[go: up one dir, main page]

HK1026702B - Frangible compounds for pathogen inactivation - Google Patents

Frangible compounds for pathogen inactivation Download PDF

Info

Publication number
HK1026702B
HK1026702B HK00105911.5A HK00105911A HK1026702B HK 1026702 B HK1026702 B HK 1026702B HK 00105911 A HK00105911 A HK 00105911A HK 1026702 B HK1026702 B HK 1026702B
Authority
HK
Hong Kong
Prior art keywords
alkyl
heteroalkyl
compound
aryl
heteroaryl
Prior art date
Application number
HK00105911.5A
Other languages
Chinese (zh)
Other versions
HK1026702A1 (en
Inventor
D‧库克
J‧马利特
A‧耐里欧
H‧拉伯波特
A‧斯塔西诺波洛斯
S‧沃洛维茨
J‧马提约维克
Original Assignee
塞鲁斯公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 塞鲁斯公司 filed Critical 塞鲁斯公司
Priority claimed from PCT/US1998/000532 external-priority patent/WO1998030545A1/en
Publication of HK1026702A1 publication Critical patent/HK1026702A1/en
Publication of HK1026702B publication Critical patent/HK1026702B/en

Links

Description

Friable compounds for pathogen inactivation
Cross Reference to Related Applications
The present application claims the right of U.S. provisional application serial No. 60/043,696, filed on 1997, 4, 15, the disclosure of which is incorporated herein by reference.
This application is also a continuation-in-part application of U.S. patent application serial No. 08/779,885 filed on 6/1/1997; also filed in 1997, is a continuation-in-part of U.S. patent application serial No. 08/779,830 filed on 6/1, 1997, the disclosures of which are incorporated herein by reference.
Statement of rights to invention made under federally sponsored research
The invention was made with U.S. government support and the number of grant for NHLBI was 1-RO1-HL 53380. The U.S. government has certain rights in this invention.
Technical Field
The present invention relates to compounds useful for inactivating pathogens in materials, such as blood products, and methods of using the compounds.
Background
Disease transmission through blood products and other biological materials is a serious health problem. Although screening and blood testing of donors has been a great deal of progress, viruses such as Hepatitis B (HBV), Hepatitis C (HCV) and Human Immunodeficiency Virus (HIV) may evade detection in blood products in the tests due to low levels of virus or viral antibodies. Apart from the viral hazard, there is currently no screening approval for the presence of bacteria or protozoa in blood for transfusion. In addition, since the hitherto unknown pathogens have become very prevalent in the blood supply, with the threat of disease transmission, there is a risk that has already occurred before the realization of transmission of HIV by blood transfusion.
Laboratory workers also have health hazard issues with exposure to blood or other bodily fluids. According to the estimates of the centers for disease treatment (Prevention of Transmission of Human Immunodeficiency Virus and Hepatitis B Guidelines ("Guidelines for Prevention of Transmission of Human Immunodeficiency Virus and Hepatitis B Virus" to Health-Care and Public Safety Workers "), Weekly reports of disease and Mortality (Morbidity and Mortality Weekly Report), vol.38, no.S-6, 6 months 1989), 1.2 million jobs each year involve blood-exposed medical Workers infected with Hepatitis B Virus.
In order to reduce the incidence of disease caused by blood transfusion, several methods have been proposed for additional screening and blood testing of blood donors. It has been suggested that: prior to clinical use of blood products, pathogens can be inactivated by the addition of chemical agents to the blood or plasma. In studying effective virucidal agents, nitrogen mustard CH may be added to blood components3-N(CH2CH2Cl)2. However, substantial hemolysis occurred at the concentrations necessary to inactivate one of the viruses studied, indicating that nitrogen mustard is not suitable for use in blood. LoGrippo et al, Proceedings of the six-meeting Proceedings of the six Congress of the International Society of Blood transfusions, hematology libraries (Bibliotheca Haematologica) (Hollander, ed.), 1958, pp.225-230.
Horowitz, et al, Blood (Blood) 79: 826(1992) and Horowitz, et al, Transfusion (Transfusion) 25: 516(1985) describes the "solvent/detergent" (S/D) method for virus inactivation. The method uses 1% tri (n-butyl) phosphate and 1% tyloxapol X at 30 ℃ for-100 four hours to inactivate viruses in fresh frozen plasma. To inactivate viruses in fresh frozen plasma, Piquet et al, Vox sang.63: 251(1992) 1% tri (n-butyl) phosphate and 1% octoxynol-9 were used. Another method of inactivating viruses in blood involves the addition of phenol or formaldehyde to the blood (us patent 4,833,165). Both the solvent/detergent method and the phenol/formaldehyde method require the removal of chemical additives prior to clinical use of the blood product.
The use of photoactivators to inactivate pathogens in blood products has also been described, see e.g., Wagner et al, blood transfusion, 34: 521(1994). However, since red blood cells absorb light in several regions of the ultraviolet and visible spectrum, light treatment is only limited to materials containing red blood cells. There are also indications that light treatment of red blood cells can alter the cells in some way, see Wagner et al, transfusion 33: 30(1993).
There is thus a need to develop compositions and methods for treating blood, blood-derived products, and other biological materials that can inactivate pathogens present in the product or material without rendering the product or material unsuitable for its intended use. Compositions that do not require separation from the biological material prior to use are particularly useful because equipment and materials for separating the compositions can be eliminated and the cost of processing the biological material can be reduced. However, there is a need to put additional demands on the composition, that is, if the composition remains in the biomaterial, the composition cannot have any harm when the biomaterial is used for the desired purpose. For example, highly toxic compounds capable of inactivating pathogens in blood samples should preclude the use of such blood for transfusion purposes (although such blood samples may still be used for laboratory analysis).
It is an object of the present invention to provide a composition and method of use thereof for inactivating pathogens present in biological materials without rendering the materials unsuitable for their intended use. Examples of how this can be accomplished include, but are not limited to, treating the biological material with the compound ex vivo or in vitro, and then isolating the compound prior to using the material; the use of a composition, even if it remains in the material, enables the material to be suitable for the intended purpose of use; or using a composition that is capable of breaking down into products after inactivation of pathogens in the material, wherein the breakdown products can remain in the material without rendering the material unsuitable for its intended use.
DISCLOSURE OF THE INVENTION
It is therefore an object of the present invention to provide a compound capable of inactivating a pathogen in a material, wherein such compound comprises a nucleic acid binding moiety, an effector moiety capable of reacting with a nucleic acid to form a covalent bond, and a fragile linker comprising covalently linked nucleic acid and effector moieties, wherein the fragile linker is degradable under conditions such that the material is still suitable for the desired purpose such that covalent linkage of the nucleic acid binding moiety and effector moiety is no longer possible.
It is a further object of this invention to provide such compounds for inactivating pathogens in materials wherein the nucleic acid binding moiety is selected from the group consisting of acridine, acridine derivatives, psoralen, isopsoralen, and psoralen derivatives.
It is a further object of this invention to provide such compounds for inactivating pathogens in materials wherein the frangible linker comprises a functional unit selected from the group consisting of forward esters, reverse esters, forward amides, reverse amides, forward thioesters, reverse thioesters, forward and reverse thiocarbonates, forward and reverse dithioacids, sulfates, forward and reverse sulfonates, phosphates, and forward and reverse phosphonates as defined herein.
It is a further object of this invention to provide such compounds for inactivating pathogens in materials wherein the effector group comprises an alkylating agent functional unit.
It is a further object of this invention to provide such compounds for inactivating pathogens in materials, wherein the effector group comprises a functional unit selected from the group consisting of a nitrogen mustard group, a nitrogen mustard group equivalent, an epoxide, an aldehyde, and a formaldehyde synthetic member (formaldehydesynthons).
It is another object of the present invention to provide compounds of the formula and all salts and stereoisomers (including enantiomers and diastereomers) thereof:
wherein at least R1、R2、R3、R4、R5、R6、R7、R8And R9One is-V-W-X-E as defined below, the remainder R1、R2、R3、R4、R5、R6、R7、R8And R9Independently selected from-H, -R10、-O-R10、-NO2、-NH2、-NH-R10、-N(R10)2、-F、-Cl、-Br、-I、-C(＝O)-R10、-C(＝O)-O-R10and-O-C (═ O) -R10，
wherein-R10Independently H, -C1-8Alkyl, -C1-8Heteroalkyl, -aryl, -heteroaryl, -C1-3Alkyl-aryl, -C1-3Heteroalkyl-aryl, -C1-3Alkyl-heteroaryl, -C1-3Heteroalkyl-heteroaryl, -aryl-C1-3Alkyl, -aryl-C1-3Heteroalkyl, -heteroaryl-C1-3Alkyl, -heteroaryl-C1-3Heteroalkyl, -C1-3alkyl-aryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Alkyl, -C1-3alkyl-heteroaryl-C1-3Alkyl, -C1-3alkyl-aryl-C1-3Heteroalkyl, -C1-3Heteroalkyl-heteroaryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Heteroalkyl, -C1-3alkyl-heteroaryl-C1-3Heteroalkyl group or-C1-3Heteroalkyl-heteroaryl-C1-3A heteroalkyl group;
v is independently-R11-、-NH-R11-or-N (CH)3)-R11-, wherein-R11-independently is-C1-8Alkyl-, -C1-8Heteroalkyl-, -aryl-, -heteroarylradical-C1-3Alkyl-aryl-, -C1-3Heteroalkyl-aryl-, -C1-3Alkyl-heteroaryl-, -C1-3Heteroalkyl-heteroaryl-, -aryl-C1-3Alkyl-, -aryl-C1-3Heteroalkyl, -heteroaryl-C1-3Alkyl-, -heteroaryl-C1-3Heteroalkyl-, -C1-3alkyl-aryl-C1-3Alkyl-, -C1-3Heteroalkyl-aryl-C1-3Alkyl-, -C1-3alkyl-heteroaryl-C1-3Alkyl-, -C1-3alkyl-aryl-C1-3Heteroalkyl-, -C1-3Heteroalkyl-heteroaryl-C1-3Alkyl-, -C1-3Heteroalkyl-aryl-C1-3Heteroalkyl-, -C1-3alkyl-heteroaryl-C1-3heteroalkyl-or-C1-3Heteroalkyl-heteroaryl-C1-3Heteroalkyl-;
w is independently-C (═ O) -O-, -O-C (═ O) -, -C (═ S) -O-, -O-C (═ S) -, -C (═ S) -S-, -S-C (═ S) -, -C (═ O) -S-, -S-C (═ O) -, -O-S (═ O)2-O-、-S(＝O)2-O-、-O-S(＝O)2-、-C(＝O)-NR10-、-NR10-C(＝O)-、-O-P(＝O)(-OR10)-O-、-P(＝O)(-OR10)-O-、-O-P(＝O)(-OR10)-；
X is independently-R11-; and
e is independently selected from-N (R)12)2、-N(R12)(R13)、-S-R12And
wherein-R12is-CH2CH2-G, wherein G is independently-Cl, -Br, -I, -O-S (═ O)2-CH3、-O-S(＝O)2-CH2-C6H5or-O-S (═ O)2-C6H4-CH3；
And wherein R13Independently is-C1-8Alkyl, -C1-8Heteroalkyl, -aryl, -heteroaryl, -C1-3Alkyl-aryl, -C1-3Heteroalkyl-aryl, -C1-3Alkyl-heteroaryl, -C1-3Heteroalkyl-heteroaryl, -aryl-C1-3Alkyl, -aryl-C1-3Heteroalkyl, -heteroaryl-C1-3Alkyl, -heteroaryl-C1-3Heteroalkyl, -C1-3alkyl-aryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Alkyl, -C1-3alkyl-heteroaryl-C1-3Alkyl, -C1-3alkyl-aryl-C1-3Heteroalkyl, -C1-3Heteroalkyl-heteroaryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Heteroalkyl, -C1-3alkyl-heteroaryl-C1-3Heteroalkyl group or-C1-3Heteroalkyl-heteroaryl-C1-3A heteroalkyl group.
It is another object of the present invention to provide compounds of the formula and all salts and stereoisomers (including enantiomers and diastereomers) thereof:
wherein R is1、R2、R3、R4、R5、R6、R7And R8Independently selected from-H, -R10、-O-R10、-NO2、-NH2、-NH-R10、-N(R10)2、-F、-Cl、-Br、-I、-C(＝O)-R10、-C(＝O)-O-R10and-O-C (═ O) -R10，
wherein-R10Independently H, -C1-8Alkyl, -C1-8Heteroalkyl, -aryl, -heteroaryl, -C1-3Alkyl-aryl, -C1-3Heteroalkyl-aryl, -C1-3Alkyl-heteroaryl, -C1-3Heteroalkyl-heteroaryl, -aryl-C1-3Alkyl, -aryl-C1-3Heteroalkyl, -heteroaryl-C1-3Alkyl, -heteroaryl-C1-3Heteroalkyl, -C1-3alkyl-aryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Alkyl, -C1-3alkyl-heteroaryl-C1-3Alkyl, -C1-3alkyl-aryl-C1-3Heteroalkyl, -C1-3Heteroalkyl-heteroaryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Heteroalkyl, -C1-3alkyl-heteroaryl-C1-3Heteroalkyl group or-C1-3Heteroalkyl-heteroaryl-C1-3A heteroalkyl group;
R20is-H or-CH3(ii) a And
R21is-R11-W-X-E，
wherein-R11-independently is-C1-8Alkyl-, -C1-8Heteroalkyl-, -aryl-, -heteroaryl-, -C1-3Alkyl-aryl-, -C1-3Heteroalkyl-aryl-, -C1-3Alkyl-heteroaryl-, -C1-3Heteroalkyl-heteroaryl-, -aryl-C1-3Alkyl-, -aryl-C1-3Heteroalkyl, -heteroaryl-C1-3Alkyl-, -heteroaryl-C1-3Heteroalkyl-, -C1-3alkyl-aryl-C1-3Alkyl-, -C1-3Heteroalkyl-aryl-C1-3Alkyl-, -C1-3alkyl-heteroaryl-C1-3Alkyl-, -C1-3alkyl-aryl-C1-3Heteroalkyl-, -C1-3Heteroalkyl-heteroaryl-C1-3Alkyl-, -C1-3Heteroalkyl-aryl-C1-3Heteroalkyl-, -C1-3alkyl-heteroaryl-C1-3heteroalkyl-or-C1-3Heteroalkyl-heteroaryl-C1-3Heteroalkyl-;
w is independently-C (═ O) -O-, -O-C (═ O) -, -C (═ S) -O-, -O-C (═ S) -, -C (═ S) -S-, -S-C (═ S) -, -C (═ O) -S-, -S-C (═ O) -, -O-S (═ O)2-O-、-S(＝O)2-O-、-O-S(＝O)2-、-C(＝O)-NR10-、-NR10-C(＝O)-、-O-P(＝O)(-OR10)-O-、-P(＝O)(-OR10)-O-、-O-P(＝O)(-OR10)-；
X is independently-R11-; and
e is independently selected from-N (R)12)2、-N(R12)(R13)、-S-R12And
wherein-R12is-CH2CH2-G, wherein each G is independently-Cl, -Br, -I, -O-S (═ O)2-CH3、-O-S(＝O)2-CH2-C6H5or-O-S (═ O)2-C6H4-CH3；
And wherein R13Independently is-C1-8Alkyl, -C1-8Heteroalkyl, -aryl, -heteroaryl, -C1-3Alkyl-aryl, -C1-3Heteroalkyl-aryl, -C1-3Alkyl-heteroaryl, -C1-3Heteroalkyl-heteroaryl, -aryl-C1-3Alkyl, -aryl-C1-3Heteroalkyl, -heteroaryl-C1-3Alkyl, -heteroaryl-C1-3Heteroalkyl, -C1-3alkyl-aryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Alkyl, -C1-3alkyl-heteroaryl-C1-3Alkyl, -C1-3alkyl-aryl-C1-3Heteroalkyl, -C1-3Heteroalkyl-heteroaryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Heteroalkyl, -C1-3alkyl-heteroaryl-C1-3Heteroalkyl group or-C1-3Heteroalkyl-heteroaryl-C1-3A heteroalkyl group.
It is another object of the present invention to provide compounds of the formula and all salts and stereoisomers (including enantiomers and diastereomers) thereof:
wherein at least R44、R55、R3、R4、R5And R8One of which is-V-W-X-E and the remainder R44、R55、R3、R4、R5And R8Independently selected from-H, -R10、-O-R10、-NO2、-NH2、-NH-R10、-N(R10)2、-F、-Cl、-Br、-I、-C(＝O)-R10、-C(＝O)-O-R10and-O-C (═ O) -R10，
wherein-R10Independently H, -C1-8Alkyl, -C1-8Heteroalkyl, -aryl, -heteroaryl, -C1-3Alkyl-aryl, -C1-3Heteroalkyl-aryl, -C1-3Alkyl-heteroaryl, -C1-3Heteroalkyl-heteroaryl, -aryl-C1-3Alkyl, -aryl-C1-3Heteroalkyl, -heteroaryl-C1-3Alkyl, -heteroaryl-C1-3Heteroalkyl, -C1-3alkyl-aryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Alkyl, -C1-3alkyl-heteroaryl-C1-3Alkyl, -C1-3alkyl-aryl-C1-3Heteroalkyl, -C1-3Heteroalkyl-heteroaryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Heteroalkyl, -C1-3alkyl-heteroaryl-C1-3Heteroalkyl group or-C1-3Heteroalkyl-heteroaryl-C1-3A heteroalkyl group;
v is independently-R11-、-NH-R11-or-N (CH)3)-R11-, wherein-R11-independently is-C1-8Alkyl-, -C1-8Heteroalkyl-, -aryl-, -heteroaryl-, -C1-3Alkyl-aryl-, -C1-3Heteroalkyl-aryl-, -C1-3Alkyl-heteroaryl-, -C1-3Heteroalkyl-heteroaromaticsRadical-, -aryl-C1-3Alkyl-, -aryl-C1-3Heteroalkyl, -heteroaryl-C1-3Alkyl-, -heteroaryl-C1-3Heteroalkyl-, -C1-3alkyl-aryl-C1-3Alkyl-, -C1-3Heteroalkyl-aryl-C1-3Alkyl-, -C1-3alkyl-heteroaryl-C1-3Alkyl-, -C1-3alkyl-aryl-C1-3Heteroalkyl-, -C1-3Heteroalkyl-heteroaryl-C1-3Alkyl-, -C1-3Heteroalkyl-aryl-C1-3Heteroalkyl-, -C1-3alkyl-heteroaryl-C1-3heteroalkyl-or-C1-3Heteroalkyl-heteroaryl-C1-3Heteroalkyl-;
w is independently-C (═ O) -O-, -O-C (═ O) -, -C (═ S) -O-, -O-C (═ S) -, -C (═ S) -S-, -S-C (═ S) -, -C (═ O) -S-, -S-C (═ O) -, -O-S (═ O)2-O-、-S(＝O)2-O-、-O-S(＝O)2-、-C(＝O)-NR10-、-NR10-C(＝O)-、-O-P(＝O)(-OR10)-O-、-P(＝O)(-OR10)-O-、-O-P(＝O)(-OR10)-；
X is independently-R11-; and
e is independently selected from-N (R)12)2、-N(R12)(R13)、-S-R12And
wherein-R12is-CH2CH2-G, wherein G is independently-Cl, -Br, -I, -O-S (═ O)2-CH3、-O-S(＝O)2-CH2-C6H5or-O-S (═ O)2-C6H4-CH3；
And wherein R13Independently is-C1-8Alkyl, -C1-8Heteroalkyl, -aryl, -heteroaryl, -C1-3Alkyl radical-aryl, -C1-3Heteroalkyl-aryl, -C1-3Alkyl-heteroaryl, -C1-3Heteroalkyl-heteroaryl, -aryl-C1-3Alkyl, -aryl-C1-3Heteroalkyl, -heteroaryl-C1-3Alkyl, -heteroaryl-C1-3Heteroalkyl, -C1-3alkyl-aryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Alkyl, -C1-3alkyl-heteroaryl-C1-3Alkyl, -C1-3alkyl-aryl-C1-3Heteroalkyl, -C1-3Heteroalkyl-heteroaryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Heteroalkyl, -C1-3alkyl-heteroaryl-C1-3Heteroalkyl group or-C1-3Heteroalkyl-heteroaryl-C1-3A heteroalkyl group.
It is a further object of the present invention to provide the compounds β -alanine, N- (2-methoxycarbonylacridin-9-yl), 2- [ bis (2-chloroethyl) amino ] ethyl ester; 4-aminobutyric acid N- (2-methoxycarbonylacridin-9-yl), 2- [ bis (2-chloroethyl) amino ] ethyl ester; n- [ (2-methoxyformamido-acridin-9-yl), 2- [ bis (2-chloroethyl) amino ] ethyl 5-aminopentanoate; beta-alanine, N- (2-methoxycarbonylacridin-9-yl), 3- [ bis (2-chloroethyl) amino ] propyl ester; beta-alanine, [ N, N-bis (2-chloroethyl) ], 3- [ (6-chloro-2-methoxyacridin-9-yl) amino ] propyl ester; beta-alanine, [ N, N-bis (2-chloroethyl) ], 2- [ (6-chloro-2-methoxyacridin-9-yl) amino ] ethyl ester; [ N, N-bis (2-chloroethyl) ] -2-aminoethyl 4, 5 ', 8-trimethyl-4' -psoralen acetate; and beta-alanine, N- (acridin-9-yl), 2- [ bis (2-chloroethyl) amino ] ethyl ester and all salts thereof.
The present invention provides a method of inactivating a pathogen in a material (e.g. a biological material), the method comprising adding one or more compounds of the invention to the material, and then incubating the material. The compound may be added to the material to form a final solution having a concentration of the compound (if a plurality of compounds are used, the total concentration of all compounds) of, for example, 1 to 500. mu.M. Biological materials that may be processed include blood, blood products, plasma, platelet preparations, red blood cells, packed red blood cells, serum, cerebrospinal fluid, saliva, urine, sweat, stool, semen, milk, tissue samples, and homogenized tissue samples produced by humans or other mammals or vertebrates.
Best mode for carrying out the invention
The present invention provides compounds for inactivating pathogens present in materials, particularly for inactivating pathogens present in biological materials, including blood or other bodily fluids. The invention also provides methods of using such compounds to inactivate pathogens in materials. The present invention also provides methods of inactivating pathogens present in or on the surface of a biological material. The compounds are useful in vitro and ex vivo. The biomaterial or material for biological use may be used in vitro, in vivo or ex vivo.
The compounds can inactivate pathogens by reacting with nucleic acids. In aqueous solution, at an appropriate pH, the compound has a period of activity when bound to and reacted with nucleic acid. After this period of time, the compound breaks down into products that can no longer bind to or react with the nucleic acid.
The chemical structure of a compound can be broadly described as an anchor covalently linked to a fragile linker, the anchor being covalently linked to an effector. An "immobilizer" is defined as a moiety that is covalently bound to a nucleic acid biopolymer (DNA or RNA). An "effector" is defined as a moiety that reacts with a nucleic acid by forming a covalent bond with the nucleic acid. A "fragile linker" is defined as a moiety that serves to covalently link an immobilizer to an effector, which moiety is capable of degrading under conditions such that the immobilizer is no longer covalently linked to the effector. The arrangement of the immobilizer-fragile linker-effector allows specific binding of the compound to the nucleic acid (due to the binding capacity of the immobilizer). This brings the effector into proximity to the nucleic acid, reacting with the nucleic acid.
The compounds are useful for inactivating pathogens present in materials, particularly pathogens present in biological materials such as blood and other bodily fluids. Both intracellular and extracellular, as well as other pathogen materials, can be inactivated. For example, when a compound of the invention is bound to a pathogen-containing red blood cell composition at physiological pH, the effector portion of the compound reacts with the pathogen nucleic acid. Effector moieties that do not react with nucleic acids are gradually hydrolyzed by the solvent. Hydrolysis of the fragile linker occurs simultaneously with the effector-nucleic acid reaction and effector hydrolysis. Thus, the fragile linker needs to break down at a sufficiently low rate to inactivate pathogens in the material; that is, the rate of cleavage of the fragile linker is lower than the rate of reaction of the compound with the nucleic acid. After a sufficiently long time has elapsed, the compound breaks down into either a immobilizer (which may still have fragments of a fragile linker) and an effector-nucleic acid breakdown product (in which fragments of a fragile linker may still be linked to an effector), or into an immobilizer (which may still have fragments of a fragile linker) and a hydrolyzed effector breakdown product (in which fragments of a fragile linker may still be linked to an effector). Additional fragments of the fragile linker may also be generated upon degradation of the compound, which are no longer attached to the immobilizer or effector. Embodiments of the compounds of the invention determine whether the immobilizer decomposition product or the effector decomposition product carries a fragile linker fragment or is capable of generating additional fragile linker fragments which are no longer bound to the immobilizer or effector decomposition product.
Preferred embodiments of the present invention include compounds that yield decomposition products with low mutagenicity upon cleavage of the fragile linker. After effector hydrolysis, the mutagenicity of the compound depends primarily on the immobilizer moiety, which interacts with the nucleic acid and has the ability to interfere with nucleic acid replication even if the effector moiety has been hydrolyzed. Preferably, the immobilizer fragment has substantially reduced mutagenicity after cleavage of the fragile linker.
Definition of
"pathogen" refers to any nucleic acid-containing agent capable of causing disease in humans, other mammals, or vertebrates. Examples include microorganisms, such as unicellular or multicellular microorganisms. Examples of pathogens are bacteria, viruses, protozoa, fungi, yeasts, molds and mycoplasmas which are capable of causing disease in humans, other mammals or vertebrates. The genetic material of the pathogen may be DNA or RNA, and the genetic material may be present in the form of single-stranded or double-stranded nucleic acid. The nucleic acid of the pathogen may be present in solution, intracellularly, extracellularly or bound to the cell. Table I lists some viruses, but does not limit the scope of the invention in any way.
TABLE I
The family name is: virus:
adenoviral vectors Adenovirus 2
Canine hepatitis virus
Arenavirus Pichinde
Lassa Virus
Benya virus Turlock
California encephalitisVirus
Herpes virus Herpes simplex virus 1
Herpes simplex virus 2
Cytomegalovirus
Pseudorabies virus
Orothomyxo Influenza virus
Papovavirus SV-40 virus
Paramyxovirus Measles virus
Mumps virus
Parainfluenza viruses 2 and 3
Picornavirus (picornavirus) Polioviruses 1 and 2
Coxsackie virus A-9
Echovirus 11
Poxviruses Vaccinia virus
Avipox virus
Reovirus
Bluetongue virus
Colorado tick fever virus
Retroviruses HIV (AIDS virus)
Avian sarcoma virus
Murine sarcoma virus
Murine leukemia virus
Rhabdovirus Vesicular stomatitis virus
Togavirus Western equine encephalitis Virus
Dengue virus 2
Dengue virus 4
St Louis encephalitis virus
Hepadnavirus Hepatitis B virus
Bacteriophage λ
T2
(Rickettsia's body) Akari (Rickett pox)
"in vivo" use of a material or compound refers to introducing the material or compound into a living body of a human, mammal, or vertebrate.
"in vitro" use of a material or compound refers to the use of the material or compound in vitro in a human, mammal, or vertebrate wherein neither the material nor the compound can be reintroduced into the living body of the human, mammal, or vertebrate. An example of an in vitro application is the analysis of blood sample components using laboratory equipment.
By "ex vivo" use of a compound is meant that the compound is used to treat a biological material outside of the living body of a human, mammal, or vertebrate, where the treated biological material is intended for use inside the living body of the human, mammal, or vertebrate. For example, blood is removed from a human body, a compound is introduced into the blood to inactivate pathogens, and if the blood is intended to be reintroduced into the human or another human body, ex vivo use of the compound may be considered. Reintroduction of human blood into the human or another person should be an in vivo application of blood, as opposed to an ex vivo application of the compound. If the compound is still present in the blood when it is reintroduced into the body, the compound is also introduced into the body in addition to ex vivo applications.
"biological material" refers to material from any type of biological organism. Examples of biological materials include, but are not limited to: blood; blood products such as plasma, platelet preparations, red blood cells, packed red blood cells, and serum; cerebrospinal fluid; saliva; urea; feces; semen; sweat, milk; a tissue sample; homogenizing the tissue sample and any other material derived from the biological organism. The biological material may also comprise a synthetic material incorporating material derived from a biological organism, such as a vaccine formulation comprising alum and a pathogen (in this case, the pathogen is a material derived from a biological organism); a sample preparation for analysis, the sample preparation being a mixture of blood and an analytical reagent; a cell culture medium; a cell culture; viral cultures and other cultures produced by living organisms.
"biological application material" refers to any material that comes into contact with or is added to the living body of a human, mammal, or vertebrate, wherein the contact carries a risk of transmitting a disease or pathogen. Such materials include, but are not limited to: medical implants, such as pacemakers and artificial joints; implants designed for sustained release of drugs; needles, venous lines, etc.; a dental instrument; dental materials such as crowns; a conduit; and any other material that presents a risk of contracting an infectious disease or pathogen when in contact with or introduced into a living body of a human, mammal, or vertebrate.
By "inactivating the pathogen" is meant rendering the pathogen incapable of replicating in the material. Inactivation is expressed as the negative log of the remaining pathogen fraction that is able to replicate. Thus, if a certain concentration of the compound renders 99% of the pathogens in the material incapable of replication, only 1% (0.01) of the pathogens are capable of replication. The negative log of 0.01 is 2 and the concentration of the compound is believed to inactivate the pathogen with 2logs, in other words, the compound has 2logs kill at this concentration.
As used herein, "alkyl" refers to a cyclic, branched or straight chain chemical group containing carbon and hydrogen, such as methyl, pentyl and adamantyl. The alkyl group may be unsubstituted or substituted with one or more substituents such as halogen, alkoxy, acyloxy, amino, hydroxy, mercapto, carboxyl, benzyloxy, phenyl, benzyl or other functional groups. The alkyl groups may be saturated or unsaturated in one or several positions (e.g.contain-C-or-C.ident.C-subunits). Typically, unless otherwise specified, an alkyl group may contain 1 to 12 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 8 carbon atoms.
As used herein, "heteroalkyl" refers to an alkyl chain incorporating one or more N, O, S or P heteroatoms in the chain. The heteroatoms may be unsubstituted or carry one or more of the above-mentioned substituents. "heteroatom" may also include heteroatom N, S and the oxidized form of P. Examples of heteroalkyl groups include (but are not limited to): methoxy, ethoxy and other alkoxy groups; an ether-containing group; amide-containing groups, such as polypeptide chains; ring systems such as piperidinyl, lactams, and lactones; and other groups incorporating heteroatoms in the carbon chain. Typically, unless otherwise indicated, in addition to heteroatoms, heteroalkyl groups will contain from 1 to 12 carbon atoms, preferably from 1 to 10 carbon atoms, more preferably from 1 to 8 carbon atoms.
"aryl" or "Ar" refers to an unsaturated aromatic carbocyclic group having one ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthracenyl) which may optionally be unsubstituted or substituted with amino, hydroxy, C1-8Alkyl, alkoxy, halogen, mercapto and other substituents.
A "heteroaryl" group refers to an unsaturated aromatic carbocyclic group having one ring (e.g., pyridyl or furyl) or multiple fused rings (e.g., acridinyl, indolyl or benzothienyl) and bearing at least one heteroatom (e.g., N, O or S) in at least one ring, which may be optionally unsubstituted or substituted with amino, hydroxyl, alkyl, alkoxy, halogen, mercapto, acyloxy, carboxyl, benzyloxy, phenyl, benzyl and other substituents.
Abbreviations
The present invention uses the following abbreviations: QM (quinacrine); hct (hematocrit); RBCs (red blood cells); LB (Luria medium); cfu (colony forming units); pfu (plaque forming unit); DMEM (Delbecco's modified eagles medium); FBS (fetal bovine serum); PRBC (packed red blood cells); rpm (revolutions per minute); TC (tissue culture); NHSP (normal human serum bank); NCS (newborn calf serum); PBS (phosphate buffered saline).
Chemical structure of the Compound
There are many groups that can be used as immobilizers, linkers, and effectors. Examples of immobilizer groups that may be used in the compounds include, but are not limited to: an intercalator; a minor groove binder; a macro groove adhesive; molecules bound by electrostatic interaction, such as polyamides; and molecules that bind through specific sequence interactions. The following groups are some non-limiting examples of possible immobilizer groups:
acridine (and acridine derivatives, e.g. proflavine sulphate, acriflavine, biacridine, acridone, benzophenonesAcridine, quinacrine), actinomycin, anthracyclines, erythromycin, daunorubicin, thioxanthone (and thioxanthone derivatives, such as milbed), antromycin, mitomycin, echinomycin (quinomycin a), trisaccharin, ellipticine (and dimers, trimers, and analogs thereof), northilin a, fluorenes (and derivatives thereof, such as fluorenone, fluorenediamine), phenazine, phenanthridine, phenothiazine (such as chlorpromazine), thiopheneOxazines, benzothiazoles,Hydrocarbons and thioxanthenes, anthraquinones, anthrapyrazoles, benzothiopyranones, 3, 4-benzopyrenes, 1-pyrenyl oxiranes, benzanthracenes, benzanalines, quinolines (e.g., chloroquine, quinine, phenylquinoline, formamide), furocoumarins (e.g., psoralen and isopsoralen), ethidium, propidium, phellodendron (coralyne), and polycyclic aromatic hydrocarbons and ethylene oxide derivatives thereof;
distamycin, fusidin, other lexitrophins, hexedrine nicotinate (Hoechst)33258 and other hexedrine nicotinate dyes, DAPI (4', 6-diamidino-2-phenylindole), diazobenzamidine acetyl glycinate and triarylmethane dyes;
aflatoxins;
spermine, spermidine and other polyamides; and
nucleic acids or analogs that bind by sequence specific actions, such as triple helix formation, D-loop formation, and direct base pairing to a single stranded target. Derivatives of these compounds are also non-limiting examples of immobilizer groups, where derivatives of compounds include, but are not limited to: compounds bearing one or more substituents of any type at any position, oxidation or reduction products of the compounds, and the like.
Examples of linkers useful in the present invention include, but are not limited to, compounds containing the following functional groups: esters(wherein the carbonyl carbon of the ester is located in the sp of the anchor and the ester3Between the oxygens, this arrangement is also referred to as "forward ester"), "reverse ester" (where the sp of the ester is3Oxygen is located between the anchor and the carbonyl carbon of the ester), thioesters (where the carbonyl carbon of the thioester is located between the anchor and the sulfur of the thioester, also known as "forward thioesters"), reverse thioesters (where the sulfur of the thioester is located between the anchor and the carbonyl carbon of the thioester, also known as "reverse thioesters"), forward and reverse thiocarbonates, forward and reverse dithioacids, sulfates, forward and reverse sulfonates, phosphates, and forward and reverse phosphonates. "thioester" refers to a group-C (═ O) -S-; "thiocarboxylate" refers to a group-C (═ S) -O-, and "dithioic acid" refers to a group-C (═ S) -S-. The fragile linker may also include an amide in which the carbonyl carbon of the amide is located between the anchor and the nitrogen of the amide (also referred to as a "normal amide"), or in which the nitrogen of the amide is located between the anchor and the carbonyl carbon of the amide (also referred to as a "reverse amide"). For groups that can be designated as "forward" and "reverse," forward orientation refers to the orientation of the following functional groups: wherein upon hydrolysis of the functional group, the resulting acidic functional group is covalently attached to the immobilizer moiety and the resulting alcohol or thiol functional group is covalently attached to the effector moiety. Reverse orientation refers to the orientation of the following functional groups: wherein upon hydrolysis of the functional group, the resulting acidic functional group is covalently attached to the effector moiety and the resulting alcohol or thiol functional group is covalently attached to the immobilizer moiety.
The fragile linker (e.g., amide moiety) can also be degraded by endogenous enzymes in the treated biological material or by enzymes added to the material under enzymatic degradation conditions.
Examples of effectors useful in the present invention include, but are not limited to: nitrogen mustard groups, nitrogen mustard group equivalents, epoxides, aldehydes, formaldehyde synthons, and other alkylating or crosslinking agents. The nitrogen mustard group is defined to include mono-or di-haloethylamine groups and monohaloethylsulfide groups. Nitrogen mustard radical equivalents are defined as radicals that react by a similar mechanism to nitrogen mustards (i.e., by reaction with nitrogen mustards)Formation of aziridinesSalt intermediates, or by carrying or forming an aziridine ring, the nitrogen mustard group equivalent being capable of reacting with a nucleophile), such as a mono or bis methylsulfonylethylamine group, a mono methylsulfonylethyl sulfide group, a mono or bis methylsulfonylethylamine group, and a mono tosylethylsulfide group. A formaldehyde-synthesizing element is defined as any compound that can decompose in aqueous solution to formaldehyde, including hydroxymethylamine, such as hydroxymethylglycine. Examples of formaldehyde synthons can be found in us patent 4,337,269 and international patent application WO 97/02028. Although the present invention is not limited to any particular mechanism, effector groups that form or are capable of forming electrophilic groups (e.g., nitrogen mustard groups) are believed to be capable of reacting with or forming covalent bonds with nucleic acids.
The following formulas I, II and III describe three embodiments of the compounds of the present invention.
Compounds of general formula I and all salts and stereoisomers (including enantiomers and diastereomers) thereof:
wherein at least R1、R2、R3、R4、R5、R6、R7、R8And R9One is-V-W-X-E as defined below, the remainder R1、R2、R3、R4、R5、R6、R7、R8And R9Independently selected from-H, -R10、-O-R10、-NO2、-NH2、-NH-R10、-N(R10)2、-F、-Cl、-Br、-I、-C(＝O)-R10、-C(＝O)-O-R10and-O-C (═ O) -R10，
wherein-R10Independently H, -C1-8Alkyl, -C1-8Heteroalkyl, -aryl, -heteroaryl, -C1-3Alkyl-aryl, -C1-3Heteroalkyl-aryl, -C1-3Alkyl-heteroaryl, -C1-3Heteroalkyl-heteroaryl, -aryl-C1-3Alkyl, -aryl-C1-3Heteroalkyl, -heteroaryl-C1-3Alkyl, -heteroaryl-C1-3Heteroalkyl, -C1-3alkyl-aryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Alkyl, -C1-3alkyl-heteroaryl-C1-3Alkyl, -C1-3alkyl-aryl-C1-3Heteroalkyl, -C1-3Heteroalkyl-heteroaryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Heteroalkyl, -C1-3alkyl-heteroaryl-C1-3Heteroalkyl group or-C1-3Heteroalkyl-heteroaryl-C1-3A heteroalkyl group;
v is independently-R11-、-NH-R11-or-N (CH)3)-R11-, wherein-R11-independently is-C1-8Alkyl-, -C1-8Heteroalkyl-, -aryl-, -heteroaryl-, -C1-3Alkyl-aryl-, -C1-3Heteroalkyl-aryl-, -C1-3Alkyl-heteroaryl-, -C1-3Heteroalkyl-heteroaryl-, -aryl-C1-3Alkyl-, -aryl-C1-3Heteroalkyl, -heteroaryl-C1-3Alkyl-, -heteroaryl-C1-3Heteroalkyl-, -C1-3alkyl-aryl-C1-3Alkyl-, -C1-3Heteroalkyl-aryl-C1-3Alkyl-, -C1-3alkyl-heteroaryl-C1-3Alkyl-, -C1-3alkyl-aryl-C1-3Heteroalkyl-, -C1-3Heteroalkyl-heteroaryl-C1-3Alkyl-, -C1-3Heteroalkyl-aryl-C1-3Heteroalkyl-, -C1-3alkyl-heteroaryl-C1-3heteroalkyl-or-C1-3Heteroalkyl-heteroaryl-C1-3Heteroalkyl-;
w is independently-C (═ O) -O-, -O-C (═ O) -, -C (═ S) -O-, -O-C (═ S) -, -C (═ S) -S-, -S-C (═ S) -, -C (═ O) -S-, -S-C (═ O)-、-O-S(＝O)2-O-、-S(＝O)2-O-、-O-S(＝O)2-、-C(＝O)-NR10-、-NR10-C(＝O)-、-O-P(＝O)(-OR10)-O-、-P(＝O)(-OR10)-O-、-O-P(＝O)(-OR10)-；
X is independently-R11-; and
e is independently selected from-N (R)12)2、-N(R12)(R13)、-S-R12And
wherein-R12is-CH2CH2-G, wherein each G is independently-Cl, -Br, -I, -O-S (═ O)2-CH3、-O-S(＝O)2-CH2-C6H5or-O-S (═ O)2-C6H4-CH3；
And wherein R13Independently is-C1-8Alkyl, -C1-8Heteroalkyl, -aryl, -heteroaryl, -C1-3Alkyl-aryl, -C1-3Heteroalkyl-aryl, -C1-3Alkyl-heteroaryl, -C1-3Heteroalkyl-heteroaryl, -aryl-C1-3Alkyl, -aryl-C1-3Heteroalkyl, -heteroaryl-C1-3Alkyl, -heteroaryl-C1-3Heteroalkyl, -C1-3alkyl-aryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Alkyl, -C1-3alkyl-heteroaryl-C1-3Alkyl, -C1-3alkyl-aryl-C1-3Heteroalkyl, -C1-3Heteroalkyl-heteroaryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Heteroalkyl, -C1-3alkyl-heteroaryl-C1-3Heteroalkyl group or-C1-3Heteroalkyl-heteroaryl-C1-3A heteroalkyl group.
Compounds of general formula II and all salts and stereoisomers (including enantiomers and diastereomers) thereof:
wherein R is1、R2、R3、R4、R5、R6、R7And R8Independently selected from-H, -R10、-O-R10、-NO2、-NH2、-NH-R10、-N(R10)2、-F、-Cl、-Br、-I、-C(＝O)-R10、-C(＝O)-O-R10and-O-C (═ O) -R10，
wherein-R10Independently H, -C1-8Alkyl, -C1-8Heteroalkyl, -aryl, -heteroaryl, -C1-3Alkyl-aryl, -C1-3Heteroalkyl-aryl, -C1-3Alkyl-heteroaryl, -C1-3Heteroalkyl-heteroaryl, -aryl-C1-3Alkyl, -aryl-C1-3Heteroalkyl, -heteroaryl-C1-3Alkyl, -heteroaryl-C1-3Heteroalkyl, -C1-3alkyl-aryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Alkyl, -C1-3alkyl-heteroaryl-C1-3Alkyl, -C1-3alkyl-aryl-C1-3Heteroalkyl, -C1-3Heteroalkyl-heteroaryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Heteroalkyl, -C1-3alkyl-heteroaryl-C1-3Heteroalkyl group or-C1-3Heteroalkyl-heteroaryl-C1-3A heteroalkyl group;
R20is-H or-CH3(ii) a And
R21is-R11-W-X-E，
wherein-R11-independently is-C1-8Alkyl-, -C1-8Heteroalkyl-, -aryl-, -heteroaryl-, -C1-3Alkyl-aryl-, -C1-3Heteroalkyl-aryl-, -C1-3Alkyl-heteroaryl-, -C1-3Heteroalkyl-heteroaryl-, -aryl-C1-3Alkyl-, -aryl-C1-3Heteroalkyl, -heteroaryl-C1-3Alkyl-, -heteroaryl-C1-3Heteroalkyl-, -C1-3alkyl-aryl-C1-3Alkyl-, -C1-3Heteroalkyl-aryl-C1-3Alkyl-, -C1-3alkyl-heteroaryl-C1-3Alkyl-, -C1-3alkyl-aryl-C1-3Heteroalkyl-, -C1-3Heteroalkyl-heteroaryl-C1-3Alkyl-, -C1-3Heteroalkyl-aryl-C1-3Heteroalkyl-, -C1-3alkyl-heteroaryl-C1-3heteroalkyl-or-C1-3Heteroalkyl-heteroaryl-C1-3Heteroalkyl-;
w is independently-C (═ O) -O-, -O-C (═ O) -, -C (═ S) -O-, -O-C (═ S) -, -C (═ S) -S-, -S-C (═ S) -, -C (═ O) -S-, -S-C (═ O) -, -O-S (═ O)2-O-、-S(＝O)2-O-、-O-S(＝O)2-、-C(＝O)-NR10-、-NR10-C(＝O)-、-O-P(＝O)(-OR10)-O-、-P(＝O)(-OR10)-O-、-O-P(＝O)(-OR10)-；
X is independently-R11-; and
e is independently selected from-N (R)12)2、-N(R12)(R13)、-S-R12And
wherein-R12is-CH2CH2-G, wherein each G is independently-Cl, -Br, -I, -O-S (═ O)2-CH3、-O-S(＝O)2-CH2-C6H5or-O-S (═ O)2-C6H4-CH3；
And whereinR13Independently is-C1-8Alkyl, -C1-8Heteroalkyl, -aryl, -heteroaryl, -C1-3Alkyl-aryl, -C1-3Heteroalkyl-aryl, -C1-3Alkyl-heteroaryl, -C1-3Heteroalkyl-heteroaryl, -aryl-C1-3Alkyl, -aryl-C1-3Heteroalkyl, -heteroaryl-C1-3Alkyl, -heteroaryl-C1-3Heteroalkyl, -C1-3alkyl-aryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Alkyl, -C1-3alkyl-heteroaryl-C1-3Alkyl, -C1-3alkyl-aryl-C1-3Heteroalkyl, -C1-3Heteroalkyl-heteroaryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Heteroalkyl, -C1-3alkyl-heteroaryl-C1-3Heteroalkyl group or-C1-3Heteroalkyl-heteroaryl-C1-3A heteroalkyl group.
Compounds of formula III and all salts and stereoisomers (including enantiomers and diastereomers) thereof:
wherein at least R44、R55、R3、R4、R5And R8One of which is-V-W-X-E and the remainder R44、R55、R3、R4、R5And R8Independently selected from-H, -R10、-O-R10、-NO2、-NH2、-NH-R10、-N(R10)2、-F、-Cl、-Br、-I、-C(＝O)-R10、-C(＝O)-O-R10and-O-C (═ O) -R10，
wherein-R10Independently H, -C1-8Alkyl, -C1-8Heteroalkyl, -aryl, -heteroaryl, -C1-3Alkyl-aryl, -C1-3Heteroalkyl-aryl, -C1-3Alkyl-heteroaryl, -C1-3Heteroalkyl-heteroaryl, -aryl-C1-3Alkyl, -aryl-C1-3Heteroalkyl, -heteroaryl-C1-3Alkyl, -heteroaryl-C1-3Heteroalkyl, -C1-3alkyl-aryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Alkyl, -C1-3alkyl-heteroaryl-C1-3Alkyl, -C1-3alkyl-aryl-C1-3Heteroalkyl, -C1-3Heteroalkyl-heteroaryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Heteroalkyl, -C1-3alkyl-heteroaryl-C1-3Heteroalkyl group or-C1-3Heteroalkyl-heteroaryl-C1-3A heteroalkyl group;
v is independently-R11-、-NH-R11-or-N (CH)3)-R11-, wherein-R11-independently is-C1-8Alkyl-, -C1-8Heteroalkyl-, -aryl-, -heteroaryl-, -C1-3Alkyl-aryl-, -C1-3Heteroalkyl-aryl-, -C1-3Alkyl-heteroaryl-, -C1-3Heteroalkyl-heteroaryl-, -aryl-C1-3Alkyl-, -aryl-C1-3Heteroalkyl, -heteroaryl-C1-3Alkyl-, -heteroaryl-C1-3Heteroalkyl-, -C1-3alkyl-aryl-C1-3Alkyl-, -C1-3Heteroalkyl-aryl-C1-3Alkyl-, -C1-3alkyl-heteroaryl-C1-3Alkyl-, -C1-3alkyl-aryl-C1-3Heteroalkyl-, -C1-3Heteroalkyl-heteroaryl-C1-3Alkyl-, -C1-3Heteroalkyl-aryl-C1-3Heteroalkyl-, -C1-3alkyl-heteroaryl-C1-3heteroalkyl-or-C1-3Heteroalkyl-heteroaryl-C1-3Heteroalkyl-;
w is independently-C (═ O) -O-, -O-C (═ O) -, -C (═ S) -O-, -O-C (═ S) -, -C (═ S) -S-, -S-C (═ S) -, -C (═ O) -S-, -S-C (═ O) -, -O-S (═ O)2-O-、-S(＝O)2-O-、-O-S(＝O)2-、-C(＝O)-NR10-、-NR10-C(＝O)-、-O-P(＝O)(-OR10)-O-、-P(＝O)(-OR10)-O-、-O-P(＝O)(-OR10)-；
X is independently-R11-; and
e is independently selected from-N (R)12)2、-N(R12)(R13)、-S-R12And
wherein-R12is-CH2CH2-G, wherein G is independently-Cl, -Br, -I, -O-S (═ O)2-CH3、-O-S(＝O)2-CH2-C6H5or-O-S (═ O)2-C6H4-CH3；
And wherein R13Independently is-C1-8Alkyl, -C1-8Heteroalkyl, -aryl, -heteroaryl, -C1-3Alkyl-aryl, -C1-3Heteroalkyl-aryl, -C1-3Alkyl-heteroaryl, -C1-3Heteroalkyl-heteroaryl, -aryl-C1-3Alkyl, -aryl-C1-3Heteroalkyl, -heteroaryl-C1-3Alkyl, -heteroaryl-C1-3Heteroalkyl, -C1-3alkyl-aryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Alkyl, -C1-3alkyl-heteroaryl-C1-3Alkyl, -C1-3alkyl-aryl-C1-3Heteroalkyl, -C1-3Heteroalkyl-heteroaryl-C1-3Alkyl, -C1-3Heteroalkyl-aryl-C1-3Heteroalkyl, -C1-3alkyl-heteroaryl-C1-3Heteroalkyl group or-C1-3Heteroalkyl-heteroaryl-C1-3A heteroalkyl group.
It will be appreciated that in formula I above, the acridine nucleus is the immobilizer moiety, the-V-W-X-group contains a fragile linker, and the E group is the effector moiety. Similarly, in formula III above, the psoralen core is the immobilizer moiety, the-V-W-X-group comprises a fragile group, and the E group is the effector group. Formula II is a subset of formula I.
Exemplary compounds of the invention are of the formula, designated as IV:
in IV, the 2-methoxycarbonylacridine ring system acts as an immobilizer moiety by intercalation. The bis (chloroethyl) amine group serves as an effector moiety capable of alkoxylating nucleic acids; if the effector does not react with the nucleic acid, the nitrogen mustard is hydrolyzed. The linking group being-NH-CH2CH2-C(＝O)-O-CH2CH2-. In aqueous solutions at physiological pH, the ester-containing linker is hydrolyzed within a few hours. Changing the solution pH can change the hydrolysis rate of the linker; for the corresponding alcohol analogue of formula IV, in which the-Cl atom of formula IV is replaced by-OH, hydrolysis of the ester bond of ≦ 1% may be observed after 100 minutes at pH 3, 37 ℃; hydrolysis of the ester bond was observed to be greater than 50% after 100 minutes at 37 ℃ at pH 8. The resulting hydrolysate of formula IV is N- (2-methoxycarbonyl-9-acridinyl) - β -alanine and triethanolamine:
wherein the 2-methoxycarboximidamide has a beta-alanine as a linker fragment and the effector cleavage product has an ethanol group as a linker fragment.
At physiological pH, the carboxylate salt of β -alanine will be negatively charged, a feature that reduces the tendency of the attached 2-methoxycarbonylacridine group to insert into negatively charged nucleic acid molecules. Thereby reducing the mutagenicity of N- (2-methoxycarbonyl-9-acridinyl) -beta-alanine relative to 9-aminoacridine. This reduction in the efficiency of the mutagenicity of the immobilizer fragment reveals an advantage provided by the fragile linker.
Another advantage of a fragile linker group similar to that in the compound of formula IV is that its rate of hydrolysis can be adjusted by varying the length of the linker arm between the 9-aminoacridine anchor and the ester functionality. As described in example 7 and tables III and IV below, for diol analogs of certain compounds of the present invention in which the-Cl atom of the nitrogen mustard is replaced by an-OH group, an increase in the number of methylene groups between the aminoacridine anchor and the ester group can result in a decrease in the amount of hydrolysis in aqueous solution at pH 8, 37 ℃.
Some examples of the compounds of the invention are given below, which are intended to illustrate the invention, but not to limit it.
Applications of
Examples of uses of the compounds of the present invention include, but are not limited to: adding a compound of the invention in solid or solution form to the biological material, thereby inactivating pathogens present in the biological material; impregnating or otherwise treating a material for biological use in a solution of a compound of the invention to inactivate pathogens present in or on the surface of the material; and entrapping the compounds of the invention in targeted liposomes to target the compounds to specific cells, thereby destroying the nucleic acid in those cells.
One should note that: when a compound of the present invention is designed to hydrolyze under certain conditions, the compound is stable under other conditions. For the brittle linker and the effector group, it is desirable to be relatively stable under certain conditions for storage. Examples of ways in which the compounds may be stored include, but are not limited to: anhydrous solids, low moisture containing oils, frozen aqueous solutions, frozen non-aqueous solutions, suspensions, and solutions that do not allow hydrolysis of the fragile or effector groups (e.g., liquid non-aqueous solutions). The compounds may be stored at temperatures below 0 ℃ (e.g. in a freezer) or above 0 ℃ (e.g. in a refrigerator or at ambient temperature). Preferably, the compounds are stable under storage conditions for the following periods of time: three days to one year, one week to one year, one month to one year, three months to one year, six months to one year, one week to six months, one month to six months, three months to six months, one week to three months, or one month to three months. The stability of a compound depends on the temperature at which it is stored and the state (e.g., non-aqueous solution, anhydrous solid) in which it is stored.
Conditions for pathogen inactivation
The conditions for treating the biological material with the pathogen inactivating compound may be selected based on the material selected and the inactivating compound. Typical concentrations of pathogen-inactivating compounds for treating biological materials (e.g., blood products) are on the order of about 0.1 μ M to 5mM, such as about 500 μ M. For example, the pathogen-inactivating compound is used at a concentration of at least about 1log, or at least about 2logs, such as at least about 3 to 6logs, sufficient to inactivate a pathogen in the sample. In one embodiment, the pathogen inactivating compound produces at least 1log kill at a concentration of no greater than 500 μ M, and more preferably at least 3logs kill at a concentration of no greater than about 500 μ M. In another non-limiting example, the pathogen inactivating compound produces at least 1log kill, preferably at least 6logs kill at a concentration of about 0.1 μ M to about 3 mM.
Incubation of the blood product with the pathogen-inactivating compound may be performed for about 5 minutes to 72 hours or more, or about 1 to 48 hours, such as about 1 to 24 hours or about 8 to 20 hours. For red blood cells, incubation may typically be performed at about 2 ℃ to 37 ℃, preferably about 18 ℃ to 25 ℃. For platelets, the temperature is preferably about 20 to 24 ℃. For plasma, the temperature may be about 0 to 60 ℃, typically about 0-24 ℃. The pH of the material to be treated is preferably about 4 to 10 c, more preferably about 6 to 8 c.
One embodiment of the invention includes the compounds and methods of their use in inactivating pathogens in blood or blood products, and preferred storage conditions for this purpose should be those that facilitate storage and use of the compounds in blood banks.
Under conditions used to inactivate pathogens in or on the surface of a material, the fragile linker and the effector group will undergo hydrolysis or reaction. The rate of hydrolysis of the fragile linker and effector group should be slow enough to inactivate the desired number of pathogens. For example, the time required for inactivation of the pathogen should be about 5 minutes to 72 hours.
Processing red blood cells
Preferably, the treatment of the material containing red blood cells with the pathogen inactivating compound should not impair the function of the red blood cells or modify the treated red blood cells. The lack of a substantial damaging effect on red blood cell function can be determined by methods well known in the art of testing red blood cell function. For example, the level of an indicator, such as intracellular ATP (adenosine 5' -triphosphate), intracellular 2, 3-DPG (glycerol 2, 3-diphosphate), or extracellular potassium, can be determined and compared to an untreated control. In addition, hemolysis, pH, hematocrit, hemoglobin, osmotic fragility, glucose consumption, and lactate production can be measured.
Methods for measuring ATP, 2, 3-DPG, glucose, hemoglobin, hemolysis, and potassium are known in the art. See, e.g., Davey et al, Transfusion,32: 52-528(1992), the disclosure of which is incorporated herein by reference. Measurement of red blood cell functionMethods of performance are also described in the following documents: greenwalt et al, Vox Sang,58: 94-99 (1990); hogman et al, Vox Sangg, 65: 271-278(1993) and Beutler et al, Blood (Blood), vol.59(1982), the disclosures of which are incorporated herein by reference. Extracellular potassium levels can be measured using a Ciba Corning model 614K +/Na + analyzer (Ciba Corning Diagnostics Corp., Medford, Mass.). The pH value can be determined using a Ciba Corning model 238 blood gas analyzer (Ciba Corning Diagnostics Corp., Medford, Mass.).
The binding of the following substances, such as IgG, albumin and IgM, to red blood cells can also be determined using methods known in the art. Binding of molecules to red blood cells can be measured using antibodies such as acridine and IgG. Antibodies for the assay are commercially available or can be prepared using methods well known in the art, which are described in the following references: harlow and Lane, "Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory", 1998, the disclosure of which is incorporated herein by reference. For example, anti-IgG (anti-IgG) is commercially available from: caltag, Burlingame, CA; sigma Chemical co., st.louis, MO and Lampire Biological Laboratory, Pipersvelle, PA.
In the method of treating a material containing red blood cells with a pathogen inactivating compound, the extracellular potassium level is preferably no more than 3-fold, more preferably no more than 2-fold, of the amount exhibited by the untreated control after one day. In another embodiment, the hemolyzed treated red blood cells after 28 days of storage preferably have a hemolysis of less than 3%, more preferably less than 2% after 42 days, and most preferably less than or equal to about 1% after 42 days of storage at 4 ℃.
Biological material
Large amounts of biological material can be treated with pathogen inactivating compounds. Biological materials include blood products, such as whole blood; compacting the red blood cells; platelets and fresh or frozen plasma. The blood product further comprises plasma protein fractions, antihemophilic factor (factor VIII), factor IX and factor IX complexes, fibrinogen, factor XIII, prothrombin and thrombin, immunoglobulins (e.g., IgG, IgA, IgD, IgE and IgM and fragments thereof), albumin, interferons and lymphokines. Also included are synthetic blood products.
Other biological materials include vaccines, recombinant DNA-generated proteins, and oligopeptide ligands. Clinical samples such as urine, sweat, saliva, stool, spinal fluid are also included. Further comprising a synthetic blood or blood product storage medium.
Reducing the concentration of compounds in the material after treatment
After treatment, the concentration of pathogen inactivating compounds in the biological material (e.g., blood product) can be reduced. Methods and devices that can be used are described in the following documents: PCT US 96/09846; U.S. serial No. 08/779,830, filed on 6/1/1997; and a method and apparatus for reducing Small Organic Compounds from Blood Products, commonly referred to as Methods and Devices for Reduction of Small Organic Compounds from Blood Products, PCT/US98/00531, attorney docket number 2000440, filed 1/6/1998, the disclosures of which are incorporated herein by reference in their entirety.
Quenching
In another embodiment, the compounds of the present invention may be used in combination with a quencher. Methods for Quenching undesirable side reactions of Pathogen inactivating compounds in Biological Materials are described in the U.S. copending application serial No. 60/070597, application date 6/1/1998, attorney docket No. 282173000600, "Methods for Quenching Pathogen Inactivators in Biological Materials," the disclosure of which is incorporated herein by reference. This co-application discloses a method of quenching undesirable side reactions of a pathogen-inactivating compound comprising a functional group that forms or is capable of forming an electrophilic group. In this embodiment, the material is treated with a pathogen inactivating compound and a quencher, wherein the quencher comprises a nucleophilic functional group capable of covalently reacting with the electrophilic group. Preferred quenchers are thiols, such as glutathione.
Examples
The following specific examples, which illustrate the preparation of representative compounds for use in the methods of the invention, provide data on the compounds useful to the practitioner; and to illustrate the manner in which the effectiveness of the compounds is determined. These examples are not intended to limit the scope of the present invention. All NMR spectra were in CDCl, unless otherwise indicated3Recorded on a Varian 200MHz instrument; chemical shifts recorded are relative to Tetramethylsilane (TMS); IR spectra were recorded using PerkinElmer FTIR; HPLC determination was carried out using a gradient YMC C8 column, mobile phase A5 mM H3PO4Aqueous solution, mobile phase B5 mM CH3CN; samples were prepared in DMSO or ethanol and maintained at ≦ 15 ℃ prior to injection.
Table II indicates the number of compounds used for each compound.
TABLE II
Example 1
Synthesis of beta-alanine, N- (2-Methoxycarbonylimidin-9-yl), 2- [ bis (2-chloroethyl) amino ] ethyl ester dihydrochloride (Compound IV)
Step A beta-alanine, N- (tert-butoxycarbonyl), 2- [ bis (2-hydroxyethyl) amino ] ethyl ester
In N2To a stirred solution of N- (tert-butoxycarbonyl) -beta-alanine (20.3g, 107mmol) and 4-methylmorpholine (13.0ml, 12.0g, 119mmol) in dry THF (200ml) at-15 deg.C under an atmosphere was added isobutyl chloroformate (13.9ml, 14.6g,107mmol) to yield a white precipitate (4-methylmorpholine HCl) formed in the middle. The reaction mixture was stirred at-15 ℃ for 5 minutes, then transferred to a flask containing a stirred solution of triethanolamine (48.3g, 324mmol) in dry THF (150ml) at-15 ℃. The reaction mixture was warmed to 23 ℃ and stirred for a further 1.5 hours, followed by isolation of the precipitate by vacuum filtration. THF was isolated from the filtrate in vacuo and the resulting viscous yellow oil was partitioned between water (500ml) and EtOAc (5X 150 ml). In Na2SO4The combined organic layers were dried. The solvent was separated in vacuo to give 25.8g (75%) of the desired product beta-alanine, N- (tert-butoxycarbonyl), 2- [ bis (2-hydroxyethyl) amino%]Ethyl ester as light yellow oil.1H NMR: δ 5.32(br s, 1H), 4.18(t, J ═ 5.4Hz, 2H), 3.58(t, J ═ 5.1Hz, 4H), 3.37-3.23(m, 2H), 2.80(t, J ═ 5.4Hz, 2H), 2.69(t, J ═ 5.1Hz, 4H), 2.51(t, J ═ 6.0Hz, 2H), 1.41(s, 9H) no hydroxyl protons were observed.13C NMR：δ173.0，156.4，79.8，63.3，60.2，57.3，54.1，36.7，35.3，28.8.
Step B beta-alanine, N- (tert-butoxycarbonyl), 2- [ bis (2-tert-butyldimethylsilyloxyethyl) amino ] ethyl ester
In N2Then, the beta-alanine, N- (tert-butoxycarbonyl), 2- [ bis (2-hydroxyethyl) amino group obtained in step A was stirred]A solution of ethyl ester (22.7g, 70.9mmol) and imidazole (11.1g, 163mmol) in acetonitrile (70ml) was cooled to 0 ℃. Tert-butyldimethylsilyl chloride (534mg, 3.54mmol) was then added and the reaction mixture was stirred at 0 ℃ for a further 5 minutes. The reaction mixture was warmed to 23 ℃ and stirred for 2 hours, followed by isolation of the resulting white precipitate (imidazole. HCl) by vacuum filtration. The acetonitrile in the filtrate was removed in vacuo and the remaining material partitioned between saturated brine (600ml) and EtOAc (3X 200 ml). In Na2SO4The combined organic layers were dried. The solvent is separated off in vacuo to yield 35.2g (90%) of the desired product beta-alanine, N- (tert-butoxycarbonyl), 2- [ bis (2-tert-butyldimethylsilyloxyethyl) amino]Ethyl ester as yellow oil.1H NMR：δ5.29(br s，1H)，4.14(t，J＝6.0Hz，2H) 3.65(t, J ═ 6.3Hz, 4H), 3.37 (apparent q, 2H), 2.85(t, J ═ 6.0Hz, 2H), 2.71(t, J ═ 6.3Hz, 4H), 2.49(t, J ═ 5.9Hz, 2H), 1.42(s, 9H), 0.88(s, 18H), 0.03(s, 12H);13C NMR：δ172.7，156.3，79.7，63.3，62.4，57.7，54.3，36.7，35.3，28.9，26.4，18.7，-4.9.
step C beta-alanine, 2- [ bis (2-tert-butyldimethylsilyloxyethyl) amino ] ethyl ester
To the mixture containing the beta-alanine, N- (tert-butoxycarbonyl), 2- [ bis (2-tert-butyldimethylsilyloxyethyl) amino group obtained in the step B]A flask of ethyl ester (3.01g, 5.48mmol) was charged with pure trifluoroacetic acid (5ml), and CO evolution was effected2A gas. The reaction mixture was stirred for 5 minutes and trifluoroacetic acid was isolated in vacuo. The remaining material was partitioned with saturated NaHCO3(100ml) and EtOAc (3X 30 ml). In Na2SO4The combined organic layers were dried. The solvent is separated off in vacuo to give 2.45g (100%) of the desired product beta-alanine, 2- [ bis (2-tert-butyldimethylsilyloxyethyl) amino]Ethyl ester as light yellow oil.1H NMR: δ 4.12(t, J ═ 6.0Hz, 2H), 3.63(t, J ═ 6.4Hz, 4H), 2.96(t, J ═ 6.2Hz, 2H), 2.84(t, J ═ 6.0Hz, 2H), 2.69(t, J ═ 6.4Hz, 4H), 2.44(t, J ═ 6.2Hz, 2H), 0.86(s, 18H), 0.03(s, 12H), no amine protons were observed,13C NMR(CDCl3)：δ173.0，63.4，62.6，57.9，54.4，38.4，38.1，26.4，18.7，-4.9.
step D beta-alanine, N- (2-Methoxycarbonylimidin-9-yl), 2- [ bis (2-hydroxyethyl) amino ] ethyl ester
At room temperature, by passing through 10ml of CHCl3Stirring for 12.5 h, adding beta-alanine, 2- [ bis (2-tert-butyldimethylsilyloxyethyl) amino]Ethyl ester (736mg, 1.64mmol) was reacted with methyl 9-methoxyacridine-2-carboxylate (669mg, 2.50 mmol). The precipitate (acridone) was filtered and the filtrate was partitioned with saturated NaHCO3(100ml) and CHCl3(3X 35 ml). In Na2SO4The combined organic layers were dried and concentrated in vacuo to give 1.61gA viscous brown oil. Deprotection of the resulting diol can be carried out by the following method: in N2The crude intermediate was dissolved in 3.0ml THF under an atmosphere and treated with HF/pyridine (1.0ml) while cooling to 0 deg.C. The solution was warmed to room temperature with stirring for 1 hour. Volatiles were removed in vacuo and the residue was partitioned with saturated NaHCO3Aqueous solution (100ml) and CHCl3(3X 35 ml). The combined organic layers were dried and concentrated to give 649mg of a tan solid. Preparative TLC (C-18, CH)3CN) to give the desired diol, beta-alanine, N- (2-methoxycarbonylacridin-9-yl), 2- [ bis (2-hydroxyethyl) amino]Ethyl ester (> 80% purity by HPLC);1h NMR: Δ 8.82(s, 1H), 8.21-7.94(m, 2H), 7.94-7.72(m, 2H), 7.59 (apparent t, 1H), 7.23 (apparent t, 1H), 4.30-4.18(m, 2H), 4.18-4.05(m, 2H), 3.89(s, 3H), 3.69-3.50(m, 4H), 2.92-2.73(m, 4H), 2.73-2.55(m, 4H)
No amine and hydroxyl protons were observed.
Step E beta-alanine, N- (2-Methoxycarbonylimidin-9-yl), 2- [ bis (2-chloroethyl) amino ] ethyl ester dihydrochloride
Beta-alanine, N- (2-methoxyformylacridin-9-yl), 2- [ bis (2-hydroxyethyl) amino ] was synthesized by a method similar to that described by Peck, et al (J.Am.chem.Soc.) -1959, 81: 3984)]The ethyl ester is converted to the dichloro compound. The product of step D (41mg, 0.090mmol) was stirred at room temperature in pure SOCl2(6ml) yellow solution for 20 h. Vacuum removal of SOCl2To give a yellow solid (dihydrochloride). Partition material into saturated NaHCO3(50ml) and CH2Cl2(3X 20 ml). In Na2SO4The combined organic layers were dried. The solvent was removed in vacuo to give 35.4mg of the dichloro compound as a yellow-orange gum.
1H NMR: δ 8.82(s, 1H), 8.20-7.83(m, 4H), 7.5 (apparent t, 1H), 7.25 (apparent t, 1H), 4.36-4.15(m, 4H), 3.93(s, 3H), 3.48(t, J ═ 6.9Hz, 4H)No amine protons were observed 3.06-2.77(m, 4H), 2.86(t, J ═ 6.9Hz, 4H).13C NMR：δ172.3，166.6，155.2，146.5，144.6，133.1，131.6，128.7，124.6，124.3，116.1，114.3，63.7，57.2，53.5，52.9，46.3，42.5，35.2.
No other carbons were observed. By dropwise addition of 1M HCl in ether from CH2Cl2Precipitating HCl salt to obtain beta-alanine, N- (2-methoxy formyl acridin-9-yl) and 2- [ bis (2-chloroethyl) amino]Ethyl ester dihydrochloride (compound IV) as a yellow solid (81% purity by HPLC).
According to a similar method, beta-alanine, N- (acridin-9-yl), 2- [ bis (2-chloroethyl) amino ] ethyl ester dihydrochloride (compound V) can be prepared. Thus, in step D, 9-methoxyacridine was used instead of methyl 9-methoxyacridine-2-carboxylate to give the intermediate diol (7.1%) as a yellow oil (74% purity by HPLC).
1H NMR: δ 8.14(d, J ═ 7.5Hz, 2H), 7.93(d, J ═ 8.6Hz, 2H), 7.52 (apparent t, 2H), 7.23 (apparent t, 2H), 4.36-4.08(m, 4H), 3.76-3.5(m, 4H), 3.08-2.60(m, 8H)
No amine and hydroxyl protons were observed.
A solution of intermediate diol (37.3mg, 0.0793mmol) in thionyl chloride (4.0ml) was stirred at 23 ℃ for 7.5 h. Thionyl chloride was removed in vacuo to give a yellow oil. This material was dissolved in ethanol (. about.4 ml) and the solvent was removed in vacuo. This material was then dissolved in CH2Cl2(4ml), the solvent was removed in vacuo; this step was repeated twice. This material was then triturated with hexane (3X 4ml) to give 40.0mg (42% purity by HGPLC) of product as a yellow hygroscopic glassy solid. For analysis, some substances can be converted to free amines by the following method: it was partitioned with saturated NaHCO3And CH2Cl2In Na2SO4The combined organic layers were dried and the solvent was removed in vacuo.
1H NMR: δ 8.21-8.00(m, 4H), 7.66 (apparent t, 2H), 7.38 (apparent t, 2H), 4.26-4.12(m, 2H), 4.12-3.98(m, 2H), 3.43(t, J ═ 6.9Hz, 4H), 2.96-2.68(m, 8H)
No amine protons were observed.
4-Aminobutanoic acid N- (2-methoxycarbonylacridin-9-yl), 2- [ bis (2-chloroethyl) amino ] can be prepared according to the above procedure except that N- (tert-butoxycarbonyl) -4-aminobutyric acid is used instead of N- (tert-butoxycarbonyl) -beta-alanine]Ethyl ester dihydrochloride, compound VI (78% purity by HPLC).1H NMR: δ 8.89(s, 1), 8.12 (apparent t, 2), 7.93-7.80(m, 2), 7.59 (apparent q, 1), 7.36-7.20(m, 1), 4.16(t, 2, J ═ 5.7Hz), 4.07-3.92(m, 2), 3.97(s, 3), 3.46(t, 4, J ═ 6.9Hz), 2.93-2.80(m, 6), 2.60(t, 2, J ═ 6.5Hz), 2.29-2.12(m, 2).
No amine protons were observed.
Example 2
Beta-alanine, N- (2-methoxycarbonylacridin-9-yl), 3- [ bis (2-chloroethyl) amino ] propyl ester dihydrochloride, compound VIII (63% purity by HPLC) was obtained using 3- [ N, N-bis (2-tert-butyldimethylsilyloxyethyl) ] aminopropanol instead of triethanolamine in step A of example 1 and then proceeding to step C.
1H NMR: δ 8.91(s, 1), 8.20-7.93(m, 4), 7.18 (apparent t, 1), 7.39 (apparent t, 1), 4.30(m, 4), 3.96(s, 3), 3.48(t, 4, J ═ 6.9Hz), 2.88-2.60(m, 2), 2.83(t, 4, J ═ 6.9Hz), 2.62(t, 2, J ═ 6.7Hz), 1.85-1.68(m, 2)
No amine protons were observed.
Example 3
The compound synthesized in example 1 can also be prepared by the following method:
synthesis of beta-alanine, N- (acridin-9-yl), 2- [ bis (2-chloroethyl) amino ] ethyl ester dihydrochloride (Compound V): method II
Step A β -alanine, N- (acridin-9-yl), methyl ester hydrochloride
Combine 9-chloroacridine (11.7g, Organic Synthesis, col. Vol III, pg57), beta-alanine methyl ester hydrochloride (9.9g) and sodium methoxide (3.26g) and add 60ml of methanol. The mixture was stirred with a magnetic stirrer and refluxed for 5.5 hours. The heating device is removed and the suspension is filtered while hot (35 ℃ C. or less). The solid salt was rinsed by adding about 10ml of methanol and the combined dark green filtrate was concentrated to give 21g of an aqueous green-yellow solid.
The solid was dissolved in 350ml of boiling 2-propanol and crystallized at room temperature. The resulting crystals were rinsed with about 15ml of 2-propanol and 15ml of hexane and then air dried to give 15.5g of bright yellow product, β -alanine, N- (acridin-9-yl), methyl ester hydrochloride (yield 78.5%).
1H NMR：δ1.9(brs，2H)；3.24(t，J＝7.0Hz，2H)；3.76(s，3H)；4.45(br s，2H)；7.23(app.t，J＝8Hz，2H)；7.49(app.t，J＝8Hz，2H)；8.11(d，J＝8.4Hz，2H)；8.30(d，J＝8.4Hz，2H)；9.68(br s，0.5H).IR：1574(s)，1691(s)，1726(s)，2336(m)，2361(m)，3227(m).
Step B beta-alanine, N- (acridin-9-yl), 2- [ bis (2-hydroxyethyl) amino ] ethyl ester dihydrochloride
The β -alanine, N- (acridin-9-yl), methyl ester hydrochloride from step A (5.00g) was partitioned between toluene (750ml), saturated Na2CO3(200ml) and water (50 ml). The aqueous layer was re-extracted with toluene (3X 250ml), the organic layers were combined and taken up with saturated Na2CO3Aqueous solution (50ml) was washed. The toluene volume was reduced to about 100ml by rotary evaporation. Then theTriethanolamine (30ml) was added to form part of the immiscible system. A solution of NaOMe (50mg) in MeOH (2ml) was then added. The solvent was rapidly removed from the reaction mixture at room temperature by rotary evaporation with stirring. After removal of the solvent, the reaction mixture was left under vacuum for a further 1-1.5 hours to give a syrupy solution.
To separate the excess triethanolamine, the crude mixture was partitioned between CH2Cl2(200ml) and saline (200 ml). By CH2Cl2The brine layer was extracted (5X 100ml) and the organic layers were combined, washed with brine (50ml) and then extracted with 0.5M HCl (2X 100 ml). Combining the aqueous acid layers with CH2Cl2(50ml) washing. In CH2Cl2(200ml) in the presence of powdery K2CO3(s)The acid solution is rendered alkaline. Separating the organic layer and reusing CH2Cl2The aqueous layer was extracted (5X 100 ml). The combined organic phases were washed with brine (50ml) and anhydrous Na2SO4(s)Drying and stripping gave the crude diol free amine (5.02g) as a viscous yellow gum. The material was identical to that prepared by another method in example 1 as determined by NMR.
A portion of the above crude material (0.400g) was stirred vigorously with isopropanol (100ml) and acidified with 1M HCl in ether. The gum was quenched to remove the first precipitate. After removal of half of the solvent, the second crystal yielded beta-alanine, N- (acridin-9-yl), 2- [ bis (2-hydroxyethyl) amino]Ethyl ester dihydrochloride as a bright yellow crystalline solid (0.200g, > 95% purity by HPLC).1H NMR: δ 8.11 (apparent t, 4H), 7.69 (apparent t, 2H), 7.41 (apparent t, 2H), 4.23(t, J ═ 5.4Hz, 2H), 4.03(t, J ═ 5.9Hz, 2H), 3.58(t, J ═ 5.2Hz, 4H), 2.73(t, J ═ 5.4Hz, 2H), 2.70(t, J ═ 5.9Hz, 2H)2.68(t, J ═ 5.2Hz, 4H), no amine and hydroxyl protons were observed
13C NMR：δ173.3，151.7，149.4，130.5，129.5，124.0，123.4，118.4，63.5，60.1，57.3，54.0，46.6，35.8.
Step C beta-alanine, N- (acridin-9-yl), 2- [ bis (2-chloroethyl) amino ] ethyl ester dihydrochloride
Adding SOCl2(0.5ml) was added to the stirred beta-alanine, N- (acridin-9-yl), 2- [ bis (2-hydroxyethyl) amino group from step B]Ethyl ester dihydrochloride (113mg, 0.24mmol) in CH3CN (0.5ml) suspension. The resulting yellow solution was stirred at 23 ℃ for 16 h, followed by removal of volatiles in vacuo. The remaining orange oil was dissolved in EtOH (. about.2 ml) and the EtOH was removed in vacuo to give a yellow solid. The material was triturated with hexane (2X 3ml) and the residual solvent removed in vacuo to give 123mg of the desired material β -alanine, N- (acridin-9-yl), 2- [ bis (2-chloroethyl) amino]Ethyl ester dihydrochloride (93% purity by HPLC) as a yellow solid.1H NMR: δ 8.09 (apparent t, J ═ 8.8Hz, 4H), 7.66 (apparent t, J ═ 7.6Hz, 2H), 7.38 (apparent t, J ═ 7.7Hz, 2H), 4.14(t, J ═ 5.9Hz, 2H), 4.00(t, J ═ 5.8Hz, 2H), 3.43(t, J ═ 6.9Hz, 4H), 2.87(t, J ═ 6.9Hz, 4H), 2.77(t, J ═ 5.9Hz, 2H), 2.69(t, J ═ 5.8Hz, 2H), no protons were observed.
13C NMR: δ 173.0, 151.5, 149.4, 130.5, 129.6, 124.1, 123.4, 118.6, 63.5, 57.3, 53.5, 46.7, 42.5, 35.7.IR (KBr pellets of HCl salt): 3423, 3236, 2939, 2879, 1736, 1634, 1586, 1572, 1540, 1473, 1272, 1173cm-1.
Example 4
Beta-alanine, N- (4-methoxyacridin-9-yl), 2- [ bis (2-chloroethyl) amino ] ethyl ester dihydrochloride, compound IX
Preparation of beta-alanine, N- (4-methoxyacridin-9-yl), methyl ester by the following method: 1.4g (5.84mmol) of 4, 9-dimethoxyacridine, 0.89g (6.42mmol) of beta-alanine methyl ester hydrochloride and 20ml of methanol are mixed and the mixture is stirred under N2Heated to reflux under atmosphere for 12 hours. The reaction was then concentrated in vacuo and dissolved in CHCl3-isopropyl alcohol(50ml, 4: 1v/v), using 50% NH4OH (2X 25ml) and brine (1X 25 ml). By using Na2SO4The organic layer was dried and concentrated in vacuo to give 1.24g (68%) of methyl ester (> 74% purity by HPLC) as a yellow oil; rf(SiO2Ethyl acetate) ═ 0.25; IR (film):
3363，2947，1730，1611，1573，1518，1484，1463，1423，1420，1246，1170，1081cm-1；1H NMR：δ2.70(t，2H，J＝5.7Hz)，3.74(s，3H)，4.00(t，2H，J＝6.3Hz)，4.11(s，3H)，6.98(d，1H，J＝7.4Hz)，7.36(m，2H)，7.65(m，2H)，8.12(d，2H，J＝8.5Hz)；13CNMR)：δ35.7，46.9，52.3，56.5，107.2，115.3，119.8，123.5，124.1，130.0，151.4，173.6.
this was converted to the diol under the conditions described in example 3, step B, to give 647mg of a yellow oil. HPLC analysis of the crude mixture indicated a yield of 85% (λ 278 nm); rf(SiO220% methanol-ethyl acetate) ═ 0.17; IR (film): 3337, 2947, 2828, 1726, 1616, 1569, 1522, 1484, 1463, 1420, 1348, 1250, 1174, 1127, 1081, 1043cm-1；1H NMR：δ2.7(m，8H)，3.55(m，4H)，3.97-4.08(m，2H)，4.08(s，3H)，4.19(t，2H，J＝5.5Hz)，6.96(d，1H，J＝7.4Hz)，7.29(m，2H)，7.61(m，2H)，8.10(m，2H)；13CNMR：δ36.0，46.9，53.7，56.4，57.1，60.1，63.3，107.4，115.7，119.1，119.6，123.2，123.5，123.9，128.5，130.0，140.8，147.4，151.6，151.7，154.3，173.3.
As described in example 3, step C, thionyl chloride was converted to beta-alanine, N- (4-methoxyacridin-9-yl), 2- [ bis (2-chloroethyl) amino]Ethyl ester dihydrochloride. Rapid filtration with ethyl acetate (SiO)2) The crude product was then filtered through 10% methanol-ethyl acetate; degradation of some product in the column gave 58mg of a yellow oil; rf(SiO2Ethyl acetate) ═ 0.26; IR (film):
3405，2955，2828，1726，1616，1577，1518，1463，1416，1348，1246，1174，1123，1081，1013cm-1；1H NMR：δ2：69-2.99(m，8H)，3.45(t，4H，J＝6.7Hz)，4.03(m，2H)，4.09(s，3H)，4.16(t，2H，J＝5.9Hz)，6.97(d，1H，J＝7.7Hz)，7.32(m，2H)，7.65(m，2H)，8.12(d，2H，J＝8.7Hz).
the crude dihydrochloride salt can be isolated in crude form by azeotropic separation of the excess thionyl chloride (2X 5ml toluene) and concentration of the reaction in vacuo. HPLC showed complete exhaustion of the starting material, 4-methoxyazapyridinone (R)T22.3 minutes) as the major impurity.1H NMR(CD3OD)：δ3.18(t，2H，J＝6.4Hz)，3.71(m，6H)，4.04(m，4H)，4.18(s，3H)，4.51(m，2H)，7.17(m，2H)，7.56(m，2H)，7.91-8.15(m，2H)，8.55(d，1H，J＝8.8Hz).
Preparation of beta-alanine, N- (3-chloro-4-methylacridin-9-yl), 2- [ bis (2-chloroethyl) amino ] from 3-chloro-9-methoxy-4-methylacridine according to an analogous procedure]Ethyl ester dihydrochloride, compound X. Of the free base1H NMR is: δ 7.96-8.17(m, 3H), 7.29-7.52(m, 3H), 4.19(t, J ═ 5.8Hz, 2H), 4.00(s, 3H), 3.89(t, J ═ 5.1Hz, 2H), 3.47(t, J ═ 6.8Hz, 4H), 2.91(t, J ═ 6.8Hz, 4H), 2.83(t, J ═ 5.8Hz, 2H), 2.67(t, J ═ 5.5Hz, 2H).
Preparation of beta-alanine, N- (6-chloro-2-methoxyacridin-9-yl), 2- [ bis (2-chloroethyl) amino ] beta-alanine from 6-chloro-2, 9-dimethoxyacridine according to an analogous procedure]Ethyl ester dihydrochloride, compound XIII. Of the free base1H NMR is: δ 7.96-8.17(m, 3H), 7.29-7.52(m, 3H), 4.19(t, J ═ 5.8Hz, 2H), 4.00(s, 3H), 3.89(t, J ═ 5.1Hz, 2H), 3.47(t, J ═ 6.8Hz, 4H), 2.91(t, J ═ 6.8Hz, 4H), 2.83(t, J ═ 5.8Hz, 2H), 2.67(t, J ═ 5.5Hz, 2H).
Example 5
Beta-alanine, [ N, N-bis (2-chloroethyl) ], 3- [ (6-chloro-2-methoxyacridin-9-yl) amino ] propyl ester dihydrochloride, compound XI
Step A beta-alanine, [ N, N-bis (2-triisopropylsilanyloxy) ethyl ] ethyl ester
Beta-alanine ethyl ester hydrochloride (1.99g, 12.9mmol), K2CO3(6.0g, 43.4mmol) and iodoethyl triisopropylsilyl ether (9.47g, 28.9mmol) were refluxed for 5-7 days in a slurry of acetonitrile (175 ml). After evaporation of the solvent in vacuo, CH is used2Cl2The solid was ground. Using dilute Na2CO3The organic layer was washed with aqueous solution, then brine, over anhydrous Na2SO4And drying. The crude product was purified by chromatography on silica gel (1: 4 EtOAc/hexane) to give 5.60g of beta-alanine, [ N, N-bis (2-triisopropylsiloxy) ethyl ] oil]Ethyl ester (83.1%).1H NMR: δ 4.12(q, J ═ 7.1Hz, 2H), 3.73(t, J ═ 6.8Hz, 4H), 2.92(t, J ═ 7.3Hz, 2H), 2.70(t, J ═ 6.6Hz, 4H), 2.46(t, J ═ 7.4Hz, 2H), 1.4-0.9(m, 45H, including trimodal 1.25(3H) and unimodal 1.06 and 1.05).
Step B beta-alanine N, N-bis (2-triisopropylsiloxy) ethyl ester
The beta-alanine, [ N, N-bis (2-triisopropylsiloxy) ethyl group obtained in the step A is added]Ethyl ester (5.60g, 10.8mmol) and lithium hydroxide (0.59g, 14.1mmol) were stirred in ethanol and refluxed for 3 hours. Separating the solvent and partitioning the crude product into CH2Cl2And dilute NaHCO3Between aqueous solutions. The organic layer was washed with brine and dried over anhydrous Na2SO4The residue was dried and stripped to give beta-alanine N, N-bis (2-triisopropylsiloxy) ethyl ester as a pale yellow oil (5.03g, yield 95.1%).1H NMR：δ3.90(t，J＝5.5Hz，4H)，3.04(t，J＝6.2Hz，2H)，2.92(t，J＝5.5Hz，4H)，2.50(t，J ＝6.1Hz，2H)，1.06(s，42H).
Step C beta-alanine, [ N, N-bis (2-hydroxyethyl) ], 3- [ (6-chloro-2-methoxyacridin-9-yl) amino ] propyl ester
In N2In CH under atmosphere2Cl2To (1ml) was stirred β -alanine N, N-bis (2-triisopropylsilanyloxy) ethyl ester (51.0mg, 0.104mmol) obtained in the above-mentioned step B. While quenching in an ice bath, SOCl was added dropwise2(0.5ml), the reaction was stirred for 2.25 hours. Stripping the reaction mixture to remove excess SOCl2Then, anhydrous CH is added2Cl2(0.5ml) in N2The solution was quenched on an ice bath under an atmosphere. Quenched 9- (3-hydroxy) propylamino-6-chloro-2-methoxyacridine (29.0mg, 91.5mmol) in CH was added2Cl2(1ml) slurry. After 0.5 hour, the mixture was partitioned in CH2Cl2And NaHCO3Between aqueous solutions. The organic layer was washed with brine and dried over anhydrous Na2SO4Dried and stripped. The resulting gum was triturated with hexane and the hexane extract stripped to give a very crude mixture of triisopropylsilyl protected starting material and product (53.5 mg).
To remove the triisopropylsilyl group, a portion of the crude protected diol (33.1mg) was stirred in ice-cooled THF (1 ml). After addition of HF/pyridine (0.5ml), the flask was filled with N2The mixture was stirred at ambient temperature under air for 2.5 hours. The reaction mixture was partitioned between CH2Cl2And NaHCO3Between the aqueous solutions, dilute NaHCO is used3The organic layer was washed several times with aqueous solution to remove excess HF/pyridine. After primary drying with brine, anhydrous Na is reused2SO4Drying and solvent stripping gave the crude diol (13.1 mg).
It was combined with additional crude diol (5.0mg) as 95 CH2Cl2The/5 iPA/1 TFA was purified by C-18 preparative TLC as eluent to give the diol TFA salt. In the presence of the salt in CH2Cl2And NaHCO3After the aqueous solution was separated, the organic layer was dried with brine and then anhydrous Na was used2SO4Drying and stripping to obtain the diol free base beta-alanine, [ N, N-bis (2-hydroxyethyl)]3- [ (6-chloro-2-methoxyacridin-9-yl) amino]Propyl ester (5.0 mg).
1H NMR: δ 7.92-8.25(m, 3H), 7.23-7.47(m, 3H), 4.30(t, J ═ 5.7Hz, 2H)), 3.98(s, 3H), 3.81(t, J ═ 6.2Hz, 2H), 3.64(t, J ═ 4.9Hz, 4H), 2.86(t, J ═ 6.1Hz, 2H), 2.67(t, J ═ 4.9Hz, 4H), 2.51(t, J ═ 5.9Hz, 2H), 2.04 (apparent quintuple, 2H).
Step D beta-alanine, [ N, N-bis (2-chloroethyl) ], 3- [ (6-chloro-2-methoxyacridin-9-yl) amino ] propyl ester dihydrochloride, Compound XI
The beta-alanine, [ N, N-bis (2-hydroxyethyl group) obtained above was used]3- [ (6-chloro-2-methoxyacridin-9-yl) amino]Propyl ester (4.0mg, 0.0073mmol) was dissolved in CH2Cl2(1ml) and quenched on an ice/water bath. Adding ice-cold SOCl2(0.1ml), the reaction was stirred at room temperature for 4 hours. Stripping the reaction mixture, removing the solvent, triturating with hexane, partitioning it into CH2Cl2And NaHCO3Between aqueous solutions. The organic layer was dried with brine and then anhydrous Na2SO4Drying and stripping gave the dichloro-compound as a yellow gum.1H NMR: δ 7.8-8.2(m, 3H), 7.2-7.5(m, 3H), 4.35(t, J ═ 5.9Hz, 2H), 3.85-4.10(3.99, s, OMe and 3.9-4.0, m, NHCH 2 Total of 5H), 3.48(t, J ═ 6.9Hz, 4H), 2.9-3.0(m, 6H), 2.49(t, J ═ 6.6Hz, 2H), 2.1-2.3(m, 2H).
In quenched CH2Cl2Stirring free amine, acidifying with 1M HCl ether solution, dripping several drops of methanol, and stripping to obtain beta-alanine, [ N, N-bis (2-chloroethyl)]3- [ (6-chloro-2-methoxyacridin-9-yl) amino]Propyl ester dihydrochloride (2.5mg), (3.5mg, 81%) as a yellow solid.
Similar diols can be prepared using the same procedure given in step C above, except that 6-chloro-9- (2-hydroxy) ethylamino-2-methoxy-acridine is used instead of 6-chloro-9- (3-hydroxy) propylamino-2-methoxy-acridine.
1H NMR：δ7.96-8.13(m，3H)，7.20-7.47(m，3H)，4.76(t，J＝4.9Hz，2H)，3.99(s，3H)，3.92-4.14(m，2H)，3.60(t，J＝5.1Hz，4H)，2.78(t，J＝6.1H2，2H)，2.63(t，J＝5.1Hz，4H)，2.45(t，J＝6.0Hz，2H).
It is converted into beta-alanine, [ N, N-bis (2-chloroethyl) by a method similar to that of step D]2- [ (6-chloro-2-methoxyacridin-9-yl) amino]Ethyl ester dihydrochloride, compound XII.1H NMR：δ7.94-8.20(m)，7.20-7.50(m)，4.42(CH 2OC＝O)，3.90-4.10(OCH 3，NHCH 2)，3.46(CH 2Cl)，2.82(N(CH 2)3)，2.39-2.56(CH 2C＝O).
Example 6
[ N, N-bis (2-chloroethyl) ] -2-aminoethyl 4, 5 ', 8-trimethyl-4' -psoralen acetate hydrochloride, Compound XIV
Step A [ N, N-bis (2-hydroxyethyl) ] -2-aminoethyl 4, 5 ', 8-trimethyl-4' -psoralen acetate
A slurry of methyl 4, 5 ', 8-trimethyl-4' -psoralen acetate (250mg, 0.832mmol), triethanolamine (12ml) and 1M HCl in diethyl ether (2ml) was stirred at 100 ℃ for 2 hours. The resulting light brown solution was cooled to room temperature and partitioned in CH2Cl2And saturated NaHCO3Between aqueous solutions. With saturated NaHCO3Rinsing the organic layer several times with aqueous solution using anhydrous Na2SO4After drying, the solvent was removed in vacuo and the residue was partitioned between CH2Cl2And 1M aqueous HCl. By means of CH2Cl2Rinsing the aqueous layer several times and then using K in the presence of an organic solvent2CO3(s) rendering it basic. The organic layer containing the neutral product was rinsed with water, then dried and concentrated. Repeating the acid-base extraction step to obtainTo the desired product as a beige solid (84.3mg, 24.3%):1H NMR：δ7.53(s，1H)，6.24(s，1H)，4.23(t，J＝5.4Hz，2H)，3.69(s，2H)，3.56(t，J＝5.3Hz，4H)，2.82(t，J＝5.4Hz，2H)，2.69(t，J＝5.3Hz，4H)，2.57(s，3H)，2.51(d，J＝1.1Hz，3H)，2.47(s，3H).
step B [ N, N-bis (2-chloroethyl) ] -2-aminoethyl 4, 5 ', 8-trimethyl-4' -psoralen acetate hydrochloride
Thionyl chloride (0.2ml) was added to the CH of the above diol (9.8mg, 0.023mmol)2Cl2(1ml) the mixture was ice-cooled and stirred overnight at room temperature under a nitrogen atmosphere. The resulting slurry was concentrated and then triturated with hexanes to give the desired product (6.2mg, 53.9%) as an off-white solid:1H NMR(CD3OD)：δ7.71(s，1H)，6.28(s，1H)，4.56(t，J＝4.8Hz，2H)，3.95(t，J ＝6.1Hz，4H)，3.89(s，2H)，3.60-3.83(m，6H)，2.54(s，3H)，2.53(s，3H)，2.50(s，3H).
example 7
Synthesis of beta-alanine, N- (acridin-9-yl), 2- [ bis (2-chloroethyl) amino ] acetamide (Compound XV)
Step A2- [ N ', N' -bis (2-hydroxyethyl) ] -N- (tert-butoxycarbonyl) ethylenediamine
To N- (tert-butoxycarbonyl) ethanolamine (1.21g, 7.5mmol) and triethylamine (1.57ml, 1.1g, 11mmol) at 0 deg.C in anhydrous CH2Cl2To the solution (25ml) was added dropwise methanesulfonyl chloride (0.64ml, 0.95g, 8.3 mmol). The reaction was stirred at 0 ℃ for 1 hour, warmed to 23 ℃ and stirred overnight. Volatiles were removed in vacuo to give the mesylate salt as a white solid. Diethanolamine (7.2ml, 7.9g, 75mmol) was added and the reaction mixture was heated to 75 ℃ for 6 hours with stirring. The crude reaction mixture was partitioned between water (60ml) and CH3Cl (4X 20 ml). The combined organics were washed with brine (20ml)Layer of Na2SO4And drying. The solvent was removed in vacuo to give 1.21g (65%) of diol as a viscous yellow oil.
1H NMR：δ5.51-5.39(m，1H)，3.61(t，J＝4.9Hz，4H)，3.29-3.13(m，2H)，2.68-2.52(m，6H)，1-44(s，9H).
No hydroxyl protons were observed.
Step B2- [ N ', N' -bis (2-tert-butyldimethylsilyloxyethyl) ] -N- (tert-butoxycarbonyl) ethylenediamine
Diol obtained in step A above (1.21g, 4.87mmol) and pyridine (1.59ml, 1.55g, 19.6mmol) in anhydrous CH at 0 deg.C2Cl2(12ml) to the stirred solution was added tert-butyldimethylsilyl chloride (2.21g, 14.7 mmol). The reaction mixture was warmed to 23 ℃ and stirred for 2 days. By means of CH2Cl2The reaction mixture was diluted (80ml) and washed first with water (3X 25ml) and then with brine (3X 25 ml). In Na2SO4The organic layer was dried. The solvent was removed in vacuo to give 2.26g (97%) of a pale yellow oil.
1H NMR：δ5.37-5.22(m，1H)，3.62(t，J＝6.2Hz，4H)，3.19-3.08(m，2H)2.63(t，J＝6.2Hz，6H)，1.42(s，9H)，0.873(s，18H)，0.04(s，12H).
Step C2- [ N, N-bis (2-tert-butyldimethylsilyloxyethyl) ] ethylenediamine
To a flask containing the protected amine of step B (4.24g, 8.89mmol) was added 5ml of trifluoroacetic acid at 23 ℃. The reaction mixture was stirred at 23 ℃ for 15 minutes, and then trifluoroacetic acid was removed in vacuo. The crude product was partitioned between 2N NaOH (100ml) and CH2Cl2(3X 35 ml). In Na2SO4The combined organic layers were dried. The solvent was removed in vacuo to yield 1.76g (53%) of a yellow oil.1H NMR：δ3.66(t，J＝6.5Hz，4H)，2.72-2.53(m，8H)，1.72-1.63(m，2H)，0.87(s，18H)，0.02(s，12H).
Step D beta-alanine, N- (tert-butoxycarbonyl), 2- [ bis (2-tert-butyldimethylsilyloxyethyl) amino ] acetamide
To a solution of 3- (N-tert-butoxycarbonyl) aminopropionic acid (822.0mg, 4.34mmol) and 4-methylmorpholine (442.0mg, 4.37mmol) in 14ml of anhydrous THF at-15 deg.C was added isobutyl chloroformate (0.53ml, 0.56g, 4.1 mmol). The reaction mixture was stirred at-15 ℃ for 1 min, followed by the addition of the amine from step C (1.72g, 4.57 mmol). The reaction mixture was warmed to 23 ℃ and stirred for 1 hour. The mixture was then filtered, the precipitate was washed with THF (5ml) and the filtrate was concentrated in vacuo. The residue was partitioned between 2N NaOH (50ml) and CH2Cl2(3X 20 ml). In Na2SO4The combined organic layers were dried. The solvent was removed in vacuo to give 2.25g of a brown yellow gum. By medium pressure liquid chromatography (silica gel, 1: 1 CHCl)3EtOAc) to yield 627.0mg (26%) of a light yellow oil.1H NMR：δ3.63(t，J＝6.2Hz， 4H)，3.54-3.35(m，4H)，3.20-3.19(m，2H)，2.71-2.50(m，6H)，1.43(s，9H)，0.89(s，18H)，0.05(s，12H).
Amide and carbamate protons were not observed.
Step E beta-alanine, 2- [ bis (2-tert-butyldimethylsilyloxyethyl) amino ] acetamide
The protected amine from step D above (627.0mg, 1.14mmol) was dissolved in trifluoroacetic acid (5ml) at 23 ℃. The resulting solution was stirred for 5 minutes (until evolution of CO ceased2) The trifluoroacetic acid is then removed in vacuo. The residue was partitioned with saturated NaHCO3(50ml) and CH2Cl2(3X 20 ml). In Na2SO4The combined organic layers were dried and the solvent removed in vacuo to yield 203.4mg (40%) of a pale yellow oil.
Step F beta-alanine, N- (acridin-9-yl), 2- [ bis (2-tert-butyldimethylsilyloxyethyl) amino ] acetamide
A mixture of the crude amine from step E (203.4mg, 0.45mmol), 9-methoxyacridine (96.8mg, 0.46mmol) and methanol (10ml) was heated to reflux for 4 h. The reaction mixture was cooled to 23 ℃ and stirred for an additional 2.5 days. Methanol was removed in vacuo and the residue was partitioned between 2N NaOH (50ml) and CH2Cl2(3X 20 ml). In Na2SO4The combined organic layers were dried and the solvent removed in vacuo to yield 69.6mg of a yellow oil. By TLC (silica gel, 1: 1 CHCl)3EtOAc) purified the crude product (69.6mg) to give 23.4 (8.3%) as a yellow oil.
1H NMR: δ 8.19(d, J ═ 8.8Hz, 2H), 8.06(d, J ═ 8.8Hz, 2H), 7.65(br t, J ═ 7.6Hz, 2H), 7.36(br t, J ═ 7-6Hz, 2H), 6.8-6.7(m, 1H), 4.06(t, J ═ 5.6Hz, 2H), 3.61(t, J ═ 5.8Hz, 4H), 3.37-3.32(m, 2H), 2.72-2.61(m, 6H), 2.51(t, J ═ 5.5Hz, 2H), 0.86(s, 18H), 0.02(s, 12H), no amine protons were observed,13C NMR：δ172.1，152.4，149.3，130.5，129.3，123.7，118.0，112.8，62.3，57.4，54.1，47，4，38.2，36.5，26.4，18.8，-4.8.
step G beta-alanine, N- (acridin-9-yl), 2- [ bis (2-hydroxyethyl) amino ] acetamide dihydrochloride
To a stirred solution of bis-protected diol (22.0mg, 0.04mmol) from step F in isopropanol (1.0ml) was added a 5-6N HCl/isopropanol solution (0.05ml) at 23 ℃. The reaction mixture was stirred at 23 ℃ for 17 hours and the resulting yellow precipitate was collected by vacuum filtration. The yellow solid was rinsed with an additional 1.0ml of isopropanol. The residual isopropanol was removed in vacuo (overnight) to give 11.4mg (69%) of diol hydrochloride as a yellow solid.
1H NMR(CD3OD)：δ8.52(d，J＝8.8Hz，2H)，7.96(br t，J＝7.5Hz，2H)，7.82(d，J＝8.4Hz，2H)，7.57(br t，J＝7.5Hz，2H)，4.49(t，J＝6.2Hz，2H)，3.91(t，J＝4.8Hz，4H)，3.74-3.56(m，2H)，3.53-3.38(m，6H)，2.97(t，J＝6.1Hz，2H).
No amide, amine and hydroxyl protons were observed.
Step H beta-alanine, N- (acridin-9-yl), 2- [ bis (2-chloroethyl) amino ] acetamide dihydrochloride
To the diol obtained in step G (11.4mg, 0.024mmol) in CH at 23 deg.C3CN (1.0ml) to the stirred suspension SOCl was added2(0.12ml, 200mg, 1.7 mmol). The reaction mixture was stirred at 23 ℃ for 15 minutes and the solution was heated to 50 ℃ for 3.5 hours. The resulting yellow precipitate was collected by vacuum filtration using CH3CN (3X 1.0ml) was rinsed and dried in vacuo to give 8.3mg (67%) of a yellow powder (95% purity by HPLC).
1H NMR(CD3OD)：δ8.55(d，J＝8.7Hz，2H)，8.00(br t，J＝7.7Hz，2H)，7.84(d，J＝8.7Hz，2H)，7.59(br t，J＝7.7Hz，2H)，4.51(t，J＝6.2Hz，2H)，3.98(t，J＝5.7Hz，4H)，3.71(t，J＝5，7Hz，4H)，3.65-3.55(m，2H)，3.55-3.42(m，2H)，2.99(t，J＝6.2Hz，2H).
No amide and amine protons were observed.
Example 8
Hydrolysis of brittle compounds
For brittle compounds containing ester groups ("forward" and "reverse" esters) in the brittle linkage, the present inventors investigated some typical compounds in order to determine the amount of hydrolysis of the ester.
The present invention studies the following reactions:
AcrNH-(CH2)n-C(＝O)-OR→AcrNH-(CH2)n-CO2H+HO-R
("Forward ester") ("acridinoic acid")
Wherein AcrNH represents a 9-aminoacridine bearing the substituents indicated in the table below, and n and R are also indicated in the table. Table III shows the increase in ester hydrolysis rate when the ester linkage between the acridine ring and the alkylamino group is saturated. The rate of hydrolysis is rapid whether the acridine moiety is located at the acid or alcohol terminus of the ester.
TABLE III
For the reaction:
AcrNH-(CH2)n-O-C(＝O)-R→AcrNH-(CH2)n-OH+HOC(＝O)-R
("reverse ester") ("acridinol")
Where AcrNH represents a 9-aminoacridine bearing the substituents indicated in the table below and n and R are also indicated in the table, the following conclusions can be reached:
TABLE IV
All compounds in tables III and IV hydrolyzed ≦ 1% at 100 minutes when pH was 3.
Since nitrogen mustard compounds undergo multiple degradation reactions simultaneously, they cannot be evaluated by the same method. Nevertheless, when compound VIII was incubated under the same conditions as in tables III and IV, the main product after prolonged incubation was acridinic acid (. gtoreq.95%) and formed 40% at 100 min. This is comparable to the record for similar diols in the table (57% hydrolysis at 100 minutes).
As is apparent from the data in tables III and IV, the hydrolysis rate of the ester linkage is inversely proportional to the linker arm length of the 9-aminoacridine moiety and the ester group (in tables III and IV, as n increases, the amount of hydrolysis at 100 minutes decreases). This provides a means to adjust the rate of hydrolysis of the compound. This ability to adjust the rate of linker decomposition allows the degree of compound reactivity to be adjusted as desired for various application conditions.
Material
The following materials may be used in the following examples.
Although commercially available from Baxter Healthcare corp., Deerfield, IL, Adsol used in this and the following experiments was prepared by sterile filtration of the following mixture: 22g glucose, 9g NaCl, 7.5g mannitol and 0.27g adenine in 1l distilled water.
Quinacrine is available from Aldrich Chemical co, st.
Whole Blood is available from Sacramento Blood Center (Sacramento CA).
Example 9
Inactivated Vesicular Stomatitis Virus (VSV)
Stock solutions (typically 10-30mM) of each compound were prepared by the following method: dissolving the appropriate amount of material previously treated with 2mM H3PO4Acidified blood bank saline, then quickly frozen into 1ml aliquots. In use, aliquots were warmed to ≦ 10 ℃ and used within 1 hour.
To prepare Packed Red Blood Cells (PRBC), Hct-assayed whole blood was centrifuged at 3800rpm for 6 minutes. Supernatant plasma was separated and assayed. Add Adsol to obtain 60% Hct PRBC. The plasma concentration is 15-20%.
Mixing VSV (stock solution, about 10)9pfu/ml, obtained from ATCC American Type Cell Culture, Rockville, Md.) was diluted 1: 10 into tissue Culture medium (DMEM with 10% NCS) or PBRC to obtain the test medium as an aliquot (1ml) in a 2ml sterile O-ring tube.
Sufficient test compound solution is added to each tube to give a test compound concentration of 10-300. mu.M. The various samples were mixed rapidly by thoroughly pumping the mixture several times. The suspension was incubated at ambient temperature for 4 hours. Following incubation of the treated medium in BHK (baby rat kidney) host cells, virus titers were determined. PRBC was used directly, not just the supernatant. Viral killing is inversely proportional to the amount of plaque present in the cell culture fluid. The difference between the titer of untreated medium and the titer of treated samples can give compound 1og killing at this concentration. Detection limit of 101.4pfu/ml。
Quinacrine Mustard (QM) and compounds IV, VI, XI, VII, and VIII inactivated > 3logs VSV in tissue culture media at < 50 μ M test compound. Compound XII inactivated 2logs at approximately 200. mu.M. The compound is considered to be particularly unstable due to hydrolysis of the ester. As shown in the first entry in Table IV of example 8, the corresponding diol compound (. beta. -alanine, [ N, N-bis (2-hydroxyethyl) ], 2- [ (6-chloro-2-methoxyacridin-9-yl) amino ] ethyl ester) was hydrolyzed 99% after 100 minutes at pH 8, 37 ℃. The nitrogen mustard compound also appears to undergo rapid hydrolysis. This illustrates the importance of directing the immobilizer portion of the molecular effector moiety to the nucleic acid and the importance of adjusting the reactivity of the 9-aminoacridines, thereby making them functional under practical use conditions. In the inactivation protocol described, hydrolysis of compound XII is expected to compete with inactivation.
In PRBC, QM and compounds IV, VI, VIII, V and XIII were inactivated by > 2logs VSV at test compound concentrations < 150. mu.M.
Example 10
Inactivated yersinia enterocolitica
PRBC and stock solutions were prepared according to the procedure for VSV preparation of example 9.
Yersinia genus (California Department of Health Services, microbiological Disease Laboratory, Berkeley, Calif.) was cultured on LB-broth medium at 37 ℃ on a shaker. A portion (10ml) was centrifuged in a 15ml conical tube at 2500rpm for 10 minutes. The pellet was resuspended in 1ml Adsol to give approximately 109Bacteria/ml. To determine the titre, a 1: 100 dilution was measured in Adsol (10)8OD at bacteria/ml610Optical density of 0.2). The bacterial stock was then diluted 1: 100 into saline or PRBC to give the test medium as an aliquot (1ml) filled into a 2ml sterile 0-loop test tube.
Sufficient test compound solution is added to each tube to give a test compound concentration of 10-300. mu.M. The various samples were mixed rapidly by thoroughly pumping the mixture several times. It was incubated at ambient temperature for 2 hours and then placed on LB-agar plates initially containing 100. mu.l of sample, initially diluted to a concentration of 10-1Then continuously diluting to 10-8. The plate was incubated overnight at 37 ℃ and the number of clones counted. The difference between the titer of untreated medium and the titer of treated samples can give log kill of compound at this concentration. The detection limit was 10 bacteria/ml.
Quinacrine Mustard (QM) and compounds IV, VI, VIII, V, IX and X inactivated > 2logs Yersinia at concentrations ≦ 200 μ M in saline.
In PSBC, QM and compounds VI, VIII, V, X and XIII inactivated > 2logs Yersinia at concentrations ≦ 200. mu.M.
Examples11
Blood function determination after introduction of the compounds of the invention
One use contemplated for the compounds of the present invention includes the addition of one or more compounds of the present invention to blood or blood products for transfusion. Blood or blood products must remain suitable for infusion after treatment with the compound. To evaluate the effect of the compounds on red blood cell function, the compounds were subjected to the following assays.
Packed red blood cells having a 50% hematocrit (Hct) of 50% were prepared by centrifuging whole blood of known Hct at 2500rpm for 6 minutes. The supernatant was separated and assayed. The suspension was diluted with a sufficient volume of Adsol to give the desired Hct.
1.5ml of PRBC was placed in respective 2ml O-ring tubes and sufficient stock solution of test compound was added to give the desired concentration. The samples were incubated at ambient temperature for 4 hours and then stored overnight at 4 ℃. According to Hogman et al, Transfusion, 31: 26-29(1991) to determine hemolysis.
Lysis standards for the various samples were prepared by diluting the 10. mu.M incubation mixture in two steps with water to give a final dilution of 1: 4000.
For the assay, the samples were removed from the 4 ℃ storage location and warmed < 15 minutes. After direct vortex mixing, aliquots were separated and spun at 14,000rpm for 2 minutes. The supernatant was separated and spun at 14000rpm for 10 minutes. The supernatant was separated and diluted in water as needed. The absorbance at 414nm of the lysis standard and the diluted supernatant were recorded relative to the water blank. Percent hemolysis was calculated:
(100% -50% Hct) × (sample A)414X dilution factor)/(lysis standard A414×4000)
Sample A due to the presence of the Compound of the invention414Any absorbance was uncorrected. The results are shown in Table Va.
TABLE Va
Hemolysis data of day one
Compounds and concentrations Percentage of hemolysis Number of sample to be tested
BBS* 0.066 14
ABBS** 0.065 8
150μM QM*** 0.220 14
300μM QM 0.320 14
150μM IV 0.091 14
300μM IV 0.109 14
150μM VI 0.103 14
300μM VI 0.140 14
150μM VIII 0.110 6
300μM VIII 0.135 6
150μM V 0.136 2
300μM V 0.149 2
150μM IX 0.116 2
300μM IX 0.099 2
150μM X 0.121 2
300μM X 0.153 2
BBS ═ blood pool saline
ABBS (acidic blood pool) saline
QM quinacrine nitrogen mustard
Extracellular potassium can be derived from Ciba Corning type 614K+/Na+The assay was performed by an analyzer (Ciba Corning Diagnostics Corp., Medford, MA). ATP can be measured using Sigma method No.366(Sigma, St. Louis, Mo.).
Table Vb shows the relative values of extracellular potassium in this experiment relative to the control value for the untreated PRBC sample. For example, a relative value of 1.03 indicates that the extracellular potassium concentration of the treated sample was 3% greater than the untreated control.
TABLE Vb relative extracellular Potassium levels (parallel measurements)
Compound (I) Concentration (μ M) Day 1 Day 7 Day 14
IV 100 1.01(1) 0.98(1) 1.03(1)
200 1.05(1) 1.15(1) 1.01(1)
300 1.03(1) 1.15(1) 1.15(1)
V 300 1.04-1.46(4) 0.96-1.01(4) 0.95-1.01(4)
[ K + ] (treated)/[ K + ] (untreated)
Table Vc shows the relative values of ATP in this experiment relative to the control values of the untreated PRBC sample. For example, a relative value of 1.03 indicates that the ATP for the treated sample was 3% greater than the untreated control.
Table Vc ATP relative levels (parallel measurements).)
Compound (I) Concentration (μ M) Day 1 Day 7 Day 14
IV 100 1.01(1) 0.93(1) 0.94(1)
200 1.05(1) 0.94(1) 0.94(1)
300 1.03(1) 0.93(1) 0.92(1)
V 300 0.96-1.00(4) 0.91-1.01(4) 0.94-1.01(4)
[ ATP ] (treated)/[ ATP ] (untreated)
Example 12
Inactivation of HIV by Compounds of the invention
Cell-associated HIV in TC medium (Popovic et al, Science),224: 497(1984): H9-IIIb cells are suspended in TC culture medium to obtain the titer which is about equal to or more than 106pfu/ml suspension. To a 2ml aliquot of the test medium, a sufficient amount of the test compound solution is added to give the desired concentration of active material in a 15ml conical tube. The suspension was mixed immediately by complete aspiration several times, then vortexed rapidly. The samples were incubated at ambient temperature for 2-4 hours and then centrifuged. The pellet was suspended in 1ml of plaque assay dilution, then rapidly frozen at-80 ℃ and the titer was determined by microetch. (Hanson et al, J.Clin.Micro., 28: 2030 (1990)).
At test compound concentrations < 25 μ M, the compounds quinacrine, IV and VI inactivate > 3logs HIV.
Cell-associated HIV in PRBC: for assays performed in PRBC, packed cells were prepared according to the method described in the VSV assay. HIV9-IIIb cells were added to Adsol prior to dilution of the centrifuged cells. The resulting suspension was mixed by thoroughly pumping all the material. Upon completion of the test compound incubation, 3ml of 1: 1 plasma containing 5 μ l heparin was used: DMEM solution diluted the samples. Infected cells were then separated using a fycol-hypaque gradient, resuspended in 1ml of diluent and frozen for later titration.
At test compound concentrations of 200 μ M or less, the compounds quinacrine, VI and V inactivate > 3logs HIV.
Cell-free HIV in PRBC: the procedure is similar to that described above except that cell-free HIV is added directly to PRBC after preparation. After incubation the medium was centrifuged and the supernatant frozen for later titration.
At test compound concentrations of 100 μ M or less, the compounds quinacrine, IV, V and VI inactivate > 3logs HIV.
Although the foregoing invention has been described in some detail by way of illustration and example, it will be readily apparent to those skilled in the art that: certain changes and modifications may be made. Accordingly, the specification and examples should not be used to limit the scope of the invention, which is described in the following claims. U.S. patent nos. 5,559,250 and 5,399,719 are incorporated herein by reference in their entirety. All other patents and references cited herein are incorporated herein by reference.

Claims (19)

1. A compound selected from the group consisting of:
2. the compound according to claim 1, which is a compound of the formula:
3. the compound according to claim 1, which is a compound of the formula:
4. the compound according to claim 1, which is a compound of the formula:
5. the compound according to claim 1, which is a compound of the formula:
6. the compound according to claim 1, which is a compound of the formula:
7. the compound according to claim 1, which is a compound of the formula:
8. the compound according to claim 1, which is a compound of the formula:
9. the compound according to claim 1, which is a compound of the formula:
10. the compound according to claim 1, which is a compound of the formula:
11. the compound according to claim 1, which is a compound of the formula:
12. a method of inactivating a pathogen in a material, the method comprising:
adding a compound according to any one of claims 1 to 11 to a material; and
incubating the material.
13. A method according to claim 12, wherein the compound is added to the material to form a final solution having a compound concentration of 1 to 500 μ M.
14. The method of claim 12, wherein the material is a biological material.
15. The method according to claim 14, wherein the biological material comprises a component selected from the group consisting of: blood, blood products, plasma, platelet preparations, red blood cells, packed red blood cells, serum, sweat, cerebrospinal fluid, saliva, urine, stool, semen, milk, tissue samples, homogenized tissue samples, cell culture media; a cell culture; viral cultures and cultures incorporating materials produced by living organisms.
16. The method of claim 14, wherein the material comprises a blood product.
17. The method of claim 14, wherein the material comprises red blood cells.
18. The method of claim 12, wherein the method comprises adding an effective amount of the compound to the material to inactivate pathogens in at least 2logs of the material.
19. A method according to claim 12, wherein the incubation time is from 1 to 48 hours.
HK00105911.5A 1997-01-06 1998-01-06 Frangible compounds for pathogen inactivation HK1026702B (en)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US77988597A 1997-01-06 1997-01-06
US08/779,885 1997-01-06
US77983097A 1997-01-07 1997-01-07
US08/779,830 1997-01-07
US4369697P 1997-04-15 1997-04-15
US60/043,696 1997-04-15
PCT/US1998/000532 WO1998030545A1 (en) 1997-01-06 1998-01-06 Frangible compounds for pathogen inactivation

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
HK10109160.3A Division HK1188441B (en) 1997-01-06 2000-09-20 Frangible compounds for pathogen inactivation

Related Child Applications (1)

Application Number Title Priority Date Filing Date
HK10109160.3A Addition HK1188441B (en) 1997-01-06 2000-09-20 Frangible compounds for pathogen inactivation

Publications (2)

Publication Number Publication Date
HK1026702A1 HK1026702A1 (en) 2000-12-22
HK1026702B true HK1026702B (en) 2013-04-19

Family

ID=

Similar Documents

Publication Publication Date Title
CN1248245B (en) Fragile compounds for pathogen inactivation
US6093725A (en) Frangible compounds for pathogen inactivation
US6514987B1 (en) Frangible compounds for pathogen inactivation
WO1998030545A9 (en) Frangible compounds for pathogen inactivation
ES2210846T3 (en) NEW PSORALENES TO ACTIVATE PATHOGEN AGENTS.
US6143490A (en) Treating blood or blood product with a compound having a mustard and a nucleic acid binding moiety
JP4643004B2 (en) Method for quenching pathogen inactivating agents in biomaterials
AU722811B2 (en) Treating red blood cell solutions with anti-viral agents
CA2221605C (en) Methods and devices for the removal of psoralens from blood products
US6544727B1 (en) Methods and devices for the removal of psoralens from blood products
US20020192632A1 (en) Method and devices for the removal of psoralens from blood products
JP2002509518A5 (en)
HK1026702B (en) Frangible compounds for pathogen inactivation
HK1188441B (en) Frangible compounds for pathogen inactivation
EP1364944A1 (en) Frangible Compounds for Pathogen Inactivation
JP2012067126A (en) Method and device for removing psoralen from blood products
AU757224B2 (en) Method and devices for the removal of psoralens from blood products
AU7153800A (en) Treating red blood cell solutions with anti-viral agents