Chemistry and Structure-Activity Relationship of HIV-1 Integrase Inhibitor Integracide B and Related Natural Products

Article abstract of DOI:10.1021/np030211s

Integracides, 4,4-dimethylergostane triterpenoids, are inhibitors of HIV-1 integrase, a critical enzyme in replication of HIV-1. The chemistry and structure-activity relationship of integracide B and related natural products are described. A charged group, e.g., a sulfate, carboxyl, or amino, is required for the HIV-1 integrase activity. These compounds showed HIV-1 integrase activity with IC₅₀ values in the range 4.8-15μM and exhibited antiviral activity in a viral spread assay, but with only a small or no therapeutic window.

Full text of DOI:10.1021/np030211s

1338
J . Nat. Prod. 2003, 66, 1338-1344
Ch em istr y a n d Str u ctu r e-Activity Rela tion sh ip of HIV-1 In tegr a se In h ibitor
In tegr a cid e B a n d Rela ted Na tu r a l P r od u cts
Sheo B. Singh,*,† J ohn G. Ondeyka,† William A. Schleif,‡ Peter Felock,‡ and Daria J . Hazuda‡
Merck Research Laboratories, P.O. Box 2000, Rahway, New J ersey 07065, and Merck Research Laboratories,
West Point, Pennsylvania 19486
Received May 9, 2003
Integracides, 4,4-dimethylergostane triterpenoids, are inhibitors of HIV-1 integrase, a critical enzyme in
replication of HIV-1. The chemistry and structure-activity relationship of integracide B and related
natural products are described. A charged group, e.g., a sulfate, carboxyl, or amino, is required for the
HIV-1 integrase activity. These compounds showed HIV-1 integrase activity with IC50 values in the range
4.8-15 µM and exhibited antiviral activity in a viral spread assay, but with only a small or no therapeutic
window.
Integration is an essential step catalyzed by HIV-1 inte-
grase, an enzyme responsible for three distinct steps includ-
ing assembly of proviral DNA onto integrase, endonucle-
olytic cleavage of the proviral DNA, and strand transfer of
the proviral DNA into the host cell DNA.1 This distinctive
retroviral process is absent in its mammalian host cells
and, therefore, presents a safe target for development of
anti-HIV therapy. This concept appears to have recently
been validated with the discovery of some diketo acid
(DKA)-based inhibitors,2a,b and at least two inhibitors are
in clinical development.2c
Over the past few years we have reported a number of
natural product integrase inhibitors from the screening of
extracts derived from microbial, terrestrial, and marine
organisms. These include equisetin,3 integric acid,4 com-
plestatin,5 integracins,6 integrastatins,7 integramycin,8
chaetochromin and derivatives,9 and integracides.10
Integracides are 4,4-dimethylergostane derivatives that
are moderately potent and selective inhibitors of HIV-1
integrase. Integracide A (1), a 3-sulfate ester of integracide
B (2), is the most active member of the five natural
products reported.10 To explore the structure-activity
relationship of this class of compounds, we undertook
additional studies including evaluation of related com-
pounds from our natural products collection and selective
derivatization of 2. We describe herein the syntheses and
HIV-1 integrase structure-activity relationship of these
derivatives and related natural products.
Resu lts a n d Discu ssion
Sou r ces of In h ibitor s. (a ) Na tu r a l P r od u cts a n d
Rela ted Com p ou n d s fr om Sa m p le Collection . Com-
(b) Ch em ica l Der iva tiza tion of 2. Acetylation of 2
pounds 3-6 were obtained from our natural products
with acetic anhydride exclusively produced tetraacetate 14
collection. Compounds 3-5 were originally isolated from
(Scheme 1). Base hydrolysis of 2 yielded the 12-hydroxy
a Fusarium sp. as inhibitors of elastase.11 Compound 6 was
derivative 15 in >70% yield. Reaction of 2 with 2,2-
dimethoxypropane under pyridinium para-toluenesulfonate
discovered as an antifungal agent. Clavaric acid (7), its
methyl ester 8, and clavarinone (9) were discovered as Ras
(PPTS) catalysis afforded acetonide 16 (80% yield). Reac-
FPTase inhibitors.12 Compounds 10-13 were obtained as
tion of 2 with methane sulfonyl chloride selectively pro-
duced the 2-mesylate, which was heated with DBU in
toluene at 50 °C to give the 2,3-epoxide 17 in 70% yield.
Benzoylation of 2 with benzoic anhydride at room temper-
side products of the sulfate hydrolysis of 1, and their
preparation and characterization were reported earlier.13
The structure and purity of these compounds were verified
by LCMS analysis.
ature afforded a 2:2:3 mixture of 2,3-di, 3-, and 2-mono
benzoates, 18, 19, and 20, respectively, in 80% combined
yield after silica gel chromatography. A similar reaction
of 2 with MOM chloride produced three MOM ethers, 21-
23, in analogous ratios. Heating of 2 with succinic anhy-
* Corresponding author. Fax: (732) 594-6880, E-mail: sheo_singh@
merck.com.
† Merck Research Laboratories, Rahway.
‡ Merck Research Laboratories, West Point.
10.1021/np030211s CCC: $25.00
© 2003 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/02/2003

HIV-1 Integrase Inhibitor Integracide B
J ournal of Natural Products, 2003, Vol. 66, No. 10 1339
Sch em e 1a
dride at 50 °C afforded a mixture of the two mono-
hemisuccinates 24 and 25 in a ratio of 1:3. These compounds
were purified by reversed-phase HPLC under acetic condi-
tions. Methylation of these hemisuccinates with diaz-
omethane furnished their respective methyl esters 26 and
27. The standard mixed anhydride activation of the car-
boxyl group of 25 followed by reaction with hydroxylamine
and reversed-phase HPLC yielded the hydroxymate 28 in
a 60% yield. Acylation of 2 with N-t-Boc-Gly succinimide
ester surprisingly afforded 2,3-diester and only the 2-mo-
noester 29 and 30 in a 2:1 ratio. A similar product
distribution [2,3-di (31) and 2-mono (32)] was also observed
upon the reaction of 2 with FMOC-Gly-OPFP ester. The
product distribution in the last two acylation reactions was
different from the benzoylation reaction. Standard depro-
tection of the Boc group with TFA caused decomposition
of the desired products 33 and 34. However, deprotection
of the FMOC protected esters 31 and 32 with piperidine
in DMF followed by purification by reversed-phase HPLC
afforded 33 and 34, respectively. During the deprotection
of the monoester 32, about 20% of the 3-monoester 35 was
also recovered as a result of acyl transfer from C-2 to C-3.
A similar C-2 to C-3 acyl transfer was also observed in the
hemisuccinate series wherein 24 was produced from 25
after several months of storage.
HIV-1 In tegr a se Str u ctu r e-Activity Rela tion sh ip .
All compounds described in this paper were first evaluated
for their ability to inhibit the coupled and strand transfer
reactions of recombinant HIV-1 integrase by an assay
protocol described earlier,2a and the data are presented in
Table 1. Like integracide A (1), all natural products with
a sulfate ester at the C-3 position (cf. 3-5) were essentially
equipotent and exhibited IC50 values of 5-6 µM in the
coupled assay. The 2-deoxy compound 6 showed slightly
better activity in this assay and displayed an IC50 value of
3.2 µM. A bulky ester group (cf. 4 and 5) at C-2 did not
have any impact on the coupled reaction inhibitory activity.
While these compounds were essentially equipotent in their
ability to inhibit the coupled reaction, they did show
significant differences in their ability to inhibit the strand
transfer reaction. The 2-deoxy compound 6 was the best
inhibitor (IC50 ) 4.8 µM) of the strand transfer reaction in
this series with a 2-fold better potency than integracide A
(1). Compound 4 was slightly less active (IC50 ) 14 µM)
than 1 in the strand transfer assay, but the dihydro
analogue 5 was ∼3-fold more active than 4 and was
equipotent in both coupled and strand transfer assays. The
dihydro analogue 3 (IC50 ) 15 µM) was slightly less active
than 1 (IC50 ) 9 µM) in the strand transfer assay. The
a
Reagents: (i) Ac2O, C5H5N; (ii) LiOH, dioxane-H2O; (iii) 2,2-dimethox-
ypropane, PPTS, CH2Cl2; (iv) (a) MsCl, DIPEA, DMAP, CH2Cl2; (b) DBU,
toluene, 50 °C; (v) (C6H5CO)2O, TEA, DMAP, THF; (vi) MOMCl, DIPEA,
CH2Cl2; (vii) succinic anhydride, TEA, DMAP, CH2Cl2, 50 °C; (viii) N-t-Boc-
Gly-Osu, or FMOC-Gly-OPFP, DIPEA, DMAP, THF-CH2Cl2, (24 f 26 and
25 f 27) CH2N2, CH2Cl2, (25 f 28) isobutyl chloroformate, CH2Cl2, NH2OH.
structure-activity relationship of these compounds indi-
cated that oxygenation (e.g., C-25 hydroxy and 23,24-
epoxide in 4 and 5) of the C-17 side chain plays no role in
the activity of these compounds. Clavaric acid (7) was
significantly less active than the sulfated compounds and
showed IC50 values of 47 and 85 µM in the coupled and
the strand transfer assays, respectively. The methyl ester
8 and clavarinone 9 were inactive in both assays. The
neutral compounds 10-23 (i.e., the compounds without
sulfate or any other charged group) were either signifi-
cantly less active or inactive in both assays. The only
exception was the 15-keto analogue 13, which showed IC50
values of 25 and 40 µM in the coupled and the strand
transfer assays, respectively.
Like the sulfated esters, compounds with either a free
carboxyl group, for example, hemisuccinates 24 and 25, or
a free amino group, for example, the glycine esters 33-
35, showed variable levels of activities in the coupled and

1340 J ournal of Natural Products, 2003, Vol. 66, No. 10
Singh et al.
Ta ble 1. HIV-1 Integrase Activity of Integracides and Analoguesa
coupledb IC50
STb,c IC50
(µM)
3′-end processingb
IC50 (µM)
antiviral activityb
MTT toxicityb,d
Hela cells (µM)
assemblyb
(IC50, µM)
PICb
(IC50, µM)
compd
(µM)
CIC95 (µM)
1
4 ((1)
82
9 ((3)
>100
15 ((3)
14 ((3)
5.6 ((2)
4.8 ((1)
85
>100
>100
>100
>100
>100
40
5
25
25
50
30
50
2
3
5 ((2)
5 ((2)
5.6 ((2)
3.1 ((1)
47
5
10
2
50
6
>50
50
12
50
10
12
4
5
6
7
8
70
9
68
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
50
50
75
25
>50
>50
48
>50
>50
>50
>69
>80
>80
>80
>80
>80
3 ((1)
3 ((1)
>50
>50
8
>50
>50
>69
>80
>80
>80
>80
>80
12 ((3)
12 ((3)
>50
5
6
6
12.5
6
12.5
6
>50
>50
>50
>50
>50
>50
1.7
23
18
>50
>50
>50
>50
11.5
>50
>50
a
Blank spaces indicate that the compounds were not tested in that assay. The IC50 values without any error limits were obtained from
the average of a duplicate or triplicate data set. See ref 2a and references therein. c ST ) strand transfer. MTT.
b
d
the strand transfer assays. Both 2- and 3-hemisuccinates
exhibited indistinguishable activities and each displayed
IC50 values of 3 and 12 µM, respectively, in the coupled
and the strand transfer assays. The monoamino esters 34
and 35 were both weakly active in the coupled assay and
were essentially inactive in the strand transfer assay at
50 µM. In contrast, the bis-amino derivative 33 was found
to be one of the most potent compounds of this series in
the coupled assay and showed an IC50 value of 1.7 µM. This
compound also showed good activity in the strand transfer
assay (IC50 ) 11.5 µM). Compounds with a protected acid
group (26 and 27) or protected amino groups, 29-32, were
completely inactive in both assays. The hydroxymate 28
showed weak activity against the coupled reaction with an
IC50 ) 8 µM but was inactive in the strand transfer assay.
The activity observed by the charged compounds would
indicate that these compounds may potentially interact
with bivalent metal (e.g., Mg2+) at the active site. However,
the lack of the strand transfer activity of the hydroxymate
28 contradicts this hypothesis.
apart from each other. The dihydro compound 5 exhibited
an IC50 of 2 µM, whereas the IC50 of compound 4 was 10
µM.
Compounds 3 and 5 inhibited the assembly formation2a,3b
with IC50 values of 10 and 12 µM, respectively. A few of
these compounds, such as 1, 3, and 5, were tested for their
effect against preintegration complex. These compounds
exhibited IC50 values of 50, 30, and 50 µM, respectively, in
the PIC assay.2a,3b
Selected compounds of the series were evaluated in a
multiple cycle antiviral assay2a using HIV-1-infected H9
T-lymphoid cells, and their CIC95 values (concentration of
the inhibitor required for >95% protection from viral
infection) are reported in Table 1. In addition, these
compounds were also tested for their cytotoxicity in cell
viability MTT staining assay using H9 cells, and these data
are also reported in Table 1. Many of these compounds
exhibited CIC95 values of 6-50 µM in this antiviral viral
spread assay, but unfortunately, they also showed toxicity
at about the same levels and therefore provided only a
small or no therapeutic window.
All of these compounds were tested in generic DNA
cleavage assays such as DNAse or ECOR1.3b All compounds
were inactive in these assays with measured IC50 > 50 µM,
except for the bis-amino compound 33, which inhibited the
DNAse assay with an IC50 value of 47 µM, and indicated
that HIV-1 integrase inhibition by most of these compounds
is selective.
In summary, we have described in this paper a series of
natural products and derivatives of integracide B with a
range of HIV-1 integrase activities including activity
against preintegration complex formation.
Exp er im en ta l Section
Many of the potent compounds from this series were
further evaluated for their ability to inhibit the 3′-end
processing reaction of HIV-1 integrase.2a,3b Integracide A
(1) and compounds 3 and 24 each inhibited this reaction
with an IC50 value of 5 µM. The potencies of the compounds
with a 2-ester and a 3-sulfate group (4 and 5) were 5-fold
For general experimental procedures see ref 14.
HIV-1 In tegr ase Cou pled, Assem bly, an d Str an d Tr an s-
fer Assa ys. In the coupled assay, an unprocessed donor DNA
and inhibitor are incubated with the HIV-1 integrase at the
same time for 30 min followed by addition of target DNA. In
the assembly assay, integrase and inhibitor were incubated

HIV-1 Integrase Inhibitor Integracide B
J ournal of Natural Products, 2003, Vol. 66, No. 10 1341
with preprocessed donor DNA followed by washing of unbound
integrase and addition of target DNA. In the strand transfer
assay, the reaction is repeated like the assembly assay with a
preprocessed donor DNA except that the target DNA and
inhibitor are added after washing of excess integrase. In both
assays, the final product that is measured is the result of the
strand transfer reaction. For coupled and strand transfer
HIV-1 integrase assays see ref 3b. All reactions were per-
formed in a 96-well plate in a final concentration of 10%
DMSO. IC50 values were determined using a series of 2-fold
dilutions in duplicate or triplicate.
3′-En d P r ocessin g Assa y. The assay for specific 3′-end
processing of LTR donor sequence oligonucleotide substrates
was performed as detailed in ref 15, except the reactions were
performed in the presence of 25 nM MnCl2 and 250 µM
integrase and are summarized in ref 3b.
(3H, s, COCH3), 2.03 (3H, s, COCH3), 1.95 (1H, dd, J ) 12, 4
Hz, H-1), 1.86 (1H, m, H-23), 1.85 (1H, m, H-23), 1.82 (1H, m,
H-5), 1.81 (1H, m, H-6), 1.70 (1H, m, H-6), 1.61 (1H, m, H-20),
1.56 (1H, m, H-22), 1.41 (1H, t, J ) 12 Hz, H-1R), 1.38 (1H,
m, H-5), 1.27 (3H, s, H3-19), 1.16 (2H, m, H-22), 1.05 (3H, s,
CH3), 1.04, 1.03 (6H, d, J ) 6.8 Hz, H3-26, H3-27), 0.99 (3H, s,
CH3), 0.95 (3H, s, CH3), 0.89 (3H, d, J ) 6.4 Hz, H3-21); ESIMS
(m/z) 658 (M + NH4)+; HREIMS (m/z) 598.3837 (M - COCH2,
calcd for C36H54O7, 598.3869).
12-Desa cetyl Com p ou n d (15). To a solution of 2 (10 mg)
in dioxane-water (2:1, 1.5 mL) was added LiOH (13.4 mg),
and the yellowish solution was stirred at room temperature
overnight. EtOAc (50 mL) was added, and the solution was
washed with 2 × 20 mL of water. The EtOAc layer was dried
(Na2SO4), evaporated under reduced pressure, and chromato-
graphed on preparative TLC (SiO2, hexane-EtOAc, 3:7).
Elution of the major band gave 2.3 mg of desacetyl compound
15 as a colorless amorphous powder: 1H NMR (CDCl3 + CD3-
OD, 10:1) δ 5.59 (1H, brs, H-15), 4.66 (1H, brs, H-28), 4.60
(1H, brs, H-28), 4.23 (1H, brs, H-11), 3.69 (1H, dt, J ) 11.2, 4
Hz, H-2), 3.69 (1H, d, J ) 1.6 Hz, H-12), 2.90 (1H, d, J ) 9.2
Hz, H-3), 2.36 (2H, m, H-16, H-7â), 2.27 (1H, dd, J ) 11, 6
Hz, H-1â), 2.20 (1H, m, H-7R), 2.20 (1H, m, H-17), 2.19 (1H,
m, H-25), 2.07 (1H, m, H-23), 1.97 (1H, m, H-16), 1.86 (1H, m,
H-23), 1.69 (1H, m, H-6â), 1.61 (1H, m, H-6R), 1.61 (1H, m,
H-20), 1.51 (1H, m, H-22), 1.27 (1H, t, J ) 12 Hz, H-1R), 1.23
(3H, s, H3-19), 1.20 (2H, m, H-5, H-22), 0.97 (3H, s, H3-18),
0.97 (3H, d, J ) 6.4 Hz, H3-21), 0.96 (3H, s, H3-30), 0.96, 0.95
(6H, d, J ) 5.6 Hz, H3-26, H3-27), 0.81 (3H, s, H3-29); HREIMS
(m/z) 472.3543 (M+, calcd for C30H48O4, 472.3552).
P r ein tegr a tion Com p lex Assa y. PICs were isolated from
HIV-1 infected cells and partially purified, and reaction was
performed as described in ref 16. The integration reaction was
analyzed by Southern blotting, and reaction products were
quantified by PhosphorImager analysis.
In tegr a cid e B (2). A solution of 1 (160 mg) in dioxane (16
mL) was heated at 66 °C for 5 min. After addition of sodium
bicarbonate (300 mg) the reaction mixture was filtered through
a bed of sodium sulfate and washed with ethyl acetate (150
mL). The combined filtrate was washed once each with 50 mL
of 10% aqueous sodium bicarbonate and 50 mL of water, dried
over sodium sulfate, concentrated under reduced pressure, and
chromatographed over a silica gel column. Elution with 50%
ethyl acetate in hexane gave 13 mg of fraction A, 27 mg of
fraction B, and 72 mg (33%) of 2 as a gum. Lyophilization of
2 from acetonitrile-water gave a colorless powder: 1H NMR
(CDCl3) δ 5.61 (1H, t, J ) 2.5 Hz, H-15), 4.97 (1H, d, J ) 2.0
Hz, H-12), 4.73 (1H, brs, H-28), 4.66 (1H, d, J ) 1.0 Hz, H-28),
4.24 (1H, brs, H-11), 3.81 (1H, ddd, J ) 11.5, 10, 4 Hz, H-2),
3.20 (1H, brs, OH), 3.05 (1H, d, J ) 9.5 Hz, H-3), 2.45 (2H, m,
H-16, H-7â), 2.37 (1H, dd, J ) 12, 5 Hz, H-1â), 2.32 (1H, dd,
J ) 17.5, 7 Hz, H-7R), 2.23 (1H, heptet, J ) 7 Hz, H-25), 2.10
(1H, m, H-23), 2.06 (1H, m, H-16), 2.05 (3H, s, H3-32), 1.97
(H, dt, J ) 10.5, 7.5 Hz, H-17), 1.89 (1H, m, H-23), 1.77 (1H,
brdd, J ) 13.5, 7.5 Hz, H-6â), 1.69 (1H, m, H-6R), 1.65 (1H,
m, H-20), 1.57 (1H, m, H-22), 1.31 (3H, s, H3-19), 1.27 (1H, t,
J ) 12 Hz, H-1R), 1.27 (1H, dd, J ) 12.5, 3.0 Hz, H-5), 1.15
(1H, m, H-22), 1.08 (3H, s, H3-18), 1.06 (3H, s, H3-30), 1.03,
1.01 (6H, d, J ) 7 Hz, H3-26, H3-27), 0.89 (3H, s, H3-29), 0.88
(3H, d, J ) 7 Hz, H3-21); 13C NMR (CDCl3) δ 171.19 (C-31),
156.58 (C-24), 146.73 (C-14), 138.03 (C-9), 125.77 (C-8), 121.45
(C-15), 106.09 (C-28), 83.40 (C-3), 79.23 (C-12), 69.13 (C-2),
68.88 (C-11), 50.11 (C-5), 49.10 (C-17), 46.58 (C-13), 42.76 (C-
1), 39.31 (C-4), 38.37 (C-10), 35.30 (C-16), 34.45 (C-22), 33.79
(C-25), 33.25 (C-20), 30.90 (C-23), 28.66 (C-30), 26.77 (C-7),
23.24 (C-19), 21.98, 21.85 (C-26, C-27), 21.28 (C-32), 18.19 (C-
21), 18.00 (C-6), 16.74 (C-18), 16.69 (C-29); ESIMS (m/z) 1046
[2M + NH4]+, 532 [M + NH4]+, 497 [M + H]+, 437 [M + H -
H2O - AcOH]+, 1141 [2M + CF3CO2]-, 627 [M + CF3CO2]-,
HREIMS m/z 454.3448 ([M -AcOH]+, calcd for C30H46O3,
454.3447), 439.3219 ([M - AcOH - CH3]+, calcd for C29H43O3,
439.3212), 311.1990 ([M - AcOH - H2O - C-17 side chain]+,
calcd for C21H27O2, 311.2010).
Tetr a a ceta te 14. To a solution of 2 (10 mg) in pyridine (0.5
mL) was added acetic anhydride (0.3 mL), and the solution
was stirred at room temperature overnight under an inert
atmosphere followed by heating at 50 °C for 3 h. Methanol
was added to consume excess acetic anhydride. The solvent
and volatile material was removed under a stream of N2. The
product was purified by preparative TLC (SiO2, hexane-
EtOAc, 7:3). The band was eluted with EtOAc. Evaporation
of EtOAc under reduced pressure afforded the tetraacetate 14
as a colorless foam: 1H NMR (CDCl3) δ 5.73 (1H, brs, H-15),
5.33 (1H, brs, H-11), 5.18 (1H, brdt, J ) 11.6, 4.4 Hz, H-2),
5.13 (1H, d, J ) 2.0 Hz, H-12), 4.77 1H, d, J ) 10 Hz, H-3),
4.74 (1H, brs, H-28), 4.68 (1H, brs, H-28), 2.50 (1H, m, H-16),
2.48 (1H, m, H-7â), 2.40 (1H, brdd, J ) 18.4, 6.8 Hz, H-7R),
2.24 (1H, doublet of heptet, J ) 6.4, 0.8 Hz, H-25), 2.13 (2H,
m, H-23, H-16), 2.10 (3H, s, COCH3), 2.09 (3H, s, COCH3), 2.08
2,3-Aceton id e (16). To a solution of 2 (10 mg) in CH2Cl2 (1
mL) was added 2,2-dimethoxypropane (0.1 mL) and pyri-
dinium p-toluenesulfonic acid (5 mg), and the solution was
stirred at room temperature for 30 min. Water (20 mL) and
EtOAc (50 mL) were added and the layers were separated.
The organic layer was sequentially washed with 20 mL each
of 10% aqueous citric acid, water, 10% aqueous NaHCO3, and
water. The EtOAc extract was dried (Na2SO4), concentrated
under reduced pressure, and chromatographed over prepara-
tive TLC (SiO2, hexane-EtOAc, 7:3). Elution of the band with
EtOAc and evaporation of the solvent gave acetonide 16 (4.2
1
mg) as an amorphous powder: H NMR (CDCl3) δ 5.65 (1H,
brs, H-15), 5.01 (1H, d, J ) 2.0 Hz, H-12), 4.75 (1H, brs, H-28),
4.69 (1H, d, J ) 1.6 Hz, H-28),4.28 (1H, brd, J H,OH ) 4.8 HZ,
H-11), 3.85 (1H, ddd, J ) 12.8, 9.6, 3.6 Hz, H-2), 3.11 (1H, d,
J ) 9.6 Hz, H-3), 2.54 (1H, dd, J ) 10.8, 3.2 Hz, H-1), 2.49
(1H, m, H-7), 2.47 (1H, m, H-16), 2.37 (1H, brdd, J ) 18, 7.2
Hz, H-7R), 2.25 (1H, heptet, J ) 6.8 Hz, H-25), 2.09 (1H, m,
H-16), 2.08 (3H, s, COCH3), 2.06 (1H, m, H-23), 1.95 (1H, m,
H-17), 1.92 (1H,m, H-23), 1.91 (1H, d, J ) 5.2 Hz, OH), 1.83
(1H, m, H-6), 1.71 (1H, m, H-6), 1.65 (1H, m, H-20), 1.57 (1H,
m, H-22), 1.47, 1.45 (3H each, s, 2 × CH3), 1.45 (1H, t, J )
13.2 Hz, H-1R), 1.36 (3H, s, CH3), 1.29 (1H, dd, J ) 12.8, 2.4
Hz, H-5), 1.20 (1H, m, H-22), 1.10 (6H, s, 2 × CH3), 1.05, 1.04
(6H, d, J ) 6.8 Hz, H3-26, H3-27), 0.98 (3H, s, CH3), 0.91 (3H,
d, J ) 6.4 Hz, H3-21); HREIMS (m/z) 554.3947 (M+, calcd for
C
35H54O5, 554.3971).
2,3-Ep oxid e (17). To a cooled (-40 °C) solution of 2 (42
mg, 0.08 mmol) in CH2Cl2 (1 mL) was added diisopropylethy-
lamine (33 µL), (dimethylamino)pyridine (5 mg), and methane
sulfonyl chloride (14 µL). The reaction mixture was stirred for
20 min and was allowed to warm to room temperature and
quenched by addition of ice. EtOAc (50 mL) was added, and
the layers were separated. The organic layer was sequentially
washed with 20 mL each of water, 10% aqueous citric acid,
water, 10% aqueous NaHCO3, and finally water, dried (Na2-
SO4), and evaporated under reduced pressure to give clean
2-mesylate (40 mg) as a foam: 1H NMR (CDCl3) δ only distinct
signals are listed, 5.64 (1H, brs, H-15), 5.00 (1H, d, J ) 0.8
Hz, H-12), 4.81 (1H, ddd, J ) 11.6, 9.6, 4.0 Hz, H-2), 4.75 (1H,
brs, H-28), 4.68 (1H, d, J ) 0.2 Hz, H-28), 4.19 (1H, brs, H-11),
3.27 (1H, d, J ) 9.6 Hz, H-3), 3.16 (3H, brs, SOCH3), 2.11 (3H,
s, COCH3), 1.30 (3H, s, CH3), 1.11 (3H, s, CH3), 1.09 (3H, s,
CH3), 1.05, 1.04 (6H, d, J ) 6.8 Hz, H3-26, H3-27), 0.96 (3H, s,
CH3), 0.90 (3H, d, J ) 6.4 Hz, H3-21); ESIMS (m/z) 610 (M +

1342 J ournal of Natural Products, 2003, Vol. 66, No. 10
Singh et al.
NH4)+. The mesylate (12 mg) in 1 mL of toluene and 50 µL of
DBU was heated at 50 °C for 30 min. Ice followed by EtOAc
(50 mL) was added to the reaction after it was cooled to room
temperature. The EtOAc layer was sequentially washed with
20 mL each of water, 10% aqueous citric acid, water, 10%
aqueous NaHCO3, and finally water, dried (Na2SO4), evapo-
rated under reduced pressure, and chromatographed over
preparative TLC (SiO2, hexane-EtOAc, 7:3). The band was
eluted with EtOAc to give 2,3-epoxide 17 (4 mg) as an
amorphous powder: 1H NMR (CDCl3) δ 5.62 (1H, t, J ) 2 Hz,
H-15), 5.01 (1H, d, J ) 2.4 Hz, H-12), 4.73 (1H, brs, H-28),
4.67 (1H, d, J ) 2 Hz, H-28), 4.31 (1H,brd, J H,OH ) 6.4 HZ,
H-11), 3.34 (1H, dt, J ) 4, 2 Hz, H-2), 2.86 (1H, d, J ) 4.4 Hz,
H-3), 2.51 (1H, dd, J ) 14.4, 2 Hz, H-1), 2.45 (1H, ddd, J )
16, 6.8, 3.2 Hz, H-16), 2.32 (2H, m, H-7), 2.23 (1H, heptet, J )
7.2 Hz, H-25), 2.10 (1H, m, H-16), 2.07 (1H, m, H-23), 2.06
(3H, s, COCH3), 1.99 (1H, m, H-17), 1.89 (1H, m, H-23), 1.79
(1H, d, J ) 5.6 Hz, OH), 1.65 (1H, m, H-20), 1.61 (2H, m, H-6),
1.60, 1.54 (2H, m, H-22), 1.48 (1H, d, J ) 14 Hz, H-1R), 1.39
(3H, s, CH3), 1.16 (1H, m, H-22), 1.084 (6H, s, 2 × CH3), 1.078
(3H, s, CH3), 1.03, 1.02 (6H, d, J ) 6.8 Hz, H3-26, H3-27), 0.89
(3H, d, J ) 6.4 Hz, H3-21); ESIMS m/z 514 (M + NH4)+;
HREIMS (m/z) 436.3344 ([M - AcOH]+, calcd for C30H44O2,
436.3341).
sequentially with 50 mL each of water, 10% aqueous citric acid,
water, 10% aqueous NaHCO3, and then water and dried (Na2-
SO4). The solvent was removed under reduced pressure, and
products were purified by preparative TLC (SiO2) using
hexane-EtOAc (1:1). The three bands were eluted with EtOAc
to give bis-MOM ether 21 (1.5 mg), 3-MOM ether 22 (3.4 mg),
and 2-MOM ether 23 (2.9 mg) as amorphous powders. 21: 1H
NMR (CDCl3) (only distinct signals are presented) δ 5.65 (1H,
brs, H-15), 5.01 (1H, d, J ) 2.0 Hz, H-12), 4.95 (1H, d, J ) 6.4
Hz, OCH2O), 4.77 (2H, brs, OCH2O), 4.76 (1H, d, J ) 6.4 Hz,
OCH2O), 4.75 (1H, brs, H-28), 4.69 (1H, d, J ) 1.0 Hz, H-28),
4.26 (1H, bd, J H,OH ) 5.6 Hz, H-11), 3.86 (1H, ddd, J ) 12, 10,
4 Hz, H-2), 3.46 (3H, s, OCH3), 3.43 (3H, s, OCH3), 3.06 (1H,
d, J ) 10 Hz, H-3), 2.07 (3H, s, H3-32), 1.77 (1H, d, J H,OH
)
5.6 Hz, 11-OH), 1.32 (3H, s, H3-19), 1.10 (3H, s, H3-18), 1.08
(3H, s, H3-30), 1.05, 1.04 (6H, d, J ) 6.8 Hz, H3-26, H3-27),
0.94 (3H, s, H3-29), 0.92 (3H, d, J ) 6.4 Hz, H3-21); ESIMS
(m/z) 620 (100%, M + NH4)+, 603 (5%, M + H)+, 585 (30%, M
1
- H2O + H)+. 22: H NMR (CDCl3) (only distinct signals are
presented) δ 5.64 (1H, t, J ) 2.0 Hz, H-15), 5.02 (1H, d, J )
1.2 Hz, H-12), 4.87 (1H, d, J ) 6.4 Hz, OCH2O), 4.75 (1H, brs,
H-28), 4.69 (1H, brs, H-28), 4.66 (1H, d, J ) 6.4 Hz, OCH2O),
4.31 (1H, bd, J H,OH ) 4.4 Hz, H-11), 3.84 (1H, ddd, J ) 12.8,
9.6, 4 Hz, H-2), 3.49 (3H, s, OCH3), 2.85 (1H, dd, J 2,3 ) 9.6
Hz, J H,OH ) 2 Hz, H-3), 2.05 (3H, s, H3-32), 1.95 (1H, d, J H,OH
) 5.2 Hz, 11-OH), 1.33 (3H, s, H3-19), 1.10 (3H, s, H3-18), 1.05,
1.04 (6H, d, J ) 6.8 Hz, H3-26, H3-27), 1.02 (3H, s, H3-30),
0.93 (3H, s, H3-29), 0.92 (3H, d, J ) 6.4 Hz, H3-21); ESIMS
(m/z): 576 (60%, M + NH4)+, 541 (90%, M - H2O + H)+, 481
Ben zoa tes (18-20). Triethylamine (20 µL), DMAP (5 mg),
and benzoic anhydride (17.2 mg, 0.076 mmol) were added to a
stirred solution of 2 (10 mg, 0.019 mmol) in anhydrous THF
(1 mL). The reaction mixture was stirred overnight under
nitrogen. Ice (5 g) was added to quench the reaction, and the
mixture was diluted with EtOAc (50 mL). The organic layer
was separated and sequentially washed with 20 mL each of
water, 10% aqueous citric acid, water, 10% aqueous NaHCO3,
and water and dried (Na2SO4), and EtOAc was evaporated
under reduced pressure. The mixture was chromatographed
by preparative TLC (SiO2, hexane-EtOAc, 7:3) to give 18 (3.4
mg), 19 (3.4 mg), and 20 (4.6 mg), all as colorless amorphous
powders. 18: 1H NMR (CDCl3) (only distinct signals are
presented) δ 7.99 (2H, dd, J ) 7.6, 1.6 Hz, ArH), 7.92 (2H, dd,
J ) 8.4, 1.2 Hz, ArH), 7.47 (2H, m, ArH), 7.37 (2H, t, J ) 8
Hz, ArH), 7.35 (2H, t, J ) 7.6 Hz, ArH), 5.68 (1H, brs, H-15),
5.62 (1H, dt, J ) 11.6, 4.4 Hz, H-2), 5.26 (1H, d, J ) 10.4 Hz,
H-3), 4.99 (1H, d, J ) 2.0 Hz, H-12), 4.75 (1H, brs, H-28), 4.69
(1H, d, J ) 1.0 Hz, H-28), 4.23 (1H, bd, J H,OH ) 5.0 Hz, H-11),
2.10 (3H, s, H3-32), 1.83 (1H, d, J H,OH ) 5.6 Hz, 11-OH), 1.52
(3H, s, CH3), 1.20 (3H, s, CH3), 1.11 (3H, s, CH3), 1.06 (3H, s,
CH3), 1.06, 1.05 (6H, d, J ) 6.8 Hz, H3-26, H3-27), 0.92 (3H, d,
J ) 6.4 Hz, H3-21); ESIMS (m/z) 1462 (85%, 2M + NH4)+, 740
1
(100%, M + H - H2O - AcOH)+. 23: H NMR (CDCl3) (only
distinct signals are presented) δ 5.65 (1H, t, J ) 2.0 Hz, H-15),
5.01 (1H, d, J ) 1.2 Hz, H-12), 4.83 (1H, d, J ) 6.8 Hz,
OCH2O), 4.76 (1H, d, J ) 6.8 Hz, OCH2O), 4.75 (1H, brs, H-28),
4.69 (1H, d, J ) 1.2 Hz, H-28), 4.27 (1H, bd, J H,OH ) 5.2 Hz,
H-11), 3.68 (1H, ddd, J ) 13.6, 9.6, 4 Hz, H-2), 3.47 (3H, s,
OCH3), 3.31 (1H, d, J H,OH ) 2 Hz, 3-OH), 3.12 (1H, dd, J 2,3
)
10 Hz, J H,OH ) 2 Hz, H-3), 2.07 (3H, s, H3-32), 1.80 (1H, d,
J H,OH ) 6.0 Hz, 11-OH), 1.31 (3H, s, H3-19), 1.12 (3H, s, H3-
18), 1.10 (3H, s, H3-30), 1.05, 1.04 (6H, d, J ) 6.8 Hz, H3-26,
H3-27), 0.93 (3H, s, H3-29), 0.91 (3H, d, J ) 6.4 Hz, H3-21);
ESIMS (m/z) 576 (100%, M + NH4)+, 541 (40%, M - H2O +
H)+.
Hem isu ccin a tes (24, 25). To a THF (2 mL) solution of 2
(10 mg, 0.019 mmol) was added triethylamine (60 µL), DMAP
(5 mg), and succinic anhydride (22 mg). The mixture was
stirred at room temperature overnight and heated at 50 °C
for 2 h. The reaction mixture was allowed to cool and then ice
followed by EtOAc (50 mL) was added. The layers were
separated, and the organic layer was sequentially washed with
2 × 20 mL each of water, 10% aqueous citric acid, and water
and then dried (Na2SO4). EtOAc was removed under reduced
pressure. Chromatography of the mixture on reversed-phase
HPLC (Zorbax RX C-8, 22 × 250 mm, gradient of 60% to 75%
CH3CN in H2O, both containing 0.05% TFA, flow rate 8 mL/
min) followed by lyophilization gave 24 (2 mg) and 25 (6 mg)
as colorless amorphous powders. 24: 1H NMR (CDCl3) (only
distinct signals are presented) δ 5.64 (1H, brs, H-15), 5.04 (1H,
brs, H-12), 4.75 (1H, brs, H-28), 4.69 (1H, d, J ) 1.0 Hz, H-28),
4.65 (1H, d, J ) 9.6 Hz, H-3), 4.23 (1H, brs, H-11), 4.00 (1H,
m, H-2), 2.73 (4H, m, 2 x CH2CO), 2.07 (3H, s, H3-32), 1.34
(3H, s, CH3), 1.11 (3H, s, CH3), 1.05, 1.04 (6H, d, J ) 6.8 Hz,
H3-26, H3-27), 0.97 (3H, s, CH3), 0.93 (3H, s, CH3), 0.91 (3H,
d, J ) 6.4 Hz, H3-21); ESIMS (m/z) 1246 (10%, 2M + NH4)+,
632 (100%, M + NH4)+, 597 (35%, M - H2O + H)+. 25: 1H
NMR (CDCl3) (only distinct signals are presented) δ 5.65 (1H,
brs, H-15), 5.15 (1H, dt, J ) 11.6, 4.4 Hz, H-2), 4.98 (1H, brs,
H-12), 4.76 (1H, brs, H-28), 4.69 (1H, d, J ) 1.0 Hz, H-28),
4.22 (1H, brs, H-11), 3.29 (1H, d, J ) 10 Hz, H-3), 2.76 (2H,
m, 2 × CH2CO), 2.68 (2H, m, 2 × CH2CO), 2.10 (3H, s, H3-32),
1.36 (3H, s, CH3), 1.10 (3H, s, CH3), 1.09 (3H, s, CH3), 1.05,
1.04 (6H, d, J ) 6.8 Hz, H3-26, H3-27), 0.95 (3H, s, CH3), 0.91
(3H, d, J ) 6.4 Hz, H3-21); ESIMS (m/z) 1246 (10%, 2M +
NH4)+, 632 (100%, M + NH4)+, 597 (35%, M - H2O + H)+.
Hem isu ccin a te Meth yl Ester s (26, 27). A mixture of 16
mg of hemisuccinates, described in the previous experiment,
1
(100%, M + NH4)+, 705 (35%, M - H2O + H)+. 19: H NMR
(CDCl3) (only distinct signals are presented) δ 8.12 (2H, d, J
) 7.2, ArH), 7.61 (1H, t, J ) 7.6 Hz, ArH), 7.49 (2H, t, J ) 8
Hz, ArH), 7.35 (2H, t, J ) 7.6 Hz, ArH), 5.67 (1H, brs, H-15),
5.02 (1H, brs, H-12), 4.84 (1H, d, J ) 10 Hz, H-3), 4.76 (1H,
brs, H-28), 4.70 (1H, d, J ) 1.0 Hz, H-28), 4.32 (1H, brs, H-11),
4.11 (1H, dt, J ) 10, 3.6 Hz, H-2), 2.10 (3H, s, H3-32), 1.39
(3H, s, CH3), 1.12 (3H, s, CH3), 1.11 (3H, s, CH3), 1.06, 1.05
(6H, d, J ) 6.8 Hz, H3-26, H3-27), 1.01 (3H, s, CH3), 0.92 (3H,
d, J ) 6.4 Hz, H3-21); ESIMS (m/z) 1254 (10%, 2M + NH4)+,
636 (100%, M + NH4)+, 601 (30%, M - H2O + H)+. 20: 1H
NMR (CDCl3) (only distinct signals are presented) δ 8.10 (2H,
dd, J ) 7.6, 1.6 Hz, ArH), 7.60 (1H, t, J ) 7.6 Hz, ArH), 7.48
(2H, t, J ) 8 Hz, ArH), 7.35 (2H, t, J ) 7.6 Hz, ArH), 5.67
(1H, brs, H-15), 5.36 (1H, dt, J ) 11.2, 4.4 Hz, H-2), 4.99 (1H,
brs, H-12), 4.75 (1H, brs, H-28), 4.69 (1H, d, J ) 1.0 Hz, H-28),
4.23 (1H, brs, H-11), 3.41 (1H, d, J ) 10 Hz, H-3), 2.09 (3H, s,
H3-32), 1.43 (3H, s, CH3), 1.14 (3H, s, CH3), 1.10 (3H, s, CH3),
1.05, 1.04 (6H, d, J ) 6.8 Hz, H3-26, H3-27), 1.02 (3H, s, CH3),
0.91 (3H, d, J ) 6.4 Hz, H3-21); ESIMS (m/z) 1254 (10%, 2M
+ NH4)+, 636 (100%, M + NH4)+, 601 (30%, M - H2O + H)+.
Meth oxym eth yl Eth er s (21-23). To a cold (0 °C) solution
of 2 (10 mg, 0.019 mmol) in 1 mL of CH2Cl2 were added
diisopropylethylamine (15.5 µL, 0.114 mmol) and methoxym-
ethyl chloride (7.4 µL, 0.095 mmol). The solution was stirred
at 0 °C for 2 h followed by stirring at room temperature
overnight. The reaction mixture was quenched with ice, and
50 mL of EtOAc was added. The organic layer was washed

HIV-1 Integrase Inhibitor Integracide B
J ournal of Natural Products, 2003, Vol. 66, No. 10 1343
was dissolved in 0.1 mL of CH2Cl2 and cooled to 0 °C. An
ethereal solution of freshly prepared diazomethane was added,
and the solution was kept at 0 °C overnight. Volatile material
was evaporated under a stream of nitrogen, and the methyl
esters were purified by preparative TLC (SiO2, hexane-EtOAc,
3:1). Elution of the bands with EtOAc gave monomethyl esters
26 (3 mg) and 27 (12 mg) as amorphous powders. 26: 1H NMR
(CDCl3) (only distinct signals are presented) δ 5.63 (1H, t, J
) 2.8 Hz, H-15), 4.98 (1H, d, J ) 1.6 Hz, H-12), 4.73 (1H, brs,
H-28), 4.67 (1H, d, J ) 1.6 Hz, H-28), 4.62 (1H, d, J ) 9.6 Hz,
H-3), 4.27 (1H, brs, H-11), 3.95 (1H, ddd, J ) 11.6, 10, 4.4 Hz,
H-2), 3.70 (3H, s, OCH3), 2.68 (4H, m, 2 × CH2CO), 2.05 (3H,
s, H3-32), 1.32 (3H, s, CH3), 1.08 (3H, s, CH3), 1.03, 1.02 (6H,
d, J ) 6.8 Hz, H3-26, H3-27), 0.94 (3H, s, CH3), 0.92 (3H, s,
CH3), 0.89 (3H, d, J ) 6.4 Hz, H3-21); ESIMS (m/z) 629 (50%,
M + H)+; HREIMS (m/z) 628.4009 (calcd for C37H56O8,
428.3975). 27: 1H NMR (CDCl3) (only distinct signals are
presented) δ 5.63 (1H, t, J ) 2.8 Hz, H-15), 5.15 (1H, ddd,
11.6, 10, 4.4 Hz, H-2), 4.97 (1H, d, J ) 1.6 Hz, H-12), 4.73
(1H, brs, H-28), 4.67 (1H, d, J ) 1.0 Hz, H-28), 4.18 (1H, brs,
H-11), 3.70 (3H, s, OCH3), 3.23 (1H, d, 10 Hz, H-3), 2.68 (4H,
m, 2 × CH2CO), 2.07 (3H, s, H3-32), 1.34 (3H, s, CH3), 1.09
(3H, s, CH3), 1.07 (3H, s, CH3), 1.03, 1.02 (6H, d, J ) 6.8 Hz,
H3-26, H3-27), 0.94 (3H, s, CH3), 0.89 (3H, d, J ) 6.4 Hz, H3-
21); ESIMS (m/z) 629 (100%, M + H)+; HREIMS (m/z) 628.3978
(calcd for C37H56O8, 428.3975).
Su ccin a te Hyd r oxim a te (28). To a cooled (-40 °C) solu-
tion of 25 (10 mg, 0.016 mmol) in THF (0.5 mL) was added
N-methylmorpholine (15 µL) followed by allyl chloroformate
(10 µL). The reaction mixture was allowed to warm to room
temperature. After stirring for 30 min under nitrogen it was
recooled at -23 °C and an aqueous solution of hydroxylamine
was added via a syringe. The mixture was stirred at 0 °C for
30 min and then quenched with ice and diluted with EtOAc
(50 mL). The ethyl acetate layer was sequentially washed with
20 mL each of 10% aqueous citric acid, water, 10% aqueous
NaHCO3, and water. The EtOAc extract was dried (Na2SO4),
concentrated under reduced pressure, and chomatographed by
reversed-phase HPLC (Zorbax RX C-8, 22 × 250 mm, 20 to
80% CH3CN in H2O (+0.1% TFA) gradient in 40 min, at 8 mL/
min). The fractions containing the product were lyophilized
to give hydroxymate 28 (4 mg) as an amorphous powder. 28:
1H NMR (CDCl3) δ (only distinct signals are listed, spectrum
was very broad) 5.64 (1H, brs, H-15), 5.14 (1H, m, H-2), 4.98
(1H, brs, H-12), 4.75 (1H, brs, H-28), 4.69 (1H, brs, H-28), 4.19
(1H, brs, H-11), 3.30 (4H, m, 2 x CH2), 2.75 (1H, H-3), 2.23
(1H, heptet, J ) 7.2 Hz, H-25), 2.09 (3H, s, COCH3), 1.35 (3H,
s, CH3), 1.09 (6H, s, 2 × CH3), 1.05, 1.04 (6H, d, J ) 6.8 Hz,
H3-26, H3-27), 0.94 (3H, s, CH3), 0.90 (3H, d, J ) 6 Hz, H3-21);
ESIMS m/z 630 (M + H)+.
(M + NH4)+. 30: 1H NMR (CDCl3) δ (only distinct signals are
listed) 5.66 (1H, brs, H-15), 5.18 (1H, dt, J ) 11.6, 4 Hz, H-2),
5.10 (1H, t, J ) 6 Hz, NH), 4.99 (1H, d, J ) 0.8 Hz, H-12),
4.76 (1H, brs, H-28), 4.69 (1H, d, J ) 1.6 Hz, H-28), 4.19 (1H,
brd, J H,OH ) 5.2 Hz, H-11), 3.97 (1H, dd, J ) 17.6, 5.0 Hz,
gly-CH), 3.92 (1H, dd, J ) 17.6, 5.6 Hz, gly-CH), 3.22 (1H, d,
J ) 7.6 Hz, H-3), 2.26 (1H, heptet, J ) 7.2 Hz, H-25), 2.10
(3H, s, COCH3), 1.93 (1H, d, J ) 5.2 Hz, OH), 1.48 (9H, s,
C(CH3)3), 1.37 (3H, s, CH3), 1.10 (3H, s, CH3), 1.09 (3H, s, CH3),
1.06, 1.05 (6H, d, J ) 6.8 Hz, H3-26, H3-27), 0.96 (3H, s, CH3),
0.91 (3H, d, J ) 6.8 Hz, H3-21); ESIMS m/z 689 (M + NH4)+.
F m oc-Glycin e Ester s (31, 32). Diisopropylethylamine (128
µL, 0.7 mmol) and (dimethylamino)pyridine (10 mg) followed
by Fmoc-glycine-pentafluorophenyl ester were added to a
solution of 2 (80 mg, 0.14 mmol) in a 2:3 mixture of CH2Cl2-
THF (5 mL). The solution was stirred overnight under
nitrogen. Water followed by EtOAc (50 mL) was added after
completion of the reaction, and the layers were separated. The
organic layer was sequentially washed with 2 × 20 mL each
of water, 10% aqueous citric acid, water, 10% aqueous NaH-
CO3, and finally water, dried (Na2SO4), evaporated under
reduced pressure, and chromatographed over preparative TLC
(SiO2, hexane-EtOAc, 7:3). The two bands were eluted with
EtOAc to give diester 31 (23.9 mg) and monoester 32 (19.9
mg) both as amorphous pale powders. 31: 1H NMR (CDCl3) δ
(only distinct signals are listed) 7.76 (4H, d, J ) 7.5 Hz, ArH),
7.60 (4H, t, J ) 8 Hz, ArH), 7.38 (4H, t, J ) 7.5 Hz, ArH),
7.26 (4H, m, ArH), 5.85 (1H, t, J ) 6 Hz, NH), 5.72 (1H, t, J
) 6.5 Hz, NH), 5.67 (1H, t, J ) 2.5 Hz, H-15), 5.37 (1H, ddd,
J ) 12, 10.5, 4.5 Hz, H-2), 4.99 (1H, d, J ) 1.5 Hz, H-12), 4.82
(1H, d, J ) 10.5 Hz, H-3), 4.74 (1H, brs, H-28), 4.69 (1H, d, J
) 1.5 Hz, H-28), 4.40-4.30 (4H,m, 2 × CH2O-), 4.24-4.16
(3H, m, Fmoc-CH, H-11), 3.90 (4H, m, 2 × gly-CH2), 2.25 (1H,
doublet of heptet, J ) 7, 1 Hz, H-25), 2.04 (3H, s, COCH3),
1.37 (3H, s, CH3), 1.08 (3H, s, CH3), 1.04, 1.03 (6H, d, J ) 6.8
Hz, H3-26, H3-27), 0.98 (3H, s, CH3), 0.94 (3H, s, CH3), 0.91
(3H, d, J ) 6.8 Hz, H3-21); HRFABMS m/z 1095.5364 (calcd
1
for C66H76N2O11Na, 1095.5347). 32: H NMR (CDCl3) δ (only
distinct signals are listed) 7.81 (2H, d, J ) 7.5 Hz, ArH), 7.65
(2H,m, ArH), 7.43 (2H, brt, J ) 8 Hz, ArH), 7.36 (2H, m, ArH),
5.67 (1H, t, J ) 2.5 Hz, H-15), 5.51(1H, t, J ) 6.0 Hz, NH),
5.14 (1H, ddd, J ) 14, 11.5, 4.0 Hz, H-2), 5.00 (1H, d, J ) 1.0
Hz, H-12), 4.76 (1H, d, J ) 1.5 Hz, H-28), 4.70 (1H, q, J ) 1.5
Hz, H-28), 4.45 (2H, d, J ) 7.5 Hz, Fmoc-CH2), 4.28 (1H, t, J
) 7.5 Hz, Fmoc-CH), 4.00 (2H, m, gly-CH2), 3.19 (1H, d, J )
10 Hz, H-3), 2.27 (1H, doublet of heptet, J ) 7, 1 Hz, H-25),
2.05 (3H, s, COCH3), 1.37 (3H, s, CH3), 1.10 (3H, s, CH3), 1.06
(3H, s, CH3), 1.06, 1.05 (6H, d, J ) 6.8 Hz, H3-26, H3-27), 0.94
(3H, s, CH3), 0.92 (3H, d, J ) 6.5 Hz, H3-21); HRFABMS m/z
816.4488 (calcd for C49H63NO8Na, 816.4452).
t-Boc-Glycin e Ester s (29, 30). To an anhydrous solution
of 2 (40 mg, 0.078 mmol) in a 2:1 mixture of CH2Cl2-THF (1.5
mL) was added N-t-Boc-glycine-succinimide ester (103 mg, 0.39
mmol) followed by diisopropylethylamine (64 µL) and (dim-
ethylamino)pyridine (5 mg). The homogeneous mixture was
stirred overninght under nitrogen followed by heating at 50
°C for 2 h. The reaction mixture was quenched by addition of
ice and was diluted with EtOAc (50 mL). The organic layer
was sequentially washed with 20 mL each of water, 10%
aqueous citric acid, water, 10% aqueous NaHCO3, and finally
water, dried (Na2SO4), evaporated under reduced pressure, and
chromatographed over a preparative TLC column (SiO2, hex-
ane-EtOAc, 7:3). The two bands were eluted with EtOAc to
give diester 29 (15.9 mg) and monoester 30 (7.6 mg) both as
amorphous powders. 29: 1H NMR (CDCl3) δ (only distinct
signals are listed) 5.66 (1H, brs, H-15), 5.48 (1H, brt, J ) 6
Hz, NH), 5.37 (1H, dt, J ) 11.6, 3.6 Hz, H-2), 5.30 (1H, t, J )
6 Hz, NH), 4.99 (1H, d, J ) 0.8 Hz, H-12), 4.81 (1H, d, J ) 10
Hz, H-3), 4.75 (1H, brs, H-28), 4.69 (1H, d, J ) 1.2 Hz, H-28),
4.20 (1H, brd, J H,OH ) 4.8 Hz, H-11), 3.93 (2H, m, gly-CH2),
3.83 (2H, m, gly-CH2), 2.26 (1H, heptet, J ) 7.2 Hz, H-25),
2.10 (3H, s, COCH3), 1.94 (1H, d, J ) 5.2 Hz, OH), 1.48, 1.46
(9H each, s, C(CH3)3), 1.38 (3H, s, CH3), 1.09 (3H, s, CH3), 1.05,
1.04 (6H, d, J ) 6.8 Hz, H3-26, H3-27), 1.00 (3H, s, CH3), 0.95
(3H, s, CH3), 0.92 (3H, d, J ) 6.8 Hz, H3-21); ESIMS m/z 846
Glycin e Ester (33). Piperidine (20 µL) was added to a
solution of 31 (12 mg) in dimethylformamide (200 µL), and
the solution was stirred at room temperature for 5 min.
Volatile material was removed under a stream of nitrogen and
chromatographed over a reversed-phase HPLC column (Zorbax
RX C-8, 22 × 250 mm, gradient of 20 to 70% aqueous CH3CN
containing 0.1% TFA, flow rate 8 mL/min). The product eluted
from 50 to 58 min. The combined fractions were directly
lyophilized to yield a colorless powder of the trifluoroacetate
salt of 33 (4 mg): 1H NMR (CD3CN-CDCl3, 1:1) δ (only
distinct signals are listed) 5.37 (1H, t, J ) 2 Hz, H-15), 5.05
(1H, dt, J ) 12, 4.5 Hz, H-2), 4.77 (1H, d, J ) 0.8 Hz, H-12),
4.63 (1H, d, J ) 10 Hz, H-3), 4.48 (1H, brs, H-28), 4.43(1H, d,
J ) 1.2 Hz, H-28), 3.86 (1H, brs, H-11), 3.65 (4H, m, 2 × gly-
CH2), 2.01 (1H, heptet, J ) 7.2 Hz, H-25), 1.81 (3H, s, COCH3),
1.10 (3H, s, CH3), 0.81 (3H, s, CH3), 0.79, 0.78 (6H, d, J ) 7
Hz, H3-26, H3-27), 0.75 (3H, s, CH3), 0.71 (3H, s, CH3), 0.65
(3H, d, J ) 6.5 Hz, H3-21); ESIMS m/z 629 (M + H)+; HREIMS
(m/z) 628.4012 (calcd for C36H56N2O7: 628.4087).
Glycin e Ester s (34, 35). 32 (8 mg) was reacted in DMF
(100 µL) with piperidine (20 µL). The products were chromato-
graphed and lyophilized in a manner similar to the procedure
described above to give 0.8 mg of the trifluoroacetate salt of
35 as an amorphous powder: 1H NMR (CD3CN-CDCl3, 1:1)
δ (only distinct signals are listed) 5.35 (1H, brs, H-15), 4.76

1344 J ournal of Natural Products, 2003, Vol. 66, No. 10
Singh et al.
(4) Singh, S. B.; Zink, D.; Polishook, J .; Valentino, D.; Shafiee, A.;
Silverman, K.; Felock, P.; Teran, A.; Vilella, D.; Hazuda, D. J .;
Lingham, R. B. Tetrahedron Lett. 1999, 40, 8775-8779. (b) Singh, S.
B.; Felock, P.; Hazuda, D. J . Bioorg. Med. Chem. Lett. 2000, 10, 235-
237.
(5) Singh, S. B.; J ayasuriya, H.; Salituro, G. M., Zink, D. L.; Shafiee, A.;
Heimbuch, B.; Silverman, K. C.; Lingham, R. B.; Genilloud, O.; Teran,
A.; Vilella, D.; Felock, P.; Hazuda, D. J . Nat. Prod. 2001, 64, 874-
882.
(1H, brs, H-12), 4.48 (1H, brs, H-28), 4.43 (1H, brs, H-28), 4.40
(1H, d, J ) 9.5 Hz, H-3), 3.97 (1H, brs, H-11), 3.66 (3H, m,
H-2, gly-CH2), 2.01 (1H, heptet, J ) 7.2 Hz, H-25), 1.76 (3H,
s, COCH3), 1.06 (3H, s, CH3), 1.03 (3H, s, CH3), 0.82 (3H, s,
CH3), 0.79, 0.78 (6H, d, J ) 7 Hz, H3-26, H3-27), 0.70 (3H, s,
CH3), 0.66 (3H, d, J ) 6.5 Hz, H3-21); ESIMS m/z 572 (M +
H)+.
(6) Singh, S. B.; Zink, D.; Bills, G. F.; Pelaez, F.; Teran, A.; Collado, J .;
Silverman, K.; Lingham, R. B.; Felock, P.; Hazuda, D. J . Tetrahedron
Lett. 2002, 43, 1617-1620.
(7) Singh, S. B.; Zink, D.; Quamina, D. S.; Pelaez, F.; Teran, A.; Felock,
P.; Hazuda, D. J . Tetrahedron Lett. 2002, 43, 2351-2354.
(8) Singh, S. B.; Zink, D.; Heimbach, B.; Genilloud, O.; Teran, A.;
Silverman, K.; Lingham, R. B.; Felock, P.; Hazuda, D. J . Org. Lett.
2002, 4, 1123-1126.
(9) Singh, S. B.; Zink, D.; Bills, G. F.; Teran, A.; Silverman, K.; Lingham,
R. B.; Felock, P.; Hazuda, D. J . Bioorg. Med. Chem. Lett. 2003, 13,
713-717.
(10) Singh, S. B.; Zink, D.; Dombrowski, A. W.; Polishook, J . D.; Ondeyka,
J . G.; Hirshfield, J .; Felock, P.; Hazuda, D. J . Bioorg. Med. Chem.
2003, 11, 1577-1582.
(11) (a) Burg, R. W.; Dulaney, E. L.; Hensens, O. D.; Liesch, J . M.;
Ondeyka, J . G.; Wichman, C. F. (Merck & Co., Inc.) US 4871727
(October 3, 1989). (b) Ashe, B. M.; Fletcher, D. S. (Merck & Co., Inc.)
US 4874755 (October 17, 1989).
(12) J ayasuriya, H. J .; Silverman, K. C.; Zink, D. L.; J enkins, R. G.;
Sanchez, M.; Pelaez, F.; Vilella, D.; Lingham, R. B.; Singh, S. B. J .
Nat. Prod. 1998, 61, 1568-1570.
(13) Singh, S. B. Tetrahedron Lett. 2000, 41, 6973-6976.
(14) Singh, S. B.; Zink, D. L.; Liesch, J . M.; Moseley, R. T.; Dombrowski,
A. W.; Bills, G. F.; Darkin-Rattray, S. J .; Schmatz, D. M.; Goetz, M.
A. J . Org. Chem. 2002, 67, 815-825.
Ack n ow led gm en t. The authors are grateful to Drs. San-
dra Morris and Robert Schwartz for providing compound 6 and
Debbie Zink for providing mass spectral data.
Refer en ces a n d Notes
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