Antitumor agents 286. Design, synthesis, and structure-activity relationships of 3'R,4'R -disubstituted-2',2'-dimethyldihydropyrano[2,3- f ]chromone (DSP) analogues as potent chemosensitizers to overcome multidrug resistance

Article abstract of DOI:10.1021/jm101249z

In this study, various 3'R,4'R-disubstituted-2'2'-dimethydihydropyrano[2,3- f]chromone (DSP) derivatives were discovered as potent chemosensitizers in the treatment of multidrug resistant cancer cells. Twenty-four DSP analogues (5-28) were synthesized and

Full text of DOI:10.1021/jm101249z

8700 J. Med. Chem. 2010, 53, 8700–8708
DOI: 10.1021/jm101249z
Antitumor Agents 286. Design, Synthesis, and Structure-Activity Relationships of 30R,40R-
Disubstituted-20,20-dimethyldihydropyrano[2,3-f ]chromone (DSP) Analogues as Potent
Chemosensitizers to Overcome Multidrug Resistance
Ting Zhou,† Qian Shi,† Kenneth F. Bastow,*,‡ and Kuo-Hsiung Lee*,†,§
†Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7568,
United States, ‡Division of Medicinal Chemistry & Natural Products, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill,
North Carolina 27599-7568, United States, and §Chinese Medicine Research and Development Center, China Medical University and Hospital,
Taichung, Taiwan
Received September 27, 2010
In this study, various 30R,40R-disubstituted-20,20-dimethydihydropyrano[2,3-f ]chromone (DSP) deri-
vatives were discovered as potent chemosensitizers in the treatment of multidrug resistant cancer cells.
Twenty-four DSP analogues (5-28) were synthesized and evaluated against a multidrug resistant
(MDR) cell line (KB-Vin) with and without vincristine (VCR). All DSP analogues exhibited low
intrinsic cytotoxicity. However, in combination treatment, most DSPs reversed resistance to VCR and
lowered the GI50 value of VCR by 12-349-fold. At a concentration of 1 μg/mL, three compounds, 11,
14, and 21, fully reversed resistance to VCR in KB-Vin cancer cells, a 2-fold increase compared to
verapamil, a first-generation chemosensitizer. Detailed structure-activity relationship (SAR) conclu-
sions were established based on 30 and 40 substitutions. Moreover, a preliminary mechanism study
indicated that the chemosensitizing activity of DSP analogues results from inhibition of P-glycoprotein
(P-gp) overexpressed in MDR cancer cells.
Introduction
toxic at the doses required to attenuate P-gp function.4 Since
then, several potent and selective next-generation inhibitors
have been developed and investigated, but none of them have
yet shown significant clinical benefit. Inhibition of P-gp by
chemosensitizers may also be problematic because drug dis-
tribution and elimination can be affected by these agents.4,5
However, it is still possible that the ideal chemotype for a
chemosensitizer has yet to be identified and developed.
It has been reported that the naturally existing pyranocou-
marin (()-praeruptorin A (PA) and its khellactone analogues
with 30,40-modifications (Figure 2) can reverse P-gp-mediat-
ed MDR.6,7 (()-30-O-40-O-Bis(3,4-dimethoxycinnamoyl)-cis-
khellactone (DMDCK, 3) showed the highest potency among
these analogues. At a concentration of 4 μM, 3 was able to
increase the activity of the chemotherapy drug vinblastine over
110-fold.7 However, even at such a high concentration, 3 was
unable to completely reverse vinblastine resistance in the liver
cancer cell system used.7 In addition, it was also reported that
the side chains at the 30 and 40 positions of the khellactone
system play an important role in maintaining high activity.
Unfortunately, only a few khellactone analogues with 30,40
modifications have been reported to show potent chemosensi-
tizing activity, so detailed SAR information is unavailable.
Therefore, continuing research to develop novel active ana-
logues is merited and forms the basis for the work on the DSP
series reported herein.
Resistance of cancer cells to anticancer drugs remains one
of the major obstacles in achieving an effective treatment for
cancer. Three primary mechanisms of anticancer drug resis-
tance have been identified: decreased uptake of water-soluble
drugs, various changes in cells that affect the capacity of
cytotoxic drugs to kill cells, and the most commonly encoun-
tered, increased energy-dependent efflux of hydrophobic cy-
totoxic drugs by one of a family of energy-dependent trans-
porters, such as P-gp (also known as MDR1a).1 One approach
used in clinical research to reverse MDR is the development of
chemosensitizers, especially drugs that target P-gp and inhibit
its activity. Verapamil and cyclosporine A (Figure 1) are
examples of first-generation chemosensitizers that inhibit
the activity of P-gp and were evaluated clinically as adjuvants
for chemotherapy. Both compounds are able to increase
the intracellular concentration of cytotoxic agents, such as
VCR, cyclophosphamide, dexamethasone, and doxorubicin,
in MDR cells.2,3 Moreover, both verapamil and cyclospor-
ine A are widely used medications, verapamil as a calcium
channel blocker to treat hypertension and cyclosporine A as
an immunosuppressant to reduce the activity of the patient0s
immune system in postallogeneic organ transplant. However,
clinical studies showed that both compounds were generally
*To whom correspondence should be addressed. For K.F.B.: phone,
919-966-7633; fax, 919-966-0204; E-mail, ken_bastow@unc.edu. For K.
H.L.: phone, 919-962-0066; fax, 919-966-3893; E-mail, khlee@unc.edu.
a Abbreviations: DSP, 30R,40R-disubstituted-20,20-dimethyldihydro-
pyrano[2,3-f ]chrmone; MDR, multidrug resistance; VCR, vincristine;
P-gp, P-glycoprotein; PA, (()-Praeruptorin A; DMDCK, (()-30-O-40-
O-bis(3,4-dimethoxycinnamoyl)-cis-khellactone; HB, hydrogen bond.
Design
Even before the structure of P-gp protein was resolved,8 a
general pharmacophore model for P-gp binding and inhibition
r
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Published on Web 11/17/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8701
was proposed based on verapamil.9 The pharmacophore con-
sists of a butterfly shaped molecule with two hydrophobic
motifs (aromatic ring system) connected with a linker that
contains hydrogen bond (HB) acceptors and/or donors.
DMDCK (3) was subsequently shown to fit the pharmaco-
phore model and could be readily docked into verapamil0s
hypothetical binding site on the protein subunit of P-gp.7,9
DSPs (Figure 2) share certain structural similarities to
DMDCK (3), which prompted us to design and synthesize a
series of 30,40-disubstituted-pyranochromone derivatives as
new chemosensitizing agents. It is likely that the pyranochro-
monemoietyhassimilarHB propertiestothoseofkhellactone
in DMDCK. Incorporation of two side chains at the 30 and
40 positions with substitutions containing aromatic moieties
could mimic the hydrophobic moieties in verapamil and
DMDCK (3). On the basis of this rationale, a series of 30,40-
disubstituted-pyranochromone derivatives with varied 30 and
40 side chains were designed, synthesized, and screened against
the MDR human esophageal cancer cell line (KB-Vin) with or
without combination of the anticancer drug VCR. This ap-
proach allowed usto evaluate the intrinsiccytotoxicity of DSP
analogues as well as their ability to reverse MDR.
30,40-Dibenzyloxy substituted DSP (22) was generated by
reacting 4 with benzyl bromide in the presence of NaH in
DMF under N2 atmosphere (Scheme 1).12
The synthesis of carbamates 24-28 was accomplished by
treatment of 4 with N,N-dialkylcarbamylchloride or pyrroli-
dine and piperidine carbonyl chloride, respectively, in anhy-
drous THF in the presence of NaH (Scheme 1).13
Results and Discussion
Chemosensitization Activity in Vitro. VCR is a potent
mitotic inhibitor and is widely used in combination chemo-
therapy of acute leukemias and some solid tumors. The GI50
value of VCR against the KB (drug-sensitive) cell line was
0.009 μg/mL (Table 1). Against the KB-Vin cell line, a multi-
drug resistant cancer cell line, the cytotoxic effect of VCR
decreases dramatically, over 270-fold, and its GI50 value is
2.44 μg/mL (Table 1). To determine the ability of DSPs to
reverse MDR in KB-Vin cells, the cytotoxic effect of VCR
was evaluated with or without the addition of each newly
synthesized DSP analogue.
To exclude the possibility that cytotoxicity of the combi-
nation treatment with VCR and DSP might result from the
cytotoxic effect of DSPs, all newly synthesized DSPs were
also screened individually against KB-Vin cells, and the
results are shown in Table 1. In general, all DSP analogues
exhibited low intrinsic cytotoxic activity, similar to that of
the prototype first-generation P-gp inhibitor verapamil.
The chemosensitizing activity of DSPs was then evaluated
by measuring the cytotoxic effect of VCR in the presence of
DSP analogues at 1 μg/mL, using verapamil as the positive
control. As seen from the data in Table 1, most of the DSP
analogues lowered the GI50 value of VCR and thus reversed
VCR resistance in the range of 12-349-fold. In comparison,
verapamil increased VCR sensitivity by 141-fold.
Compounds 5-16 are pyranochromones with benzoyloxy
groups at the 30 and 40 positions. Different functional groups
were selectively introduced on the o-, m-, or p-position of the
benzoyl rings. As shown in Table 1, except for 10, nonsub-
stituted, o-, and m-substituted benzoyloxy-DSPs (5-9) ex-
hibited lower chemosensitizing activity than corresponding
p-substituted compounds. Compounds 5, 6, and 8 failed to
decrease the GI50 of VCR below 0.2 μg/mL, while o- and m-
methoxy-substituted benzoyloxy-DSPs (7 and 9) exhibited
only moderate chemosensitizing activity by increasing the
potency of VCR around 28-fold. Compound 10, with an m-
CN substituent, exhibited better chemosensitizing activity
(160-fold).
Chemistry
Compound 4, 30R,40R-20,20-dimethyl-dihydropyranochro-
mone, is the core structure of all target compounds and a key
intermediate in all synthetic pathways. The synthesis of 4 has
been reported previously.10,11 Side chains with varied chain
lengths and aromatic or nonaromatic functional groups at the
chain terminus wereintroduced atthe 30 and 40 positions of the
core scaffold (Scheme 1).
Compound 4 was stirred with substituted benzoyl chloride
or cyclohexyl carbonyl chloride in the presence of DMAP at
room temperature to afford 5-16 and 23. Reaction of phe-
nylacetyl chlorides or 3-phenylpropanoyl chloride with 4 gave
17-19, while reaction of 4 with trans-cinnamoyl chlorides
afforded 20-21.
DSP analogues with p-substituted benzoyloxy rings (11-16)
showed substituent-dependent activity. Compounds 11 and 14
with methyl and methoxy substituents, respectively, showed
Figure 1
Figure 2. Structures of (() Praeruptorin A, DCK, MMDCK, DMDCK, and DSP.

8702 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Zhou et al.
Scheme 1. Synthesis of 30,40-Dibenzoyl-pyranodihydrochromone Derivatives (5-28)a
a Reagents and conditions: (i) diverse benzoyl chlorides, DMAP, anhydrous CH2Cl2, rt; (ii) phenylacetyl chlorides or 3-phenylpropanoyl chloride,
DMAP, anhydrous CH2Cl2, rt; (iii) trans-cinnamoyl chlorides, DMAP, CH2Cl2, rt; (iv) cyclohexanecarbonyl chloride, DMAP, anhydrous CH2Cl2, rt;
(v) benzyl bromide, NaH/DMF, N2, 0 °C; (vi) diverse dialkylcarbamyl chlorides or cyclocarbamyl chlorides, NaH, rt.
the most promising chemosensitizing activity. GI50 values of
the combinations of 11 and 14 with VCR were 0.007 and
0.0095 μg/mL, respectively, corresponding to 349- and 257-
fold increased potency relative to VCR alone. The improved
potency was greater than that with verapamil. Interestingly,
12, with an ethyl group, had a substantially lower effect (20.3-
fold VCR increase), and 13, substituted with a trifluoromethyl
group, improved VCR activity by only 16.3-fold. Compounds
15 and 16, with electron-withdrawing groups at the p-position,
showed enhancements of approximately 200-fold, which was
better than that of verapamil. These results suggested that
p-substituents are generally preferred over o- or m-substituents
for chemosensitizing activity. Substituents with extended con-
jugation, e.g., CN or NO2 groups, and smaller less sterically
hindered groups may benefit the efficacy of 30,40-disubstituted
DSPs as new potent chemosensitizers.
Compounds 17-21 were designed, synthesized, and eval-
uated in order to understand the effects of adding a linker
between the ester carbonyl group and the aromatic substituents
in the side chains. Introducing a saturated linker slightly in-
creased the chemosensitizing ability, with improved GI50 values
of the VCR-DSP combination from over 0.2 μg/mL for 5 (-O2-
CC6H5), 0.03 μg/mL for 17 (-O2CCH2C6H5), and 0.023 μg/
mL for 19 (-O2CCH2CH2C6H5). Unexpectedly, replacing the
phenyl group of 17 with a p-methoxyphenyl in 18 resulted in a
2.4-fold reduction in chemosensitizing efficacy, and 7.5 times
reduction compared with 14, which also contains a p-methox-
yphenyl group, but linked directly to the ester carbonyl moiety.
Compounds 20 and 21 have a conjugated double bond between
the ester carbonyl group and the phenyl ring. Interestingly,
combination of either compound with VCR led to significantly
enhanced activity. Compound 20, with an unsaturated vinyl
group, showed 2-fold increased efficacy compared to 19, with a
saturated ethylene group. Compound 21, with p-methoxycin-
namoyloxy 30,40 side chains, was able to fully restore the cyto-
toxcitiy of VCR. The GI50 value of the VCR-21 combination
was 0.0085 μg/mL, which was comparable to the values with 11
and 14, Thus, cinnamic and p-methoxycinnamic esters linked to
the core dihydropyranochromone structure favored MDR re-
versal activity.
To investigate the impact of the carbonyl group in the
active analogues, 22 was synthesized. In this compound, the
carbonyl moieties in the 30 and 40 side chains have been
replaced with a methylene group. With an ether rather than
ester side chain, 22 will likely be chemically and metabolically
more stable than its analogue 5. Interestingly, 22 showed
better capability to reverse MDR than 5. This finding shows
that the carbonyl group may not be essential for activity.
Further SAR studies are needed to confirm this hypothesis.
To further investigate the importance of the 30,40-side
chains, compounds 23-28, with various 30,40-carbamate
groups, were designed and synthesized. Most of these com-
pounds, which do not have an aromatic substituent in the
side chains, showed either no or marginal chemosensitizing
activity. The exception was 25 (N,N-diethylcarbamoyloxy),
which showed 10 times better efficacy than its counterparts

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8703
Table 1. Intrinsic Cytotoxicity of Indicated Compounds and Cytotoxicity of DSP þ VCR Formula

8704 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Table 1. Continued
Zhou et al.
a Cytotoxicity of single compound as GI50 values for KB-Vin cell line or as indicated, the concentration that caused 50% reduction in absorbance at
562 nm relative to untreated cells using the sulforhodamine B assay. b Mean GI50 value of VCR þ indicated compounds (1 μg/mL), from 2 or more
independent tests (SD listed in Supporting Information). c Cytotoxicity fold increase = 2.44 (GI50 of VCR in KB-Vin)/GI50 of VCR þ DSP formula.
d Molar concentration of DSP and verapamil that were used in the combination formula. Except for compounds 22, 24, 25, and 27, all other DSPs were
added at the same or lower molar concentration as verapamil in this assay.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8705
24 (N,N-dimethylcarbamoyloxy) and 26 (N,N-dibutylarba-
moyloxy). 25 also presented 5-fold more potency than its
cyclic analogues 27 and 28, suggesting that the orientation of
the N-alkyl substitutions at 30 and 40 positions affects the
interactions of the drug molecules and the binding pocket in
the target protein, for example, the different orientation
between chain alkyl groups (25) and cyclic groups (27, 28),
which, in turn, affects the activity.
In summary, three compounds, 11, 14, and 21, were found
to be the most potent chemosensitizers of VCR among 24
newly synthesized compounds. At a nontoxic concentration,
they are able to fully restore the cytotoxicity of VCR against
KB-Vin so that the MDR cell line was as sensitive as the
parent KB cell line. They were also over two times better than
the positive control verapamil (Table 1). While most of the
active compounds contain 30,40-ester side chains ending in an
aromatic ring, 25, which contains two carbamate side chains
and no aromatic system, also exhibited considerable potency.
The dicarbamate 25 should also exhibit increased polarity
and different physicochemical properties, which may benefit
further development ofnew analogues withinteresting biolog-
ical activity.
The SAR study on 30,40 side chains suggested the following
conclusions: (1) A terminal aromatic ring is important but
not necessary to maintain high chemosensitizing capability.
(2) Substituents on the aromatic rings can significant impact
the activity. Moieties that have increased electron density,
extended conjugation systems, or HB effects can benefit
efficacy. (3) The characteristic of the linker between the core
structure and the aromatic ring system can also affect the
chemosensitizing potential. Extending the conjugation sys-
tem by adding double bond(s) benefits the activity.
Previous modification studies of PA and DCK (1) reveal-
ed that aromatic rings in the 30 and 40 side chains of the
khellactone benefit the chemosensitizing activity, and in
addition, methoxy groups substituted on cinnamoyl side
chains could impact the activity.7 Those results are mostly,
but not fully, consistent with those reported here for pyr-
anochromone-based DSP active analogues. For example, we
found that 25, without an aromatic ring in the 30,40-side
chains, displayed good chemosensitizing ability, and thus an
aromatic ring is not essential for activity. In addition, our
studies showed both positive (14 vs 5, 21 vs 20) and negative
(18 vs 17) effects for the addition of p-methoxy group on
a phenyl ring. Fong et al. also reported that p-methoxycin-
namoyl-khellactone (MMDCK, 2) exhibited decreased po-
tency compared with DCK (1).7
Because of the different cell lines and MDR substrates, it is
difficult to directly compare the activities between the prior
khellactone compounds and the DSP analogues reported
here. However, in both HepG2/Dox and KB-V1 cell lines,
the best reported khellactone compound, DMDCK (3) at
4 μM, could not completely restore the cytotoxicity of vin-
blastine, an alkaloid analogue of VCR. In contrast, DSP
analogues 11, 14, and 21 completely restored the cytotoxic
effect of VCR, suggesting they are more efficacious and more
potent than DMDCK (3).
Figure 3
P-gp substrate calcein-AM was investigated using the estab-
lished and widely used assay for cellular P-gp function.15
Inhibition of P-gp leads to the accumulation of calcein-AM
in the cytoplasm and concomitant degradation by esterase
action to give a fluorescent derivative that can be conve-
niently monitored as a quantitative measure of P-gp inhibi-
tion. Four DSP analogues (8, 13-15) were selected and their
ability to facilitate calcein-AM influx was examined and
compared to that of verapamil as the positive inhibitor control.
The results are shown in Figure 3. Compounds 15 and 14,
which showed improved activity versus verapamil in the
chemosensitization assay (Table 1), also exerted more potent
inhibition of cellular P-gp based on the functional assay results.
Compound 13, which showed weaker chemosensitizing activ-
ity versus verapamil (16- and 142-fold respectively, Table 1),
was correspondingly less active as a P-gp inhibitor. Compound
8 exerted no effect on calcein-AM efflux, which is consistent
with its lack of chemosensitization activity. These results
demonstrate that the rank order of chemosensitizing efficacy
of DSP analogues is consistent with their relative inhibitory
activity toward cellular P-gp. Thus, DSP analogues most likely
act as pump inhibitors in the KB-MDR cell system.
Conclusions
Novel DSP analogues with significant chemosensitizing
activity in a MDR human cancer cell line were discovered.
An SAR study on the 30 and 40 side chains of pyranochromone
ring was conducted with the following conclusions. p-Sub-
stitution on the aromatic ring in the side chain is better than
o- and m-substitution. Small substitutions that can increase
electron density or provide hydrogen-bonding capacity at the
putative interaction domain on P-gp benefit the activity. The
length and identity of the linker is also important and a
conjugated double-bond is favorable. Compared with
verapamil, a prototype first generation chemosensitizer, nine
DSPs exhibited improved or comparable chemosensitizing
potential. Compounds 11, 14, and 21 fully overcame VCR
resistance in KB-Vin cancer cells at a concentration of 1 μg/mL
(1.64-1.90 μM range). Compared to recent work on khellac-
tone-based chemosensitizers, our study provides a more com-
prehensive SAR and discovered DSP analogues with more
promising potency at lower concentration. Preliminary work
indicates that the chemosensitizing activity of DSP analogues
results from P-gp inhibition. Detailed mechanistic studies are
currently ongoing.
Effect on Cellular P-gp Activity. The acquired VCR-
resistance in the KB-Vin cell line is due to enhanced drug
efflux via P-gp overexpression.14 Our research results de-
monstrate that addition of DSPs could restore VCR’s cyto-
toxity againt KB-Vin, suggesting that DSP analogues might
function like verapamil as P-gp inhibitors. To test this
hypothesis, the effect of DSP analogues on efflux of the
Experimental Section
Chemistry. Melting points were measured with a Fisher Johns
melting apparatus without correction. The proton nuclear magnetic

8706 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Zhou et al.
resonance (1H NMR) spectra were measured on a 300 MHz
Varian Gemini 2000 spectrometer and a Varian Inova 400 MHz
spectrometer using TMS as internal standard. The solvent used
was CDCl3 unless indicated. Microwave reactions were per-
formed with a Biotage initiator EXP US. Mass spectra were
measured on a Shimazu LCMS-2010 (ESI-MS). Optical rota-
tion was measured with a Jasco Dip-2000 digital polarimeter at
20 °C at the sodium D line. Thin-layer chromatography (TLC)
was performed on PLC silica gel 60 F254 plate (20 ꢀ 20, Merck).
Biotage Flash and Isco companion systems were used for
medium-pressure column chromatography. Silica 40 μM col-
umns from Grace Inc. were used for column chromatography.
All final compounds are >95% pure on the basis of HPLC.
Synthesis of Compounds 5-21 and 23. A mixture of com-
pound 4 (1 equiv), substituted benzoyl cholorde, cyclohenxal
chloride, phenylacetyl chlorides, 3-phenylpropanoyl chloride,
or trans-cinnamoyl chlorides (3 equiv) and DMAP (4 equiv)
were stirred in CH2Cl2 for 1-2 h at rt, monitored by TLC. At
completion, the mixture was diluted with CH2Cl2 and washed
with water and brine. The solvent was then removed under
reduced pressure, and the residue was purified by PTLC with
hexane:EtOAc = 3:2 to afford the appropriate compounds
(5-21 and 23).
(1H, d, J = 4.8 Hz, H-40), 5.41 (1H, d, J = 4.8 Hz, H-30), 3.87,
3.85 (each 3H, s, 3-methoxybenzoyl-OCH3-30,40), 2.65 (2H,
q, J = 7.6 Hz, CH2CH3-2), 1.61, 1.49 (each 3H, s, CH3-20),
1.30 (3H, t, J = 7.6 Hz, CH2CH3-2). [R]D -116.3° (c=0.0008,
CHCl3). HRMS for (Mþ þ H) calcd m/z 559.1963; found
559.1986.
30R,40R-Di-(3-cyanobenzoyloxy)-20,20-dimethyl-2-ethyl-dihydro-
pyrano[2,3-f ]chromone (10). Yield 65% (starting from 100 mg
of 4); white solid; mp =253-254 °C. 1NMR δ 8.19 (1H, d, J =
9.0 Hz, H-5), 8.11 (4H, m, benzoyl-30,40), 7.86, 7.58 (each 2H, m,
benzoyl-30,40), 7.02 (1H, d, J = 9.0 Hz, H-6), 7.00 (1H, d, J = 5.4
Hz, H-40), 6.16 (1H, s, H-3), 5.66 (1H, d, J = 5.4 Hz, H-30), 2.36
(2H, q, J = 7.8 Hz, CH2CH3-2), 1.69, 1.57 (each 3H, s, CH3-20),
1.03 (3H, t, J = 7.8 Hz, CH2CH3-2). [R]D -125.0° (c = 0.0004,
CHCl3). HRMS for (Mþ þ H) calcd m/z 549.1662; found
549.1666.
30R,40R-Di-(4-methylbenzoyloxy)-20,20-dimethyl-2-ethyldihydro-
pyrano[2,3-f ]chromone (11). Yield 80% (starting from 25 mg
of 4); white solid; mp =144-145 °C. 1NMR δ 8.13 (1H, d, J =
9.0 Hz, H-5), 7.78, 7.78 (each 2H, d, J = 8.1 Hz, COOC-
(CHCH)2CCH3-30,40), 7.17, 7.17 (each 2H, d, J = 8.1 Hz,
COOC(CHCH)2CCH3-30,40), 6.97 (1H, d, J = 9.0 Hz, H-6),
6.96 (1H, d, J = 4.8 Hz, H-40), 6.07 (1H, s, H-3), 5.62 (1H, d, J =
4.8 Hz, H-30), 2.39, 2.38 (each 3H, s, methylbenzoyl-CH3-30,40),
2.34 (2H, q, J=7.5 Hz, CH2CH3-2), 1.67, 1.53 (each 3H, s, CH3-
20), 1.02 (3H, t, J = 7.5 Hz, CH2CH3-2). [R]D -81.3° (c =
0.0015, CHCl3). HRMS for (Mþ þ H) calcd m/z 527.2070;
found 527.2063.
30R,40R-Dibenzoyloxy-20,20-dimethyl-2-ethyldihydropyrano[2,3-
f ]chromone (5). Yield 85% (starting from 30 mg of 4); white solid;
mp 63-65 °C. 1NMR δ 8.15 (1H, d, J = 8.8 Hz, H-5), 8.10-8.13
(1H, m, benzoyl-30,40), 7.87-7.90, 7.46-7.61, 7.32-7.46 (each
3H, m, bezoyl-30,40), 7.01 (1H, d, J=4.8 Hz, H-40), 6.99 (1H,
d, J = 8.8 Hz, H-6), 6.08 (1H, s, H-3), 5.66 (1H, d, J = 4.8 Hz,
H-30), 2.34 (2H, q, J = 8.0 Hz, CH2CH3-2), 1.69, 1.54 (each 3H,
s, CH3-20), 1.02 (3H, t, J = 8.0 Hz, CH2CH3-2). [R]D -82.9 o (c=
0.0035, CHCl3). HRMS for (Mþ þ H) calcd m/z 499.1757; found
499.1735.
30R,40R-Di-(2-trifluoromethylbenzoyloxy)-20,20-dimethyl-
2-ethyldihydropyrano[2,3-f ]chromone (6). Yield 88% (starting from
30 mg of 4); white solid; mp = 104-106 °C. 1NMR δ 7.95 (1H, d,
J = 8.8 Hz, H-5), 7.50-7.80 (8H, m, benzoyl-30,40), 6.65 (1H, d,
J = 8.8 Hz, H-6), 6.30 (1H, s, H-3), 5.85 (1H, d, J = 4.8 Hz, H-40),
5.50 (1H, d, J = 4.8 Hz, H-30), 2.34 (2H, q, J = 8.0 Hz, CH2-
CH3-2), 1.69, 1.54 (each 3H, s, CH3-20), 1.02 (3H, t, J = 8.0 Hz,
CH2CH3-2). [R]D -34.7° (c = 0.0019, CHCl3). HRMS for (Mþ þ
H)calcd m/z 635.1504; found 635.1493.
30R,40R-Di-(2-methoxybenzoyloxy)-20,20-dimethyl-2-ethyldi-
hydropyrano[2,3-f]chromone (7). Yield 80% (starting from 30 mg
of 4); white solid; mp = 104-106 °C. 1NMR δ 8.18, 7.81, 7.63,
7.07 (each 1H, d, J = 7.6 Hz, methoxybenzoyl-30,40), 8.11 (1H, d,
J = 8.8 Hz, H-5), 7.44, 7,42, 6.93, 6.90 (each 1H, t, J = 7.6 Hz,
methoxybenzoyl-30,40), 7.00 (1H, d, J = 4.8 Hz, H-40), 6.94 (1H, d,
J = 9.0, H-5), 6.09 (1H, s, H-3), 5.62 (1H, d, J = 4.8 Hz, H-30),
3.77, 3.70 (each 3H, s, methoxybenzoyl-OCH3-30,40), 2.48 (2H,
q, J = 7.5 Hz, CH2CH3-2), 1.63, 1.56 (each 3H, s, CH3-20), 1.14
(3H, t, J = 7.5 Hz, CH2CH3-2). [R]D -40.0° (c = 0.0007, CHCl3).
HRMS for (Mþ þ H) calcd m/z 559.1963; found 559.1937.
30R,40R-Di-(3-trifluoromethylbenzoyloxy)-20,20-dimethyl-
2-ethyldihydropyrano[2,3-f ]chromone (8). Yield 80% (starting from
100 mg of 4); white solid; mp=124-126 °C. 1NMR δ 8.15 (1H, d,
J=8.8 Hz, H-5), 8.02-8.07 (4H, m, benzoyl-30,40), 7.77-7.82 (2H,
m, benzoyl-30,40), 7.49-7.55 (2H, m, benzoyl-30,40), 6.99 (1H, d,
J = 4.2 Hz, H-40), 6.98 (1H, d, J = 8.8 Hz, H-6), 6.06 (1H, s, H-3),
5.67 (1H, d, J=4.2 Hz, H-30), 2.33 (2H, q, J=7.6 Hz, CH2CH3-2),
1.68, 1.54 (each 3H, s, CH3-20), 1.02 (3H, t, J=7.6Hz, CH2CH3-2).
[R]D -74.6° (c=0.0013, CHCl3). HRMS for (Mþ þ H) calcd m/z
635.1504; found 635.1514.
30R,40R-Di-(4-ethylbenzoyloxy)-20,20-dimethyl-2-ethyldihydro-
pyrano[2,3-f ]chromone (12). Yield 70% (starting from 65 mg
1
of 4); white powder; mp =109-102 °C. NMR δ 8.13 (1H, d,
J = 9.0 Hz, H-5), 7.80, 7.80 (each 2H, d, J= 7.2 Hz, COOC-
(CHCH)2CCH2CH3-30,40), 7.20, 7.17 (2H, d, J=7.2 Hz, COOC-
(CHCH)2CCH2CH3-30,40), 6.98 (1H, d, J=9.0 Hz, H-6), 6.97 (1H,
d, J = 4.8 Hz, H-40), 6.07 (1H, s, H-3), 5.63 (1H, d, J = 4.8 Hz,
H-30), 2.69, 2.68 (each 2H, q, J=7.5 Hz, ethylbenzoyl-CH2CH3-
30,40), 2.32 (2H, q, J = 7.5 Hz, CH2CH3-2), 1.68, 1.53 (each 3H, s,
H-20), 1.24(3H, t, J=7.5 Hz, CH2CH3-2), 1.23, 1.03 (each 3H, t, J=
7.2 Hz ethylbenzoyl-CH2CH3-30,40). [R]D -93.6° (c=0.01, CHCl3).
HRMS for (Mþ þ H) calcd m/z 555.2383; found 555.2366.
30R,40R-Di-(4-trifluoromethylbenzoyloxy)-20,20-dimethyl-
2-ethyl-dihydropyrano[2,3-f ] chromone (13). Yield 60% (starting
from 25 mg of 4); white powder; mp=114-115 °C. 1NMR δ 8.22
(2H, d, J = 8.1 Hz, trifluoromethylbenzoyl-H), 8.16 (1H, d, J = 8.7
Hz, H-5), 8.99 (2H, d, J=8.4 Hz, trifluoromethylbenzoyl-H), 7.74,
(2H, d, J=8.1 Hz, trifluoromethylbenzoyl-C(CHCH)2CCF3-30,40),
7.65 (2H, d, J = 8.4 Hz, trifuoromethylbenzoyl-C(CHCH)2CCF3-
30,40), 7.00 (1H, d, J = 8.7 Hz, H-6), 7.00 (1H, d, J = 4.8 Hz, H-40),
6.12 (1H, s, H-3), 5.67 (1H, d, J=4.8 Hz, H-30), 2.33(2H, q, J=7.8
Hz, CH2CH3-2), 1.68, 1.56 (each 3H, s, CH3-20), 1.02 (3H, t, J =
7.8 Hz, CH2CH3-2). [R]D -74.0° (c=0.0025, CHCl3). HRMS for
(Mþ þ H) calcd m/z 635.1504; found 635.1488.
30R,40R-Di-(4-methoxybenzoyloxy)-20,20-dimethyl-2-ethyl-
dihydropyrano[2,3-f]chromone (14). Yield 80% (starting from
100 mg of 4); yellow oil. 1NMR δ 8.13 (1H, d, J = 8.7 Hz, H-5),
7.85, 7.85 (each 2H, d, J = 8.7 Hz, COOC(CHCH)2COCH3-
30,40), 6.97 (1H, d, J = 8.7 Hz, H-6), 6.95 (1H, d, J = 4.8 Hz,
H-40), 6.85, 6.82 (each 2H, d, J = 8.7 Hz, COOC(CH-
CH)2COCH3-30,40), 6.06 (1H, s, H-3), 5.61 (1H, d, J=4.8 Hz,
H-30), 3.85, 3.84 (each 3H, s, methoxybenzoyl-OCH3-30,40), 2.33
(2H, q, J = 7.2 Hz, CH2CH3-2), 1.66, 1.53 (each 3H, s, CH3-20),
1.03 (3H, t, J = 7.2 Hz, CH2CH3-2). [R]D -105.0° (c=0.0012,
CHCl3). HRMSfor(Mþ þ H) calcd m/z559.1968; found 559.1954.
30R,40R-Di-(4-cyanobenzoyloxy)-20,20-dimethyl-2-ethyl-dihy-
dropyrano[2,3-f]chromone (15). Yield 80% (starting from 100 mg
of 4); white powder; mp=253-254 °C. 1NMR δ 8.17 (1H, d, J =
8.7 Hz, H-5), 7.98, 7.97 (each 2H, d, J = 8.7 Hz, benzoyl-
COC(CHCH)2CCN-30,40), 7.71, 7.68 (each 2H, d, J = 8.7 Hz,
benzoyl-COC(CHCH)2CCN-30,40), 6.99 (1H, d, J = 8.7 Hz,
H-6), 6.98 (1H, d, J = 5.1 Hz, H-40), 6.08 (1H, s, H-3), 5.66
30R,40R-Di-(3-methoxybenzoyloxy)-20,20-dimethyl-2-ethyldi-
hydropyrano[2,3-f ]chromone (9). Yield 80% (starting from 30
mg of4); white solid; mp = 90-92 °C; MS-ESI þ (m/z,%) 559(Mþ þ
1, 100). 1NMR δ 8.09 (1H, d, J = 8.4 Hz, H-5), 7.71, 7.68 (each 1H, d,
J= 7.6 Hz, benzoyl-30,40), 7.61 (2H, s, benzoyl-30,40), 7.38 (2H, t,
J = 8.4, 7.6 Hz, benzoyl-30,40), 7.15 (2H, J = 8.4 Hz, benzoyl-
30,40), 6.93 (1H, d, J = 8.4 Hz, H-6), 6.17 (1H, s, H-3), 5.53

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8707
(1H, d, J=5.1 Hz, H-30), 2.34 (2H, t, J=7.8 Hz, CH2CH3-2), 1.67,
1.56 (each 3H, s, CH3-20), 1.01 (3H, t, J = 7.8 Hz, CH2CH3-2).
[R]D -104.8° (c = 0.0014, CHCl3). HRMS for (Mþ þ H) calcd
m/z 549.1662; found 549.1648.
30R,40R-Di(cyclohexanecarbonyloxy)-20,20-dimethyl-2-ethyl-
dihydropyrano[2,3-f ]chromone (23). Yield 20% (starting from
25 mg of 4); white solid; mp=72-74 °C. 1NMR δ 8.08 (1H, d,
J = 9.0 Hz, H-5), 6.88 (1H, d, J = 9.0 Hz, H-6), 6.61 (1H, d, J =
4.5 Hz, H-40), 6.12 (1H, s, H-3), 5.27 (1H, d, J = 4.5 Hz, H-30),
2.56 (2H, q, J = 7.5 Hz, CH2CH3-2), 2.35 (2H, m, cyclohex-
anecarbonyl-30,40), 2.80 (12H, m, cyclohexanecarbonyl-30,40),
1.49, 1.42 (each 3H, s, CH3-20), 1.35 (8H, m, cyclohexanecarbo-
nyl-30,40), 1.23 (3H, t, J = 7.5 Hz, CH2CH3-2). [R]D -15.4° (c =
0.005, CHCl3). HRMS for (Mþ þ H) calcd m/z 511.2696; found
511.2713.
Synthesis of 30R,40R-Dibenzyloxy-20,20-dimethyl-2-ethyl-dihydro-
pyrano[2,3-f ]chromone (22). NaH (20.4 mg, 0.51 mmoL) was
suspended in dry DMF. To this suspension were added at 0 °C
a solution of compound 4 (50 mg, 0.17 mmoL) in dry DMF
(0.5 mL) and benzyl bromide (0.05 mL, 0.43 mmoL), under the
protection of N2. After 1 h, the mixture was poured into 5%
aqueous NaHCO3 solution and extracted with Et2O three times.
The organic layers were combined and dried over Na2SO4. The
crude product was purified by preparative TLC with hexane:
EtOAc = 1:1 to afford 21 (16.2 mg, white solid); 57.3% yield;
mp=77-79 °C. 1NMR δ 8.00 (1H, d, J = 8.7 Hz, H-5), 7.41,
7.28 (each 5H, m, benzyl-30,40), 6.81(1H, d, J = 8.7 Hz, H-6),
6.13 (1H, s, H-3), 5.10, 4.95, 4.85, 4.75 (each 1H, d, J = 11.4 Hz,
benzyl-CH2-30,40)5.09 (1H, d, J = 4.2 Hz, H-40), 3.75 (1H, d,
J = 4.2 Hz, H-30), 2.60 (2H, q, J = 7.5 Hz, CH2CH3-2), 1.57,
1.50 (each 3H, s, CH3-20), 1.28 (3H, t, J = 7.5 Hz, CH2CH3-2).
[R]D -40.9° (c = 0.01, CHCl3). HRMS for (Mþ þ H) calcd m/z
471.2171; found 471.2155.
30R,40R-Di-(4-nitrobenzoyloxy)-20,20-dimethyl-2-ethyl-dihydro-
pyrano[2,3-f ]chromone (16). Yield 50% (starting from 50 mg of 4);
white solid; mp=109-110 °C. 1NMR δ 8.23, 8.22 (each 2H, d,
J = 9.0 Hz, COOC(CHCH)2CNO2-30,40), 8.14 (1H, d, J = 8.4 Hz,
H-5), 8.06, 8.04 (each 2H, d, J = 9.0 Hz, COOC(CHCH)2CNO2-
30,40), 6.99 (1H, d, J = 8.4 Hz, H-6), 6.98 (1H, d, J = 4.8 Hz, H-40),
6.06 (1H, s, H-3), 5.67 (1H, d, J = 4.8 Hz, H-30), 2.33 (2H, q, J =
7.2 Hz, CH2CH3-2), 1.68, 1.57 (each 3H, s, CH3-20), 1.00 (3H,
t, J = 7.2 Hz, CH2CH3-2). [R]D -74.7° (c = 0.0015, CHCl3).
HRMS for (Mþ þ H) calcd m/z 589.1458; found 589.1437.
30R,40R-Di-(2-phenylacetoxy)-20,20-dimethyl-2-ethyl-dihydro-
pyrano[2,3-f ]chromone (17). Yield 53% (starting from 30 mg of
4); solid powder; mp = 75-77 °C. 1NMR δ 8.07 (1H, d,
J = 8.8 Hz, H-5), 7.30 (10H, m, phenyl-30,40), 6.85 (1H, d, J =
8.8 Hz, H-6), 6.58 (1H, d, J = 4.8 Hz, H-40), 6.05 (1H, s, H-3),
5.20 (1H, d, J = 4.8 Hz, H-30), 3.53, 3.46 (each 2H, d, J = 15.9 Hz,
acetoxy-COOCH2-30,40), 2.25 (2H, q, J = 7.6 Hz, CH2CH3-2),
1.32, 1.25 (each 3H, s, CH3-20), 1.12 (3H, t, J=7.6Hz, CH2CH3-2).
[R]D -60.0° (c = 0.0015, CHCl3). HRMS for (Mþ þ H) calcd m/z
527.2070; found 527.2060.
30R,40R-Di-[2-(4-methoxyphenylacetoxy)]-20,20-dimethyl-
2-ethyl-dihydropyrano[2,3-f ] chromone (18). Yield 50% (starting
1
from 25 mg of 4); yellow oil. NMR δ 8.07 (1H, d, J = 9.0 Hz,
H-5), 7.20, 7.17 (each 2H, d, J= 8.7 Hz, phenyl-C(CHCH)2COCH3-
30,40), 6.87, 6.85 (each, 2H, d, J = 8.7 Hz, phenyl-C(CH-
CH)2COCH3-30,40), 6.78 (1H, d J = 9.0 Hz, H-6), 6.44 (1H, d,
J = 4.8 Hz, H-40), 6.10 (1H, s, H-3), 4.03 (1H, d, J = 4.8 Hz, H-30),
2.25 (2H, q, J = 7.5 Hz, CH2CH3-2), 1.49, 1.34 (each 3H, s, CH3-20),
1.13 (3H, t, J = 7.5 Hz, CH2CH3-2). [R]D -47.7° (c = 0.0013,
CHCl3). HRMS for (Mþ þ H) calcd m/z 587.2281; found 587.2266.
30R,40R-Di-(3-phenylpropanoyloxy)-20,20-dimethyl-2-ethyl-
dihydropyrano[2,3-f ]chromone (19). Yield 70% (starting from
25 mg of 4); clear oil. 1NMR δ 8.09 (1H, d, J = 9.0 Hz, H-5), 7.22
(10H, m, phenyl-C(CHCH)2CH-30,40), 6.88 (1H, d, J = 9.0 Hz,
H-6), 6.59 (1H, d, J = 4.8 Hz, H-40), 6.09 (1H, s, H-3), 5.26 (1H,
d, J = 4.8 Hz, H-30), 2.95, 2.95 (4H, t, J = 7.6 Hz, propanoyl-
CH2CH2-30.40), 2.67, 2.58 (each 2H, t, J = 7.6 Hz, propanoyl-
CH2CH2-30,40), 2.46 (2H, q, J = 7.5 Hz, CH2CH3-2), 1.43,
1.33 (each 3H, s, CH3-20), 1.67 (3H, t, J = 7.5 Hz, CH2CH3-2).
[R]D -74.8° (c = 0.0023, CHCl3). HRMS for (Mþ þ H) calcd
m/z 555.2393; found 555.2378.
Synthesis of Compounds 24-28. Sodium hydride (5 equiv) was
added to a solution of compound 4 in dry THF at ice-bath
condition and diverse carbamyl chlorides (3 equiv) were added.
The reaction mixture was stirred at rt and monitored by TLC.
At completion, the reaction mixture was poured carefully onto
EtOAc and saturated aqueous NaHCO3. The organic layer
was washed with water for three times, dried over anhydrous
MgSO4, and evaporated under reduced pressure. The crude pro-
duct was purifed by prepare TLC.
30R,40R-Di(dimethylcarbamoyloxy)-20,20-dimethyl-2-ethyl-
dihydropyrano[2,3-f]chromone (24). Yield 30% (starting from
100 mg of 4); clear oil. 1NMR δ 8.07 (1H, d, J = 8.8 Hz, H-5),
6.88 (1H, d, J = 8.8 Hz, H-6), 6.52 (1H, d, J = 4.8 Hz, H-40),
6.11 (1H, s, H-3), 5.22 (1H, d, J = 4.8 Hz, H-30), 2.97, 2.85 (each
6H, m, dimethylcarbamoyl-CH2-30,40), 2.55 (2H, q, J = 7.2 Hz,
CH2CH3-2), 1.49, 1.46 (each 3H, s, CH3-20), 1.24 (3H, t, J = 7.2
Hz, CH2CH3-2). [R]D -28.6° (c = 0.01, CHCl3). HRMS for
(Mþ þ H) calcd m/z 433.1957; found 433.1951.
30R,40R-Di-(E)- phenylacryloyloxy-20,20-dimethyl-2-ethyl-di-
hydropyrano[2,3f ]chromone (20). Yield 65% (starting from 50 mg
of 4); white solid; mp = 104-106 °C. 1NMR δ 8.05 (1H, d,
J = 9.0 Hz, H-5), 7.63, 7.63 (each 1H, d, J = 15.9 Hz COO-
CHCH-30,40), 7.36-1.40 (5H, m, phenyl-30,40), 1.23-1.29 (5H, m,
phenyl-30,40), 6.88 (1H, d, J = 9.0 Hz, H-6), 6.75 (1H, d,
J = 4.8 Hz, H-40), 6.38 (2H, d, J = 15.9 Hz, COOCHCH-30.40),
6.02 (1H, s, H-3), 5.42 (1H, d, J = 4.8 Hz, H-30), 2.42 (2H, q, J =
6.9 Hz, CH2CH3-2), 1.52, 1.43 (each 3H, s, CH3-20), 1.17 (3H, t,
J=6.9 Hz, CH2CH3-2). [R]D -75.3° (c = 0.0017, CHCl3). HRMS
for (Mþ þ H) calcd m/z 551.2070; found 551.2074.
30R,40R-Di(diethylcarbamoyloxy)-20,20-dimethyl-2-ethyl-dihydro-
pyrano[2,3-f ]chromone (25). Yield 25% (starting from 25 mg
of 4); white solid; mp = 125-126 °C. 1NMR δ 8.07 (1H, d, J =
8.8 Hz, H-5), 6.88 (1H, d, J = 8.8 Hz, H-6), 6.59 (1H, d, J = 4.8
Hz, H-40), 6.11 (1H, s, H-3), 5.25 (1H, d, J = 4.8 Hz, H-30), 3.35,
3.35, 3.18, 3.18 (each 2H, q, J = 6.8 Hz, diethylcarbamoyl-CH2-
30,40), 2.55 (2H, q, J = 7.6 Hz, CH2CH3-2), 1.49, 1.47 (each 3H, s,
CH3-20), 1.23 (3H, t, J = 7.6 Hz, CH2CH3-2), 1.16 (6H, t, J = 6.8
Hz, diethylcarbamoyl-CH3-40), 1.09, 0.98 (each 3H, t, J = 6.8 Hz,
diethylcarbamoyl-CH3-30). [R]D -30.0° (c = 0.001, CHCl3). HR-
MS for (MþþH) calcd m/z 489.2601; found 489.2594.
30R,40R-Di-(E)-4-methoxyphenylacryloyloxy-20,20-dimethyl-
2-ethyl-dihydropyrano[2,3f ] chromone (21). Yield 50% (starting
from 25 mg of 4); white powder; mp = 94-96 °C. 1NMR δ 8.10
(1H, d, J = 9.0 Hz, H-5), 7.66, 7.65 (each 1H, d, J = 15.3 Hz,
(E)-acryloxy-OCOCHCH-30,40), 7.40 (4H, dd, J = 8.7, 3.0 Hz,
4-methoxyphenyl-C(CHCH)2COCH3-30,40), 6.93 (1H, d, J =
9.0 Hz, H-6), 6.85 (4H, dd, J = 8.7, 3.0 Hz, 4-methoxyphenyl-
C(CHCH)2COCH3-30,40), 6.80 (1H, d, J = 4.8 Hz, H-40), 6.33,
6.32 (each 1H, d, J = 15.3 Hz, (E)-acryloxy-OCOCHCH-30,40),
6.10 (1H, s, H-3), 5.47 (1H, d, J = 4.8 Hz, H-30), 6.65 (6H, s,
OCH3-30,40), 2.47 (2H, q, J = 7.5 Hz, CH2CH3-2), 1.58,
1.49 (each 3H, s, CH3-20), 1.59 (3H, t, J = 7.5 Hz, CH2CH3-
2). [R]D -70.0° (c=0.0043, CHCl3). HRMS for (Mþ þ H) calcd
m/z 611.2281; found 611.2260.
30R,40R-Di(dibutylcarbamoyloxy)-20,20-dimethyl-2-ethyl-dihydro-
pyrano[2,3-f ]chromone (26). Yield 30% (starting from 50 mg of 4);
clear oil. 1NMR δ8.06 (1H, d, J=8.8Hz,H-5),6.86(1H,d,J=8.8
Hz, H-6), 6.58 (1H, d, J = 4.8 Hz, H-40), 6.11 (1H, s, H-3), 5.23
(1H, d, J = 4.8 Hz, H-30), 3.25 (8H, m, dibutylcarbamoyl-30,40),
2.56 (2H, q, J = 7.6 Hz, CH2CH3-2), 1.57 (6H, m, dibutylcarba-
moyl-CH2), 1.47, 1.46 (each 3H, s, CH3-20), 1.33 (8H, m, dibutyl-
carbamoyl-CH2), 1.25 (3H, t, J = 7.6 Hz, CH2CH3-2), 1.08
(2H, q, J = 7.2 Hz, dibutylcarbamoyl-CH2-30), 0.95, 0.94 (each
3H, t, J=7.6 Hz, dibutylcarbamoyl-CH3-40), 0.86, 0.72 (each 3H, t,
J = 7.2 Hz, dibutylcarbamoyl-CH3-30). [R]D -13.5° (c = 0.012,
CHCl3). HRMS for (Mþ þ H) calcd m/z 601.3853; found 601.3833.

8708 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Zhou et al.
30R,40R-Di(pyrrolidine-1-carbonyloxy)-20,20-dimethyl-2-ethyl-
dihydropyrano[2,3-f ] chromone (27). Yield 35% (starting from
25 mg of 4); white powder; mp = 104-106 °C. NMR δ 8.05
(1H, d, J = 8.8 Hz, H-5), 6.87 (1H, d, J = 8.8 Hz, H-6), 6.54 (1H,
d, J = 4.8 Hz, H-40), 6.11 (1H, s, H-3), 5.23 (1H, d, J = 4.8 Hz,
H-30), 3.48, 3.26 (each 4H, m, pyrrolidine-N(CH2)2(CH2)2-30,40),
2.55 (2H, q, J = 7.6 Hz, CH2CH3-2), 1.86 (8H, m, pyrrolidine-
N(CH2)2(CH2)2-30,40), 1.50, 1.47 (each 3H, s, CH3-20), 1.21 (3H, t,
J=7.6 Hz, CH2CH3-2). [R]D -23.8° (c=0.011, CHCl3). HRMS
for (Mþ þ H) calcd m/z 485.2288; found 485.2277.
(2) Hindenburg, A. A.; Baker, M. A.; Gleyzer, E.; Stewart, V. J.; Case,
N.; Taub, R. N. Effect of verapamil and other agents on the
distribution of anthracyclines and on reversal of drug resistance.
Cancer Res. 1987, 47 (5), 1421–1425.
(3) Thomas, H.; Coley, H. Overcoming multidrug resistance in cancer:
an updated on the clinical strategy of inhibitinf P-glycoprotein.
Cancer Control 2003, 10, 159–165.
(4) Szakacs, G.; Paterson, J. K.; Ludwig, J. A.; Booth-Genthe, C.;
Gottesman, M. M. Targeting multidrug resistance in cancer.
Nature Rev Drug Discovery 2006, 5 (3), 219–234.
(5) Hollt, V.; Kouba, M.; Dietel, M.; Vogt, G. Stereoisomers of
calcium antagonists which differ markedly in their potencies as
calcium blockers are equally effective in modulating drug transport
by P-glycoprotein. Biochem. Pharmacol. 1992, 43 (12), 2601–2608.
(6) Wu, J. Y.; Fong, W. F.; Zhang, J. X.; Leung, C. H.; Kwong, H. L.;
Yang, M. S.; Li, D.; Cheung, H. Y. Reversal of multidrug
resistance in cancer cells by pyranocoumarins isolated from Radix
peucedani. Eur. J. Pharmacol. 2003, 473 (1), 9–17.
1
30R,40R-Di(piperidine-1-carbonyloxy)-20,20-dimethyl-2-ethyl-
dihydropyrano[2,3-f ]chromone (28). Yield 32% (starting from 25
mg of 4); white solid; mp = 107-109 °C. 1NMR δ 8.06 (1H, d,
J = 8.8 Hz, H-5), 6.87 (1H, d, J = 8.8 Hz, H-6), 6.56 (1H, d, J =
4.8 Hz, H-40), 6.11 (1H, s, H-3), 5.24 (1H, d, J = 4.8 Hz, H-30),
3.77 (8H, m, piperidine-N(CH2)2(CH2)2CH2-30,40), 2.57 (2H, q,
J = 7.6 Hz, CH2CH3-2), 1.58 (12H, m, piperidine-N(CH2)2-
(CH2)2CH2-30,40), 1.47, 1.46 (each 3H, s, CH3-20), 1.26 (3H, t,
J = 7.6 Hz, CH2CH3-2). [R]D -28.2° (c = 0.0017, CHCl3).
HRMS for (Mþ þ H) calcd m/z 513.2601; found 513.2620.
Chemosensitization (MDR Modulation) Assay. Three thou-
sand drug-resistant cells, KB-Vin cells (MDR-1) selected using
VCR,16 were treated for two or three days with test agent in the
presence or absence of VCR. The end-point is cellular protein
measured using the sulforhodamine B (SRB) method developed
at NIH-NCI for in vitro anticancer drug screening.17 The degree
of chemoreversal was determined by fixing the concentration
of test agent and measuring the alteration in dose-response of
cells to VCR toxicity. Verapamil was used as chemosensitizer
control. Data from replicate experiments were plotted, and the
apparent GI50 value was determined using statistical software
(GraphPad Prizm, CA).
(7) Fong, W. F.; Shen, X. L.; Globisch, C.; Wiese, M.; Chen, G. Y.;
Zhu, G. Y.; Yu, Z. L.; Tse, A. K.; Hu, Y. J. Methoxylation of 30,40-
aromatic side chains improves P-glycoprotein inhibitory and multi-
drug resistance reversal activities of 7,8-pyranocoumarin against
cancer cells. Bioorg. Med. Chem. 2008, 16 (7), 3694–3703.
(8) Aller, S. G.; Yu, J.; Ward, A.; Weng, Y.; Chittaboina, S.; Zhuo, R.;
Harrell, P. M.; Trinh, Y. T.; Zhang, Q.; Urbatsch, I. L.; Chang, G.
Structure of P-glycoprotein reveals a molecular basis for poly-
specific drug binding. Science 2009, 323 (5922), 1718–1722.
(9) Pajeva, I. K.; Wiese, M. Pharmacophore model of drugs involved
in P-glycoprotein multidrug resistance: explanation of structural
variety (hypothesis). J. Med. Chem. 2002, 45, 5671–5686.
(10) Zhou, T.; Shi, Q.; Chen, C. H.; Zhu, H.; Huang, L.; Ho, P.; Lee,
K. H. Anti-AIDS agents 79. Design, synthesis, molecular modeling
and structure-activity relationships of novel dicamphanoyl-20,-
20-dimethyldihydropyranochromone (DCP) analogs as potent
anti-HIV agents. Bioorg. Med. Chem. 2010, 18 (18), 6678–6689.
(11) Zhou, T.; Shi, Q.; Lee, K. H. Anti-AIDS agent 83. Efficient
microwave-assisted one-pot preparation of augular 2,2-dimethyl-
2H-chromone containing compounds. Tetrahedron Lett. 2010, 51
(33), 4382–4386.
(12) Baba, T.; Huang, G.; MInoru, I. Synthesis of the JKLM-ring
Fragment of Ciguatoxin. Tetrahedron 2003, 59, 6851–6872.
(13) Nguyen, T. M.; Sittisombut, C.; Boutefnouchet, S.; Lallemand, M.-
C.; Michel, S.; Koch, M.; Tillequin, F.; Mizinghien, R.; Lansiaux, A.;
David-Cordonnier, M.-H.; Pfeiffer, B.; Kraus-Berthier, L.; Leonce,
S.; Pierre, A. Synthesis, antitumor activity, and mechanism of action
of benzo[a]pyrano[3,2-h]acridin-7-one analogues of acronycine.
J. Med. Chem. 2006, 49, 3383–3394.
(14) Gouaze, V.; Yu, J. Y.; Bleicher, R. J.; Han, T. Y.; Liu, Y. Y.; Wang,
H.; Gottesman, M. M.; Bitterman, A.; Giuliano, A. E.; Cabot,
M. C. Overexpression of glucosylceramide synthase and P-glyco-
protein in cancer cells selected for resistance to natural product
chemotherapy. Mol. Cancer Ther. 2004, 3 (5), 633–639.
(15) Armstrong, L.; Sullivan, R.; Mechetner, E., New functional assays
for multidrug resistance. http://www.biosciencetechnology.com/
ShowPR.aspx?PUBCODE=090&ACCT=9000009885&ISSUE=
0404&RELTYPE=PR&Cat=SubCat=1& PRODCODE=
00005949&PRODLETT=A&CALLFROM=PR&CommonCount=0,
2004, (accessed April 28, 2004) .
(16) Lunney, E. A.; Hagen, S. E.; Domagala, J. M.; Humblet, C.;
Kosinski, J.; Tait, B. D.; Warmus, J. S.; Wilson, M.; Ferguson,
D.; Hupe, D.; Tummino, P. J.; Baldwin, E. T.; Bhat, T. N.; Liu, B.;
Erickson, J. W. A novel nonpeptide HIV-1 protease inhibitor:
elucidation of the binding mode and its application in the design of
related analogs. J. Med. Chem. 1994, 37 (17), 2664–2677.
(17) Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.; Simon, R. M.;
Tosini, S.; Skehan, P.; Scudiero, D. A.; Monks, A.; Boyd, M. R.
Comparison of in vitro anticancer-drug-screening data generated
with a tetrazolium assay versus a protein assay against a diverse
panel of human tumor cell lines. J. Natl. Cancer Inst. 1990, 82 (13),
1113–1118.
Calcein-AM Loading Assay. KB-Vin (MDR) cells (10000 per
well) were seeded into 96-well plates with medium containing
5% FBS and incubated for one day for adhesion and equilibra-
tion. Treatment involved supplementing cultures with distinct
concentrations of test compounds for 30 min at 37 °C. After
removing medium, fresh medium with both 1 μM calcein-AM
(Alexis Biochemicals), and test compounds as needed was then
replaced. Following incubation for 30 min at 37 °C, medium was
removed and wells were washed gently and quickly with cold
PBS buffer in low ambient light conditions. Cells were lysed
using 20 mM Tris-HCl buffer, and fluorescence was measured
using a BioTek TLx800 ELISA reader set to Ex. 494 nm and Em.
517 nm. Composite data obtained from two or more indepen-
dent experiments were analyzed using Prizm (Graphpad Soft-
ware, San Diego, CA).15
Acknowledgment. This investigation was supported by NIH
grant CA17625 awarded to K. H. Lee.
Supporting Information Available: HPLC condition and
summary of HPLC purity data for final compounds; statistical
analysis for cell assay. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
(1) Dalton, W. S. Is p-glycoprotein a potential target for reversing
clinical drug resistance? Curr. Opin. Oncol. 1994, 6 (6), 595–600.