A general solution- and solid-phase synthetic procedure for incorporating three contiguous imidazole moieties into DNA sequence reading polyamides

Full text of DOI:10.1021/jo001034s

1030
J . Org. Chem. 2001, 66, 1030-1034
studies of Pelton and Wemmer11-13 showed that at
A Gen er a l Solu tion - a n d Solid -P h a se
sufficiently high drug to DNA ratios two distamycin
molecules can be located simultaneously in the minor
groove in a highly overlapped antiparallel side by side
manner. The distamycin units can be linked in two
different ways as a hairpin14 (I) or in a cross-linked
(stapled)15,16 (II) manner (Figure 1). Alternative ap-
proaches developed by Dervan17 and Lown18 for the
design of sequence-specific DNA binding molecules that
recognize longer sequences of double-helical DNA is to
couple DNA binding units of similar or diverse base pair
specification as extended bispolyamides tethered by
linkers of appropriate shape and dimensions III (Figure
1). These extended bispolyamides can then address the
problems of mismatching and phasing since by selecting
a linker of appropriate length the semantophoric moieties
may be brought into register. A number of pyrrolobis-
polyamides tethered by various linkers with varying
geometry and length which have diverse activities have
been synthesized in our group.18 For example, a number
of them were active inhibitors of HIV-1 integrase.18b The
biological activities of pyrrolobispolyamides differed mark-
edly depending on the geometrical configuration of the
two polyamides units, and the agents bearing a para
configuration were considerably more potent than those
with the meta configuration. Recently it has been deter-
mined that geometrically constrained bis- distamycins
are inhibitors of HIV-1 reverse transcription RNA di-
rected DNA polymerization18e and bind selectively to
Okazaki fragments with a specific orientation of the
linkers.18c It was evident from results of Pommier et al.18b
that the effective inhibitory concentrations of the tripyr-
rolobispyrroloamides were approximately 200-fold higher
for the artificial control GC containing sequences than
with the natural sequences containing the conserved AT
stretch.
Syn th etic P r oced u r e for In cor p or a tin g
Th r ee Con tigu ou s Im id a zole Moieties in to
DNA Sequ en ce Rea d in g P olya m id es
Sanjay K. Sharma, Manju Tandon, and
J . William Lown*
Department of Chemistry, University of Alberta,
Edmonton, AB, Canada T6G 2G2
annabelle.wiseman@ualberta.ca
Received J uly 10, 2000
In tr od u ction
Efforts to discover a universal set of chemical rules for
the digital readout of information from double helical
DNA by synthetic agents have met with encouraging
success.1,2 Small molecules that target specific DNA
sequences have the potential to control gene expression.3
Polyamides containing N-methylpyrrole and N-meth-
ylimidazole moieties, related to the naturally occurring
oligopeptidic antibiotics netropsin4 and distamycin,5,6 are
synthetic ligands that have an affinity for DNA compa-
rable with naturally occurring DNA binding proteins.2,7
The DNA sequence specificity of these small molecules
is controlled, in part, by the linear sequence of pyrrole
and imidazole units. The natural products netropsin and
distamycin bind tightly and specifically to regions with
four and five AT base pairs,8 respectively. However in
an attempt to recognize and bind longer sequences of
DNA the empirical n + 1 rule9 cannot be extended
indefinitely because as the number of repeating units
increases the mismatching becomes more severe (called
the phasing problem) and this is manifested by a trend
of weaker binding with higher homologues.10 The NMR
Dickerson19 and Lown20 first conceived the idea of
“lexitropsin” longer chain analogues of netropsin which
retain pyrrole groups at those positions where an AT base
pair was to be read but substituted an imidazole group
(1) (a) Mosier, E. D.; Dervan, P. B. Science 1987, 238, 645. (b)
LeDoan, T.; Perrouault, L.; Praseuth, D.; Habhoula, N.; Decout, J . L.;
Thoung, N. T.; Chomme, J .; Helene, C. Nucleic Acids Res. 1987, 15,
1749. (c) Thoung, N. T.; Helene, C. Angew. Chem., Int. Ed. Engl. 1993,
32, 666.
(2) (a) Wade, W. S.; Mrksich, M.; Dervan, P. B. J . Am. Chem. Soc.,
1992, 114, 8783. (b) Mrksich, M.; Wade, W. S.; Dwyer, T. J .; Geier-
stanger, B. H.; Wemmer, D. E.; Dervan, P. B. Proc. Natl. Acad. Sci.
U.S.A. 1992, 89, 7586. (c) Wade, W. S.; Mrksich, M.; Dervan, P. B.
Biochemistry 1993, 32, 11385. (d) Trauger, J . W.; Baird, E. E.; Dervan,
P. B. Nature 1996, 382, 559. (e) Swalley, S. E.; Baird, E. E.; Dervan,
P. B. J . Am. Chem. Soc. 1997, 119, 6953. (f) Turner, J . M.; Baird, E.
E.; Dervan, P. B. J . Am. Chem. Soc. 1997, 119, 7636. (g) Trauger, J .
W.; Baird, E. E.; Dervan, P. B. Angew. Chem. 1998, 37, 1421. (h)
Turner, J . M.; Swalley, S. E.; Baird, E. E.; Dervan, P. B. J . Am. Chem.
Soc. 1998, 120, 6219. (i) White, S.; Szewczyk, J . W.; Turner, J . M.;
Baird, E. E.; Dervan, P. B. Nature 1998, 391, 468. (j) Kielkopf, C. L.;
White, S.; Szewczyk, J . W.; Turner, J . M.; Baird, E. E.; Dervan, P. B.;
Rees, D. C. Science 1998, 282, 111.
(3) (a) Gottesfeld, J . M.; Nealy, L.; Trauger, J . W.; Baird, E. E.;
Dervan, P. B. Nature 1997, 387, 202. (b) Dickinson, L. A.; Gulizia, R.
J .; Trauger, J . W.; Baird, E. E.; Mosier, E. D.; Gottesfeld, J . M.; Dervan,
P. B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12890.
(4) J ulia, M.; Preau-J oseph, N. Bull. Soc. Chim. Fr. 1967, 4348.
(5) Arcamone, F.; Orezzi, P. G.; Barbieri, W.; Nicolella, V.; Penco,
S. Gazz. Chim. Ital. 1967, 97, 1097.
(11) Pelton, J . G.; Wemmer, D. E. Proc. Natl. Acad. Sci. U.S.A. 1989,
86, 5723.
(12) Pelton, J . G.; Wemmer, D. E. J . Am. Chem. Soc. 1990, 112, 1393.
(13) Fagan, P.; Wemmer, D. E. J . Am. Chem. Soc. 1992, 114, 1080.
(14) Mrksich, M.; Parks, M. E.; Dervan, P. B. J . Am. Chem. Soc.
1994, 116, 7983.
(15) (a) Chen, Y. H.; Lown, J . W. J . Am. Chem. Soc. 1994, 116, 6995.
(b) Al-Said, N. H.; Lown, J . W. Tetrahedron Lett. 1994, 35, 7577. (c)
Al-Said, N. H.; Lown, J . W. Synthetic Commun. 1995, 25, 1059. (d)
Chen, Y. H.; Yang, Y.; Lown, J . W. J . Biomol. Struct. Dyn. 1996, 14,
3663. (e) Sharma, S. K.; Tandon, M.; Lown, J . W. J . Org. Chem. 2000,
65, 1102.
(16) Mrkisch, M.; Dervan, P. B. J . Am. Chem. Soc. 1994, 116, 3663.
(17) Youngquist, R. S.; Dervan, P. B. J . Am. Chem. Soc. 1985, 107,
5528.
(18) (a) Wang, W.; Lown, J . W. J . Med. Chem. 1992, 35, 2890. (b)
Neenamati, N.; Majumdar, A.; Sunder, S.; Owen, J . M.; Tandon, M.;
Lown, J . W.; Pommier, Y. Mol. Pharmacol. 1998, 54, 2890. (c) Gmiener,
W. H.; Cui, W.; Konerding, D. E.; Keifer, P. A.; Sharma, S. K.; Soto, A.
M.; Marky, L. A.; Lown, J . W. J . Biomol. Struct. Dyn. 1999, 17, 507.
(d) Guo, D.; Gupta, R.; Lown, J . W. Anti Cancer Drug Design 1993, 8,
369. (e) Felipowsky, M. E.; Kopka, M. L.; Brazil-Zison, M.; Lown, J .
W.; Dickerson, R. E. Biochemistry 1999, 35, 15397. (f) Sharma, S. K.;
Tandon, M.; Lown, J . W. Eur. J . Org. Chem. 2000, 2095. (g) Krowicki,
K.; Lown, J . W. J . Org. Chem. 1987, 52, 3493.
(6) Hahn, F. F. In Antibiotics III Mechanism of Action of Antimi-
crobial and Antitumor Agents; Corcoran, J . W., Hahn, F.E., Eds.;
Springer-Verlag: New York, 1975; p 79.
(7) Trauqer, J . W.; Barird, E. E.; Dervan, P. B. Nature 1996, 382,
559.
(8) Zimmer, C.; Wahnert, U. Prog. Biophy. Mol. Biol. 1986, 47, 31.
(9) VanDyke, M. W.; Dervan, P. B. Nucleic Acids. Res. 1983, 11,
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(19) Kopka, M. L.; Yoon, C.; Doodsell, D. S.; Pjura, P.; Dickerson,
R. E. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 1376.
(10) Kissinger, K. L.; Dabrowiak, J . C.; Lown, J . W. Chem. Res.
Toxicol. 1990, 3, 162.
(20) Lown, J . W.; Krowicki, K.; Bhat, U. G.; Skorbogaty, A.; Ward,
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10.1021/jo001034s CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/11/2001

Notes
J . Org. Chem., Vol. 66, No. 3, 2001 1031
Sch em e 1
F igu r e 1. Hairpin (I), cross-linked (stapled) (II), extended
bispolyamide (III).
at sites where GC pair reading was desired. Imidazole
provides space for a guanine-2-amine group and provides
an acceptor for a new hydrogen bond from G-2-NH2
groups, thereby permitting a change in the base site
recognition from that shown by pyrrole. It remained to
be determined that given the structural requirement for
accommodating the exocyclic 2-amine group of contiguous
GC pairs sequences, whether one can design polyamide
sequences in a such a way that these GC rich sequences
could be read out with very high fidelity. Discrimination
of GC from CG by the Im/Py pair requires precise
positioning of the key hydrogen bond between the imi-
dazole N3 and the exocyclic amine of guanine.21 Dervan
et al.22 have synthesized hairpins with a maximum of four
imidazole units and extended polyamides with a maxi-
mum of two contiguous imidazole units on each side of
the linker. Encouraged by these and other preliminary
results and with the results of the potent inhibitory
effects of pyrrolobispolyamides on HIV-1 integrase (in
nano molar concentrations), murine leukemic and of
Okazaki fragments, we have designed a general protocol
for the synthesis of imidazole polyamides which could
recognize extended GC sequences with very high affinity.
We have linked the three imidazole units by both flexible
and rigid linkers. The problem of cellular uptake and
plasma membrane permeability posed by polyamides
bearing unnatural dimethyl(aminopropyl)amino termini
has been addressed by incorporating the natural amidi-
num end group 13-15. The corresponding triimidazobis-
polyamides bearing the unnatural end group, i.e., di-
methylaminopropyl 7-9 were, however, also synthesized
for comparison purposes against various targets. As the
number of imidazole units increases in the sequence the
solubility of the polyamide intermediate decreases and
hence the purification of the compounds become difficult.
To overcome this problem we have synthesized the
triimidazole-polyamide precursor 16 for these bispoly-
amides by a solid-phase method.We report here a general
procedure for the introduction of several contiguous
imidazole moieties into the DNA reading polyamides by
a combined solution and solid-phase method.
Sch em e 2a
a
Key: (a) DCC/HOBt, rt, 24 h, 30% yield; (b) DMF/THF (1:1),
Et3N, rt, 10 h, 50% yield.
synthesized from 3 and 1.18g The triimidazotricarbox-
amide 4 was reduced by Pd/C to give an unstable amine
which was coupled with fumaric acid in 2:1 stoichiom-
etery (Scheme 2), catalyzed by DCC/HOBt (path a,
Scheme 2) to give the desired compound 7 in 30% yield
in 24 h. The yields were better (50%) when the reduced
amine of 4 was coupled with the fumaryl chloride with
triethylamine (path b, Scheme 2). Since the yields were
better with acid chloride coupling, therefore we converted
the diacid 2,5-pyridine dicarboxylic acid into acid chlo-
rides by treating them with SOCl2/DMF while tereph-
thaloyl chloride and fumaryl chlorides were used directly.
These diacid chlorides were coupled with the reduced
amine of 4 to afford compounds 8 and 9 (Scheme 2, Figure
2). Trimidazotricarboxamide compound 5 bearing a pro-
pionitrile terminus was converted to the amidinium
terminus by Pinner reaction24 with HCl in ethanol
followed by ammonia which gave an excellent yield (70%)
of the triimidazotricarboxamide 6 bearing an amidinium
terminus. Attempts were made to couple the reduced
amine of 6 with the acid chlorides of the linkers,
Resu lts a n d Discu ssion
Compound 1 was synthesized by the reported proce-
dure.23 Subsequently, condensation with 3-(dimethylami-
no)propylamine or 3-aminopropionitrile fumarate in THF
gave 2 and 3, respectively (Scheme 1). A Pd/C-facilated
reduction of 2 yielded an unstable amine which on
coupling with 1 resulted in a diimidazole unit which was
again reduced by Pd/C and coupled with 1 to give a
triimidazotricarboxamide 4. Similarly, compound 5 was
(21) Pilch, D. S.; Poklar, N. A.; Gelfand, C. A.; Law, S. M.; Breslauer,
K. J .; Baird, E. E.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 1996,
93, 8306.
(22) Swalley, S. E.; Baird, E. E.; Dervan, P. B. J . Am. Chem. Soc.
1997, 119, 6953.
(23) Nishiwaki, E.; Tanaka, S.; Lee, H.; Shibuya, M. Heterocycles
1988, 27, 1945.

1032 J . Org. Chem., Vol. 66, No. 3, 2001
Notes
F igu r e 2. Triimidazolo bispolyamide.
but this did not work well because of the contamination
with the inorganic salts generated by the Pinner reaction.
Therefore we synthesized compounds 10-12 by coupling
the reduced amine of 5 with the acid chlorides of the
linkers in a similar way as described for compounds 7-9.
Finally the bispolyamides 10-12 with propionitrile ter-
mini were subjected to modified Pinner reaction condi-
tions to give compounds 13-15, respectively, in pure form
bearing natural amidinium termini. Our observations
agree with those of Baksheev et al.14c that the first step
of the reaction of the cyano group, i.e., formation of the
imino ester with an alcohol in the presence of hydrogen
chloride, is completed in 90 min and that longer reaction
times promote side reactions resulting in lower yields.
The imino ester reacts readily with ammonia in ethanolic
solution within 1h at ambient temperature to afford
amidinium termini.
The triimidazole polyamide 16 having three contiguous
imidazole moieties was synthesized by a solid-phase
method. A considerable amount of work by Dervan’s
group has been reported on the synthesis of hairpins by
using a Boc-â-alanine-Pam- resin with Boc chemistry.25
In all the hairpins reported so far they have used a
pyrrole moiety as the first unit linked to the resin and
then subsequently either pyrrole or imidazole or hy-
droxypyrrole units are incorporated for increasing the
chain length of hairpin, thereby leading to a terminus of
â-alanine-N,N′-dimethylaminopropyl always with a pyr-
role unit after cleaving the hairpin from the solid supprt
with N,N′-dimethylaminopropylamine. Since we were
interested in the synthesis of contiguous imidazole
introduction in the DNA sequence reading polyamide we
adapted the reported methodologies25 for the synthesis
of a triimidazole polyamide.
The free amine from the resin was obtained by de-
blocking the Boc group with 80% TFA then coupling the
freshly prepared Boc-Im-COOBt ester from the Boc-Im-
COOH in dry DMF with DIEA base. The reaction vessel
was shaken for 2h, the resin was washed and dried, then
a small sample of this resin was taken for analysis, while
with the rest of the resin was used for another cycle. The
shaking for the rest of the cycle was continued for 90 min.
Finally the sequence was cleaved from the resin with
N,N′-dimethylaminopropylamine to afford 16 (Scheme 3).
The terminal imidazole moiety bears a protected amino
functionality which can be used for further chain elonga-
tion of the sequence of imidazole or pyrrole or could be
converted into the formyl group as required.
Certain polyamides bearing contiguous imidazole groups
have shown pronounced RNase A type cleavage activity
at significantly low concentrations. RNase promotes
hydrolysis at neutral pH by locally increased concentra-
tions of acid and base. The optimal pH of the RNase like
activity of the imidazole bearing polyamides was found
to be 7.0 which is at or near the pKa of the imidazole
ring. Cleavage activity of the imidazole polyamides is
significantly enhanced by salt concentrations (especially
magnesium ions) in the low millimolar concentration
(24) Pinner, A.; Klein, F. Chem. Ber. 1877, 10, 1889.
(25) Baird, E. E.; Dervan, P. B. J . Am. Chem. Soc. 1996, 118, 6141.

Notes
J . Org. Chem., Vol. 66, No. 3, 2001 1033
Sch em e 3
N,N′-Bis[[(3-(dim eth ylam in o)pr opyl]-1-[m eth yl-2-[1-m eth -
yl-2-(1-m eth yl-2-ca r boxa m id o-4-im id a zole)ca r boxa m id o-4-
im id a zole]ca r boxa m id o-4-im id a zol-yl]]fu m a r yl Dica r box-
a m id e (7). Gen er a l P r oced u r e. Compound 4 (0.50 g, 0.99
mmol) was dissolved in DMF and methanol (10 mL, 1:1, v/v)
and hydrogenated in the presence of Pd-C (10%, 0.15 g) for 2 h
at room temperature. The catalyst was removed by filtration
and washed with methanol. The solvent was evaporated in
vacuo, and the residue was dried under high vacuum to remove
traces of the solvent. The amino compound so obtained was
redissolved in anhydrous DMF (10 mL) and fumaryl chloride
(75 mg, 0.49 mmol) in DMF/THF (1:1, 5 mL) was added to this
solution at 0 °C under an atmosphere of argon. The reaction
mixture was gradually brought to room temperaure. The reac-
tion mixture was maintained under an atmosphere of argon and
stirred at 22 °C for 18 h. At this time, the TLC showed
completion of the reaction. The solvent was removed in vacuo
and the crude product was purified on a silica gel column using
CH2Cl2/MeOH/NH4OH (8:2:0.2) as eluent to afford pure 7 (158
mg, 50% yield). Mp: >300 °C. IR (film): 3386, 2956, 2714, 1672,
range. Activity is weak in the absence of salt and
inhibited at high concentrations of magnesium chloride
or sodium chloride. The cleavage activity shows a bell-
shaped dependence on the concentrations of the imidazole
polyamide. Appreciable cleavage activity is already seen
at a concentration of 0.3 mM of the compounds and
activity is significantly decreased at concentration higher
than 3.0 mM. RNA cleavage is structure and site specific
and cleavage occurs preferentially in the single stranded
regions of the different tRNA substrates.26 The new
synthetic procedure described herein will permit, inter
alia, systematic exploration of this novel property of
polyamides.
Exp er im en ta l Section
Gen er a l Meth od s. Melting points were determined with an
electrothermal melting point apparatus and are uncorrected. All
the chemicals used were of reagent grade. Dimethylformamide
(DMF), methanol (MeOH), and tetrahydrofuran (THF) were of
anhydrous grade procured from Aldrich Chemical Co. and were
used without further purification. Freshly distilled dichlo-
romethane (DCM) was used. The progress of the reactions was
monitored by thin-layer chromatography using precoated silica
gel 60F 254, E. Merck TLC plates visualizing under UV light.
1H NMR spectra were recorded with a Brucker (300 MHz)
spectrometer with tetramethylsilane (TMS) as internal standard
on the ppm scale (δ). Multiplicity of resonance peaks are
indicated as singlet (s),broad singlet (bs), doublet (d), quartet
(q), triplet (t), and multiplet (m). Mass spectrometric analysis
was performed by positive mode elctrospray ionization with
Micromass ZapSpec Hybrid Sector -TOF.
1
1538, 1473, 1335, 1125, 1019, 907, 794 cm-1. H NMR (DMSO-
d6): δ 1.76 (m, J ) 7.0 Hz, 4H), 2.50 (s, 12H), 2.70 (t, J ) 7.0
Hz, 4H), 3.32 (t, J ) 6.0 Hz, 4H), 3.90 (s, 6H), 3.94 (s, 6H),4.10
(s, 6H), 7.27 (s, 2H), 7.55 (s, 2H), 7.65 (s, 2H), 7.68 (s, 2H), 8.48
(t, J ) 6.0 Hz, 2H, exchanged with D2O), 9.68 (s, 2H, exchanged
with D2O), 9.80 (s, 2H, exchanged with D2O), 11.06 (s, 2H,
exchanged with D2O). HRMS: calcd for C44H59N22O8 1023.488,
found 1023.489 (M+ + H, 100).
N,N′-Bis[[(3-(dim eth ylam in o)pr opyl]-1-[m eth yl-2-[1-m eth -
yl-2-(1-m eth yl-2-ca r boxa m id o-4-im id a zole)ca r boxa m id o-4-
im id a zole]ca r b oxa m id o-4-im id a zol-yl]]p h en yl-1,4-d ica r -
boxa m id e (8). The title compound was synthesized similarly
by coupling the reduced amine of 4 with terephthaloyl chloride
to give 8 in 53% yield. Mp: 243-245 °C. IR (film): 3388, 2959,
1668, 1605, 1549, 1481, 1368, 1025, 908, 765 cm-1 1H NMR
.
(DMSO-d6): δ 1.80 (m, J ) 7.0 Hz, 4H), 2.56 (s, 12H), 2.78 (t, J
) 7.0 Hz, 4H) 3.20 (t, J ) 6.0 Hz, 4H), 3.95 (s, 6H), 4.00 (s, 6H),
(26) Helm, M.; Kopka, M. L.; Sharma, S. K.; Lown, J . W.; Giege, R.
Bioorg. Med. Chem. 2000, submitted.

1034 J . Org. Chem., Vol. 66, No. 3, 2001
Notes
4.10 (s, 6H), 7.54 (s, 2H), 7.68 (s, 2H), 7.75 (s, 2H), 8.12 (s, 4H),
8.46 (t, J ) 6.0 Hz, 2H, exchanged with D2O), 9.63 (s, 2H,
exchanged with D2O), 9.80 (s, 2H, exchanged with D2O), 11.14
(s, 2H, exchanged with D2O). HRMS: calcd for C48H61N22O8
1072.504, found 1073.504 (M+ + H, 100).
3.53 (q, 4H), 4.03 (s, 6H), 4.05 (s, 6H), 4.10 (s, 6H), 7.42 (s, 2H),
7.64 (s, 2H), 7.67 (s, 2H), 8.52 (t, J ) 6.0 Hz, 2H), 8.90 (brs,
4H), 9.01 (brs, 2H), 9.70 (s, 2H), 10.62 (s, 2H), 11.10 (s, 2H).
HRMS: calcd for C40H49N24O8 993.416, found 993.419 (M+ + H,
100).
N,N′-Bis[[(3-(dim eth ylam in o)pr opyl]-1-[m eth yl-2-[1-m eth -
yl-2-(1-m eth yl-2-ca r boxa m id o-4-im id a zole)ca r boxa m id o-4-
im id a zole]ca r boxa m id o-4-im id a zol-yl]]p yr id in e-2,5-d ica r -
boxa m id e (9). The title compound was synthesized in a similar
manner by coupling the reduced amine of 4 with the 2,5-diacid
chloride of pyridine to give 9 in 50% yield. Mp: 198-200 °C. IR
N,N′-Bis[(3-p r op ia m id in e)-1-[m et h yl-2-[1-m et h yl-2-(1-
m eth yl-2-ca r boxa m id o-4-im id a zole)ca r boxa m id o-4-im id a -
zole]ca r b oxa m id o-4-im id a zolyl]p h en yl-1,4-d ica r b oxa m -
id e Dih yd r och lor id e (14). Yield: 65%. Mp: >300 °C. IR
(film): 3344, 1666, 1605, 1542, 1477, 1407, 1125, 1019, 907, 750
cm-1 1H NMR (DMSO-d6): δ 2.79 (t, J ) 6.0 Hz, 4H), 3.50 (q,
.
(film): 3387, 2954, 1666, 1543, 1476, 1367, 1020, 908, 765 cm-1
.
4H), 4.05 (s, 6H), 4.08 (s, 6H), 4.13 (s, 6H), 7.67 (s, 2H), 7.70 (s,
2H), 7.80 (s, 2H), 8.25 (s, 4H), 8.56 (t, J ) 6.0 Hz, 2H), 9.84 (s,
2H), 8.91 (brs, 4H), 9.15 (brs, 2H), 10.89 (s, 2H), 11.12 (s, 2H).
1H NMR (DMSO-d6): δ 1.88 (m, J ) 7.0 Hz, 4H), 2.72 (s, 12H),
3.04 (t, J ) 7.0 Hz, 4H) 3.30 (t, J ) 6.0 Hz, 4H), 3.92 (s, 6H),
3.97 (s, 6H),4.08 (s, 6H), 7.50 (s, 2H), 7.65 (s, 2H), 7.75 (s, 2H),
8.28 (d, J ) 7.0 Hz, 1H), 8.56 (t, J ) 6.0 Hz, 3H, 2H exchanged
with D2O), 8.59 (m,1H), 9.26 (d, J ) 2.0 Hz, 1H) , 9.90 (s, 2H,
exchanged with D2O), 10.01 (s, 2H, exchanged with D2O), 10.08
(s, 2H, exchanged with D2O). HRMS: calcd for C47H60N23O8
1074.499, found 1074.501 (M+ + H, 100).
HRMS: calcd for C44H51N24O8 1043.432, found 1043.434 (M+
H).
+
N,N′-Bis[(3-p r op ia m id in e)-1-[m et h yl-2-[1-m et h yl-2-(1-
m eth yl-2-ca r boxa m id o-4-im id a zole)ca r boxa m id o-4-im id a -
zole]ca r boxa m id o-4-im id a zolyl]p yr id in e-2,5-d ica r boxa m -
id e Dih yd r och lor id e (15). Yield: 65%. Mp: >300 °C. IR
(film): 3338, 1671, 1603, 1542, 1474, 1367, 1125, 1019, 907, 797,
N,N′-Bis[(3-(3-p r op ion itr ile)-1-[m eth yl-2-[1-m eth yl-2-(1-
m eth yl-2-ca r boxa m id o-4-im id a zole)ca r boxa m id o-4-im id a -
zole]ca r b oxa m id o-4-im id a zolyl]fu m a r yl Dica r b oxa m id e
(10). Compound 5 was reduced with 10% Pd/C in DMF similarly,
and the reduced amine was allowed to react with fumaryl
chloride in a similar manner to give 10 in 45% yield. Mp: 259-
261 °C. IR (film): 3380, 2982, 1665, 1640, 1543, 1480, 1021, 910,
766 cm-1 1H NMR (DMSOd6): δ2.83 (t, J ) 6.0 Hz,4H), 3.54
.
(q, 4H), 4.01 (s, 6H), 4.08 (s, 6H), 4.16 (s, 6H), 7.43 (s, 2H), 7.62
(s, 2H), 7.70 (s, 2H), 8.29 (d, J ) 7.0 Hz, 1H), 8.57 (t, J ) 6.0
Hz, 3H), 9.10 (brs, 5H), 9.45 (brs, 2H), 9.69 (s, 2H), 10.80 (s,
2H), 11.08 (s, 2H). HRMS: calcd for C43H50N25O8 1044.427, found
1044.424 (M+ + H).
764 cm-1 1H NMR (DMSO-d6): δ2.78 (t, J ) 6.0 Hz,4H), 3.48
.
Syn th esis of Tr iim idazole Moiety by Solid-P h ase Meth od
(16). Boc-â-alanine-Pam-resin25 (230 mg, 0.046 mmol) was
packed in a 20 mL filtration column fitted with a filter and
stopper at both ends. A volume of 2 mL of dry DMF was added
to the resin, the mixture was shaken for 5 min, and then the
solvent was drained; this procedure was repeated two more
times. Then the resin was washed with dry DCM (2 × 5 mL).
The resin was dried by vacuum in the column and after drying
it was treated with 2 mL of 80% TFA/DCM and 1 mL of 0.5 M
PhSH and shaken (2 × 30 min.). The resin was washed
thoroughly with DMF (3 × 5 mL) and then with DCM (5 × 5
mL). The activated ester of imidazole was prepared25 by stirring
the mixture of 50 mg of Boc-Im-COOH (0.2 mol), HOBt (27 mg,
0.2 mmol), and DCC (41 mg, 0.2 mmol) in DMF (2 mL) for 15
min. This activated ester was then transferred to the column
containing free NH2 group on the resin with DMF (1 mL) and
0.1 mL of DIEA. This mixture was shaken for 2 h. A small
portion of the resin was taken for analysis while the rest of the
resin was subjected to two more cycles of coupling. After three
cycles of imidazole coupling the resin was washed with DMF (3
× 5 mL) and DCM (5 × 5 mL), the resin was dried and then
placed in a small glass vial (5 mL). To the resin was added 1.5
mL of 3-dimethylaminopropylamine, and this mixture was
heated at 55 °C for 18 h. The resin was collected washed with
DCM, and the filtrate was concentrated and purified by column
chromatography.The pure compound 16 was eluted with 80:20:
0.2 (CH2Cl2/MeOH/NH4OH) in 55% yield as a white colored solid.
Mp: 159-162 °C. IR (film): 3269, 2953, 2702, 1715, 1667, 1571,
(q, 4H,), 3.97 (s, 6H), 4.00 (s, 6H), 4.05 (s, 6H), 7.55 (s, 3H), 7.66
(s, 2H), 7.68 (s, 2H), 7.71(s, 2H), 8.50 (t, J ) 6.0 Hz, 2H,
exchanged with D2O), 9.64 (s, 2H, exchanged with D2O), 9.72
(s, 2H, exchanged with D2O), 11.05 (s, 2H, exchanged with D2O).
HRMS: calcd for C40H43N22O8 959.363, found 959.362 (M+ + H).
The following compounds were synthesized in
manner:
a similar
N,N′-Bis[(3-(3-p r op ion itr ile)-1-[m eth yl-2-[1-m eth yl-2-(1-
m eth yl-2-ca r boxa m id o-4-im id a zole)ca r boxa m id o-4-im id a -
zole]ca r b oxa m id o-4-im id a zolyl]p h en yl-1,4-d ica r b oxa m -
id e (11). Yield: 50%. Mp: 223-225 °C. IR (film): 3380, 2980,
1661, 1642, 1601, 1543, 1475, 1020, 910, 763 cm-1 1H NMR
.
(DMSO-d6): δ 2.79 (t, J ) 6.0 Hz, 4H), 3.49 (q, 4H), 3.95 (s, 6H),
3.97 (s, 6H), 4.02 (s, 6H), 7.02 (s, 2H), 7.58 (s, 2H), 7.66 (s, 2H),
8.02 (s, 4H), 8.57 (t, J ) 6.0 Hz, 2H, exchanged with D2O), 9.59
(s, 2H, exchanged with D2O), 9.90 (s, 2H, exchanged with D2O),
10.45 (s, 2H, exchanged with D2O). HRMS: calcd for C44H45N22O8
1009.379, found 1009.379 (M+ + H).
N,N′-Bis[(3-(3-p r op ion itr ile)-1-[m eth yl-2-[1-m eth yl-2-(1-
m eth yl-2-ca r boxa m id o-4-im id a zole)ca r boxa m id o-4-im id a -
zole]ca r boxa m id o-4-im id a zolyl]p yr id in e-2,5-d ica r boxa m -
id e (12). Yield: 43% yield. Mp: 225-227 °C. IR (film) 3383,
2923, 1665, 1638, 1601, 1550, 1463, 1019, 908, 764 cm-1 1H
.
NMR (DMSO-d6): δ2.75 (t, J ) 6.0 Hz, 4H), 3.49 (q, 4H), 3.91
(s, 6H), 3.99 (s, 6H), 4.00 (s, 6H), 7.51 (s, 2H), 7.61 (s, 2H), 7.72
(s, 2H), 8.19 (d, J ) 7.0 Hz, 1H), 8.49 (t, J ) 6.0 Hz, 3H, 2H
exchanged with D2O), 9.10 (m, 1H), 10.01 (s, 2H, exchanged with
D2O), 10.45 (s, 2H, exchanged with D2O), 11.31 (s, 2H, exchanged
with D2O). HRMS: calcd for C43H44N23O8 1010.374, found
1010.373(M++H)
1543, 1234, 1166, 1009, 907, 764 cm-1 1H NMR (DMSO-d6) δ:
.
1.45 (s, 9H), 1.74 (m, 2H), 2.36 (t, J ) 7.0 Hz, 2H), 2.52 (s, 6H),
2.85 (t, J ) 4.9 Hz, 2H), 3.09 (m, 2H), 3.24-3.46 (m, 8H), 3.94
(s, 6H), 3.98 (s, 3H), 7.27 (s, 1H), 7.50 (s, 1H), 7.61 (s, 1H), 8.18
(2t, J ) 6.0 Hz, 2H, exchanged with D2O), 9.53 (s, 1H), 9.60 (brs,
1H, exchanged with D2O), 9.82 (s, 1H, exchanged with D2O).
13CNMR (DMSO-d6) δ: 170.74, 158.24, 155.46, 155.35, 152.67,
136.97, 135.01, 134.58, 134.18, 133.30, 132.75, 114.19, 113.68,
54.76, 42.43, 35.79, 35.18, 35.07, 34.95, 34.89, 28.06, 24.61, 21.46.
HRMS: calcd for C28H43N12O6 643.342, found 643.342 (M+ + H,
100).
N,N′-Bis[(3-p r op ia m id in e)-1-[m et h yl-2-[1-m et h yl-2-(1-
m et h yl-2-ca r b oxa m i-d o-4-im id a zole)ca r b oxa m id o-4-im i-
d a zole]ca r boxa m id o-4-im id a zolyl]fu m a r yl d ica r boxa m id e
Dih yd r och lor id e (13). Gen er a l P r oced u r e. Compound 10
(500 mg, 0.52 mmol) in 25 mL of anhydrous ethanol was
saturated with dry HCl with cooling.14c Upon saturation, the
compound dissolved in the ethanol solution. After 90 min at room
temperature the solvent was evaporated to dryness and the
residue was washed with dry ethyl ether (2 × 50 mL) and then
dried. The residue was again dissolved in dry ethanol followed
by the treatment with NH3 condensed (4.5 mL) into the reaction
vessel. The reaction mixture was stirred at room temperature
for 1 h. The solvent was then removed. The residue was
redissolved in methanol (100 mL), and the impurities were
collected by filtration. The filtrate was concentrated to a small
volume (10 mL), and the pure compound was collected as the
dihydrochloride salt 13 (336 mg,65% yield). Mp: >300 °C. IR
(film): 3269, 2953, 2719, 1667, 1538, 1474, 1370, 1125, 1020,
Ack n ow led gm en t. We are grateful for a research
grant (to J .W.L) from the Natural Sciences and Engi-
neering Research Council of Canada.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
compounds 4, 5, 7-12, and 16, HRMS of compound 10-16,
and 13CNMR spectra of compound 16. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
1
967, 766 cm-1. H NMR (DMSO-d6): δ 2.80 (t, J ) 6.0 Hz, 4H),
J O001034S