U.S. patent application number 10/208583 was filed with the patent office on 2003-05-01 for psoralens for pathogen inactivation.
This patent application is currently assigned to Cerus Corporation. Invention is credited to Nerio, Aileen, Wollowitz, Susan.
Application Number | 20030082510 10/208583 |
Document ID | / |
Family ID | 22068105 |
Filed Date | 2003-05-01 |
United States Patent
Application |
20030082510 |
Kind Code |
A1 |
Wollowitz, Susan ; et
al. |
May 1, 2003 |
Psoralens for pathogen inactivation
Abstract
Psoralen compound compositions are synthesized which have
primaryamino substitutions on the 3-, 4-, 5-, and 8-positions of
the psoralen, which yet permit their binding to nucleic acid of
pathogens. Reaction conditions that photoactivate these psoralens
result in the inactivation of pathogens which contain nucleic acid.
The compounds show similar activity in test systems to 4' and 5'
derivatives of psoralen useful for inactivation of pathogens in
blood products. In addition to the psoralen compositions, the
invention contemplates such inactivating methods using the new
psoralens.
Inventors: |
Wollowitz, Susan;
(Lafayette, CA) ; Nerio, Aileen; (Fremont,
CA) |
Correspondence
Address: |
John W. Tessman
Cerus Corporation
Suite 300
2525 Stanwell Drive
Concord
CA
94520
US
|
Assignee: |
Cerus Corporation
Concord
CA
|
Family ID: |
22068105 |
Appl. No.: |
10/208583 |
Filed: |
July 30, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10208583 |
Jul 30, 2002 |
|
|
|
09500680 |
Feb 9, 2000 |
|
|
|
6455286 |
|
|
|
|
09500680 |
Feb 9, 2000 |
|
|
|
09196935 |
Nov 20, 1998 |
|
|
|
6133460 |
|
|
|
|
60066224 |
Nov 20, 1997 |
|
|
|
Current U.S.
Class: |
435/2 |
Current CPC
Class: |
A61M 1/3683 20140204;
A61L 2/08 20130101; A61P 31/00 20180101; A61P 43/00 20180101; C07D
493/04 20130101; C12N 7/04 20130101; A61M 1/3686 20140204; A61L
2/0011 20130101; A61M 1/3681 20130101 |
Class at
Publication: |
435/2 |
International
Class: |
A01N 001/02 |
Goverment Interests
[0002] This invention was made with United States government
support under SBIR Grant No. 1 R43 HL51796-01 from the NIH. The
United States Government has certain rights in this invention.
Claims
We claim:
1. A method of treating a biological composition suspected of
containing a pathogen, comprising, in the following order: a)
providing, in any order, i) a compound of the formula selected from
the group consisting of 15I) a substituent A on the benzene ring is
selected from the group consisting of:
--(CH.sub.2).sub.u--NH.sub.2,
--(CH.sub.2).sub.w--J--(CH.sub.2).sub.z- --NH.sub.2,
--(CH.sub.2).sub.w--J--(CH.sub.2).sub.x--K--(CH.sub.2).sub.z---
NH.sub.2, and
--(CH.sub.2).sub.w--J--(CH.sub.2).sub.x--K--(CH.sub.2).sub.y-
--L--(CH.sub.2).sub.z--NH.sub.2; wherein J, K, and L are
independently selected from the group consisting of O and NH, in
which u is a whole number from 1 to 10, w is a whole number from 1
to 5, x is a whole number from 2 to 5, y is a whole number from 2
to 5, and z is a whole number from 2 to 6; and II) substituents B,
R.sub.1, R.sub.2, R.sub.4, and R.sub.5 on the benzene ring, 3-, 4-,
4'- and 5'-carbon atoms respectively, are independently selected
from the group consisting of --H and --(CH.sub.2).sub.vCH.sub.3,
where v is a whole number from 0 to 5; and all salts thereof; ii) a
light source for photoactivating said compound; and iii) a
biological composition suspected of containing a pathogen; b)
adding said compound to said biological composition; and c)
photoactivating said compound, so as to inactivate the pathogen, if
present.
2. The method of claims 1, wherein said biological composition is a
blood product.
3. The method of claim 2, wherein said blood product comprises
plasma.
4. The method of claim 2, wherein said blood product comprises a
plasma fraction.
5. The method of claim 2, wherein said blood product comprises
platelets.
6. The method of claim 2, wherein said pathogen is selected from
the group consisting of viruses and bacteria.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
09/500,680, filed Feb. 9, 2000, which is a continuation of
application Ser. No. 09/196,935, filed on Nov. 20, 1998, which
issued as U.S. Pat. No. 6,133,460, which claims priority to U.S.
Provisional Patent Application Serial No. 60/066,224, filed Nov.
20, 1997; the disclosure of which is hereby incorporated by
reference.
FIELD OF THE INVENTION
[0003] The present invention provides new psoralens having enhanced
ability to inactivate pathogens in the presence of ultraviolet
light. The present invention also provides methods of using new
psoralens to inactivate pathogens in health related products to be
used in vivo and in vitro, and in particular, blood products.
BACKGROUND
[0004] Psoralens are tricyclic compounds formed by the linear
fusion of a furan ring with a coumarin. Psoralens can intercalate
between the base pairs of double-stranded nucleic acids
BACKGROUND
[0005] Psoralens are tricyclic compounds formed by the linear
fusion of a furan ring with a coumarin. Psoralens can intercalate
between the base pairs of double-stranded nucleic acids (or base
paired regions of single-stranded nucleic acids), forming covalent
adducts to pyrimidine bases upon absorption of long wave
ultraviolet light (UVA). G. D. Cimino et al., Ann. Rev. Biochem.
54:1151 (1985); Hearst et al., Quart. Rev. Biophys. 17:1 (1984). If
there is a second pyrimidine adjacent to a psoralen-pyrimidine
monoadduct and on the opposite strand, absorption of a second
photon can lead to formation of a diadduct which functions as an
interstrand crosslink [S. T. Isaacs et al., Biochemistry 16:1058
(1977); S. T. Isaacs et al., Trends in Photobiology (Plenum) pp.
279-294 (1982); J. Tessman et al., Biochem. 24:1669 (1985); Hearst
et al., U.S. Pat. Nos. 4,124,598, 4,169,204, and 4,196,281, hereby
incorporated by reference].
[0006] The photoreaction of psoralens with nucleic acid has been
useful in the study of nucleic acid folding, the attachment of
diagnostic probes to nucleic acids, the attachment of nucleic acids
to surfaces and materials, the blocking of polymerase reactions and
the inactivation of organisms and cells that require nucleic acid
replication to proliferate, e.g., bacteria, viruses, leukocytes and
overproliferating cells, such as those resulting in psoriasis,
restenosis, or cancer. The inactivation of a virus can also be
applied to preparation of vaccines. The level of reaction with
cellular nucleic acid can be modulated to stop proliferation of the
cell yet maintain cell functions such as protein synthesis. This
can be applied to the treatment of T-cell lymphocytes as a means of
preventing graft vs. host disease in, for example, bone marrow
transplants.
[0007] The use of psoralens for pathogen inactivation in blood
products is of particular interest as the safety of the blood
supply is an issue of universal concern. While transfusion
associated viral infections have been considerably reduced by
testing, transmission of human immunodeficiency virus (HIV),
hepatitis B virus (HBV) and hepatitis C virus (HCV) continue to
occur in 1/450,000 to 660,000 units, 1/200,000 units and 1/3000
units respectively [R. Dodd, Blood Supply: Risks, Perceptions, and
Prospects for the Future, S. J. Nance, ed., p.1 (1994); E. Lackritz
et al., New Eng. J. Med. 333: 1721 (1995)]. Testing is not an
option for some viruses. Cytomegalovirus is commonly found within
the blood supply yet is of clinical importance only to immune
compromised patients for which infection can be fatal [R. Bowden,
Blood Safety: Current Challenges, S. J. Nance, ed., p.201 (1992)].
Universal screening for CMV would lead to a serious reduction in
eligible donors and thus a reduction in the national blood supply.
Special donor pools must be used for these patients at present. It
is also recognized that other unknown viruses or new strains of
known viruses may find their way into the blood supply and will not
be identified until morbidity or mortality is noted, nor will they
be able to be screened out until tests become available. The
identification of hepatitis G in blood units is the most recent
example of such an occurrence [H. Alter, Transfusion 37: 569
(1997)]. Bacterial contamination, especially of platelet
concentrates (PC) has been increasingly recognized as a problem as
well. It is estimated that 1/1,000 to 2,000 PC units show levels of
contamination that results in a septic response [J. Morrow et al.,
JAMA 266: 555 (1991); M. Blajchman, Blood Safety: Current
Challenges, S. J. Nance, ed., p.213 (1992); E. Chiu et al.,
Transfusion 34: 950 (1994)]. There are at present no screening
tests available for blood units for any of the ten or so bacteria
that have been associated with fatal transfusion associated sepsis
in the United States. While there are potential methods for storage
of platelets at lower temperatures to alleviate this problem [U.S.
Pat. Nos. 5,827,640 and 5,827,741], inactivation of the bacteria by
psoralen would have far less impact on the routine storage of
platelets.
[0008] Psoralens are ideal candidates for photosensitized,
decontamination of platelet concentrates [H. Alter et al., Lancet
ii:1446 (1988); L. Lin et al., Blood 74: 517 (1989); C. Hanson,
Blood Cells 18: 7 (1992)]. For example, 8-methoxypsoralen (8-MOP)
is quite effective at deactivation of a number of bacteria found in
platelet concentrates. However, it is not sufficiently active to
inactive pathogens with small genomes (i.e., viruses) without using
concentrations and irradiation times which damage platelets. The
highly active psoralen, 4'-aminomethyl-4, 5', 8-trimethylpsoralen
(AMT), exhibits excellent photochemical inactivation properties but
is highly mutagenic in the absence of light in some bacterial
assays [S. Wagner et al., Photochem. Photobio. Meeting Abstract,
55: 113S (1992)]. Other 4'- and 5'-aminomethyl substituted
psoralens have been developed which show excellent photochemical
inactivation properties with considerable reduction in
mutagenicity.
[0009] Several patents are directed toward psoralen inactivation of
pathogens in blood products [G. Wiesehahn et al., U.S. Pat. Nos.
4,727,027 and 4,748,120, L. Lin et al., U.S. Pat. Nos. 5,288,605,
5,482,828, and 5,709,991, and S. Wollowitz et al., U.S. Pat. No.
5,593,823, hereby incorporated by reference]. P. Morel et al.,
Blood Cells 18:27 (1992) show that 300 .mu.g/mL of 8-MOP together
with ten hours of irradiation with ultraviolet light can
effectively inactivate viruses in human serum. Similar studies
using 8-MOP and AMT have been reported by other investigators [Dodd
R Y, et al., Transfusion 31:483-490 (1991); Margolis-Nunno, H., et
al., Thromb Haemostas 65: 1162 (Abstract)(1991)]. Indeed, the
photoinactivation of a broad spectrum of microorganisms has been
established, including HBV, HCV, and HIV. [Hanson C. V., Blood
Cells: 18: 7-24 (1992); Alter, H. J., et al., The Lancet ii:1446
(1988); Margolis-Nunno H. et al., Thromb Haemostas 65: 1162
(Abstract) (1991); Lin et al. Transfusion 37: 423 (1997)]. There
are clearly a broad class of psoralen compounds effective in the
inactivation of pathogens in general and particularly in blood
products.
[0010] The most highly active psoralen compounds useful for
inactivation have amino derivatives on the 4' and 5' positions
[Wollowitz et al., U.S. Pat. Nos. 5,578,736 and 5,654,443, Kaufman
U.S. Pat. No. 4,294,822]. 5-alkoxy and 8-alkoxypsoralens with amino
substituents at the 8 or 5 position, respectively, as well as
8-aminomethyl psoralen and 8-aminomethyl-4-methylpsoralen are known
[J. Hansen et al., J. Med. Chem. 28: 1001-1010 (1985); Kaufman U.S.
Pat. Nos. 4,269,851 and 4,328,239]. The limited data provided for
these latter compounds suggested that even the amino substituted
alkoxypsoralens have relatively poor photoactivity and that amino
substitution at the furan ring is important for high photoactivity.
Also, these compounds are formed by methods which offer little
flexibility in modifying the ring functionality.
SUMMARY OF THE INVENTION
[0011] The present invention provides new aminopsoralens with
unpredicted photoreactivity with nucleic acids that can be used for
nucleic acid probe preparations, preparation of conjugates,
inhibition of cell proliferation, inactivation of virus for vaccine
preparation, and in particular, for the inactivation of pathogens
in blood products. The present invention also provides new routes
to the synthesis of aminopsoralens and intermediates that may be
useful for providing psoralens conjugated to a variety of other
functional groups.
[0012] With respect to new compounds, some of the new psoralens are
primaryamino-pyrone-linked psoralens comprising a primaryamino
group (i.e. --NH.sub.2 group) linked to the pyrone ring of the
psoralen (3- and 4-carbon atoms) via an alkyl chain optionally
containing oxygen and nitrogen atoms, and wherein the psoralen ring
may have one or more alkyl groups at other positions. The present
invention further contemplates psoralen compounds with a
primaryamino substituent on the pyrone ring, comprising: a) a
substituent A on the pyrone ring, selected from the group
consisting of: --(CH.sub.2).sub.u--NH.sub.2,
--(CH.sub.2).sub.w--J--(CH.sub.2).sub.z--NH.sub.2,
--(CH.sub.2).sub.w--J--(CH.sub.2).sub.x--K--(CH.sub.2).sub.z--NH.sub.2,
and
--(CH.sub.2).sub.w--J--(CH.sub.2).sub.x--K--(CH.sub.2).sub.y--L--(CH.-
sub.2).sub.z--NH.sub.2; wherein J, K, and L are independently
selected from the group consisting of O and NH, in which u is a
whole number from 1 to 10, w is a whole number from 1 to 5, x is a
whole number from 2 to 5, y is a whole number from 2 to 5, and z is
a whole number from 2 to 6; and b) substituents B, R.sub.3,
R.sub.4, R.sub.5, and R.sub.6 on the pyrone ring (the 3- or
4-carbon atom which does not have the primaryamino substituent),
5-, 4'-, 5'- and 8-carbon atoms respectively, independently
selected from the group consisting of --H and
--(CH.sub.2).sub.vCH.sub.3, where v is a whole number from 0 to 5;
or a salt thereof. Where an element is "independently selected"
from a group, it means that the element need not be the same as
other elements chosen from the same group. The structure of these
compounds is as follows. 1
[0013] The present invention further contemplates compounds of the
above structure with a primaryamino substituent on the pyrone ring,
wherein A is at the 3-carbon atom and B is at the 4-carbon atom,
preferably wherein A is selected from the group consisting of
--CH.sub.2--NH.sub.2 and --CH.sub.2--O--(CH.sub.2).sub.2--NH2. More
specifically, the invention contemplates compounds wherein A is
--CH.sub.2--NH.sub.2, and wherein B, R.sub.3, R.sub.5, and R.sub.6
are --H, and wherein R.sub.4 is --CH.sub.3; wherein A is
--CH.sub.2--NH.sub.2, and wherein R.sub.3 and R.sub.5 are --H, and
wherein B, R.sub.4, and R.sub.6 are --CH.sub.3; wherein A is
--CH.sub.2--NH.sub.2, and wherein R.sub.3 and R.sub.4 are --H, and
wherein B, R.sub.5, and R.sub.6 are --CH.sub.3; wherein A is
--CH.sub.2--NH.sub.2, and wherein R.sub.3 is --H, and wherein B,
R.sub.4, R.sub.5, and R.sub.6 are --CH.sub.3; wherein A is
--CH.sub.2--O--(CH.sub.- 2).sub.2--NH.sub.2, and wherein R.sub.3
and R.sub.5 are --H, and wherein B, R.sub.4, and R.sub.6 are
--CH.sub.3. The structure of these compounds is as follows. 2
[0014] The present invention further contemplates compounds of the
above structure with a primaryamino substituent on the pyrone ring,
wherein A is at the 4-carbon atom and B is at the 3-carbon atom,
preferably wherein A is selected from the group consisting of
--CH.sub.2--NH.sub.2 and --CH.sub.2--O--(CH.sub.2).sub.2--NH.sub.2.
More specifically, the invention contemplates compounds wherein A
is --CH.sub.2--NH.sub.2, and wherein B, R.sub.3, R.sub.5, and
R.sub.6 are --H, and wherein R.sub.4 is --CH.sub.3; wherein A is
--CH.sub.2--NH.sub.2, and wherein B and R.sub.3 are --H, and
wherein R.sub.4, R.sub.5, and R.sub.6 are --CH.sub.3; wherein A is
--CH.sub.2--O--(CH.sub.2).sub.2--NH.sub.2, and wherein B and
R.sub.3 are --H, and wherein R.sub.4, R.sub.5, and R.sub.6 are
--CH.sub.3. The structure of these compounds is as follows. 3
[0015] Additionally some of the new psoralens are
primaryamino-benzene-lin- ked psoralens comprising a primaryamino
group linked to the benzene ring of the psoralen (5- and 8-carbon
atoms) via an alkyl chain optionally containing oxygen and nitrogen
atoms, and wherein the psoralen ring may have one or more alkyl
groups at other positions. The present invention further
contemplates psoralen compounds with a primaryamino substituent on
the benzene ring, comprising: a) a substituent A on the benzene
ring, selected from the group consisting of:
--(CH.sub.2).sub.u--NH.sub.2,
--(CH.sub.2).sub.w--J--(CH.sub.2).sub.z--NH.sub.2,
--(CH.sub.2).sub.w--J--(CH.sub.2).sub.x--K--(CH.sub.2).sub.z--NH.sub.2,
and
--(CH.sub.2).sub.w--J--(CH.sub.2).sub.x--K--(CH.sub.2).sub.y--L--(CH.-
sub.2).sub.z--NH.sub.2; wherein J, K, and L are independently
selected from the group consisting of O and NH, in which u is a
whole number from 1 to 10, w is a whole number from 1 to 5, x is a
whole number from 2 to 5, y is a whole number from 2 to 5, and z is
a whole number from 2 to 6; and b) substituents B, R.sub.1,
R.sub.2, R.sub.4, and R.sub.5 on the benzene ring (the 5- or
8-carbon atom which does not have the primaryamino substituent),
3-, 4-, 4'-, and 5'-carbon atoms respectively, independently
selected from the group consisting of --H and
--(CH.sub.2).sub.vCH.sub.3, where v is a whole number from 0 to 5,
or a salt thereof; except c) when A is --CH.sub.2--NH.sub.2 and at
the 8-carbon atom, one of B, R.sub.1, R.sub.4, and R.sub.5 must be
--(CH.sub.2).sub.vCH.sub.3. Where an element is "independently
selected" from a group, it means that the element need not be the
same as other elements chosen from the same group. The structure of
these compounds is as follows. 4
[0016] The present invention further contemplates compounds of the
above structure with a primaryamino substituent on the benzene
ring, wherein A is at the 5-carbon atom and B is at the 8-carbon
atom, preferably wherein A is selected from the group consisting of
--CH.sub.2--NH.sub.2 and --CH.sub.2--O--(CH.sub.2).sub.2--NH.sub.2.
More specifically, the invention contemplates compounds wherein A
is --CH.sub.2--NH.sub.2, and wherein B, R.sub.1, R.sub.2, R.sub.4,
and R.sub.5 are --H; wherein A is
--CH.sub.2--O--(CH.sub.2).sub.2--NH.sub.2, and wherein B, R.sub.1,
and R.sub.2 are --H, and wherein R.sub.4 and R.sub.5 are
--CH.sub.3. The structure of these compounds is as follows. 5
[0017] The present invention further contemplates compounds of the
above structure with a primaryamino substituent on the benzene
ring, wherein A is at the 8-carbon atom and B is at the 5-carbon
atom, preferably wherein A is selected from the group consisting of
--CH.sub.2--NH.sub.2 and --CH.sub.2--O--(CH.sub.2).sub.2--NH.sub.2,
and wherein when A is --CH.sub.2--NH.sub.2, at least one of B,
R.sub.1, R.sub.4, and R.sub.5 are --(CH.sub.2).sub.vCH.sub.3. More
specifically, the invention contemplates compounds wherein A is
--CH.sub.2--NH.sub.2, and wherein B, R.sub.1, and R.sub.4 are --H,
and wherein R.sub.2 and R.sub.5 are --CH.sub.3; wherein A is
--CH.sub.2--NH.sub.2, and wherein B and R.sub.1 are --H, and
wherein R.sub.2, R.sub.4, and R.sub.5 are --CH.sub.3; wherein A is
--CH.sub.2--O--(CH.sub.2).sub.2--NH.sub.2, and wherein B and
R.sub.1 are --H, and wherein R.sub.2, R.sub.4, and R.sub.5 are
--CH.sub.3. The structure of these compounds is as follows. 6
[0018] The present invention also provides new routes to the
synthesis of aminopsoralens and intermediates that may be useful
for providing psoralens conjugated to a variety of other functional
groups.
[0019] Without intending to be limited to any method of synthesis,
the compounds of the present invention can be prepared by
introduction of the amino functionality (or of a building block for
said primaryamino group) in a protected form early on in the
synthesis before the psoralen ring is fully constructed. This
method provides new routes to the synthesis of
primaryamino-pyrone-linked and benzene-linked psoralens that allow
more flexibility in the ring substituents than existing methods.
This is exemplified in the synthesis of
3-aminomethyl-4'-methylpsoralen,
3-aminomethyl-4,4',8-trimethylpsoralen, 3-aminomethyl-4, 5',
8-trimethylpsoralen, 3-aminomethyl-4,4',5',8-tetramethylpsoralen,
3-(4-amino-2-oxa)butyl-4,4',8 trimethylpsoralen,
4-aminomethyl-4'-methylp- soralen, 4-aminomethyl-4', 5',
8-trimethylpsoralen, 4-(4-amino-2-oxa)butyl-4', 5',
8-trimethylpsoralen, 5-aminomethylpsoralen,
5-(4-amino-2-oxa)butyl-4', 5' dimethylpsoralen,
8-aminomethyl-4,4',5'-trimethylpsoralen,
8-aminomethyl-4,5'-dimethylpsora- len), and
8-(4-amino-2-oxa)butyl-4,4',5'-trimethylpsoralen (described in the
examples below).
[0020] The present invention contemplates methods of inactivating
pathogens in a biological composition, comprising, in the following
order: a) providing, in any order, i) a compound selected from the
group consisting of primaryamino-pyrone-linked psoralens and
primaryamino-benzene-linked psoralens; ii) photoactivating means
for photoactivating said compounds; and iii) a biological
composition suspected of being contaminated with a pathogen which
contains nucleic acid; b) adding said compound to said biological
composition; and c) photoactivating said compound, so as to
inactivate said pathogen. In one embodiment, the biological
composition is a blood product. In a preferred embodiment, the
blood product is either platelets or plasma. A preferred method of
the present invention is performed in a blood bank or similar
setting, wherein said compound is formulated in solution and said
solution is contained in a blood compatible bag, and wherein said
compound is added to said biological composition by flowing said
biological composition through said bag. After treatment with the
method, the blood product is suitable for its intended use.
[0021] A biological composition is defined as a composition
originating from a biological organism of any type. Examples of
biological compositions 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, tissue samples,
homogenized tissue samples, and any other substance having its
origin in a biological organism. Biological compositions 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 being the substance
having its origin in a biological organism), cell culture medium,
cell cultures, viral cultures, and other cultures derived from a
biological organism.
[0022] A pathogen is defined as any agent which contains nucleic
acid and is capable of causing disease in a human, other mammals,
or vertebrates. Examples include microorganisms such as unicellular
or multicellular microorganisms including but not limited to
bacteria, viruses, protozoa, fungi, yeasts, molds, and mycoplasmas.
The pathogen can comprise either DNA or RNA and this nucleic acid
can be single stranded or double stranded.
[0023] The present invention contemplates that the photoactivating
means comprises a photoactivation device capable of emitting a
given intensity of a spectrum of electromagnetic radiation
comprising wavelengths between 180 nm and 400 nm, preferably
between 300 nm and 400 nm, and in particular, between 320 nm and
380 nm. It is preferred that the intensity is between 1 and 30
mW/cm.sup.2 and that the mixture is exposed to this intensity for
between one second and thirty minutes [U.S. Pat. No.
5,593,823].
[0024] The present invention contemplates embodiments wherein said
blood preparation is in a synthetic media. In one embodiment, the
concentration of compound is between 0.1 .mu.M and 1000 .mu.M,
preferably between 1 .mu.M and 500 .mu.M. In a preferred
embodiment, the compound is added to said blood preparation at a
concentration of between 10 .mu.M and 250 .mu.M.
[0025] The present invention contemplates embodiments of the
methods where inactivation is performed without limiting (e.g.
reducing) the concentration of molecular oxygen. Preferably,
inactivation is performed without limiting the concentration of
singlet oxygen that may be formed during the photoreaction step.
Furthermore, there is no need for the use of cosolvents (e.g.
dimethyl sulphoxide (DMSO)) to increase compound solubility.
[0026] In one embodiment, the present invention contemplates
methods of inactivating microorganisms in a blood product, wherein
the compound is a primaryamino-pyrone-linked psoralen, comprising:
a) a substituent A on the pyrone ring, selected from the group
consisting of: --(CH.sub.2).sub.u--NH.sub.2,
--(CH.sub.2).sub.w--J--(CH.sub.2).sub.z--NH- .sub.2,
--(CH.sub.2).sub.w--J--(CH.sub.2).sub.x--K--(CH.sub.2).sub.z--NH.s-
ub.2, and
--(CH.sub.2).sub.w--J--(CH.sub.2).sub.x--K--(CH.sub.2).sub.y--L--
-(CH.sub.2).sub.z--NH.sub.2; wherein J, K, and L are independently
selected from the group consisting of O and NH, in which u is a
whole number from 1 to 10, w is a whole number from 1 to 5, x is a
whole number from 2 to 5, y is a whole number from 2 to 5, and z is
a whole number from 2 to 6; and b) substituents B, R.sub.3,
R.sub.4, R.sub.5, and R.sub.6 on the pyrone ring, 5-, 4'-, 5'- and
8-carbon atoms respectively, independently selected from the group
consisting of --H and --(CH.sub.2).sub.vCH.sub.3, where v is a
whole number from 0 to 5; or a salt thereof.
[0027] Alternatively, the present invention contemplates
embodiments of the method of inactivation, wherein the compound is
a primaryamino-benzene-linked psoralen comprising: a) a substituent
A on the benzene ring, selected from the group consisting of:
--(CH.sub.2).sub.u--NH.sub.2,
--(CH.sub.2).sub.w--J--(CH.sub.2).sub.z--NH- .sub.2,
--(CH.sub.2).sub.w--J--(CH.sub.2).sub.x--K--(CH.sub.2).sub.z--NH.s-
ub.2, and
--(CH.sub.2).sub.w--J--(CH.sub.2).sub.x--K--(CH.sub.2).sub.y--L--
-(CH.sub.2).sub.z--NH.sub.2; wherein J, K, and L are independently
selected from the group consisting of O and NH, in which u is a
whole number from 1 to 10, w is a whole number from 1 to 5, x is a
whole number from 2 to 5, y is a whole number from 2 to 5, and z is
a whole number from 2 to 6; and b) substituents B, R.sub.1,
R.sub.2, R.sub.4, and R.sub.5 on the benzene ring, 3-, 4-,
[0028] 4'-, and 5'-carbon atoms respectively, independently
selected from the group consisting of --H and
--(CH.sub.2).sub.vCH.sub.3, where v is a whole number from 0 to 5;
or a salt thereof, except c) when A is --CH.sub.2--NH.sub.2 and at
the 8-carbon atom, one of B, R.sub.1, R.sub.4, and R.sub.5 must be
--(CH.sub.2).sub.vCH.sub.3.
[0029] In one embodiment of the method of inactivation, at least
two of the compounds are present. The present invention
contemplates embodiments where the compound is introduced either in
aqueous solutions, such as water, saline, or a synthetic media,
preferably a phosphate buffered media, non aqueous solutions such
as alcohols, polyethylene glycols, or solvent mixtures with water,
or in a dry formulation in which additives may be present. In one
embodiment, the present invention contemplates a synthetic platelet
storage media, comprising a glucose and magnesium free aqueous
solution of: 45-120 mM sodium chloride; 5-15 mM sodium citrate;
20-40 mM sodium acetate; and 20-30 mM sodium phosphate. In a
preferred embodiment, the aqueous solution comprises: approximately
86 mM sodium chloride; approximately 10 mM sodium citrate;
approximately 30 mM sodium acetate; and approximately 26 mM sodium
phosphate. The solution has a pH of approximately 300
milliosmolar/Kg. By not containing glucose or magnesium, the media
is readily autoclaved.
[0030] The present invention contemplates embodiments wherein the
compound may be introduced to the reaction vessel at the point of
manufacture. Alternatively, the compound may be added to the
reaction vessel at some point after the manufacture of, for
example, a blood product. In one embodiment, a solution of the
psoralen is provided in a biocompatible container that is attached
to a disposable plastic set containing a unit of platelets or
plasma. The psoralen solution is mixed with the blood product by
passing said blood product through the container of psoralen and
the resultant mixture is photoactivated with an illumination device
suitable for uniform irradiation of blood bags [U.S. Pat. No.
5,593,823]. In a further embodiment, the residual psoralen and any
low molecular weight psoralen photoproducts are removed from the
solution [PCT publication WO 98/30327, hereby incorporated by
reference].
[0031] In one embodiment, the blood product is admixed with the
psoralen and the mixture is passed through a flow system where it
is passed over a static light source resulting in photoactivation
of said psoralen. The means of passing through said flow system
includes but is not limited to gravity flow or metered flow using a
pump system.
DESCRIPTION OF THE INVENTION
[0032] The present invention provides new psoralens and methods of
synthesis of new psoralens having enhanced ability to inactivate
pathogens in the presence of ultraviolet light. The new psoralens
are potentially effective against a wide variety of pathogens. The
present invention also provides methods of using new and known
compounds to inactivate pathogens in biological products to be used
in vivo and in vitro, and in particular, blood products.
[0033] The inactivation methods of the present invention provide ex
vivo methods of inactivating pathogens, and in particular, viruses,
in blood products prior to use in vitro or in vivo. In contrast
with previous approaches, the method requires only short
irradiation times and there is no need to limit (e.g. reduce) the
concentration of molecular oxygen or of singlet oxygen present or
generated in the system.
[0034] In vivo use of a material is defined as introduction of the
material or compound into a living human, mammal, or vertebrate. In
vitro use of 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 a component of
a blood sample using laboratory equipment. Ex vivo use of a
compound is defined as using a compound for treatment of a
biological material such as a blood product outside of 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.
[0035] The description of the invention is divided into the
following sections: I) Compound Synthesis, II) Photoactivation
Devices, III) Binding of Compounds to Nucleic Acid, IV)
Inactivation of Nucleic Acid Containing Materials V) Preservation
of Biochemical Properties of Material Treated, VI) Psoralen
Conjugates and Other uses of Psoralens.
I. Compound Synthesis
[0036] A. Psoralens as Photoactivation Compounds
[0037] The present invention contemplates those compounds described
as psoralens: [7H-furo(3,2-g)-(1)-benzopyran-7-one, or b-lactone of
6-hydroxy-5-benzofuranacrylic acid], which are linear: 7
[0038] and in which the two oxygen residues appended to the central
aromatic moiety have a 1, 3 orientation, and further in which the
furan ring moiety is linked to the 6-position of the two ring
coumarin system. Psoralen derivatives are derived from substitution
of the linear furocoumarin at the 3-, 4-, 5-, 8-, 4'-, or 5'-carbon
atoms indicated in the above structure. For the purpose of this
invention, substituents on the psoralen ring will be designated by
the three ring structure consisting of the furan ring substituents
(substituents linked to the 4'- and 5'-carbon atoms), the central
benzene ring substituents (substituents linked to the 5- and
8-carbon atoms) and the pyrone ring substituents (substituents
linked to the 3- and 4-carbon atoms). More specifically, the
present invention contemplates compounds with a primaryamino
substituent on the 3- or 4-carbon atom herein referred to as
primaryamino-pyrone-linked psoralens and compounds with a
primaryamino substituent on the 5- or 8-carbon atom herein referred
to as primaryamino-benzene-linked psoralens. In addition, the other
of the 3- or 4-carbon atom of a primaryamino-pyrone-linked psoralen
may contain an alkyl substituent. Similarly, the other of the 5- or
8-carbon atom of a primaryamino-benzene-linked psoralen may contain
an alkyl substituent.
[0039] 8-Methoxypsoralen (known in the literature under various
names, e.g., xanthotoxin, methoxsalen, 8-MOP) is a naturally
occurring psoralen with relatively low photoactivated binding to
nucleic acids and low mutagenicity in an Ames assay.
4'-Aminomethyl-4,5',8-trimethylpsoralen (AMT) is one of the most
reactive nucleic acid binding psoralen derivatives, providing up to
1 AMT adduct per 3.5 DNA base pairs [S. T. Isaacs, G. Wiesehahn and
L. M. Hallick, NCI Monograph 66: 21 (1984)]. However, AMT also
exhibits significant levels of mutagenicity. A new group of
psoralens was desired which would have the best characteristics of
both 8-MOP and AMT: low mutagenicity and high nucleic acid binding
affinity, to ensure safe and thorough inactivation of pathogens.
One group of psoralens that has been synthesized and studied are
primaryamino-furan-linked psoralens which are discussed in detail
in U.S. Pat. No. 5,593,823. The compounds of the present invention
are primaryamino-pyrone and primaryamino-benzene-linked analogs
shown to be very effective at inactivation of R17, suggesting very
high nucleic acid binding affinity.
[0040] Primaryamino-pyrone-linked psoralens are defined as psoralen
compounds which have an --NH.sub.2 group linked to the 3- or
4-carbon atom of the psoralen by a hydrocarbon chain having a total
length of 1 to 24 carbons, where 0 to 3 of those carbons are
independently replaced by NH or O, and each point of replacement is
separated from each other point of replacement by at least two
carbons, and is separated from the psoralen by at least one carbon.
Primaryamino-pyrone-linked psoralens may have additional
substituents on the other of the 3- or 4-carbon atom and on the 5-,
8-, 4'-, and 5'-carbon atoms. Said substituents include but are not
limited to --H and --(CH.sub.2).sub.vCH.sub.3, where v is a whole
number from 0 to 5. Compound I above gives the structure of
primaryamino-pyrone-linked psoralens.
[0041] Primaryamino-benzene-linked psoralens are defined as
psoralen compounds which have an --NH.sub.2 group linked to the 5-
or 8-carbon atom of the psoralen by a hydrocarbon chain having a
total length of 1 to 24 carbons, where 0 to 3 of those carbons are
independently replaced by NH or O, and each point of replacement is
separated from each other point of replacement by at least two
carbons, and is separated from the psoralen by at least one carbon.
Primaryamino-benzene-linked psoralens may have additional
substituents on the other of the 5- or 8-carbon atom and on the 3-,
4-, 4'-, and 5'-carbon atoms. Said substituents include but are not
limited to --H and --(CH.sub.2).sub.vCH.sub.3, where v is a whole
number from 0 to 5. When the primaryamino substituent is on the
8-carbon atom, the present invention is limited in that at least
one of the 3-, 5-, 4'- or 5'-substituents is
--(CH.sub.2).sub.vCH.sub.3. Compound IV above gives the structure
of primaryamino-benzene-linked psoralens.
[0042] B. Synthesis of the Primaryamino-Pyrone-Linked Psoralens
[0043] Scheme 1 shows a method of synthesis of
3-halomethylcoumarins (1) and 4-halomethylcoumarins (4) useful for
the preparation of the primaryamino-pyrone-linked compounds of the
present invention. The compounds can be prepared from commercially
available materials and converted to phthalimidomethyl-coumarins (2
and 5) and on to aminomethyl-pyrone-linked psoralens (3 and 6) by
applying previously described methods [McLeod et al. Tetrahedron
Lett. (1972) p. 237; Isaacs et al., Biochem. 16: 1058 (1977)] and
further detailed in the examples. 8
[0044] Longer chain aminoalkyl-pyrone linked psoralens can be
prepared from the analogous haloalkylcoumarins (7 and 9 as shown in
Scheme 2). While there are many ways of making the desired
coumarins, many of them can most conveniently be prepared by the
Pechman reaction [Organic Reactions, Vol VII, Chap 1, ed. Adams et
al., Wiley, N.Y., (1953)] of resorcinols with the functionalized
beta-keto esters. The desired beta-keto esters having a halide or
halide synthon can be prepared by known methods [e.g., J. March,
Advance Organic Chemistry, 3rd Ed., Wiley, (1985) pp437-440 and
824]. For example, Lambert et al., J. Org. Chem., 1985, 50, 5352;
Gupta et al., J. Organomet. Chem. 1993, 444,1; Crombie et al., J.
Chem. Soc Perk Trans I, 1987,333; Tremul Lozano, Span. Pat. 549788
A1 describe the preparation of desirable beta-keto-esters. The
synthesis of such haloalkyl and hydroxyalkylcoumarins have been
previously described [Zaniuk et al, Pol. Pat. PL 144435 B1, Fall et
al, Heterocycles, (1995) 41, 647]. 9
[0045] Alternatively, one can carry the protected alkylcoumarin
(e.g. 7 where Y=halo, OH, OMe) through to a haloalkyl-pyrone linked
psoralen and prepare the compounds of the present invention by
applying some methods known in the art to functionalize
haloalkylpsoralens. Examples of such functionalizations include the
reaction of 4'-bromomethyl or chloromethylpsoralens (4'-BMT and
4'-CMT respectively) with ammonia, or potassium phthalimide (KPhth)
followed by hydrazine to give AMT, as well as the reaction of the
4'-BMT and 4'-CMT with a variety of other amines. The identical
reaction of 5'-bromomethyl or chloromethylpsoralen with KPhth is
known [Kaufman U.S. Pat. No 4,294,822]. The reaction of
5-chloromethyl-8-methoxypsoralen and
8-chloromethyl-5-methoxypsoralen with amines is known.
[0046] For the preparation of compounds of the present invention in
which the linker between the primary amine and the psoralen
contains one or more oxygen, one or more nitrogen, or both oxygen
and nitrogen atoms, the preparation of such functionalized systems
from haloalkylpsoralens has been thoroughly described previously
for 4'- and 5'-primaryamino substituted psoralens [U.S. Pat. No.
5,654,443, incorporated by reference herein]. The same synthetic
methods can be applied to the 3- and 4-primaryamino substituted
psoralens as shown in the examples. Finally, the use of pseudo
halides such as the methanesulfonyl group has been described such
as in the reaction of 4'-(4-methanosulfonyloxy-2-oxa)butyl-
-4,5',8-trimethylpsoralen with sodium azide and subsequently
converted into 4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen.
The same synthetic methods can be applied to the 3- and
4-primaryamino substituted psoralens as shown in the examples and
typified in scheme 3 for the synthesis of 13 and 16, which have an
oxygen atom in the primaryamino substituent chain. The synthesis
starts from methoxyalkylcoumarins 7 and 9 above (where Y.dbd.OMe)
which may be prepared from 7 and 9 (Y=halo, OH) or via direct
coumarin synthesis. Conversion to psoralens 11a and 14a follows the
same procedures described here in Scheme 1 and elsewhere. The
psoralens are then de-methylated in a procedure that provides
either the haloalkylpsoralen directly, or a hydroxyalkylpsoralen
that is converted to a pseudohaloalkylpsoralen (11b and 14b below).
By known procedures, set forth in the examples and elsewhere [U.S.
Pat. No. 5,654,443] the haloalkylpsoralens are converted to the
primaryamino-pyrone-linked substituted psoralens 13 and 16. 10
[0047] C. Synthesis of Primaryamino-Benzene-Linked Psoralens
[0048] As described above for the primaryamino-pyrone-linked
compounds, the desired primaryamino-benzene-linked compounds can be
prepared by initial functionalization prior to formation of the
psoralen ring system. Halomethyl coumarins (17 and 19 below) can be
prepared by bromination of the appropriate coumarin as shown in
Scheme 4 and then converted by procedures described in the examples
and elsewhere into the desired aminomethylpsoralens (18 and 20
below) of the present invention. For primaryamino compounds linked
to the 8 carbon atom, the 8-aminoloweralkylpsoralens and
8-aminoloweralkyl-4-loweralkylpsoralens are described in U.S. Pat.
Nos. 4,328,239 and 4,269,851, hereby incorporated by reference.
11
[0049] The above aminomethyl-benzene linked compounds and longer
chain aminoalkyl-benzene linked psoralens (23 and 26 below) can be
prepared by pre forming the analogous haloalkylcoumarins (22 and 25
as shown in Scheme 5). While there are many ways of making the
desired coumarins, many of them can most conveniently be prepared
by the Pechman reaction [Organic Reactions, Vol VII, Chap 1, ed.
Adams et al., Wiley, N.Y., (1953)] of functionalized resorcinols
with beta-keto esters. The desired resorcinols having a halide or
halide synthon (21 and 24 below) can be prepared by known methods
[see for example Makriyannis et al, U.S. Pat. No. 5,440,052;
Seebach et al, Helv. Chim. Acta (1994) 77, 1673; Charalambous et
al, J. Med. Chem (1992) 35, 3076; Elix et al, Aust. J. Chem. (1987)
40, 1841]. The haloalkylcoumarins can then be converted to
protected aminoalkylcoumarins and taken on to the psoralens by
methods described in the examples. 12
[0050] Alternatively, one can carry the protected alkylcoumarin
(e.g. 22 and 25 where Y=halo, OH, OMe) through to a
haloalkylpyrone-linked psoralen and prepare the compounds of the
present invention by methods known in the art to functionalize
haloalkylpsoralens as shown in Scheme 6 for the synthesis of 28 and
30 below, which have an oxygen atom in the primaryamino substituent
chain. The synthesis starts from methoxyalkylcoumarins 22 and 25
above (where Y.dbd.OMe) which may be prepared from 22 and 25
(Y=halo, OH) or via direct coumarin synthesis. Conversion to the
psoralens, 27 and 29, follows the same procedures described here in
Scheme 3 and elsewhere. By known procedures, set forth in the
examples, the haloalkylpsoralens are converted to 28 and 30.
[0051] For the preparation of primaryamino-benzene-linked psoralens
in which the linker between the primary amine and the psoralen
contains one or more oxygen, one or more nitrogen, or both oxygen
and nitrogen atoms, methods discussed above for the
primaryamino-pyrone-linked psoralens can be used. 13
[0052] D. Synthesis of Psoralen Conjugates.
[0053] The preparation of psoralen conjugates where the psoralen is
linked to nucleotides, biotin, other intercalators, etc. has been
described for the 4'-linked psoralens, for the 5-methyl
linked-8-methoxypsoralen and for the 8-methyl
linked-5-methoxypsoralen. While not being limited to any synthetic
method to conjugate the psoralen onto another small molecule,
protein, nucleic acid or material surface, typical methods for
linking psoralens often entail one of three methods: 1) the
bromomethylpsoralen is reacted with an amino or hydroxy function on
the conjugated moiety; 2) the aminomethylpsoralen is reacted with
an amide, urea or carbamate precursor (e.g., a succinamido group,
or an isocyanate) on the conjugating moiety; and 3) the
hydroxymethylpsoralen is reacted with an ester, or carbamate
precursor on the conjugating moiety.
[0054] By the use of such known methods of conjugation, or
alternative methods that may provide greater ease of preparation,
one can prepare compositions comprising a psoralen linked through
the pyrone ring to other nucleic acid binding moieties, to
proteins, to nucleic acids, to fluorescent probes or other small
molecules useful in diagnostics and to material surfaces.
[0055] Likewise, by the use of such known methods of conjugation,
or alternative methods that may provide greater ease of
preparation, one can prepare compositions comprising a psoralen
linked through the benzene ring to other nucleic acid binding
moieties, to proteins, to nucleic acids, to fluorescent probes or
other small molecules useful in diagnostics and to material
surfaces.
II. Photoactivation Devices
[0056] A variety of light devices may be useful for the
photoactivation of compounds of the present invention and may be
useful in the present methodology. Features of possible devices may
be found in U.S. Pat. Nos. 5,593,823 and 5,683,661, hereby
incorporated by reference. Additional features for possible uses of
the compounds of this invention would include a means for passing a
solution for inactivation through a light device such that the
solution is sufficiently illuminated so as to inactivate pathogens
within the solution. Said means may include gravity flow or metered
flow, such as through a peristaltic pump or similar flow
apparatus.
III. Binding of Compounds to Nucleic Acid
[0057] The present invention contemplates binding new and known
compounds to nucleic acid, including (but not limited to) viral
nucleic acid, bacterial nucleic acid, nucleic acid of lymphocytes,
and nucleic acid of tissue cells such as smooth muscle cells. One
approach of the present invention to binding photoactivation
compounds to nucleic acid is photobinding. Photobinding is defined
as the binding of photobinding compounds in the presence of
photoactivating wavelengths of light. Photobinding compounds are
compounds that bind to nucleic acid in the presence of
photoactivating wavelengths of light. The present invention
contemplates methods of photobinding with compounds of the present
invention.
[0058] One embodiment of the method of the present invention for
photobinding involves the steps: a) providing a photobinding
compound of the present invention; and b) mixing the photobinding
compound with nucleic acid in the presence of photoactivation
wavelengths of electromagnetic radiation.
[0059] The invention further contemplates a method for modifying
nucleic acid, comprising the steps: a) providing photobinding
compound of the present invention and nucleic acid; and b)
photobinding the photobinding compound to the nucleic acid, so that
a compound:nucleic acid complex is formed.
IV. Inactivation of Nucleic Acid Containing Materials
[0060] The present invention contemplates treating a blood product
with a photoactivation compound and irradiating to inactivate
contaminating pathogen nucleic acid sequences before using the
blood product. The present invention could also be applied to
inactivation of other nucleic acid containing materials, such as
lymphocytes, tissue cells, and solutions containing nucleic acids,
for example solutions which have been amplified by polymerase chain
reaction or a similar nucleic acid amplification technique.
[0061] A. Inactivation in General
[0062] The term "inactivation" is here defined as the altering of
the nucleic acid in a material so as to render the nucleic acid
incapable of replication. When the nucleic acid is that of a
pathogen, the inactivation of the nucleic acid renders the pathogen
incapable of replication. The inactivation of pathogens is detailed
in U.S. Pat. No. 5,593,823. In addition, inactivation may occur in
any cell and the level of inactivation within a cell may be
controlled by the level of photobinding of the psoralen to the
nucleic acid. The level of photobinding can be controlled by
varying either the dose of light used or the dose of the psoralen.
The level of inactivation can be controlled, ranging from
completely shutting down all cellular functions (high levels of
photobinding) to shutting down proliferation of the cell while
maintaining cellular functions (low levels of photobinding), i.e.
the cell is still capable of transcribing the nucleic acid for the
production of proteins. For example, a lymphocyte or tissue cell
may be inactivated in that it can not replicate yet can still
produce proteins and maintain biological function. This may also be
referred to as inhibition of cellular proliferation rather than
inactivation.
[0063] B. Inactivation of Potential Pathogens
[0064] In the case of inactivation methods for material to be used
by humans, whether in vivo or in vitro, the detection method can
theoretically be taken to be the measurement of the level of
infection with a disease as a result of exposure to the material.
The threshold below which the inactivation method is complete is
then taken to be the level of inactivation which is sufficient to
prevent disease from occurring due to contact with the material. It
is recognized that in this practical scenario, it is not essential
that the methods of the present invention result in "total
inactivation". That is to say, "substantial inactivation" will be
adequate as long as the viable portion is insufficient to cause
disease. The inactivation method of the present invention renders
nucleic acid in pathogens substantially inactivated. In one
embodiment, the inactivation method renders pathogen nucleic acid
in blood preparations substantially inactivated.
[0065] Without intending to be limited to any method by which the
compounds of the present invention inactivate pathogens, it is
believed that inactivation results from light induced binding of
psoralens to pathogen nucleic acid. Further, while it is not
intended that the inactivation method of the present invention be
limited by the nature of the nucleic acid; it is contemplated that
the inactivation method render all forms of nucleic acid (whether
DNA, mRNA, etc.) substantially inactivated.
[0066] In the case of photoactivation compounds modifying nucleic
acid, it is preferred that interaction of the pathogen nucleic acid
(whether DNA, mRNA, etc.) with the photoactivation compound causes
the pathogen to be unable to replicate, such that, should a human
be exposed to the treated pathogen, infection will not result.
[0067] "Synthetic media" is herein defined as an aqueous synthetic
blood or blood product storage media. In one embodiment, the
present invention contemplates inactivating blood products in
synthetic media. This method may reduce product degradation during
storage and permits the use of lower concentrations of
photoactivation compounds.
[0068] The psoralen photoinactivation method inactivates nucleic
acid based pathogens present in blood through a single procedure.
Thus, it has the potential to eliminate bacteria, protozoa, and
viruses as well. Had an effective decontamination method been
available prior to the advent of the AIDS pandemic, no transfusion
associated HIV transmission would have occurred. Psoralen-based
decontamination has the potential to eliminate all infectious
agents from the blood supply, regardless of the pathogen involved.
Additionally, psoralen-based decontamination has the ability to
sterilize blood products after collection and processing, which in
the case of platelet concentrates could solve the problem of low
level bacterial contamination and result in extended storage life.
[J. Morrow et al., JAMA 266: 555-558 (1991); F. Bertolini et al.,
Transfusion 32: 152-156 (1992)].
[0069] A list of viruses which have been photochemically
inactivated by one or more psoralen derivatives appears in Table 2
[From Table 1 of Hanson, C. V., Blood Cells 18:7 (1992)]. This list
is not exhaustive, and is merely representative of the great
variety of pathogens psoralens can inactivate. The present
invention contemplates the inactivation of these and other viruses
by the compounds described herein. The compounds of the present
invention are particularly well suited for inactivating envelope
viruses, such as the HIV virus.
1TABLE 2 Viruses Photochemically Inactivated by Psoralens 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.sup.1 Poliovirus 1 and 2 Coxsackie A-9 Echo 11 Pox Vaccinia
Fowl Pox Reo Reovirus 3 Blue tongue Colorado tick fever Retro HIV
Avian sarcoma Murine sarcome Murine leukemia Rhabdo Vesticular
stomatitis virus Toga Western equine encephalitis Dengue 2 Dengue 4
St. Louis encephalitis Hepadna hepatitis B Bacteriophage Lambda T2
(Rickettsia) R. akari (rickettsialpox)
[0070] C. Selecting Photoactivation Compounds for Inactivation of
Pathogens
[0071] In order to evaluate a compound to decide if it would be
useful in the methods of the present invention, two important
properties should be considered: the compound's ability to
inactivate pathogens and the compounds effect on the suitability of
the treated product for its intended use. A discussion of
inactivation of pathogens other than the R17 model discussed below
can be found in U.S. Pat. No. 5,593,823. This reference enables the
selection criteria for use in pathogen inactivation of blood
products for the compounds of the present invention. The screening
technique used to evaluate the compounds of the present invention
is to perform a bacteriophage screen; an assay which determines
nucleic acid binding of test compounds. A screen of this type, an
R17 screen, is described in detail in EXAMPLE 13, below.
[0072] The R17 bacteriophage screen is believed to be predictive of
HIV inactivation efficiency, as well as the efficiency of compounds
against many other viruses. It is a small, single stranded RNA
phage. Without intending to be limited to any means by which the
present invention operates, it is expected that shorter pieces of
nucleic acid are harder to inactivate because they require a higher
frequency of formation of psoralen adducts than do longer pieces of
nucleic acid. Further, single stranded RNA pathogens are more
difficult to inactivate because psoralens can neither intercalate
between base pairs, as with double-stranded nucleic acids, nor form
diadducts which function as interstrand crosslinks. Thus it is
expected that when inactivation of R17 is achieved, these same
conditions will cause the inactivation of many viruses and
bacteria. More specifically, those compounds which exhibit >1
log inactivation of R17 (i.e. >90% kill) at a compound
concentration in a test medium of 320 .mu.M or less are expected to
be reasonable candidates for inactivation of pathogens in blood
products.
V. Preservation of Biochemical Properties of Material Treated
[0073] Psoralens are useful in inactivation procedures because the
reaction can be carried out at temperatures compatible with
retaining biochemical properties of blood and blood products
[Hanson, C. V., Blood Cells 18:7 (1992)]. The inactivation
compounds and methods of the present invention are especially
useful because they provide a means to inactivate pathogens while
potentially retaining the suitability of the product for its
intended use. The suitability of plasma may be measured by
functionality of its protein components, either in whole plasma or
after separation into plasma fractions. The suitability of
platelets may be determined by methods and criteria similar to
those used for establishing the suitability of storage and handling
protocols.
VI. Psoralen Conjugates and Other Uses of Psoralens
[0074] Because of their affinity for nucleic acids and ability to
covalently bind to nucleic acids, compounds of the present
invention could be very useful when conjugated to other molecules.
The conjugates may be formed by either chemical or photochemical
attachment of the psoralen to another molecule, molecular fragment,
or ligand. Materials to which psoralen may be conjugated include,
but are not limited to, other nucleic acid binding moieties such as
acridines or lexitropsins, proteins such as antibodies or receptor
ligands, nucleic acids, small molecules useful in diagnostics such
as fluorescent probes and biotin, and material surfaces.
[0075] The amino terminated chain of the psoralens of the present
invention, or the halogen substituted intermediates indicated in
EXAMPLES 6-9, are particularly suited to chemical attachment to
other molecules. Such amino terminated compounds could be
substituted for AMT in the following examples. In Wang Z, et al.,
J. Am. Chem. Soc. 117(20): 5438-5444 (1995), AMT is conjugated to a
protected amino acid and subsequently used to prepare a
psoralen:peptide conjugate. Such conjugates could be used to probe
sequence specific protein--nucleic acid interactions, or perhaps to
selectively control gene expression. Similarly, psoralen may be
conjugated to a nucleic acid oligonucleotide which is directed to a
specific nucleic acid sequence (Vaghefi et al., PCT publication WO
92/02641). Such conjugates could be used as a control of gene
expression or as a site specific probe of the nucleic acid
sequence. The conjugation of the psoralen allows for a covalent
photochemical attachment of the oligonucleotide to the target
sequence. Other molecules conjugated to a psoralen starting from an
amino terminal chain of the psoralen include, but are not limited
to, biotin, fluorescent dyes, insulin, and lexitropsin, the uses of
which are discussed in the references [U.S. Pat. Nos. 4,737,454,
and 4,599,303, Biochem. and Biophys. Res. Comm. 141(2): 502-509
(1986), Anti-cancer Drug Design 9: 221-237 (1994)].
[0076] Any psoralen could potentially be conjugated to a nucleic
acid photochemically. Such photochemical conjugates could be used
as probes to specific nucleic acid targets. The photochemically
conjugated psoralen could be prepared such that when the modified
oligonucleotide pairs with the complementary target nucleic acid,
the psoralen can crosslink the probe to the target strand with an
additional UV light dose. Preparation and uses of such
psoralen-oligonucleotide photochemical conjugates are described in
U.S. Pat. Nos. 4,737,454, 4,599,303, and 5,532,146. Similarly, any
material with which psoralen can interact and photobind could be
photochemically conjugated to psoralen. For example, Bioconjugate
Chem. 5(5): 463-467 (1994) give a method of photoreacting a
psoralen compound with a polystyrene surface such as a microtiter
plate. The psoralen can then be conjugated to another molecule such
as an oligonucleotide, peptide, or biotin. While this reference
uses psoralens containing a secondary amine, the primaryamino
compounds of the present invention would also be useful using the
conjugation schemes discussed in the references above.
[0077] Other possible uses of the compounds of the present
invention include the inactivation of viruses for the purpose of
preparing a vaccine, the inhibition of leukocytes to control
proliferation yet maintain some function as a means of preventing
graft vs. host disease in bone marrow transplants, and the
inhibition of smooth muscle cells to control proliferation after
injury, for example to prevent restenosis after balloon
angioplasty. A discussion of the use of psoralens in vaccine
preparation can be found in U.S. Pat. No. 5,106,619. A discussion
of the use of 8-Methoxypsoralen for prevention of restenosis can be
found in U.S. Pat. No. 5,354,774. These references are herein
incorporated by reference.
Experimental
[0078] The following examples serve to illustrate certain preferred
embodiments and aspects of the present invention and are not to be
construed as limiting the scope thereof.
[0079] In the experimental disclosure which follows, the following
abbreviations apply: J (Joules); TLC (Thin Layer Chromatography);
NMR (Nuclear Magnetic Resonance; spectra obtained at room
temperature on a Varian Gemini 200 MHz Fourier Transform
Spectrometer); THF (tetrahydrofuran); DMF (N,N-dimethylformamide);
DMEM (Dulbecco's Modified Eagles Medium); FBS (fetal bovine serum);
LB (Luria Broth); EDTA (ethelenediaminetetraacetic acid). Starting
materials for the synthesis examples are obtained from common
suppliers such as Aldrich Chemicals, Milwaukee, Wis.
[0080] When isolating compounds of the present invention in the
form of an acid addition salt, the acid is preferably selected so
as to contain an anion which is non-toxic and pharmacologically
acceptable, at least in usual therapeutic doses. Representative
salts which are included in this preferred group are the
hydrochlorides, hydrobromides, sulphates, acetates, phosphates,
nitrates, methanesulphonates, ethanesulphonates, lactates,
citrates, tartrates or bitartrates, and maleates. Other acids are
likewise suitable and may be employed as desired. For example,
fumaric, benzoic, ascorbic, succinic, salicylic,
bismethylenesalicylic, propionic, gluconic, malic, malonic,
mandelic, cinnamic, citraconic, stearic, palmitic, itaconic,
glycolic, benzenesulphonic, and sulphamic acids may also be
employed as acid addition salt-forming acids.
[0081] The following examples serve to illustrate certain preferred
embodiments and aspects of the present invention and are not to be
construed as limiting the scope thereof.
EXAMPLE 1
Synthesis of 3-aminomethyl-4,4',8-trimethylpsoralen
Hydrochloride
Compound 31
[0082] Step 1:
[0083] A solution of 7-hydroxy-3,4,8-trimethylcoumarin (5.19 g,
25.4 mmol) was refluxed in acetic anhydride (10 mL) for 1.5 hours.
The solution was slowly poured into ice water (200 mL) and the
resulting solid was filtered, then rinsed with water to yield
7-acetoxy-3,4,8-trimethylcoumar- in, a beige solid (6.22 g, 99.5%).
.sup.1H NMR (CDCl.sub.3): (7.47 (d, J=8.6 Hz, 1H), 7.00 (d, J=8.7
Hz, 1H), 2.40 (s, 3H), 2.37 (s, 3H), 2.29 (s, 3H), 2.22 (s,
3H).
[0084] Step 2:
[0085] A mixture of 7-acetoxy-3,4,8-trimethylcoumarin (6.22 g, 25.3
mmol), N-bromosuccinimide (4.61 g, 25.9 mmol) and benzoyl peroxide
(30 mg) were refluxed in carbon tetrachloride for 3.5 hours. The
mixture was cooled to room temperature and partitioned between
CH.sub.2Cl.sub.2 and water. The organic layer was separated and
washed with water several times, then washed with brine. The
organic layer was then dried with anhydrous sodium sulfate and
evaporated to give crude product (11.8 g) which was recrystallized
twice in toluene to give 7-acetoxy-3-bromomethyl-4,8-dimet-
hylcoumarin, a white crystalline solid (4.86 g, 59%). .sup.1H NMR
(CDCl.sub.3): (7.54 (d, J=8.8 Hz, 1H), 7.05 (d, J=8.8 Hz, 1H), 4.57
(s, 2H), 2.50 (s, 3H), 2.37 (s, 3H), 2.28 (s, 3H).
[0086] Step 3:
[0087] A mixture of 7-acetoxy-3-bromomethyl-4,8-dimethylcoumarin
(200 mg, 0.617 mmol) and potassium phthalimide (126 mg, 0.680 mmol)
was stirred overnight at room temperature in DMF (3 mL). The slurry
was poured into ice water, filtered and washed with a copious
amount of water to remove traces of DMF to give
7-acetoxy-4,8-dimethyl-3-phthalimidomethylcoumarin, a creamy white
solid after drying (221 mg, 91.7%). .sup.1H NMR (CDCl.sub.3):
(7.84-7.67 (m, 4H), 7.55 (d, J=8.8 Hz, 1H), 7.02 (d, J=8.8 Hz, 1H),
4.92 (s, 2H), 2.62 (s, 3H), 2.36 (s, 3H), 2.25 (s, 3H).
[0088] Step 4:
[0089] A solution of
7-acetoxy-4,8-dimethyl-3-phthalimidomethylcoumarin (15.9 g, 40.6
mol) was stirred in methanol (2000 mL) while concentrated
H.sub.2SO.sub.4 (75 mL) was added dropwise. The resulting mixture
was refluxed for 3 hours, allowed to cool to room temperature, then
chilled in an ice water bath. The precipitate was collected in a
Buchner funnel and rinsed with ice cold methanol to give
7-hydroxy-4,8-dimethyl-3-phthal- imidomethylcoumarin, a white solid
(12.1 g, 85.6%). .sup.1H NMR (CD.sub.3OD): (7.87-7.78 (m, 4H), 7.55
(d, J=8.7 Hz, 1H), 6.84 (d, J=8.8 Hz, 1H), 2.61 (s, 3H), 2.23 (s,
3H), the methylene peak is presumably obscured by solvent hydroxy
peak at 4.88.
[0090] Step 5:
[0091] A slurry of
7-hydroxy-4,8-dimethyl-3-phthalimidomethylcoumarin (4.00 g, 11.5
mmol), potassium carbonate (4.5 g, 36.9 mmol), chloroacetone (1 mL,
11.5 mmol) and acetone (200 mL) were refluxed overnight. After the
solution was allowed to cool to room temperature, CH.sub.2Cl.sub.2
(200 mL) was added and the resulting solid was filtered off. The
remaining solution was cold decolorized with charcoal and the
solvent evaporated. The resulting solid was stirred with water,
vacuum filtered, and washed with water. After air drying,
4,8-dimethyl-7-(2-oxo)propyloxy-3-(phthalimidomethyl)coumarin (4.06
g, 87.1%) was obtained as an off-white solid. .sup.1H NMR
(CDCl.sub.3): (7.66-7.83 (m, 4H), 7.48 (d, J=8.8 Hz, 1H), 6.65 (d,
J=8.8 Hz, 1H), 4.91 (s, 2H), 4.62 (s, 2H), 2.59 (s, 3H), 2.35 (s,
3H), 2.32 (s, 3H).
[0092] Step 6:
[0093] Compound
4,8-dimethyl-7-(2-oxo)propyloxy-3-(phthalimidomethyl)couma- rin
(655 mg, 1.62 mmol) was stirred in concentrated NaOH (30 mL)
overnight. The brown solution obtained was poured into ice water
(150 mL) and acidified with concentrated H.sub.2SO.sub.4 to pH 1.
The white solid obtained was allowed to stir for several hours,
filtered, and rinsed with water. After drying in a vacuum
dessicator with phosphorus pentoxide,
3-(o-carboxybenzamido)methyl-4,4',8-trimethylpsoralen (68.3 mg,
102%) was obtained as a white solid. .sup.1H NMR (DMSO-d.sub.6):
(8.54 (m, 1H), 7.41-7.93 (m, 6H), 4.45 (d, J=4.8 Hz, 2H), 2.65 (s,
3H), 2.30 (s, 3H).
[0094] Step 7:
[0095] A slurry of the carboxylic acid
3-(o-carboxybenzamido)methyl-4,4',8- -trimethylpsoralen (304 mg,
0.751 mmol) in 6N HCl (50 mL) was refluxed overnight. The reaction
mixture was extracted with CH.sub.2Cl.sub.2. The aqueous acid layer
was made basic with solid K.sub.2CO.sub.3 and extracted with
CH.sub.2Cl.sub.2. The organic layer was dried and evaporated to
give 3-aminomethyl-4,4',8-trimethylpsoralen (129 mg, 66.8%) as a
yellow solid. .sup.1H NMR (CDCl.sub.3): (7.58 (s, 1H), 7.48 (app.
quartet, J=1.3 Hz, 1H), 3.91 (s, 2H), 2.59 (s, 6H), 2.29 (d, J=1.2
Hz, 3H). .sup.13C NMR (CD.sub.3OD): 8.35, 8.89, 15.57, 39.47,
109.72, 112.54, 116.35, 117.04, 124.85, 125.78, 143.18, 147.91,
148.80, 155.86, 162.55.
[0096] Step 8:
[0097] The free amine 3-aminomethyl-4,4',8-trimethylpsoralen (129
mg, 0.502 mmol) was dissolved in a minimum amount of warm ethanol
and acidified with 1M HCl in ether (0.7 mL). After refluxing for a
few minutes to expel ether, the solution was allowed to cool to
room temperature and chilled in an ice bath. The solid formed was
filtered and rinsed with ice cold ethanol to give
3-aminomethyl-4,4',8-trimethylpsoral- en hydrochloride (28.9 mg,
19.7%), a yellow solid.
EXAMPLE 2
Synthesis of 8-aminomethyl-4,4',5'-trimethylpsoralen
Compound 32
[0098] Step 1:
[0099] Hydroxymethylphthalimide (1.59 g, 8.98 mmol) was added in
portions approximately every 10 minutes to a solution of
7-hydroxy-4-methylcoumari- n (1.50 g, 8.55 mmol) dissolved in
concentrated H.sub.2SO.sub.4 (18 mL). More H.sub.2SO.sub.4 (4 mL)
was added and the slurry was stirred for 1 hour at room
temperature. The clear solution was poured into 50 mL of ice water
and stirred until the ice melted. The resulting white precipitate
was filtered off, rinsed with water and allowed to air dry. After
the crude solid was triturated (2.times.100 mL) with chloroform to
remove soluble impurities,
7-hydroxy-4-methyl-8-phthalimidomethylcoumarin, a white solid, was
obtained (1.96 g, 68.5%). .sup.1H NMR (CDCl.sub.3): (7.70-7.96 (m,
4H), 7.50 (d, J=8.8 Hz, 1H), 6.97 (d, J=8.9 Hz, 1H), 6.17 (s, 1H),
5.13 (s, 2H), 2.38 (s, 3H).
[0100] Step 2:
[0101] In the same manner as step 5 of EXAMPLE 1 above using
3-chloro-2-butanone instead of chloroacetone,
7-hydroxy-4-methyl-8-phthal- imidomethylcoumarin was reacted to
form 4-methyl-7-(1-methyl-2-oxo)propylo-
xy-8-(phthalimidomethyl)coumarin, a pale yellow solid. .sup.1H NMR
(CDCl.sub.3): (7.64-7.88 (m, 4H), 7.46 (d, J=8.9 Hz, 1H), 6.58 (d,
J=9.0 Hz, 1H), 6.19 (s, 1H), 5.21 (s, 2H), 4.67 (q, J=6.9 Hz, 1H),
2.37 (s, 3H), 2.02 (s, 3H), 1.42 (d, J=6.8 Hz, 3H).
[0102] Step 3:
[0103]
4-Methyl-7-(1-methyl-2-oxo)propyloxy-8-phthalimidomethylcoumarin
(2.99 g, 8.92 mmol) was refluxed in 10% NaOH (135 mL) for 30 min.
The olive green solution was allowed to cool to room temperature,
chilled in an ice bath and acidified with HCl to pH 1. The
precipitate obtained was filtered off, washed with water and dried
to give 8-(o-carboxybenzamido)m- ethyl-4,4',5'-trimethylpsoralen,
an off-white solid (3.15 g, 87.3%). .sup.1H NMR (DMSO-d.sub.6):
(8.73-8.86 (m, 1H), 7.83 (s, 1H), 7.40-7.79 (m, 4H), 6.38 (s, 1H),
4.82 (d, J=4.5 Hz, 2H), 2.45 (s, 3H), 2.23 (s, 3H), 3rd methyl
group presumably obscured by DMSO peak.
[0104] Step 4:
[0105] In the same manner as step 7 of EXAMPLE 1,
8-(o-carboxybenzamido)me- thyl-4,4',5'-trimethylpsoralen was
reacted to form 8-aminomethyl-4,4',5'-t- rimethylpsoralen, a light
yellow solid. .sup.1H NMR (CDCl.sub.3): (7.47 (s, 1H), 6.25 (s,
1H), 4.32 (s, 2H), 2.51 (s, 3H), 2.43 (s, 3H), 2.19 (s, 3H).
[0106] Step 5:
[0107] In the same manner as step 8 of EXAMPLE 1,
8-Aminomethyl-4,4',5'-tr- imethylpsoralen was converted to
8-Aminomethyl-4,4',5'-trimethylpsoralen hydrochloride, an off-white
solid. .sup.1H NMR (DMSO-d.sub.6): (8.46 (s, 3H), 7.97 (s, 1H),
6.44 (s, 1H), 4.38 (s, 2H), 2.57 (s, 3H), 2.47 (s, 3H), 2.24 (s,
3H).
[0108] .sup.13C NMR (CD.sub.3OD): 7.96, 12.10, 19.53, 19.61, 33.48,
104.92, 112.18, 113.64, 117.22, 117.66, 129.50, 155.30, 156.62,
162.64.
[0109] By the same method but using chloroacetone in step 2,
8-aminomethyl-4,5'-dimethylpsoralen hydrochloride (Compound 33) may
be prepared.
EXAMPLE 3
Synthesis of 3-aminomethyl-4,4',5',8-tetramethylpsoralen
Compound 34
[0110] Step 1:
[0111] In the same manner as step 5 of EXAMPLE 1 but using
3-chloro-2-butanone in place of chloroacetone,
7-hydroxy-4,8-dimethyl-3-p- hthalimidomethylcoumarin was reacted to
form 4,8-dimethyl-7-(1-methyl-2-ox-
o)propyloxy-3-phthalimidomethylcoumaran, a yellow solid. .sup.1H
NMR (CDCl.sub.3): (7.67-7.83 (m, 4H), 7.45 (d, J=8.8 Hz, 1H), 6.63
(d, J=9.0 Hz, 1H), 4.91 (s, 2H), 4.72 (q, J=6.8 Hz, 1H), 2.58 (s,
3H), 2.35 (s, 3H), 2.17 (s, 3H), 1.55 (d, J=6.8 Hz, 3H).
[0112] Step 2:
[0113] In the same manner as step 3 of EXAMPLE 2,
4,8-dimethyl-7-(1-methyl-
-2-oxo)propyloxy-3-phthalimidomethylcoumarin was reacted to form
3-(o-carboxybenzamido)methyl-4,4',5',8-tetramethylpsoralen, an
off-white solid. .sup.1H NMR (DMSO-d.sub.6): (8.53 (t, J=1.2 Hz,
1H), 7.80 (s, 1H), 7.37-7.79 (m, 4H), 4.45 (d, J=4.6 Hz, 2H), 2.64
(s, 3H), 2.44 (s, 3H), 2.22 (s, 3H), 4th methyl group presumably
obscured by DMSO peak.
[0114] Step 3:
[0115] In the same manner as step 7 of EXAMPLE 1,
3-(o-carboxybenzamido)me- thyl-4,4',5',8-tetramethylpsoralen was
reacted to form 3-aminomethyl-4,4',5',8-tetramethylpsoralen, a pale
yellow solid. .sup.1H NMR (CDCl.sub.3): (7.44 (s, 1H), 3.90 (s,
2H), 2.57 (s, 6H), 2.42 (s, 3H), 2.19 (s, 3H).
[0116] Step 4:
[0117] In the same manner as step 8 of EXAMPLE 1,
3-aminomethyl-4,4',5',8-- tetramethylpsoralen was converted to
3-aminomethyl-4,4',5',8-tetramethylps- oralen hydrochloride, a
yellow solid. .sup.1H NMR (DMSO-d.sub.6): (8.00-8.22 (m, 3H), 7.90
(s, 1H), 4.09 (s, 2H), 2.67 (s, 3H), 2.45 (s, 3H), 2.23 (s, 3H),
4th methyl group presumably obscured by DMSO peak. .sup.13C NMR
(DMSO): 7.82, 8.46, 12.01, 16.18, 35.38, 107.71, 110.38, 113.40,
115.62, 115.79, 126.98, 147.78, 152.83, 153.78, 154.06, 160.85.
EXAMPLE 4
Synthesis of 3-aminomethyl-4,5',8-trimethylpsoralen
Compound 35
[0118] Step 1:
[0119] A slurry of
7-hydroxy-4,8-dimethyl-3-phthalimidomethylcoumarin (649 mg, 1.86
mmol), potassium carbonate (320 mg, 2.60 mmol), potassium iodide
(15 mg, 0.093 mmol), and 2,3-dichloro-1-propene (0.20 mL, 2.23
mmol) in DMF (15 mL) was stirred at 55-65.degree. C. for 8 hours,
allowed to cool to room temperature, then chilled in an ice water
bath. The solid was filtered off to obtain the first crop of crude
product (1.09 g). Half of the solvent was removed from the filtrate
and after chilling, a second crop of crystals was obtained (33 mg).
The solids were combined, dissolved in CH.sub.2Cl.sub.2, and washed
with water several times. The organic layer was dried and
evaporated to give 7-(beta-chloroallyloxy)-4,-
8-dimethyl-3-phthalimidomethylcoumarin, a white solid (690 mg,
87%). .sup.1H NMR (CDCl.sub.3): (7.66-7.83 (m, 4H), 7.49 (d, J=9.1
Hz, 1H), 6.79 (d, J=8.8 Hz, 1H), 5.56 (s, 1H), 5.47 (s, 1H), 4.91
(s, 2H), 4.67 (s, 2H), 2.59 (s, 3H), 2.32 (s, 3H).
[0120] Step 2:
[0121]
7-(beta-chloroallyloxy)-4,8-dimethyl-3-phthalimidomethylcoumarin
(487 mg, 1.15 mmol) was refluxed in 1,4-diisopropylbenzene (20 mL)
for 17 hours. After cooling to room temperature, the beige
precipitate (347 mg) consisting of the intermediate
6-(beta-chloroallyl)-4,8-dimethyl-7-hydrox-
y-3-phthalimidomethylcoumarin and the decomposition product
4,8-dimethyl-7-hydroxy-3-phthalimidomethylcoumarin and residual
solvent were collected and rinsed with hexane.
[0122] Concentrated H.sub.2SO.sub.4 (2.5 mL) was added dropwise to
an ice cold slurry of crude product (296 mg) in 70% H.sub.2SO.sub.4
and stirred for 25 minutes. The resulting clear solution was poured
into ice water (100 mL). The white precipitate was collected by
vacuum filtration, rinsed with water and partitioned between
CH.sub.2Cl.sub.2 (200 mL) and 10% NaOH (50 mL). The aqueous base
layer was washed with CH.sub.2Cl.sub.2 (2.times.50 mL) and the
organic layers were combined, washed with water (3.times.100 mL),
dried with brine, then with anhydrous sodium sulfate and
evaporated. After trituration with ether to remove residual
1,4-diisopropylbenzene,
3-phthalimidomethyl-4,5',8-trimethylpsoralen, a white solid (114
mg, 27.3%) was obtained with 93% purity. .sup.1H NMR (CDCl.sub.3):
(7.65-7.86 (m, 4H), 7.60 (s, 1H), 6.41 (s, 1H), 4.96 (s, 2H), 2.68
(s, 3H), 2.55 (s, 3H), 2.49 (s, 3H).
[0123] Step 3:
[0124] The 3-phthalimidomethyl-4,5',8-trimethylpsoralen (29.8 mg,
0.0704 mmol) was stirred in THF (2 mL), then 40% aqueous
methylamine was added (1 mL). After 20 minutes, the resulting clear
yellow solution was evaporated and partitioned between chloroform
and 0.3 N HCl. The aqueous acid layer was made basic with solid
K.sub.2CO.sub.3 and extracted with CH.sub.2Cl.sub.2, which was then
dried and evaporated to give 3-aminomethyl-4,5',8-trimethylpsoralen
(17 mg, 94%), a light yellow solid. .sup.1H NMR (CDCl.sub.3): (7.54
(s, 1H), 6.40 (s, 1H), 3.87 (s, 2H), 2.57 (s, 3H), 2.53 (s, 3H),
2.48 (s, 3H). .sup.13C NMR (CDCl.sub.3): 8.97, 14.65, 15.55, 39.45,
103.12, 109.18, 113.04, 117.02, 125.92, 148.07, 148.18, 155.45,
157.73, 162.72.
[0125] Step 4:
[0126] In the same manner as step 8 of EXAMPLE 1,
3-aminomethyl-4,5',8-tri- methylpsoralen was converted to
3-aminomethyl-4,5',8-trimethylpsoralen hydrochloride, a yellow
solid. .sup.1H NMR (CD.sub.3OD): (7.90 (s, 1H), 6.61 (s, 1H), 4.22
(s, 2H), 2.68 (s, 3H), 2.58 (s, 3H), 2.51 (s, 3H).
EXAMPLE 5
Synthesis of 4-aminomethyl-4',5',8-trimethylpsoralen
Compound 36
[0127] Step 1:
[0128] In the same manner as step 3 of EXAMPLE 1,
7-acetoxy-4-chloromethyl- -8-methylcoumarin (prepared from
7-hydroxy-4-chloromethyl-8-methylcoumarin (obtained as per Zagotto
et al., Photochem. Photobio. 58: 486 (1993)) similarly to step 1 of
EXAMPLE 1) is reacted to form
7-acetoxy-4-phthalimidomethyl-8-methylcoumarin, a beige solid, was
obtained. .sup.1H NMR (CDCl.sub.3): (7.76-7.99 (m, 4H), 7.67 (d,
J=8.5 Hz, 1H), 7.08 (d, J=8.8 Hz, 1H), 6.23 (s, 1H), 5.00 (s, 2H),
2.38 (s, 3H), 2.29 (s, 3H).
[0129] Step 2:
[0130] In the same manner as step 4 of EXAMPLE 1,
7-acetoxy-4-phthalimidom- ethyl-8-methylcoumarin was reacted to
form 7-hydroxy-4-phthalimidomethyl-8- -methylcoumarin, a pale
yellow solid. .sup.1H NMR (DMSO-d.sub.6): (10.58 (s, 1H), 7.87-8.06
(m, 4H), 7.66 (d, J=8.7 Hz, 1H), 6.93 (d, J=8.8 Hz, 1H), 6.08 (s,
1H), 4.97 (s, 2H), 2.19 (s, 3H).
[0131] Step 3:
[0132] In the same manner as step 5 of EXAMPLE 1, using
3-chloro-2-butanone instead of chloroacetone,
7-hydroxy-4-phthalimidometh- yl-8-methylcoumarin was reacted to
form 8-methyl-7-(1-methyl-2-oxo)propylo-
xy-4-phthalimidomethylcoumarin a white solid. .sup.1H NMR
(CDCl.sub.3): (7.73-7.97 (m, 4H), 7.56 (d, J=8.8 Hz, 1H), 6.68 (d,
J=8.8 Hz, 1H), 6.09 (s, 1H), 4.96 (s, 2H), 4.75 (q, J=6.9 Hz, 1H),
2.38 (s, 3H), 2.19 (s, 3H), 1.59 (s, 3H).
[0133] Step 4:
[0134] A solution of
8-methyl-7-(1-methyl-2-oxo)propyloxy-4-phthalimidomet- hylcoumarin
(500 mg, 1.23 mmol), 10% NaOH (1.09 mL, 2.46 mmol) and water (25
mL) was heated at 50-60.degree. C. for 4 hours. The slurry was
dissolved in water (200 mL) and washed with chloroform. The aqueous
acid layer was acidified with 6 N HCl, chilled and filtered to give
4-(o-carboxybenzamido)methyl-4',5',8-trimethylpsoralen, a crude
yellow precipitate (470 mg, 93.9% yield of >90% purity). .sup.1H
NMR (DMSO-d.sub.6): (9.02 (apparent t, J=4.7 Hz, 1H), 7.45-7.98 (m,
5H), 6.56 (s, 1H), 4.78 (s, 2H), 2.45 (s, 3H), 2.22 (s, 3H), third
methyl group obscured by DMSO peak.
[0135] Step 5:
[0136] A mixture of
4-(o-carboxybenzamido)methyl-4',5',8-trimethylpsoralen (251 mg,
0.620 mmol), 6 N HCl (30 mL) and water (20 mL) were refluxed for
several hours, cooled to room temperature and filtered. The
precipitate obtained was a mixture (88 mg) of
4-phthalamidomethyl-4',5',8-trimethylps- oralen and
4-aminomethyl-4',5',8-trimethylpsoralen hydrochloride. A second
precipitate formed in the mother liquor and was collected to give
4-aminomethyl-4',5',8-trimethylpsoralen hydrochloride, a yellow
solid (41 mg). .sup.1H NMR (DMSO-d.sub.6): (8.73 (broad s, 3H),
7.82 (s, 1H), 6.52 (s, 1H), 4.50 (s, 2H), 2.52 (s, 3H), 2.45 (s,
3H), 2.22 (s, 3H).
EXAMPLE 6
Synthesis of 3-(4-amino-2-oxa)butyl-4,4',8-trimethylpsoralen
Compound 37
[0137] Step 1:
[0138] 7-acetoxy-3-bromomethyl-4,8-dimethylcoumarin (2.50 g, 8.90
mmol) was refluxed in methanol (300 mL) overnight, cooled to room
temperature and concentrated under vacuum to give crude
4,8-dimethyl-7-hydroxy-3-meth- oxymethylcoumarin. .sup.1H NMR
(CD.sub.3OD): (7.52 (d, J=8.8 Hz, 1H), 6.83 (d, J=8.8 Hz, 1H), 4.50
(s, 2H), 3.41 (s, 3H), 2.49 (s, 3H), 2.25 (s, 3H).
[0139] Step 2:
[0140] In the same manner as step 5 of EXAMPLE 1,
4,8-dimethyl-7-hydroxy-3- -methoxymethylcoumarin was reacted to
form 4,8-dimethyl-3-methoxymethyl-7-- (2-oxo)propyloxycoumarin, a
light yellow solid. .sup.1H NMR (CDCl.sub.3): (7.47 (d, J=8.9 Hz,
1H), 6.66 (d, J=8.9 Hz, 1H), 4.63 (s, 2H), 4.53 (s, 2H), 3.43 (s,
3H), 2.48 (s, 3H), 2.39 (s, 3H), 2.34 (s, 3H).
[0141] Step 3:
[0142] Crude
4,8-dimethyl-3-methoxymethyl-7-(2-oxo)propyloxycoumarin was
refluxed with water (200 mL) and 10% NaOH (3.5 mL) overnight. The
basic slurry was chilled, acidified with a few drops of
concentrated HCl to pH 1, filtered and rinsed with water. The crude
light brown solid was purified by preparative TLC on silica gel
with 1% acetonitrile in CH.sub.2Cl.sub.2 to give
3-methoxymethyl-4,4',8-trimethylpsoralen, a light yellow solid
(172.1 mg, 6% yield from 7-acetoxy-3-bromomethyl-4,8-d-
imethylcoumarin). .sup.1H NMR (CDCl.sub.3): (7.60 (s, 1H), 7.47 (s,
1H), 4.58 (s, 2H), 3.45 (s, 3H), 2.61 (s, 3H), 2.58 (s, 3H), 2.28
(s, 3H).
[0143] Step 4:
[0144] Sodium iodide (275 mg, 1.84 mmol) was added to a slurry of
3-methoxymethyl-4,4',8-trimethylpsoralen (250 mg, 0.919 mmol) in
acetonitrile. Under nitrogen, trimethylsilyl chloride (0.23 mL,
1.84 mmol) was added and the solution was refluxed for 3 hours. The
reaction solvent was evaporated and the reaction partitioned
between CH.sub.2Cl.sub.2 and aqueous sodium thiosulfate. The
organic layer was dried with brine, then with anhydrous sodium
sulfate and the solvent was removed under vacuum to give
3-iodomethyl-4,4',8-trimethylpsoralen (283 mg, 83.7% crude yield,
>90% purity), a beige solid. .sup.1H NMR (CDCl.sub.3): (7.61 (s,
1H), 7.49 (s, 1H), 4.54 (s, 2H), 2.59 (s, 3H), 2.48 (s, 3H), 2.29
(s, 3H).
[0145] Step 5:
[0146] A solution of crude 3-iodomethyl-4,4',8-trimethylpsoralen
(843 mg) was refluxed with ethylene glycol (35 mL, 650 mmol) in
acetone (90 mL) overnight. After the solvent was evaporated, the
viscous liquid was partitioned between CH.sub.2Cl.sub.2 and water
to remove excess diol. After several washings with water, the
organic layer was dried and evaporated. The crude product was
purified on two silica gel preparative TLC plate first eluted with
CH.sub.2Cl.sub.2, then eluted with 2% 2-propanol in
CH.sub.2Cl.sub.2, to give 3-(4-hydroxy-2-oxa)butyl-4,4',8-t-
rimethylpsoralen, a yellow solid (224 mg). .sup.1H NMR
(CDCl.sub.3): (7.60 (s, 1H),7.47 (s, 1H), 4.69 (s, 2H), 3.68-3.90
(m, 4H), 2.63 (s, 3H), 2.57 (s, 3H), 2.29 (s, 3H).
[0147] Step 6:
[0148] A mixture of
3-(4-hydroxy-2-oxa)butyl-4,4',8-trimethylpsoralen (214 mg, 0.709
mmol), TEA (0.37 mL, 2.65 mmol), and methanesulfonyl chloride
(0.150 mL, 1.94 mmol) in CH.sub.2Cl.sub.2 (5 mL) was stirred
overnight under nitrogen. The reaction mixture was then partitioned
between CH.sub.2Cl.sub.2 and water. The organic layer was washed
with water, dried and stripped to give
3-(4-methanesulfonyloxy-2-oxa)butyl-4,4',8-tri- methylpsoralen, a
yellow solid (220 mg, >90% purity). .sup.1H NMR (CDCl.sub.3):
(7.63 (s, 1H), 7.49 (app q, J=1.2 Hz, 1H), 4.71 (s, 2H), 4.34-4.47
(m, 2H), 3.79-3.94 (m, 2H), 3.05 (s, 3H), 2.64 (s, 3H), 2.59 (s,
3H), 2.30 (d, J=1.2 Hz, 3H).
[0149] Step 7:
[0150] A mixture of crude
3-(4-methanesulfonyloxy-2-oxa)butyl-4,4',8-trime- thylpsoralen (220
mg) and sodium azide (75.3 mg) in ethanol (10 mL) and water (1 mL)
was refluxed overnight, evaporated, and azeotroped with toluene.
The residue was triturated with CH.sub.2Cl.sub.2 and filtered to
remove insoluble solids. The mother liquor was stripped to give
3-(4-azido-2-oxa)butyl-4,4',8-trimethylpsoralen, a yellow solid
(171 mg, >90% purity). .sup.1H NMR (CDCl.sub.3): (7.61 (s, 1H),
7.47 (s, 1H), 4.70 (s, 2H), 3.72-3.82 (m, 2H), 3.35-3.46 (m, 2H),
2.64 (s, 3H), 2.58 (s, 3H), 2.29 (s, 3H). .sup.13C NMR
(CDCl.sub.3): 8.32, 8.85, 16.00, 51.27, 65.24, 69.72, 109.66,
112.71, 116.40, 116.80, 119.63, 125.81, 143.29, 149.18, 153.34,
156.26, 162.46.
[0151] Step 8:
[0152] A mixture of crude
3-(4-azido-2-oxa)butyl-4,4',8-trimethylpsoralen (171 mg),
triphenylphosphine (205 mg), and water (10 drops) was stirred in
THF (9 mL) overnight, evaporated and partitioned between
CH.sub.2Cl.sub.2 and 1M HCl. The aqueous acid layer was made basic
with K.sub.2CO.sub.3 and extracted with CH.sub.2Cl.sub.2. The
organic layer was dried and evaporated to give
3-(4-amino-2-oxa)butyl-4,4',8-trimethylp- soralen, an orange solid
(114 mg). .sup.1H NMR (CDCl.sub.3): (7.60 (s, 1H), 7.47 (s, 1H),
4.65 (s, 2H), 3.60 (t, J=5.1 Hz, 2H), 2.84-2.97 (m, 2H), 2.62 (s,
3H), 2.57 (s, 3H), 2.28 (s, 3H).
[0153] Step 9:
[0154] Crude 3-(4-amino-2-oxa)butyl-4,4',8-trimethylpsoralen was
acidified with 5-6N HCl in isopropanol and evaporated. The salt was
recrystallized in isopropanol and the resulting precipitate was
washed with hexane to give
3-(4-amino-2-oxa)butyl-4,4',8-trimethylpsoralen hydrochloride, an
off white solid (64.5 mg). .sup.1H NMR (CD.sub.3OD): (7.89 (s, 1H),
7.67 (app. q, J=1.1 Hz, 1H), 4.70 (s, 2H), 3.74-3.88 (m, 2H),
3.13-3.26 (m, 2H), 2.70 (s, 3H), 2.57 (s, 3H), 2.32 (d, J=1.2 Hz,
3H). .sup.13C NMR (CD.sub.3OD): 8.11, 8.63, 16.22, 40.98, 66.07,
67.60, 110.02, 114.52, 117.59, 117.75, 119.88, 127.20, 145.00,
149.82, 155.43, 157.24, 164.14.
EXAMPLE 7
4-(4-Amino-2-oxa)butyl-4',5',8-trimethylpsoralen
Compound 38
[0155] Step 1:
[0156] A mixture of 4-chloromethyl-7-hydroxy-8-methylcoumarin (2.00
g, 8.90 mmol) and sodium methoxide (12.0 g, 222 mmol) in methanol
(400 mL) was refluxed overnight. The solution was allowed to cool
to room temperature, acidified to pH 0-1 with 5-6N HCl in
isopropanol and evaporated. The residue was azeotroped with toluene
several times to give crude product
7-hydroxy-4-methoxymethyl-8-methylcoumarin, a yellow oil. .sup.1H
NMR (CD.sub.3OD): (7.38 (d, J=8.5 Hz, 1H), 6.82 (d, J=8.8 Hz, 1H),
6.29 (s, 1H), 4.66 (s, 2H), 3.51 (s, 3H), 2.26 (s, 3H).
[0157] Step 2:
[0158] In the same manner as step 5 of EXAMPLE 1 but using
3-chloro-2-butanone instead of chloroacetone,
7-hydroxy-4-methoxymethyl-8- -methylcoumarin was reacted to form
8-methyl-4-methoxymethyl-7-(1-methyl-2- -oxo)propyloxycoumarin, an
off-white solid. .sup.1H NMR (CDCl.sub.3): (7.34 (d, J=8.8 Hz, 1H),
6.61 (d, J=8.9 Hz, 1H), 6.40 (s, 1H), 4.72 (q, J=6.8 Hz, 1H), 4.57
(d, J=1.3 Hz, 2H), 3.49 (s, 3H), 2.39 (s, 3H), 2.19 (s, 3H), 1.56
(d, J=6.8 Hz, 2H).
[0159] Step 3:
[0160] Crude
8-methyl-4-methoxymethyl-7-(1-methyl-2-oxo)propyloxycoumarin was
refluxed in water (200 mL) and 10% NaOH (2.8 mL) overnight. The
basic slurry was chilled, acidified with a few drops of
concentrated HCl to pH 1, filtered and rinsed with water to give
4-methoxymethyl-4',5',8-trimeth- ylpsoralen (1.58 g), an off-white
solid. .sup.1H NMR (CDCl.sub.3): (7.35 (s, 1H), 6.49 (s, 1H), 4.72
(s, 2H), 3.54 (s, 3H), 2.58 (s, 3H), 2.42 (s, 3H), 2.18 (s, 3H).
.sup.13C NMR (CDCl.sub.3): d 8.38, 8.94, 12.43, 59.48, 71.21,
109.62, 110.07, 110.26, 111.13, 113.67, 127.39, 149.73, 152.61,
152.96, 155.00, 162.07.
[0161] Step 4:
[0162] A mixture of 4-methoxymethyl-4',5',8-trimethylpsoralen (500
mg, 1.84 mmol) in methylene chloride (20 mL) was chilled to
-78.degree. C. A 1M solution of boron tribromide in methylene
chloride (2.6 mL, 2.58 mmol) was then added dropwise. The reaction
mixture was allowed to stir overnight under a serum cap, and
partitioned between methylene chloride and water. The organic layer
was dried with brine, then with anhydrous sodium sulfate and
evaporated to give 4-hydroxymethyl-4',5',8-trimethylps- oralen (500
mg, 105%), an olive green solid. .sup.1H NMR (CDCl.sub.3): (7.30
(s, 1H), 6.57 (s, 1H), 5.00 (s, 2H), 2.53 (s, 3H), 2.42 (s, 3H),
2.17 (s, 3H).
[0163] Step 5:
[0164] In the same manner as step 6 of EXAMPLE 6,
4-hydroxymethyl-4',5',8-- trimethylpsoralen was reacted to form
4-methanesulfonyloxymethyl-4',5',8-t- rimethylpsoralen. .sup.1H NMR
(CDCl.sub.3): (7.31 (s, 1H), 6.51 (s, 1H), 5.49 (d, J=1.1 Hz, 2H),
3.16 (s, 3H), 2.58 (s, 3H), 2.43 (s, 3H), 2.19 (s, 3H).
[0165] Step 6:
[0166] In the same manner as step 5 of EXAMPLE 6,
4-methanesulfonyloxymeth- yl-4',5',8-trimethylpsoralen was reacted
to form 4-(4-hydroxy-2-oxa)butyl-- 4',5',8-trimethylpsoralen. The
product was eluted with 80% ethyl acetate, 20% hexane instead of 2%
2-propanol in CH.sub.2Cl.sub.2. .sup.1H NMR (CDCl.sub.3): (7.32 (s,
1H), 6.52 (s, 1H), 4.83 (s, 2H), 3.72-3.93 (m, 4H), 2.55 (s, 3H),
2.41 (s, 3H), 2.17 (s, 3H).
[0167] Step 7:
[0168] In the same manner as step 7 of EXAMPLE 6,
4-(4-hydroxy-2-oxa)butyl- -4',5',8-trimethylpsoralen was reacted to
form 4-(4-methanesulfonyloxy-2-o-
xa)butyl-4',5',8-trimethylpsoralen was prepared. This crude
mesylate (105 mg, approx. 70% pure) was refluxed 1-2 days with
sodium azide (90 mg, 1.38 mmol) in ethanol (5 mL) and water (0.5
mL), then evaporated. The residue was triturated with
CH.sub.2Cl.sub.2 and the insoluble solids were filtered off to give
4-(4-azido-2-oxa)butyl-4',5,8-trimethylpsoralen (71.6 mg). .sup.1H
NMR (CDCl.sub.3): (7.38 (s, 1H), 6.50 (s, 1H), 4.83 (s, 2H), 3.80
(t, J=4.9, 2H), 3.49 (t, J=4.9, 2H), 2.57 (S, 3H), 2.41 (S, 3H),
2.17 (S, 3H).
[0169] Step 8:
[0170] In the same manner as step 8 of EXAMPLE 6,
4-(4-azido-2-oxa)butyl-4- ',5,8-trimethylpsoralen was reacted to
form 4-(4-amino-2-oxa)butyl-4',5,8-- trimethylpsoralen. .sup.1H NMR
(CDCl.sub.3): (7.34 (s, 1H), 6.52 (s, 1H), 4.80 (s, 2H), 3.64-3.75
(m, 3H), 2.92-3.08 (m, 2H), 2.57 (s, 3H), 2.42 (s, 3H), 2.17 (s,
3H).
[0171] Step 9:
[0172] Crude 4-(4-Amino-2-oxa)butyl-4',5,8-trimethylpsoralen was
dissolved in boiling isopropanol and hot filtered through fluted
filter paper. The hot mother liquor was acidified with 5-6 N HCl in
isopropanol to pH 1 and allowed to cool first to room temperature,
then in an ice water bath. The solid product was filtered off,
rinsed with ice cold isopropanol, then with hexane to give
4-(4-amino-2-oxa)butyl-4',5,8-trimethylpsoralen hydrochloride.
.sup.1H NMR (CD.sub.3OD): (7.55 (s, 1H), 6.61 (s, 1H), 5.00 (s,
2H), 3.93 (m, 2H), 2.55 (s, 3H), 2.44 (s, 3H), 2.22 (s, 3H). The
CH2NH2 peaks lie under solvent peaks at 3.3 ppm.
EXAMPLE 8
Synthesis of 5-(4-amino-2-oxa)butyl-4',5'-dimethylpsoralen
Compound 39
[0173] STEP 1:
[0174] In the same manner as step 1 of EXAMPLE 1,
7-hydroxy-5-methylcoumar- in was reacted to form
7-acetoxy-5-methylcoumarin. .sup.1H NMR (CDCl.sub.3): (7.88 (d,
J=9.9 Hz, 1H), 6.96 (s, 1H), 6.89 (s, 1H), 6.40 (d, J=9.8 Hz, 1H),
2.52 (s, 3H), 2.33 (s, 3H).
[0175] Step 2:
[0176] In the same manner as step 2 of EXAMPLE 1,
7-acetoxy-5-methylcoumar- in was reacted to form
7-acetoxy-5-bromomethylcoumarin without recrystallizing (75-85%
purity). .sup.1H NMR (CDCl.sub.3): (8.00 (d, J=9.9 Hz, 1H), 7.11
(s, 1H), 7.09 (s, 1H), 6.51 (d, J=9.9 Hz, 1H), 4.62 (s, 2H), 2.34
(s, 3H). .sup.13C NMR (CDCl.sub.3): 21.56, 28.44, 111.68, 115.50,
116.78, 120.05, 136.61, 139.29, 153.05, 156.01, 160.25, 168.92.
[0177] Step 3:
[0178] In the same manner as step 1 of EXAMPLE 6,
7-acetoxy-5-bromomethylc- oumarin was reacted to form crude
7-hydroxy-5-methoxymethylcoumarin. .sup.1H NMR (CD.sub.3OD): (8.10
(d, J=9.5 Hz, 1H), 6.82 (s, 1H), 6.68 (s, 1H), 6.22 (d, J=9.7 Hz,
1H), 4.64 (s, 2H), 3.40 (s, 3H).
[0179] Step 4:
[0180] In the same manner as step 5 of EXAMPLE 1, using
3-chloro-2-butanone instead of chloroacetone,
7-hydroxy-5-methoxymethylco- umarin was reacted to form
5-methoxymethyl-7-(1-methyl-2-oxo)propyloxycoum- arin. .sup.1H NMR
(CDCl.sub.3): (7.92 (d, J=9.8 Hz, 1H), 6.84 (s, 1H), 6.66 (s, 1H),
6.29 (d, J=9.8 Hz, 1H), 4.71 (q, J=6.8 Hz, 1H), 4.60 (s, 2H), 3.41
(s, 3H), 2.20 (s, 3H), 1.54 (d, J=6.9 Hz, 3H).
[0181] Step 5:
[0182] 5-Methoxymethyl-4',5'-dimethylpsoralen. Crude
5-methoxymethyl-7-(1-methyl-2-oxo)propyloxycoumarin (12.2 g,
approx. 90% purity) was refluxed in water (500 mL) and 10% NaOH (17
mL) for 6 hours. The aqueous solution was partitioned between
CH.sub.2Cl.sub.2 and water. The organic layer was washed with
water, dried with brine, then with anhydrous sodium sulfate and
evaporated to give 5-methoxymethyl-4',5'-dim- ethylpsoralen, a
beige solid (6.87 g, >95% purity). .sup.1H NMR (CDCl.sub.3):
(8.17 (d, J=9.9 Hz, 1H), 7.33 (s, 1H), 6.40 (d, J=9.9 Hz, 1H), 4.92
(s, 2H), 3.46 (s, 3H), 2.40 (s, 3H), 2.36 (s, 3H).
[0183] Step 6:
[0184] A mixture of 5-methoxymethyl-4',5'-dimethylpsoralen (6.87 g,
26.6 mmol) in methylene chloride (300 mL) was chilled to
-78.degree. C., then a 1M solution of boron tribromide in methylene
chloride (37.2 mL, 37.2 mmol) was added dropwise under nitrogen.
The reaction mixture was allowed to stir overnight under nitrogen,
and partitioned between methylene chloride and water. The organic
layer was dried with brine, then with anhydrous sodium sulfate and
evaporated to give 5-bromomethyl-4',5'-dimet- hylpsoralen (5.89 g,
72.1% yield), a black solid. .sup.1H NMR (CDCl.sub.3): (8.11 (d,
J=9.9 Hz, 1H), 7.33 (s, 1H), 6.49 (d, J=9.9 Hz, 1H), 5.02 (s, 2H),
2.46 (s, 3H), 2.43 (s, 3H).
[0185] Step 7:
[0186] In the same manner as step 5 of EXAMPLE 6,
5-bromomethyl-4',5'-dime- thylpsoralen was reacted to form
5-(4-hydroxy-2-oxa)butyl-4',5'-dimethylps- oralen without
chromatographic purification (>80% purity). .sup.1H NMR
(CDCl.sub.3): (8.17 (d, J=9.9 Hz, 1H), 7.33 (s, 1H), 6.40 (d, J=9.9
Hz, 1H), 5.04 (s, 2H), 3.63-3.85 (m, 4H), 2.41 (s, 3H), 2.36 (s,
3H). .sup.13C NMR (CDCl.sub.3): 11.17, 12.30,62.37, 65.16, 72.10,
99.83, 109.97, 114.58, 114.71, 126.75, 127.49, 141.31, 152.31,
154.08, 156.04, 161.47.
[0187] Step 8:
[0188] In the same manner as step 6 of EXAMPLE 6,
5-(4-hydroxy-2-oxa)butyl- -4',5'-dimethylpsoralen was reacted to
form 5-(4-methanesulfonyloxy-2-oxa)- butyl-4',5'-dimethylpsoralen.
.sup.1H NMR (CDCl.sub.3): (8.18 (d, J=9.9 Hz, 1H), 7.35 (s, 1H),
6.42 (d, J=9.9 Hz, 1H), 5.07 (s, 2H), 4.34-4.43 (m, 2H), 3.81-3.90
(m, 2H), 2.97 (s, 3H), 2.41 (s, 3H), 2.36 (s, 3H).
[0189] Step 9:
[0190] In the same manner as step 7 of EXAMPLE 6,
5-(4-methanesulfonyloxy-- 2-oxa)butyl-4',5'-dimethylpsoralen was
reacted to form 5-(4-azido-2-oxa)butyl-4',5'-dimethylpsoralen, a
beige solid. .sup.1H NMR (CDCl.sub.3): (8.19 (d, J=9.9 Hz, 1H),
7.34 (s, 1H), 6.41 (d, J=9.9 Hz, 1H), 5.06 (s, 2H), 3.72 (t, J=5.0
Hz, 2H), 3.42 (t, J=4.9 Hz, 2H), 2.41 (s, 3H), 2.37 (s, 3H).
[0191] Step 10:
[0192] In the same manner as step 8 of EXAMPLE 6,
5-(4-azido-2-oxa)butyl-4- ',5'-dimethylpsoralen was reacted to form
5-(4-amino-2-oxa)butyl-4',5'-dim- ethylpsoralen, a light yellow
solid. .sup.1H NMR (CDCl.sub.3): d 8.18 (d, J=9.9 Hz, 1H), 7.32 (s,
1H), 6.39 (d, J=9.9 Hz, 1H), 5.00 (s, 2H), 3.61 (t, J=5.2 Hz, 2H),
2.90 (t, J=5.2 Hz, 2H), 2.40 (s, 3H), 2.35 (s, 3H).
[0193] Step 11:
[0194] 5-(4-amino-2-oxa)butyl-4',5'-dimethylpsoralen (232 mg, 0.956
mmol) was dissolved in hot isopropanol and 5-6 N HCl in isopropanol
was added until pH 1 was reached. A solid precipitated out and the
slurry was allowed to cool to room temperature, then chilled in an
ice bath. The precipitate was filtered off with a Buchner funnel
and washed first with ice cold isopropanol then with hexane to give
5-(4-amino-2-oxa)butyl-4',5- '-dimethylpsoralen hydrochloride (194
mg, 72.6% yield), a beige solid. .sup.1H NMR (CD.sub.3OD): (8.40
(d, J=9.9 Hz, 1H), 7.40 (s, 1H), 6.41 (d, J=9.9 Hz, 1H), 5.16 (s,
2H), 3.80 (t, J=4.9 Hz, 2H), 3.16 (t, J=5.0, 2H), 2.42 (s, 3H),
2.40 (s, 3H). .sup.13C NMR: 11.05, 11.90, 41.05, 65.81, 67.27,
100.48, 111.29, 114.85, 116.01, 128.12, 128.81, 143.44, 153.35,
155.47, 157.27, 163.19.
EXAMPLE 9
Synthesis of 8-(4-amino-2-oxa)butyl-4,4',5'-trimethylpsoralen
Compound 40
[0195] Step 1:
[0196] In the same manner as step 2 of EXAMPLE 1,
7-acetoxy-4,8-dimethylco- umarin was reacted to form
7-acetoxy-8-bromomethyl-4-methylcoumarin. The crude yellow solid
was not recrystallized (75-85% purity). .sup.1H NMR (CDCl.sub.3):
(7.61 (d, J=8.8 Hz, 1H), 7.15 (d, J=8.8 Hz, 1H), 6.31 (s, 1H), 4.68
(s, 2H), 2.43 (s, 6H).
[0197] Step 2:
[0198] In the same manner as step 2 of EXAMPLE 6,
7-acetoxy-8-bromomethyl-- 4-methylcoumarin is reacted to form
7-hydroxy-8-methoxymethyl-4-methylcoum- arin as a yellow solid (85%
purity). .sup.1H NMR (CD.sub.3OD): (7.44 (d, J=8.8 Hz, 1H), 6.84
(d, J=8.8 Hz, 1H), 6.11 (app. q, J=1.1 Hz, 1H), 5.05 (s, 2H), 3.55
(s, 3H), 2.40 (d, J=1.1 Hz, 3H).
[0199] Step 3:
[0200] In the same manner as step 5 of EXAMPLE 1, using
3-chloro-2-butanone instead of chloroacetone,
7-hydroxy-8-methoxymethyl-4- -methylcoumarin was reacted to form
8-methoxymethyl-7-(1-methyl-2-oxo)prop- yloxy-4-methylcoumarin as a
yellow solid (>90% purity). .sup.1H NMR (CDCl.sub.3): (7.51 (d,
J=8.8 Hz, 1H), 6.73 (d, J=8.8 Hz, 1H), 6.18 (s, 1H), 4.65-4.88 (m,
3H), 3.47 (s, 3H), 2.39 (s, 3H), 2.36 (s, 3H), 2.22 (s, 3H), 1.58
(d, J=6.8 Hz, 3H).
[0201] Step 4:
[0202] In the same manner as step 5 of example 8,
8-methoxymethyl-7-(1-met- hyl-2-oxo)propyloxy-4-methylcoumarin was
reacted to form 8-methoxymethyl-4,4',5'-trimethylpsoralen as a
light yellow solid (>93% purity). .sup.1H NMR (CDCl.sub.3):
(7.55 (s, 1H), 6.26 (s, 1H), 4.98 (s, 2H), 3.50 (s, 3H), 2.52 (s,
3H), 2.44 (s, 3H), 2.20 (s, 3H).
[0203] Step 5:
[0204] In the same manner as step 6 of EXAMPLE 8,
8-methoxymethyl-4,4',5'-- trimethylpsoralen was reacted to form
8-bromomethyl-4,4',5'-trimethylpsora- len as a light yellow solid
(>93% purity). .sup.1H NMR (CDCl.sub.3): (7.54 (s, 1H), 6.28 (s,
1H), 4.97 (s, 2H), 2.52 (s, 3H), 2.46 (s, 3H), 2.20 (s, 3H).
.sup.13C NMR (CDCl.sub.3): 8.32, 12.50, 19.76, 20.18, 109.57,
110.57, 113.47, 114.35, 116.44, 127.93, 148.95, 153.43, 153.64,
153.96, 161.04
[0205] Step 6:
[0206] In the same manner as step 5 of EXAMPLE 6,
8-bromomethyl-4,4',5'-tr- imethylpsoralen was reacted to form
8-(4-hydroxy-2-oxa)butyl-4,4',5'-trime- thylpsoralen without
chromatographic purification. The crude product (>85% purity)
was prepared as a mustard yellow solid. .sup.1H NMR (CDCl.sub.3):
(7.55 (s, 1H), 6.26 (s, 1H), 5.09 (s, 2H), 3.76 (s, 4H), 2.52 (s,
3H), 2.43 (s, 3H), 2.20 (s, 3H).
[0207] Step 7:
[0208] In the same manner as step 6 of EXAMPLE 6,
8-(4-hydroxy-2-oxa)butyl- -4,4',5'-trimethylpsoralen was reacted to
form 8-(4-methanesulfonyloxy-2-o-
xa)butyl-4,4',5'-trimethylpsoralen (>85% purity) as a
yellow-brown solid. .sup.1H NMR (CDCl.sub.3): (7.55 (s, 1H), 6.25
(s, 1H), 5.06 (s, 2H), 4.40 (t, J=4.4 Hz, 2H), 3.87 (t, J=4.4 Hz,
2H), 3.03 (s, 3H), 2.52 (s, 3H), 2.43 (s, 3H), 2.20 (s, 3H).
[0209] Step 8:
[0210] In the same manner as step 7 of EXAMPLE 6,
8-(4-methanesulfonyloxy-- 2-oxa)butyl-4,4',5'-trimethylpsoralen was
reacted to form 8-(4-azido-2-oxa)butyl-4,4',5'-trimethylpsoralen, a
beige solid (>85% purity). .sup.1H NMR (CDCl.sub.3): (7.55 (s,
1H), 6.26 (s, 1H), 5.08 (s, 2H), 3.81 (t, J=5.1 Hz, 2H), 3.41 (t,
J=5.1 Hz, 2H), 2.52 (s, 3H), 2.43 (s, 3H), 2.20 (s, 3H).
[0211] Step 9:
[0212] In the same manner as step 8 of EXAMPLE 6,
8-(4-azido-2-oxa)butyl-4- ,4',5'-trimethylpsoralen was reacted to
form 8-(4-amino-2-oxa)butyl-4,4',5- '-trimethylpsoralen, a light
yellow solid. .sup.1H NMR (CDCl.sub.3): (7.53 (s, 1H), 6.25 (s,
1H), 5.04 (s, 2H), 3.66 (t, J=5.1 Hz, 2H), 2.89 (t, J=5.1 Hz, 2H),
2.51 (s, 3H), 2.43 (s, 3H), 2.19 (s, 3H).
[0213] Step 10:
[0214] In the same manner as step 9 of EXAMPLE 7,
8-(4-amino-2-oxa)butyl-4- ,4',5'-trimethylpsoralen (784 mg) was
reacted to form 8-(4-amino-2-oxa)butyl-4,4',5'-trimethylpsoralen
hydrochloride (483 mg, 54.9% yield), an off-white solid.
[0215] .sup.1H NMR (CD.sub.3OD): (7.85 (s, 1H), 6.33 (s, 1H), 5.09
(s, 2H), 3.83 (t, J=5.0 Hz, 2H), 3.16 (t, J=5.0 Hz, 2H), 2.59 (s,
3H), 2.45 (s, 3H), 2.25 (s, 3H). .sup.13C NMR (CD.sub.3OD): 8.03,
12.14, 19.68,40.95, 62.58, 67.76, 109.12, 111.74, 113.19, 115.87,
117.22, 129.15, 150.61, 154.70, 155.90, 156.54, 163.30.
EXAMPLE 10
Synthesis of 3-aminomethyl-4'-methylpsoralen
Compound 41
[0216] Step 1:
[0217] In the same manner as step 1 of EXAMPLE 1,
7-hydroxy-3-methylcoumar- in was reacted to form
7-acetoxy-3-methylcoumarin, a beige solid. .sup.1H NMR
(CDCl.sub.3): (7.51 (s, 1H), 7.42 (d, J=8.4 Hz, 1H), 6.99-7.14 (m,
2H), 2.34 (s, 3H), 2.22 (d, J=1.2 Hz, 3H).
[0218] Step 2:
[0219] In a similar manner as step 2 of EXAMPLE 1,
7-acetoxy-3-methylcouma- rin was reacted to form
7-acetoxy-3-bromomethylcoumarin (70% purity), a beige solid.
.sup.1H NMR (CDCl.sub.3): (7.84 (s, 1H), 7.51 (d, J=8.3 Hz, 1H),
7.03-7.20 (m, 2H), 4.42 (s, 2H), 2.34 (s, 3H).
[0220] Step 3:
[0221] 7-acetoxy-3-bromomethylcoumarin (630 mg) was stirred
overnight with potassium phthalimide (432 mg, 2.33 mmol) in DMF
(100 mL). The reaction solvent was evaporated and the reaction
partitioned between CH.sub.2Cl.sub.2 and water, then washed several
times with water, then with aqueous NaHCO.sub.3. The organic layer
was dried with brine, then with anhydrous sodium sulfate and
evaporated. The crude product was recrystallized in acetic acid to
give 7-acetoxy-3-phthalimidomethylcoumar- in (375 mg, >95%
purity), an off-white solid. .sup.1H NMR (CDCl.sub.3): (7.73-7.97
(m, 4H), 7.53 (s, 1H), 7.43 (d, J=8.4 Hz, 1H), 7.13 (s, 1H), 7.03
(dd, J=7.5, 2.1, 1H), 4.82 (s, 2H), 2.33 (s, 3H).
[0222] Step 4:
[0223] In a similar manner as step 4 of EXAMPLE 1,
7-acetoxy-3-phthalimido- methylcoumarin was reacted to form
7-hydroxy-3-phthalimidomethylcoumarin, a beige solid. .sup.1H NMR
(DMSO-d.sub.6): (7.85-7.98 (m, 5H), 7.51 (d, J=8.3 Hz, 1H),
6.72-6.83 (m, 2H), 4.58 (s, 2H).
[0224] Step 5:
[0225] In a similar manner as step 5 of EXAMPLE 1,
7-hydroxy-3-phthalimido- methylcoumarin was reacted to form
7-(2-oxo)propyloxy-3-phthalimidomethylc- oumarin, an off-white
solid. .sup.1H NMR (DMSO-d.sub.6): (7.83-8.04 (m, 5H), 7.60 (d,
J=8.8 Hz, 1H), 7.05 (s, 1H), 6.95 (d, J=8.8 Hz, 1H), 4.99 (s, 2H),
4.61 (s, 2H), 2.18 (s, 3H).
[0226] Step 6:
[0227] In a similar manner as step 3 of EXAMPLE 2,
7-(2-oxo)propyloxy-3-ph- thalimidomethylcoumarin was reacted to
form 3-(o-carboxybenzamido)methyl-4- '-methylpsoralen, a beige
solid. .sup.1H NMR (DMSO-d.sub.6): (8.89 (m, 1H), 8.15 (s, 1H),
7.45-8.04 (m, 7H), 4.28 (d, J=5.3 Hz, 2H), 2.29 (s, 3H).
[0228] Step 7:
[0229] In a similar manner as step 7 of EXAMPLE 1,
3-(o-carboxybenzamido)m- ethyl-4'-methylpsoralen, was reacted to
form 3-aminomethyl-4'-methylpsoral- en, a light yellow solid.
.sup.1H NMR (CDCl.sub.3): (7.80 (s, 1H), 7.57 (s, 1H), 7.47 (app.
q, J=1.3 Hz, 1H), 7.42 (s, 1H), 3.81 (s, 2H), 2.28 (d, J=1.3 Hz,
3H).
[0230] Step 8:
[0231] 3-aminomethyl-4'-methylpsoralen hydrochloride. In the
similar manner as step 8 of EXAMPLE 1,
3-aminomethyl-4'-methylpsoralen was converted to
3-aminomethyl-4'-methylpsoralen hydrochloride, an off-white solid.
.sup.1H NMR (CD.sub.3OD): (8.27 (s, 1H), 7.92 (s, 1H), 7.69 (app.
q, J=1.3 Hz, 1H), 7.54 (s, 1H), 4.10 (s, 2H), 2.31 (d, J=1.4 Hz,
3H).
EXAMPLE 11
Synthesis of 4-aminomethyl-4'-methylpsoralen
Compound 42
[0232] Step 1:
[0233] In the same manner as step 1 of EXAMPLE 1,
7-hydroxy-4-methylcoumar- in was reacted to form
7-acetoxy-4-methylcoumarin, a white solid. .sup.1H NMR
(CDCl.sub.3): (7.61 (d, J=8.4 Hz, 1H), 7.03-7.16 (m, 2H), 6.27 (s,
1H), 2.43 (s, 3H), 2.35 (s, 3H).
[0234] Step 2:
[0235] In a similar manner as step 2 of EXAMPLE 1,
7-acetoxy-4-methylcouma- rin was reacted to form crude
7-acetoxy-4-bromomethylcoumarin (60% purity), a beige solid.
[0236] Step 3:
[0237] In a similar manner as step 3 of EXAMPLE 10,
7-acetoxy-4-bromomethylcoumarin was reacted to form
7-acetoxy-4-phthalimidomethylcoumarin (>93% purity), a yellow
solid. .sup.1H NMR (CDCl.sub.3): (7.74-8.00 (m, 5H), 7.10-7.22 (m,
2H), 6.25 (s, 1H), 5.00 (s, 2H), 2.35 (s, 3H).
[0238] Step 4:
[0239] In a similar manner as step 4 of EXAMPLE 1,
7-acetoxy-4-phthalimido- methylcoumarin was reacted to form
7-hydroxy-4-phthalimidomethylcoumarin, a yellow solid. .sup.1H NMR
(DMSO-d.sub.6): (7.83-8.05 (m, 4H), 7.80 (d, J=8.8 Hz, 1H), 6.87
(dd, J=8.7, 2.1 Hz, 1H), 6.78 (s, 1H), 6.09 (s, 1H), 4.97 (s,
2H).
[0240] Step 5:
[0241] In a similar manner as step 5 of EXAMPLE 1,
7-hydroxy-4-phthalimido- methylcoumarin was reacted to form
7-(2-oxo)propyloxy-4-phthalimidomethylc- oumarin, a pale yellow
solid. .sup.1H NMR (CDCl.sub.3): (7.76-7.98 (m, 4H), 7.73 (d, J=9.1
Hz, 1H), 6.94 (dd, J=8.9, 2.5 Hz, 1H), 6.80 (d, J=2.5 Hz, 1H), 6.12
(s, 1H), 4.98 (s, 2H), 4.65 (s, 2H), 2.31 (s, 3H).
[0242] Step 6:
[0243] In a similar manner as step 4 of EXAMPLE 5,
7-(2-oxo)propyloxy-4-ph- thalimidomethylcoumarin is reacted to form
4-(o-carboxybenzamido)methyl-4'- -methylpsoralen, a beige
solid.
[0244] Step 7:
[0245] In a similar manner as step 7 of EXAMPLE 1,
4-(o-carboxybenzamido)m- ethyl-4'-methylpsoralen was reacted to
form 4-aminomethyl-4'-methylpsorale- n, a light yellow solid.
.sup.1H NMR (CDCl.sub.3): (7.70 (s, 1H), 7.48 (s, 1H), 7.45 (s,
1H), 6.57 (s, 1H), 4.21 (s, 2H), 2.29 (s, 3H).
[0246] Step 8:
[0247] 4-aminomethyl-4'-methylpsoralen hydrochloride. In a similar
manner as step 9 of EXAMPLE 6, 4-aminomethyl-4'-methylpsoralen was
converted to 4-aminomethyl-4'-methylpsoralen hydrochloride, an
off-white solid. .sup.1H NMR (CD.sub.3OD):
[0248] (8.00 (s, 1H), 7.71 (s, 1H), 7.56 (s, 1H), 6.45 (s, 1H),
4.61 (s, 2H), 2.35 (s, 3H).
EXAMPLE 12
Synthesis of 5-aminomethylpsoralen
Compound 43
[0249] Step 1:
[0250] In a similar manner as step 3 of EXAMPLE 1,
5-bromomethylpsoralen is reacted to form
5-phthalimidomethylpsoralen, a white solid. .sup.1H NMR
(CDCl.sub.3): (8.73 (d, J=10.0 Hz, 1H), 7.67-7.89 (m, 5H), 7.47 (s,
1H), 7.38 (d, J=2.2 Hz, 1H), 6.50 (d, J=9.9 Hz, 1H), 5.27 (s,
2H).
[0251] Step 2:
[0252] A slurry of 5-phthalimidomethylpsoralen (300 mg, 0.879 mmol)
and hydrazine acetate (648 mg, 7.03 mmol) in ethanol (15 mL) was
refluxed for 2 hours. The black solution was evaporated and the
residue was partitioned between methylene chloride and dilute
aqueous HCl. The aqueous acid layer was washed with methylene
chloride, then made basic with solid K.sub.2CO.sub.3 and extracted
with methylene chloride. The final organic layer was dried and
evaporated to give 5-aminomethylpsoralen. .sup.1H NMR (CDCl.sub.3):
(8.25 (d, J=9.9 Hz, 1H), 7.71 (d, J=2.2 Hz, 1H), 7.42 (s, 1H), 6.95
(d, J=1.4 Hz, 1H), 6.45 (d, J=9.9 Hz, 1H), 4.32 (s, 2H).
[0253] Step 3:
[0254] In the similar manner as step 9 of EXAMPLE 6,
5-aminomethylpsoralen was converted to 5-aminomethylpsoralen
hydrochloride, an off-white solid. .sup.1H NMR (CD.sub.3OD): (8.40
(d, J=9.9 Hz, 1H), 8.02 (d, J=2.2 Hz, 1H), 7.69 (s, 1H), 7.26 (d,
1.5 Hz, 1H), 6.54 (d, J=10 Hz, 1H), 4.74 (s, 2H).
EXAMPLE 13
[0255] The R17 was grown up in Hfr 3000 bacteria, approximate titer
5.times.10.sup.11. (R17 and Hfr 3000 were obtained from American
Tissue Culture Collection (ATCC), Washington, D.C.) The R17 phage
stock was added to a solution of 15% fetal bovine serum in DMEM to
a final phage concentration of 10.sup.8/mL. An aliquot (0.5 mL) was
transferred to a 1.5 mL snap-top polyethylene tube. An aliquot
(0.004-0.040 mL) of the test compound stock solution prepared in
water, ethanol or dimethylsulfoxide at 0.5 mM was added to the
tube. Compounds were tested at concentrations between 4 .mu.M and
32 .mu.M. (AMT is commercially available from HRI, Inc., Concord,
Calif.; 8-MOP is commercially available from Sigma, St. Louis,
Mo.). The tubes were placed in a light device similar to that
described in U.S. Pat. No. 5,593,823 (Baxter Ultraviolet
Irradiation System, modified #4R4440) and irradiated at a dose
setting of 1 J/cm.sup.2. Sterile 13 mL dilution tubes were
prepared; each test compound required one tube with 0.4 mL of LB
broth and five tubes containing 0.5 mL of LB broth. To make the
dilutions, a 0.100 mL aliquot of the irradiated solution of phage
and test compound was added to the first dilution tube of 0.4 mL of
media then 0.020 mL of this solution was added to the second tube
of 0.5 mL medium (1:25). The second solution was then diluted
serially (1:25) into the remaining tubes. To each diluted sample
was added 0.050 mL of Hfr 3000 bacteria cultured overnight and 3 mL
of molten LB top agar and the mixed materials were poured onto LB
broth plates. After the top agar hardened, the plates were
incubated at 37.degree. C. overnight. The plaque forming units were
then counted the following morning and the titer of the phage
remaining after phototreatment was calculated based on the dilution
factors.
[0256] The following controls were run: the "phage only" in which
phage was not treated with test compound and not irradiated (listed
as "starting titer" in the tables below); and the "dark" control in
which the phage/test compound solution was not irradiated before it
was diluted and plated. A "UV only" control in which the phage was
illuminated without treatment with test compound was not run with
these experiments. This control has been shown to have no
significant inactivation of R17 in various other studies (data not
shown). The dark control was not run on all compounds tested but no
significant inactivation of R17 was observed in the compounds
tested. Similar controls have been run on other psoralens, mostly
4' and 5' amino substituted psoralens with no significant
inactivation of R17 as well (data not shown).
[0257] TABLE 3, below, shows the results of various experiments
which tested a number of compounds of the present invention
according to the R17 protocol just described. AMT was also run for
comparison. The number of replicate experiments done for each
compound is indicated. The results indicate that all of the
compounds selected are likely to meet the selection criteria of
>1 log inactivation at 320 .mu.M. Most of the compounds tested
were at least as effective as AMT while
3-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen was at least twice
as effective as AMT (i.e. similar levels of inactivation at a given
concentration of AMT were achieved at less than half of the
concentration, data not shown). The structure of
3-(4-amino-2-oxa)butyl-4- ,5',8-trimethylpsoralen is shown below.
14
2TABLE 3 Log inactivation of R17 with compound at 4 .mu.M and 32
.mu.M Compound Number Average log Inactivation.sup.a Average (per
EXAMPLES 1-12) 4 .mu.M 32 .mu.M Log Titer.sup.b AMT 1.6 >6.5 7.5
41 0 0.6 8.0 31 3.1 N/A N/A 35 1.6 N/A N/A 34 1.4 N/A N/A 37 4.3
>6.5 7.5 42 0 1.2 8.0 36 0.1 1.1 7.1 38 2.2 .gtoreq.4.0 7.5 39
1.6 >6.6 7.6 33 0.4 1.7 7.1 32 1.1 N/A N/A 40 2.1 >6.3 7.3
.sup.aAverage of 1-4 replicates. .sup.bThis is the log titer of R17
prior to inactivation.
EXAMPLE 14
[0258] Pathogen inactivation efficiency of three compounds tested
in the previous example were evaluated by examining the ability of
the compounds to inactivate cell-free virus (HIV). Inactivation of
cell-free HIV was performed as follows.
[0259] Small aliquots of the compounds were added to stock HIV-1 to
a compound concentration of 32 .mu.M in a total of 0.5 mL. The
stock HIV (10.sup.5-10.sup.7 plaque forming units/mL) was in
DMEM/15% FBS. The 0.5 mL test aliquots were placed in 24 well
polystyrene tissue culture plates and irradiated with 320-400 nm
(20 mW/cm.sup.2) for 1 minute on a device similar to the device of
EXAMPLE 13. Controls included HIV-1 stock only, HIV-1 plus UVA
only, and HIV-1 plus the highest concentration of each psoralen
tested, with no UVA. Post irradiation, all samples were stored
frozen at -70.degree. C. until assayed for infectivity by a
microtiter plaque assay. Aliquots for measurement of residual HIV
infectivity in the samples were withdrawn and cultured.
[0260] Residual HIV infectivity was assayed using an MT-2
infectivity assay. (Previously described in Hanson, C. V.,
Crowford-Miksza, L. and Sheppard, H. W., J. Clin. Micro 28:2030
(1990)). The assay medium was 85% DMEM (with a high glucose
concentration) containing 100 .mu.g of streptomycin, 100 U of
penicillin, 50 .mu.g of gentamicin, and 1 .mu.g of amphotericin B
per mL, 15% FBS and 2 .mu.g of Polybrene (Sigma Chemical Co., St.
Louis, Mo.) per mL. Test and control samples from the inactivation
procedure were diluted in a mixture of 50% assay medium and 50%
normal human serum. The samples were serially diluted directly in
96-well plates (Corning Glass Works, corning, N.Y.). The plates
were mixed on an oscillatory shaker for 30 seconds and incubated at
37.degree. C. in a 5% CO.sub.2 atmosphere for 1 to 18 hours. MT-2
cells (0.025 mL) [clone alpha-4, available (catalog number 237)
from the National Institutes of Health AIDS Research and Reference
Reagent Program, Rockville, Md.] were added to each well to give a
concentration of 80,000 cells per well. After an additional 1 hour
of incubation at 37.degree. C. in 5% CO.sub.2, 0.075 mL of assay
medium containing 1.6% SeaPlaque agarose (FMC Bioproducts,
Rockland, Me.) and pre warmed to 38.5.degree. C. was added to each
well. The plates were kept at 37.degree. C. for a few minutes until
several plates had accumulated and then centrifuged in plate
carriers at 600.times. g for 20 minutes. In the centrifuge, cell
monolayers formed prior to gelling of the agarose layer. The plates
were incubated for 5 days at 37.degree. C. in 5% CO.sub.2 and
stained by the addition of 0.05 mL of 50 .mu.g/mL propidium iodide
(Sigma Chemical Co.) in phosphate-buffered saline (pH 7.4) to each
well. After 24 to 48 hours, the orange fluorescence-stained
microplaques were visualized by placing the plates on an 8,000
.mu.W/cm.sup.2 304 nm UV light box (Fotodyne, Inc., New Berlin,
Wis.). The plaques were counted at a magnification of .times.20 to
.times.25 through a stereomicroscope. The results are shown in
TABLE 4, below.
[0261] The results support that the compounds of the present
invention are effective in inactivating HIV. In fact, the data for
these compounds is comparable to levels of inactivation observed
for AMT (data not shown).
3TABLE 4 Log kill of cell-free HIV with 1 minute irradiation with
compound at 32 .mu.M Compound Number Log Kill Log Titer 31 1.9 5.3
34 3.5 5.5 32 1.1 5.4
[0262] It is to be understood that the invention is not to be
limited to the exact details of operation or exact compounds,
composition, methods, or procedures shown and described, as
modifications and equivalents will be apparent to one skilled in
the art.
* * * * *