Asymmetric syntheses of piperidino-benzodiazepines through 'cation-pool' host/guest supramolecular approach and their DNA-binding studies

Article abstract of DOI:10.1016/j.tetasy.2010.10.030

The asymmetric synthetic approach to piperidino-benzodiazepine 4a (a homolog of DC-81) has been developed. The absolute stereochemistry of 4 and 5 has been assigned to be (S) at C-12a position. This procedure features the use of a 'cation-pool' strategy and also a host/guest supramolecular co-catalysis approach. In this study, the chloroformate of 8-phenylmenthyl has been employed as a chiral auxiliary and includes one-pot conditions for anodic oxidation, which are followed by nucleophilic addition to an N-acyliminium ion. In addition, intramolecular azido reductive-cyclization and nitro reductive dithioacetal deprotective tandem-cyclization approaches have also been utilized for the syntheses of these compounds 4a,b and 5a,b. Some of the representative compounds exhibited an enhanced DNA-binding ability in comparison to the natural product DC-81.

Full text of DOI:10.1016/j.tetasy.2010.10.030

Tetrahedron: Asymmetry 21 (2010) 2625–2630
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Asymmetric syntheses of piperidino-benzodiazepines through ‘cation-pool’
host/guest supramolecular approach and their DNA-binding studies
Nagula Markandeya b, Nagula Shankaraiah c, Ch. Sanjeeva Reddy b, , Leonardo Silva Santos d,
⇑
Ahmed Kamal a,
⇑
a Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 607, India
b Department of Chemistry, Kakatiya University, Warangal 506 009, India
c National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
d Laboratory of Asymmetric Synthesis, Chemistry Institute of Natural Resources, University of Talca, Talca, PO Box 747, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
The asymmetric synthetic approach to piperidino-benzodiazepine 4a (a homolog of DC-81) has been
developed. The absolute stereochemistry of 4 and 5 has been assigned to be (S) at C-12a position. This
procedure features the use of a ‘cation-pool’ strategy and also a host/guest supramolecular co-catalysis
approach. In this study, the chloroformate of 8-phenylmenthyl has been employed as a chiral auxiliary
and includes one-pot conditions for anodic oxidation, which are followed by nucleophilic addition to
an N-acyliminium ion. In addition, intramolecular azido reductive-cyclization and nitro reductive dithio-
acetal deprotective tandem-cyclization approaches have also been utilized for the syntheses of these
compounds 4a,b and 5a,b. Some of the representative compounds exhibited an enhanced DNA-binding
ability in comparison to the natural product DC-81.
Received 10 August 2010
Accepted 27 October 2010
Available online 20 November 2010
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Streptomycin species,1,2 which include anthramycin 1, tomaymy-
cin 2, and DC-81 3 (Fig. 1). A stereogenic center at the C11a position
A growing interest in the development of new synthetic routes
for the construction of chiral N-heterocycles continues to be essen-
tial for accessing natural and unnatural products, particularly in a
stereoselective manner. Nowadays, chirality in organic molecules
plays an enormous role in areas ranging from medicine to material
science, yet the synthesis of such entities in one enantiomeric form
is considered as one of the most difficult challenges that need to be
addressed by the synthetic chemists. However, inspite of many
developments in synthetic organic chemistry, still there is scope
for the development of newer methods that could provide impor-
tant insights in employing chiral auxiliaries for the stereoselective
construction of predetermined moieties in certain classes of com-
pounds. As part of our efforts in the field of biologically relevant
N-moieties, we became interested in the development of a new
asymmetric synthetic route for the preparation of imine-contain-
ing piperidino-benzodiazepines and their dilactams.
and a DNA-reactive imine moiety at the N10–C11 position, are char-
acteristics of these tricyclic PBD’s that have been extensively inves-
tigated and rationalized to obtain a snug fit into the minor groove of
duplex DNA. The interaction of PBD with DNA is due to a covalent
aminalbondwiththeN2–aminogroupoftheguaninebase3 thatpro-
vides a preference for Pu–G–Pu sequences.1 Interestingly, these
compounds have also been shown to inhibit both endonuclease4
and RNA polymerase5 enzymes in a sequence-dependent manner.
A large number of PBDs have been designed and synthesized with
a view to understand their structure–activity relationship6–8 apart
from the development of a variety of synthetic approaches9,10 both
in solution-phase and on the solid-phase.11,12
Based on this analogy we have replaced the pyrrole (five-mem-
bered ring) with piperidine (six-membered ring) to evaluate their
potential for DNA-binding interactions with duplex DNA by modi-
fying the stereogenic position from C11a to C12a. Herein, we have
developed a new asymmetric synthetic route for the piperidino-
benzodiazepines (PiBDs) through a b-CD-host/guest supramolecu-
lar approach using 8-phenylmenthyl chloroformate as a chiral aux-
iliary for the construction of the (S)-pipecolic moiety. Takaya
et al.13 have reported the synthesis of racemic ( )-piperidino-ben-
There has been increasing interest in the synthesis of DNA se-
quence selective binding agents, particularly by low molecular
weight antitumor antibiotics. Among them, the pyrrolo[2,1-
c][1,4]benzodiazepines (PBDs) are a well known class of DNA-inter-
active potent antitumor agents derived from Streptomycin species.
zodiazepine dilactam 5a from D,L-pipecolic acid. However, only a
few methods have been reported for the synthesis of optically ac-
tive pipecolic acid derivatives, mainly involving enzymatic
routes,14 alkylation of chiral glycine enolates,15 from natural amino
⇑
Corresponding authors. Tel.: +91 40 27193157; fax: +91 40 27193189 (A.K.).
E-mail addresses: chsrkuc@yahoo.co.in (C.S. Reddy), ahmedkamal@iict.res.in (A.
Kamal).
0957-4166/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2010.10.030

2626
N. Markandeya et al. / Tetrahedron: Asymmetry 21 (2010) 2625–2630
O
O
O
NH2
N
H3CO
HO
N
H
OCH3
H3CO
N
H
H
N
OH
1
3
2
O
O
O
6
4
3
4
5
N
12a
7
6
7
H3CO
RO
5
2
N
H3CO
HO
H3CO
8
9
N
2
C
11a
A
B
1
1
H
O
N
8
H
H
RO
N
N
11
9
10
12
H
10
11
R = H; 4a
R = Bn; 4b
R = H; 5a
3
R = Bn; 5b
Figure 1. Representative chemical structures of anthramycin 1, tomaymycin 2, DC-81 3, a homolog of DC-81 4a, and its derivatives 4b and 5a,b.
acids16, and other enantioselective synthetic processes.17 Recently,
we developed a synthetic strategy that was based on the diastereo-
selective addition of NCꢀ to an N-acyliminium ion bearing a chiral
auxiliary (8-phenylmenthyl) for the introduction of asymmetry
into pipecolic acid.18 In this context, we became interested in
applying this protocol to the synthesis of optically active PiBDs
and to evaluate their DNA-binding ability.
we determined the synthesis of dithioacetal protected key inter-
mediate 11 from 9 as depicted in Scheme 2.
The selective reduction of ester 9 to the corresponding aldehyde
employing DIBAL-H in CH2Cl2 at ꢀ78 °C for 45 min did not proceed
well. Therefore, the ester group was reduced completely to the
alcohol (80%), and the aldehyde was obtained in very low yield
(10%). Moreover, the remaining starting material 9 was recovered
(ꢂ10%) and the reaction was monitored by GC analysis. We
decided to employ the Dess–Martin periodinate for the oxidation
of the alcohol to an aldehyde. It was observed that the ester reduc-
tion followed by periodinate oxidation afforded the aldehyde in
excellent yield (92%). The aldehyde (without purification) was
immediately protected with EtSH/TMSCl in CH2Cl2 at room tem-
perature for 6 h to give 11 in 95% yields. Finally, the chiral interme-
diates 10/12 were obtained by the removal of the chiral auxiliary
using HCl/CHCl3 (6.0 M) at reflux for 48 h. These intermediates
are directly utilized for the next step without any further purifica-
tion. It should be noted that when CHCl3 was used as the solvent
with no excess of water, ester 10 was isolated as its hydrochloride
salt accompanied by the chiral auxiliary (95%). When the reaction
was performed in H2O, pipecolic acid was obtained in 99% yield.
The enantiomeric excess of compounds 10 and 12 were deter-
mined from the phenylamide derivatives of 10 and 12, which were
subjected to HPLC analysis by employing a chiral Welch-01 column
(n-hexane/isopropanol = 9:1, 1.0 mL/min, 254 nm UV detector), the
enantiomeric excess for 10 and 12 were determined as >95%.
The substituted azido benzoic acids 13a,b were coupled with
methyl (2S)-piperidinoate 10 using EDCI/HOBt in CH2Cl2 by stirring
them overnight to provide 15a,b in good yields (75% and 86%,
respectively). Another protocol was also employed for the coupling
of nitro acids with (2S)-piperidino-2-carboxaldehyde diethyl
dithioacetal 12. The nitro acids were initially treated with SOCl2
in dry benzene with a catalytic amount of DMF (added for solubil-
ity). Then, after evaporation it was added dropwise to a stirred
solution of 12 employing Et3N as a base taken in anhydrous THF
2. Results and discussion
The synthetic strategy for the preparation of compounds 4a,b
and 5a,b is outlined in Scheme 1. The key intermediates 10/12
were synthesized from L-pipecolic acid, which was obtained from
the N-carbamate of 8-phenylmenthyl piperidine 6 by anodic oxida-
tion using a ‘cation-pool’ technique. Next, we attempted to con-
struct the tricyclic framework based on two approaches: (i) a
nitro reduction followed by diethanethiol deprotective-cycliza-
tion19 for the synthesis of PiBD imines 4a,b; and (ii) azido reductive
tandem-cyclization20 for PiBD dilactams 5a,b. We also attempted
the reduction of azido ester 15a to the corresponding aldehyde
with DIBAL-H, however, this reaction produced a complicated mix-
ture even at very low temperatures (ꢀ78 °C).
According to our previous model experiments,18 the best condi-
tions were obtained by nucleophilic addition of TMSCN in the pres-
ence of b-CD to N-acyliminium ion 6 by using TMSOTf as a Lewis
acid. Furthermore, the use of the chloroformate of 8-phenylmen-
thyl as a chiral auxiliary affords the corresponding a-methoxycar-
bamate, which reacts in situ with TMSCN catalyzed by TMSOTf in
CH2Cl2 at ꢀ40 °C. The protocol provided 8 in 65% yields and 91%
de. The diastereomeric excess was determined by chiral HPLC anal-
ysis using a ChiralPack OD column and the enantiomeric excess
was determined after hydrolysis of 8 as (S)-(ꢀ)-pipecolic acid,
½
a 3D1
ꢁ
¼ ꢀ26 (c 1.0, H2O), mp: 271–272 °C (lit. mp: 272 °C). Com-
pound 8 was hydrolyzed with 1 M HCl and taken up for further
esterification with SOCl2 in MeOH to give 9 in 80% yield. Next,
O
H3CO
(S)-4a,b
(S)-5a,b
N
N
Y
N
OR*
O
OR*
RO
Y
X
O
X = N3/NO2
R = H, Bn
6
14a,b 15a,b
/
Y = CO2CH3/CH(SEt)2
9/11
Scheme 1. Retrosynthetic analysis for piperidino-benzodiazepines (4a,b and 5a,b).

N. Markandeya et al. / Tetrahedron: Asymmetry 21 (2010) 2625–2630
2627
ref 18
NC−
+
N
N
N
CN
OR*
O
OR*
O
OR*
O
*R = 8-phenylmenthyl, 6
8
7
i, ii
iv, v, vi
iii
N
CH(SEt)2
OR*
N
CO2CH3
OR*
CH(SEt)2
N
H
O
O
11
9
iii
N
H
CO2CH3
10
Scheme 2. Reagents and conditions: (i) 1 M HCl, rt, 5 h; (ii) SOCl2, MeOH, rt, 12 h, 80% (two steps); (iii) 6 M HCl/CHCl3, 48 h, reflux; (iv) DIBAL-H, CH2Cl2, ꢀ78 °C, 45 min, 80%;
(v) Dess–Martin periodinate, rt, 30 min, 92%; (vi) TMSCl/EtSH, CH2Cl2, rt, 6 h, 95%.
at 0 °C in about 30 min. The reaction mixture was then brought to
ambient temperature and stirred for another 2 h to give 14a,b in
88% yield. The reduction of nitro dithioacetal intermediates 14a,b
with SnCl2ꢃ2H2O followed by deprotective-cyclization using
HgCl2/CaCO3 in CH3CN/H2O (4:1) affords the piperidino-benzodi-
azepines 4a,b. Compound 4a was obtained in 68% overall yield
from these two steps, and no racemization was observed in the
tained throughout the synthesis. We also carried out an intramo-
lecular azido reductive tandem-cyclization process employing
Al(OTf)3/NaI for the reduction of azides 15a,b. It was observed that
the substituted 2-azidobenzoyl piperidine esters 15a,b reduced
selectively with Al(OTf)3 (20 mol %) and NaI (3 equiv) by using
CH3CN as a solvent to provide the desired dilactams 5a,b in good
yields (85–88%) as shown in Scheme 3.
process, ½a 3D1
¼ þ256 (c 1.0, CHCl3), mp: 119–121 °C; (natural
ꢁ
product DC-81) [a]D = +135 (c 0.2, CHCl3). Analogously, compound
3. DNA-binding ability studies
4b was also obtained in 75% yield, ½a D31
¼ þ295 (c 1.0 in CHCl3);
ꢁ
mp: 69–71 °C. All the compounds were characterized by NMR
and MS spectroscopic studies. The specific rotation values for these
final compounds suggest that the stereochemistry at (S)-C12a is re-
The DNA-binding ability of PiBDs 4a,b was examined by ther-
mal denaturation studies using calf-thymus (CT) DNA.21 Melting
studies showed that these compounds stabilize the thermal he-
O
O
H3CO
RO
CO2H
i, 12
H3CO
RO
H3CO
RO
N
N
iii, iv
CH(SEt)2
(S)
X
H
NO2
N
X = N3/NO2
R = H; 13a
13b
14a
R = H;
4a
R = H;
R = Bn; 14b
R = Bn; 4b
R = Bn;
i, 10
O
O
H3CO
RO
H3CO
RO
N
N
(
)
S
ii
H
N3 O
15a
N
OCH3
H
O
5a
R = H;
R = Bn; 15b
R = H;
R = Bn; 5b
Scheme 3. Reagents and conditions: (i) EDCI/HOBt, CH2Cl2, 0 °C to rt, overnight, 75–86% for azides and (a) SOCl2, benzene, 1–2 drops DMF, 6 h, (b) Et3N, dry THF, 0 °C to rt,
2 h, 88–90% for nitro group; (ii) Al(OTf)3/NaI, CH3CN, 20 min, 85–88% for azides; (iii) SnCl2ꢃ2H2O, MeOH, reflux, 5 h for nitro group; (iv) HgCl2–CaCO3, CH3CN/H2O (4:1), 6 h,
68–75% (two steps overall yield).

2628
N. Markandeya et al. / Tetrahedron: Asymmetry 21 (2010) 2625–2630
Table 1
azidobenzoic acids by wearing safety glasses; facemask, gloves,
and reactions were performed in a fume hood. IR spectroscopy,
FT-IR Nicolet Nexus 470 equipment and KCl cell. Infrared spectra
were recorded and the wave numbers are expressed in cmꢀ1. Melt-
ing points (uncorrected) were measured with an Electrothermal
apparatus. Platinum plate anode (4.0 cm2) and tungsten wire as
cathode was used for the ‘cation-pool’ technique. Thermal denatur-
ation (DNA-binding) studies were evaluated by BECKMAN COUL-
TER-DU 800 spectrophotometer. Specific rotations were recorded
on SEPA-300 (Horiba high sensitive polarimeter) fixed with a so-
dium lamp of wavelength 589 nm. 1H and 13C NMR spectra were
recorded on Gemini 200, Avance 300, Inova 400, Inova 500, and
Bruker 600 MHz spectrometers using tetramethyl silane (TMS) as
the internal standard. Chemical shifts are reported in parts per mil-
lion (ppm) downfield from tetramethyl silane. Spin multiplicities
are described as s (singlet), br s (broad singlet), d (doublet), dd
(double doublet), t (triplet), q (quartet), and or m (multiplet). Cou-
pling constants are reported in Hertz (Hz). Mass spectra were re-
Thermal denaturation data for piperidino-benzodiazepines 4a,b and 5a,b with calf
thymus CT-DNA
Compounds [PiBD]/[DNA] molar ratioa
D
Tm (°C) after incubation at
b
37 °C for
0 h
18 h
4a
4b
5a
5b
1:5
1:5
1:5
1:5
1:5
1.2
0.9
1.2
0.4
0.3
2.2
1.3
1.8
0.6
0.7
DC-81 (3)
a
For CT-DNA alone at pH 7.00 0.01, Tm = 69.6 °C 0.01 (mean value from
eight separate determinations), all Tm values are 0.05 to 0.15 °C.
b
For
tion = 100
buffer [10 mM sodium phosphate + 1 mM EDTA, pH 7.00 0.01].
a
1:5 molar ratio of [PiBD]/[DNA], where CT-DNA concentra-
M and ligand concentration = 20 M in aqueous sodium phosphate
l
l
lix-coil or melting stabilization (
7.0, incubated at 37 °C, where PiBD/DNA molar ratio was 1:5. These
studies show the melting stabilization ( Tm) for the CT-DNA du-
plex at pH 7.0, incubated at 37 °C, where the PiBD/DNA molar ratio
was 1:5. An increase in helix melting temperature ( Tm) up to
DTm) for the CT-DNA duplex at pH
corded on
a Quattro-LC, (ESI). Column chromatography was
D
performed using silica gel 60–120 and 100–200 mesh. TLC analyses
were performed with silica gel plates using iodine, KMnO4 and UV-
lamp for visualization.
D
2.2 °C for 4a was observed compared to an untreated control
DNA after 18 h incubation at 37 °C. However, in the same experi-
5.1.1. (S)-2-Methyl 1-((1R,2S,5R)-5-methyl-2-(2-phenylpropan-
ment, the natural product DC-81 3 exhibited a
D
Tm of 0.7 °C, con-
2-yl)cyclohexyl) piperidine-1,2-dicarboxylate 9
sistent with the notion that the homolog of DC-81 4a forms a more
stable DNA-binding complex by replacing the (five-membered)
pyrrole with a piperidine ring (six-membered ring). Analogously,
compound 4b elevates the helix melting temperature of CT-DNA
by 0.9 °C at 0 h, which did not show a significant difference in
the melting temperatures even after incubation for 18 h (1.3 °C).
Furthermore, the non-covalent DNA-interactive PiBD dilactams
5a,b were also evaluated for their DNA-binding ability by using a
similar procedure. The dilactams 5a,b exhibited moderate binding
White crystalline solid, mp: 60–62 °C; ½a D31
¼ ꢀ49:9 (c 1.0,
ꢁ
CHCl3); 1H NMR (500 MHz, CDCl3): d 7.27 (m, 4H), 7.13 (t, 1H,
J = 5.84, 6.57 Hz), 4.71–4.78 (dd, 1H, J = 3.65, 4.38 Hz), 3.94 (dt,
1H, J = 2.19, 11.68 Hz), 3.63 (s, 3H), 3.36 (d, 1H, J = 5.11 Hz), 2.72
(t, 1H, J = 9.49, 10.95 Hz), 1.95–2.04 (m, 2H), 1.85–1.88 (m, 3H),
1.77 (m, 1H), 1.55–1.67 (m 6H), 1.17 (s, 3H), 0.95 (m, 2H), 0.84
(s, 6H). 13C NMR (75 MHz, CDCl3): d 172.4, 152.4, 127.8, 127. 6,
125.0, 124.8, 124.6, 54.0, 53.6, 51.6, 50.7, 50.5, 42.2, 41.5, 39.2,
34.5, 31.1, 29.2, 26.9, 26.4, 24.6, 24.3, 23.1, 21.7, 20.7. (ESI) HRMS:
m/z calcd for C24H35NO4Na 424.2463, found 424.2452 [M]+Å.
to the DNA as
DTm values ranged from 0.4 to 1.8 °C. These results
are illustrated in Table 1.
5.1.2. (S)-((1R,2S,5R)-5-Methyl-2-(2-phenylpropan-2-yl)cyclo-
hexyl) 2-(bis(ethylthio)methyl)piperidine-1-carboxylate 11
Diisobutylaluminiumhydride (DIBAL-H) solution (0.91 mL of
1.0 M solution in hexane) was added dropwise to a vigorously stir-
red solution of the compound 9 (230 mg, 0.573 mmol) in anhy-
drous CH2Cl2 (5 mL) under dry nitrogen at ꢀ78 °C. Next, the
reaction mixture was stirred for an additional 30 min, unfortu-
nately the ester group was reduced completely to the alcohol
(171 mg, 80%), and the aldehyde obtained in low yield (10%). More-
over, the starting material 9 was recovered in around 10%. The fol-
lowing reaction was analyzed by GC analysis, a ratio of 8:1:1 from
alcohol/aldehyde/9 was observed. Next, the obtained correspond-
ing alcohol (110 mg, 0.295 mmol) in CH2Cl2 (3 mL) was treated
with Dess–Martin periodinate (150 mg, 0.353 mmol) oxidation to
produce the aldehyde in excellent yield (100 mg, 92%). This reac-
tion mixture was quenched with saturated aqueous solutions of
Na2S2O3/NaHCO3 (1:1, 10 mL) and stirred for another 15 min. The
organic layer was separated, washed with brine, and then dried
in anhydrous Na2SO4 followed by evaporation under reduced pres-
sure. The aldehyde (100 mg, 0.269 mmol) was promptly (without
column chromatographic purification) protected with EtSH
(0.05 mL, 0.673 mmol), TMSCl (0.085 mL, 0.673 mmol) in CH2Cl2
at room temperature for 6 h to give 11 (122 mg) in 95% yield as
4. Conclusion
In conclusion, we have reported a facile asymmetric synthetic
route to an important class of piperidino-benzodiazepines from
commercially available piperidine and easily accessible aromatic
azido/nitro acid derivatives. In this protocol, we employed a one-
pot procedure for the generation of the required stereogenic center
in the N-acyliminum ion by anodic oxidation through a ‘cation-
pool’ technique using host/guest supramolecular diasteroselective
approach, with significantly improved % de. Furthermore, an intra-
molecular azido reductive-cyclization and dithioacetal deprotec-
tion tandem-cyclization approach has also been applied to the
synthesis of these heterocycles. Interestingly, some of these mole-
cules exhibit an enhanced DNA-binding ability in comparison to
DC-81. This new strategy provides potential applications for the
syntheses of both natural as well as medicinally important hetero-
cyclic compounds.
5. Experimental
5.1. General methods
a colorless liquid. ½a D32
ꢁ
¼ ꢀ85:7 (c 1.0, CHCl3); 1H NMR (500 MHz,
Purchased chemical reagents were used without further purifi-
cation. Anhydrous THF, CH2Cl2, CH3CN, MeOH, and DMF were pre-
pared by distillation under a nitrogen atmosphere over sodium/
benzophenone, CaH2, sodium/P2O5, and CaH2/molecular sieves,
respectively, and were used for reactions. Solvents for extraction
and column chromatography were distilled prior to use. Sodium
azide was handled with care for the preparation of substituted 2-
CDCl3): d 7.28 (m, 4H), 7.11 (t, 1H, J = 4.34, 6.98 Hz), 4.66–4.75
(ddd, 1H, J = 3.77, 3.96 Hz), 4.37 (d, 1H, J = 10.19 Hz), 4.05–4.17
(dd, 1H, J = 9.82, 10.57 Hz), 3.67 (m, 1H), 3.29 (d, 1H,
J = 10.57 Hz), 2.51–2.82 (m, 5H), 2.53 (t, 2H, J = 11.27, 13.03 Hz),
2.27 (d, 1H, J = 12.08 Hz), 1.92–2.10 (m, 2H), 1.58 (s, 3H), 1.20–
1.33 (m, 13H), 0.90–1.07 (m, 1H), 0.86 (s, 3H), 0.84 (s, 3H). 13C

N. Markandeya et al. / Tetrahedron: Asymmetry 21 (2010) 2625–2630
2629
NMR (75 MHz, CDCl3): d 151.8, 127.9, 125.5, 125.2, 125.0, 124.6,
52.9, 52.3, 50.6, 42.3, 39.8, 39.0, 34.6, 31.2, 27.2, 26.9, 26.3, 25.8,
24.7, 23.7, 21.7, 18.9, 14.4. (ESI) HRMS: m/z calcd for C27H43NO2S2-
Na 500.2632, found 500.2622 [M]+Å.
3.75 (m, 1H), 3.20–3.27 (m, 1H), 2.07–2.10 (m, 1H), 1.93–2.00
(m, 1H), 1.82–1.87 (m, 3H), 1.67–1.73 (m, 1H). 13C NMR
(125 MHz, CDCl3): d 167.4, 162.5, 150.2, 145.6, 142.2, 135.2,
121.9, 112.0, 56.41, 49.8, 39.9, 24.4, 22.9, 18.2. (ESI) HRMS: m/z
calcd for C14H16N2O3 261.1239, found 261.1234 [M+H]+Å.
5.1.3. (S)-(2-(Bis(ethylthio)methyl)piperidin-1-yl)(4-hydroxy-5-
methoxy-2-nitrophenyl)methanone 14a
5.1.6. (S)-3-(Benzyloxy)-2-methoxy-7,8,9,10-tetrahydrobenzo-
[e]pyrido[1,2-a][1,4]diazepin-12(6aH)-one 4b
To a stirred solution of 4-hydroxy-5-methoxy-2-nitrobenzoic
acid 13a (300 mg, 1.408 mmol) and thionyl chloride (0.30 mL,
4.22 mmol) in dry benzene (10 mL), 1–2 drops of DMF were added
and the stirring was continued for 6 h. Then, the benzene was
evaporated in vacuo. The resultant oil was dissolved in dry THF
(10 mL), and then added dropwise over a period of 30 min to a stir-
red suspension of (2S)-piperidino-2-carboxaldehyde diethyl
dithioacetal 12 (308 mg, 1.408 mmol), Et3N (0.60 mL, 4.224 mmol),
and THF (5 mL) cooled in an ice bath. After the completion of addi-
tion, the reaction mixture was brought to ambient temperature
and stirred for an additional 2 h. Next, the THF solvent was evapo-
rated, the aqueous phase adjusted to pH 4 using 4 M HCl, and then
extracted with ethyl acetate followed by washing with brine and
dried over Na2SO4. This was further purified by column chromatog-
raphy (80% EtOAc–hexane) to afford compound 14a (364 mg, 88%)
as a yellowish liquid. 1H NMR (400 MHz, CDCl3): d 7.73 (s, 1H), 6.98
(s, 1H), 6.05 (br s, 1H), 4.71 (d, 1H, J = 12.41 Hz), 4.27–4.35 (m, 1H),
3.97 (s, 3H), 3.20 (d, 1H, J = 14.18 Hz), 2.97–3.03 (m, 1H), 2.69–2.83
(m, 4H), 2.34 (t, 4H, J = 7.09 Hz), 1.52–1.71 (m, 2H), 1.25 (m, 6H).
13C NMR (50 MHz, CDCl3): d 167.2, 152.3, 146.0, 137.6, 126.3,
111.2, 108.9, 58.2, 56.5, 51.7, 50.2, 43.7, 38.2, 26.2, 25.1, 23.4,
18.9, 14.2. (ESI) MS: m/z 415 [M]+Å.
Compound 14b (65 mg, 0.129 mmol) dissolved in MeOH (5 mL).
Next, SnCl2ꢃ2H2O (145 mg, 0.645 mmol) was added and the mix-
ture refluxed for 6 h or until TLC indicated that the reaction was
complete. The methanol was evaporated by vacuum and the aque-
ous layer was then carefully adjusted to pH 8 with 10% NaHCO3
solution and the tin salts separated through a Celite bed. Extraction
was carried out with ethyl acetate (3 ꢄ 20 mL). The combined or-
ganic phase was dried over anhydrous Na2SO4 and evaporated un-
der vacuum to afford the amino diethyl thioacetal. Next, the
reaction mixture (59 mg, 0.124 mmol) was dissolved in CH3CN/
H2O (4:1, 10 mL), after which HgCl2 (84 mg, 0.311 mmol), and
CaCO3 (31 mg, 0.311 mmol) were added and stirred at room tem-
perature for 6 h until TLC (ethyl acetate) indicated the complete
loss of starting material. The reaction mixture was further diluted
with EtOAc (10 mL) and filtered through a Celite bed. The clear yel-
low organic supernatant was extracted with saturated 5% NaHCO3
(10 mL) and the combined organic phase was dried over Na2SO4.
The organic layer was evaporated in vacuo and purified by column
chromatography to afford compound 4b (column chromatography,
EtOAc 100%, 32 mg, 75%). Mp: 69–71 °C, ½a D31
¼ þ295 (c 1.0,
ꢁ
CHCl3). 1H NMR (400 MHz, CDCl3): d 7.87 (d, 1H, J = 5.93 Hz),
7.30–7.45 (m, 6H), 6.82 (s, 1H), 5.18 (m, 2H), 4.23 (d, 1H,
J = 14.41 Hz), 3.92 (s, 3H), 3.74 (q, 2H, J = 7.28 Hz), 3.18–3.25 (m,
1H), 2.04–2.09 (m, 1H), 1.35–1.99 (m, 1H), 1.27 (t, 2H,
J = 7.63 Hz). 13C NMR (50 MHz, CDCl3): d 167.4, 163.0, 150.4,
148.1, 139.7, 136.1, 128.6, 128.0, 127.2, 121.4, 111.6, 110.6, 70.7,
56.1, 49.5, 39.6, 24.4, 22.9, 18.3. (ESI) HRMS: m/z calcd for
5.1.4. (S)-(4-(Benzyloxy)-5-methoxy-2-nitrophenyl)(2-
(bis(ethylthio)methyl)piperidin-1-yl)methanone 14b
Yield (333 mg, 90%). 1H NMR (400 MHz, CDCl3): d 7.73 (s, 1H),
7.34–7.46 (m, 5H), 6.99 (s, 1H), 5.20 (s, 2H), 4.98–5.05 (m, 1H),
4.26–4.34 (m, 1H), 3.95 (s, 3H), 3.30 (br s, 1H), 3.21 (d, 1H,
J = 15.53 Hz), 2.91–3.00 (m, 2H), 2.68–2.82 (m, 4H), 2.31–2.47 (m,
1H), 4.54 (m, 1H), 1.52–1.70 (m, 2H), 1.25 (m, 6H). 13C NMR
(50 MHz, CDCl3): d 166.5, 154.5, 147.7, 137.3, 135.3, 128.7, 128.4,
127.4, 111.6, 109.5, 109.0, 71.2, 58.0, 56.4, 51.8, 49.9, 43.6, 38.0,
29.6, 26.3, 25.6, 23.3, 22.1, 19.4, 14.3. EI-MS: m/z 505 [M]+Å.
C
21H22N2O3Na 373.1528, found 373.1547 [M]+Å.
5.1.7. (S)-Methyl 1-(2-azido-4-hydroxy-5-methoxybenzoyl)-
piperidine-2-carboxylate 15a
To a stirred solution of 4-hydroxy-5-methoxy-2-azidobenzoic
acid 13a (146 mg, 0699 mmol) in CH2Cl2 (5 mL), EDCI (200 mg,
1.048 mmol), and HOBt (141 mg, 1.048 mmol) were added and
stirred for 30 min at 0 °C. Methyl (2S)-piperidinoate 10 (100 mg,
0.699 mmol) in CH2Cl2 (5 mL) was then added to the reaction mix-
ture and stirring was continued at room temperature overnight.
The reaction mixture was isolated in CH2Cl2 (3 ꢄ 20 mL), washed
with NaHCO3 solution (1 ꢄ 20 mL), brine (1 ꢄ 20 mL), and then
dried over anhydrous Na2SO4. The crude product was further puri-
fied through column chromatography using (silica-gel 60–
120 mesh) EtOAc–hexane (8:2) as eluent and the pure product
15a (174 mg) was obtained in 75% yield. FT-IR: (cmꢀ1) 3455,
2943, 2860, 2111, 1739, 1637, 1511, 1428, 1393, 1363, 1247,
1210, 1168, 1072, 1014, 860, 814, 747, 698, 637. 1H NMR
(300 MHz, CDCl3): d 6.79 (s, 1H), 6.75 (s, 1H), 5.98 (br s, 1H),
5.51 (d, 1H, J = 6.04 Hz), 3.88 (s, 3H), 3.78 (s, 3H), 3.39–3.43 (m,
1H), 3.10–3.19 (m, 1H), 2.20–2.36 (m, 2H), 1.55–1.82 (m, 4H). 13C
NMR (75 MHz, CDCl3): d 171.1, 168.4, 147.7, 144.5, 128.9, 118.7,
110.2, 105.2, 56.1, 52.2, 45.4, 39.7, 26.4, 25.1, 20.9. (ESI) HRMS:
m/z calcd for C15H18N4O5Na 357.1174, found 357.1164 [M]+Å.
5.1.5. (S)-3-Hydroxy-2-methoxy-7,8,9,10-tetrahydrobenzo[e]-
pyrido[1,2-a][1,4]diazepin-12(6aH)-one 4a (homolog of DC-81)
Compound 14a (80 mg, 0.193 mmol) was dissolved in MeOH
(5 mL). Next SnCl2ꢃ2H2O (217 mg, 0.966 mmol) was added and
the mixture refluxed for 5 h or until TLC indicated that the reaction
was complete. The methanol was evaporated by vacuum and the
aqueous layer was then carefully adjusted to pH 8 with 10% NaH-
CO3 solution and the tin salts separated through a Celite bed.
Extraction was carried out with ethyl acetate (3 ꢄ 20 mL). The
combined organic phase was dried over anhydrous Na2SO4 and
evaporated under vacuum to afford the amino diethyl thioacetal.
Next, this reaction mixture (70 mg, 0.182 mmol) was dissolved in
CH3CN/H2O (4:1, 10 mL), after which HgCl2 (123 mg, 0.455 mmol)
and CaCO3 (45 mg, 0.455 mmol) were added and stirred at room
temperature for 6 h until TLC (ethyl acetate) indicated the com-
plete loss of the starting material. The reaction mixture was further
diluted with EtOAc (10 mL) and filtered through a Celite bed. The
clear yellow organic supernatant was extracted with saturated
5% NaHCO3 (10 mL) and the combined organic phase was dried
over Na2SO4. The organic layer was evaporated in vacuo and puri-
fied by column chromatography (95% EtOAc–MeOH) affords the
5.1.8. (S)-Methyl 1-(2-azido-4-(benzyloxy)-5-methoxybenzoyl)-
piperidine-2-carboxylate 15b
To a stirred solution of 4-benzyloxy-5-methoxy-2-azidobenzoic
acid 13b (229 mg, 0.769 mmol) in CH2Cl2 (10 mL), EDCI (200 mg,
1.048 mmol), and HOBt (141 mg, 1.048 mmol) were added and
stirred for 30 min at 0 °C. Methyl (2S)-piperidinoate 10 (100 mg,
compound 4a (32 mg, 68%). Mp: 119–121 °C, ½a D31
ꢁ
¼ þ256 (c 1.0,
CHCl3); (natural product DC-81) [a]
D = +135 (c 0.2, CHCl3). 1H
NMR (600 MHz, CDCl3): d 7.92 (d, 1H, J = 6.07 Hz), 7.39 (s, 1H),
7.25 (s, 1H), 5.29 (br s, 1H), 4.19–4.23 (m, 1H), 3.93 (s, 3H), 3.73–

2630
N. Markandeya et al. / Tetrahedron: Asymmetry 21 (2010) 2625–2630
0.699 mmol) in CH2Cl2 (5 mL) was added to the reaction mixture at
the same temperature and stirring was continued at room temper-
ature overnight. The crude product was further purified through
column chromatography using (silica-gel 60–120 mesh) EtOAc–
hexane (3:7) as eluent and the pure product 15b (278 mg) was ob-
tained in 86% yield. FT-IR: (cmꢀ1) 2946, 2859, 2114, 1740, 1617,
1516, 1436, 1312, 1256, 1212, 1168, 1073, 1044, 1010, 930, 866,
809, 755, 639. 1H NMR (400 MHz, CDCl3): d 7.33–7.45 (m, 5H),
6.78 (m, 1H), 6.66 (m, 1H), 5.51 (s, 2H), 4.67 (d, 1H, J = 13.5 Hz),
4.22 (s, 1H), 3.87 (s, 3H), 3.78 (s, 3H), 3.37 (d, 1H, J = 11.4 Hz),
3.15 (t, 1H, J = 8.32, 14.5 Hz), 2.79 (t, 1H, J = 11.4, 15.6 Hz), 2.31
(d, 1H, J = 13.5 Hz), 2.21 (d, 1H, J = 13.5 Hz), 1.75 (m, 2H). 13C
NMR (50 MHz, CDCl3): d 171.1, 167.9, 162.5, 149.5, 147.1, 136.0,
128.5, 128.1, 127.2, 111.2, 104,4, 71.2, 56.2, 52.1, 52.0, 45.3, 39.5,
27.2, 26.4, 25.2, 24.3, 21.0. (ESI) HRMS: m/z calcd for C22H24N4O5Na
447.1644, found 447.1631 [M]+Å.
References
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½
a 3D1
ꢁ
¼ þ172 (c 1.0, CHCl3). 1H NMR (300 MHz, CDCl3): d 8.06 (br
s, 1H), 7.36 (s, 1H), 6.51 (s, 1H), 6.34 (br s, 1H), 4.50 (d, 1H,
J = 13.59 Hz), 4.13–4.16 (m, 1H), 3.93 (s, 3H), 2.97 (t, 1H,
J = 9.82 Hz), 2.17–2.26 (m, 1H), 1.79–1.97 (m, 1H), 1.53–1.97 (m,
4H). 13C NMR (75 MHz, CDCl3): d 164.1, 155.2, 144.9, 134.9,
129.0, 128.2, 121.2, 116.1, 55.6, 42.3, 39.3, 36.1, 30.4, 29.2. (ESI)
HRMS: m/z calcd for C14H16N2O4Na 300.1193, found 300.1202
[M]+Å.
5.1.10. (S)-3-(Benzyloxy)-2-methoxy-7,8,9,10-tetrahydrobenzo-
[e]pyrido[1,2-a][1,4]diazepine-6,12(5H,6aH)-dione 5b
White solid (38 mg, 88%), mp: 195–197 °C, ½a D32
¼ þ248 (c 1.0,
ꢁ
CHCl3). 1H NMR (200 MHz, CDCl3): d 8.12 (br s, 1H), 7.31–7.41
(m, 6H), 6.42 (s, 1H), 5.15 (s, 2H), 4.49 (d, 1H, J = 13.91 Hz), 4.13–
4.16 (m, 1H), 3.92 (s, 3H), 2.96 (t, 1H, J = 9.52 Hz), 2.17–2.23 (m,
1H), 1.89–1.95 (m, 1H), 1.54–1.73 (m, 4H). 13C NMR (75 MHz,
CDCl3): d 171.6, 168.1, 151.1, 147.0, 135.8, 129.9, 128.6, 128.1,
127.1, 119.7, 112.6, 105.1, 70.8, 56.1, 51.2, 40.1, 23.1, 22.6, 19.0.
20. (a) Kamal, A.; Markandeya, N.; Shankaraiah, N.; Reddy, Ch. R.; Prabhakar, S.;
Reddy, Ch. S.; Eberlin, M. N.; Santos, L. S. Chem. Eur. J. 2009, 15, 7214–7224; (b)
Kamal, A.; Reddy, K. L.; Devaiah, V.; Shankaraiah, N.; Reddy, G. S. K.; Raghavan,
S. J. Comb. Chem. 2007, 9, 29–42; (c) Kamal, A.; Shankaraiah, N.; Reddy, K. L.;
Devaiah, V. Tetrahedron Lett. 2006, 47, 4253–4257.
(ESI) HRMS: m/z calcd for
C21H22N2O4Na 389.1477, found
389.1471 [M]+Å.
21. (a) Bose, D. S.; Thompson, A. S.; Ching, J. A.; Hartley, J. A.; Berardini, M. D.;
Jenkins, T. C.; Neidle, S.; Hurley, L. H.; Thurston, D. E. J. Am. Chem. Soc. 1992,
114, 4939–4941; (b) Bose, D. S.; Thompson, A. S.; Smellie, M.; Berardini, M. D.;
Hartley, J. A.; Jenkins, T. C.; Neidle, S.; Thurston, D. E. J. Chem. Soc., Chem.
Commun. 1992, 1518–1520.
Acknowledgments
This work has been supported by the Facility for Chemical Biol-
ogy (Project SIP-0011, N. Shankaraiah) and FONDECYT (Project
1085308, L.S.S.).