U.S. patent application number 10/341917 was filed with the patent office on 2004-02-12 for frangible compounds for pathogen inactivation.
This patent application is currently assigned to Cerus Corporation. Invention is credited to Cook, David, Merritt, John E., Nerio, Aileen, Rapoport, Henry, Stassinopoulos, Adonis, Wollowitz, Susan.
Application Number | 20040029897 10/341917 |
Document ID | / |
Family ID | 27485246 |
Filed Date | 2004-02-12 |
United States Patent
Application |
20040029897 |
Kind Code |
A1 |
Cook, David ; et
al. |
February 12, 2004 |
Frangible compounds for pathogen inactivation
Abstract
Compounds and methods for inactivating pathogens in materials
are described, including compositions and methods for inactivating
pathogens in biological materials such as red blood cell
preparations and plasma. The compounds and methods may be used to
treat materials intended for in vitro or in vivo use, such as
clinical testing or transfusion. The compounds are designed to
specifically bind to and react with nucleic acid, and then to
degrade to form breakdown products. The degradation reaction is
preferably slower than the reaction with nucleic acid.
Inventors: |
Cook, David; (Lafayette,
CA) ; Merritt, John E.; (Walnut Creek, CA) ;
Nerio, Aileen; (Fremont, CA) ; Rapoport, Henry;
(Berkeley, CA) ; Stassinopoulos, Adonis; (Dublin,
CA) ; Wollowitz, Susan; (Lafayette, CA) |
Correspondence
Address: |
John W. Tessman
Cerus Corporation
Suite 300
2525 Stanwell Drive
Concord
CA
94520
US
|
Assignee: |
Cerus Corporation
Concord
CA
|
Family ID: |
27485246 |
Appl. No.: |
10/341917 |
Filed: |
January 13, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10341917 |
Jan 13, 2003 |
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09610652 |
Jul 2, 2000 |
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6514987 |
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09610652 |
Jul 2, 2000 |
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09003115 |
Jan 6, 1998 |
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6093725 |
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10341917 |
Jan 13, 2003 |
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08779885 |
Jan 6, 1997 |
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10341917 |
Jan 13, 2003 |
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08779830 |
Jan 6, 1997 |
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60043696 |
Apr 15, 1997 |
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Current U.S.
Class: |
514/255.05 ;
514/338; 514/422; 514/455; 544/405; 546/283.1; 548/525;
549/282 |
Current CPC
Class: |
C07D 219/10 20130101;
B01J 20/28 20130101; C07D 493/04 20130101; A61L 2/0082 20130101;
A61M 1/0209 20130101; C07D 405/12 20130101; C07F 9/64 20130101 |
Class at
Publication: |
514/255.05 ;
514/338; 514/422; 514/455; 544/405; 546/283.1; 548/525;
549/282 |
International
Class: |
A61K 031/497; A61K
031/4433; A61K 031/4025; A61K 031/366; C07D 493/02 |
Goverment Interests
[0003] This invention was made with United States government
support under Grant 1-RO1-HL53380 from the NHLBI. The United States
Government has certain rights in this invention.
Claims
What is claimed is:
1. A compound having the formula: 20wherein at least one of
R.sub.44, R.sub.55, R.sub.3, R.sub.4, R.sub.5, and R.sub.8 is
--V--W--X--E, and the remainder of R.sub.44, R.sub.55, R.sub.3,
R.sub.4, R.sub.5, and R.sub.8 are independently selected from the
group consisting of --H, --R.sub.10, --O--R.sub.10, --NO.sub.2,
--NH.sub.2, --NH--R.sub.10, --N(R.sub.10).sub.2, --F, --Cl, --Br,
--I, --C(.dbd.O)--R.sub.10, --C(.dbd.O)--O--R.sub.10, and
--O--C(.dbd.O)--R.sub.10, where --R.sub.10 is independently H,
--C.sub.1-8 alkyl, --C.sub.1-8 heteroalkyl, -aryl, -heteroaryl,
--C.sub.1-3alkyl-aryl, --C.sub.1-3heteroalkyl-aryl,
--C.sub.1-3alkyl-heteroaryl, --C.sub.1-3heteroalkyl-heteroaryl,
-aryl-C.sub.1-3alkyl, -aryl-C.sub.1-3heteroalkyl,
-heteroaryl-C.sub.1-3al- kyl, -heteroaryl-C.sub.1-3heteroalkyl,
--C.sub.1-3alkyl-aryl-C.sub.1-3 alkyl,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 alkyl,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 alkyl,
--C.sub.1-3alkyl-aryl-C.sub.- 1-3 heteroalkyl,
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 alkyl,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl; V is
independently --R.sub.11--, --NH--R.sub.11-- or
--N(CH.sub.3)--R.sub.11--- , where --R.sub.11-- is independently
--C.sub.1-8alkyl-, --C.sub.1-8heteroalkyl-, -aryl-, -heteroaryl-,
--C.sub.1-3alkyl-aryl-, --C.sub.1-3heteroalkyl-aryl-,
--C.sub.1-3alkyl-heteroaryl-, --C.sub.1-3heteroalkyl-heteroaryl-,
-aryl-C.sub.1-3alkyl-, -aryl-C.sub.1-3 heteroalkyl-,
-heteroaryl-C.sub.1-3alkyl-, -heteroaryl-C.sub.1-3heteroalkyl-,
--C.sub.1-3alkyl-aryl-C.sub.1-3 alkyl-,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 alkyl-,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 alkyl-,
--C.sub.1-3alkyl-aryl-C.sub- .1-3 heteroalkyl-,
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 alkyl-,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl-,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl-, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl-; W is
independently --C(.dbd.O)--O--, --O--C(.dbd.O)--, --C(.dbd.S)--O--,
--O--C(.dbd.S)--, --C(.dbd.S)--S--, --S--C(.dbd.S)--,
--C(.dbd.O)--, --S--C(.dbd.O)--, --O--S(.dbd.O).sub.2--O,
--S(.dbd.O).sub.2--O--, --O--S(.dbd.O).sub.2--,
--C(.dbd.O)--NR.sub.10--, --NR.sub.10--C(.dbd.O)-- -,
--O--P(.dbd.O)(--OR.sub.10)--O--, --P(.dbd.O)(--OR.sub.10)--O--,
--O--P(.dbd.O)(--OR.sub.10)--; X is independently --R.sub.11--; and
E is independently selected from the group consisting of
--N(R.sub.12).sub.2, --N(R.sub.12)(R.sub.13), --S--R.sub.12, and
21where --R.sub.12 is --CH.sub.2CH.sub.2--G, where each G is
independently --Cl, --Br, --I, --O--S(.dbd.O).sub.2--CH.sub.3,
--O--S(.dbd.O).sub.2--CH.sub.2--C.sub.6H.- sub.5, or
--O--S(.dbd.O).sub.2--C.sub.6H.sub.4--CH.sub.3; and where R.sub.13
is independently --C.sub.1-8 alkyl, --C.sub.1-8 heteroalkyl, -aryl,
-heteroaryl, --C.sub.1-3alkyl-aryl, --C.sub.1-3heteroalkyl-aryl,
--C.sub.1-3alkyl-heteroaryl, --C.sub.1-3heteroalkyl-heteroaryl,
-aryl-C.sub.1-3alkyl, -aryl-C.sub.1-3heteroalkyl,
-heteroaryl-C.sub.1-3al- kyl, -heteroaryl-C.sub.1-3heteroalkyl,
--C.sub.1-3alkyl-aryl-C.sub.1-3 alkyl,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 alkyl,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 alkyl,
--C.sub.1-3alkyl-aryl-C.sub.- 1-3 heteroalkyl,
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 alkyl,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl, or --C.sub.1-3
heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl; and all salts and
stereoisomers (including enantiomers and diastereomers)
thereof.
2. A compound having the formula: 22
3. A method of making a compound of claim 1, wherein the method
comprises the steps of: a) transesterifying a psoralenacetate to
provide an aminoalkyl psoralenacetate; and, b) converting the
aminoalkyl psoralenacetate into a (chloroalkyl)-aminoalkyl
psoralenacetate.
4. A method according to claim 3, wherein the psoralenacetate is a
methyl-substituted psoralenacetate.
5. A method according to claim 3, wherein the psoralenacetate is a
4'-psoralenacetate.
6. A method according to claim 3, wherein the aminoalkyl
psoralenacetate is an aminoethyl psoralenacetate.
7. A method according to claim 3, wherein the conversion of the
aminoalkyl psoralenacetate comprises a reaction that converts a
hydroxyl group into a chloride group.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisonal of U.S. patent application
Ser. No. 09/610,652, the contents of which are hereby incorporated
by reference herein in their entirety, which is a continuation of
U.S. patent application Ser. No. 09/003,115, which issued as U.S.
Pat. No. 6,093,725 on Jul. 25, 2000, the contents of which are
hereby incorporated by reference herein in their entirety, which
claims the benefit of U.S. Provisional Application Serial No.
60/043,696, filed Apr. 15, 1997, the disclosure of which is
incorporated herein by reference.
[0002] This application also is a continuation-in-part of U.S.
patent application Ser. No. 08/779,885, filed Jan. 6, 1997,
abandoned; and is a continuation-in-part of U.S. patent application
Ser. No. 08/779,830, filed Jan. 6, 1997, abandoned, the disclosures
of which are incorporated herein by reference.
TECHNICAL FIELD
[0004] This invention relates to compounds which are useful for
inactivating pathogens in a material, such as a blood product, and
to methods of use of the compounds.
BACKGROUND ART
[0005] The transmission of disease by blood products and other
biological materials remains a serious health problem. While
significant advances in blood donor screening and blood testing
have occurred, viruses such as hepatitis B (HBV), hepatitis C
(HCV), and human immunodeficiency virus (HIV) may escape detection
in blood products during testing due to low levels of virus or
viral antibodies. In addition to the viral hazard, there are
currently no licensed tests to screen for the presence of bacteria
or protozoans in blood intended for use in transfusions. The risk
also exists that a hitherto unknown pathogen may become prevalent
in the blood supply and present a threat of disease transmission,
as in fact occurred before the recognition of the risk of HIV
transmission via blood transfusions.
[0006] Exposure of laboratory workers to blood or other body fluids
also presents a health hazard. Twelve thousand health-care workers
whose jobs involve exposure to blood are infected with hepatitis B
virus each year, according to estimates from the Centers for
Disease Control ("Guidelines for Prevention of Transmission of
Human Immunodeficiency Virus and Hepatitis B Virus to Health-Care
and Public-Safety Workers," Morbidity and Mortality Weekly Report,
vol. 38, no. S-6, June 1989).
[0007] Several methods have been proposed to complement donor
screening and blood testing to decrease the incidence of disease
due to transfusions. The introduction of chemical agents into blood
or blood plasma has been suggested to inactivate pathogens prior to
clinical use of the blood product. Nitrogen mustard,
CH.sub.3--N(CH.sub.2CH.sub.2Cl).s- ub.2, was added to blood
components in an investigation of potential virucidal agents.
However, substantial hemolysis occurred at the concentrations
necessary to inactivate one of the viruses studied, rendering
nitrogen mustard unsuitable for use in blood. LoGrippo et al.,
Proceedings of the Sixth Congress of the International Society of
Blood Transfusion, Bibliotheca Haematologica (Hollander, ed.),
1958, pp. 225-230.
[0008] A "solvent/detergent" (S/D) method for inactivating viruses
was described in Horowitz et al., Blood 79:826 (1992) and in
Horowitz et al., Transfusion 25:516 (1985). This method utilized 1%
tri(n-butyl)phosphate and 1% Triton X-100 at 30.degree. C. for 4
hours to inactivate viruses in fresh frozen plasma. Piquet et al.,
Vox Sang. 63:251 (1992), used 1% tri(n-butyl)phosphate and 1%
Octoxynol-9 to inactivate viruses in fresh frozen plasma. Another
method for inactivating viruses in blood involves the addition of
phenol or formaldehyde to the blood. U.S. Pat. No. 4,833,165.
However, both the solvent/detergent method and the
phenol/formaldehyde method require removal of the chemical
additives prior to clinical use of the blood product.
[0009] Inactivation of pathogens in blood products using
photoactivated agents has also been described; see, e.g., Wagner et
al., Transfusion, 34:521 (1994). However, due to the absorption of
light by hemoglobin in several regions in the ultraviolet and
visible spectrum, phototreatment is limited in its application to
materials containing red blood cells. There is also some indication
that phototreatment of red blood cells alters the cells in some
manner; see Wagner et al., Transfusion 33:30 (1993).
[0010] There is thus a need for compositions and methods for
treating blood, blood-derived products, and other biological
materials, which will inactivate pathogens present in the products
or materials without rendering the products or materials unsuitable
for their intended use. Compositions which do not need to be
removed from the biological material prior to its use would be
particularly useful, as equipment and supplies needed to remove the
compositions would be obviated and the costs of handling the
biological material would be reduced. This places an additional
requirement on the composition, however, in that if the composition
remains in the biological material, it must not pose a hazard when
the biological material is used for its intended purpose. For
example, a highly toxic compound which inactivates pathogens in a
blood sample would preclude the use of that blood for transfusion
purposes (although the blood sample may still be suitable for
laboratory analysis).
[0011] It is one intention of this invention to provide
compositions and methods of use of the compositions for
inactivating pathogens in biological materials, without rendering
the materials unsuitable for their intended purpose. Examples of
how this may be accomplished include, but are not limited to, using
the compounds in an ex vivo or in vitro treatment of the biological
materials and then removing the compounds prior to the use of the
material; by using a composition which, even though it remains in
the material, does not render the material unsuitable for its
intended use; or by using a composition which, after inactivating
pathogens in the material, will break down to products, where the
breakdown products can remain in the material without rendering the
material unsuitable for its intended use.
DISCLOSURE OF THE INVENTION
[0012] Accordingly, it is an object of this invention to provide
compounds for inactivating pathogens in a material, where such
compounds comprise a nucleic acid binding moiety; an effector
moiety, capable of forming a covalent bond with nucleic acid; and a
frangible linker covalently linking the nucleic acid moiety and the
effector moiety; wherein the frangible linker degrades so as to no
longer covalently link the nucleic acid binding moiety and the
effector moiety, under conditions which do not render the material
unsuitable for its intended purpose.
[0013] It is an additional object of this invention to provide such
compounds for inactivating pathogens in a material, wherein the
nucleic acid binding moiety is selected from the group consisting
of acridine, acridine derivatives, psoralen, isopsoralen and
psoralen derivatives.
[0014] It is an additional object of this invention to provide such
compounds for inactivating pathogens in a material, wherein the
frangible linker comprises a functional unit selected from the
group consisting of forward esters, reverse esters, forward amides,
reverse amides, forward thioesters, reverse thioesters, forward and
reverse thionoesters, forward and reverse dithioic acids, sulfates,
forward and reverse sulfonates, phosphates, and forward and reverse
phosphonate groups, as defined herein.
[0015] It is an additional object of this invention to provide such
compounds for inactivating pathogens in a material, wherein the
effector group comprises a functional unit which is an alkylating
agent.
[0016] It is an additional object of this invention to provide such
compounds for inactivating pathogens in a material, wherein the
effector group comprises a functional unit selected from the group
consisting of mustard groups, mustard group equivalents, epoxides,
aldehydes, and formaldehyde synthons.
[0017] It is an additional object of this invention to provide
compounds of the formula: 1
[0018] wherein at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 is --V--W--X--E as
defined below, and the remainder of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are
independently selected from the group consisting of --H,
--R.sub.10, --O--R.sub.10, --NO.sub.2, --NH.sub.2, --NH--R.sub.10,
--N(R.sub.10).sub.2, --F, --Cl, --Br, --I, --C(.dbd.O)--R.sub.10,
--C(.dbd.O)--O--R.sub.10, and --O--C(.dbd.O)--R.sub.10,
[0019] where --R.sub.10 is independently H, --C.sub.1-8 alkyl,
--C.sub.1-8 heteroalkyl, -aryl, -heteroaryl, --C.sub.1-3alkyl-aryl,
--C.sub.1-3 heteroalkyl-aryl, --C.sub.1-3alkyl-heteroaryl,
--C.sub.1-3 heteroalkyl-heteroaryl, -aryl-C.sub.1-3alkyl,
-aryl-C.sub.1-3heteroalkyl, -heteroaryl-C.sub.1-3alkyl,
-heteroaryl-C.sub.1-3heteroalkyl, --C.sub.1-3alkyl-aryl-C.sub.1-3
alkyl, --C.sub.1-3heteroalkyl-aryl-C.sub.- 1-3 alkyl,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 alkyl,
--C.sub.1-3alkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3heteroalkyl-heter- oaryl-C.sub.1-3 alkyl,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl;
[0020] V is independently --R.sub.11--, --NH--R.sub.11-- or
--N(CH.sub.3)--R.sub.11--, where --R.sub.11-- is independently
--C.sub.1-8alkyl-, --C.sub.1-8heteroalkyl-, -aryl-, -heteroaryl-,
--C.sub.1-3alkyl-aryl-, --C.sub.1-3heteroalkyl-aryl-,
--C.sub.1-3alkyl-heteroaryl-, --C.sub.1-3heteroalkyl-heteroaryl-,
-aryl-C.sub.1-3alkyl-, -aryl-C.sub.1-3heteroalkyl-,
-heteroaryl-C.sub.1-3alkyl-, -heteroaryl-C.sub.1-3heteroalkyl-,
--C.sub.1-3alkyl-aryl-C.sub.1-3 alkyl-,
--C.sub.1-3heteroalkyl-aryl-C.sub- .1-3 alkyl-,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 alkyl-,
--C.sub.1-3alkyl-aryl-C.sub.1-3 heteroalkyl-,
--C.sub.1-3heteroalkyl-hete- roaryl-C.sub.1-3 alkyl-,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl-,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3heteroalkyl-, or
-C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl-;
[0021] W is independently --C(.dbd.O)--O--, --O--C(.dbd.O)--,
--C(.dbd.S)--O--, --O--C(.dbd.S)--, --C(.dbd.S)--S--,
--S--C(.dbd.S)--, --C(.dbd.O)--S--, --S--C(.dbd.O)--,
--O--S(.dbd.O).sub.2--O--, --S(.dbd.O).sub.2--O--,
--O--S(.dbd.O).sub.2--, --C(.dbd.O)--NR.sub.10--,
--NR.sub.10--C(.dbd.O)--, --O--P(.dbd.O)(--OR.sub.10)--O--,
--P(.dbd.O)(--OR.sub.10)--O--, --O--P(.dbd.O)(--OR.sub.10)--;
[0022] X is independently --R.sub.11--; and
[0023] E is independently selected from the group consisting of
--N(R.sub.12).sub.2, --N(R.sub.12)(R.sub.13), --S--R.sub.12,
[0024] and 2
[0025] where --R.sub.12 is --CH.sub.2CH.sub.2--G, where each G is
independently --Cl, --Br, --I, --O--S(.dbd.O).sub.2--CH.sub.3,
--O--S(.dbd.O).sub.2--CH.sub.2--C.sub.6H.sub.5, or
--O--S(.dbd.O).sub.2--C.sub.6H.sub.4--CH.sub.3;
[0026] and where R.sub.13 is independently --C.sub.1-8 alkyl,
--C.sub.1-8 heteroalkyl, -aryl, -heteroaryl, --C.sub.1-3alkyl-aryl,
--C.sub.1-3heteroalkyl-aryl, --C.sub.1-3alkyl-heteroaryl,
--C.sub.1-3heteroalkyl-heteroaryl, -aryl-C.sub.1-3alkyl,
-aryl-C.sub.1-3 heteroalkyl, -heteroaryl-C.sub.1-3alkyl,
-heteroaryl-C.sub.1-3heteroalkyl- , --C.sub.1-3alkyl-aryl-C.sub.1-3
alkyl, --C.sub.1-3heteroalkyl-aryl-C.sub- .1-3 alkyl,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 alkyl,
--C.sub.1-3alkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3heteroalkyl-heter- oaryl-C.sub.1-3 alkyl,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl;
[0027] and all salts and stereoisomers (including enantiomers and
diastereomers) thereof.
[0028] It is another object of this invention to provide compounds
of the formula: 3
[0029] where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.7, and R.sub.8 are independently selected from the group
consisting of --H, --R.sub.10, --O--R.sub.10, --NO.sub.2,
--NH.sub.2, --NH--R.sub.10, --N(R.sub.10).sub.2, --F, --Cl, --Br,
--I, --C(.dbd.O)--R.sub.10, --C(.dbd.O)--O--R.sub.10, and
--O--C(.dbd.O)--R.sub.10,
[0030] where --R.sub.10 is independently H, --C.sub.1-8 alkyl,
--C.sub.1-8 heteroalkyl, -aryl, -heteroaryl, --C.sub.1-3alkyl-aryl,
--C.sub.1-3heteroalkyl-aryl, --C.sub.1-3alkyl-heteroaryl,
--C.sub.1-3heteroalkyl-heteroaryl, -aryl-C.sub.1-3alkyl,
-aryl-C.sub.1-3heteroalkyl, -heteroaryl-C.sub.1-3alkyl,
-heteroaryl-C.sub.1-3heteroalkyl, --C.sub.1-3alkyl-aryl-C.sub.1-3
alkyl, --C.sub.1-3heteroalkyl-aryl-C.sub.1-3 alkyl,
--C.sub.1-3alkyl-heteroaryl-- C.sub.1-3 alkyl,
--C.sub.1-3alkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 alkyl,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl;
[0031] R.sub.20 is --H or --CH.sub.3; and
[0032] R.sub.21 is --R.sub.11--W--X--E,
[0033] where --R.sub.11-- is independently --C.sub.1-8alkyl-,
--C.sub.1-8heteroalkyl-, -aryl-, -heteroaryl-,
--C.sub.1-3alkyl-aryl-, --C.sub.1-3heteroalkyl-aryl-,
--C.sub.1-3alkyl-heteroaryl-, --C.sub.1-3heteroalkyl-heteroaryl-,
-aryl-C .sub.1-3alkyl-, -aryl-C.sub.1-3heteroalkyl-,
-heteroaryl-C.sub.1-3alkyl-, -heteroaryl-C.sub.1-3heteroalkyl-,
--C.sub.1-3alkyl-aryl-C.sub.1-3 alkyl-,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 alkyl-,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 alkyl-,
--C.sub.1-3alkyl-aryl-C.sub- .1-3 heteroalkyl-,
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 alkyl-,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl-,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl-, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl-;
[0034] W is independently --C(.dbd.O)--O--, --O--C(.dbd.O)--,
--C(.dbd.S)--O--, --O--C(.dbd.S)--, --C(.dbd.S)--S--,
--S--C(.dbd.S)--, --C(.dbd.O)--, --S--C(.dbd.O)--,
--O--S(.dbd.O).sub.2--O--, --S(.dbd.O).sub.2--O--,
--O--S(.dbd.O).sub.2--, --C(.dbd.O)--NR.sub.10--,
--NR.sub.10--C(.dbd.O)--, --O--P(.dbd.O)(--OR.sub.10)--O--,
--P(.dbd.O)(--OR.sub.10)--O--, --O--P(.dbd.O)(--OR.sub.10)--;
[0035] X is independently --R.sub.11--; and
[0036] E is independently selected from the group consisting of
--N(R.sub.12).sub.2, --N(R.sub.12)(R.sub.13), --S--R.sub.12,
[0037] and 4
[0038] where --R.sub.12 is --CH.sub.2CH.sub.2--G, where each G is
independently --Cl, --Br, --I, --O--S(.dbd.O).sub.2--CH.sub.3,
--O--S(.dbd.O).sub.2--CH.sub.2--C.sub.6H.sub.5, or
--O--S(.dbd.O).sub.2--C.sub.6H.sub.4--CH.sub.3;
[0039] and where R.sub.13 is independently --C.sub.1-8 alkyl,
--C.sub.1-8 heteroalkyl, -aryl, -heteroaryl, --C.sub.1-3alkyl-aryl,
--C.sub.1-3heteroalkyl-aryl, --C.sub.1-3alkyl-heteroaryl,
--C.sub.1-3heteroalkyl-heteroaryl, -aryl-C.sub.1-3alkyl,
-aryl-C.sub.1-3heteroalkyl, -heteroaryl-C.sub.1-3alkyl,
-heteroaryl-C.sub.1-3heteroalkyl, --C.sub.1-3alkyl-aryl-C.sub.1-3
alkyl, --C.sub.1-3heteroalkyl-aryl-C.sub.1-3 alkyl,
--C.sub.1-3alkyl-heteroaryl-- C.sub.1-3 alkyl,
--C.sub.1-3alkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 alkyl,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl;
[0040] and all salts and stereoisomers (including enantiomers and
diastereomers) thereof.
[0041] It is another object of this invention to provide compounds
of the formula: 5
[0042] wherein at least one of R.sub.44, R.sub.55, R.sub.3,
R.sub.4, R.sub.5, and R.sub.8 is --V--W--X--E, and the remainder of
R.sub.44, R.sub.55, R.sub.3, R.sub.4, R.sub.5, and R.sub.8 are
independently selected from the group consisting of --H,
--R.sub.10, --O--R.sub.10, --NO.sub.2, --NH.sub.2, --NH--R.sub.10,
--N(R.sub.10).sub.2, --F, --Cl, --Br, --I, --C(.dbd.O)--R.sub.10,
--C(.dbd.O)--O--R.sub.10, and --O--C(.dbd.O)--R.sub.10,
[0043] where --R.sub.10 is independently H, --C.sub.1-8 alkyl,
--C.sub.1-8 heteroalkyl, -aryl, -heteroaryl, --C.sub.1-3alkyl-aryl,
--C.sub.1-3heteroalkyl-aryl, --C.sub.1-3alkyl-heteroaryl,
--C.sub.1-3heteroalkyl-heteroaryl, -aryl-C.sub.1-3alkyl,
-aryl-C.sub.1-3heteroalkyl, -heteroaryl-C.sub.1-3alkyl,
-heteroaryl-C.sub.1-3heteroalkyl, --C.sub.1-3alkyl-aryl-C.sub.1-3
alkyl, --C.sub.1-3heteroalkyl-aryl-C.sub.1-3 alkyl,
--C.sub.1-3alkyl-heteroaryl-- C.sub.1-3 alkyl,
--C.sub.1-3alkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 alkyl,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl;
[0044] V is independently --R.sub.11, --NH--R.sub.11-- or
--N(CH.sub.3)--R.sub.11--, where --R.sub.11-- is independently
--C.sub.1-8alkyl-, --C.sub.1-8heteroalkyl-, -aryl-, -heteroaryl-,
--C.sub.1-3alkyl-aryl-, --C.sub.1-3heteroalkyl-aryl-,
--C.sub.1-3alkyl-heteroaryl-, --C.sub.1-3heteroalkyl-heteroaryl-,
-aryl-C.sub.1-3alkyl-, -aryl-C.sub.1-3heteroalkyl-,
-heteroaryl-C.sub.1-3alkyl-, -heteroaryl-C.sub.1-3heteroalkyl-,
--C.sub.1-3alkyl-aryl-C.sub.1-3 alkyl-,
--C.sub.1-3heteroalkyl-aryl-C.sub- .1-3 alkyl-,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 alkyl-,
--C.sub.1-3alkyl-aryl-C.sub.1-3 heteroalkyl-,
--C.sub.1-3heteroalkyl-hete- roaryl-C.sub.1-3 alkyl-,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl-,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl-, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl-;
[0045] W is independently --C(.dbd.O)--O--, --O--C(.dbd.O)--,
--C(.dbd.S)--O--, --O--C(.dbd.S)--, --C(.dbd.S)--S--,
--S--C(.dbd.S)--, --C(.dbd.O)--S--, --S--C(.dbd.O)--,
--O--S(.dbd.O).sub.2--O--, --S(.dbd.O).sub.2--O--,
--O--S(.dbd.O).sub.2--, --C(.dbd.O)--NR.sub.10--,
--NR.sub.10--C(.dbd.O)--, --O--P(.dbd.O)(--OR.sub.10)--O--,
--P(.dbd.O)(--OR.sub.10)--O--, --O--P(.dbd.O)(--OR.sub.10)--;
[0046] X is independently --R.sub.11--; and
[0047] E is independently selected from the group consisting of
--N(R.sub.12).sub.2, --N(R.sub.12)(R.sub.13), --S--R.sub.12,
[0048] and 6
[0049] where --R.sub.12 is --CH.sub.2CH.sub.2--G, where each G is
independently --Cl, --Br, --I, --O--S(.dbd.O).sub.2--CH.sub.3,
--O--S(.dbd.O).sub.2--CH.sub.2--C.sub.6H.sub.5, or
--O--S(.dbd.O).sub.2--C.sub.6H.sub.4--CH.sub.3;
[0050] and where R.sub.13 is independently --C.sub.1-8 alkyl,
--C.sub.1-8 heteroalkyl, -aryl, -heteroaryl, --C.sub.1-3alkyl-aryl,
--C.sub.1-3heteroalkyl-aryl, --C.sub.1-3alkyl-heteroaryl,
--C.sub.1-3heteroalkyl-heteroaryl, -aryl-C.sub.1-3alkyl,
-aryl-C.sub.1-3heteroalkyl, -heteroaryl-C.sub.1-3alkyl,
-heteroaryl-C.sub.1-3heteroalkyl, --C.sub.1-3alkyl-aryl-C.sub.1-3
alkyl, --C.sub.1-3heteroalkyl-aryl-C.sub.1-3 alkyl,
--C.sub.1-3alkyl-heteroaryl-- C.sub.1-3 alkyl,
--C.sub.1-3alkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 alkyl,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl;
[0051] and all salts and stereoisomers (including enantiomers and
diastereomers) thereof.
[0052] It is yet another object of this invention to provide the
compounds .beta.-alanine, N-(2-carbomethoxyacridin-9-yl),
2-[bis(2-chloroethyl)amin- o]ethyl ester; 4-aminobutyric acid
N-[(2-carbomethoxyacridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl
ester; 5-aminovaleric acid N-[(2-carbomethoxyacridin-9-yl),
2-[bis(2-chloroethyl)amino]ethyl ester; .beta.-alanine,
N-(2-carbomethoxyacridin-9-yl), 3-[bis(2-chloroethyl)amin- o]propyl
ester; .beta.-alanine, [N,N-bis(2-chloroethyl)],
3-[(6-chloro-2-methoxyacridin-9-yl)amino]propyl ester;
.beta.-alanine, [N,N-bis(2-chloroethyl)],
2-[(6-chloro-2-methoxyacridin-9-yl)amino]ethyl ester;
[N,N-bis(2-chloroethyl)]-2-aminoethyl 4,5',8-trimethyl-4'-psoralen-
acetate; and .beta.-alanine, N-(acridin-9-yl),
2-[bis(2-chloroethyl)amino]- ethyl ester; and all salts
thereof.
[0053] Provided are methods for inactivating pathogens in a
material, such as a biological material, the methods comprising
adding one or more compounds of the invention to the material; and
incubating the material. The compound may be added to the material
to form a final solution having a concentration of the compound (or
total concentration of all compounds, if more than one is used),
for example, of between 1 and 500 .mu.M. Biological materials which
may be treated include blood, blood products, plasma, platelet
preparations, red blood cells, packed red blood cells, serum,
cerebrospinal fluid, saliva, urine, sweat, feces, semen, milk,
tissue samples, and homogenized tissue samples, derived from human
or other mammalian or vertebrate sources.
BEST MODE FOR CARRYING OUT THE INVENTION
[0054] This invention provides for compounds useful for
inactivating pathogens found in materials, particularly for
inactivating pathogens found in biological materials such as blood
or other body fluids. This invention also provides for methods of
use of such compounds for inactivating pathogens in materials. The
invention also provides for inactivating pathogens found in or on
materials for biological use. The compounds may be used in vitro
and ex vivo. The biological materials or materials for biological
use may be intended for use in vitro, in vivo, or ex vivo.
[0055] The compounds are designed to inactivate pathogens by
reacting with nucleic acid. In aqueous solution, at appropriate pH
values, the compounds have a period of activity during which they
can bind to and react with nucleic acid. After this period, the
compounds break down to products which are no longer able to bind
to nor react with nucleic acid.
[0056] The chemical organization of the compounds can be broadly
described as an anchor, covalently bonded to a frangible linker,
which is covalently bonded to an effector. "Anchor" is defined as a
moiety which binds non-covalently to a nucleic acid biopolymer (DNA
or RNA). "Effector" is defined as a moiety which reacts with
nucleic acid by a mechanism which forms a covalent bond with the
nucleic acid. "Frangible linker" is defined as a moiety which
serves to covalently link the anchor and effector, and which will
degrade under certain conditions so that the anchor and effector
are no longer linked covalently. The anchor-frangible
linker-effector arrangement enables the compounds to bind
specifically to nucleic acid (due to the anchor's binding ability).
This brings the effector into proximity for reaction with the
nucleic acid.
[0057] The compounds are useful for inactivating pathogens found in
materials, particularly biological materials such as blood and
other body fluids. Intracellular and extracellular and or other
pathogen materials may be inactivated. For example, when a compound
of the invention is combined with a pathogen-containing red blood
cell composition at physiological pH, the effector portion of the
compound reacts with pathogen nucleic acid. Effector moieties which
do not react with nucleic acid are gradually hydrolyzed by the
solvent. Hydrolysis of the frangible linker occurs concurrently
with the effector-nucleic acid reaction and effector hydrolysis. It
is desirable that the frangible linker break down at a rate slow
enough to permit inactivation of pathogens in the material; that
is, the rate of breakdown of the frangible linker is slower than
the rate at which the compound reacts with nucleic acid. After a
sufficient amount of time has passed, the compound has broken down
into the anchor (which may also bear fragments of the frangible
linker) and the effector-nucleic acid breakdown products (where
fragments of the frangible linker may also remain attached to the
effector), or into the anchor (which may also bear fragments of the
frangible linker) and the hydrolyzed effector breakdown products
(where fragments of the frangible linker may also remain attached
to the effector). Additional fragments of the frangible linker may
also be generated upon degradation of the compound which do not
remain bonded to either the anchor or the effector. The exact
embodiment of the compound of the invention determines whether the
anchor breakdown product or the effector breakdown product bears
fragments of the frangible linker, or whether additional fragments
of the frangible linker are generated which do not remain bonded to
either the anchor or the effector breakdown products.
[0058] A preferred embodiment of the invention comprises compounds
which, upon cleavage of the frangible linker, result in breakdown
products of low mutagenicity. Mutagenicity of the compounds, after
hydrolysis of the effector, is due primarily to the anchor moiety,
as the anchor interacts with nucleic acid and may have the
potential to interfere with nucleic acid replication, even if the
effector moiety has been hydrolyzed. Preferably, after cleavage of
the frangible linker, the anchor fragment has substantially reduced
mutagenicity.
Definitions
[0059] "Pathogen" is defined as any nucleic acid containing agent
capable of causing disease in a human, other mammals, or
vertebrates. Examples include microorganisms such as unicellular or
multicellular microorganisms. Examples of pathogens are bacteria,
viruses, protozoa, fungi, yeasts, molds, and mycoplasmas which
cause disease in humans, other mammals, or vertebrates. The genetic
material of the pathogen may be DNA or RNA, and the genetic
material may be present as single-stranded or double-stranded
nucleic acid. The nucleic acid of the pathogen may be in solution,
intracellular, extracellular, or bound to cells. Table I lists
examples of viruses, and is not intended to limit the invention in
any manner.
1 TABLE I Family: Virus: Adeno Adenovirus 2 Canine hepatitis Arena
Pichinde Lassa Bunya Turlock California encephalitis Herpes Herpes
simplex 1 Herpes simplex 2 Cytomegalovirus Pseudorabies Orothomyxo
Influenza Papova SV-40 Paramyxo Measles Mumps Parainfluenza 2 and 3
Picorna Poliovirus 1 and 2 Coxsackie A-9 Echo 11 Pox Vaccinia Fowl
Pox Reo Blue tongue Colorado tick fever Retro HIV Avian sarcoma
Murine sarcoma Murine leukemia Rhabdo Vesicular stomatitis virus
Toga Western equine encephalitis Dengue 2 Dengue 4 St. Louis
encephalitis Hepadna hepatitis B Bacteriophage Lambda T2
(Rickettsia) R. akari (rickettsialpox)
[0060] "In vivo" use of a material or compound is defined as
introduction of the material or compound into a living human,
mammal, or vertebrate.
[0061] "In vitro" use of a material or compound is defined as a use
of the material or compound outside a living human, mammal, or
vertebrate, where neither the material nor compound is intended for
reintroduction into a living human, mammal, or vertebrate. An
example of an in vitro use would be the analysis of components of a
blood sample using laboratory equipment.
[0062] "Ex vivo" use of a compound is defined as using a compound
for treatment of a biological material outside a living human,
mammal, or vertebrate, where that treated biological material is
intended for use inside a living human, mammal, or vertebrate. For
example, removal of blood from a human, and introduction of a
compound into that blood to inactivate pathogens, is defined as an
ex vivo use of that compound if the blood is intended for
reintroduction into that human or another human. Reintroduction of
the human blood into that human or another human would be in vivo
use of the blood, as opposed to the ex vivo use of the compound. If
the compound is still present in the blood when it is reintroduced
into the human, then the compound, in addition to its ex vivo use,
is also introduced in vivo.
[0063] "Biological material" is defined as a material originating
from a biological organism of any type. Examples of biological
materials include, but are not limited to, blood, blood products
such as plasma, platelet preparations, red blood cells, packed red
blood cells, and serum, cerebrospinal fluid, saliva, urine, feces,
semen, sweat, milk, tissue samples, homogenized tissue samples, and
any other substance having its origin in a biological organism.
Biological materials also include synthetic material incorporating
a substance having its origin in a biological organism, such as a
vaccine preparation comprised of alum and a pathogen (the pathogen,
in this case, being the substance having its origin in a biological
organism), a sample prepared for analysis which is a mixture of
blood and analytical reagents, cell culture medium, cell cultures,
viral cultures, and other cultures derived from a living
organism.
[0064] "Material for biological use" is defined as any material
that will come into contact with, or be introduced into, a living
human, mammal, or vertebrate, where such contact carries a risk of
transmitting disease or pathogens. Such materials include, but are
not limited to, medical implants such as pacemakers and artificial
joints; implants designed for sustained drug release; needles,
intravenous lines, and the like; dental tools; dental materials
such as tooth crowns; catheters; and any other material which, when
in contact with or introduced into a living human, mammal, or
vertebrate, entails risk of transmitting disease or pathogens.
[0065] "Inactivation of pathogens" is defined as rendering
pathogens in a material incapable of reproducing. Inactivation is
expressed as the negative logarithm of the fraction of remaining
pathogens capable of reproducing. Thus, if a compound at a certain
concentration renders 99% of the pathogens in a material incapable
of reproduction, 1% or one-one hundredth (0.01) of the pathogens
remain capable of reproduction. The negative logarithm of 0.01 is
2, and that concentration of that compound is said to have
inactivated the pathogens present by 2 logs. Alternatively, the
compound is said to have 2 logs kill at that concentration.
[0066] "Alkyl" as used herein refers to a cyclic, branched, or
straight chain chemical group containing carbon and hydrogen, such
as methyl, pentyl, and adamantyl. Alkyl groups can either be
unsubstituted or substituted with one or more substituents, e.g.,
halogen, alkoxy, acyloxy, amino, hydroxyl, thiol, carboxy,
benzyloxy, phenyl, benzyl, or other functionality. Alkyl groups can
be saturated or unsaturated (e.g., containing --C.dbd.C-- or
--C.ident.C-- subunits), at one or several positions. Typically,
alkyl groups will comprise 1 to 12 carbon atoms, preferably 1 to 10
carbon atoms, and more preferably 1 to 8 carbon atoms, unless
otherwise specified.
[0067] "Heteroalkyl" as used herein are alkyl chains with one or
more N, O, S, or P heteroatoms incorporated into the chain. The
heteroatom(s) may bear none, one, or more than one of the
substituents described above. "Heteroatoms" also includes oxidized
forms of the heteroatoms N, S and P. Examples of heteroalkyl groups
include (but are not limited to) methoxy, ethoxy, and other
alkyloxy groups; ether-containing groups; amide containing groups
such as polypeptide chains; ring systems such as piperidinyl,
lactam and lactone; and other groups which incorporate heteroatoms
into the carbon chain. Typically, heteroalkyl groups will comprise,
in addition to the heteroatom(s), 1 to 12 carbon atoms, preferably
1 to 10 carbon atoms, and more preferably 1 to 8 carbon atoms,
unless otherwise specified.
[0068] "Aryl" or "Ar" refers to an unsaturated aromatic carbocyclic
group having a single ring (e.g., phenyl) or multiple condensed
rings (e.g., naphthyl or anthryl), which can be optionally
unsubstituted or substituted with amino, hydroxyl, C.sub.1-8 alkyl,
alkoxy, halo, thiol, and other substituents.
[0069] "Heteroaryl" groups are unsaturated aromatic carbocyclic
groups having a single ring (e.g., pyridyl or furyl) or multiple
condensed rings (e.g., acridinyl, indolyl or benzothienyl) and
having at least one hetero atom, such as N, O, or S, within at
least one of the rings. The ring(s) can optionally be unsubstituted
or substituted with amino, hydroxyl, alkyl, alkoxy, halo, thiol,
acyloxy, carboxy, benzyloxy, phenyl, benzyl, and other
substituents.
[0070] Abbreviations
[0071] The following abbreviations are used: QM (quinacrine
mustard); Hct (hematocrit); RBC (red blood cell); LB (Luria Broth);
cfu (colony forming units); pfu (plaque forming units); DMEM
(Delbecco's modified eagles medium); FBS (fetal bovine serum); PRBC
(packed red blood cells); rpm (revolutions per minute); TC (tissue
culture); NHSP (normal human serum pool); NCS (newborn calf serum);
PBS (phosphate buffered saline).
[0072] Chemical Structure of the Compounds
[0073] A wide variety of groups are available for use as the
anchors, linkers, and effectors. Examples of anchor groups which
can be used in the compound include, but are not limited to,
intercalators, minor groove binders, major groove binders,
molecules which bind by electrostatic interactions such as
polyamines, and molecules which bind by sequence specific
interactions. The following is a non-limiting list of possible
anchor groups:
[0074] acridines (and acridine derivatives, e.g. proflavine,
acriflavine, diacridines, acridones, benzacridines, quinacrines),
actinomycins, anthracyclinones, rhodomycins, daunomycin,
thioxanthenones (and thioxanthenone derivatives, e.g. miracil D),
anthramycin, mitomycins, echinomycin (quinomycin A), triostins,
ellipticine (and dimers, trimers and analogs thereof), norphilin A,
fluorenes (and derivatives, e.g. flourenones, fluorenodiamines),
phenazines, phenanthridines, phenothiazines (e.g., chlorpromazine),
phenoxazines, benzothiazoles, xanthenes and thioxanthenes,
anthraquinones, anthrapyrazoles, benzothiopyranoindoles,
3,4-benzopyrene, 1-pyrenyloxirane, benzanthracenes, benzodipyrones,
quinolines (e.g., chloroquine, quinine, phenylquinoline
carboxamides), furocoumarins (e.g., psoralens and isopsoralens),
ethidium, propidium, coralyne, and polycyclic aromatic hydrocarbons
and their oxirane derivatives;
[0075] distamycin, netropsin, other lexitropsins, Hoechst 33258 and
other Hoechst dyes, DAPI (4',6-diamidino-2-phenylindole), berenil,
and triarylmethane dyes;
[0076] aflatoxins;
[0077] spermine, spermidine, and other polyamines; and
[0078] nucleic acids or analogs which bind by sequence specific
interactions such as triple helix formation, D-loop formation, and
direct base pairing to single stranded targets. Derivatives of
these compounds are also non-limiting examples of anchor groups,
where a derivative of a compound includes, but is not limited to, a
compound which bears one or more substituent of any type at any
location, oxidation or reduction products of the compound, etc.
[0079] Examples of linkers which can be used in the invention are,
but are not limited to, compounds which include functional groups
such as ester (where the carbonyl carbon of the ester is between
the anchor and the Sp.sup.3 oxygen of the ester; this arrangement
is also called "forward ester"), "reverse ester" (where the
Sp.sup.3 oxygen of the ester is between the anchor and the carbonyl
carbon of the ester), thioester (where the carbonyl carbon of the
thioester is between the anchor and the sulfur of the thioester,
also called "forward thioester"), reverse thioester (where the
sulfur of the thioester is between the anchor and the carbonyl
carbon of the thioester, also called "reverse thioester"), forward
and reverse thionoester, forward and reverse dithioic acid,
sulfate, forward and reverse sulfonates, phosphate, and forward and
reverse phosphonate groups. "Thioester" designates the
--C(.dbd.O)--S--group; "thionoester" designates the
--C(.dbd.S)--O-- group, and "dithioic acid" designates the
--C(.dbd.S)--S-- 6tgroup. The frangible linker also may include an
amide, where the carbonyl carbon of the amide is between the anchor
and the nitrogen of the amide (also called a "forward amide"), or
where the nitrogen of the amide is between the anchor and the
carbonyl carbon of the amide (also called a "reverse amide"). For
groups which can be designated as "forward" and "reverse", the
forward orientation is that orientation of the functional groups
wherein, after hydrolysis of the functional group, the resulting
acidic function is covalently linked to the anchor moiety and the
resulting alcohol or thiol function is covalently linked to the
effector moiety. The reverse orientation is that orientation of the
functional groups wherein, after hydrolysis of the functional
group, the resulting acidic function is covalently linked to the
effector moiety and the resulting alcohol or thiol function is
covalently linked to the anchor moiety.
[0080] The frangible linker, such as an amide moiety, also may be
capable of degrading under conditions of enzymatic degradation, by
endogenous enzymes in the biological material being treated, or by
enzymes added to the material.
[0081] Examples of effectors which can be used in the invention
are, but are not limited to, mustard groups, mustard group
equivalents, epoxides, aldehydes, formaldehyde synthons, and other
alkylating and cross-linking agents. Mustard groups are defined as
including mono or bis haloethylamine groups, and mono
haloethylsulfide groups. Mustard group equivalents are defined by
groups that react by a mechanism similar to the mustards (that is,
by forming an aziridinium intermediate, or by having or by forming
an aziridine ring, which can react with a nucleophile), such as
mono or bis mesylethylamine groups, mono mesylethylsulfide groups,
mono or bis tosylethylamine groups, and mono tosylethylsulfide
groups. Formaldehyde synthons are defined as any compound that
breaks down to formaldehyde in aqueous solution, including
hydroxymethylamines such as hydroxymethylglycine. Examples of
formaldehyde synthons are given in U.S. Pat. No. 4,337,269 and in
International Patent Application WO 97/02028. While the invention
is not limited to any specific mechanism, the effector groups,
which are, or are capable of forming an electrophilic group, such
as a mustard group, are believed to react with and form a covalent
bond to nucleic acid.
[0082] Three embodiments of the compounds of this invention are
described by the following general formulas I, II, and III.
[0083] General formula I is: 7
[0084] wherein at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 is --V--W--X--E as
defined below, and the remainder of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are
independently selected from the group consisting of --H,
--R.sub.10, --O--R.sub.10, --NO.sub.2, --NH.sub.2, --NH--R.sub.10,
--N(R.sub.10).sub.2, --F, --Cl, --Br, --I, --C(.dbd.O)--R.sub.10,
--C(.dbd.O)--O--R.sub.10, and --O--C(.dbd.O)--R.sub.10,
[0085] where --R.sub.10 is independently H, --C.sub.1-8 alkyl,
--C.sub.1-8 heteroalkyl, -aryl, -heteroaryl, --C.sub.1-3alkyl-aryl,
--C.sub.1-3heteroalkyl-aryl, --C.sub.1-3alkyl-heteroaryl,
--C.sub.1-3heteroalkyl-heteroaryl, -aryl-C.sub.1-3alkyl,
-aryl-C.sub.1-3heteroalkyl, -heteroaryl-C.sub.1-3alkyl,
-heteroaryl-C.sub.1-3heteroalkyl, --C.sub.1-3alkyl-aryl-C.sub.1-3
alkyl, --C.sub.1-3heteroalkyl-aryl-C.sub.1-3 alkyl,
--C.sub.1-3alkyl-heteroaryl-- C.sub.1-3 alkyl,
--C.sub.1-3alkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 alkyl,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl;
[0086] V is independently --R.sub.11--, --NH--R.sub.11-- or
--N(CH.sub.3)--R.sub.11--, where --R.sub.11-- is independently
--C.sub.1-8alkyl-, --C.sub.1-8heteroalkyl-, -aryl-, -heteroaryl-,
--C.sub.1-3alkyl-aryl-, --C.sub.1-3heteroalkyl-aryl-,
--C.sub.1-3alkyl-heteroaryl-, --C.sub.1-3heteroalkyl-heteroaryl-,
-aryl-C.sub.1-3alkyl-, -aryl-C.sub.1-3heteroalkyl-,
-heteroaryl-C.sub.1-3alkyl-, -heteroaryl-C.sub.1-3heteroalkyl-,
--C.sub.1-3alkyl-aryl-C.sub.1-3 alkyl-,
--C.sub.1-3heteroalkyl-aryl-C.sub- .1-3 alkyl-,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 alkyl-,
--C.sub.1-3alkyl-aryl-C.sub.1-3 heteroalkyl-,
--C.sub.1-3heteroalkyl-hete- roaryl-C.sub.1-3 alkyl-,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl-,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl-, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl-;
[0087] W is independently --C(.dbd.O)--O--, --O--C(.dbd.O)--,
--C(.dbd.S)--O--, --O--C(.dbd.S)--, --C(.dbd.S)--S--,
--S--C(.dbd.S)--, --C(.dbd.O)--S--, --S--C(.dbd.O)--,
--O--S(.dbd.O).sub.2--O--, --S(.dbd.O).sub.2--O--,
--O--S(.dbd.O).sub.2--, --C(.dbd.O)--NR.sub.10--,
--NR.sub.10--C(.dbd.O)--, --O--P(.dbd.O)(--OR.sub.10)--O--,
--P(.dbd.O)(--OR.sub.10)--O--, --O--P(.dbd.O)(--OR.sub.10)--;
[0088] X is independently --R.sub.11--; and
[0089] E is independently selected from the group consisting of
--N(R.sub.12).sub.2, --N(R.sub.12)(R.sub.13), --S--R.sub.12,
[0090] and 8
[0091] where --R.sub.12 is --CH.sub.2CH.sub.2--G, where each G is
independently --Cl, --Br, --I, --O--S(.dbd.O).sub.2--CH.sub.3,
--O--S(.dbd.O).sub.2--CH.sub.2--C.sub.6H.sub.5, or --O--S
(.dbd.O).sub.2--C.sub.6H.sub.4--CH.sub.3;
[0092] and where R.sub.13 is independently --C.sub.1-8 alkyl,
--C.sub.1-8 heteroalkyl, -aryl, -heteroaryl, --C.sub.1-3alkyl-aryl,
--C.sub.1-3heteroalkyl-aryl, --C.sub.1-3alkyl-heteroaryl,
--C.sub.1-3heteroalkyl-heteroaryl, -aryl-C.sub.1-3alkyl,
-aryl-C.sub.1-3heteroalkyl, -heteroaryl-C.sub.1-3alkyl,
-heteroaryl-C.sub.1-3heteroalkyl, --C.sub.1-3alkyl-aryl-C.sub.1-3
alkyl, --C.sub.1-3heteroalkyl-aryl-C.sub.1-3 alkyl,
--C.sub.1-3alkyl-heteroaryl-- C.sub.1-3 alkyl,
--C.sub.1-3alkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 alkyl,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl;
[0093] and all salts and stereoisomers (including enantiomers and
diastereomers) thereof.
[0094] General formula II is: 9
[0095] where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.7, and R.sub.8 are independently selected from the group
consisting of --H, --R.sub.10, --O--R.sub.10, --NO.sub.2,
--NH.sub.2, --NH--R.sub.10, --N(R.sub.10).sub.2, --F, --Cl, --Br,
--I, --C(.dbd.O)--R.sub.10, --C(.dbd.O)--O--R.sub.10, and
--O--C(.dbd.O)--R.sub.10,
[0096] where --R.sub.10 is independently H, --C.sub.1-8 alkyl,
--C.sub.1-8 heteroalkyl, -aryl, -heteroaryl, --C.sub.1-3alkyl-aryl,
--C.sub.1-3heteroalkyl-aryl, --C.sub.1-3alkyl-heteroaryl,
--C.sub.1-3heteroalkyl-heteroaryl, -aryl-C.sub.1-3alkyl,
-aryl-C.sub.1-3heteroalkyl, -heteroaryl-C.sub.1-3alkyl,
-heteroaryl-C.sub.1-3heteroalkyl, --C.sub.1-3alkyl-aryl-C.sub.1-3
alkyl, --C.sub.1-3heteroalkyl-aryl-C.sub.1-3 alkyl,
--C.sub.1-3alkyl-heteroaryl-- C.sub.1-3 alkyl,
--C.sub.1-3alkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 alkyl,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl;
[0097] R.sub.20 is --H or --CH.sub.3; and
[0098] R.sub.21 is --R.sub.11--W--X--E,
[0099] where --R.sub.11-- is independently --C.sub.1-8alkyl-,
--C.sub.1-8heteroalkyl-, -aryl-, -heteroaryl-,
--C.sub.1-3alkyl-aryl-, --C.sub.1-3heteroalkyl-aryl-,
--C.sub.1-3alkyl-heteroaryl-, --C.sub.1-3heteroalkyl-heteroaryl-,
-aryl-C .sub.1-3alkyl-, -aryl-C.sub.1-3heteroalkyl-,
-heteroaryl-C.sub.1-3alkyl-, -heteroaryl-C.sub.1-3heteroalkyl-,
--C.sub.1-3alkyl-aryl-C.sub.1-3 alkyl-,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 alkyl-,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 alkyl-,
--C.sub.1-3alkyl-aryl-C.sub- .1-3 heteroalkyl-,
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 alkyl-,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl-,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl-, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl-;
[0100] W is independently --C(.dbd.O)--O--, --O--C(.dbd.O)--,
--C(.dbd.S)--O--, --O--C(.dbd.S)--, --C(.dbd.S)--S--,
--S--C(.dbd.S)--, --C(.dbd.O)--S--, --S--C(.dbd.O)--,
--O--S(.dbd.O).sub.2--O--, --S(.dbd.O).sub.2--O--,
--O--S(.dbd.O).sub.2--, --C(.dbd.O)--NR.sub.10--,
--NR.sub.10--C(.dbd.O)--, --O--P(.dbd.O)(--OR.sub.10)--O--,
--P(.dbd.O)(--OR.sub.10)--O--, --O--P(.dbd.O)(--OR.sub.10)--;
[0101] X is independently --R.sub.11--; and
[0102] E is independently selected from the group consisting of
--N(R.sub.12).sub.2, --N(R.sub.12)(R.sub.13), --S--R.sub.12,
[0103] and 10
[0104] where --R.sub.12 is --CH.sub.2CH.sub.2--G, where each G is
independently --Cl, --Br, --I, --O--S(.dbd.O).sub.2--CH.sub.3,
--O--S(.dbd.O).sub.2--CH.sub.2--C.sub.6H.sub.5, or
--O--S(.dbd.O).sub.2--C.sub.6H.sub.4--CH.sub.3;
[0105] and where R.sub.13 is independently --C.sub.1-8 alkyl,
--C.sub.1-8 heteroalkyl, -aryl, -heteroaryl, --C.sub.1-3alkyl-aryl,
--C.sub.1-3heteroalkyl-aryl, --C.sub.1-3alkyl-heteroaryl,
--C.sub.1-3heteroalkyl-heteroaryl, -aryl-C.sub.1-3alkyl,
-aryl-C.sub.1-3heteroalkyl, -heteroaryl-C.sub.1-3alkyl,
-heteroaryl-C.sub.1-3heteroalkyl, --C.sub.1-3alkyl-aryl-C.sub.1-3
alkyl, --C.sub.1-3heteroalkyl-aryl-C.sub.1-3 alkyl,
--C.sub.1-3alkyl-heteroaryl-- C.sub.1-3 alkyl,
--C.sub.1-3alkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 alkyl,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl;
[0106] and all salts and stereoisomers (including enantiomers and
diastereomers) thereof.
[0107] General formula III is: 11
[0108] wherein at least one of R.sub.44, R.sub.55, R.sub.3,
R.sub.4, R.sub.5, and R.sub.8 is --V--W--X--E, and the remainder of
R.sub.44, R.sub.55, R.sub.3, R.sub.4, R.sub.5, and R.sub.8 are
independently selected from the group consisting of --H,
--R.sub.10, --O--R.sub.10, --NO.sub.2, --NH.sub.2, --NH--R.sub.10,
--N(R.sub.10).sub.2, --F, --Cl, --Br, --I, --C(.dbd.O)--R.sub.10,
--C(.dbd.O)--O--R.sub.10, and --O--C(.dbd.O)--R.sub.10,
[0109] where --R.sub.10 is independently H, --C.sub.1-8 alkyl,
--C.sub.1-8 heteroalkyl, -aryl, -heteroaryl, --C.sub.1-3alkyl-aryl,
--C.sub.1-3heteroalkyl-aryl, --C.sub.1-3alkyl-heteroaryl,
--C.sub.1-3heteroalkyl-heteroaryl, -aryl-C.sub.1-3alkyl,
-aryl-C.sub.1-3heteroalkyl, -heteroaryl-C.sub.1-3alkyl,
-heteroaryl-C.sub.1-3heteroalkyl, --C.sub.1-3alkyl-aryl-C.sub.1-3
alkyl, --C.sub.1-3heteroalkyl-aryl-C.sub.1-3 alkyl,
--C.sub.1-3alkyl-heteroaryl-- C.sub.1-3 alkyl,
--C.sub.1-3alkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 alkyl,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl;
[0110] V is independently --R.sub.11--, --NH--R.sub.11-- or
--N(CH.sub.3)--R.sub.11--, where --R.sub.11-- is independently
--C.sub.1-8alkyl-, --C.sub.1-8heteroalkyl-, -aryl-, -heteroaryl-,
--C.sub.1-3alkyl-aryl-, --C.sub.1-3heteroalkyl-aryl-,
--C.sub.1-3alkyl-heteroaryl-, --C.sub.1-3heteroalkyl-heteroaryl-,
-aryl-C.sub.1-3alkyl-, -aryl-C.sub.1-3heteroalkyl-,
-heteroaryl-C.sub.1-3alkyl-, -heteroaryl-C.sub.1-3heteroalkyl-,
--C.sub.1-3alkyl-aryl-C.sub.1-3 alkyl-,
--C.sub.1-3heteroalkyl-aryl-C.sub- .1-3 alkyl-,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 alkyl-,
--C.sub.1-3alkyl-aryl-C.sub.1-3 heteroalkyl-,
--C.sub.1-3heteroalkyl-hete- roaryl-C.sub.1-3 alkyl-, --C.sub.1-3
heteroalkyl-aryl-C.sub.1-3 heteroalkyl-,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl-, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl-;
[0111] W is independently --C(.dbd.O)--O--, --O--C(.dbd.O)--,
--C(.dbd.S)--O--, --O--C(.dbd.S)--, --C(.dbd.S)--S--,
--S--C(.dbd.S)--, --C(.dbd.O)--S--, --S--C(.dbd.O)--,
--O--S(.dbd.O).sub.2--O--, --S(.dbd.O).sub.2--O--,
--O--S(.dbd.O).sub.2--, --C(.dbd.O)--NR.sub.10--,
--NR.sub.10--C(.dbd.O)--, --O--P(.dbd.O)(--OR.sub.10)--O--,
--P(.dbd.O)(--OR.sub.10)--O--, --O--P(.dbd.O)(--OR.sub.10)--;
[0112] X is independently --R.sub.11--; and
[0113] E is independently selected from the group consisting of
--N(R.sub.12).sub.2, --N(R.sub.12)(R.sub.13), --S--R.sub.12,
[0114] and 12
[0115] where --R.sub.12 is --CH.sub.2CH.sub.2--G, where each G is
independently --Cl, --Br, --I, --O--S(.dbd.O).sub.2--CH.sub.3,
--O--S(.dbd.O).sub.2--CH.sub.2--C.sub.6H.sub.5, or
--O--S(.dbd.O).sub.2--C.sub.6H.sub.4--CH.sub.3;
[0116] and where R.sub.13 is independently --C.sub.1-8 alkyl,
--C.sub.1-8 heteroalkyl, -aryl, -heteroaryl, --C.sub.1-3alkyl-aryl,
--C.sub.1-3heteroalkyl-aryl, --C.sub.1-3alkyl-heteroaryl,
--C.sub.1-3heteroalkyl-heteroaryl, -aryl-C.sub.1-3alkyl,
-aryl-C.sub.1-3heteroalkyl, -heteroaryl-C.sub.1-3alkyl,
-heteroaryl-C.sub.1-3heteroalkyl, --C.sub.1-3alkyl-aryl-C.sub.1-3
alkyl, --C.sub.1-3heteroalkyl-aryl-C.sub.1-3 alkyl,
--C.sub.1-3alkyl-heteroaryl-- C.sub.1-3 alkyl,
--C.sub.1-3alkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 alkyl,
--C.sub.1-3heteroalkyl-aryl-C.sub.1-3 heteroalkyl,
--C.sub.1-3alkyl-heteroaryl-C.sub.1-3 heteroalkyl, or
--C.sub.1-3heteroalkyl-heteroaryl-C.sub.1-3 heteroalkyl;
[0117] and all salts and stereoisomers (including enantiomers and
diastereomers) thereof.
[0118] It will be appreciated that, in general formula I above, the
acridine nucleus is the anchor moiety, the --V--W--X-- group(s)
comprises the frangible linker, and the E group(s) is the effector
group. Similarly, in general formula III above, the psoralen
nucleus is the anchor moiety, the --V--W--X-- group(s) comprises
the frangible linker, and the E group(s) is the effector group.
General formula II is a subset of general formula I.
[0119] An exemplary compound of the invention is the structure
below, designated IV: 13
[0120] In IV, a 2-carbomethoxyacridine ring system serves as the
anchor moiety via intercalation. A bis (chloroethyl) amine group
serves as the effector moiety, which can alkylate nucleic acid; the
nitrogen mustard hydrolyzes if it does not react with nucleic acid.
The linker is
--NH--CH.sub.2CH.sub.2--C(.dbd.O)--O--CH.sub.2CH.sub.2--. In
aqueous solution at physiological pH, this ester-containing linker
hydrolyzes within hours. Changing the pH of the solution changes
the rate at which the linker hydrolyzes; for the corresponding
alcohol analog of IV (where the --Cl atoms of IV are replaced with
--OH groups), .ltoreq.1% hydrolysis of the ester linkage is
observed at pH 3 after 100 minutes at 37.degree. C.; at pH 8, more
than 50% hydrolysis of the ester linkage is observed after 100
minutes at 37.degree. C. The resulting hydrolysis products of IV
are N-(2-carbomethoxy-9-acridinyl)-.beta.-alanine and
triethanolamine: 14
[0121] where the 2-carbomethoxyacridine bears .beta.-alanine as a
linker fragment, and the effector breakdown product bears an
ethanol group as a linker fragment.
[0122] At physiological pH values, the carboxylate of the
.beta.-alanine will be negatively charged, a feature which
decreases the tendency of the attached 2-carbomethoxyacridine group
to intercalate into a negatively charged nucleic acid molecule.
This lowers the mutagenicity of
N-(2-carbomethoxy-9-acridinyl)-.beta.-alanine relative to
9-aminoacridine. This potential for lowering the mutagenicity of
the anchor fragment illustrates one advantage provided by the
frangible linker.
[0123] Another advantage of the frangible linker in compounds
similar to IV is that the hydrolysis rate can be adjusted by
varying the length of the linker arm between the 9-aminoacridine
anchor moiety and the ester function. As described in Example 7 and
Tables III and IV below, an increase in the number of methylene
groups between the aminoacridine anchor and the ester group results
in a decrease in the amount of hydrolysis seen in aqueous solution,
at pH 8, 37.degree. C., for diol analogs of certain compounds of
the invention (where the --Cl atoms of the mustards are replaced
with --OH groups).
[0124] Examples of the compounds of the invention are given below,
as illustration and not as any limitation on the invention.
151617
[0125] Applications
[0126] Examples of uses of the compounds of the invention include,
but are not limited to: addition of the compounds of the invention
in solid or solution form to biological materials, to inactivate
pathogens present in the biological materials; immersion or other
treatment of a material for biological use in a solution of the
compounds of the invention, to inactivate pathogens present in or
on the material; and inclusion of compounds of the invention in
targeted liposomes, to direct the compounds to particular cells in
order to damage the nucleic acid of those cells.
[0127] It should be noted that while the compounds of the invention
are designed to hydrolyze under certain conditions, they are stable
under other conditions. It is desirable for the frangible linker
and the effector group(s) to be relatively stable under certain
conditions used for storage. Examples of manners in which the
compounds may be stored include, but are not limited to, dry
solids, oils with low water content, frozen aqueous solutions,
frozen non-aqueous solutions, suspensions, and solutions which do
not permit hydrolysis of the frangible linker or the effector
group(s), for example liquid non-aqueous solutions. The compounds
may be stored at temperatures at or below 0.degree. C. (e.g., in a
freezer), or at temperatures above 0.degree. C. (e.g., in a
refrigerator or at ambient temperatures). The compounds preferably
are stable under the storage conditions for a period of between
three days and one year, between one week and one year, between one
month and one year, between three months and one year, between six
months and one year, between one week and six months, between one
month and six months, between three months and six months, between
one week and three months, or between one month and three months.
The stability of the compounds will be determined both by the
temperature at which they are stored, and by the state in which
they are stored (e.g., non-aqueous solution, dry solid).
[0128] Conditions for Pathogen Inactivation
[0129] Conditions for treating biological materials with a pathogen
inactivating compound may be selected based on the selected
material and the inactivating compound. Typical concentrations of
pathogen inactivating compound for the treatment of biological
materials such as blood products are on the order of about 0.1 M to
5 mM, for example about 500 M. For example, a concentration of
pathogen inactivating compound may be used which is sufficient to
inactivate at least about 1 log, or at least about 2 logs, or for
example, at least about 3 to 6 logs of a pathogen in the sample. In
one embodiment, the pathogen inactivating compound produces at
least 1 log kill at a concentration of no greater than about 500
.mu.M, more preferably at least 3 logs kill at no greater than 500
.mu.M concentration. In another non-limiting example, the pathogen
inactivating compound may have at least 1 log kill, and preferably
at least 6 logs kill at a concentration of about 0.1 M to about 3
mM.
[0130] Incubation of blood products with the pathogen inactivating
compound can be conducted for example, for about 5 minutes to 72
hours or more, or about 1 to 48 hours, for example, about 1 to 24
hours, or, for example, about 8 to 20 hours. For red blood cells,
the incubation is typically conducted at a temperature of about
2.degree. C. to 37.degree. C., preferably about 18.degree. C. to
25.degree. C. For platelets, the temperature is preferably about 20
to 24.degree. C. For plasma, the temperature may be about 0 to
60.degree. C., typically about 0-24.degree. C. The pH of the
material being treated is preferably about 4 to 10, more preferably
about 6 to 8.
[0131] One embodiment of the invention encompasses compounds and
methods for use in inactivating pathogens in blood or blood
products, and a preferred set of storage conditions for this
purpose would be those conditions that allow the convenient storage
and use of the compounds at blood banks.
[0132] Under the conditions used for pathogen inactivation in or on
a material, the frangible linker and effector group(s) will undergo
hydrolysis or reaction. The hydrolysis, of both the frangible
linker and the effector groups(s), preferably is slow enough to
enable the desired amount of pathogen inactivation to take place.
The time required for pathogen inactivation may be, for example,
about 5 minutes to 72 hours.
[0133] Treatment of Red Blood Cells
[0134] Preferably, treatment of red blood cell containing materials
with the pathogen inactivating compound does not damage red blood
cell function or modify red blood cells after treatment. The lack
of a substantially damaging effect on red blood cell function may
be measured by methods known in the art for testing red blood cell
function. For example, the levels of indicators such as
intracellular ATP (adenosine 5'-triphosphate), intracellular
2,3-DPG (2,3-diphosphoglycerol) or extracellular potassium may be
measured, and compared to an untreated control. Additionally
hemolysis, pH, hematocrit, hemoglobin, osmotic fragility, glucose
consumption and lactate production may be measured.
[0135] Methods for determining ATP, 2,3-DPG, glucose, hemoglobin,
hemolysis, and potassium are available in the art. See for example,
Davey et al., Transfusion, 32:525-528 (1992), the disclosure of
which is incorporated herein. Methods for determining red blood
cell function are also described in Greenwalt et al., Vox Sang,
58:94-99 (1990); Hogman et al., Vox Sang, 65:271-278 (1993); and
Beutler et al., Blood, Vol. 59 (1982) the disclosures of which are
incorporated herein by reference. Extracellular potassium levels
may be measured using a Ciba Corning Model 614 K.sup.+/Na.sup.+
Analyzer (Ciba Corning Diagnostics Corp., Medford, Mass.). The pH
can be measured using a Ciba Corning Model 238 Blood Gas Analyzer
(Ciba Coming Diagnostics Corp., Medford, Mass.).
[0136] Binding of species such as IgG, albumin, and IgM to red
blood cells also may be measured using methods available in the
art. Binding of molecules to red blood cells can be detected using
antibodies, for example to acridine and IgG. Antibodies for use in
assays can be obtained commercially, or can be made using methods
available in the art, for example as described in Harlow and Lane,
"Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory,"
1988, the disclosure of which is incorporated herein. For example,
anti-IgG is commercially available from Caltag, Burlingame, Calif.;
Sigma Chemical Co., St. Louis, Mo. and Lampire Biological
Laboratory, Pipersvelle, Pa.
[0137] In a method of treatment of a material comprising red blood
cells with the pathogen inactivating compound, preferably the level
of extracellular potassium is not greater than 3 times, more
preferably no more than 2 times the amount exhibited in the
untreated control after 1 day. In another embodiment, preferably,
hemolysis of the treated red blood cells is less than 3% after 28
day storage, more preferably less than 2% after 42 day storage, and
most preferably less than or equal to about 1% after 42 day storage
at 4.degree. C.
[0138] Biological Materials
[0139] A variety of biological materials may be treated with a
pathogen inactivating compound. Biological materials include blood
products such as whole blood, packed red blood cells, platelets and
fresh or frozen plasma. Blood products further encompass plasma
protein portion, antihemophilic factor (Factor VIII), Factor IX and
Factor IX complex, fibrinogens, Factor XIII, prothrombin and
thrombin, immunoglobulins (such as IgG, IgA, IgD, IgE and IgM and
fragments thereof), albumin, interferon, and lymphokines. Also
contemplated are synthetic blood products.
[0140] Other biological materials include vaccines, recombinant DNA
produced proteins and oligopeptide ligands. Also encompassed are
clinical samples such as urine, sweat, sputum, feces, spinal fluid.
Further encompassed are synthetic blood or blood product storage
media.
[0141] Reducing the Concentration of Compounds in Materials After
Treatment
[0142] The concentration of the pathogen inactivating compound in a
biological material, such as a blood product, can be reduced after
the treatment. Methods and devices which may be used are described
in PCT/US96/09846; U.S. Ser. No. 08/779,830, filed Jan. 6, 1997;
and in the co-filed application, "Methods and Devices for the
Reduction of Small Organic Compounds from Blood Products",
PCT/US98/00531, filed Jan. 6, 1998, the disclosures of each of
which are incorporated herein by reference in their entirety.
[0143] Quenching
[0144] In another embodiment the compounds of the invention may be
used in combination with a quencher. Methods for quenching
undesired side reactions of pathogen inactivating compounds in
biological materials are described in the cofiled U.S. Provisional
Application Serial No. 60/070,597, filed Jan. 6, 1998, Attorney
Docket No. 282173000600, "Methods for Quenching Pathogen
Inactivators in Biological Materials," the disclosure of which is
incorporated herein. Disclosed in the cofiled application are
methods for quenching undesired side reactions of a pathogen
inactivating compound that includes a functional group which is, or
which is capable of forming, an electrophilic group. In this
embodiment, the material is treated with the pathogen inactivating
compound and a quencher, wherein the quencher comprises a
nucleophilic functional group that is capable of covalently
reacting with the electrophilic group. Preferred quenchers are
thiols, such as glutathione.
EXAMPLES
[0145] The following specific examples are presented to illustrate
the preparative methods for representative compounds useful in the
method of this invention, to provide relevant data regarding the
compounds useful to the practitioner, and to illustrate the manner
in which the effectivity of the compounds is determined, and are
not to be construed as limiting the scope of the invention. All NMR
spectra were recorded on a Varian 200 MHz instrument in CDCl.sub.3
unless otherwise noted; chemical shifts are reported versus
tetramethylsilane (TMS). IR spectra were recorded with a Perkin
Elmer FTIR. HPLC was carried out with a YMC C8 column in a gradient
mode using 5 mM aq. H.sub.3PO.sub.4 as mobile phase A and 5 mM
CH.sub.3CN as mobile phase B. Samples were prepared in DMSO or
ethanol and kept at .ltoreq.15.degree. C. prior to injection.
[0146] Table II indicates the designation of compound number used
for the various compounds.
2TABLE II COMPOUND NUMBER CHEMICAL NAME IV .beta.-alanine,
N-(2-carbomethoxyacridin-9-yl), 2-[bis(2- chloroethyl)amino]ethyl
ester V .beta.-alanine, N-(acridin-9-yl),
2-[bis(2-chloroethyl)amino] ethyl ester VI 4-aminobutyric acid
N-[(2-carbomethoxyacridin-9-yl), 2- [bis(2-chloroethyl)amino]ethyl
ester VII 5-aminovaleric acid N-[(2-carbomethoxyacridin-9-yl), 2-
[bis(2-chloroethyl)amino]ethy- l ester VIII .beta.-alanine,
N-(2-carbomethoxyacridin-9-yl), 3-[bis(2- chloroethyl)amino]propyl
ester IX .beta.-alanine, N-(4-methoxyacridin-9-yl), 2-[bis(2-
chloroethyl)amino]ethyl ester X .beta.-alanine,
N-(3-chloro-4-methylacridin-9-yl), 2-[bis(2-
chloroethyl)amino]ethyl ester XI .beta.-alanine,
[N,N-bis(2-chloroethyl)], 3-[(6-chloro-2- methoxyacridin-9-yl)ami-
no]propyl ester XII .beta.-alanine, [N,N-bis(2-chloroethyl)],
2-[(6-chloro-2- methoxyacridin-9-yl)amino]ethyl ester XIII
.beta.-alanine, N-(6-chloro-2-methoxyacridin-9-yl), 2-[bis(2-
chloroethyl)amino]ethyl ester XIV [N,N-bis(2-chloroethyl)]-2-amino-
ethyl 4,5', 8-trimethyl-4'-psoralenacetate XV .beta.-alanine,
N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino] ethyl amide
Example 1
Synthesis of .beta.-Alanine, N-(2-Carbomethoxyacridin-9-yl),
2-[bis(2-Chloroethyl)amino]ethyl Ester Dihydrochloride (Compound
IV,)
[0147] Step A. .beta.-Alanine, N-(tert-Butoxycarbonyl),
2-[bis(2-Hydroxyethyl)amino]ethyl Ester
[0148] To a stirred solution of
N-(tert-butoxycarbonyl)-.beta.-alanine (20.3 g, 107 mmol) and
4-methylmorpholine (13.0 mL, 12.0 g, 119 mmol) in dry THF (200 mL)
at -15.degree. C. under N.sub.2 was added isobutyl chloroformate
(13.9 mL, 14.6 g, 107 mmol) resulting in the immediate formation of
a white precipitate (4-methylmorpholine.HCl). The reaction mixture
was stirred at -15.degree. C. for 5 min. followed by the transfer
of the reaction mixture to a flask containing a stirred solution of
triethanolamine (48.3 g, 324 mmol) in dry THF (150 mL) at
-15.degree. C. The reaction mixture was allowed to warm to
23.degree. C. and stirred for an additional 1.5 h. followed by
removal of the precipitate by vacuum filtration. The THF was then
removed in vacuo from the filtrate and the remaining viscous yellow
oil was partitioned between water (500 mL) and EtOAc (5.times.150
mL). The combined organic layers were dried over Na.sub.2SO.sub.4.
Removal of solvent in vacuo gave 25.8 g (75%) of the desired
product, .beta.-alanine, N-(tert-butoxycarbonyl),
2-[bis(2-hydroxyethyl)amino]ethyl ester, as a pale yellow oil.
.sup.1H NMR: .delta. 5.32 (br s, 1H), 4.18 (t, J=5.4 Hz, 2H), 3.58
(t, J=5.1 Hz, 4H), 3.37-3.23 (m, 2H), 2.80 (t, J=5.4 Hz, 2H), 2.69
(t, J=5.1 Hz, 4H), 2.51 (t, J=6.0 Hz, 2H), 1.41 (s, 9H) The
hydroxyl protons were not observed. .sup.13C NMR: .delta. 173.0,
156.4, 79.8, 63.3, 60.2, 57.3, 54.1, 36.7, 35.3, 28.8.
[0149] Step B. .beta.-Alanine, N-(tert-Butoxycarbonyl),
2-[bis(2-tert-Butyldimethylsilyloxyethyl)amino]ethyl Ester
[0150] A stirred solution of the .beta.-alanine,
N-(tert-butoxycarbonyl), 2-[bis(2-hydroxyethyl)amino]ethyl ester
from step A (22.7 g, 70.9 mmol) and imidazole (11.1 g, 163 mmol) in
acetonitrile (70 mL) under N.sub.2 was cooled to 0.degree. C.
Tert-butyldimethylsilyl chloride (534 mg, 3.54 mmol) was then added
and the reaction mixture was stirred for an additional 5 min. at
0.degree. C. The reaction mixture was allowed to warm to 23.degree.
C. and stirred for 2 h followed by removal of the resultant white
precipitate (imidazole.HCl by vacuum filtration. The acetonitrile
was removed in vacuo from the filtrate and the remaining material
was partitioned between saturated brine (600 mL) and EtOAc
(3.times.200 mL). The combined organic layers were dried over
Na.sub.2SO.sub.4. Removal of solvent in vacuo gave 35.2 g (90%) of
the desired product, .beta.-alanine, N-(tert-butoxycarbonyl),
2-[bis(2-tert-butyldimethylsilyloxyethyl)amino]ethyl ester, as a
yellow oil. .sup.1H NMR: .delta. 5.29 (br s, 1H), 4.14 (t, J=6.0
Hz, 2H), 3.65 (t, J=6.3 Hz, 4H), 3.37 (apparent q, 2H), 2.85 (t,
J=6.0 Hz, 2H), 2.71 (t, J=6.3 Hz, 4H), 2.49 (t, J=5.9 Hz, 2H), 1.42
(s, 9H), 0.88 (s, 18H), 0.03 (s, 12H); .sup.13C NMR: .delta. 172.7,
156.3, 79.7, 63.3, 62.4, 57.7, 54.3, 36.7, 35.3, 28.9, 26.4, 18.7,
-4.9.
[0151] Step C. .beta.-Alanine,
2-[bis(2-tert-Butyldimethylsilyloxyethyl)am- ino]ethyl Ester
[0152] To a flask containing .beta.-alanine,
N-(tert-butoxycarbonyl),
2-[bis(2-tert-butyldimethylsilyloxyethyl)amino]ethyl ester from
step B (3.01 g, 5.48 mmol) was added neat trifluoroacetic acid (5
mL) resulting in the evolution of CO.sub.2 gas. The reaction
mixture was stirred for 5 min. and the trifluoroacetic acid was
removed in vacuo. The remaining material was partitioned between
saturated NaHCO.sub.3 (100 mL) and EtOAc (3.times.30 mL). The
combined organic layers were dried over Na.sub.2SO.sub.4. Removal
of solvent in vacuo gave 2.45 g (100%) of the desired product,
.beta.-alanine, 2-[bis(2-tert-butyldimethylsilyloxyethyl-
)amino]ethyl ester, as a pale yellow oil. .sup.1H NMR: .delta. 4.12
(t, J=6.0 Hz, 2H), 3.63 (t, J=6.4 Hz, 4H), 2.96 (t, J=6.2 Hz, 2H),
2.84 (t, J=6.0 Hz, 2H), 2.69 (t, J=6.4 Hz, 4H), 2.44 (t, J=6.2 Hz),
0.86 (s, 18H), 0.03 (s, 12H). The amine protons were not observed.
.sup.13C NMR (CDCl.sub.3): .delta. 173.0, 63.4, 62.6, 57.9, 54.4,
38.4, 38.1, 26.4, 18.7, -4.9.
[0153] Step D. .beta.-Alanine, N-(2-Carbomethoxyacridin-9-yl),
2-[bis(2-Hydroxyethyl)amino]ethyl Ester
[0154] The .beta.-alanine,
2-[bis(2-tert-butyldimethylsilyloxyethyl)amino]- ethyl ester (736
mg, 1.64 mmol) was reacted with methyl
9-methoxyacridine-2-carboxylate (669 mg, 2.50 mmol) by stirring in
10 mL of CHCl.sub.3 for 12.5 h at room temperature. The precipitate
(acridone) was then filtered off and the filtrate partitioned
between saturated aqueous NaHCO.sub.3 (100 mL) and CHCl.sub.3
(3.times.35 mL). The combined organic layers were dried over
Na.sub.2SO.sub.4 and concentrated in vacuo to give 1.61 g of
viscous brown oil. Deprotection of the resultant diol was carried
out by dissolving the crude intermediate in 3.0 mL of THF under
N.sub.2 and, upon cooling to 0.degree. C., treating with
HF/pyridine (1.0 mL). The solution was allowed to warm to room
temperature with stirring for 1 h. The volatiles were removed in
vacuo and the residue was partitioned between saturated aqueous
NaHCO.sub.3 (100 mL) and CHCl.sub.3 (3.times.35 mL). The combined
organic layers were dried and concentrated to give 649 mg of a
brownish yellow solid. Preparative TLC (C-18, CH.sub.3CN) gave a
20% yield of the desired diol, .beta.-alanine,
N-(2-carbomethoxyacridin-9-yl), 2-[bis(2-hydroxyethyl)ami- no]ethyl
ester (>80% pure by HPLC);
[0155] .sup.1H NMR: .delta. 8.82 (s, 1H), 8.21-7.94 (m, 2H),
7.94-7.72 (m, 2H), 7.59 (apparent t, 1H), 7.23 (apparent t, 1H),
4.30-4.18 (m, 2H), 4.18-4.05 (m, 2H), 3.89 (s, 3H), 3.69-3.50 (m,
4H), 2.92-2.73 (m, 4H), 2.73-2.55 (m, 4H) The amine and hydroxyl
protons were not observed.
[0156] Step E. .beta.-Alanine, N-(2-Carbomethoxyacridin-9-yl),
2-[bis(2-Chloroethyl)amino]ethyl Ester Dihydrochloride
[0157] Conversion of .beta.-alanine,
N-(2-carbomethoxyacridin-9-yl), 2-[bis(2-hydroxyethyl)amino]ethyl
ester to the dichloro compound was achieved by a method similar to
that of Peck, et al. (J. Am. Chem. Soc. 1959, 81: 3984). A yellow
solution of the product from step D (41 mg, 0.090 mmol) in neat
SOCl.sub.2 (6 mL) was stirred at room temperature for 20 hours. The
SOCl.sub.2 was then removed in vacuo to give a yellow solid
(dihydrochloride salt). The material was then partitioned between
saturated NaHCO.sub.3 (50 mL) and CH.sub.2Cl.sub.2 (3.times.20 mL).
The combined organic layers were dried over Na.sub.2SO.sub.4.
Removal of solvent in vacuo gave 35.4 mg of the dichloro compound
free base as an orange gum. .sup.1H NMR: .delta. 8.82 (s, 1H),
8.20-7.83 (m, 4H), 7.5 (apparent t, 1H), 7.25 (apparent t, 1H),
4.36-4.15 (m, 4H), 3.39 (s, 3H), 3.48 (t, J=6.9 Hz, 4H), 3.06-2.77
(m, 4H), 2.86 (t, J=6.9 Hz, 4H) The amine proton was not observed.
.sup.13C NMR: .delta. 172.3, 166.6, 155.2, 146.5, 144.6, 133.1,
131.6, 128.7, 124.6, 124.3, 116.1, 114.3, 63.7, 57.2, 53.5, 52.9,
46.3, 42.5, 35.2. No other carbons were observed. The HCl salt was
precipitated from CH.sub.2Cl.sub.2 by addition of 1 M HCl in ether
to give .beta.-alanine, N-(2-carbomethoxyacridin-9-yl),
2-[bis(2-chloroethyl)amino]ethyl ester dihydrochloride (Compound
IV,) as a yellow solid (81% pure by HPLC).
[0158] .beta.-Alanine, N-(acridin-9-yl),
2-[bis(2-chloroethyl)amino]ethyl ester dihydrochloride, (Compound
V) was prepared in a similar manner. Thus using 9-methoxyacridine
in place of methyl 9-methoxyacridine-2-carbo- xylate in Step D, the
intermediate diol was obtained (7.1%) as a yellow oil (74% pure by
HPLC). .sup.1H NMR: .delta. 8.14 (d, J=7.5 Hz, 2H), 7.93 (d, J=8.6
Hz, 2H), 7.52 (apparent t, 2H), 7.23 (apparent t, 2H), 4.36-4.08
(m, 4H), 3.76-3.5 (m, 4H), 3.08-2.60 (m, 8H) The amine and hydroxyl
protons were not observed.
[0159] A solution of the intermediate diol (37.3 mg, 0.0793 mmol)
in thionyl chloride (4.0 mL) was stirred at 23.degree. C. for 7.5
h. The thionyl chloride was removed in vacuo to give a yellow oil.
The material was dissolved in ethanol (.about.4 mL) and the solvent
removed in vacuo. The material was then dissolved in
CH.sub.2Cl.sub.2 (4 mL) and solvent removed in vacuo; this step was
repeated twice. The material was then triturated with hexane
(3.times.4 mL) to give 40.0 mg (42% pure by HPLC) of the product in
the form of a yellow hydroscopic glassy solid. Some of the material
was converted to the free amine for analytical purposes by
partitioning between saturated NaHCO.sub.3 and CH.sub.2Cl.sub.2
followed by drying the combined organic layers over
Na.sub.2SO.sub.4 and removal of the solvent in vacuo. .sup.1H NMR:
.delta. 8.21-8.00 (m, 4H), 7.66 (apparent t, 2H), 7.38 (apparent t,
2H), 4.26-4.12 (m, 2H), 4.12-3.98 (m, 2H), 3.43 (t, J=6.9 Hz, 4H),
2.96-2.68 (m, 8H) The amine proton was not observed.
[0160] Following the above procedure but replacing
N-(tert-butoxycarbonyl)- -.beta.-alanine with
N-(tert-butoxycarbonyl)-4-aminobutyric acid led to the preparation
of 4-aminobutyric acid N-[(2-carbomethoxyacridin-9-yl),
2-[bis(2-chloroethyl)amino]ethyl ester dihydrochloride, Compound VI
(78% pure by HPLC). .sup.1H NMR: .delta. 8.89 (s, 1), 8.12
(apparent t, 2), 7.93-7.80 (m, 2), 7.59 (apparent q, 1), 7.36-7.20
(m, 1), 4.16 (t, 2, J=5.7 Hz), 4.07-3.92 (m, 2), 3.97 (s, 3), 3.46
(t, 4, J=6.9 Hz), 2.93-2.80 (m, 6), 2.60 (t, 2, J=6.5 Hz),
2.29-2.12 (m, 2). The amine proton was not observed.
Example 2
[0161] Substituting the triethanolamine in Example 1, Step A with
3-[N,N-Bis(2-tert-butyldimethylsilyloxyethyl)]aminopropanol, and
then continuing from step C, led to the preparation of
.beta.-alanine, N-(2-carbomethoxy-acridin-9-yl),
3-[bis(2-chloroethyl)amino]propyl ester dihydrochloride, Compound
VIII, (63% pure by HPLC).
[0162] .sup.1H NMR: .delta. 8.91 (s, 1), 8.20-7.93 (m, 4), 7.18
(apparent t, 1), 7.39 (apparent t, 1), 4.30 (m, 4), 3.96 (s, 3),
3.48 (t, 4, J=6.9 Hz), 2.88-2.60 (m, 2), 2.83 (t, 4, J=6.9 Hz),
2.62 (t, 2, J=6.7 Hz), 1.85-1.68 (m, 2) The amine proton was not
observed.
Example 3
[0163] The compounds synthesized in Example 1 can also be prepared
by the following method:
Synthesis of .beta.-Alanine, N-(Acridin-9-yl),
2-[bis(2-Chloroethyl)amino]- ethyl Ester Dihydrochloride (Compound
V): Method II
[0164] Step A: .beta.-Alanine, N-(Acridin-9-yl), Methyl Ester
Hydrochloride
[0165] 9-Chloroacridine (11.7 g, Organic Synthesis, Coll. Vol III,
pg 57), .beta.-alanine methyl ester hydrochloride (9.9 g) and
sodium methoxide (3.26 g) were combined and 60 mL of methanol was
added. The mixture was stirred with a magnetic stirrer and refluxed
for 5.5 h. Heat was removed and the suspension was filtered while
warm (.ltoreq.35.degree. C.). The solid salts were rinsed with
about 10 mL of additional methanol and the combined dark green
filtrate was concentrated to give 21 g of a moist greenish-yellow
solid.
[0166] The solid was dissolved in 350 mL of boiling 2-propanol and
allowed to crystallize at room temperature. The resulting crystals
were rinsed with about 15 mL of 2-propanol and 15 mL of hexane,
then air dried to give 15.5 g of bright yellow product,
.beta.-alanine, N-(acridin-9-yl), methylester hydrochloride, (yield
78.5%). .sup.1H NMR: .delta. 1.9 (br s, 2H); 3.24 (t, J=7.0 Hz,
2H); 3.76 (s, 3H); 4.45 (br s, 2H); 7.23 (app. t, J=8 Hz, 2H); 7.49
(app. t, J=8 Hz, 2H); 8.11 (d, J=8.4 Hz, 2H); 8.30 (d, J=8.4 Hz,
2H); 9.68 (br s, 0.5H). IR: 1574 (s), 1691 (s), 1726 (s), 2336 (m),
2361 (m), 3227 (m).
[0167] Step B: .beta.-Alanine, N-(Acridin-9-yl),
2-[bis(2-Hydroxyethyl)ami- no]ethyl Ester Dihydrochloride
[0168] The .beta.-alanine, N-(acridin-9-yl), methyl ester
hydrochloride, from Step A, (5.00 g) was partitioned between
toluene (750 mL), saturated aqueous Na.sub.2CO.sub.3 (200 mL) and
H.sub.2O (50 mL). The aqueous layer was extracted again with
toluene (3.times.250 mL) and the organic layers were combined and
washed with saturated aqueous Na.sub.2CO.sub.3 (50 mL). The volume
of toluene was reduced to about 100 mL by rotary evaporation.
Triethanolamine (30 mL) was then added to form a partially
immiscible system. A solution of NaOMe (50 mg) in MeOH (2 mL) was
then added. Solvents were quickly removed from the reaction mixture
by rotary evaporation with agitation at room temperature. After the
solvent was removed the reaction mixture was left under vacuum for
another 1-1.5 h to give a syrupy solution.
[0169] The crude mixture was partitioned between CH.sub.2Cl.sub.2
(200 mL) and brine (200 mL) to remove excess triethanolamine. The
brine layer was extracted with CH.sub.2Cl.sub.2 (5.times.100 mL).
The organic layers were combined and washed with brine (50 mL) then
extracted with 0.5M HCl (2.times.100 mL). The aqueous acid layers
were combined and washed with CH.sub.2Cl.sub.2 (50 mL). The acid
solution was made basic with powdered K.sub.2CO.sub.3 (s) in the
presence of CH.sub.2Cl.sub.2 (200 mL). The organic layer was
separated and the aqueous layer was extracted again with
CH.sub.2Cl.sub.2 (5.times.100 mL). The combined organic layers were
washed with brine (50 mL), dried with anhydrous
Na.sub.2SO.sub.4(s), and stripped to give crude diol free amine
(5.02 g), a sticky yellow gum. This material was identical by NMR
to that prepared in Example 1 by an alternate procedure.
[0170] A portion of the above crude (0.400 g) was vigorously
stirred with isopropanol (100 mL) and acidified with 1 M HCl in
ether. The slurry was chilled and the first precipitate was
discarded. After removing half the solvent the second set of
crystals gave .beta.-alanine, N-(acridin-9-yl),
2-[bis(2-hydroxyethyl)amino]ethyl ester dihydrochloride as a bright
yellow crystalline solid (0.200 g) >95% pure by HPLC. .sup.1H
NMR: .delta. 8.11 (apparent t, 4H), 7.69 (apparent t, 2H), 7.41
(apparent t, 2H), 4.23 (t, J=5.4 Hz, 2H), 4.03 (t, J=5.9 Hz, 2H),
3.58 (t, J=5.2 Hz, 4H), 2.73 (t, J=5.4 Hz, 2H), 2.70 (t, J=5.9 Hz,
2H), 2.68 (t, J=5.2 Hz, 4H). The amine and hydroxyl protons were
not observed. .sup.13C NMR: .delta. 173.3, 151.7, 149.4, 130.5,
129.5, 124.0, 123.4, 118.4, 63.5, 60.1, 57.3, 54.0, 46.6, 35.8.
[0171] Step C: .beta.-Alanine, N-(Acridin-9-yl),
2-[bis(2-Chloroethyl)amin- o]ethyl Ester Dihydrochloride
[0172] SOCl.sub.2 (0.5 ml) was added to a stirred suspension of
.beta.-alanine, N-(acridin-9-yl), 2-[bis(2-hydroxyethyl)amino]ethyl
ester dihydrochloride from Step B (113 mg, 0.24 mmol) in CH.sub.3CN
(0.5 mL). The resultant yellow solution was stirred at 23.degree.
C. for 16 h followed by removal of the volatiles in vacuo. The
remaining orange oil was dissolved in EtOH (.about.2 mL) and the
EtOH was removed in vacuo to give a yellow solid. The material was
then triturated with hexane (2.times.3 mL). Removal of residual
solvents in vacuo gave 123 mg of the desired material,
.beta.-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl
ester dihydrochloride, (93% pure by HPLC) as a yellow solid.
.sup.1H NMR: .delta. 8.09 (apparent t, J=8.8 Hz, 4H), 7.66
(apparent t, J=7.6 Hz, 2H), 7.38 (apparent t, J=7.7 Hz, 2H), 4.14
(t, J=5.9 Hz, 2H), 4.00 (t, J=5.8 Hz, 2H), 3.43 (t, J=6.9 Hz, 4H),
2.87 (t, J=6.9 Hz, 4H), 2.77 (t, J=5.9 Hz, 2H), 2.69 (t, J=5.8 Hz,
2H). The amine proton was not observed. .sup.13C NMR: .delta.
173.0, 151.5, 149.4, 130.5, 129.6, 124.1, 123.4, 118.6, 63.5, 57.3,
53.5, 46.7, 42.5, 35.7. IR (KBr pellet of HCl salt): 3423, 3236,
2939, 2879, 1736, 1634, 1586, 1572, 1540, 1473, 1272, 1173
cm.sup.-1.
Example 4
.beta.-Alanine, N-(4-Methoxy-acridin-9-yl),
2-[bis(2-Chloroethyl)amino]eth- yl Ester Dihydrochloride, Compound
IX
[0173] .beta.-alanine, N-(4-methoxy-acridin-9-yl), methyl ester was
prepared by mixing 1.4 g (5.84 mmol) of 4,9-dimethoxyacridine, 0.89
g (6.42 mmol) of .beta.-alanine methyl ester hydrochloride and 20
ml of methanol and then heating to reflux for 12 h under N.sub.2.
The reaction was then concentrated in vacuo, dissolved in
CHCl3-isopropanol (50 ml, 4:1 .nu./.nu.), and washed with 50%
NH.sub.4OH (2.times.25 ml) and brine (1.times.25 ml). The organic
layer was dried with Na.sub.2SO.sub.4 and concentrated in vacuo to
yield 1.24 g (68%) of the methyl ester (>74% purity by HPLC) as
a yellow oil; R.sub.f (SiO2, ethyl acetate)=0.25; IR (thin film):
3363, 2947, 1730, 1611, 1573, 1518, 1484, 1463, 1423, 1420, 1246,
1170, 1081 cm.sup.-1; .sup.1H NMR: .delta. 2.70 (t, 2H, J=5.7 Hz),
3.74 (s, 3H), 4.00 (t, 2H, J=6.3 Hz), 4.11 (s, 3H), 6.98 (d, 1H,
J=7.4 Hz), 7.36 (m, 2H), 7.65 (m, 2H), 8.12 (d, 2H, J=8.5 Hz);
.sup.13C NMR): 635.7, 46.9, 52.3, 56.5, 107.2, 115.3, 119.8, 123.5,
124.1, 130.0, 151.4, 173.6.
[0174] This was converted to the diol under conditions described in
Example 3, Step B to afford 647 mg of a yellow oil. HPLC analysis
of the crude mixture indicated a yield of 85%(.lambda.=278 nm);
R.sub.f (SiO2, 20% methanol-ethyl acetate)=0.17; IR (thin film):
3337, 2947, 2828, 1726, 1616, 1569, 1522, 1484, 1463, 1420, 1348,
1250, 1174, 1127, 1081, 1043 cm.sup.-1; .sup.1H NMR: .delta. 2.7
(m, 8H), 3.55 (m, 4H), 3.97-4.08 (m, 2H), 4.08 (s, 3H), 4.19 (t,
2H, J=5.5 Hz), 6.96 (d, 1H, J=7.4 Hz), 7.29 (m, 2H), 7.61 (m, 2H),
8.10 (m, 2H); .sup.13C NMR: .delta. 36.0, 46.9, 53.7, 56.4, 57.1,
60.1, 63.3, 107.4, 115.7, 119.1, 119.6, 123.2, 123.5, 123.9, 128.5,
130.0, 140.8, 147.4, 151.6, 151.7, 154.3, 173.3.
[0175] This was converted to .beta.-alanine,
N-(4-methoxy-acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester
dihydrochloride with thionyl chloride as described in Example 3,
Step C. Flash filtration (SiO.sub.2) of the crude product using
ethyl acetate followed by 10% methanol-ethyl acetate gave 58 mg of
a yellow oil after apparent on-column degradation of some product;
R.sub.f (SiO2, ethyl acetate)=0.26; IR (thin film): 3405, 2955,
2828, 1726, 1616, 1577, 1518, 1463, 1416, 1348, 1246, 1174, 1123,
1081, 1013 cm.sup.-1; .sup.1H NMR: .delta. 2.69-2.99 (m, 8H), 3.45
(t, 4H, J=6.7 Hz), 4.03 (m, 2H), 4.09 (s, 3H), 4.16 (t, 2H, J=5.9
Hz), 6.97 (d, 1H, J=7.7 Hz), 7.32 (m, 2H), 7.65 (m, 2H), 8.12 (d,
2H, J=8.7 Hz).
[0176] The dihydrochloride salt could be isolated in crude form by
concentrating the reaction in vacuo with azeotropic removal of
excess thionyl chloride (2.times.5 ml toluene). HPLC analysis
indicated complete consumption of the starting material and
4-methoxy acridone (R.sub.T=22.3 min) to be the major impurity.
.sup.1H NMR (CD.sub.3OD): .delta. 3.18 (t, 2H, J=6.4 Hz), 3.71 (m,
6H), 4.04 (m, 4H), 4.18 (s, 3H), 4.51 (m, 2H), 7.17 (m, 2H), 7.56
(m, 2H), 7.91-8.15 (m, 2H), 8.55 (d, 1H, J=8.8 Hz).
[0177] Similarly prepared from 3-chloro-9-methoxy-4-methylacridine
was .beta.-alanine, N-(3-chloro-4-methylacridin-9-yl),
2-[bis(2-chloroethyl)amino]ethyl ester dihydrochloride, Compound X.
.sup.1H NMR of the free base: .delta. 7.96-8.17 (m, 3H), 7.29-7.52
(m, 3H), 4.19 (t, J=5.8 Hz, 2H), 4.00 (s, 3H), 3.89 (t, J=5.1 Hz,
2H), 3.47 (t, J=6.8 Hz, 4H), 2.91 (t, J=6.8 Hz, 4H), 2.83 (t, J=5.8
Hz, 2H), 2.67 (t, J=5.5 Hz, 2H).
[0178] Similarly prepared from 6-chloro-2,9-dimethoxyacridine was
.beta.-alanine, N-(6-chloro-2-methoxyacridin-9-yl),
2-[bis(2-chloroethyl)amino]ethyl ester dihydrochloride, Compound
XIII. .sup.1H NMR of the free base: .delta. 7.96-8.17 (m, 3H),
7.29-7.52 (m, 3H), 4.19 (t, J=5.8 Hz, 2H), 4.00 (s, 3H), 3.89 (t,
J=5.1 Hz, 2H), 3.47 (t, J=6.8 Hz, 4H), 2.91 (t, J=6.8 Hz, 4H), 2.83
(t, J=5.8 Hz, 2H), 2.67 (t, J=5.5 Hz, 2H).
Example 5
.beta.-Alanine, [N,N-bis(2-Chloroethyl)],
3-[(6-Chloro-2-methoxyacridin-9-- yl)amino]propyl Ester
Dihydrochloride, Compound XI
[0179] Step A .beta.-Alanine,
[N,N-bis(2-Triisopropylsilyloxy)ethyl]ethyl Ester
[0180] A slurry of .beta.-alanine ethyl ester hydrochloride (1.99
g, 12.9 mmol), K.sub.2CO.sub.3 (6.0 g, 43.4 mmol) and iodoethyl
triisopropylsilyl ether (9.47 g, 28.9 mmol) in acetonitrile (175
mL) were refluxed for 5-7 days. After vacuum evaporation of the
solvent, the solid was triturated with CH.sub.2Cl.sub.2. The
organic layer was washed with dilute Na.sub.2CO.sub.3(aq), then
with brine and dried over anhydrous Na.sub.2SO.sub.4. The crude
product was purified by silica gel chromatography (1:4
EtOAc/hexane) to provide 5.60 g of the oil, .beta.-alanine,
[N,N-bis(2triisopropylsilyloxy)ethyl]ethyl ester, (83.1%). .sup.1H
NMR: .delta. 4.12 (q, J=7.1 Hz, 2H), 3.73 (t, J=6.8 Hz, 4H), 2.92
(t, J=7.3 Hz, 2H), 2.70 (t, J=6.6 Hz, 4H), 2.46 (t, J=7.4 Hz, 2H),
1.4-0.9 (m, 45H, includes triplet at 1.25 (3H) and singlets at 1.06
and 1.05).
[0181] Step B .beta.-Alanine,
N,N-bis(2-Triisopropylsilyloxy)ethyl
[0182] The .beta.-alanine,
[N,N-bis(2-triisopropylsilyloxy)-ethyl]ethyl ester from Step A
above (5.60 g, 10.8 mmol) and lithium hydroxide (0.59 g, 14.1 mmol)
were stirred in ethanol and refluxed for 3 h. The solvent was
removed and the crude product was partitioned between
CH.sub.2Cl.sub.2 and dilute NaHCO.sub.3(aq). The organic layer was
washed with brine, dried over anhydrous Na.sub.2SO.sub.4, and
stripped to give .beta.-alanine,
N,N-bis(2-triisopropylsilyloxy)ethyl as a pale yellow oil (5.03 g,
95.1% yield). .sup.1H NMR: .delta. 3.90 (t, J=5.5 Hz, 4H), 3.04 (t,
J=6.2 Hz, 2H), 2.92 (t, J=5.5 Hz, 4H), 2.50 (t, J=6.1 Hz, 2H), 1.06
(s, 42H).
[0183] Step C .beta.-Alanine, [N,N-bis(2-Hydroxyethyl)],
3-[(6-Chloro-2-methoxyacridin-9-yl)amino]propyl Ester
[0184] The .beta.-alanine, N,N-bis(2-triisopropylsilyloxy)ethyl
from Step B above (51.0 mg, 0.104 mmol) was stirred under N.sub.2
in CH.sub.2Cl.sub.2 (1 mL). While chilling on an ice bath,
SOCl.sub.2 (0.5 mL) was added dropwise and the reaction was stirred
for 2.25 h. After stripping the reaction mixture to remove excess
SOCl.sub.2, dry CH.sub.2Cl.sub.2 (0.5 mL) was added and the
solution was chilled in an ice bath while under N.sub.2. A chilled
slurry of 9-(3-hydroxy)propylamin- o-6-chloro-2-methoxy-acridine
(29.0 mg, 91.5 mmol) in CH.sub.2Cl.sub.2 (1 mL) was added. After
0.5 h the mixture was partitioned between CH.sub.2Cl.sub.2 and
aqueous NaHCO.sub.3. The organic layer was washed with brine, dried
with anhydrous Na.sub.2SO.sub.4, and stripped. The gum obtained was
triturated with hexane and the hexane extract was stripped to
obtain a very crude mixture (53.5 mg) of triisopropylsilyl
protected starting material and product.
[0185] To remove the triisopropylsilyl groups, a portion of the
crude protected diol (33.1 mg) was stirred in ice cold THF (1 mL).
After the addition of HF/pyridine (0.5 mL) the mixture was stirred
at ambient temperature under a N.sub.2 filled balloon for 2.5 h.
The reaction mix was partitioned between CH.sub.2Cl.sub.2 and
NaHCO.sub.3(aq) and the organic layer was washed several times with
dilute NaHCO.sub.3 (aq) to remove excess HF/pyridine. After
preliminary drying with brine, then with anhydrous
Na.sub.2SO.sub.4, the solvent was stripped off to give crude diol
(13.1 mg).
[0186] This was combined with additional crude diol (5.0 mg) and
purified by C-18 preparative TLC with 95 CH.sub.2Cl.sub.2/5 iPA/1
TFA as eluent to obtain the diol TFA salt. After partitioning the
salt between CH.sub.2Cl.sub.2 and NaHCO.sub.3(aq), the organic
layer was dried with brine, then with anhydrous Na.sub.2SO.sub.4,
and stripped to give the free base of the diol, .beta.-alanine,
[N,N-bis(2-hydroxyethyl)],
3-[(6-chloro-2-methoxyacridin-9-yl)amino]propyl ester, (5.0
mg).
[0187] .sup.1H NMR: .delta. 7.92-8.25 (m, 3H), 7.23-7.47 (m, 3H),
4.30 (t, J=5.7 Hz, 2H)), 3.98 (s, 3H), 3.81 (t, J=6.2 Hz, 2H), 3.64
(t, J=4.9 Hz, 4H), 2.86 (t, J=6.1 Hz, 2H), 2.67 (t, J=4.9 Hz, 4H),
2.51 (t, J=5.9 Hz, 2H), 2.04 (apparent quintet, 2H).
[0188] Step D .beta.-Alanine, [N,N-bis(2-Chloroethyl)],
3-[(6-Chloro-2-methoxyacridin-9-yl)amino]propyl Ester
Dihydrochloride, Compound XI.
[0189] The .beta.-alanine, [N,N-bis(2-hydroxyethyl)],
3-[(6-chloro-2-methoxyacridin-9yl)amino]propyl ester from above
(4.0 mg, 0.0073 mmol) was dissolved in CH.sub.2Cl.sub.2 (1 mL) and
chilled in an ice/water bath. Ice cold SOCl.sub.2 (0.1 mL) was
added and the reaction was allowed to stir for 4 h at room
temperature. The reaction mixture was stripped to remove solvent,
triturated with hexane, and partitioned between CH.sub.2Cl.sub.2
and NaHCO.sub.3(aq). After the organic layer was dried with brine,
then with anhydrous Na.sub.2SO.sub.4 and stripped, the
dichloro-compound was obtained as a yellow gum. 1H NMR: .delta.
7.8-8.2 (m, 3H), 7.2-7.5 (m, 3H), 4.35 (t, J=5.9 Hz, 2H), 3.85-4.10
(3.99, s, OMe and 3.9-4.0, m, NHCH.sub.2, total 5H), 3.48 (t, J=6.9
Hz, 4H), 2.9-3.0 (m, 6H), 2.49 (t, J=6.6 Hz, 2H), 2.1-2.3 (m,
2H).
[0190] The free amine was stirred in chilled CH.sub.2Cl.sub.2,
acidified with 1M HCl in ether and stripped with a few drops of
methanol to obtain the desired compound, .beta.-alanine,
[N,N-bis(2-chloroethyl)],
3-[(6-chloro-2-methoxyacridin-9-yl)amino]propyl ester
dihydrochloride (2.5 mg), (3.5 mg, 81%), as a yellow solid.
[0191] In the same manner as given in the foregoing Step C, but
using 6-chloro-9-(2-hydroxy)ethylamino-2-methoxy-acridine instead
of 6-chloro-9-(3-hydroxy)propylamino-2-methoxy-acridine, was
prepared the analogous diol. .sup.1H NMR: .delta. 7.96-8.13 (m,
3H), 7.20-7.47 (m, 3H), 4.76 (t, J=4.9 Hz, 2H), 3.99 (s, 3H),
3.92-4.14 (m, 2H), 3.60 (t, J=5.1 Hz, 4H), 2.78 (t, J=6.1 Hz, 2H),
2.63 (t, J=5.1 Hz, 4H), 2.45 (t, J=6.0 Hz, 2H). By analogy to Step
D this was converted to .beta.-alanine, [N,N-bis(2-chloroethyl)],
2-[(6-chloro-2-methoxyacridin-9-yl)amino]ethyl ester
dihydrochloride, Compound XII. .sup.1H NMR: .delta. 7.94-8.20 (m),
7.20-7.50 (m), 4.42 (CH.sub.2OC.dbd.O), 3.90-4.10 (OCH.sub.3,
NHCH.sub.2), 3.46 (CH.sub.2Cl), 2.82 (N(CH.sub.2).sub.3), 2.39-2.56
(CH.sub.2C.dbd.O).
Example 6
[N,N-bis(2-Chloroethyl)]-2-aminoethyl
4,5',8-Trimethyl-4'-psoralenacetate Hydrochloride, Compound XIV
[0192] Step A: [N,N-bis(2-Hydroxyethyl)]-2-aminoethyl
4,5',8-Trimethyl-4'-psoralenacetate
[0193] A slurry of methyl 4,5',8-trimethyl-4'-psoralenacetate (250
mg, 0.832 mmol), triethanolamine (12 mL) and 1M HCl in ether (2 mL)
were stirred at 100.degree. C. for 2 h. The resulting clear brown
solution was allowed to cool to room temperature and partitioned
between CH.sub.2Cl.sub.2 and saturated NaHCO.sub.3(aq). The organic
layer was rinsed several times with saturated NaHCO.sub.3 (aq).
After drying with anhydrous Na.sub.2SO.sub.4, solvent was removed
in vacuo and the residue was partitioned between CH.sub.2Cl.sub.2
and 1M aq. HCl. The aqueous layer was rinsed several times with
CH.sub.2Cl.sub.2 and then made basic with K.sub.2CO.sub.3(s) in the
presence of the organic solvent. The organic layer containing the
neutral product was rinsed with water several times, then dried and
concentrated. A repetition of the acid-base extraction procedure
gave the desired product as a beige solid (84.3 mg, 24.3%): .sup.1H
NMR: .delta. 7.53 (s, 1H), 6.24 (s, 1H), 4.23 (t, J=5.4 Hz, 2H),
3.69 (s, 2H), 3.56 (t, J=5.3 Hz, 4H), 2.82 (t, J=5.4 Hz, 2), 2.69
(t, J=5.3 Hz, 4H), 2.57 (s, 3H), 2.51 (d, J=1.1 Hz, 3H), 2.47 (s,
3H).
[0194] Step B: [N,N-bis(2-Chloroethyl)]-2-aminoethyl
4,5',8-Trimethyl-4'-psoralenacetate Hydrochloride
[0195] Thionyl chloride (0.2 mL) was added to an ice cold mixture
of the above diol (9.8 mg, 0.023 mmol) in CH.sub.2Cl.sub.2 (1 mL)
and stirred at room temperature overnight under nitrogen. The
resulting slurry was concentrated then triturated with hexane to
give the desired product (6.2 mg, 53.9%) as an off-white solid:
.sup.1H NMR (CD.sub.3OD): .delta. 7.71 (s, 1H), 6.28 (s, 1H), 4.56
(t, J=4.8 Hz, 2H), 3.95 (t, J=6.1 Hz, 4H), 3.89 (s, 2H), 3.60-3.83
(m, 6H), 2.54 (s, 3H), 2.53 (s, 3H), 2.50 (s, 3H).
Example 7
Synthesis of .beta.-Alanine, N-(Acridin-9-yl),
2-[bis(2-Chloroethyl)amino]- ethyl Amide (Compound XV)
[0196] Step A
2-[N',N'-bis(2-Hydroxyethyl)]-N-(tert-butoxycarbonyl)ethylen-
ediamine
[0197] To a solution of N-(tert-butoxycarbonyl)ethanolamine (1.21
g, 7.5 mmol) and triethylamine (1.57 mL, 1.1 g, 11 mmol) in dry
CH.sub.2Cl.sub.2 (25 mL) at 0.degree. C. was added methanesulfonyl
chloride (0.64 mL, 0.95 g, 8.3 mmol) dropwise. The reaction was
stirred at 0.degree. C. for 1 h, allowed to warm to 23.degree. C.
and was stirred overnight. The volatiles were removed in vacuo to
give the mesylate as a white solid. Diethanolamine (7.2 mL, 7.9 g,
75 mmol) was added and the reaction mixture was heated to
75.degree. C. with stirring for 6 h. The crude reaction mixture was
partitioned between H.sub.2O (60 mL) and CHCl.sub.3 (4.times.20
mL). The combined organic layers were washed with brine (20 mL) and
dried over Na.sub.2SO.sub.4. Removal of solvent in vacuo gave 1.21
g (65%) of the diol as a thick-yellow oil, .sup.1H NMR: .delta.
5.51-5.39 (m, 1H), 3.61 (t, J=4.9 Hz, 4H), 3.29-3.13 (m, 2H),
2.68-2.52 (m, 6H), 1-44 (s, 9H). The hydroxyl protons were not
observed,
[0198] Step B
2-[N',N'-bis(2-tert-Butyldimethylsilyloxyethyl)]-N-(tert-but-
oxycarbonyl)ethylenediamine
[0199] To a stirred solution of the diol from step A (1.21 g, 4.87
mmol) and pyridine (1.59 mL, 1.55 g, 19.6 mmol) in dry
CH.sub.2CL.sub.2 (12 mL) at 0.degree. C. was added
tert-butyldimethylsilyl chloride (2.21 g, 14.7 mmol). The reaction
mixture was allowed to warm to 23.degree. C. and was stirred for 2
d. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (80 mL)
and washed with H.sub.2O (3.times.25 mL) and then brine (3.times.25
mL). The organic layer was dried over Na.sub.2SO.sub.4. Removal of
solvent in vacuo) gave 2.26 g (97%) of a pale yellow oil. 1H NMR:
.delta. 5.37-5.22 (m, 1H), 3.62 (t, J=6.2 Hz, 4H), 3.19-3.08 (m,
2H) 2.63 (t, J=6.2 Hz, 6H), 1.42 (s, 9H), 0.873 (s, 18H), 0.04 (s,
12H).
[0200] Step C
2-[N,N,bis(2,tert-Butyldimethylsilyloxyethyl)]ethylenediamin- e
[0201] To a flask containing the protected amine from step B (4.24
g, 8.89 mmol) was added 5 mL of trifluoroacetic acid at 23.degree.
C. The reaction mixture was stirred for 15 minutes at 23.degree. C.
followed by removal of the trifluoracetic acid in vacuo. The crude
product was partitioned between 2 N NaOH (100 mL) and
CH.sub.2Cl.sub.2 (3.times.35 mL). The combined organic layers were
dried over Na.sub.2SO.sub.4. Removal of solvent in vacuo gave 1.76
g (53%) of a yellow oil. .sup.1H NMR: .delta. 3.66 (t, J=6.5 Hz,
4H), 2.72-2.53 (m, 8H), 1.72-1.63 (m, 2H), 0.87 (s, 18H), 0.02 (s,
12H).
[0202] Step D .beta.-4Alanine, N-(tert-Butoxycarbonyl),
2-[bis(2-tert-Butyldimethylsilyloxyethyl)amino]ethyl Amide
[0203] To a solution of 3-(N-tert-butoxycarbonyl)aminopropanoic
acid (822.0 mg, 4.34 mmol) and 4-methylmorpholine (442.0 mg, 4.37
mmol) in 14 mL of dry THF at -15.degree. C. was added
isobutylchloroformate (0.53 mL, 0.56 g, 4.1 mmol). The reaction
mixture was stirred at -15.degree. C. for 1 min followed by the
addition of the amine from step C (1.72 g, 4.57 mmol). The reaction
mixture was allowed to warm to 23.degree. C. and was stirred for 1
h. The mixture was then filtered, the precipitate was washed with
THF (5 mL) and the filtrate was concentrated in vacuo. The
remaining material was partitioned between 2 N NaOH (50 mL) and
CH.sub.2Cl.sub.2 (3.times.20 mL). The combined organic layers were
dried over Na.sub.2SO.sub.4 and the solvent removed in vocuo to
give 2.25 g of a brownish yellow gum. Purification of the crude
material (2.25 g) by medium pressure liquid chromatography (silica
gel, 1:1 CHCl.sub.3/EtOAc) gave 627.0 mg (26%) of a pale yellow
oil. .sup.1H NMR: .delta. 3.63 (t, J=6.2 Hz, 4H), 3.54-3.35 (m,
4H), 3.20-3.19 (m, 2H), 2.71-2.50 (m, 6H), 1.43 (s, 9H), 0.89 (s,
18H), 0.05 (s, 12H). The amide and carbamate protons were not
observed.
[0204] Step E .beta.-Alanine,
2-(bis(2-tert-Butyldimethylsilyloxyethyl)ami- no]ethyl Amide
[0205] The protected amine formed in step D (627.0 mg, 1.14 mmol)
was dissolved in trifluoroacetic acid (5 mL) at 23.degree. C. The
resulting solution was stirred for 5 min (until CO.sub.2 evolution
ceased) followed by removal of the trifluoroacetic acid in vacuo.
The remaining material was partitioned between saturated
NaHCO.sub.3 (50 mL) and CH.sub.2Cl.sub.2 (3.times.20 mL). The
combined organic layers were dried over Na.sub.2SO.sub.4 and the
solvent removed in vacuo to give 203.4 mg (40%) of a pale yellow
oil.
[0206] Step F .beta.-Alanine, N-(Acridin-9-yl),
2-[bis(2-tert-Butyldimethy- lsilyloxyethyl)amino]ethyl Amide
[0207] A mixture of the crude amine from step E (203.4 mg, 0.45
mmol), 9-methoxyacridine (96.8 mg, 0.46 mmol) and methanol (10 mL)
was heated to reflux for 4 h. The reaction mixture was allowed to
cool to 23.degree. C. and was stirred for an additional 2.5 days.
The methanol was removed in vacuo and the remaining material was
partitioned between 2 N NaOH (50 mL) and CH.sub.2Cl.sub.2
(3.times.20 mL). The combined organic layers were dried over
Na.sub.2SO.sub.4 and the solvent removed in vacuo to give 69.6 mg
of a yellow oil. Purification of the crude material (69.6 mg) by
TLC (silica gel, 1:1 CHCl.sub.3/EtOAC) gave 23.4 mg (8.3%) of a
yellow oil. .sup.1H NMR: .delta. 8.19 (d, J=8.8 Hz, 2H), 8.06 (d,
J=8.8 Hz, 2H), 7.65 (br t, J=7.6 Hz, 2H), 7.36 (br t, J=7-6 Hz,
2H), 6.8-6.7 (m, 1H), 4.06 (t, J=5.6 Hz, 2H), 3.61 (t, J=5.8 Hz,
4H), 3.37-3.32 (m, 2H), 2.72-2.61 (m, 6H), 2.51 (t, J=5.5 Hz, 2H),
0.86 (s, 18H), 0.02 (s, 12H). The amine proton was not observed,
.sup.13C NMR: .delta. 172.1, 152.4, 149.3, 130.5, 129.3, 123.7,
118.0, 112.8, 62.3, 57.4, 54.1, 47,4, 38.2, 36.5, 26.4, 18.8,
-4.8.
[0208] Step G .beta.-Alanine, N-(Acridin-9-yl),
2-[bis(2-Hydroxyethyl)amin- o]ethyl Amide Dihydrochloride
[0209] To a stirred solution of the bis-protected diol from step F
(22.0 mg, 0.04 mmol) in isopropanol (1.0 mL) was added a 5-6 N
HCl/isopropanol solution (0.05 mL) at 23.degree. C. The reaction
mixture was stirred at 23.degree. C. for 17 h and the resultant
yellow precipitate was collected by vacuum filtration. The yellow
solid was rinsed with an additional 1.0 mL of isopropanol. Residual
isopropanol was removed in vacuo (overnight) to give 11.4 mg (69%)
of the diol dihydrochloride salt as a yellow solid. .sup.1H NMR
(CD.sub.3OD): .delta. 8.52 (d, J=8.8 Hz, 2H), 7.96 (br t, J=7.5 Hz,
2H), 7.82 (d, J=8.4 Hz, 2H), 7.57 (br t, J=7.5 Hz, 2H), 4.49 (t,
J=6.2 Hz, 2H), 3.91 (t, J=4.8 Hz, 4H), 3.74-3.56 (m, 2H), 3.53-3.38
(m, 6H), 2.97 (t, 6.1 Hz, 2H). The amide, amine and hydroxyl
protons were not observed.
[0210] Step H .beta.-Alanine, N-(Acridin-9-yl),
2-[bis(2-Chloroethyl)amino- ]ethyl Amide Dihydrochloride
[0211] To a stirred suspension of the diol from step G (11.4 mg,
0.024 mmol) in CH.sub.3CN (1.0 mL) was added SOCL.sub.2 (0.12 mL,
200 mg, 1.7 mmol) at 23.degree. C. The reaction mixture was stirred
at 23.degree. C. for 15 minutes and the solution was heated to
50.degree. C. for 3.5 h. The resultant yellow precipitate was
collected via vacuum filtration and was rinsed with CH.sub.3CN
(3.times.1.0 mL) and dried in vacuo to give 8.3 mg (67%) of a
yellow powder (95% pure by HPLC). .sup.1H NMR (CD.sub.3OD): .delta.
8.55 (d, J=8.7 Hz, 2H), 8.00 (br t, J=7.7 Hz, 2H), 7.84 (d, J=8.7
Hz, 2H), 7.59 (br t, J=7.7 Hz, 2H), 4.51 (t, J=6.2 Hz, 2H), 3.98
(t, J=5.7 Hz, 4H), 3.71 (t, J=5,7 Hz, 4H), 3.65-3.55 (m, 2H),
3.55-3.42 (m, 2H), 2.99 (t, J=6.2 Hz, 2H). The amide and amine
protons were not observed.
Example 8
Hydrolysis of the Frangible Compounds
[0212] For the frangible compounds incorporating an ester group
("forward" and "reverse" esters) in the frangible linker, model
compounds were studied to determine the amount of ester
hydrolysis.
[0213] The reaction 18
[0214] where AcrNH indicates a 9-amino acridine bearing
substituents as indicated in the following table, and n and R are
as indicated, was studied. Table III shows the rate enhancement for
ester hydrolysis when the ester linkage is situated between, and in
proximity to, an acridine ring and an alkylamino group. The
hydrolysis rate is rapid regardless of whether the acridine moiety
is positioned at the acid terminus of the ester, or at the alcohol
terminus.
3 TABLE III Percent Hydrolysis at 100 minutes (aqueous solution, pH
8, 37.degree. C.) Acridine R = R = R = Substituent(s) n = methyl
--(CH.sub.2).sub.2N(CH.sub.2CH.sub.2OH).- sub.2
--(CH.sub.2).sub.3N(CH.sub.2CH.sub.2OH).sub.2 6-Cl, 2-OMe 1 28%
6-Cl, 2-OMe 2 22% 6-Cl, 2-OMe 3 5% 6-Cl, 2-OMe 4 2% 6-Cl, 2-OMe 7
<1% 2-CO.sub.2CH.sub.3 2 9% 55% 57% 2-CO.sub.2CH.sub.3 3 18%
2-CO.sub.2CH.sub.3 4 17%
[0215] For the reaction 19
[0216] where AcrNH indicates a 9-amino acridine bearing
substituents as indicated in the following table, and n and R are
as indicated, the following results were obtained:
4 TABLE IV Percent Hydrolysis at 100 minutes (aqueous solution, pH
8, 37.degree. C.) Acridine R = R = R = Substituent(s) n = methyl
--(CH.sub.2).sub.2N(CH.sub.2CH.sub.2OH).- sub.2
--(CH.sub.2).sub.3N(CH.sub.2CH.sub.2OH).sub.2 6-Cl, 2-OMe 2 2% 99%
6-Cl, 2-OMe 3 2% 65%
[0217] At pH 3, all compounds in Tables III and IV showed
.ltoreq.1% hydrolysis at 100 min.
[0218] The mustard compounds cannot be evaluated in the same manner
since multiple degradation pathways occur simultaneously.
Nevertheless, when Compound VIII is incubated under the same
conditions as in Tables III and IV, the acridine acid is the major
product (.ltoreq.95%) after long incubation times and 40% is formed
at 100 minutes. This compares favorably to the table entry for the
analogous diol (57% hydrolysis at 100 minutes).
[0219] It will be appreciated from the data in Tables III and IV
that the hydrolysis rate of the ester linkage varies inversely with
the length of the linker arm between the 9-aminoacridine moiety and
the ester group (in Tables III and IV, as n increases, the amount
of hydrolysis at 100 minutes decreases). This provides a method of
tuning the hydrolysis rate of the compounds. This ability to tune
the breakdown of the linker allows compound reactivity to be
adjusted for various applications, as desired.
MATERIALS
[0220] The following materials were used in the following
Examples:
[0221] While it is commercially available from Baxter Healthcare
Corp., Deerfield, Ill., Adsol used in this and the following
experiments was made by sterile filtering the following mixture: 22
g glucose, 9 g NaCl, 7.5 g mannitol, and 0.27 g adenine in 1 liter
of distilled water.
[0222] Quinacrine mustard was obtained from Aldrich Chemical Co.,
St. Louis, Mo.
[0223] Whole blood was obtained from the Sacramento Blood Center
(Sacramento Calif.).
Example 9
Inactivation of Vesicular Stomatitis Virus (VSV)
[0224] Stock solutions (typically 10-30 mM) of each compound are
prepared by dissolving an appropriate amount of material in blood
bank saline previously acidified with 2 mM H.sub.3PO.sub.4, then
quickly frozen in 1 mL aliquots. At the time of use, aliquots are
warmed to .ltoreq.10.degree. C. and used within one hour.
[0225] For preparation of packed red blood cells (PRBC), whole
blood, with measured Hct, is centrifuged at 3800 rpm for 6 min.
Supernatant plasma is removed and measured. Adsol is added to
provide PRBC with 60% Hct. Plasma concentration is 15-20%.
[0226] A VSV (stock solution, approx. 10.sup.9 pfu/mL, obtained
from ATCC American Type Cell Culture, Rockville, Md.) is diluted
1:10 into tissue culture medium (DMEM with 10% NCS) or into PBRC to
provide the test medium which is aliquoted (1 mL) into 2 mL sterile
o-ring tubes.
[0227] To each tube is added sufficient test compound solution to
provide a test compound concentration of 10-300 .mu.M. Each sample
is quickly mixed by fully pipetting the mixture several times.
Suspensions are incubated at ambient temperature for 4 h. Virus
titer was ascertained following incubation of the treated medium in
BHK (baby hamster kidney) host cells. PRBC was used directly rather
than the supernatant alone. Virus kill was inversely proportional
to the appearance of plaques in the cell cultures. The difference
between the titer of the untreated test medium and that of a
treated sample provides the log kill for the compound at that
concentration. The detection limit is 10.sup.1.4 pfu/mL.
[0228] In tissue culture medium, quinacrine mustard (QM), and
Compounds IV, VI, XI, VII, and VIII inactivated >3 logs of VSV
at <50 .mu.M test compound. Compound XII inactivated 2 logs at
approx. 200 .mu.M. This compound is believed to be particularly
unstable with respect to ester hydrolysis. As indicated in the
first entry in Example 8, Table IV, the corresponding diol compound
.beta.-alanine, [N,N-bis(2-hydroxyethyl)],
2-[(6-chloro-2-methoxyacridin-9-yl)amino]ethyl ester) was 99%
hydrolyzed after 100 minutes at pH 8, 37.degree. C. It is likely
that the mustard compound also underwent rapid hydrolysis. This
illustrates the importance of the anchor moiety for directing the
effector portion of the molecule to nucleic acid, and the
importance of tuning the reactivity of the 9-aminoacridine class of
compounds so that they are effective under conditions of actual
use. Under the described inactivation protocol, hydrolysis of
Compound XII is expected to be competitive with inactivation.
[0229] In PRBC, QM and Compounds IV, VI, VIII, V, and XIII
inactivated >2 logs of VSV at <150 .mu.M of test
compound.
Example 10
Inactivation of Yersinia enterocolitica
[0230] PRBC and stock solutions were prepared as for VSV in Example
9. Yersinia (California Department of Health Services, Microbial
Disease Laboratory, Berkeley, Calif.) is cultured in LB-broth at
37.degree. C. overnight on a shaker. A portion (10 mL) is
centrifuged at 2500 rpm for 10 min in a 15 mL conical tube. The
pellet is resuspended in 1 mL of Adsol to provide approx. 10.sup.9
bacteria/mL. To measure the titer, the optical density is measured
of a 1:100 dilution in Adsol (OD.sub.610=0.2 at 10.sup.8
bacterial/mL). The bacterial stock is then diluted 1:100 into
saline or PRBC to provide the test medium which is aliquoted (1 mL)
into 2 mL o-ring sterile tubes.
[0231] To each tube is added a sufficient amount of the test
compound solution to provide a test compound concentration of
10-300 .mu.M. Each sample is quickly mixed by fully pipetting the
mixture several times. It is then incubated for two hours at
ambient temperature, then plated out on LB-agar starting with 100
.mu.L sample starting at 10.sup.-1 dilution and continuing
dilutions to 10.sup.-8. The plates are incubated overnight at
37.degree. C. and the colonies are counted. The difference between
the titer of the untreated test medium and that of a treated sample
provides the log kill for the compound at that concentration. The
detection limit is 10 bacteria/mL.
[0232] In saline, quinacrine mustard (QM) and Compounds IV, VI,
VIII, V, IX, and X inactivated >2 logs of Yersinia at
concentrations .ltoreq.200 .mu.M.
[0233] In PRBC, QM and Compounds VI, VIII, V, X, and XIII
inactivated >2 logs of Yersinia at concentrations .ltoreq.200
.mu.M.
Example 11
Blood Function Assay After Introduction of a Compound of the
Invention
[0234] One of the contemplated uses of the compounds of the
invention involves the introduction of one or more of the compounds
of the invention into blood or blood products intended for
transfusion. The blood or blood product must remain suitable for
transfusion after treatment with the compounds. To evaluate the
effect of the compounds on red blood cell function, the compounds
were tested as described below.
[0235] Packed red blood cells with a 50% hematocrit (Hct) is
prepared by spinning whole blood with a known Hct at 2500 rpm for 6
min. The supernate is removed and measured. The suspension is
diluted with a sufficient volume of Adsol to achieve the desired
Hct.
[0236] 1.5 mL of PRBC is placed in each 2 ml o-ring tube and enough
of the stock solution of the test compound is added to achieve the
desired concentration. The samples are incubated at ambient
temperature for 4 hours, then stored overnight at 4.degree. C.
Hemolysis was determined as described in Hogman et al.,
Transfusion, 31:26-29 (1991).
[0237] A lysis standard is prepared for each sample by diluting 10
.mu.M of the incubated mixture in two steps with water to give a
final 1:4000 dilution.
[0238] For the assay, samples were removed from 4.degree. C.
storage and warmed for <15 minutes. After vortexing briefly to
mix, an aliquot was removed and spun for 2 minutes at 14,000 rpm.
The supernate was removed and spun for 10 minutes at 14,000 rpm.
The supernate was removed and diluted as needed in water. The
absorbance at 414 nm of the lysis standards and the diluted
supernates were read against a water blank. Percent hemolysis was
calculated as:
[0239] (100%-50%Hct).times.(A.sub.414 of sample.times.dilution
factor)/(A.sub.414 of lysis standard.times.4000)
[0240] The A.sub.414 of the samples is uncorrected for any
absorbance due to the presence of the compound of the invention.
The results are given in Table Va.
5TABLE Va Hemolysis Data at Day 1 Percent Compound and
Concentration Hemolysis Number of Samples Tested BBS* 0.066 14
ABBS** 0.065 8 150 .mu.M QM*** 0.220 14 300 .mu.M QM 0.320 14 150
.mu.M IV 0.091 14 300 .mu.M IV 0.109 14 150 .mu.M VI 0.103 14 300
.mu.M VI 0.140 14 150 .mu.M VIII 0.110 6 300 .mu.M VIII 0.135 6 150
.mu.M V 0.136 2 300 .mu.M V 0.149 2 150 .mu.M IX 0.116 2 300 .mu.M
IX 0.099 2 150 .mu.M X 0.121 2 300 .mu.M X 0.153 2 *BBS = blood
bank saline **ABBS = acidic blood bank saline ***QM = quinacrine
mustard
[0241] Extracellular potassium was measured using a Ciba Corning
Model 614 K.sup.+/Na.sup.+ Analyzer (Ciba Corning Diagnostics
Corp., Medford, Mass.). ATP was measured using Sigma procedures No.
366 (Sigma, St. Louis, Mo.).
[0242] Table Vb. shows the relative values of extracellular
potassium relative to the control values of the untreated PRBC
samples for that experiment. For example, a relative value of 1.03
meant that the treated sample has 3% more extracellular potassium
concentration than the untreated control.
6TABLE Vb Relative Extracellular Potassium Levels (replicates)*
Concentration Compound (.mu.M) Day 1 Day 7 Day 14 IV 100 1.01 (1)
0.98 (1) 1.03 (1) 200 1.05 (1) 1.15 (1) 1.01 (1) 300 1.03 (1) 1.15
(1) 1.15 (1) V 300 1.04- 0.96- 0.95- 1.46 (4) 1.01 (4) 1.01 (4)
*[K+] (treated)/[K+] (untreated)
[0243] Table Vc. shows the relative values ATP relative to the
control values of the untreated PRBC samples for that experiment.
For example, a relative value of 1.03 meant that the treated sample
has 3% more ATP than the untreated control.
7TABLE Vc Relative ATP Levels (replicates)* Compound Concentration
(.mu.M) Day 1 Day 7 Day 14 IV 100 1.01 (1) 0.93 (1) 0.94 (1) 200
1.05 (1) 0.94 (1) 0.94 (1) 300 1.03 (1) 0.93 (1) 0.92 (1) V 300
0.96-1.00 (4) 0.91- 0.94- 1.01 (4) 1.01 (4) *[ATP] (treated)/[ATP]
(untreated)
Example 12
Inactivation of HIV by Compounds of the Invention
[0244] Cell associated HIV in TC Medium (Popovic et al., Science,
224:497 (1984): H9-IIIb cells are suspended in TC Medium to provide
a suspension with a titer of approximately .ltoreq.10.sup.6 pfu/mL.
To 2 mL aliquots of the test medium in 15 mL conical tubes is added
a sufficient amount of test compound solution to achieve the
desired concentration of active material. The suspensions are
immediately mixed by fully pipetting several times, then vortexing
briefly. The samples are incubated at ambient temperature for 2-4
h, then centrifuged. The pellets are resuspended in 1 mL of plaque
assay diluent, then quickly frozen at -80.degree. C. and titrated
by a microplaque assay. (Hanson et al., J. Clin. Micro., 28:2030
(1990)).
[0245] Compounds quinacrine mustard, IV and VI inactivated >3
logs of HIV at .ltoreq.25 .mu.M of test compound.
[0246] Cell-associated HIV in PRBC: For assays run in PRBC, the
packed cells are prepared as described in the VSV assay. The
1-HV9-IIIb cells are added to the Adsol prior to dilution of the
centrifuged cells. The resultant suspension is mixed by fully
pipetting all the material. Upon completion of incubation of the
test compound, the samples are diluted with 3 mL of a 1:1
plasma:DMEM solution containing 5 .mu.L of heparin. The infected
cells are then isolated using a fycol-hypaque gradient, resuspended
in 1 mL of the diluent, and frozen for later titration.
[0247] Compounds quinacrine mustard, VI and V inactivated >3
logs of HIV at .ltoreq.200 .mu.M of test compound.
[0248] Cell-free HIV in PRBC: The protocol is similar to that
described above, except that cell-free HIV is added directly to the
PRBC after preparation. After incubation, the medium is centrifuged
and the supernate is frozen for later titration.
[0249] Compounds quinacrine mustard, IV, V and VI inactivated >3
logs of HIV at .ltoreq.100 .mu.M of test compound.
[0250] Although the forgoing invention has been described in some
detail by way of illustration and example for purposes of clarity
and understanding, it will be apparent to those skilled in the art
that certain changes and modifications may be practical. Therefore,
the description and examples should not be construed as limiting
the scope of the invention, which is delineated by the appended
claims. The entirety of U.S. Pat. Nos. 5,559,250 and 5,399,719 are
hereby incorporated by reference. All other patents and references
cited herein are hereby incorporated by reference.
* * * * *