U.S. patent application number 10/222028 was filed with the patent office on 2003-08-28 for colormetric sensor compositions and methods.
Invention is credited to Andrioletti, Bruno, Black, Christopher, Sessler, Jonathan, Try, Andrew.
Application Number | 20030162960 10/222028 |
Document ID | / |
Family ID | 26834328 |
Filed Date | 2003-08-28 |
United States Patent
Application |
20030162960 |
Kind Code |
A1 |
Sessler, Jonathan ; et
al. |
August 28, 2003 |
Colormetric sensor compositions and methods
Abstract
The present invention provides novel compounds exemplified by
pyrrolic nitrogens used as anion and neutral species recognition
elements with an aromatic core as a signal group. Described are
methods for the synthesis of various pyrrole aryl compounds as well
as various applications for these compounds. Methods of use include
the binding and detection of specific analytes in a mixture and, in
some examples, the separation of the analyte from the mixture.
Additional methods of use include the transport of therapeutic
agents and the sensing of components, degradants, and impurities in
foodstuffs.
Inventors: |
Sessler, Jonathan; (Austin,
TX) ; Andrioletti, Bruno; (Paris, FR) ; Try,
Andrew; (New South Wales, AU) ; Black,
Christopher; (Austin, TX) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
1301 MCKINNEY
SUITE 5100
HOUSTON
TX
77010-3095
US
|
Family ID: |
26834328 |
Appl. No.: |
10/222028 |
Filed: |
August 16, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10222028 |
Aug 16, 2002 |
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09579040 |
May 26, 2000 |
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6482949 |
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60136467 |
May 28, 1999 |
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Current U.S.
Class: |
540/145 ;
544/338; 544/343; 544/353; 548/518 |
Current CPC
Class: |
C07D 207/333 20130101;
C07D 471/14 20130101; C07D 487/22 20130101; C07D 403/14
20130101 |
Class at
Publication: |
540/145 ;
544/353; 544/338; 544/343; 548/518 |
International
Class: |
C07D 487/22; C07D
43/14 |
Goverment Interests
[0002] The government owns rights in the present invention pursuant
to National Institutes of Health (grant no. GM 58907 to Jonathon L.
Sessler), the National Science Foundation (CHE-9725399 to Jonathan
L. Sessler), the Texas ARP (grant 003658-102 to JLS) and a NIH
Postdoctoral Fellowship to Christopher B. Black.
Claims
What is claimed is:
1. A pyrrole-aryl compound of the general formulas, 30wherein Ar is
an aryl group, wherein R.sub.1-R.sub.6, individually at each
occurrence, are the same or different, and are hydrogen, alkyl,
hydroxyalkyl, glycol, polyglycol, amino, nitro, halo, cyano, aryl,
heteroaryl, thio, thioalkyl, amide, ester, acyl, aldehyde, or
carboxy, and provided, however, that if Ar is an unsubstituted
quinoxaline group then R.sub.1-R.sub.6 cannot each be hydrogen.
2. A pyrrole-aryl compound of the general formula: 31wherein
R.sub.1-R.sub.10, individually at each occurrence, are the same or
different and are hydrogen, alkyl, hydroxyalkyl, glycol,
polyglycol, amino, nitro, halo, cyano, aryl, heteroaryl, thio,
thioalkyl, amide, ester, acyl, aldehyde, or carboxy, provided,
however, that R.sub.1-R.sub.10 cannot each be hydrogen.
3. The compound of claim 2, wherein
R.sub.1.dbd.R.sub.2.dbd.OCH.sub.3.
4. The compound of claim 2, wherein
R.sub.1.dbd.R.sub.2.dbd.NO.sub.2.
5. The compound of claim 2, wherein
R.sub.1.dbd.R.sub.2.dbd.CH.sub.3.
6. The compound of claim 2, wherein
R.sub.1.dbd.R.sub.2.dbd.O(CH.sub.2CH.s- ub.2O).sub.3CH.sub.3.
7. The compound of claim 2, wherein
R.sub.1.dbd.R.sub.2.dbd.O(CH.sub.2).su- b.nCH.sub.3, and wherein n
is 0-20.
8. The compound of claim 2, wherein R.sub.1.dbd.H,
R.sub.2.dbd.NO.sub.2.
9. The compound of claim 2, wherein R.sub.1.dbd.NH.sub.2,
R.sub.2.dbd.NO.sub.2.
10. The compound of claim 2, wherein R.sub.1.dbd.NH.sub.2,
R.sub.2.dbd.NH.sub.2.
11. The compound of claim 2 wherein R.sub.1 and R.sub.2 are part of
a cyclic group and further wherein said compound is selected from
the group consisting of: 32
12. The compound of any one of claims 1-11 in which one or more of
the pyrrole nitrogens exist in anionic form.
13. A compound of the general formulas, 33wherein individually at
each occurrence, each of R.sub.1, -R.sub.6 are the same or
different, and are hydrogen, alkyl, hydroxyalkyl, glycol,
polyglycol, amino, nitro, halo, cyano, aryl, heteroaryl, thio,
thioalkyl, amide, ester, acyl, aldehyde, or carboxy.
14. The compound of claim 13, wherein
R.sub.1.dbd.CO.sub.2CH.sub.2CH.sub.3- , R.sub.2.dbd.CH.sub.3,
R.sub.3.dbd.CH.sub.3.
15. The compound of claim 13, wherein
R.sub.1.dbd.CO.sub.2CH.sub.2CH.sub.3- ,
R.sub.2.dbd.CH.sub.2CH.sub.3, R.sub.3.dbd.CH.sub.2CH.sub.3.
16. The compound of claim 13, wherein R.sub.1.dbd.CH.sub.3,
R.sub.2.dbd.COCH.sub.3, R.sub.3.dbd.CH.sub.3.
17. The compound of claim 13, wherein R.sub.1.dbd.CH.sub.3,
R.sub.2.dbd.CO(CH.sub.2).sub.nCH.sub.3, R.sub.3.dbd.CH.sub.3, and
wherein n is 0-20.
18. The compound of claim 13, wherein R.sub.1.dbd.H,
R.sub.2.dbd.CH.sub.3, R.sub.3.dbd.CH.sub.3.
19. The compound of claim 13, wherein R.sub.1.dbd.H,
R.sub.2.dbd.CH.sub.2CH.sub.3, R.sub.3.dbd.CH.sub.2CH.sub.3.
20. The compound of claim 13, wherein R.sub.1.dbd.H,
R.sub.2.dbd.R.sub.3.dbd.CH.sub.2(CH.sub.2).sub.2CH.sub.3.
21. The compound of any one of claims 13-20 in which one or more of
the pyrrole nitrogens exist in anionic form.
22. A compound of the general formula, 34wherein individually at
each occurrence, each of R.sub.1, -R.sub.7 are the same or
different and are hydrogen, alkyl, hydroxyalkyl, glycol,
polyglycol, amino, nitro, halo, cyano, aryl, heteroaryl, thio,
thioalkyl, amide, ester, acyl, aldehyde, or carboxy, and n=0-10,
and further wherein each of R.sub.1-R.sub.7 on either side of the
axis of the diketone bridge may be the same or different from the
corresponding R.sub.1-R.sub.7 on the opposite side of the diketone
bridge and further wherein each R.sub.4 and R.sub.5 of the n
subunits of pyrrole may be the same or different from corresponding
R.sub.4 and R.sub.5 of any other subunits of pyrrole.
23. The compound of claim 22, wherein each of R is H and wherein
n=0.
24. A compound of the general formula: 35wherein individually at
each occurrence, each of R.sub.1, -R.sub.11 are the same or
different and are hydrogen, alkyl, hydroxyalkyl, glycol,
polyglycol, amino, nitro, halo, cyano, aryl, heteroaryl, thio,
thioalkyl, amide, ester, acyl, aldehyde, or carboxy, and n=0-10,
and further wherein each of R.sub.1-R.sub.7 on either side of the
axis of the quinoxaline bridge may be the same or different from
the corresponding R.sub.1-R.sub.7 on the opposite side of the
quinoxaline bridge and further wherein each R.sub.4 and R.sub.5 of
the n subunits of pyrrole may be the same or different from
corresponding R.sub.4 and R.sub.5 of any other subunits of
pyrrole.
25. The compound of claim 24 wherein n=1.
26. The compound of claim 24 wherein n=2.
27. A pyrrole-aryl compound of the general structure: 36wherein
individually at each occurrence, R.sub.1, -R.sub.10 are the same or
different and are hydrogen, alkyl, hydroxyalkyl, glycol,
polyglycol, amino, nitro, halo, cyano, aryl, heteroaryl, thio,
thioalkyl, amide, ester, acyl, aldehyde, or carboxy and M is Li, B,
Na, Mg, Al, Si, K, As, Rb, Sb, Cs, La, Hf, Bi, Pr, Eu, Yb, or
Th.
28. A pyrrole-aryl compound of the general structure: 37wherein
R.sub.1, -R.sub.8 individually at each occurrence, are the same or
different and are hydrogen, alkyl, hydroxyalkyl, glycol,
polyglycol, amino, nitro, halo, cyano, aryl, heteroaryl, thio,
thioalkyl, amide, ester, acyl, aldehyde, or carboxy; and further
wherein Ar is selected from the group consisting of: 38 and further
wherein X, Y, and Z are selected from the group consisting of
hydrogen, aldehyde, nitro, and amino.
29. A pyrrole-quinoxaline compound of the general structure:
39wherein R.sub.1, -R.sub.8 individually at each occurrence, are
the same or different and are hydrogen, alkyl, hydroxyalkyl,
glycol, polyglycol, amino, nitro, halo, cyano, aryl, heteroaryl,
thio, thioalkyl, amide, ester, acyl, aldehyde, or carboxy, and
wherein TMS is a trimethylsilyl group.
30. The compound of claim 1, 2, 24, 27, or 28 in which one or more
of the pyrrole nitrogens exist in anionic form.
31. The compound of claim 1, 2, 24, 27, or 28 incorporated into a
macrocyclic molecule.
32. The compound of claim 31 selected from the group consisting of
the following structures: 40wherein all R, individually at each
occurrence, are the same or different and are hydrogen, alkyl,
hydroxyalkyl, glycol, polyglycol, amino, nitro, halo, cyano, aryl,
heteroaryl, thio, thioalkyl, amide, ester, acyl, aldehyde, or
carboxy and n=1-10.
33. A pyrrole-aryl compound of the following general formulas:
41wherein R.sub.1-R.sub.6, individually at each occurrence, are the
same or different and are hydrogen, alkyl, hydroxyalkyl, glycol,
polyglycol, amino, nitro, halo, cyano, aryl, heteroaryl, thio,
thioalkyl, amide, ester, acyl, aldehyde, or carboxy.
34. A method for the analysis of an anion or neutral species
comprising: (a) obtaining a pyrrole-aryl compound; (b) contacting
the pyrrole-aryl compound with a sample containing an anion or
neutral species; and (c) optically monitoring the pyrrole-aryl
compound in the presence of the sample.
35. The method of claim 34 wherein said pyrrole-aryl compound is a
pyrrole-quinoxaline compound.
36. The method of claim 34 or 35, wherein the pyrrole-aryl compound
is the compound of claim 2.
37. The method of claim 36, wherein
R.sub.1.dbd.R.sub.2.dbd.NO.sub.2.
38. The method of claim 36, wherein R.sub.1.dbd.H,
R.sub.2.dbd.NO.sub.2.
39. The method of claim 34 or 35 wherein the anion is an anion of
fluoride, cyanide, phenolate, carboxylate, sulfate, sulfite,
sulfide, sulfonate, nitrate, nitrite, bromide, iodide,
pertechtenate, perrhenate, phosphate, phosphonates, nucleobase,
nucleotide or oligonucleotide.
40. The method of claim 39, wherein the anion is fluoride or
chloride.
41. The method of claim 34 or 35 wherein the neutral species is
cis-3-hexenal.
42. The method of claim 34 or 35, wherein the optically monitoring
is fluorescence excitation, fluorescence emission, visual detection
or ultraviolet or visible absorption.
43. The method of claim 34 or 35, wherein the pyrrole-aryl compound
is attached to a solid support.
44. The method of claim 34 or 35, wherein the optical monitoring is
performed by the means of a spectroscopic apparatus comprising a
fiber optic.
45. The method of claim 34 or 35, in which the analysis is carried
out in vivo, in vitro, or in situ.
46. A method of using a pyrrole-aryl compound in vivo, in vitro, or
in situ to selectivlely transport therapeutic agents.
47. A method of using a pyrrole-aryl compound as a sensing element
in the analysis of foodstuffs.
Description
[0001] This application claims priority to U.S. Provisional
Application No. 60/136,467 filed May 28, 1999.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The invention relates generally to compositions and methods
for binding and optically sensing anions, cations, and neutral
species. Analytical methods for such species is the primary goal of
optical sensing. These methods may be qualitative or quantitative.
In particular, compositions containing pyrroles as the key
recognition element and a quinoxaline backbone as part of the
compound, are shown to provide a system with a built-in optical
probe for selective sensing.
[0005] 2. Description of Related Art.
[0006] In the recent decades, supramolecular chemists have devoted
considerable effort to developing systems capable of recognizing,
sensing, and transporting anions (Dietrich, et al., 1997). This is
an area of effort that is considered both timely and important.
Indeed, some 70 to 75% of all natural biological processes are
thought to involve a negatively charged species (Schmidtchen,
1988).
[0007] Anion recognition constitutes an important problem area
within the generalized field of supramolecular chemistry. Not
surprisingly, therefore, it has been pursued extensively,
particularly within the calixarene domain. Indeed, most attention
has focused on calixarene systems that have been modified, via
attachment to, or reaction with, electron deficient metal centers,
so as to make more electrophilic the normally .pi.-electron rich
calixarene moiety.
[0008] Anions constitute key components in food stuffs (e.g.,
fluoride, citrate and benzoate) and are products for, and
pollutants from, modem agriculture (e.g., phosphate and nitrate)
and can also act as potent toxins (e.g. cyanide). One anion,
pertechnetate, is critical to radio-diagnostic and therapy
procedures and, in a different isotopic form, is a major
radioactive pollutant. Given these few examples, it is clearly
important that we have a means to readily monitor the presence of
these species in our everyday environment.
[0009] Among the range of biologically important anions, fluoride
is of particular interest due to its established role in preventing
dental caries (Kirk, 1991) Fluoride anion is also being explored
extensively as a treatment for osteoporosis, (Riggs, 1984 and
Kleerekoper, 1998) and, on a less salubrious level, can lead to
fluorosis, (Wiseman, 1970 and Gale, et al., 1996) a type of
fluoride toxicity that generally manifests itself clinically in
terms of increasing bone density. This diversity of function, both
beneficial and otherwise, makes the problem of fluoride anion
detection one of considerable current interest. Thus, while
traditional methods of fluoride anion analysis, involving, e.g.,
ion selective electrodes and .sup.19F-NMR spectroscopy remain
important, there is an increasing incentive to find alternative
means of analysis, including those based on the use of specific
chemosensors. Particularly useful would be systems that can
recognize fluoride anion in solution and signal its presence via an
easy-to-detect optical signature.
[0010] In the past few years, a wide range of anion sensors have
been proposed (sapphyrins, Sessler, et al. 1997; calixpyrroles,
Gale, et al. 1996 and Sessler, et al., 1998; cyclic polyamines,
Dietrich, et al., 1981; Hoseini and Lehn, 1988) guanidinium
(Dietrich, et al., 1981 and Metzger, et al., 1997)) that present
varying degrees of affinity (and selectivity) toward anions such as
F.sup.-, Cl.sup.-, H.sub.2PO.sub.4.sup.- and/or carboxylates.
Unfortunately, and in spite of considerable effort, a need for good
anion sensors remains. The number of anion sensors which can select
for one biological anion over a range of anions present in vivo
(phosphate, chloride, fluoride, etc.) remains at best, very
limited. While there exists small molecule sensors which can bind
anions relatively well, they do so with little or no specificity.
This is particularly true in the case of fluoride anion where few,
if any, easy-to-use signaling agents exist.
[0011] In addition to anion sensing, it is also desirable to
develop sensing elements capable of sensing cations and neutral
species. The presence or absence, as well as the level of, various
neutral molecular species is a useful diagnostic tool that can
signal chemcial decomposition. One example is the sensing of
cis-3-hexenal (or chemical derivatives thereof), a metabolite of
the bacterial E. Coli, Salmonela, and Lysteria. Such sensors would
find applicability in the food industry as detectors of food
contamination and spoilage. They could, for instance, be
incorporated into food packaging materials.
[0012] Therefore, a need exists to develop methods and compositions
for the selective detection of anions, cations, and neutral species
in general, and for fluoride in particular. A motivation for the
preparation of new sensors is to obtain sensor compounds designed
to recognize selectively a particular analyte within a range of
species and produce an easily detected signal.
SUMMARY OF THE INVENTION
[0013] The present invention provides novel compounds containing
both pyrrole-derived anion and neutral species recognition subunits
and an aromatic core as the optical or visual signaling group to
provide chemosensors that allow for the convenient, color-based
sensing of anions. Most commonly, the aromatic core will be a
quinoxaline moiety, but may be any aryl system having two pyrroles
covalently bound to neighboring (but not necessarily directly
ajacent) carbons on an aryl moiety through a C--C single bond
connecting pyrroles and the aromatic moiety.
[0014] Formula I illustrates the general pyrrole-aryl systems
(.alpha.,.alpha. and .beta.,.beta. substitution on the pyrrole
rings) along with the specific pyrrole-quinoxaline analog shown
directly below. Note that the pyrrole substitution may also be
mixed, i.e., .alpha.,.beta. or .beta.,.alpha.. As used herein,
"aryl" means any aromatic system consisting of one or more rings
which may be homonuclear or heteronuclear, and which may or may not
contain aromatic or non-aromatic side groups (substitution), and
which may be further complexed to one or more metals. The present
invention further provides methods of use and synthetic schemes for
these novel compounds. 1
[0015] The present invention provides novel compounds exemplified
by the pyrrolic nitrogens used as anion recognition elements with
an aromatic core as a signal group. The compounds of the present
invention are termed pyrrole-aryls, and as used herein, the
compounds of the present invention which, at least, combine these
two elements will be referred to as such. Although not shown above,
the pyrrole carbon atoms may also be substituted. The aryls may or
may not contain heteroatoms. Subsituents may include, but are not
limited to, hydrogen, alkyl, hydroxyalkyl, glycol, polyglycol,
amino, nitro, halo, cyano, aryl, heteroaryl, thio, thioalkyl,
amide, ester, acyl, or carboxy and may be the same or different at
each occurrence.
[0016] Compounds of the present invention may be prepared by a
condensation between a 1,2-diamine and a 2,3-dipyrryl ethanedione
as shown in Scheme 1. 2
[0017] While specific substituents are listed above, the
quinoxaline analogs may have a wider variability of substituent
groups. R.sub.1 and R.sub.2 may be, individually at each
occurrence, hydrogen, alkyl, hydroxyalkyl, glycol, polyglycol,
amino, nitro, halo, cyano, aryl, heteroaryl, thio, thioalkyl,
amide, ester, acyl, or carboxy. Although not shown above, any or
all of these possible substitutions may be present on the remaining
available carbon atoms of the quinoxaline. Additionally, any or all
of these same possible substitution combinations may also be
present on the .alpha. or .beta. positions, or on both the .alpha.
and .beta. positions (relative to nitrogen) of the pyrrole
rings.
[0018] Oxalyl chloride, o-phenylenediamine,
4-nitro-1,2-diaminobenzene were purchased from Aldrich and used
without further purification. 4,5-Diamino-1,2-dimethoxybenzene was
prepared according to the method of Sessler, 1992.
4,5-Dinitro-1,2-diaminobenzene was prepared according to the method
of Cheeseman, 1962.
[0019] Thus, in a second respect, the present invention is the
2,3-dipyrryl-ethanediones used to produce the pyrrole-aryls. In
this aspect of the invention the dipyrryl-ethanediones are of
Formula II: 3
[0020] wherein individually at each occurrence, each of R.sub.1,-
R.sub.6 are the same or different and are hydrogen, alkyl,
hydroxyalkyl, glycol, polyglycol, amino, nitro, halo, cyano, aryl,
heteroaryl, thio, thioalkyl, amide, ester, acyl, or carboxy. Though
not shown the di-.beta.-linked diketone (bridging group attached to
pyrroles in position .beta. to nitrogen atoms) is within this
family, as is the mixed .alpha., .beta.-linked diketone.
[0021] These dipyrryl-ethanediones may be produced by reaction of a
pyrrol either commercially available or obtainable through
synthetic methods known to one of skill in the art, with oxalyl
chloride as represented in Scheme 1 to generate a variety of
dipyrryl-ethanediones.
[0022] Further to this, the present invention provides a new set of
novel dione compounds generated from the reaction of bipyrroles,
terpyrroles etc. with oxalyl chloride to generate the compounds of
Formula III: 4
[0023] wherein individually at each occurrence, each of
R.sub.1-R.sub.7 are the same or different and are hydrogen, alkyl,
hydroxyalkyl, glycol, polyglycol, amino, nitro, halo, cyano, aryl,
heteroaryl, thio, thioalkyl, amide, ester, acyl, or carboxy and
n=0-10. The analogous R.sub.x groups on either side of the diketone
bond may be the same or different (i.e., R.sub.1, R.sub.2, . . .
R.sub.x on one side of the diketone bond may be the same or
different from the corresponding R.sub.1, R.sub.2, . . . R.sub.x on
the opposite side, etc.; additionally, the R.sub.4 and R.sub.5
groups may have variability amongst individual pyrrole subunits;
e.g. R.sub.4 on any given subunit may be the same or different from
a corresponding R.sub.4 on any other subunit). Symmetry in
substitution along the axis bisecting the diketone bond or among
any pyrrole subunit is not required and maximum variability in
substitution is possible so long as the general formula is
followed.
[0024] These novel diones may then be used to generate novel
pyrrole-aryl compounds such as 2,3-di(bipyrryl)quinoxalines (n=0),
2,3-di(terpyrrylquinoxalines (n=1), 2,3-di(tetrapyrrylquinoxaline
(n=2) etc. The preferred route is via a condensation reaction
involving the two ketones with an aryl compound.
[0025] It is further contemplated that the
2,3-dipyrryl-ethanediones may undergo reaction with any 1,2-diamine
under conditions outlined in Scheme 1 to generate a variety of new
compounds for anion sensing as represented by Formula IV
(functionalized quinoxaline analogs) and Formula V (functionalized
pyrrole, functionalized quinoxaline analogs), respectively. 5
[0026] In Formula IV and V, respectively, individually at each
occurrence, each of R.sub.1-R.sub.4 and R.sub.1-R.sub.10 are the
same or different and are hyrdogen, alkyl, hydroxyalkyl, glycol,
polyglycol, amino, nitro, halo, cyano, aryl, heteroaryl, thio,
thioalkyl, amide, ester, acyl or carboxy. Note that the quinoxaline
analogs are used for illustrative purposes in the above examples.
It is readily apparent to one of ordinary skill in the art that an
appropriate aryl or substituted aryl may be used in place of
quinoxaline for the more generalized pyrrole-aryl aryl compounds.
As earlier discussed, the only requirement is orbital overlap of
the ring systems comprising the aryl and pyrrole groups which are
altered by bond rotation upon binding of substrate (anion, cation,
or neutral atom or molecule): 6
[0027] The pyrrole groups are preferably on, but need not be on,
adjacent carbons of the aryl moiety. Note that the nitrogen atoms
may be deprotonated to afford cation-binding systems.
[0028] 2,3-dipyrrol-2'yl-5,6-dicyanopyrazine (Example 30) is an
example of an analogous pyrrole-aryl sensing compound that does not
contain the quinoxaline moiety. The present invention provides a
solution to the needs described herein above by producing compounds
and methods for selective sensing. In particular, the preferred
pyrrole-aryl compounds of the present invention have the ability to
selectively bind fluoride anion over biologically important
competitors such as chloride and phosphate and in doing so, produce
a color change from yellow to orange in the case of 1 and from
orange to purple for 3 and 8 which is, in some circumstances,
visible to the naked-eye. It was further found that for these
particular analogues, organic solvents encourage fluoride binding
while polar solvents, such as methanol or water, lead to fluoride
dissociation. This property would allow for the original sensor to
be regenerated by changing solvents once the sensing is
complete.
[0029] The compounds of the present invention are particularly
contemplated for use in fluoride sensing, especially in the
presence of other biologically common anion species. While
analogues such as 3 may display other anion sensitivities, the
ability to selectively sense fluoride anion would be particularly
useful for many purposes as further discussed in Examples 40 and
42.
[0030] Therefore, an aspect of the present invention is the
development of analytical methods for species which are selectively
bound by the pyrrole-aryl compounds. As used herein, "analysis"
means both quantitative and qualitative analysis. As used herein,
optical methods included instrumental spectroscopic methods as well
as visual observation. While the focus is on optical and visual
analytical methods, electrochemical methods employing the
pyrrole-aryls as sensing elements are also envisioned. Time-based
analytical methods, such as those monitoring changes in
fluorescence lifetimes (as well as other photophysical temporal
phenomena) measurements are also envisioned. Either of these
analytical examples would be sensitive to the modification of the
molecular electronic structure of the sensing compound which would
be caused upon substrate binding. Many other analytical
measurements sensitive to such changes in electronic structure and
which are known to those of skill in the art would be applicable in
the present invention.
[0031] It is contemplated that the pyrrole-aryls of the present
invention have a wide variety of uses. A range of compounds with a
large number of substituents fall within the scope of the present
invention. The precursor molecules, the starting pyrrole or dione,
may be derivatized as desired or the pyrrole-aryl may be modified
post synthetically to yield compounds with desired substituents.
Therefore, it is contemplated that the selectivity of the
pyrrole-aryl compounds of the present invention will have a number
of different selectivities achieved by variation of substituents
within the structure.
[0032] It is also envisioned that the binding and sensing
capabilities of the pyrrole-aryls can be further exploited by
surface immobilization. Functionalization of the pyrrole-aryl with
reactive groups would afford the ability to attach them to solid
phases. Polymer phases, silica and polystyrene, among others, are
solid surfaces which find applicability in this embodiment of the
invention. Surface immobilization is useful in the separation
sciences, in the fabrication of sensors such as fiber optic probes,
as well as other applications. In the field of separation science,
surface immobilization can be used to fabricate novel stationary
phases. In fiber optic sensing application, immobilization of the
sensing element on the distal end of a fiber optic tip can be used
to construct sensors useful for remote analyses. Fiber optic
sensors are known to be amenable to remote sensing, such as in
vivo, in vitro or in-situ sensing. In vivo applications would
involve miniaturization of the fiber optic tip, thus the high
sensitivity achievable with the pyrrole-aryls is particularly
advantageous. In the area of foodstuff analysis, surface
immobilization of the pyrrole-aryls onto fiber optic sensors, or
alternatively, onto packaging components of foodstuffs is
envisioned to afford a quick, convenient way to monitor
spoilage.
[0033] Additionally, the pyrrole-aryls of the present invention may
be modified to increase aqueous solubility for use analytically or
as therapeutic agents. In sensing applications, modification of
solubility may be used to optimize a sensing element for the
particular environment to be interrogated. This is performed
through functionalization of the pyrrole-aryl compounds with groups
that impart water solubility. Polar groups, especially those which
readily carry a charge under various conditions, are candidates for
such functionalization. Carboxy, hydroxy, and amine groups are most
obvious but others are possible. Enhancing water solubility is
useful therapeutically by enhancing bioavailability.
[0034] Additional modifications are envisioned in which the
pyrrole-aryls may be incorporated into macrocyclic compound.
Porphyrin-type complexes are but one example further described
below. By incorporating the binding site into a macrocyclic
compound, novel compounds may be made to optimize transport of
therapeutic agents, or to tailor the sensing element for a specific
application. Metal-linked systems of pyrrole-aryls are another
aspect of the present invention. These may be prepared by first
preparing silyl derivatives having, for example, TMS groups
appended. Subsequent deprotection and reaction with a metal salt
will afford metal linked systems.
[0035] Therapeutic uses of the compounds of the invention are also
described. The binding capabilities may be exploited for uses as
transporting agents. Anionic, cationic, and neutral species,
through binding to the appropriate pyrrole-aryl, can be directed in
vivo to areas where their therapeutic effect is optimally realized.
The high affinity for a number of these species for chloride ion
has potential applicability in the treatment of cystic fibrosis.
Cystic fibrosis is characterized by a reduced ability to effect
chloride ion transport at the cellular level. involves the
localized introduction of chloride ion. A means for enhancing the
transport of chloride ion is therefore useful in such treatments.
While this is one specific example, the ability to transport
therapeutically active agents is expected to have wider
applicability. This has the beneficial advantages of allowing for
more efficient and lower dosages, which minimizes side and toxicity
effects.
[0036] The compounds of the present invention provide a further
advantage in the ease with which the pyrrole-aryls can, in light of
the present disclosure, be modified. The synthetic steps are
relatively simple and inexpensive to carry out. As the optical and
binding properties can be controlled by the types of substituents
present, this allows enough flexibility to accommodate a number of
applications as well as the fine tuning of desired properties, for
application in a specific environment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0038] FIG. 1 provides a view of the hydrogen bonded complex of
[1.cndot.F].sup.- as it extends along the a axis. Hydrogen bonding
interactions are indicated by dashed lines. The relevant geometry
for these interactions are: N1-H1N . . . F1 (related by x-1, y, z),
N F 2.629(2) .ANG., H . . . F 1.63(3) .ANG., N--H . . . F
169(2).degree.; N17-H17 F1, N . . . F 2.640(2) .ANG., H F 1.69(3)
.ANG., N--H . . . F 168(2).degree.; O1w-H1w F1, O . . . F 2.590(2)
.ANG., H F 1.70(3) .ANG., N--H O 177(3).degree..
[0039] FIG. 2 is a view of the 2,3-dipyrrylquinoxaline unit 1 in
its anion-free water complex.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0040] The present invention generally relates to methods and
compositions for anion and neutral species binding, analysis and
recognition. As used herein, "analysis" encompasses both
quantitative or qualitative analysis. In particular, the compounds
and methods of the present invention utilize pyrroles as the key
recognition-inducing component and aryl groups as optical reporting
group. Many of the disclosed compounds contain quinoxalines as the
aryl as part of the backbone structure to provide systems with
built in chromophores and/or fluorophores for optical sensing.
While quinoxaline is preferred, the invention is not limited to
sensing compounds with this aryl component.
[0041] With reference to the substituents contemplated for use in
accordance with the present invention, alkyl may be of the
repeating unit --(CH.sub.2).sub.nCH.sub.3. The number of repeating
units within an alkyl substituent may be up to fifty, preferably up
to 20 and more preferably from 0-10. Representative examples of
alkanes include methane, ethane, straight chain branched or cyclic
isomers of propane, butane pentane hexane, octane, nonane and
decane. Representative examples of substituted alkyls include
alkyls substituted by two or more functional groups as described
herein. Hydroxyalkyls includes alcohols of alkyl groups as defined
previously. Representative examples of hydroxyalkyls include
alcohols of methane, ethane, straight chain branched or cyclic
isomers of propane, butane, pentane hexane, octane, nonane and
decane. Hydroxyalkyl is meant to include glycols and polyglycols.
Representative examples of glycols include diols of ethane,
straight-chain, branched or cyclic isomers of propane, butane,
pentane hexane, octane, nonane and decane. Representative examples
of polyglycols include polyethylene glycol, polypropylene glycol,
polypropylene diol and polybutylene diol. Representative examples
of oxyalkyls include the alkyl groups defined herein above having
ether linkages.
[0042] Representative examples of thioalkyls include thiols of an
alkyl as described herein above including thiols of ethane, thiols
of straight-chain, branched or cyclic isomers of propane, butane,
pentane, hexane, heptane, octane, nonane and decane. Sulfate
substituted alkyls include alkyls as described above substituted by
one or more sulfate groups, a representative example of which is
diethyl sulfate ((C.sub.2H.sub.5).sub.2SO.sub.4); they also include
simple anionic sulfate or sulfonate substituents such as
--C.sub.2H.sub.5SO.sub.3.sup.-.
[0043] As used herein, aryl refers to a compound whose molecules
having either the pi-conjugated ring of benzene or the condensed
rings of the other aromatic derivatives including
heteroatom-containing aromatic derivatives (heteroaryls). They may
additionally contain non-aromatic subsituents as side groups or be
linked to metals. Representative examples include benzene,
naphthalene, phenanthrene, phenanthroline, and anthracene. A
heteroaryl compound, as used herein, refers to a compound which
contains more than one kind of atom in an aromatic ring.
Representative examples include pyridine, pyrimidine, furan,
thiophene, pyrrole and imidazole.
[0044] Representative examples of amines include primary, secondary
and tertiary amines of an alkyl as described herein above.
[0045] Representative examples of carboxy groups include carboxylic
acids of the alkyls described above as well as aryl carboxylic
acids such as benzoic acid. Carboxy groups also include derivatives
of carboxylic acids such as esters, amides, acyl halides,
anhydides, and nitriles. Representative examples of carboxyamides
include primary carboxyamides (RCONH.sub.2), secondary (RCONHR')
and tertiary (RCONR'R") carboxyamides where each of R' and R" is a
functional group as described herein and the carboxy group is as
defined herein above.
[0046] Representative examples of ester groups include compounds of
the form RCOR' where the R group is an alkyl as described herein
above and where R' is a functional group as described herein.
Representative examples of acyl groups include acyl derivatives
RCO- or ArCO-, wherein R is an alkyl as described herein above and
Ar is an aryl group as defined herein.
[0047] The choice of metal ion for complexing to analogues of the
present invention will generally be dependent on the use or
intended use of the analogue. For example, representative metals
for the porphyrin derivative include Zn, Cu, Pd, Ni, Fe, Co, Ru,
Rh, and Os.
[0048] In one embodiment a method for anion sensing is disclosed
using compounds of general Formula I shown herein above.
Importantly, the general structures may possess substituent (R)
groups at any carbon capable of accommodating such substituents
(i.e., those bound to at least one hydrogen atom in the general
structures) each R being defined as above.
[0049] The synthesis of compounds represented in Formula I is
outlined in Scheme 1. In this embodiment the substituents R.sub.1
and R.sub.2 are introduced during the synthesis on the aryl
1,2-diamine as shown. It would be well appreciated by one of skill
in the art, in light of the present disclosure, that a wide variety
of aryl 1,2-diamines may be condensed with a 1,2-dipyrryl
ethanedione to provide a vast array of different compounds and that
any or all of the remaining positions on the 1,2-diamine may bear
substituents as exemplified by Formula IV.
[0050] Additionally, compounds derived from those represented by
Formula I are contemplated for the formation of metal complexes.
For example, structure 9 may be condensed with
1,10-phenanthroline-5,6-dione to form compound 13. An alternate
condensation of structure 9 with 5,6-diamino-1,10-phenanthroline
affords structure 14. Both of these examples are shown in Example
34 where the metal binds through the phenanthroline nitrogens.
Furthermore, compounds with altered fluorescence, represented by 15
and 16, may be synthesized using conditions similar to those
provided in Example 34. Additionally, those syntheses detailed in
other Examples could be effected using the appropriate dione.
[0051] Further variants may be produced by using a porphyrin dione
in the condensation to form a compound exemplified by structure 17
(Example 34), bearing a range of substituents at the meso and
.beta.-pyrrolic positions. The porphyrin may be metallated using
standard techniques to generate a range of compounds containing
various metals (for example Zn, Cu, Pd, Ni, Fe, Co, Ru, Rh, Os).
For the substituents shown in structure 17, individually at each
occurrence, each of R.sub.1, R.sub.4, R.sub.7, R.sub.10, is a
hydrogen atom, alkyl, aryl or heteroaryl and individually at each
occurrence, each of R.sub.2, R.sub.3, R.sub.5, R.sub.6, R.sub.8,
R.sub.9 is a hydrogen atom, alkyl, aryl or heteroaryl, halo, cyano,
acyl, carboxyl, carboxy ester, carboxyamide, nitro or amino. While
these are the preferred substituents, a wider range of substitution
is possible and may even be desirable for given applications. One
of skill in the art can appreciate the wide range of substitution
possible. These porphyrin containing compounds are contemplated for
analytical applications, particularly as redox-based and/or optical
sensors.
[0052] Other possible metal-containing analogs include pyrrole-aryl
tethered metallocenes such as ferrocene. Additionally, crown
ether-derivatized pyrrole-aryls can be used as metal bearing
agents. Other host-guest species such as cyclodextrins may be
coupled covalently to the pyrrole-aryls. Metallocenes,
crown-ethers, and cyclodextrins act as substrate binders and
catalysts in their own distinctive chemistries. Binding to the
pyrrole-aryls will produce novel, synergistic binding effects.
These are expected to further extend the usefulness of these novel
species to electrochemical applications, particularly for
analytical sensing. It is envisioned that the metal-complexed
derivatives will be useful as ion channels. The selective binding
and transport capabilities of such "pore-forming" species will have
application as transport agents therapeutically and as sensors
analytically.
[0053] The dipyrrole quinoxalines of the present invention may also
be modified post-synthetically to introduce a variety of
substituents at the .alpha. and/or .beta. pyrrolic positions,
R.sub.3 and R.sub.4, as shown in compound 18 (Example 35). The
substituents R.sub.1 and R.sub.2 are as previously described,
R.sub.3 and R.sub.4 may be the same or different, and may be
halides, including, iodo and bromo, alkyl, aryl, heteroaryl, acyl,
nitrile, carboxy amide, carboxy ester or sulfide. The introduction
of these substituents allows for further modification of properties
for a given compound. Using protocols well known in the art, the
dibromo or diiodo compounds may be used to generate a further range
of .alpha. substituted compounds such as those provided by
structure 19. Compounds such as 20, may be used in subsequent
reaction with a metal salt, such as [PdCl.sub.2(PEt.sub.3).sub.2],
{i.e., bis(triethylphosphine) palladium(II) dichloride, or
phosphino-platinum dichloride complexes, following removal of the
TMS groups to afford metal linked systems.
[0054] The pyrroles used in accordance with the present invention
to prepare the pyrryl-quinoxalines, may be derivatized as
represented in Scheme 5, structure 21 (Example 36). The pyrrole
units with R.sub.1-R.sub.3 substituents may, in some cases, be
commercially available or in other instances may be prepared
through synthetic methods well known to one of skill in the
art.
[0055] The 1,2-dipyrryl-ethanediones used in the present invention
may be generated using the above-described pyrroles in a reaction
with oxalyl chloride to provide many different 1,2
dipyrryl-ethanediones as shown in Scheme 5. Substituents R.sub.1,
R.sub.2 and R.sub.3 are alkyls, hydroxyalkyls, substituted alkyls,
amines, halo, cyano, aryl, heteroaryl, thio, thioalkyl, amide,
ester, acyl and carboxy. Representative examples include those
described previously with the preferred substituents as listed in
Example 36 for compound 22.
[0056] Additionally, several other compounds are contemplated for
use in the reaction with oxalyl chloride to generate a series of
diones. In particular, many different polypyrroles, bipyrroles
(n=0), terpyrroles (n=1), up to and including n=10 may be employed
as shown in Scheme 6 (Example 36) to generate compounds exemplified
by structure 24. R.sub.1 through R.sub.7 are alkyl, hydroxyalkyl,
glycol, polyglycol, amino, nitro, aryl, heteroaryl, acyl, halo,
carboxy ester, carboxy amide and carboxy as previously described,
such that R.sub.1-R.sub.7 may be the same or different at each
occurrence and n is 0-10, with R.sub.1 through R.sub.7 as H and n=0
being preferred.
[0057] It is further contemplated that quinoxaline compounds of the
present invention with a free .alpha.-position may undergo further
reaction as shown in Schemes 7A-7D (Example 37) for incorporation
into macrocycles for use as sensing/transporting agents, and
optical display devices. A wide variety of conditions known to one
of skill in the art, may be employed to promote reaction of the
remaining .alpha.-free position on the pyrrole rings as shown in
Example 37. These compounds are as represented by structures 25-32,
and thus fall within the scope of the present invention.
[0058] Further sources of synthetic variation may be generated
using diones comprised of various heterocyclic rings as shown in
Example 38. These compounds may provide alternate selectivity for a
variety of other analytes, such as metal ions in the case of the
furan, thiophene, pyridine and pyridine N-oxide derived
systems.
[0059] Another source of variation involves the use of dianions of
either the 1,2-di-pyrrylethanedione or the
2,3-di-pyrrylquinoxalines, as represented in Example 39, in
accordance with the present invention. These dianions are
contemplated for use as ligands for the generation of metal
complexes. Such compounds would find use in the areas of molecular
wires and display devices.
[0060] It is particularly contemplated that the pyrrole quinoxaline
compounds of the present invention will be of use as anion sensors.
Example 40 provides a further discussion. It is further
contemplated that the preferred compounds will find utility as
novel fluoride anion sensors and receptors, and other examples will
find use as chloride and phosphate sensors and receptors. The
unsubstituted pyrrolic nitrogens provide anion binding sites as
detailed in Examples 31-33. Furthermore, the pyrrole quinoxaline
compounds of the present invention provide a quinoxaline core as a
chromophoric signal group for the color-based sensing of
anions.
[0061] Any one of a wide variety of anions may be detected using
the pyrrole-aryls or alternate heterocyclic quinoxalines in
accordance herewith. These anions include, but are not limited to,
cyanide anion, phenolate anion, carboxylate anion, sulfate anion,
sulfonate anion, nitrate anion, nitrite anion, bromide anion,
pertechnetate anion, perrhenate anion, chloride anion, phosphates
and phosphonates.
[0062] Additionally, the disclosed methods are contemplated for
binding or complexing a range of analytes, including anion, cations
and neutral species, but particularly anions and neutrals.
Therefore, also contemplated are phosphate-containing compounds,
including simple alkyl or aryl phosphates, alkyl phosphonates,
nucleotides, oligo- and polynucleotides such as DNA, RNA and
anti-sense constructs and nucleotide analogues. Representative
examples of phosphates include phosphate or polyphosphate groups.
Representative examples of phosphate substituted alkyls include
alkyls as described above substituted by one or more phosphate or
polyphosphate groups. Representative examples of phosphonate
substituted alkyls include alkyls as described above substituted by
one or more phosphonate groups.
[0063] The term nucleotide, as used herein, refers generally to any
moiety that includes within its structure a purine, pyrimidine, or
nucleic acid with a ribose group and at least one phosphate group,
or any derivative of these such as a protected nucleotide. Thus the
term nucleotide includes adenosine tri-, di- and monophosphate,
guanosine tri-, di- and monophosphate, cytidine tri-, di- and
monophosphate, thymidine tri-, di- and monophosphate, uridine tri-,
di- and monophosphate and xylo-guanosine monophosphate a well as
any nucleotide derivative based upon these or related
structures.
[0064] As discussed previously, the increased specificity and
optical analysis makes this class of anion sensors considerably
more effective at anion recognition and detection than other
classes of molecules. The capacity of pyrrole-aryls and analogues
thereof, to effect specific sensing of anions in general, and
fluoride in particular, is contemplated to be advantageous for use
in vitro and in vivo. For example, the disclosed compounds may be
used to effect the construction of electrodes (analogous to pH
sensing) or to fiber optic cables for analysis of drinking water,
in vitro, or for in vivo fluoride sensing in the analysis of bone
density. In addition to optical sensors, other applications are
contemplated such as chromatography. Here for example, compounds
1-3 are coupled to a solid support. The pyrryl-quinoxalines can be
attached to solid supports, via condensation reaction between
complementary functional groups on the quinoxaline and the solid
support, e.g., amine on quinoxaline with carboxyl on solid support
to yield an amide linkage, or a carboxyl group on the quinoxaline
with an amine or hydroxyl on the solid support to give an amide or
ester linkage respectively. These coupled compounds may be used to
separate various anions from each other and from other species in
the mixture.
[0065] Additionally, the binding and/or sensing of fluorinated
compounds is also contemplated. It is well established that
fluorinated hydrocarbons are damaging to the atmosphere, as well as
to humans, and that fluorinated phosphates are extremely toxic when
ingested, for instance from chemical weapons. In order to increase
solubility in aqueous solutions, derivatives containing additional
pyrroles in the pyrrole-aryl system as outlined in Example 41. This
would allow for analysis of fluoride levels in blood samples as
well as a treatment for any detected fluorosis.
[0066] In the detection of fluoride anion as described in Examples
31-33, the preferred solvent is an aprotic one with dichloromethane
particularly preferred. However, other solvents are contemplated
for the appropriate analogues as described previously.
Immobilization on Solid Supports
[0067] The target pyrrole-aryl sensing compound can be covalently
attached to a solid support using any of the number of methods
commonly employed in the art to immobilize molecular species to
solid supports. Covalent attachment of the sensing compound to the
solid support may occur by reaction between a reactive site or a
binding moiety on the solid support and a reactive site or another
binding moiety attached to the sensing compound or via intervening
linkers or spacer molecules, where the two binding moieties can
react to form a covalent bond. For example, binding of a sensing
compound to a solid support can be carried out by reacting a free
amino group of an amino-functionalized sensing compound with a
reactive carboxy of the solid support. Similarly the reaction of
alcohol groups and the derivatized and native SiOH groups of silica
can afford immobilization.
[0068] Coupling of a sensing compound to a solid support in this
way may be carried out through a variety of covalent attachment
functional groups. Any suitable functional group may be used to
attach the sensing compound to the solid support, including but not
limited to disulfide, carbamate, hydrazone, ester, N-functionalized
thiourea, functionalized maleimide, mercuric-sulfide, gold-sulfide,
amide, thiolester, azo, ether and amino. The solid support for use
in separation science or sensor technologies may be made from a
wide variety of materials, such as silica, cellulose,
nitrocellulose, nylon membranes, controlled-pore glass beads,
acrylamide gels, polystyrene, activated dextran, agarose,
polyethylene, functionalized plastics, glass, silicon, brominated
Wang resin, Merrifield resin, agarobiose, carboxypolystyrene HL,
and TG-amino resin. Some solid support materials may require
functionalization prior to attachment of the sensing compound.
Solid supports that may require such surface modification include
aluminum, steel, iron, copper, nickel, gold, silicon, and
nonfunctionalized polymers. In the area of sensing of components,
degradants, and impurities in foodstuffs, solid support materials
include polyethylenes such as LDPE and ULLDPE, EVA and others. In
addition to solid surface immobilization through covalent
interactions, noncovalent interactions (such as that of biotin with
streptavidin, for example), as well as other similar chemistries
that are well-known to those of skill in the art are applicable in
the present invention. Example 43 describes the ability of a
surface bound sensing compound to detect fluoride ion.
[0069] While the above examples recite solid support immobilization
via covalent interactions, binding moieties may also include
functional groups that attach to the solid support via a
high-affinity, noncovalent interaction (such as biotin with
streptavidin), as well as other means that are well-known to those
of skill in the art.
[0070] The selective binding capabilities of the sensing elements
described herein may be useful in a number of applications wherein
it will be advantageous to have such binding on a solid support.
For example, binding to silica-based supports would have utility in
the separation sciences. These have additional utility in the area
of fiber optic sensing. Functionalization of the sensing clement
with silyl alcohols groups, among others, will allows for
attachment onto silica. Additionally, it will be beneficial for
similar purposes to incorporate such sensing elements onto a
polymer support. The molecule shown at right in the example below
may be incorporated into a polymerizing chemistry known to one of
skill in the art to afford a material that has the ability to
behave as a sponge toward analytes of interest. 7
[0071] In the above example, R.sub.1-R.sub.3 is hydrogen, alkyl,
hydroxyalkyl, glycol, polyglycol, amino, nitro, halo, cyano, aryl,
heteroaryl, thio, thioalkyl, amide, ester, acyl, aldehyde, or
carboxy.
Enhancement of Water Solubility
[0072] The unsubstituted pyrrole-aryls have relatively low
solubility. In order to enhance further the general utility of
these species as sensing compounds, it is desirable to afford one
the capability of readily enhancing water solubility. Such
solubility characteristics may be tailored through
functionalzation. Water solubility is most markedly enhanced
through the functionalization of these sensing compounds with
groups that readily carry a formal charge in aqueous solutions.
Additionally, groups of varying polarity may be used to modify
water solubility. Combinations of these groups can be used
depending upon the level of water solubility sought to be imparted
to the sensing compound.
[0073] Functional groups which find applicability in this regard
include carboxy, amino, sulfonate, alcohols. Oxyhydroxyalkyl and
oxycarboxy groups such a hydroxypropoxy and carboxyethoxy are
useful in this regard. Multiply carboxylated derivatives may be
used in their native form or they may be converted to ester or
amide products that can be used to further append hydroxylated
substituents. Polyether-linked polyhydroxylated alkyl groups,
polyethylene glycols and other multi-hydroxy containing groups will
also afford enhanced water solubility while allowing the sensing
element to retain lipophilicity.
Pharmaceutical Compositions and Routes of Administration
[0074] Apart from the application of the subject sensing elements
as in vivo and in vitro sensors, it is envisioned that they may be
used as therapeutic agents. They are particularly useful in
applications were anion and neutral molecule transport are
necessary. One specific example involves the use of chloride ion
transport for the treatment of cystic fibrosis. Cystic fibrosis is
a hereditary disease characterized by the production of defective
chloride channel proteins in epithelial cells, particularly the
lungs. Established treatments target the symptoms of the disease
but do not compensate for the poor chloride ion transport. An
effective, biocompatible carrier that functions in vivo to augment
cell permeability for chloride anion could provide a conceptually
simple, potentially new approach to cystic fibrosis treatment.
These sensing elements have therapeutic uses in any situation
wherein the selective transport of an anion or neutral species is
desired. One variation on the class of compounds described herein
involves the derivatization with crown ethers. Such compounds would
enhance the role of ion or neutral species transport.
[0075] Where clinical application of controlled release
compositions is undertaken, it will be necessary to prepare the
composition as a pharmaceutical composition appropriate for the
intended application. Generally, this will entail preparing a
sterile, physiologically compatible pharmaceutical composition that
is essentially free of pathogens, as well as any other impurities
that could be harmful to humans or animals. One also will generally
desire to employ appropriate buffers to render the complex stable
and allow for uptake by target cells.
[0076] Aqueous compositions of the present invention comprise an
effective amount of a controlled release composition as discussed
above dispersed in a pharmaceutically acceptable carrier or aqueous
medium. Such compositions also are referred to as inocula. The
phrases "pharmaceutically" or "pharmacologically acceptable" refer
to compositions that do not produce an adverse, allergic or other
untoward reaction when administered to an animal, or a human, as
appropriate.
[0077] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients also can be
incorporated into the compositions.
[0078] Solutions of therapeutic compositions can be prepared in
water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions also can be prepared in
glycerol, liquid polyethylene glycols, mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0079] The therapeutic compositions of the present invention are
advantageously administered in the form of injectable compositions
either as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid prior to injection may also
be prepared. These preparations also may be emulsified. A typical
composition for such purpose comprises a pharmaceutically
acceptable carrier. For instance, the composition may contain 10
mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per
milliliter of phosphate buffered saline. Other pharmaceutically
acceptable carriers include aqueous solutions, non-toxic
excipients, including salts, preservatives, buffers and the
like.
[0080] Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oil and injectable organic esters
such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, saline solutions, parenteral vehicles
such as sodium chloride, Ringer's dextrose, etc. Intravenous
vehicles include fluid and nutrient replenishers. Preservatives
include antimicrobial agents, anti-oxidants, chelating agents and
inert gases. The pH and exact concentration of the various
components in the pharmaceutical composition are adjusted according
to well known parameters.
[0081] Additional formulations are suitable for oral
administration. Oral formulations include such typical excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and the like. The compositions take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations or powders. When the route is topical, the form may be
a cream, ointment, salve or spray.
[0082] The therapeutic compositions of the present invention may
include classic pharmaceutical preparations. Administration of
therapeutic compositions according to the present invention will be
via any common route so long as the target tissue is available via
that route. This includes oral, nasal, buccal, rectal, vaginal or
topical. Topical administration would be particularly advantageous
for the treatment of skin cancers, to prevent chemotherapy-induced
alopecia or other dermal hyperproliferative disorder.
Alternatively, administration may be by orthotopic, intradermal
subcutaneous, intramuscular, intraperitoneal or intravenous
injection. Such compositions would normally be administered as
pharmaceutically acceptable compositions that include
physiologically acceptable carriers, buffers or other excipients.
For treatment of conditions of the lungs, the preferred route is
aerosol delivery to the lung. Volume of the aerosol is between
about 0.01 ml and 0.5 ml. Similarly, a preferred method for
treatment of colon-associated disease would be via enema. Volume of
the enema is between about 1 ml and 100 ml.
[0083] An effective amount of the therapeutic composition is
determined based on the intended goal. The term "unit dose" or
"dosage" refers to physically discrete units suitable for use in a
subject, each unit containing a predetermined-quantity of the
therapeutic composition calculated to produce the desired
responses, discussed above, in association with its administration,
i.e., the appropriate route and treatment regimen. The quantity to
be administered, both according to number of treatments and unit
dose, depends on the protection desired.
[0084] Precise amounts of the therapeutic composition also depend
on the judgment of the practitioner and are peculiar to each
individual. Factors affecting the dose include the physical and
clinical state of the patient, the route of administration, the
intended goal of treatment (alleviation of symptoms versus cure)
and the potency, stability and toxicity of the particular
therapeutic substance. Example 42 discusses anion binding compounds
as therapeutic agents.
[0085] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
[0086] The following examples are merely illustrative and not
exhaustive. Examples of compositions useful in the present
invention, along with their preparation are given. It is expected
that one skilled in the art may appreciate that minor deviations
may be incorporated without deviating from the scope of the present
invention.
EXAMPLE 1
Synthesis of 1,2-Dipyrryl ethanedione
[0087] Oxalyl chloride (6.4 g, 0.05 mol) and dichloromethane (25
mL) were placed together under an argon atmosphere and stirred.
Upon cooling to -78.degree. C. in an acetone/CO.sub.2 bath, dry
pyridine (10 g, 0.12 mol) was added, resulting in the formation of
a yellow precipitate. To this cooled suspension was added a
solution of freshly distilled pyrrole (6.7 g, 0.1 mol) in
dichloromethane (25 mL) by use of a canula. Immediately, the
reaction mixture turns from yellow to brown. The reaction was
allowed to stir for an additional 15 minutes at -60.degree. C.,
after which time hydrochloric acid (5 M, 100 mL) was added to
quench the reaction. The biphasic system is then separated and the
organic phase was collected. The aqueous phase was extracted with
dichloromethane (3.times.30 mL), and the combined organic phases
were washed with water (100 mL), dried over anhydrous sodium
sulfate, filtered and evaporated to dryness. This afforded a green
precipitate. The crude product was further purified by silica gel
column chromatography (acetone/hexanes (80/20 v/v)) to afford 10
(1.81g, 38%) as a yellow powder.
Chracterization Data for 10
[0088] 1H NMR (250 MHz, DMSO d.sub.6) .delta.6.26 (2H, dd,
J.sub.1=3.7 Hz, J.sub.2=2.4 Hz, H.sub..beta.), 6.89 (2H, dd,
J.sub.1=3.7 Hz, J.sub.2=0.5 Hz, H.sub..beta.pyrr), 7.30 (broad s,
H.sub..alpha.pyrr), 12.27 (2H, s, NH); .sup.13C NMR (125 MHz, DMSO
d.sub.6) .delta.111.0, 120.9, 128.4, 128.6, 181.4.
EXAMPLE 2
Synthesis of 2,3-dypyrrylquinoxaline (1)
[0089] The synthesis of 2,3-dipyrrylquinoxaline (1) was carried out
as follows. The diketone 10 (570 mg, 3.03 mmol) was dissolved in
glacial acetic acid (70 mL) and to this was added a solution of
ortho-phenylenediamine (715 mg, 6.62 mmol) in acetic acid (30 mL)
with stirring. The resultant mixture was then brought to reflux for
90 min under an atmosphere of argon. After this time majority of
the acetic acid was removed under vacuum and the residue was taken
up in a mixture of water (30 mL) and dichloromethane (30 mL). The
organic phase was separated off and the aqueous phase was extracted
with further dichloromethane (2.times.20 mL). The organic phases
were combined and washed with saturated aqueous sodium bicarbonate
solution (50 mL), water and (50 mL) and brine (50 mL). After drying
over anhydrous sodium sulfate, filtered and evaporated to dryness.
The residue obtained was the purified using silica gel column
chromatography (dichloromethane) to afford 1 (730 mg, 94%) as a
yellow powder.
[0090] Oxalyl chloride, o-phenylenediamine,
4-nitro-1,2-diaminobenzene were purchased from Aldrich and used
without further purification. 4,5-Diamino-1,2-dimethoxybenzene was
prepared according to the method of Sessler, 1992.
4-5,-Dinitro-1,2-diaminobenzene was prepared according to the
method of Cheeseman, 1962.
Characterization Data for 1:
[0091] .sup.1H NMR (250 MHz, CDCl.sub.3) .delta.6.16 (2H, m,
H.sub..beta.2), 6.82 (2H, m, H.sub..beta.1), 6.89 (2H, m,
H.sub..alpha.), 7.46 (2H, dd, J.sub.o=12.5 Hz, J.sub.m=2.9 Hz,
CH.sub.benz), 7.78 (2H, dd, J.sub.o=12.5 Hz, J.sub.m=2.9 Hz,
CH.sub.benz), 9.54 (2H, broad s, NH); .sup.13C NMR (125 MHz,
CDCl.sub.3) .delta.109.9, 112.8, 121.1, 128.0, 128.8, 129.0, 139.6,
143.6; HRMS (CI+) m/z calcd for C.sub.16H.sub.13N.sub.4: 261.11402,
found: 261.11343; Anal. Calcd for
C.sub.16H.sub.13N.sub.4.0.5H.sub.2O: C, 71.35; H, 4.87; N, 20.80.
Found C, 71.23; H, 4.79; N, 20.48.
EXAMPLE 3
Synthesis of 2,3-dipyrrol-2'-yl-6,7-dimethoxyquinoxaline (2)
[0092] 4,5-Diamino-1,2-dimethoxybenzene (1.25 g, 7.45 mmol) and
1,2-dipyrrol-2'-ylethanedione (2 g, 10.65 mmol) were dissolved in
acetic acid (250 mL) and heated at reflux under an atmosphere of
argon overnight. The solvent was removed under vacuum and the
residue was taken up in a mixture of water (50 mL) and
dichloromethane (100 mL). The organic phase was separated off and
the aqueous phase was extracted with further dichloromethane
(2.times.40 mL). The organic phases were combined and washed with
saturated aqueous sodium bicarbonate solution (50 mL), water (50
mL), then brine (50 mL). After drying over anhydrous sodium
sulfate, the solution was filtered and evaporated to dryness. Final
purification was then effected using silica gel column
chromatography (methanol/chloroform, 2:98) to afford
2,3-dipyrrol-2'-yl-6,7-dimethoxyqui- noxaline (2.05 g, 86%) as a
yellow/green powder: m.p. 196-198.degree. C.; .sup.1H NMR (250 MHz,
DMSO d6) .delta.3.98 (6H, s), 6.05-6.09 (2H, m), 6.11-6.15 (2H, m),
6.92-6.96 (2H, m), 7.32 (2H, s), 11.35 (2H, br s, NH); .sup.13C NMR
(62.5 MHz, CDCl.sub.3) .delta.56.7, 106.5, 110.4, 112.1, 120.8,
129.7, 137.3, 142.3, 152.7; HRMS (CI+) m/z (M+1) calcd. for
C.sub.18H.sub.17N.sub.4O.sub.2: 321.1352, found: 321.1358; UV-vis
(CH.sub.2CI.sub.2) .lambda..sub.max [nm] (.epsilon.): 272 (23 890),
292sh (17 850), 412 (15 900).
Characterization Data for 2
[0093] m.p. 196-198.degree. C.; .sup.1H NMR (250 MHz, DMSO d.sub.6)
.delta.3.98 (6H, s), 6.05-6.09 (2H, m), 6.11-6.15 (2H, m),
6.92-6.96 (2H, m), 7.32 (2H, s), 11.35 (2H, br, s, NH); .sup.13C
NMR (125 MHz, CDCl.sub.3) .delta.56.7, 106.5, 110.4, 112.1, 120.8,
129.7, 137.3, 142.3, 152.7; HRMS (CI+) m/z (M+1) calcd. for
C.sub.18H.sub.17N.sub.4O.sub.2: 321.1352, found: 321.1358.
EXAMPLE 4
Synthesis of 2,3-dipyrrol-2'-yl-6,7-dinitroquinoxaline (3)
[0094] 1,2-Dipyrrol-2'-ylethanedione (200 mg, 1.06 mmol) and
1,2-diamino-4,5-dinitrobenzene (265 mg, 1.34 mmol) were dissolved
in glacial acetic acid (40 mL) and the resultant solution was
heated at reflux in the dark for 4 h under an atmosphere of argon.
The solution was allowed to cool and evaporated to dryness in
vaccuo. The residue was taken up in dichloromethane (50 mL) and
washed with sodium hydrogen carbonate solution (sat., 2.times.50
mL), brine (50 mL), dried over anhydrous sodium sulfate, filtered
and evaporated to dryness. The residue was chromatographed over
silica (chloroform) and the front running band was collected to
afford 2,3-dipyrrol-2'-yl-6,7-dinitroquinoxaline (297 mg, 80%) as a
red powder: m.p. 215-217.degree. C.; 1H NMR (250 MHz, DMSO d.sub.6)
.delta.6.20-6.28 (2H, m, pyrrole H), 6.68-6.77 (2H, m, pyrrole H),
7.15-7.22 (2H, m, pyrrole H), 8.50 (2H, s, quinoxline H), 11.94
(2H, br s, NH); .sup.13C NMR (62.5 MHz, DMSO d.sub.6) .delta.109.9,
114.9, 124.6, 125.7, 127.9, 140.0, 140.7, 147.7; HRMS (CI+) m/z
(M+1) calcd. for C.sub.16H.sub.11N.sub.6O.sub.4 351.0842, found
351.0852; UV-vis (CH.sub.2Cl.sub.2) .lambda..sub.max [nm]
(.epsilon.): 340 (29 100), 460 (29 200).
EXAMPLE 5
Synthesis of 6-nitro-2-3-dipyrrylquinoxaline (7)
[0095] 1,2-Dipyrrol-2'-ylethanedione (175 mg, 0.93 mmol) and
4-nitro-1,2-diaminobenzene (245 mg, 1.60 mmol) were dissolved in
glacial acetic acid (30 mL) and the resultant solution was heated
at reflux in the dark overnight under an atmosphere of argon. The
solution was allowed to cool and evaporated to dryness in vaccuo.
The residue was taken up in dichloromethane (50 mL) and washed with
sodium hydrogen carbonate solution (sat., 2.times.50 mL), brine (50
mL), dried over anhydrous sodium sulfate, filtered and evaporated
to dryness. The residue was chromatographed over silica
(chloroform) and the front running band was collected to afford
2,3-dipyrrol-2'-yl-6-nitroquinoxaline (240 mg, 85%) as a red
powder: m.p. 206-208.degree. C.; .sup.1H NMR (250 MHz, DMSO
d.sub.6) .delta.6.15-6.25 (2H, m, pyrrole H), 6.45-6.60 (2H, m,
pyrrole H), 7.07-7.19 (2H, m, pyrrole H), 8.05 (1H, d, J 9.1 Hz, H8
quinoxaline), 8.48 (1H, dd, J 9.1, 2.5 Hz, H7 quinoxaline), 8.65
(1H, d, J 2.5 Hz, H5 quinoxaline), 11.75 (1H, br s, NH), 11.86 (1H,
br s, NH); .sup.13C NMR (62.5 MHz, DMSO d.sub.6) .delta.109.2,
109.6, 112.9, 114.1, 122.3, 122.6, 123.6, 123.7, 128.1, 128.3,
129.2, 137.8, 142.4, 146.3, 146.5, 147.0; HRMS (CI+) m/z (M+1)
Calcd for C.sub.16H.sub.12N.sub.5O.sub.2 306.0991, found 306.0996;
UV-vis (CH.sub.2Cl.sub.2) .lambda..sub.max [nm] (.epsilon.): 325
(22 000), 370 (13 650), 450 (18 730); Anal. Calc. for
C.sub.16H.sub.11N.sub.5O.sub.2: C, 62.95; H, 3.61; N, 22.95. Found
C, 62.87; H, 3.67; N, 22.96.
EXAMPLE 6
Synthesis of Mono-2-(trimethylsilyl)ethoxymethyl (SEM) protected
derivative (12).
[0096] 2,3-Dipyrrylquinoxaline (400 mg, 1.54 mmol) was dissolved in
N,N-dimethylformamide (30 ml) under an atmosphere of argon. Sodium
hydride (60% dispersion in mineral oil, 100 mg) was added and the
resultant mixture was allowed to stir at room temperature for 1 h.
2-(trimethylsilyl)ethoxymethyl chloride (SEM-Cl) (205 mg, 1.23
mmol) was then added and the mixture was stirred at room
temperature for a further 90 min. The solvent was removed under
vacuum and the residue was taken up in dichloromethane (150 ml) and
washed with water (2.times.100 ml), and brine (100 ml). The organic
phase was dried over sodium sulfate, filtered and evaporated to
dryness. Final purification was then effected using silica gel
column chromatography (toluene initially, then
toluene/ethylacetate, 95/5) to afford 7 (40 mg, 6.6%) as a brown
gum: .sup.1H NMR (500 MHz, CD.sub.2Cl.sub.2) .delta.-0.70 (9H, s),
0.56-0.60 (2H, m), 3.23-3.27 (2H, m), 5.15 (2H, s), 5.78-5.80 (1H,
m), 6.12-6.14 (1H, m), 6.34-6.37 (1H, m), 6.48-6.51 (1H, m),
6.96-6.98 (1H, m), 7.02-7.04 (1H, m), 7.63-7.67 (1H, m), 7.71-7.75
(1H, m), 7.98-7.82 (1H, m), 9.90-10.00 (1H, br s); .sup.13C NMR
(125 MHz, CD.sub.2Cl.sub.2) .delta.-1.6, 17.8, 30.1, 66.1, 77.0,
109.0, 111.0, 112.3, 113.1, 122.0, 123.4, 128.4, 128.8, 129.4,
130.6, 140.4, 141.2, 145.3, 146.0.
EXAMPLE 7
Preparation of 2,3-di-5'-bromopyrrol-2'-ylquinoxaline
[0097] 2,3-Dipyrrol-2'-ylquinoxaline (1.5 g, 5.77 mmol) was
dissolved in carbon tetrachloride (150 ml) and recrystallised
N-bromosuccinimde (2.25 mg, 12.64 mmol) and benzoyl peroxide (40
mg) were added and the resultant mixture was heated at reflux under
an atmosphere of argon in the dark overnight. The solvent was then
removed under vacuum and the residue was chromatographed over
silica (ethyl acetate/dichloromethane 5:95) and the major band was
collected to afford 2,3-di-5'-bromopyrrol-2'-ylquinoxaline (1.7 g,
70%) as a light brown solid: m.p. 204-206.degree. C.; .sup.1H NMR
(250 MHz, DMSO d.sub.6) .delta.6.16-6.24 (4H, m, .beta.-pyrrolic
H), 7.73-7.81 (2H, m, aryl H), 7.95-8.03 (2H, m, aryl H), 12.3 (2H,
broad s, NH); .sup.13C NMR (62.5 MHz, DMSO d.sub.6) .delta.101.9,
111.1, 112.8, 128.1, 129.6, 130.4, 139.5, 143.9; HRMS (CI+) m/z
calcd for C.sub.16H.sub.11N.sub.4Br.sub.2: 416.93504, found:
416.93557; Anal. Calcd for C.sub.16H.sub.10N.sub.4Br.sub.2: C,
45.93; H, 2.39; N, 13.40. Found C, 46.09; H, 2.56; N, 13.49.
EXAMPLE 8
Preparation of 2,3-di-5'-iodopyrrol-2'-ylquinoxaline
[0098] 2,3-Dipyrrol-2'-ylquinoxaline (500 mg, 1.92 mmol) and sodium
acetate (1.04 g, 7.68 mmol) were dissolved in acetic acid (100 mL)
and cooled to 0.degree. C. in an ice bath. As the acetic acid began
to crystallise, a solution of iodine monochloride (592 mg, 3.65
mmol) in acetic acid (10 mL) was added to the quinoxaline solution
and the mixture was stirred for 10 min. A saturated solution of
sodium thiosulfate (30 mL) was then added and the mixture was
stirred at room temperature for 1 h. The mixture was then washed
with ethyl acetate (100 mL) and the organic phase was separated
off. The organic phase was washed with water (2.times.100 mL) and
brine (100 mL), dried over anhydrous sodium sulfate, filtered and
evaporated to dryness. The residue ws achromatographed over silica
(dichloromethane/hexane 1:1, v/v) to afford
2,3-di-5'-iodopyrrol-2'-ylquinoxaline (830 mg, 84%).
EXAMPLE 9
Preparation of 2,3-di(5'-formylpyrrol-2'-yl)quinoxaline
[0099] Phosphorus oxychloride (240 .mu.L, 2.57 mmol) was added to
DMF (454 .mu.L, 5.86 mmol) at 0.degree. C. under an atmosphere of
argon. This mixture was then allowed to warm to room temperature
and stirred for 10 min before 1,2-dichloroethane (3 mL) was added.
To this mixture was then added a solution of
2,3-dipyrrol-2'-ylquinoxaline (260 mg, 1.00 mmol) in
1,2-dichloroethane (3 mL) over a period of 10 min. The resulting
mixture was heated at reflux for 30 min before being cooled to
0.degree. C. A saturated aqueous solution of sodium acetate (3 mL)
was then added and the mixture was heated at reflux for a further
30 min. Upon cooling, the mixture was washed with chloroform
(2.times.50 mL) and the combined organic phases were then washed
with water (2.times.50 mL), brine (50 mL), dried over anhydrous
sodium sulfate, filtered and evaporated to dryness. The residue was
then chromatographed over silica (dichloromethane initially, then
dichloromethane/methanol, 99.5:0.5 v/v) to afford
2,3-di(5'-formylpyrrol-2'-yl)quinoxaline (216 mg, 68%) as a yellow
solid.
EXAMPLE 10
Preparation of 2,3-di(5'-benzoylpyrrol-2'-yl)quinoxaline
[0100] Under an atmosphere of argon, NN-dimethylbenzamide (1.20 g,
8.0 mmol) was added to 1,2-dichloroethane (5 mL) followed by the
addition of phosphorus oxychloride (368 .mu.L, 3.96 mmol) and and
the mixture was stirred at room temperature for 30 min. To this
mixture was added a solution of 2,3-dipyrrol-2'-ylquinoxaline (400
mg, 1.54 mmol) in 1,2-dichloroethane (50 mL) over a period of 20
min. The resulting mixture was heated at reflux for 24 h before
being allowed to cool. An aqueous solution of sodium acetate (20
mL) was then added and the mixture was heated at reflux for a
further 30 min. Upon cooling, the mixture was washed with
dichloromethane (2.times.100 mL) and the combined organic phases
were then washed with water (2.times.150 mL), brine (150 mL), dried
over anhydrous sodium sulfate, filtered and evaporated to dryness.
The residue was then chromatographed over silica (dichloromethane)
to afford 2,3-di(5'-benzoylpyrrol-2'-yl)quinoxaline (314 mg,
44%).
EXAMPLE 11
Preparation of 2,3-dipyrrol-2'-yl-6,7-dipentoxyquinoxaline
[0101] 4,5-Dipentoxy-1,2-diaminobenzene (1.12 g, 4.0 mmol) and
1,2-dipyrrol-2'-ylethanedione (500 mg, 2.66 mmol) were dissolved in
acetic acid (80 ml) and the resulting mixture was first evacuated,
and then placed under an atmosphere of argon and heated at reflux
in the dark overnight. The mixture was allowed to cool and the
solvent was removed under vacuum. The residue was then dissolved in
dichlormethane (100 ml) and washed with sodium carbonate solution
(100 ml), brine (100 ml), dried over anhydrous sodium sulfate and
evaporated to dryness. The residue was then chromatographed over
silica (dichloromethane) and the front running band was collected
to afford 2,3-dipyrrol-2'-yl-6,7-dipentoxyquinoxaline (768 mg, 67%)
as a yellow-green powder: m.p. 142-144.degree. C.; .sup.1H NMR (250
MHz, DMSO d.sub.6) .delta.0.94 (6H, t, J 7.0 Hz, CH.sub.3),
1.33-1.56 (8H, m, .gamma. and .delta. CH.sub.2), 1.84 (4H, app
quin., .beta. CH.sub.2), 4.17 (4H, t, J 6.3 Hz, .alpha. CH.sub.2),
6.03-6.08 (2H, m, pyrrole H), 6.09-6.14 (2H, m, pyrrole H),
6.90-6.95 (2H, m, pyrrole H), 7.25 (2H, s, quinoxaline H), 11.38
(2H, br s, NH); .sup.13C NMR (62.5 MHz, DMSO d.sub.6) .delta.13.9,
21.9, 27.8, 28.1, 68.4, 106.8, 108.6, 110.1, 120.2, 128.9, 151.4;
HRMS (CI+) m/z (M+1) calcd. for C.sub.26H.sub.33N.sub.4O.sub.2
433.2603, found 433.2613.
EXAMPLE 12
Preparation of 2,3-di(5'-bromopyrrol-2'-yl)-6-nitroquinoxaline
[0102] 2,3-Dipyrrol-2'-yl-6-nitroquinoxaline (500 mg, 1.64 mmol)
was dissolved in carbon tetrachloride (150 mL) and recrystallised
NBS (613 mg, 3.44 mmol, 2.1 equiv.) and benzoyl peroxide (10 mg)
were added. The resultant mixture was heated at reflux under an
atmosphere of argon in the dark overnight. The mixture was allowed
to cool and then evaporated to dryess. The residue was
chrommatographed over silica (dichloromethane) to afford
2,3-di(5'-bromopyrrol-2'-yl)-6-nitroquinoxaline (226 mg, 30%) as a
red solid.
EXAMPLE 13
Preparation of 2,3-dipyrrol-2'-yl-6-aminoquinoxaline
[0103] 2,3-Dipyrrol-2'-yl-6-nitroquinoxaline (107 mg, 0.35 mmol)
was dissolved in ethanol (20 mL) and palladium on carbon (10%, 10
mg) was added. The mixture was placed under an atmosphere of
hydrogen and stirred overnight in the dark. The mixture was then
filtered through celite and the filtrate was evaporated to dryness
to afford 2,3-dipyrrol-2'-yl-6-ami- noquinoxaline (90 mg, 93 %) as
an orange solid.
EXAMPLE 14
Preparation of 2,3-dipyrrol-2'-yl-6,7-diaminoquinoxaline
[0104] 2,3-Dipyrrol-2'-yl-6,7-dinitroquinoxaline (90 mg, 0.25 mmol)
was dissolved in absolute ethanol (40 mL) and 10% palladium on
carbon (15 mg) was added. The resulting mixture was stirred under
an atmosphere of hydrogen overnight in the dark. The solution was
then filtered through celite and evaporated to dryness to afford
crude 2,3-dipyrrol-2'-yl-6,7-d- iaminoquinoxaline (72 mg, 100%) and
used without further purification.
EXAMPLE 15
Preparation of 2,3-dipyrrol-2'-yl-5-nitroquinoxaline
[0105] 1,2-Dipyrrol-2'-ylethanedione (500 mg, 2.66 mmol) and
3-nitro-1,2-phenylenediamine (680 mg, 4.45 mmol) were dissolved in
acetic acid (100 mL) and heated at reflux in the dark under an
atmosphere of argon overnight. The solvent was removed under vacuum
and the residue was dissolved in dichloromethane (150 mL), washed
with sodium carbonate solution (2.times.100 mL) and brine (100 mL),
dried over anhydrous sodium sulfate, filtered and evaporated to
dryness. The crude material was then chromatographed over silica
(dichloromethane) and the major front running band was collected to
afford 2,3-dipyrrol-2'-yl-5-nitroquinoxaline (480 mg, 59%) as an
orange solid: m.p. 183-185.degree. C.; .sup.1H NMR (250 MHz, DMSO
d.sub.6) .delta.6.15-6.22 (2H, m, pyrrole H), 6.39-6.47 (2H, m,
pyrrole H), 7.05-7.10 (2H, m, pyrrole H), 7.76-7.84 (1H, m,
quinoxaline H), 8.14-8.22 (2H, m, quinoxaline H), 11.41 (1H, br s,
NH), 11.77 (1H, br s, NH); .sup.13C NMR (62.5 MHz, DMSO d.sub.6)
.delta.109.3, 113.0, 113.4, 122.7, 123.0, 123.2, 0127.6, 128.0,
128.1, 131.0, 131.9, 139.3, 145.9, 146.2; HRMS (CI+) m/z (M+1)
Calcd for Cl.sub.6H.sub.12N.sub.5O.sub.2 306.0991, found
306.0995.
EXAMPLE 16
Preparation of 2,3-dipyrrol-2'-yl-6-bromoquinoxaline
[0106] 1,2-Dipyrrol-2'-ylethanedione (440 mg, 2.34 mmol) and
4-bromo-1,2-phenylenediamine (560 mg, 3.00 mmol) were dissolved in
acetic acid (70 mL) and heated at reflux in the dark under an
atmosphere of argon overnight. The solvent was removed under vacuum
and the residue was dissolved in dichloromethane (150 mL), washed
with sodium carbonate solution (2.times.100 mL) and brine (100 mL),
dried over anhydrous sodium sulfate, filtered and evaporated to
dryness. The crude material was then chromatographed over silica
(dichloromethane) and the major front running band was collected to
afford 2,3-dipyrrol-2'-yl-6-bromoquinoxaline (510 mg, 65%) as a
brown/green solid: m.p. 144-146.degree. C.; .sup.1H NMR (500 MHz,
DMSO d6) .delta.6.12-6.15 (2H, m, pyrrole H), 6.28-6.30 (2H, m,
pyrrole H), 6.98-7.02 (2H, m, pyrrole H), 7.80 (1H, dd, J 2.1, J
8.8 Hz, quinoxaline H), 7.85 (1H, d, J 8.8 Hz, quinoxaline H), 8.07
(1H, d, J 2.1 Hz, quinoxaline H), 11.57 (2H, br s, NH); .sup.13C
NMR (125 MHz, DMSO d.sub.6) .delta.108.9, 109.1, 112.0, 112.2,
121.5, 121.8, 122.0, 128.3, 128.4, 129.1, 130.0, 132.0, 138.2,
140.1, 145.3, 145.7; MS (CI+) m/z (M+1) Calcd for
C.sub.16H.sub.12N.sub.4Br 339.02453, found 339.02430.
EXAMPLE 17
Preparation of 2,3-diaminonaphthalene adduct
[0107] 1,2-Dipyrrol-2'-yl ethanedione (200 mg, 1.06 mmol) and
2,3-diaminonaphthalene (252 mg, 1.59 mmol) were dissolved in
glacial acetic acid (30 mL) and the resultant solution was heated
at reflux under an atmosphere of argon in the dark overnight. The
solution was allowed to cool and evaporated to dryness in vaccuo.
The residue was taken up in dichloromethane (50 mL) and washed with
sodium hydrogen carbonate solution (sat., 2.times.50 mL), brine (50
mL), dried over anhydrous sodium sulfate, filtered and evaporated
to dryness. The residue was chromatographed over silica
(dichloromethane) and the front running band was collected to
afford the 2,3-diaminonaphthalene adduct (156 mg, 95%) as a light
brown solid: m.p. 184-186.degree. C.; .sup.1H NMR (250 MHz, DMSO
d.sub.6) .delta.6.15-6.20 (2H, m, pyrrole H), 6.34-6.39 (2H, m,
pyrrole H), 7.03-7.08 (2H, m, pyrrole H), 7.55-7.64 (2H, m),
8.15-8.23 (2H, m), 8.54 (2H, s), 11.71 (2H, br s, NH); .sup.13C NMR
(62.5 MHz, DMSO d.sub.6) .delta.108.9, 112.6, 122.0, 125.6, 126.3,
128.2, 129.0, 133.0, 136.7, 145.8; HRMS (CI+) m/z (M+1) Calcd for
C.sub.20H.sub.15N.sub.4 311.12967, found 311.12963.
EXAMPLE 18
Preparation of 9,10-diaminophenanthrene adduct
[0108] 1,2-Dipyrrol-2'-yl ethanedione (500 mg, 2.66 mmol) and
9,10-diaminophenanthrene (830 mg, 3.99 mmol) were dissolved in
glacial acetic acid (150 mL) and the resultant mixture was heated
at reflux under an atmosphere of argon in the dark overnight. The
mixture was allowed to cool and evaporated to dryness in vaccuo.
The residue was chromatographed over silica (diclhoromethane) and
the front running band was collected to afford the
9,10-diaminophenanthrene adduct (687 mg, 72%) as a light brown
solid. HRMS (CI+) m/z (M+1) Calcd for C.sub.24H.sub.17N.sub.4
361.14532, found 361.14525.
EXAMPLE 19
Preparation of 2,3-dipyrrol-2'-yl-6-carboxyquinoxaline
[0109] 1,2-Dipyrrol-2'-ylethanedione (500 mg, 2.66 mmol) and
3,4-diaminobenzoic acid (404 mg, 2.66 mmol) were dissolved in
glacial acetic acid (80 mL) and the resultant solution was heated
at reflux in the dark overnight. The solution was allowed to cool
and evaporated to dryness in vaccuo. The residue was taken up in
ethyl acetate (150 mL) and washed with hydrochloric acid (3 M, 80
mL), brine (80 mL), dried over anhydrous sodium sulfate, filtered
and evaporated to dryness. The residue was chromatographed over
silica (ethyl acetate/dichloromethane, 3:2 v/v) and the major band
was collected to afford 2,3-dipyrrol-2'-yl-6-carboxyqu- inoxaline
(270 mg, 33%). .sup.1H NMR (250 MHz, DMSO d.sub.6) .delta.6.14-6.21
(2H, m, pyrrole H), 6.35-6.43 (2H, m, pyrrole H), 7.02-7.09 (2H, m,
pyrrole H), 8.00 (1H, d, J 8.7 Hz, H8 quinoxaline), 8.17 (1H, dd, J
8.7, 1.9 Hz, H7 quinoxaline), 8.49 (1H, d, J 2.5 Hz, H5
quinoxaline), 11.66 (1H, br s, NH), 11.73 (1H, br s, NH), 13.40
(1H, br s, CO.sub.2H); .sup.13C NMR (62.5 MHz, DMSO d.sub.6)
.delta.109.0, 109.3, 112.1, 112.8, 121.8, 122.5, 128.1, 128.4,
128.5, 129.9, 130.6, 138.5, 141.5, 145.8, 146.3, 166.8; HRMS (CI+)
m/z (M+1) Calcd for C.sub.17H.sub.13N.sub.4O.sub.2 305.1039, found
305.1035.
EXAMPLE 20
Preparation of 2,3-dipyrrol-2'-yl-6-carboxyquinoxaline
octylester
[0110] 2,3-Dipyrrol-2'-yl-6-carboxyquinoxaline (100 mg, 0.33 mmol)
and octanol (47 mg, 0.36 mmol) were dissolved in dichloromethane
(20 mL). To this solution was added DCC (82 mg, 0.40 mmol) and DMAP
(2.5 mg, 0.02 mg)in dichloromethane (10 mL) over a 10 min period
and the resulting mixture was stirred at room temperature for 4 h.
The mixture was then evaporated to dryness and chromographed over
silica (dichloromethane) to afford
2,3-dipyrrol-2'-yl-6-carboxyquinoxaline octyl ester (76 mg, 55%) as
a yellow/green solid.
EXAMPLE 21
Preparation of
2,3-dipyrrol-2'-yl-6-carbxyamidoaquinoxaline-4"-benzo-18-cr-
own-6
[0111] 2,3-Dipyrrol-2'-yl-6-carboxyquinoxaline (100 mg, 0.33 mmol)
was dissolved in a mixture of dichloromethane (10 mL) and
N,N-diisopropylethylamine (100 mg, 0.724 mmol) and HBTU (137 mg,
0.362 mmol) was added. The resulting mixture was stirred for 5 min
prior to the addition of a solution of 4-aminobenzo-18-crown-6 (118
mg, 0.36 mmol) in dichloromethane (6 mL). The reaction mixture was
allowed to stir under an atmosphere of argon in the dark overnight.
The mixture was then evaporated to dryness and the residue was
chromatographed over silica (ethyl acetate initially, then
methanol/ethyl acetate, 5:95 v/v) to afford
2,3-dipyrrol-2'-yl-6-carbxyamidoaquinoxaline-4"-benzo-18-crown-6
(150 mg, 74%).
EXAMPLE 22
Preparation of 2,3-dipyrrol-2'-yl-6-carboxyquinoxaline coupled to a
bead
[0112] 2,3-Dipyrrol-2'-yl-6-carboxyquinoxaline (45 mg, 0.14 mmol),
HBTU (62 mg, 0.16 mmol), N,N-diisopropylethylamine (50 mg, 0.36
mmol) and DMF (0.5 mL) were added to dichloromethane (7 mL). This
solution was then added to pre-swelled TG-amino resin (the resin
was successively rinsed with DMF, then dichloromethane and then
methanol several times). The mixture was stirred overnight and then
filtered under vacuum and washed successively with dichoromethane,
DMF, dichoromethane, DMF, dichloromethane, methanol,
dichloromethane, and finally two rinses with methanol. After the
third wash the solutions were colourless. The beads were then dried
under vacuum for 4 days without further purification.
EXAMPLE 23
1,2-Di(3',4'-difluoropyrrol-2'-yl)ethanedione
[0113] Oxalyl chloride (800 .mu.L, 8.64 mmol) and dichloromethane
(15 mL) were placed together under an argon atmosphere and stirred.
Upon cooling to -78.degree. C. in an acetone/CO.sub.2 bath, dry
pyridine (1.28 mL, 15.8 mmol) was added, resulting in the formation
of a yellow precipitate. To this cooled suspension was added a
solution of 3,4-difluoropyrrole (1.49 g, 14.4 mmol) in
dichloromethane (3 mL) via syringe. The reaction was allowed to
stir for 3 h at -60.degree. C., and then warmed to 0.degree. C.
over a 4 h period. The solution was then diluted with
dichloromethane (20 mL) and washed with hydrochloric acid (3 M,
2.times.50 mL). The biphasic system was separated off and the
organic phase was collected and washed with water (50 mL) and brine
(50 mL). The organic phase was then dried over anhydrous sodium
sulfate, filtered and evaporated to dryness. The acidic aqueous
phase from the initial extraction was extracted with ethyl acetate
(50 mL). The organic phase was separated off and washed with brine
(100 ml), dried over anhydrous sodium sulfate, filtered and
evaporated to dryness. This afforded a green precipitate which was
purified by silica gel column chromatography (dichloromethane/ethyl
acetate, 95:5 to 90:10 (v/v) as eluent) to afford
1,2-di(3',4'-difluoropyrrol-2'-yl)ethanedione (405 mg, 21%) as a
yellow powder: m.p. 260-263.degree. C. decomposed; .sup.1H NMR (500
MHz, DMSO d.sub.6) .delta.7.43-7.46 (2H, m), 12.36 (2H, br s, NH);
.sup.13C NMR (125 MHz, DMSO d.sub.6, .sup.19F decoupled)
.delta.110.7 (m), 113.3 (d, J 192 Hz), 136.8 (dd, J 9.1, 1.5 Hz),
141.0 (t, J 8.2 Hz), 178.8; .sup.19F NMR (470 MHz, DMSO d.sub.6)
.delta.-164.1, -177.9; HRMS (CI+) m/z (M+1) calcd for
C.sub.10H.sub.5N.sub.2O.sub.2F.sub.4: 261.0287, found:
261.0288.
EXAMPLE 24
2,3-Di(3',4'-difluoropyrrol-2'-yl)quinoxaline
[0114] 1,2-Di(3',4'-difluoropyrrol-2'-yl)ethanedione (112 mg, 0.43
mmol) and ortho-phenylenediamine (125 mg, 1.15 mmol) were dissolved
in glacial acetic acid (20 mL). The resultant mixture was then
heated at reflux under an atmosphere of argon in the dark
overnight. The reaction mixture was evaporated to dryness under
vacuum and the residue obtained was purified using silica gel
column chromatography (dichloromethane eluent) to afford
2,3-di(3',4'-difluoropyrrol-2'-yl)quinoxaline (133 mg, 93%) as a
yellow-green powder: m.p. 188-192.degree. C.; .sup.1H NMR (500 MHz,
DMSO d.sub.6) .delta.6.94-6.98 (2H, m, pyrrole H), 7.80-7.84 (2H,
m, quinoxaline H), 8.01-8.05 (2H, m, quinoxaline H), 11.47 (2H,
broad s, NH); .sup.13C NMR (125 MHz, DMSO d.sub.6, .sup.19F
decoupled) .delta.104.0 (d, J 22 Hz), 111.1 (d, J 16 Hz), 128.3,
130.3, 136.3 (dd, J 244, 11 Hz), 137.6 (dd, J 235, 11 Hz), 139.7,
142.4; .sup.19F NMR (470 MHz, DMSO d.sub.6) .delta.-172.6 (dt, J
12.2, 3.3 Hz), -180.5 (dt, J 12.6, 3.3 Hz); HRMS (CI+) m/z (M+1)
calcd for C.sub.16H.sub.9N.sub.4F.sub- .4: 333.0763, found:
333.0754; Anal. Calcd for C.sub.16H.sub.8N.sub.4F.sub- .4: C,
57.84; H, 2.43; N, 16.86. Found C, 57.71; H, 2.50; N, 16.81.
EXAMPLE 25
2,3-Di(3',4'-difluoropyrrol-2'-yl)-6-nitroquinoxaline
[0115] 1,2-Di(3',4'-difluoropyrrol-2'-yl)ethanedione (98 mg, 0.38
mmol) and 4-nitro-1,2-diaminobenzene (115 mg, 0.75 mmol) were
dissolved in glacial acetic acid (30 mL) and the resultant solution
was heated at reflux in the dark overnight under an atmosphere of
argon. The solution was allowed to cool and evaporated to dryness
in vaccuo. The residue was chromatographed over silica (chloroform)
and the front running band was collected to afford
2,3-di(3',4'-difluoropyrrol-2'-yl)-6-nitroquinoxaline (120 mg, 84%)
as a red powder: m.p. 217-219.degree. C. with decomposition; HRMS
(CI+) m/z (M+1) Calcd for C.sub.16H.sub.7N.sub.5F.sub- .4O.sub.2
378.06141, found 378.06206.
EXAMPLE 26
Preparation of
1,2-di(5'-ethoxycarbonyl-3',4'-dimethylpyrrol-2'-yl)ethaned-
ione
[0116] A solution of 5'-ethoxycarbonyl-3',4'-dimethylpyrrole (500
mg, 2.99 mmol) in dry dichloromethane (10 mL) was cooled in an ice
bath under an atmosphere of argon. To this solution was added
oxalyl chloride (220 mg, 1.79 mmol) and the solution was allowed to
cool prior to the addition of tin(IV) chloride (857 mg, 380 .mu.L,
3.29 mmol). The resultant mixture was stirred at 0.degree. C. for 1
h and then allowed to warm to room temperature over 1 h. The
solution was then washed with water (2.times.50 mL), brine (50 mL),
dried over anhydrous sodium sulfate, filtered and evaporated to
dryness. The crude residue was then recrystallised from a mixture
of ethyl acetate/hexane, 1:9 v/v to afford
1,2-di(5'-ethoxycarbonyl-3',4'-dimethylpyrrol-2'-yl)ethanedione as
a light green powder (128 mg, 22%): m.p. 210-212.degree. C.;
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta.1.39 (6H, t, J 7.1 Hz),
2.27 (6H, s), 2.34 (6H, s), 4.38 (4H, q, J 7.1 Hz), 10.93 (2H, br
s, NH); .sup.13C NMR (125 MHz, CDCl.sub.3) .delta.9.7, 11.2, 14.4,
61.0, 125.1, 126.9, 127.6, 133.4, 160.7, 179.5; HRMS (CI+) m/z
(M+1) calcd for C.sub.20H.sub.25N.sub.2O.sub.6: 389.17126, found:
389.17092.
EXAMPLE 27
Preparation of
1,2-di(4'-acetyl-3',5'-dimethylpyrrol-2'-yl)ethanedione
[0117] A solution of 2,4-dimethyl-3-acetylpyrrole (1.48 g, 10.8
mmol) in dry ether (180 mL) was cooled in an ice bath under an
atmosphere of argon. To this solution was added oxalyl chloride
(720 .mu.L, 8.1 mmol) and the solution was then stirred at
0.degree. C. for 1 h, then allowed to warm to room temperature. The
resultant dark-red-purple precipitate was then collected on a frit
and the filtrate was removed and the precipitate was then washed
with dichloromethane (30 mL). Mass spectral analysis revealed that
the initial filtrate contained very little of the desired compound,
however peaks at m/z (M+1) 210 and 228 can be attributed to
mono-condensation with oxalyl chloride, followed by hydrolysis of
the acid chloride to the acid, and mono-condensation product with
oxalyl chloride respectively. The dichloromethane washings of the
precipitate contained essentially only the desired product. The
precipitate itself also contained essentially only the desired
1,2-di(4'-acetyl-3',5'-dimethylpyrrol-2'-yl)ethanedione (420 mg,
24%) as a purple solid. .sup.1H NMR (250 MHz, DMSO d.sub.6)
.delta.2.28 (3H, s), 2.40 (3H, s), 2.53 (3H, s), 11.74 (2H, br s,
NH); .sup.13C NMR (125 MHz, DMSO d.sub.6) .delta.12.3, 14.5, 31.3,
123.5, 124.9, 132.0, 143.1, 183.4, 194.6; HRMS (CI+) m/z (M+1)
calcd for C.sub.18H.sub.21N.sub.2O.sub.4: 329.15013, found:
329.15075.
EXAMPLE 28
2,3-Di(3',5'-dimethyl-4'acetylpyrrol-2'-yl)-6-nitroquinoxaline
[0118] 1,2-Di(4'-acetyl-3',5'-dimethylpyrrol-2'-yl)ethanedione (100
mg, 0.30 mmol) and 4-nitro-1,2-diaminobenzene (79 mg, 0.52 mmol)
were dissolved in glacial acetic acid (15 mL) and the resultant
solution was heated at reflux in the dark overnight under an
atmosphere of argon. The solution was allowed to cool and the
evaporated to dryness in vaccuo. The residue was then taken up in
chloroform (70 mL) and washed with hydrochloric acid (3 M,
3.times.40 mL), water (50 mL), sodium bicarbonate solution (60 mL),
brine (70 mL). The organic phase was then dried over anhydrous
sodium sulfate, filtered and evaporated to dryness to afford 34 mg
of crude material.
EXAMPLE 29
Preparation of
1,2-di(4'-heptanoyl-3',5'-dimethylpyrrol-2'-yl)ethanedione
[0119] A solution of 2,4-dimethyl-3-heptanoylpyrrole (500 mg, 2.41
mmol) in dry dichloromethane (10 mL) was cooled in an ice bath
under an atmosphere of argon. To this solution was added oxalyl
chloride (183 mg, 1.44 mmol, 125 .mu.L) and the solution was then
stirred at 0.degree. C. for 1 h, then at r.t. for 2 h. The reaction
mixture was diluted with dichloromethane (50 mL), and then washed
with water (2.times.30 mL), brine (30 mL), dried over anhydrous
sodium sulfate, filtered, and evaporated to dryness. Mass spec
analysis of the crude product indicated the presence of the desired
compound, 1,2-di(4'-heptanoyl-3',5'-dimethylp-
yrrol-2'-yl)ethanedione. No further purification was attempted.
(CI+) m/z (M+1) C.sub.28H.sub.41N.sub.2O.sub.4 requires 469; 208
(70), 252 (40), 280 (100), 298 (45), 469 (45). (CI-) m/z
C.sub.28H.sub.40N.sub.2O.sub.4 requires 468; 278 (28), 468
(100).
EXAMPLE 30
Preparation of 2,3-dipyrrol-2'yl-5,6-dicyanopyrazine
[0120] Diaminomaleonitrile (348 mg, 3.22 mmol) and
1,2-dipyrrol-2'-yl ethanedione (500 g, 2.66 mmol) were dissolved in
acetic acid (70 mL) and heated at reflux under an atmosphere of
argon for overnight in the dark. The solvent was removed under
vacuum and the residue was taken up in a mixture of water (50 mL)
and dichloromethane (100 mL). The organic phase was separated off
and the aqueous phase was extracted with further dichloromethane
(2.times.40 mL). The organic phases were combined and washed with
saturated aqueous sodium bicarbonate solution (50 mL), water (50
mL), then brine (50 mL). After drying over anhydrous sodium
sulfate, the organic phase was filtered and evaporated to dryness.
Final purification was then effected using silica gel column
chromatography (dichloromethane) to afford
2,3-dipyrrol-2'yl-5,6-dicyanopyrazine (65 mg. 10%). .sup.1H NMR
(250 MHz, DMSO d.sub.6) .delta.6.17 6-6.25 (2H, m), 6.82-6.88 *2H,
m), 7.12-7.18 (2H, m), 11.96 (2H, br s, NH; .sup.13C NMR (62.5 MHz,
CDCl.sub.3) .delta.110.2, 114.5, 114.7m, 125.4, 126.3, 144.0; HRMS
(CI+) m/z (M+1) calcd. For C.sub.14H.sub.9N.sub.6: 261.08887,
found: 261.08869.
EXAMPLE 31
Structure Determination of Fluoride Complexes
X-ray Structure Analysis
[0121] The molecular structure of compound 1 as well as its
corresponding tetrabutylammonium fluoride complex ([1.cndot.F]-)
were deduced from single crystal X-ray diffraction analyses. The
requisite single crystals were obtained from the slow evaporation
of dichloromethane/methanol (90/10: v/v) and neat dichloromethane
solutions of 1 and [1F].sup.-.cndot.(Bu).sub.4N.sup.+ respectively.
In both cases, the quinoxaline moiety was found to possess the
expected planar structure. As shown in FIG. 1 and FIG. 2, the two
pyrrole subunits were found to be rotated in such a way that they
point out, in opposite directions, towards the exterior of the
system.
[0122] While the structures of the fluoride complex and anion-free
receptor are quite similar, a major difference involves the
hydrogen bonding network. The fluoride complex [1.cndot.F].sup.-
presents a network that involves 1) two pyrrolic NH subunits
derived from two distinct dipyrrylquinoxaline units, 2) a fluoride
anion and 3) a molecule of water. The net result is a planar
network, wherein two identical planes are separated by a layer of
tetrabulammonium cations, as shown in FIG. 1. By contrast, the
structure of the fluoride-free system reveals a hydrogen bonding
network that serves to arrange the dipyrrylquinoxaline moieties
into layers wherein a molecule of water bridges two pyrrolic NH
groups from two distinct molecules. In this instance, it is also
worth noting that, in addition to the conventional N . . . O
hydrogen bonding interactions, one hydrogen atom of the bridging
water molecule is directed at the centroid of a pyrrole ring. The
result of this interaction is that the molecules from two different
planes are related by a two-fold screw axis.
[0123] In the case of the fluoride complex [1.cndot.F].sup.-, the
tightness of the hydrogen bonds is highlighted by the short F . . .
N distance: These distances are 2.629(2) and 2.640(2) .ANG.
respectively, and are, in fact, shorter than the corresponding F N
distances (2.790(2) .ANG.) observed in the case of calixpyrrole
fluoride anion complex (Dietrich, et al., 1981).
EXAMPLE 32
Spectroscopic Studies-Colormetric Assay for Anion Binding
[0124] The conclusion that 1 binds fluoride anion in
dichloromethane solution was further supported by mass
spectrometric analyses and titration experiments made using
UV-visible absorption and fluorescence emission methods (Tables 1
and 2). The latter studies, which provided K.sub.a values (Tables 1
and 2), were complemented by molar ratio analyses (Job plots) with
1:1 binding stoichiometries observed in all cases.
[0125] In order to analyze compounds having only one pyrrolic
nitrogen, a mono SEM protected analogue (12) was prepared as
described in Example 6. Additionally, quinoxaline (11) was used in
the analysis. 8
[0126] The results are as shown in the following tables:
1TABLE 1 Spectroscopic properties of 2,3-dipyrrylquinoxaline 1,
dipyrryl ethanedione 10, 6,7-dimethoxy-2,3-dipyrrylquinoxaline 2,
6,7-dinitro-2,3- dipyrrylquinoxaline 3,
6-nitro-2,3-dipyrrylquinoxaline 7, and quinoxaline 11. All values
measured in dichloromethane. MonoSE 2,3-di- Dipyrryl M-2,3-di-
pyrryl ethane- Di- Mono- pyrrylquin- Quin- quinoxaline dione
methoxy nitro Dinitro oxaline oxaline 1 10 2 7 3 12 11
.lambda..sub.max(ex) 412 nm 341 nm 414 nm 450 nm 460 nm 396 nm 315
nm .lambda..sub.max(em).sup.a 490 nm 458 nm 475 nm 600 nm 620 nm
492 nm 403 nm .epsilon. 17,110 16,200 15,900 18,730 29,200 14860
6,222 (at .lambda..sub.max) M.sup.-1cm.sup.-1 M.sup.-1cm.sup.-1
M.sup.-1cm.sup.-1 M.sup.-1cm.sup.-1 M.sup.-1cm.sup.-1
M.sup.-1cm.sup.-1 M.sup.-1cm.sup.-1 .sup.aFluorescence emission
scan parameters: excitation at .lambda..sub.max, 240 nm/min.,
emission slit width = 5 .mu.m, excitation slit width = 5 .mu.m.
[0127]
2TABLE 2 Anion binding constants (K.sub.a) for
2,3-dipyrrylquinoxalines 2, 3 and 7 and control compounds 1, 10 and
11.sup.a 2,3- Mono SEM- dipyrrylquin- Dipyrryl 2,3-dipyrryl oxaline
ethanedione Dimethoxy Mononitro Dinitro quinoxaline Anion 1.sup.b
10.sup.c 2 7 3 12 F 18,200 M.sup.-1 23,000 M.sup.-1 2,300 M.sup.-1
118,000 M.sup.-1 117,000 M.sup.-1 2,300 M.sup.-1
H.sub.2PO.sub.4.sup.- 60 M.sup.-1 170 M.sup.-1 <50 M.sup.-1 80
M.sup.-1 55 M.sup.-1 <50 M.sup.-1 Cl.sup.- 50 M.sup.-1 <50
M.sup.-1 <50 M.sup.-1 45 M.sup.-1 45 M.sup.-1 <50 M.sup.-1
.sup.aAll errors are .+-. 10%. All binding constants are reported
as the average of 2-4 trials. .sup.bBinding constants determined by
fluorescence quenching: .lambda..sub.ex = 412 nm, .lambda..sub.em=
420-750 nm, 240 nm/min, emission slit width = 5 .mu.m, excitation
slit width = 5 .mu.m. .sup.cBinding constants were determined from
UV-vis absorbance titration measurements monitoring the spectral
changes occurring at 341 nm.
[0128] In terms of specifics, we found that in dichloromethane
solution, the diketone precursor, 10, displays a relatively large
extinction coefficient whereas its fluorescence emission is
minimal. Under the same conditions, quinoxaline itself, 11,
displays a relatively low extinction coefficient. Nonetheless, even
with these control systems the addition of tetrabutylammonium salts
of various anions (F.sup.-, Cl.sup.- and
H.sub.2PO.sub.4.sub..sup.-), gave rise to results that were quite
interesting. In the case of 11 (in dichloromethane), the addition
of F.sup.-, Cl.sup.- and H.sub.2PO.sub.4.sub..sup.- did not induce
any significant change in the absorbance or emission spectra. By
contrast, in the case of 10 remarkable changes were noticed in the
emission spectra when either F.sup.- or H.sub.2PO.sub.4.sub..sup.-
were added. As a matter of fact, in the presence of F.sup.-, a
significant decrease and a shift in the absorbance intensity could
be observed visually with the color of the solution changing from
yellow-green to orange. Such effects can be rationalized in terms
of the electron withdrawing nature of the two carbonyl groups,
which serve to pull the electrons from the pyrrole units and thus
act to increase the acidity of the pyrrolic NH protons. As a
result, hydrogen bonding interactions with F.sup.- are favored. In
the particular case of inorganic phosphate, we speculate that the
two carbonyl groups may also be involved in binding, stabilizing
the complex via ancillary hydrogen bonding interactions involving
the two HOP-phosphate protons.
[0129] Unfortunately, the fluorescence quantum yield of 10 is low.
Thus we considered that 2,3-dipyrrylquinoxaline system 1 derived
from it as well as its analogues 2, 3, and 7 might prove to be far
better sensors. Not only should they display high affinities toward
fluoride anion in dichloromethane solution, but they were expected
to exhibit even more dramatic fluoride anion induced color changes
as measured by absorption and emission spectroscopy, as well as
naked eye color detection. As summarized in Table 2, this
expectation is indeed realized in the case of 1, 3, and 7, with the
latter two systems showing very dramatic yellow to purple fluoride
anion-induced colorimetric responses.
[0130] The greater "success" of 3 and 7 relative to 1 and 2, a
system that hardly "works" at all in terms of colorimetric F.sup.-
signaling, is not really surprising considering that the relative
electron deficiency of the mono- and dinitro derivatives should
lead to increase in their hydrogen bonding donating character.
Indeed, both 3 and 7 display affinity constants (K.sub.a), for
F.sup.- binding in dichloromethane (ca 10.sup.5 M.sup.-1), that are
quite high. By contrast, 1 (K.sub.a=2.times.10.sup.4 M.sup.-1) and
2 (K.sub.a=2.times.10.sup.3 M.sup.-1) display affinity constant
that are much lower. Interestingly, even in the case of the high
affinity systems 3 and 7 an excellent selectivity for fluoride
anion is maintained; indeed, the selectivity ratio for F.sup.- over
Cl.sup.- is more than 360.
[0131] In addition to the specific pyrrole-quinoxalines, the
usefulness of the general pyrrole-aryl structures as sensing
compounds has been demonstrated. Using
2,3-dipyrrol-2'yl-5,6dicyanopyrazine (preparation is described in
Example 30) as a sensing compound, a vivid yellow-to-red color
change in solution observed upon addition of fluoride ion verifies
that the quinoxaline backbone is not essential for chemical sensing
in this family of compounds. This is consistent with the theory
that changes in orbital overlap between the pi systems of
conjugated bridging unit and the pyrrole rings account for the
sensitivity of the sensing elements to analyte species.
EXAMPLE 33
Mechanistic Studies of Fluoride Anion Binding
[0132] In order to understand more fully the above results, studies
were performed to map out the mode of fluoride anion binding.
Firstly, it was verified that two pyrrolic nitrogens are required
to observe the anion-induced color change. Toward this end, the
mono protected 2,3-dipyrrylquinoxaline 12 was prepared by reacting
0.8 equivalents of SEMCl with one equivalent of
2,3-dipyrrylquinoxaline. (See Scheme 2 illustrating mono SEM
protection of 2,3-dipyrrylquinoxaline.) In dichloromethane, this
mono-protected adduct showed no color change when treated with
tetrabutyl ammonium fluoride. 9
[0133] The next series of mechanistic studies involved carrying out
temperature dependent NMR measurements (room temperature to
-80.degree. C.). These revealed little out of the ordinary (i.e.,
no temperature dependent chemical shift changes were observed) and
are consistent with a single atropisomer of 1 being present in
solution. Further studies involve X-ray diffraction analysis with
the realties as shown in Table 3.
3TABLE 3 Selected bond distances (.ANG.) and bond angles from X-ray
1 [1 .multidot. F] N--H . . . H--N 6.562 6.892 N . . . N 5.766
5.763 H.sub..alpha.. . . H.sub..alpha. 8.795 8.746 H.sub..beta.2. .
. H.sub..beta.2 2.607 2.612 F . . . H--N1 -- 1.639 F . . . H--N2 --
8.196
EXAMPLE 34
Analogues of Pyrrole-Aryls for Metal Binding and Altered
Fluorescence Properties
[0134] Analogues of the pyrrole-aryls may be prepared to form metal
complexes as shown below. By reaction of 9 with a
1,10-phenanthroline-5,6- -dione, a compound represented by 13 may
be synthesized. Compounds exemplified by 13 and 14 are contemplated
for the ability to complex a metal through the phenanthroline unit.
10 11
[0135] Similar analogues may be synthesized using the appropriate
1,2-diamines to produce compounds such as 15 and 16 for altered
fluorescence/binding properties. 12
[0136] Additionally, using a porphyrin 1,2-diones, compounds
exemplified by structure 17 can be generated as shown below: 13
[0137] wherein R.sub.1-R.sub.10 are as described previously and
representative metals include Li, B, Na, Mg, Al, Si, K--As, Rb--Sb,
Cs--La, Hf--Bi, pr, Eu, Yb and Th.
EXAMPLE 35
Post-Synthetic Modification of the `Parent`
2,3-Dipyrrol-2'-yl-quinoxaline
[0138] A wide variety of substituents may be introduced to the
pyrrole quinoxaline post-synthetically at the .alpha.-pyrrolic
positions as shown with the following selected examples. 14
[0139] with any of R.sub.1-R.sub.2, R.sub.3-R.sub.4 may be as
follows:
R.sub.3.dbd.R.sub.4.dbd.I, R.sub.3.dbd.R.sub.4.dbd.Br,
R.sub.3.dbd.R.sub.4.dbd.COCH.sub.3, R.sub.3.dbd.R.sub.4.dbd.CHO
[0140] As shown in Scheme 4 below. 15
[0141] One of skill in the art will further recognize that the
diiodo or the dibromo compounds may be modified through known
synthetic means to generate a range of .alpha.-substituted
compounds as exemplified by structures 19. 16
[0142] Methods are also available for the synthesis of a TMS
derivative as shown below: 17
[0143] Removal of the TMS group and subsequent reaction with a
metal salt, as described previously, will afford metal linked
systems.
EXAMPLE 36
The Starting Pyrrole Unit as a Source of Variation
[0144] It will be apparent to one of skill in the art that many
pyrrolylquinoxalines may be obtained within the context of the
present invention. For example, as shown below, various
substituents may be introduced on the starting pyrrole to generate
a series of diones. These diones are then used in the synthesis of
pyrrole-aryls to effect desired properties, such as a particular
anion specificity, or increased solubility in a specific solvent.
Accordingly it is contemplated that variety of approaches may be
employed, in accordance with the present invention, to prepare an
array of diones with a wide variety of substituents as represented
in Scheme 5 below. 18
[0145] It is further envisioned that different combinations of
polypyrroles, in particular bipyrroles and terpyrroles, may be
employed in the reaction with oxalyl chloride to produce various
dione analogues as represented below. 19
EXAMPLE 37
Incorporation into Macrocycles
[0146] It is contemplated that the remaining .alpha.-free position
on the pyrrole rings may react under a variety of conditions as
shown below, to provide a number of novel compounds with a
pyrrole-aryl and a macrocyclic component. These macrocycles may be
used in fields such as anion binding (26), cation binding (28, 31,
32) and in optical devices (26, 28, 31, 32) and molecular wires
(28, 31, 32). In the following examples, R.sub.1-R.sub.4 are as
previously described, n=0-10 with the preferred compound(s) as
indicated within the synthetic scheme. 20 21 22 23
[0147] R.sub.4.dbd.H, n=1
[0148] It should again be stressed that although not shown above,
all of the reaction schemes can be applied to analogues having
widely substituted pyrrole rings. The pyrroles may be substituted
both .alpha. and .beta., or in both ways to the pyrrole nitrogen
atoms. The above reaction schemes are not intended to be limited to
unsubstituted pyrroles or to the substitutions shown.
EXAMPLE 38
Alternate Heterocycles in the Starting Diketone
[0149] It is contemplated that alternate heterocyclic diones may be
used in a condensation reaction with aryl 1,2-diamines, as outlined
in previous examples, to produce 2,3-heteroaryl quinoxaline
analogues. It is believed that these compounds will act as
effective sensors for a variety of cations or neutrals. Compounds
33-38 below represent some proposed alternative diones contemplated
for use in accordance with the present invention. 24
EXAMPLE 39
Dianions as Ligands
[0150] The pyrrole-aryls of the present invention may be used as
sensors for anions, cations or neutral molecules provided the
compound is of the appropriate charge of polarization. For
instance, pyrrole-aryls of the present invention are contemplated
for use as metal cation chelants by removing protons from the
pyrrolic nitrogens as shown below. 25
EXAMPLE 40
Pyrrole-Aryls As Anion Sensing Agents
[0151] The compounds of the present invention are used as anion
sensors in a variety of applications including, chromatography,
anion quantification, ion-selective electrodes and fiber-optics. A
preferred example for use of the present compounds is for selective
anion sensing, in particular fluoride sensing. Fluoride sensing has
largely been complicated by competition from other biologically
common species such as hydroxide, chloride and phosphate. The
ability to sense fluoride is important for the analysis of drinking
water, as well as ground water, in biological systems such as teeth
and bones and in certain disease states such as fluorosis. In vitro
sensing for fluoride is also important, for example to determine
the amount of damaging fluorocarbons in the atmosphere and even the
presence of fluorinated phosphates which can be used as chemical
weapons due to their toxicity when ingested. The compounds of the
present invention provide a distinct advantage over other sensors
due to the selectivity for fluoride ion as demonstrated in Examples
7-9 and the dramatic color change produced upon binding. This makes
the compounds particularly amenable for use in paper based sensing
such as litmus paper, solid support sensing and as a coating on
either fiber optic wires or electrodes. Selective sensing and
separation are also contemplated using chromatographic methods. For
example the present compounds may be coupled to a solid- support
and used to separate various anions from each other and from other
species in the mixture. In certain circumstances a desired anion
may even be collected as the system is entirely reversible based on
the environment. Washing under appropriate conditions completely
removes fluoride anion and returns the molecule to its original
state.
[0152] It will also be apparent to one of skill in the art that
many derivatives and analogues may be obtained within the context
of the disclosed methods and compounds.
[0153] Once a range of pyrrole-aryl or alternate heterocyclic
analogues have been generated, as described herein in the foregoing
detailed examples, the specificity, kinetics and thermodynamics of
anion binding under a range of conditions and with an array of
different anions may be determined. The structure and function of
the most promising compounds may then be optimized so that they
bind the desired anion under the appropriate conditions, solvent,
temperature, affinity pH etc., to function as an efficient
sensor.
EXAMPLE 41
Synthesis of Water Soluble Pyrrole-Aryl Analogues
[0154] It is contemplated that the addition of charged groups
and/or polar groups such as glycols or polyglycols to the
2,3-dipyrrylquinoxaline core will impart solubility in aqueous
solutions. The synthesis of such a compound is outlined in the
scheme below. The compound is that of 5 from Example 1. 26
[0155] It is further contemplated that the addition of further
pyrroles to the quinoxaline subunit will result in the increase of
solubility in aqueous solutions. The synthesis of such compounds is
illustrated in the following schemes.
[0156] It is expected that these compounds will serve as sensors
and therapeutic agents in aqueous solutions as described in the
next example. 27 28 29
EXAMPLE 42
Anion Binding Compounds as Therapeutic Agents
[0157] The compounds of the present invention, in particular those
compounds with increased solubility in aqueous solutions are
contemplated to be of use as in vivo anion sensors, for example
blood samples, and as therapeutic agents to bind excess anion in
certain disease states such as fluorosis.
[0158] To develop pyrrole-aryl compounds of the present invention
for use as therapeutic agents, in vitro tests will be first
conducted to determine the efficiency of binding, retention and
selectivity in aqueous solutions. These will follow similar
protocols as previously described for screening in organic
solvents.
[0159] Following such in vitro tests, the binding activity of
promising compounds will be followed up, in for example biological
fluids such as blood, and then in in vivo animal studies. These
studies will be conducted according to the standard practice for
such animal trials, the execution of which will be ell known to one
of skill in the art.
[0160] During the animal trials, the compounds may be further
modified if required. They may be modified to increase solubility
or to overcome in vivo degradation. Alternatively, if such problems
occur, the compounds may be enveloped within a bio-compatible
liposome and then administered intravenously.
[0161] Toxicity studies will also be carried out at this stage. The
methods for determining both acute and chronic toxicity will be
well known to one of skill in the art. Toxicity can be investigated
in relation to solubility, net charge at physiological pH and
changes in substituents.
[0162] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
EXAMPLE 43
Sensing Compound Immobilized on a Solid Support
[0163] The usefulness of the pyrrole-aryl compounds as sensing
agents when bound to solid supports was demonstrated by visual
color change in the presence of tetrabutylammonium fluoride.
2,3-Dipyrrol-2'-yl-6-carboxyquin- oxaline (preparation is described
in Example 19) was immobilized on a polystyrene beads
functionalized with polyethylene glycol groups that terminate in an
amine. The quinoxaline is linked to TG-amino resin through an amide
bond through a condensation reaction. An acetyl derivatized bead
was used a blank.
[0164] The presence of fluoride ion was signaled by a dramatic
visual color change from yellow to red for the
2,3-dipyrrol-2'-yl-6-carboxyquino- xaline-fuctionalized bead. The
blank bead was initially colorless and remained so upon addition of
fluoride ion.
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