Dissecting the complex recognition interfaces of potent tetrazole- and pyrrole-based anion binders

Article abstract of DOI:10.1021/jo400372c

Tetrazoles are potent anion binders. We report here a new family of tetrazole-pyrrole-amide hosts that arise when a tetrazole is incorporated as a new binding element alongside the well-known amidopyrrole anion-binding scaffold. In addition to reporting three new, modular synthetic routes that can be used to access these structures, we also report that the new hosts are highly potent binders of chloride. Along the way, we carried out studies of a pyrrole ester control compound that, surprisingly, bound anions almost as strongly as did the amide analogues. This led us to investigate further the relative importance of the amide NH in halide binding. We report that, despite the regular appearance of this close amide NH - -Cl contact in calculated and experimental X-ray structures, the amide NH in this family of anion hosts does not hydrogen bond strongly to chloride in solution.

Full text of DOI:10.1021/jo400372c

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Featured Article
Dissecting the complex recognition interfaces of
potent tetrazole- and pyrrole-based anion binders
Thomas Pinter, Chakravarthi Simhadri, and Fraser Hof
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo400372c • Publication Date (Web): 30 Apr 2013
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Dissecting the complex recognition interfaces of potent tetrazole- and pyrrole-based anion
binders
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Thomas Pinter, Chakravarthi Simhadri and Fraser Hof*
Department of Chemistry, University of Victoria, Victoria, Canada V8W 3V6
fhof@uvic.ca
Abstract
Tetrazoles are potent anion binders. We report here a new family of tetrazoleꢀpyrroleꢀamide
hosts that arise when a tetrazole is incorporated as a new binding element alongside the wellꢀ
known amidoꢀpyrrole anionꢀbinding scaffold. In addition to reporting three new, modular
synthetic routes that can be used to access these structures, we also report that the new hosts are
highly potent binders of chloride. Along the way, we carried out studies of a pyrroleꢀester control
compound that, surprisingly, bound anions almost as strongly as did the amide analogs. This led
us to investigate further the relative importance of the amide NH in halide binding. We report
that, despite the regular appearance of this close amide NHꢀꢀꢀCl contact in calculated and
experimental Xꢀray structures, the amide NH in this family of anion hosts does not hydrogen
bond strongly to chloride in solution.
Introduction
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Anions are ubiquitous in living organisms, where maintenance of intraꢀ and extracellular
anion concentrations is a vital control mechanism. As such, a variety of natural and synthetic ion
carriers have been discovered,1 many of which have therapeutic potential.2ꢀ4 Synthetic anion
sensors for the monitoring of ion concentrations in both biological and environmental samples
are also of growing interest.5ꢀ9
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Despite the broad importance of anion recognition the vast majority of anion receptors
are constructed by assembly of only a few different, well understood anionꢀbinding functional
groups that include amides,10ꢀ12 sulfonamides,13, 14 and (thio)ureas.15ꢀ17 Pyrrole (1) is a dominant
player in anion recognition, represented by many derivatized pyrroles18ꢀ20 and related pyrrolic
ring systems.21ꢀ24 Triazoles, easily assembled by “click” reactions of alkynes and azides,25, 26
have recently been added to this toolkit as agents that bind anions via their electronꢀdeficient CH
group.27, 28 A related heterocycle, tetrazole, is relatively underꢀused as a neutral binder of anions
but is attractive for many reasons. Tetrazoles are easily assembled by variants of the “click”
reaction that involve almost any organic nitrile being treated with NaN3 under a variety of
conditions.29ꢀ32 Further, tetrazoles are potent anionꢀbinding elements that operate well in a
variety of structural contexts because their acidic NH bears a much larger partial positive charge
than other amideꢀlike groups and heterocycles.33ꢀ35 In fact, a single unadorned tetrazole (2) is a
stronger anion binder34 than many more elaborate, multiꢀdentate hydrogen bond donating hosts
(e.g. 3, 4, and 5) (Figure 1).35, 36 One tetrazoleꢀcontaining anionꢀbinding motif that we have
previously reported is represented by the pyrroleꢀtetrazole hybrids 6 and 7, which are some of the
most potent and simple anion recognition motifs in the pyrrole family. Monoꢀtetrazole 6 binds
chloride 120ꢀfold stronger than does analogous monoamide 4, and bisꢀtetrazole 7 binds chloride
almost 200ꢀfold stronger than does its closely analogous bisꢀamide 8 (Figure 1).36 We report here
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a new family of pyrroleꢀbased hosts that contain both amides and tetrazoles (hosts 13 and 14), as
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well as esterꢀfunctionalized host 11. These hosts show generally high affinities for HSO4 , and
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even higher affinities for Cl–, and allow us to dissect out energetic influences of different groups
at the recognition interface. Surprisingly, we uncover evidence that the amide NH is a spectator
that is not required for the very strong halide binding in these systems, in contrast to previous
results reported for various pyrroloꢀamide hosts.37ꢀ40
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Figure 1. Structures of pyrrole (1) and related anion receptors 2ꢀ8, along with the 1:1 binding
constants for the complexation of Cl– in CD3CN that have been previously reported in the
literature (see text). (Bn = Benzyl)
Results
Synthesis
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We developed multiple routes to the selective installation of both tetrazole and
ester/amide functionality at the 2ꢀ and 5ꢀpositions of pyrrole (1). The first synthetic strategy
(Scheme 1a) began with ethyl pyrroleꢀ2ꢀcarboxylate (9), which was cyanated with chlorosulfonyl
isocyanate to give 10. This was followed by tetrazole formation upon treatment with NaN3 and
NH4Cl (generating HN3 in situ) to give esterꢀfunctionalized host 11. Hydrolysis of the ester
provided highly polar carboxylic acid 12, and subsequent EDCꢀmediated coupling to pꢀtoluidine
or pꢀmethoxybenzylamine gave amideꢀfunctionalized hosts 13 and 14, respectively. One
shortcoming in this route was the poor regioselectivity of the cyanation of 9, where substitutions
at the 4ꢀposition (undesired) and 5ꢀposition (desired) were observed at approximately a 3:2 ratio
that persisted despite efforts to optimize conditions. During the course of these investigations we
found the ethoxy group in 10 could be displaced directly by certain primary amines at high
temperature, raising the possibility of a more direct synthetic route.
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Scheme 1. Synthetic approaches to tetrazolyl amidopyrroles.
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To take advantage of the higher reactivity for methyl esters in direct esterꢀtoꢀamide
conversions, we switched to a route starting with methylꢀ2ꢀpyrrole carboxylate 15, and cyanated
as before to give 16 (Scheme 1b). Direct displacement of the methoxy group was achieved by
stirring 16 in neat pꢀmethoxybenzylamine at 120oC for 2 days, which cleanly provided amide 17
in >80% yield. Standard tetrazole forming conditions gave the final product 14 in only four steps
from commercially available material 15. Attempting synthesis of host 13 via the direct
amidation met with disappointing results, as stirring precursor 16 in molten pꢀtoluidine even at
temperatures in excess of 150oC for several days resulted only in recovery of starting material 16.
In a general sense, our studies have taught us that the tetrazole formation is best left as the last
step when possible, as tetrazoleꢀcontaining intermediates such as 12 can be difficult to purify
and/or dissolve for subsequent reactions. We used this information to develop a simple
alternative route to 13 (Scheme 1c), which avoids the formation of problematic intermediate 12.
Hydrolysis of 16 gave the cyanoꢀacid 18, and EDC coupling of 18 with pꢀtoluidine was followed
by tetrazole formation as described above to complete an efficient synthesis of 13. This pathway
proved superior to our original strategy providing easier to handle intermediates and higher
yields.
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NMR-based binding studies
1H NMR titrations were used to determine the anionꢀbinding capabilities of hosts 11, 13,
and 14. Studies were carried out in CD3CN, as this solvent allows comparisons to the broadest
set of values for other pyrroleꢀcontaining hosts reported in the literature. Briefly, anionic guests
were added as their Bu4N+ salts, and the resulting host chemical shift changes were fitted to 1:1
or 2:1 (H:G) binding isotherms using HypNMR (Protonic Software, 2008). Binding
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stoichiometries under these conditions were cleanly 1:1 for all complexes except those of host 14
and Cl– (the strongest complexation pair observed in this study), which showed a small
contribution from 2:1 (H2G) complex formation. Our choices of binding stoichiometries for
curve fitting were confirmed by Job plot42 data for all three hosts with Cl–, and for hosts 11 and
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–
14 additionally with Br– and HSO4 (see ESI). The resulting association constants between the
hosts studied and various anionic guests are given in Table 1. Representative stacked plots and
binding curves are shown in Figure 2.
Discussion
Halide binding
All hosts showed similar high affinities for chloride and lowest for iodide. The ability of
11 to bind Cl– and Br– as well as do amides 13 and 14 was unexpected, as its ester oxygen atom
lone pairs must be in close proximity to anions engaged by the central pyrrole NH. Even more
surprising, when picturing a repulsive, close OꢀꢀꢀCl– contact, is that 11 is 5.5ꢀfold more potent
than its unsubstituted parent compound 6.35 The best interpretations of these data are that a) the
OꢀꢀꢀCl– contact for 11 is long enough not to destabilize the complex significantly, and b) the
electronꢀwithdrawing nature of the ester acidifies the pyrrole NH and thereby increases the
strength of pyrrole NHꢀꢀꢀCl– hydrogen bond. The pyrrole NH in free 11 is 0.8 ppm downfield of
the chemical shift of the same NH in parent host 6, giving further support to this line of
reasoning.
So what are the amide NH’s in 13 and 14 in fact doing in the exceptionally stable 1:1
complexes of each host with Cl–? Comparison to host 11, which has no amide NH’s but has
similar Cl– affinity, would suggest that they aren’t strongly involved in hydrogen bonding to the
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anion. Upon binding Cl– the amide NH’s in 13 and 14 shift downfield by only 0.5 and 0.2 ppm,
respectively, as fitted ꢀδmax values; host 13 in particular shows a barely detectable experimental
downfield shift (Figure 2). Much larger downfield shifts of ~2 ppm are normally observed upon
formation of NHꢀꢀꢀCl– hydrogen bonds. The answer then, would seem to be that the amides serve
mainly as electron withdrawing groups that increase the strength of pyrrole NHꢀꢀꢀCl– hydrogen
bonding in a manner analogous to the ester in host 11. Given the similarity of the amidoꢀpyrrole
motifs in 13 and 14 with the large number of previously published amidoꢀpyrrole hosts in the
literature, we wondered if this lesson could tell us something about this broader set of hosts.
Literature hosts 3,35 4,36 and 836 bind Cl– with affinities of 28–138 M–1 in CD3CN. Some
substantial part of these affinities are routinely attributed to amide NHꢀꢀꢀCl– hydrogen bonds.
Close contacts between amide NH and anionic guest are always observed in calculated hostꢀ
guest structures, and are sometimes also observed in xꢀray coꢀcrystal structures of the hostꢀguest
complexes.40 To understand these motifs better, we carried out control titrations that revealed
that even unsubstituted pyrrole (1) and ethylꢀ2ꢀpyrrole carboxylate (9) bind to Cl– with
significant affinity (Kassoc ≥ 10 M–1; see ESI). More importantly, the pyrrole NꢀH signals in
compounds 1 and 9 experience downfield shifts of ≥2 ppm when saturated with chloride, while
smaller shifts are observed for amide protons in 3, 4, or 8 that resemble more the small shifts we
detect for 13 and 14. When considering all lines of evidence, it is clear that the amides in 13 and
14 don’t contribute strong Hꢀbonds to halide guests, and that a similar interpretation is probably
justified for most of the many amidoꢀpyrrole hosts that have been reported.39,40
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Oxyanion binding
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But the amides do not always remain innocent… host 13 shows moderately strong
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that of esterꢀfunctionalized host 11. Host 13 shows fourꢀfold stronger binding for TsO– than
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–
–
NO3 . Conversely, 14 displayed an approximately 1.5ꢀfold weaker binding for TsO– than NO3 .
In a general sense, amides 13 and 14 show better aptitude for engaging the varied geometries of
oxyanions than does ester 11. The amide NH chemical shifts inform us on the possible formation
of NHꢀꢀꢀO hydrogen bonds in these various hostꢀoxyanion complexes. The largest complexationꢀ
induced shifts of the amide NH are seen for TsO–, while insignificant shifts are seen for NO3 .
–
These shifts offer direct experimental evidence of amide NHꢀꢀꢀanion hydrogen bonding (or lack
thereof), but we can’t draw simple connections between observed affinities and the presence or
absence of the aforementioned hydrogen bonds. Again, these results raise questions about the
roles of amide NH’s in oxyanion binding by previously reported amidoꢀpyrrole hosts. An Xꢀray
crystal structure of a bisꢀamidopyrrole (8) in complex with benzoate reveals all Hꢀbond donors
engaging the guest. In this complex, one benzoate oxygen is engaged by both the pyrrole NꢀH
and one amide NꢀH, with HꢀꢀꢀꢀO bond lengths varying by less than 1 Å. The remaining amide
NꢀH and guest oxygen are separated by a distance virtually identical to the pyrroleꢀguest bond
length demonstrating that each Hꢀbond donor contributes nearly equally to guest stabilization. In
an indoleꢀbased system that included pendant urea and amide groups, a similarly weak
participation of the amide NꢀH in guest binding was also observed.41 The binding constants of 8
with chloride and benzoate were determined to be 138 and 2500 Mꢀ1 respectively. Conversely,
monoꢀamide 4 shows affinities of 28 and 202 Mꢀ1, respectively, for the same two guests, a clear
indication that a third hydrogen bond donor is necessary for strong binding of oxyanions.
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2:1 complexation by 14
Cl– complex, indicated by Job plot to be a 2:1 host:guest binding event, was fit to
The 14
ꢀ
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a 2:1 (H2G) binding isotherm using HypNMR. The results show a K11 value of 2 x 104 M–1,
similar to those seen for 11•Cl– and 13•Cl–, and a K21 value ~two orders of magnitude weaker.
No other titration data collected in this study could be fit well to any analogous 2:1 isotherm.
The 2:1 complex only exists when a large excess of host is present, and only about 2% of it is
present in solution after 1 equivalent of guest is added (Figure 2).
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Table 1: Binding constants for the hosts studied obtained via 1H NMR titrations in CD3CN.a)
-
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Host
11
Cl-
Br-
I-
HSO4
OTs-
NO3
18, 000 ± 2300
31, 000 ± 4600
K11 = 23, 000 ±
4700
1700 ± 260
1800 ± 100
85 ± 21
71 ± 11
130 ± 13
950 ± 7
440 ± 35
1500 ± 230 3000 ± 98 750 ± 85
1200 ± 58 770 ± 49 1120 ± 12
13
1300 ± 700 150 ± 23
14
K21 = 820 ± 11
a) All values are for K11 unless otherwise noted. All titrations were done in duplicate or triplicate,
and the errors reported are standard deviations. Host solutions of 0.5–1 mM were first prepared,
and then also used as solvent to make the titrant solution (containing 8–15 mM of each guest).
The guest solutions were titrated into the host until a point of saturation was reached. See
Supplementary Information for details.
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Figure 2. Left: Excerpts of stacked H NMR plots following pyrrole (downfield singlet) and
amide (upfield singlet) signals for each host in this study. Titrations in these examples were
performed in CD3CN with Bu4N+Cl– as the guest (see experimental section for details). Right:
Representative binding curves following the pyrrole NꢀH and speciation plots (see text) (black
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points = experimental chemical shift data, black line = fitted chemical shift data, red line = [1:1
complex], blue line = [free host], brown line = [2:1 host:guest complex]).
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Molecular modeling
Molecular modeling was conducted to further investigate the conclusions drawn from
solution phase data (Figure 3). Local minimum energy structures were identified for the chloride
complexes of 11, 13, and 14, including the H2G complex posited for 14. All structures have
reasonable bond lengths and angles, and notably, all complexes of amideꢀcontaining hosts have
local minima with amide NH groups forming hydrogen bonds to Cl–. Heavy atom (NꢀꢀꢀCl)
separations with respect to guest and tetrazole NH were 3.35 and 3.32 Å for hosts 13 and 14
respectively; guest and pyrrole nitrogen were observed to be 3.21 and 3.18 Å for hosts 13 and 14,
respectively; guest and amide nitrogen were observed to be 3.55 and 3.58 Å for hosts 13 and 14,
respectively. As with many other previously reported amidoꢀpyrrole examples,39,40 the calculated
NꢀꢀꢀCl contacts for amides, while moderately long, would suggest an energetically favorable
contact between these groups that the NMR data tell us must not exist in solution.
Modeling the H2G complex for 14 revealed a local minimum in which two of the benzyl
amide functionalized hosts (14) bound chloride in their hydrogen bond donating clefts
orthogonally to one another, but this structure could not be identified as a local minimum for the
other hosts. It can be seen in the model (Figure 3a) that an edgeꢀtoꢀface interaction between the
two aromatic rings is occurring. The methylene linker in 14 allows for an extra degree of
rotational freedom relative to the more rigid host 13. It is possible that this additional aromaticꢀ
aromatic contact is the reason why 14 forms weak, but measurable 2:1 complexes with Cl– while
13 does not, but the data in hand don’t definitively rule out other explanations.
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Figure 3. Local minima identified for the hostꢀguest complexes with Cl– by calculations at the
HF/6ꢀ31+G* level of theory. a) The 2:1 complex observed between host 11 and chloride. b)
The 1:1 complex between host 11 and chloride. c, d) The 1:1 complexes of the other two hosts
with chloride. Hydrogen bonds that are suggested by calculated structures but whose energetic
importance is refuted (or diminished) by solutionꢀphase data are marked with an asterisk (*).
Conclusion
We have synthesized a new class of anion recognition elements containing tetrazole and
amide functionalities at the 2ꢀ and 5ꢀpositions of pyrrole. These compounds were able to
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outperform common bisꢀamidopyrroles such as 8 in chloride recognition by a wide margin.
Further, a 2:1 host:guest complex was observed between 14 and chloride due to a key edgeꢀtoꢀ
face interaction between the appended pꢀmethoxybenzyl moieties. Of particular importance, we
found that an extra amideꢀtype hydrogen bond donor does not increase halide affinity
significantly in this family of hosts, and probably does not make strong hydrogen bonds to
halides in solution. Previous studies have shown this to be true in similar systems such as indole
analogues of amidopyrroles.41 Also to our surprise, we observed that the association constants of
hosts 11, 13, and 14 for chloride are relatively comparable, and also similar to that of bisꢀ
tetrazole 7. These data suggest that adding a third Hꢀbond donor does not significantly affect the
stability of the complex, but that it is the electron withdrawing nature of tetrazole, ester, or amide
functionalities at the 2ꢀposition of a pyrrole that can have a profound, favorable influence on
binding affinities. In any case, the introduction of tetrazoles clearly produces some of the most
potent halideꢀbinding hosts in the pyrrole family. In other areas of the chemical sciences, authors
extol the virtues of tetrazoles’ high stability in biological systems and high degree of usefulness
as pharmacological agents.29 We continue to explore the possibility that tetrazoles might find
utility as anionꢀbinding therapeutic and/or sensing agents in biological settings.
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Experimental Section
Proton (1H) NMR spectra were recorded on 500 MHz, 360 MHz, or 300 MHz
spectrometers, as indicated in each case. Carbon (13C) NMR spectra were recorded at 125 MHz,
90 MHz, or 75 MHz as indicated in each case. All NMR binding studies were performed on a
500 MHz spectrometer. HRꢀESIꢀMS was obtained at the UVic Genome BC Proteomics Centre
on a LTQ Orbitrap in positive ionization mode unless otherwise indicated. Melting points are
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uncorrected. All molecular modeling was performed using Spartan '04 or Spartan '06
(Wavefunction, Inc.) at the HF/ 6ꢀ31+G* level of theory. Microwave reactions were carried out
in a Biotage Initiator 2.5 microwave reactor at the temperatures indicated.
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General procedure for pyrrole cyanation:
Ethyl-5-cyano-1H-pyrrole-2-carboxylate (10). Ethylꢀ1Hꢀpyrroleꢀ2ꢀcarboxylate (500 mg, 3.6
mmol) dissolved in 10 ml : 7 ml anhydrous MeCN : DMF was cooled (ꢀ40oC) Chlorosulfonyl
isocyanate (0.94 ml, 10.8 mmol) was added dropwise and the reaction allowed to warm to
ambient temperature. After 24 h, poured over ca. 100 g of ice containing 20 ml of 2 M NaOH.
The ice was allowed to melt and the aqueous layer extracted with DCM (3 x 50 ml). The
combined organic phases were dried over MgSO4, filtered and concentrated. The crude brown
solid was purified (SiO2, 2:1 Hexanes: EtOAc) yielding 126 mg of 10 (0.77 mmol, 21%) as a
pale brown solid. mp: 82ꢀ84 °C; IR(KBr, thin film): 3349s, 3132w, 2990w, 2921w, 2233s,
1689s, 1568s, 1270s, 1205w, 1107; 1H NMR (CDCl3, 300 MHz): δ 1.37 (t, 3H, J = 7.11Hz), 4.35
(q, 2H, J = 7.11 Hz), 7.12 (m, 1H), 7.42 (m, 2H), 10.40 (s, 1H); 13C NMR (CDCl3, 75 MHz): δ
14.4, 61.6, 95.2, 115.5, 117.8, 124.4, 129.2, 160.6; HRꢀESIꢀMS: 187.04810 (MNa+,
C8H8N2O2Na+; calc. 187.04781).
Methyl 5-cyano-1H-pyrrole-2-carboxylate (16). The general procedure for pyrrole cyanation
was applied to methylꢀ1Hꢀpyrroleꢀ2ꢀcarboxylate (15). mp: 140ꢀ142 °C; IR(KBr, thin film):
1
3300s, 3100w, 2990w, 2325w, 2150s, 1701s, 1568s, 1495s, 1270s, 1205w, 750s; H NMR
(CDCl3, 300 MHz): δ 3.90 (s, 3H), 7.12 (dd, J = 2.52 Hz, 1.50 Hz, 1H), 7.41 (dd, J = 3.21 Hz,
1.46 Hz, 1H), 9.57 (s, 1H); 13C NMR (Acetoneꢀd6, 75 MHz): δ 52.1, 95.3, 116.0, 118.0, 125.1,
130.9, 160.7; HRꢀESIꢀMS: 173.03222 (MNa+, C7H6N2O2Na+; calc. 173.03211).
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5-cyano-N-(4-methoxybenzyl)-1H-pyrrole-2-carboxamide (17). A mixture of compound 10
(50 mg) dissolved in pꢀmethoxybenzylamine (3 ml) was heated to 120oC and stirred for 2 days.
The reaction was allowed to cool and 20 ml of EtOAc was added. The organic phase was
washed with 1 M HCl (5 x 15 ml), dried (MgSO4) and concentrated leaving pure 17 (45 mg,
89%) as a pale brown solid. mp: 235 °C (dec); IR(KBr, thin film): 3370m, 3174s, br, 2225s,
1634s, 1538w, 1512m, 1436w, 1253m, 1150w; 1H NMR (DMSOꢀd6): δ 3.69 (s, 3H), 4.33 (d, J =
6.01 Hz, 2H), 6.85 (d, J = 6.84 Hz, 2H), 7.14 (d, J = 1.19 Hz, 1H), 7.17 (d, J = 7.17 Hz, 2H),
7.64 (d, J = 1.11 Hz, 1H), 8.72 (t, J = 5.95 Hz, 1H), 12.43 (s, 1H). 13C (DMSOꢀ d6): 41.5, 55.1,
91.9, 112.3, 113.7, 116.5, 127.9, 128.6, 129.1, 131.3, 158.3, 159.2. HRꢀESIꢀMS: (ꢀve):
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ꢀ
254.09353 (MꢀH, C14H12N3O2 , calc’d: 254.09359)
General procedure for tetrazole formation:
Ethyl-5-(5’-tetrazolyl)-1H-pyrrole-2-carboxylate (11).
Ethyl 5ꢀcyanoꢀ1Hꢀpyrroleꢀ2ꢀ
carboxylate 10 (25 mg, 0.15 mmol), NaN3 (19.2 mg, 3.2 mmol), NH4Cl (17.1 mg, 3.2 mmol) and
anhydrous DMF (1 ml) were added to a microwave vial. The vessel was purged with argon,
sealed, vortexed at maximum speed for 1 min and placed in a microwave reactor at 110oC for 1 h.
The mixture was transferred to a separatory funnel with 30 ml of saturated NaHCO3, the aqueous
layer washed with 30 ml EtOAc and subsequently acidified to pH<1 with conc. HCl. The
aqueous layer was then extracted with EtOAc (3 x 20 ml) and the combined organic extracts
dried (MgSO4), filtered and concentrated. The crude brown solid was triturated in CHCl3 and
the insolubles filtered and airꢀdried yielding 28 mg (90%) of 11 as a pale brown solid. mp:
220 °C (dec.); IR(KBr, thin film): 3279m, 2993w, 2981w, 1722s, 1611w, 1475w, 1290m,
1
1503m, 1763m; H NMR (CDCl3, 300 MHz): δ 1.39 (t, 3H, J = 7.11Hz), 4.36 (q, 2H, J = 7.11
Hz), 6.86 (d, 1H, J = 4.05 Hz), 6.98 (d, 1H, J = 4.11 Hz); 13C NMR (MeOD, 90 MHz): δ 14.7,
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calc. 208.08287).
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Compound 14. The general procedure for tetrazole formation was applied to compound 17. mp:
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235 °C (dec); IR(KBr, thin film): 3291s, br, 2932w, 1615s, 1514s, 1568m, 1249m; H NMR
(MeOD, 300 MHz): δ 3.78 (s, 3H), 4.49 (s, 2H), 6.83ꢀ6.98 (m, 4H), 7.28 (m, 2H); 13C NMR
(CDCl3, 90 MHz): δ 43.5, 66.7, 102.9, 112.9, 113.2, 114.9, 120.4, 129.9, 131.0, 132.0, 160.5,
162.4; HRꢀESIꢀMS: 321.10692 (MNa+, C14H14N6O2Na+; calc. 321.10701).
Compound 13. The general procedure for tetrazole formation was applied to compound 19. mp:
190 °C (dec); IR(KBr, thin film): 3180s, br, 1654s, 1625s, 1602s, 1535s, 1449m, 1332m, 815m;
1H NMR (DMSOꢀd6, 300 MHz): δ 2.28 (s, 3H), 7.15 (d, J = 8.40 Hz, 2H), 7.50ꢀ7.79 (m, 4H),
10.00 (s, 1H), 12.30 (s, 1H); 13C NMR (DMSOꢀd6), 90 MHz): δ 20.5, 108.2, 109.6, 120.0, 122.6,
128.1, 129.1, 132.3, 136.5, 151.1, 158.4; HRꢀESIꢀMS: 291.09652 (MNa+, C13H12N6ONa+; calc.
291.09651).
5-carboxy-1H-pyrrole-2-carbonitrile (18). To a mixture of compound 16 (50 mg, 0.4 mmol) in
H2O/EtOH (1 ml/2ml) was added LiOH (47.9 mg, 2 mmol). The mixture was heated at reflux
with stirring for 2 h then cooled to room temperature. 10 ml of EtOAc was added and the
organic layer washed with 1 M HCl (3 x 10 ml). The organic layer was dried (MgSO4), filtered
and concentrated leaving pure 18 in quantitative yield. mp: 185°C (dec); IR(KBr, thin film):
1
3239s, br, 3131s, 2920m, 2236s, 1674s, 1454m, 1121s; H NMR (DMSOꢀd6, 300 MHz): δ 7.11
(s, 1H), 7.77 (s, 1H), 12.67 (s, 1H); 13C NMR (DMSOꢀd6, 90 MHz): δ 93.7, 115.5, 117.3, 125.2,
–
129.7, 161.5; HRꢀESIꢀMS: 135.02021 (MꢀH–, C6H3N2O2 ; calc. 135.01945).
5-cyano-N-(p-tolyl)-1H-pyrrole-2-carboxamide (19). Compound 18 (40 mg, 0.29 mmol),
EDC
ꢀ
(1ꢀEthylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide HCl) (90 mg, 0.58 mmol) HOBT
ꢀ
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(Hydroxybenzotriazole) (60 mg, 0.44 mmol) and pꢀtoluidine (38 mg, 0.35 mmol) were dissolved
in anhydrous DMF (5 ml) and stirred at room temperature for 18 h. Ethyl acetate (15 ml) was
added and the organic phase washed with 1m HCl (3 x 10 ml). The organic layers were
combined, dried (MgSO4) and concentrated. The product was purified (SiO2, 2:1 EtOAc:Hex )
yielding 58 mg (89%) of 19 as a brown solid. mp: 185 (dec); IR(KBr, thin film): 3239s, br,
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3131s, 2920m, 2236s, 1674s, 1454m, 1121s; H NMR (Acetoneꢀd6, 300 MHz): δ 2.29 (s, 3H),
13
7.15 (m, 2H), 7.30 (m, 2H), 7.62 (m, 2H), 7.72 (m, 2H), 9.34 (s, 1H), 11.64 (s, 1H); C NMR
(Acetoneꢀd6, 920 MHz): δ 20.0, 93.9, 112.5, 115.5, 120.1, 128.4, 129.0, 129.2, 136.4, 157.7;
HRꢀESIꢀMS: 226.09748 (MH+, C13H11N3OH+; calc. 226.09748).
Acknowledgements. This work was supported by NSERC and the University of Victoria. FH is
a Canada Research Chair and Michael Smith Foundation for Health Research Scholar.
Supporting Information. Supplementary NMR titration data and Job plots, NMR spectra of
new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.
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