CN116731015A - Imidazoquinoline compounds, methods of preparation, use and compositions thereof - Google Patents

Imidazoquinoline compounds, methods of preparation, use and compositions thereof Download PDF

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CN116731015A
CN116731015A CN202310566830.XA CN202310566830A CN116731015A CN 116731015 A CN116731015 A CN 116731015A CN 202310566830 A CN202310566830 A CN 202310566830A CN 116731015 A CN116731015 A CN 116731015A
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tumor
imidazoquinoline
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刘慧涛
韩志强
万娜
邢风云
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Shenzhen Weikesen Biomedical Technology Co ltd
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    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
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Abstract

The embodiment of the application provides an imidazoquinoline compound, and a preparation method, application and composition thereof. The imidazoquinoline compound is a compound of a formula I or a pharmaceutically acceptable salt thereof, and X comprises any one of n-propyl, n-butyl, n-butylamine, n-amyl and 2-methoxyethyl; r is R 1 Comprises C 1 ‑C 36 Alkyl, C 1 ‑C 36 Any one of the alkoxy groups; r is R 2 Comprising hydrogen, substituted or unsubstituted aryl, fused ring aryl, heteropolycyclic groups, -S-R 3 Any one of, wherein R 3 Including substituted or unsubstitutedAny one of substituted aryl, condensed ring aryl, heteropolycyclic groups. The imidazoquinoline compound selectively releases molecules with activity of exciting TLR7/8 receptors in tumors, acts on immune cells in the tumors, reduces the activity of the immune cells outside the tumors, and has the characteristic of low systemic immune toxicity while having anti-tumor activity.

Description

Imidazoquinoline compounds, methods of preparation, use and compositions thereof
Technical Field
The application belongs to the technical field of medicines, and particularly relates to an imidazoquinoline compound, and a preparation method, application and composition thereof.
Background
Toll-like receptors (TLRs) are widely expressed in immune cell profiles, involved in nonspecific immunity and mediating specific immunity. TLR is the first barrier of the natural immune system to monitor and recognize a variety of different disease-related molecular patterns (PAMPs) against infectious diseases. Currently 13 TLRs (TLR 1-TLR 13) have been identified in mammals, including human TLR1-TLR11, mouse TLR1-TLR9 and TLR11-TLR13. Activation of TLRs can induce MyD88 or TRIF dependent signaling pathways, and thus NF- κb, induce cytokine and chemokine secretion, activate innate immune responses, and mediate adaptive immune responses. TLR agonists are therefore useful as immunoadjuvants for anti-tumour and for the treatment of infectious diseases.
TLR7/8 agonists are important components of TLR agonists, TLR7/8 agonists include the imidazoquinoline family IMQ, such as imimod (R837) and raschimod (R848), and the like. When these TLR7/8 agonists such as R848 are used as adjuvants, they are small molecule compounds, which have no specific targeting property, short half-life, and intravenous injection can cause systemic immune system to be excessive, thereby causing cytokine release syndrome (cytokine release syndrome, CRS), which is difficult to tolerate in vivo. Thus, TLR7/8 agonists are widely recognized as effective adjuvants for topical antitumor therapy.
Although TLR7/8 agonists are effective adjuvants for local anti-tumor therapy, the serious toxic side effects of TLR7/8 agonists are an important issue facing current clinical applications. The reason for the toxic side effects of TLR7/8 agonists is that in addition to the large number of tumor cells, there are also a large number of non-tumor cells, including various immune cells, fibroblasts, stromal cells, etc., on the cellular constitution of the solid tumor microenvironment. Unlike conventional Antibody Drug Conjugate (ADC) and polypeptide conjugate drug (PDC) and other cytotoxin drugs, the target cell of the TLR7/8 agonist acts on tumor cells after targeting release to the tumor cells, wherein the target cell of the TLR7/8 agonist is an immune cell in the tumor, and the immune cell in the tumor comprises an original immune cell in the tumor and an immune cell induced to infiltrate by the TLR7/8 agonist. When TLR7/8 agonists are delivered into tumors and phagocytosed by immune cells, particularly Antigen Presenting Cells (APCs) such as Dendritic Cells (DCs), activating TLR7 and TLR8 receptors on the endosome, activating inflammatory cytokine expression through MYD 88-mediated NF-KB signaling pathway, inducing secretion of cytokines IFN- α, IFN- γ, TNF- α, IL-1β, IL-12p40 and IP-10, the activated APC cells present antigen to T cells, mediating antigen-specific T cell anti-tumor immunity. Therefore, after the TLR7/8 receptor is activated by the TLR7/8 agonist of the small molecular medicine, the release of the tumor antigen and the mediation of the specific T cell immunity of the tumor antigen can be promoted, the T cell circulation is activated to kill the distant metastasis tumor in a whole body, the in-situ immunity activation is realized, and the whole body anti-tumor immunity effect is induced. However, because small molecule TLR7/8 agonists do not have specific targeting, even if such small molecule TLR7/8 agonists are administered by intratumoral injection, it is difficult to overcome systemic immune toxicity due to rapid absorption.
Disclosure of Invention
The application aims to provide an imidazoquinoline compound, and a preparation method, application and composition thereof, so as to solve the problem that a small molecular TLR7/8 agonist in the prior art has no specific targeting and acts on immune cells in and out of tumors to cause systemic immune toxicity.
In a first aspect, embodiments of the present application provide an imidazoquinoline compound which is a compound of formula I:
x comprises any one of n-propyl, n-butyl, n-butylamine, n-amyl and 2-methoxyethyl;
R 1 comprises C 1 -C 36 Alkyl, C 1 -C 36 Any one of the alkoxy groups;
R 2 comprising hydrogen, substituted or unsubstituted aryl, fused ring aryl, heteropolycyclic groups, -S-R 3 Any one of, wherein R 3 Including any of substituted or unsubstituted aryl, fused ring aryl, heteropolycyclic groups.
The imidazoquinoline compounds of embodiments of the application are TLR7/8 agonists or derivatives thereof. The imidazoquinoline compound selectively releases molecules having activity to agonize the TLR7/8 receptor in a tumor in a targeted manner after administration, while releasing molecules having activity to agonize the TLR7/8 receptor in an environment outside the tumor, such as plasma. Therefore, the imidazoquinoline compound of the embodiment of the application selectively releases TLR7/8 agonists in tumors, mainly acts on immune cells in tumors, and activates TLR7 and TLR8 receptors on cytoplasmic endosomes.
According to the molecules with activity of exciting TLR7/8 receptors, which are selectively released in tumors, of the imidazoquinoline compounds disclosed by the embodiment of the application, the molecules selectively act on immune cells in the tumors, activate TLR7 and TLR8 receptors on cytoplasm endosomes, activate inflammatory cytokine expression through a MYD 88-mediated NF-KB signaling pathway, induce inflammatory cytokines such as TNF-alpha secretion, and mediate antigen-specific T cell anti-tumor immunity. On the basis, the imidazoquinoline compound effectively reduces the content of TLR7/8 agonist outside the tumor, reduces or avoids the effect on immune cells outside the tumor, and reduces the nonspecific immune toxicity caused by cytokine storm such as TNF-alpha and the like as much as possible while realizing the anti-tumor effect.
In a second aspect, the present application provides a process for the preparation of an imidazoquinoline compound. The preparation method comprises the following steps:
providing compound a and compound B:
carrying out a first condensation reaction on the compound A and the compound B in a first reaction system to obtain an imidazoquinoline compound shown in a formula I;
wherein Y comprises-COOH,Any one of them;
z comprises hydrogen, substituted or unsubstituted aryl, condensed ring aryl, heteropolycyclic group,Any one of, wherein R 4 Comprises C 1 -C 36 Alkyl, C 1 -C 36 Alkoxy groups, X 2 Comprising hydrogen, & gt>Any one of them.
According to the preparation method provided by the embodiment of the application, the compound A and the compound B are subjected to condensation reaction, so that the amino group at the para position of the benzene ring of the compound A and the compound B are subjected to condensation reaction, and the amino group at the para position of the benzene ring of the compound A is acylated to form an amide bond, so that the imidazoquinoline compound is prepared. The preparation method has high yield and less byproducts. The imidazoquinoline compound can be a prodrug, can selectively release active molecules with the functions of exciting TLR7/8 receptors in tumors in vivo, can induce systemic anti-tumor immunity after in-situ activation of immunity of the tumors, and can reduce the effect on immune cells outside the tumors and reduce systemic immune toxicity.
The third aspect of the embodiment of the application provides application of the imidazoquinoline compound in preparing antitumor drugs and/or antiviral drugs.
According to the imidazoquinoline compound disclosed by the embodiment of the application, the TLR7/8 agonist is selectively released in tumors, so that the TLR7/8 agonist molecules mainly act on immune cells in the tumors, antigen-specific T cell anti-tumor immunity is mediated, in-situ immune activation is realized, meanwhile, the agonist activity on the TLR7/8 receptor of the immune cells outside the tumors is effectively reduced, and further, the systemic immune toxicity is reduced, and the imidazoquinoline compound is applied to anti-tumor medicines, and has the beneficial effects of low systemic immune toxicity while having the anti-tumor activity. In addition, the imidazoquinoline compound provided by the embodiment of the application has a good antiviral effect, and can be used for preparing antiviral drugs.
In a fourth aspect, embodiments of the present application provide a composition. The composition comprises the imidazoquinoline compound of the embodiment of the present application described above.
The composition provided by the embodiment of the application is based on the imidazoquinoline compound which acts on the immune cells in the tumor in a targeting way, so that the composition has the antitumor activity, reduces or avoids the effect on the immune cells outside the tumor, reduces the expression of inflammatory cytokines in blood plasma, and effectively reduces the systemic immunotoxicity.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present application, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a process flow diagram of a method for preparing an imidazoquinoline compound according to an embodiment of the present application;
FIG. 2 is a process flow diagram showing a second condensation reaction step in the method for preparing an imidazoquinoline compound according to the embodiment of the present application;
FIG. 3 is a nuclear magnetic hydrogen spectrum of 78A 1-SH;
FIG. 4 is a nuclear magnetic hydrogen spectrum of 78A 1-S-Trityl;
FIG. 5 is a nuclear magnetic resonance hydrogen spectrum of 78A1-SS-78A 1;
FIG. 6 is a mass spectrum of 78A1-SS-78A 1;
FIG. 7 is a schematic diagram showing the results of detection of the inducible expression activity of R848, 78A1-SH, 78A1-S-Trityl and 78A1-SS-78A1 on lymphocyte TNF- α;
FIG. 8 is a graph showing the results of detecting the TNF- α content of serum after subcutaneous injections 78A1 and 78A1-SS-78A1, wherein A in FIG. 8 is a line graph and B in FIG. 8 is a bar graph;
FIG. 9 is a schematic diagram showing the results of detection of the TNF-. Alpha.content of serum after 1h of subcutaneous injections of R848, 78A1-SH, 78A1-S-Trityl and 78A1-SS-78A 1;
FIG. 10 is a graph showing the results of serum and intratumoral TNF- α levels at various time points of intratumoral injections 78A1 and 78A1-SS-78A1, wherein A in FIG. 10 is the level of TNF- α in the serum and B in FIG. 10 is the level of intratumoral TNF- α;
FIG. 11 is a graph showing the results of detecting the TNF- α content in serum and tumor after 1h of intratumoral injection of R848, 78A1-S-Trityl and 78A1-SS-78A1, wherein A in FIG. 11 is the TNF- α content in serum and B in FIG. 11 is the intratumoral TNF- α content;
FIG. 12 is a schematic representation of the change in body weight of mice;
FIG. 13 is a schematic diagram showing tumor growth curves of intratumoral fast injection PBS, 78A1-S-Trityl 10. Mu.g, 78A1-SH 10. Mu.g, 78A1-SS-78A 110. Mu.g, 78A1-SS-78A1 20. Mu.g and microinjection pump injection 78A1-SS-78A1 20. Mu.g;
FIG. 14 is a schematic summary of tumor growth curves;
FIG. 15 is a graphical representation of tumor weight results;
fig. 16 is a schematic representation of experimental endpoint tumor size.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
In a first aspect, embodiments of the present application provide an Imidazoquinoline (IMQ) compound which is a compound of formula I:
x comprises any one of n-propyl, n-butyl, n-butylamine, n-amyl and 2-methoxyethyl;
R 1 comprises C 1 -C 36 Alkyl, C 1 -C 36 Any one of the alkoxy groups;
R 2 comprising hydrogen, substituted or unsubstituted aryl, fused ring aryl, heteropolycyclic groups, -S-R 3 Any one of, wherein R 3 Including any of substituted or unsubstituted aryl, fused ring aryl, heteropolycyclic groups.
The small molecule TLR7/8 agonist can activate TLR7 and TLR8 receptors on the cytoplasmic endosome after being phagocytized by immune cells, and mediate antigen-specific T cell anti-tumor immunity. Because small molecule TLR7/8 agonists are not targeted, they are not only distributed within the tumor but also outside the tumor after administration. TLR7/8 agonist molecules distributed in tumors can promote tumor antigen release and mediate tumor antigen-specific T cell immunity, but the action of TLR7/8 agonist molecules distributed outside tumors on immune cells outside tumors can cause immune responses outside tumors of the body, resulting in increased cytokines such as TNF- α, and even the occurrence of cytokine storm and systemic immune system hyperactivity, leading to systemic immunotoxicity.
The imidazoquinoline compound of the embodiment of the application is a compound obtained by acylating amino on the benzene ring para position of a TLR7/8 agonist, contains an amide bond and also comprises at least one of sulfhydryl, thioether bond and disulfide bond. The imidazoquinoline compound can selectively release molecules with activity of exciting TLR7/8 receptors in tumors, activate TLR7 and TLR8 receptors of immune cells in the tumors, activate inflammatory cytokine expression, promote release of tumor antigens and mediate tumor antigen-specific T cell immunity, realize in-situ immune activation and induce anti-tumor immune effects. Meanwhile, the imidazoquinoline compound disclosed by the embodiment of the application can reduce or avoid the release of molecules with activity of exciting TLR7/8 receptors outside tumors, reduce or avoid the activation of immune cells outside tumors, and greatly reduce the possibility of occurrence of systemic immune system hyperfunction and cytokine release syndrome (cytokine release syndrome, CRS). According to the imidazoquinoline compound disclosed by the embodiment of the application, molecules with activity of exciting TLR7/8 receptors are selectively released in tumors, so that the imidazoquinoline compound has anti-tumor activity and simultaneously greatly reduces toxic and side effects such as systemic immune toxicity to organisms.
In some embodiments, X is 2-methoxyethyl (-CH) 2 OCH 2 CH 3 )。
In some embodiments, X is n-butyl (-CH) 2 CH 2 CH 2 CH 3 ) At this time, the structural formula of the imidazoquinoline compound of the embodiment of the application is shown as formula I 1 As shown in the drawing,
x is n-butyl, as described above in formula I 1 After administration of the imidazoquinoline compounds shown, I 1 The amide bond of the molecule shown hydrolyzes, releasing the 78A1 molecule, the structure of the 78A1 molecule is shown below:
78A1 has CAS number 1258457-59-8, is a small molecular LTR7/8 agonist and has good anti-tumor activity, but after 78A1 is administrated, immune response in tumor is activated, cytokines such as TNF-alpha in tumor are increased, antigen-specific T cell anti-tumor immunity is mediated, and 78A1 receptor is distributed outside tumor such as blood plasma, so that the content of the extracellular cytokines in the body is increased, and further the immune toxicity of the body is caused. I is a kind of 1 The compound can be selectively hydrolyzed to release 78A1 molecules in tumor after administration, specifically increase the secretion of cytokines and chemokines in tumor, mediate antigen-specific T cell anti-tumor immunity, have good anti-tumor effect, simultaneously reduce or avoid the activation of TLR7 and TLR8 receptors of immune cells outside tumor, and avoid the activation of TN outside tumor of organism The secretion of cytokines and chemokines such as F-alpha is increased, and the nonspecific immune toxicity caused by the storm of cytokines such as TNF-alpha is reduced.
In some embodiments, R 1 May be- (CH) 2 CH 2 O) n -wherein n = 1-36, further n = 1-20, further n = 1-10, still further n = 1-5, in particular n may be typical but not limiting values of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 26, 30, 35, etc.
In a specific example, R 1 May include-CH 2 CH 2 O-、-CH 2 OCH 2 -、-CH 2 OCH 2 CH 2 -、-CH 2 CH 2 OCH 2 -、-CH 2 CH 2 OCH 2 CH 2 -any one of the following.
In some embodiments, R 1 May be- (CH) 2 ) m -wherein m = 1-36, further m = 1-20, further m = 1-10, still further m = 1-5, in particular m may be typical but not limiting values of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 26, 30, 35, etc.
In a specific example, R 1 May include-CH 2 CH 2 -、-CH 2 -、-(H 2 CH 2 ) 2 -、-(CH 2 CH 2 ) 3 -、-(CH 2 CH 2 ) 4 -any one of the following.
In some embodiments, R 2 In the case of hydrogen, the structural formula of the imidazoquinoline compound of the embodiment of the application is shown as the formula I 2 As shown in the drawing,
i is a kind of 2 The thiol derivative of the molecular TLR7/8 agonist contains thiol-SH in the molecule, so that the molecule is easy to oxidize, but can recover the structure of the-SH group in a reducing environment after oxidation, so that the molecule can be used for preparing the compound So as to have certain activity of exciting the TLR7/8 receptor. Meanwhile, due to lower concentration of glutathione (r-glutamyl cysteingl +glycine, GSH) in blood, the GSH content in tumor is higher, and the formula I in tumor is shown in the specification 2 The molecules shown tend to exist in a form in which-SH is reduced. Therefore, even after the administration by intraperitoneal injection, the levels of pro-inflammatory cytokines such as TNF-. Alpha.in the blood are not too high while the levels of pro-inflammatory cytokines in the tumor are increased.
In addition, when R 1 Is C 1 -C 36 Alkyl or C 1 -C 36 Alkoxy radicals, e.g. R 1 Is- (CH) 2 CH 2 O) n -or- (CH) 2 ) m -when R 1 Is different in carbon chain length, I 2 The activities of the molecules shown differ, I when the length of the carbon chain is 36 2 The potency of the indicated molecules is reduced.
In some embodiments, when R 2 Is C 1 -C 36 In the case of alkyl radicals, R 2 May include-CH 2 CH 2 、-CH 2 、-(CH 2 CH 2 ) 2 、-(CH 2 CH 2 ) 3 、-(CH 2 CH 2 ) 4 And (3) any one of tertiary butyl groups.
In some embodiments, when R 2 Is C 1 -C 36 In the case of alkoxy, R 2 May include-OCH 2 CH 2 、-CH 2 OCH 2 、-CH 2 OCH 2 CH 2 、-CH 2 CH 2 OCH 2 、-CH 2 CH 2 OCH 2 CH 2 Any one of them.
In some embodiments, when R 2 When substituted or unsubstituted aryl, the substituted or unsubstituted aryl may include at least one of triphenylmethyl and methoxytrityl. The structure of triphenylmethyl is shown below:
in some embodiments, R 2 Any one of acetamidomethyl and trimethylacetamidomethyl may also be included.
When R is 2 When the imidazoquinoline compound is a larger sterically hindered group such as triphenylmethyl, methoxytrityl, tert-butyl, acetamidomethyl, trimethylacetamidomethyl and the like, the imidazoquinoline compound does not have activity of exciting a TLR7/8 receptor, but can selectively release molecules with activity of exciting the TLR7/8 receptor in tumors, thereby realizing specific activation of the TLR7/8 receptor of the immune cells in the tumors and reducing the effect on the immune cells outside the tumors.
In some embodiments, R 2 is-S-R 3 In this case, the imidazoquinoline compound has a disulfide bond in its molecular structure.
Wherein, in the embodiment, R 3 Any of substituted or unsubstituted aryl, fused ring aryl, heteropolycyclic groups may be included.
R 3 When substituted or unsubstituted aryl, condensed ring aryl, heteropolycyclic, R 3 The substituted or unsubstituted aryl, fused ring aryl, heteropolycyclic groups shown may be the same as those described above for R 2 The substituted or unsubstituted aryl, condensed ring aryl, heteropolycyclic groups shown are the same.
In some embodiments, R 3 Can be a group of formula II:
Wherein X is 1 Comprises any one of n-propyl, n-butyl, n-butylamine, n-amyl and 2-methoxyethyl,
R 4 comprises C 1 -C 36 Alkyl, C 1 -C 36 Any one of the alkoxy groups.
In some embodiments, X 1 And X is the same or different. In a specific example, X 1 N-butyl, in which case R 3 Is of the formula II 1 Shown in the figureThe group:
in a specific example, when X and X 1 When both are n-butyl, the imidazoquinoline compound is shown as the formula I 3 The following is shown:
in some embodiments, the R' s 4 C shown 1 -C 36 Alkyl, C 1 -C 36 Alkoxy groups may be independently substituted with R as described above 1 C shown 1 -C 36 Alkyl, C 1 -C 36 The alkoxy groups are the same.
In embodiments, R 4 May be- (CH) 2 CH 2 O) n -wherein n = 1-36, further n = 1-20, further n = 1-10, still further n = 1-5, in particular n may be typical but not limiting values of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 26, 30, 35, etc. In a specific example, R 4 May include-CH 2 CH 2 O-、-CH 2 OCH 2 -、-CH 2 OCH 2 CH 2 -、-CH 2 CH 2 OCH 2 -、-CH 2 CH 2 OCH 2 CH 2 -any one of the following.
In embodiments, R 4 May be- (CH) 2 ) m -wherein m = 1-36, further m = 1-20, further m = 1-10, still further m = 1-5, in particular m may be typical but not limiting values of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 26, 30, 35, etc.
In a specific example, R 4 May include-CH 2 CH 2 -、-CH 2 -、-(H 2 CH 2 ) 2 -、-(CH 2 CH 2 ) 3 -、-(CH 2 CH 2 ) 4 -any one of the following.
As shown in I 3 The imidazoquinoline compounds shown contain disulfide bonds which are sensitive linkers to GSH cleavage sites and are therefore of formula I 3 The imidazoquinoline compounds shown can be cleaved to the formula I under the action of GSH in tumor tissue and in high concentration in cytoplasm of tumor cells 2 And I 31 The molecules shown act.
Because of the low levels of GSH in lymphocytes and plasma, the lymphocyte and plasma are of formula I 3 The imidazoquinoline compounds shown are not effective in releasing formula I 2 And I 31 The molecules shown, which do not bind sufficiently to the TLR7/8 receptor, cause a strong immune activation effect, thus making the levels of cytokines such as TNF- α in the plasma unchanged significantly.
In some embodiments, based on X as described above 1 、R 1 、R 2 、R 3 、R 4 And X 2 The imidazoquinoline compound shown in the formula I of the embodiment of the present application may include at least one of the formulas (1) to (4):
the imidazoquinoline compound represented by the above formula (1) is a mercapto derivative of 78 A1. On the one hand, the imidazoquinoline compound shown in the formula (1) contains an amide bond, the lysosome content in the tumor is high, the internal environment of the lysosome is acidic, and the acidic environment can hydrolyze the amide bond, release 78A1 and activate a TLR7/8 receptor. On the other hand, the molecule shown in the formula (1) can still be combined with the TLR7/8 receptor to activate the TLR7/8 receptor, but the molecular structure shown in the formula (1) contains sulfhydryl-SH, so that the molecule is easy to oxidize and loses the capability of combining with the TLR7/8 receptor after oxidation. But it can restore the structure of the-SH group in a reducing environment after oxidation, so that the activity of the molecular structure shown in the formula (1) for exciting the TLR7/8 receptor is influenced by the environment due to the-SH group. Because of the low concentration of glutathione (r-glutamyl cysteingl glycine, GSH) in blood, the GSH content in tumors is high, and the molecules shown in the formula (1) in the tumors tend to exist in a form that-SH is reduced, so that the imidazoquinoline compound shown in the formula (1) has activity of exciting TLR7/8 receptors. Based on the effects of the two aspects, the compound shown in the formula (1) can selectively release the compound with the activity of activating the TLR7/8 receptor in tumors.
The imidazoquinoline compound represented by the above formula (2) is a thioether compound of 78A1, wherein the Trityl thio (Tr-SH) structure formed by the triphenylmethyl group and the sulfur atom is a protective structure of-SH, and can be deprotected in a weakly acidic environment (e.g., trifluoroacetic acid, tumor microenvironment) to form the imidazoquinoline compound represented by the formula (1). Meanwhile, the Trityl thio structure may provide a steric hindrance effect, so that the imidazoquinoline compound shown in the formula (2) cannot efficiently activate the TLR7/8 receptor in blood.
The imidazoquinoline compound represented by the above formula (3) is a disulfide compound of 78A1, which has a disulfide bond. Since disulfide bonds belong to GSH cleavage site sensitive linkers, the imidazoquinoline compound represented by the formula (3) can be cleaved into the compound represented by the above formula (1) under the action of GSH at a high concentration in tumor tissues and cytoplasm of tumor cells to act. Because the level of GSH in lymphocyte and blood plasma is lower, the lymphocyte and blood plasma imidazoquinoline compound can not release molecules with activity of activating TLR7/8 receptor with high efficiency, and can not fully bind TLR7/8 receptor to cause strong immune activation effect, so that the level of cytokines such as TNF-alpha in blood plasma is not changed obviously.
The imidazoquinoline compound represented by the above formula (4) is a disulfide compound of 78A1, and has a disulfide bond. The environment in the tumor easily causes the disulfide bond, the carbon-oxygen bond and the amide bond in the molecule to be broken, so that the 78A1 proto-structure of the medicine is released, the molecular structure shown in the formula (4) selectively releases 78A1 in the tumor, and the TLR7/8 receptor of immune cells in the tumor is specifically activated.
The imidazoquinoline compounds can selectively release compounds with TLR7/8 receptor agonistic activity in tumors after being administered, so that the imidazoquinoline compounds have good anti-tumor activity, and meanwhile, the imidazoquinoline compounds have low immune toxicity due to low content of the compounds with TLR7/8 receptor agonistic activity released outside the tumors.
In a second aspect, embodiments of the present application provide a method for preparing the above imidazoquinoline compound. The synthetic flow of the preparation method of the imidazoquinoline compound is shown in the figure 1, and the preparation method comprises the following steps:
step S01: providing compound a and compound B:
step S02:
carrying out a first condensation reaction on the compound A and the compound B in a first reaction system to obtain an imidazoquinoline compound shown in a formula I;
Wherein Y comprises-COOH,Any one of them;
z comprises hydrogen, substituted or unsubstituted aryl, condensed ring aryl, heteropolycyclic group,Any one of, wherein R 4 Comprises C 1 -C 36 Alkyl, C 1 -C 36 Alkoxy groups, X 2 Comprising hydrogen, & gt>Any one of them.
According to the preparation method provided by the embodiment of the application, the compound A and the compound B are subjected to a first condensation reaction, so that the amino at the para position of the benzene ring of the compound A is acylated to form an amide bond, and the imidazoquinoline compound shown in the formula I is prepared. The preparation method has the advantages of less process steps and high reaction yield. Meanwhile, the prepared imidazoquinoline compound is a prodrug, and can selectively release compound A or a derivative of compound A in tumors after administration, so that TLR7/8 receptors in the tumors are excited. The imidazoquinoline compound has good selectivity in the release process of exciting TLR7/8 receptor exciting active molecules, has anti-tumor activity, and simultaneously effectively avoids the increase of the content of extracellular cytokines and chemokines and effectively reduces the extracellular immune response.
Step S01:
providing a compound A and a compound B through a substrate of the imidazoquinoline compound reaction shown in the formula I in the step S01, wherein the compound A is a TLR7/8 agonist, the compound A contains benzylamine in a molecule, the benzylamine and the compound B are subjected to condensation reaction, and the compound A is acylated to form the compound shown in the formula I.
Wherein X in the compound A is the same as X in the imidazoquinoline compound shown in the formula I and R in the compound B 1 And R is 2 R in the imidazoquinoline compound of the above formula I 1 And R is 2 And will not be described in detail herein. Compound a and compound B are commercially available.
Step S02:
through the step S02, the compound A and the compound B are subjected to a first condensation reaction in a first reaction system, so that an amidation reaction of an amino group at a benzyl position of the compound A, for example, a dehydration condensation reaction of benzylamine of the compound A and carboxyl of the compound B, is carried out, and an imidazoquinoline compound shown in the formula I is formed, wherein the reaction is as follows:
in some embodiments, when Z comprises any of hydrogen, substituted or unsubstituted aryl, fused ring aryl, heteropolycyclic groups, Z may beTo be as same as R 2 The same or different groups.
In some embodiments, when Y isZ is->In the process, the compound B is shown as B1, and the compound prepared by the preparation method of the embodiment of the application is shown as follows:
at this time I 4 The structural formula and formula I are shown 3 The structure shown differs in that I 3 X in (2) 1 X is the same group as that of I 4 The structure shown.
In some embodiments, the reaction solvent that may control the first condensation reaction includes at least one of tetrahydrofuran, triethylamine, diisopropylethylamine, 4-dimethylaminopyridine, N-dimethylformamide, and dichloromethane, and the compound a and the compound B have better solubility in these solvents.
In some embodiments, the molar ratio of compound a to compound B can be controlled to be 1:1 to 1:10, further may be 1:1 to 1:5, may specifically be 1: 2. 1: 3. 1: 4. 1:5, etc., typically but not limited to molar ratios.
In some embodiments, the concentration of compound a in the reaction solution system of the first condensation reaction may be controlled to be 5 to 20mg/ml. In specific examples, the concentration of compound A may be, but is not limited to, typical 6mg/ml, 7mg/ml, 8mg/ml, 9mg/ml, 10mg/ml, 11mg/ml, 12mg/ml, 13mg/ml, 14mg/ml, 15mg/ml, 16mg/ml, 17mg/ml, 18mg/ml, 19mg/ml, etc.
In some embodiments, the reaction system of the first condensation reaction may be controlled to contain a condensing agent. In particular examples, the controllable condensing agent includes at least one of 2- (7-azabenzotriazol) -N, N, N ', N ' -tetramethyluronium Hexafluorophosphate (HATU), N, N-Diisopropylethylamine (DIEA), N, N ' -Diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazol, 1-propylphosphoric anhydride, dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide. In a further embodiment, the condensing agent may be dissolved in a solvent such as 50% ethyl acetate solution and then added to the first reaction system.
The molar ratio of the compound A to the compound B is controlled, the concentration of the compound A in the concentration of the reaction solution is controlled, and the condensation reagent is controlled, so that the occurrence of side reaction is effectively reduced, and the yield and purity of the imidazoquinoline compound shown in the formula I are improved.
In some embodiments, the molar ratio of compound a to the condensing agent may also be controlled, and further the molar ratio of compound a to the condensing agent may be controlled to be 1: 3-1: 15, further may be 1: 3-1: 10. for example, a specific example may be 1: 4. 1: 5. 1: 6. 1: 7. 1: 8. 1: 9. 1:10, etc. typical but non-limiting molar ratio. The molar ratio of compound a and the condensing agent is controlled within this range to further reduce side reactions. In a specific example, the molar ratio of compound a to compound B may be controlled to be 1:5, the molar ratio of the compound A to the T3P is 1: at the moment, no obvious by-product is found in the reaction product, and a cleaner target product can be obtained after the substrate is extracted.
In some embodiments, the reaction temperature of the first condensation reaction may be controlled to be 20 to 25 ℃. Specifically, the temperature may be, but is not limited to, typical temperatures of 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃ and the like.
In some embodiments, the reaction time of the first condensation reaction may be controlled to be 2 to 18 hours. Specifically, typical but non-limiting reaction times may be 2h, 5h, 10h, 15h, 18h, etc.
The reaction temperature and the reaction time of the first condensation reaction are controlled within the range, so that the occurrence of side reactions is effectively reduced, the yield of target products is improved, the energy consumption is reduced, and the preparation efficiency is improved.
In some embodiments, when Z in compound B is hydrogen, the product of the first condensation reaction has the molecular structural formula I as described above 2 As shown in the drawing,
i is a kind of 2 The molecular structure contains sulfhydryl group, and can control two formulas I 2 The molecule is condensed by dehydrogenation and oxidation between sulfhydryl groups, and as shown in fig. 2, after the first condensation reaction, the preparation method of the embodiment of the application can also be controlled to include:
step S021: the product of the first condensation reaction itself is subjected to a second condensation reaction.
By step S021, the compound of formula I 2 The mercapto groups between molecules of the first condensation reaction product are dehydrogenated and oxidized to form disulfide bonds, and the two disulfide bonds have the formula I 2 The molecule is formed as shown in formula I 5 The dimers shown.
I is a kind of 5 The structural formula is shown as formula I 3 The structural formulae shown differ in that: when formula I 3 X in (2) 1 And X is the same group, R 1 And R is 4 When the same groups are present, i.e. of formula I 5 The structural formula is shown.
In some embodiments, after the first condensation reaction and/or the second condensation reaction, the solvent in the reaction solution may be removed, and then the target product in the concentrated reaction solution is extracted with an oil-water mixture, separated, and the oil phase is dried, and then the oil phase solvent is removed to obtain the imidazoquinoline compound shown in formula I. In a further embodiment, the oil-water mixed solution is a mixed solution of ethyl acetate and water, and the imidazoquinoline compound has good solubility in the ethyl acetate, so that the extraction rate is improved, and meanwhile, the ethyl acetate is easy to volatilize, so that the subsequent removal of the oil phase solvent is facilitated.
In a third aspect, the embodiment of the application provides an application of the imidazoquinoline compound in preparing an anti-tumor drug and/or an antiviral drug.
According to the imidazoquinoline compound disclosed by the embodiment of the application, molecules with activity of exciting TLR7/8 receptors are selectively released in tumors, the content of inflammatory mediators in the tumors is improved, and the rise of pro-inflammatory cytokines such as TNF-alpha in blood plasma is effectively avoided, so that the imidazoquinoline compound has great application potential in preparing antitumor drugs. In addition, the imidazoquinoline compound has good antiviral effect due to the activation of the imidazoquinoline compound to the immune system of a human body, so that the imidazoquinoline compound can be applied to the preparation of antiviral drugs.
In some embodiments, in preparing an anti-tumor drug, the tumor may comprise a solid tumor. In further embodiments, the solid tumor may include at least one of T cell lymphoma, melanoma, breast cancer, rectal cancer, lung cancer, pancreatic cancer, prostate cancer, ovarian cancer, bone cancer, brain tumor. The solid tumor is easier to be administrated by intratumoral injection, so that the enrichment degree of molecules with activity of exciting TLR7/8 receptors in the tumor is further improved, and the anti-tumor effect of the imidazoquinoline compound is further improved.
In some embodiments, in preparing an antiviral drug, the virus may include hepatitis b virus (Hepatitis B virus, HBV).
In a fourth aspect, embodiments of the present application provide a composition comprising an imidazoquinoline compound as described above.
After the composition is administrated, the imidazoquinoline compound contained in the composition can be used as a prodrug, selectively release molecules with activity of exciting TLR7/8 receptors in tumors, activate immune cells in the tumors, promote the rise of pro-inflammatory cytokines such as TNF-alpha and the like, and have good selectivity.
In some embodiments, the compositions of the present application may be pharmaceutical compositions, and further, the pharmaceutical compositions may further include pharmaceutically acceptable excipients.
In some embodiments, the mode of administration of the composition may be controlled to be injection, further, subcutaneous injection and/or intratumoral injection, further, may include at least one of intratumoral bolus injection, subcutaneous bolus injection. The intratumoral quick injection is to inject the medicine into the tumor body tissue in a short time by a conventional injector, and the intratumoral control injection is to control the flow by using a microinjection pump through an indwelling needle, so that the concentration of the medicine in the tumor body tissue is maintained in an effective concentration range in the continuous injection time.
The imidazoquinoline compounds of the present application and methods for their preparation, use, compositions and the like are illustrated by the following examples.
1. Imidazoquinoline Compounds and examples of methods for their preparation
Example 11
This example provides an imidazoquinoline compound of formula (1) which is 78A1-SH,78A1-SH prepared as follows:
a100 mL single-port bottle was taken, 78A1 (200 mg,0.557 mmol), mercaptopropionic acid (295 mg,2.78 mmol) and THF (10 mL) were added, dissolved by magnetic stirring, 50% solution of T3P ethyl acetate (1.77 g,5.57 mmol) was added, mixed, triethylamine (169 mg,1.67 mmol) was then slowly added dropwise, and after mixing, the reaction mixture was stirred at room temperature for 2 hours to conduct the first condensation reaction. After the reaction was completed, the reaction solution was concentrated to remove THF, ethyl acetate and water were added thereto, the mixture was stirred and separated, the aqueous phase was extracted twice with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate and concentrated to obtain a crude product, and the crude product was purified to obtain 40mg of the objective product, wherein the purity of 78A1-SH in the objective product was 91.5%. The nuclear magnetic hydrogen spectrum of 78A1-SH is shown in FIG. 3.
Example 12
The imidazoquinoline compound shown in the formula (2) provided in the embodiment is 78A1-S-Trity, and the preparation process is as follows:
A100 mL single-necked flask was charged with 78A1 (200 mg,0.557 mmol), 3- (tritylthio) propionic acid (967 mg,2.78 mmol) and THF (10 mL), dissolved by magnetic stirring, a 50% solution of T3P ethyl acetate (1.77 g,5.57 mmol) was added and stirred to mix, triethylamine (169 mg,1.67 mmol) was then slowly added dropwise, stirred and mixed, and the reaction mixture was stirred at room temperature for 2 hours to carry out the first condensation reaction. After the reaction was completed, THF was removed by concentrating the reaction solution, ethyl acetate and water were added thereto, the mixture was separated by stirring, the aqueous phase was extracted twice with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated to obtain a crude product, and purified to obtain 50mg of the objective product. The nuclear magnetic hydrogen spectrum of 78A1-S-Trity is shown in FIG. 4.
Example 13
This example provides an imidazoquinoline compound of formula (3) which is 78A1-SS-78A1, prepared as follows:
to a 10mL vial containing a magneton was added compound 78A1 (200 mg, 0.552 mmol), dithiobissuccinimide propionate (103.60 mg,0.278 mmol), then 3mL of N, N-dimethylformamide was added and dissolved, triethylamine (84.45 mg,0.835 mmol) was slowly added dropwise, and after the addition was completed, the reaction was stirred at room temperature overnight. After LCMS confirmed the end of the reaction, the reaction was purified by HPLC to give 110mg. The nuclear magnetic hydrogen spectrum of 78A1-SS-78A1 is shown in FIG. 5, and the mass spectrum is shown in FIG. 6.
2. Imidazoquinoline Compound Activity assay
Example 21: detection of lymphocyte TNF-alpha-inducible expression Activity
SPF-grade BALB/C,5-6 week old, 18-20 g female mice, was taken, and whole blood of the mice was extracted by eyeball extraction for lymphocyte extraction from peripheral blood. The same volume of whole blood dilution was added to whole blood and mixed thoroughly, and added to the same volume of mouse peripheral blood lymphocyte separation solution, the running parameters of the centrifuge were set to 1000g, centrifuged for 20min, the lymphocyte layer was carefully taken, washed with 10mL of Phosphate Buffer (PBS), and centrifuged for 10min at 250 g. The supernatant was discarded and the lymphocytes resuspended in complete RPMI-1640 cell culture medium. The whole blood dilution and lymphocyte separation liquid of the mice used in the experiment are purchased from Beijing Soy Bao technology Co., ltd, wherein the kit of the peripheral blood lymphocyte separation liquid of the mice is provided with the product number P8620.
R848 (control), 78A1-SH, 78A1-S-Trityl and 78A1-SS-78A1 were taken, and RPMI-1640 cell culture solutions of three concentrations of low, medium and high were prepared for each compound. Wherein 78A1-SH readily polymerize into dimers, so that 78A1-SH exists as dimers in the storage state, or in the solution state, of the portions 78A 1-SH. R848 and 78A1 were used as control groups, 78A1-SH, 78A1-S-Trityl and 78A1-SS-78A1 were used as experimental groups, and RPMI-1640 cell culture solution was used as blank control group.
Wherein, the structural formula of R848 is as follows:
mu.L of RPMI-1640 cell culture medium from each of the above groups was added to 100. Mu.L of 1.5X 10 5 In each of the normal lymphocyte wells, each group of peripheral lymphocyte culture solutions was prepared, and the concentrations of the different compounds in each peripheral lymphocyte culture solution were as shown in table 1. Since 78A1-SS-78A1 is a dimer, 78A1-SS-78A1 is present in a molar concentration of one half of 78A 1.
TABLE 1
The peripheral lymphocyte culture solutions of each group were placed at 37℃with 5% CO 2 After incubation for 24 hours in a cell culture box, centrifuging at 1000rpm for 5 minutes, collecting cell supernatant, and detecting the concentration of TNF-alpha in the supernatant of the peripheral blood mononuclear cell culture solution according to ELISA specification operation, wherein the manufacturer of the mouse TNF-alpha double-antibody sandwich ELISA detection kit for detection is Wuhan Sanying, and the product number isKE10002。
As shown in FIG. 7, the TNF-. Alpha.content of the 78A1 group was significantly increased over the dose range tested, and the TNF-. Alpha.contents of the 78A1-SH group, 78A1-S-Trityl group and 78A1-SS-78A1 group were not significantly different from those of the blank group, while the TNF-. Alpha.content was significantly increased in the medium and high concentration dose groups of R848. This indicates that compounds 78A1-SH, 78A1-S-Trityl and 78A1-SS-78A1 have no significant activity for inducing lymphocyte TNF-alpha expression. Among them, since 78A1-SH was unstable during the cryopreservation, it was examined using LC-MS for one week of storage, and it was observed that most of it was a dimer structure, and a smaller amount was present as a monomer. Comparing the results of the detection of the induced expression activity of lymphocyte TNF-alpha, the 78A1-SH group has no obvious difference with the 78A1-SS-78A1 group, and has no obvious activity on the induced lymphocyte TNF-alpha expression.
Example 22: detection of induced expression Activity of TNF-alpha in mouse serum by subcutaneous injection
Taking 78A1 and 78A1-SS-78A1, and respectively preparing PBS solutions with proper concentrations of 78A1 and 78A1-SS-78A1 by taking Phosphate Buffer Solution (PBS) as a solvent.
SPF-grade BALB/C,5-6 week old, 18-20 g female mice were purchased from Henan province laboratory animal center. The mice were divided into 3 groups, A1 group, A2 group and A3 group, respectively, wherein the A1 group and the A2 group are 12 each, and the A3 group is 3.
Group A1 mice were subcutaneously injected with 100. Mu.L of the 78A1 solution, group A2 mice were subcutaneously injected with 100. Mu.L of the 78A1-SS-78A1 solution, and group A3 mice were subcutaneously injected with 100. Mu.L of PBS solution. And the injection dose of each group of mice was set as shown in table 2.
TABLE 2
Grouping Injected compounds Dosage of administration
Group A1 78A1 The molar amount is consistent with that of 10 mu g R848
A2 group 78A1-SS-78A1 The molar amount is half of the molar amount of 10 mu g R848
A3 group PBS ——
3 mice were bled from the orbit 5min, 30min, 60min and 240min post-subcutaneous injection for groups A1 and A2, respectively, and 3 mice from group A3 were bled from the orbit 1h post-subcutaneous injection. After the orbit was collected, the collected mouse blood was centrifuged at 3800rpm for 10min to separate serum, and the concentration of TNF-. Alpha.in the serum was measured in the same manner as in example 21.
The results of the experiment are shown in fig. 8, wherein the significance of the differences in the present description was analyzed using one/two way ANOVA (ns: p >0.05, p <0.01, p <0.001, p < 0.0001).
The results show that the TNF-alpha content in serum of the mice in group A1 of the subcutaneous bolus 78A1 is obviously increased, and the TNF-alpha content in serum of the mice reaches a peak value after 1h injection. The TNF- α content in serum of mice injected with 78A1 was up to 10-30 fold higher relative to mice injected with PBS. The A2 group of mice injected with 78A1-SS-78A1 subcutaneously had only A1-3 fold small increase in serum TNF- α levels over 4h of injection compared to the PBS-injected mice, and the A2 group injected with 78A1-SS-78A1 had a significantly reduced increase compared to the A1 group injected with 78A 1.
Example 23: detection of induced expression Activity of TNF-alpha in mouse serum after 1h of subcutaneous injection
R848, 78A1-S-Trityl and 78A1-SS-78A1, 78A1-SH compounds are taken, PBS is taken as solvent to prepare compound solutions with proper concentration.
The same mice as in example 22 were divided into five groups, respectively, group B1 to group B5, each group having 3 mice. Groups B1 to B5 mice were subcutaneously injected with 100. Mu.L of each of the above-mentioned compounds having the appropriate solutions and PBS, and the amounts of each group of mice administered are shown in Table 3.
TABLE 3 Table 3
A mouse Subcutaneous injection solution Dosage of administration
B1 group R848 solution 10μg
B2 group 78A1 solution The molar amount is consistent with the molar amount of R848 of 10 mug
B3 group 78A1-SH solution The molar amount is consistent with the molar amount of R848 of 10 mug
Group B4 78A1-S-Trityl solution The molar amount is consistent with the molar amount of R848 of 10 mug
Group B5 78A1-SS-78A1 solution The molar amount is half of the molar amount of 10 mu g R848
B6 group PBS ——
60min after subcutaneous injection of mice from groups B1 to B6, orbital blood was collected, centrifuged at 3800rpm for 10min, serum was isolated, and the concentration of TNF- α in the serum was measured, and the method for measuring the concentration of TNF- α was the same as in example 22.
The results of the experiment are shown in FIG. 9, and the results show that 1h after subcutaneous R848 and 78A1, the TNF-alpha content in the serum of the mice is obviously increased and is obviously higher than that of the serum of the mice after PBS is injected subcutaneously. However, 1h after subcutaneous injections of 78A1-SH, 78A1-S-Trityl and 78A1-SS-78A1, the TNF- α content in serum was not significantly different from that in the PBS group, indicating a significant improvement in the safety of 78A1-SH, 78A1-S-Trityl and 78A1-SS-78A1 at the tested doses.
Example 24: detection of TNF-alpha-induced expression Activity in intratumoral injected tumors and serum
Mice identical to example 22 were divided into three groups, C1, C2 and C3, wherein 8 groups of C1 and C2 each, and 2 groups of C3 are blank groups.
CT-26 colon cancer cells purchased from ATCC were cultured at 37℃with 5% CO 2 In the incubator, subcultured with DMEM medium containing 10% fetal bovine serum. The specific method comprises discarding culture supernatant, and washing cells with PBS without calcium and magnesium ions for 1-2 times. Adding 3-5ml of digestive juice (0.25% Trypsin-0.53mM EDTA) into a culture flask, placing into a 37 ℃ incubator for digestion for 1-2min, observing the digestion condition of cells under a microscope, if most of the cells become round and fall off, rapidly taking back to a clean bench, and adding 5ml of complete culture medium containing 10% serum to stop digestion. Gently blowing the cells until the cells fall off completely, sucking out, centrifuging under the condition of 250g8-10 min, discarding supernatant, adding 1-2mL culture solution, and blowing uniformly. The culture solution is added according to 5-6 ml/bottle, and the cell suspension is added according to 1:2 to 1:4 to a new T75 cm containing 5-6ml of culture medium 2 In a cell culture flask. And selecting cells with good growth conditions in the logarithmic growth stage, grafting tumors on the left armpits of the mice in the groups C1 to C3, and constructing a mouse tumor model. On the day of inoculation, the tumor cell solution was centrifuged at 1000rpm for 5min, and the supernatant was discarded, and the final cell concentration was adjusted to 5X 10 5 Mice were engrafted with tumor subcutaneously at the left underarm at 100 μl.
About one week after inoculation, tumors grow to 100mm 3 . 78A1 and 78A1-SS-78A1 are taken, and 78A1 solution and 78A1-SS-78A1 solution with proper concentrations are prepared by taking PBS as a solvent respectively. The PBS, 78A1 solution and 78A1-SS-78A1 solution were injected into the tumors of the mice, respectively, with A1 mL syringe, at 50. Mu.L. The doses administered to each group of mice are shown in Table 4.
TABLE 4 Table 4
A mouse Injection liquid Dosage for administration
C1 group 78A1 solution The molar amount is consistent with the molar amount of R848 of 10 mug
C2 group 78A1-SS-78A1 solution Half the molar amount of R848 of 10. Mu.g
Group C3 PBS ——
After 5min, 30min, 60min and 240min of intratumoral injection, 2 mice were taken and orbital blood was taken, and after 1h of intratumoral injection, 2 mice were taken and orbital blood was taken from group C3. After orbital blood was collected from each group of mice, the serum was separated by centrifugation at 3800rpm for 10min, tumor tissue was simultaneously dissected and collected, and after weighing the tumor tissue, 1.0mL of PBS was added to 0.1g of tumor tissue for homogenization, and the concentration of TNF- α in the serum and tumor homogenate was measured, and the measurement method was the same as in example 22.
The experimental results are shown in fig. 10 (a and B), and the results show that after intratumoral rapid injection of 78A1, the TNF-alpha content in the serum and in the tumor of the mice is obviously increased, and the TNF-alpha content in the serum and in the tumor of the mice reaches the peak value and the amplification reaches 10-30 times 1 hour after injection. In addition, the trend of the TNF-alpha content in the serum of the intratumoral bolus 78A1 and the intratumoral bolus is basically the same.
Within 4h of intratumoral bolus injection of 78A1-SS-78A1, the TNF-alpha level in serum was only 1-2 times as much increased, which was greatly reduced compared with 10-30 times as much as the TNF-alpha level in serum of mice injected with 78A1, indicating a greatly improved safety of 78A1-SS-78A 1. Meanwhile, the change trend of TNF-a level in tumors of two groups of mice injected with 78A1 and 78A1-SS-78A1 is basically consistent, compared with the C3 group injected with PBS, the TNF-a in tumors reaches a peak value at 1h after injection.
Example 25: detection of TNF-alpha-induced expression Activity in intratumoral injected tumors and serum
The same mice as in example 24 were divided into five groups, D1 to D5, respectively, 3 each.
Mouse tumor molding was performed by the same molding method as in example 24.
About one week after inoculation, tumors grow to 100mm 3 . R848, 78A1-S-Trityl and 78A1-SS-78A1 are taken, and PBS is taken as solvent to prepare R848, 78A1-S-Trityl and 78A1-SS-78A1 solutions with proper concentrations. Using a 1mL syringeThe above PBS, R848, 78A1-S-Trityl and 78A1-SS-78A1 solutions were injected in the tumors of the mice, respectively, at 50. Mu.L. The doses administered to each group of mice are shown in Table 5.
TABLE 5
After 60min of injection, 3 mice were taken from each group, orbital blood was taken from each group, centrifuged at 3800rpm for 10min, serum was separated, tumor tissue was dissected and taken, and after weighing the tumor tissue, 1.0mL of PBS was added to 0.1g of tumor tissue for homogenization, and the concentration of TNF- α in the serum and tumor homogenate was detected in the same manner as in example 22.
The experimental results are shown in fig. 11 (a and B), and the results show that after intratumoral rapid injection of R848 and 78A1, the TNF- α content in the serum of the mice is significantly increased, and the TNF- α in the serum of the mice is 10-30 times as high as that in the PBS control group 1h after injection. The intratumoral bolus 78A1-S-Trityl and 78A1-SS-78A1 serum showed only A1-2 fold small increase in TNF- α levels, with a significant difference compared to the 78A1 group, as compared to the 10-30 fold increase in TNF-a content in the serum of mice injected with 78A 1. Meanwhile, there was no significant difference in TNF-a content in tumors of the two groups of mice injected with 78A1 and 78A1-SS-78A1, and there was a significant increase compared to the group D5 injected with PBS.
Example 26: antitumor Activity assay
The same mice as in example 24 were divided into 7 groups, each of which was E1 to E7, and 6 mice each were subjected to tumor molding by the same molding method as in example 24.
About one week after inoculation, tumors grow to 100mm 3 PBS is taken as a solvent, and R848, 78A1-S-Trityl and 78A1-SS-78A1 are taken to prepare a solution with proper concentration, and the intratumoral injection administration is carried out on mice. Wherein, the E1 group to the E6 group are injected rapidly in tumor by adopting a 1mL syringe, and the volume of the injected solution is 50 mu L; e7 groupThe administration was continued using a microinjection pump for 25 hours, with a volume of 500. Mu.L. The doses of drug and compound administered for injection into mice of groups E1 through E7 are shown in Table 6.
TABLE 6
Mice in groups E1 to E7 of this example were dosed once every 3 days for a total of 2 doses. The change in body weight and tumor volume of mice was measured every 2 days during the course of dosing until 22 days after dosing, wherein the tumor volume was measured in the following manner: the size of the long diameter (l) and the short diameter (w) of the tumor is measured by a vernier caliper, and the volume formula is adopted: v=0.5×l×w 2 Tumor volumes were calculated. After 22 days, the treatment was completed, the mice were euthanized, the dissected tumor was taken and the results were analyzed. The experimental results are shown in fig. 12 to 16.
As shown in fig. 12, mice in the intratumoral bolus 78A1 group showed a significant trend of decrease in body weight at the early stage of treatment compared to the PBS injection group. While the intratumoral bolus 78A1-SS-78A1 10 μg group, 78A1-SS-78A1 20 μg group and 78A1-SH mice had no significant change in body weight compared to the PBS injection group.
As shown in FIGS. 13 to 16, intratumoral bolus doses of 10. Mu.g of 78A1-SH, 78A1-S-Trityl and 78A1-SS-78A1, and a dose of 20. Mu.g of 78A1-SS-78A1 were effective in inhibiting tumor growth, with no significant difference from the experimental results of injection of 78A 1. In addition, under the condition that 78A1-SS-78A1 is matched with a microinjection pump for controlling injection (78A 1-SS-78A1 10 mug CI), although 2 mice have tumors to grow up, 4 mice can observe the tumor clearing at the earliest 10 th day, and the final result has no obvious difference compared with other groups, so that 78A1-SS-78A1 is matched with the microinjection pump and has practical clinical research value.
The foregoing examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims.

Claims (10)

1. An imidazoquinoline compound which is a compound of formula I:
the X comprises any one of n-propyl, n-butyl, n-butylamine, n-amyl and 2-methoxyethyl;
the R is 1 Comprises C 1 -C 36 Alkyl, C 1 -C 36 Any one of the alkoxy groups;
the R is 2 Comprising hydrogen, substituted or unsubstituted aryl, fused ring aryl, heteropolycyclic groups, -S-R 3 Any one of the above, wherein the R 3 Including any of substituted or unsubstituted aryl, fused ring aryl, heteropolycyclic groups.
2. The imidazoquinoline compound of claim 1, wherein:
x is n-butyl; and/or
The R is 3 Is represented by formula II:
wherein the X is 1 Comprises any one of n-propyl, n-butyl, n-butylamine, n-amyl, 2-methoxyethyl and R 4 Comprises C 1 -C 36 Alkyl, C 1 -C 36 Any one of the alkoxy groups; and/or
The R is 4 Comprises C 1 -C 36 Alkyl, C 1 -C 36 Any one of the alkoxy groups.
3. The imidazoquinoline compound of any one of claims 1-2, wherein the structural formula of the imidazoquinoline compound comprises at least one of the following formulae (1) - (4):
4. a process for the preparation of an imidazoquinoline compound according to any one of claims 1 to 3, comprising the steps of:
providing compound a and compound B:
carrying out a first condensation reaction on the compound A and the compound B to obtain an imidazoquinoline compound shown in the formula I;
wherein said Y comprises-COOH,Any one of them;
the Z comprises hydrogen, substituted or unsubstituted aryl, condensed ring aryl, heteropolycyclic group,Any one of the above, wherein the R 4 Comprises C 1 -C 36 Alkyl, C 1 -C 36 Alkoxy, said X 2 Comprising hydrogen, & gt>Any one of them.
5. The production method according to claim 4, wherein the conditions for the first condensation reaction include at least one of the following conditions (1) to (6):
(1) The reaction solvent comprises at least one of tetrahydrofuran, triethylamine, diisopropylethylamine, 4-dimethylaminopyridine, N-dimethylformamide and dichloromethane;
(2) The molar ratio of the compound A to the compound B is 1:1 to 1:10;
(3) The concentration of the compound A in the reaction solution system of the first condensation reaction is 5-20 mg/ml;
(4) The reaction solution system of the first condensation reaction contains a condensation reagent, and the molar ratio of the compound A to the condensation reagent is 1: 3-1: 15;
(5) The reaction temperature is 20-25 ℃;
(6) The reaction time is 2-18 h.
6. The method of claim 5, wherein the condensing agent comprises at least one of 2- (7-azabenzotriazol) -N, N '-tetramethylurea hexafluorophosphate, N-diisopropylethylamine, N' -diisopropylcarbodiimide, 1-hydroxybenzotriazole, 1-propylphosphoric anhydride, dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide.
7. The method of any one of claims 4-6, wherein when Z is hydrogen, after the first condensation reaction, the method further comprises subjecting the product molecules of the first condensation reaction to a second condensation reaction, the second condensation reaction being a sulfhydryl dehydrogenation oxidation reaction; wherein the sulfhydryl is the sulfhydryl contained in the imidazoquinoline compound shown in the formula I.
8. Use of an imidazoquinoline compound according to any one of claims 1-3 for the manufacture of an antitumor drug and/or for the manufacture of an antiviral drug.
9. The use according to claim 8, wherein:
the tumor comprises solid tumor, and the solid tumor comprises at least one of skin T cell lymphoma, melanoma, breast cancer, rectal cancer, lung cancer, pancreatic cancer, prostatic cancer, ovarian cancer, bone cancer and brain tumor; and/or
The virus includes hepatitis b virus.
10. A composition comprising an imidazoquinoline compound according to any one of claims 1 to 3 or an imidazoquinoline compound obtainable by a process according to any one of claims 4 to 6.
CN202310566830.XA 2023-05-18 2023-05-18 Imidazoquinoline compounds, methods of preparation, use and compositions thereof Pending CN116731015A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101824034A (en) * 2004-09-14 2010-09-08 诺华疫苗和诊断公司 Imidazoquinolie compounds
CN101980707A (en) * 2008-03-24 2011-02-23 4Sc股份有限公司 Novel substituted imidazoquinolines
CN108368140A (en) * 2015-12-14 2018-08-03 葛兰素史密丝克莱恩生物有限公司 PEGylated imidazoquinolines as TLR7 and TLR8 agonists
CN114599360A (en) * 2019-10-29 2022-06-07 骏达贸易有限公司 4-amino-imidazoquinoline compounds and uses thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101824034A (en) * 2004-09-14 2010-09-08 诺华疫苗和诊断公司 Imidazoquinolie compounds
CN101980707A (en) * 2008-03-24 2011-02-23 4Sc股份有限公司 Novel substituted imidazoquinolines
CN108368140A (en) * 2015-12-14 2018-08-03 葛兰素史密丝克莱恩生物有限公司 PEGylated imidazoquinolines as TLR7 and TLR8 agonists
CN114599360A (en) * 2019-10-29 2022-06-07 骏达贸易有限公司 4-amino-imidazoquinoline compounds and uses thereof

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