Chemical library and structure-activity relationships of 11-demethyl-12-oxo calanolide A analogues as anti-HIV-1 agents

Article abstract of DOI:10.1021/jm701405p

(+)-Calanolide A (1) as a natural product was previously found as an inhibitor of HIV-1 reverse transcriptase. In our further investigation of its template, racemic 11-demethyl-12-oxo calanolide A (15), which had two fewer chiral carbon centers at the C-11 and C-12 positions than (+)-calanolide A, had a comparably inhibitory activity and better therapeutic index (EC₅₀ = 0.11 μM, TI = 818) against HIV-1 in vitro. A library based on its structural core was then designed and synthesized with introduction of nine diversity points in this article. The evaluations of anti-HIV-1 activity in vitro concluded their structure-activity relationships (SARs). A novel compound (10-bromomethyl-11-demethyl-12-oxo calanolide A, 123) was identified to have much higher inhibitory potency and therapeutic index (EC₅₀ = 2.85 nM, TI > 10,526) than those of the class compound against HIV-1. This finding provided a very important clue that modifications of the C ring at the C-10 position may be conducted to obtain drug candidates with better activity against HIV-1.

Full text of DOI:10.1021/jm701405p

1432
J. Med. Chem. 2008, 51, 1432–1446
Chemical Library and Structure–Activity Relationships of 11-Demethyl-12-oxo Calanolide A
Analogues as Anti-HIV-1 Agents
Tao Ma,† Li Liu,‡ Hai Xue,† Li Li,† Chunyan Han,† Lin Wang,† Zhiwei Chen,‡ and Gang Liu*,†
Department of Synthetic Medicinal Chemistry, Institute of Materia Medica, Chinese Academy of Medical Sciences, and Peking Union Medical
College, 2# Nan Wei Road, Beijing 100050, China, and AIDS Institute, Li Ka Shing Faculty of Medicine, the UniVersity of Hong Kong, Room
45, 5/F, Laboratory Block, 21 Sassoon Road, Pokfulam, Hong Kong, China
ReceiVed NoVember 8, 2007
(+)-Calanolide A (1) as a natural product was previously found as an inhibitor of HIV-1 reverse transcriptase.
In our further investigation of its template, racemic 11-demethyl-12-oxo calanolide A (15), which had two
fewer chiral carbon centers at the C-11 and C-12 positions than (+)-calanolide A, had a comparably inhibitory
activity and better therapeutic index (EC50 ) 0.11 µM, TI ) 818) against HIV-1 in vitro. A library based
on its structural core was then designed and synthesized with introduction of nine diversity points in this
article. The evaluations of anti-HIV-1 activity in vitro concluded their structure–activity relationships (SARs).
A novel compound (10-bromomethyl-11-demethyl-12-oxo calanolide A, 123) was identified to have much
higher inhibitory potency and therapeutic index (EC50 ) 2.85 nM, TI > 10,526) than those of the class
compound against HIV-1. This finding provided a very important clue that modifications of the C ring at
the C-10 position may be conducted to obtain drug candidates with better activity against HIV-1.
Introduction
clinically isolated resistant strains such as A17 (Y181C mutant).
Studies (phase I) of single doses and multiple escalating doses
of (+)-calanolide A have demonstrated its safety and a
pharmacokinetic profile, which could significantly reduce viral
load in HIV patients with a dose of 600 mg (bid) in a
monotherapeutic trial, worthy of further clinical study.8,9
Interestingly, it was demonstrated that (+)-calanolide A also
inhibited Mycobacterium tuberculosis (TB) at a maximum
inhibitory concentration (MIC) of 3.13 µg/mL.10 The data
generated in these studies have shed light on the application of
natural products as therapeutic agents for AIDS and/or TB.
(+)-Calanolide A is the first natural product identified as
active against HIV-1 and has recently been investigated continu-
ously in phase II/III clinical trials. However, no final report or
other communication has been evaluated by the US FDA since
the beginning of clinical trials in 1997. Its lower inhibitory
potency may be the reason accounting for such long-term
clinical studies.
Since then, some other coumarin analogues, such as (+)-
inophyllum B (2)11 and (+)-cordatolide A (3, Chart 1),12 have
also been isolated from plants of the genus Calophyllum and
identified to be specific HIV-1 reverse transcriptase inhibitors.
They have the common scaffold, tetracyclic dipyranocoumarin,
but different groups substituted at the C-4 position. Apparently,
there are three chiral carbon centers in the scaffold at the C-10,
-11, and -12 positions. Because of their limited availability from
natural resources, the total synthesis of these compounds and
their derivatives has already been undertaken.7,13–23
Human immunodeficiency virus (HIVa) is the cause of
acquired immunodeficiency syndrome (AIDS). Since its first
report in 1981,1 AIDS has spread rapidly through the human
population worldwide. The development of chemotherapeutic
strategies for AIDS has recently been considered one of the
most challenging scientific projects.2 The United States Food
and Drug Administration (FDA) has approved several anti-HIV
drugs, including seven nucleoside reverse transcriptase inhibitors
(NRTIs), one nucleotide reverse transcriptase inhibitor (NtRTI),
three nonnucleoside reverse transcriptase inhibitors (NNRTIs),
nine protease inhibitors (PIs), and one fusion inhibitor.3
Although highly active antiretroviral therapy (HAART) using
a combination of anti-HIV drugs has been highly effective in
suppressing the HIV load and decreasing mortality in AIDS
patients, the emergence of drug resistance among HIV carriers,
the unavoidable viral recurrency after drug treatments, and the
toxicity of the therapies have made the continued search for
novel anti-HIV drugs necessary.4,5
In 1992, Kashman6 reported (+)-calanolide A (1, Chart 1),
isolated from a tropical rainforest plant of the species Calo-
phyllum lanigerum, to be active against HIV-1, with a half-
maximal effective concentration (EC50) of 0.1 µM and thera-
peutic index in the range of 16–279 in various cell lines.7 (+)
-Calanolide A inhibits not only wild-type HIV-1 but also
* To whom correspondence should be addressed. Phone/Fax: +86 10
63167165. E-mail: gangliu27@yahoo.com.
†
In our previous studies15,16 (Scheme 1) using phloroglucinol
(6) as the starting material, racemic calanolide A was obtained
through the consecutive construction of the three skeletal rings,
coumarin (rings A and B, 7), 2,3-dimethylchromanone (ring C,
8), and 2,2-dimethylchromene (ring D, 10). The acylation of
5,7-dihydroxy-4-propyl-2H-chromen-2-one (7) and ring closure
were achieved simultaneously to produce 8 with the Friedel–
Crafts reaction by using tigloyl chloride in the presence of AlCl3.
Compound 10 was then obtained by condensation of 8 with
1,1-diethoxy-3-methyl-2-butene (11) under pyridine catalysis.
The 10,11-trans and -cis isomers were separated from the
Department of Synthetic Medicinal Chemistry, IMM, CAMS, and
PUMC.
‡
AIDS Institute, the University of Hong Kong.
a Abbreviations: HIV-1, human immunodeficiency virus (type I); SARs,
structure–activity relationships; AIDS, acquired immunodeficiency syn-
drome; FDA, Food and Drug Administration (USA); NRTIs, nucleoside
reverse transcriptase inhibitors; NtRTI, nucleotide reverse transcriptase
inhibitor; NNRTIs, nonnucleoside reverse transcriptase inhibitors; PIs,
protease inhibitors; HAART, highly active antiretroviral therapy; EC50, half-
maximal effective concentration; TB, Mycobacterium tuberculosis; MIC,
maximum inhibitory concentration; PPA, polyphosphoric acid; TBAF,
tetrabutyl ammonium fluoride; THF, tetrahydrofuran; TLC, thin-layer
chromatography; tosyl, 4-toluenesulfonyl; EDC ·HCl, 1-ethyl-(3-dimethyl-
aminopropyl)carbodiimide hydrochloride.
10.1021/jm701405p CCC: $40.75
2008 American Chemical Society
Published on Web 02/20/2008

SARs of 11-Demethyl-12-oxo Calanolide A Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1433
Chart 1. Chemical Structures of the Biologically Active Natural Dipyranocoumarins
Scheme 1. Synthesis of Racemic Calanolide A by Zhoua
a Reagents and conditions: (a) C3H7COCH2COOC2H5, H2SO4; (b) (i) tiglic acid, SOCl2, reflux, (ii) AlCl3, PhNO2/CS2, reflux, 16 h, (iii) column
chromatography; (c) pyridine, toluene, reflux, 6 h; (d) NaBH4, CeCl3 ·7H2O, 0 °C.
Scheme 2. Optimized Synthetic Route of the Racemic Calophyllum Coumarinsa
a Reagents and conditions: (a) RCOCH2COOC2H5, HCl/CH3OH; (b) tiglic acid (or crotonic acid), PPA; (c) 1,1-diethoxy-3-methyl-2-butene (11), pyridine/
toluene, microwave irradiation, 140 °C, 150 W, 120 psi, 20 min; (d) NaBH4, CeCl3 ·7H2O, 0 °C.
mixture by silica gel column chromatography. Racemic ino-
phyllum B and cordatolide A were also prepared by using this
synthetic strategy.24,25 The chemical resolution of racemic
calanolide A and cordatolide A was developed.26
Further improvements of the reaction conditions were made.
For instance, polyphosphoric acid (PPA) was first used as both
catalyst and solvent to obtain the angular chromanone 8,27 and
microwave irradiation was recently used to significantly ac-
celerate the construction of ring D (10). The PPA method gave
the highest ratio of the anticipated angular compound 8 (70%)
to the unavoidable linear compound 9 (9%). Obviously, such
improvements markedly facilitated the total synthesis of cal-
anolide A, inophyllum B, cordatolide A, and their 11-demethyl
analogues with fewer reaction steps and higher overall yields
(Scheme 2).28
Previous studies have demonstrated that (()-11-demethyl
calanolide A (12) also has inhibitory activity against HIV-1,
with an EC50 value of 0.31 µM and a range of therapeutic index
from 21 to 169.29 It inhibited different HIV-1 strains in cell
culture, especially the NNRTI (L697661)-resistant strain, con-

1434 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Ma et al.
Figure 1. Identification of the lead compound and construction of the library.
Scheme 3. Synthetic Route for the Tetracyclicdipyranocourmines Library Constructiona
a Reagents and conditions: (a) R1COCH(R2)COOC2H5, HCl/CH3OH; (b) R,ꢀ-unsaturated acids, PPA; (c) R,ꢀ-unsaturated acetals, microwave irradiation.
Scheme 4. Synthesis of the Thio-Substituted Target Compounda
a Reagents and conditions: (a) p-methylbenzenesulfonyl chloride, pyridine; (b) crotonic acid, PPA; (c) P2S5, xylene, reflux, 5 h; (d) TBAF, THF, reflux;
(e) compound 11, pyridine/toluene, microwave irradiation, 140 °C, 150 W, 120 psi, 20 min.
taining the clinic Y181C mutant. In particular, compound 12
also exerted synergistic effects in combination with indinavir,
AZT, and T-20.29Obviously, the removal of the methyl group
at the C-11 position made it an attractive candidate for drug
development because it could be synthesized much more
conveniently without consideration of trans/cis configurations
in ring C. Unfortunately, this research was terminated because
of the toxicity of the compound for cells tested both in vitro
and in mice because of its relatively lower inhibitory
potency.29
Fortunately, 11-demethyl-12-oxo calanolide A (15), which
is the precursor of (()-11-demethyl calanolide A synthesis, also
has similar inhibitory activity against HIV-1, with an EC50 value
of 0.11 µM and a better therapeutic index (818). Because there
is only one chiral carbon center remaining in the structure, this
result encouraged us to pursue further studies of its structure–
activity relationships (SARs) by using our combinatorial
chemistry technology. Therefore, a chemical library based on
tetracyclic dipyranocoumarin (15) was constructed (Figure 1,
Scheme 3).28 In all, nine diversity points were finally introduced
through structural modifications, focusing on rings B, C, and
D. A total of 85 aimed compounds were synthesized in parallel
and then biologically evaluated against HIV-1 in this study.
Chemistry
Diversity of Ring B. The replacement of the CdO of the
ester with CdS in ring B was first explored. We successfully
replaced the CdO with a CdS in the presence of thionation
reagent.30 Scheme 4 illustrates the practical strategies used to
achieve this replacement. The hydroxyl group(s) was protected
by a tosyl31 group when the oxygen atom was substituted under
P2S5 or Lawesson’s reagent in either method.
The selective removal of the 5,7-ditosyl protective groups of
compound 18 in the presence of tetrabutyl ammonium fluoride
(TBAF) was also observed in our experiments. The tosyl group
at the 5-O position was selectively removed in the presence of
TBAF at room temperature, whereas the other tosyl protecting

SARs of 11-Demethyl-12-oxo Calanolide A Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1435
Chart 2. Diversities of R1 and R2 in Coumarins
a
Scheme 5. Synthetic Route of Chloro-Substituted Coumarin at R2
a Reagents and conditions: (a) SO2Cl2, ether; (b) compound 6, HCl/CH3OH.
a
Scheme 6. Introduction of the CH2CH2CF3 Group of R1
a Reagents and conditions: (a) acetone, concentrated H2SO4; (b) CF3CH2CH2COOH, EDC ·HCl; (c) CH3OH, reflux; (d) compound 6, HCl/CH3OH.
group at 7-O had to be removed at a higher reaction temperature,
such as under solvent refluxing conditions or microwave
irradiation. This selective deprotection of the two protective
groups was used as an alternative strategy during our following
structural modifications focusing on ring C.
When compound 42 was reacted with crotonic acid in the
presence of PPA to produce its chromanone, the 7-O-acylated
(71) and 5,7-di-O-acylated (72, Chart 5) intermediates were also
observed.
Compound 52 was further modified through the nucleophilic
substitutions to attain compounds 73 and 74. Compound 52 also
could be dimerized to offer 75 through piperazine as a linker
(Chart 6).
The purified angular chromanones (Chart 3) were condensed
with compound 11 under parallel microwave irradiation to
produce their target tetracyclic dipyranocoumarin compounds
(76–91). Compound 82 was further modified through the
nucleophilic substitutions to attain compounds 92 and 93.
Compound 86 was reduced by SnCl2 ·2H2O/HCl that converted
the electron-withdrawing nitro group into an electron-donating
amino group (94, Table 1).
To explore the diversities of R1 and R2 on ring B, besides
commercially available ꢀ-oxo esters, two methods of varying
ꢀ-oxo esters were used to construct 3,4-substituted coumarins
(Chart 2) through Pechmann condensation, as outlined in
Schemes 532 and 6.33
The corresponding coumarins diversified at the R1 and R2
positions (26, 31–45) were illustrated in Chart 2.
The coumarins were reacted with crotonic acid in the presence
of PPA to offer their corresponding angular chromanones,
respectively (46–61, Chart 3), and also to produce some linear
byproducts (62–70, Chart 4).

1436 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Chart 3. Diversities of R1 and R2 in Angular Chromanones
Ma et al.
Chart 4. Diversities of R1 and R2 in Linear Chromanones
Diversity of Ring C. The following commercially available
or premade R,ꢀ-unsaturated acids (Chart 7) were used as the
building blocks for the introduction of diversities at the R3 and
R4 positions on ring C.
Compound 7 was reacted with the R,ꢀ-unsaturated acids
mentioned above in the presence of PPA to produce their
corresponding angular chromanones, respectively (95–103), and
also some linear byproducts (104–106, Chart 8).
Further eliminating the last chiral center of 15 was also
investigated in this article. The anticipated compound should
be 10,11-didemethyl-12-oxo-calanolide A. According to the
method described above, acrylic acid was used to construct the
target angular chromanone in PPA. However, only three
byproducts were identified, 7-O-acylated (107), 5-O-acylated
(108), and 5,7-di-O-acylated (109) intermediates from the
reactant mixture, instead of the anticipated closed-ring com-
pound. The replacement of PPA with CF3SO3H was also tested
but only produced a novel byproduct (110) that was 7-O-
acylated with a closed ring at the C-8 position (Scheme 7).
Interestingly, when compound 7 was reacted with crotonic
acid in the presence of CF3SO3H, another closed-ring product
(111) with 6-acylation was produced, together with a linear
byproduct (112, Scheme 8). Unfortunately, we could not produce
the corresponding tetracyclic product from compound 111 by
direct condensation with compound 11, which would have been
an analogue of pseudocalanolide C34 (4, Chart 1). The chemical
shift of the hydroxyl group in compound 111 was 12.483 ppm,
which was downfield when compared with that of compound
16 (11.760 ppm). This indicates that a hydrogen bond formed
between the adjacent carbonyl and hydroxyl groups. Perhaps,

SARs of 11-Demethyl-12-oxo Calanolide A Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1437
Chart 5. 7-O-Acylated and 5,7-Di-O-acylated Coumarins as
Side Products
Under parallel microwave irradiation, the angular chromanone
compounds were reacted with acetal 131 to offer the 7-methyl-
substituted tetracyclic dipyranocoumarin compounds at the R7
positions (174–178, Table 9), along with their side products
with ∆7,8-saturation by EtOH (179–181, Table 10). When
compound 16 was reacted with acetal 132 under microwave
irradiation, the anticipated o-NO2-phenyl-substituted tetracyclic
dipyranocoumarin compound (182, Table 9) along with a
byproduct (183, Table 10) was yielded, which was also further
reduced by SnCl2 ·2H2O to offer a o-NH2-phenyl-substituted
product (184, Table 9).
Novel Synthetic Strategy for the Construction of Ring
D and Ring C. As mentioned above, the selective removal of
the two protective tosyl groups at O-5 and O-7 of compound
17 was able to be adopted (Scheme 9). When the tosyl group
at the O-5 position was primarily deprotected in the presence
of TBAF at room temperature (185), the chromene ring (ring
D) was able to be constructed through subsequent condensation
with compound 11 (186). Afterward, the remaining tosyl group
at the O-7 position could be removed by TBAF under solvent
refluxing or microwave irradiation conditions to attain 187.
Finally, the chromanone ring (ring C) was constructed through
the reaction with acyl chloride in the presence of a strong Lewis
acid, such as BF3/Et2O.35 This strategy allowed us to introduce
the molecular diversities into ring C at the last reaction step in
a parallel manner, which made the synthesis and purification
of the final products much easier.
this hydrogen bond reduced the reactant activity of the hydroxyl
group, rendering the construction of the chromene ring (D ring)
unsuccessful.
Surprisingly, when the following R,ꢀ-unsaturated acids were
reacted with compound 7 in the presence of PPA, halogen atoms
and substituted phenyl groups were all removed. Only the
common 8,9-unsaturated chromanone (113, Chart 9) was
obtained in each reaction. The detailed mechanisms are currently
under further investigation.
The angular chromanone compounds produced (95–103, 110,
and 113) were successfully condensed with compound 11 under
parallel microwave irradiation to generate their corresponding
tetracyclic dipyranocoumarin compounds (114–124, Table 2).
Diversity of Ring D. To introduce diversity at points R5,
R6, and R7, the selected building blocks, R,ꢀ-unsaturated acetals
(125–132, Chart 10), were freshly synthesized with the corre-
sponding commercially available R,ꢀ-unsaturated aldehydes and
EtOH in the presence of HC(OEt)3.15
Under parallel microwave irradiation, the angular chromanone
compounds were reacted with acetal 125 to generate 6-methyl-
substituted tetracyclic dipyranocoumarin compounds at the R5
(or R6) positions (133–139, Table 3).
With 126 as the building block, 6-ethyl-substituted tetracyclic
dipyranocoumarin compounds at the R5 (or R6) positions were
obtained (140–144, Table 4).
With 127 as the building block, 6-n-propyl-substituted
tetracyclic dipyranocoumarin compounds at the R5 (or R6)
positions were produced (145–151, Table 5).
With 128 as the building block, 6-propene-substituted tetra-
cyclic dipyranocoumarin compounds at the R5 (or R6) positions
were obtained (152–157, Table 6).
With 129 as the building block, 6-n-butyl-substituted tetra-
cyclic dipyranocoumarin compounds at the R5 (or R6) positions
were produced (158–165, Table 7).
Structure–Activity Relationships (SARs) of 11-Demethyl-
12-oxo Calanolide A Analogues against HIV-1. The inhibitory
activities against HIV-1 of all compounds were tested in vitro
by using a pseudotyped viral assay, as described previously.36
Zembower et al.37 previously reported that 10,11-trans or 10,11-
cis 12-ketones of calanolide A analogues exerted an inhibitory
activity against HIV-1 but with a lower potency than that of
(+)-calanolide A. Here, we report for the first time that 11-
demethyl-12-oxo calanolide A (15) is active against HIV-1, with
an EC50 value of 0.11 µM and a higher therapeutic index (818)
than that of (+)-calanolide A. This result indicates that the chiral
carbon centers at the 11 and 12 positions are unnecessary for
its anti-HIV-1 activity. This result encouraged us to prepare a
chemical library of compound 15 to further study the struc-
ture–activity relationships of compound 15. Thus, a chemical
library with a maximum of nine diversity points on the scaffold
was designed and synthesized in this study.
The CdO group was first replaced with a CdS group on
ring B, according to the isostere principle. This modification
resulted in an inactive compound (23). Position 4 (R1) on ring
B was linked with a short alkyl chain. A two- or three-carbon
chain was required to maintain a similar level of activity, such
as an ethyl (76, EC50 ) 0.27 µM, TI > 1000) or an n-propyl
(15, EC50 ) 0.11 µM, TI ) 818) substitution. However, a longer
(n-butyl group) or shorter (methyl group) chain at R1 was
With 130 as the building block, 6-phenyl-substituted tetra-
cyclic dipyranocoumarin compounds at the R5 (or R6) positions
were obtained (166–173, Table 8).
Chart 6. Nucleophilic Substitutions in the Angular Chromanones

1438 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Table 1. Diversities of R1 and R2 in Target Compounds
Ma et al.
a “+”, “++”, and “+++” mean that <10%, 10–80%, and >80% of the host cells of the virus died, respectively. b Therapeutic index (TI) was calculated
by a formula of IC50/EC50. c Not detectable.
Chart 7. Diversities of R3 and R4

SARs of 11-Demethyl-12-oxo Calanolide A Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1439
Chart 8. Diversities of R3 and R4 in Chromanones
Scheme 7. Side Products with Acrylic Acid as the Building Block
unfavorable. The replacement of R1 with the aromatic groups
(phenyl, substituted phenyl, or aromatic heterocycle) or pip-
erazine (or N-methyl piperazine) made the compounds inactive.
Furthermore, halogenations of 15 at R1, such as with CF3,
CH2Cl, or (CH2)2CF3, also caused a loss of activity. Cyclization
at R1 and R2 with a five- or six-membered ring to constrain the
conformation did not improve its biological activity. From such
multiple modifications and their results, we concluded that a
hydrophobic R1 of a proper length is necessary and is thought
to engage in a compact hydrophobic interaction with the reverse
transcriptase of HIV-1.
R2 of ring B was modified with halogenation (F or Cl atom).
Chlorine substitution (79) produced a similar level of activity
(EC50 ) 0.37 µM, TI ) 243–730) to that of (+)-calanolide A,
whereas fluorine substitution (77, 78) was both useless and
cytotoxic at 10 µM. An alternative strategy to modify R2 with
hydrophobic methyl or benzyl groups also did not result in the
anticipated anti-HIV-1 activity. Thus, modifications at the R2

1440 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Ma et al.
Scheme 8. Crotonic Acid in the Presence of CF3SO3H Offered a Novel Side Product
Chart 9. Some Special R,ꢀ-Unsaturated Acids and Their Common Chromanone
Table 2. Diversities of R3 and R4 in Target Compounds
compd
R3
R4
yield (%)
inhibition (%, 1.0 µM)
toxicity (10 µM)a
EC50 (TI)b
114
115
116
117
118
119
120
121
122
123
124
di-CH3
H
n-C3H7
n-C5H11
C6H5
p-CH3C6H4
trans-cyclohexyl
cis-cyclohexyl
H ∆10,11
BrCH2
carbonyl,12-CH2
H
CH3
H
H
H
80
74
81
80
82
85
80
84
71
88
72
68.3
38.9
61.9
46.4
62.7
57
c
0.64 µM (47–140)
++
+
+
+
c
+
+
c
c
32.5 nM (102–308)
c
c
c
c
c
c
H
40
59.3
64.9
93.5
c
H
H
H
c
c
2.85 nM (>10,526)
c
a “+”, “++”, and “+++” mean that <10%, 10–80%, and >80% of host cells of the virus died, respectively. b Therapeutic index was calculated by a
formula of IC50/EC50. c Not detectable.
position of ring B were not useful in the search for novel highly
active agents against HIV-1.
C-10 and C-11 positions (122), and reversal of the direction of
ring C (124), did not cause significant biological improvement.
C-10 bromomethylated 15 (123) was originally designed as
an intermediate for further modification at the C-10 position
through nucleophilic substitutions. Interestingly, 123 showed
greater anti-HIV-1 activity (2.85 nM) and therapeutic index
(>10 526). Although it may serve the toxic potentialities to the
molecule during long-term treatment because the bromine easily
generates an alkylating reactive intermediate, this finding
provided a very important clue that modifications of the C ring
at the C-10 position may be conducted to obtain drug candidates
with activity against HIV-1. Thus, mimicking bromine at the
R3 and R4 of ring C were modified by various substitutions.
10,10-Dimethyl-11-demethyl-12-oxo calanolide A (114) was
still active against HIV-1, with an EC50 value of 0.64 µM.
However, its therapeutic index was in a lower range of 47–140.
Although an extended hydrophobic chain increased the activity
to the nanomolar level (EC50 ) 32.5 nM), the novel compound
(116) still had a lower therapeutic index (102–308). Other
modifications of ring C at R3 and R4, such as the movement of
the methyl group from C-10 to C-11 (115), aromatization of
C-10 (118 and 119), cyclization (120 and 121), alkenes at the

SARs of 11-Demethyl-12-oxo Calanolide A Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1441
Chart 10. Diversities of R5, R6, and R7
Table 3. Target Compounds With R5 (or R6) ) CH3
compd
R1
R2
R3
R4
yield (%)
inhibition (%, 1.0 µM)
toxicity (10 µM)a
EC50 (TI)b
133
134
135
136
137
138
139
n-C3H7
C2H5
n-C3H7
n-C3H7
n-C4H9
C2H5
H
H
H
Cl
H
F
H
CH3
H
H
H
H
79
59
62
64
61
65
66
62.4
57.4
c
55.7
75.4
39.5
c
++
c
c
c
c
c
c
c
c
c
c
c
CH3
CH3
CH3
CH3
CH3
CH3
H
H
c
c
n-C3H7
F
a “+”, “++”, and “+++” mean that <10%, 10–80%, and >80% of the host cells of the virus died, respectively. b Therapeutic index was calculated by
a formula of IC50/EC50. c Not detectable.
Table 4. Target Compounds with R5 (or R6) ) C2H5
Table 5. Target Compounds with R5 (or R6) ) n-C3H7
yield
(%)
inhibition
(%, 1.0 µM)
toxicity
EC50
(TI)b
compd
R1
R2
(10 µM)a
yield inhibition
toxicity EC50
140
141
142
143
144
C2H5
H
H
Cl
F
64
68
71
70
65
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
compd
R1
R2 R3
R4 (%) (%, 1.0 µM) (10 µM)a (TI)b
n-C3H7
n-C3H7
C2H5
145 n-C3H7
146 C2H5
147 n-C3H7
148 n-C3H7 Cl CH3
149 n-C4H9
150 C2H5
H
H
H
H
CH3
CH3
CH3 69
c
c
c
c
c
c
c
c
c
H
H
H
H
H
H
72
55
73
76
68
62
97
54.1
c
c
c
++
n-C3H7
F
c
c
a “+”, “++”, and “+++” mean that <10%, 10–80%, and >80% of
the host cells of the virus died, respectively. b Therapeutic index was
calculated by a formula of IC50/EC50. c Not detectable.
H
F
F
CH3
CH3
CH3
c
c
c
151 n-C3H7
c
a “+”, “++”, and “+++” mean that <10%, 10–80%, and >80% of
the host cells of the virus died, respectively. b Therapeutic index was
calculated by a formula of IC50/EC50. c Not detectable.
C-10 position is being undertaken in our laboratory with the
aims of producing potential druggable properties and tolerated
toxicity.
R5 or R6 of ring D was diversified with lipophilic or alkene
chains or phenyl groups in this study. Many combinations of
R5 or R6 modifications together with diversity at R1, R2, R3,
and R4 were synthesized. In a short summary, modifications
only at R5 or R6 of ring D did not improve the compounds’
activities against HIV-1. A longer lipophilic chain and aroma-
tization at R5 or R6 of ring D significantly increased the
cytotoxicity of the compounds in vitro, for example compounds
159, 167, 169, and 170.
the C-7 and C-8 positions of ring D (compounds 179–181 and
183) caused a loss of activity against HIV-1.
In summary, a chemical library of 11-demethyl-12-oxo
calanolide A was designed and synthesized with nine diversity
points. Evaluation of the anti-HIV-1 activities of the compounds
in vitro indicated that (1), in ring B, a hydrophobic R1 of the
proper length is necessary and modifications at the R2 position
of ring B are not useful in the search for novel highly active
agents against HIV-1; (2) at R3 and R4 of ring C, an extended
hydrophobic chain at the C-10 position results in a compound
(116) active against HIV-1 at nanomolar concentrations (EC50
The movement of the 6,6-dimethyl group to the C-7 position
(compounds 174–178) and the saturation of the bond between

1442 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Table 6. Target Compounds with R5 (or R6) ) (CHdCHCH3)
Ma et al.
Table 8. Target Compounds with R5 (or R6) ) C6H5
yield inhibition
toxicity EC50
compd
R1
R2 R3 R4 (%) (%, 1.0 µM) (10 µM)a (TI)b
152 n-C3H7
153 n-C3H7
154 n-C3H7
155 C2H5
156 C2H5
157 CH2CH2CF3
a “+”, “++”, and “+++” mean that <10%, 10–80%, and >80% of
the host cells of the virus died, respectively. b Therapeutic index was
calculated by a formula of IC50/EC50. c Not detectable.
H
H
H
CH3
CH3 65
37.8
c
57.3
56.4
46.2
c
c
c
c
c
c
c
c
c
c
c
c
c
yield inhibition
toxicity EC50
H
H
H
H
H
63
58
67
66
56
compd
R1
R2 R3 R4 (%) (%, 1.0 µM) (10 µM)a (TI)b
Cl CH3
H
F
166 n-C3H7
167 C2H5
168 n-C3H7
169 n-C3H7
170 n-C4H9
171 C2H5
H
H
H
H
CH3
CH3
CH3 70
c
+
c
c
c
c
c
c
c
c
CH3
CH3
CH3
H
H
H
H
H
H
H
64
66
69
62
75
68
58
96.4
c
96.3
96.4
c
++
c
H
Cl CH3
+++
H
F
F
CH3
CH3
CH3
CH3
++
c
c
c
172 n-C3H7
173 CH2CH2CF3
c
47.6
H
Table 7. Target Compounds with R5 (or R6) ) n-C4H9
a “+”, “++”, and “+++” mean that <10%, 10–80%, and >80% of
the host cells of the virus died, respectively. b Therapeutic index was
calculated by a formula of IC50/EC50. c Not detectable.
commercially available kits. Antiviral data were reported as the
quantity of drug required to inhibit production by percentage.
Controls included (1) infected cells without adding any drugs as a
baseline; (2) pseudovirus with a nonspecific envelope (e.g., HCV)
that cannot infect TZM. CD4+. CCR5+ cells; and (3) an HIV-
specific RT inhibitor as a positive control (e.g., AZT).
The cell toxicity of the compounds against HIV was evaluated
by similar microtiter assays with TZM. CD4+. CCR5+ and Vero
cells. Serial diluted drugs (10 mM, 1 mM, 100 µM, 10 µM, 1 µM,
and 0.1 µM) were tested in cells by using the cell viability assay,
which is commercially available (Promega). The toxicity data are
reported as the quantity of a drug required to reduce cell viability
by percentage.
yield inhibition
toxicity EC50
compd
R1
R2 R3 R4 (%) (%, 1.0 µM) (10 µM)a (TI)b
158 n-C3H7
159 C2H5
160 n-C3H7
161 n-C3H7
162 n-C4H9
163 C2H5
H
H
H
H
CH3
CH3
CH3 71
35.2
+
++
c
c
c
c
c
c
c
c
c
c
c
c
c
c
H
H
H
H
H
H
H
78
56
71
79
76
58
74
96.7
c
c
c
c
c
53.6
General Synthetic Strategy. All the reagents and chemicals were
obtained from commercial suppliers and used as received. Melting
points are uncorrected and were determined without correction with
a Yanaco micromelting point apparatus. Reaction progress was
monitored by thin-layer chromatography (TLC) on Qing Dao silica
gel 60 F-254 with UV detection. Flash column chromatography
was performed with silica gel 60 (160–200 mesh) from Qing Dao
Haiyang Chemical Factory with petroleum and ethyl acetate as the
eluent solvents. 1H NMR spectra were recorded with Mercury-
300, -400, and INOVA-500 spectrometers in DMSO-d6 solution
unless otherwise indicated. Chemical shifts are reported in parts
per million (δ) relative to the solvent signal, and coupling constant
(J) values are reported in hertz. The spin multiplicities were
indicated by the symbols s (singlet), d (doublet), t (triplet), m
(multiplet), and b (broad). Auto HPLC-MS analysis was performed
on a ThermoFinnigan LCQ-Advantage mass spectrometer equipped
with an Agilent pump, an Agilent detector, an Agilent liquid
handler, and a fluent splitter. The employed column was a Kromasil
C18 column (4.6 µm, 4.6 mm × 50 mm) from DIKMA. The eluent
was a mixture of acetonitrile and water containing 0.05% HCOOH
with a linear gradient from 5:95 (v:v) acetonitrile-H2O to 95:5
(v:v) acetonitrile-H2O over 5 min at a 1 mL/min flow rate. A
portion of the eluent (5%) was split into the MS system. The UV
detection was carried out at a UV wavelength of 254 nm. HR-MS
data were recorded with an Agilent LC-MSD/TOF mass spectrom-
eter. Infrared spectra were recorded with an Impaco 400 FT-IR
spectrophotometer. Elemental analyses were performed with a Carlo
Erba 1106 elemental analyzer, where analyses are indicated by the
symbols of the elements; the analytical results obtained were within
Cl CH3
H
F
F
CH3
CH3
CH3
CH3
164 n-C3H7
165 CH2CH2CF3
H
a “+”, “++”, and “+++” mean that <10%, 10–80%, and >80% of
the host cells of the virus died, respectively. b Therapeutic index was
calculated by a formula of IC50/EC50. c Not detectable.
) 32.5 nM), although the therapeutic index is still low
(102–308); (3) a longer lipophilic chain and aromatization at
R5 or R6 of ring D significantly increase its cytotoxicity in vitro;
and (4) however, 10-bromomethyl-11-demethyl-12-oxo calano-
lide A (123) showed the greater anti-HIV-1 activity (EC50
)
2.85 nM) and therapeutic index (>10 526). This finding
provided a very important clue that modifications of the C ring
at the C-10 position may be conducted to obtain drug candidates
with activity against HIV-1.
Experimental Section
Cell-Based Assays. Serial diluted drugs (10 µM, 1 µM, 100 nM,
10 nM, 1 nM, and 0.1 nM) were tested against 100 TCID50
HIVADA infection in a pseudovirus-based assay, as we previously
described.36 The assay quantifies the activity of a drug to inhibit
HIV-induced reporter luciferase activity. The viral infection was
determined on the third day by measuring the reporter luciferase
activity in TZM. CD4+. CCR5+ cells postinfection by using

SARs of 11-Demethyl-12-oxo Calanolide A Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1443
Table 9. Diversities of R5 (6) and R7 in the Target Compounds
yield
(%)
inhibition
(%, 1.0 µM)
toxicity
EC50
(TI)b
compd
R1
R2
R5 (6)
R7
(10 µM)a
174
175
176
177
178
182
184
C2H5
H
H
Cl
F
F
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
H
49
53
51
47
49
44
81
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
n-C3H7
n-C3H7
n-C3H7
C2H5
n-C3H7
n-C3H7
49.1
47.1
c
52.6
c
o-NO2-C6H4
o-NH2-C6H4
H
a “+”, “++”, and “+++” mean that <10%, 10–80%, and >80% of the host cells of the virus died, respectively. b Therapeutic index was calculated by
a formula of IC50/EC50. c Not detectable.
Table 10. Side Products with ∆7,8-Saturation by EtOH
yield
(%)
inhibition
(%, 1.0 µM)
toxicity
EC50
(TI)b
compd
R1
R2
R5 (6)
R7
(10 µM)a
179
180
181
183
n-C3H7
C2H5
n-C3H7
n-C3H7
H
H
Cl
H
H
H
H
CH3
CH3
CH3
H
26
19
21
25
c
c
51.7
61
c
c
c
+
c
c
c
c
o-NO2-C6H4
a “+”, “++”, and “+++” mean that <10%, 10–80%, and >80% of the host cells of the virus died, respectively. b Therapeutic index was calculated by
a formula of IC50/EC50. c Not detectable.
Scheme 9. Novel Synthetic Strategy of the Target Compoundsa
a Reagents and conditions: (a) 1.5 equiv of TBAF, THF, rt, 3 h; (b) compound 11, microwave irradiation in toluene/pyridine, 140 °C, 150 W, 120 psi,
20 min; (c) 10 equiv of TBAF, THF, reflux (or microwave irradiation); (d) (i) crotonic acid (or 2-methyl acrylic acid), SOCl2, reflux, (ii) BF3/Et2O, reflux.
(0.4% of the theoretical values. Microwave heating was carried
out with a Discover microwave synthesizer (CEM Corporation,
NC).
Typical procedures and compounds are only described in the
article. All characterizations of synthesized compounds are detailed
in the Supporting Information. The original spectra of various
compounds can be obtained from the authors.
General Method for the Synthesis of Coumarins. To an
equimolar mixture of pholoroglucinol (6) and ꢀ-keto-ethyl acetate
was added 50 mL of methanol saturated with dry hydrochloride
gas under stirring until it was completely dissolved at room
temperature. The precitipate was then collected and dried. With
this method, compounds 26 and 31–45 were prepared, respectively.
5-Hydroxy-8-methyl-4-propyl-8,9-dihydro-2H,10H-pyrano[2,3-
f]chromene-2,10-dione (16) and 5-Hydroxy-8-methyl-4-propyl-
7,8-dihydro-2H,6H-pyrano[3,2-g]chromene-2,6-dione (112). Com-
pound 7 (2.20 g, 10 mmol) and crotonic acid (1.29 g, 15 mmol)
were dissolved in 100 mL of PPA and were mixed with mechanical
stirring at 90 °C for 20 min. The reactant mixture was poured into
200 mL of ice–water and stirred; a yellow solid was precipitated,
subsequently. The solid was filtered and washed with water. The
obtained mixture was purified by column chromatography to give
angular compound 16 (2.01 g, 70%) as target and linear compound
112 (260 mg, 9%) as byproduct. 16: 1H NMR (300 MHz, DMSO-
d6, ppm): 11.760 (s, 1H, OH), 6.282 (s, 1H, 6-H), 6.025 (s, 1H,
3-H), 4.635 (m, 1H, 8-H), 2.863 (t, 2H, J ) 7.5 Hz, 4-CH2CH2CH3),

1444 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Ma et al.
2.648 (dd, 1H, J ) 11.1 Hz, 16.5 Hz, 9-He), 2.562 (dd, 1H, J )
4.2 Hz, 16.5 Hz, 9-Ha), 1.552 (sex, 2H, J ) 7.5 Hz, 7.2 Hz,
4-CH2CH2CH3), 1.385 (d, 3H, J ) 6.0 Hz, 8-CH3), 0.924 (t, 3H, J
) 7.2 Hz, 4-CH2CH2CH3). ESI-MS (m/z): 289.1 [M + H]+ (calcd
MW ) 288.30). 112: 1H NMR (300 MHz, DMSO-d6, ppm): 13.900
(s, 1H, OH), 6.450 (s, 1H, 10-H), 6.042 (s, 1H, 3-H), 4.753 (m,
1H, 8-H), 2.971 (dd, 1H, J ) 12.3 Hz, 17.4 Hz, 7-He), 2.875 (t,
2H, J ) 7.5 Hz, 4-CH2CH2CH3), 2.793 (dd, 1H, J ) 3.0 Hz, 17.4
Hz, 7-Ha), 1.584 (sex, 2H, J ) 7.5 Hz, 7.2 Hz, 4-CH2CH2CH3),
1.437 (d, 3H, J ) 6.3 Hz, 8-CH3), 0.948 (t, 3H, J ) 7.2 Hz,
4-CH2CH2CH3). ESI-MS (m/z): 289.1 [M + H]+ (calcd MW )
288.30).
under reduced pressure. The residue was redissolved again in ethyl
acetate. The excess TBAF was then washed off with water. The
organic layer was dried over MgSO4 and concentrated under
reduced pressure. The residue was purified to yield 266 mg of
1
compound 19 in 75% (yellow powder) with mp 204–206 °C. H
NMR (300 MHz, DMSO-d6): 10.901 (s, 1H, 7-OH), 10.634 (s, 1H,
5-OH), 6.766 (s, 1H, 3-H), 6.358 (d, 1H, J ) 2.4 Hz, 8-H), 6.344
(d, 1H, J ) 2.4 Hz, 6-H), 2.826 (t, 2H, J ) 7.5 Hz, 9-CH2), 1.578
(sex, 2H, J ) 7.5 Hz, 7.2 Hz, 10-CH2), 0.932 (t, 3H, J ) 7.2 Hz,
11-CH3). ESI-MS (m/z): 237.1 [M + H]+ (calcd MW ) 236.29).
Anal. Calcd for C12H12O3S: C, 61.00; H, 5.12. Found: C, 60.84;
H, 5.14.
With this method, compounds 20, 46–72, 95–109, and 113 were
prepared, respectively.
With this method, compound 187 was also prepared.
Ethyl 2-chloro-3-oxohexanoate (25) was prepared according to
1,1-Diethoxy-3-methyl-2-butene (11) was prepared as previously
described.15,16 Compounds 125–132 were prepared by the same
method.
ref 31 as a light yellow liquid with a yield of 90%.
2,2-Dimethyl-1,3-dioxane-4,6-dione (28) was prepared according
to ref 32 as a white needle crystal with a yield of 62%.
2,2-Dimethyl-5-(4,4,4-trifluorobutanoyl)-1,3-dioxane-4,6-di-
one (29). Compound 28 (288 mg, 2 mmol) and 4,4,4-trifluorobu-
tanoic acid (284 mg, 2 mmol) were dissolved in 5 mL of THF.
EDC ·HCl (340 mg, 2.2 mmol) was then added to the mixture. The
reactants were reacted at room temperature for 5 h. The solvent
was removed through evaporation. The residue was dissolved with
20 mL of ethyl acetate and then washed with 1 M HCl solution (3
× 15 mL). The organic layer was dried with anhydrous Na2SO4.
The solvent was removed, and the residue was then ready for the
next synthesis.
Methyl 6,6,6-Trifluoro-3-oxohexanoate (30). The residue of 29
was dissolved in 10 mL of methanol, and 4-methylbenzenesulfonic
acid in catalytical amount was added. The mixture was reflux for
4 h and then concentrated through evaporation. The residue was
ready for the next synthesis.
6,6,10-Trimethyl-4-propyl-10,11-dihydro-2H,6H,12H-dipyr-
ano[2,3-f:2′,3′-h]chromene-2,12-dione (15). To the compound 16
(14 mg, 0.05 mmol) and 11 (0.2 mmol) in a microwave reaction
tube was added 2 mL of toluene and 2 drops of pyridine. The
reaction was continued for 20 min under the conditions of 150 W
and 140 °C. The reactants were purified by silica gel chromography
to gain compound 15 as an off-white powder with mp 114–116
1
°C. The yield was 86%. H NMR (300 MHz, DMSO-d6, ppm):
6.566 (d, 1H, J ) 9.9 Hz, 8-H), 6.086 (s, 1H, 3-H), 5.774 (d, 1H,
J ) 9.9 Hz, 7-H), 4.707 (m, 1H, J ) 6.3 Hz, 12.0 Hz, 3.9 Hz,
10-H), 2.837 (t, 2H, J ) 7.5 Hz, 4-CH2CH2CH3), 2.708 (dd, 1H, J
) 12.0 Hz, 16.5 Hz, 11-He), 2.606 (dd, 1H, J ) 3.9 Hz, 16.5 Hz,
11-Ha), 1.537 (sex, 2H, J ) 7.5 Hz, 7.2 Hz, 4-CH2CH2CH3), 1.488,
1.450 (2s, 6H, CH3), 1.439 (d, 3H, J ) 6.3 Hz, 10-CH3), 0.963 (t,
3H, J ) 7.2 Hz, 4-CH2CH2CH3). ESI-MS (m/z): 355.1 [M + H]+
(calcd MW ) 354.41).
5-Hydroxy-8-methyl-4-(piperazin-1-ylmethyl)-8,9-dihydro-
2H,10H-pyrano[2,3-f]chromene-2,10-dione (73). Compound 52
(0.05 mmol) was dissolved in 10 mL of THF, and 2 mL of
piperazine was added to the solution. The reaction mixture was
refluxed for 4 h and then purified by column chromatography.
Compound 73 was obtained as a light yellow powder with mp
256–258 °C. The yield was 68%. 1H NMR (400 MHz, DMSO-d6,
ppm): 12.820 (s, 1H, 5-OH), 9.520 (s, 1H, NH), 7.652 (s, 1H, 6-H),
6.643 (s, 1H, 3-H), 4.605 (m, 1H, J ) 6.0 Hz, 12.4 Hz, 2.4 Hz,
8-H), 3.810 (m, 4H, CH2), 3.121 (s, 2H, 4-CH2), 3.029 (m, 4H,
CH2), 2.869 (dd, 1H, J ) 12.4 Hz, 17.2 Hz, 9-He), 2.709 (dd, 1H,
J ) 2.4 Hz, 17.2 Hz, 9-Ha), 1.409 (d, 3H, J ) 6.0 Hz, 8-CH3).
ESI-MS (m/z): 345.2 [M + H]+ (calcd MW ) 344.37). HRESIMS
With this method, compounds 23, 76–91, 114–124, 133–183,
and 186 were prepared, respectively.
2-Oxo-4-propyl-2H-chromene-5,7-diyl Bis(4-methylbenzene-
sulfonate) (17). Compound 7 (5 mmol) was dissolved in pyridine
and cooled down in an ice bath. p-Methylbenzenesulfonyl chloride
(6 equiv) was then added with stirring. The reaction was continued
at room temperature overnight. The reactants were poured into
ice–water under strong stirring. The precipitate was filtered and
washed with water. After completely drying, a white solid (17)
was obtained as 2.45 g, yield 93%, mp 239–241 °C. 1H NMR (300
MHz, DMSO-d6): 7.762 (m, 4H, Ar-H), 7.499 (m, 4H, Ar-H), 7.150
(d, 1H, J ) 2.4 Hz, 8-H), 6.781 (d, 1H, J ) 2.4 Hz, 6-H), 6.322 (s,
1H, 3-H), 2.691 (t, 2H, J ) 7.5 Hz, 9-CH2), 2.429, 2.408 (2s, 6H,
14, 17-CH3), 1.413 (sex, 2H, J ) 7.5 Hz, 7.2 Hz, 10-CH2), 0.792
(t, 3H, J ) 7.2 Hz, 11-CH3). ESI-MS (m/z): 529.1 [M + H]+ (calcd
MW ) 528.60). IR (KBr) cm-1: 3087, 2962, 2877, 1743, 1612,
1421, 1379, 1194.
+
obsd m/z 345.1454, calcd for C18H21N2O5 345.1450.
With this method, compounds 74, 75, 92, and 93 were prepared,
respectively.
4-(4-Aminophenyl)-6,6,10-trimethyl-10,11-dihydro-2H,6H,12H-
dipyrano[2,3-f:2′,3′-h]chromene-2,12-dione (94). To a solution
of compound 86 (0.02 mmol) in a sufficient volume of ethanol
was added 5 equiv of 38% HCl and 15 equiv of SnCl2 ·2H2O. After
refluxing for 2 h, the reactants were poured into a 40% NaOH
solution under stirring. The product was extracted with DCM (3 ×
20 mL) and purificed by silica gel chromography to gain 5.1 mg
of the final product (white powder, mp 149–151 °C). The yield
With this method, compound 21 was also prepared.
4-Propyl-2-thioxo-2H-chromene-5,7-diyl Bis(4-methylben-
zenesulfonate) (18). Compound 17 (3 mmol) was dissolved in
xylene, and 5 equiv of P2S5 was added. The reaction was continued
for 4 h at room temperature. The excess P2S5 was then filtered off.
The filtrate was concentrated under reduced pressure. The residue
was finally purified by silica gel chromatography to attain 1.14 g
of compound 18 with mp 198–201 °C. The yield is 70%. 1H NMR
(300 MHz, DMSO-d6): 7.768 (dd, 4H, J ) 8.4 Hz, 6.9 Hz, Ar-H),
7.496 (dd, 4H, J ) 8.1 Hz, 6.6 Hz, Ar-H), 7.345 (d, 1H, J ) 2.7
Hz, 8-H), 7.082 (s, 1H, 3-H), 6.859 (d, 1H, J ) 2.4 Hz, 6-H), 2.637
(t, 2H, J ) 7.5 Hz, 9-CH2), 2.426, 2.403 (2s, 6H, 14, 17-CH3),
1.403 (sex, 2H, J ) 7.5 Hz, 7.2 Hz, 10-CH2), 0.785 (t, 3H, J ) 7.2
Hz, 11-CH3). ESI-MS (m/z): 545.1 [M + H]+ (calcd MW )
544.67). IR (KBr) cm-1: 3087, 2974, 2881, 1614, 1597, 1385, 1144.
Anal. Calcd for C26H24O7S3 ·0.5H2O: C, 56.40; H, 4.55. Found: C,
56.52; H, 4.40.
1
was 63%. H NMR (400 MHz, DMSO-d6): 7.089 (m, 2H, 4-Ar-
H), 6.492 (m, 2H, 4-Ar-H), 6.477 (d, 1H, J ) 10.0 Hz, 8-H), 6.275
(d, 1H, J ) 8.4 Hz, 3-H), 5.577 (d, 1H, J ) 10.0 Hz, 7-H), 5.495
(d, 2H, J ) 6.0 Hz, NH2), 4.647 (m, 1H, J ) 6.0 Hz, 3.2 Hz, 12.4
Hz, 10-H), 2.840 (dd, 1H, J ) 12.4 Hz, 15.6 Hz, 11-He), 2.677
(dd, 1H, J ) 3.2 Hz, 15.6 Hz, 11-Ha), 1.453 (d, 3H, J ) 6.0 Hz,
10-CH3), 1.273, 1.224 (2s, 6H, 6-CH3). ESI-MS (m/z): 404.1 [M
+ H]+ (MW ) 403.44). HRESIMS obsd m/z 404.1495, calcd for
+
C24H22NO5 404.1498.
With this method, compound 184 was also prepared.
5-Hydroxy-4-propyl-9,10-dihydro-2H,8H-pyrano[2,3-
f]chromene-2,8-dione (110). To the mixture of compound 7 (0.2
mmol) and acrylic acid (0.2 mmol) was added 5 mL of CF3SO3H.
After the reaction was continued for 4 h at 60 °C, the reactants
were poured into ice–water under stirring. The crude was then
With this method, compound 22 was also prepared.
5,7-Dihydroxy-4-propyl-2H-chromene-2-thione (19). Com-
pound 18 (1.5 mmol) was dissolved in THF, and 3 equiv of TBAF
was added. The reaction was refluxed for 10 h and then concentrated

SARs of 11-Demethyl-12-oxo Calanolide A Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1445
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collected after extraction with ethyl acetate (3 × 15 mL) and drying
with MgSO4, and then it was concentrated under reduced pressure.
A white powder (110, 35.6 mg) was finally attained by column
1
chromography with mp 166–167 °C in a yield of 65%. H NMR
(400 MHz, DMSO-d6, ppm): 11.034 (s, 1H, 5-OH), 6.600 (s, 1H,
6-H), 6.053 (s, 1H, 3-H), 2.852 (m, 6H, 4, 9, 10-CH2), 1.571 (m,
2H, 4-CH2CH2CH3), 0.969 (t, 3H, J ) 7.2 Hz, 4-CH2CH2CH3).
ESI-MS (m/z): 275.1 [M + H]+ (calcd MW ) 274.27). Anal. Calcd
for C15H14O5: C, 65.69; H, 5.15. Found: C, 65.45; H, 5.20.
+
HRESIMS obsd m/z 275.0918, calcd for C15H15O5 275.0919.
With this method, compound 111 was also prepared.
5-Hydroxy-2-oxo-4-propyl-2H-chromen-7-yl 4-Methylbenze-
nesulfonate (185). Compound 17 (1 mmol) was dissolved in 10
mL of THF, and TBAF (1.5 mmol) was added. The mixture was
stirred at room temperature and monitored by TLC. The solvent
was evaporated in vacuo, and the residue was dissolved in ethyl
acetate (3 × 15 mL) and then washed with water (3 × 50 mL).
The organic layer was dried with anhydrous Na2SO4 and then
concentrated. The residue was purified by column chromatography.
The final product was obtained with a yield of 88% and mp 155–157
1
°C. H NMR (300 MHz, DMSO-d6, ppm): 11.261 (s, 1H, OH),
7.790 (m, 2H, Ar-H), 7.499 (m, 2H, Ar-H), 6.521 (d, 1H, J ) 2.4
Hz, 8-H), 6.472 (d, 1H, J ) 2.4 Hz, 6-H), 6.104 (s, 1H, 3-H), 2.859
(t, 2H, J ) 7.5 Hz, CH2), 2.422 (s, 3H, CH3), 1.547 (sex, 2H, J )
7.5 Hz, 7.2 Hz, CH2), 0.921 (t, 3H, J ) 7.2 Hz, CH3). ESI-MS
(m/z): 375.1 [M + H]+ (MW ) 374.42).
6,6,11-Trimethyl-4-propyl-10,11-dihydro-2H,6H,12H-dipyr-
ano[2,3-f:2′,3′-h]chromene-2,12-dione (115) under the Novel
Strategy. Compound 187 (0.2 mmol) was dissolved in 5 mL of
BF3/Et2O with an argon bubble, and crotonic acyl chloride (0.3
mmol) was added dropwise. The reaction was stirred and heated
at 50 °C and monitored by TLC. The mixture was poured into
ice–water and extracted with ethyl acetate (3 × 20 mL). The organic
layer was dried by anhydrous Na2SO4 and concentrated. The residue
was purified by column chromatography. The target compound was
obtained with a yield of 82% and mp 108–109 °C. 1H NMR (300
MHz, DMSO-d6, ppm): 6.565 (d, 1H, J ) 9.9 Hz, 8-H), 6.105 (s,
1H, 3-H), 5.789 (d, 1H, J ) 9.9 Hz, 7-H), 4.630 (dd, 1H, J ) 5.1
Hz, 11.1 Hz, 10-He), 4.256 (t, 1H, J ) 11.1 Hz, 10-Ha), 2.846 (m,
3H, 11-CH, 4-CH2CH2CH3), 1.562 (sex, 2H, J ) 7.5 Hz, 7.2 Hz,
4-CH2CH2CH3), 1.492, 1.469 (2s, 6H, CH3), 1.052 (d, 3H, J ) 6.9
Hz, 11-CH3), 0.968 (t, 3H, J ) 7.2 Hz, 4-CH2CH2CH3). ESI-MS
(m/z): 355.1 [M + H]+ (calcd MW ) 354.41).
(15) Zhou, C. M.; Wang, L.; Zhang, M. L.; Zhao, Z. Z.; Zhang, X. Q.;
Chen, X. H.; Chen, H. S. Total synthesis of (()-calanolide A and its
anti-HIV activity. Chin. Chem. Lett. 1998, 9, 433–434.
(16) Zhou, C. M.; Wang, L.; Zhang, M. L.; Zhao, Z. Z.; Zhang, X. Q.;
Chen, X. H.; Chen, H. S. Synthesis and anti-HIV activity of (()-
calanolide A and its analogues. Acta Pharm. Sin. 1999, 34, 673–678.
(17) Palmer, C. J.; Josephs, J. L. Synthesis of the Calophyllum coumarins.
Tetrahedron Lett. 1994, 35, 5363–5366.
(18) Deshpande, P. P.; Tagliaferri, F.; Victory, S. F.; Yan, S.; Baker, D. C.
Synthesis of optically active calanolide A and B. J. Org. Chem. 1995,
60, 2964–2965.
(19) Trost, B. M.; Toste, F. D. A catalytic enantioselective approach to
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9075.
With this method, compound 15 was also resynthesized.
Conclusion
A library was designed and synthesized in this article. A novel
compound (10-bromo-methyl-11-demethyl-12-oxo calanolide A,
123) was identified to have much higher inhibitory potency and
therapeutic index (EC50 ) 2.85 nM, TI > 10,526) than that of
(+)-calanolide A against HIV-1 through the assays in vitro.
Acknowledgment. We are grateful to Dr. Xiaoming Yu for
his kindly discussion and suggestion on chemistry. This research
was financially supported by the National Natural Science
Foundation of China (No. 39970868).
(20) Tanaka, T.; Kumanoto, T.; Ishikawa, T. Enantioselective total synthesis
of anti HIV-1 active (+)-calanolide A through a quinine-catalyzed
asymmetric intromolecular oxo-Michael addition. Tetrahedron Lett.
2000, 41, 10229–10232.
(21) Sekino, E.; Kumamoto, T.; Tanaka, T.; Ikeda, T.; Ishikawa, T. Concise
synthesis of anti-HIV-1 active (+)-inophyllum B and (+)-calanolide
A by application of (-)-quinine-catalyzed intramolecular oxo-Michael
addition. J. Org. Chem. 2004, 69, 2760–2767.
(22) Khilevich, A.; Mar, A.; Flavin, M. T.; Rizzo, J. D.; Lin, L.; Dzekhtser,
S.; Brankovic, D.; Zhang, H.; Chen, W.; Liao, S.; Zembower, D. E.;
Xu, Z. Q. Synthesis of (+)-calanolide A, an anti-HIV agent, via
enzyme-catalyzed resolution of the aldol products. Tetrahedron:
Asymmetry 1996, 7, 3315–3326.
(23) Cardellina, J. H., II; Bokesch, H. R.; McKee, T. C.; Boyd, M. R.
Resolution and comparative anti-HIV evaluation of the enantiomers
of canlanolide A and B. Bioorg. Med. Chem. Lett. 1995, 5, 1011–
1014.
Supporting Information Available: Complete spectroscopic and
analytical data for the preparation of all intermediates and target
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
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