Synthesis of bis-1,2,3-triazolo-bridged unsymmetrical pyrrolobenzodiazepine trimers via 'click' chemistry and their DNA-binding studies

Article abstract of DOI:10.1016/j.tet.2010.05.003

New conceivable synthetic approach for the construction of nitrogen-rich 1,2,3-triazolo-pyrrolo[2,1-c] [1,4]benzodiazepine (TPBD, 3ac) trimers has been developed.The first example of a bis-1,2,3-triazolo-bridged unsymmetrical PBD trimer has been successfully synthesized by employing a CuAAC type 'click' chemistry protocol.This efficient route generates tri-imine functionality in a single molecule.It has been envisaged that such tri-imine functionalities could bring in efficient interaction with DNA in a sequence-selective manner in the minor groove of duplex DNA.One of the representative analogues 3c has shown improved DNA-binding ability (DT_m 23.7 °C) by thermal denaturation studies using CT-DNA and this data is also supported by molecular modeling (MD) studies.

Full text of DOI:10.1016/j.tet.2010.05.003

Tetrahedron 66 (2010) 5498e5506
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of bis-1,2,3-triazolo-bridged unsymmetrical pyrrolobenzodiazepine
trimers via ‘click’ chemistry and their DNA-binding studies
,
b
a
a
a
Ahmed Kamal a , Nagula Shankaraiah , Ch. Ratna Reddy , S. Prabhakar , Nagula Markandeya ,
*
Hemant Kumar Srivastava c, G. Narahari Sastry c
,
*
a Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 607, Andhra Pradesh, India
b National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, Andhra Pradesh, India
c Molecular Modeling Group, Indian Institute of Chemical Technology, Hyderabad 500 607, Andhra Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
New conceivable synthetic approach for the construction of nitrogen-rich 1,2,3-triazolo-pyrrolo[2,1-c]
[1,4]benzodiazepine (TPBD, 3aec) trimers has been developed. The first example of a bis-1,2,3-triazolo-
bridged unsymmetrical PBD trimer has been successfully synthesized by employing a CuAAC type ‘click’
chemistry protocol. This efficient route generates tri-imine functionality in a single molecule. It has been
envisaged that such tri-imine functionalities could bring in efficient interaction with DNA in a sequence-
selective manner in the minor groove of duplex DNA. One of the representative analogues 3c has shown
Received 12 February 2010
Received in revised form 28 April 2010
Accepted 1 May 2010
Available online 7 May 2010
Keywords:
Pyrrolobenzodiazepine trimers
1,2,3-Triazoles
improved DNA-binding ability (
is also supported by molecular modeling (MD) studies.
D
Tm 23.7 ꢀC) by thermal denaturation studies using CT-DNA and this data
Ó 2010 Elsevier Ltd. All rights reserved.
Click chemistry
Cycloadditions
DNA-binding affinity
Molecular modeling
1. Introduction
synthesis through an interplay of non-covalent and covalent in-
teractions in either the minor or major groove of the double helix.
In addition, the DNA interstrand cross-linking (ISC) agents play an
important role in cancer therapy by disrupting cell maintenance
and replication. Some of the anticancer drugs (e.g., cisplatin,
chlorambucil, and mitomycin C) have already been employed in
clinical medicine.10 Among them, over the years scaffolds like
pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) have gained consider-
able interest in the area of medicinal chemistry with respect to the
development of synthetic strategies and preclinical studies, par-
ticularly as potential antitumor and gene-targeting compounds.11
These tricyclic antitumor antibiotics are derived from Streptomy-
ces bacteria,12 members of which include DC-81, tomaymycin, and
anthramycin. They exert biological activity by selective covalent
binding between the imine functionality and the N2 of guanine base
in the minor groove of DNA.13
The development and construction of highly nitrogen-rich DNA-
interactive new heterocyclic compounds continue to be an essen-
tial aspect for producing bioactive natural products and their
structural analogues. ‘Click’ chemistry represents a modular ap-
proach toward the synthesis of low as well as high molecular
weight compounds that access only the most practical trans-
formations to make connections with excellent fidelity.1 Huisgen
1,3-dipolar cycloaddition of terminal alkynes and organic azides to
give five-membered 1,2,3-triazoles has emerged as a powerful
linking reaction and found widespread applications ranging from
combinatorial drug research2 and material science3 to bioconjugate
chemistry.4 Moreover, these triazoles display a broad spectrum of
biological activities like antibacterial, antifungal, and antihelmintic
activities,5,6 including anticancer activity.7
It is well-known that DNA has long been recognized as an im-
portant cellular target in the rational design of potential chemo-
therapeutic and gene targeting agents.8,9 DNA-reactive drugs exert
their biological profile by inhibiting nucleic acid or protein
Thurston and co-workers have synthesized C8/C80-linked PBD
dimer (DSB-120 2, in Fig. 1) comprising of two DC-81 (1) subunits
joined through their C8-positions by an inert propyldioxy spacer.14
These have been examined for their cytotoxic effect, DNA cross-
linking potency,15 and cellular pharmacology.13d,16e19 Investigation
in our laboratory has led to a number of monomeric,20 dimeric21
PBD hybrids with enhanced DNA-binding affinity and promising
anticancer activity. It has been observed most of these PBD dimers
* Corresponding authors: Tel.: þ91 40 27193157; fax: þ91 40 27193189; e-mail
addresses: ahmedkamal@iict.res.in (A. Kamal), gnsastry@gmail.com (G.N. Sastry).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.05.003

A. Kamal et al. / Tetrahedron 66 (2010) 5498e5506
5499
Etherification of the corresponding compound 6 by different
dibromoalkane spacers in DMF with K2CO3 affords the desired in-
termediates 7aec (82e85%).
Herein, we report a new step in which CeC2/AeC8-diazido
substituted 2-nitrobenzoyl prolinemethyl ester (8aec) is obtained
in one-pot manner by the in situ azidation of both CeC2-mesyl
(bimolecular nucleophilic substitution reaction SN2) and AeC8-
bromo (elimination reaction) groups in presence of excess sodium
azide (10 equiv) in anhydrous DMF at 60e70 ꢀC for 6 h (80e88%).
Next, the selective reduction of ester functionalities (8aec) with
DIBAL-H at ꢁ78 ꢀC followed by aldehyde protection with ethyl-
mercaptane has yielded the desired key diazido intermediates
9aec (90e95%). Another key intermediate 10 with terminal alkyne
has been prepared by the earlier reported method.22 Herein, the
‘click’ reaction has been employed for the construction of the
bridged bis-1,2,3-triazolo-PBD trimer framework. Thus, trimeriza-
tion reaction has been accomplished in a single step by the mixing
of organic azides (9aec, 1 mmol) and terminal alkyne (10, 2 mmol)
in H2O/t-BuOH (1:1) with 1 mol % of CuSO4$5H2O and 5 mol % of
sodium ascorbate. The corresponding unsymmetrical bis-1,2,3-tri-
azlolo-bridged 2-nitrobenzoyl ethylmercaptane prolinaldehyde
precursors 11aec have been obtained in good yields (78e88%).
Finally, the nitro groups have been reduced with excess of
SnCl2$2H2O followed by ethylmercaptane deprotective tandem
cyclization reaction using HgCl2/CaCO3 to provide the desired title
1,2,3-triazolo-PBD trimers (3aec) in moderate to good yields
(68e75%) as depicted in Scheme 1.
Figure 1. Representative structures of bioactive pyrrolo[2,1-c][1,4]benzodiazepines,
DC-81 (1), DSB-120 (2), and triazolo-PBD trimer analogues (3aec).
that the synthesis is usually problematic during the reaction work-
up and chromatographic purifications by using high polar solvents.
To overcome this complexity, recently we have developed a method
based on ‘click’ chemistry protocol that has been applied for the
synthesis of C8eC8/C2eC8-linked 1,2,3-triazolo-PBD dimer conju-
gates.22 Based on the above findings, it is considered of interest to
prepare the PBD trimers that could interact within the minor
groove of DNA in a sequence-selective manner. In this context, the
‘click’ protocol has been applied for the synthesis of pyrrolo[2,1-c]
[1,4]benzodiazepine (PBD) trimers (3aec) that are joined through
a triazole moiety to explore their DNA-binding ability.
2. Results and discussion
3. DNA-binding affinity studies
The synthetic strategy reveals that the skeleton of triazole-
containing PBD trimers 3aec has been assembled from the key
precursors 9aec and 10. This framework represents that one of the
1,2,3-triazole moiety is sandwiched between the two monomeric
DC-81 subunits at their C8-positions of the aromatic A-rings via
diether linkage by using alkane spacers and the other 1,2,3-triazole
moiety is joined to the pyrrolidine C-ring through its C2-position
and C8-position of the other aromatic A-ring through a monoether
linkage. The earlier research relating to the synthesis and biological
evaluation of PBD dimers like DSB-120 (2),23 SJG-136,24 and mixed
imineeamides25 have exhibited immense potential in the de-
velopment of molecules of this type. Many attempts have been
made previously in our laboratory to synthesize PBD trimers
through diether linkages using simple dibromoalkane spacers in
both solution-phase as well as on solid-support, but such studies
have given unsatisfactory results probably owing to their in-
solubility problems. However, in this investigation by taking ad-
vantage of the copper-catalyzed azideealkyne cycloaddition
(CuAAC) protocol, we decided to apply such a ‘click’ process for the
preparation of PBD trimers. This has also prompted us to explore
the DNA-binding potential of such PBD trimer analogues linked
through 1,2,3-triazole moieties by using the diazido functionality of
9aec and terminal alkyne group of 10. Interestingly, it has been
observed that the solubility of these PBD trimers is enhanced in
most of the organic solvents and this may be attributed to the
presence of two 1,2,3-triazole rings.
The DNA-binding affinity of these new triazolo-PBD trimers
(3aec) has been examined by thermal denaturation studies using
calf thymus (CT) DNA.15 Melting studies show that these com-
pounds stabilize the thermal helix-coil or melting stabilization
(D
Tm) for the CT-DNA duplex at pH 7.0, incubated at 37 ꢀC, wherein
PBD/DNA molar ratio is 1:5. In this assay, the helix melting tem-
perature changes ( Tm) for each compound has been studied at 0 h
D
and after 18 h of incubation at 37 ꢀC. Data for DC-81 (1) and DSB-
120 (2) are incorporated in Table 1 for comparison. In this study,
PBD trimer compounds 3aec have shown elevated melting tem-
peratures ranging from 5.6 to 23.7 ꢀC (Table 1). Interestingly, the
D
Tm of compound 3c is 18.3 ꢀC at 0 h while the melting tempera-
ture increases to 23.7 ꢀC upon incubation for 18 h at 37 ꢀC. Whereas
for compound 3a the
Tm is 10.7 ꢀC at 0 h, which increases to
D
14.1 ꢀC after incubation for 18 h. Compound 3b elevates the helix
melting temperature of CT-DNA by 5.6 ꢀC at 0 h, however, there is
not much difference in the melting temperatures after incubation
for 18 h (7.9 ꢀC) in comparison to 3a and 3c. In the same experiment
the naturally occurring DC-81 (1) and the dimer DSB-120 (2) ele-
vate the helix melting temperature of CT-DNA by 0.7 ꢀC and 15.1 ꢀC,
respectively, after incubation for 18 h.
4. Molecular modeling studies
Docking of the 1,2,3-triazolo-PBD trimers (3aec) into the minor
groove of the DNA duplex was initially carried out using GOLD3.2,26
with default settings and the binding is effectively guided by non-
covalent interaction. On inspection of docked poses, all three N10-
C11 imine functionalities of PBD trimer are close to the C2-amino
group of three ‘G’ residues of 15-mer DNA. The 15-mer sequence 50-
GGGGCGAGAGAGGGG-30 having the preferred binding site Pu-G-Pu
for the PBD molecules was chosen for modeling the B-DNA duplex
structure, which was prepared and minimized with OPL2005 force
field using Maestro program of Schrodinger package.27 This DNA
sequence is chosen because it provided good results in the previous
study and it has the Pu-G-Pu binding site for the covalent bond
The synthetic approach began with the preparation of 4-(ben-
zyloxy)-5-methoxy-2-nitrobenzoic acid (4), which has been syn-
thesized from commercially available vanillin from three steps. This
has been converted to its acid chloride with SOCl2, and then cou-
pled to trans-4-hydroxyl-L-proline methylester hydrochloride in
the presence Et3N to give the corresponding compound 5 in 84%
overall yield from two steps. Mesylated product has been obtained
in 88% yield by using mesylchloride and Et3N at 0 ꢀC to room
temperature for 6 h, and then it has been successfully converted to
its debenzylated product
6 (83%) employing BF3$OEt2/EtSH.

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A. Kamal et al. / Tetrahedron 66 (2010) 5498e5506
Scheme 1. Synthesis of unsymmetrical bis-triazolo-PBD trimer conjugates (3aec).
formation with the PBD trimer.25 These calculations un-
ambiguously revealed the extreme of covalent bonds. Thus three
covalent bonds are enforced on the initial structure and the re-
sultant complex minimized. Going by the precedence of earlier
results there is a possibility of covalent bond formation. However,
the current line of docking methods could not consider covalent
docking, with default settings. The docking results of PBD trimer
(3aec) conjugates are presented in Table 2 along with their
experimental
docking fitness scores indicating that the complex formation to the
DNA groove is quite facile. Compound with highest Tm value also
has highest GOLD score while compound with lowest Tm value
also has lowest GOLD score. The docked poses presented in Figure 2
showing that these conjugates bound to DNA with three covalent
bonds and mainly in the minor groove region of DNA. The clearer
picture of all three covalent bonding of nitrogen atom of guanine
DTm values. All the compounds have fairly substantial
D
D

A. Kamal et al. / Tetrahedron 66 (2010) 5498e5506
5501
Table 1
Thermal denaturation data for 1,2,3-triazole-PBD trimer (3aec) conjugates with calf
thymus CT-DNA
PBD compounds
[PBD]/[DNA]
molar ratioa
D
Tm (ꢀC)b after incubation at 37 ꢀC for
0 h
18 h
3a
3b
3c
1:5
1:5
1:5
1:5
1:5
10.7
5.6
18.3
0.3
14.1
7.9
23.7
0.7
DC-81 (1)
DSB-120 (2)
10.2
15.1
a
For CT-DNA alone at pH 7.00ꢂ0.01, Tm¼69.6ꢂ0.01 ꢀC (mean value from 10
separate determinations), all Tm values are ꢂ0.05e0.15 ꢀC.
b
For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentration¼100
mM and
ligand concentration¼20
mM in aq sodium phosphate buffer [10 mM sodium
phosphateþ1 mM EDTA, pH 7.00ꢂ0.01].
Table 2
GOLD fitness score and IE of 1,2,3-triazolo-PBD trimer (3aec) conjugates with their
experimental
DTm values
Compounds
GOLD scorea
IEb
D
Tm (ꢀC) at 18 h
3a
3b
3c
85.83
78.29
91.25
ꢁ128.03
ꢁ127.45
ꢁ146.51
14.1
7.9
23.7
Figure 3. Covalent bonding of PBD trimer 3c with DNA (guanine residues involved in
the bonding are written in red color, C-atom of PBD and N-atom of guanine are shown
in CPK).
a
GOLD score is the best fitness score obtained in GOLD docking.
b
‘IE’ is the interaction energy calculated by using Generalized Born method after
2 ns MD simulation.
appears to be a bit longer compared to the optimal length. The
docking score also reveals that trimer binding is stronger compared
to that of tetramer. It may be noted that even in tetramer the spacer
chain length of five carbon units (see 3c, where n¼3) is preferred.
Therefore, the current study, for the first time, reveals that trimer
has the most optimal binding. These studies indicate that there is
no structural variation between the double helical strands of DNA
upon changing the linker length of the PBD trimer.
Figure 2. Docked poses of 3aec with DNA sequence 50-GGGGCGAGAGAGGGG-30.
Figure 4. The three best score poses of tetramer PBD docked in the DNA groove (part
of the PBD, which is not docked in the minor groove are shown in circles).
and carbon atom of PBD trimer is shown in Figure 3. Compound 3c
has shown the best GOLD score probably due to five carbon chain in
between the monomers, which provide the flexibility to rotate and
fit in the DNA groove without unfavorable close contacts.
Table 3
In order to investigate whether trimer is an optimum oligomeric
unit, we resort to a docking analysis of the monomer, dimer, and
tetramer along with trimer by choosing the same sequence and
same region of DNA minor groove to estimate the structural and
energetic compatibility of the oligomeric units of different length.
This analysis brings out very clearly that the trimer is the most
optimal unit with maximum binding strength and with highest
structural compatibility as can be gauged from Figure 4 and Table 3.
In conformity with current and earlier studies, mononer and dimer
units nicely fit into the DNA minor groove region. An exhaustive
study has been carried out to assess the suitability of tetramer unit
to the DNA and three best among the several putative attempts
were taken (Fig. 4). The results show that in none of the orienta-
tions, the tetramer is fully fitting into the DNA groove and thus it
Comparative GOLD fitness score of various PBDs
PBD
GOLD scorea
Monomer
Dimer
Trimer (3c)
Tetramer (Pose 1)
Tetramer (Pose 2)
Tetramer (Pose 3)
44.54
73.56
91.25
84.75
76.82
76.73
a
GOLD SCORE is the best fitness score obtained in GOLD docking.
After establishing the mode of binding of the novel trimer to
DNA we performed more rigorous MD simulation. The MD simu-
lation with explicit solvent, involving w10,000 water molecules
having performed up to 2 ns. Figure 5 is the snapshots of the

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A. Kamal et al. / Tetrahedron 66 (2010) 5498e5506
prepared by distillation under 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 by wearing safety glasses, facemask,
gloves, and reactions were performed in a fume hood. IR spec-
troscopy: FT-IR Nicolet Nexus 470 equipment and KCl cell. Infrared
spectra were recorded and the wave numbers are expressed in
cmꢁ1. Melting points (uncorrected) were measured with an Ele-
trothermal apparatus. Thermal denaturation (DNA-binding) studies
were evaluated by BECKMAN COULTER-DU 800 spectrophotometer.
Specific rotations were recorded on SEPA-300 fixed with sodium
lamp of wavelength 589 nm. 1H and 13C NMR spectra were recorded
on 200, 300, 400, and 500 MHz spectrometers using tetramethyl
silane (TMS) as the internal standard. Chemical shifts are reported
in parts per million (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). Coupling constants are reported in hertz (Hz). Mass
spectra were recorded on a Quattro-LC (ESI). Column chromatog-
raphy was performed using silica gel 60e120 and 100e200 mesh.
TLC analyses were performed with silica gel plates using iodine,
KMnO4, and UV-lamp for visualization.
Figure 5. Snapshots of docked poses of 3aec at 2 ns MD simulation.
docked poses of compounds 3aec at 2 ns MD simulation. The
simulation reveals that all the three PBD trimers (3aec) are fairly
rigid up to 2 ns and the most stable and active among them 3c
showed a very rigid structure. Thus, the MD calculations help un-
ambiguously establish the complex formation and its stability. To
see the deviation of the docked complexes during MD simulation,
we plotted root-mean-square deviation (RMSD) as a function of
time. RMSD of complex as well as of DNA and ligand is presented
separately to show a systematic picture of deviation. It is very clear
from the RMSDs that the docked poses of all the compounds are
stable in 2 ns MD simulation.
6.2. Computational details
Sybyl 6.9.2 program (Tripos Inc., St. Louis, MO) has been used for
preparing the PBD conjugates. All the compounds were minimized
ꢀꢁ1
to 0.001 kcal molꢁ1
A
root-mean-square gradients by using Gas-
Irrespective of the ligand and the simulation conditions of all the
compounds remain bound to the DNA near the preferential binding
position and do not experience substantial fluctuations with respect
to their initial placements in the DNA groove. The interaction energy
(IE), which provides a quantitative estimate of average binding en-
ergy, between the DNA and the PBDs is calculated to confirm their
bindingstabilityand presented inTable 2.28,29 Generalized Born(GB)
method, which is semi-analytical approximations to continuum
electrostatics is used for the calculation of interaction energies.30
Interaction energies of all the compounds considered, points to
a stable complex formation and these trends are in excellent
agreement with the experimental and docking results. Thus, the
modeling studies establish that these compounds interact with the
minor groove of DNA, which is not only due to strong non-covalent
forces but also due to the formation of three covalent bonds.
teigereHückel partial atomic charges and Tripos force field. Radius
of 30 A from the middle atom of DNA was used as a central atom to
ꢀ
scan the entire DNA sequences in molecular docking. All the PBD
conjugates were subjected to a conformational search by using
molecular mechanics method and minimum energy conformation
was selected for final study. Previously 50-GGGGCGAGAGAGGGG-30
DNA sequence provide better results in the docking study of similar
compounds25 thus, we have used this sequence in the current
study. Our ongoing studies indicate that the GOLD docking on DNA
sequences is considered to be the best docking protocol, which
reliably reproduces the crystallographic poses of DNAeligand
complex and it provided good results in our earlier studies.31 Thus
in the present study we have performed the docking calculations
using GOLD3.2 program. The default parameters in GOLD3.2
(number of islands 5, population size of 100, number of operations
was 100,000, a niche size of 2, and a selection pressure of 1.1, and
the van der Waals and hydrogen bonding were set to 4.0 and 2.5,
respectively) have been used. The 10 best conformations have been
generated for each compound and the best score pose was chosen
for MD simulation using AMBER 8.0.32 The final input files were
created by merging DNA and ligand in a complex as docked pose.
Three covalent bonds were made in the docked poses and the
structure was minimized by using impact minimization on Maestro
package. The ‘leaprc.gaff’ (generalized amber force filed) was used
to prepare the ligands while ‘leaprc.ff03’ was used for DNA. The
‘addions’ command implemented in ‘xleap’ of AMBER 8.0 was used
to add the Naþ ions explicitly to neutralize the system. The ‘Sol-
5. Conclusion
The unsymmetrical bis-1,2,3-triazolo-PBD trimers have been
designed and synthesized by employing ‘click’ chemistry process.
Interestingly, by using this ‘click’ chemistry protocol the solubility
aspects have been improved that facilitated the purification and
isolation of the target compounds. These new PBD trimers have
shown significant DNA-binding ability. Molecular modeling studies
substantiate the formation of three covalent bonds with the PBD
trimer and guanine. Compound 3c appears to be the optimal binder
as further increase in linker or chain length decreases the binding
strength of these compounds with DNA. The structureeactivity
relationship and cytotoxicity studies of these new compounds are
under in progress.
ꢀ
vateOct’ command was used to solvate the complex in a 10 A water
box with TIP3P water. Equilibration of the solvated complex has
been done by carrying out a short minimization (500 steps of each
steepest descent and conjugate gradient method), 50 ps of heating
and 50 ps of density equilibration with weak restraints on the
complex followed by 500 ps of constant pressure equilibration at
300 K. The final production run is performed for the 2 ns and the
coordinates are recorded in every 10 ps. Before submitting for the
MM-PBSA production run we verified that the system has equili-
brated. We extracted 200 snapshots from production runs by using
6. Experimental section
6.1. General methods
Purchased chemical reagents were used without further puri-
fication. Anhydrous THF, CH2Cl2, CH3CN, MeOH, and DMF were

A. Kamal et al. / Tetrahedron 66 (2010) 5498e5506
5503
‘extract_coords.mmpbsa’ script and calculated the interaction en-
ergies by using ‘binding_energy.mmpbsa’ script. Both the scripts
are available on AMBER web site. The final reported interaction
energies are the average of all 200 snapshots.
compound 6 (1.50 g, 3.58 mmol) in dry DMF (20 mL), K2CO3 (2.47 g,
17.93 mmol) and 1,3-dibromopropane (0.86 g, 4.29 mmol) were
added and the reaction mixture was stirred at room temperature
for 12 h. This reaction mixture was poured into ice-cold water and
extracted with ether and the organic layer was separated dried over
anhydrous Na2SO4 and evaporated in vacuo. This was purified by
column chromatography with ethyl acetate/hexane (6:4) as eluent
to afford compound 7a (1.60 g, 83%). 1H NMR (200 MHz, CDCl3):
6.3. Reaction procedures and spectral data
6.3.1. Methyl-(2S,4R)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4-
hydroxypyrrolidine-2-carboxylate (5). To a stirred suspension of
compound 4 (3.03 g, 10.0 mmol) and thionyl chloride (2.15 mL,
30.0 mmol) in dry benzene (40 mL), 4e5 drops of DMF was added
and the stirring was continued for 6 h. The benzene was evaporated
in vacuum and the resultant oil dissolved in dry THF (40 mL) and
added drop wise over a period of 30 min to a stirred suspension of
d
¼7.69 (s, 1H), 6.78 (s, 1H), 5.22 (m, 1H), 4.84 (t, J¼8.30 Hz, 1H), 4.24
(t, J¼5.26, 6.04 Hz, 2H), 3.97 (s, 3H), 3.84 (s, 3H), 3.61 (m, 2H), 3.57
(m, 1H), 3.07 (s, 3H), 2.66e2.73 (m, 1H), 2.23e2.44 ppm (m, 4H);
ꢃ
(ESI) MS: m/z 539 [M]þ
.
6.3.4. (2S,4R)-Methyl
1-(4-(4-bromobutoxy)-5-methoxy-2-nitro-
4-hydroxy-L-proline methylester hydrochloride (2.14 g, 15.0 mmol),
benzoyl)-4-(methylsulfonyloxy)pyrrolidine-2-carboxylate (7b). To
Et3N (4.17 mL, 30 mmol), and THF/H2O (25-5 mL) cooled in an ice
bath. After the completion of addition, the temperature was
allowed to rise to room temperature and stirred for an additional
1 h. THF was evaporated in vacuum and the aqueous layer was
washed with ethyl acetate (50 mL). The aqueous phase was then
adjusted to pH 3 using 6 N HCl and extracted with ethyl acetate, and
then washed with brine dried over Na2SO4. The crude product was
purified by column chromatography (30% ethyl acetate/hexane)
afforded compound 5 as yellow oil (3.60 g, 84%). 1H NMR (200 MHz,
compound 6 (1.20 g, 2.63 mmol) in dry DMF (20 mL), K2CO3 (1.81 g,
13.15 mmol) and 1,4-dibromopropane (0.68 g, 3.15 mmol) were
added and the reaction mixture was stirred at room temperature for
12 h to afford compound 7b (1.30 g, 82%).1H NMR (200 MHz, CDCl3):
d
¼7.64 (s, 1H), 6.78 (s, 1H), 5.21 (m, 1H), 4.83 (t, J¼7.81, 8.59 Hz, 1H),
4.12 (t, J¼5.46 Hz, 2H), 3.98 (s, 3H), 3.84 (s, 3H), 3.42 (t, J¼6.25,
7.03 Hz, 2H), 3.07 (s, 3H), 2.64e2.80 (m, 1H), 2.22e2.36 (m, 1H),
ꢃ
1.85e2.05 (m, 4H), 1.62e1.74 ppm (m, 2H); (ESI) MS: m/z 553 [M]þ
.
CDCl3):
d
¼7.75 (s, 1H), 7.30e7.45 (m, 5H), 6.90 (s, 1H), 5.20 (s, 2H),
6.3.5. (2S,4R)-Methyl 1-(4-(5-bromopentyloxy)-5-methoxy-2-nitro-
benzoyl)-4-(methylsulfonyloxy)pyrrolidine-2-carboxylate (7c). To
compound 6 (0.82 g, 1.96 mmol) in dry DMF (10 mL), K2CO3 (1.35 g,
9.80 mmol) and 1,4-dibromopropane (0.54 g, 2.35 mmol) were
added and the reaction mixture was stirred at room temperature
for 12 h to afford compound 7c (0.94 g, 85%). 1H NMR (200 MHz,
4.70e4.80 (m, 1H), 4.40 (m, 1H), 3.96 (s, 3H), 3.85 (s, 3H), 3.50e3.60
ꢃ
(m, 3H), 2.05e2.30 ppm (m, 2H); (ESI) MS: m/z 431 [M]þ
.
6.3.2. (2S,4R)-Methyl 1-(4-hydroxy-5-methoxy-2-nitrobenzoyl)-4-
(methylsulfonyloxy)pyrrolidine-2-carboxylate (6). To a stirred solu-
tion of compound 5 (4.30 g, 10.0 mmol) in CH2Cl2 (40 mL), Et3N
(4.17 mL, 30.0 mmol) was added at 0 ꢀC and the reaction mixture
was stirred for 30 min and mesylchloride (5.02 mL, 30.0 mmol) was
added drop wisely. Then, the reaction mixture was brought to
ambient temperature and stirred for an overnight to afford mesy-
lated product. The solvent was evaporated and excess of mesyl-
chloride was quenched with aq NaHCO3 solution (40 mL). The
combined reaction mixture was extracted with ethyl acetate
(3ꢄ50 mL) and dried over Na2SO4. The organic layer was evapo-
rated in vacuum and purified by column chromatography using
ethyl acetate/hexane (6:4) to afford the methyl-(2S,4R)-N-(4-ben-
zyloxy-5-methoxy-2-nitrobenzoyl)-4-[(methylsulfonyl)oxy]pyrro-
lidine-2-carboxylate in 88% (4.47 g) yield. 1H NMR (200 MHz,
CDCl3):
d
¼7.64 (s, 1H), 6.78 (s, 1H), 5.21 (m, 1H), 4.83 (t, J¼7.81,
8.59 Hz, 1H), 4.10 (t, J¼6.25 Hz, 2H), 3.98 (s, 3H), 3.84 (s, 3H), 3.59
(m, 2H), 3.42 (t, J¼6.75, 7.03 Hz, 2H), 3.07 (s, 3H), 2.64e2.76 (m,1H),
2.22e2.36 (m, 1H), 1.85e2.05 (m, 4H), 1.62e1.74 ppm (m, 2H); (ESI)
ꢃ
MS: m/z 567 [M]þ
.
6.3.6. (2S,4S)-Methyl
4-azido-1-(4-(3-azidopropoxy)-5-methoxy-
2-nitrobenzoyl)pyrrolidine-2-carboxylate (8a). To (2S,4R)-methyl
1-(4-(3-bromopropoxy)-5-methoxy-2-nitrobenzoyl)-4-(methyl-
sulfonyloxy)pyrrolidine-2-carboxylate (7a,1.50 g, 2.78 mmol) in dry
DMF (15 mL), NaN3 (1.81 g, 27.88 mmol) was added and stirred at
50e60 ꢀC for 6 h, in this transformation both the bromo- and mesyl-
functionalities can take place into azidation. Later, this reaction
mixture was poured into ice-cold water and extracted with ethyl
acetate (3ꢄ50 mL). The organic layer was dried over Na2SO4 and
evaporated in vacuo, and then purified by column chromatography
with ethyl acetate/hexane (7:3) to afford compound 8a (1.28 g, 85%).
CDCl3):
d¼7.79 (s, 1H), 7.30e7.50 (m, 5H), 6.80 (s, 1H), 5.20 (s, 2H),
4.80e4.90 (t, J¼8.60 Hz, 1H), 3.80e4.10 (s, 6H), 3.42e3.60 (s, 2H),
3.09e3.20 (m, 3H), 2.60e2.80 (m, 1H), 2.20e2.40 (m, 1H),
ꢃ
2.20e2.25 ppm (m, 1H); ESI (MS): m/z 509 [M]þ . This above com-
pound (4.40 g, 8.66 mmol) was taken in CH2Cl2 (40 mL), EtSH
(12.35 mL, 173.2 mmol) and BF3$OEt2 (9.39 mL, 86.6 mmol) were
added drop wise at room temperature. Stirring was continued until
TLC indicated completion of the reaction. The solvent was evapo-
rated under vacuum. The residue was quenched with aq NaHCO3
solution (1ꢄ100 mL) and then extracted with ethyl acetate
(3ꢄ50 mL). Later, the combined organic phase was washed with
brine (1ꢄ100 mL), dried over Na2SO4, and the solvent was evapo-
rated in vacuum to afford the crude product. This was further pu-
rified by column chromatography using ethyl acetate/hexane (5:5)
as eluent to give (2S,4R)-methyl 1-(4-hydroxy-5-methoxy-2-
nitrobenzoyl)-4-(methylsulfonyloxy)pyrrolidine-2-carboxylate (6,
1H NMR (300 MHz, CDCl3):
d
¼7.68 (s,1H), 6.91 (s,1H), 4.89 (t, J¼3.77,
5.28 Hz,1H), 4.41 (m,1H), 4.13 (m, 2H), 3.99 (s, 3H), 3.84 (s, 3H), 3.33
(m, 2H), 2.63 (m,1H), 2.42 (m,1H), 2.28 (m,1H),1.95 ppm (m, 3H); IR
(KBr): n
ꢁ 2950, 2860, 2101, 1746, 1651, 1577, 1520, 1421, 1336, 1276,
1215, 1059, 1013, 870, 812, 756, 620, 557 cmꢁ1; (ESI) HRMS: m/z
ꢃ
calcd for C17H20N8O7 449.3386, found 449.3408 [MþH]þ
.
6.3.7. (2S,4S)-Methyl 4-azido-1-(4-(4-azidobutoxy)-5-methoxy-2-
nitrobenzoyl)pyrrolidine-2-carboxylate (8b). Yield 80% (1.01 g). 1H
NMR (300 MHz, CDCl3):
d
¼7.56 (s, 1H), 6.81 (s, 1H), 4.75 (m, 1H),
4.33 (m, 1H), 4.12 (t, J¼5.28 Hz, 2H), 3.93 (s, 3H), 3.77 (s, 3H), 3.34
(m, 2H), 3.31(dd, J¼3.77, 7.55 Hz, 1H), 2.53e2.63 (m, 1H), 2.28e2.44
(m, 1H), 2.09e2.17 (m, 1H), 1.88 (m, 2H), 1.75 ppm (m, 2H); IR (KBr):
nꢁ 2950, 2878, 2104, 1747, 1653, 1577, 1521, 1423, 1337, 1278, 1217,
3.0 g, 83%). 1H NMR (200 MHz, CDCl3þDMSO):
¼7.62 (s, 1H), 6.77
d
(s, 1H), 5.27 (br s, 1H), 4.69 (t, J¼8.05, 8.78 Hz, 1H), 3.97 (s, 3H), 3.79
(s, 3H), 3.65 (dd, J¼3.66, 8.78 Hz, 1H), 3.09 (s, 3H), 2.53e2.72 (m,
1062, 1014, 921, 872, 843, 806, 756, 622, 558 cmꢁ1; (ESI) HRMS: m/z
ꢃ
ꢃ
2H), 2.26e2.40 ppm (m, 2H); (ESI) MS: m/z 419 [M]þ
.
calcd for C18H22N8O7 463.1601, found 463.1633 [MþH]þ
.
6.3.3. (2S,4R)-Methyl 1-(4-(3-bromopropoxy)-5-methoxy-2-nitro-
benzoyl)-4-(methylsulfonyloxy)pyrrolidine-2-carboxylate (7a). To
6.3.8. (2S,4S)-Methyl 4-azido-1-(4-(5-azidopentyloxy)-5-methoxy-
2-nitrobenzoyl)pyrrolidine-2-carboxylate (8c). Yield 88% (0.73 g). 1H

5504
A. Kamal et al. / Tetrahedron 66 (2010) 5498e5506
NMR (300 MHz, CDCl3):
4.91 Hz, 1H), 4.38 (m, 1H), 4.10 (m, 2H), 4.00 (s, 3H), 3.84 (s, 3H),
3.47 (m, 1H), 3.30 (m, 2H), 2.63 (m, 1H), 2.42 (m, 1H), 2.27 (m, 1H),
d
¼7.61 (s,1H), 6.87 (s,1H), 4.85 (dd, J¼4.53,
753 cmꢁ1; (ESI) HRMS: m/z calcd for C22H32N8O5 553.2010, found
ꢃ
553.2026 [MþH]þ
.
1.92 (m, 4H), 1.70 ppm (m, 2H); IR (KBr):
n
ꢁ 2944, 2869, 2100, 1744,
6.3.12. (4-((1-((3S,5S)-5-(Bis(ethylthio)methyl)-1-(4-(3-(4-((4-((S)-
2-(bis(ethylthio)methyl)pyrrolidine-1-carbonyl)-2-methoxy-5-nitro-
phenoxy)methyl)-1H-1,2,3-triazol-1-yl)propoxy)-5-methoxy-2-nitro-
benzoyl)pyrrolidin-3-yl)-1H-1,2,3-triazol-4-yl)methoxy)-5-methoxy-
2-nitrophenyl)((S)-2-(bis(ethylthio)methyl)pyrrolidin-1-yl)
methanone (11a). (2S)-N-[(4-propargyloxy)-5-methoxy-2-nitro-
benzoyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal (9a,
0.50 g, 0.95 mmol) and (2S,4S)-N-[4-(3-azido-1-propoxy)-5-
methoxy-2-nitrobenzoyl]-4-azidopyrrolidine-2-carboxaldehyde
diethyl thioacetal (10, 0.92 g, 2.10 mmol) were suspended in a 1:1
mixture of H2O and t-BuOH (30 mL). Freshly prepared sodium
ascorbate solution (19.0 mg, 5 mol %) was added followed by
CuSO4$5H2O (4.84 mg, 1 mol %). The heterogeneous mixture was
stirred vigorously for 12 h, at which point it cleared and TLC anal-
ysis indicated complete consumption of the reactants. To this re-
action mixture, 2 mL of 3% ammonia solution was added for
quenching of excess CuSO4$5H2O and stirred for further 10 min.
t-BuOH was rotavaporated under reduced pressure, the aqueous
layer was diluted with CHCl3 (50 mL), stirred for another
10e15 min, and then filtered through a Celite bed. The combined
reaction mixture was extracted with CHCl3 (3ꢄ50 mL), washed
with brine, and dried over anhydrous Na2SO4. This was further
purified by column chromatography using ethyl acetate (100%)
afforded 11a in 1.08 g (82%) of pure product as an off-white powder.
1651, 1575, 1518, 1421, 1334, 1275, 1213, 1058, 1011, 871, 809, 757,
619 cmꢁ1; (ESI) HRMS: m/z calcd for C19H24N8O7 499.1927, found
ꢃ
499.1955 [MþNa]þ
.
6.3.9. ((2S,4S)-4-Azido-2-(bis(ethylthio)methyl)pyrrolidin-1-yl)(4-
(3-azidopropoxy)-5-methoxy-2-nitrophenyl)methanone (9a). Diiso-
butylaluminiumhydride solution (6 mL of 1.0 M solution in hexane)
was added drop wise to a vigorously stirred solution of the (2S,4S)-
methyl 4-azido-1-(4-(3-azidopropoxy)-5-methoxy-2-nitrobenzoyl)
pyrrolidine-2-carboxylate (8a, 1.20 g, 2.67 mmol) in dry CH2Cl2
(20 mL) under nitrogen atmosphere at ꢁ78 ꢀC. The reaction mix-
ture was stirred for an additional 45 min and the excess of reagent
was decomposed by careful addition of MeOH (4 mL) followed by
5% HCl (4 mL). The resulting mixture was allowed to warm to room
temperature and the organic layer was removed in vacuum. The
aqueous layer was extracted with ethyl acetate (4ꢄ20 mL), the
organic layers were combined, and dried over Na2SO4. The solvent
was evaporated in vacuum to afford the crude aldehyde approxi-
mately 75% (0.83 g). Without any further purifications, EtSH
(0.35 mL, 4.96 mmol) was added to a stirred solution of nitro-
aldehyde (0.83 g, 1.98 mmol) in dry CH2Cl2 (15 mL) under nitrogen
atmosphere. The mixture was stirred for 30 min followed by the
addition of TMSCl (0.61 mL, 4.95 mmol). After a further 12 h of
stirring, the reaction mixture, when TLC indicated that the reaction
was completed, and then the reaction mixture was carefully
neutralized with aq NaHCO3 (10 mL) solution and extracted with
CHCl3 (2ꢄ10 mL). The combined organic phase was dried over
Na2SO4, evaporated in vacuum, which was further purified by col-
umn chromatography (ethyl acetate) to give compound 9a (0.98 g,
Mp: 125e127 ꢀC. 1H NMR (300 MHz, CDCl3):
d
¼7.88 (s, 1H), 7.87 (s,
1H), 7.78 (s, 1H), 7.73 (s, 1H), 7.64 (s, 1H), 6.84 (s, 3H), 5.33 (s, 2H),
5.30 (s, 2H), 4.91 (m, 1H), 4.86e5.09 (m, 5H), 4.62e4.73 (m, 4H),
4.13 (m, 2H), 3.96 (s, 3H), 3.93 (s, 6H), 3.19e3.32 (m, 4H), 2.97e3.07
(m, 1H), 2.65e2.87 (m, 11H), 2.48e2.57 (m, 2H), 2.21e2.32 (m, 2H),
2.04e2.16 (m, 2H), 1.90e2.01 (m, 1H), 1.75e1.84 (m, 1H), 1.61 (m,
95%). 1H NMR (300 MHz, CDCl3):
d
¼7.69 (s, 1H), 6.81 (s, 1H), 4.78
5H), 1.35 ppm (m, 18H); 13C NMR (125 MHz, CDCl3):
154.4, 154.2, 148.4, 147.4, 137.2, 128.7, 127.1, 109.5, 109.4, 109.1, 108.4,
96.1, 68.8, 63.0, 62.7, 60.9, 59.7, 56.9, 56.2, 52.9, 52.7, 50.0, 49.7, 49.2,
d
¼166.5, 166.1,
(m, 2H), 4.73e4.83 (m, 2H), 4.19 (t, J¼6.04 Hz, 2H), 3.95 (s, 3H), 3.57
(t, J¼6.79, 6.04 Hz, 2H), 3.47 (dd, J¼7.55, 3.02 Hz, 1H), 3.21 (t,
J¼10.57, 9.82 Hz, 1H), 2.65e2.85 (m, 5H), 2.10e2.18 (m, 2H),
27.2, 26.4, 26.1, 25.4, 24.6, 15.0, 14.9 ppm; (ESI) HRMS: m/z calcd for
ꢃ
1.34 ppm (t, J¼7.55 Hz, 6H); 13C NMR (75 MHz, CDCl3):
d
¼166.6,
C60H80N12O15S6 1401.4263, found 1401.4277 [MþH]þ
.
154.7, 148.4, 137.1, 127.5, 109.1, 108.5, 66.2, 59.7, 57.5, 56.4, 53.8, 52.7,
48.0, 32.8, 28.3, 26.3, 15.1 ppm; IR (KBr):
n
ꢁ 2952, 2929, 2873, 2102,
6.3.13. (4-((1-((3S,5S)-5-(Bis(ethylthio)methyl)-1-(4-(4-(4-((4-((S)-
2-(bis(ethylthio)methyl)pyrrolidine-1-carbonyl)-2-methoxy-5-nitro-
phenoxy)methyl)-1H-1,2,3-triazol-1-yl)butoxy)-5-methoxy-2-nitro-
benzoyl)pyrrolidin-3-yl)-1H-1,2,3-triazol-4-yl)methoxy)-5-methoxy-
2-nitrophenyl)((S)-2-(bis(ethylthio)methyl)pyrrolidin-1-yl)
methanone (11b). Yield 78% (0.61 g). Mp: 120e122 ꢀC. 1H NMR
1645, 1577, 1520, 1421, 1337, 1276, 1217, 1065, 868, 812, 755 cmꢁ1
;
(ESI) HRMS: m/z calcd for C20H28N8NaO5 547.1516, found 547.1540
ꢃ
[MþNa]þ
.
6.3.10. ((2S,4S)-4-Azido-2-(bis(ethylthio)methyl)pyrrolidin-1-yl)(4-
(4-azidobutoxy)-5-methoxy-2-nitrophenyl)methanone (9b). Yield
90% (0.53 g). 1H NMR (300 MHz, CDCl3):
¼7.61 (s, 1H), 6.75 (s, 1H),
(300 MHz, CDCl3):
d
¼7.76e7.82 (m, 4H), 7.55 (s, 1H), 6.78 (s, 3H),
d
5.29 (s, 2H), 5.21 (s, 2H), 4.79e5.04 (m, 4H), 4.86e5.09 (m, 5H), 4.64
(m, 2H), 4.49 (m, 2H), 4.10 (m, 2H), 3.95 (s, 3H), 3.92 (s, 3H), 3.90 (s,
3H), 3.37 (t, J¼6.79, 7.55 Hz, 1H), 3.23 (m, 4H), 2.66e2.86 (m, 11H),
1.92e2.36 (m, 7H), 1.74e1.86 (m, 3H), 1.50 (m, 2H), 1.35 ppm (m,
4.77 (m, 2H), 4.08e4.15 (m, 2H), 3.95 (s, 3H), 3.41 (t, J¼6.79, 6.04 Hz,
2H), 3.16 (m, 2H), 2.66e2.81 (m, 5H), 1.93e2.02 (m, 2H), 1.79e1.87
(m, 4H), 1.35 ppm (t, J¼7.55 Hz, 6H); 13C NMR (100 MHz, CDCl3):
d
¼167.2, 155.3, 148.7, 137.3, 128.7, 109.1, 108.6, 68.8, 59.2, 58.8, 56.1,
18H); 13C NMR (75 MHz, CDCl3):
d¼167.1, 166.7, 155.1, 148.5, 147.9,
53.6, 52.9, 52.1, 32.8, 28.6, 26.0, 22.8, 18.2, 14.9 ppm; IR (KBr): nꢁ
146.4, 138.4, 137.1, 128.7, 127.8, 126.2, 111.7, 109.8, 108.1, 67.7, 63.5,
62.8, 61.2, 58.8, 57.1, 56.2, 55.6, 52.8, 50.4, 49.8, 34.5, 28.3, 27.2, 26.2,
2929, 2871, 2102, 1646, 1577, 1520, 1421, 1337, 1275, 1217, 1064, 869,
803, 753 cmꢁ1; (ESI) HRMS: m/z calcd for C21H30N8NaO5 561.1673,
25.4, 24.6, 22.0, 18.4, 15.2, 14.8 ppm; (ESI) HRMS: m/z calcd for
ꢃ
ꢃ
found 561.1697 [MþNa]þ
.
C61H82N12O15S6 1415.4420, found 1415.4407 [MþH]þ
.
6.3.11. ((2S,4S)-4-Azido-2-(bis(ethylthio)methyl)pyrrolidin-1-yl)(4-
(5-azidopentyloxy)-5-methoxy-2-nitrophenyl)methanone (9c). Yield
6.3.14. (4-((1-((3S,5S)-5-(Bis(ethylthio)methyl)-1-(4-(5-(4-((4-((S)-
2-(bis(ethylthio)methyl)pyrrolidine-1-carbonyl)-2-methoxy-5-nitro-
phenoxy)methyl)-1H-1,2,3-triazol-1-yl)pentyloxy)-5-methoxy-2-ni-
trobenzoyl)pyrrolidin-3-yl)-1H-1,2,3-triazol-4-yl)methoxy)-5-me-
thoxy-2-nitrophenyl)((S)-2-(bis(ethylthio)methyl)pyrrolidin-1-yl)
methanone (11c). Yield 88% (0.77 g). Mp: 113e115 ꢀC. 1H NMR
93% (0.67 g). 1H NMR (300 MHz, CDCl3):
d
¼7.60 (s, 1H), 6.74 (s, 1H),
4.66e4.79 (m, 2H), 4.09 (t, J¼5.14, 6.61 Hz, 2H), 3.94 (s, 3H),
3.40e3.49 (m, 1H), 3.32 (t, J¼6.42, 6.61 Hz, 2H), 3.17 (t, J¼10.0 Hz,
1H), 2.62e2.85 (m, 5H), 1.85e1.95 (m, 2H), 1.50e1.74 (m, 6H),
1.35 ppm (t, J¼7.34 Hz, 6H); 13C NMR (75 MHz, CDCl3):
d
¼166.6,
(300 MHz, CDCl3):
d
¼7.79 (m, 2H), 7.66 (s, 2H), 7.50 (s, 1H), 6.76 (m,
154.4, 148.6,137.1,127.1,109.0, 108.1, 69.2, 59.6, 57.4, 56.4, 53.6, 52.6,
51.1, 32.8, 28.5, 26.3, 23.2, 15.1, 14.8 ppm; IR (KBr): nꢁ 2931, 2870,
2102, 1645, 1576, 1520, 1421, 1337, 1276, 1218, 1065, 869, 810,
3H), 5.16e5.30 (m, 5H), 4.77e4.83 (m, 5H), 4.62 (m, 2H), 4.37 (m,
3H), 4.26 (m, 2H), 3.94 (s, 3H), 3.90 (s, 3H), 3.88 (s, 3H), 3.21 (m, 5H),
2.64e2.84 (m, 11H), 2.20e2.34 (m, 4H), 1.66e2.09 (m, 10H), 1.59 (m,

A. Kamal et al. / Tetrahedron 66 (2010) 5498e5506
5505
2H), 1.33 ppm (m, 18H); 13C NMR (75 MHz, CDCl3):
154.5,148.6,147.3,142.6,142.3,138.2,137.0,128.8,127.1,123.0,110.6,
109.3, 108.9, 69.0, 63.0, 60.9, 59.6, 56.4, 53.9, 52.7, 50.1, 29.8, 28.0,
d
¼166.7, 166.3,
(s, 1H), 6.89 (s, 1H), 4.53 (m, 6H), 4.23 (m, 1H), 4.10 (t, J¼6.59,
7.32 Hz, 1H), 3.95e4.02 (m, 2H), 3.90 (s, 3H), 3.87 (s, 6H),
3.69e3.73 (m, 4H), 3.33 (m, 2H), 3.07 (t, J¼7.32, 8.05 Hz, 2H),
2.47e2.54 (m, 2H), 2.32e2.36 (m, 2H), 2.06 (m, 2H), 1.79e1.91 (m,
27.1, 26.4, 26.1, 25.4, 24.5, 23.0, 22.7, 18.3, 14.9 ppm; (ESI) HRMS: m/
ꢃ
z calcd for C62H84N12O15S6 1429.4576, found 1429.4593 [MþH]þ
.
6H), 1.40e1.44 ppm (m, 4H); 13C NMR (100 MHz, CDCl3):
d
¼167.0,
160.2, 159.2, 158.6, 153.9, 151.3, 149.3, 148.5, 147.4, 144.2, 139.6,
133.8, 129.6, 118.7, 114.8, 111.4, 110.0, 107.3, 105.5, 104.9, 68.1, 56.6,
56.1, 55.9, 53.2, 52.4, 50.1, 46.6, 33.0, 31.9, 31.6, 29.5, 27.8, 22.7, 22.3,
6.3.15. (2S,11aS)-7-Methoxy-2-(4-(((S)-7-methoxy-5-oxo-2,3,5,11a-
tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)methyl)-
1H-1,2,3-triazol-1-yl)-8-(3-(4-(((S)-7-methoxy-5-oxo-2,3,5,11a-
tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)methyl)-
1H-1,2,3-triazol-1-yl)propoxy)-2,3-dihydro-1H-benzo[e]pyrrolo
[1,2-a][1,4]diazepin-5(11aH)-one (3a). Compound 11a (100 mg,
0.071 mmol) was dissolved in MeOH (20 mL) by sonication at 25 ꢀC
for 10 min, SnCl2$2H2O (237 mg, 1.07 mmol) was added and
refluxed for 1.0 h or until the TLC indicated that reaction was
complete. The methanol was evaporated by vacuum and the
aqueous layer was then carefully adjusted to pH 8 with 10% NaHCO3
(20 mL) solution and separated the tin salts through Celite which
was extracted with ethyl acetate (3ꢄ30 mL). The combined organic
phase was dried over anhydrous Na2SO4 and evaporated under
vacuum to afford the crude amino diethyl thioacetal 12a (74 mg,
85%). Next step, the diethanthiol deprotective-cyclization reaction
was performed without any further purification. A solution of 12a
(70 mg, 0.53 mmol), HgCl2 (108 mg, 0.40 mmol), and CaCO3 (40 mg,
0.40 mmol) in CH3CN/H2O (4:1, 15 mL) was stirred at room tem-
perature for 12 h until TLC (ethyl acetate) indicates complete loss of
starting material. The reaction mixture was diluted with CHCl3
(20 mL), stirred for another 10 min, and filtered through a Celite
bed. The clear yellow organic supernatant was extracted with sat-
urated 5% NaHCO3 (10 mL) and the combined organic phase was
dried in anhydrous Na2SO4. The organic layer was evaporated in
vacuum and the final crude product was purified by preparative
14.0 ppm; (ESI) HRMS: m/z calcd for C50H56N12O9 999.4477, found
ꢃ
999.4500 [MþCH3OHþH]þ
.
Acknowledgements
The authors Ch.R.R., S.P.R., and N.M.K. are grateful to CSIR, New
Delhi, for the award of Research Fellowships.
References and notes
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TLC, CHCl3/MeOH (95:5) as eluent affords the desired target com-
33
pound 3a (38 mg, 72%). [
a
]
þ307 (c 1.0, CHCl3). 1H NMR
D
(400 MHz, CDCl3):
d
¼7.65e7.82 (m, 5H), 7.49e7.51 (m, 3H), 6.98 (s,
1H), 6.80 (m, 2H), 5.22e5.34 (m, 4H), 4.94 (m, 2H), 4.68 (m, 1H),
4.58 (m, 3H), 3.95 (s, 3H), 3.92 (s, 6H), 3.72e3.84 (m, 4H), 3.52e3.60
(m, 2H), 2.62e2.92 (m, 4H), 2.29e2.43 (m, 4H), 1.85e2.11 ppm (m,
6H); 13C NMR (100 MHz, CDCl3):
d
¼167.2, 163.2, 158.4, 154.2, 152.0,
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46.6, 33.0, 32.4, 29.6, 26.9, 22.5,18.4 ppm; (ESI) HRMS: m/z calcd for
ꢃ
C48H50N12O9 971.4079, found 971.4122 [MþCH3OHþH]þ
.
6.3.16. (2S,11aS)-7-Methoxy-2-(4-(((S)-7-methoxy-5-oxo-2,3,5,11a-
tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)methyl)-
1H-1,2,3-triazol-1-yl)-8-(4-(4-(((S)-7-methoxy-5-oxo-2,3,5,11a-
tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)methyl)-
1H-1,2,3-triazol-1-yl)butoxy)-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a]
[1,4]diazepin-5(11aH)-one (3b). Yield (35 mg, 68%). [
33
a
]
þ291 (c
D
1.0, CHCl3). 1H NMR (400 MHz, CDCl3):
d
¼7.73e7.92 (m, 5H), 7.65 (s,
2H), 7.49 (s, 1H), 6.97 (m, 1H), 6.80 (s, 1H), 6.73 (s, 1H), 5.21e5.32
(m, 4H), 4.93 (m, 2H), 4.67 (m, 2H), 4.46 (m, 2H), 3.94 (s, 3H), 3.91
(s, 6H), 3.70e3.83 (m, 6H), 3.51e3.59 (m, 2H), 2.63e2.90 (m, 4H),
2.29 (t, J¼5.12, 7.32 Hz, 2H), 2.01e2.11 (m, 6H), 1.80 ppm (m, 2H);
17. Jenkins, T. C.; Hurley, L. H.; Neidle, S.; Thurston, D. E. J. Med. Chem. 1994, 37,
4529e4537.
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(ESI) HRMS: m/z calcd for C49H52N12O9 985.4248, found 985.4307
ꢃ
[MþCH3OHþH]þ
.
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tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)methyl)-
1H-1,2,3-triazol-1-yl)-8-(5-(4-(((S)-7-methoxy-5-oxo-2,3,5,11a-
tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)methyl)-
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1H-1,2,3-triazol-1-yl)pentyloxy)-2,3-dihydro-1H-benzo[e]pyrrolo
33
[1,2-a][1,4]diazepin-5(11aH)-one (3c). Yield (42 mg, 75%).
[a]
D
þ312 (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3):
¼8.27 (m, 2H),
d
8.17 (s, 1H), 8.01 (s, 1H), 7.96 (s, 1H), 7.46 (m, 3H), 7.05 (s, 1H), 6.96

5506
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