Synthesis of the new oligopeptide pyrrole derivative isonetropsin and its one pyrrole unit analogue

Article abstract of DOI:10.1016/j.tet.2013.01.076

We have designed and synthesized isonetropsin and its one pyrrole unit analogue in which the amine and carbonyl groups have been switched in positions 2 and 4, respectively instead of 4 and 2 positions of the natural antibiotic netropsin.

Full text of DOI:10.1016/j.tet.2013.01.076

Tetrahedron 69 (2013) 2550e2554
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of the new oligopeptide pyrrole derivative isonetropsin
and its one pyrrole unit analogue
Alessandra Montalbano, Barbara Parrino, Patrizia Diana, Paola Barraja, Anna Carbone,
*
ꢀ
Virginia Spano, Girolamo Cirrincione
Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, STEMBIO, via Archirafi 32, 90123-Palermo, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We have designed and synthesized isonetropsin and its one pyrrole unit analogue in which the amine
and carbonyl groups have been switched in positions 2 and 4, respectively instead of 4 and 2 positions of
the natural antibiotic netropsin.
Received 29 October 2012
Received in revised form 30 December 2012
Accepted 21 January 2013
Ó 2013 Published by Elsevier Ltd.
Available online 1 February 2013
Keywords:
Netropsin
DNA minor groove binders
Isonetropsin
Oligopeptide pyrrole
1. Introduction
On this basis, we have designed and synthesized netropsin
isomers in which the amine and carbonyl groups are switched in
Netropsin (1), formerly named congocidine, is a natural occur-
ring oligopeptide antibiotic isolated in 1951 from Streptomyces
position 2 and 4, respectively instead of the 4 and 2 positions of the
natural antibiotic. In order to evaluate the influence of the position
of these two functional groups, as well as the number of pyrrole
units, we planned the synthesis of the one pyrrole unit derivative 2,
‘isonetropsin analogue’, and of the two pyrrole units derivative,
‘isonetropsin’ 3 (Fig.1).
netropsis.1 Its first total synthesis was reported in 1963 by Julia and
2
ꢁ
Preau-Joseph; a new and more efficient synthesis was described in
1985 by Lown and Krowicki,3 who obtained netropsin through
a seven step procedure in 11% overall yield.
Netropsin strongly binds to the DNA minor groove, showing
high A/T selectivity.4 Sequence specificity and high affinity are due
to a combination of interactions including hydrogen bonding be-
tween the amines and carbonyls of A/T bases and the amides of
netropsin, van der Waals contacts and electrostatic interactions
with the DNA phosphate backbone.5
Netropsin is able to inhibit some DNA-binding enzymes as
bacterial RNA and DNA polymerases, DNAase I, gyrase and mam-
malian topoisomerase I and II,6 and shows antiviral properties. The
antiviral activity is strictly connected to the position of the peptidic
bond. In fact, derivatives bearing the carboxamide groups in posi-
tions 2 and 5 of the pyrrole ring were revealed to be less active than
compounds in which the peptidic bonds were in positions 2 and 4
of the pyrrole ring.7
A great number of analogues of the natural antibiotic were
synthesized and their biological properties were investigated.8
* Corresponding author. Tel.: þ39 09123896810; fax: þ39 09123860854; e-mail
address: girolamo.cirrincione@unipa.it (G. Cirrincione).
Fig. 1. Netropsin 1, isonetropsin analogue 2 and isonetropsin 3.
0040-4020/$ e see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.tet.2013.01.076

A. Montalbano et al. / Tetrahedron 69 (2013) 2550e2554
2551
Table 2
2. Docking
In order to investigate the potential interaction of netropsin
Schematic view of analogue 2 interactions with DNA
Analogue 2
DNA bases
N1
N3
N6
N3 Adenine 15
O2 Thymine 6
O2 Thymine 17
N3 Guanosine 18
N3 Guanosine 18
analogues with the DNA minor groove, docking modelling studies
were performed. The X-ray structure with code 261D9 was selected
from the Protein Data Bank; the DNA fragment was prepared using
Protein Preparation Wizard. Docking was carried out using Glide
software XP mode default parameters.10
N8
Both analogues overlap well with netropsin but analogue 3
seems to be more efficient at interacting with the DNA minor
groove (Fig. 2). Comparing docking score results, isonetropsin an-
alogue 2 shows a lower affinity towards the DNA fragment than
Netropsin whereas isonetropsin 3 shows a docking score value
comparable with that of Netropsin.
3. Results and discussion
The crucial step of the synthetic pathway was the isolation
of the 2-aminopyrrole derivatives. In fact in comparison with
the 3-aminopyrroles used for the total synthesis of netropsin, 2-
aminopyrroles are much more unstable and their isolation and
handling are very difficult. They are liable to oxidation and often
decompose in acid medium, and even during purification processes
on silica gel.11a,b
The key intermediate in the synthesis of the netropsin analogue
is 1-methyl-2-nitropyrrole-4-carboxamidopropionamidine hydro-
bromide (12) (Scheme 1). This synthetic pathway takes place from
1,1,1-trichloro-4-[(2,2-dimethoxyethyl)-amino]but-3-en-2-one (6)
obtained in quantitative yields by reacting aminoacetaldehyde di-
methyl acetal (4) with 4-ethoxy-1,1,1-trichloro-3-buten-2-one (5)
prepared according to the procedure previously reported.12
Derivative 6 was refluxed in acetic acid for 2 h, affording 1H-3-
trichloroacetylpyrrole (7) that was nitrated at ꢀ50 ꢁC with a mix-
ture of nitric acid and acetic anhydride allowing the isolation of
compound 8 in good yield.
Methylation of the pyrrole nitrogen was achieved through re-
action with potassium tert-butoxide, tris[2-(2-methoxyethoxy)
ethyl]amine (TDA-1) and iodomethane; the N-methyl derivative 9
was then condensed with the amidine side chain synthesized as
previously reported;13 after an appropriate work-up of the reaction
mixture it was possible to isolate derivative 12 in good yield.
Derivative 12 was dissolved in ethanol and hydrogenated over
10% Pd on charcoal. The work-up of this reaction was carried out
under argon atmosphere in a glove box. The amine thus obtained
was very unstable turning into a black tar very quickly, so it was
treated with ethanol saturated with gaseous HCl to obtain the
corresponding hydrochloride. Nevertheless, the hydrochloride was
also quite unstable and it decomposed during NMR acquisition.
Therefore the amine 13 was used as formed immediately,
without further purifications and it was condensed with guanidi-
neacetic acid hydrochloride, previously activated with carbon-
yldiimidazole (CDI), affording the one pyrrole unit isonetropsin
analogue 2.
Fig. 2. Superimposition of analogues 2, 3 and Netropsin (red) in the DNA minor groove
fragment.
Analyzing the binding mode of the two analogues, the amide
groups of analogue 3 form bifurcated hydrogen bonds to bases on
opposite strands of two adjacent base pairs in a similar way to
netropsin (Table 1).
Table 1
Schematic view of analogue 3 interactions with DNA
The key intermediate in the synthesis of isonetropsin 3 is
compound 14, which derives from condensation of the amine 13
with another pyrrole unit.
Analogue 3
DNA bases
N1
N3
N3 Adenine 14
O2 Thymine 7
N3 Adenine 15
O2 Thymine 6
O2 Thymine 16
O2 Thymine 17
N3 Guanosine 18
N3 Guanosine 18
First of all we reduced 12, as previously mentioned and we
carried out the reaction between the amine with trichloro de-
rivative 9 but the reaction was not successful; so we tried the
condensation with 1-methyl-2-nitro-1H-pyrrole-4-carboxylic acid
(10) properly activated. This latter was synthesized through alka-
line hydrolysis of derivative 9. Unfortunately the attempt to con-
dense 10, activated with CDI, and the amine 13 was not successful.
Instead carboxylic acid 10, reacted with SOCl2 in refluxing toluene,
to give the acid chloride 11, which was directly condensed with
amine 13 affording the dimer 14 in good yield.
N5
N9
N10
Analogue 2 interacts through hydrogen bonds with the DNA
bases, and due to its reduced length, it is not able to fit well along
the minor groove (Table 2).
On the basis of these docking virtual screening results we
planned the synthesis of both derivatives to test them as new
DNA-interactive compounds.
Derivative 14 was hydrogenated over 10% Pd on charcoal to give
the amine 15, which, without further purifications, was reacted
with guanidineacetic acid hydrochloride as previously described
for derivative 2, affording isonetropsin 3.

2552
A. Montalbano et al. / Tetrahedron 69 (2013) 2550e2554
O
O
O
O
O
O
CCl3
CCl3
O
O
CCl3
i
ii
CCl3
iii
+
O2N
N
H
8
N
H
6
N
NH2
O
H
7
4
5
iv
O
O
NH2
O
CCl3
O
N
H
NH2
R
viii
x
O2N
N
N
H
N
O2N
N
CH3
CH3
R
N
CH3
9
CH3
14 R=NO2
15 R=NH2
10 R=OH
11 R=Cl
xi
ix
v
R=NH2
xii
Br
O
NH2
N
NH2
R=NH2
3
2
H
R
vii
N
CH3
12 R=NO2
13 R=NH2
vi
Scheme 1. Synthesis of isonetropsin analogue 2 and isonetropsin 3. Reagents and conditions: (i) acetonitrile, rt, 24 h, 100%; (ii) acetic acid, reflux, 2 h, 65%; (iii) acetic anhydride,
HNO3 (70%), ꢀ50 ꢁC, 2 h, 60%; (iv) t-BuOꢀKþ, TDA-1, CH3I, toluene, reflux, 8 h, 97%; (v) NaHCO3, (1-amino-3-ammonio-propylidene)ammonium dihydrobromide, water/dioxane
reflux, 9 h, 60%; (vi) H2/PdeC, EtOH; (vii) guanidineacetic acid hydrochloride, CDI, THF, rt, overnight, 48%; (viii) NaOH, EtOH/H2O, then HCl, 61%; (ix) SOCl2, toluene, reflux, 1 h; (x)
13, NaHCO3, dioxane/H2O, 72%; (xi) H2/PdeC, EtOH; (xii) guanidineacetic acid hydrochloride, CDI, THF, rt, overnight, 45%.
Condensation with guanidineacetic acid was confirmed by 1H
NMR measured in DMSO/D2O and by MS spectra. In particular, the
1H NMR spectrum, besides the aromatic pyrrole protons, shows the
N-methyl singlets at 3.00 and 3.01 ppm, two triplets related to the
propionamidine side chain at 2.18 and 2.86 ppm and the singlet due
to the guanidineacetic methylene at 3.17 ppm.
compounds were tested at the NCI of Bethesda against a panel of 60
human tumour cell lines. Unfortunately both derivatives, at a con-
centration of 10ꢀ5 M, showed no significant antiproliferative activity.
5. Experimental section
The MS spectra were recorded infusing a methanol/water (1:1)
solution of derivative 3 (0.01 mg/cm3) into a Triple Quadrupole
thermo TSQ Quantum Ultra EMR equipped with an electrospray
source (ESI). The ESI/MS spectrum shows a peak at m/z¼449 cor-
responding to the hydrated quasi molecular ion [MþH2OþH]þ of
the conjugated base of 3. The presence of water is not surprising, as
ESI is a soft ionization process, and there are fully documented
problems related to high netropsin hygroscopicity; in fact many
formulae were assigned to netropsin and the differences among
them were due to hygroscopicity of the natural antibiotic.11a
The MSeMS spectrum of the [MþH2OþH]þ ion is quite simple
and only shows a fragment ion at m/z¼236 formally corresponding
to an ion having composition C10H14N5O2þ, probably deriving from
hydrogen transposition and cleavage of the carbonylepyrrole bond.
This peak is also accompanied by the corresponding hydrated ion at
m/z¼254 ðC10H16N5O3þÞ.
5.1. Reagents and techniques
Anhydrous organic solvents were prepared by the appropriate
procedurespriortouse. Theotherorganicsolventswerereagentgrade
and used as received. Analytical TLC was performed on Merck Kie-
selgel 60-F254 plates. Column chromatography was performed with
€
Merck silica gel 230e400 mesh ASTM or with a Buchi Sepacor chro-
matography module (prepacked cartridge system). All melting points
were taken ona Buchi-Tottoli capillaryapparatusand are uncorrected;
IR spectra were determined in bromoform with a Jasco FT/IR 5300
spectrophotometer; 1H and 13C NMR spectra were measured in
DMSO-d6 or CDCl3 (Me4Si as internal reference), at 200 and 50.3 MHz,
using a Bruker AC-E series 200 MHz spectrometer. ESI mass spectra
were recorded on a triple-quadrupole TSQ quantum access. Elemental
analyses (C,H,N) were within ꢂ0.4% of the theoretical values.
5.2. Synthetic procedure for the preparation of 2 and 3
4. Conclusion
5.2.1. 1,1,1-Trichloro-4-[(2,2-dimethoxyethyl)amino]but-3-en-2-one
(6). A solution of 4-ethoxy-1,1,1-trichloro-3-buten-2-one (5)
(3.45 g, 16 mmol) and aminoacetaldehyde dimethyl acetal (4)
In summary we have synthesized both isonetropsin, the isomer of
the natural antibiotic, as well as its one-pyrrole unit analogue. Both

A. Montalbano et al. / Tetrahedron 69 (2013) 2550e2554
2553
(1.74 mL, 16 mmol) in acetonitrile (40 mL) was stirred at room
temperature for 24 h. After removing the solvent under reduced
pressure, the title compound 6 was obtained in quantitative yield
(4.42 g, 100%) as a yellow oil [Found: C, 34.50; H, 4.44; N, 5.13.
C8H12Cl3NO3 requires C, 34.75; H, 4.37; N 5.06%]; Rf (CH2Cl2) 0.20;
dC (DMSO-d6) 32.3 (t), 36.0 (t), 38.1 (q), 111.9 (d), 118.0 (s), 132.3 (d),
136.9 (s), 161.8 (s), 168.9 (s).
5.2.6. 1-Methyl-2-aminopyrrole-4-carboxamidopropionamidine hy-
drobromide (13). 1-Methyl-2-nitropyrrole-4-carboxamidopropio-
namidine dihydrobromide (12) (0.75 g, 3.1 mmol) was dissolved in
anhydrous ethanol (40 mL) and hydrogenated over 10% Pd on
charcoal. The catalyst was removed by filtration under argon and
the solvent was removed in vacuo. Because of its instability, the
amine was used immediately, without further purifications.
ymax(liquid film): 3415 (NH) and 1646 (C]O) cmꢀ1
; dH (CDCl3)
3.38e3.43 (8H, m), 4.42 (1H, t, J 5.2 Hz), 5.70 (1H, d, J 7.4 Hz), 7.11
(1H, dd, J 7.4 and 5.2 Hz), 9.61 (1H, br s, NH); dC (CDCl3) 51.2 (t), 54.9
(qꢃ2), 96.7 (s), 85.3 (d), 103.1 (d), 158.0 (d), 182.3 (s).
5.2.2. 1H-3-Trichloroacetylpyrrole (7). A solution of (6) (4.42 g,
16 mmol) was refluxed in acetic acid (16 mL) for 2 h. The reaction
mixture was poured into crushed ice, filtered off and dried to afford
the desired product (2.21 g, 65%) as a green solid [Found: C, 33.68;
H, 2.06; N, 6.41. C6H4Cl3NO requires C, 33.92; H, 1.90; N, 6.59%]; Rf
(CH2Cl2) 0.32; mp 119 ꢁC; ymax(liquid film): 3446 (NH) and 1683
5.2.7. 1-Methyl-2-(guanidineacetamido)pyrrole-4-carboxamido-
propionamidine hydrobromide hydrochloride (2). Guanidineacetic
acid hydrochloride (0.23 g, 1.7 mmol) was dissolved in anhydrous
THF (10 mL) and CDI (0.27 g, 1.7 mmol) was added. The mixture was
stirred at room temperature over night under nitrogen. A solution of
1-methyl-2-aminopyrrole-4-carboxamidopropionamidine hydro-
bromide (13) (2.6 mmol) prepared as mentioned above in THF/di-
oxane (1:1) (10 mL) was added and the mixture was stirred at room
temperature overnight. The crude material was filtered off under
argon atmosphere in a glove box to afford the desired product
(0.35 g, 48%) as a brownish solid [Found: C, 33.62; H, 5.34; N, 26.45.
C12H22BrClN8O2 requires C, 33.86; H, 5.21; N, 26.32%]; Rf (CH2Cl2/
MeOH¼1:1) 0.37; mp 211.5 ꢁC; ymax(liquid film): 3386 (NH2) and
(C]O) cmꢀ1
; dH (CDCl3) 6.81e6.84 (1H, m), 6.88e6.91 (1H, m),
7.80e7.82 (1H, m), 9.06 (1H, br s, NH); dC (CDCl3) 96.4 (s), 111.9 (d),
115.4 (s), 119.4 (d), 127.8 (d), 177.6 (s).
5.2.3. 1H-2-Nitro-4-trichloroacetylpyrrole (8). To a solution of 1H-
3-trichloroacetylpyrrole (7) (1.00 g, 4.7 mmol) in acetic anhydride
(6 mL) a mixture of nitric acid (70%, 0.6 mL) and acetic anhydride
(2.7 mL) was added dropwise at ꢀ50 ꢁC. The solution was stirred for
2 h, allowing the temperature reach 0 ꢁC, then it was poured into
crushed ice, extracted with CH2Cl2 (3ꢃ50 mL) and dried over
Na2SO4. The solvent was evaporated in vacuo and the residue ob-
tained was purified by Sepacor chromatography apparatus using
CH2Cl2 as eluant, to yield the title compound 8 (0.73 g, 60%) as
a yellow solid [Found: C, 28.13; H, 1.25; N, 10.70. C6H3Cl3N2O3 re-
quires C, 27.99; H, 1.17; N, 10.88%]; Rf (CH2Cl2) 0.20; mp 113 ꢁC;
1671 (C]O) cmꢀ1
; dH (DMSO-d6) 2.16 (2H, t, J 2.0 Hz, CH2), 2.84 (2H,
t, J 2.0 Hz, CH2), 3.88 (2H, d, J 6.0 Hz, CH2), 4.02 (3H, s, CH3), 7.19e9.53
(13H, m, H-3, H-5, 2ꢃNH2, 3ꢃNH, 2 ꢃ NH2þ). dC (DMSO-d6) 32.4 (q),
43.4 (t), 60.0 (t), 72.2 (t),121.2 (s), 126.6 (s), 129.7 (d),135.0 (d),157.8
(s), 166.6 (s), 170.2 (s), 172.2 (s).
5.2.8. 1-Methyl-2-nitro-1H-pyrrole-4-carboxylic acid (10). A solu-
tion of NaOH (0.22 g, 5.3 mmol) in 25 mL of water was added to 1-
methyl-2-nitro-4-trichloroacetylpyrrole (9) (0.78 g, 2.9 mmol) in
ethanol (6 mL). The reaction mixture was stirred for 2 h at room
temperature. After removing ethanol, HCl was added until pH 2.0.
The desired product was filtered off, dried into the desiccator, to
afford the desired product as a brownish solid (0.30 g, 61%)
[Found: C, 42.04; H, 3.70; N, 16.21. C6H6N2O4 requires C, 42.36;
H, 3.55; N, 16.47%]; Rf (CH2Cl2/MeOH¼8:2) 0.30; mp 223 ꢁC;
ymax(liquid film): 3305 (NH) and 1702 (C]O) cmꢀ1
; dH (CDCl3) 7.75
(1H, s), 7.89 (1H, s), 10.31 (1H, br s, NH); dC (CDCl3) 95.1 (s), 112.6 (d),
116.8 (s), 129.5 (d), 137.8 (s), 176.4 (s).
5.2.4. 1-Methyl-2-nitro-4-trichloroacetylpyrrole (9). 1H-2-Nitro-4-
trichloroacetylpyrrole (8) (0.50 g, 1.9 mmol) was dissolved in an-
hydrous toluene (80 mL) and potassium tert-butoxide (0.30 g,
2.7 mmol) and a catalytic amount of TDA-1 were added. The re-
action mixture was stirred 2 h at room temperature. Iodomethane
(0.36 mL, 2.9 mmol) was added and the mixture was refluxed for
8 h. The solvent was removed under reduced pressure, the crude
solid was washed with CH2Cl2 and filtered off. The organic layer
was evaporated and the oil obtained was purified by column
chromatography on silica gel using CH2Cl2 as eluant to yield the title
compound 9 (0.51 g, 97%) as a brown solid [Found: C, 30.70; H, 2.13;
N, 10.40. C7H5Cl3N2O3 requires C, 30.97; H, 1.86; N, 10.32%]; Rf
ymax(liquid film): 3500, 3100 (OH) and 1687 (C]O) cmꢀ1
; dH
(DMSO-d6) 3.98 (3H, s, CH3), 7.40 (1H, d, J 2.2 Hz), 7.86 (1H, d, J
2.2 Hz), 12.67 (1H, s, OH); dC (DMSO-d6) 38.1 (q), 113.4 (d), 114.9
(s), 133.7 (d), 137.5 (s), 163.6 (s).
5.2.9. 1-Methyl-2-(1-methyl-2-nitropyrrole-4-carboxamido)-pyr-
role-4-carboxamidopropionamidine hydrochloride (14). To a solution
of 1-methyl-2-nitro-1H-pyrrole-4-carboxylic acid (10) (0.51 g,
3.0 mmol) in anhydrous toluene (5 mL), thionyl chloride (0.7 mL,
10 mmol) was added and the reaction mixture was refluxed for 1 h.
After removing the solvent under reduced pressure, the acyl chloride
was afforded as a brown solid that was used without further puri-
fication. 1-Methyl-2-nitropyrrole-4-carboxamidopropionamidine
hydrobromide (12) (0.99 g, 3.1 mmol) was dissolved in anhydrous
ethanol (40 mL) and hydrogenated over 10% Pd on charcoal. The
catalyst was removed by filtration under argon and the solvent was
evaporated in vacuo; because of its instability the amine was used
immediately without further purification.
The acyl chloride was dissolved in dioxane (5 mL) and added
dropwise to the solution of the amine and NaHCO3 (0.5 g, 6.0 mmol)
in water (5 mL). The reaction mixture was stirred under nitrogen
overnight at room temperature; 1 M HCl was added until reaching
pH¼3.0, the solvent was evaporated under reduced pressure and the
crude product was purified by Sepacor chromatography apparatus
using CH2Cl2/MeOH¼9:1 as eluant. The title compound 14 was
afforded as a yellow solid (0.85 g, 72%) [Found: C, 45.51; H, 4.96;
N, 24.53. C15H20ClN7O4 requires C, 45.29; H, 5.07; N, 24.65%];
(CH2Cl2) 0.52; mp 78 ꢁC; ymax(liquid film): 1698 (C]O) cmꢀ1
; dH
(CDCl3) 4.11 (3H, s, CH3), 7.70 (1H, br s), 7.81 (1H, br s); dC (CDCl3)
39.2 (q), 95.1 (s), 113.4 (s), 115.6 (d), 134.7 (d), 138.1 (s), 175.8 (s).
5.2.5. 1-Methyl-2-nitropyrrole-4-carboxamidopropionamidine hy-
drobromide (12). (1-Amino-3-ammoniopropylidene)ammonium
dihydrobromide13 (0.47 g, 1.9 mmol) and NaHCO3 (0.16 g, 1.9 mmol)
in water (7 mL) and dioxane (10 mL) were added, dropwise, to
a solution of 1-methyl-2-nitro-4-trichloroacetylpyrrole (9) (0.51 g,
1.9 mmol) in dioxane (20 mL). The mixture was refluxed for 9 h and
after removal of the solvent under reduced pressure, the residue
was purified by Sepacor chromatography apparatus using CH2Cl2/
MeOH¼9:1 as eluant. The title compound 12 (0.36 g, 60%) was ob-
tained as a white solid [Found: C, 33.96; H, 4.56; N, 21.70.
C9H14BrN5O3 requires: C, 33.76; H, 4.41; N, 21.88%]; Rf (CH2Cl2/
MeOH¼7:3) 0.36; mp 235 ꢁC; ymax(liquid film): 3228e3127 (NH,
NH2), and 1704 (C]O) cmꢀ1
; dH (DMSO-d6) 2.64e2.70 (2H, m, CH2),
3.53e3.61 (2H, m, CH2), 3.98 (3H, s, CH3), 7.76 (1H, s), 7.84
(1H, s), 8.58e8.69 (3H, m, NH2 and NH), 9.06 (2H, br s, NH2þ);

2554
A. Montalbano et al. / Tetrahedron 69 (2013) 2550e2554
Rf (CH2Cl2/MeOH¼8:2) 0.25; mp >300 ꢁC; ymax(liquid film):
(MHþþH2O-42%); ESI/MS-MS m/z 449 (MHþþH2O-20%), m/z 254
ðC10H16N5O3þ ꢀ 1%Þ, m/z 236 ðC10H14N5O2þ ꢀ 100%Þ.
3237e3020 (NH and NH2) and 1641 (C]O) cmꢀ1
; dH (DMSO-d6) 2.74
(2H, br s, CH2), 3.08 (2H, br s, CH2), 3.46 (3H, s, CH3), 4.02 (3H, s, CH3),
6.43 (1H, s), 7.36 (1H, s), 7.95 (1H, s), 8.07 (1H, s), 8.15 (1H, t, J 5.7 Hz,
NH), 8.70 (2H, br s, NH2), 9.13 (2H, br s, NH2þ), 9.98 (1H, s, NH). dC
(DMSO-d6) 32.4 (t), 33.2 (q), 35.8 (t), 38.3 (q),103.3 (d),112.6 (d),116.6
(s), 117.2 (s), 122.4 (d), 126.1 (s), 133.1 (d),137.2 (s), 161.8 (s), 164.0 (s),
169.3 (s).
5.3. Docking
The reported X-ray structure, in which a decamer DNA fragment
is cocrystalized with Netropsin, was downloaded from Protein Data
Bank (http://www.rcsb.org/pdb; PDB code 261D).9
Docking experiments were performed using GLIDE 15 soft-
5.2.10. 1-Methyl-2-(1-methyl-2-aminopyrrole-4carboxamido)-pyr-
role-4-carboxamidopropionamidine hydrochloride (15). 1-Methyl-2-
(1-methyl-2-nitropyrrole-4carboxamido)-pyrrole-4-carboxamido-
propionamidine hydrochloride (14) (0.45 g, 1.1 mmol) was dis-
solved in anhydrous ethanol (40 mL) and hydrogenated over 10%
Pd on charcoal. The catalyst was removed by filtration under
argon and the solvent was evaporated in vacuo. Because of its
instability the amine 15 was used immediately without further
purifications.
ware,10 using its XP default parameters.
To validate the Glide docking protocol, Netropsin, the co-
crystallized ligand, was docked into the minor groove binding
site. The docked structure was compared with the crystal structure
showing that this protocol successfully reproduces the crystal DNA-
Netropsin complex.
References and notes
1. Finlay, A. C.; Hochstein, F. A.; Sobin, B. A.; Murphy, F. X. J. Am. Chem. Soc. 1951,
73, 341.
2. Julia, M. M.; Preau-Joseph, N. Comp. Rendus 1963, 257, 1115.
5.2.11. 1-Methyl-2-(1-methyl-2-(guanidineacetamido)pyrrole-4car-
boxamido)-pyrrole-4-carboxamidopropionamidine dihydrochloride
(3). Guanidineacetic acid hydrochloride (0.10 g, 0.7 mmol) was
dissolved in anhydrous THF (5 mL) and CDI (0.25 g, 1.7 mmol) was
added. The mixture was stirred at room temperature over night
under nitrogen; the solvent was removed under reduced pressure.
The crude material was dissolved in anhydrous dioxane (30 mL)
and the solution was added dropwise, to the amine 15 prepared as
above, dissolved in dioxane (15 mL) and methanol (6 mL), under
nitrogen. The reaction mixture was stirred at room temperature
overnight and then the solid filtered off, washed with ethyl acetate
under nitrogen to afford the title compound 3 as a brown solid
(0.17 g, 45%) [Found: C, 42.75; H, 5.83; N, 27.60. C18H28Cl2N10O3
requires C, 42.95; H, 5.61; N, 27.82%]; Rf (CH2Cl2/MeOH¼1:1) 0.56;
mp>300 ꢁC; ymax(liquid film): 3417 (br s NH2), 1697 (C]O) and
ꢁ
3. Lown, J. W.; Krowicki, K. J. Org. Chem. 1985, 50, 3774.
€
€
4. Zimmer, C.; Reinert, K. E.; Luck, G.; Wahnert, U.; Lober, G.; Thrum, H. J. Mol. Biol.
1971, 58, 329.
5. Degtyareva, N. N.; Fresia, M. J.; Petty, J. T. Biochemistry 2007, 46, 15136.
6. Sukhanova, A.; Grokhvsky, S.; Ermishov, M.; Mochalov, K.; Zhuze, A.; Oleinikov,
V.; Nabiev, I. Biochem. Pharmacol. 2002, 64, 79.
7. Bialer, M.; Yagen, B.; Mechoulam, R.; Becker, Y. J. Pharm. Sci. 1980, 69, 1334.
8. For example, see: (a) Bailly, C.; Chaires, B. J. Bioconjug. Chem. 1998, 9, 513; (b)
Andronova, V. L.; Grokhovsky, S. L.; Surovaya, A. N.; Arkhipova, V. S.; Gursky, G. V.;
Galegov, G.A. Biochem.Biophys. 2008, 422, 296; (c) Bielawski, K.; Bielawska, A. Chem.
Med. Chem. 2008, 3, 536; (d) Anthony, N. G.; Breen, D.; Clarke, J.; Donoghue, G.;
Drummond, A. J.; Ellis, E. M.; Gemmell, C. G.; Helesbeux, J.; Hunter, I. S.; Khalaf, A. I.;
Mackay, S. P.;Parkinson, J. A.; Suckling, C. J.;Waigh, R. D. J. Med. Chem. 2007, 50, 6116.
9. Nunn, C. M.; Garman, E.; Neidle, S. Biochemistry 1997, 36, 4792.
€
10. 15 GLIDE Version 4.5; Schrodinger LLC: New York, NY, 2007.
11. (a) Cirrincione, G.; Almerico, A. M.; Aiello, E.; Dattolo, G. Chem. Heterocycl.
Compd.dPyrroles 1992, 48 part II, 299; (b) Cirrincione, G.; Almerico, A. M.;
Diana, P.; Barraja, P.; Mingoia, F.; Grimaudo, S.; Dattolo, G.; Aiello, E. J. Hetero-
cycl. Chem. 1996, 33, 161.
12. Tietze, L.; Meier, H.; Vob, E. Synthesis 1988, 274.
13. Hilgetag, G.; Paul, H.; Gunther, J.; Witt, M. Chem. Ber. 1964, 97, 704.
1633 (C]O) cmꢀ1
; dH (DMSO-d6 and D2O) 2.18 (2H, t, J 2.0 Hz, CH2),
2.86 (2H, t, J 2.0 Hz, CH2), 3.00 (3H, s, CH3), 3.01 (3H, s, CH3), 3.17
(2H, s), 7.03 (2H, s), 7.67 (1H, s), 8.39 (1H, s). ESI/MS m/z¼449