Structure-activity modifications of the HIV-1 inhibitors (+)-calanolide A and (-)-calanolide B

Article abstract of DOI:10.1021/jm9602827

The Δ^7,8 olefinic linkages within (+)-calanolide A (1) and (-)- calanolide B (2) were catalytically reduced to determine impact on the anti- HIV activity of the parent compounds. In addition, a series of structure modifications of the C-12 hydroxyl group in (-)-calanolide B was made to investigate the importance of that substituent to the HIV-1 inhibitory activity of these coumarins. A total of 14 analogs were isolated or prepared and compared to (+)-calanolide A and (-)-calanolide B in the NCI primary anti-HIV assay. While none of the compounds showed activity superior to the two unmodified leads, some structure-activity requirements were apparent from the relative anti-HIV potencies of the various analogs.

Full text of DOI:10.1021/jm9602827

J . Med. Chem. 1996, 39, 4507-4510
4507
Notes
Str u ctu r e-Activity Mod ifica tion s of th e HIV-1 In h ibitor s (+)-Ca la n olid e A a n d
(-)-Ca la n olid e B1
Deborah L. Galinis,2 Richard W. Fuller, Tawnya C. McKee,* J ohn H. Cardellina II, Robert J . Gulakowski,
J ames B. McMahon, and Michael R. Boyd*
Laboratory of Drug Discovery Research and Development, Developmental Therapeutics Program, Division of Cancer Treatment,
Diagnosis and Centers, National Cancer Institute, Building 1052, Room 121, Frederick, Maryland 21702-1201
Received April 12, 1996X
The ∆7,8 olefinic linkages within (+)-calanolide A (1) and (-)-calanolide B (2) were catalytically
reduced to determine impact on the anti-HIV activity of the parent compounds. In addition,
a series of structure modifications of the C-12 hydroxyl group in (-)-calanolide B was made to
investigate the importance of that substituent to the HIV-1 inhibitory activity of these
coumarins. A total of 14 analogs were isolated or prepared and compared to (+)-calanolide A
and (-)-calanolide B in the NCI primary anti-HIV assay. While none of the compounds showed
activity superior to the two unmodified leads, some structure-activity requirements were
apparent from the relative anti-HIV potencies of the various analogs.
Ch a r t 1
In tr od u ction
(+)-Calanolide A (1, Chart 1), isolated from the
tropical rainforest tree Calophyllym lanigerum var.
austrocoriaceum,3 represents a novel subclass of HIV-1
specific reverse transcriptase inhibitor.4 It provides
comparable cytoprotection to several host cell lines
against three laboratory and eight clinical strains of
HIV-1.4d Recent data from this laboratory point to a
complex mechanism involving two binding sites on
HIV-1 RT.4e During efforts to identify a sufficient
source of 1 for preclinical drug development, (-)-
calanolide B (2) was obtained in relatively high yield
(g15%) from latex of C. teysmannii var. inophylloide.5
(-)-Calanolide B (2) is active against HIV-1 with a
potency similar to that of 1. The immediate availability
of significant quantities of 2 provided us with the
opportunity to explore some structure-activity relation-
ships of these molecules.
Our efforts focused on two sites within the calanolide
structure motif: the ∆7,8 olefin and the C-12 hydroxyl
group. Reduction of the ∆7,8 olefin would permit evalu-
ation of its impact on in vivo toxicity and potential
access to radiolabeled analogs for use in preclinical
development studies. Our interest in the relationship
of C-12 functionality and stereochemistry with HIV-
inhibitory activity stemmed from prior observations that
the 12-acetate (3) and 12-methoxyl (4) derivatives of (+)-
calanolide A were weakly active and inactive, respec-
tively,3 and that (+)-calanolide A and (-)-calanolide B
were potent inhibitors of HIV-1, while their respective
enantiomers were devoid of AIDS-antiviral activity.6
NH4OH-poisoned PtO2 gave (+)-7,8-dihydrocalanolide
A (6) and (-)-7,8-dihydrocalanolide B (7), respectively.
Turning to the C-12 functionality, we first sought to
invert the stereochemistry at C-12 in (-)-calanolide B
to obtain the enantiomer of natural (+)-calanolide A.
We used the oxidation-reduction approach, since this
would also provide the C-12 ketone for biological evalu-
ation. J ones oxidation afforded the best yields (up to
70%) of (-)-12-oxocalanolide B (8). Reduction of 8 with
NaBH4 in the presence of CeCl3 gave a 73% diastereo-
meric excess of (-)-calanolide A (9) in a mixture with
Resu lts
Ch em istr y. Initial efforts to reduce the ∆7,8-olefin
in 1 utilizing Pd/C yielded only 12-deoxy-7,8-dihydro-
calanolide A (5), the result of hydrogenolysis of the
benzylic alcohol. However, reduction of 1 and 2 with
X Abstract published in Advance ACS Abstracts, October 15, 1996.
S0022-2623(96)00282-8
This article not subject to U.S. Copyright. Published 1996 by the American Chemical Society

4508 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 22
Notes
Ta ble 1. HIV-Inhibitory Activities of (+)-Calanolide A (1),
2. A similar approach was used in the final step of a
recently reported synthesis of racemic calanolide A.8 The
J ones oxidation was also used to produce the ketones
of (+)-calanolide A (10), and (-)-7,8-dihydrocalanolide
B (11) for comparative testing.
(-)-Calanolide B (2), and Derivatives in the NCI Primary
Anti-HIV Screening Assay
compd
EC50 (µM)a
IC50 (µM)a
TIb
1
2
6
7
8
0.18 ( 0.01
0.2 ( 0.1
0.2 ( 0.1
0.2 ( 0.02
0.4 ( 0.1
c
7.3 ( 1.8
5.9 ( 1.9
5.2 ( 0.4
8.2 ( 1.8
8.5 ( 4.2
c
40
31
23
41
24
c
The 12-fluoro analog 12 was prepared by reacting (-)-
calanolide B directly with DAST. Complete 1H-NMR
analysis of the product revealed that the H-12 resonance
had shifted downfield to δ 5.53 and had J HF ) 51 Hz
and established that the reaction had proceeded with
retention of stereochemistry. Purification of 12 was
problematic, due to degradation on exposure to silica
gel and/or CDCl3. Rapid vacuum liquid chromatography
(VLC) on cyano-bonded phase provided adequate quan-
tities of 12 for complete characterization, but incubation
of 12 in cell culture media at 37 °C for 24 h gave
complete conversion to (-)-calanolide B (2). Therefore,
12 could not be independently evaluated in the available
anti-HIV assay. The silica gel and CDCl3-promoted
decomposition of 12 gave a new product, calanolene (13),
by elimination of HF.
Nucleophilic displacement of the derived triflate ester
was used for the preparation of the azide and thiol
analogs. The triflate was generated using standard
conditions (Tf2O/pyr, -78 °C) and was added rapidly by
cannula to the nucleophile in THF at -78 °C. NaSH
was used to obtain the 12-thio derivative 14. The
reaction of the triflate with NaN3 provided 12-azido-
calanolide B (15) in 61% yield. NMR analyses of 14 and
15 revealed a 3.5 Hz coupling constant for H-12,
indicating retention of stereochemistry.
9
10
11
12
13
14
15
16
0.15 ( 0.02
0.37 ( 0.6
d
9.3 ( 0.6
7.9 ( 0.6
d
62
21
d
c
c
c
c
4
8.4 ( 1.1
1.6 ( 0.1
0.6 ( 0.1
>150e
5.9 ( 2.2
5.8 ( 0.6
10
a
b
Averages of quadruplicate determinations. In vitro thera-
peutic indices (IC50/EC50). c Not measurable. Not independently
d
evaluated; not stable under assay conditions; rapidly converts to
active compound 2 under assay conditions.
relative to the amino derivative (16) was also consistent
with this conclusion. The inactivity of the (-) enantio-
mer of calanolide A6 indicated a critical stereochemistry
requirement, despite the potency of the derived ketone
(8), i.e., the oxygen substituent must lie in the plane of
the aromatic system or possess the S configuration.
Given the rather similar potencies of (+)-calanolide A
and (-)-calanolide B, the stereochemistry at C-11 rela-
tive to C-12 must underlie an essential conformation of
the dihydropyran ring. Reduction of the ∆7,8 double
bond conferred only a slight reduction in potency.
In summary, we have prepared a concise set of
analogs of (-)-calanolide B which revealed some inter-
esting structure-activity requirements in the molecule.
None of the derivatives was superior in potency to the
underivatized natural products (+)-calanolide A or (-)-
calanolide B. Racemic calanolide A is now accessible
by synthesis,8,10 but the inactivity of the unnatural
isomer indicates that a large-scale stereospecific syn-
thesis of the natural isomer would be desirable.11 In
the meantime, the presently more abundant5 natural
(-)-calanolide B represents a feasible alternative for
further preclinical research and development.
We had planned to prepare the 12-amino analog of
(-)-calanolide B from the azide, but attempts at selec-
tive reduction of the azido group in the presence of the
∆
7,8-olefin with Ph3P, CrCl2, and SnCl2 all failed.
Overreduction (or decomposition) was a problem in
reducing the azide (15) with the poisoned PtO2 catalyst;
therefore, the reduction was stopped prior to completion,
thus diminishing yields of 12-amino-7,8-dihydrocalano-
lide B, 16.
Biology. Using the XTT assay9 for cell viability, we
ranked compounds 1-16 with respect to their protection
of human lymphoblastoid CEM-SS cells from the cyto-
pathic effects of HIV-1 (RF strain). In those experi-
ments, the three dihydro derivatives (6, 7, and 16) and
the two ketones 8 and 10 were generally comparable in
potency to one another and to 1 and 2. The azide (15)
was somewhat less potent than the latter group, and
the thiol (14) was considerably less potent than 15. The
acetate of calanolide A (3) had only marginal activity,3
while 12-methoxycalanolide A (4), the 12-deoxy calano-
lide A derivative 5, and the alkene 13 were all inactive
against HIV-1. (-)-Calanolide A (9) was also devoid of
HIV inhibitory activity. These results are summarized
in Table 1.
Exp er im en ta l Section
Gen er a l. NMR spectra were acquired with a Varian VXR
500 spectrometer, using CDCl
as solvent and internal
3
standard. All triflate displacement reactions were carried out
in oven-dried glassware under a positive flow of argon. THF
and CH2Cl2 were distilled from CaH2 and stored over 4 Å
molecular sieves under argon. Salts were dried over P2O5 at
56 °C prior to use. All other commercial reagents were used
as received.
(+)-7,8-Dih yd r oca la n olid e A (6). Newly purchased PtO2
was exposed to ammonia vapors by setting an open vial
containing PtO2 in a covered beaker containing concentrated
NH4OH for 2 days. Traces of excess NH4OH were removed
from the catalyst under vacuum (24 h). Racemic synthetic
calanolide A (40 mg, 0.11 mmol) was reduced in 10 mL of
MeOH with H2 and 22 mg (0.1 mmol) of poisoned PtO2 for 60
min under positive atmospheric pressure (balloon). The crude
racemic mixture was separated into enantiomers by chiral
HPLC (Regis Whelk-O, 1 × 25 cm, hexane-iPrOH, 9:1, 6 mL/
min), monitored at 270 nm, to yield (+)-7,8-dihydrocalanolide
A (6) 17.5 mg (42.7%) and (-)-7,8-dihydrocalanolide A 19.0
Discu ssion
The inactivity of 5 and 13 indicated that a heteroatom
is required at C-12 for activity. The relative potency of
the ketones (8 and 10) and azide (15) strongly suggested
that the C-12 functionality acts primarily as a hydrogen
bond acceptor, while the decreasing potency in the series
8 > 15 > 3 indicated that there is a fairly stringent
spatial limitation and/or stereoelectronic requirement
around C-12. The decreased potency of the thiol (14)
1
mg (46.3%); 16 [R]D +88° (c 0.2, CHCl3); H NMR (CDCl3) δ
5.29 (s, 1H, H-3), 4.74 (d, J ) 7.5 Hz, 1H, H-12), 3.91 (dq, J )
9.5, 6.3 Hz, 1H, H-10), 2.88 (m, 2H, H-13), 2.62 (m, 2H, H-8),
1.90 (m, 1H, H-11), 1.77 (m, 2H, H-7), 1.61 (m, 2H, H-14), 1.44

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 22 4509
(d, J ) 6.3 Hz, 1H, H-18), 1.39 (s, 3H, H-16 or H-17), 1.35 (s,
3H, H-16 or H-17), 1.14 (d, J ) 7.0 Hz, 3H, H-19), 1.01 (t, J )
7.0 Hz, 2H, H-15); 13C NMR (CDCl3) δ 160.8 (C-2), 159.5 (C-
4), 155.6 (C-8b), 153.6 (C-12b), 151.9 (C-4b), 109.9 (C-3), 105.3,
105.2 (C-8a, C-12a), 103.9 (C-4a), 76.9 (C-6), 75.7 (C-10), 67.3
(C-12), 40.5 (C-11), 39.2 (C-13), 31.4 (C-8), 27.1 (C-16 or C-17),
26.1 (C-16 or C-17), 23.4 (C-14), 19.0 (C-7), 16.9 (C-7), 15.1
(C-19), 13.9 (C-15); HRFABMS m/ z 373.1995 (calcd for
C22H29O5, 373.2015).
(-)-7,8-Dih yd r oca la n olid e B (7). (-)-Calanolide B (500
mg, 1.35 mmol) was reduced in 10 mL of MeOH with H2 and
32 mg (0.15 mmol) of poisoned PtO2 for 1 h. Catalyst was
removed by filtration, and the crude reaction mixture was
purified by HPLC on Rainin Dynamax silica (1 × 25cm, eluted
with 3:2 hexane-EtOAc) to yield 346 mg of 7 (69%): [R]D -28°
(c ) 0.6, CHCl3); 1H NMR (CDCl3) δ 5.92 (s, 1H, H-3), 4.99 (d,
J ) 3.0 Hz, 1H, H-12), 4.24 (dq, J ) 6.5, 4.5 Hz, 1H, H-10),
2.88 (m, 2H, H-13), 2.63 (q, J ) 6.8 Hz, 2H, H-8), 1.77 (t, J )
6.8 Hz, 2H, H-7), 1.73 (m, 1H, H-11), 1.62 (m, 1H, H-14), 1.42
(d, J ) 6.5 Hz, 3H, H-18), 1.37 (s, 6H, H-16,17), 1.14 (d, J )
7.0 Hz, 3H, H-19), 1.01 (t, J ) 9.0 Hz, 3H, H-15); 13C NMR
(CDCl3) δ 161.5 (C-2), 159.3 (C-4), 155.6 (C-8b), 153.1 (12b),
152.1 (C-4b), 109.9 (C-3), 105.2 (C-8a), 104.9 (12a), 103.4 (4a),
75.8 (C-6), 72.8 (C-10), 61.8 (C-12), 39.0 (C-11), 38.3 (C-13),
31.5 (C-8), 26.6 (C-16), 26.5 (C-17), 23.4 (C-14), 18.9 (C-18),
16.9 (C-7), 13.9 (C-15), 12.5 (C-19); HREIMS 372.1919 (calcd
for C22H28O5, 372.1936)
H-19), 1.02 (t, J ) 7.0 Hz, 3H, H-15); 13C NMR (CDCl3) δ 189.9
(C-12), 159.1 (C-2), 157.0 (C-4, C-4b), 155.9 (C-8b), 155.5 (C-
12b), 127.0 (C-7), 115.8 (C-8), 112.1 (C-3), 105.4 (C-8a), 104.5
(C-12a), 103.5 (C-4a), 79.6 (C-10), 79.2 (C-6), 47.3 (C-11), 38.8
(C-13), 28.3, 28.0 (C-16, C-17), 23.2 (C-14), 19.6 (C-18), 13.9
(C-15), 10.5 (C-19); HRFABMS m/ z 369.1698 (calcd for
C22H29O5, 369.1702).
(-)-7,8-Dih yd r o-12-oxoca la n olid e B (11). (-)-7,8-Dihy-
drocalanolide B (40 mg, 0.11 mmol) was oxidized, as described
above, to yield 26.8 mg of 11 (68%): [R]D -58° (c ) 0.5, CHCl3);
1H NMR (CDCl3) δ 6.02 (s, 1H, H-3), 4.27 (dq, J ) 6.5, 5.0 Hz,
1H, H-10), 2.87 (dd, J ) 8.0, 7.5 Hz, 2H, H-13), 2.68 (m, 2H,
H-8), 2.52 (m, 1H, H-11), 1.84 (ddd, J ) 13.5, 7.0, 6.5 Hz, 2H,
H-7), 1.60 (sextet, J ) 8.0 Hz, 2H, H-14), 1.52 (d, J ) 6.5 Hz,
3H, H-18), 1.43 (s, 3H, H-16 or H-17), 1.41 (s, 3H, H-16 or
H-17), 1.21 (d, J ) 7.0 Hz, 3H, H-15), 1.08 (t, J ) 7.5 Hz, 3H,
H-19); 13C NMR (CDCl3) δ 190.1 (C-12), 161.9 (C-2), 160.1 (C-
4), 157.6 (C-8b), 156.7 (C-12b), 154.5 (C-4b), 111.8 (C-3), 104.7
(C-8a) 104.4 (C-12a), 102.8 (C-4a), 79.3 (C-6), 77.2 (C-10), 47.1
(C-11), 39.2 (C-13), 31.1 (C-8) 26.9, 26.3 (C-16, C-17), 23.3 (C-
14), 19.6 (C-18), 16.7 (C-7), 13.8 (C-15), 10.5 (C-19); HRFABMS
m/ z 371.1827 (calcd for C22H27O5, 371.1858).
(-)-12-F lu or oca la n olid e B (12). (-)-Calanolide B (37.8
mg, 0.1 mmol) in CH2Cl2 (1 mL) was slowly added to a solution
of DAST (8.5 mg, 0.05 mmol) and powdered, oven-dried
Na2CO3 in CH2Cl2 (4 mL) at -78 °C and allowed to warm
slowly to room temperature. After 1 h, the reaction mixture
was extracted with water and the organic layer was subjected
to vacuum liquid chromatography on cyano-bonded phase,
eluting with an EtOAc-hexane gradient (5-30%), to give 12-
fluorocalanolide B (12) (10 mg, 27%): [R]D -98° (c ) 0.015,
C6H6); 1H NMR (C6D6) δ 6.67 (d, J ) 9.8 Hz, 1H, H-8), 5.82 (s,
1H, H-3), 5.53 (dd, J ) 2.4, 51.3 Hz, 1H, H-12), 5.09 (d, J )
10.2 Hz, 1H, H-7), 3.96, (m, 1H, H-10), 2.51 (m, 3H, H-11, H-13,
H-13′), 1.35 (sextet, J ) 7.3 Hz, 2H, H-14, H-14′), 1.12 (s, 6H,
H-16, H-17), 1.02 (d, J ) 6.3 Hz, 3H, H-18), 0.79 (t, J ) 7.3
Hz, 3H, H-15), 0.77 (d, J ) 6.8 Hz, 3H, H-19); 13C NMR (C6D6)
δ 159.3 (C-2), 156.7 (C-4), 155.5 (C-12b), 153.7 (C-8b), 152.7
(C-4b), 126.3 (C-7), 116.8 (C-8), 111.9 (C-3), 105.7 (C-8a), 103.9
(C-4a), 102.6 (d, J C,F ) 20 Hz, C-12a), 81.8 (d, J C,F ) 173 Hz,
C-12), 77.6 (C-6), 73.2 (C-10), 38.5 (C-13), 37.9 (d, J C,F ) 20
Hz, C-11), 27.5 (C-16 and C-17), 23.3 (C-14), 18.6 (C-18), 14.1
(-)-12-Oxoca la n olid e B (8). (-)-Calanolide B (20 mg, 0.05
mmol) was dissolved in 5 mL acetone and stirred at 0 °C. J ones
reagent (100 µL) was added slowly, and the solution was
stirred for 10 min. After 10 min, an additional 100 µL J ones
reagent was slowly added and the solution was stirred at 0
°C for 40 min. The reaction mixture was diluted with 15 mL
of H2O and extracted with CH2Cl2 (3 × 15 mL). The organic
layer was washed with saturated NaHCO3 and then dried over
anhydrous MgSO4 and filtered. The crude product was puri-
fied by HPLC (Rainin Dynamax silica, 2.1 × 25 cm), using
hexane-EtOAc (13:7, 20 mL/min) to give 12.4 mg of (-)-12-
1
oxocalanolide B (61% yield): [R]D -55° (c ) 1.27, CHCl3); H
NMR (CDCl3) δ 6.59 (d, J ) 9.8 Hz, 1H, H-8), 5.98 (s, 1H, H-3),
5.55 (d, J ) 9.8 Hz, 1H, H-7), 4.25 (dq, J ) 11.2, 6.9 Hz, 1H,
H-10), 2.83 (dt, J ) 7.8, 3.0 Hz, 2H, H-13, H-13′), 2.50 (dq, J
) 11.2, 6.8 Hz, 1H, H-11), 1.59 (sextet, J ) 7.8 Hz, 2H, H-14,
H-14′), 1.50 (s, 3H, H-16), 1.49 (d, J ) 6.9 Hz, 3H, H-18), 1.47
(s, 3H, H-17), 1.16 (d, J ) 6.8 Hz, 3H, H-19), 0.97 (t, J ) 7.3
Hz, 3H, H-15); 13C NMR (CDCl3) δ 189.9 (C-12), 159.6 (C-2),
159.0 (C-4), 157.0, 155.9, 155.4 (C-4b, C-8b, C-12b), 126.9 (C-
7), 115.8 (C-8), 112.0 (C-3), 105.4, 104.4 (C-8a, C-12a), 103.5
(C-4a), 79.5 (C-10), 79.2 (C-6), 47.2 (C-11), 38.7 (C-13), 28.3
(C-16), 27.9 (C-17), 23.1 (C-14), 19.6 (C-18), 13.9 (C-15), 10.4
(C-19); HRElMS m/z 368.1591 (calcd for C22H24O5, 368.1624).
(-)-Ca la n olid e A (9). A solution of (-)-12-oxocalanolide
B (8) (18.4 mg, 0.05 mmol) in EtOH (1 mL) was added to a
slurry of NaBH4 (5.0 mg, 0.13 mmol) and CeCl3‚(H2O)7 (18.4
mg, 0.05 mmol) in EtOH (4 mL). The resulting mixture was
stirred at 25 °C for 0.5 h. Water (2 mL) was added to the
reaction mixture, which was then extracted with EtOAc (3 ×
5 mL). The combined organic layers were concentrated and
purified by silica HPLC, using the same conditions reported
above, to give (-)-calanolide B (2) (2.6 mg, 13%) and the
enantiomer of natural calanolide A (9) (16.3 mg, 87%): [R]D
-68° (c ) 1.36, CHCl3); HREIMS m/z 370.1770 (calcd for
(C-15), 12.1 (C-19); HREIMS m/z 372.1738 (calcd for C22H25
-
FO4, 372.1737).
12-Th ioca la n olid e B (14). A solution of (-)-calanolide B
(75 mg, 0.2 mmol) and pyridine (12 µL) in CH2Cl2 (2 mL) was
added slowly (dropwise) to a solution of triflic anhydride (50
µL, 0.3 mmol) in CH2Cl2 (2 mL) at -78 °C. After 1 h, the
reaction mixture was transferred via cannula to a flask
containing excess NaSH in THF (2 mL) at -78 °C. After
slowly warming overnight, the reaction mixture was extracted
with water (3 mL) and the organic layer concentrated. Vacuum-
liquid chromatography on silica, using an EtOAc-hexane
gradient (5-30%), followed by HPLC on a column (2.1 × 25
cm), eluting with hexane-EtOAc (7:3) at 15 mL/min, gave
calanolene (13) (20.1 mg, 29%) and 12-thiocalanolide B (14)
(14.5 mg, 19%).
1
13: [R]D +28.7° (c ) 0.28, CHCl3); H-NMR (CDCl3) δ 6.61
(s, 1H, H-12), 6.59 (d, J ) 10.2 Hz, 1H, H-8), 5.90 (s, 1H, H-3),
5.43 (d, J ) 10.2 Hz, 1H, H-7), 4.86 (q, J ) 6.3 Hz, 1H, H-10),
2.85 (m, 2H, H-13, H-13′), 1.82 (br s, 3H, H-19), 1.62 (sextet,
J ) 7.3 Hz, 2H, H-14, H-14′), 1.46 (s, 3H, H-16), 1.44 (s, 3H,
H-17), 1.36 (d, J ) 6.8 Hz, 3H, H-18), 1.00 (t, J ) 7.3 Hz, 3H,
H-15); 13C NMR (CDCl3) δ 161.0 (C-2), 158.4 (C-4), 150.7, 150.0
(C-8b, C-12b), 149.7 (C-4b), 132.2 (C-12), 127.2 (C-7), 116.4 (C-
8), 112.0 (C-11), 110.8 (C-3), 106.4 (C-12a), 103.8 (C-4a and
C-8a), 77.7 (C-6), 76.0 (C-10), 38.6 (C-13), 28.0 (C-16), 27.6 (C-
17), 23.2 (C-14), 19.3 (C-18 and C-19), 14.0 (C-15); HREIMS
m/z 352.1674 (calcd for C22H24O4, 352.1675).
1
C22H26O6, 370.1780); H and 13C NMR spectra were identical
to those reported for natural (+)-calanolide A.3
(+)-12-Oxoca la n olid e A (10). (()-Calanolide A (40 mg,
0.11 mmol) was oxidized using two 100 µL portions of J ones
reagent as described above. The crude reaction mixture was
resolved by chiral HPLC (Regis Whelk-O, 1 × 25 cm, hexane-
iPrOH, 9:1) to yield 2.0 mg of 10 and 2.1 mg of its enantiomer
(8) (10% overall yield).
14: [R]D +203.4° (c ) 0.71, CHCl3); 1H NMR (CDCl3) δ 6.55
(d, J ) 9.7 Hz, 1H, H-8), 5.91 (s, 1H, H-3), 5.45 (d, J ) 9.7 Hz,
1H, H-7), 4.99 (d, J ) 3.4 Hz, 1H, H-12), 4.56 (dq, J ) 10.2,
6.8 Hz, 1H, H-10), 2.85 (dd, J ) 7.8, 7.8 Hz, 2H, H-13, H-13′),
2.08 (m, 1H, H-11), 1.62 (sexet, J ) 7.3 Hz, 2H, H-14, H-14′),
1.46 (d, J ) 6.8 Hz, 1H, SH), 1.44 (s, 3H, H-16), 1.42 (d, J )
6.8 Hz, 3H, H-19), 1.40 (s, 3H, H-17), 1.38 (d, J ) 7.3 z, 3H,
10: [R ]D +56° (c ) 0.5, CHCl3); 1H NMR (CDCl3) δ 6.62 (d,
J ) 9.5 Hz, 1H, H-8), 6.02 (s, 1H, H-3), 5.57 (d, J ) 9.5 Hz,
1H, H-7), 4.27 (dq, J ) 11.5, 6.3 Hz, 1H, H-10), 2.85 (dt, J )
7.3, 2.5 Hz, 2H, H-13, H-13′), 2.52 (m, 1H, H-11), 1.61 (sextet,
J ) 7.3 Hz, 2H, H-14, H-14′), 1.53 (s, 3H, H-16), 1.51 (d, J )
6.3 Hz, 3H, H-18), 1.49 (s, 3H, H-17), 1.21 (d, J ) 7.0 Hz, 3H,

4510 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 22
Notes
H-18), 1.00 (t, J ) 7.3 Hz, 3H, H-15); 13C NMR (CDCl3) δ 160.7
(C-2), 158.2 (C-4), 152.4 (C-12b), 152.3 (C-8b), 150.7 (C-4b),
126.5 (C-7), 116.7 (C-8), 110.4 (C-3), 106.0 (C-8a), 105.9 (C-
12a), 103.4 (C-4a), 77.4 (C-6), 74.3 (C-10), 43.5 (C-12), 39.2 (C-
11), 38.6 (C-13), 27.7 (C-17), 27.6 (C-16), 23.2 (C-14), 19.1 (C-
18), 15.1 (C-19), 14.0 (C-15); HREIMS m/z 386.1530 (calcd for
C22H26O4S, 386.1552).
12-Azid oca la n olid e B (15). Generation of the triflate,
displacement with NaN3, and purification as described above
resulted in the formation of calanolene (13) (9.8 mg, 28%) and
12-azidocalanolide B (15) (24.2 mg, 61%). 15: [R]D +105° (c
) 0.5, CHCl3); 1H NMR (CDCl3) δ 6.58 (d, J ) 9.8 Hz, 1H,
H-8), 5.94 (s, 3H, H-3), 5.51 (d, J ) 9.8 Hz, 1H, H-7), 4.95 (d,
J ) 3.4 Hz, 1H, H-12), 4.12 (dq, J ) 10.7, 6.4 Hz, 1H, H-10),
2.87 (dd, J ) 6.4, 8.9 Hz, 2H, H-13, H-13′), 1.80 (m, 1H, H-11),
1.64 (sextet, J ) 7.3 Hz, 2H, H-14, H-14′), 1.48 (s, 3H, H-16),
1.46 (s, 3H, H-17), 1.38 (d, J ) 6.4 Hz, 3H, H-18), 1.10 (d, J )
6.8 Hz, 3H, H-19), 1.01 (t, J ) 7.3 Hz, 3H, H-15); 13C NMR
(CDCl3) δ 160.1 (C-2), 158.2 (C-4), 153.6 (C-8b), 152.8 (C-12b),
151.8 (C-4b), 126.9 (C-7), 116.2 (C-8), 110.7 (C-3), 106.0 (C-
12a), 103.4 (C-8a), 102.0 (C-4a), 77.8 (C-6), 73.4 (C-10), 55.3
(C-12), 38.5 (C-11), 37.1 (C-13), 27.8 (C-16 and C-17), 23.1 (C-
14), 18.8 (C-18), 14.0 (C-15), 13.5 (C-19); HRMS m/z 395.1836
(calcd for C22H25N3O4, 395.1845).
(2) Current address: American Cyanamid Co., Agricultural Prod-
ucts Research Division, P.O. Box 400, Princeton, NJ 08543-0400.
(3) Kashman, Y.; Gustafson, K. R.; Fuller, R. W.; Cardellina, J . H.,
II; McMahon, J . B.; Currens, M. J .; Buckheit, R. W., J r.; Hughes,
S. H.; Cragg, G. M.; Boyd, M. R. The calanolides, a novel HIV-
inhibitory class of coumarin derivatives from the tropical rain-
forest tree, Calophyllum lanigerum. J . Med. Chem. 1992, 35,
2735-2743.
(4) (a) Hizi, A.; Tal, R.; Shaharabany, M.; Currens, M. J .; Boyd, M.
R.; Hughes, S. H.; McMahon, J . B. Specific inhibition of the
reverse transcriptase of human immunodeficiency virus type 1
and the chimeric enzymes of human immunodeficiency virus
type 1 and type 2 by nonnucleoside inhibitors. Antimicrob.
Agents Chemother. 1993, 37, 1037-1042. (b) Boyer, P. L.;
Currens, M. J .; McMahon, J . B.; Boyd, M. R.; Hughes, S. H.
Analysis of nonnucleoside drug-resistant variants of human
immunodeficiency virus type 1 reverse transcriptase. J . Virol.
1993, 67, 2412-2420. (c) Buckheit, R. W., J r.; Fliakas-Boltz, V.;
Decker, W. D.; Roberson, J . L.; Pyle, C. A.; White, E. L.; Bowden,
B. J .; McMahon, J . B.; Boyd, M. R.; Bader, J . P.; Nickell, D. G.;
Barth, H.; Antonucci, T. K. Biological and biochemical anti-HIV
activity of the benzothiadiazine class of nonnucleoside reverse
transcriptase inhibitors. Antiviral Res. 1994, 25, 43-56. (d)
Currens, M. J .; Gulakowski, R. J .; Mariner, J . M.; Moran, R. A.;
Buckheit, R. W., J r.; Gustafson, K. R.; McMahon, J . B.; Boyd,
M. R. Antiviral activity and mechanism of action of calanolide
A against the human immunodeficiency virus. J . Pharmacol.
Exp. Ther., in press. (e) Currens, M. J .; Mariner, J . M.;
McMahon, J . B.; Boyd, M. R. Kinetic analysis of inhibition of
HIV-1 reverse transcriptase by calanolide A. J . Pharmacol. Exp.
Ther., in press.
12-Am in o-7,8-d ih yd r oca la n olid e B (16). Poisoned plati-
num oxide catalyst (PtO2, 13.0 mg, 0.06 mmol) was added to
(-)-12-azidocalanolide B (15, 23.4 mg, 0.06 mmol) in EtOAc
(5 mL). The mixture was stirred under a hydrogen atmo-
sphere for 20 min and then filtered rapidly through a 20 µm
filter. Concentration of the EtOAc solution yielded a mixture
of starting material and desired product. HPLC purification
on an amino-bonded phase column (Rainin, 1 × 25 cm), eluting
with hexane-EtOAc (17:3) at 4 mL/min and monitoring at 290
nm, gave 12-amino-7,8-dihydrocalanolide B (16, 6.4 mg,
(5) Fuller, R. W.; Bokesch, H. R.; Gustafson, K. R.; McKee, T. C.;
Cardellina, J . H., II; McMahon, J . B.; Cragg, G. M.; Soejarto, D.
D.; Boyd, M. R. HIV-Inhibitory coumarins from latex of the
tropical rainforest tree Calophyllum teysmannii var. inophyl-
loide. BioMed. Chem. Lett. 1994, 4, 1961-2964.
(6) Cardellina, J . H., II; Bokesch, H. R.; McKee, T. C.; Boyd, M. R.
Resolution and comparative anti-HIV evaluation of the enanti-
omers of calanolides A and B. BioMed. Chem. Lett. 1995, 5,
1011-1014.
1
28%): [R]D +101.2° (c ) 0.4, CHCl3); H NMR (CDCl3) δ 5.94
(s, 1H, H-3), 5.01 (d, J ) 2.9 Hz, 1H, H-12), 4.13 (dq, J ) 10.7,
6.3 Hz, 1H, H-10), 2.88 (dt, J ) 2.9, 8.3 Hz, 2H, H-8), 2.63 (m,
2H, H-13, H-13′), 1.82 (m, 1H, H-11), 1.78 (t, J ) 6.3 Hz, 2H,
H-7), 1.63 (m, 4H, H-14, 12-NH2), 1.40 (d, J ) 6.3 Hz, 3H,
H-18), 1.38 (s, 3H, H-16), 1.39 (s, 3H, H-17), 1.12 (d, J ) 6.8
Hz, 3H, H-19), 1.02 (t, J ) 7.3 Hz, 3H, H-15); 13C NMR (CDCl3)
δ 160.6 (C-2), 158.8 (C-4), 155.3 (C-8b), 152.8, 152.6 (C-4b and
C-12b), 110.5 (C-3), 104.9 (C-12a), 103.3 (C-4a), 100.8 (C-8a),
75.9 (C-6), 73.3 (C-10), 55.5 (C-12), 39.0 (C-13), 37.2 (C-11),
31.4 (C-8), 26.7 (C-17 and C-18), 23.3 (C-14), 18.9 (C-18), 16.9
(C-7), 13.9 (C-15), 13.5 (C-19); HREIMS m/z 371.2083 (calcd
for C22H29NO4, 371.2097).
(7) Gemal, A. L.; Luche, J .-L. Lanthanoids in organic synthesis. 6.
The reduction of R-enones by sodium borohydride in the presence
of lanthanoid chlorides: synthetic and mechanistic aspects. J .
Am. Chem. Soc. 1983, 103, 5454-5459.
(8) Chenera, B.; West, M. L.; Finkelstein, J . A.; Dreyer, G. B. Total
synthesis of (+)-calanolide A, a non-nucleoside inhibitor of HIV-1
reverse transcriptase. J . Org. Chem. 1993, 58, 5605-5606.
(9) Gulakowski, R. J .; McMahon, J . B.; Staley, P. G.; Moran R. A.;
Boyd, M. R. A semiautomated multiparameter approach for anti-
HIV drug screening. J . Virol. Methods 1991, 33, 87-100.
(10) (a) Kucherenko, A.; Flavin, M. T.; Boulanger, W. A.; Khilevich,
A.; Shone, R. L.; Rizzo, J . D.; Sheinkman, A. K.; Xu, Z-Q. Novel
approach for synthesis of (()-calanolide A and its anti-HIV
activity. Tetrahedron Lett. 1995, 36, 5475-5478. (b)Palmer, C.
J .; J osephs, J . L. Synthesis of Calophyllum coumarins. Tetra-
hedron Lett. 1994, 35, 5363-5366.
(11) Since the completion of this work, several approaches to chiral
syntheses of the calanolides have been published: (a) Rama Rao,
A. V.; Gaitonde, A. S.; Prakash, K. R. C.; Prahlada Rao, S. A
concise synthesis of chiral 2-methyl chroman-4-ones: stereo-
selective build-up of the chromanol moiety of anti-HIV agent,
calanolide A. Tetrahedron Lett. 1994, 35, 6347-6450. (b) Desh-
pande, P. P.; Baker, D. C. A simple approach to the synthesis of
the chiral substituted chroman ring of Calophyllum coumarins.
Synthesis 1995, 630-632. ( c) 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.
Ack n ow led gm en t. We thank A. Kozikowsi and D.
Torok for helpful suggestions, L. Pannell and G. Gray
for mass spectral analyses, G. Cragg for coordinating
the supply of C. teysmannii latex, and M. Serit, H.
Bokesch, and B. Krepps for technical assistance.
Refer en ces
(1) Part 30 in the series HIV-Inhibitory Natural Products. For part
29, see: McKee, T. C.; Fuller, R. W.; Covington, C. D.; Cardellina,
J . H., II; Gulakowski, R. J .; Krepps, B. L.; McMahon, J . B.; Boyd,
M. R. New pyranocoumarins isolated from Calophyllum lani-
gerum and Calophyllum teysmannii. J . Nat. Prod. 1996, 59,
754-758.
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