Synthesis of carbocyclic analogs of dehydroaltenusin: Identification of a stable inhibitor of calf DNA polymerase α

Article abstract of DOI:10.1016/j.tet.2015.08.005

Syntheses of carbocyclic analogs of dehydroaltenusin tautomers are reported. Both target compounds, cDHA (2,8-dihydroxy-6-methoxy-10a-methyl-10,10a-dihydrophenanthrene-3,9-dione) and cDHAs (4′,5-dihydroxy-6′-methoxy-2-methylspiro[cyclohexa[2,5]diene-1,1′-

Full text of DOI:10.1016/j.tet.2015.08.005

Tetrahedron 71 (2015) 7575e7582
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of carbocyclic analogs of dehydroaltenusin: identification
of a stable inhibitor of calf DNA polymerase
a
a
a
b
ꢀꢁ ꢀ a
ꢀꢁ
Silvia Kovacova , Santosh Kumar Adla , Lukas Maier , Michal Babiak ,
,
Yoshiyuki Mizushina c, Kamil Paruch a
*
a Department of Chemistry, Faculty of Science Masaryk University, Kamenice 5, 62500 Brno, Czech Republic
b Central-European Institute of Technology, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic
c Graduate School of Agriculture, Shinshu University, Minamiminowa-mura, Kamiina-gun, Nagano 399-4598, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 May 2015
Received in revised form 24 July 2015
Accepted 3 August 2015
Available online 7 August 2015
Syntheses of carbocyclic analogs of dehydroaltenusin tautomers are reported. Both target compounds,
cDHA (2,8-dihydroxy-6-methoxy-10a-methyl-10,10a-dihydrophenanthrene-3,9-dione) and cDHAs (40,5-
dihydroxy-60-methoxy-2-methylspiro[cyclohexa[2,5]diene-1,10-indene]-30,4(20H)-dione), were prepared
from 3,5-dimethoxybenzaldehyde in 11 and 13 steps, respectively. Unlike dehydroaltenusin, both cDHA
and cDHAs are stable and their structures were confirmed by X-ray crystallography. Compound cDHA
was found to be active against calf DNA polymerase
analog cDHAs was inactive.
a
but not related isozymes, while the spirocyclic
Keywords:
Dehydroaltenusin
DNA polymerase
Ó 2015 Elsevier Ltd. All rights reserved.
DNA damage repair
Spirocyclic compounds
Regioselective aldol condensation
1. Introduction
the inhibitory activity.8 In order to help elucidate this issue, we
decided to carry out bioisosteric replacement of the lactone ring
oxygen by methylene group and prepare the carbocyclic analogs of
the spirocyclic and non-spirocyclic forms of dehydroaltenusin e
cDHAs and cDHA, respectively (Scheme 1). These compounds
were envisioned to be significantly more stable and resistant to
the rearrangement.
Proteins and pathways involved in DNA damage/repair con-
tinue to be intensely studied, especially in the context of de-
velopment of new cancer therapeutic agents.1 The inhibition of
DNA polymerases (pols) seems to be a proper component of syn-
thetic lethal combination therapy.2 Specifically, deletion of pol
a
was found to be synthetic lethal with elimination or inhibition of
CHK1 kinase, but elimination of additional pols (delta and epsilon)
resulted in significantly weaker synthetic lethal response.3 Selec-
tive inhibitors of pol
a could therefore be of considerable thera-
peutic potential. Of the compounds that are known to inhibit this
enzyme,1 only two seem to be sufficiently selective: nucleoside
analog BuPdGTP and its derivatives,4 and dehydroaltenusin.5 Both
substances possess suboptimal pharmacological properties:
BuPdGTP exhibits only weak activity in the cell,6 while chemical
stability of dehydroaltenusin is limited, as it undergoes a rear-
rangement in aqueous solutions to give a mixture of the spirocyclic
and non-spirocyclic forms (structures DHAs and DHA in Scheme 1;
absolute configurations are not known).7 It is not clear which of
these forms is active and it has been suggested that the rear-
rangement o-quinone intermediate might be also responsible for
Scheme 1. Spirocyclic and non-spirocyclic isomers of dehydroaltenusin and their
carbocyclic analogs. Of note, the central scaffolds of both cDHA and cDHAs are only
sporadically documented in the literature.
* Corresponding author. E-mail address: paruch@chemi.muni.cz (K. Paruch).
http://dx.doi.org/10.1016/j.tet.2015.08.005
0040-4020/Ó 2015 Elsevier Ltd. All rights reserved.

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2. Results and discussion
(10% and 35%, respectively). Best result (55%) was obtained with
dirhodium caprolactamate plus tert-butylhydroperoxide in aceto-
The retrosynthetic analysis of cDHA, depicted in Scheme 2, re-
lies on benzylic oxidation and alkylation of protected tetralone 3 to
provide intermediate 2 possessing the required quaternary center,
aldol cyclization forming the tricyclic scaffold 1, and final oxidation
and deprotection to yield the target compound (cDHA).
nitrile, buffered with NaHCO3.11
Methylation (t-BuOK, MeI) of 5 led to a mixture of mono- and di-
methylated ketones, along with the unreacted starting material.
Reasonable yield (55%) of the desired mono-methylated product was
obtained when the alkylation was carried out in the presence of
DMPU. Subsequent reaction with methyl vinyl ketone (MVK) proved
difficult and out of many conditions tried, only the method by von
Doering12 afforded the desired diketone 2 in acceptable (61%) yield.
Intramolecular aldol condensation of 2 using t-BuOH/t-BuOK
provided tricyclic ketone 1. Subsequent oxidation with oxygen
under basic conditions, followed by acidic cleavage of the ketal
provided compound 6 in good overall yield (Scheme 3).
Finally, selective demethylation of 6 using BBr3 gave the desired
target compound cDHA, whose structure was confirmed by X-ray
crystallography (Scheme 3).
The retrosynthetic analysis of cDHAs (depicted in Scheme 4)
relies on double alkylation of ketone 9, regioselective ring closure of
8 leading to the spirocyclic intermediate 7, and final oxidation and
selective demethylation to afford cDHAs.
Scheme 2. Retrosynthetic analysis of cDHA. Construction of the quaternary center and
the tricyclic scaffold.
Starting from 3,5-dimethoxybenzaldehyde, Wittig reaction fol-
lowed by hydrogenation afforded acid 4, whose intramolecular Frie-
deleCrafts cyclization gave the required tetralone (not shown).9
Attempted protections of the tetralone using ethanediol, 2-methoxy-
1,3-dioxolane, ethanediol plus CH(OCH3)3, or 2-methoxy-1,3-dioxolane
in the presence of TsOH did not provide the desired product 3 in ac-
ceptable yields, likely due to the sensitivity of the substance to strong
acids. However, reaction with ethanediol in the presence of PPTS in
refluxing benzene afforded 3 in satisfactory yield (Scheme 3).
Scheme 4. Retrosynthetic analysis of cDHAs. Construction of the spirocyclic core.
Ketone 9 was prepared in two steps by condensation of 3,5-
dimethoxybenzaldehyde with nitroethane, followed by reduction
with iron in AcOH.13 Because our subsequent attempts to add MVK
to 9 failed under a variety of conditions and in most cases lead only
to the recovery of the starting material, we attempted alkylations
with MVK equivalents. Best results were obtained with iodide 11,
which we prepared in 80% yield by modification of the procedure
published for the analogous bromide.14 This way we obtained in-
termediate 10 that underwent second alkylation with t-butyl-2-
iodoacetate.15 The resulting ester 12 was then deprotected to give
diketone 8, which was used for the subsequent intramolecular aldol
closure (Scheme 5).
The regioselectivity of the aldol closure proved very dependent
on the reaction conditions. Condensation using piperidine/AcOH in
toluene,16 which may proceed via enamine formation at the less
sterically hindered carbonyl, afforded the desired enone 13a (81%).
In contrast, when carried out under basic conditions (t-BuOK/THF),
the condensation yielded exclusively the isomer 13b (Scheme 5).
Construction of the spirocyclic scaffold was completed by intra-
molecular FriedeleCrafts reaction of 13a in the presence of MsOH,
which provided the spirocyclic enone 7 in good yield (Scheme 5).
In contrast to the non-spirocyclic series, we were not able to
oxidize enone 7 directly. We thus attempted to convert it into a silyl
enol ether that could be used for subsequent Rubottom oxidation.
In accordance with the literature,17 regioselective formation of the
desired silyl enol ether proved non-trivial: numerous attempts to
carry out mono-silylation of 7 yielded only mixtures of the desired
product, often along with its exocyclic isomer, bis-silyl enol ether 14
Scheme 3. Synthesis and structural assignment of cDHA.
Benzylic oxidation of 3 using Cr(CO)6 or PDC with tert-butyl-
hydroperoxide10 afforded the desired ketone 5 in modest yields

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7577
underwent clean mono-silylation to afford enol ether 18, which was
then oxidized to hydroxyketone 19. Exposure of methanolic solu-
tion of 19 to air and K2CO3, which effected the desired oxidation
and cleavage of the pivalate, afforded the target compound cDHAs,
whose structure was again unambiguously confirmed by X-ray
crystallography (Scheme 7).
Scheme 7. Completion of synthesis of cDHAs.
In the biochemical assay we developed previously,18 we tested
both cDHA and cDHAs against the following mammalian pols: calf
Scheme 5. Construction of the spirocyclic scaffold.
pol
a and human pols g, k, and l, which belong to the B-, A-, Y-, and
X-family of pols, respectively. As shown in Table 1, cDHA specifically
inhibits pol a. In contrast, cDHAs inhibits none of the tested pols.
and unreacted 7. However, with excess of TBSOTf the bis-silylation
of 7 proceeded cleanly and the subsequent epoxidation occurred
predominantly at the six membered ring (Scheme 6). The resulting
hydroxyketone 15 (as ca. 5:1 mixture of diastereomers) was oxi-
dized to 16. However, all our attempts to cleave the phenolic methyl
ether in the vicinity of indanone carbonyl (e.g., using BBr3 or BCl3)
failed and yielded complex mixtures that contained only traces of
the desired product cDHAs.
Table 1
IC50 values of cDHA and cDHAs for mammalian DNA polymerasesa
Polymerase
IC50 value (
cDHA
mM)
cDHAs
Calf pol
a
5.5ꢀ0.29
>100
>100
>100
>100
>100
>100
Human pol
Human pol
Human pol
g
k
l
>100
a
Data are shown as the means ꢀ SE of three independent experiments. De-
scription of the assay is given in Experimental section.
3. Conclusions
In conclusion, we prepared racemic novel carbocyclic analogs of
dehydroaltenusin cDHA and cDHAs in 11 and 13 steps, respectively,
starting from 3,5-dimethoxybenzyldehyde. While cDHA is ap-
proximately eight times weaker inhibitor than dehydroaltenusin
itself (previously reported IC50 value for dehydroaltenusin against
calf pol a is 0.68 m
M),5 it is stable and can be stored in the solid state
as well as in aqueous DMSO solution for at least four weeks without
noticeable decomposition. In addition, the enantiomers of cDHA
can be efficiently separated by HPLC on chiral stationary phase
(details given in Supplementary data). Bioisosteric replacement of
oxygen atom by the methylene group is commonly used in me-
dicinal chemistry;19 however, in the context of this project, it en-
abled not only identification of more stable analogs, but also
provided significant (albeit indirect) information on which of the
dehydroaltenusin isomers is responsible for its biological activity.
Our observation of the dramatic difference in activities of cDHA and
cDHAs strongly suggests that dehydroaltenusin’s inhibitory activity
Scheme 6. Attempted conversion of enone 7 into cDHAs.
Fortunately, selective demethylation was possible at the stage of
spirocyclic enone 7, which enabled us to introduce an alternative
protecting group that could be cleaved more easily at the end of the
synthesis (Scheme 7). Along this line we decided to prepare piv-
aloyl ester 17. Interestingly (and in contrast to 7), ester 17

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S. Kovacova et al. / Tetrahedron 71 (2015) 7575e7582
toward pols is due to its non-spirocyclic form DHA and while the
presence reactive o-quinone intermediate may contribute to
dehydroaltenusin’s inhibitory activity, it may not be absolutely
required.
We believe the methodology presented above will enable
preparation of additional analogs possessing the rare and non-
trivial tricyclic central motifs. In addition, our results could be
used to guide further development of potent and selective in-
4-(3,5-dimethoxyphenyl)but-3-enoic acid as a yellow solid (1.80 g,
66%). Mp 58e60 ꢂC; 1H NMR (300 MHz, CDCl3)
2H), 6.47 (d, J¼15.9 Hz, 1H), 6.39 (t, J¼2.3 Hz, 1H), 6.28 (dt,
J¼15.9 Hz, J¼7.2 Hz, 1H), 3.80 (s, 6H), 3.30 (d, J¼7.2 Hz, 2H).1H NMR
spectrum was identical with the published literature.21
d
¼6.55 (d, J¼2.3 Hz,
4.3. 4-(3,5-Dimethoxyphenyl)butanoic acid (4)
hibitors of mammalian polymerases, namely pol
a
, exploiting the
A solution of 4-(3,5-dimethoxyphenyl)but-3-enoic acid (2.00 g,
9.0 mmol) in EtOH (24 mL) was added to Pd/C (10% Pd, 478 mg) and
ammonium formate (1.70 g, 27.0 mmol). The reaction mixture was
stirred at 60 ꢂC for 14 h, then cooled, filtered through a pad of Celite,
and concentrated under reduced pressure. The residue was diluted
with H2O (100 mL), acidified with conc. HCl to pH¼4, and extracted
with EtOAc (3ꢃ50 mL). The combined organic extracts were dried
over Na2SO4 and concentrated. The residue was purified by column
chromatography (silica gel, EtOAc/hexane (1:1)) to afford 4 as
a white solid (1.44 g, 71%). Mp 63e65 ꢂC; 1H NMR (300 MHz, CDCl3)
recently published crystal structure.20
4. Experimental section
4.1. General
All reagents and solvents were of reagent grade and used
without further purification. Anhydrous solvents (THF, dichloro-
methane, CH3CN) were purchased from commercial suppliers
ꢂ
(Aldrich, Acros) and stored over 4 A molecular sieves. All reactions
d
¼6.35 (d, J¼2.0 Hz, 2H), 6.32 (dd, J¼2.0 Hz,1H), 3.78 (s, 6H), 2.62 (t,
were carried out in oven-dried glassware and under N2 atmosphere
unless stated otherwise. Column chromatography was carried on
silica gel (230e400 mesh). TLC plates were visualized under UV and
stained with phosphomolybdic acid or KMnO4 solution or Vaughn’s
reagent ((NH4)6Mo7O24$4H2O/Ce(SO4)2$4H2O/conc. H2SO4/H2O).
NMR spectra were recorded on Bruker Avance 300 and 500 MHz
spectrometers, with operating frequencies 300.13, 500.13 MHz for
1H and 75.48, 125.77 MHz for 13C. The 1H and 13C NMR chemical
J¼7.6 Hz, 2H), 2.38 (t, J¼7.4 Hz, 2H), 2.02e1.90 (m, 2H).
4.4. 6,8-Dimethoxy-3,4-dihydronaphthalen-1(2H)-one
A mixture of 4 (1.44 g, 6.42 mmol) and polyphosphoric acid
(14 mL) was stirred at 90 ꢂC for 4 h, then it was poured into icee-
water (250 mL) and extracted with EtOAc (3ꢃ100 mL). The com-
bined organic extracts were washed with saturated aqueous
solution of NaHCO3 (2ꢃ50 mL) and dried over Na2SO4. Removal of
the solvent under reduced pressure afforded the product as a yel-
low solid (1.24 g, 93%). Mp 62e63 ꢂC; 1H NMR (300 MHz, CDCl3)
shifts (d in ppm) were referenced to the residual signals of solvents:
CDCl3 [7.24 (1H) and 77.23 (13C)]. Structural assignments of reso-
nances have been performed with the help of 2D NMR gradients
experiments (HSQC, HMBC, NOESY).
d
¼6.35e6.30 (m, 3H), 3.87 (s, 2H), 3.84 (s, 3H), 2.87 (t, J¼6.0 Hz,
High resolution mass spectra were measured on Agilent 6224
Accurate-Mass TOF LC-MS with dual electrospray/chemical ioni-
zation mode with mass accuracy greater than 2 ppm, applied mass
range was from 25 to 20,000 Da.
2H), 2.57 (t, J¼6.5 Hz, 2H), 2.07e1.96 (m, 2H). 1H NMR spectrum
was identical with the published literature.22
4.5. 60,80-Dimethoxy-30,40-dihydro-20H-spiro[[1,3]dioxolane-
IR spectra (4000e400 cmꢁ1) were collected on an EQUINOX 55/
S/NIR FTIR spectrometer. Samples were prepared as KBr pellets.
The diffraction data for sample cDHA (CCDC number 1012667)
2,10-naphthalene] (3)
A mixture of 6,8-dimethoxy-3,4-dihydronaphthalen-1(2H)-one
(206 mg, 1.00 mmol), pyridinium p-toluenesulfonate (25 mg,
were collected with a KUMA KM-4
k-axis diffractometer equipped
with a Sapphire2 CCD detector and a Cryostream Cooler (Oxford
0.01 mmol), and ethylene glycol (170 mL, 3.00 mmol) in benzene
ꢂ
Cryosystems, UK). Mo K
tube, graphite monochromator) was used.
a
radiation (
l
¼0.71073 A, fine-focus sealed
(25 mL) was heated at reflux in a flask equipped with a Dean-Stark
apparatus for 14 h. The mixture was cooled to 25 ꢂC, K2CO3
(100 mg) was added, and the solvent was evaporated. The residue
was purified by column chromatography (silica gel, EtOAc/hexane/
Et3N (100:50:1)) to afford 3 as a white solid (200 mg, 80%), which
was used immediately in the next step. Mp 85 ꢂC (dec); 1H NMR
The diffraction data for sample cDHAs (CCDC number 1012668)
were collected with a Rigaku partial
c geometry diffractometer
equipped with a Saturn 944þ HG CCD detector and a Cryostream
ꢂ
Cooler (Oxford Cryosystems, UK). Cu K
a
radiation (
l
¼1.54184 A,
MicroMax-007HF rotating anode source, multilayer optic VariMax)
was used. Data reduction and final cell refinement were carried out
using the CrysAlisPro software ([CrysAlisPro] CrysAlisPRO, Agilent
Technologies UK Ltd).
(500 MHz, CDCl3)
d
¼6.33 (d, J¼2.4 Hz, 1H), 6.21 (d, J¼2.4 Hz, 1H),
4.25e4.18 (m, 2H), 4.08e4.01 (m, 2H), 3.80 (s, 3H), 3.76 (s, 3H),
2.75e2.70 (m, 2H), 1.96e1.91 (m, 2H) 1.86e1.79 (m, 2H); 13C NMR
(126 MHz, CDCl3)
d
¼160.2,160.0, 142.3, 117.8, 108.1, 104.3, 98.0, 65.3
(2C), 55.8, 55.1, 36.8, 31.1, 21.1; HRMS (APCI): calcd for C14H19O4
4.2. 4-(3,5-Dimethoxyphenyl)but-3-enoic acid
[MþH]þ: 251.1283, found: 251.1348.
To NaH (60% suspension in mineral oil, 1.90 g, 49.7 mmol) was
added dry DMSO (36 mL) and the mixture was stirred and heated at
70 ꢂC. After the evolution of bubbles ceased, the mixture was cooled
to 10 ꢂC. To this mixture, a solution of (2-carboethoxy)triphenyl-
phosphonium bromide (11.00 g, 26.5 mmol) in DMSO (26 mL) was
added dropwise. To the resulting dark red reaction mixture, a so-
lution of 3,5-dimethoxybenzaldehyde (2.0 g, 12.0 mmol) in DMSO
(5 mL) was added. The reaction mixture was stirred at 50 ꢂC for
16 h, then cooled, poured into iceewater (200 mL), acidified with
conc. HCl to pH<4, and extracted with EtOAc (4ꢃ50 mL). The
combined organic layers were washed with H2O (2ꢃ50 mL), dried
over Na2SO4 and concentrated. The product was purified by column
chromatography (silica gel, EtOAc/hexane (1:1)) to obtain the title
4.6. 60,80-Dimethoxy-20H-spiro[[1,3]dioxolane-2,10-naph-
thalen]-40(30H)-one (5)
Rh2(cap)4 (9 mg, 0.01 mol) and t-BuOOH (5.5 M in decane,
900
mL, 4.95 mmol) were added to a mixture of 3 (250 mg,
1.00 mmol) and NaHCO3 (42 mg, 0.50 mmol) in CH3CN (2 mL). The
reaction mixture was stirred at 25 ꢂC for 16 h, then concentrated,
and purified by column chromatography (silica gel, EtOAc/hexane
(1:2)) to yield 5 as a white solid (145 mg, 55%). Mp 81 ꢂC (dec); 1H
NMR (300 MHz, CDCl3)
1H), 4.31e4.23 (m, 2H), 4.13e4.05 (m, 2H), 3.86 (s, 3H), 3.84 (s, 3H),
2.79 (m, 2H), 2.28 (m, 2H); 13C NMR (75 MHz, CDCl3)
¼197.3, 161.0,
159.1, 135.2, 123.4, 106.8, 106.0, 100.9, 65.8 (2C), 56.3, 55.6, 36.2,
d
¼7.11 (d, J¼2.5 Hz, 1H), 6.71 (d, J¼2.5 Hz,
d

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7579
34.4; IR (KBr): ṽ¼2962 cmꢁ1 (m), 2939 (w), 2887 (w), 1685 (s), 1603
(s), 1466 (m), 1430 (w), 1354 (s), 1321 (s), 1300 (s), 1217 (s), 1163 (s),
1114 (s), 1040 (s), 980 (w), 916 (m), 842 (m); HRMS (APCI): calcd for
49.3, 36.8, 35.6, 33.2, 21.7; IR (KBr): ṽ¼2950 cmꢁ1 (m), 2922 (w),
2890 (w), 1660 (s), 1603 (s), 1579 (s), 1466 (s), 1425 (m), 1356 (s),
1327 (s), 1296 (s), 1275 (s), 1207 (s), 1157 (s), 1134 (s), 1101 (s), 1147
(s), 1020 (m), 989 (m), 950 (m), 849 (w), 837 (m), 821 (w); HRMS
(APCI): calcd for C19H23O5 [MþH]þ: 331.1540, found: 331.1543.
C
14H17O5 [MþH]þ: 265.1071, found: 265.1068.
4.7. 60,80-Dimethoxy-30-methyl-20H-spiro[[1,3]dioxolane-2,10-
naphthalen]-40(30H)-one
4.10. 2-Hydroxy-6,8-dimethoxy-10a-methyl-10,10a-dihy-
drophenanthrene-3,9-dione (6)
A solution of compound 5 (100 mg, 0.38 mmol) and 1,3-
dimethyltetrahydropyrimidin-2(1H)-one (330
m
L) in THF (1 mL)
t-BuOK (110 mg, 0.30 mmol) was added to a solution of 1
(186 mg, 0.33 mmol) in t-BuOH (2 mL) and THF (2 mL) and the
mixture was stirred under O2 atmosphere at 25 ꢂC for 4 h. The
reaction mixture was diluted with saturated aqueous solution of
NH4Cl (30 mL) and extracted with CH2Cl2 (3ꢃ20 mL). The combined
organic phases were dried over Na2SO4, filtered, and the solvent
was evaporated. The residue was dissolved in acetone (5 mL), TsOH
(10 mg, 0.05 mmol) was added, and the mixture was stirred at 25 ꢂC
for 16 h. The solvent was removed under reduced pressure and the
residue was purified by column chromatography (silica gel, EtOAc/
CH2Cl2 (1:1)) to afford 6 (71 mg, 72%) as a white solid. Mp
was cooled to ꢁ78 ꢂC, t-BuOK (46.8 mg, 0.42 mmol) was added, and
the mixture was stirred at ꢁ78 ꢂC for 45 min. Iodomethane (30
mL,
0.48 mmol) was added, the mixture was allowed to warm to 25 ꢂC
and stirred for 16 h, then diluted with H2O (50 mL), and extracted
with CH2Cl2 (3ꢃ20 mL). The combined organic extracts were dried
over Na2SO4 and concentrated. The residue was purified by column
chromatography (silica gel, EtOAc/hexane (1:2)) to yield 60,80-
dimethoxy-30-methyl-20H-spiro[[1,3]dioxolane-2,10-naphthalen]-
40(30H)-one as a white solid (58 mg, 55%). Mp 99 ꢂC (dec); 1H NMR
(500 MHz, CDCl3)
d
¼7.11 (d, J¼2.6 Hz, 1H), 6.70 (d, J¼2.6 Hz, 1H),
4.33e4.24 (m, 2H), 4.17e4.13 (m, 1H), 4.11e4.06 (m, 1H), 3.86 (s,
3H), 3.85 (s, 3H), 3.00e2.92 (m, 1H) 2.25 (m, 1H), 2.12 (m, 1H), 1.22
218e220 ꢂC; 1H NMR (300 MHz, CDCl3)
d
¼6.84 (s, 1H), 6.73 (d,
J¼2.3 Hz, 1H), 6.61 (d, J¼2.3 Hz, 1H), 6.37 (br s, 1H), 6.13 (s, 1H), 3.95
(s, 3H), 3.94 (s, 3H), 2.83 (dAB, J¼16.1 Hz, 1H), 2.57 (dAB, J¼16.1 Hz,
(d, J¼6.7 Hz, 3H); 13C NMR (126 MHz, CDCl3)
¼199.6, 161.0, 159.1,
d
135.4,123.0,106.7,105.5,100.9, 65.9, 65.8, 56.1, 55.5, 42.7, 39.9,14.6;
IR (KBr): ṽ¼2987 cmꢁ1 (w), 2968 (m), 2893 (m), 2860 (m), 2839
(w), 1680 (s), 1603 (s), 1470 (m), 1456 (s), 1365 (m), 1323 (s), 1300
(s), 1229 (s), 1207 (s), 1157 (m),1115 (s),1082 (m),1041 (m), 964 (m),
949 (w), 941 (w), 839 (s); HRMS (APCI): calcd for C15H19O5 [MþH]þ:
279.1227, found: 279.1227.
1H), 1.34 (s, 3H); 13C NMR (126 MHz, CDCl3)
d¼192.0, 181.7, 164.8,
162.6, 162.3, 146.5, 141.8, 123.8, 122.3, 114.0, 102.5, 101.1, 56.3, 55.7,
51.0, 42.0, 25.8; IR (KBr): ṽ¼3234 cmꢁ1 (w), 3018 (w), 2972 (w),
2931 (w), 1657 (s), 1606 (s), 1589 (s), 1558 (s), 1477 (m), 1454 (m),
1407 (m), 1377 (w), 1342 (s), 1306 (w), 1265 (s), 1245 (s), 1215 (s),
1207 (s), 1165 (s), 1115 (s), 1026 (w), 999 (m), 957 (w), 895 (w), 850
(w), 839 (w); HRMS (APCI): calcd for C17H17O5 [MþH]þ: 301.1071,
found: 301.1067.
4.8. 60,80-Dimethoxy-30-methyl-30-(3-oxobutyl)-20H-spiro[[1,3]
dioxolane-2,10-naphthalen]-40(30H)-one (2)
4.11. 2,8-Dihydroxy-6-methoxy-10a-methyl-10,10a-dihy-
drophenanthrene-3,9-dione (cDHA)
A stream of nitrogen saturated by MVK was bubbled through
a solution of 60,80-dimethoxy-30-methyl-20H-spiro[[1,3]dioxolane-
2,10-naphthalen]-40(30H)-one (40 mg, 0.14 mmol) and DBU (22
mL,
BBr3 (1 M in CH2Cl2, 0.2 mL, 0.2 mmol) was added dropwise to
a solution of 6 (30 mg, 0.1 mmol) in anhydrous CH2Cl2 (3 mL) at
ꢁ78 ꢂC. The reaction mixture was stirred at ꢁ78 ꢂC for 1 h, then
allowed to warm to 0 ꢂC, and quenched with aqueous 10% NaOH
(1 mL). The mixture was acidified with 1 M HCl to pH¼6 and
extracted with CH2Cl2 (2ꢃ10 mL). The combined organic extracts
were dried over Na2SO4, filtered, and concentrated. The residue was
purified by preparative TLC (CH2Cl2/EtOAc (1:1)) to afford cDHA
(15 mg, 52%) as a pale yellow solid. Mp 199 ꢂC (dec); 1H NMR
0.14 mmol) in toluene (1 mL). Upon completion, indicated by TLC
(silica gel, EtOAc/hexane (1:2)), the solvent was evaporated. The
crude residue was purified by column chromatography (silica gel,
EtOAc/hexane (1:2)) to afford 2 as a colorless wax (29.7 mg, 61%). 1H
NMR (300 MHz, CDCl3)
d
¼7.10 (d, J¼2.5 Hz, 1H), 6.71 (d, J¼2.5 Hz,
1H), 4.33e4.20 (m, 2H), 4.16e4.05 (m, 2H), 3.84 (s, 6H), 2.57e2.44
(m, 1H), 2.39e2.27 (m, 1H), 2.22 (dAB, J¼14.4 Hz, 1H), 2.12 (s, 3H),
2.10 (dAB J¼14.4 Hz, 1H) 2.06e1.85 (m, 2H), 1.20 (s, 3H); 13C NMR
(75 MHz, CDCl3)
d
¼208.1, 201.5, 161.2, 159.1, 133.9, 122.4, 105.8,
(300 MHz, CDCl3)
d
¼12.61 (s,1H), 6.84 (s,1H), 6.72 (d, J¼2.3 Hz,1H),
105.6, 101.3, 65.64, 65.56, 56.1, 55.5, 44.9, 44.5, 38.6, 31.3, 29.8, 23.1;
IR (KBr): ṽ¼2964 cmꢁ1 (w), 2937 (w), 2899 (w), 1713 (s), 1687 (s),
1601 (s), 1464 (m), 1356 (m), 1323 (s), 1296 (s), 1221 (m), 1159 (s),
1115 (m), 1090 (m), 1045 (m), 1011 (w), 948 (w), 848 (w); HRMS
(APCI): calcd for C19H25O6 [MþH]þ: 349.1646, found: 349.1647.
6.54 (d, J¼2.3 Hz,1H), 6.38 (s,1H), 6.13 (s,1H), 3.90 (s, 3H), 2.85 (dAB
,
J¼17.0 Hz, 1H), 2.67 (dAB, J¼17.0 Hz, 1H), 1.35 (s, 3H); 13C NMR
(126 MHz, CDCl3)
d
¼199.1, 181.5, 166.5, 165.5, 160.4, 146.6, 139.6,
123.0, 122.3, 109.1, 105.5, 102.8, 55.9, 48.8, 41.6, 26.3; IR (KBr):
ṽ¼3375 cmꢁ1 (m), 2964 (w), 2926 (w), 1632 (s), 1564 (w), 1479 (w),
1454 (w), 1429 (m), 1389 (m), 1365 (m), 1343 (w), 1292 (s), 1238
(m), 1203 (s), 1159 (s), 1126 (w), 1101 (w), 1067 (w), 978 (w), 903
(w), 879 (w); HRMS (APCI): calcd for C16H15O5 [MþH]þ: 287.0914,
found: 287.0912.
4.9. 60,80-Dimethoxy-10a0-methyl-100,10a0-dihydro-10H-spiro
[[1,3]dioxolane-2,90-phenanthren]-30(20H)-one (1)
t-BuOK (57.9 mg, 0.52 mmol) was added to a solution of 2
(150 mg, 0.43 mmol) in t-BuOH (2 mL), the reaction mixture was
stirred at 25 ꢂC for 3 h H2O (5 mL) was added and the mixture was
extracted with EtOAc (3ꢃ10 mL). The combined organic extracts
were dried over Na2SO4, filtered, and concentrated in a vacuum to
afford 1 as a white solid (110 mg, 77%), which was used in the next
step without further purification. Mp 126 ꢂC (dec); 1H NMR
Crystal data for cDHA: CCDC ref. No. 1012667. Crystallized from
CHCl3.
4.12. 1,3-Dimethoxy-5-(2-nitroprop-1-en-1-yl)benzene
A mixture of 3,5-dimethoxybenzaldehyde (10.0 g, 60.18 mmol),
nitroethane (45 mL, 630.68 mmol) and NH4OAc (3.36 g,
43.57 mmol) was stirred and heated at reflux for 4 h (oil bath
130 ꢂC). The reaction mixture was cooled to 25 ꢂC, diluted with Et2O
(200 mL), and washed with brine (4ꢃ150 mL). The organic layer
was dried over Na2SO4, filtered, and the solvent was evaporated.
1,3-Dimethoxy-5-(2-nitroprop-1-en-1-yl)benzene (13.04 g, 97%)
(300 MHz, CDCl3)
d
¼6.70 (d, J¼2.3 Hz, 1H), 6.60 (d, J¼2.3 Hz, 1H),
6.47 (br s, 1H), 4.37e4.27 (m, 1H), 4.23e4.04 (m, 3H), 3.86 (s, 3H),
3.82 (s, 3H), 2.70e2.54 (m, 1H), 2.48e1.39 (m, 1H), 2.14e1.85 (m,
4H), 1.30 (s, 3H); 13C NMR (75 MHz, CDCl3)
d¼199.6, 162.2, 160.9,
160.0, 135.6, 121.9, 118.7, 106.3, 102.9, 101.3, 65.8, 65.2, 56.0, 55.4,

ꢀꢁ
ꢀ
7580
S. Kovacova et al. / Tetrahedron 71 (2015) 7575e7582
was obtained as a yellow solid. Mp 86e87 ꢂC; 1H NMR (300 MHz,
CDCl3)
1H), 3.81 (s, 6H), 2.43 (d, J¼0.9 Hz, 3H).
170.2, 161.0 (2C), 142.6, 109.8, 105.2 (2C), 98.9, 81.0, 64.6, 64.5, 57.4,
55.3 (2C), 39.5, 33.2, 27.8 (3C), 27.5, 25.4, 23.7; IR (KBr):
ṽ¼2978 cmꢁ1 (w), 2937 (w), 2885 (w), 1726 (s), 1713 (s), 1598 (s),
1458 (m), 1425 (m), 1369 (m), 1352 (m), 1296 (w), 1205 (s), 1157 (s),
1064 (m), 858 (w), 845 (w); HRMS (ESI): calcd for C23H34O7Na
[MþNa]þ: 445.2197, found: 445.2198.
d
¼7.99 (br s, 1H), 6.54 (d, J¼2.2 Hz, 2H), 6.51 (d, J¼2.2 Hz,
4.13. 1-(3,5-Dimethoxyphenyl)propan-2-one (9)
Fe powder (1.60 g, 28.7 mmol) was added to a solution of 1,3-
dimethoxy-5-(2-nitroprop-1-en-1-yl)benzene
(493
mg,
4.17. tert-Butyl 3-acetyl-3-(3,5-dimethoxyphenyl)-6-
2.21 mmol) in AcOH (13.9 mL, 221.0 mmol) and the mixture was
heated at reflux for 3 h. The mixture was cooled to 25 ꢂC, filtered,
diluted with water (70 mL), and extracted with CH2Cl2 (3ꢃ30 mL).
Combined organic extracts were washed with aqueous 5% NaOH
(2ꢃ30 mL), brine (2ꢃ30 mL), dried over Na2SO4, and concentrated.
The crude residue was purified by column chromatography (silica
gel, EtOAc/hexane (2:1)) to provide 9 (303 mg, 71%) as a colorless
oxoheptanoate (8)
TsOH (75 mg, 0.17 mmol) was added to a solution of 12 (721 mg,
1.71 mmol) in acetone (10 mL) and the mixture was stirred at 25 ꢂC
for 8 h. The solvent was evaporated, the residue was dissolved in
Et2O (20 mL) and washed with saturated aqueous solution of
NaHCO3 (10 mL). The aqueous phase was re-extracted with Et2O
(2ꢃ10 mL). The combined organic extracts were washed with H2O
(10 mL), dried over Na2SO4 and concentrated in a vacuum to yield 8
(641 mg, 100%) as a white solid. Mp 94e97 ꢂC; 1H NMR (500 MHz,
oil. 1H NMR (300 MHz, CDCl3)
3.57 (s, 2H), 2.11 (s, 3H).
d
¼6.37e6.31 (m, 3H), 3.74 (s, 6H),
4.14. 2-(2-Iodoethyl)-2-methyl-1,3-dioxolane (11)
CDCl3)
d
¼6.36 (t, J¼2.2 Hz, 1H), 6.30 (d, J¼2.2 Hz, 2H), 3.76 (s, 6H),
2.98 (d, J¼14.3 Hz, 1H), 2.79 (d, J¼14.3 Hz, 1H), 2.48e2.42 (m, 1H),
Iodotrimethylsilane (1.7 mL,12 mmol) was added dropwise at 0 ꢂC
2.29e2.15 (m, 3H), 2.07 (s, 3H), 1.97 (s, 3H), 1.35 (s, 9H); 13C NMR
to a mixture of methyl vinyl ketone (90%, 833
m
L, 10 mmol) and eth-
(126 MHz, CDCl3)
d
¼207.6, 207.4, 170.0, 161.2 (2C), 142.2, 105.0 (2C),
ylene glycol (1.24 mL, 22 mmol). The reaction mixture was stirred for
2 h while allowed to warm to 25 ꢂC, then it was diluted with pentane
(20 mL) and washed successively with 5% aqueous Na2CO3 (10 mL)
and 5% aqueous Na2S2O3 (10 mL). The organic layer was dried over
K2CO3 and concentrated under reduced pressure to provide11 (1.93 g,
80%) as a pale yellow oil, which was used directly in the next step
99.0, 81.4, 57.1, 55.4 (2C), 39.8, 38.5, 29.8, 27.8 (3C), 27.5, 25.6; IR
(KBr): ṽ¼2975 cmꢁ1 (w), 2939 (w), 1720 (s), 1707 (s), 1593 (s), 1466
(m), 1427 (m), 1365 (m), 1352 (s), 1311 (s), 1292 (m), 1211 (s), 1146
(s), 1068 (m), 1051 (m), 924 (w), 840 (w); HRMS (ESI): calcd for
C
21H30O6Na [MþNa]þ: 401.1935, found: 401.1935.
without further purification. 1H NMR (300 MHz, CDCl3)
(m, 4H), 3.21e3.12 (m, 2H), 2.35e2.24 (m, 2H), 1.30 (s, 3H).
d¼4.01e3.87
4.18. tert-Butyl 2-(30,50-dimethoxy-6-methyl-4-oxo-1,2,3,4-
tetrahydro-[1,10-biphenyl]-1-yl)acetate (13a)
4.15. 3-(3,5-Dimethoxyphenyl)-5-(2-methyl-1,3-dioxolan-2-
yl)pentan-2-one (10)
AcOH (0.12 mL, 2.13 mmol) and piperidine (0.19 mL, 1.93 mmol)
were added to a solution of 8 (732 mg, 1.93 mmol) in toluene
(10 mL) and the mixture was heated at reflux (oil bath 110 ꢂC) for
18 h. The solvent was evaporated in a vacuum, the residue was
dissolved in EtOAc (150 mL) and washed with H2O (100 mL) and
then with brine (100 mL). The organic layer was dried over Na2SO4,
concentrated, and the residue was purified by column chroma-
tography (silica gel, EtOAc/hexane (1:2)) to afford 13a as a white
t-BuOK (677 mg, 6.04 mmol) was added to a solution of 9 (1.02 g,
5.24 mmol) in anhydrous THF (12 mL) and the reaction mixture was
stirredat 25 ꢂC for 1 h.11 (1.7 g, 7.02 mmol) was added dropwise over
the period of 5 min and the reaction mixture was stirred at 25 ꢂC for
16 h. Saturated aqueous solution of NaHCO3 (1 mL) was added, the
solvents were evaporated and the residue was directly loaded onto
a column and purified by column chromatography (silica gel, EtOAc/
hexane (1:2)). 10 (1.37 g, 85%) was obtained as a pale yellow oil. 1H
semi-solid (563 mg, 81%). 1H NMR (300 MHz, CDCl3)
d¼6.40 (d,
J¼2.1 Hz, 2H), 6.36 (t, J¼2.1 Hz, 1H), 6.16 (br s, 1H), 3.76 (s, 6H), 2.91
(s, 2H), 2.72e2.60 (m, 1H), 2.32e2.01 (m, 3H), 1.98 (d, J¼1.2 Hz, 3H),
NMR (300 MHz, CDCl3)
d
¼6.35 (s, 3H), 3.94e3.84 (m, 4H), 3.77 (s,
1.43 (s, 9H); 13C NMR (75 MHz, CDCl3)
d
¼198.7, 169.8, 163.5, 161.0
6H), 3.54 (t, J¼7.4 Hz, 1H), 2.16e2.02 (m, 1H), 2.05 (s, 3H), 1.86e1.71
(2C), 144.7, 130.4, 105.7 (2C), 96.1, 81.4, 55.3 (2C), 46.8, 43.8, 35.7,
34.2, 28.0 (3C), 21.7; IR (KBr): ṽ¼2976 cmꢁ1 (w), 2937 (w), 1724 (s),
1672 (s), 1601 (s), 1456 (s), 1425 (m), 1369 (m), 1348 (m), 1310 (m),
1207 (s), 1159 (s), 1068 (w), 1043 (w), 841 (w); HRMS (ESI): calcd for
(m, 1H), 1.64e1.43 (m, 2H), 1.29 (s, 3H); 13C NMR (CDCl3, 75 MHz)
d
¼207.9, 161.2 (2C), 141.2, 109.9, 106.4 (2C), 99.2, 64.61, 64.58, 59.7,
55.3 (2C), 36.6, 28.8, 26.0, 23.8; IR (KBr): ṽ¼2958 cmꢁ1 (w), 2941 (w),
1712 (s), 1670 (w), 1605 (s), 1595 (s), 1460 (m), 1431 (m), 1352 (w),
1205 (s), 1157 (s), 1065 (s), 858 (w), 837 (w); HRMS (ESI): calcd for
C
21H28O5Na [MþNa]þ: 383.1829, found: 383.1829.
C
17H24O5Na [MþNa]þ: 331.1516, found: 331.1517.
4.19. tert-Butyl 2-(30,50-dimethoxy-4-methyl-6-oxo-1,2,3,6-
tetrahydro-[1,10-biphenyl]-1-yl)acetate (13b)
4.16. tert-Butyl 3-acetyl-3-(3,5-dimethoxyphenyl)-5-(2-
methyl-1,3-dioxolan-2-yl)pentanoate (12)
t-BuOK (14.6 mg, 0.13 mmol) was added to a solution of 8 (50 mg,
0.13 mmol) in THF (1 mL) at 0 ꢂC. The reaction mixture was stirred at
0 ꢂC for 1 h, then saturated aqueous solution of NaHCO3 (1 mL) was
added. The solvent was evaporated and the residue was loaded di-
rectly onto a column and purified by column chromatography (silica
gel, EtOAc/hexane (1:2)) to give 13b as a colorless oil (25.2 mg, 54%).
t-BuOK (580 mg, 5.17 mmol) was added to a solution of 10
(1.39 g, 4.50 mmol) in THF (16 mL) at 0 ꢂC and the resulting yellow
mixture was stirred at 0 ꢂC for 1 h. Then, t-butyl-2-iodoacetate
(2.0 g, 5.85 mmol) was added, the mixture was allowed to warm
to 25 ꢂC and stirred for 20 h. Reaction was quenched by saturated
solution of NaHCO3 (1 mL). The solvents were evaporated and the
residue was purified by column chromatography (silica gel, EtOAc/
hexane (1:4 to 1:1)) to yield 12 (924 mg, 62%) as a colorless oil. 1H
1H NMR (500 MHz, CDCl3)
d¼6.40 (d, J¼2.2 Hz, 2H), 6.34 (t, J¼2.1 Hz,
1H), 5.95 (br s,1H), 3.75 (s, 6H), 2.93 (dAB, J¼16.2,1H), 2.73e2.66 (m,
1H), 2.56 (dAB, J¼16.2, 1H), 2.51e2.45 (m, 1H), 2.34e2.24 (m, 1H),
2.17e2.10 (m, 1H), 1.84 (s, 3H), 1.36 (s, 9H); 13C NMR (126 MHz,
NMR (300 MHz, CDCl3)
d
¼6.36e6.33 (m, 3H), 3.93e3.80 (m, 2H),
CDCl3)
d
¼199.2, 170.6, 161.4, 160.8 (2C), 141.8,126.4,105.3 (2C), 96.7,
3.76 (s, 6H), 2.96 (dAB, J¼14.7 Hz, 1H), 2.81 (dAB, J¼14.7 Hz, 1H),
80.4, 55.3 (2C), 50.9, 45.1, 31.3, 28.7 (3C), 28.2, 24.1; IR (KBr):
ṽ¼2974 cmꢁ1 (w), 2931 (w), 1730 (s), 1668 (s), 1637 (w), 1595 (s),
1458 (s), 1425 (m), 1367 (m), 1348 (m), 1308 (m), 1294 (w), 1205 (s),
2.40e2.27 (m, 1H), 2.07e1.96 (m, 3H), 1.98 (s, 3H), 1.45e1.37 (m,
2H), 1.35 (s, 9H), 1.30 (s, 3H); 13C NMR (75 MHz, CDCl3)
d
¼207.8,

ꢀꢁ
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S. Kovacova et al. / Tetrahedron 71 (2015) 7575e7582
7581
1159 (s), 1080 (w), 1063 (w), 1049 (w), 843 (w); HRMS (APCI): calcd
(5 mL) was added to NaH (60% dispersion in mineral oil, 65 mg,
1.63 mmol) and the mixture was stirred at 25 ꢂC for 45 min. Pivaloyl
chloride (189 mL,1.52 mmol) was added and the mixture was stirred
for additional 1 h. Water (50 mL) was added and the mixture was
extracted with EtOAc (3ꢃ50 mL). The combined organic extracts
were dried over Na2SO4, filtered, and concentrated. The residue was
purified by column chromatography (silica gel, EtOAc/hexane (1:1))
to afford 17 (352 mg, 92%) as a white solid. Mp 136e138 ꢂC; 1H NMR
for C21H29O5 [MþH]þ: 361.2010, found: 361.2004.
4.20. 40,60-Dimethoxy-2-methylspiro[cyclohex[2]ene-1,10-in-
dene]-30,4(20H)-dione (7)
A solution of 13a (415 mg, 1.15 mmol) in MsOH (4.2 mL) was
heated at 50 ꢂC for 15 h. The mixture was cooled to 25 ꢂC, poured
into cold saturated solution of NaHCO3 (30 mL), and extracted with
EtOAc (5ꢃ30 mL). The combined organic extracts were dried over
Na2SO4, evaporated, and the residue was purified by gradient col-
umn chromatography (silica gel, EtOAc/hexane (1:2 to pure EtOAc))
to afford 7 (275 mg, 84%) as a pale brown solid. Mp 159e164 ꢂC; 1H
(300 MHz, CDCl3)
d
¼6.63 (br s, 1H), 6.59 (br s, 1H), 6.00 (br s, 1H),
3.86 (s, 3H), 2.81 (dAB, J¼18.7 Hz, 1H), 2.64 (dAB, J¼18.9 Hz, 1H),
2.57e2.30 (m, 3H), 2.11e2.01 (m, 1H), 1.64 (br s, 3H); 1.40 (s, 9H);
13C NMR (75 MHz, CDCl3)
d
¼198.2, 197.6, 176.0, 166.5, 162.14,
162.09, 149.5, 128.5, 121.9, 109.1, 106.6, 55.1, 47.4, 46.8, 39.1, 37.1,
34.9, 27.1 (3C), 20.4; IR (KBr): ṽ¼2978 cmꢁ1 (w), 2956 (w), 2931 (w),
1759 (s), 1711 (s), 1670 (s), 1591 (m), 1481 (m), 1438 (m), 1340 (w),
1332 (w), 1310 (s), 1261 (m), 1244 (m), 1142 (s), 1109 (s), 1084 (s),
1036 (m), 1009 (m), 897 (w), 856 (w); HRMS (APCI): calcd for
NMR (300 MHz, CDCl3)
d
¼6.37 (d, J¼1.9 Hz, 2H), 6.32 (d, J¼1.9 Hz,
1H), 6.00 (br s, 1H), 3.93 (s, 3H), 2.86 (s, 3H), 2.82 (dAB, J¼18.5, 1H),
2.65 (dAB, J¼18.5, 1H), 2.56e2.48 (m, 2H), 2.46e2.29 (m, 1H),
2.06e1.97 (m, 1H), 1.64 (d, J¼1.2 Hz, 3H); 13C NMR (75 MHz, CDCl3)
d
¼199.2, 197.9, 167.5, 163.8, 162.5, 159.5, 128.4, 118.8, 100.4, 98.3,
C
21H25O5 [MþH]þ: 357.1697, found: 357.1695.
55.91, 55.89, 47.4, 46.7, 37.2, 34.9, 20.4; IR (KBr): ṽ¼2953 cmꢁ1 (w),
2927 (w), 1697 (s), 1664 (s), 1576 (s), 1460 (m), 1439 (w), 1427 (w),
1350 (w), 1315 (s), 1234 (s), 1203 (s), 1159 (s), 1107 (w), 1053 (m),
1026 (w), 860 (m); HRMS (APCI): calcd for C17H19O4 [MþH]þ:
287.1278, found: 287.1279.
4.23. 4-((tert-Butyldimethylsilyl)oxy)-60-methoxy-2-methyl-
30-oxo-20,30-dihydrospiro-[cyclohexa[2,4]diene-1,10-inden]-40-
yl pivalate (18)
TBSOTf (143
mL, 0.61 mmol) was added to a solution of 17
4.21. 40-Hydroxy-60-methoxy-2-methylspiro[cyclohex[2]ene-
(135 mg, 0.38 mmol) in CH2Cl2 (1 mL) at ꢁ78 ꢂC, then DBU (92
mL,
1,10-indene]-30,4(20H)-dione
0.61 mmol) was added dropwise and the reaction mixture was
stirred at ꢁ78 ꢂC for 30 min, filtered through a pad of silica gel (1 cm
height, 2 cm diameter), and the pad was quickly washed with EtOAc
(100 mL). After evaporation of the solvents from the filtrate, 18
(152 mg, 85%) was obtained as a colorless semi-solid, which was
used in the next step without any further purification. Analytically
pure sample was obtained by preparative TLC (silica gel, EtOAc/
hexane (1:2)); 1H NMR (300 MHz, CDCl3)¼6.84 (d, J¼2.0 Hz, 1H),
6.54 (d, J¼2.0 Hz,1H), 5.67 (br s,1H), 4.85e4.79 (m,1H), 3.88 (s, 3H),
2.81 (dAB, J¼18.3 Hz, 1H), 2.74 (dd, J¼16.9 Hz, J¼2.6 Hz, 1H), 2.45
(dAB, J¼18.3 Hz, 1H), 2.26 (dd, J¼16.9 Hz, J¼5.8 Hz, 1H), 1.53 (s, 3H),
1.41 (s, 9H), 0.95 (s, 9H), 0.16 (s, 6H); 13C NMR (126 MHz, CDCl3)
BCl3 (1 M solution in CH2Cl2, 0.96 mL, 0.96 mmol) was added
dropwise to a solution of 7 (275 mg, 0.96 mmol) in CH2Cl2 (10 mL) at
0
ꢂC and the mixture was stirred at 0 ꢂC. Additional five 0.96 mL
portions of 1 M BCl3 in CH2Cl2 (total volume of 4.8 mL, 4.8 mmol)
were added in one hour intervals and then the mixture was stirred
for 16 h while allowed to warm to 25 ꢂC. Then the reaction mixture
was cooled to 0 ꢂC, additional four 2.88 mL portions of 1 M BCl3 in
CH2Cl2 (total volume of 11.5 mL, 11.5 mmol) were added in three
hour intervals and the mixture was stirred for additional 16 h while
allowed to warm to 25 ꢂC. Then it was quenched with cold saturated
aqueous solution of NaHCO3 (50 mL), and extracted with CH2Cl2
(3ꢃ50 mL). The combined organic extracts were dried over Na2SO4,
filtered, and concentrated. The residue was purified by column
chromatography (silica gel, EtOAc/hexane (2:1)) to afford 40-hy-
droxy-60-methoxy-2-methylspiro[cyclohex[2]ene-1,10-indene]-
30,4(20H)-dione (170 mg, 65%) as a white solid. Mp 128e129 ꢂC; 1H
d
¼200.3, 176.2, 166.1, 163.9, 149.1, 148.9, 141.0, 124.2, 122.0, 108.6,
107.3, 99.0, 56.0 (2C), 48.4, 46.4, 39.1, 38.6, 27.2 (3C), 25.7 (3C), 19.5,
18.1, ꢁ4.4; IR (KBr): ṽ¼2958 cmꢁ1 (m), 2931 (m), 2856 (w), 1761 (s),
1711 (s), 1674 (s), 1610 (s), 1589 (s), 1479 (m), 1462 (m), 1461 (m),
1441 (m), 1396 (w), 1340 (m), 1310 (s), 1273 (m), 1244 (m), 1194 (w),
1144 (s), 1109 (s),1186 (s), 1036 (m), 862 (w), 839 (w); HRMS (APCI):
calcd for C27H39O5Si [MþH]-: 471.2561, found: 471.2560.
NMR (300 MHz, CDCl3)
d
¼9.07 (s, 1H), 6.34 (d, J¼1.9 Hz 1H), 6.31 (d,
J¼1.9 Hz, 1H), 5.98 (br s, 1H), 3.82 (s, 3H), 2.85 (dAB, J¼18.9 Hz, 1H),
2.69 (dAB, J¼18.9 Hz, 1H), 2.61e2.46 (m, 2H), 2.44e2.28 (m, 1H),
2.10e2.00 (m, 1H), 1.64 (d, J¼1.2 Hz, 3H); 13C NMR (75 MHz, CDCl3)
4.24. 5-Hydroxy-60-methoxy-2-methyl-30,4-dioxo-20,30-dihy-
drospiro[cyclohex[2]ene-1,10-inden]-40-yl pivalate (19)
d
¼204.0, 197.7, 168.4, 162.0, 160.5, 159.1, 128.3, 116.2, 102.8, 100.1,
55.9, 47.6, 46.9, 36.7, 34.8, 20.3; IR (KBr): ṽ¼3433 cmꢁ1 (w), 2976 (w),
2928 (w), 2918 (w), 1672 (s), 1659 (s), 1624 (s), 1597 (s), 1491 (m),
1443 (m), 1367 (s), 1325 (m), 1254 (m), 1205 (m), 1151 (s), 1022 (w),
989 (w), 962 (w), 864 (w), 849 (w); HRMS (APCI): calcd for C16H17O4
[MþH]þ: 273.1121, found: 273.1125.
mCPBA (77%, 110 mg, 0.49 mmol) was added to a mixture of 18
(155 mg, 0.33 mmol) and KHCO3 (77 mg, 0.77 mmol) in CH2Cl2,
(5 mL) at 0 ꢂC and the reaction mixture was stirred at 25 ꢂC for 16 h.
EtOAc (50 mL) was added and the mixture was washed with sat-
urated aqueous solution of NaHCO3 (20 mL), then with H2O (30 mL),
dried over Na2SO4, filtered, and concentrated in a vacuum. The
residue was dissolved in CH2Cl2 (10 mL), pyr HF (1 mL) was added,
and the mixture was stirred at 25 ꢂC for 16 h. The mixture was
diluted with H2O (10 mL) and extracted with EtOAc (3ꢃ30 mL). The
combined organic extracts were dried over Na2SO4, filtered, and
concentrated under reduced pressure. The residue was purified by
preparative TLC (silica gel, EtOAc/hexane (2:1) to afford 19 (45 mg,
4.22. 60-Methoxy-2-methyl-30,4-dioxo-20,30-dihydrospiro[cy-
clohex[2]ene-1,10-inden]-40-yl pivalate (17)
37%) as a colorless semi solid. 1H NMR (500 MHz, CDCl3)
d
¼6.71 (d,
J¼2.0 Hz, 1H), 6.63 (d, J¼2.0 Hz, 1H), 6.16e6.15 (m, 1H), 4.57 (dd,
J¼12.9 Hz, J¼5.8 Hz, 1H), 3.87 (s, 3H), 3.08 (dAB, J¼18.7 Hz, 1H), 2.56
(dAB, J¼18.7 Hz, 1H), 2.50 (dd, J¼13.2 Hz, J¼5.8 Hz, 1H), 2.27 (dd,
J¼13.2 Hz, J¼12.9 Hz, 1H), 1.78 (d, J¼1.2 Hz, 3H), 1.43 (s, 9H); 13C
A solution of 40-hydroxy-60-methoxy-2-methylspiro[cyclohex
[2]ene-1,10-indene]-30,4(20H)-dione (295 mg, 1.08 mmol) in dry THF
NMR (126 MHz, CDCl3)
d
¼198.4, 198.1, 176.1, 166.3, 163.5, 159.0,

ꢀꢁ
ꢀ
7582
S. Kovacova et al. / Tetrahedron 71 (2015) 7575e7582
150.1, 125.9, 121.3, 109.0, 107.6, 69.0, 56.2, 51.1, 48.2, 44.1, 39.2, 27.2
(3C), 20.1; IR (KBr): ṽ¼2976 cmꢁ1 (w), 2933 (w), 1759 (s), 1711 (s),
1684 (s), 1608 (s), 1587 (s), 1479 (w), 1440 (w), 1340 (m), 1308 (m),
1244 (m), 1142 (s), 1109 (s), 1053 (w), 1032 (w), 899 (w), 867 (w);
curve and examining the effect of different concentrations of in-
hibitor on reversing enzyme activity (functional antagonist assay).
Acknowledgements
HRMS (APCI): calcd for
373.1641.
C
21H25O6 [MþH]þ: 373.1646, found:
The project was funded from the SoMoPro Programme
(SRGA771). Research leading to these results has received a finan-
cial contribution from the European Community within the Sev-
enth Framework Programme (FP/2007e2013) under Grant
Agreement No. 229603. The research was also co-financed by the
South Moravian Region. Authors would like to thank Dr. Jakub
4.25. 40,5-Dihydroxy-60-methoxy-2-methylspiro[cyclohexa
[2,5]diene-1,10-indene]-30,4(20H)-dione (cDHAs)
K2CO3 (44.5 mg, 0.322 mmol) was added to a solution of 19
(38 mg, 0.107 mmol) in MeOH (4 mL), and the mixture was stirred
in an open flask at 50 ꢂC for 6 h. MeOH was then evaporated and the
residue was dissolved in H2O (10 mL) and the pH was adjusted to 7
with 1 M HCl. The mixture was extracted with EtOAc (4ꢃ10 mL), the
combined organic extracts were dried over Na2SO4, filtered, and the
solvent was evaporated. The residue was purified by preparative
TLC (silica gel, CH2Cl2/EtOAc (2:1)) to afford cDHAs (14.5 mg, 50%)
ꢁ
Svenda for helpful comments and review of the manuscript and Dr.
ꢁ
Marek Necas for help with X-ray crystallography.
Supplementary data
Supplementary data (1H, 13C NMR, IR and HRMS spectra plus
conditions for separation of the enantiomers of cDHA are available.)
associated with this article can be found in the online version, at
http://dx.doi.org/10.1016/j.tet.2015.08.005.
as a white solid. Mp 177 ꢂC (dec); 1H NMR (300 MHz, CDCl3)
d¼9.02
(br s, 1H), 6.42e6.33 (m, 3H), 6.14 (d, J¼1.7 Hz, 1H), 6.03, (s, 1H),
3.83 (s, 3H), 2.92 (s, 2H), 1.75 (s, 3H); 13C NMR (126 MHz, CDCl3)
d
¼203.4, 181.6, 168.8, 162.8, 159.3, 156.8, 146.0, 125.5, 120.0, 116.0,
References and notes
102.6, 101.0, 56.1, 50.8, 47.3, 19.5; IR (KBr): ṽ¼3387 cmꢁ1 (w), 2955
(w), 2922 (m), 2850 (w), 1680 (s), 1639 (s), 1628 (s), 1433 (w), 1420
(w), 1377 (m), 1311 (m), 1205 (s), 1176 (m), 1148 (s), 1105 (w), 1022
(w), 887 (w); HRMS (APCI): calcd for C16H15O5 [MþH]þ: 287.0914,
found: 287.0912.
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The four mammalian pols a, g, k, and l were prepared, and the
reaction mixtures for these pols, as described previously.23 Briefly,
poly(dA)/oligo(dT)18 (A/T, 2:1) and [3H]-dTTP (100 cpm/pmol) were
used as the DNA template-primer substrate and nucleotide (dNTP;
20-deoxynucleotide-50-triphosphate) substrate, respectively. The
tested compounds were dissolved in distilled dimethyl sulfoxide
(DMSO) at concentrations 0e200
mM. Subsequently, 4-mL aliquots
were mixed with 16 L of each enzyme (0.05 units) in 50 mM
m
TriseHCl (at pH 7.5) containing 1 mM dithiothreitol, 50% glycerol
(v/v), and 0.1 mM ethylenediaminetetraacetic acid, and were held
at 0 ꢂC for 10 min. Subsequently, 8
m
L of these inhibitor-enzyme
L aliquots of enzyme standard re-
action mixture containing 50 mM TriseHCl (at pH 7.5), 1 mM
dithiothreitol, 1 mM MgCl2, 15% glycerol, 5 M poly(dA)/oligo(dT)18
(A/T, 2:1) and 10
M [3H]-dTTP, and were incubated at 37 ꢂC for
mixtures were added to 16-
m
m
m
60 min. The enzyme activity in the absence of inhibitor was taken
as 100% (the activity without the enzyme was considered 0%), and
the inhibitory activity was determined for each inhibitor concen-
tration. One unit of pol activity was defined as the amount of each
enzyme that catalyzed incorporation of 1 nmol dTTP into synthetic
DNA template-primers in 60 min at 37 ꢂC, under standard reaction
conditions.24 The 50% inhibitory concentration (IC50 value) of the
enzyme inhibitor was determined by constructing a doseeresponse
23. (a) Mizushina, Y.; Tanaka, N.; Yagi, H.; Kurosawa, T.; Onoue, M.; Seto, H.; Horie,
T.; Ayoagi, N.; Yamaoka, M.; Matsukage, A.; Yoshida, S.; Sakaguchi, K. Biochim.
Biophys. Acta 1996, 1308, 256e262; (b) Mizushina, Y.; Yoshida, S.; Matsukage,
A.; Sakaguchi, K. Biochim. Biophys. Acta 1997, 1336, 509e521.