U.S. patent application number 09/770015 was filed with the patent office on 2001-10-18 for processes and synthetic intermediates for preparing n-arylacridancarboxylic acid derivatives.
Invention is credited to Akhavan-Tafti, Hashem, Eickholt, Robert A., Handley, Richard S..
Application Number | 20010031869 09/770015 |
Document ID | / |
Family ID | 25087207 |
Filed Date | 2001-10-18 |
United States Patent
Application |
20010031869 |
Kind Code |
A1 |
Akhavan-Tafti, Hashem ; et
al. |
October 18, 2001 |
Processes and synthetic intermediates for preparing
N-arylacridancarboxylic acid derivatives
Abstract
Synthetic processes and intermediates are disclosed for the
preparation of N-arylacridancarboxylic acid derivatives. The
derivatives are esters, thioesters, amides and sulfonimides. A key
feature of the processes is the preparation of N-aryl substituted
intermendiates by formation of a bond between the nitrogen atom of
the acridan ring and a carbon atom of another aromatic or
heteroaromatic ring compound. The arylation reaction is catalyzed
by a palladium catalyst. The N-arylacridancarboxylic acid
derivatives are useful in methods for producing light and in assays
for peroxidase enzymes and enzyme inhibitors and in assays
employing enzyme-labeled specific binding pairs.
Inventors: |
Akhavan-Tafti, Hashem;
(Howell, MI) ; Eickholt, Robert A.; (Troy, MI)
; Handley, Richard S.; (Canton, MI) |
Correspondence
Address: |
LUMIGEN, INC.
22900 W. EIGHT MILE ROAD
SOUTHFIELD
MI
48034
US
|
Family ID: |
25087207 |
Appl. No.: |
09/770015 |
Filed: |
January 25, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09770015 |
Jan 25, 2001 |
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09557726 |
Apr 26, 2000 |
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09557726 |
Apr 26, 2000 |
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09358002 |
Jul 21, 1999 |
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6090571 |
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09358002 |
Jul 21, 1999 |
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08894143 |
Aug 13, 1997 |
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6045727 |
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08894143 |
Aug 13, 1997 |
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08585090 |
Jan 16, 1996 |
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08894143 |
Aug 13, 1997 |
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08683927 |
Jul 19, 1996 |
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Current U.S.
Class: |
546/104 ;
546/23 |
Current CPC
Class: |
C07D 219/06 20130101;
C07D 219/04 20130101; C07D 417/04 20130101 |
Class at
Publication: |
546/104 ;
546/23 |
International
Class: |
C07D 219/06; C07D
219/08 |
Claims
We claim:
1. A process for preparing an N-arylacridancarboxylic acid
derivative of the formula (1): 14wherein R.sup.1 is selected from
alkyl, substituted alkyl, heteroalkyl, aralkyl, substituted
aralkyl, aryl, substituted aryl and heteroaryl groups, Z is
selected from O and S atoms or the group ZR.sup.1 is an
--NR.sup.10R.sup.11 group wherein R.sup.10 and R.sup.11 are
independently selected from alkyl, substituted alkyl, aryl,
substituted aryl, aralkyl, alkylsulfonyl and arylsulfonyl groups,
and wherein R.sup.10 and R.sup.11 can be combined with N into a
heterocycle with leaving group ability, each of R.sup.2-R.sup.9 is
independently selected from substituents which contain from 1 to 50
atoms selected from C, H, N, O, S, P and halogen atoms and wherein
Ar is selected from aryl, substituted aryl and heteroaryl groups,
the process comprising the steps of: a) reducing an acridone
compound (2) having the formula: 15wherein R.sup.2-R.sup.9 are as
defined above to an acridan compound (3) having the formula:
16wherein R.sup.2-R.sup.9 are as defined above; b) converting the
acridan compound (3) to an N-arylacridan compound (4) having the
formula: 17wherein R.sup.2-R.sup.9 are as defined above and Ar is
an aryl, substituted aryl or heteroaryl ring group by reacting the
acridan compound with an arylating compound selected from aryl,
substituted aryl and heteroaryl halides and aryl, substituted aryl
and heteroaryl sulfonate esters in an inert solvent in the presence
of a base and a palladium catalyst; and c) converting the
N-arylacridan (4) to the N-arylacridancarboxylic acid derivative by
a carboxylation reaction in which a carbonyl-containing group is
attached to the 9-position of the acridan ring.
2. The process of claim 1 wherein the palladium catalyst is
prepared from a divalent palladium compound PdL.sub.2 and a
tertiary phosphine PR.sub.3, wherein each L is a labile ligand and
wherein each R is independently selected from alkyl and aryl
groups.
3. The process of claim 2 wherein the tertiary phosphine is
selected from P(t-Bu).sub.3, BINAP, DPPE, DPPF, DPPB and DPPP.
4. The process of claim 2 wherein the ligand L is selected from
carboxylate esters, halogens and ketones.
5. The process of claim 1 wherein acridone compound (2) is reduced
by reaction with a reducing agent selected from Na/Hg amalgam,
Al/Hg amalgam, copper chromite, NH.sub.2NH.sub.2 and a hydride
reducing agent.
6. The process of claim 1 wherein the group Ar is selected from
phenyl, naphthyl, biphenyl, anthryl, pyrenyl, pyridyl, quinolyl,
acridinyl, furyl, xanthenyl, thienyl, thioxanthyl, thiazolyl,
benzothiazolyl, indolyl, imidazolyl and pyrrolyl groups any of
which can contain one or more substituents selected from halogen,
trihalomethyl, nitro, nitroso, cyano, ammonium, hydrazinyl,
carboxyl, carboxamide, carboalkoxy, --CHO, keto, amino, substituted
amino, imino, imido, aryl, alkyl, perfluoroalkyl, alkenyl, alkynyl,
alkoxy, hydroxy, sulfhydryl, alkylthio, sulfate, sulfonate,
phosphonium, phosphate and phosphonate groups.
7. The process of claim 1 wherein the group --NR.sup.10OR.sup.11 is
a cyclic group selected from pyrazole, imidazole, benzimidazole,
triazole, benzotriazole, tetrazole, oxazole, benzoxazole, thiazole
and benzothiazole.
8. The process of claim 1 wherein in the group 'NR.sup.10OR.sup.11
one of R.sup.10 and R.sup.11 is alkylsulfonyl or arylsulfonyl and
the other of R.sup.10 or R.sup.11 is an alkyl, phenyl or
substituted phenyl group.
9. The process of claim 1 wherein Z is O or S and R.sup.1 is
selected from substituted alkyl and substituted aryl groups which
are substituted with at least one electron withdrawing group.
10. The process of claim 9 wherein the electron withdrawing group
is a halogen atom.
11. The process of claim 9 wherein R.sup.1 is a trifluorophenyl
group.
12. The process of claim 1 wherein each of R.sup.2 to R.sup.9 is
hydrogen.
13. The process of claim 1 wherein step c comprises: (i) reacting
the N-arylacridan (4) with a base to generate an anion of the
N-arylacridan; (ii) capturing the anion with CO.sub.2 to produce an
N-arylacridancarboxylic acid (5) having the formula: 18and (iii)
reacting the N-arylacridancarboxylic acid with a compound
HZ--R.sup.1 to form compound (1).
14. The process of claim 13 wherein a coupling agent is used in the
reaction to convert the N-arylacridancarboxylic acid to the acid
derivative and the coupling agent is selected from thionyl
chloride, PCl.sub.3, a carbodiimide, carbonyl diimidazole, strong
acids and bases.
15. The process of claim 1 wherein step c comprises: (i) reacting
the N-arylacridan (4) with a base to generate an anion of the
N-arylacridan; (ii) reacting the anion with a reagent having the
formula X--CO--ZR.sup.1 wherein X is a leaving group.
16. A process for preparing an N-arylacridancarboxylic acid
derivative of the formula (1): 19wherein R.sup.1 is selected from
alkyl, substituted alkyl, heteroalkyl, aralkyl, substituted
aralkyl, aryl, substituted aryl and heteroaryl groups, Z is
selected from O and S atoms or the group ZR.sup.1 is an
--NR.sup.10OR.sup.11 group wherein R.sup.10 and R.sup.11 are
independently selected from alkyl, substituted alkyl, aryl,
substituted aryl, aralkyl, alkylsulfonyl and arylsulfonyl groups,
and wherein R.sup.10 and R.sup.11 can be combined with N into a
heterocycle with leaving group ability, each of R.sup.2-R.sup.9 is
independently selected from substituents which contain from 1 to 50
atoms selected from C, H, N, O, S, P and halogen atoms and wherein
Ar is selected from aryl, substituted aryl and heteroaryl groups,
the process comprising converting an acridancarboxylic acid
derivative (6) 20having groups Z and R-R.sup.9 as defined above to
the N-arylacridancarboxylic acid derivative (1) by reacting the
compound (6) with an arylating compound selected from aryl,
substituted aryl and heteroaryl halides and aryl, substituted aryl
and heteroaryl sulfonate esters in an inert solvent in the presence
of a base and a palladium catalyst.
17. The process of claim 16 wherein the palladium catalyst is
prepared from a divalent palladium compound PdL.sub.2 and a
tertiary phosphine PR.sub.3, wherein each L is a labile ligand and
wherein each R is independently selected from alkyl and aryl
groups.
18. The process of claim 17 wherein the tertiary phosphine is
selected from P(t-Bu).sub.3, BINAP, DPPE, DPPF, DPPB and DPPP.
19. The process of claim 17 wherein the ligand L is selected from
carboxylate esters, halogens and ketones.
20. The process of claim 16 wherein the group Ar is selected from
phenyl, naphthyl, biphenyl, anthryl, pyrenyl, pyridyl, quinolyl,
acridinyl, furyl, xanthenyl, thienyl, thioxanthyl, thiazolyl,
benzothiazolyl, indolyl, imidazolyl and pyrrolyl groups any of
which can contain one or more substituents selected from halogen,
trihalomethyl, nitro, nitroso, cyano, ammonium, hydrazinyl,
carboxyl, carboxamide, carboalkoxy, --CHO, keto, amino, substituted
amino, imino, imido, aryl, alkyl, perfluoroalkyl, alkenyl, alkynyl,
alkoxy, hydroxy, sulfhydryl, alkylthio, sulfate, sulfonate,
phosphonium, phosphate and phosphonate groups.
21. A process for preparing an N-arylacridancarboxylic acid
derivative of the formula (1): 21wherein R.sup.1 is selected from
alkyl, substituted alkyl, heteroalkyl, aralkyl, substituted
aralkyl, aryl, substituted aryl and heteroaryl groups, Z is
selected from O and S atoms or the group ZR.sup.1 is an
--NR.sup.10R.sup.11 group wherein R.sup.10 and R.sup.11 are
independently selected from alkyl, substituted alkyl, aryl,
substituted aryl, aralkyl, alkylsulfonyl and arylsulfonyl groups,
and wherein R.sup.10 and R.sup.11 can be combined with N into a
heterocycle with leaving group ability, each of R.sup.2-R.sup.9 is
independently selected from substituents which contain from 1 to 50
atoms selected from C, H, N, O, S, P and halogen atoms and wherein
Ar is selected from aryl, substituted aryl and heteroaryl groups,
the process comprising the steps of: a) converting an
acridancarboxylic acid (7) 22to an N-arylacridancarboxylic acid (5)
by reacting the acridancarboxylic acid (7) with an arylating
compound selected from aryl, substituted aryl and heteroaryl
halides and aryl, substituted aryl and heteroaryl sulfonate esters
in an inert solvent in the presence of a base and a palladium
catalyst; and b) reacting the N-arylacridancarboxylic acid (5) with
a compound HZ--R.sup.1 wherein Z and R.sup.1 are as defined above
to form compound (1).
22. The process of claim 21 wherein a coupling agent is used in the
reaction to convert the N-arylacridancarboxylic acid to the acid
derivative and the coupling agent is selected from thionyl
chloride, PCl.sub.3 a carbodiimide, carbonyl diimidazole, strong
acids and bases.
23. The process of claim 21 wherein the palladium catalyst is
prepared from a divalent palladium compound PdL.sub.2 and a
tertiary phosphine PR.sub.3, wherein each L is a labile ligand and
wherein each R is independently selected from alkyl and aryl
groups.
24. The process of claim 23 wherein the tertiary phosphine is
selected from P(t-Bu).sub.3, BINAP, DPPE, DPPF, DPPB and DPPP.
25. The process of claim 23 wherein the ligand L is selected from
carboxylate esters, halogens and ketones.
26. The process of claim 21 wherein the group Ar is selected from
phenyl, naphthyl, biphenyl, anthryl, pyrenyl, pyridyl, quinolyl,
acridinyl, furyl, xanthenyl, thienyl, thioxanthyl, thiazolyl,
benzothiazolyl, indolyl, imidazolyl and pyrrolyl groups any of
which can contain one or more substituents selected from halogen,
trihalomethyl, nitro, nitroso, cyano, ammonium, hydrazinyl,
carboxyl, carboxamide, carboalkoxy, --CHO, keto, amino, substituted
amino, imino, imido, aryl, alkyl, perfluoroalkyl, alkenyl, alkynyl,
alkoxy, hydroxy, sulfhydryl, alkylthio, sulfate, sulfonate,
phosphonium, phosphate and phosphonate groups.
27. A process for preparing an N-arylacridancarboxylic acid
derivative of the formula (1): 23wherein R.sup.1 is selected from
alkyl, substituted alkyl, heteroalkyl, aralkyl, substituted
aralkyl, aryl, substituted aryl and heteroaryl groups, Z is
selected from O and S atoms or the group ZR.sup.1 is an
--NR.sup.10R.sup.11 group wherein R.sup.10 and R.sup.11 are
independently selected from alkyl, substituted alkyl, aryl,
substituted aryl, aralkyl, alkylsulfonyl and arylsulfonyl groups,
and wherein R.sup.10 and R.sup.11 can be combined with N into a
heterocycle with leaving group ability, each of R.sup.2-R.sup.9 is
independently selected from substituents which contain from 1 to 50
atoms selected from C, H, N, O, S, P and halogen atoms and wherein
Ar is selected from aryl, substituted aryl and heteroaryl groups,
comprising the steps of: a) converting an acridone compound (2)
having the formula: 24wherein R.sup.2-R.sup.9 are as defined above
to an N-arylacridone compound (8) having the formula: 25wherein Ar
and R.sup.2-R.sup.9 are as defined above with an arylating compound
selected from aryl, substituted aryl and heteroaryl halides and
aryl, substituted aryl and heteroaryl sulfonate esters in an inert
solvent in the presence of a base and a palladium catalyst wherein
R.sup.2-R.sup.9 are as defined above; b) reducing the
N-arylacridone compound (8) to an N-arylacridan compound (4) having
the formula: 26wherein Ar and R.sup.2-R.sup.9 are as defined above;
and c) converting the N-arylacridan (4) to the
N-arylacridancarboxylic acid derivative by a carboxylation reaction
in which a carbonyl-containing group is attached to the 9-position
of the acridan ring.
28. The process of claim 27 wherein the palladium catalyst is
prepared from a divalent palladium compound PdL.sub.2 and a
tertiary phosphine PR.sub.3, wherein each L is a labile ligand and
wherein each R is independently selected from alkyl and aryl
groups.
29. The process of claim 28 wherein the tertiary phosphine is
selected from P(t-Bu).sub.3, BINAP, DPPE, DPPF, DPPB and DPPP.
30. The process of claim 28 wherein the ligand L is selected from
carboxylate esters, halogens and ketones.
31. The process of claim 27 wherein the group Ar is selected from
phenyl, naphthyl, biphenyl, anthryl, pyrenyl, pyridyl, quinolyl,
acridinyl, furyl, xanthenyl, thienyl, thioxanthyl, thiazolyl,
benzothiazolyl, indolyl, imidazolyl and pyrrolyl groups any of
which can contain one or more substituents selected from halogen,
trihalomethyl, nitro, nitroso, cyano, ammonium, hydrazinyl,
carboxyl, carboxamide, carboalkoxy, --CHO, keto, amino, substituted
amino, imino, imido, aryl, alkyl, perfluoroalkyl, alkenyl, alkynyl,
alkoxy, hydroxy, sulfhydryl, alkylthio, sulfate, sulfonate,
phosphonium, phosphate and phosphonate groups.
32. A compound having the formula: 27or a salt thereof wherein each
of R.sup.2-R.sup.9 is independently selected from substituents
which contain from 1 to 50 atoms selected from C, H, N, O, S, P and
halogen atoms and wherein Ar is selected from aryl, substituted
aryl and heteroaryl groups.
33. The compound of claim 32 wherein the group Ar is selected from
phenyl, naphthyl, biphenyl, anthryl, pyrenyl, pyridyl, quinolyl,
acridinyl, furyl, xanthenyl, thienyl, thioxanthyl, thiazolyl,
benzothiazolyl, indolyl, imidazolyl and pyrrolyl groups any of
which can contain one or more substituents selected from halogen,
trihalomethyl, nitro, nitroso, cyano, ammonium, hydrazinyl,
carboxyl, carboxamide, carboalkoxy, --CHO, keto, amino, substituted
amino, imino, imido, aryl, alkyl, perfluoroalkyl, alkenyl, alkynyl,
alkoxy, hydroxy, sulfhydryl, alkylthio, sulfate, sulfonate,
phosphonium, phosphate and phosphonate groups.
34. The compound of claim 33 wherein Ar is selected from phenyl,
substituted phenyl, naphthyl and substituted naphthyl.
35. The compound of claim 32 wherein each of R.sup.2-R.sup.9 is a
hydrogen.
36. The compound of claim 34 wherein each of R.sup.2-R.sup.9 is a
hydrogen.
37. The compound of claim 32 wherein the salt is selected from
alkali metal salts.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of applicant's
application Ser. No. 09/557,726 filed on Apr. 26, 2000 which is a
continuation of allowed U.S. application Ser. No. 09/358,002 filed
on Jul. 21, 1999 which is a division of U.S. application Ser. No.
08/894,143 filed on Aug. 13, 1997 as a result of National Stage
entry from PCT application U.S. Ser. No. 97/00015, now U.S. Pat.
No. 6,045,727 which is a continuation-in-part of U.S. application
Ser. No. 08/585,090, abandoned and 08/683,927, abandoned.
FIELD OF THE INVENTION
[0002] This invention relates to synthetic processes and
intermediates useful for preparing N-arylacridancarboxylic acid
derivatives. The N-arylacridancarboxylic acid derivatives are
useful in methods to produce chemiluminescence, for example by
reaction with a peroxide and a peroxidase. The chemiluminescent
reaction is useful in methods of analysis for detecting peroxidase
enzymes or hydrogen peroxide. It is also useful in methods to
detect and quantify various biological molecules wherein a
peroxidase is used as a label and in methods to detect oxidase
enzymes which generate hydrogen peroxide.
BACKGROUND OF THE INVENTION
[0003] The detection and quantitation of biological molecules has
been accomplished historically with excellent sensitivity by the
use of radiolabeled reporter molecules. Recently numerous
non-radioactive methods have been developed to avoid the hazards
and inconvenience posed by these materials. Methods based on
enzyme-linked analytes offer the best sensitivity since the ability
to catalytically turn over substrate to produce a detectable change
achieves an amplification. Substrates which generate color,
fluorescence or chemiluminescence have been developed, the latter
achieving the best sensitivity.
[0004] Further increases in assay sensitivity will expand the range
of utility of chemiluminescence-based methods by permitting the
detection of analytes present in smaller quantities or reducing the
amount of time and/or reagents required to perform the assay. A way
to increase the speed and sensitivity of detection in an enzymatic
chemiluminescent assay is through the use of substrates which
generate light with a higher efficiency or for a greater length of
time.
[0005] Among the enzymes used in enzyme-linked detection methods
such as immunoassays, detection of oligonucleotides and nucleic
acid hybridization techniques, the most extensively used to date
has been horseradish peroxidase. Chemiluminescent reagents known in
the art do not permit full advantage to be taken of the beneficial
properties of this enzyme in analysis mainly due to sensitivity
limitations. A reagent which permits the detection of lower amounts
of enzyme is needed to enable the use of peroxidase conjugates in
applications requiring ultrasensitive detection. Specifically,
reagents are required which generate higher levels of
chemiluminescence without an accompanying increase in the
background or non-specific chemiluminescence. The increased
chemiluminescence can be accomplished via either a higher maximum
intensity or a longer duration than compounds known in the art.
[0006] a. Enzymatic Oxidation of N-Alkylacridancarboxylic Acid
Derivatives
[0007] Applicants' U.S. Pat. Nos. 5,491,072, 5,523,212, 5,593,845,
5,670,644, 5,723,295 and 5,750,698 disclose the use of a peroxidase
enzyme to oxidize substituted and unsubstituted
N-alkylacridancarboxylic acid derivatives to generate
chemiluminescence. In the presence of a peroxidase enzyme and a
peroxide, N-alkylacridancarboxylic acid derivatives are efficiently
oxidized to produce the N-alkylacridone and blue chemiluminescence.
N-aryl-substituted acridan-carboxylic acid derivatives are not
disclosed.
[0008] U.S. Pat. No. 6,030,803 discloses a group of
acridancarboxylic acid derivatives having a substituted alkoxy or
alkylthio leaving group but not aryloxy or arylthio leaving groups
as chemiluminescent substrate for peroxidase enzymes. N-aryl
acridan compounds are claimed but no examples of N-aryl compounds
are provided. All exemplary compounds contain a methyl group as the
substituent on the acridan ring nitrogen atom.
[0009] U.S. Pat. No. 6,162,610 discloses a group of
acridancarboxylic acid derivatives having a substituted alkoxy,
alkylthio or amide leaving group as chemiluminescent substrate for
peroxidase enzymes. The claimed compounds bear a group designated
--OX alleged to be a triggering group. No examples of N-aryl
compounds are provided nor is a basis for the alleged triggering
effect.
[0010] N-arylacridancarboxylic acid derivatives having a
heteroaromatic group bound to the nitrogen atom are not taught or
suggested in this or any of the cited publications nor in any other
publication prior to the present invention.
OBJECTS
[0011] It is therefore an object of the present invention to
provide processes for the synthesis of N-arylacridan-carboxylic
acid derivatives for use in generating chemiluminescence. It is
another object of the present invention to provide synthetic
intermediates useful in methods for preparing
N-arylacridancarboxylic acid derivatives. It is still another
object of the present invention to provide N-arylacridancarboxylic
acid derivatives for use in generating chemiluminescence. It is
also an object of the present invention to provide
N-arylacridancarboxylic acid derivatives for use in methods of
analysis and detection. It is a further object to provide
chemiluminescent methods for the detection of biological materials
and compounds. It is also an object of the present invention to
provide a chemiluminescent method for detecting peroxidase enzymes
and enzyme-conjugates. Additionally, it is an object of the present
invention to provide improved methods for use in solution or on
surfaces in nucleic acid assays, protein-binding assays, Western
blots, Southern blots and other DNA and RNA hybridization assays
and for detection of haptens, proteins and antibodies in enzyme
immunoassays.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0012] Definitions
[0013] The term "substituted" when used to describe an organic
moiety such as a chain or ring group refers to the replacement of
one or more hydrogen atoms on the chain or ring with another atom
or group. Exemplary groups include halogen, trihalomethyl, nitro,
nitroso, cyano, ammonium, hydrazinyl, carboxyl, carboxamide,
carboalkoxy, formyl (--CHO), keto, amino, substituted amino, imino,
imido, aryl, alkyl, perfluoroalkyl, alkenyl, alkynyl, alkoxy,
hydroxy, sulfhydryl, alkylthio, sulfate, sulfonate, phosphonium,
phosphate and phosphonate groups.
[0014] The term "leaving group ability" as used herein refers to
the propensity for a group when attached to the carbonyl group of
the acridancarboxylic acid derivative to be displaced in the
nucleophilic reaction of the invention involving a peroxide or
hydroperoxide or its anion.
[0015] The present invention relates to N-arylacridancarboxylic
acid derivatives of the formula: 1
[0016] wherein R.sup.1 is selected from alkyl, substituted alkyl,
heteroalkyl, aralkyl, substituted aralkyl, aryl, substituted aryl
and heteroaryl groups, R.sup.2 to R.sup.9 are independently
selected from substituents which contain from 1 to 50 atoms
selected from C, H, N, O, S, P and halogen atoms, wherein Ar is an
aryl, substituted aryl or heteroaryl group and Z is selected from O
and S atoms or the group ZR.sup.1 is an --NR.sup.10R.sup.11 group
wherein R.sup.10 and R.sup.11 are independently selected from
alkyl, substituted alkyl, aryl, substituted aryl, aralkyl,
alkylsulfonyl and arylsulfonyl groups, and wherein R.sup.10 and
R.sup.11 can be combined with N into a heterocycle with leaving
group ability including pyrazole, imidazole, benzimidazole,
triazole, benzotriazole, tetrazole, oxazole, benzoxazole, thiazole
and benzothiazole.
[0017] When R.sup.10 or R.sup.11 is alkylsulfonyl or arylsulfonyl
it is preferably selected from methanesulfonyl,
trifluoromethanesulfonyl, benzenesulfonyl or substituted
benzenesulfonyl and the other of R.sup.10 or R.sup.11 is preferably
an alkyl, phenyl or substituted phenyl group. When R.sup.1 is a
substituted alkyl or substituted aryl group the group is preferably
substituted with one or more electron withdrawing groups,
preferably halogen atoms and most preferably with fluorine.
[0018] Compounds having Formula I which contain an N-aryl group are
useful in methods for producing chemiluminescence. These compounds
distinguish from known prior art compounds which all have an alkyl
group substituted on the ring nitrogen atom, usually a methyl
group. In contrast, the compounds of the present invention bear an
aromatic or heteroaromatic ring group on the ring nitrogen.
Representative aryl groups include phenyl, naphthyl, biphenyl,
anthryl, pyrenyl, pyridyl, quinolyl, acridinyl, furyl, xanthenyl,
thienyl, thioxanthyl, thiazolyl, benzothiazolyl, indolyl,
imidazolyl and pyrrolyl groups. The aryl or heteroaryl group can be
substituted with one or more substituents as defined above and
which allows or does not interfere with or prevent the production
of light from reaction of the N-arylacridan-carboxylic acid
derivative with an oxidant. Representative substituents which can
be present on the aryl group include without limitation, halogen,
trihalomethyl, nitro, nitroso, cyano, ammonium, hydrazinyl,
carboxyl, carboxamide, carboalkoxy, formyl (--CHO), keto, amino,
substituted amino, imino, imido, aryl, alkyl, perfluoroalkyl,
alkenyl, alkynyl, alkoxy, hydroxy, sulfhydryl, alkylthio, sulfate,
sulfonate, phosphonium, phosphate and phosphonate.
[0019] Examples of some preferred compounds are: 2
[0020] wherein X is an electron withdrawing group, Y is a
non-hydrogen substituent and wherein m and n are each integers from
0 to 5 and R.sup.10 and R.sup.11 are as defined above. Preferably
R.sub.10 is alkylsulfonyl or arylsulfonyl and R.sup.11 is alkyl or
aryl. Preferred electron withdrawing groups are halogen atoms, more
preferably chlorine or fluorine atoms. The number of such electron
withdrawing groups m is preferably at least one and more desirably
at least two.
[0021] Another class of preferred compounds is: 3
[0022] wherein X, m, R.sup.10 and R.sup.11 are as defined above and
Np is a naphthyl group.
[0023] Yet other preferred compounds have the formula: 4
[0024] wherein alkyl-X.sub.m is a substituted alkyl and X, Y and m
and n are as defined above and Z is O or S.
[0025] The present invention further relates to novel processes and
synthetic intermediates which are used to prepare the
N-arylacridancarboxylic acid derivatives. Applicants have
discovered new processes for preparing N-arylacridancarboxylic acid
derivatives (I) 5
[0026] beginning from acridone or a ring-substituted acridone
compound having the formula: 6
[0027] Where not commercially available, acridones can be prepared
by art-known methods including 1) cyclization of
2-amino-2'-halobenzophenone derivatives, 2) cyclization of
diphenylamine-2-carboxylic acids, 3) hydrolysis of
9-chloroacridines and 9-methoxyacridines and 4) rearrangement of
3-phenylanthranils as described in Acridines, R. M. Acheson, ed.
Wiley, (1973) Chapter III, pp. 143-196. The acridone compound is
reduced to the corresponding acridan by reduction of the ketone
moiety. Reduction can be achieved with known reagents for ketone
reductions as disclosed In the aforementioned Acridines, pp. 201-2,
including Na/Hg amalgam, Al/Hg amalgam, copper chromite,
NH.sub.2NH.sub.2, base and ethylene glycol. In another method the
acridone is converted to the 9-chloroacridine compound and then
reduced with Raney nickel. In addition, the ketone can be reduced
with hydride reducing agents such as LiAlH.sub.4.
[0028] The substituted or unsubstituted acridan compound thus
formed is converted to the N-arylacridancarboxylic acid derivative
by a process involving an N-arylation reaction and a reaction
process for attaching the carboxylic acid derivative moiety
--C(.dbd.O)ZR.sup.1. The arylation and carboxylate attaching steps
can be performed at different points in the synthetic scheme as
described in more detail below.
[0029] In a first embodiment of the synthetic process, the acridan
compound is reacted with an arylating compound selected from aryl
halides and sulfonate esters in an inert solvent in the presence of
a base and a palladium catalyst to form an N-arylacridan compound.
7
[0030] In this context aryl includes both aromatic and
heteroaromatic ring compounds. Halides include iodide, bromide and
chloride. Sulfonate esters include trifluoromethanesulfonates
(triflates) and other esters active as leaving groups. Preferred
palladium catalysts are prepared from a divalent palladium compound
PdL.sub.2 and a tertiary phosphine PR.sub.3 wherein each R is
independently selected from alkyl and aryl groups Suitable divalent
compounds PdL.sub.2 include any divalent palladium compound with
labile ligands selected from carboxylate esters, halogens and
ketones and include palladium acetate, palladium chloride,
palladium bis(dibenzylideneacetone) Pd(dba).sub.2 and
Pd.sub.2(dba).sub.3. Suitable tertiary phosphines include
trialkylphosphines such as P(t-Bu).sub.3, triaryl phosphines such
as BINAP and mixed alkylarylphosphines such as DPPE, DPPF, DPPB and
DPPP. Bases include KHPO.sub.4, CsCO.sub.3, and alkoxide salts such
as sodium t-butoxide. Inert solvents useful in this step include
toluene, benzene, THF, DME, diglyme and the like. The solvent
preferably has a boiling point above about 50.degree. C. to enable
heating of the reaction. However the reaction can be performed at
room temperature or elevated temperatures.
[0031] The N-arylacridan compound is then converted to an
N-arylacridancarboxylic acid derivative by a carboxylation reaction
in which a carbonyl-containing group is attached to the 9-position
of the acridan ring. The carbonyl-containing group can be a
carboxyl group (--COOH) or its salt, a carboxyl ester group
(--COOR.sup.1), a thioester group (--COSR.sup.1) or an amide group
(--CONR.sup.10R.sup.11). Conversion of the N-arylacridan to the
carboxylic acid derivative involves formation of the acridan anion
at the 9-position by treatment with a strong base and then reaction
with a reagent to attach the carbonyl-containing group.
[0032] This conversion can be accomplished in two steps by reaction
of the N-arylacridan with a base to generate the anion at the
9-position and capture of the anion with CO.sub.2 to produce the
N-arylacridancarboxylic acid or its salt. The
N-arylacridancarboxylic acid is subsequently converted to any of
the various acid derivatives (I) having the group Z--R.sup.1 by
reaction of the N-arylacridancarboxylic acid with a compound
HZ--R.sup.1 where Z and R.sup.1 are as defined above. Preferably a
coupling agent is used to promote the conversion of the acid to the
acid derivative. In one embodiment the N-arylacridancarboxylic acid
is first converted to the acid chloride by methods generally known
in the art such as by use of thionyl chloride (SOCl.sub.2) or
PCl.sub.3. The acid chloride is reacted with a compound of the
formula HZ--R.sup.1 in the presence of a base or with a salt of the
compound HZ--R.sup.1. In another embodiment the acid is coupled to
the compound HZ--R.sup.1 with the aid of a carbodiimide coupling
agent such as dicyclohexylcarbodiimide or with carbonyl
diimidazole, CDI. In other embodiments strong acids or bases are
used as the coupling agent to catalyze the formation of the acid
derivative.
[0033] Conversion of the N-arylacridan to the
N-arylacridancarboxylic acid derivative can also be accomplished in
one step by reaction of the 9-position anion described above with a
reagent having the formula X--CO--ZR.sup.1 which attaches one of
the ester (--COOR.sup.1), thioester (--COSR.sup.1) or amide
(--CONR.sup.10R.sup.11) groups directly. Suitable reagents have a
leaving group X such as a halogen attached to the carbonyl group of
the carboxylating agent. Suitable reagents would therefore include
chloroformate esters (Cl--COOR.sup.1), chlorothioformate esters
(Cl--COSR.sup.1) and carbamoyl chlorides
(Cl--CONR.sup.10R.sup.11).
[0034] The present invention further relates, in a second
embodiment, to a synthetic process for preparing an
N-arylacridancarboxylic acid derivative (I) in which the
N-arylation step is performed after formation of the
acridancarboxylic acid or acid derivative. In an exemplary process
(a) an acridan-9-carboxylic acid derivative or an
acridan-9-carboxylic acid is reacted with an arylating agent and a
palladium catalyst as described above to effect N-arylation. When
the N-arylation reaction is performed on the carboxylic acid, the
product N-arylacridancarboxylic acid is then converted to the acid
derivative (I) by reaction with a compound HZ--R.sup.1 where Z and
R.sup.1 are as defined above. Preferably a coupling agent as
described above is used to promote the conversion of the acid to
the acid derivative. 8
[0035] Acridine-9-carboxylic acid is available commercially and can
be prepared using methods known to one of skill in the art of
organic chemistry by consultation of the scientific literature.
Ring substituted acridan-9-carboxylic acids can similarly be
prepared by like methods.
[0036] The present invention further relates, in a third
embodiment, to a synthetic process for preparing an
N-arylacridancarboxylic acid derivative (I) in which the acridone
or ring-substituted acridone undergoes the N-arylation step
according to the above-described N-arylation reaction method. The
N-arylacridone intermediate is then reduced to the corresponding
N-arylacridan by reduction of the ketone moiety according to one of
the methods described above. The N-arylacridan compound is then
converted to an N-arylacridancarboxylic acid derivative by a
carboxylation reaction in which a carbonyl-containing group is
attached to the 9-position of the acridan ring via either the one
step or two step processes as described above 9
[0037] Another aspect of the present invention relates to synthetic
intermediates used in preparing N-arylacridancarboxylic acid
derivatives. In particular, N-arylacridancarboxylic acid compounds
(II) and their carboxylate salts are claimed wherein R.sup.2 to
R.sup.9 are independently selected from substituents which contain
from 1 to 50 atoms selected from C, H, N, O, S, P and halogen
atoms, wherein Ar is an aryl, substituted aryl or heteroaryl group.
10
[0038] Representative aryl groups include phenyl, naphthyl,
biphenyl, anthryl, pyrenyl, pyridyl, quinolyl, acridinyl, furyl,
xanthenyl, thienyl, thioxanthyl, thiazolyl, benzothiazolyl,
indolyl, imidazolyl and pyrrolyl groups. The aryl or heteroaryl
group can be substituted with one or more substituents defined as
defined above and which allows or does not interfere with or
prevent the production of light from the N-arylacridancarboxylic
acid derivative when it is reacted with a peroxide and a
peroxidase. Preferably, Ar is selected from phenyl, substituted
phenyl and naphthyl groups. Representative substituents which can
be present on the aryl group include without limitation, halogen,
trihalomethyl, nitro, nitroso, cyano, ammonium, hydrazinyl,
carboxyl, carboxamide, carboalkoxy, formyl (--CHO), keto, amino,
substituted amino, imino, imido, aryl, alkyl, perfluoroalkyl,
alkenyl, alkynyl, alkoxy, hydroxy, sulfhydryl, alkylthio, sulfate,
sulfonate, phosphonium, phosphate and phosphonate. In preferred
compounds of formula (II) each of R.sup.2 to R.sup.9 are hydrogen
or one of R.sup.2 to R.sup.9 is an alkoxy group and each of the
others is hydrogen. When compound (II) is present in the form of a
salt, the counter ion will be the same as the counter ion of the
strong base used in the carboxylation reaction. Preferred counter
ions include alkali metal ions.
[0039] Another aspect of the present invention relates to reaction
of N-arylacridancarboxylic acid derivatives (I) of the present
invention with an oxidant to generate visible chemiluminescence. In
one embodiment, reaction of an N-arylacridancarboxylic acid
derivative with a base which can remove the proton at the
9-position of the acridan ring, i.e. the proton .alpha. to the
carbonyl, in the presence of molecular oxygen in an aprotic solvent
produces chemiluminescence. Suitable bases include, hydroxide
salts, alkoxide salts such as sodium methoxide and potassium
t-butoxide, and tetraalkylammonium fluoride. Aprotic solvents
useful include dimethyl sulfoxide, dimethylformamide,
dimethylacetamide and tetrahydrofuran.
[0040] In another method, reaction of an N-arylacridancarboxylic
acid derivative with an oxidant system comprising a peroxide and a
peroxidase enzyme produces chemiluminescence. This reaction system
is highly useful for assay applications. The chemiluminescence is
believed to arise from the excited state of N-arylacridone or the
substituted N-arylacridone product as shown in the generalized
reaction below. 11
[0041] Compounds of the present invention typically produce light
over a 100-200 nm wide band of emission, which exhibits a maximum
intensity at wavelengths in the near ultraviolet to the visible
region of the electromagnetic spectrum. Typical wavelengths of
maximum intensity .lambda..sub.max are in the range of 350-500 nm.
It is contemplated that compounds of formula I bearing a covalently
linked fluorophore could undergo intramolecular energy transfer
resulting in emission at longer wavelengths from the excited state
of the fluorophore.
[0042] The peroxidase which can undergo the chemiluminescent
reaction include lactoperoxidase, microperoxidase, myeloperoxidase,
haloperoxidase, e.g. vanadium bromoperoxidase, horseradish
peroxidase, fungal peroxidases such as lignin peroxidase and
peroxidase from Arthromyces ramosus and Mn-dependent peroxidase
produced in white rot fungi, and soybean peroxidase. Other
peroxidase mimetic compounds which are not enzymes but possess
peroxidase-like activity including iron complexes and Mn-TPPS.sub.4
(Y.-X. Ci, et al., Mikrochem. J., 52, 257-62 (1995)) are explicitly
considered to be within the scope of the meaning of peroxidase as
used herein. Conjugates or complexes of a peroxidase and a
biological molecule can also be used in the method for producing
chemiluminescence, the only proviso being that the conjugate
display peroxidase activity. Biological molecules which can be
conjugated to one or more molecules of a peroxidase include DNA,
RNA, oligonucleotides, antibodies, antibody fragments, antibody-DNA
chimeras, antigens, haptens, proteins, lectins, avidin,
streptavidin and biotin. Complexes including or incorporating a
peroxidase such as liposomes, micelles, vesicles and polymers which
are functionalized for attachment to biological molecules can also
be used in the methods of the present invention.
[0043] Compounds of the present invention are useful in a reagent
composition which generates light in the presence of a peroxidase.
Compositions comprise the acridan and a peroxide compound in
aqueous solution, preferably a buffer solution, wherein the
peroxide participates in the reaction of the acridan with the
peroxidase. Optionally the composition can comprise any or all of
the following additional components:
[0044] a compound which enhances light production from the
chemiluminescent reaction;
[0045] a chelating agent which prevents the peroxide compound from
reacting prior to addition of the peroxidase to the composition;
and/or
[0046] a surfactant including nonionic, anionic and cationic
compounds including monomeric or polymeric compounds.
[0047] The peroxide component is any peroxide or alkyl
hydroperoxide capable of reacting with the peroxidase. Preferred
peroxides include hydrogen peroxide, urea peroxide, and perborate
salts.
[0048] Suitable buffers include any of the commonly used buffers
capable of maintaining a pH in the range of about 6 to about 10 for
example, phosphate, borate, carbonate,
tris(hydroxymethylamino)methane, glycine, tricine,
2-amino-2-methyl-1-propanol, diethanolamine and the like.
[0049] Chemiluminescence enhancing compounds usable include
art-known compounds which promote the reactivity of the enzyme.
Included among these enhancers are phenolic compounds and aromatic
amines known to enhance other peroxidase reactions as described in
G. Thorpe, L. Kricka, in Bioluminescence and Chemiluminescence, New
Perspectives, J. Scholmerich, et al, Eds., pp. 199-208 (1987), M.
Ii, H. Yoshida, Y. Aramaki, H. Masuya, T. Hada, M. Terada, M.
Hatanaka, Y. Ichimori, Biochem. Biophys. Res. Comm., 193(2), 540-5
(1993), and in U.S. Pat. Nos. 5,171,668 and 5,206,149 which are
incorporated herein by reference. Substituted and unsubstituted
arylboronic acid compounds and their ester and anhydride
derivatives as disclosed in U.S. Pat. Nos. 5,512,451 and 5,629,168,
incorporated herein by reference, are also considered to be within
the scope of enhancers useful in the present invention. Yet other
enhancer compounds are taught in U.S. Pat. Nos. 5,171,668 and
5,206,149. Also included are phenothiazine and phenoxazine
compounds as taught in PCT Publication WO97/39142. Preferred
enhancers include but are not limited to: p-phenylphenol,
p-iodophenol, p-bromophenol, p-hydroxycinnamic acid,
p-imidazolylphenol, acetaminophen, 2,4-dichlorophenol, 2-naphthol
and 6-bromo-2-naphthol. Mixtures of more than one enhancer from
those classes mentioned above can also be employed. These enhancer
compounds are thought to act as co-substrates for the peroxidase
and undergo a reversible oxidation.
[0050] Chelating agents include cation complexing agents wherein
the agent can be selected from the group consisting of chelating
agents such as ethylenediaminetetraacetic acid (EDTA),
diethylenetriaminepentaacetic acid (DTPA), or
ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) and their
salts.
[0051] Additives which suppress the generation of chemiluminescence
from the reaction of hydrogen peroxide and N-arylacridancarboxylic
acid derivatives in the absence of peroxidase enzymes are employed
to further improve the utility of the invention. It has also been
found that certain surfactants including anionic surfactants such
as sodium dodecyl sulfate (SDS), cationic surfactants and nonionic
surfactants such as polyoxyethylenated alkylphenols,
polyoxyethylenated alcohols, polyoxyethylenated ethers,
polyoxyethylenated sorbitol esters and the like improve the
sensitivity of detection of the peroxidase enzyme in assays of the
present invention by providing a larger chemiluminescence signal
and a better signal to background ratio. The improvement occurs
through minimizing the background chemiluminescence in the absence
of added peroxidase, possibly due to a slowing of the autoxidative
decomposition of the acridan derivative. The preferred amounts of
the various components of a composition of the present invention
are set forth below.
1TABLE 1 Reagent Compositions for Producing Chemiluminescence with
a Peroxidase Enzyme. Acridan 0.01-10 mM Enhancer 0.001-10 mM
Surfactant 0.005-5% Peroxide 0.01-10 mM Chelating agent 0.01-5
mM
[0052] This solution is contacted with the peroxidase enzyme which
can either be in solution or adhered to a solid support. Optimum
concentrations of reagents must be determined individually for each
composition. The concentration of acridan compound and enhancer in
particular should be optimized with care for each case in order to
produce the maximum enhancement of light emission. The detection
reaction can be performed over a range of temperatures including at
least the range 20-40.degree. C. Detection can be conveniently and
advantageously carried out at ambient temperature.
[0053] Light emitted by the present method can be detected by any
suitable known means such as a luminometer, x-ray film, high speed
photographic film, a CCD camera, a scintillation counter, a
chemical actinometer or visually. Each detection means has a
different spectral sensitivity. The human eye is optimally
sensitive to green light, CCD cameras display maximum sensitivity
to red light, x-ray films with maximum response to either UV to
blue light or green light are available. Choice of the detection
device will be governed by the application and considerations of
cost, convenience, and whether creation of a permanent record is
required.
[0054] Another aspect of the present invention is a method for
detecting a peroxidase enzyme or an analyte linked to or capable of
being linked to a peroxidase enzyme in an assay procedure by a
chemiluminescent reaction. The method comprises reacting an acridan
of formula I with a peroxide and the peroxidase enzyme to produce
chemiluminescence, detecting the amount of chemiluminescence and
relating the amount of chemiluminescence to the amount of the
analyte or enzyme.
[0055] The present invention also relates to a method for detecting
hydrogen peroxide in an assay procedure by a chemiluminescent
reaction. The method comprises reacting hydrogen peroxide and a
peroxidase enzyme with an acridan of formula I to produce
chemiluminescence and relating the amount of chemiluminescence to
the amount of peroxide.
[0056] Further, the invention relates to the use of the method to
detect and quantify various biological molecules which are bound to
this enzyme by chemical bonds or through physical interactions.
Further, the invention relates to the use of the method to detect
and quantify various biological molecules which have been or are
capable of being bound to peroxidase, for example, by using a
biotin-labeled analyte and streptavidin-peroxidase conjugate. Other
high affinity binding pairs well known in the art such as
fluorescein and anti-fluorescein, digoxigenin and anti-digoxigenin
or complementary nucleic acid sequences can also be readily
employed as a means of linking a peroxidase enzyme to an analyte
for the purpose of practicing this invention. The intensity of the
resulting chemiluminescence provides a direct measure of the
quantity of labeled organic or biological molecule. For example,
the method can be used to detect haptens, antigens and antibodies
by the technique of immunoassay, proteins by Western blotting, and
DNA and RNA by Southern and Northern blotting, respectively. The
method can also be used to detect DNA in DNA sequencing
applications.
[0057] The method can additionally be used to detect hydrogen
peroxide generated by enzymes such as cholesterol oxidase, glucose
oxidase, glucose-6-phosphate dehydrogenase, galactose oxidase,
galactose-6-phosphate dehydrogenase, and amino acid oxidase. The
method can also therefore be used as a means to detect the enzymes
mentioned above which generate hydrogen peroxide.
[0058] In a further embodiment the methods of the present invention
can be used for the detection and measurement of enzyme inhibitors.
Inhibitors can act reversibly or irreversibly by denaturing the
enzyme, irreversibly binding to the enzyme, or by reversibly
binding to the enzyme and competing with substrate. For example,
peroxidase inhibitors include cyanide, sulfide and high
concentrations of hydrogen peroxide. Further it is recognized that
some substances are only inhibitory at certain concentrations and
can be only partially inhibitory. In a method of detecting an
enzyme inhibitor according to the present invention, a compound of
formula (1) is reacted with a peroxidase and a peroxide in the
presence and in the absence of the inhibitor and the results are
compared to determine the presence or amount of the inhibitor. The
effect of the inhibitor can decrease the light intensity, slow the
rate of rise of light intensity or cause a delay period before
light emission begins or any combination of these effects.
EXAMPLES
Synthesis of Acridan Derivatives
[0059] Acridancarboxylic acid derivatives 1a-q were synthesized in
accordance with the methods of the present invention.
2TABLE 2 12 N-Arylacridancarboxylic acid derivatives Prepared.
Compound Ar X Z R.sup.1 1a C.sub.6H.sub.5 H O C.sub.6H.sub.5 1b
C.sub.6H.sub.5 H S 4-Cl--C.sub.6H.sub.4 1c C.sub.6H.sub.5 H O
2,3,6-C.sub.6H.sub.2F.sub.3 1d C.sub.6H.sub.5 H S 2-C.sub.10H.sub.7
1e 2,6-di-Me-C.sub.6H.sub.3 H S 4-Cl--C.sub.6H.sub.4 1f
2,6-di-Me-C.sub.6H.sub.3 H S 2-C.sub.10H.sub.7 1g
4-MeO-C.sub.6H.sub.4 H S 4-Cl--C.sub.6H.sub.4 1h 2-C.sub.10H.sub.7
(naphthyl) H S 4-Cl--C.sub.6H.sub.4 1i 2-C.sub.10H.sub.7 H S
2-C.sub.10H.sub.7 1j 4-C.sub.6H.sub.5--C.sub- .6H.sub.4 H S
4-Cl-C.sub.6H.sub.4 1k 4-C.sub.6H.sub.5--C.sub.6H.sub- .4 H O
2,3,6-C.sub.6H.sub.2F.sub.3 1l C.sub.6H.sub.5 H S
2,6-di-Me--C.sub.6H.sub.3 1m benzothiazol-2-yl H O
2,3,6-C.sub.6H.sub.2F.sub.3 1n benzothiazol-2-yl H S
4-Cl--C.sub.6H.sub.4 1o C.sub.6H.sub.5 H O CH.sub.3 1p
C.sub.6H.sub.5 OCH.sub.3 S 4-Cl--C.sub.6H.sub.4 1q C.sub.6H.sub.5
OCH.sub.3 O 2,3,6-C.sub.6H.sub.2F.sub.3
Example 1
Compound 1a
[0060] A 1 L round bottom flask was charged with 16.80 g of lithium
aluminum hydride (0.4427 mol) and 300 mL of diethyl ether. After
hydrogen evolution had ceased, 25.00 g of 9(10H)-acridone (0.1281
mol) was added to the flask in portions with 300 mL toluene. The
grey slurry was then purged with argon and the reaction was heated
to reflux. The mixture was allowed to stir for 3 h when the
resultant mixture, containing a yellow solid, was cooled to
0.degree. C. and treated dropwise with 16.8 mL of water via
addition funnel. Hydrogen evolution occured and 100 mL additional
diethyl ether was added to the mixture. Once the bubbling ceased,
16.8 mL of a solution of 15% NaOH (weight %) was added dropwise to
the mixture, followed by 50.4 mL of water. The resultant granular
precipitate was isolated by suction filtration and washed throughly
with 2 L of CH.sub.2Cl.sub.2. The solid was discarded and the
organic filtrate was concentrated to dryness. The residue was
chromatographed on silica gel with CH.sub.2Cl.sub.2 to afford 21.09
g of the acridan as a white solid (90.8%).
[0061] .sup.1H-NMR: (CDCl.sub.3) .delta.4.05 (s, 2H), 5.94 (s, 1H),
6.66 (d, 2H), 6.85 (t, 2H), 7.05-7.11 (m, 4H).
[0062] 18.12 g of acridan (0.10 mol), 17.30 g of bromobenzene (0.11
mol), 14.40 g of sodium t-butoxide (0.15 mol), 1.12 g of
Pd(OAc).sub.2 (5 mmol), and 0.81 g of tri-t-butylphosphine (4 mmol)
were taken up in 100 mL of dry toluene and this reaction mixture
was allowed to stir under inert atmosphere. The exothermic reaction
heated to reflux, then cooled to room temperature and was allowed
to stir for 1 h. TLC analysis showed the reaction had gone to
completion, so the reaction mixture was passed through a short plug
of silica gel, which was subsequently washed with 1 L of
CH.sub.2Cl.sub.2. The filtrate was concentrated to dryness and
purified by column chromatography to afford 25.21 g of the
N-phenylacridan (98.0%).
[0063] .sup.1H-NMR: (CDCl.sub.3) .delta.4.23 (s, 2H), 6.18 - 6.20
(d, 2H), 6.82-6.96 (m, 4H), 7.13 - 7.15 (d, 2H), 7.32 - 7.34 (d,
2H), 7.47 - 7.52 (t, 1H), 7.59 - 7.64 (t, 2H).
[0064] A solution of N-phenylacridan (14.15 g, 55 mmol) in 200 mL
of dry THF under inert atmosphere was cooled to -78.degree. C. and
33.0 mL of 2.5 M n-butyllithium in hexanes (82.5 mmol) was added
dropwise over 20 minutes. The solution was stirred at -78.degree.
C. for 30 minutes and then warmed to room temperature over 35
minutes. The reaction mixture was again cooled to -78.degree. C.
and 350 g of crushed dry ice was added causing a precipitate to
form. The reaction was allowed to slowly warm to room temperature
causing the reaction to become homogeneous. Stirring under argon
was continued at room temperature. The resultant precipitate was
collected by suction filtration. The solid was dissolved in 100 mL
of water, which was acidified to pH 3 with 2N HCl and extracted
with 4.times.200 mL of ethyl acetate. The combined organics were
dried over sodium sulfate and concentrated to dryness to afford
8.72 g of crude product. The reaction mixture filtrate was also
concentrated to dryness to afford a solid that was taken up in 300
mL of water. The aqueous solution was acidified, extracted with
3.times.200 mL of methylene chloride and the combined organics were
dried over sodium sulfate and concentrated to afford a second crop
of 3.44 g of N-phenylacridan-9-carbo- xylic acid (73.4%).
[0065] .sup.1H-NMR: (CDCl.sub.3) .delta.5.12 (s, 1H) , 6.30 - 6.33
(d, 2H) , 6.88 - 6.93 (t, 2H), 7.02 - 7.07 (t, 2H), 7.25 - 7.34 (m,
4H), 7.47 - 7.52 (t, 1H), 7.58 - 7.63 (t, 2H).
[0066] To a solution of 3.00 g of the acid (9.96 mmol) in 100 mL of
dry acetonitrile (MgSO.sub.4) under inert atmosphere was added 2.10
g of carbonyl diimidazole (12.9 mmol). After stirring for 10
minutes, 1.41 g of phenol (14.9 mmol) was added to the reaction
which was allowed to stir for 18 hours. An additional 1.05 g of
carbonyl diimidazole (6.48 mmol) and 0.71 g of phenol (7.55 mmol)
was added to the reaction, which was stirred 72 hours. The reaction
mixture was concentrated in vacuo and the resultant brown oil was
chromatographed on silica gel with 5% ethyl acetate in hexanes to
afford 2.28 g of the ester (1a) as a brown oil. (60.6%).
[0067] .sup.1H-NMR: (CDCl.sub.3) .delta.5.37 (s, 1H), 6.34 - 6.37
(d, 2H), 6.92 - 6.96 (m, 4H), 7.04 - 7.10 (m, 2H), 7.14 - 7.19 (t,
1H), 7.26 - 7.52 (m, 6H), 7.58 - 7.63 (t, 2H).
Alternate Synthesis of 1a by Reaction with Phenyl Chloroformate
[0068] 1.00 g of N-phenylacridan (3.9 mmol) in 30 mL of anhydrous
THF was cooled to 0.degree. C. under inert atmosphere and treated
with a solution of 2.3 mL (3.8 mmol) of 2.5 M n-butyllithium in 10
mL of hexanes dropwise over 15 min. The resultant dark brown
solution was warmed to room temperature over 15 min, followed by
rapid addition of 1.46 mL of phenyl chloroformate (0.0117 mol).
After 30 min, TLC analysis showed several products had formed.
Additional stirring of the reaction mixture led to no further
change by TLC, so the reaction mixture was concentrated in vacuo to
a brown oil and separated on a column of silica gel eluted with
5-10% ethyl acetate in hexanes.
Example 2
Compound 1b
[0069] To a solution of 6.09 g of N-phenylacridan-9-carboxylic acid
(20.2 mmol), prepared as described in Example 1, in 100 mL of dry
acetonitrile (MgSO4) under inert atmosphere was added 4.26 g of
carbonyl diimidazole, CDI (26.3 mmol). After stirring for 10 min,
4.38 g of 4-chlorothiophenol (30.3 mmol) was added to the reaction
which was allowed to stir for 1 h. The reaction mixture was
concentrated in vacuo and the resultant brown solid was
chromatographed on silica gel with 5%-20% ethyl acetate in hexanes
to afford a yellow solid that was recrystallized in ethyl acetate
and hexanes to afford 4.40 g of the thioester as a white solid.
(55.3%).
[0070] .sup.1H-NMR: (CDCl.sub.3) .delta.5.30 (s, 1H), 6.35 - 6.38
(d, 2H), 6.93 - 6.98 (t, 2H), 7.08 - 7.13 (t, 2H), 7.19 -7.22 (d,
2H), 7.29 - 7.34 (m, 4H), 7.38 - 7.41 (d, 2H), 7.50 - 7.55 (t, 1H),
7.61 - 7.66 (t, 2H).
Example 3
Compound 1c
[0071] A solution of N-phenylacridan-9-carboxylic acid (0.36 g, 1.2
mmol) and 252 mg of CDI (1.55 mmol) was prepared in 30 mL of dry
acetonitrile under inert atmosphere. After stirring for 15 min,
265.4 mg of 2,3,6-trifluorophenol (1.8 mmol) was added and stirring
continued for 1 h. The reaction mixture was concentrated in vacuo
and the crude product was chromatographed on a column of silica gel
with 5% ethyl acetate in hexanes to afford 171.3 mg of the ester
product as an oily residue. (39.7%).
[0072] .sup.1H-NMR: (CDCl.sub.3) .delta.5.51 (s, 1H), 6.34 - 6.37
(d, 2H), 6.81 - 6.89 (m, 1H), 6.93 - 7.00 (m, 3H), 7.06 - 7.11 (m,
2H), 7.34 - 7.41 (m, 4H), 7.48 - 7.53 (t, 1H), 7.59 - 7.64 (t,
2H).
Example 4
Compound 1d
[0073] A solution of 3.00 g of N-phenylacridan-9-carboxylic acid
(10 mmol) and 2.10 g of CDI (12.9 mmol) was prepared in 100 mL of
dry acetonitrile under inert atmosphere. After stirring for 10 min,
2.39 g of 2-naphthalenethiol (14.9 mmol) was added to the reaction
and the solution was allowed to stir for 1 h. TLC analysis showed
product had formed, so the reaction mixture was filtered and the
filtrate was concentrated in vacuo to afford a gummy brown solid.
The solid was resuspended in diethyl ether, filtered and purified
by column chromatography, eluting with 25-100% CH.sub.2Cl.sub.2 in
hexanes to afford 2.16 g of pure thioester product as a white
solid. (48.9%).
[0074] .sup.1H-NMR: (CDCl.sub.3) .delta.5.34 (s, 1H), 6.37 - 6.39
(d, 2H), 6.94 - 6.99 (t, 2H), 7.09 - 7.14 (t, 2H), 7.291 - 7.33 (d,
1H), 7.36 - 7.55 (m, 7H), 7.61 - 7.66 (t, 2H), 7.74 - 7.83 (m,
4H).
Example 5
Compound 1e
[0075] A mixture containing 5.0 g of acridan (0.027 mol), 186 mg of
Pd(OAc).sub.2 (8.2.times.10.sup.-4 mol), 186 mg of
tri-t-butylphosphine (8.2.times.10.sup.-4 mol), 3.97 g of sodium
t-butoxide (0.041 mol), and 6.12 g of 1-bromo-2,6-dimethylbenzene
(0.033 mol) in 70 mL of dry toluene was stirred under argon at room
temperature for 16 h. Since TLC analysis still showed the presence
of starting material, the reaction mixture was heated at 85.degree.
C. for 3 h. The reaction mixture was cooled, filtered and the
precipitate was washed with diethyl ether. The filtrate was passed
through a 2" plug of celite and concentrated to a brown solid. The
crude product was chromatographed on silica gel with 5% - 20%
CH.sub.2Cl.sub.2 in hexanes to afford 5.1 g (66.2%) of
N-(2,6-dimethylphenyl)acridan.
[0076] .sup.1H-NMR: (CDCl.sub.3) .delta.2.06 (s, 6H), 4.32 (s, 2H),
6.01-6.03 (d, 2H), 6.81-7.29 (m, 10H) .
[0077] A solution of 4.28 g of N-(2,6-dimethylphenyl)acridan (15
mmol) in 100 mL of THF under argon was cooled to -78.degree. C.
After 10 min, 7.6 mL of n-BuLi (18.7 mmol) was added. The black
solution stirred 30 min and then the temperature was raised to
0.degree. C. for 40 min. The reaction was again cooled to
-78.degree. C. and 270 g of crushed dry ice was added to the
reaction mixture to form a yellow solution with a white precipitate
that dissolved once the reaction was warmed to room temperature.
The reaction was stirred for 16 h at room temperature, and then
concentrated and taken up in a mixture of water and diethyl ether.
The aqueous portion was acidified with HCl to afford a white
precipitate, which was filtered and dried to give 4.8 g of
N-(2,6-dimethylphenyl)-acri- dan-9-carboxylic acid as a white solid
(97%).
[0078] .sup.1H-NMR: (DMSO-d.sub.6) .delta.1.83 (s, 3H), 2.03 (s,
3H), 5.12 (s, 1H), 5.98-6.01 (d, 2H), 6.87-7.37 (m, 10H), 12.47
(bs, 1H).
[0079] A solution of the preceding acid (6.1 mmol) and 1.03 g of
CDI (6.4 mmol) in 30 mL of THF was stirred for 10 min under inert
atmosphere when 1.1 g of p-chlorothiophenol (7.6 mmol) was added to
the reaction mixture. After 20 min, the reaction mixture was
chromatographed on silica gel in 5% - 40% CH.sub.2Cl.sub.2 in
hexanes to afford 2.1 g of white solid (75.5%). .sup.1H-NMR
analysis confirmed the white solid as the desired thioester.
[0080] .sup.1H-NMR: (CDCl.sub.3) .delta.1.92 (s, 3H), 2.22 (s, 3H),
5.37 (s,1H), 6.16-6.19 (d, 2H),6.92-7.37 (m, 13H).
Example 6
Compound 1f
[0081] A solution of 2.0 g of
N-(2,6-dimethylphenyl)-acridan-9-carboxylic acid (6.1 mmol)
prepared as described in Example 5, and 1.03 g of CDI (6.4 mmol) in
30 mL of THF was stirred for 10 min under inert atmosphere when
1.21 g of 2-naphthalenethiol (7.6 mmol) was added to the reaction
mixture. The reaction was stirred at room temperature for 2 h. The
reaction mixture was then chromatographed on silica gel with 30%
CH.sub.2Cl.sub.2 in hexanes to afford 1.4 g of the desired
thioester (49%).
[0082] .sup.1H-NMR: (CDCl.sub.3) .delta.1.94 (s, 3H), 2.26 (s,
(3H), 5.42 (s, 1H), 6.17 - 6.20 (d, 2H), 6.94 - 6.99 (t, 2H), 7.09
- 7.15 (t, 2H), 7.26 - 7.49 (m, 8H), 7.74 - 7.82 (m, 4H).
Example 7
Compound 1g
[0083] A mixture of 10.00 g of acridan (55.2 mmol), 0.260 g of
Pd(OAc).sub.2 (1.16 mmol), 0.223 g of tri-t-butylphosphine (1.1
mol), 8.75 g of sodium t-butoxide (91 mmol), and 6.90 mL of
bromoanisole (60.7 mmol) in 55 mL of dry toluene was purged with
argon and the black solution was allowed to stir at room
temperature. After 10 min, 20 mL additional toluene was added to
the thick solution and the reaction was allowed to stir for 1 h.
The reaction mixture was filtered and the collected precipitate was
washed with 50 mL of CH.sub.2Cl.sub.2, followed by 50 mL of THF.
The precipitate was then triturated in water for 1 h, collected by
suction filtration, and allowed to air dry. NMR analysis showed
11.5 g of N-(4-methoxyphenyl)acridan was isolated in pure form
(72.5%).
[0084] .sup.1H-NMR: (CDCl.sub.3) .delta.3.90 (s, 3H), 4.22 (s, 2H),
6.23 (d, 2H), 6.84 (t, 2H), 6.95 (t, 2H), 7.12 (m, 4H), 7.24 (d,
2H).
[0085] N-(4-Methoxyphenyl)acridan (9.23 g, 32.1 mmol) in 220 mL of
dry THF was cooled to -78.degree. C. under inert atmosphere and
treated dropwise with a solution of 20 mL of 2.5 M n-butyllithium
(48.2 mmol). The reaction mixture was warmed to room temperature to
afford a dark brown solution which was stirred for 20 min. The
reaction was again cooled to -78.degree. C., and 350 g of dry ice
was added to the solution. The reaction was warmed to room
temperature and was stirred for 12 h. The resultant slurry was
separated by suction filtration, and the solid was washed with 30
mL of THF and dissolved in 300 mL of H.sub.2O. The aqueous solution
was acidified to pH 2 and a dark oil separated out of the milky
white solution. The solution was decanted, extracted with
3.times.100 mL of ethyl acetate, and the combined organics were
dried over sodium sulfate and concentrated to dryness to afford 1.8
g of the desired product. The dark oil was extracted into ethyl
acetate and the organic layer was dried over sodium sulfate and
concentrated to dryness to afford 5.95 g of a solid containing 1:1
carboxylic acid product:impurity. Total yield: 7.75 g.
[0086] .sup.1H-NMR: (CDCl.sub.3) .delta.3.90 (s, 3H), 5.11 (s, 1H),
6.35 (d, 2H), 6.91 (t, 2H), 7.05 (t, 2H), 7.10 (d, 2H), 7.24 (d,
4H).
[0087] To a solution of 0.92 g of the acid (2.78 mmol) in 20 mL of
dry THF under inert atmosphere was added 0.59 g of CDI (3.62 mmol).
After stirring for 10 min, 0.60 g of 4-chlorothiophenol (4.18 mol)
was added to the reaction which was allowed to stir for 3 h. The
reaction mixture was chromatographed on silica gel with 5% ethyl
acetate in hexanes to afford 310 mg of the thioester. (24%).
[0088] .sup.1H-NMR: (CDCl.sub.3) .delta.3.89 (s, 3H), 5.28 (s, 1H),
6.40 (d, 2H), 6.93 (t, 2H), 7.07-7.13 (m, 4H), 7.18 (d, 2H),
7.26-7.32 (m, 6H).
Example 8
Compound 1h
[0089] 10.02 g of acridan (55.3 mmol), 12.67 g of
2-bromonaphthalene (61.2 mmol), 8.02 g of sodium t-butoxide (83.5
mmol), 0.25 g of Pd(OAc).sub.2 (1.11 mmol), and 0.19 g of
tri-t-butylphosphine (0.94 mmol) were taken up in 100 mL of dry
toluene and the reaction mixture was allowed to stir for 18 h under
inert atmosphere. TLC and NMR analysis showed starting material
present, so 0.19 g of tri-t-butylphosphine (0.94 mol) and
Pd(OAc).sub.2 were added to the reaction mixture, causing an
exothermic reaction that warmed the reaction mixture above room
temperature. After stirring an additional 2 h, TLC analysis showed
the reaction had gone to completion. The reaction mixture was
concentrated to a black oil and chromatographed on silica gel with
4% ethyl acetate in hexanes to afford 14.02 g of a white solid. NMR
analysis confirmed the desired N-(2-naphthyl)acridan was isolated
in 82.6% yield.
[0090] .sup.1H-NMR: (CDCl.sub.3) .delta.4.27 (s, 2H), 6.22 (d, 2H),
6.82-6.94 (m, 4H), 7.17 (d, 2H), 7.39 (dd, 1H), 7.52-7.64 (m, 2H),
7.87-7.90 (m, 2H), 7.97 (d, 1H), 8.10 (d, 1H).
[0091] N-(2-naphthyl)acridan (9.00 g, 29.3 mmol) in 200 mL of dry
THF was cooled to -78.degree. C. under inert atmosphere and 18 mL
of 2.5 M n-BuLi in hexanes (45 mmol) was added. The solution was
stirred at -78.degree. C. for 30 min and then warmed to room
temperature. The reaction mixture was added to a separate,
stoppered flask containing solid CO2 via syringe and was allowed to
slowly warm to room temperature. The reaction mixture was then
concentrated in vacuo to afford a yellow solid which was washed
with acetone to remove impurities. The remaining white solids were
dissolved in water, acidified to pH 2-3 and extracted with ethyl
acetate. The combined organics were dried over magnesium sulfate
and concentrated to a brown gummy solid (8.21 g). NMR analysis
showed the solid contained both the desired acid and some impurity.
This product was taken directly to the esterification reaction
without further purification.
[0092] To a solution of crude N-(2-naphthyl)acridan-9-carboxylic
acid (8.21 g , ca. 23.4 mmol) in 80 mL of dry DMF under inert
atmosphere was added 5.31 g of CDI (32.7 mmol). After stirring for
15 min, 5.41 g of 4-chlorothiophenol (37.4 mmol) was added to the
reaction which was allowed to stir for 18 h. The reaction mixture
was concentrated in vacuo and chromatographed on silica gel with 3%
ethyl acetate in hexanes to afford an impure, off-white solid (6.57
g). This solid was purified again by column chromatography (silica
gel, 2 % ethyl acetate in hexanes) to afford 4.12 g of a solid
containing 20-25% of the desired product by NMR analysis, and 0.96
g of a solid containing 75-80% of the desired product by NMR
analysis. Each of these solids were rechromatographed in 5% ethyl
acetate in hexanes to afford a combined amount of 1.04 g of the
desired ester as a white solid (24.4% over two steps).
[0093] .sup.1H-NMR: (CDCl.sub.3) .delta.5.34 (s, 1H), 6.39 (d, 2H),
6.92-7.00 (m, 2H), 7.03-7.12 (m, 2H), 7.23-7.26 (m, 1H), 7.30-7.40
(m, 4H), 7.45 (dd, 1H), 7.54-7.66 (m, 2H), 7.88-7.93 (m, 2H), 7.99
(d, 1H) , 8.12 (d, 1H).
Example 9
Compound 1i
[0094] N-(2-naphthyl)acridan-9-carboxylic acid (8.04 g, ca. 22.9
mmol) prepared as described in Example 8 in 50 mL of dry DMF under
inert atmosphere was added 4.89 g of CDI (30.1 mmol). After
stirring for 5 minutes, 5.69 g of 2-naphthalenethiol (35.5 mmol)
was added to the reaction which was allowed to stir for 18 hours.
The reaction mixture was concentrated in vacuo and the residue was
taken up in CH.sub.2Cl.sub.2 and extracted with water. The material
obtained from the extracted CH.sub.2Cl.sub.2 was chromatographed on
silica gel with 10% ethyl acetate in hexanes to afford a crude
solid (5.85 g). This solid was purified again by column
chromatography (silica gel, 5% ethyl acetate in hexanes) to afford
a combined total of 4.40 g of slightly impure product (.about.65%).
One of the fractions containing 1.07 g of material was .about.90%
pure by NMR analysis.
[0095] .sup.1H-NMR: (CDCl.sub.3) .delta.5.38 (s, 1H), 6.40 - 6.42
(d, 2H), 6.96 - 7.00 (t, 2H), 7.06 - 7.12 (t, 2H), 7.33 - 7.61 (m,
8H), 7.76 - 7.80 (m, 7H), 8.07 - 8.13 (m, 1H).
Example 10
Compound 1j
[0096] 10.00 g of acridan (55.2 mmol), 14.15 g of 4-bromo-biphenyl
(60.7 mmol), 7.95 g of sodium t-butoxide (82.7 mmol), 0.25 g of
Pd(OAc).sub.2 (1.10 mmol), and 0.18 g of tri-t-butylphosphine (0.88
mmol) were taken up in 50 mL of dry toluene and this reaction
mixture was allowed to stir under inert atmosphere for 1 h. TLC
analysis showed the reaction had gone to completion, so 300 mL of
CH.sub.2Cl.sub.2 and 100 g of silica gel were added to the reaction
mixture. This mixture was stirred and filtered, and the solid
material was washed with an addition 1.5 L of CH.sub.2Cl.sub.2. The
filtrate was concentrated in vacuo to afford a thick liquid
containing a precipitate. The precipitate was collected by
filtration, washed with hexanes, and dried to afford 13.09 g of the
desired product by .sup.1H-NMR analysis. The mother liquor was
concentrated to a yellow solid which was triturated in hexanes. The
solid was collected by filtration to afford a second crop of
product. The filtrate was again concentrated, triturated with
hexanes and filtered to afford a third crop of product containing
.about.10% impurity by .sup.1H-NMR analysis. The three lots of
product were combined to afford 18.60 g of N-biphenylacridan.
[0097] .sup.1H-NMR: (CDCl.sub.3) .delta.4.25 (s, 2H), 6.28 - 6.31
(d, 2H), 6.84 - 6.89 (t, 2H), 6.95 - 6.99 (t, 2H), 7.15 - 7.17 (d,
2H), 7.38 - 7.43 (m, 3H), 7.48 - 7.53 (t, 2H), 7.68 - 7.71 (d, 2H),
7.82 - 7.85 (d, 2H).
[0098] N-biphenylacridan (13.09 g, 39 mmol) was dissolved in 200 mL
of dry THF under inert atmosphere. The solution was cooled to
-78.degree. C. and 23.6 mL of 2.5 M n-BuLi in hexanes (59 mmol) was
added dropwise over 15 min. The solution was stirred at -78.degree.
C. for 30 min and then warmed to room temperature over 30 min. The
reaction mixture was again cooled to -78.degree. C. and 400 g of
crushed dry ice was added, causing a precipitate to form. The
reaction was allowed to slowly warm to room temperature causing the
reaction to become homogeneous. Stirring under argon was continued
at room temperature and the reaction mixture again developed a
precipitate. After stirring 18 h, the precipitate was collected by
suction filtration and washed with 300 mL of diethyl ether. The
solid was stirred in 500 mL of water, which was then treated with
50 mL of 15% NaOH (aq), but a homogeneous solution was not
obtained. The mixture was acidified to pH 3 with 2N hydrochloric
acid and extracted with 3.times.800 mL of ethyl acetate. The
combined organics were dried over sodium sulfate and concentrated
to dryness to afford a light yellow solid. This solid was
triturated in hexanes, filtered and dried to afford 5.79 g of the
carboxylic acid. The filtrate from the hexanes wash was also
concentrated to dryness to afford a second crop of product. Total
yield was 10.97 g (74.0%).
[0099] .sup.1H-NMR: (CDCl.sub.3) .delta.5.06 (s, 1H), 6.37 - 6.39
(d, 2H), 6.88 - 6.90 (m, 2H), 7.01 - 7.03 (m, 2H), 7.28 - 7.30 (m,
2H), 7.38 - 7.40 (m, 3H), 7.46 - 7.48 (m, 2H), 7.67 - 7.69 (m, 2H),
7.80 - 7.83 (m, 2H).
[0100] To a solution of 2.20 g of the acid (5.8 mmol) in 100 mL of
dry THF (MgSO.sub.4) under inert atmosphere was added 1.23 g of CDI
(7.6 mmol). After stirring for 15 min, 1.26 g of 4-chlorothiophenol
(8.7 mmol) was added to the reaction which was allowed to stir for
18 h. The reaction mixture was concentrated in vacuo and the
resultant brown oil was chromatographed on silica gel with 5% ethyl
acetate in hexanes to afford 1.90 g (64.6%)of the thioester as a
white solid.
[0101] .sup.1H-NMR: (CDCl.sub.3) .delta.5.32 (s, 1H), 6.45 - 6.48
(d, 2H), 6.95 - 6.99 (t, 2H), 7.11 - 7.16 (t, 2H), 7.20 - 7.23 (d,
2H), 7.29 - 7.36 (m, 4H), 7.42 - 7.53 (m, 5H), 7.68 - 7.71 (d, 2H),
7.83 - 7.86 (d, 2H).
Example 11
Compound 1k
[0102] A solution of N-biphenylacridan-9-carboxylic acid (1.50 g, 4
mmol) as prepared in Example 10 in 100 mL of dry THF (MgSO.sub.4)
under inert atmosphere was treated with 0.84 g of CDI (5.2 mmol).
After stirring for 15 min, 0.89 g of 2,3,6-trifluorophenol (6 mmol)
was added to the reaction which was allowed to stir for 90 h. The
reaction mixture was concentrated in vacuo and the resultant brown
residue was chromatographed on silica gel with 5%-10% ethyl acetate
in hexanes to afford a white solid. The impure product was again
chromatographed on silica gel with 50% CH.sub.2Cl.sub.2 in hexanes
to afford 1.30 g (64.7%) of the ester as a white solid.
[0103] .sup.1H-NMR: (CDCl.sub.3) .delta.5.53 (s, 1H) , 6.45 - 6.48
(d, 2H) , 6.81 - 6.89 (m, 1H), 6.94 - 7.05 (m, 3H), 7.09 - 7.15 (t,
2H), 7.38 - 7.53 (m, 7H), 7.68 - 7.70 (d, 2H), 7.82 - 7.84 (d,
2H).
Example 12
Compound 1l
[0104] A solution of 8.00 g of N-phenylacridan-9-carboxylic acid
(26.6 mmol) and 5.60 g of CDI (34.5 mmol) in 300 mL of dry
CH.sub.3CN (MgSO.sub.4) was stirred under inert atmosphere for 1 h.
2,6-dimethylthiophenol (5.50 g, 39.8 mmol) was added to the
reaction which was allowed to stir for 18 h. The reaction mixture
was concentrated in vacuo and the resultant brown solid was
chromatographed on silica gel with 5% ethyl acetate in hexanes to
afford 6.49 g of 2',6'-dimethylthiophenyl
10-phenylacridan-9-carboxylate (58%).
[0105] .sup.1H-NMR: (CDCl.sub.3) .delta.2.09 (s, 6H), 5.31 (s, 1H),
6.33 - 6.36 (d, 2H), 6.90 - 6.95 (t, 2H), 7.04 - 7.17 (m, 5H), 7.32
- 7.34 (d, 2H), 7.39 - 7.41 (d, 2H), 7.49 - 7.54 (t, 1H), 7.60
-7.65 (t, 2H).
Example 13
Compound 1m
[0106] A mixture of 16.2 g of acridan (89.2 mmol), 1.00 g of
Pd(OAc).sub.2 (4.46 mmol), 0.72 g of tri-t-butylphosphine (3.56
mmol), 12.9 g of sodium t-butoxide (134 mmol), and 16.6 g of
2-chlorobenzothiazole (98.1 mmol) in 200 mL of dry toluene was
allowed to stir under inert atmosphere at room temperature for 72
h. The reaction mixture was poured into a mixture of 300 mL of
CH.sub.2Cl.sub.2 and 100 g of silica gel. The mixture was filtered,
washed with an additional 1 L of CH.sub.2Cl.sub.2, and the solids
were discarded. The filtrate was concentrated in vacuo to afford a
brown solid. The solid was triturated in 2.times.200 mL of hexanes,
filtered, washed with an additional 100 mL of hexanes, and dried to
afford a light brown solid containing 2:1 desired product:starting
material by NMR analysis. The solid was taken up in 500 mL of
CH.sub.2Cl.sub.2 and treated with 7.8 mL of oxalyl chloride (89.4
mmol) to convert the starting material to a more polar derivative
to facilitate separation. The mixture stirred 6 h and the resultant
precipitate was collected by fitration, washed with 100 mL
CH.sub.2Cl.sub.2, and analysed by NMR to show the pure HCl salt of
N-benzothiazolylacridan was obtained. This solid was treated with
7.5 mL triethylamine in 500 mL CH.sub.2Cl.sub.2. After stirring 1
h, the mixture was washed with 3.times.400 mL of water, dried over
sodium sulfate, and concentrated to afford 11.47 g of a yellow
solid. NMR analysis confirmed the solid was the pure
N-benzothiazolylacridan (41%).
[0107] .sup.1H-NMR: (CDCl.sub.3) .delta.3.91 (s, 2H), 7.16 - 7.23
(m, 3H), 7.30 - 7.37 (m, 5H), 7.60 - 7.63 (d, 1H), 7.70 - 7.73 (d,
1H), 7.95 - 7.98 (d, 2H).
[0108] N-benzothiazolylacridan (11.47 g, 36.5 mmol) was dissolved
in 500 mL of dry THF under inert atmosphere. The solution was
cooled to -78.degree. C. and 21.9 mL of 2.5 M n-BuLi in hexanes
(54.7 mmol) was added dropwise over 15 minutes. The solution was
stirred at -78.degree. C. for 30 min and then warmed to room
temperature over 30 min. The reaction mixture was again cooled to
-78.degree. C. and 300 g of crushed dry ice was added. The reaction
was allowed to slowly warm to room temperature. After stirring 18
h, the reaction mixture was concentrated in vacuo. The residue was
dissolved in 300 mL of water, acidified to pH 6 with concentrated
HCl. After stirring 15 min, the resultant precipitate was collected
by filtration, washed with 100 mL water, and dried to afford 8.93 g
of solid. The filtrate was acidified to pH 3 with concentrated HCl
and formed a precipitate while stirring at room temperature. The
precipitate was collected by filtration and dried to afford 2.65 g
of solid. NMR analysis of both crops confirmed
N-benzothiazolylacridan-9-carboxylic acid had been isolated (11.58
g, 89%).
[0109] .sup.1H-NMR: (CDCl.sub.3) .delta.4.82 (s, 1H), 7.17 - 7.26
(m, 3H), 7.32 - 7.43 (m, 5H), 7.62 - 7.70 (m, 2H), 7.94 - 7.96 (d,
2H).
[0110] A solution of 2.00 g of the acid (5.58 mmol) and 1.18 g of
CDI (7.25 mmol) in 100 mL of dry THF was stirred under inert
atmosphere for 15 min. 1.24 g of 2,3,6-trifluorophenol (8.37 mmol)
was added to the reaction which was allowed to stir for 18 h. The
reaction mixture was concentrated in vacuo and the resultant yellow
oil was chromatographed twice (silica gel, 5% ethyl acetate in
hexanes and 50% CH.sub.2Cl.sub.2 in hexanes) to afford 0.63 g of a
white solid (23%). .sup.1H-NMR analysis confirmed isolation of
2',3',6'-trifluorophenyl 10-benzothiazolylacridan--
9-carboxylate.
[0111] .sup.1H-NMR: (CDCl.sub.3) .delta.5.29 (s, 1H), 6.71 - 6.80
(m, 1H), 6.86 - 6.97 (m, 1H), 7.24 - 7.28 (m, 3H), 7.38 - 7.43 (t,
3H), 7.48 - 7.51 (d, 2H), 7.64 - 7.67 (d, 1H), 7.84 - 7.93 (m,
3H).
Example 14
Compound 1n
[0112] A solution of 4.00 g of N-benzothiazolylacridan-9-carboxylic
acid (11.2 mmol) and 2.35 g of CDI (14.5 mmol) in 250 mL of dry THF
was stirred under inert atmosphere for 15 min. 2.42 g of
p-chlorothiophenol (16.7 mmol) was added to the reaction which was
allowed to stir for 18 h. The reaction mixture was concentrated in
vacuo and the resultant yellow oil was chromatographed on silica
gel with 50% CH.sub.2Cl.sub.2 in hexanes to afford 2.45 g of a
white solid (45%) . .sup.1H-NMR analysis confirmed isolation of
4'-chlorothiophenyl 10-benzothiazolylacridan-9-car- boxylate.
[0113] .sup.1HMR: (CDCl.sub.3) .delta.5.07 (s, 1H), 7.02 - 7.05 (d,
2H), 7.18 7.21 (d, 2H), 7.23 - 7.30 (m, 3H), 7.39 - 7.45 (m, 5H),
7.67 - 7.70 (d, 1H), 7.85 - 7.93 (m, 3H).
Example 15
Compound 1o
[0114] A solution of 1.00 g of acridan-9-carboxylic acid methyl
ester (4.18 mmol), 0.44 mL of bromobenzene (4.18 mmol), 0.60 g of
sodium t-butoxide (6.27 mmol), 23 mg of Pd(OAc).sub.2 (0.105 mmol),
and 17 mg of tri-t-butylphosphine (0.084 mmol) in 15 mL of toluene
was stirred under inert atmosphere at room temperature. After 2.5
h, 5 g of silica gel in 15 mL of CH.sub.2Cl.sub.2 was added to the
reaction mixture. The slurry was filtered, washed with excess
CH.sub.2Cl.sub.2 and the filtrate was concentrated to a thick
solution. 20 mL of hexanes was added to this solution, causing a
white precipitate to form. The precipitate was filtered, washed
with hexanes, and dried to afford 0.96 g of
N-phenylacridan-9-carboxylic acid methyl ester (72%).
[0115] .sup.1H-NMR: (CDCl.sub.3) .delta.3.66 (s, 3H), 5.17 (s, 1H),
6.30 - 6.33 (d, 2H), 6.89 (t, 2H), 7.04 (t, 2H), 7.25 - 7.27 (m,
2H), 7.34 - 7.36 (d, 2H), 7.50 (t, 1H), 7.59 - 7.62 (t, 2H).
Example 16
Compound 1p
[0116] A solution of 60.0 g of o-chlorobenzoic acid (0.38 mol),
60.0 g of potassium carbonate (0.43 mol), 49.8 g of m-anisidine
(0.40 mol), and 1.98 g of copper powder (0.031 mol) in 600 mL of
pentanol was stirred at reflux for 27 h. The reaction mixture was
concentrated in vacuo to afford a black solid. The solid was
dissolved in 600 mL of water and poured into 3 L of water
containing 300 mL of concentrated HCl. The resultant black
precipitate was collected by filtration, washed with 2 L of water
and dried to afford 91.25 g of solid N-(2-carboxyphenyl)anisidine
(98%).
[0117] .sup.1H-NMR: (CD.sub.3OCD.sub.3) .delta.3.80 (s, 3H), 6.65 -
6.68 (d, 1H), 6.78 - 6.86 (m, 3H), 7.21 - 7.44 (m, 3H), 8.01 - 8.04
(d, 1H), 9.6 (s, 1H).
[0118] An solution of 90.25 g of the acid (0.37 mol) in 250 mL of
POCl.sub.3 (exothermic) was stirred for several minutes, then
heated to reflux and allowed to stir 6 h. The reaction mixture was
cooled to room temperature and concentrated in vacuo. The brown
residue was dissolved in 1 L of CH.sub.2Cl.sub.2. A 2 L solution of
5% NH.sub.4OH in water was slowly added with rapid stirring. Once
the mixture stopped bubbling, the organic layer was separated,
dried over sodium sulfate, and concentrated to a dark brown solid.
The solid was triturated in 1 L of hexanes, collected by
filtration, and dried to afford 94.34 g of a yellow/brown solid
3-methoxy-9-chloroacridine (104%).
[0119] .sup.1H-NMR: (CDCl.sub.3) .delta.4.02 (s, 3H), 7.29 - 7.33
(d, 1H), 7.44 - 7.45 (S, 1H), 7.56 - 7.61 (m, 1H), 7.77 - 7.82 (t,
1H), 8.14 - 8.17 (d, 1H), 8.30 - 8.33 (d, 1H), 8.38 - 8.41 (d,
1H).
[0120] A solution of 93.34 g of 3-methoxy-9-chloroacridine (0.38
mol) in 2 L of 5 N HCl was stirred at reflux for 40 h. The reaction
mixture was cooled to room temperature and the precipitate was
collected by suction filtration, washed with 1 L of water, and
dried. The solid was stirred in 1 L of methanol and the insoluble
precipitate was collected by filtration to afford 15.3 g of the
desired 3-methoxyacridine. The filtrate was concentrated in vacuo
and chromatographed on silica gel in 20% methanol in
CH.sub.2Cl.sub.2 to afford a dark brown solid. The solid was
triturated in 200 mL of methanol and the insoluble precipitate was
collected by filtration to afford a second crop of product. The
filtrate was concentrated to a thick solution, and a third crop of
product was collected by filtration. The second and third crop of
product were combined to give 2.6 g of 3-methoxyacridone, for a
combined total of 17.9 g (21%).
[0121] .sup.1H-NMR: (CDCl.sub.3) .delta.3.93 (s, 3H), 6.80 - 6.87
(m, 2H), 7.18 - 7.23 (t, 1H), 7.43 - 7.46 (d, 1H), 7.58 - 7.64 (t,
1H), 8.28 - 8.31 (d, 1H), 8.35 - 8.38 (d, 1H), 11.0 (s, 1H).
[0122] To a stirred solution of 15.29 g of 3-methoxyacridone (67.9
mmol) in 200 mL of diethyl ether was added 7.73 g of LiAlH.sub.4
slowly portionwise. Once evolution of H.sub.2 ceased, 200 mL of dry
toluene was added under inert atmosphere and the reaction was
stirred at reflux for 2 h. The reaction mixture was cooled to
0.degree. C. and dropwise addition of 8.0 mL of water, 8.0 mL of
15% NaOH (aq) and 24.0 mL of water were performed sequentially. 500
mL of CH.sub.2Cl.sub.2 was added and the resultant precipitate was
filtered, washed with 500 mL of CH.sub.2Cl.sub.2 and discarded. The
filtrate was concentrated in vacuo, resuspended in hexanes, and
again concentrated to afford 14.06 g of 3-methoxyacridan (98%).
[0123] .sup.1H-NMR: (CDCl.sub.3) .delta.3.77 (s, 3H), 4.00 (s, 2H),
5.94 (bs, 1H), 6.23 (s, 1H), 6.41 - 6.44 (d, 1H), 6.63 - 6.66 (d,
1H), 6.82 - 6.87 (t, 1H), 6.98 - 7.10 (m, 3H).
[0124] A mixture of 13.0 g of 3-methoxyacridan (57.7 mmol), 259 mg
of Pd(OAc).sub.2 (1.15 mmol)! 187 g of tri-t-butylphosphine (0.92
mmol), 8.32 g of sodium t-butoxide (86.6 mmol), and 10.9 g of
bromobenzene (69.2 mmol) in 100 mL of dry toluene was stirred under
inert atmosphere at room temperature for 18 h. The reaction mixture
was poured into a mixture of 300 mL of CH.sub.2Cl.sub.2 and 100 g
of silica gel. The mixture was filtered, washed with an additional
500 mL of CH.sub.2Cl.sub.2, and the solids were discarded. The
filtrate was concentrated in vacuo to afford a yellow solid. The
solid was triturated in 100 mL of hexanes, filtered, washed with an
additional 100 mL of hexanes, and dried to afford 13.8 g of
N-phenyl-3-methoxyacridan, by NMR analysis (83%).
[0125] .sup.1H-NMR: (CDCl3) .delta.3.62 (s, 3H), 4.17 (s, 2H), 5.77
(s, 1H), 6.16 - 6.18 (d, 1H), 6.40 - 6.43 (d, 1H), 6.82 - 6.86 (t,
1H), 6.90 - 6.95 (t, 1H), 7.03 - 7.06 (d, 1H), 7.12 - 7.14 (d, 1H),
7.30 - 7.33 (d, 2H), 7.45 - 7.50 (t, 1H), 7.57 -7.62 (t, 2H).
[0126] N-phenyl-3-methoxyacridan (12.5 g, 43.5 mmol) was dissolved
in 500 mL of dry THF under inert atmosphere. The solution was
cooled to -78.degree. C. and 26.1 mL of 2.5 M n-BuLi in hexanes
(65.3 mmol) was added dropwise over 15 min. The solution was
stirred at -78.degree. C. for 30 min and then warmed to room
temperature. The reaction mixture was again cooled to -78.degree.
C. and 300 g of crushed dry ice was added. The reaction was allowed
to slowly warm to room temperature. After stirring 18 h, the
reaction mixture was concentrated in vacuo. The residue was
dissolved in 300 mL of water, acidified to pH 6 with concentrated
HCl, and allowed to stir 15 min. The resultant precipitate was
collected by filtration, washed with 100 mL water, and dried to
afford 2.91 g of product, by NMR analysis. The filtrate was
acidified to pH 4 with concentrated HCl and formed a precipitate
while stirring at room temperature. The precipitate was collected
by filtration, washed with 100 mL of water and dried to afford a
second crop of product. A third and fourth crop of product were
isolated by acidifying and filtering in the same manner, to afford
a total of 10.97 g of product. NMR analysis of each crop confirmed
the crude acid had been isolated (13.9 g, 96%).
[0127] .sup.1H-NMR: (DMSO-d) .delta.3.54 (s, 3H), 4.92 (s, 1H),
5.64 (s, 1H), 6.13 - 6.15 (d, 1H), 6.46 - 6.49 (d, 1H), 6.82 - 6.87
(t, 1H), 6.97 - 7.01 (t, 1H), 7.15 - 7.18 (d, 1H), 7.22 - 7.24 (d,
1H), 7.30 - 7.35 (d, 2H), 7.54 - 7.59 (t, 1H), 7.66 -7.71 (t,
2H).
[0128] A solution of 4.00 g of
3-methoxy-10-phenylacridan-9-carboxylic acid (12.1 mmol) and 2.54 g
of CDI (15.7 mmol) in 200 mL of dry THF was stirred under inert
atmosphere for 15 min. 2.62 g of 4-chlorothiophenol (18.1 mmol) was
added to the reaction which was allowed to stir for 18 h. The
reaction mixture was concentrated in vacuo and the resultant brown
oil was chromatographed on silica gel in 50% CH.sub.2Cl.sub.2 in
hexanes to afford 4.06 g of a gummy white solid (73%). .sup.1H-NMR
analysis confirmed isolation of 4'-chlorothiophenyl
3-methoxy-10-phenylacridan-9-c- arboxylate.
[0129] .sup.1H-NMR: (CDCl.sub.3) .delta.3.66 (s, 3H), 5.24 (s, 1H),
5.90 (s, 1H), 6.32 - 6.35 (d, 1H), 6.51 - 6.54 (d, 1H), 6.91 - 6.96
(t, 1H), 7.06 - 7.12 (t, 1H), 7.19 - 7.33 (m, 6H), 7.37 - 7.40 (d,
2H), 7.49 - 7.54 (t, 1H), 7.60 - 7.65 (t, 2H).
Example 17
Compound 1q
[0130] A solution of 2.00 g of
3-methoxy-10-phenylacridan-9-carboxylic acid (6.04 mmol) and 1.27 g
of CDI (7.85 mmol) in 100 mL of dry THF was stirred under inert
atmosphere for 15 min. 1.34 g of 2,3,6-trifluorophenol (9.05 mmol)
was added to the reaction which was allowed to stir for 18 h. The
reaction mixture was concentrated in vacuo and the resultant brown
oil was chromatographed on silica gel with 50% CH.sub.2Cl.sub.2 in
hexanes to afford a yellow liquid. The liquid was stirred in
diethyl ether and concentrated in vacuo three times to afford a
brown solid. The solid was triturated in 25 mL of diethyl ether,
collected by filtration, and dried to afford 578 mg of
2',3',6'-trifluorophenyl
3-methoxy-10-phenylacridan-9-carboxylate.
[0131] .sup.1H-NMR: (CDCl.sub.3) .delta.3.64 (s, 3H), 5.46 (s, 1H),
5.90 (s, 1H), 6.32 - 6.35 (d, 1H), 6.51 - 6.54 (d, 1H), 6.80 - 6.88
(m, 1H), 6.92 - 7.10 (m, 3H), 7.30 - 7.39 (m, 4H). 7.47 - 7.52 (t,
1H), 7.57 - 7.62 (t, 2H).
[0132] Synthesis of Acridans
[0133] The following additional N-aryl-acridans (3) were
synthesized by Pd-catalyzed coupling of acridan and an aromatic
compound in accordance with the methods of the present invention
and can be converted to N-arylacridancarboxylic acids and acid
derivatives (1) in accordance with the methods of the present
invention.
3TABLE 3 13 Additional N-Arylacridans Synthesized Ar
4-PivO--C.sub.6H.sub.4 4-NO.sub.2--C.sub.6H.sub.4
3-PivO--C.sub.6H.sub.4 3-HO--C.sub.6H.sub.4 4-F--C.sub.6H.sub.4
4-(Benzothiazol-2-yl)-phenyl anthryl 2-pyrenyl 4-CN--C.sub.6H.sub.4
4-PivO--C.sub.6H.sub.4--C.sub.6H.sub.4 2-C.sub.6H.sub.4N
(pyridyl)
Example 18
Synthesis of N-(4'-Pivalovloxyphenyl)Acridan
[0134] A solution of 20.02 g of 4-bromophenol (0.116 mol) in 100 mL
of dry THF was treated with 15.0 mL of pivaloyl chloride (0.126
mol), followed by 35.5 mL of triethylamine (0.255 mol). A large
amount of white solid precipitated so 50 mL of additional THF was
added to the mixture to afford a free-flowing slurry. The reaction
mixture was filtered and the precipitate was washed with 200 mL THF
and then discarded. The filtrate was concentrated in vacuo to a
liquid which solidified when dried under reduced pressure to yield
30.5 g (100%) of 4-bromophenyl pivalate.
[0135] .sup.1H-NMR: (CDCl.sub.3) .delta.1.35 (s, 9H), 6.95 (d, 2H),
7.81 (d, 2H).
[0136] A mixture of 7.05 g of acridan (38.9 mmol), 0.218 g of
Pd(OAc).sub.2 (0.97 mmol), 0.157 g of tri-t-butyl-phosphine (0.78
mmol), 5.61 g of sodium t-butoxide (58.3 mmol), and 10.0 g of
4-bromophenyl pivalate (38.9 mmol) in 60 mL of dry toluene was
stirred at room temperature under inert atmosphere for 18 h. The
reaction mixture was filtered and the collected precipitate was
washed with CH.sub.2Cl.sub.2 and discarded. The filtrate was
concentrated in vacuo and the residue was chromatographed on silica
gel with 5% ethyl acetate in hexanes to afford 8.6 g of pure
N-(4'-pivaloyloxyphenyl)acridan.
[0137] .sup.1H-NMR: (CDCl.sub.3) .delta.1.41 (s, 9H), 4.22 (s, 2H),
6.22 (d, 2H), 6.85 (t, 2H), 6.95 (t, 2H), 7.14 (d, 2H), 7.33 (s,
4H).
Example 19
Synthesis of N-(4'-Nitrophenyl)Acridan
[0138] A mixture of 10.00 g of acridan (55.2 mmol), 0.26 g of
Pd(OAc).sub.2 (0.11 mmol), 0.20 g of tri-t-butylphosphine (0.88
mmol), 7.96 g of sodium t-butoxide (82.8 mmol), and 12.39 g of
1-bromo-4-nitrobenzene (61.3 mmol) in 150 mL of dry toluene (MgSO4)
was stirred at room temperature under inert atmosphere for 18 h.
The reaction mixture was extracted with 1 L of CH.sub.2Cl.sub.2 and
filtered through silica gel. The filtrate chromatographed on silica
gel with 2.5-5% ethyl acetate in hexanes to afford white solid that
was triturated in hexanes, filtered and dried to yield 4.00 g (24%)
of pure N-(4-nitrophenyl)acridan- .
[0139] .sup.1H-NMR: (CDCl.sub.3) .delta.4.15 (s, 2H), 6.35-6.38 (d,
2H), 6.94-7.06 (m, 4H), 7.20-7.26 (d, 2H), 7.50-7.54 (d, 2H),
8.40-8.45 (d, 2H).
Example 20
Synthesis of N-(3'-Pivaloyl-Oxyphenyl)Acridan
[0140] A mixture of 1.0 g of acridan (5.5 mmol), 795.4 mg of sodium
t-butoxide (8.3 mmol), 64.1 mg of Pd(OAc).sub.2 (0.29 mmol), 1.56 g
of 3-bromophenyl pivalate (6.07 mmol), and 47.2 mg of
tri-t-butylphosphine (0.23 mmol) in 30 mL of dry toluene was
allowed to stir at room temperature for 2 h under inert atmosphere.
The reaction mixture was filtered through a plug of silica gel
which was washed with 500 mL of CH.sub.2Cl.sub.2. The filtrate was
concentrated in vacuo and chromatographed on silica gel in 2.5%
ethyl acetate in hexanes to afford a solid, which triturated in
hexanes, filtered and dried to afford 829.9 mg of
N-(3'-pivaloyl-oxyphenyl)acridan (42%).
[0141] .sup.1H-NMR: (CDCl.sub.3) .delta.1.35 (s, 9H), 4.22 (s, 2H),
6.25-6.27 (d, 2H), 6.84-6.88 (t, 2H), 6.95-6.99 (t, 2H), 7.07-7.08
(s, 1H), 7.13-7.15 (d, 2H), 7.20-7.26 (m, 2H), 7.59-7.65 (t,
1H).
Example 21
Synthesis of N-(3'-Hydroxyphenyl)Acridan
[0142] A solution of 621.9 mg of N-(3'-pivaloyloxyphenyl)-acridan
in 20 mL of methanol containing 2.61 g of 1 M NaOH (2.6 mmol) was
refluxed until TLC analysis showed the starting material had been
consumed. The solvent was concentrated and the residue was
dissolved in water and acidified with 1 M hydrochloric acid. The
resulting precipitate was collected via suction filtration, taken
up with methylene chloride, dried over sodium sulfate and
concentrated to a crude oil. The oil was chromatographed on silica
gel in 5% ethyl acetate in hexanes. The purified product was
treated with hexanes and the resultant solid was collected by
filtration to afford 315.8 mg of N-(3'-hydroxyphenyl)acridan
(67%).
[0143] .sup.1H-NMR: (CDCl.sub.3) .delta.4.22 (s, 2H), 6.25-6.28 (d,
2H), 6.80-6.98 (m, 7H), 7.12-7.15 (d, 2H), 7.44-7.50 (t, 1H).
Example 22
Synthesis of N-(4'-Fluorophenyl)Acridan
[0144] 9.0 g of acridan (49.6 mmol), 446 mg of Pd(OAc).sub.2 (2
mmol), 321 mg of tri-t-butylphosphine, 7.16 g of sodium t-butoxide
(74 mmol), and 9.6 g of 1-bromo-4-fluorobenzene (54 mmol) in 90 mL
of dry toluene was stirred under argon at room temperature for 2 h.
TLC showed the absence of starting material. The reaction mixture
was filtered through a Buchner funnel and the solid washed with
CH.sub.2Cl.sub.2. The filtrate was then concentrated in vacuo and
the residue was taken up in CH.sub.2Cl.sub.2 and refiltered. The
filtrate was concentrated to dryness and chromatographed on silica
gel in 5% ethyl acetate in hexanes, changing to CH.sub.2Cl.sub.2.
Fractions containing product were combined and concentrated to
dryness to afford N-(4'-fluorophenyl)acridan.
[0145] .sup.1H-NMR: (CDCl.sub.3) .delta.4.22 (s, 2H), 6.16-6.19 (d,
2H), 6.84-6.88 (t, 2H), 6.93-6.98 (t, 2H), 7.13-7.16, (d, 2H),
7.29-7.31 (d, 4H).
Example 23
Synthesis of N-[4-(Benzothiazol-2-yl)-Phenyl]Acridan
[0146] A solution of 15.0 g of 4-bromobenzoic acid (74 mmol) and
13.9 g of 2-aminothiophenol (110 mmol) in 250 mL of dry toluene was
heated to 45.degree. C. and 10.1 mL of phosphorus trichloride (110
mmol) was slowly added to the reaction mixture via syringe. The
mixture was allowed to reflux for 4 h. The reaction mixture was
cooled to room temperature, filtered and the filtrate was
concentrated under reduced pressure to afford a yellow solid. The
solid was slowly added to 500 mL of saturated aq. NaHCO.sub.3 and
the aqueous solution was extracted with 2.times.200 mL of ethyl
acetate. The combined organic solutions were washed with water
(3.times.), dried over sodium sulfate and concentrated to dryness.
The crude product was purified by column chromatography to afford
6.0 g of 2-(4-bromophenyl)benzothiazole.
[0147] .sup.1H-NMR: (CDCl.sub.3) .delta.7.38-7.43 (t, 1H),
7.48-7.54 (t, 1H), 7.62-7.65 (d, 2H), 7.90-7.93 (d, 1H), 7.96-7.98
(d, 2H), 8.06-8.09 (d, 1H).
[0148] A mixture of 3.74 g of acridan (20 mmol), 69 mg of
Pd(OAc).sub.2 (0.3 mmol), 44 mg of tri-t-butylphosphine (0.22
mmol), 2.88 g of sodium t-butoxide (30 mmol), and 6.0 g of
2-(4-bromophenyl)benzothiazole (20 mmol) in 60 mL of toluene was
stirred at room temperature for 1 h under inert atmosphere,
followed by heating at 50.degree. C. for 1 h. The reaction mixture
was cooled to room temperature, filtered and concentrated under
reduced pressure. The crude product was purified by column
chromatography with 15% ethyl acetate in hexanes. NMR analysis
confirmed that 3.1 g of the N-arylated acridan product was
isolated.
[0149] .sup.1H-NMR: (CDCl.sub.3) .delta.4.25 (s, 2H), 6.27-6.30 (d,
2H), 6.86-6.91 (t, 2H), 6.95-7.00 (t, 2H), 7.16-7.18 (d, 2H),
7.41-7.57 (m, 4H), 7.95-7.97 (d, 1H), 8.11-8.14 (d, 1H), 8.33-8.36
(d, 2H).
Example 24
Synthesis of N-Anthracenylacridan
[0150] A mixture of 10.00 g of acridan (55.2 mmol), 247.7 mg of
Pd(OAc)2 (1.10 mmol), 178.6 mg of tri-t-butylphosphine (0.88 mmol),
7.95 g of sodium t-butoxide (82.7 mmol), and 15.61 g of
9-bromoanthracene (60.7 mmol) in 100 mL of dry toluene was allowed
to stir under inert atmosphere for 1 h. An additional 200 mL of
toluene was added to the mixture and stirring was continued at room
temperature for 30 min. The reaction mixture was poured into a
mixture of 600 mL of CH.sub.2Cl.sub.2 and 100 g of silica gel.
After stirring 15 min, the mixture was filtered, and the solid
material was washed with an additional 600 mL of CH.sub.2Cl.sub.2.
The filtrate was concentrated in vacuo to afford a thick liquid,
which was treated with 500 mL of hexanes. After stirring 15 min,
the precipitate was collected by filtration, washed with 500 mL of
hexanes, and dried. The solid was then triturated in 500 mL of
hexanes for 1 h, filtered and dried to afford 14.39 g of
N-anthracenylacridan by .sup.1H-NMR analysis. The mother liquor was
concentrated to a yellow solid which was triturated in 300 mL of
hexanes for 15 min. The precipitate was collected by filtration,
washed with 200 mL of hexanes and dried to afford 5.40 g of solid.
.sup.1H-NMR analysis showed the second crop of product was slightly
less pure than the first. The two lots were combined to afford
19.79 g of the N-anthracenylacridan (100%).
[0151] .sup.1H-NMR: (CDCl.sub.3) .delta.4.52 (s, 2H), 5.70 - 5.73
(d, 2H), 6.69 - 6.74 (t, 2H), 6.80 - 6.85 (t, 2H), 7.22 - 7.25 (d,
2H), 7.35 - 7.40 (t.sub.7 2H).sub.1 7.47 - 7.51 (t, 2H), 7.91 -
7.94 (d, 2H), 8.12 - 8.15 (d, 2H), 8.63 (s, 1H).
Example 25
Synthesis of N-Pyrenylacridan
[0152] A mixture of 10.00 g of acridan (55.2 mmol), 247.7 mg of
Pd(OAc).sub.2 (1.10 mmol), 178.6 mg of tri-t-butylphosphine (0.88
mmol), 7.95 g of sodium t-butoxide (82.7 mmol), and 17.06 g of
1-bromopyrene (60.7 mmol) in 100 mL of dry toluene was allowed to
stir under inert atmosphere. The exothermic reaction heated to
reflux, causing a precipitate to form. An additional 200 mL of
toluene was added to the mixture and stirring was continued at room
temperature for 1 h. The reaction mixture was poured into a mixture
of 600 mL of CH.sub.2Cl.sub.2 and 100 g of silica gel. The mixture
was filtered, and the solid material was washed with 600 mL of
CH.sub.2Cl.sub.2. The filtrate was concentrated in vacuo to afford
a thick liquid containing a precipitate. The precipitate was
collected by filtration, washed with 300 mL of toluene and 500 mL
of hexanes, and dried to afford 12.69 g of N-pyrenylacridan by
.sup.1H-NMR analysis. The mother liquor was concentrated to a
yellow solid which was triturated in 300 mL of hexanes for 30 min.
The precipitate was collected by filtration, washed with 200 mL of
hexanes and dried to afford 7.46 g of solid. .sup.1H-NMR analysis
showed the second crop of product was slightly less pure than the
first. The two lots were combined to afford 20.15 g of the
N-pyrenylacridan (95.7%).
[0153] .sup.1H-NMR: (CDCl.sub.3) .delta.4.45 (s, 2H), 5.93 - 5.96
(d, 2H), 6.76 - 6.87 (m, 4H) , 7.22 - 7.25 (d, 2H), 7.98 - 8.06 (m,
4H), 8.17 - 8.27 (m, 4H), 8.37 - 8.40 (d, 1H).
Example 26
Synthesis of N-(4'-Cyanophenyl)Acridan
[0154] A solution of 10.00 g of acridan (55.2 mmol), 10.48 g of
4-bromobenzonitrile (57.6 mmol), 7.89 g of sodium t-butoxide (82.1
mmol), 0.18 g of Pd(OAc).sub.2 (0.80 mmol), and 0.1245 g of
tri-t-butylphosphine (0.62 mmol) in 100 mL of dry toluene was
stirred for 18 h at room temperature and 7 h at reflux under inert
atmosphere. 0.12 g of Pd(OAc).sub.2 (0.53 mmol) and 0.09 g of
tri-t-butylphosphine (0.44 mmol) were added to the reaction mixture
which was refluxed for an additional 4 h. The reaction mixture was
concentrated to a thick slurry and was chromatographed on silica
gel with 5% ethyl acetate in hexanes to afford 11.67 g of impure
product. A solution of the combined solids in 100 mL of
CH.sub.2Cl.sub.2 was treated with 5 mL of oxalyl chloride and
allowed to stir for 1 h. The reaction mixture was extracted with
aqueous NaHCO.sub.3, and the organic layer was concentrated to an
orange solid. The solid was purified by chromatography on silica
with 2% ethyl acetate in hexanes to afford 4.81 g of
N-(4'-cyanophenyl)acridan (30.9%).
[0155] .sup.1H-NMR: (CDCl.sub.3) .delta.4.19 (s, 2H), 6.19 - 6.22
(d, 2H), 6.90 - 7.01 (m, 4H), 7.18 - 7.26 (d, 2H), 7.46 - 7.49 (d,
2H), 7.89 - 7.92 (d, 2H).
Example 27
Synthesis of N-(4'-(4'-(4"-Pivaloyl)Biohenyl)-Acridan
[0156] A solution of 20.00 g of N-(4'-(4"-Hydroxybiphenyl))-acridan
(80.3 mmol) in 200 mL of dry THF under inert atmosphere was treated
with 10.4 mL of pivaloyl chloride (84.3 mmol), followed by 24.0 mL
of dry triethylamine (KOH) (169 mmol). The solution was stirred 30
min at room temperature, forming a precipitate. 1.0 mL of pivaloyl
chloride was added to the reaction, and after 15 min, the
precipitate was collected by vacuum filtration, washed with 100 mL
of THF and discarded. The filtrate was concentrated in vacuo and
dried to afford 27.33 g of a white solid. .sup.1H-NMR analysis
confirms that 4-(4'-bromophenyl)phenyl pivalate was isolated
(102%).
[0157] .sup.1H-NMR: (CDCl.sub.3) .delta.1.38 (s, 9H), 7.11 - 7.14
(d, 2H), 7.41 - 7.44 (d, 2H), 7.53 - 7.57 (m, 4H).
[0158] A mixture of 10.00 g of acridan (55.2 mmol), 247.7 mg of
Pd(OAc)2 (1.10 mmol), 178.6 mg of tri-t-butylphosphine (0.88 mmol),
7.95 g of sodium t-butoxide (82.7 mmol), and 20.22 g of
4-(4'-bromophenyl)phenyl pivalate (60.7 mmol) in 60 mL of dry
toluene was placed under inert atmosphere. The exothermic reaction
mixture stirred at room temperature for 1 h, when TLC analysis
showed the starting material was consumed. 600 mL of
CH.sub.2Cl.sub.2 and 100 g of silica gel was added to the reaction
mixture. After stirring 15 min, the mixture was filtered, and the
solid material was washed with an additional 600 mL of
CH.sub.2Cl.sub.2. The filtrate was concentrated in vacuo to afford
a thick liquid, which was treated with 500 mL of hexanes. After
stirring 18 h, the precipitate was collected by filtration, washed
with 300 mL of hexanes, and dried to afford 21.63 g of
N-(4'-(4"-pivaloyl)biphenyl)acridan (90%).
[0159] .sup.1H-NMR: (CDCl.sub.3) .delta.1.40 (s, 9H), 4.24 (s, 2H),
6.27 - 6.30 (d, 2H), 6.84 - 6.89 (t, 2H), 6.94 - 6.99 (t, 2H), 7.14
- 7.21 (m, 4H), 7.37 - 7.40 (d, 2H), 7.67 - 7.70 (d, 2H), 7.79 -
7.81 (d, 2H).
Example 28
Synthesis of N-Pyridylacridan
[0160] A mixture of 10.00 g of acridan (55.2 mmol), 247.7 mg of
Pd(OAc)2 (1.10 mmol), 178.6 mg of tri-t-butylphosphine (0.88 mmol),
7.95 g of sodium t-butoxide (82.7 mmol), and 6.3 mL of
2-bromopyridine (66.2 mmol) in 100 mL of dry toluene was allowed to
stir under inert atmosphere for 18 h. The reaction mixture was
poured into a mixture of 300 mL of CH.sub.2Cl.sub.2 and 100 g of
silica gel with stirring. The mixture was filtered, and the solid
material was washed with 1 L of CH.sub.2Cl.sub.2. The filtrate was
concentrated in vacuo to afford a yellow oil, which was taken up in
hexanes. The semi-solution was again concentrated in vacuo to
afford a yellow solid. The solid was triturated in 100 mL of
hexanes for several hours, filtered and dried to afford 12.50 g of
N-pyridylacridan (88%).
[0161] .sup.1H-NMR: (CDCl3) .delta.4.12 (s, 2H), 6.59 - 6.62 (d,
2H), 6.91 - 6.96 (t, 2H), 7.00 - 7.05 (t, 2H), 7.17 - 7.20 (d, 2H),
7.28 - 7.35 (m, 2H), 7.81 - 7.87 (m, 1H), 8.69 - 8.70 (d, 1H).
Chemiluminescence Measurements
[0162] The experiments in the following examples were performed
using either a Turner Designs TD-20e (Sunnyvale, Calif.)
luminometer fitted with neutral density filter for light
attenuation or a Labsystems Luminoskan (Helsinki, Finland)
luminometer. Data collection, analysis and display were software
controlled.
Example 29
Chemiluminescent Formulations for Peroxidase Detection
[0163] A preferred formulation for producing chemiluminescence
using an N-arylacridancarboxylic acid derivative of the present
invention in a reaction with a peroxidase enzyme contains the
following components:
[0164] (1) 0.01 M tris buffer, pH 8.0
[0165] (2) 0.05 mM acridan compound 1
[0166] (3) 0.5 mM urea peroxide
[0167] (4) 0.1 mM p-phenylphenol
[0168] (5) 0.025% TWEEN 20
[0169] (6) 1 mM EDTA.
[0170] The reagent in final form also contained p-Dioxane 1.25% and
Ethanol 1.25 % used for solubilization purposes.
Example 30
Comparison of the Light Intensity-Time Profile for Detection of HRP
with Compounds 1b-1,p,g
[0171] In separate experiments, 100 .mu.L volumes of solutions of
each of compound 1b-1, 1p and 1q were reacted with 1 .mu.L of a
solution containing 1.4.times.10.sup.-16 mol of HRP in water. The
formulations were prepared according to Example 29. Table 4 gives
the maximum light intensity values obtained under these
conditions.
4TABLE 4 Relative Intensities and other tests on N-arylacridans.
Compound I.sub.max @ Time (min) 1b 344 60 1c 1014 60 1d 78 60 1e
110 30 1f 61 60 1g 35 60 1h 32 60 1i 15 30 1j 11.5 60 1k 100 60 1l
2.3 60 1p 335 30 1q 135 40
Example 31
Improved Chemiluminescent Detection of Proteins by Western Blot
[0172] Chemiluminescent western blots were performed to demonstrate
the utility of N-arylacridan-carboxylic acid derivatives in methods
for detecting peroxidase conjugates of a biomolecule. Detection
reagents containing each of Compounds 1b-1e were used.
.beta.-Galactosidase, mouse anti-.beta.-galactosidase IgG and sheep
anti-mouse IgG peroxidase conjugated Fab fragments were obtained
from Roche Molecular Products (Indianapolis, Ind.). The IgG sample
was centrifuged at 10,000.times.g for 2 min and the supernatant was
used in the immunological reaction.
[0173] SDS-PAGE was performed utilizing the buffer system described
by LaemmLi (U. K. Laemmli, Nature (London), 227, 680 (1970)). The
stacking gel was 4.38% acrylamide:0.12% bisacrylamide. The
separating gel was 6.81% acrylamide: 0.19% bisacrylamide. Following
electrophoresis the gel was equilibrated for 7-8 min with the
transfer buffer which contained 20 mM Tris, 153 mM glycine and 20%
(v/v) methanol. The gel, sandwiched between a sheet of PVDF
transfer membrane and a sheet of chromatography paper 3MM
(Whatman), was placed in the transfer unit (Bio-Rad Laboratories,
Richmond, Calif.). The proteins in the gel were electroeluted for
25 min at 4.degree. C. at a 100 V constant voltage. The membrane
was then placed in 50 mM Tris-HCl buffered saline at pH 7.4 (TBS)
at 4.degree. C. overnight. After this period the membrane was
washed with TBS for 15 min.
[0174] The membrane was treated with 0.05% TWEEN-20 in 50 mM
Tris-HCl buffered saline at pH 7.4 (T-TBS) containing 1% non-fat
powdered milk (NFM) for 1 hr at room temperature. This blocked
membrane was incubated for 75 min at room temperature with primary
antibody (1:1500 dilution of 3.3 .mu.g/mL mouse
anti-g-galactosidase IgG fraction) using T-TBS containing 1%
NFM.
[0175] The membrane was then rinsed and washed twice for 10 min
each with T-TBS at room temperature and then twice for 10 min each
with 1% NFM in T-TBS. The washed membrane was incubated for 30 min
at room temperature with secondary antibody (67 mU/mL, 1:600
dilution of anti-mouse IgG peroxidase conjugated Fab fragments)
using T-TBS containing 1% NFM. The membrane was rinsed and washed
four times for 15 min each with T-TBS.
[0176] The washed membrane was soaked in a detection reagent for 3
min, drained and placed between sheets of transparency film. The
X-ray film was exposed to the membrane for varying periods of time
and developed. The composition of detection reagent solution
containing the acridan compounds was:
5 Tris buffer, pH 8.0 0.025 M Acridan 1b, c, d or e 0.05 mM
p-Iodophenol 2 mM TWEEN 20 0.1% (w/w) Urea peroxide 2.5 mM EDTA 0.5
mM p-Dioxane 1.25% Ethanol 1.25%
[0177] The .beta.-galactosidase standards utilized (5 ng to 5 pg)
were clearly visible down to 5 pg/slot over the background. It was
possible to make several exposures of the membrane during a period
of 24 h as the membrane continued to emit light.
[0178] It is intended that the foregoing description be only
illustrative of the present invention and that the present
invention be limited only by the appended claims.
* * * * *