Metal-chelating inhibitors of a zinc finger protein HIV-EP1. Remarkable potentiation of inhibitory activity by introduction of SH groups

Article abstract of DOI:10.1021/jm950831t

HIV-EP1 is a C₂H₂ type zinc finger protein which binds to DNA κB site present in the long terminal repeat of HIV provirus. Previously we have reported zinc chelators having histidine-pyridine-histidine skeleton and were successful in inhibiting the DNA binding of HIV-EP1 by removing zinc from the zinc finger domain. Aiming at the potentiation of the inhibitory activity of our previous zinc chelators, herein synthesized were novel chelators comprising pyridine and aminoalkanethiol. These showed marked inhibitory activity on the DNA binding of HIV-EP1. In particular, one of them having a bis(2-mercaptoethyl)amino side chain showed inhibitory activity (IC₅₀, ~4 μM) 10 times stronger than that of the strongest inhibitor that we reported previously. It appeared that these inhibited the DNA binding of HIV-EP1 by a mechanism distinct from that of the previous histidine-based inhibitors.

Full text of DOI:10.1021/jm950831t

J . Med. Chem. 1996, 39, 503-507
503
Meta l-Ch ela tin g In h ibitor s of a Zin c F in ger P r otein HIV-EP 1. Rem a r k a ble
P oten tia tion of In h ibitor y Activity by In tr od u ction of SH Gr ou p s
Mikako Fujita, Masami Otsuka,* and Yukio Sugiura*
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611, J apan
Received November 10, 1995X
HIV-EP1 is a C2H2 type zinc finger protein which binds to DNA κB site present in the long
terminal repeat of HIV provirus. Previously we have reported zinc chelators having histidine-
pyridine-histidine skeleton and were successful in inhibiting the DNA binding of HIV-EP1
by removing zinc from the zinc finger domain. Aiming at the potentiation of the inhibitory
activity of our previous zinc chelators, herein synthesized were novel chelators comprising
pyridine and aminoalkanethiol. These showed marked inhibitory activity on the DNA binding
of HIV-EP1. In particular, one of them having a bis(2-mercaptoethyl)amino side chain showed
inhibitory activity (IC50, ∼4 µM) 10 times stronger than that of the strongest inhibitor that we
reported previously. It appeared that these inhibited the DNA binding of HIV-EP1 by a
mechanism distinct from that of the previous histidine-based inhibitors.
In tr od u ction
Zinc finger proteins constitute a major group of
transcription factor and play important roles in the gene
expression at the terminus of cellular signal transduc-
tion. Our interest has been focused on a C2H2 type zinc
finger protein HIV-EP11 (also designated as PRDII-BF12
or MBP13) which binds to DNA κB site (5′-GGGAC-
TTTCC-3′)4 present in the long terminal repeat of HIV
provirus to activate the HIV-1 gene expression.5 Inhibi-
tion of HIV-EP1 would lead to the interference of the
replication of AIDS virus. Recently we presented a new
strategy for the inhibition of zinc finger proteins, i.e.
removal of zinc from the finger domain by use of a
chelator, and demonstrated this concept to be applicable
to the case of HIV-EP1.6 Thus, heterocyclic ligands
comprising (dimethylamino)pyridine and histidine units
such as 1 (Figure 1) were designed and synthesized.6
These compounds exhibited remarkable zinc-binding
capability and showed marked inhibitory effect on the
DNA binding activity of HIV-EP1.6 Indeed, the inhibi-
tion seemed to be due to the abstraction of zinc from
the zinc finger sites of HIV-EP1 because the DNA-
F igu r e 1. Structure of metal-binding inhibitors of HIV-EP1.
binding capability of HIV-EP1 was restored by adding
extra zinc during or after the treatment with the
inhibitor.6
into each imidazole ring and hydrolysis of the ester
groups resulted in the marked improvement of zinc-
binding affinity of the ligand.6 However, the increase
of zinc-binding affinity did not always lead to the
enhancement in the inhibition against HIV-EP1, pre-
sumably because of the bulkiness and hydrophobicity
of the molecule arising from the introduction of two
trityl groups onto the imidazoles. In order to overcome
this constraint in the imidazole-based ligands, we
needed a new system less bulky and less hydrophobic.
To this end, we intended to replace the imidazole by a
mercapto group considering that all known zinc finger
proteins contain mercapto group as a key ligating
residue. Mercapto compounds, in fact, display unique
and strong metal binding property due to back-donation
of electron from metal dπ orbital to sulfur dπ or pπ
orbital8 as exemplified by cystein-zinc complex which
has stability constant greater than that of other amino
acids, e.g. histidine.9,10 Thus, it was considered that
replacement of the imidazole moiety of our previous
synthetic chelators by a mercapto group would alter the
For the regulation of actual cellular biochemical
process, it was desired to gain specificity and efficiency
of the inhibitors. We made some progress in the issue
of specificity by developing inhibitors which discriminate
two κB site binding proteins HIV-EP1 and NF-κB both
being induced by the same extracellular stimuli.7 Ef-
ficiency problem is the subject of the present study, and
described herein is our success in the remarkable
potentiation of activity of the inhibitor.
Resu lts a n d Discu ssion
Ch em istr y. Our previous metal chelators contain
imidazole, secondary amine, pyridine, and carboxylic
ester groups as potential metal chelating sites in a
common histidine-pyridine-histidine skeleton as en-
visaged in the inhibitor 1. Introduction of a trityl group
* To whom correspondence should be addressed.
X Abstract published in Advance ACS Abstracts, December 15, 1995.
0022-2623/96/1839-0503$12.00/0 © 1996 American Chemical Society

504 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Fujita et al.
formation of disulfides whose main constituents were
those assignable to 19 and 20, respectively, based on
the HRMS data (19, HRMS calcd for C13H22N4S2
298.1286, found 298.1307; 20, HRMS calcd for
C15H26N4S2 326.1598, found 326.1573). Special care had
to be taken to prevent 2 and 3 from the autoxidation
and to maintain their reduced form. We found disul-
fides 17-20 to be stable in air, easy to handle, and
quantitatively reducible by dithiothreitol (DTT) to gen-
erate either 2 or 3 in situ.16 Therefore, we employed
17-20 as practical equivalents for 2 and 3 in the
biochemical experiments described below.
Im p r oved In h ibition of DNA Bin d in g of HIV-
EP 1. Figures 3 and 4 show the comparison of inhibitory
effects of imidazole compound 1, mercapto compounds
2 and 3, alkylthio compounds 4-8, and other mercapto
compounds on the DNA binding of HIV-EP1 as demon-
strated by electrophoretic mobility shift assay. Com-
pound 2 or 3 generated from 17 or 18 was indistin-
guishable from that obtained from 19 or 20 in terms of
the inhibitory activity. Mercapto compounds 2 and 3
exhibited remarkable inhibitory effect (Figure 3, lanes
3-6), much stronger than that of imidazole compound
1 (lane 2). The most potent was compound 2, which
inhibited DNA binding of HIV-EP1 almost completely
at 30 µM concentration, whereas 300 µM of 1 was
required for the effective inhibition (Figure 4).6 The IC50
of 2 was ∼4 µM (Figure 4, second column). Thus,
inhibitory activity of compound 2 was shown to be 10
times stronger than that of 1. tert-Butylthio, tritylthio,
and methylthio analogues 4-8, and other thiols, e.g.
2-aminoethanethiol, glutathione, and DTT, showed
markedly lowered inhibitory effect at 30 µM concentra-
tion (Figure 4).17 It should be noted that the inhibitory
effect of 2-aminoethanethiol was small but significant
because this constitutes the side chain of the inhibitor
2, demonstrating the effect of assembling the 2-amino-
ethanethiol units on a pyridine ring in potentiating the
inhibitory activity.
F igu r e 2. Synthetic intermediates for inhibitors and related
compounds.
fundament of the metal-binding characteristics, and
hence we designed novel chelators 2 and 3 and some
related sulfur-containing ligands (Figure 1).
These were prepared as follows (Figure 2). S-Alkyl
ligands 4-8 were obtained by the Schiff base formation
between dialdehyde 911 and (alkylthio)ethylamine 10-
1412-15 followed by in situ reduction with NaBH3CN.
Compounds 4 and 5 were further transformed to mer-
capto ligands 2 and 3, respectively. Treatment of 4 with
2-nitrobenzenesulfenyl chloride (Nps-Cl) afforded 15 in
77% yield (similarly, compound 6 also gave 15 in 70%
yield). The 2-nitrophenylthio (Nps) group on the sec-
ondary amino nitrogen of 15 was selectively removed
by the treatment with aqueous HCl-3-methylindole to
give disulfide 17 in 88% yield. Reduction of 17 with
NaBH4 proceeded with color change from yellow to
orange, and subsequent careful extractive workup at pH
6-7 afforded the desired thiol 2 in 75% yield. Com-
pound 3 was prepared by the same procedure starting
with 5.
As previously reported,6,7 compound 1 was shown to
abstract zinc from the zinc finger site of HIV-EP1
because the DNA-HIV-EP1 binding was restored by the
Compounds 2 and 3 were found to be easily autoxi-
dized under basic conditions (pH > 7), resulting in the
F igu r e 3. Effect of synthetic chelators (30 µM) on the DNA binding of HIV-EP1. After HIV-EP1 was incubated with each chelator
in the presence of poly(dI-dC) at room temperature for 30 min, a radioactive DNA probe containing a κB site from the mouse κ
light-chain enhancer was added. Sample was loaded onto a polyacrylamide band shift gel, and the gel electrophoresis was run.
b
c
d
aGenerated from 20. Generated from 18. Generated from 19. Generated from 17.

Metal-Chelating Inhibitors of HIV-EP1
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 505
Ta ble 1. Effect of Zinc in the Inhibition of the DNA Binding of
HIV-EP1 by Compounds 1-3
DNA-bound
no.
compound
Zn
HIV-EP1 (%)a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
1 (0.7 mM)
1 (0.7 mM)
1 (0.7 mM)
1 (1.0 mM)
1 (1.0 mM)
1 (1.0 mM)
1 (1.0 mM)
2d (30 µM)
2d (30 µM)
2d (300 µM)
2d (300 µM)
2d (1.0 mM)
2d (1.0 mM)
2d (1.0 mM)
2d (1.0 mM)
3e (1.0 mM)
3e (1.0 mM)
0f
100f
100f
0
99
77
94
4g
23
1g
22
0
0.7 mMb
2.1 mMc
1.0 mMb
1.0 mMc
2.0 mMc
90 µMc
900 µMc
1.0 mMb
2.0 mMc
3.0 mMc
4
0
2
0
3.0 mMc
12
a
Quantitation of radioactivity of the electrophoretic band was
conducted using an image analyzer. Zn2+ was added before
b
addition of HIV-EP1. c Zn2+ was added after addition of HIV-EP1.
Generated from 19. e Generated from 20. f Reference 6. Figure
d
g
4.
F igu r e 4. Effect of synthetic chelators and mercapto com-
pounds on the DNA binding of HIV-EP1. aQuantitation of
radioactivity of the electrophoretic band was conducted using
b
c
an image analyzer. Generated from 17. Generated from 19.
dGenerated from 18. Generated from 20.
e
addition of zinc before or after the inhibition reaction
(Table 1, numbers 1-7). In contrast, when zinc was
introduced after the DNA binding inhibition reaction
with 2, virtually no or limited recovery of HIV-EP1-
DNA complex was observed depending on the dose of 2
(30 µM, 300 µM, and 1.0 mM) (Table 1, numbers 8-12,
14, 15; Figure 5, lane 4). Addition of zinc prior to the
inhibition reaction also resulted in no recovery of DNA
binding (Table 1. number 13; Figure 5, lane 3). Com-
pound 3 gave the same result (Table 1, numbers 16, 17).
These results can hardly be explained solely by the zinc
abstraction mechanism,18 and alternate consistency
could be attained by invoking, for example, literature
precedents that certain mercapto compounds inhibit
zinc enzymes not by abstracting zinc but by binding to
the zinc site of the enzymes.19,20
F igu r e 5. Effect of zinc in the inhibition of the DNA binding
of HIV-EP1 by compound 2 (1 mM) generated from 19. Zn2+
was introduced before (lane 3) or after (lane 4) the addition of
HIV-EP1.
new clue to the specificity issue to distinguish zinc finger
proteins involved in the cellular signal crosstalk, which
is our next subgoal.
Exp er im en ta l Section
Gen er a l P r oced u r es. Melting points were determined
with a Yanaco MP-J 3 apparatus and are uncorrected. NMR
spectra were measured on Varian VXR-200 specteometers
using TMS as the internal standard. IR spectra were obtained
using a Hitachi 260-50. Mass spectra were measured on a
J EOL J MS-DX300. Fast atom bombardment high-resolution
mass spectra were determined on a J EOL J MS-SX102A mass
spectrometer by courtesy of J EOL Ltd. Elemental analyses
were performed with a Yanaco CORDER MT-5. Homogeneity
of new compounds 2-8 and 15-18 were confirmed by HPLC,
TLC, and 1H NMR.
Con clu sion
Herein we prepared several sulfur-containing ligands
by replacing the histidine moiety of our previous imi-
dazole-based compounds. Thus, we were successful in
the 10-fold potentiation of the inhibitory activity of
synthetic inhibitor of HIV-EP1 by introducing mercapto
groups. The mechanism of the inhibition, seemingly
distinct from that of our previous inhibitors, could be a
[2-(ter t-Bu tylth io)eth yl]a m in e (10).12 To a solution of
2-aminoethanethiol hydrochloride (3.97 g, 34.9 mmol) in 2 N
aqueous HCl (16 mL) was added tert-butyl alcohol (4.3 mL,

506 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Fujita et al.
46 mmol). The mixture was heated under reflux for 11 h and
concentrated in vacuo. The residue was dried over P2O5,
crushed, and washed with acetone (20 mL × 4). The solid was
crystallized from 4 N aqueous HCl to afford white crystal 10‚-
HCl‚1/3H2O (1.19 g, 19%): mp 190-193 °C; 1H NMR (CD3OD)
δ 1.32 (s, 9H), 2.79 (t, J ) 7.0 Hz, 2H), 3.07 (t, J ) 7.0 Hz,
2H); IR (KBr) 2950, 1600, 1450, 1360, 1250, 1160, 1130, 1070,
930, 910, 730 cm-1; MS(FAB) m/ z 134 (M + H)+. Anal. Calcd
for C6H15NS‚HCl‚1/3H2O: C, 41.01; H, 9.56; N, 7.97. Found:
C, 41.32; H, 9.47; N, 8.21.
4-(Dim eth ylam in o)-2,6-bis[[[2-(ter t-bu tylth io)eth yl]am i-
n o]m eth yl]p yr id in e (4). To a solution of 4-(dimethylamino)-
pyridine-2,6-dicarbaldehyde (9)11 (102 mg, 0.572 mmol) and
10‚HCl‚1/3H2O (195 mg, 1.11mmol) in methanol (3.0 mL) was
added sodium cyanoborohydride (95%, 91.0 mg, 1.38 mmol).
The mixture was stirred at room temperature for 36 h. Water
(2 mL) was added, and then the mixture was stirred at room
temperature for 5 h and concentrated in vacuo. The residue
was dried over P2O5 and chromatographed on silica gel (eluted
with CH2Cl2:MeOH ) 30:1 f 5:1) to afford colorless oil 4 (113
mg, 49%): 1H NMR (CDCl3) δ 1.30 (s, 18H), 2.76 (t, J ) 6.0
Hz, 4H), 2.90 (t, J ) 6.0 Hz, 4H), 3.19 (s, 6H), 4.01 (s, 4H),
5.92 (brs, 2H), 6.65 (s, 2H); 13C NMR (CD3OD) δ 28.7, 32.2,
40.6, 44.1, 50.5, 53.3, 106.3, 155.6, 158.9; IR (KBr) 2960, 2320,
1610, 1550, 1460, 1390, 1360, 1160, 1120 cm-1; HRMS (FAB)
calcd for C21H41N4S2 413.2773, found 413.2740.
mixture. 2: 1H NMR (CDCl3) δ 2.65 (t, J ) 6.5 Hz, 4H), 2.89
(t, J ) 6.5 Hz, 4H), 3.01 (s, 6H), 3.84 (s, 4H), 6.34 (s, 2H); 13
C
NMR (CD3OD) δ 25.4, 40.3, 53.6, 55.1, 105.9, 158.4, 159.2;
HRMS calcd for C13H24N4S2 300.1442, found 300.1432. HCl
salt of 2: 1H NMR (CD3OD) δ 2.93 (t, J ) 7.0 Hz, 4H), 3.30 (s,
6H), 3.39 (t, J ) 7.0 Hz, 4H), 4.48 (s, 4H), 7.29 (s, 2H); 13C
NMR (CD3OD) δ 21.8, 41.7, 49.5, 52.7, 110.6, 145.9, 159.9; IR
(KBr) 3390, 2920, 2740, 1640, 1570, 1410 cm-1; HRMS (FAB)
calcd for C13H25N4S2 301.1521, found 301.1528.
4-(Dim eth yla m in o)-2,6-bis[[N-[2-[(tr ip h en ylm eth yl)th -
io]eth yl]a m in o]m eth yl]p yr id in e (6). Compound 6 was
synthesized from [2-[(triphenylmethyl)thio]ethyl]amine hy-
drochloride hemihydrate (12)14 according to the same proce-
dure as that for 4. Compound 6 was obtained as white solid
(41% yield): mp 65-67 °C; 1H NMR (CDCl3) δ 2.35 (t, J ) 7.5
Hz, 4H), 2.38 (t, J ) 7.5 Hz, 4H), 3.14 (s, 6H), 3.70 (s, 4H),
4.72 (br s, 2H), 6.57 (s, 2H), 7.0-7.5 (m, 30H); 13C NMR (CD3-
OD) δ 33.3, 40.8, 49.5, 52.4, 68.8, 106.0, 128.7, 129.8, 131.5,
146.9, 155.2, 159.4; IR (KBr) 3430, 1630, 1600, 1440, 1120,
1030, 740, 700, 450 cm-1; HRMS (FAB) calcd for C51H53N4S2
785.3712, found 785.3712.
[3-(ter t-Bu tylth io)p r op yl]a m in e (11).13 tert-Butanethiol
(9.3 mL, 82 mmol) and a solution of sodium hydroxide (5.7 g,
143 mmol) in methanol (5 mL) and water (10 mL) were
successively added to a solution of 3-chloropropylamine hy-
drochloride (1.08 g, 8.31 mmol) in methanol (10 mL) and water
(10 mL) at 5 °C. The mixture was stirred at room temperature
for 1 d, and methanol was removed in vacuo. The pH of the
solution was adjusted to 8 with 1 N aqueous HCl, and the
resulting mixture was extracted with dichloromethane (200
mL × 2). The organic layer was dried over MgSO4, acidified
with 1.0 M HCl solution in diethyl ether (20 mL), and
concentrated in vacuo to afford white solid 11‚HCl‚1/3H2O
4-(Dim eth yla m in o)-2,6-bis[[N-[(2-n itr op h en yl)th io]-N-
[2-[(2-n itr op h en yl)d ith io]eth yl]a m in o]m eth yl]p yr id in e
(15). To a solution of 4 (10.5 mg, 0.0254 mmol) in acetic acid
(0.9 mL) was added 2-nitrobenzenesulfenyl chloride (58.0 mg,
0.306 mmol). The mixture was stirred at room temperature
for 16 h and concentrated in vacuo. Dichloromethane (10 mL)
and water (5 mL) were added, and the mixture was neutralized
with aqueous NaHCO3. The organic layer was separated and
concentrated in vacuo. The residue was chromatographed on
silica gel (eluted with hexane:EtOAc ) 10:1 f EtOAc f CH2-
Cl2:MeOH ) 10:1) to afford yellow solid 15 (17.8 mg, 77%).
Compound 15 was also obtained from 6 according to the same
1
(0.984 g, 62%): mp 142-143 °C; H NMR (CD3OD) δ 1.33 (s,
9H), 1.91 (quint, J ) 7.0 Hz, 2H), 2.63 (t, J ) 7.0 Hz, 2H),
3.02 (t, J ) 7.0 Hz, 2H); IR (KBr) 2960, 1600, 1490, 1440, 1360,
1170, 1010, 960, 840 cm-1; MS(FAB) m/ z 148 (M + H)+. Anal
calcd for C7H17NS‚HCl‚1/3H2O: C, 44.31; H, 9.92; N, 7.38.
Found: C, 44.60; H, 9.81; N, 7.07.
1
procedure: mp 88-91 °C; H NMR (CDCl3) δ 2.93 (t, J ) 6.0
Hz, 4H), 3.03 (s, 6H), 3.51 (br, 4H), 4.34 (br, 4H), 6.50 (s, 2H),
4-(Dim eth yla m in o)-2,6-bis[[[3-(ter t-bu tylth io)p r op yl]-
a m in o]m eth yl]p yr id in e (5). Compound 5 was synthesized
from 11‚HCl‚1/3H2O according to the same procedure as that
for 4. Compound 5 was obtained as pale purple oil (39%
yield): 1H NMR (CDCl3) δ 1.29 (s, 18H), 1.95 (quint, J ) 7.0
Hz, 4H), 2.62 (t, J ) 7.0 Hz, 4H), 2.95 (t, J ) 7.0 Hz, 4H), 3.12
(s, 6H), 4.03 (s, 4H), 6.54 (s, 2H), 6.69 (br s, 2H); 13C NMR
(CD3OD) δ 27.1, 29.5, 32.2, 40.4, 43.9, 49.3, 53.6, 106.7, 154.6,
158.4; IR (KBr) 2950, 2310, 1610, 1540, 1460, 1390, 1360, 1160,
1110 cm-1; HRMS (FAB) calcd for C23H45N4S2 441.3086, found
441.3081.
7.19-7.39 (m, 4H), 7.53-7.76 (m, 4H), 8.01-8.33 (m, 8H); 13
C
NMR (CDCl3) δ 36.7, 39.3, 55.6, 63.6, 104.6, 124.8, 125.1, 125.9,
126.0, 126.1, 127.1, 133.9, 134.1, 137.2, 142.1, 145.1, 145.3,
155.6, 156.4; IR (KBr) 3420, 1590, 1510, 1330, 1300, 730 cm-1
;
HRMS (FAB) calcd for C37H37O8N8S6 913.1059, found 913.1068.
4-(Dim eth ylam in o)-2,6-bis[[N-[2-[(2-n itr oph en yl)dith io]-
eth yl]a m in o]m eth yl]p yr id in e (17). 3-Methylindole (17.9
mg, 0.136 mmol) and 0.5 N aqueous HCl (0.6 mL, 0.3 mmol)
were successively added to a solution of 15 (17.8 mg, 0.0195
mmol) in dichloromethane-methanol (2:3, 2.5 mL) at 5 °C.
The mixture was stirred at room temperature for 9 h, neutral-
ized with aqueous NaHCO3, and concentrated in vacuo.
Dichloromethane (30 mL) and water (5 mL) were added to the
mixture. The organic layer was separated and concentrated
in vacuo. The residue was chromatographed on silica gel
(eluted with EtOAc f CH2Cl2:MeOH ) 10:1) to afford yellow
solid 17 (10.4 mg, 88%): 1H NMR (CDCl3) δ 2.90 (t, J ) 7.0
Hz, 4H), 2.92 (t, J ) 7.0 Hz, 4H), 3.06 (s, 6H), 3.57 (brs, 2H),
3.84 (s, 4H), 6.46 (s, 2H), 7.26-7.42 (m, 2H), 7.62-7.75 (m,
2H), 8.20-8.39 (m, 4H); 13C NMR (CD3OD) δ 39.3, 40.3, 48.8,
55.5, 105.9, 127.9, 128.5, 129.3, 136.2, 138.8, 147.8, 158.2,
4-(Dim eth yla m in o)-2,6-bis[[N-[(2-n itr op h en yl)th io]-N-
[3-[(2-n i t r o p h e n y l)d i t h i o ]p r o p y l]a m i n o ]m e t h y l]-
p yr id in e (16). Compound 16 was synthesized from 5 accord-
ing to the same procedure as that for 15. Compound 16 was
1
obtained as yellow solid (61% yield): mp 51-54 °C; H NMR
(CDCl3) δ 1.99 (quint, J ) 6.5 Hz, 4H), 2.72 (t, J ) 6.5 Hz,
4H), 2.97 (s, 6H), 3.32 (br, 4H), 4.27 (br, 4H), 6.42 (s, 2H), 7.17-
7.35 (m, 4H), 7.52-7.67 (m, 4H), 7.93-8.04 (m, 2H), 8.13-
8.32 (m, 6H); 13C NMR (CDCl3) δ 28.1, 35.8, 39.3, 55.7, 64.2,
104.4, 124.7, 125.1, 126.0, 126.1, 127.2, 133.7, 134.0, 137.6,
142.2, 145.5, 145.9, 155.5, 157.4; IR (KBr) 3410, 1590, 1510,
1330, 1300, 730 cm-1; HRMS (FAB) calcd for C39H40N8S6O8
941.1372, found 941.1368.
159.7; IR (KBr) 3420, 2920, 1600, 1510, 1340, 1300, 730 cm-1
;
HRMS (FAB) calcd for C25H31O4N6S4 607.1290, found 607.1264.
4-(Dim eth yla m in o)-2,6-bis[[(2-m er ca p toeth yl)a m in o]-
m eth yl]p yr id in e (2). To a solution of 17 (43.6 mg, 0.0718
mmol) in methanol (11 mL) was added sodium borohydride
(40.9 mg, 1.08 mmol). The mixture was stirred at room
temperature for 1 h and concentrated in vacuo. Water (14 mL)
was added, and then the solution was acidified to pH 1 with 1
N aqueous HCl and washed with dichloromethane (45 mL ×
4). The aqueous solution was neutralized (pH 6-7) with
aqueous NaHCO3, and the mixture was extracted with dichlo-
romethane (45 mL × 5). The organic layer was concentrated
in vacuo to afford colorless oil 2 (16.3 mg, 75%). HCl salt of 2
(pale yellow solid, hygroscopic) was also prepared by concen-
trating the acidic aqueous solution obtained after the acidifica-
tion and dichloromethane washing of the above reaction
4-(Dim eth ylam in o)-2,6-bis[[N-[3-[(2-n itr oph en yl)dith io]-
p r op yl]a m in o]m eth yl]p yr id in e (18). Compound 18 was
synthesized from 16 according to the same procedure as that
for 17. Compound 18 was obtained as yellow solid (90%
yield): 1H NMR (CDCl3) δ 1.90 (quint, J ) 7.0 Hz, 4H), 2.74
(t, J ) 7.0 Hz, 4H), 2.82 (t, J ) 7.0 Hz, 4H), 2.91 (br s, 2H),
3.00 (s, 6H), 3.76 (s, 4H), 6.39 (s, 2H), 7.27-7.42 (m, 2H), 7.62-
7.76 (m, 2H), 8.21-8.34 (m, 4H); 13C NMR (CD3OD) δ 30.5,
37.5, 40.3, 48.9, 55.6, 105.9, 127.9, 128.5, 129.3, 136.2, 138.8,
147.8, 158.3, 159.2; IR (KBr) 3430, 2920, 1600, 1510, 1330,
1300, 730 cm-1; HRMS (FAB) calcd for C27H35O4N6S4 635.
1602, found 635.1594.
4-(Dim eth ylam in o)-2,6-bis[[(3-m er captopr opyl)am in o]-
m eth yl]p yr id in e (3). Compound 3 was synthesized from 18

Metal-Chelating Inhibitors of HIV-EP1
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 507
according to the same procedure as that for 2 except that
dithiothreitol (2.3 equiv) was used instead of sodium borohy-
dride. Compound 3 was obtained as a colorless oil (67% yield).
3: 1H NMR (CDCl3) δ 1.85 (quint, J ) 7.0 Hz, 4H), 2.63 (t, J
) 7.0 Hz, 4H), 2.76 (t, J ) 7.0 Hz, 4H), 3.00 (s, 6H), 3.75 (s,
4H), 6.42 (s, 2H); 13C NMR (CD3OD) δ 23.5, 35.0, 40.3, 49.1,
55.5, 106.0, 158.3, 158.8; HRMS calcd for C15H28N4S2 328.1755,
found 328.1743. HCl salt of 3: pale yellow solid, hygroscopic;
1H NMR (CD3OD) δ 2.11 (quint, J ) 6.5 Hz, 4H), 2.67 (t, J )
6.5 Hz, 4H), 3.3-3.4 (m, 4H), 3.33 (s, 6H), 4.47 (s, 4H), 7.34
(s, 2H); 13C NMR (CD3OD) δ 22.7, 32.1, 41.8, 48.9, 49.6, 110.7,
Refer en ces
(1) Maekawa, T.; Sakura, H.; Sudo, T.; Ishii, S. Putative Metal
Finger Structure of the Human Immunodeficiency Virus Type
1 Enhancer Binding Protein HIV-EP1. J . Biol. Chem. 1989, 264,
14591-14593.
(2) Fan, C.-M.; Maniatis, T. A DNA-Binding Protein Containing Two
Widely Separated Zinc Finger Motifs That Recognize the Same
DNA Sequence. Genes Dev. 1990, 4, 29-42.
(3) Baldwin, A. S., J r.; Leclair, K. P.; Singh, H.; Sharp, P. A. A Large
Protein Containing Zinc Finger Domains Binds to Related
Sequence Elements in the Enhancers of the Class I Major
Histocompatibility Complex and Kappa Immunoglobuline Genes.
Mol. Cell. Biol. 1990, 10, 1406-1414.
(4) Baeuerle, P. A. The Inducible Transcription Activator NF-κB:
Regulation by Distinct Protein Subunits. Biochim. Biophys. Acta
1991, 1072, 63-80.
(5) Seeler, J .-S.; Muchardt C.; Suessle A.; Gaynor, R. B. Transcrip-
tion Factor PRDII-BF1 Activates Human Immunodeficiency
Virus Type 1 Gene Expression. J . Virol. 1994, 68, 1002-1009.
(6) Otsuka, M.; Fujita, M.; Sugiura, Y.; Ishii, S.; Aoki, T.; Yamamoto,
T.; Inoue, J . Novel Zinc Chelators Which Inhibit the Binding of
HIV-EP1 (HIV Enhancer Binding Protein) to NF-κB Recognition
Sequence. J . Med. Chem. 1994, 37, 4267-4269.
(7) Otsuka, M.; Fujita, M.; Aoki, T.; Ishii, S.; Sugiura, Y.; Yamamoto,
T.; Inoue, J . Novel Zinc Chelators with Dual Activity in the
Inhibition of the κB Site-Binding Proteins HIV-EP1 and NF-
κB. J . Med. Chem. 1995, 38, 3264-3270.
(8) Glusker, J . P. Structural Aspects of Metal Liganding to Func-
tional Groups in Proteins. Adv. Protein Chem. 1991, 42, 1-76.
(9) Hallman, P. S.; Perrin, D. D.; Watt, A. E. The Computed
Distribution of Copper (II) and Zinc (II) Ions Among Seventeen
Amino Acids Present in Human Blood Plasma. Biochem. J . 1971,
121, 549-555.
(10) Giroux, E. L.; Henkin, R. I. Competition for Zinc Among Serum
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145.7, 160.1; IR (KBr) 3390, 2920, 2760, 1640, 1570, 1420 cm-1
;
HRMS (FAB) calcd for C15H29N4S2 329.1834, found 329.1840.
4-(Dim eth yla m in o)-2,6-bis[[[2-(m eth ylth io)eth yl]a m i-
n o]m eth yl]p yr id in e (7). Compound 7 was synthesized from
[2-(methylthio)ethyl]amine hydrochloride (13)15 according to
the same procedure as that for 4. Compound 7 was obtained
as pale pink oil (19% yield): 1H NMR (CDCl3) δ 2.12 (s, 6H),
2.75 (t, J ) 6.0 Hz, 4H), 2.97 (t, J ) 6.0 Hz, 4H), 3.21 (s, 6H),
4.07 (s, 4H), 5.13 (br s, 2H), 6.66 (s, 2H); 13C NMR (CD3OD) δ
15.9, 34.2, 40.5, 48.8, 53.4, 106.3, 155.9, 158.9; IR (KBr) 2910,
2320, 1610, 1540, 1430, 1390, 1120 cm-1; HRMS (FAB) calcd
for C15H29N4S2 329.1834, found 329.1821.
4-(Dim eth yla m in o)-2,6-bis[[[3-(m eth ylth io)p r op yl]a m i-
n o]m eth yl]p yr id in e (8). Compound 8 was synthesized from
[3-(methylthio)propyl]amine hydrochloride (14) according to
the same procedure as that for 4. Compound 8 was obtained
as pale blue oil (13% yield): 1H NMR (CDCl3) δ 2.03 (quint, J
) 6.0 Hz, 4H), 2.10 (s, 6H), 2.61 (t, J ) 6.0 Hz, 4H), 3.02 (t, J
) 6.0 Hz, 4H), 3.10 (s, 6H), 4.09 (s, 4H), 5.94 (br s, 2H), 6.53
(s, 2H); 13C NMR (CD3OD) δ 16.0, 28.3, 32.8, 40.4, 50.2, 53.6,
106.6, 154.7, 158.5; IR (KBr) 2910, 2310, 1610, 1540, 1430,
1390, 1110 cm-1; HRMS calcd for C17H32N4S2 356.2068, found
356.2050.
(11) Otsuka, M; Satake, H.; Sugiura, Y.; Murakami, S.; Shibasaki,
M; Kobayashi, S. Restructuring of the Bleomycin Metal Core.
Novel Oxygen-Activating Ligands with Symmetrized Structure.
Tetrahedron Lett. 1993, 34, 8497-8500.
Electr op h or etic Mobility Sh ift Assa y (EMSA). Double-
stranded oligonucleotide containing a κB site from the mouse
immunoglobulin κ light-chain enhancer
(12) Tombeux, J . J .; Goeminne, A. M.; Eeckhaut, Z. A Potentiometric
Study of the Silver(I) Complexes of Some Sulfur-Containing
Amines. J . Inorg. Nucl. Chem. 1977, 39, 1655-1658.
(13) Papaioannou, G. New Mannich Bases Derived from Certain
Tetracyclines. Chem. Chron. 1981, 10, 243-252; Chem. Abstr.
1983, 98, 53479t.
(14) Carroll, F. I.; Dickson, H. M.; Wall, M. E. Organic Sulfur
Compounds. III. Synthesis of 2-(Substitutedalkylamino)et-
hanethiols. J . Org. Chem. 1965, 30, 33-38.
(15) Burfield, D. R.; Gan, S.-N.; Smithers, R. H. Reactions of a Mono-
and a Tri-substituted Epoxide with Some Simple and â-Substi-
tuted Primary Amines; Novel Examples of Electrophilic Anchi-
meric Assistance. J . Chem. Soc., Perkin Trans. 1 1977, 666-
671.
(16) Reduction of 17 or 19 with 3 equiv of DTT proceeded immediately
and completely to afford 2. Similarly, 18 or 20 gave 3.
(17) Compounds 4, 5, 7, and 8 at 300 µM concentration and compound
6 at 30 µM concentration also showed some inhibitory effect on
the DNA binding of HIV-EP1 (Figure 4).
(18) NMR monitoring of zinc titration of compound 2 in the binding
buffer used in the EMSA assay suggested that compound 2
evidently forms a zinc complex when equimolar zinc was added.
On the basis of the NMR spectra, it was not likely that compound
2 tightly bound 3 equiv of extra zinc added after the inhibition
reaction to interfere the recovery of DNA binding.
(19) Monzingo, A. F.; Matthews, B. W. Structure of a Mercaptan-
Thermolysin Complex Illustrates Mode of Inhibition of Zinc
Proteases by Substrate-Analogue Mercaptans. Biochemistry
1982, 21, 3390-3394.
(20) Roderick, S. L.; Fournie-Zaluski, M. C.; Roques, B. P.; Matthews,
B. W. Thiorphan and retro-Thiorphan Display Equivalent In-
teractions When Bound to Crystalline Thermolysin. Biochem-
istry 1989, 28, 1493-1497.
5′-AGCTTCAGAGGGGACTTTCCGAGAGG-3′
3′-AGTCTCCCCTGAAAGGCTCTCCAGCT-5′
was phosphorylated with polynucleotide kinase in the presence
of [γ-32P]ATP (Du Pont, >5000 Ci/mmol) and purified by a G-50
Sephadex spin column. The DNA binding domain of HIV-EP1
was expressed as a fusion protein with â-galactosidase in
bacteria. About 500 ng of HIV-EP1 was used for EMSA. After
incubation of the each reaction mixture containing pH 7.0
binding buffer (15 mM Tris‚HCl, pH 7.0, 75 mM NaCl, 1.5 mM
EDTA,7 1.5 mM dithiothreitol, 7.5% glycerol, 0.3% NP-40, 1
µg/µL BSA), 4% methanol, 2.4 µg of poly(dI-dC), HIV-EP1, and
each compound at room temperature for 30 min, labeled DNA
probe (30 000 cpm) was added, and the mixture was further
incubated at room temperature for 15 min. The sample in a
volume of 24 µL was loaded onto 4% polyacrylamide gels and
electrophoresed at 150 CV.
Ack n ow led gm en t. The authors thank Dr. S. Ishii,
Tsukuba Life Science Center, The Institute of Physical
and Chemical Research (RIKEN), for kind gift of HIV-
EP1 and also Drs. K. Tanaka and A. Kusai, J EOL Ltd.
for the measurement of fast atom bombardment high-
resolution mass spectra. The present study was finan-
cially supported in part by a Grant-in-Aid for Scientific
Research (No. 07672420) from the Ministry of Educa-
tion, Science, and Culture, J apan. A fellowship of the
J apan Society for the Promotion of Science to M.F. is
acknowledged.
J M950831T