Synthesis, chromatographic resolution, and anti-human immunodeficiency virus activity of (±)-calanolide A and its enantiomers

Article abstract of DOI:10.1021/jm950797i

The anti-HIV agent (±)-calanolide A (1) has been synthesized in a five- step approach starting with phloroglucinol [→ 5 → 6 → 11 → 18 → (±)- 1], which includes Pechmann reaction, Friedel-Crafts acylation, chromenylation with 4,4-dimethoxy-2-methylbutan-2-ol, cyclization, and Luche reduction. Cyclization of chromene 11 to chromanone 18 was achieved by employing either acetaldehyde diethyl acetal or paraldehyde in the presence of trifluoroacetic acid and pyridine or PPTS. Luche reduction of chromanone 18 at lower temperature preferably yielded (±)-1. Reduction of chromone 12, synthesized by Kostanecki-Robinson reaction from chromene 11, failed to afford (±)-1. The synthetic (±)-1 has been chromatographically resolved into its optically active forms, (+)- and (-)-1. The anti-HIV activities for synthetic (±)-1, as well as resultant (+)- and (-)-1, have been determined. Only (+)-1 accounted for anti-HIV activity, which was similar to the data reported for the natural product, and (-)-1 was inactive.

Full text of DOI:10.1021/jm950797i

J . Med. Chem. 1996, 39, 1303-1313
1303
Syn th esis, Ch r om a togr a p h ic Resolu tion , a n d An ti-Hu m a n Im m u n od eficien cy
Vir u s Activity of (()-Ca la n olid e A a n d Its En a n tiom er s
Michael T. Flavin, J ohn D. Rizzo, Albert Khilevich, Alla Kucherenko, Abram K. Sheinkman,
†
†
†
Vilayphone Vilaychack, Lin Lin, Wei Chen, Eugenia Mata Greenwood, Thitima Pengsuparp, J ohn M. Pezzuto,
‡
Stephen H. Hughes, Thomas M. Flavin, Michael Cibulski, William A. Boulanger, Robert L. Shone, and
Ze-Qi Xu*
MediChem Research, Inc., 12305 South New Avenue, Lemont, Illinois 60439, Program for Collaborative Research in the
Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago,
8
33 South Wood Street, Chicago, Illinois 60612, and ABL-Basic Research Program, NCI-Frederick Cancer Research and
Development Center, Frederick, Maryland 21702
X
Received October 27, 1995
The anti-HIV agent (()-calanolide A (1) has been synthesized in a five-step approach starting
with phloroglucinol [f 5 f 6 f 11 f 18 f (()-1], which includes Pechmann reaction, Friedel-
Crafts acylation, chromenylation with 4,4-dimethoxy-2-methylbutan-2-ol, cyclization, and Luche
reduction. Cyclization of chromene 11 to chromanone 18 was achieved by employing either
acetaldehyde diethyl acetal or paraldehyde in the presence of trifluoroacetic acid and pyridine
or PPTS. Luche reduction of chromanone 18 at lower temperature preferably yielded (()-1.
Reduction of chromone 12, synthesized by Kostanecki-Robinson reaction from chromene 11,
failed to afford (()-1. The synthetic (()-1 has been chromatographically resolved into its
optically active forms, (+)- and (-)-1. The anti-HIV activities for synthetic (()-1, as well as
resultant (+)- and (-)-1, have been determined. Only (+)-1 accounted for anti-HIV activity,
which was similar to the data reported for the natural product, and (-)-1 was inactive.
In tr od u ction
to identify compounds targeting different stages of
HIV replication for therapeutic intervention such as
Saquinavir, a protease inhibitor, which has been ap-
Since human immunodeficiency virus (HIV) was
identified as the etiological cause of acquired immune
deficiency syndrome (AIDS) about 1 decade ago, the
1
0,13-17
proved by the FDA very recently.
1
As with other retroviruses, the genetic information
of HIV is encoded in RNA and must be converted into
DNA in the infected host cell. Only then can the viral
genes be transcribed and translated into proteins in the
provision for useful chemotherapeutic agents has been
very challenging. Four antiviral nucleoside agents,
AZT, ddC, ddI, and d4T, have been approved for clinical
treatment of AIDS and AIDS-related complex. Al-
though these drugs can extend the life of AIDS patients,
none are capable of curing the disease, and serious side
effects are induced. For example, treatment with AZT
leads to a suppression of bone-marrow formation which
often causes anemia and leukopenia, resulting in the
need for frequent blood transfusions.2 The use of ddI,
ddC, and d4T is associated with painful sensory-motor
peripheral neuropathy, as well as acute pancreatitis and
1
8
usual sequence.
The virally encoded reverse tran-
scriptase (RT) is the sole enzyme responsible for the
catalytic formation of unintegrated viral DNA from the
single-stranded RNA viral genome and, accordingly,
1
8-20
plays a key role in the life cycle of HIV.
It is
therefore logical to focus on this enzyme, which is not
required for normal cellular processes, as a target for
1
0,13,16-20
the development of anti-HIV drugs.
hepatotoxicity in some cases.3,4 Also, AZT, the standard
The nucleoside analogues approved for drug therapy
of AIDS target the viral RT enzyme and inhibit its
enzymatic activity by competing with the normal sub-
strates, deoxynucleoside triphosphates, after being ac-
tivated in the cell into triphosphates. These nucleoside
analogues lack a 2′-hydroxyl group and, when incorpo-
rated into the growing DNA chain, act as chain termi-
nators. Recently, a series of structurally diverse non-
nucleoside analogues, such as TIBO, nevirapine, pyridi-
none, BHAP, HEPT, TSAO, and R-APA, have been
antiviral therapy for initiating treatment of patients
with HIV infection, has a very short half-life in the body,
and high doses (250 mg) must be ingested every 4 h to
maintain a constant level of drug in the body. More
importantly, long-term treatment of patients with all
these drugs has led to emergence of drug-resistant HIV
strains;5-7 primary infections have also been found with
AZT-resistant HIV strains.8
The pandemic of AIDS is alarming with over 17
million people worldwide having been infected with
1
0,13,21
identified as HIV RT inhibitors.
Unlike nucleoside
analogues, these nonnucleoside compounds are highly
specific and selective, targeting only HIV-1 RT but not
cellular DNA or other DNA polymerases such as HIV-2
RT, thereby being relatively nontoxic to human cells.
9
HIV, and four million people have already progressed
1
0
to AIDS.
The need for other promising AIDS drug
candidates having improved selectivity, and activity
against HIV is extremely urgent.
9
,11,12
Several ap-
Although the mechanism of RT inhibition by HIV-1-
specific compounds is not clear, it is suggested that
binding of these compounds to RT is slow, reversible,
and noncompetitive with respect to template-primer and
deoxynucleoside triphosphates. A hydrophobic pocket,
as indicated by the X-ray crystal structures of HIV-1
proaches, including chemical synthesis, natural prod-
ucts screening, and biotechnology, have been utilized
†
University of Illinois at Chicago.
NCI-Frederick Cancer Research and Development Center.
‡
X
Abstract published in Advance ACS Abstracts, February 15, 1996.
0
022-2623/96/1839-1303$12.00/0 © 1996 American Chemical Society

1
304 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6
Flavin et al.
RT complexed with various nonnucleoside analogues
Sch em e 1
such as nevirapine,2
2-24
TIBO,
25,26
and R-APA, is
27
generally believed to be the binding site for the
HIV-1-specific inhibitors to interact with HIV-1 RT.
Such binding may result in conformational changes
of RT to significantly slow the rate of the chemical
reaction catalyzed by the enzyme2
8-30
or to interfere
with interaction between the enzyme and template-
2
2,23,25,27
primer,
thus suppressing translocation of the
template-primer following nucleotide incorporation. How-
ever, as in the case of the nucleoside analogues, the
rapid emergence of HIV strains,3 both in vitro and in
vivo, which are resistant to these nonnucleoside inhibi-
tors, has become a major concern that may affect the
further development of these compounds as monothera-
pies.
1
Certain coumarin derivatives, isolated from several
tropical plants of the genus Calophyllum, were recently
identified as HIV-1-specific nonnucleoside inhibitors,
among which (+)-calanolide A (1) and inophyllum B (2)
are the most potent.3
2,33
Since these compounds are
active not only against the AZT-resistant strain of HIV-1
3
4-37
but also against the pyridinone-resistant strain A17,
they belong to a second generation of HIV-1-specific
nonnucleoside inhibitors,38 a pharmacological class dif-
ferent from that of the previously known nonnucleoside
HIV-1 RT inhibitory drugs, and may represent a novel
anti-HIV chemotype for drug development. The A17
strain contains Tyr 181 Cys and Lys 103 Asn amino acid
changes and is cross-resistant to most of the other
nonnucleoside inhibitors such as TIBO, pyridinone,
nevirapine, HEPT, and diphenyl sulfone.3 Combina-
tions of (+)-calanolide A and AZT have been demon-
strated in vitro to synergistically inhibit virus strain
A17, and it has also been suggested that combination
of (+)-calanolide A with other HIV-1-specific nonnucleo-
side inhibitors such as HEPT and diphenyl sulfone
(
+)-, and (-)-calanolide A, which will be compared with
the results reported for the natural product, (+)-cal-
anolide A.
Ch em istr y
Calanolide A has three different rings, coumarin,
chromene, and chromane, built around a phloroglucinol
core. Accordingly, our synthetic strategy was to utilize
phloroglucinol as a starting material and then build the
coumarin ring followed by the chromene ring. The
chromane ring is introduced last, the stereochemistry
of which plays a very important role in maintaining
7
3
7
would be beneficial.
3
2,33
antiviral activity.
4
4
Thus, Pechmann reaction on phloroglucinol with
ethyl butyrylacetate in the presence of concentrated
sulfuric acid afforded 5,7-dihydroxy-4-propylcoumarin
(5) quantitatively (Scheme 1). Product yield and purity
were dependent on the amount of sulfuric acid used; 1
mL of H2SO4/g of ethyl butyrylacetate led to optimal
results in a 100 g scale reaction. When polyphosphoric
acid was used, only a 50% yield of coumarin 5 was
obtained.
Acylation of coumarin 5 with propionyl chloride in the
presence of AlCl3 led to a mixture of 8-acylated coumarin
6 along with 6-acylated coumarin 7 and 6,8-bis-acylated
coumarin 8 in a ratio of 1:1:1 in 50% yield (Scheme 1).
The desired 8-acylated product 6 was separated from 7
and 8 by silica gel chromatography. Structural assign-
ments of the 8- or 6-acyl derivatives 6 and 7 were made
Supplies of the compound from plant material are
extremely limited and difficult to obtain, since the
original natural source of (+)-calanolide A was destroyed
and other nearby members of the same species did not
contain the same compound. Further pharmacological
investigation of this class of compounds, as well as
related analogues, is dependent upon the ready avail-
ability of significant amounts of material. Thus, a
practical total synthesis of calanolide A is of great
4
5
by comparison of UV spectra with analogues and
4
6
confirmed by Gibbs color reaction and spectroscopic
data. A route developed for the synthesis of Mammea
4
5
coumarins was initially attempted for preparation of
compound 6, but it proved too awkward and low
yielding.
importance. Total synthesis of (()-calanolide A has
been recently reported by us39 and others;
40,41
chiral
4
2
synthesis of (+)-calanolide A has also been suggested
Since various factors can affect the Friedel-Crafts
4
3
47
and developed. Herein we wish to communicate a full
account of our synthesis of (()-calanolide A, as well as
the chromatographic resolution of the synthesized race-
mate into its enantiomers and further characteriza-
tion of the antiviral properties of the three entities, (()-,
acylation, further investigations to improve the selec-
tivity and yield for the 8-acylated product 6 were
conducted. It was found that 8-acylated coumarin 6
could be obtained as a single product in 45% yield by
replacing propionyl chloride with propionic anhydride

Synthesis of Anti-HIV Agent (()-Calanolide A
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6 1305
under modified conditions. No trace of 7 or 8 was
Sch em e 2
observed, although a small amount of the 7-monoester
9
did form. The desired product 6 was purified without
the use of column chromatography. Temperature was
determined to be an important factor in this reaction.
Addition of a mixture of propionic anhydride and AlCl3
into a preheated (75 °C) mixture of coumarin 5 and
AlCl3 was essential for the selective formation of 6. At
ambient temperature, the reaction of propionic anhy-
dride with the coumarin 5 in the presence of AlCl3 led
to formation of 7-monoester 9 as a single product when
either dichloromethane, 1,2-dichloroethane, a mixture
of dichloromethane and cyclohexane, or THF was used
as solvent. Excessive propionic anhydride resulted in
formation of the 5,7-diester 10 (Scheme 1). The order
of addition of the reagents (AlCl3, propionic anhydride,
and coumarin 5) did not affect the outcome of the
reaction at ambient temperature. The structure of
7
-monoester 9 was in full agreement with all of the
spectroscopic data and further confirmed by a positive
Gibb’s test.46
Attempts to convert both 9 and 10 under Fries
conditions into the corresponding 8-acylated coumarin
6
4
were unsuccessful. It was reported that 5,7-diacetoxy-
-phenylcoumarin underwent Fries rearrangement to
4
8
form 6,8-diacetyl-5,7-dihydroxy-4-phenylcoumarin. The
same was observed for the 5,7-diester 10. In refluxing
triglyme, 5,7-diester 10 was decomposed into the start-
ing coumarin in the presence of AlCl3. The 7-monoester
9
was stable in refluxing pyridine, but in 1,2-dichloro-
ethane with AlCl3, it afforded both the 8- or 6-acylated
products 6 and 7 in a ratio varying from 1:1 to 3:7,
depending upon the reaction temperature (Scheme 1).
The chromene ring was then introduced upon treat-
ment of the 8-acylated coumarin 6 with 4,4-dimethoxy-
2
-methylbutan-2-ol.45,49 The reaction proceeded readily
in the presence of pyridine to provide chromene 11
Scheme 2). Azeotropic removal of water and MeOH
(
formed during the reaction by using toluene as solvent
helped to facilitate the reaction upon scaleup (see the
Experimental Section). The H NMR signals for
1
2 could be reduced to a saturated alcohol, that is, (()-
1
calanolide A (1) and its diastereoisomers. However,
results indicated that the γ-pyrone in chromone 12 is
quite resistant to reduction of the double bond and/or
the keto group, and the reduction failed to afford (()-1.
Instead, the R-pyrone system (coumarin) in chromone
chromene 11 were solvent-dependent. Thus, H-7 in the
chromene ring and H-3 in the coumarin ring were
shifted downfield from 5.58 ppm in CDCl3 to 5.79 ppm
in DMSO-d6 and 6.00 to 6.12 ppm, respectively; how-
ever, H-8 and the CH2 group in the propionyl side chain
were shifted upfield from 6.73 ppm in CDCl3 to 6.62 ppm
in DMSO-d6 and 3.35 to 3.19 ppm, respectively. It has
been reported that the chemical shifts for H-8 of the
1
2 proved to be more reactive. A variety of reduced
products were isolated depending upon the reducing
agent used and the reaction conditions utilized (Scheme
2
er’s yeast, L-Selectride, and DIBAL preferentially re-
acted at the lactone position to lead to the formation of
diol 13 as the major product. In contrast, n-Bu3SnH
produced chromene ring-opened compound 14 in a 15%
yield and a mixture which contained unsaturated
alcohol 15, as indicated by H NMR spectroscopy. The
use of SmI2/HMPA and 9-BBN resulted in a similar
mixture to that produced by n-Bu3SnH, while [(Ph3P)-
CuH]6 did not react with chromone 12. It was as-
sumed that the γ-pyrone in chromone 12, under acidic
conditions, might form a pyrylium salt (16) which might,
in turn, be readily reduced. Thus, chromone 12 was
52
53
). For example, NaBH4/CeCl3, NaBH4/CuCl2, Bak-
chromene ring are characteristic for 8-acylchromeno-
coumarins, which appear at 6.62-6.67 ppm.4
1,45
In
contrast, in 6-acylchromenocoumarins, the correspond-
54
ing olefinic protons appear at 6.75-6.85 ppm.4
1,45
There-
fore, the chemical shifts for H-8 of chromene 11 in both
CDCl3 and DMSO-d6 (6.73 and 6.62 ppm) are consistent
1
4
1,45
with the range reported for 8-acylchromenocoumarins
55
and further confirm the structure of chromene 11,
which, in turn, implies that the structure of compound
56
6
was correctly assigned.
Conversion of chromene 11 to form chromone 12
under Kostanecki-Robinson5
0,51
conditions was initially
57
carried out, since chromene 11 contains an o-hydroxy-
aryl ketone moiety. Thus, in the presence of acetic
anhydride and sodium acetate, the Kostanecki-Robin-
son reaction proceeded smoothly to generate chromone
first treated with various acids such as perchloric acid,
HCl, and trifluoroacetic acid followed by a variety of
reducing agents such as NaCNBH3, NaBH4, and bo-
rane-pyridine complex. In alcoholic solvents such as
MeOH and EtOH, lactone-opened ester 17 was obtained
1
2 in 62% yield (Scheme 2). It was hoped that chromone

1
306 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6
Flavin et al.
Sch em e 3
(()-calanolide B (3) were separated by semipreparative
HPLC, and (()-calanolide A (1) thus obtained exhibited
initial mp 52-54 °C (lit. mp 56-58 °C) which in-
creased to 102 °C after being thoroughly dried.
4
0
Attempts to resolve the synthetic (()-1 into its enan-
tiomers by enzyme-catalyzed acylation or hydrolysis met
with only limited success. Enzymes that have been in-
vestigated include lipase CC (Candida cylindracea),
lipase AK (C. cylindracea), lipase AY (C. cylindracea),
lipase PS (Pseudomonas species), lipase AP (Aspergillus
niger), lipase N (Rhizopus nieveus), lipase FAP (Rhizo-
pus nieveus), lipase PP (porcine pancrease), pig (porcine)
liver esterase (PLE), pig liver acetone powder (PLAP),
subtilisin, and their immobilized forms on Celite, mo-
lecular sieves, or ion exchange resin. Among the en-
zymes mentioned above, only lipase PS-13 (Sigma)
enantioselectively acylated (-)-1, with the (+)-enanti-
omer left unreacted. However, reaction was very slow
and impractical. For example, after 20 days, 21% of (-)-
as the major product of these reactions. Attempts to
reduce the γ-pyrone in ester 17 by utilizing NaBH4 in
MeOH failed to produce any products, with only recov-
ery of ester 17 observed. The results are understand-
able if one considers that chromone 12 contains both R-
and γ-pyrone moieties and that the carbonyl group of
γ-pyrones is generally less active than a normal ketone,
due to aromatic character, while R-pyrones behave as
R,â,γ,δ-unsaturated lactones.58 Consequently, the double
bond in the γ-pyrone of chromone 12 might not be
electron-deficient (or electrophilic) enough to coordinate
1
, as detected by chiral HPLC, was converted into its
butyrate ester when vinyl butyrate was used as acylat-
ing agent. There were no significant changes observed
for different vinyl esters such as acetate, butyrate, and
propionate. These results imply that the hydroxyl group
in (()-1 might be so sterically hindered that it does not
fit into the catalytic pocket of the enzymes examined
62,63
above.
Practically, the synthetic (()-1 was resolved into its
enantiomers, (+)- and (-)-1, by using a semipreparative
5
6
55,59
with copper or samarium,
which is crucial for
initiating the conjugate reduction of R,â-unsaturated
carbonyl compounds. It has been reported that reduc-
tion of benzo-R-pyrones (coumarins) with LiAlH4 led to
64
chiral HPLC column. Thus, on a normal phase silica
gel HPLC column, only one peak was observed with a
retention time of 10.15 min when hexane/ethyl acetate
5
8,60
diol derivatives.
As to benzo-γ-pyrones (chromones),
(70:30) was used as the mobile phase at a flow rate of
the reduction of the ketone and double bond to form
γ-pyranols and 2,3-dihydro-γ-pyranols (chromanones)
has also been reported.58
1
.5 mL/min and a wavelength of 290 nm was used as
the UV detector setting. However, on a chiral HPLC
column packed with amylose carbamate, two peaks with
retention times of 6.39 and 7.15 min in a ratio of 1:1
were observed. The mobile phase was hexane/ethanol
Attention was then shifted to cyclization of chromene
1
1 into chromanone 18, which could be readily reduced
to (()-1. In the presence of a base such as K2CO3 or
LDA, reaction of chromene 11 with acetaldehyde was
unsuccessful in forming 18.61 However, treatment of
chromene 11 with acetaldehyde diethyl acetal in the
presence of trifluoroacetic acid and pyridine formed
racemic chromanone 18 with the desired stereochemical
arrangement, characterized by spectroscopic data and
consistent with previously reported structures40 (Scheme
(
2
95:5) and the UV detector was set at a wavelength of
54 nm. These two components were then separated
using a semipreparative chiral HPLC column packed
with the same material as the analytical column to
provide the enantiomers of calanolide A. The chemical
structures of the separated enantiomers, which were
assigned based on their optical rotations and compared
with the reported natural and synthetic product,
were further characterized by spectroscopic data.
In conclusion, (()-calanolide A (1) has been synthe-
sized in a five-step approach (overall yield ca. 6.6%)
starting with phloroglucinol [f 5 f 6 f 11 f 18 f
32
43
3
). The yield for this reaction, however, was somewhat
3
9
inconsistent, with the best being ca. 30%. The process
was improved by replacing acetaldehyde diethyl acetal
with paraldehyde as the acetaldehyde equivalent and
under modified conditions (for details, see the Experi-
mental Section); the formation of racemic chromanone
(
()-1] and has been chromatographically resolved into
its optically active forms, (+)- and (-)-1.
1
8 (30% yield), along with the cis-isomer (()-19 (cal-
Biologica l Activity
anolide D) as a minor product, was reproducible and
scalable.
The synthetic (()-1 and resolved enantiomers [(+)-
and (-)-1] were tested in vitro against both laboratory
strains and clinical isolates of HIV. The results are
shown in Table 1. Essentially, (+)- and (-)-1 exhibit
the same level of cytotoxicity in the cell lines examined
and do not exhibit synergistic toxicity, since the cyto-
toxicity of (()-1 is approximately the same. However,
only (+)-1 accounted for the anti-HIV activity, which
was similar to the data reported for the natural prod-
uct, and (-)-1 was inactive. Both (()- and (+)-1
showed the same relative activity against RFII and IIIB
strains of HIV-1 as well as clinical isolates H112-2 in
CEM or MT2 cell lines; however, (+)-1 was generally
Reduction of (()-ketone 18 with NaBH4 at room temp-
erature afforded the hydroxyl epimers in ca. a 1:1 ratio.
5
2
Luche reduction of ketone (()-18 using NaBH4/CeCl3
at -10 to -40 °C was highly selective in affording 90%
(
3
()-calanolide A (1) over (()-calanolide B (3)39 (Scheme
), while at room temperature the selectivity for cal-
anolide A over calanolide B was only 60:40. On a rela-
tive larger scale, selection of calanolide A was slightly
decreased to 80% and unreacted starting material re-
mained, due in part to the incomplete solubility of (()-
3
2
1
8 in EtOH at the reaction temperature (see the Experi-
mental Section for details). (()-Calanolide A (1) and

Synthesis of Anti-HIV Agent (()-Calanolide A
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6 1307
Ta ble 1. Anti-HIV Activity of (()-, (+)-, and (-)-Calanolide A (1)
strain/cell
(()-1
(+)-1
(-)-1
18.70
AZT
0.023
301.60
13 113
0.029
51.64
1780
ddC
RFII/CEM
IIIB/MT2
EC50 (µM)
IC50 (µM)
IC50/EC50 (TI)
EC50 (µM)
IC50 (µM)
IC50/EC50 (TI)
EC50 (µM)
IC50 (µM)
IC50/EC50 (TI)
EC50 (µM)
IC50 (µM)
0.486
22.81
47
0.108
6.86
64
0.135
6.53
48
0.108
7.42
69
0.267
22.96
86
0.053
14.80
279
0.107
7.15
67
0.027
7.17
266
0.427
6.99
0.189
47.34
250
0.900
83.80
93
1.562
258.97
166
0.994
212.10
213
0.331
134.93
408
7.31
d
(2.67a)
H112-2 /MT2
0.037
119.84
3236
b
6.21 (4.05 )
c
(2 )
(0.747 )
e
a
G910-6 /MT2
b
6.16 (3.97 )
131.71
c
IC50/EC50 (TI)
EC50 (µM)
IC50 (µM)
IC50/EC50 (TI)
(5 )
f
A17 /MT2
0.297
6.94
23
0.014
83.44
5960
5.89
16
a
EC25 (µM). b IC25 (µM). c IC25/EC25. d Pre-AZT treatment isolate. e Post-AZT treatment isolate. f Pyridinone-resistant isolate.
Ta ble 2. Inhibitory Effects of (()-, (+)-, and (-)-Calanolide A (1) on Nucleic Acid Polymerasea
HIV-1 RTb
DNA polymerases
TIBO-resistant
HIV-1 RT
c
compd
DDDP
RDDP
HIV-2 RT
AMV RT
R
â
RNA polymerases
(+)-1
(()-1
(-)-1
0.38
83.9%)
0.7
0.32
(97.4%)
0.97
(97.6%)
27.8
inactive
(0%)
inactive
(10%)
inactive
(5%)
10.1
(91.5%)
17.6
(81.2%)
inactive
(0%)
inactive
(20.6%)
inactive
(13.3%)
inactive
(16.8%)
inactive
(22.9%)
inactive
(24.9%)
inactive
(22.6%)
205
(62%)
279
(61%)
214
(77.1%)
inactive
(16%)
inactive
(27.8%)
inactive
(12.1%)
(
(
80.1%)
155.7
52.7%)
(
(68.1%)
a
Values are IC50 (µM); percent inhibition values at 200 µg/mL are shown in the parentheses. b HIV RT was assessed for DNA-dependent
DNA polymerase (DDDP) and RNA-dependent DNA polymerase (RDDP) activities. IC50 value for AZT-TP against RDDP activity of
HIV-1 RT was 0.051 µΜ. AMV RT: avian myeloblastosis virus reverse transcriptase.
c
2
-fold more active, consistent with expectations if only
moderately active against the HIV-1 RT enzyme with
IC50 ) 155.7 µM for DNA-dependent DNA polymerase
and IC50 ) 27.8 µM for RNA-dependent DNA poly-
merase (Table 2).
one enantiomer is active. Both the AZT-resistant strain
G910-6 and the pyridinone-resistant strain A17 were
fully inhibited by (()- and (+)-1.3
2,37
The (+)-1 was
more active than (()-1 against the AZT-resistant strain
G910-6 with EC50 values of 0.03 and 0.1 µM, respec-
tively, while, surprisingly, the activity of (()-1 against
the pyridinone-resistant strain A17 was more compa-
rable with that of (+)-1 (EC50 ) 0.3 and 0.4 µM,
respectively) (Table 1).
Direct cytotoxicity of (+)-1 upon the target cells was
apparent only at concentrations of ca. 100- or 200-fold
efficacy; the concentrations of (()-1 leading to cytotox-
icity were lower. Although the separation of antiviral
and cytotoxicity concentrations (therapeutic index) for
It may be anticipated that cytotoxicity associated with
calanolide A is due to its relatively nonspecific inhibition
of polymerases. A number of studies have reported that
cellular DNA polymerase R, the enzyme primarily
involved in cell replication, is resistant to nucleoside
analogues, but both polymerases â and γ are sensitive
to nucleoside analogues (e.g., IC50’s for AZT with
polymerases â and γ were 11 and 16 µM, respectively).
This has been suggested to account for the toxicity of
66
AZT and the resulting serious limitation of clinical
67
usefulness.
The potential of calanolide A to inhibit
(
()- and (+)-1 is relatively narrow compared with other
nucleic acid polymerases was therefore investigated
Table 2). Among the cellular polymerases tested, DNA
1
3,21
nonnucleoside inhibitors
such as TIBO, pyridinone,
(
and nevirapine, it is reasonable when compared with
ddC.
polymerase â seems to be the most sensitive to all three
entities of calanolide A, (()-, (+)-, and (-)-1, but activity
was weak and only observed at higher concentrations
To further investigate the activity of the calanolides,
inhibition profiles have been evaluated with HIV-1 RT
and a variety of other nucleic acid polymerases. As
expected,32 both (()- and (+)-1 specifically inhibited
HIV-1 RT and were not active (<30% inhibition at 200
µg/mL) against HIV-2 RT and avian myeloblastosis
virus (AMV) RT (Table 2). Therefore, calanolide A does
function as a nonnucleoside HIV-1-specific RT inhibitor.
Compounds (()- and (+)-1 were effective inhibitors of
the DNA-dependent DNA polymerase (IC50 ) 0.7 and
(i.e., 60% inhibition for (()- and (+)-1 at 279 and 205
µM and 77% inhibition for (-)-1 at 214 µM). Neither
DNA polymerase R nor RNA polymerase was inhibited
significantly in the presence of high concentrations of
calanolide A (Table 2). Comparison of the IC50 values
obtained with HIV-1 RT (0.3-1.0 µM) with the IC50
values obtained with polymerase â (around 200 µM)
may indicate that the cytotoxicity of calanolide A could
be, at least in part, due to the inhibition of this cellular
enzyme. Similarly, other coumarin compounds such as
costatolide (3) and soulattrolide (4) have been deter-
mined to very weakly inhibit DNA polymerase â with
0
.38 µM, respectively) and RNA-dependent DNA poly-
merase (IC50 ) 0.97 and 0.32 µM, respectively) activities
of HIV-1 RT. Accordingly, calanolide A may be consid-
ered a potent inhibitor of the polymerase activity of
HIV-1 RT, since the activity of (+)-1 is only 6-fold less
68
IC50 values being >500 µM.
6
5
than that observed with AZT-TP (IC50 ) 0.051 µM).
Str u ctu r e-Activity Rela tion sh ip s
Furthermore, calanolide A [both (()- and (+)-1] inhib-
ited TIBO-resistant HIV-1 RT. Although (-)-1 was not
active against HIV-1 in cell culture assays, it was
On the basis of the published preliminary SAR studies
related to calanolides and inophyllums, one can specu-

1
308 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6
Flavin et al.
late that the saturated lower pyran ring plays a very
important role in maintaining antiviral activity for this
series of compounds because the stereochemistry of the
methyl groups at C10 and C11 and the existence of the
hydroxyl group at C12 are essential. For example, the
methyl groups at C10 and C11 must be trans to each
other in order to observe the maximum anti-HIV activ-
light and/or stained with iodine. Column chromatography was
performed with silica gel 60 (70-230 mesh from EM Science).
An isocratic liquid chromatograph (Hitachi Instruments, Inc.,
Naperville, IL) was used for HPLC which consisted of a
L-6200A pump, a L-4000H/L-4200H UV/UV-vis detector, and
a D-2500 chromatointegrator. The analytical normal phase
silica gel HPLC column was 250 mm × 4.6 mm i.d. Zorbasil
with 5 µm particle size (MAC-MOD Analytical, Inc., PA), and
the analytical chiral HPLC column was packed with amylose
carbamate (250 mm × 4.6 mm i.d. Chiralpak AD, 10 µm
particle size; Chiral Technologies, Inc., PA). The semiprepara-
tive normal phase HPLC column was 250 mm × 22 mm i.d.
Econosil silica gel with 10 µm particle size (Alltech Associates
Inc., IL), and the semipreparative chiral HPLC column was
packed with the same material as the chiral analytical column
but with a column size of 250 mm × 20 mm i.d. All chemical
reagents and anhydrous solvents were purchased from Aldrich
Chemical Co. The reagent 4,4-dimethoxy-2-methylbutan-2-
3
2
ity as evidenced by (+)-calanolide A (1), (+)-inophyl-
3
3
64
lum B (2), costatolide [or (-)-calanolide B] (3), and
soulattrolide6 [or (-)-inophyllum P] (4). The anti-HIV
activity for compounds with cis-methyl groups at C10
9
70
3
3
and C11 such as (+)-inophyllum A and (+)-inophyllum
3
3
D
is dramatically decreased. The compound (-)-
calanolide C was first reported to be very weakly active
against HIV,32 but its structure was later revised to
pseudocalanolide C.71 The corresponding 12-keto com-
4
5
ol was prepared according to the literature procedure.
3
3,72
pounds, e.g., (+)-inophyllum C
and (+)-inophyllum
5
,7-Dih yd r oxy-4-p r op ylcou m a r in (5). Concentrated sul-
3
3
E, were inactive, as was the 12-methoxy (+)-calanolide
furic acid (200 mL) was added into a mixture of phloroglucinol
dihydrate (150 g, 0.926 mol) and ethyl butyrylacetate (161 g,
1.02 mol). The resulting mixture was mechanically stirred at
90 °C for 2 h, after which it was poured onto ice. The solid
product was collected by filtration and then dissolved in ethyl
acetate. The solution was washed with brine and dried over
3
2
However, the 12-acetoxy derivatives32,33 of (+)-
A.
calanolide A (1) and (+)-inophyllum B (2) exhibited anti-
HIV-1 activity, although their antiviral activity was
significantly lower as compared to that of the corre-
sponding free hydroxyl compounds. These results sug-
gest that much of the antiviral activity of the calanolides
and inophyllums is associated with a free 12-hydroxyl
group or a functional group which can be hydrolyzed to
a hydroxyl group.
2 4
Na SO . After removal of the solvent in vacuo, the residue
was triturated with hexane to provide essentially pure 5 (203
40
g) in near quantitative yield, mp 233-235 °C (lit. mp 236-
238 °C): 1H NMR (300 MHz, DMSO-d ) δ 0.95 (3H, t, J ) 7.3
6
Hz, CH
3
2
), 1.59 (2H, apparent sextet, J ) 7.4 Hz, CH ), 2.85
(
2H, t, J ) 7.5 Hz, CH ), 5.84 (1H, s, H-3), 6.19 (1H, d, J ) 2.3
2
The stereochemistry of 12-OH in the calanolides and
inophyllums was originally believed not to be critical
for anti-HIV activity because (+)-calanolide A (1), (+)-
inophyllum B (2), and their 12-OH epimers, (+)-calano-
Hz, H-6), 6.28 (1H, d, J ) 2.3 Hz, H-8), 10.32 (1H, s, OH),
+
1
0.60 (1H, s, OH); MS (EI) 220 (100, M ), 205 (37.9, M - CH
92 (65.8, M - C ), 177 (24.8, M - C ), 164 (60.9, M -
+ 1), 163 (59.6, M - CHCO ); IR (KBr) 3210 (vs, br,
OH), 1649 (vs, sh), 1617 (vs, sh), 1554 (s) cm . Anal. Calcd
for C12 : C, 65.45; H, 5.49. Found: C, 65.04; H, 5.43.
3
),
1
2
H
4
3 7
H
CHCO
2
2
-
1
lide B and (+)-inophyllum P, were all reported to be
active against HIV.3
2,33
Recently, it was found that the
12 4
H O
naturally occurring (+)-calanolide B was a partially
racemized mixture containing 45% (-)-calanolide B
Acyla tion of 5,7-Dih yd r oxy-4-p r op ylcou m a r in (5): A.
With P r op ion yl Ch lor id e. A three-necked flask (500 mL)
equipped with an efficient mechanical stirrer, thermometer,
and addition funnel was charged with 4-propyl-5,7-dihydroxy-
coumarin (5) (25.0 g, 0.113 mol) and aluminum chloride (62.1
g, 0.466 mol). Nitrobenzene (150 mL) was then added, and
the mixture was stirred until a solution was obtained, which
was then cooled to 0 °C in an ice bath. A solution of propionyl
chloride (15.2 g, 0.165 mol) in carbon disulfide (50 mL) was
added dropwise at such a rate that the reaction temperature
was held at 8-10 °C. Addition was completed over a period
of 1 h with vigorous stirring. The reaction was monitored by
TLC using a mobile phase of 50% ethyl acetate/hexane. After
3 h, an additional portion of propionyl chloride (2.10 g, 0.0227
mol) in carbon disulfide (10 mL) was added, and the mixture
was stirred for an additional 30 min, whereupon the reaction
mixture was poured onto ice and allowed to stand overnight.
The nitrobenzene was removed by steam distillation, and
the remaining solution was extracted several times with
6
4
(
3). It was further discovered that only (-)-calanolide
B (3) was active against HIV and the (+)-enantiomer
was inactive.6 This result, combined with the fact that
4
(
(
-)-calanolide A (Tables 1 and 2), the 12-OH epimer of
-)-calanolide B (3), is inactive against HIV, implies
that the 12-OH must be in the â-position in order to
confer the maximum anti-HIV activity. This may be
also true for the inophyllums. Although both (+)- and
7
0
(
-)-inophyllum P were reported to be active against
3
3,69
HIV,
the fact that the optical rotation for (+)-
3
3
inophyllum P (+19.8°) was lower than that of (-)-
inophyllum P (4)70 (-29.6°)73 led us to believe that the
3
3
naturally occurring (+)-inophyllum P was also par-
6
4
tially racemized (ca. 67% ee), as with (+)-calanolide B.
Consequently, only (-)-inophyllum P (4)70 possesses the
â-hydroxyl group at the C12 position for activity against
HIV,69 while (+)-inophyllum P with the R-hydroxyl
group is predicted to be inactive. Ultimate proof of this
hypothesis must await synthesis of both (+)- and (-)-
inophyllum P or resolution of the racemate followed by
the appropriate biological assays.
ethyl acetate. The extracts were combined and dried over Na
SO The crude product obtained by evaporation in vacuo
2
-
4
.
was purified by chromatography on a silica gel column eluting
with 50% ether/hexane to provide compounds 8, 6, and 7,
successively.
5
,7-Dih yd r oxy-8-p r op ion yl-4-p r op ylcou m a r in (6): 5.3
1
g (17% yield) was obtained, mp 244-246 °C; H NMR (300
MHz, DMSO-d
J ) 7.1 Hz, CH
6
) δ 0.96 (3H, t, J ) 7.3 Hz, CH
3
), 1.09 (3H, t,
3
), 1.57 (2H, apparent sextet, J ) 7.3 Hz, CH
2
),
),
Exp er im en ta l Section
2
.86 (2H, t, J ) 7.6 Hz, CH ), 3.03 (2H, q, J ) 7.1 Hz, CH
2
2
Ch em istr y. All melting points are corrected. NMR spectra
were run on either a Hitachi 60 MHz R-1200 NMR or a Varian
VX-300 NMR spectrometer. The chemical shifts are reported
in parts per million (δ, ppm) downfield from tetramethylsilane
5.95 (1H, s, H-3), 6.28 (1H, s, H-6), 11.50 and 12.75 (2H, 2s, 2
+
OH); MS (EI) 277 (6.6, M + 1), 276 (39.0, M ), 247 (100, M -
C H ); IR (KBr) 3239 (s, br, OH), 1693 (s, CdO), 1625 and 1593
2 5
(s) cm ; UV λmax (MeOH): 219, 288, 319 nm. Anal. Calcd
for C15 : C, 65.21; H, 5.84. Found: C, 64.92; H, 5.83.
-
1
(TMS), which was used as an internal standard. IR spectra
16 5
H O
were obtained using a Midac M series FT-IR spectrometer.
Mass spectral data were recorded using a Finnegan MAT 90
mass spectrometer. Analytical thin-layer chromatography
5,7-Dih yd r oxy-6-p r op ion yl-4-p r op ylcou m a r in (7): 5.0
g (16% yield) was obtained, mp 252-253 °C; H NMR (300
1
MHz, DMSO-d
J ) 7.1 Hz, CH
6
) δ 0.95 (3H, t, J ) 7.3 Hz, CH
3
), 1.09 (3H, t,
(TLC) was carried out on precoated plates (silica gel 60 F254
3
), 1.57 (2H, apparent sextet, J ) 7.4 Hz, CH
), 3.14 (2H, q, J ) 7.1 Hz, CH
2
),
),
from EM Science), and components were visualized with UV
2.86 (2H, t, J ) 7.4 Hz, CH
2
2

Synthesis of Anti-HIV Agent (()-Calanolide A
.93 (1H, s, H-3), 6.29 (1H, s, H-6), 11.94 and 15.55 (2H, 2s, 2
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6 1309
5
(s) cm-1. Anal. Calcd for C18
Found: C, 64.79; H, 5.95.
20 6
H O : C, 65.05; H, 6.07.
+
OH); MS (CI) 277 (21.7, M + 1), 276 (92.6, M ), 261 (15.7, M
CH ), 247 (100, M - C ), 233 (32.6, M - C ), 219 (57.6,
M - C CO); IR (KBr) 3241 (m, br, OH), 1696 (s, CdO), 1626
s) cm ; UV λmax (MeOH) 201, 278, 324 nm. Anal. Calcd for
: C, 65.21; H, 5.84. Found: C, 64.79; H, 5.89.
,7-Dih yd r oxy-6,8-d ip r op ion yl-4-p r op ylcou m a r in (8):
-
3
2
H
5
3
H
7
6,6-Dim eth yl-9-h yd r oxy-10-p r op ion yl-4-p r op yl-2H,6H-
ben zo[1,2-b:3,4-b′]d ip yr a n -2-on e (11): A. In P yr id in e. A
mixture of compound 6 (2.60 g, 9.42 mmol) and 4,4-dimethoxy-
2-methylbutan-2-ol45 (5.54 g, 37.7 mmol) was dissolved in dry
pyridine (6.5 mL). This mixture was refluxed under nitrogen
for 3 days. After removal of the solvent in vacuo, the residue
was dissolved in ethyl acetate. The ethyl acetate solution was
washed several times with 1 N HCl and brine. It was then
2
H
5
-
1
(
15 16 5
C H O
5
1
6
.1 g (16% yield) was obtained, mp 128-129 °C; H NMR (300
MHz, CDCl ) δ 1.03 (3H, t, J ) 7.3 Hz, CH ), 1.21 (3H, t, J )
.1 Hz, CH ), 1.24 (3H, t, J ) 7.1 Hz, CH ), 1.64 (2H, apparent
sextet, J ) 7.5 Hz, CH
2H, q, J ) 7.1 Hz, CH
1H, s, H-3), 16.59 and 16.67 (2H, 2s, 2 OH); MS (CI) 333 (100,
M + 1), 332 (11.5, M ), 305 (9.6, M - C
C
CdO), 1630 (s), 1588 (vs), 1551 (s) cm
3
3
7
3
3
2
), 2.96 (2H, t, J ) 7.6 Hz, CH
), 3.34 (2H, q, J ) 7.1 Hz, CH
2
), 3.23
), 6.00
2 4
dried over Na SO . The crude product obtained by evaporation
in vacuo was purified by chromatography on a silica gel column
eluting with 25% ethyl acetate/hexane to afford 2.55 g of 11
(
(
2
2
+
1
2
H
4
+ 1), 277 (12.4, M
in 78% yield, mp 96-98 °C: H NMR (300 MHz, CDCl
3
) δ 1.05
), 1.53
), 2.90 (2H, t, J )
), 5.58 (1H, d, J )
10.2 Hz, H-7), 6.00 (1H, s, H-3), 6.73 (1H, d, J ) 10.2 Hz, H-8),
-
2
H
4
CO + 1); IR (KBr) 3453 and 2461 (br-w, OH), 1746 (s,
(3H, t, J ) 7.3 Hz, CH
(6H, s, 2 CH ), 1.66 (2H, m, J ) 7.6 Hz, CH
7.7 Hz, CH ), 3.35 (2H, q, J ) 7.1 Hz, CH
3
), 1.23 (3H, t, J ) 7.1 Hz, CH
3
-
1
.
Anal. Calcd for
3
2
1
C
18
H
20
O
6
‚ /
B. With P r op ion ic An h yd r id e. 5,7-Dihydroxy-4-propyl-
coumarin (5) (5 g, 22.7 mmol) and AlCl (6 g, 45 mmol) were
3
H
2
O: C, 63.90; H, 6.16. Found: C, 64.25; H, 5.89.
2
2
1
14.50 (1H, s, OH); H NMR (300 MHz, DMSO-d
t, J ) 7.3 Hz, CH ), 1.11 (3H, t, J ) 7.1 Hz, CH
2 CH ), 1.59 (2H, m, J ) 7.8 Hz, CH ), 2.88 (2H, t, J ) 7.7 Hz,
CH ), 3.19 (2H, q, J ) 7.0 Hz, CH ), 5.79 (1H, d, J ) 10.1 Hz,
6
) δ 1.00 (3H,
3
mixed in 1,2-dichloroethane (60 mL). The resulting orange
suspension was stirred and heated to 75 °C for 30 min, after
which a solution was obtained. Then, a mixture of propionic
3
3
), 1.49 (6H, s,
3
2
2
2
anhydride (3.3 g, 22.7 mmol) and AlCl
3
(6 g, 45 mmol) in 1,2-
H-7), 6.12 (1H, s, H-3), 6.62 (1H, d, J ) 10.1 Hz, H-8); MS
+
dichloroethane (30 mL) was added dropwise over 1 h. The
reaction mixture was allowed to stir at 75 °C for an additional
(EI) 343 (5.7, M + 1), 342 (22.5, M ), 327 (100, M - CH
3
); IR
: C,
-
1
(KBr) 1728 (vs, CdO) cm
22
. Anal. Calcd for C20H O
5
1
h. After the mixture was cooled to room temperature, the
70.16; H, 6.48. Found: C, 70.45; H, 6.92.
reaction was quenched with ice water and 1 N HCl. The
precipitated product was taken into ethyl acetate, and the
aqueous solution was extracted with the same solvent (400
mL × 3). The combined ethyl acetate solutions were dried over
B. In Tolu en e/P yr id in e. A mixture of compound 6 (510.6
g, 1.85 mol) and 4,4-dimethoxy-2-methylbutan-2-ol (305.6 g,
2.06 mol) was dissolved in a mixture of toluene (1.5 L) and
dry pyridine (51 mL). This mixture was stirred and refluxed;
water and MeOH formed during the reaction were removed
azeotropically via a Dean-Stark trap. The reaction was
monitored by TLC. After 6 days, the reaction had proceeded
to completion. The mixture was then cooled to ambient
temperature and diluted with ethyl acetate (1 L) and 1 N HCl
(1 L). The ethyl acetate solution was separated and washed
with 1 N HCl (500 mL) and brine (1 L). After being dried over
2 4
Na SO and concentrated in vacuo to provide a solid product
which was taken up into acetone and cooled at -40 °C. Pure
compound 6 (total 2.8 g, 45%) was collected by filtration. The
product was identical with that obtained from the propionyl
chloride reaction described above.
5
-Hydr oxy-7-(pr opion yloxy)-4-pr opylcou m ar in (9). Into
a suspension of coumarin 5 (5 g, 22.7 mmol) in dichlo-
romethane (25 mL) was added dropwise a solution of propionic
anhydride (3.3 g, 22.7 mmol) and AlCl (6 g, 45 mmol) in
3
dichloromethane (25 mL) at ambient temperature with stir-
ring. After 16 h, the reaction was quenched with ice water.
The organic layer was separated and washed with 1 N HCl,
Na2SO
4
and evaporated in vacuo, a quantity of 590 g (93%
yield) of compound 11 was obtained which was >95% pure
without further purification and compared with an authentic
sample by TLC and spectroscopic data.
6,6,10,11-Tetr a m eth yl-4-p r op yl-2H,6H,12H-ben zo[1,2-
b:3,4-b′:5,6-b′′]t r ip yr a n -2,12-d ion e (12). A mixture of
chromene 11 (1.76 g, 5.11 mmol) and sodium acetate (0.419 g,
5.11 mmol) in acetic anhydride (12 mL) was refluxed for 4 h,
and the solvent was then removed in vacuo. The residue was
purified by chromatography on a silica gel column eluting first
with 25% ethyl acetate/hexane followed by 50% ethyl acetate/
hexane to provide 1.16 g of chromone 12 (62% yield) as a yellow
solid, mp 209-209.5 °C after recrystallization from ethyl
2 4
water, and brine. After being dried over Na SO , the crude
product was obtained from rotary evaporation and contained
only the starting material and a product as shown by TLC.
Recrystallization from ethyl acetate and hexane afforded 0.6
g (10% yield) of 7-monoester 9, which led to a greenish blue
1
color on Gibbs test, mp 170-171 °C: H NMR (300 MHz,
DMSO-d
Hz, CH
6
) δ 0.96 (3H, t, J ) 7.2 Hz, CH ), 1.13 (3H, t, J ) 7.2
3
3
), 1.61 (2H, apparent sextet, J ) 7.4 Hz, CH
2H, q, J ) 7.5 Hz, CH ), 2.92 (2H, t, J ) 7.5 Hz, CH
2
), 2.62
), 6.10
acetate: 1H NMR (300 MHz, DMSO-d
Hz, CH ), 1.51 (6H, s, 2 CH ), 1.60 (2H, apparent sextet, J )
7.6 Hz, CH ), 1.88 (3H, s, CH ), 2.39 (3H, s, CH ), 2.90 (2H, t,
J ) 7.7 Hz, CH ), 5.89 (1H, d, J ) 10.0 Hz, H-7), 6.26 (1H, s,
H-3), 6.75 (1H, d, J ) 10.2 Hz, H-8); MS (EI) 366 (29.6, M ),
351 (100, M - CH ), 323 (16.5, M - C ); IR (KBr) 1734 (vs,
CdO), 1657, 1640, 1610, 1562 (s) cm
: C, 72.12; H, 6.05. Found: C, 72.14; H, 6.15.
Red u ction of Ch r om on e 12 u n d er Lu ch e Con d ition s.
) δ 1.00 (3H, t, J ) 7.2
(
(
2
2
6
1H, s, H-3), 6.56 (1H, d, J ) 2.3 Hz, H-6 or H-8), 6.67 (1H, d,
3
3
J ) 2.3 Hz, H-8 or H-6), 11.11 (1H, s, OH); MS (CI) 277 (100,
2
3
3
+
M ), 249 (16.4, M - C
2
H
5
+ 1), 221 (21.9, M - C
2
H
5
CO + 1);
2
+
IR (KBr) 3218 (w, OH), 1769 (m, CdO), 1680 (vs, C)O), 1612
-
1
(
s) cm
Found: C, 64.57; H, 5.77.
,7-Bis(p r op ion yloxy)-4-p r op ylcou m a r in (10). Into a
solution of coumarin 5 (0.5 g, 2.3 mmol) in THF (5 mL) at -78
C was added a precooled (-78 °C) suspension of AlCl (0.3 g,
.2 mmol) in THF (5 mL) followed by addition of propionic
.
Anal. Calcd for C15
H
16
O
5
:
C, 65.21; H, 5.84.
3
3 7
H
-
1
.
Anal. Calcd for
22 22 5
C H O
5
°
2
3
To a solution of chromone 12 (50 mg, 0.14 mmol) in EtOH (1.8
mL) were added sodium borohydride (20 mg, 0.52 mmol) and
CeCl (H O) (51.7 mg, 0.14 mmol) at ambient temperature.
3 2 7
After stirring for 30 min, the mixture was diluted with water
anhydride (1.0 g, 7.8 mmol). The resulting mixture was
warmed to ambient temperature and refluxed overnight (∼16
h), whereupon the reaction was quenched with ice water and
the mixture extracted with ethyl acetate. The organic layer
(5 mL) and extracted with ethyl acetate (3 × 10 mL). The
2 4
combined extracts were dried over Na SO and concentrated
was separated and washed with 1 N HCl, H
being dried over Na SO , the crude solid product was obtained
from evaporation and reprecipitated from cold ether to afford
2
O, and brine. After
in vacuo. The crude product was purified by preparative TLC
using ethyl acetate/hexane (1:1) as the mobile phase to afford
2
4
1
the diol 13 (40 mg, 77%), mp 109-110 °C: H NMR (300 MHz,
1
0
.44 g (57% yield) of 5,7-diester 10, mp 118-120 °C: H NMR
CDCl
1.40 (3H, s, CH
2.35 (2H, t, J ) 7.6 Hz, CH
CH ), 5.56 (1H, d, J ) 9.6 Hz, H-3), 5.97 (1H, t, J ) 7.6 Hz,
CdCHCH OH), 6.70 (1H, d, J ) 10.0 Hz, H-4), 13.51 (1H, s,
OH); MS (EI) 370 (23.7, M ), 339 (100, M - CH
3
) δ 0.91 (3H, t, J ) 7.9 Hz, CH
3
), 1.39 (2H, m, CH
), 2.01 (4H, s, CH + OH),
), 2.42 (3H, s, CH ), 3.84 (2H, m,
2
),
(300 MHz, CDCl
3
) δ 1.04 (3H, t, J ) 7.3 Hz, CH
), 1.30 (3H, t, J ) 7.5 Hz, CH
), 2.62 (2H, q, J ) 7.5 Hz,
), 2.78 (2H, t, J ) 7.6 Hz,
), 6.21 (1H, s, H-3), 6.87 (1H, d, J ) 2.4 Hz, H-6 or H-8),
3
), 1.27 (3H, t,
3
), 1.47 (3H, s, CH
3
3
J ) 7.6 Hz, CH
apparent sextet, J ) 7.5 Hz, CH
CH
CH
7
1
3
3
), 1.67 (2H,
2
3
2
2
2
), 2.66 (2H, q, J ) 7.5 Hz, CH
2
2
+
2
OH); IR (KBr)
2
-
1
.06 (1H, d, J ) 2.3 Hz, H-8 or H-6); MS (CI) 333 (100, M +
), 277 (85.0, M - C CO + 1), 221 (19.9, M - C CO -
CO + 1); IR (KBr) 1767 (s, CdO), 1730 (s, CdO), 1612
1649 (vs), 1584, 1437 (s) cm
Red u ction of Ch r om on e 12 w ith n -Bu
of chromone 12 (300 mg, 0.82 mmol) and tri-n-butyltin hydride
.
2
H
4
2
H
4
3
Sn H. A mixture
2 5
C H

1
310 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6
Flavin et al.
(
600 mg, 2.05 mmol) in dry dioxane (2.4 mL) was refluxed
1.63 (2H, apparent sextet, J ) 7.6 Hz, CH
2
), 2.57 (1H, dq, J )
under nitrogen for 24 h, whereupon the reaction was quenched
with water (20 mL). The mixture was extracted with ethyl
acetate (3 × 10 mL). The combined extracts were dried over
2
6.9, 11.0 Hz, H-11), 2.91 (2H, t, J ) 7.6 Hz, CH ), 4.33 (1H,
dq, J ) 6.3, 11.1 Hz, H-10), 5.64 (1H, d, J ) 10.1 Hz, H-7),
6.05 (1H, s, H-3), 6.66 (1H, d, J ) 10.1 Hz, H-8); MS (CI) 369
(100, M + 1); IR 1736 (vs, CdO), 1686 (s, CdC-CdO), 1645
2 4
Na SO . The crude product (500 mg) obtained by evaporation
-
1
in vacuo was chromatographed on a silica gel column. Elution
with 30% ethyl acetate in hexane afforded 45 mg (15% yield)
of compound 14, mp 137-138 °C: H NMR (300 MHz, CDCl
(m), 1606 (m), 1578 (s), 1557 (vs) cm
: C, 71.72; H, 6.57. Found: C, 71.71; H, 6.70.
B. F r om P a r a ld eh yd e. To a stirring solution of chromene
. Anal. Calcd for
22 24 5
C H O
1
3
)
δ 1.04 (3H, t, J ) 7.3 Hz, CH
apparent sextet, J ) 8.1 Hz, CH
3H, s, CH ), 2.47 (3H, s, CH ), 3.01 (2H, t, J ) 7.6 Hz, CH
.59 (2H, d, J ) 7.3 Hz, CdCCH ), 5.21 (1H, t, J ) 7.4 Hz,
CdCH), 6.06 (1H, s, H-3), 14.78 (1H, s, OH); MS (EI) 368 (89.3,
3
), 1.67 (3H, s, CH
3
), 1.68 (2H,
), 2.05
),
11 (350 mg, 1.0 mmol) and PPTS (250 mg, 1.0 mmol) in 1,2-
2
), 1.86 (3H, s, CH
3
dichloroethane (2 mL) at ambient temperature under N was
2
added 3 mL of paraldehyde (22.5 mmol). The resulting
mixture was refluxed for 7 h. Then, CF CO H (1 mL), an
(
3
3
3
2
2
3
2
additional 1 equiv of PPTS, and 1 mL of paraldehyde were
added; the mixture was refluxed overnight. The reaction
+
M ), 339 (17.8, M - CH
IR (KBr) 1740 (vs, CdO), 1649, 1591, 1451 (s) cm . Anal.
Calcd for C22
H, 6.72.
Continuing elution with 80% ethyl acetate in hexane af-
forded 80 mg of an oil, which was subjected to further
purification using preparative TLC. The fraction with an R
of ca. 0.4 was collected and concentrated to afford 13.3 mg of
an oil, which was still a mixture, with unsaturated alcohol 15
2
CH
3
), 325 (100, M - CH
2 2 3
CH CH );
-
1
mixture was neutralized with saturated aqueous NaHCO and
3
extracted with ethyl acetate (50 mL × 3). The crude product
obtained by evaporation under reduced pressure was washed
with hexane. The residue was purified by column chroma-
tography eluting with ethyl acetate/hexane (1:2) to afford 100
mg (27% yield) of chromanone 18, which was identical with
an authentic sample by comparison of TLC, HPLC, and
spectroscopic data, and 30 mg (8% yield) of 19, the analytical
data of which are shown below.
1
H
24
O
5
6 2
‚ / H O: C, 71.14; H, 6.60. Found: C, 71.14;
f
1
being a predominant component: H NMR (60 MHz, CDCl
3
)
δ 0.92 (3H, t, J ) 6.0 Hz, CH
CH ), 1.63 (2H, m, CH ), 1.96 (3H, s, CH
.45 (2H, t, J ) 6.0 Hz, CH
3
), 1.26 (3H, s, CH
3
), 1.39 (3H, s,
10,11-ci s-D ih y d r o -6,6,10,11-t e t r a m e t h y l-4-p r o p y l-
2H,6H,12H-ben zo[1,2-b:3,4-b′:5,6-b′′]tr ipyr an -2,12-dion e or
(()-ca la n olid e D (19): mp 122-124 °C after recrystallization
from ethyl acetate (lit.40 mp 130-132 °C); H NMR (300 MHz,
3
2
3
3
), 2.36 (3H, s, CH ),
2
2
), 3.65 (1H, s, H-12), 5.51 (1H, d,
J ) 10.0 Hz, H-7), 6.06 (1H, s, H-3), 6.67 (1H, d, J ) 10.0 Hz,
1
H-8), 13.25 (1H, br s, OH); MS (EI) 369 (3.8, M + 1), 368 (4.4,
CDCl
Hz, CH
1.64 (2H, apparent sextet, J ) 7.7 Hz, CH
3
) δ 1.03 (3H, t, J ) 7.3 Hz, CH
3
), 1.16 (3H, d, J ) 7.2
), 1.54 (6H, 2s, 2 CH ),
), 2.68 (1H, dq, J )
),
+
M ), 367 (8.3, M - 1), 366 (28.4, M - 2), 351 (100, M - OH);
3
), 1.42 (3H, d, J ) 6.6 Hz, CH
3
3
-
1
IR (KBr) 1651 (s), 1589 (m) cm
.
2
Red u ction of Ch r om on e 12 w ith Na BH
en ce of HClO . To a stirred solution of chromone 12 (50 mg,
.14 mmol) in THF (2 mL) at ambient temperature and under
4
in th e P r es-
3.4, 7.2 Hz, H-11), 2.88 (2H, apparent dd, J ) 6.2, 9.1 Hz, CH
2
4.70 (1H, dq, J ) 3.4, 6.6 Hz, H-10), 5.60 (1H, d, J ) 10.2 Hz,
H-7), 6.04 (1H, s, H-3), 6.66 (1H, d, J ) 10.2 Hz, H-8); MS
4
0
+
N
2
was added 1 equiv of HClO
4
dropwise. After 24 h, NaBH
4
(EI) 368 (41.9, M ), 353 (100, M - CH
3
), 325 (7.1, M - C
-C ), 269 (15.3, M
O); IR 1738 (vs, CdO), 1718 (s, CdC-CdO),
3 7
H ),
(10.3 mg, 0.27 mmol) was added, and the suspension was
312 (7.7, M - C
- CH - C
1677 (m), 1624 (m), 1557 (vs) cm
: C, 71.72; H, 6.57. Found: C, 71.75; H, 6.53.
Red u ction of Ch r om a n on e 18 u n d er Lu ch e Con d i-
tion s. To a stirring solution of chromanone 18 (51.5 g, 0.14
mol) in EtOH (1.5 L) was added CeCl (H O) (102 g, 274 mmol).
The mixture was stirred for 1.5 h at room temperature under
and then cooled to -30 °C with an ethylene glycol/H O (1:
2, w/w) dry ice bath. After the temperature was equilibrated
to -30 °C, NaBH (21.3 g, 563 mmol) was added and the
4
H
8
), 297 (45.8, M - CH
3
4 8
H
stirred at ambient temperature for 3 days. Analysis of the
reaction mixture by TLC indicated that no reaction took place.
The precipitates were dissolved by adding EtOH (2 mL). Then
3
5
H
8
-
1
.
Anal. Calcd for
22 24 5
C H O
4
4 4
equiv of HClO and an additional 1 equiv of NaBH were
added. The resulting solution was stirred at ambient tem-
perature for an additional 24 h. Analysis by TLC indicated
that a product had formed. However, the TLC profile did not
change after prolonged reaction time (total 7 days) and
elevated temperature (at 55 °C for 10 h). The reaction was
quenched with water (10 mL) and the mixture extracted with
ethyl acetate (3 × 10 mL). The combined extracts were dried
3
2
7
N
2
2
4
mixture stirred at the same temperature for 8.5 h, at which
time the reaction was quenched with water (2 L) and extracted
with ethyl acetate (2 L × 3). The extracts were combined,
2 4
over Na SO . The crude product (35 mg) obtained by evapora-
tion in vacuo was chromatographed on a silica gel column.
Elution with 5% MeOH in CH Cl afforded 11 mg (30% yield)
2
2
2 4
washed with brine (2 L), and dried over Na SO . The crude
of compound 17 (R ) Et) and 22 mg (60%) of the starting
product obtained by removal of solvent under reduced pressure
was passed through a short silica gel column to provide 53 g
of mixture which contained 68% (()-calanolide A, 14% (()-
calanolide B, and 13% starting chromanone 18 as shown by
HPLC. This material was subjected to further purification by
semipreparative HPLC. The mobile phase for the semipre-
parative HPLC was hexane/ethyl acetate (70:30) at a flow rate
of 9.0 mL/min, and the UV detector was set at a wavelength
of 290 nm. The fractions for (()-calanolide A (1) and (()-
1
chromone 12. Compound 17 (R ) Et): mp 140-141 °C; H
NMR (300 MHz, CD
3H, t, J ) 7.2 Hz, CH
.50 (2H, m, CH ), 2.01 (3H, s, CH
2H, m, CH ), 3.98 (2H, q, J ) 7.1 Hz, OCH
0.2 Hz, H-3), 6.03 (1H, s, CdCH), 6.72 (1H, d, J ) 10.1 Hz,
3
OD) δ 0.97 (3H, t, J ) 7.3 Hz, CH
3
), 1.08
), 1.43 (3H, s, CH ),
), 2.46 (3H, s, CH ), 2.48
), 5.60 (1H, d, J )
(
1
(
1
3
), 1.39 (3H, s, CH
3
3
2
3
3
2
2
+
H-4); MS (CI) 413 (100, M + 1), 412 (19.6, M ), 367 (34.1, M
OCH CH ); IR (KBr) 3387 (w, br, OH), 1726 (s, CdO), 1645,
586 and 1443 (s) cm
0,11-tr a n s-Dih yd r o-6,6,10,11-t et r a m et h yl-4-p r op yl-
-
1
2
3
-
1
.
calanolide B (3) were pooled, combined, and concentrated.
(()-Ca la n olid e A (1):3
2,40
mp 52-54 °C, which increased
1
40
2
1
H,6H,12H-ben zo[1,2-b:3,4-b′:5,6-b′′]tr ipyr an -2,12-dion e or
2-Oxoca la n olid e A (18): A. F r om Aceta ld eh yd e Dieth yl
to 102 °C after it was dried thoroughly (lit. mp 56-58 °C);
1
H NMR (300 MHz, CDCl
3
) δ 1.03 (3H, t, J ) 7.3 Hz, CH
), 1.46 (3H, d, J ) 6.8 Hz, CH
), 1.51 (3H, s, CH
), 1.93 (1H, apparent sextet, J ) 7.8 Hz, H-11),
),
3
),
Aceta l. A solution containing chromene 11 (344 mg, 1.0
mmol), acetaldehyde diethyl acetal (473 mg, 4.0 mmol), pyri-
dinium tosylate (35 mg, 0.14 mmol), trifluoroactic acid (1.5
mL), and dry pyridine (0.7 mL) was heated at 140 °C under
1.15 (3H, d, J ) 6.8 Hz, CH
3
3
),
1.47 (3H, s, CH
3
3
), 1.65 (2H, apparent sextet,
J ) 7.4 Hz, CH
2
2.86, 2.92 (2H, t-AB type, J ) 8.0 Hz, J AB ) 21.3 Hz, CH
2
N
2
. The reaction was monitored by TLC analysis. After 4 h,
3.60 (1H, br-s, OH), 3.93 (1H, dq, J ) 6.4, 9.0 Hz, H-10), 4.72
(1H, d, J ) 8.0 Hz, H-12), 5.54 (1H, d, J ) 9.9 Hz, H-7), 5.94
(1H, s, H-3), 6.62 (1H, d, J ) 9.9 Hz, H-8); MS (CI) 371 (75.4,
the reaction mixture was cooled to room temperature, diluted
with ethyl acetate, and washed several times with 10%
aqueous NaHCO
+
and brine. The organic layer was separated
SO . The solvent was removed in vacuo,
M + 1), 370 (16.1, M ), 353 (100, M - OH); IR 3590 (w), 3449
3
and dried over Na
2
4
(m, br, OH), 1726 (sh), 1701 (vs, CdO), 1640 (m), 1609 (m),
-
1
and the crude product was purified by chromatography on a
silica gel column eluting with ethyl acetate/hexane (2:3).
Chromanone 18 (110 mg, 30% yield) was obtained, mp 176-
26 5
1581 (vs) cm . Anal. Calcd for C22H O : C, 71.33; H, 7.07.
Found: C, 71.63; H, 7.21.
(()-Ca la n olid e B (3):32 liquid; H NMR (300 MHz, CDCl
1
)
3
4
0
1
1
1
1
77 °C (lit. mp 130-132 °C): H NMR (300 MHz, CDCl
.05 (3H, t, J ) 7.3 Hz, CH
.56 (3H, d, J ) 6.3 Hz, CH
3
) δ
δ 1.03 (3H, t, J ) 7.3 Hz, CH
1.43 (3H, d, J ) 6.3 Hz, CH
CH ), 1.65 (2H, apparent sextet, J ) 7.5 Hz, CH
3
), 1.14 (3H, d, J ) 7.0 Hz, CH
), 1.48 (3H, s, CH ), 1.49 (3H, s,
), 1.73 (1H,
3
),
3
), 1.22 (3H, d, J ) 6.9 Hz, CH
), 1.54 and 1.57 (6H, 2 s, 2 CH
3
),
),
3
3
3
3
3
2

Synthesis of Anti-HIV Agent (()-Calanolide A
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6 1311
6
m, H-11), 2.53 (1H, br-s, OH), 2.86, 2.92 (2H, t-AB type, J )
cell on MT-2 cells in a volume of 1 mL/10 cells and ca. 0.12
6
7
.5 Hz, J AB ) 21.1 Hz, CH
2
), 4.26 (1H, dq, J ) 6.3, 10.7 Hz,
TCID50/cell on CEM-SS cells in a volume of 1 mL/10 cells.
H-10), 4.98 (1H, br, H-12), 5.53 (1H, d, J ) 10.0 Hz, H-7), 5.95
The cell-virus suspension was incubated at 37 °C for 4 h,
whereupon the cells were centrifuged and the virus superna-
tant was discarded. Medium was added to the cells to attain
a cell density of 105 cells/mL. The infected cells as well as
uninfected cells, which were prepared in the same manner but
without the addition of virus, were added to a 96-well micro-
titer plate in the amount of 100 µL/well to give a starting cell
number of 104 cells/mL. After being incubated for 6 days
(CEM-SS cells) and 7 days (MT-2 cells), respectively, the
cellular viability was then measured with a tetrazolium dye,
MTT (5 mg/mL), added to the test plates, as described in the
literature. Antiviral and toxicity data are reported as the
quantity of drug required to inhibit 50% of virus-induced cell
killing or virus production (EC ) and the quantity of drug
required to inhibit virus-induced cell viability by 50% (IC ).
HIV Rever se Tr a n scr ip ta se (RT) En zym e In h ibition
Assa ys. [Methyl- H]TTP (15 Ci/mmol) and [methyl- H]UTP
(6.5 Ci/mmol) were purchased from ICN Radiochemicals
(Irvine, CA), [ H]poly(rA) was from Amersham (IL), DNA
polymerases R and â were from Molecular Biology Resources,
Inc. (Milwaukee, WI), RNA polymerase (Escherichia coli) and
poly(dT) were from Pharmacia (Piscataway, NJ ), and DEAE-
cellulose filter discs (DE-81, Whatman) were from VWR
Scientific (Batavia, IL). Activated calf thymus DNA, native
DNA, herring sperm DNA, TTP, ATP, CTP, GTP, dATP, dCTP,
dGTP, poly(rA), oligo(dT), dithiothreitol, glutathione, gelatin,
and BSA were purchased from Sigma Chemical Co. (St. Louis,
MO).
Dimeric HIV-1 (p66/p51) RT was purified by modification
of the literature procedures.75 HIV-2 (p66/p51) RT is a
recombinant enzyme consisting of two polypeptides subunits,
which was synthesized in an E. coli expression system using
a genetically engineered plasmid.76 The enzyme possesses
DNA-dependent DNA polymerase (DDDP), RNA-dependent
DNA polymerases (RDDP), and ribonuclease H (RNase H)
activities typical of retroviral RT’s.
(1H, s, H-3), 6.63 (1H, d, J ) 9.9 Hz, H-8).
Resolu tion of th e Syn th etic (()-1 by Ch ir a l HP LC.
The synthetic (()-1 was resolved using a semipreparative
chiral HPLC column packed with amylose carbamate. The
mobile phase was hexane/ethanol (95:5) at a flow rate of 6.0
mL/min, and the UV detector was set at a wavelength of 254
nm. The enantiomers of calanolide A, (+)- and (-)-1, were
collected, and their chemical structures were assigned based
on their optical rotations and spectroscopic data, which were
3
2
in full agreement with the data reported for the natural and
4
3
74
synthetic products.
3
2,43
32
43
(
+)-Ca la n olid e A (1):
mp 47-50 °C (lit. oil, lit. mp
2
5
32
25
4
(
(
5-48 °C); [R]
CHCl
300 MHz, CDCl
J ) 6.8 Hz, CH
CH ), 1.51 (3H, s, CH
Hz, CH
D
) +68.8° (CHCl
3
, c 0.7) {(lit. [R]
D
1
) +60°
50
4
3
25
, c 0.5), lit. [R]
D
) +66° (CHCl
3
, c 0.5)}; H NMR
50
3
3
) δ 1.03 (3H, t, J ) 7.3 Hz, CH
3
), 1.15 (3H, d,
), 1.47 (3H, s,
), 1.66 (2H, apparent sextet, J ) 7.5
), 1.93 (1H, apparent sextet, J ) 7.8 Hz, H-11), 2.87,
.92 (2H, t-AB type, J ) 7.9 Hz, J AB ) 21.7 Hz, CH ), 3.52
1H, d, J ) 2.9 Hz, OH), 3.93 (1H, dq, J ) 6.4, 9.1 Hz, H-10),
.72 (1H, dd, J ) 2.7, 7.8 Hz, H-12), 5.54 (1H, d, J ) 9.9 Hz,
3
3
3
), 1.46 (3H, d, J ) 6.4 Hz, CH
3
3
3
3
2
2
(
4
2
1
3
H-7), 5.95 (1H, s, H-3), 6.62 (1H, d, J ) 9.9 Hz, H-8); C NMR
CDCl ) δ 13.99 (CH ), 15.10 (CH ), 18.93 (CH ), 23.26 (CH ),
7.38 (CH ), 28.02 (CH ), 38.66 (CH ), 40.42 (CH), 67.19 (CH-
(
2
3
3
3
3
2
3
3
2
OH), 77.15 (CH-O), 77.67 (C-O), 104.04 (C-4a), 106.36 (C-8a,
C-12a), 110.14 (C-3), 116.51 (C-8), 126.97 (C-7), 151.14 (C-4b),
1
53.10 (C-8b), 154.50 (C-12b), 158.88 (C-4), 160.42 (CdO); MS
+
(
3
CI) 371 (100, M + 1), 370 (23.6, M ), 353 (66.2, M - OH); IR
611 (w), 3426 (m, br, OH), 1734 (vs, CdO), 1643 (m), 1606
-1
(
2
m), 1587 (vs) cm ; UV λmax (MeOH) 204 (32 100), 228 (23 200),
1
83 (22 200), 325 (12 700) nm. Anal. Calcd for C22
H
26
O
5
‚ /
4
H
2
O: C, 70.47; H, 7.12. Found: C, 70.64; H, 7.12.
3
2,43
25
(
-)-Ca la n olid e A (1):
mp 47-50 °C; [R]
) -66° (CHCl , c 0.5)}; H NMR
) δ 1.03 (3H, t, J ) 7.4 Hz, CH
), 1.46 (3H, d, J ) 6.3 Hz, CH
.51 (3H, s, CH ), 1.66 (2H, apparent sextet, J ) 7.4 Hz, CH
D
) -75.6°
4
3
25
1
(CHCl
3
, c 0.7) {(lit. [R]
D
3
(CDCl
3
3
), 1.15 (3H, d, J ) 6.8
Hz, CH
1
1
3
3
), 1.47 (3H, s, CH
3
),
),
HIV-2, mutant (TIBO-resistant) HIV-1, and AMV RT’s were
assessed for RDDP activities, and HIV-1 RT was assessed for
DDDP and RDDP activities. All RT assays were performed
For RDDP assays, the
reaction mixture contained the following: 50 mM Tris‚HCl
buffer (pH 8.0), 150 mM KCl, 5 mM MgCl , 5 mM dithiothrei-
3
2
.93 (1H, apparent sextet, J ) 7.3 Hz, H-11), 2.87, 2.92 (2H,
), 3.50 (1H, d, J )
.9 Hz, OH), 3.93 (1H, dq, J ) 6.4, 9.0 Hz, H-10), 4.72 (1H,
dd, J ) 2.7, 7.8 Hz, H-12), 5.54 (1H, d, J ) 10.0 Hz, H-7), 5.94
following a standard protocol.7
7,78
t-AB type, J ) 7.8 Hz, J AB ) 22.3 Hz, CH
2
2
2
1
3
(
1H, s, H-3), 6.62 (1H, d, J ) 10.0 Hz, H-8); C NMR (CDCl
3
)
tol, 0.3 mM glutathione, 0.5 mM ethylene glycol bis(â-amino-
ethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 250 µg/mL
bovine serum albumin, 41 µM poly(rA) [ꢀ260 (mM) ) 7.8], 9.5
δ 13.99 (CH
(
3
), 15.10 (CH
3
), 18.93 (CH
3
), 23.36 (CH
2
), 27.38
CH
3
), 28.02 (CH
3
), 38.66 (CH
2
), 40.42 (CH), 67.19 (CH-OH),
3
7
1
7.15 (CH-O), 77.67 (C-O), 104.04 (C-4a), 106.36 (C-8a, C-12a),
10.14 (C-3), 116.51 (C-8), 126.97 (C-7), 151.14 (C-4b), 153.11
µM oligo(dT) [ꢀ260 (mM) ) 5.6], 20 µM TTP, and 0.5 µCi [ H]-
TTP. The required amount of each test compound in DMSO
(10 µL) was added to the reaction mixture (80 µL) in 96-well
microtiter plates (2 wells/concentration). The reaction was
started by the addition of 10 µL (0.08 µg) of RT followed by
incubation at 37 °C for 1 h. The reaction was terminated by
heating to 80 °C (5 min) and then chilling on ice (15 min).
Aliquots of each reaction mixture (90 µL) were spotted
uniformly onto circular 2.5 cm DE-81 (Whatman) filters and
kept at ambient temperature for 15 min before washing four
(
3
(
C-8b), 154.50 (C-12b), 158.90 (C-4), 160.44 (CdO); MS (CI)
+
71 (95.2, M + 1), 370 (41.8, M ), 353 (100, M - OH); IR 3443
m, br, OH), 1732 (vs, CdO), 1643 (m), 1606 (m), 1584 (vs)
-1
cm ; UV λmax (MeOH) 200 (20 500), 230 (19 400), 283 (22 500),
3
H, 7.12. Found: C, 70.27; H, 7.21.
1
26 (12 500) nm. Anal. Calcd for C22
H
26
O
5
4 2
‚ / H O: C, 70.47;
An tivir a l Assa ys. Antiviral assays were performed at
Southern Research Institute, Birmingham, AL. Materials and
methods employed were the same as described in the litera-
times with 5% aqueous Na
2
HPO
4
2
‚7H O. This was followed
by two more washings with distilled H O. Finally, the filters
were thoroughly dried and subjected to scintillation counting
in a nonaqueous scintillation fluid.
3
7
ture. CEM-SS and MT-2 cells used for the screening were
maintained in RPMI 1640 medium supplemented with 10%
2
(v/v) heat-inactivated fetal calf serum, 100 units/mL penicillin,
1
00 µg/mL streptomycin, 25 mM HEPES, and 20 µg/mL
Each test compound was prescreened at 200 µg/mL. If
inhibition at 200 µg/mL was >50%, 50% inhibition concentra-
tion (IC50) values were determined by assaying at 10 different
concentrations (in duplicate) prepared by half-log serial dilu-
tions. Fagaronine chloride (IC50 ) 13 µM) and DMSO (10 µL)
were used as positive and negative controls, respectively.
Percentage inhibition was calculated as (1 - test/negative
control) × 100%, and IC50 values were estimated from dose-
response curves.
gentamicin. The medium used for dilution of compounds and
maintenance of cultures during the assay was the same as
above. Cultures were maintained in disposable tissue culture
2
labware at 37 °C in a humidified atmosphere of 5% CO in
air. The appropriate amounts of the compounds for anti-HIV
evaluations were dissolved in 100% DMSO and then diluted
with the medium to the desired initial concentration. Each
dilution was added to plates in the amount of 100 µL/well.
Compounds were tested in triplicate wells per dilution with
infected cells and in duplicate wells per dilution with unin-
fected cells for evaluation of cytotoxicity. After addition of cells
to the plates, the high drug concentration was 100 µg/mL and
the high DMSO concentration was 0.25%.
DDDP assays with HIV-1 RT were performed using similar
procedures, except activated calf thymus DNA (final concen-
tration, 20 µg/mL), dGTP, dCTP, and dATP (each at a final
3
concentration of 50 µM), and 5 µCi [ H]TTP were used.
DNA P olym er a se Assa ys. These assays were performed
using the same procedure as the RT assays. The reaction
mixture contained the following: 80 mM Tris‚HCl buffer (pH
Infected cells were charged with HIV-1 virus to yield an
approximate multiplicity of infection (MOI) of 0.03 TCID50
/

1
312 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6
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Flavin et al.
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and 2.0 µCi [methyl- H]TTP. The required amount of each test
compound in DMSO (10 µL) was added to 80 µL of the reaction
mixture, and the reaction was initiated by adding 0.5 unit of
either DNA polymerase R or â. One unit is the amount of
enzyme required to incorporate 1 nmol of total nucleotide into
acid-insoluble form in 60 min at 37 °C.
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RNA P olym er a se Assa ys. These assays were performed
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mixture contained the following: 40 mM Tris‚HCl buffer (pH
(
7
.9), 150 mM KCl, 10 mM MgCl
2
, 0.1 mM dithiothreitol, 25
µg/mL bovine serum albumin, 41 µM native calf thymus DNA,
3
8
0 µM each of ATP, CTP, and GTP, and 1.5 µCi [methyl- H]-
UTP. The required amount of each test compound in DMSO
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nmol of AMP into acid-insoluble product using poly(dA)‚poly-
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the synthetic (()-1 in the XTT assay was carried out
by Dr. Michael Boyd’s group at NCI and Dr. Robert W.
Buckheit’s group at Southern Research Institute. Dis-
cussion with Dr. Buckheit regarding antiviral assays
is gratefully acknowledged. We also thank Dr. Kyaw
Zaw and Mr. Richard Dvorak of the Department of
Medicinal Chemistry and Pharmacognosy, University
of Illinois at Chicago, for assistance with the NMR and
MS analyses. The work described herein was supported
in part by phase I and phase II Small Business Innova-
tion Research (SBIR) grants (1R43 AI34805 and 2R44
AI34805) from the National Institutes of Health, Be-
thesda, MD, and by the National Cancer Institute,
DHHS, under contract with ABL.
(
(
1
Su p p or tin g In for m a tion Ava ila ble: Copies of H NMR
spectra of (()-, (+)-, and (-)-1, (()-3, 5-15, and 17-19 and
2
, 407-415.
1
3
copies of C NMR spectra of (+)- and (-)-1 (20 pages).
(
26) Ren, J .; Esnouf, R.; Hopkins, A.; Ross, C.; J ones, Y.; Stammers,
D.; Stuart, D. The Structure of HIV-1 Reverse Transcriptase
Complexed with 9-Chloro-TIBO: Lessons for Inhibitor Design.
Structure 1995, 3, 915-926.
Ordering information is given on any current masthead page.
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