3.79 (2H, s, H-3a), 2.48 (2H, d, J ¼ 7:3 Hz,
NCH2CHMe2), 1.79 (1H, m, NCH2CHMe2), 0.94 (6H,
d, J ¼ 6:6 Hz, NCH2CHMe2), and for the rest of the
data see above; 13C NMR (CDCl3) d 135.8 (s, C-1),
136.8 (s, C-2), 59.5 (t, C-2a), 59.1 (t, C-3a), 65.0 (t,
NCH2CHMe2), 27.3 (d, NCH2CHMe2), 20.9 (2C, q,
NCH2CHMe2), and for the rest of the data see above;
HRMS m=z [M]þ 421.2209 (calcd for C26H31NO4,
421.2253); EIMS m=z (rel. int. %) 421 (18, [M]þ), 419
(28), 378 (100), 349 (56), 318 (18), 189 (20).
Common 1H NMR data for 20–23: d (CDCl3) 7.48 (1H, s,
H-4), 7.09–7.10 (1H, s, H-5), 6.94–6.98 (1H, s, H-8),
6.86–6.88 (1H, d, J ¼ 1:9 Hz, H-20), 6.92–6.97 (1H, d,
J ¼ 8:0 Hz, H-50), 6.88–6.90 (1H, dd, J ¼ 1:9, 8.0 Hz, H-
60), (dH-5 > dH-6 > dH-2 ), 3.96–3.99 (3H, s, 6-OMe),
3.73–3.75 (3H, s, 7-OMe), 3.83–3.86 (3H, s, 30-OMe),
3.94–3.96 (3H, s, 40-OMe).
0
0
0
1
Compound 24: Rf 0.42 (5% MeOH–CHCl3); H NMR
(CDCl3) d 7.99 (1H, s, H-4), 6.99 (1H, s, H-8), 6.97 (1H,
d, J ¼ 8:0 Hz, H-50), 6.85 (1H, dd, J ¼ 8:0, 1.8 Hz, H-
60), 6.82 (1H, d, J ¼ 1:8 Hz, H-20), 4.14 (1H) and 4.09
(1H) (each d, J ¼ 12:8 Hz) (H-2a), 3.95 (3H, s, 6-OCH3),
3.93 (3H, s, 30-OCH3), 3.84 (3H, s, 40-OCH3), 3.83 (2H,
s, H-3a), 3.72 (3H, s, 7-OCH3), 2.59 (3H, s, NCH3);
EIMS m=z (rel. int. %) 457 (100, [M]þ), 377 (75), 262
(10).
13
Common C NMR data for 20–23: d (CDCl3) 148.8–
149.4 (s, C-6), 148.8–149.2 (s, C-7), 148.7–148.9 (s, C-30),
148.1–148.6 (s, C-40), 131.8–132.3 (s, C-8a), 130.9–131.4
(s, C-3), 129.1–129.4 (s, C-4a), 127.2–127.5 (s, C-10),
121.7–121.8 (d, C-60), 118.5–118.9 (d, C-4), 112.7–112.8
(d, C-20), 111.2–111.5 (d, C-50), 106.5–106.6 (d, C-5),
104.8–104.9 (d, C-8), 55.9–56.0 (q, 6-OMe), 55.82–55.9
(q, 40-OMe), 55.7–55.8 (q, 7-OMe), 55.6–55.7 (q, 30-
OMe), (d6-OMe > d4 -OMe > d7-OMe > d3 -OMe).
3.15. 4,5,30,40,90-Pentamethoxy-9-alkylamino-2,70-cyclo-
ligna-7,70-dienes (25: alkyl ¼ Me; 26: alkyl ¼ nPr; 27:
alkyl ¼ iPr; 28: alkyl ¼ iBu)
0
0
Compound 20: Rf 0.41 (5% MeOH–CHCl3); IR mmax
:
2959, 1620, 1509, 1254, 1119, 1026, 953 cmꢀ1; 1H NMR
(CDCl3) d 4.08 (1H) and 4.02 (1H) (each d, J ¼ 13:1 Hz,
H-2a), 3.81 (2H, s, H-3a), 2.57 (3H, s, NMe), and for the
rest of the data see above; 13C NMR (CDCl3) d 131.9 (s,
C-1), 136.9 (s, C-2), 61.1 (t, C-2a), 60.5 (t, C-3a), 42.4 (q,
NMe), and for the rest of the data see above; HREIMS
m=z [M]þ 379.1742 (calcd for C23H25NO4, 379.1785),
[M)2H]þ 377.1619 (calcd for C23H23NO4, 377.1628);
EIMS m=z (rel. int. %) 379 (52%, [M]þ), 378 (100), 363
(14), 242 (12), 189 (8), 149 (6).
The mixture of 2 (110 mg, 0.28 mmol), methanol
(10 mL), and methylamine (64 lL, 0.56 mmol) was stir-
red for 10 min and then sodium borohydride (30 mg,
3 equiv) was added portionwise. The mixture was stirred
for additional 30 min at rt and was evaporated to give a
residue, which was partitioned between H2O (10 mL)
and CHCl3 (10 mL · 3). The combined organic layers
were dried over anhyd Na2SO4 and evaporated to give a
residue (124 mg), which was chromatographed on silica
gel (4 g), eluted with 1% MeOH–CHCl3, to give com-
pound 25 (113 mg, 99%).
1
Compound 21: Rf 0.42 (5% MeOH–CHCl3); H NMR
(CDCl3) d 4.24 (1H) and 4.17 (1H) (each d, J ¼ 12:8 Hz,
H-2a), 3.97 (2H, s, H-3a), 2.75 (2H, m, NCH2C2H5),
1.64 (2H, m, NCH2CH2CH3), 0.93 (3H, t, J ¼ 7:3 Hz,
NCH2CH2CH3), and for the rest of the data see above;
13C NMR (CDCl3) d 133.9 (s, C-1), 134.7 (s, C-2), 59.0
(t, C-2a), 58.6 (t, C-3a), 58.2 (t, NCH2C2H5), 21.1 (t,
NCH2CH2CH3), 11.7 (q, NC2H4CH3), and for the rest
of the data see above; HREIMS m=z [M]þ 407.2055
(calcd for C25H29NO4, 407.2096), [M)2H]þ 405.1931
(calcd for C25H27NO4, 407.1941); EIMS m=z (rel. int. %)
407 (58, [M]þ), 406 (100), 378 (68), 349 (36), 189 (13).
Under similar reaction conditions and workup to the
preparation of 25, compounds 26 (109 mg, 98%), 27
(109 mg, 98%), and 28 (112 mg, 98%) were produced
from 2 (each 100 mg, 0.25 mmol) and alkylamines (n-
propylamine, 41 lL, 0.50 mmol, 2 equiv for 26; isoprop-
ylamine, 43 lL, 2 equiv for 27; isobutylamine, 50 lL,
0.5 mmol, 2 equiv for 28) by reduction with sodium
borohydride (each 28 mg, 3 equiv).
Common 1H NMR data for 25–28: d 6.73–6.75 (1H, s, H-
3), 7.11–7.12 (1H, s, H-6), 7.72–7.74 (1H, s, H-7), 6.85–
6.86 (1H, d, J ¼ 2:0 Hz, H-20), 6.97 (1H, d, J ¼ 8:0 Hz,
H-50), 6.83–6.85 (1H, dd, J ¼ 2:0, 8.0 Hz, H-60), 4.33–
1
Compound 22: Rf 0.43 (5% MeOH–CHCl3); H NMR
(CDCl3) d 4.15 (1H) and 4.09 (1H) (each d, J ¼ 12:8 Hz,

K. S. Sagar et al. / Bioorg. Med. Chem. 12 (2004) 4045–4054
4053
4.35 (1H) and 4.28–4.30 (1H) (each d, J ¼ 10:0 ꢁ
10:3 Hz, H-90), 3.68–3.69 (3H, s, 4-OMe), 3.95–3.97 (6H,
s, 5-OMe and 40-OMe), 3.83–3.84 (3H, s, 30-OMe), 3.25–
3.26 (3H, s, 90-OMe).
Compounds to be analyzed were prepared as 1 mM
stock solution in 50% DMSO. HIV-1 RT was purchased
from HT Biotechnology Ltd, Cambridge, UK. The
chemicals and assay procedures were followed as de-
scribed in an earlier paper.5
Common 13C NMR data for 25–28: d 128.9–129.0 (s, C-
1), 129.9–130.0 (s, C-2), 105.9–106.0 (d, C-3), 149.5–
149.6 (s, C-4), 149.8 (s, C-5), 106.2 (d, C-6), 122.5 (d, C-
7), 128.1–128.2 (s, C-8), 58.1–58.3 (t, C-9), 127.8–128.0
(s, C-10), 113.5 (d, C-20), 148.5 (s, C-30), 148.1–148.2 (s,
C-40), 110.7 (d, C-50), 122.5 (d, C-60), 131.3–131.4 (s, C-
70), 139.9–140.0, C-80), 70.2–70.3 (t, C-90), 55.80–55.83
(q, 4-OMe), 55.86–55.90 (q, 5-OMe), 55.6 (q, 30-OMe),
0
3.17. Virus infectivity assay
A one-round complementation assay was used to mea-
sure the anti-HIV activity of the compounds. They were
tested using a R5 pseudotype virus, ConB/pNL43E-L+
in the U87-CD4-CCR5 cells. In addition, AZT, a FDA
approved, prototype reverse transcriptase inhibitor, was
also included in the analysis to provide a comparison of
the anti-HIV activity between the tested compounds and
a drug currently used in clinical therapy. Each com-
pound was dissolved in 100% DMSO to a concentration
of 10 mg/mL and diluted to 1 mg/mL as a working
concentration. Different concentrations (0.02–20 lg/mL)
were applied to the virus infectivity assay for the
determination of the IC50 and IC90, which were mea-
sured from the luciferase activity of the virus-infected
cells. The CC50 and CC90 (cytotoxicity) were also
determined.
55.83–55.86 (q, 40-OMe), (d5-OMe > d4 -OMe > d4-OMe
>
0
3 -OMe).
d
1
Compound 25: Rf 0.41 (4% MeOH–CHCl3); H NMR
(CDCl3) d 4.03 (2H, s, H-9), 2.55 (3H, s, 9-NHMe), and
for the rest of the data see above; 13C NMR (CDCl3) d
53.2 (q, 90-OMe), 34.5 (q, 9-NHMe), and for the rest of
the data see above; HREIMS m=z [M]þ 411.1996 (calcd
for C24H29NO5, 411.2046); EIMS m=z (rel. int. %) 411
(2, [M]þ), 380 (100), 349 (28), 319 (16), 174 (10).
1
Compound 26: Rf 0.42 (4% MeOH–CHCl3); H NMR
(CDCl3) d 4.09 (2H, s, H-9), 2.74 (2H, t, J ¼ 7:2 Hz, 9-
NHCH2C2H5), 1.61 (2H, m, 9-NHCH2CH2CH3), 0.93
(3H, t, J ¼ 7:4 Hz, 9-NHCH2CH2CH3), and for the rest
of the data see above; 13C NMR (CDCl3) d 51.7 (q, 90-
OMe), 50.4 (t, 9-NHCH2C2H5), 22.0 (t, 9-NHCH2-
CH2CH3), 11.5 (q, 9-NHC2H4CH3), and for the rest of
the data see above; HREIMS m=z [M)2H]þ 437.2196
(calcd for C26H31NO5, 437.2202); EIMS m=z (rel. int. %)
439 (10, [M]þ), 407 (62), 380 (100), 349 (32), 174 (20),
160(19).
Acknowledgements
This work was supported by the National Science
Council, Taiwan, ROC, under the grant NSC 91-2314-
B-002-003 and by the Ministry of Education under the
grant 89-FA01-1-4.
References and notes
1
Compound 27: Rf 0.43 (4% MeOH–CHCl3); H NMR
(CDCl3) d 4.07 (2H, s, H-9), 3.02 (1H, m, 9-NHCHMe2),
1.22 (6H, d, J ¼ 6:4 Hz, 9-NHCHMe2), and for the rest
of the data see above; 13C NMR (CDCl3) d 49.1 (q, 90-
OMe), 48.6 (d, 9-NHCHMe2), 21.6 (2C, q, 9-
NHCHMe2), and for the rest of the data see above;
HRMS m=z [M]þ 439.2309 (calcd for C26H33NO5,
439.2359); EIMS m=z (rel. int. %) 439 (8, [M]þ), 421 (85),
407 (20), 380 (100), 349 (38), 319 (16), 175 (16).
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