Tro?ger's base scaffold in racemic and chiral fashion as a spacer for bisdistamycin formation. Synthesis and DNA binding study

Article abstract of DOI:10.1016/j.tet.2006.06.056

'Head-to-head' oligo-N-methylpyrrole peptide dimers linked by a methano[1,5]diazocin scaffold are presented in racemic as well as chiral fashion. Their DNA binding activities were assayed on calf thymus DNA, poly(dA-dT)₂, and poly(dC-dG)_2<

Full text of DOI:10.1016/j.tet.2006.06.056

Tetrahedron 62 (2006) 8591–8600
Tr €o ger’s base scaffold in racemic and chiral fashion as a spacer for
bisdistamycin formation. Synthesis and DNA binding study
a
b
a
a
a
Martin Val ´ı k, Jaroslav Malina, Luk ꢀa sˇ Palivec, Jarmila Folt ꢀy nov ꢀa , Marcela Tkadlecov ꢀa ,
c
b
a,
Marie Urbanov ꢀa , Viktor Brabec and Vladim ´ı r Kr ꢀa l
*
a
Institute of Chemical Technology Prague, Dept. of Analytical Chemistry, Technick aꢀ 5, 166 28 Prague 6, Czech Republic
b
Institute of Biophysics, Academy of Sciences of the Czech Republic, Kr aꢀ lovopolsk aꢀ 135, 612 65 Brno, Czech Republic
Institute of Chemical Technology Prague, Dept. of Physics and Measurements, Technick aꢀ 5, 166 28 Prague 6, Czech Republic
c
Received 21 February 2006; revised 29 May 2006; accepted 15 June 2006
Available online 14 July 2006
Abstract—‘Head-to-head’ oligo-N-methylpyrrole peptide dimers linked by a methano[1,5]diazocin scaffold are presented in racemic as well
as chiral fashion. Their DNA binding activities were assayed on calf thymus DNA, poly(dA-dT) , and poly(dC-dG) by NMR and ECD spec-
2
2
troscopies, and fluorescence probe displacement assay. The presented dimers prefer AT sequences, but show higher affinity to poly(dC-dG)2
than distamycin A. The (4R,9R) configuration of methanodiazocin bridge was found to be better suited for interaction with ct-DNA and
poly(dA-dT)
2
than (4S,9S) configuration.
Ó 2006 Elsevier Ltd. All rights reserved.
1
. Introduction
AT base pairs. In addition, extension of the number of
pyrrole units in distamycin A analogues increased the
sequence specificity for longer tracts of AT rich DNA,
9
,10
The low molecular weight, sequence selective agents that
interact with double-stranded DNA have been studied over
four decades. These molecules are mainly based on natural
products, which bind to the minor groove of DNA. One of
the most studied minor groove binders is distamycin A
and replacing N-methylpyrrole (Py) by N-methylimidazole
(Im) ring changed the selectivity from AT to the cytosine–
1
1
1,12
13,14
guanosine (CG) base pair.
ing
NMR
experiments showed that distamycin A and its
and footprint-
1
5,16
2
1,3–5
(
Fig. 1, 1) and its analogues.
homologues can bind DNA as monomers as well as anti-
parallel dimers.
Distamycin A is naturally occurring antibiotic agent that can
reversibly bind to the minor groove of DNA by hydrogen and
van der Waals bonds, and electrostatic interactions. Solution
Based on this understanding, covalently linked oligopeptide
dimers have been prepared, which were coupled in anti-
parallel ‘head-to-tail’ fashion with various linkers.
6,7
8
4,5,17–19
NMR spectroscopy
and X-ray diffraction studies
established a strong preference of distamycin A for adenine–
thymine (AT) rich sequences containing at least four
These covalent linkages of two oligopeptides form the
ligands with a U-shape motif, which binds to DNA with
both increased affinity and specificity, as compared to the
unlinked oligopeptides. For instance, dimer ImPyPy-
NH2
0
PyPyPy linked by g-aminobutyric acid binds to 5 -TGTTA-
3 with more than 2 orders of magnitude greater affinity
+
0
than the unlinked oligopeptides, ImPyPy and PyPyPy.
NH2
H
H
N
17
HN
O
O
20
21–30
H
N
H
N
The parallel ‘tail-to-tail’ and ‘head-to-head’
linked
N
N
oligopeptide dimers have been designed as bidentate DNA
binders. In the case of ‘head-to-head’ dimeric oligopeptides,
it was found that conformationally constrained cycloalkane
O
N
O
2
8
1
and trans-alkenic linkers prove higher effectivity for their
bidentate binding to DNA than flexible polymethylene
Figure 1.
2
6,27
linked chains. In addition, the stereochemistry of the
linkers is also important and can control the binding.
To the best of our knowledge, only two examples of DNA
binding studies of ‘head-to-head’ dimeric oligopeptides
2
3,29
Keywords: Tr €o ger’s base; Distamycin dimer; Enantioselective binding;
Dissymmetry factor; VCD.
*
Corresponding author. E-mail: kralv@vscht.cz
0
040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.06.056

8592
M. Valı´k et al. / Tetrahedron 62 (2006) 8591–8600
with an optically active linker have been reported to
date. And the problem of ideal shape and stereochemis-
try of linker for bidentate binding is still unclear.
was achieved by nickel boride (Ni B) to give amine hydro-
2
2
3,29
43
chloride 4. Treatment of 4 with aqueous solution of form-
3
9
aldehyde regioselectively gave TB analogue 5. Catalytic
hydrogenation of benzyl ester groups of 5 yielded dicarb-
oxylic derivative 6 that was subsequently converted to active
ester 2 by its reaction with 1-hydroxy benzotriazole (HOBt)
In order to obtain structural information and further insights
into chiral discrimination in the binding of optically active
0
‘
head-to-head’ dimeric oligopeptides to DNA, we report in
and N,N -dicyclohexylcarbodiimide (DCC).
this study a constrained system of dimeric oligo-N-methyl-
pyrrole peptides linked by the methano[1,5]diazocin scaf-
fold, which fashions Tr €o ger’s base (TB) derivatives. The
unique V-shape geometry (81–104 ) of TB and the inherent
chirality have been used in the construction of various recep-
O N
HCl. H N
2
2
ꢀ
OBz a)
OBz
N
N
3
1–33
O
O
tors.
cluding phenanthroline and acridine interact specifically
Moreover, some heterocyclic TB derivatives in-
3
4 92%
3
4–36
and enantioselectively with DNA. The design of pre-
sented oligo-N-methylpyrrole peptide dimers also involved
the width of linkers, which was established to be approxi-
b)
5
R=OBz
c)
d)
O
˚
mately 8 A. The size of linkers should not cause steric re-
6 R=OH, 88%
N
N
straints on binding of proposed dimers to the minor groove
˚
R
2
R=O, 66%
37
R
of DNA that is 10 A wide. In addition, the groove width
in DNA–distamycin complexes was determined from 7 to
N
N
N
N
O
˚
4 A.
13,37,38
N
1
We rationalized that the interconnection of
5
R=OBz, 59%
two distamycin analogues with methanodiazocin scaffold
of TB could afford enantio-selective bidentate minor groove
binders.
Scheme 1. Reagents and conditions: (a) Ni B, aqueous concd HCl, metha-
2
nol, 70 C; (b) aqueous concd H
Pd/C, DMF, rt; (d) DCC, HOBt, DMF, CH
ꢀ
2
CO, aqueous concd HCl, ethanol, rt; (c) H
Cl , rt.
2
2
,
2
2
. Results and discussion
The active ester 2 was then used for the preparation of meth-
anodiazocin bridged dimeric oligo-N-methylpyrrole deriva-
4
0
2
.1. Synthesis
tives via amide protocol with amines 7–10 (see Scheme 2).
Products 11–14 were separated from reaction mixtures by
column chromatography on silica with 5% concd aqueous
ammonia in methanol as an eluent. The most effective DNA-
binder 14 was then prepared in both optically pure forms
(4R,9R)-14 and (4S,9S)-14 by a similar synthetic method.
3
9
As we described previously, direct preparation of such
compounds by methanodiazocin bridging of the correspond-
ing amino-monomers is unsuccessful because degradation
effects predominate. The synthetic strategy is based on the
extension of central 4,9-methano-1,6-dimethyl-4,5,9,10-
0
0
tetrahydro-1H,6H-dipyrrolo-[3,2-b:3 ,2 -f][1,5]diazocin-
,7-dicarboxylate core via amide coupling of active ester
precursor 2 and amino-arm.
Enantiomeric synthesis came from an analogue of benzyl
ester 5, 1-phenylethyl ester 15 that is easily obtainable in
2
4
2
the optically pure state as we have recently described. Con-
version of 1-phenylethyl esters 15a and 15b (Fig. 2) into ac-
tive esters (4R,9R)-2 and (4S,9S)-2 were performed without
separation of acid 6 due to racemization hazard of methano-
diazocin bridge. Note that acid (4R,9R)-6 and (4S,9S)-6
were also prepared due to better band assignment in IR
Active ester precursor 2 was obtained in four steps by reac-
tion sequences shown in Scheme 1. Benzyl 1-methyl-4-
nitropyrrole-2-carboxylate (3) was prepared as described
4
0
41,42
elsewhere. The following reduction
of nitro group
O
N
N
N
H
N
N
N
O
O
+
N
N
N
N
N
N
N
7 n=0
N
O
n
8
n=1
n=2
O
9
2
10 n=3
a)
N
O
N
N
N
N
O
n
N
N
O
N
N
11 n=0, 91%
1
N
N
2 n=1, 67%
3 n=2, 66%
O
1
n
N
14 n=3, 39-72%
ꢀ
Scheme 2. Reagents and conditions: (a) DMF, amine, 60 C.

M. Valı ´k et al. / Tetrahedron 62 (2006) 8591–8600
8593
O
N
N
N
N
O
O
N
N
N
O
1
5a
O
O
O
N
O
15b
Figure 2.
and vibrational CD (VCD) spectra of (4R,9R)-14 and
4S,9S)-14. The optical stability of active esters (4R,9R)-2
and (4S,9S)-2, as well as dimers (4R,9R)-14 and (4S,9S)-
4 was confirmed by ECD (see Fig. 3, ECD spectra of final
(
1
dimer (4R,9R)-14 and (4S,9S)-14) and optical rotation
measurements of both enantiomers.
The structures of all prepared compounds were verified by
detailed analyses of H, C, 1D NOESY, gHSQC, gHMBC,
and gCOSY NMR experiments. The optically pure acids
1
13
(
(
4R,9R)-6 and (4S,9S)-6 and dimers (4R,9R)-14 and
4S,9S)-14 were then investigated by VCD that is a powerful
tool for the description of chiral systems, like prediction of
4
4–47
conformation of macromolecules,
absolute configuration of molecules, and interaction
studies
determination of
4
8
4
9,50
51
of molecules as well as macromolecules.
Figure 4. VCD and IR absorption spectra of (A) (4S,9S)-6 (dashed line) and
(4R,9R)-6 (solid line), and (B) (4S,9S)-14 (dashed line) and (4R,9R)-14
The racemate 14 and enantiomers (4S,9S)-14 and (4R,9R)-
4 provide identical IR absorption spectra in the measured
region 1800–1100 cm (see Fig. 4B). The VCD spectra
(
3 2
solid line), and IR absorption of 14 racemate (solid thin line) in (CD ) SO.
1
ꢁ1
of (4S,9S)-14 and (4R,9R)-14 are nearly symmetrical
However, we were not able to resolve the vibration of
N-methylpyrroles that are bridged by methanodiazocin,
and the vibrations of N-methylpyrroles from the tripeptidic
arms. Moreover, the bands of the methanodiazocin bridge
are overlapped by the bands of N-methylpyrroles and N,N-
dimethylaminopropyl terminals (see Table 1 and Fig. 4B,
Peak no. 4, 7, and 8).
(mirror-image) with respect to baseline. The baseline was
obtained as VCD spectrum of racemate 14 and it was close
to zero. The characteristic vibrations of pyrroles and amide I
ꢁ
1
(1800–1350 cm ) are well separated from the characteristic
vibrations of methanodiazocin bridge (1350–1100 cm ).
5
2
ꢁ1
3 2
Table 1. Characteristic IR vibrations of acid 6 and dimer 14 in (CD ) SO,
ꢁ
1
1
100–1800 cm spectral region
-1
Band No. Wavenumbers [cm ] Characteristic vibrations
Acid 6
Dimer 14
1
1680
1643
C]O stretch: –COOH group
for 6), amide I (for 14)
(
2a
2b
2c
1591
1575
1557
C]C stretch: pyrrole
C]C stretch: pyrrole
C]C stretch:
1550
pyrrole of central core
C]C stretch: pyrrole
2
3
4
5
6
7
8
d
1531
1455
1432
1404
1376
1344
1259
1461
1435
1404
1376
1344
1241
CH
CH
3
asymmetric bend
bend (scissor)
2
Ring vibration: pyrrole
CH
CH
3
symmetric bend
and CH deformation
3
2
C–N stretch: methanodiazocin
bridge, N-methylpyrrole groups+
N,N-dimethylamino group (for 14)
C–N stretch:
methanodiazocin bridge
9
1201
1201
Figure 3. ECD spectra of final dimer (4R,9R)-14 and (4S,9S)-14.

8594
M. Valı´k et al. / Tetrahedron 62 (2006) 8591–8600
Figure 5. VCD dissymmetry factor D3/3 of selected bands of (A) 6 and (B) 14.
IR and VCD spectra of enantiomers of 6, the compound
representing the di-N-methylpyrrolomethanodiazocin core,
were studied in parallel to elucidate the assignment of vibra-
tion bands of 14 (see Fig. 4 and Table 1). Enantiomers
the same and reach about one quarter of the D3/3 value of
methanodiazocin bridge (9). For dimer 14, the D3/3 value
of amide I stretch (1) and the CH symmetrical bend (6)
3
y
are given by summation of contributions of individual
(
4S,9S)-6 and (4R,9R)-6 also provide identical IR absorption
spectra and mirror-image VCD spectra as observed for
4S,9S)-14 and (4R,9R)-14.
amide I and methyl groups, respectively. Both values, there-
fore, represent the average of individual contributions. They
reach only one fifth of the D3/3 value of methanodiazocin
bridge (9).
(
ꢁ1
The absorption band at 1557 cm (2c) was assigned to
N-methylpyrroles of the central core of 14 because this
The transfer of asymmetry from methanodiazocin bridge to
the N-methylpyrrole tripeptide arms of 14 can be clarified
by comparing the relative D3/3 values of C]O stretch (1)
ꢁ
1
band is also present in spectra of 6 at 1550 cm (2c) and
the VCD pattern corresponding to these absorption bands
are almost the same. The other bands (2a, 2b, and 2d)
were assigned to N-methylpyrroles of the tripeptide arms
of 14 and do not possess VCD signals. The bands at 1680
and 1643 cm (1) were assigned to C]O vibration of carb-
oxylic group of 6 and the amide group of 14, respectively.
The CH bend vibrations (3, 6, and 7), the CH bend vibra-
and CH symmetrical bend (6) referred to the D3/3 value of
3
band no. 9 for 14 versus the same values obtained for 6.
There are two possible extreme situations. Firstly, the
asymmetry in 14 is transferred only to the first amide group
of the tripeptidic arms, then the decrease of relative D3/3
values (1 and 6) of 14 versus 6 should be approximately
80%. Secondly, the asymmetry in 14 is transferred to all
amide groups of tripeptidic arms equally, then the relative
D3/3 values (1 and 6) should be the same for 6 and 14. The
approximately 20% reduction of relative D3/3 values (1 and
ꢁ
1
3
2
tions (4 and 7), the ring vibration of pyrroles (5), and C–N
vibrations (8 and 9) were observed in the absorption spectra
of acid 6 as well as dimer 14.
6
) of 14 compared to 6 indicates a descending but impor-
2
.2. Asymmetry transfer evolution
tant influence of the chiral methanodiazocin bridge on
the optical activity in the N-methylpyrrole tripeptidic
arms of 14. On the other hand, partially differentiated
vibrations of the N-methylpyrrole units of 14 (2a, 2b, 2c
and 2d), showing only one VCD signal for N-methyl-
pyrroles of the central core (2c) of 14, is not consistent
with the distribution of asymmetry far from the di-N-methyl-
pyrrolomethanodiazocin core.
5
3,54
Dissymmetry factor
(ratio of intensity of VCD signal
and the intensity of corresponding IR signal, D3/3) values
calculated for characteristic vibrations were used to describe
the distribution of asymmetry in both molecules 6 and 14.
Generally, D3/3 is the highest for chiral center and decreases
with increasing distance from the chiral center. In other
words, the D3/3 values of selected vibrations referred to
the D3/3 value of chiral center, relative D3/3 values represent
the transfer of asymmetry from chiral center to the rest of
molecule. Dissymmetry factors of 6 (Fig. 5A) and 14
In summary, the asymmetry in both 6 and 14 is definitely
transported up to the carboxyl and first amide, respectively.
Distribution of asymmetry to the rest of dimer 14 is then
ambiguous.
(
Fig. 5B) were calculated for characteristic vibrations of
methanodiazocin bridge, C]O, and methyl groups, which
are located in the chiral center and in a different distance
from the chiral core.
y
The IR and VCD signals of all present amide groups as well as methyl
groups are not resolved and are given by summation of all appropriate sig-
nals. This is evident from different ratio between the IR signal intensity of
the methyl groups and the methanodiazocin bridge in the spectra of 6 and
The maximal D3/3 value was observed for the C–N stretch-
ing vibrations of the chiral methanodiazocin bridge (9) in the
spectra of 6 and 14, as expected. The D3/3 values of C]O
ꢁ1
1
4. While the IR signal of 6 at 1376 cm is as intensive as the IR signal of
ꢁ ꢁ1
at 1201 cm and the IR signal of 14 at 1376 cm is three times more
1
1
6
ꢁ
stretch (1) and CH symmetrical bend (6) of 6 are almost
3
intense than IR signal of 14 at 1201 cm
.

M. Valı ´k et al. / Tetrahedron 62 (2006) 8591–8600
8595
2
.3. Binding study
to DNA increases with the number of N-methylpyrrole units
present in the oligopeptides.
2
0
DNA binding activity of dimers 11–14 was assayed on
DNAs by NMR and ECD spectroscopies, and using a fluores-
cence probe displacement assay in deutero-water and deu-
tero-water/deutero-methanol mixture (2:1), and cacodylate
buffer, respectively.
The ECD signal of ct-DNA, the positive and negative bands
5
7
at 274 and 245 nm, is typical for the B-form of DNA and
did not change on adding dimer 11–14 to ct-DNA (see
Fig. 6). So, the interaction between B-DNA and 13, and 14
does not significantly influence the structure of ct-DNA.
NMR experiments were only performed with 11 and 12,
because the solubility of the other dimers (13 and 14) is
limited and these dimers form aggregates in water as
well as in the mixture of water and methanol. Addition
of calf thymus (ct) DNA to the solution of 11 led to disten-
The following ECD spectroscopic measurements were done
for the interactions of dimer 12–14 with poly(dA-dT) and
2
poly(dC-dG) , respectively. As expected, 13-poly(dA-dT)
2
2
and 14-poly(dA-dT)2 exhibited the induced ECD band
above 300 nm. In addition, dramatic changes in the induced
1
sion (line broadening) of all signals of 11 in H spectrum.
The presence of ct-DNA has the biggest influence on the
signals of methylene protons of methanodiazocin bridge.
Distension of signals of 11 indicates an intermediate rate
of exchange between the complexed and free state of 11.
In addition, the hydrogen nuclei of 11 relaxed faster in
the presence of ct-DNA. Generally, shorter relaxation times
signify a decrease in the degree of freedom of molecules,
which is consistent with the complex formation of 11
and ct-DNA. The same results were achieved for dimer
ECD band of 14-poly(dA-dT) were observed at higher drug
2
concentration (c(14):c(poly(dA-dT) ),w1:4). This indicates
2
a two-step binding mode of 14 to poly(dA-dT) . The inten-
2
sity of the induced ECD band in the spectrum of 12-poly(dA-
dT) and 14-poly(dC-dG) was low and reached less than
one sixth of the intensity of ECD band corresponding to
2
2
13-poly(dA-dT) or 14-poly(dA-dT) . The addition of 12
2
2
and 13 to the poly(dC-dG) did not lead to induction of the
2
ECD band above 300 nm.
12 at considerable (approximately 10 times) lower ratio
of ct-DNA and ligand.
The binding stoichiometry (n , number of dimer molecules
b
bound per nucleotide base pair) of 14 to ct-DNA and
poly(dA-dT) was determined by ECD spectral titrations
of DNA with 14. The n was determined as n (the ratio of
b
c(14) to c(DNA)) at the break in the plot of the intensity of
the induced ECD signal at selected wavelength against
On the other hand, dimers 11 and 12 did not provide the
induced ECD band above 300 nm upon their addition to
ct-DNA (see Fig. 6) that is typical for binding of 1 and its
2
5
5,56
analogues to minor groove of ct-DNA.
In addition, the
intensity of the induced ECD band is proportional to the
strength of binding. In the presence of ct-DNA, only dimers
the n. The n values (w0.15 for ct-DNA and w0.12 for
b
poly(dA-dT) ) indicate that DNA saturates at about one
2
1
3 and 14 provided new ECD bands above 300 nm. The
molecule of 14 per six nucleotide base pairs in the case of
ct-DNA and one molecule of 14 per about eight nucleotide
base pairs in the case of poly(dA-dT) . The number of
base pairs bonded by dimer 14 is lower than one would
expect for bidentate binding mode of such compound to
magnitude of the positive ECD band was more than three
times higher in the case of dimer 14 compared to 13. These
results are in agreement with the previous observation that
the strength of binding of distamycin related compounds
2
DNA. In addition, a similar n value was previously obtained
b
2
8
for the interaction of 1 with poly(dA-dT)2. It appears that
dimers 12–14 bind to DNA either through one of their
N-methylpyrrole peptide arms and partial contacts of the
linker or through the terminal segments of both N-methyl-
pyrrole peptide arms only.
The quantification of binding strength of dimers 12–14 to
DNA was done by the competition between compounds 1,
5
8
1
2–14, and ethidium bromide (EB).
Displacement
of ethidium bromide from ct-DNA, poly(dA-dT) , and
2
poly(dC-dG) was accompanied by a decrease in the fluores-
2
cence intensity measured at 595 nm. The apparent binding
constants (Kapp) were calculated from KEB[EB]¼Kapp[drug],
where [drug] is the concentration of 12–14 at a 50% reduc-
5
9
tion of fluorescence and KEB is known. The Kapp values for
1 are close to the data obtained by previous measure-
ments.
2
7,60
The Kapp values for 12–14 calculated by this
way (see Table 2) are consistent with the results obtained
by ECD spectroscopy. The almost identical Kapp values of
12 to all types of studied DNAs show that dimer 12 binds
DNA without any selectivity. Dimers 13 and 14 showed
lower affinity for ct-DNA and poly(dA-dT) than 1, how-
2
ever, they bound significantly stronger to poly(dC-dG)2.
Nevertheless, both compounds exhibit a preference for
AT sequences compared to GC sequences. The strength
Figure 6. ECD spectra of 11–14 in the presence of ct-DNA (solid lines)
and the spectrum of pure ct-DNA (dashed line).

8596
M. Valı´k et al. / Tetrahedron 62 (2006) 8591–8600
5
-1
Table 2. Apparent binding constants, Kapp (ꢂ10 M )
4. Experimental
Compound ct-DNA poly(dA-dT)
2
poly(dC-dG)
2
2
poly(dA-dT) /
poly(dC-dG)
2
4.1. General
1
1
1
1
140ꢃ4
346ꢃ3
2.0ꢃ0.2
1.8ꢃ0.4
20ꢃ2
173
—
1.7
3.7
4.5
3.5
Thin-layer chromatography was performed on Merck Silica
gel 60 F254 TLC plates. For column chromatography the
2
3
4
2.0ꢃ0.5 2.4ꢃ0.5
33ꢃ3
99ꢃ4
34ꢃ4
148ꢃ4
191ꢃ4
128ꢃ3
˚
neutral silica gel SiliTech 32–63, 60 A (ICN Biomedicals)
40ꢃ3
(
(
4R,9R)-14 112ꢃ4
42ꢃ3
1
13
was used. H, C, 1D NOESY, gHSQC, gHMBC, and
gCOSY NMR spectra were obtained with Varian Gemini
4S,9S)-14
90ꢃ3
36ꢃ2
1
13
3
NMR spectra) at 23 C in CDCl or (CD ) SO. Correlation
00 HC (300.1 MHz for H NMR and 75.5 MHz for
ꢀ
C
3
3 2
of binding to DNAs and the preference for AT sequences
increases with the increasing number of pyrrole units in
the dimer. A decrease in the binding affinity and selectivity
of methanodiazocin linked distamycin analogues compared
to distamycin itself confirms the conclusion from the bind-
ing stoichiometry determination, the nonbidentate binding
of dimers 12–14 to DNA. On the other hand, distamycin A
and netropsin exhibit higher binding affinity for ct-DNA
than ‘head-to-head’ bisnetropsin analogues with a 1,2-
cyclopropanedicaraboxamide linker that binds to DNA
with a bidentate binding mode. Nevertheless, all the data
evaluation indicates that the rigid methanodiazocin bridge
is not an optimal spacer molecule for the construction of
bidentate minor groove binders based on ‘head-to-head’
bisdistamycin derivatives.
NMR techniques were applied for the assignment of chemi-
cal shifts to atoms of the molecules. The chemical shifts are
given in parts per million relative to (CH ) Si. For the sake
of clarity in the assignment of NMR spectra, the pyrrole
rings of the dimer 12–14 are numbered from methano-
diazocin bridge to N,N-dimethylaminopropyl terminal.
Mass spectra were recorded with a VG Analytical ZAB-EQ
spectrometer and Biflex mass spectrometer (MALDI-MS).
3
4
Calf thymus DNA (42% C+G, mean molecular mass
w20 MDa) was prepared and characterized as described pre-
2
8
6
1
viously. Homopolymers poly(dA-dT) and poly(dC-dG)
2
2
were purchased from Sigma. The DNA concentrations are
expressed per base pairs.
The ECD spectra were measured at a resolution of 1 nm using
Jasco-810 spectrophotometer using 0.1 cm path length and
three accumulations. The concentration of 11–14 was
0.2 mM. The DNA concentration was 1.6 mM. Cacodylate/
NaCl (0.02/0.1 M) buffer of pH 7.4 was used as a solvent.
The stereochemical control of minor groove binding by the
methanodiazocin bridge on N-methylpyrrole oligopeptide
dimer was studied with the optically pure forms of the
most effective binding dimer 14. The ECD spectral titrations
suggested a similar affinity of (4R,9R)-14 and (4S,9S)-14
to all studied DNAs. The ECD spectra of (4R,9R)-14-
poly(dC-dG) and (4S,9S)-14-poly(dC-dG) after subtrac-
tion of poly(dC-dG) contributions were nearly symmetrical
2
(mirror-image) with respect to base lines. This ‘spectral
symmetry’ explains the small intensity of the ECD band
Fluorescence measurements were performed on a Shimadzu
RF 40 spectrofluorophotometer using a 1 cm quartz cell at
room temperature. The DNA–EB complexes were excited
at 546 nm and the fluorescence was measured at 595 nm.
To the solution of EB and DNA (10 mM Tris pH 7.4,
1 mM EDTA, 1.3 mM EB, and 3.9 mM DNA) were added
aliquots of a 1 mM stock solution of 12–14 and the fluores-
cence was measured after each addition until the fluores-
cence was reduced to 50%.
2
2
above 300 nm at titration of poly(dC-dG) with racemic
2
1
4. Fluorescence probe displacement assay shows that enan-
tiomer (4R,9R)-14 has a slightly higher affinity for all
studied DNAs than enantiomer (4S,9S)-14 (Table 2). In addi-
tion, enantiomer (4R,9R)-14 also exhibited slightly higher
discrimination for poly(dA-dT) versus poly(dC-dG) se-
The VCD and IR absorption spectra were measured at a
resolution of 4 cm using Bruker FTIR IFS 66/S spectro-
2
2
ꢁ
1
quences than enantiomer (4S,9S)-14. The ‘R enantiomeric
discrimination’ has also been described by Lown and
co-workers on a netropsin dimer linked by trans-1,2-cyclo-
6
2
meter equipped with a VCD/IRRAS module PMA 37.
Demountable cell composed of 50 mm Teflon spacer and
2
9
propane.
CaF windows were used. VCD spectra were obtained as
2
the average of 25 blocks of scans, each block counts 3680
scans (20 min). Concentrations of 6 and 14 were 0.16 and
0.04 M ((CD ) SO), respectively. The concentration was
3. Conclusion
3
2
chosen so that the absorbance maxima did not exceed value
of about 0.8 for 50 mm path length in the spectral region
We have described new bisdistamycin analogues with
a TB scaffold in racemic as well as chiral fashion. DNA
binding studies of such compounds showed that the
ꢁ
1
1800–1100 cm
.
‘
head-to-head’ linking of two N-methylpyrrole oligopep-
4.2. Benzyl 1-methyl-4-nitropyrrole-2-carboxylate (3)
tides by the methanodiazocin scaffold caused decreasing
affinity and selectivity of binding to AT sequences, and
increases affinity to CG sequences compared to distamycin
A. Moreover, the directionality in the binding can be con-
trolled by the stereochemistry of our linker. In the case of
the most effective binding dimer 14, the (4R,9R)-form is
better suited for interaction with all studied DNAs than
the (4S,9S)-form.
To a solution of 1-methyl-4-nitro-2-trichloroacetylpyrrole
(30.02 g, 110.57 mmol) in THF (160 ml), a mixture of
benzyl alcohol (30 ml, 290 mmol) and natrium hydride
ꢀ
(0.50 g, 21.7 mmol) in THF (40 ml) was added at 0 C.
The resulting suspension was warmed to room temperature
and stirred overnight. The reaction mixture was neutralized
with TsOH$H O (yellow color changed to white) and the
2

M. Valı ´k et al. / Tetrahedron 62 (2006) 8591–8600
8597
insoluble part was filtered off. The filtrate was concentrated
in vacuo and the residue was diluted with petroleum ether
4.36 (2H, d, J¼16.5, exo CHHN), 5.24 (4H, s, CH Ph),
2
1
3
6.77 (2H, s, CHCCO), 7.36 (10H, m, CH-Ph); C NMR
(CDCl ) d 32.56 (NCH ), 52.21 (CCH N), 65.31 (CH Ph),
68.90 (NCH N), 110.29 (CHCCO), 119.76 (CCO), 127.18
(
300 ml), which caused crystallization of 3. Pale yellow
3
3
2
2
crystals of product 3 (25.12 g, 87%) were filtered off,
washed with petrolether (3ꢂ100 ml), cold methanol
2
(CCH N), 127.86 (o-CH of Ph), 127.97 (p-CH of Ph),
2
128.46 (m-CH of Ph), 131.24 (CNCH ), 136.50 (ipso-C of
2
Ph), 160.90 (CO).
(
2ꢂ50 ml), water (2ꢂ50 ml) and again with methanol
(
2ꢂ50 ml), and dried in vacuo.
ꢀ
d 3.97 (3H, s, NCH ), 5.29 (2H, s, CH ), 7.32–7.43 (5H,
40
ꢀ
1
Mp 109–111 C (lit. mp 112–113 C); H NMR (CDCl )
3
4.5. 4,9-Methano-1,6-dimethyl-4,5,9,10-tetrahydro-
0
0
1H,6H-dipyrrolo-[3,2-b:3 ,2 -f][1,5]diazocin-2,7-dicarb-
oxylic acid (6)
3
2
m, CH-Ph), 7.44 (1H, d, J¼2.2, CHCCO), 7.59 (1H, d,
13
J¼2.2, CHNCH ); C NMR (CDCl ) d 37.83 (NCH ),
3
3
3
6
1
1
6.38 (CH ), 112.76 (CHCCO), 128.05 (o-CH of Ph),
2
To a solution of benzyl ester 5 (0.490 g, 0.99 mmol) in dry
DMF (15 ml), a catalyst (5% Pd/C, 50 mg) was added.
The reaction mixture was treated under hydrogen atmo-
sphere (101.3 kPa) at room temperature for 3 h. The catalyst
was filtered off and the filtrate was evaporated in vacuo to
dryness. Brownish residue was stirred with methanol
(3 ml) for 3 h and the mixture was then filtered to give
acid 6 (275 mg, 88% yield) as a white powder.
22.68 (CCO), 127.66 (CHNCH ), 128.34 (p-CH of Ph),
3
28.55 (m-CH of Ph), 135.09 (CNO ), 135.31 (ipso-C of
2
Ph), 159.36 (CO).
4.3. Benzyl 1-methyl-4-aminopyrrole-2-carboxylate
hydrochloride (4)
Freshly prepared nickel boride Ni B (15.00 g) and 3
2
ꢀ
Mp>200 C (decomp.); H NMR ((CD ) SO) d 3.56 (6H,
3 2
1
(
and 1 M hydrochloric acid (130 ml). The resulting suspen-
15.00 g, 57.63 mmol) were added to methanol (390 ml)
s, NCH ), 3.89 (2H, d, J¼16.6, endo CHHN), 3.94 (2H, s,
3
ꢀ
sion was stirred at 70 C till 3 was no longer present in the
NCH N), 4.27 (2H, d, J¼16.5, exo CHHN), 6.53 (2H, s,
2
1
3
reaction mixture (followed by TLC). For each 1 h, 2 ml of
concd hydrochloric acid were slowly added to the reaction
mixture. The reaction mixture was filtered and the filter
cake was washed with methanol (3ꢂ50 ml). All liquid por-
tions were combined and concentrated in vacuo to 100 ml
CHCCO); C NMR ((CD ) SO) d 32.82 (NCH ), 51.52
3 2 3
(CCH N), 68.28 (NCH N), 109.67 (CHCCO), 119.59
2
(CCO), 127.23 (CCH N), 130.95 (CNCH ), 162.11 (CO);
2
2
2
+
MS (FAB) m/z (%): 317 (100) [M +H]; elemental analysis
calcd (%) for C H N O (316.32): C 56.96, H 5.10, N
17.71; found: C 56.87, H 5.51, N 17.81.
1
5 16 4 4
(
mainly to remove methanol), which caused crystallization
of 4 (10.52 g, 68%) as pale brown crystals. A second crop
of product 4 (3.64 g, 24%) was obtained by concentration
of mother liquor.
4.6. Bis(benzotriazol-1-yl)-4,9-methano-1,6-dimethyl-
0
0
4,5,9,10-tetrahydro-1H,6H-dipyrrolo-[3,2-b:3 ,2 -f]-
1,5]diazocin-2,7-dicarboxylate (2)
[
ꢀ
1
Mp 213–217 C (decomp.); H NMR ((CD ) SO) d 3.84
2
3
(
3H, s, NCH ), 5.23 (2H, s, CH ), 6.83 (1H, d, J¼2.2,
A solution of acid 6 (100 mg, 0.32 mmol) with HOBt
(120 mg, 0.889 mmol) and DCC (165 mg, 0.800 mmol) in
dichloromethane (15 ml) and DMF (2 ml) was stirred for
12 h at room temperature. The insoluble part was filtered off
and the filtrate was evaporated. The residue was chromato-
graphed on silica (gradient from dichloromethane/diethyl
ether 7:3 to diethyl ether) to give active ester 2 (115 mg,
66%), which was further purified by precipitation (dichloro-
methane/diethyl ether).
3
2
CHCCO), 7.27 (1H, d, J¼2.2, CHNCH ), 7.28–7.42 (5H,
3
+
13
m, CH-Ph), 10.25 (3H, br s, NH ); C NMR ((CD ) SO)
3 2
3
d 36.50 (NCH ), 65.15 (CH ), 111.63 (CHCCO), 113.88
3
(
of Ph), 127.96 (p-CH of Ph), 128.40 (m-CH of Ph), 136.13
2
CNH ), 120.56 (CCO), 123.97 (CHNCH ), 127.76 (o-CH
3 3
(ipso-C of Ph), 159.36 (CO); MS (FAB) m/z (%): 231
(100) [M ꢁCl], 243 (10) [M ꢁHCl+Na].
+
+
4
tetrahydro-1H,6H-dipyrrolo-[3,2-b:3 ,2 -f][1,5]diazo-
.4. Dibenzyl-4,9-methano-1,6-dimethyl-4,5,9,10-
0
0
ꢀ
1
Mp>220 C (decomp.); H NMR (CDCl ) d 3.70 (6H,
3
cin-2,7-dicarboxylate (5)
s, NCH ), 4.09 (2H, d, J¼16.8, endo CHHN), 4.20 (2H, s,
3
NCH N), 4.51 (2H, d, J¼16.8, exo CHHN), 7.22 (2H, s,
2
Hydrochloride 4 (5.00 g, 18.75 mmol) was dissolved in
ethanol (50 ml), and then formaldehyde (7.5 ml, 36% aque-
ous solution) and concd HCl (7.5 ml) were added. The reac-
tion mixture was stirred for 24 h at room temperature and
then concentrated under reduced pressure to one half of
the original volume, diluted with water (100 ml), and finally
basified with ammonia solution. The basic mixture (pH 14)
was extracted with dichloromethane (3ꢂ50 ml) and the
combined organic layers were dried over anhydrous
CHCCO), 7.36–7.58 (6H, m, CH-Ar), 8.06 (2H, d, J¼8.3,
1
3
CHCNO); C NMR (CDCl ) d 32.94 (NCH ), 52.60
3
3
(CCH N), 68.77 (NCH N), 108.30 (CHCHCNN), 113.16
2
2
(CHCCO), 113.86 (CCO), 120.35 (CHCNO), 124.61
(CHCHCNO), 128.48 (CHCHCNN), 128.95 (CNN),
131.47 (CCH N), 132.51 (CCH N), 143.32 (CNO), 156.50
2
2
+
(CO); MS (FAB) m/z (%): 551 (65) [M +H], 416 (100)
[M ꢁC H N O]; elemental analysis calcd (%) for
+
6
4 3
C H N O (550.54): C 58.91, H 4.03, N 25.44; found:
2
7 22 10 4
MgSO and evaporated to dryness. The residue was crystal-
4
C 58.82, H 4.18, N 25.30.
lized (dichloromethane/diethyl ether) to give benzyl ester 5
2.75 g, 59%) as colorless crystals.
(
4.6.1. Compound (4R,9R)-2. 1-Phenylethyl ester
(R)(4R,9R)(R)-15a (504 mg, 0.961 mmol) was dissolved in
DMF (10 ml), and then TEA (600 ml) and catalyst (5% Pd/C,
97 mg) were added. The reaction mixture was treated
ꢀ
1
Mp 182–185 C; H NMR (CDCl ) d 3.64 (6H, s, NCH ),
3
3
3
.92 (2H, d, J¼15.9, endo CHHN), 4.09 (2H, s, NCH N),
2

8598
M. Valı´k et al. / Tetrahedron 62 (2006) 8591–8600
0
bonyl-1-methylpyrrol-3-yl)-4,9-methano-1,6-dimethyl-
4,5,9,10-tetrahydro-1H,6H-dipyrrolo-[3,2-b:3 ,2 -f]-
[1,5]diazocin-2,7-dicarboxamide (12)
under hydrogen atmosphere (101.3 kPa) at room tempera-
ture for 3 h. The catalyst was filtered off and the filtrate
was diluted with dichloromethane (50 ml), and then HOBt
4.9. N,N -Bis(5-[3-dimethylaminopropyl]aminocar-
0
0
(
600 mg, 4.445 mmol) and DCC (800 mg, 3.879 mmol)
were added. The reaction mixture was stirred for 12 h at
room temperature and then evaporated in vacuo to dryness.
Dichloromethane (15 ml) was added to the residue and the
insoluble part was filtered off. Filtrate was chromatographed
on silica (gradient from dichloromethane/diethyl ether 7:3 to
diethyl ether) to give active ester (4R,9R)-2 (400 mg, 83%),
which was further purified by precipitation (dichloro-
methane/diethyl ether).
Reaction of amine 8 (176 mg, 787 mmol) with active ester 2
(170 mg, 309 mmol) gave dimer 12 (150 mg, 67%) as a light
yellow powder.
ꢁ
1
nmax (KBr)/cm 3400 (NH), 1636 (C]O), 1578 and 1533
(pyrrole, ring vib.), and 1198 (C–N); H NMR ((CD ) SO)
1
3
2
d 1.59 (4H, quintet, J¼6.9, CH CH N), 2.15 (12H, s,
2
2
N(CH ) ), 2.26 (4H, t, J¼6.6, CH CH N), 3.16 (4H, q,
3
2
2
2
2
0
Compound (4R,9R)-2 has identical NMR spectra as 2; [a]D
62 (c 0.156 g/100 ml DMF).
J¼6.2, CH NH), 3.58 (6H, s, NCH -Py ), 3.76 (3H, s,
2
3
0
+
NCH -Py ), 3.83 (2H, d, J¼16.2, endo CHHN), 3.95 (2H,
3 1
s, NCH N), 4.32 (2H, d, J¼16.2, exo CHHN), 6.68 (2H, s,
2
4
.6.2. Compound (4S,9S)-2. Enantiomer (4S,9S)-2 was
CHCCO-Py ), 6.76 (2H, d, J¼1.9, CHCCO-Py ), 7.12
0
1
prepared by the same synthetic procedure as (4R,9R)-2. Re-
duction of 15b (200 mg, 17.3 mmol) by 5% Pd/C (40 mg)
followed by reaction with HOBt (240 mg, 1.778 mmol)
and DCC (320 mg, 1.551 mmol) gave active ester (4S,9S)-
(2H, d, J¼1.7, CHNCH -Py ), 8.06 (2H, t, J¼5.6, CONH-
3
1
1
1 0 3 2
3
Py ), 9.65 (2H, s, CONH-Py ); C NMR ((CD ) SO)
d 27.02 (CH CH N), 32.18 (NCH -Py ), 35.95 (NCH -
2
2
3
0
3
Py ), 37.00 (CH NH), 45.01 (N(CH ) ), 51.52 (CCH N),
1
2
3 2
2
2
(150 mg, 78%).
56.95 (CH CH N), 68.50 (NCH N), 103.91 (CHCCO-
2 2 2
Py ), 105.52 (CHCCO-Py ), 117.64 (CHNCH ), 122.11
1
0
3
2
0
Compound (4S,9S)-2 has identical NMR spectra as 2; [a]D
–
(CNH-Py ), 122.65 (CCO-Py ), 123.01 (CCO-Py ), 125.00
1
0
1
59 (c 0.409 g/100 ml DMF).
(CCH N), 130.56 (CNCH ), 158.60 (CO-Py ), 161.21
2 2 0
+
(
CO-Py ); MS (MALDI) m/z (%): 729 (100) [M +H]; ele-
1
0
,6-dimethyl-4,5,9,10-tetrahydro-1H,6H-dipyrrolo-
4
.7. N,N -Bis(dimethylaminopropan-3-yl)-4,9-methano-
mental analysis calcd (%) for C H N O (728.90): C
37 52 12 4
60.97, H 7.19, N 23.06; found: C 60.84, H 7.27, N 23.02.
1
0 0
3,2-b:3 ,2 -f][1,5]diazocin-2,7-dicarboxamide (11)
[
0
carbonyl-1-methylpyrrol-3-yl}aminocarbonyl]-1-
4
.10. N,N -Bis(5-[{5-(3-dimethylaminopropyl)amino-
N,N-Dimethyl-propane-1,3-diamine 7 (2 ml) was added to
a DMF (3 ml) solution of TB active ester (150 mg,
methylpyrrol-3-yl)-4,9-methano-1,6-dimethyl-4,5,9,10-
0
0
2
72 mmol) and the mixture was stirred for 5 h at room tem-
tetrahydro-1H,6H-dipyrrolo-[3,2-b:3 ,2 -f][1,5]diazo-
cin-2,7-dicarboxamide (13)
perature. The reaction mixture was evaporated to dryness in
vacuo and the residue was separated by column chromato-
graphy (concd aqueous ammonia/methanol 5:95) to give
desired amide 11 (120 mg, 91%) as white powder.
Reaction of amine 9 (138 mg, 398 mmol) with active ester 2
(90 mg, 163 mmol) gave dimer 13 (105 mg, 66%) as a yellow
powder.
ꢀ
C]O), 1560 and 1535 (pyrrole, ring vib.), and 1198
-1
Mp>220 C (decomp.); n
(KBr)/cm 3350 (NH), 1629
max
ꢁ
1
(
(
nmax (KBr)/cm 3400 (NH), 1640 (C]O), 1581 and 1531
(pyrrole, ring vib.), and 1198 (C–N); H NMR ((CD ) SO)
1
1
C–N); H NMR ((CD ) SO) d 1.55 (4H, quintet, J¼7.2,
3
2
3 2
CH CH N), 2.12 (12H, s, N(CH ) ), 2.23 (4H, t, J¼7.2,
d 1.60 (4H, quintet, J¼7.0, CH CH N), 2.16 (12H, s,
2
2
3 2
2
2
CH CH N), 3.12 (4H, m, CH NH), 3.52 (6H, s, NCH ),
3
N(CH ) ), 2.27 (4H, t, J¼7.0, CH CH N), 3.17 (4H, m,
2
2
2
3 2
2
2
3
4
7
.75 (2H, d, J¼16.2, endo CHHN), 3.89 (2H, s, NCH N),
CH NH), 3.60 (6H, s, NCH -Py ), 3.77 (6H, s, NCH -
2 3 0 3
2
.25 (2H, d, J¼16.2, exo CHHN), 6.46 (2H, s, CHCCO),
Py ), 3.81 (6H, s, NCH -Py ), 3.85 (2H, d, J¼16.5, endo
2
3
1
1
3
.84 (2H, t, J¼5.6, NH); C NMR ((CD ) SO) d 27.17
CHHN), 3.96 (2H, s, NCH N), 4.34 (2H, d, J¼16.0, exo
3
2
2
(
(
(
(
(
CH CH N), 32.03 (NCH ), 36.78 (CH NH), 45.00
2
N(CH ) ), 51.40 (CCH N), 56.80 (CH CH N), 68.47
2
NCH N), 104.75 (CHCCO), 122.84 (CCO), 124.24
2
CCH N), 130.39 (CNCH ), 161.36 (CO); MS (FAB) m/z
2
CHHN), 6.69 (2H, s, CHCCO-Py ), 6.81 (2H, d, J¼1.8,
2
2
3
0
CHCCO-Py ), 6.98 (2H, d, J¼1.8, CHCCO-Py ), 7.16
3
2
2
2
2
1
(2H, d, J¼1.8, CHNCH -Py ), 7.18 (2H, d, J¼1.8,
3
2
CHNCH -Py ), 8.07 (2H, t, J¼5.5, CONH-Py ), 9.72 (2H,
0 1
2
3 1 2
+
13
%): 485 (100) [M +H]; elemental analysis calcd (%) for
s, CONH-Py ), 9.87 (2H, s, CONH-Py ); C NMR
((CD ) SO) d 26.98 (CH CH N), 32.22 (NCH -Py ), 35.97
3 2 2 2 3 0
C H N O (484.65): C 61.96, H 8.32, N 23.12; found: C
2
5
40
8
2
6
1.79, H 8.38, N 23.03.
(NCH -Py ), 36.10 (NCH -Py ), 36.99 (CH NH), 44.96
3 2 3 1 2
(N(CH ) ), 51.55 (CCH N), 56.92 (CH CH N), 68.52
3 2 2 2 2
4
1
.8. General protocol for the preparation of dimers
2–14
(NCH N), 104.03 (CHCCO-Py ), 104.58 (CHCCO-Py ),
2 2 1
105.57 (CHCCO-Py ), 117.77 (CHNCH -Py ), 118.31
0
3
2
(
CHNCH -Py ), 122.15 (CNH-Py +Py ), 122.64 (CCO-
3 1 1 2
The solution of amine (8–10) and TB active ester 2 in DMF
ꢀ
Py ), 122.79 (CCO-Py ), 122.99 (CCO-Py ), 125.07
0 1 2
(CCH N), 130.59 (CNCH ), 158.46 (CO-Py ), 158.65
2 2 1
was stirred for 12 h at 60 C. The reaction mixture was
evaporated to dryness in vacuo and the residue was separated
by column chromatography (concd aqueous ammonia/
methanol 5:95) to give corresponding TB distamycin dimer
(CO-Py ), 161.23 (CO-Py ); MS (MALDI) m/z (%): 973
0 2
+
+
(100) [M +H], 995 (10) [M +Na]; elemental analysis calcd
(%) for C H N O (973.16): C 60.48, H 6.63, N 23.03;
found: C 60.37, H 6.72, N 23.00.
4
9 64 16 6
(
12–14).

M. Valı ´k et al. / Tetrahedron 62 (2006) 8591–8600
8599
0
4
.11. N,N -Bis(5-[{5-[{5-(3-dimethylaminopropyl)-
NR 8355-3/2005, and EU grant ‘CIDNA’ NMP4-CT-2003-
505669 supported the work.
aminocarbonyl-1-methylpyrrol-3-yl}aminocarbonyl]-1-
methylpyrrol-3-yl}aminocarbonyl]-1-methylpyrrol-
3
1
-yl)-4,9-methano-1,6-dimethyl-4,5,9,10-tetrahydro-
0
0
H,6H-dipyrrolo-[3,2-b:3 ,2 -f][1,5]diazocin-2,7-dicarb-
Supplementary data
oxamide (14)
Supplementary data associated with this article can be found
in the online version, at doi:10.1016/j.tet.2006.06.056.
Reaction of amine 10 (310 mg, 662 mmol) with active ester 2
(150 mg, 272 mmol) gave dimer 14 (106 mg, 39%) as a
yellow powder.
References and notes
ꢁ
1
nmax (KBr)/cm 3400 (NH), 1640 (C]O), 1581 and 1531
(
1
pyrrole, ring vib.), and 1198 (C–N); H NMR ((CD ) SO)
3
1. Johnson, D. S.; Boger, D. L. Comprehensive Supramolecular
Chemistry; Murakami, Y., Atwood, J. L., Davies, J. E.,
MacNickol, D. D., V €o gtle, F., Lehn, J.-M., Eds.; Pergamon:
Oxford, 1996; pp 74–160.
2
d 1.63 (4H, quintet, J¼6.9, CH CH N), 2.22 (12H, s,
2
2
N(CH ) ), 2.35 (4H, t, J¼6.9, CH CH N), 3.19 (4H, m,
3
2
2
2
CH NH), 3.62 (6H, s, NCH -Py ), 3.80 (6H, s, NCH -
2
3
0
3
Py ), 3.87 (14H, m, 2ꢂNCH -(Py +Py )+endo CHHN),
2. Arcamone, F.; Nicolell, V.; Penco, S.; Orezzi, P.; Pirelli, A.
Nature 1964, 203, 1064–1065.
3. Baraldi, P. G.; Tabrizi, M. A.; Preti, D.; Fruttarolo, F.; Avitabile,
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3
3
1
2
3
.98 (2H, s, NCH N), 4.36 (2H, d, J¼16.0, exo CHHN),
2
6
.72 (2H, s, CHCCO-Py ), 6.84 (2H, s, CHCCO-Py ), 7.01
0
3
(
2H, s, CHCCO-Py ), 7.04 (2H, s, CHCCO-Py ), 7.18
1 2
(
2H, s, CHNCH -Py ), 7.20 (2H, s, CHNCH -Py ), 7.23
3 3 3 2
(
9
(
2H, s, CHNCH -Py ), 8.08 (2H, t, J¼5.1, CONH-Py ),
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3
1
3
.73 (2H, s, CONH-Py ), 9.89 (2H, s, CONH-Py ), 9.94
0 2
1
3
2H, s, CONH-Py1); C NMR ((CD ) SO) d 26.78
3 2
(
(
CH CH N), 32.21 (NCH -Py ), 35.96 (NCH -Py ), 36.10
3
NCH -Py +Py ), 36.84 (CH NH), 44.73 (N(CH3)2),
3 1 2 2
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2
2
3
0
3
5
1
1.53 (CCH N), 56.73 (CH CH N), 68.53 (NCH N),
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3
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2
2
2
(CHCCO-Py1 or 2), 105.58 (CHCCO-Py ), 117.81 (CHNCH -
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0
3
3
3
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1
1
(
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CCO-Py ), 122.77 (CCO-Py +Py ), 122.95 (CCO-Py ),
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0
1
2
3
1
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25.07 (CCH N), 130.58 (CNCH ), 158.48 (CO-Py1 or 2),
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58.51 (CO-Py1 or 2), 158.65 (CO-Py ), 161.27 (CO-Py );
0
3
+
MS (MALDI) m/z (%): 1217 (100) [M +H], 615 (90)
[M -C H N O ]; elemental analysis calcd (%) for
31 39 9 4
C H N O (1217.42): C 60.18, H 6.29, N 23.01; found:
+
6
1 76 20 8
C 60.04, H 6.34, N 22.84.
1
1
1
1
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4.11.1. Compound (4R,9R)-14. Enantiomer (4R,9R)-14
was prepared using general procedure. Reaction of amine
1
1
1
0 (165 mg, 352 mmol) with active ester (4R,9R)-2 (80 mg,
45 mmol) gave dimer (4R,9R)-14 (91 mg, 51%) as a yellow
powder.
1
2
Compound (4R,9R)-14 has identical NMR spectra as 14;
[
1
2
0
a] –182 (c 0.060 g/100 ml DMF).
D
1
4
.11.2. Compound (4S,9S)-14. Enantiomer (4S,9S)-14 was
prepared using general procedure. Reaction of amine 10
170 mg, 363 mmol) with active ester (4S,9S)-2 (80 mg,
45 mmol) gave dimer (4S,9S)-14 (128 mg, 72%) as a yellow
1
1
2
1
(
1
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powder.
1
465.
Compound (4S,9S)-14 has identical NMR spectra as 14;
[
2
0
21. Dyatkina, N. B.; Roberts, C. D.; Keicher, J. D.; Dai, Y.;
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a] +183 (c 0.055 g/100 ml DMF).
D
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