Highly suppressing wild-type HIV-1 and Y181C mutant HIV-1 strains by 10-chloromethyl-11-demethyl-12-oxo-calanolide A with druggable profile

Article abstract of DOI:10.1021/jm901653e

We herein report a new compound: 10-chloromethyl-11-demethyl-12-oxo- calanolide A (20, EC₅₀ = 7.4 nM, SI = 1417), which demonstrates a druggable profile with 32.7% oral bioavailability in rat, tolerated oral single dose toxicity in mice, and especially the feature of highly efficient suppression of the wild-type HIV-1 and Y181C mutant HIV-1 at an EC₅₀ = 7.4 nM and EC₅₀ = 0.46 nM, respectively.

Full text of DOI:10.1021/jm901653e

J. Med. Chem. 2010, 53, 1397–1401 1397
DOI: 10.1021/jm901653e
Highly Suppressing Wild-Type HIV-1 and Y181C Mutant HIV-1 Strains by 10-Chloromethyl-11-
demethyl-12-oxo-calanolide A with Druggable Profile
Hai Xue,†,§ Xiaofan Lu,‡,§ Purong Zheng,† Li Liu,‡ Chunyan Han,† Jinping Hu,† Zijie Liu,† Tao Ma,† Yan Li,† Lin Wang,†
Zhiwei Chen,*,‡ and Gang Liu*,†
†Department of Synthetic Medicinal Chemistry, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical
College, No. 2 Nan Wei Road, Beijing 100050, China and ‡AIDS Institute, Department of Microbiology, Li Ka Shing Faculty of Medicine, The
University of Hong Kong, Room45, 5/F Laboratory Block, 21 Sassoon Road, Pokfulam, Hong Kong. §These authors contributed to this study equally.
Received November 9, 2009
We herein report a new compound: 10-chloromethyl-11-demethyl-12-oxo-calanolide A (20, EC50 =7.4nM,
SI = 1417), which demonstrates a druggable profile with 32.7% oral bioavailability in rat, tolerated oral
single dose toxicity in mice, and especially the feature of highly efficient suppression of the wild-type HIV-1
and Y181C mutant HIV-1 at an EC50 = 7.4 nM and EC50 = 0.46 nM, respectively.
Introduction
be used in clinical settings in combination with other antiviral
drugs to suppress HIV-1 mutants. Nevertheless, its low inhibi-
tory potency against HIV-1 probably may account for the
limitation in clinical trials although it was shown to be well
tolerated in phase Ia/Ib studies.9,10 Thus, targeted modifica-
tion of (þ)-calanolide A to find new analogues with high
potency against HIV-1 has become crucial.
Over 20 antiretroviral medications have now been approved
for the treatment of HIV-infected individuals.1 Although
highly active antiretroviral therapy (HAARTa) has been very
effective in suppressing HIV load, these medications present
several limitations such as the rapid emergence of drug-
resistant mutant strains due to the narrow range of chemical
structure of the cocktail components. Because of viral resis-
tance and issues related to drug side effects, there remains a
great need to discover novel antivirals, especially ones that
function as nonnucleoside reverse transcriptase inhibitors
(NNRTIs). Today, four NNRTIs have been approved by
the FDA,2 therefore, the scarcity of unique inhibitors war-
rants the development of novel NNRTI compounds.
(þ)-CalanolideA (Figure1) was previously described asthe
first natural product that inhibits HIV-1 reverse transcrip-
tase.3 It inhibits reverse transcriptase by a mechanism invol-
ving at least two binding sites, which are distinguished as
having competitive and uncompetitive components.4 There-
fore, this type of drug could inhibit diverse HIV-1 strains
resistant to other nucleoside and nonnucleoside reverse tran-
scriptase inhibitors.5-7 In particular, (þ)-calanolide A is
unique in inhibiting HIV-1 isolates carrying the viral reverse
transcriptaseY181Caminoacid mutation, which isassociated
with high-level resistance to most current NNRTIs.6-8 In
addition, because (þ)-calanolide A displays additive to syner-
gistic anti-HIV activity with a range of nucleoside analogues,
protease inhibitors, and NNRTIs,4,5,7 this compound could
There are several papers documenting the chemical mod-
ifications of (þ)-calanolide A and related natural species in an
effort to find new analogues with higher anti-HIV-1 potency.11-16
Zembower and co-workers illustrated for the first time that
the 12-ketone form of calanolide A exhibits anti-HIV-1
activity; however, this molecule possesses a lower therapy
index than the parent natural product (þ)-calanolide A.11,12
Through a systematic analysis of structure-activity relation-
ships (SARs), we found that 10-bromomethyl-12-oxo-cala-
nolide A (3, Figure 1) has significantly enhanced antiviral
potency (EC50 = 2.85 nM).16 To address the concern that
compound 3 bearing a bromine atom at position C-10 of ring
C may confer toxicity, in this article we describe the modifica-
tions of ring C involving a replacement of the bromine atom,
with the aim of finding alternative antiviral drug candidate
presenting similar or higher anti-HIV activity against both
wild type and drug mutants with tolerated toxicity, and
acceptable oral bioavailability.
Results and Discussion
Chemistry. To replace bromine atom, we carried out a
nucleophilic substitution reaction of the three-rings bro-
mated intermediate 4 with NaN3 or NaSCH3 in DMF to
prepare the intermediates 5 and 10 followed by the construc-
tion of ring D with 1,1-diethoxy-3-methyl-2-butene to afford
the analogues 6 and 11 (Scheme 1). Moreover, when com-
pound 4 was treated with K2CO3 in anhydrous THF at room
temperature, a three-membered cycle attached to ring C in
compound 8 was formed through a intramolecular nucleo-
philic substitution. After ring closure utilizing 1,1-diethoxy-3-
methyl-2-butene, a new analogue 9 was readily obtained,
termed as 10,11-cyclopropyl-12-oxo-calanolide A. Oxidation
*To whom correspondence should be addressed. For Z.C.: phone,
(852) 2819 9825; fax, (852) 2817 7805; E-mail: zchenai@hku.hk. For
G.L.: phone, þ86 10 63167165; fax, þ86 10 63165246; E-mail:
gliu@imm.ac.cn.
a Abbreviations: HIV, human immunodeficiency virus; HAART,
highly active antiretroviral therapy; NNRTI, nonnucleoside reverse tran-
scriptase inhibitor; EC50, half maximal effective concentration; SI, selec-
tivity index; FDA, Food and Drug Administration (USA); DMF,
dimethylformamide; THF, tetrahydrofuran; DCM, dichloromethane;
DCE, 1,2-dichloroethane; m-CPBA, m-chloroperbenzoic acid; TCID50
50% tissue culture infective dose; MRT, mean residence time.
,
r
2010 American Chemical Society
Published on Web 01/05/2010
pubs.acs.org/jmc

1398 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Xue et al.
Scheme 2. Nucleophilic Substitutionwiththe Secondary Aminea
Figure 1. Chemical structure of the biologically active dipyrano-
coumarins.
Scheme 1a
a Reagents and conditions: pyrrolidine (or dimethylamine,
piperidine), THF, 12-36 h.
Scheme 3a
a Reagents and conditions: (a) 4-fluorophenyl isocyanate (or 2-meth-
oxyphenyl isothiocyanate), THF, reflux, 3 h; (b) 4-fluorobenzenesulfo-
nyl chloride (or 4-methoxybenzenesulfonyl chloride, N-acetylsulfanilyl
chloride), DCM, reflux, 3 h.
chloride, 4-methoxybenzenesulfonyl chloride, and N-acetylsul-
fanilyl chloride in dicloromethane, respectively, in the presence
of pyridine provided the corresponding amino derivatives 17b,
17c, 17d, and 17e (Scheme 3).
To obtain the Cl-mimic analogue of compound 3, 4-bromo-
substituted crotonic acid was converted into the corresponding
4-hydyoxy species,17 which was then treated with SOCl2. The
resulting acyl chloride was reacted with 4-propyl-5,7-dihydroxy
coumarin through Friedel-Crafts acylation, using anhydrous
AlCl3 as catalyst. The subsequent ring closure using sodium
acetate afforded the three-ringed chloro-intermediate 19a. The
target compound 20 was obtained after the construction of ring
D, according to the synthetic method described in Scheme 4.
Similarly, the analogue 21 containing an CH3O-substituent
at position C-10 was produced using 4-methoxy-crotonic
acid as the starting material.18 Attempts to prepare the CF3-
mimic analogue of 3 with this strategy were unsuccessful. An
alternative approach was developed which consists of first
acylation of coumarin at C-8 position to afford 22, the
subsequent classic condensation with trifluoro anhydride in
the presence of pyridine, to produce the unsaturated species 23
followed by reduction of 10,11-double bond with trime-
thylsilane catalyzed by Pd[P(Ph)3]3Cl, and the ultimate ring
closure of the above resulted intermediate 24 treated with
1,1-diethoxy-3-methyl-2-butene to obtain the title compound
25 (Scheme 5).
a Reagents and conditions: (a) NaN3, DMF, 75 °C, 1 h; (b) K2CO3,
ethanol, reflux, 3 h; (c) 1,1-diethoxy-3-methyl-2-butene, pyridine, to-
luene, 1.5 h; (d) SnCl2 H2O, ethanol, reflux,3 h; (e) NaSCH3, DMF,
3
75 °C, 0.5 h; (f) m-CPBA, THF, 0 °C, 1 h; (g) m-CPBA, THF, reflux, 3 h.
of 10 in THF with m-CPBA at different temperature
could produce the intermediate 12 (0 °C) and 14 (reflux), which
were further converted into the corresponding final sulfoxide
analogue 13 and the sulfone analogue 15, respectively. The
amine analogue 7was also synthesized through reduction of the
azide analogue 6 with SnCl2 in the presence of hydrochloride
acid. When compound 3 was reacted with secondary amines in
THF at room temperature, the classical nucleophilic substitu-
tion products (16a, 16b, and 16c) were obtained (Scheme 2) in
which hydrophilic groups were introduced at the C-10 position
of 12-oxo-calanolide A. The amine analogue 7 reacted with 4-
fluorophenyl isocyanate in THF at 65 °C to afford compound
17a. Similarly, treatment of compound 7 with 2-methoxyphe-
nyl isothiocyanate in THF, and 4-fluorobenzenesulfonyl

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1399
Scheme 4. Synthetic Route for the Cl-Mimic Analogue 20a
Table 1. Inhibitory Activities of 12-Oxo-calanolide A Analogues
against 100 TCID50 HIVADA Infection
a Reagents and conditions: (a) (i) KOH or NaOCH3, Hþ, (ii) SOCl2;
(b) 4-propyl-5,7-dihydroxy coumarin, anhydrous AlCl3, PhNO2/DCE,
75 °C, 36 h; (c) anhydrous sodium acetate, ethanol, 5 h; (d) 1,1-diethoxy-
3-methyl-2-butene, pyridine, toluene, 1.5 h.
Scheme 5a
a Reagents and conditions: (a) acetic anhydride, AlCl3, DCE/
CH3NO2, 80 °C, 16 h; (b) (i) trifluoroacetic anhydride, pyridine, rt, (ii)
HCl (2N), reflux; (c) triethylsilane, Rh[P(Ph)3]3Cl, THF, reflux, 6 h; (d)
1,1-diethoxy-3-methyl-2-butene, pyridine, toluene, 1.5 h.
a No suppression. b Selectivity index(SI) was calculated by the for-
mula: IC50/EC50 (cytotoxicity data IC50 were supplemented in Support-
ing Information). c The substituent at position C10,C11. d Not
determined.
Antiviral Activity in Vitro. All synthesized compounds
were assayed for inhibition of HIV-1 replication against
100 TCID50 HIVADA infections in a pseudovirus-based
assay, as previously described.19 Clearly, aminomethylation
at position C-10 or further various acylation of aminomethy-
lated-12-oxo calanolide A were useless for improving the
activity against HIV-1. For instance, compounds 6, 16a-16c
and 17a-17e became inactive (Table 1). These compounds
were also observed with their higher toxicity to the virus host
cells (data not show); in particular, the azide analogue 6
exhibited extreme cellular toxicity. The results imply that
positive charge groups are inappropriate at C-10 of position.
Methylthioylation at C-10 position (11) did also not give
an improved inhibitory potency against HIV-1 as compared
to (þ)-calanolide A. This included its gradually oxidated
products of sulfoxide (13) and sulfone (15). Replacement of
bromo atom of compound 3 (EC50 = 0.18 μM) with met-
hoxyl group (21) resulted in a similarinhibitoryactivity to (þ)-
calanolide A, however, with lower selectivity index (Table 1).
Significant improvement of activity compared to the
natural product (þ)-calanolide A was observed with com-
pounds cis-10,11-cyclopropyl-12-oxo-calanolide A (9) and
10-chloromethyl-12-oxo-calanolide A (20). Compound 9
yielded an EC50 value of 14.1 nM (SI = 709) and compound
20 yielded 7.4 nM (SI = 1417), respectively. C-10 halogen-
methylation of 12-oxo-calanolide A gave the variable results
with their atomic order. Trifluoromethylated compound 25
offered the antiviral activity to 14.9 μM of EC50 value,
however, chloromethylated 20 gave a remarkably higher
potency of 7.4 nM. With the observation of previous result
of bromomethylation of 12-oxo-calanolide A (3, EC50
=
2.85 nM), it is likely that the penetration ability of cell
membrane is the key factor because, in principle, the fluorine
atom is able to improve the binding ability of small molecule
through the mimic of hydrogen atom, however, the chlorine
atom could help a small molecule passing through a cell
membrane with its hydrophobic property. Furthermore, the

1400 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Xue et al.
Table 2. Biological Activity of 20 in Drug Resistant HIV-1 Strains
in this experiment was moderately high (= 32.7%). Single dose
toxicity test of compound 20 was carried out in mice. After
intragastric administration of 20 with a dose of 4640 mg/kg, no
death of the mice was observed and there was no abnormity of
the body weight increase for the animals in two weeks (seeing
Supporting Information). A long-term toxicity study is under-
way that will be reported elsewhere when it is finished.
mutants
EC50
(nM)
PNL4-3a K103N Y181C Y188L G190A T139I
20
(þ)-calanolide A
7.4
97.5
11.4
316
0.46 6127
42.6 10475 385
464 10591 7166 159.4
43.9 103.5
6151
2.2
13416
neverapine
a Wild-type HIV-1.
Conclusion
This study provided one new HIV-1 inhibitor of compound
20 with low EC50 value (7.4 nM) and high SI (1417). Compound
20 has presented a druggable profile for its 32.7% oral bioavail-
ability in rat, tolerated oral acute toxicity in mice, and extremely
high potency against the clinical Y181C single mutation of HIV-1.
Table 3. Pharmacokinetic Parameters of 20 in Rats after a Single Oral
and Intravenous Dosing (n = 3)
AUC
(μg/
mL h)
T1/
Cmax
(μg/
mL)
MRT
(h)
Tmax
(h)
2
(h)
3
intravenous
oral
1.5
4.7
1.7
9.2
1.9
3.8
0.03
8.00
0.93
0.34
Experimental Section
Preparation of the Cl-Mimic Analogue 20. In a 50 mL round-
bottom flask, 4-hydroxy-crotonic acid (250 mg) was treated in
SOCl2 (3 mL) at reflux for 2 h and then the resulting solution of
acyl chloride was condensed to an yellow oil. The mixture of
anhydrous AlCl3 (500 mg) and 5,7-dihydroxy-coumarin (330 mg)
in 3 mL of PhNO2 and 15 mL of DCE were added to the flask
and were simultaneously stirred at 75 °C for 36 h. After
hydrolysis with 100 g of ice and 8 mL of hydrochloric acid, the
mixture was extracted with EtOAc for 3 times and then the
organic phase was dried and filtered. Chromatography on silica
gel of the filtrate eluting first with petroleum and then with
petroleum EtOAc (2:1) afforded 228 mg compound 18a as a
yellow powder with the yield of 47.2%; mp 174.5-176.1 °C. 1H
NMR (300 MHz, DMSO-d6, ppm): 11.290 (s, 1H, OH), 11.196 (s,
1H, OH), 6.635-6.798 (m, 2H, alkene-H), 6.376 (s, 1H, Ar-H),
5.911 (s, 1H, 3-H), 4.442 (d, 2H, J = 8.4 Hz, Cl-CH2,), 2.861 (t,
2H, J = 7.2 Hz, 4-CH2CH2CH3), 1.592 (sex, 2H, J = 7.2 Hz,
4-CH2CH2CH3), 0.939 (t, 3H, J = 7.5 Hz, 4-CH2CH2CH3). ESI-
MS (m/z): 323.14/325.16 [M þ H]þ (MW = 322.74). HRESIMS
obsd m/z 323.0689, calcd for C16H16ClO5þ 323.0681.
much stronger electron-withdrawing ability of CF3 group
than that of CH2Br or CH2Cl might be another reason that it
significantly decreased the electronic interaction between 25
and HIV-1 reverse transcriptase.
Anti-HIV Drug Mutants Profile of Compound 20. Resis-
tance to chemotherapy can develop in a significant number
of patients during the long-term HAART that combines
three or more different drugs. Efavirenz and nevirapine are
the most widely used NNRTIs in combination regimens.
Many studies demonstrated that each of these NNRTIs is
usually associated with a single point mutation that triggers
the development of clinical resistance. For efavirenz, it is the
K103N mutation. For nevirapine, it is the Y181C muta-
tion.20 The Y181C mutation can even cause multidrug
resistance to NNRTIs.21 More importantly, a dual mutation
of Y181C and K103N has occurred, which can cause high-
level resistance to each of the available NNRTIs. Studies
indicated that compound 20 presents the similar potency as
nevirapine against the K103N mutant, but at a much higher
level than that of (þ)-calanolide A (Table 2). The compound
20 was also proved with a moderate potency against G190A
single mutation of HIV-1 (EC50 = 43.9 nM) but not for (þ)-
Anhydrous sodium acetate (10 mg) was added into the
solution of 18a in 20 mL of ethanol and 1 mL of H2O and then
stirred for 5 h at reflux. After condensation to about 3 mL, the
resulted mixture was extracted with EtOAc for 3 times. Purifica-
tion of the dried organic phase through chromatography on
silica gel eluting with petroleum-EtOAc(1:1) produced 20 mg
19a as white powder with the yield of 75%, mp 216.5-218.2 °C.
1H NMR (300 M Hz, DMSO-d6, ppm): 11.814 (s, 1H, OH),
6.315 (s, 1H, Ar-H), 6.046 (s, 1H, 3-H), 4.655-4.737 (m, 1H,
8-H), 3.602 (d, 2H, J = 7.2 Hz, ClCH2), 2.871 (t, 2H, J = 7.5 Hz,
4-CH2CH2CH3), 2.752 (m, 1H, 9-CH2), 2.562 (d, 1H, J = 3.3 Hz,
9-CH2), 1.550 (m, 2H, 4-CH2CH2CH3), 0.930 (t, 3H, J = 7.5 Hz,
4-CH2CH2CH3). ESI-MS (m/z) [M þ H]þ: 323.09/325.08 (MW =
calanolide A (EC50 = 385 nM) and nevirapine (EC50
=
159.4 nM). Compound 20 was demonstrated an in vitro
potency against wild-type HIV-1 approximately 10-fold
higher than the natural product (þ)-calanolide A, however,
exceeding much higher ability (∼90-fold) to inhibit the
Y181C mutation than (þ)-calanolide A while the nevirapine
was completely inactive. The compound 20 presents less
potency against T139I, which was indicated as a mutant site
point of (þ)-calanolide A treatment.8 The suppression was
not observed for the Y188L viral strain in the assays of
compound 20, (þ)-calanolide A, and neverapine (Table 3).
Accordingly, it is of particular interest in the inclusion of 20
in a combination regimen to treat patients who have failed
other NNRTIs and have developed the Y181C mutation or
the Y181C/K103N dual mutations. Therefore, compound 20
deserved to be selected and developed as a new drug candi-
date against the resistant HIV-1 infection.
Pharmacokinetic Study and Safety Assessment of 20. As
indicated in Table 3, when administered at 50 mg/kg orally as
a suspension in 0.5% CMC, the compound 20 was rapidly
absorbed with a Tmax of 8 h, a favorable half-life of 3.8 h, and
mean residence time (MRT) of 9.2 h. The Cmax of 20 was 0.34 μg/
mL (860 nM) that is more than 100-fold the EC50 value of anti-
HIV-1 activity in vitro (Table 1). The measured oral bioavailability
þ
322.74). HRESIMS obsd m/z 323.0685, calcd for C16H16ClO5
323.0681.
6,6,-Dimethyl-10-chloromethyl-4-propyl-2H,6H,12H-benzo-
[1,2-b:3,4-b0:5,6-b00]tripyran-2,12-dione (20). Compound 20
(white-off powder) was prepared from 19a in the procedure
described as the literature (ref 16) with the yield of 79%; mp
159.2-160.0 °C. 1H NMR (300 M Hz, DMSO-d6, ppm): 6.624 (d,
1H, J= 10.2 Hz, 8-H), 6.133 (s, 1H, 3-H), 5.852 (d, 1H, J=9.9Hz,
7-H), 4.857-4.918 (m, 1H, 10-H), 3.941-4.100 (m, 2H, ClCH2),
2.842-2.939 (m, 3H, 11-CH2, 4-CH2CH2CH3), 2.683 (dd, 1H, J =
3.0 Hz, 16.2 Hz, 11-CH2), 1.604 (m, 2H, 4-CH2CH2CH3), 1.514,
1.472 (2s, 6H, 6-CH3), 0.972 (t, 3H, J = 7.2 Hz, 4-CH2CH2CH3).
ESI-MS (m/z): 389.78 [M þ H]þ (MW = 388.84). HRESIMS
obsd m/z389.1152, calcd for C21H22ClO5þ 389.1150.
Cell-Based Anti-HIV-1 Assay. The inhibitory activities of the
compounds against HIV are evaluated as described previously
by anti-HIV assays using TZM-b1 cells.19 TZM cells contain the
HIV primary receptor CD4 and coreceptor CCR5 as well as

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1401
a reporter luciferase gene driven by the HIV promoter. These
assays quantify the activity of a drug to inhibit HIV-induced
reporter luciferase activity. Briefly, a serial diluted drug was
tested against 100 TCID50 HIV infection. The viral infection was
determined on day 3 by measuring the reporter luciferase
activity in TZM-b1 cells postinfection using commercially avail-
able kits. Antiviral data are reported as the quantity of drug
required to inhibit production by 50% (EC50).
Perno, C. F.; Gago, F.; Balzarini, J. The Role of Thr139 in
the Human Immunodeficiency Virus Type 1 Reverse Transcrip-
tase Sensitivity to (þ)-Calanolide A. Mol. Pharmacol. 2005, 68, 652–
659.
(9) (a) Creagh, T.; Ruckle, J. L.; Tolbert, D. T.; Giltner, J.; Eiznhamer,
D. A.; Dutta, B.; Flavin, M. T.; Xu, Z. Q. Safety and Pharmaco-
kinetics of Single Doses of (1)-Calanolide A, a Novel, Naturally
Occurring Nonnucleoside Reverse Transcriptase Inhibitor, in
Healthy, Human Immunodeficiency Virus-Negative Human Sub-
jects. Antiviral Chem. Chemother. 2001, 45 (5), 1379–1386. (b)
Eiznhamer, D. A.; Creagh, T.; Ruckle, J. L.; Tolbert, D. T.; Giltner, J.;
Dutta, B.; Flavin, M. T.; Jenta, T.; Xu, Z. Q. Safety and pharmacokinetic
profile of multiple escalating doses of (þ)-calanolide A, a naturally
occurring nonnucleoside reverse transcriptase inhibitor, in healthy
HIV-negative volunteers. HIV Clin. Trials 2002, 3 (6), 435–450.
(10) Sherer, R.; Dutta, B.; Anderson, R.; Laudette-Aboulhab, J.;
Kamarulzaman, A.; D’Amico, R.; Paton, N.; Abdullah, M. S.;
Pollard, R.; Cooley, T.; Flavin, M. T.; Xu, Z. Q. A phase 1B study of (þ)-
calanolide A in HIV-1-infected, antiretroviral therapy-naive patients. 7th
CROI, San Francisco, 2000, Abstract 508 .
Acknowledgment. This research is supported financially by
the National Natural Science Foundation of China (No.
90713045) and the National 863 Program of China,
No.2006AA020501. We thank HKU-UDF and HKSARG
FHB/RFCID09080772 for partial funding support of this
study. We are also grateful to Mr. Jinfeng Wei for his
assistance of single dose toxicity measurement.
(11) Zembower, D. E.; Liao, S.; Flavin, M. T.; Xu, Z.-Q.; Stup, T. L.;
Buckheit, R. W., Jr.; Khilevich, A.; Mar, A. A.; Sheinkmann, A. K.
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Supporting Information Available: Experimental details of the
synthesis of 6-17, 21-25; biological methods and detailed data
of cytotoxicity and single dose acute toxicity test. This material
is available free of charge via the Internet at http://pubs.acs.org.
(12) Xu, Z. Q.; Buckheit, R. W.; Stup, T. L.; Flavin, M. T.; Khilevich,
A.; Rizzo, J. D.; Lin, L.; Zembower, D. E. In vitro anti-HIV
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