Chemical modification of coumarin dimer and HIV-1 integrase inhibitory activity

Article abstract of DOI:10.1248/cpb.50.1634

A systematic series of chemically modified coumarin dimmers has been synthesized and tested for their inhibitory activity against HIV-1 integrase. We observed that modified coumarin dimmers containing hydrophobic moiety on the linker display potent inhibi

Full text of DOI:10.1248/cpb.50.1634

1634
Notes
Chem. Pharm. Bull. 50(12) 1634—1637 (2002)
Vol. 50, No. 12
Chemical Modification of Coumarin Dimer and HIV-1 Integrase
Inhibitory Activity
Pili Chih-Min MAO,a Jean-Francois MOUSCADET,b Herve LEH,b Christian AUCLAIR,b and
Ling-Yih HSU*,c
a Department of Pharmacy, Veterans General Hospital Kaohsiung; 386 Ta-Chung 1st RD., Kaohsiung: c School of
Pharmacy, National Defense Medical Center; P.O. Box 90048–508, Neihu, Taipei, Taiwan, ROC: and b L.B.P.A, CNRS
UMR8532, Ecole Normale Superieure de Cachan; 61, avenue du Président Wilson, F-92335, Cachan France.
Received August 30, 2002; accepted September 20, 2002
A systematic series of chemically modified coumarin dimmers has been synthesized and tested for their in-
hibitory activity against HIV-1 integrase. We observed that modified coumarin dimmers containing hydrophobic
moiety on the linker display potent inhibitory activities.
Key words chemical modification; coumarin dimmer; HIV-1 integrase; inhibitor
Integration of reversely transcribed viral DNA of the benzaldehyde (3a—f), and arylcarbonyloxybenzaldehyde
human immunodeficiency virus type 1 into chromosomal (3a—e) by using 2-chloroethanol, alkyl- and aryl-sulfonyl
DNA of newly infected cells is an essential step of the viral chloride, and 4-substituted benzoyl chloride, respectively.
replicative cycle. HIV-1 integrase (HIV-1 IN) is responsible Subsequent coupling with 4-hydroxycoumarin provided tar-
for the integration. This enzyme catalyzes two reactions re- get compounds (2), (4a—f) and (6a—e) in moderate to high
ferred to as 3Ј-processing (cleavage of a dinucleotide from yield.
each end of viral DNA) and strand transfer (insertion of the
viral DNA into the host cellular DNA).1—3) Because the inte- Results and Discussion
gration process is essential for the replication of HIV and the
HIV-1 IN inhibition assays were carried out in the pres-
enzyme appears to be absent in the mammalian host, HIV-1 ence of low amounts of purified recombinant integrase
IN represents a potential target for the development of non- (50 nM) in the presence of 7.5 mM Mg2ϩ as the cationic cofac-
toxic antiviral therapeutic agents.4,5) Systematic screening of tor, using 21-mer double-strand oligonucleotide substrate.
potential inhibitors has been undertaken using mostly puri- The compounds were screened for their inhibitory activity in
fied integrase-based assays. From such screens several inte- a 3Ј-processing assay which was performed as described pre-
grase inhibitor classes have now been identified.6—10) The viously.14) Effects against the strand transfer activity were
tetrameric 4-hydroxycoumarin (NSC 158393, I) was first re- evaluated during the same assay from the homologous inte-
ported against HIV-1 IN by Mazumder et al.11) with potent gration events and found to be no significant differences
inhibition of both 3Ј-processing (IC50ϭ1.5 mM) and strand from 3Ј-processing inhibition, thus confirming that
transfer (IC50ϭ0.8 mM). In order to delineate structural fea- coumarins affect equally both steps of the integration reac-
tures of NSC 158393 necessary for high inhibitory activity, tion. The inhibitory effects of 3Ј-processing are summarized
Zhao et al.12) disclosed the minimum active pharmacophore in Table 1. All compounds tested were found to have good to
(II), i.e. a coumarin dimmer containing an aryl substituent on potent HIV-1 IN inhibitory activity against the recombinant
the central linker methylene. We also recently reported 4-hy- wild-type enzyme. The inhibitory activity of NSC 158393 (I)
droxycoumarin dimmers bearing aniline mustard moiety and II were included for a comparison. On the basis of their
(III)13) and reasoned the mustard moiety might react with structure, the compounds tested can be roughly classified as
nearby nucleophile of the coumarin–IN complex binding site (A), compounds with aryl-substituted attached on the ben-
to form a covalent bonding and to improve the inhibitory ac- zoyloxy- or sulfonyloxy-linker (4b—f, 6a—e) and (B), com-
tivity. However, these compounds seem to fail forming cova- pounds without aryl-substituted attached on the linker (2,
lent binding at coumarin–IN complex binding site because 4a). Class A compounds exhibited potent inhibitory activity
the compounds with the appending of aniline mustard did not with IC50 value in the range of 0.5—3.9 mM. Their inhibitory
markedly increase the inhibitory activity. This finding indi- activities were superior to II and comparable to NSC 158393
cates a probably non-nucleophile or hydrophobic region at (I). However, Class B compounds showed moderate to non
the coumarin–IN complex binding site. Therefore we pay our inhibitory activity with IC50 value 23.7 and Ͼ100 mM, respec-
efforts on the synthesis of coumarin dimmer appending with tively. It is of interest to note: First that compounds with an
hydrophobic moiety (IV). In this report, we describe the syn- aryl-substituted linker (4b—f, 6a—e) possessed higher in-
thesis of chemically modified coumarin dimmer analogues hibitory activity than compounds without this substitution (2,
and their inhibitory activity on HIV-1 IN.
4a); And second, that a benzoyloxy- or sulfonyloxy-linker
seemed necessary for the inhibitory activity since com-
pounds 2 devoid of benzoyloxy or sulfonyloxy linkage did
Chemistry
The synthetic route to the target compounds (2), (4a—f) not show any activity. Based on this observation, we postu-
and (6a—e) are outlined in Chart 1. Starting 4-hydroxyben- late that an interaction formed between a hydrophobic sur-
zaldehyde was transformed to their corresponding 4-(2-hy- face located in the close vicinity of the coumarin–IN com-
droxyethoxy)benzaldehyde (1), alkyl- and aryl-sulfonyloxy- plex binding site and the aromatic moiety linked to coumarin
* To whom correspondence should be addressed. e-mail: hly@ndmctsgh.edu.tw
© 2002 Pharmaceutical Society of Japan

December 2002
1635
Chart 1
Table 1. HIV-1 Integrase Inhibitory Activities of Modified Coumarin
Dimmers
Entry
Compound
Yield (%)
3Ј-Processing (IC50 mM)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
2
4a
4b
4c
4d
4e
4f
6a
66
81
83
66
57
56
56
67
70
68
64
60
Ͼ100.0
23.7
3.9
1.9
3.1
1.5
2.1
2.6
2.9
1.1
0.5
6b
6c
6d
6e
NSC 158393 (I)
(II)
3.1
1.1
43.0
Fig. 1
molecule is important for strengthening the anchorage of the
active compound. However, this assumption needs further
verification.
In conclusion, twelve new chemically modified coumarin
dimmer analogues have been synthesized. Among these eas-
ily available coumarin dimmers, ten were found to be active
Experimental
General Melting points (mp) were taken on a BUCHI 530 apparatus
and are uncorrected. Merck Art No105554 plates precoated with Silica gel
60 containing fluorescent indicator were used for thin-layer chromatography,
and Silica gel 60 (Merck Art No 109385, 230—400 mesh) was employed for
column chromatography. Evaporations were carried out at Ͻ50 °C using a
rotary evaporator at reduced pressure (water aspirator). 1H- and 13C-NMR
molecules against HIV-1 IN. These active molecules all con- spectra were obtained at Varian 300 NMR spectrometer at 300 and 75 MHz,
respectively. Where necessary, deuterium exchange experiments were used
to obtained proton shift assignments. Mass spectra were recorded on a JEOL
J.M.S-300 spectrophotometer. Analytical samples were dried under reduced
pressure at 78 °C in the presence of P2O5 for at least 12 h unless otherwise
specified. Elemental analyses were obtained from Perkin-Elmer 2400 Ele-
mental Analyzer.
tain hydrophobic moiety on the linker, which might have
some contribution on the inhibitory activity. This finding
may be useful in future studies concerned with the develop-
ment of potent new HIV-1 IN inhibitors.

1636
4-(2-Hydroxyethoxy)-benzaldehyde15) (1) The reaction mixture of 2-
Vol. 50, No. 12
Condensation of Benzaldehydes with 4-Hydroxycoumarin (2, 4a—f,
6a—e).17,18) General Procedure The reaction mixture of 4-hydroxy-
coumarin (2 eq), benzaldehyde (1 eq) in ethanol was heated under reflux for
18 h. The mixture was concentrated under reduced pressure to furnished
product. Pure products were obtained under recrystalliztion with certain sol-
vents.
3,3Ј-[4-(2-Hydroxyethoxy)benzylidene]bis(4-hydroxy-coumarin) (2): Rf
0.16 (n-hexane/EtOAcϭ1/4). mp: 176—177 °C (EtOH). H-NMR (DMSO-
d6) d: 3.69, 3.91 (m, 2H each, OCH2CH2OH), 6.28 (s, 1H, CH), 6.78—7.91
(m, 12H, Ar-H). MS m/z: 472 (Mϩ). Anal. Calcd for C27H20O8: C, 68.64; H,
4.27. Found: C, 68.49; H, 4.51.
chloroethanol (3.3 g, 41 mmol), 4-hydroxybenzaldehyde (5.0 g, 41 mmol),
sodium carbonate (5.9 g, 56 mmol) and DMF (15 ml) was heated under re-
flux for 24 h. After cooling, the mixture was poured into water. Extraction
with ethyl acetate, drying with magnesium sulfate the mixture was concen-
trated under reduced pressure. Column chromatography of the residue on
silica gel with 1 : 1 n-hexane/EtOAc as eluent gave 1 (4.4 g, 65%) as a light
1
1
brown oil. Rf 0.45 (n-hexane/EtOAcϭ1/1). H-NMR (CDCl3)d: 3.79—4.25
(m, 4H, CH2), 6.96 (d, 2H, Jϭ8.5 Hz), 7.77 (d, 2H, Jϭ8.5 Hz), 9.81 (s, 1H,
CHO). MS m/z: 166 (Mϩ). Anal. Calcd for C9H10O3: C, 65.05; H, 6.07.
Found: C, 64.96; H, 6.20.
3,3Ј-[4-[(Methylsulfonyl)oxy]benzylidene]bis(4-hydroxy-coumarin) (4a):
Rf 0.32 (n-hexane/EtOAcϭ1/4). mp: 235—236 °C (Actone). 1H-NMR
(DMSO-d6) d: 3.34 (s, 3H, CH3), 6.30 (s, 1H, CH), 7.20—7.90 (m, H, Ar-
H). MS m/z: 506 (Mϩ). Anal. Calcd for C26H18O9S: C, 61.66; H, 3.58.
Found: C, 61.33; H, 3.65.
Sulfonic Acid 4-Formylphenyl Ester (3a—f). General Procedure
Freshly distilled methanesulfonyl chloride (2.33 g, 20 mmol) was added to a
solution of 4-hydroxybenzaldehyde (1.24 g, 10 mmol) in dry pyridine (10
ml) under ice bath. The reaction mixture was stirred at room temperature for
4 h and then poured into 10% HCl solution. Extraction with ethyl acetate,
washing with saturated sodium bicarbonate and brine, drying with magne-
sium sulfate the mixture was concentrated under reduced pressure. Column
chromatography of the residue on silica gel with 1 : 1 n-hexane/EtOAc as
eluent gave 3a (1.3 g, 64%) as a light brown oil.
3,3Ј-[4-[[(4-Methylphenyl)sulfonyl]oxy]benzylidene]bis(4-hydroxy-
coumarin) (4b): Rf 0.34 (n-hexane/EtOAcϭ1/4). mp: 240—241 °C (Ac-
1
tone). H-NMR (DMSO-d6) d: 2.39 (s, 3H, CH3), 6.27 (s, 1H, CH), 6.83—
7.87 (m, 16H, Ar-H). MS m/z: 582 (Mϩ). Anal. Calcd for C32H22O9S: C,
65.97; H, 3.81. Found: C, 66.01; H, 3.82.
3b—f were obtained under the same condition.
3,3Ј-[4-[[(4-Methoxyphenyl)sulfonyl]oxy]benzylidene]bis(4-hydroxy-
coumarin) (4c): Rf 0.41 (n-hexane/EtOAcϭ1/4). mp: 246—247 °C (Actone).
1H-NMR (DMSO-d6) d: 3.89 (s, 3H, OCH3), 6.05 (s, 1H, CH), 6.92—8.09
(m, 16H, Ar-H). MS m/z: 598 (Mϩ). Anal. Calcd for C32H22O10S: C, 64.21;
H, 3.70. Found: C, 64.26; H, 3.60.
Methanelsulfonic Acid 4-Formylphenyl Ester (3a): Rf 0.55 (n-
hexane/EtOAcϭ1/1). mp: 62—63 °C (lit.16) 60—61 °C). 1H-NMR (CDCl3)
d: 3.22 (s, 3H, CH3), 7.46, 7.76 (d, 2H each, Jϭ8.5 Hz, Ar), 10.0 (s, 1H,
CHO). MS m/z: 200 (Mϩ). Anal. Calcd for C8H8O4S: C, 47.99; H, 4.03.
Found: C, 48.02; H, 4.20.
3,3Ј-[4-[[(4-tert-Butylphenyl)sulfonyl]oxy]benzylidene]bis(4-hydroxy-
coumarin) (4d): Rf 0.27 (n-hexane/EtOAcϭ1/4). mp: 149—150 °C (Ac-
Toluenesulfonic Acid 4-Formylphenyl Ester (3b): Rf 0.67 (n-hexane/
EtOAcϭ1/1). mp: 76—77 °C. 1H-NMR (CDCl3) d: 2.45 (s, 3H, CH3),
7.15—7.84 (m, 8H, Ar-H), 9.96 (s, 1H, CHO). MS m/z: 276 (Mϩ). Anal.
Calcd for C14H12O4S: C, 60.86; H, 4.38. Found: C, 60.62; H, 4.20.
4-Methoxybenzenesulfonic Acid 4-Formylphenyl Ester (3c): Rf 0.70 (n-
hexane/EtOAcϭ1/1). mp: 86—87 °C. 1H-NMR (CDCl3) d: 3.89 (s, 3H,
OCH3), 6.97—7.85 (m, 8H, Ar-H), 9.97 (s, 1H, CHO). MS m/z: 292 (Mϩ).
Anal. Calcd for C14H12O5S: C, 57.52; H, 4.14. Found: C, 57.62; H, 4.27.
4-tert-Butylbenzenesulfonic Acid 4-Formylphenyl Ester (3d): Rf 0.63 (n-
hexane/EtOAcϭ1/1). mp: 115—116 °C. 1H-NMR (CDCl3) d: 1.35 (s, 9H,
CH3), 7.19—7.87 (m, 8H, Ar-H), 9.98 (s, 1H, CHO). MS m/z: 318 (Mϩ).
Anal. Calcd for C17H18O4S: C, 64.13; H, 5.70. Found: C, 63.98; H, 5.57.
1-Naphthalenesulfonic Acid 4-formylphenyl Ester (3e): Rf 0.54 (n-
hexane/EtOAcϭ1/1). mp: 123—124 °C. 1H-NMR (CDCl3) d: 6.69—8.20
(m, 11H, Ar-H), 9.92 (s, 1H, CHO). MS m/z: 312 (Mϩ). Anal. Calcd for
C17H12O4S: C, 65.37; H, 3.87. Found: C, 65.68; H, 3.57.
1
tone). H-NMR (DMSO-d6) d: 1.26 (s, 9H, CH3), 6.34 (s, 1H, CH), 6.89—
7.92 (m, 16H, Ar-H). MS m/z: 624 (Mϩ). Anal. Calcd for C35H28O9S: C,
67.30; H, 4.52. Found: C, 67.03; H, 4.76.
3,3Ј-[4-[[(1-Naphthalyl)sulfonyl]oxy]benzylidene]bis(4-hydroxy-
coumarin) (4e): Rf 0.21 (n-hexane/EtOAcϭ1/4). mp: Ͼ300 °C (Actone). 1H-
NMR (DMSO-d6) d: 6.20 (s, 1H, CH), 6.69—8.65 (m, 19H, Ar-H). MS m/z:
618 (Mϩ). Anal. Calcd for C35H22O9S: C, 67.95; H, 3.58. Found: C, 68.02;
H, 3.58.
3,3Ј-[4-[[(2-Naphthalyl)sulfonyl]oxy]benzylidene]bis(4-hydroxy-
coumarin) (4f): Rf 0.30 (n-hexane/EtOAcϭ1/1). mp: 230—232 °C (Actone).
1H-NMR (DMSO-d6) d: 6.29 (s, 1H, CH), 6.88—8.54 (m, 19H, Ar-H). MS
m/z: 618 (Mϩ). Anal. Calcd for C35H22O9S: C, 67.95; H, 3.58. Found: C,
67.75; H, 3.57.
3,3Ј-[4-(Benzoyloxy)benzylidene]bis(4-hydroxy-coumarin) (6a): Rf 0.43
1
(n-hexane/EtOAcϭ1/4). mp: 200—201 °C (EtOH). H-NMR (DMSO-d6) d:
2-Naphthalenesulfonic Acid 4-Formylphenyl Ester (3f): Rf 0.46 (n-
6.37 (s, 1H, CH), 7.12—8.14 (m, 17H, Ar-H). MS m/z: 532 (Mϩ). Anal.
Calcd for C32H20O8: C, 72.18; H, 3.79. Found: C, 72.16; H, 3.65.
3,3Ј-[4-(4-Chlorobenzoyloxy)benzylidene]bis(4-hydroxy-coumarin) (6b):
Rf 0.21 (n-hexane/EtOAcϭ1/4). mp: 147—148 °C (Acetone). 1H-NMR
(DMSO-d6) d: 6.41 (s, 1H, CH), 7.14—8.12 (m, 16H, Ar-H). MS m/z: 567
(Mϩ). Anal. Calcd for C32H19ClO8: C, 67.79; H, 3.38. Found: C, 67.49; H,
3.61.
1
hexane/EtOAcϭ1/1). mp: 83—84 °C. H-NMR (CDCl3) d: 7.16—8.01 (m,
9H, Ar-H), 8.37 (s, 2H, Ar-H), 9.92 (s, 1H, CHO). MS m/z: 312 (Mϩ). Anal.
Calcd for C17H12O4S: C, 65.37; H, 3.87. Found: C, 65.44; H, 3.66.
Benzoic Acid 4-Formylphenyl Ester (5a—e). General Procedure
Benzoyl chloride (2.8 g; 20 mmol) was added to a solution of 4-hydroxyben-
zaldehyde (1.24 g, 10 mmol) in dry pyridine (20 ml) under ice bath. The re-
action mixture was stirred at room temperature for 8 h. Extraction with ethyl
acetate, drying with magnesium sulfate the mixture was concentrated under
reduced pressure. Column chromatography of the residue on silica gel with
1 : 1 n-hexane/EtOAc as eluent gave 5a (1.45 g, 64%) as a yellow crystal.
5b—e were obtained under the same condition.
3,3Ј-[4-(4-Bromobenzoyloxy)benzylidene]bis(4-hydroxy-coumarin) (6c):
Rf 0.18 (n-hexane/EtOAcϭ1/4). mp: 159—160 °C (Acetone). 1H-NMR
(DMSO-d6) d: 6.38 (s, 1H, CH), 7.14—8.06 (m, H, Ar-H). MS m/z: 611
(Mϩ). Anal. Calcd for C32H19BrO8: C, 62.86; H, 3.13. Found: C, 63.02; H,
3.44.
3,3Ј-[4-(4-Methylbenzoyloxy)benzylidene]bis(4-hydroxy-coumarin) (6d):
Rf 0.26 (n-Hexane/EtOAcϭ1/4). mp: 164—165 °C (Acetone). 1H-NMR
(DMSO-d6) d: 2.50 (s, 3H, CH3), 6.32 (s, 1H, CH), 7.09—8.02 (m, 16H, Ar-
H). MS m/z: 546 (Mϩ). Anal. Calcd for C33H22O8: C, 72.52; H, 4.06. Found:
C, 72.59; H, 4.40.
3,3Ј-[4-(4-Methoxbenzoyloxy)benzylidene]bis(4-hydroxy-coumarin) (6e):
Rf 0.48 (n-hexane/EtOAcϭ1/4). mp: 159—160 °C (Acetone). 1H-NMR
(DMSO-d6) d: 3.85 (s, 3H, CH3), 6.37 (s, 1H, CH), 7.08—8.05 (m, 16H, Ar-
H). MS m/z: 562 (Mϩ). Anal. Calcd for C33H22O9: C, 70.46; H, 3.94. Found:
C, 70.28; H, 4.16.
HIV-1 Integrase Inhibitory Assay. Oligonucleotides Oligonucleotides
were purchased from Eurogentec and further purified on 18% acrylamide/
urea denaturing gel. U5B: GTGTGGAAAATCTCTAGCA; U5B-2: GT-
GTGGAAAATCTCTAG; U5A: 5Ј-ACTGCTAGAGATTTTCCACAC; ST1:
AGTGAATTAGCCCTTGGTCA-biotine; ST2: 5Ј-TGACCAAGGGCTAAT-
TCACT-biotine; U5B and U5B-2 were radiolabeled using T4 polynucleotide
kinase for respectively 3Ј-processing and strand transfer reactions.
HIV-1 Integrase Assays Wild-type HIV-1 integrase was purified as de-
scribsed previously.19) 3Ј-Processing assay was performed in a reaction vol-
Benzoic Acid 4-Formylphenyl Ester (5a): Rf 0.46 (n-hexane/EtOAcϭ1/1).
1
mp: 119—120 °C. H-NMR (CDCl3) d: 7.23—8.49 (m, 9H, Ar-H), 9.98 (s,
1H, CHO). MS m/z: 226 (Mϩ). Anal. Calcd for C14H10O3: C, 74.33; H, 4.46.
Found: C, 74.64; H, 4.46.
4-Chlorobenzoic Acid 4-Formylphenyl Ester (5b): Rf 0.38 (n-hexane/
EtOAcϭ4/1). mp: 104—105 °C. 1H-NMR (CDCl3) d: 7.39—8.14 (m, 8H,
Ar-H), 10.02 (s, 1H, CHO). MS m/z: 260 (Mϩ). Anal. Calcd for C14H9ClO3:
C, 64.51; H, 3.48. Found: C, 64.64; H, 3.46.
4-Bromobenzoic Acid 4-Formylphenyl Ester (5c): Rf 0.56 (n-hexane/
EtOAcϭ1/1). mp: 176—177 °C. 1H-NMR (CDCl3) d: 7.33—7.99 (m, 8H,
Ar-H), 9.96 (s, 1H, CHO). MS m/z: 305 (Mϩ). Anal. Calcd for C14H9BrO3:
C, 55.11; H, 2.97. Found: C, 55.44; H, 3.05.
4-Methylbenzoic Acid 4-Formylphenyl Ester (5d): Rf 0.35 (n-hexane/
EtOAcϭ4/1). mp: 74—75 °C. 1H-NMR (CDCl3) d: 2.45 (s, 3H, CH3),
7.26—8.09 (m, 8H, Ar-H), 10.01 (s, 1H, CHO). MS m/z: 240 (Mϩ). Anal.
Calcd for C15H12O3: C, 74.99; H, 5.03. Found: C, 74.64; H, 5.24.
4-Methoxybenzoic Acid 4-Formylphenyl Ester (5e): Rf 0.25 (n-hexane/
1
EtOAcϭ4/1). mp: 106—107 °C. H-NMR (CDCl3) d: 3.90 (s, 3H, OCH3),
6.98—8.13 (m, 8H, Ar-H), 10.01 (s, 1H, CHO). MS m/z: 256 (Mϩ). Anal.
Calcd for C15H12O4: C, 70.31; H, 4.72. Found: C, 70.14; H, 5.00.

December 2002
1637
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ume of 20 ml containing 0.025 pmol of labeled U5A/U5B double-stranded
DNA substrate and 1 pmol of integrase in buffer A [20 mM Hepes (pH 7.2),
10 mM MgCl2, 25 mM NaCl, 1 mM DTT]. Products were separated on a 18%
acrylamide/urea denaturing gel and quantified on a phosphoimager using
ImageQuant software (AmershamPharmaciaBiotech). Strand transfer reac-
tions were performed in triplicate in 96-well plates using 0.25 pmol of la-
beled U5A/U5B-2 double-stranded DNA substrate, 12 pmol of ST1/ST2 3Ј-
biotinylated target DNA and 2 pmol of integrase in buffer A in a final vol-
ume of 40 ml. Radiolabeled reaction products were bound to Streptavidin-
coated magnetic beads (DynaL), washed twice in buffer B (PBS buffer sup-
plemented with 0.025% tween 20 and 10 mg/ml BSA) and quantified on a
beta radiation counter. Inhibition in the presence of drugs is expressed as the
fractional product in percent of the control without drug.
Acknowledgments Financial support from National Science Council
(NSC 90-2320-B016-040) of the Republic of China is gratefully acknowl-
edged.
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