Substituted 3, 7-dihydro-1h-purine-2, 6-diones and uses thereof

Abstract

The present invention relates to a 3, 7, 8-trisubstituted 3, 7-dihydro-1H-purine-2, 6-dione compound (also named as a 3, 7, 8-trisubstituted xanthine compound of general formula (I)) or a pharmaceutically acceptable salt thereof. The invention further relates to pharmaceutical compositions comprising such compounds and to the use of said compounds or pharmaceutical compositions as medicaments, in particular in methods of treating or preventing neoplastic and / or infectious diseases and in vitro methods. These compounds activate immune cells, in particular T cells such as CD4 + and CD8 + cells. # imgabs0 #

Description

Field of the Invention

[0001] The present invention relates to a 3,7,8-trisubstituted 3,7-dihydro-1H-purine-2,6-dione compound (also named a 3,7,8-trisubstituted xanthine compound of general formula (I)) or a pharmaceutically acceptable salt thereof.

[0002]

[0003] The present invention further relates to pharmaceutical compositions comprising such compounds and their use as medicaments, in particular in methods for treating or preventing neoplastic and / or infectious diseases and in vitro methods. These compounds activate immune cells, including NK cells or T cells such as CD4+ and CD8+ cells. Background of the Invention

[0005] Malignant tumors (cancer) and infectious diseases are two of the leading causes of death worldwide. An increasing number of cases show that there is often a mutual dependence between neoplastic and infectious diseases, such as, for example, between cervical tumors and herpes simplex virus infection. Although a large number of compounds for the treatment and prevention of these diseases have been discovered, it is well known that such compounds have significant disadvantages, such as causing serious side effects. Therefore, there is still an unmet need for new compounds for the treatment and prevention of neoplastic and / or infectious diseases.

[0006] In order to overcome these shortcomings, therapeutic and preventive schemes based on regulating the patient's immune response are becoming increasingly important in current medicine. Therefore, the patient's immune activity is usually supported by drug therapy. In practice, under this background, immunotherapy for neoplastic and infectious diseases is particularly interesting. In this regard, it is well known that in vivo, several types of immune cells, such as, for example, natural killer (NK) cells, T cells, B cells, dendritic cells, monocytes and macrophages, often participate in the inactivation and removal of pathogens. Taking tumors as an example, it is well known that each mature neoplasm (tumor) carries specific antigens and / or neoantigens (for example, Sensi and Anichini, 2006, Clin Cancer Res.12:5023-5032). This can usually trigger the adaptive immune system (for example, T cells and / or B cells) and the innate immune system (for example, natural killer (NK) cells). The immune system in healthy bodies is usually enough to effectively prevent or cure the neoplastic and infectious diseases of the body.

[0007] CD4+ T cells, along with CD8+ T cells, make up the majority of T lymphocytes. After activation and differentiation into different effector subtypes, CD4+ T cells play a crucial role in mediating immune responses through the secretion of specific cytokines. CD4+ T cells have multiple functions, including activating cells of the innate immune system, B lymphocytes, cytotoxic T cells, and non-immune cells, and they play a key role in suppressing immune responses.

[0008] Cytotoxic CD8 + T cells also play a key role in eliminating intracellular infections and malignant cells and can provide long-term protective immunity. + T cell metabolism is coupled to transcriptional, translational, and epigenetic changes driven by extracellular metabolites and immune signals. These programs contribute to CD8 + T cells adapt to the diverse and dynamic metabolic environments they encounter in the circulation and in tissues.

[0009] In some cases, however, the immune system cannot eliminate such neoplastic or infectious diseases, and such diseases become chronic diseases. In these cases, particularly when a patient suffers from a malignant tumor (cancer), the immune system is often downregulated. In healthy bodies (i.e., in a non-suppressed immune environment), it is found that the appropriate expression of the major histocompatibility complex I (MHC I) of antigen presentation to immune cells (e.g., cytotoxic CD8 T cells) is reduced, but the MHC I expression in tumor cells is reduced. NK cells can resist this phenomenon, particularly identify and destroy cells with reduced MHC-I surface expression. However, in the process of vegetation maturation (particularly in the process of tumor progression), due to multiple immunosuppressive mechanisms causing immune tolerance, mature vegetation can increasingly escape the immune system, i.e., vegetation antigens are not identified as non-self, and the immune system is not activated. This mechanism is the universal principle of vegetation maturation, is neither limited to specific vegetation nor depends on specific vegetation antigens. It is noteworthy that it has been found that in most cancer patients, although tumor-associated T cells and NK cells are present, they do not produce sufficient amounts of cytokines (e.g., IL-2 and IFN-γ) or do not exert cytotoxic activity against tumors, because inhibition achieved through various mechanisms hinders effective anti-tumor immune responses (De Paola et al., 2003, British Journal of Cancer 88:320-326; Ahmadzadeh et al., 2009, Blood 114:1537-1544, especially pages 1541-1542, "PD-1+TILs display an impaired effector function" section). This is obviously one of the reasons why tumor vaccines often fail.

[0010] Similarly, it is known that a variety of infections can downregulate a patient's immune system, particularly viral infections such as, for example, human immunodeficiency virus (HIV) infection or herpes simplex virus (HSV) infection. In this context as well, the production of cytokines by T cells and other antiviral immune cells is also disrupted.

[0011] There remains an unmet need for compounds that are able to increase immunogenic activity and thereby be able to treat and / or prevent neoplastic and / or infectious diseases.

[0012] The present invention aims to provide compounds and pharmaceutical compositions that activate immune cells, particularly T cells. These compounds can be used as pharmaceutically active agents, particularly for preventing and / or treating neoplastic and infectious diseases.

[0013] The present invention provides a novel 3,7,8-trisubstituted xanthine compound of general formula (I), which can activate immune cells.

[0014] Therefore, the objects of the present invention are solved by the teaching of the independent claims. Further advantageous features, aspects and details of the present invention are apparent from the dependent claims, the description, the drawings and the examples of the present application.

[0015] Description of the Invention

[0016] Therefore, the present invention relates to compounds of formula (I):

[0017]

[0018] in

[0019] A stands for

[0020] B is -OR 3 、-O-CHR 3 R 3* 、-O-CH2-CH2-R 3 or -O-CH2-CH2-CH2-R 3 ; R 1 represent

[0021] R 2a and R 2bEach independently represents -H, -F, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CH2OCH3, -CH2CH2OCH3, -CF2-CH3, -CHF-CHF2, -CHF-CF3, -CF2-CF3; wherein R 2a Not -H;

[0022] R 3 represent

[0023] R 3* represents -H, -F, -CH3, -C2H5, -C3H7, -CH(CH3)2, -CH2F, -CHF2, -CF3, -CH2-CF3;

[0024] R 4 and R 4* represents independently of one another -H, -F, -Cl, -Br, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -OCH3, -OC2H5, -OC3H7, -OCH(CH3)2, -OCH2F, -OCHF2, -OCF3, -CH2-CF3, -CHF-CH2F, -CHF-CHF2, -CHF-CF3, -CF2-CH3, -CF2-CH2F, -CF2-CHF2, -CF2-CF3,

[0025] R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 and R 13 represents independently of each other -H, -cyclo-C3H5, -cyclo-C4H7, -cyclo-C5H9, -cyclo-C6H 11 、-cyclo-C7H 13, -cycloC3H5O, -OH, -OCH3, -OC2H5, -OC3H7, -O-cyclo-C3H5, -OCH(CH3)2, -OC(CH3)3, -OC4H9, -OPh, -OCH2-Ph, -OCPh3, -CH2-OCH3, -C2H4-OCH3, -C3H6-OCH3, -CH2-OC2H5, -C2H4-OC2H5, -C3H6-OC2H5, -CH2-OC3H7, -C2H4-OC3H7, -C3H6-OC3H7, -CH2-O-cyclo-C3H5, -C2H4-O-cyclo-C3H5, -C3H6-O-cyclo-C3H5, -CH2-OCH(CH 3)2, -C2H4-OCH(CH3)2, -C3H6-OCH(CH3)2, -CH2-OC(CH3)3, -C2H4-OC(CH3)3, -C3H6-OC(CH3)3, -CH2-OC4H9, -C2H4-OC4H9, -C3H6-OC4H9, -CH2-OPh, - C2H4-OPh, -C3H6-OPh, -CH2-OCH2-Ph, -C2H4-OCH2-Ph, -C3H6-OCH2-Ph, -SH, -SCH3, -SC2H5, -SC3H7, -S-cyclo-C3H5, -SCH(CH3)2, -SC(CH3)3, -F, -Cl, -Br , -I, -CN, -COCH3, -COC2H5, -COC3H7, -CO-cyclo-C3H5, -COCH(CH3)2, -COC(CH3)3, -COOH, -COOCH3, -COOC2H5, -COOC3H7, -COO-cyclo-C3H5, -COOCH(CH3)2, -COOC(CH3)3, -OOC-CH3, -OOC-C2H5, -OOC-C3H7, -OOC-cyclo-C3H5, -OOC-CH(CH3)2, -OOC-C(CH3)3, -CONH2, -CONHCH3, -CONHC2H5, -CONHC3H7, -CONH-cyclo-C3H 5. -CONH[CH(CH3)2], -CONH[C(CH3)3], -CON(CH3)2, -CON(C2H5)2, -CON(C3H7)2, -CON(ring-C3H5)2, -CON[CH(CH3)2]2, -CON[C(CH3)3]2, -NHCOCH3, -NH COC2H5, -NHCOC3H7, -NHCO-ring-C3H5, -NHCO-CH(CH3)2, -NHCO-C(CH3)3, -NHCO-OCH3, -NHCO-OC2H5, -NHCO-OC3H7, -NHCO-O-ring-C3H5, -NHCO-OCH(CH3)2,-NHCO-OC(CH3)3, -NH2, -NHCH3, -NHC2H5, -NHC3H7, -NH-cyclo-C3H5, -NHCH(CH3)2, -NHC(CH3)3, -N(CH3)2, -N(C2H5)2, -N(C3H7)2, -N(cyclo-C3H5)2, -N[CH(CH3)2]2, -N[C(CH3)3]2, -SOCH3, -SOC2H5, -SOC3H7, -SO-cyclo-C3H5, -SOCH(CH3)2, -SOC(CH3)3, -SO2CH3, -SO2C2H5, -SO2C3H7, -SO2-cyclo-C3H5, -SO2CH(CH3)2, -SO2C(CH3)3, -SO3H, -SO3CH3, -SO3C2H5, -SO3C3H7, -SO3-cyclo-C3H5, -SO3CH(CH3)2, -SO3C(CH3)3, -SO2NH2, -SO2NHCH3, -SO2NHC2H5, -SO2NHC3H7, -SO2NH-cyclo-C3H5, -SO2NHCH(CH3)2, -SO2NHC(CH3)3, -SO2N(CH3)2, -SO2N(C2H5)2, -SO2N(C3H7)2, -SO2N(cyclo-C3H5)2, -SO2N[CH(CH3)2]2, -SO2N[C(CH3)3]2, -O-S(=O)CH3, -O-S(=O)C2H5, -O-S(=O)C3H7, -O-S(=O)-cyclo-C3H5, -O-S(=O)CH(CH3)2, -O-S(=O)C(CH3)3, -S(=O)(=NH)CH3, -S(=O)(=NH)C2H5, -S(=O)(=NH)C3H7, -S(=O)(=NH)-cyclo-C3H5, -S(=O)(=NH)CH(CH3)2, -S(=O)(=NH)C(CH3)3, -NH-SO2-CH3, -NH-SO2-C2H5, -NH-SO2-C3H7, -NH-SO2-cyclo-C3H5, -NH-SO2-CH(CH3)2, -NH-SO2-C(CH3)3, -O-SO2-CH3, -O-SO2-C2H5, -O-SO2-C3H7, -O-SO2-cyclo-C3H5, -O-SO2-CH(CH3)2, -O-SO2-C(CH3)3, -OCH2F, -OCHF2, -OCF3, -CH2-OCF3, -C2H4-OCF3, -C3H6-OCF3, -CH2-OCHF2, -C2H4-OCHF2, -C3H6-OCHF2, -OC2F5, -CH2-OC2F5, -C2H4-OC2F5, -C3H6-OC2F5, -O-COOCH3-O-COOC2H5, -O-COOC3H7, -O-COO-cyclo-C3H5, -O-COOCH(CH3)2, -O-COOC(CH3)3, -NH-CO-NH2, -NH-CO-NHCH3, -NH-CO-NHC2H5, -NH-CO-NHC3H7, -NH-C(=NH)-NH2, -NH-CO-N(C3H7)2, -NH-CO-NH[CH(CH3)2], -NH-CO-NH[C(CH3)3], -NH-CO-N(CH3)2, -NH-CO-N(C2H5)2, -NH-CO-NH-cyclo-C3H5, -NH-CO-N(cyclo-C3H5)2, -NH-CO-N[CH(CH3)2]2, -NH-C(=NH)-NHCH3, -NH-C(=NH)-NHC2H5, -NH-C(=NH)-NHC3H7, -O-CO-NH-cyclo-C3H5, -NH-C(=NH)-NH-cyclo-C3H5, -NH-C(=NH)-NH[CH(CH3)2], -O-CO-NH[CH(CH3)2], -NH-C(=NH)-NH[C(CH3)3], -NH-C(=NH)-N(CH3)2, -NH-C(=NH)-N(C2H5)2, -NH-C(=NH)-N(C3H7)2, -NH-C(=NH)-N(cyclo-C3H5)2, -O-CO-NHC3H7, -NH-C(=NH)-N[CH(CH3)2]2, -NH-C(=NH)-N[C(CH3)3]2, -O-CO-NH2, -O-CO-NHCH3, -O-CO-NHC2H5, -O-CO-NH[C(CH3)3], -O-CO-N(CH3)2, -O-CO-N(C2H5)2, -O-CO-N(C3H7)2, -O-CO-N(cyclo-C3H5)2, -O-CO-N[CH(CH3)2]2, -O-CO-N[C(CH3)3]2, -O-CO-OCH3, -O-CO-OC2H5, -O-CO-OC3H7, -O-CO-O-cyclo-C3H5, -O-CO-OCH(CH3)2, -O-CO-OC(CH3)3, -CH2F, -CHF2, -CF3, -CH2-CH2F, -CH2-CHF2, -CH2-CF3, cyclo-C8H, 15 , -Ph, -CH2-Ph, -CH2-CH2-Ph, -CH=CH-Ph, -CPh3, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9, -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H 11、-CH(CH3)-C3H7、-CH2-CH(CH3)-C2H5、-CH(CH3)-CH(CH3)2、-C(CH3)2-C2H5、-CH2-C(CH3)3、-CH(C2H5)2、-C2H4-CH(CH3)2、-C6H 13 、-C7H 15 、-C8H 17、-C3H6-CH(CH3)2、-C2H4-CH(CH3)-C2H5、-CH(CH3)-C4H9、-CH2-CH(CH3)-C3H7 、 -CH(CH3)-CH2-CH(CH3)2 、-CH(CH3)-CH(CH3)-C2H5、 -CH2-CH(CH3)-CH(CH3)2 、-CH2-C(CH3)2-C2H5、-C(CH3)2-C3H7、-C(CH3)2-CH(CH3)2、-C2H4-C(CH3)3、-CH(CH3)-C(CH3)3、-CH=CH2、-CH2-CH=CH2、-C(CH3)=CH2、-CH=CH-CH3、-C2H4-CH=CH2、-CH2-CH=CH-CH3、-CH=CH-C2H5、-CH2-C(CH3)=CH2、-CH(CH3)-CH=CH、-CH=C(CH3)2、-C(CH3)=CH-CH3、-CH=CH-CH=CH2、-C3H6-CH=CH2、-C2H4-CH=CH-CH3、-CH2-CH=CH-C2H5、-CH=CH-C3H7、-CH=CH-CH=CH-CH3、-C2H4-C(CH3)=CH2、-CH2-CH(CH3)-CH=CH2、-CH(CH3)-CH2-CH=CH2、-CH2-CH=C(CH3)2、-CH2-C(CH3)=CH-CH3、-CH(CH3)-CH=CH-CH3、-CH=CH-CH(CH3)2、-CH=C(CH3)-C2H5、-C(CH3)=CH-C2H5、-C(CH3)=C(CH3)2、-C(CH3)2-CH=CH2、-CH(CH3)-C(CH3)=CH2、-C4H8-CH=CH2、-C3H6-CH=CH-CH3、-C2H4-CH=CH-C2H5、-CH2-CH=CH-C3H7、-CH=CH-C4H9、-C3H6-C(CH3)=CH2 、 -C2H4-CH(CH3)-CH=CH2 、-CH2-CH(CH3)-CH2-CH=CH2 、 -C2H4-CH=C(CH3)2 、-CH(CH3)-C2H4-CH=CH2 、 -C2H4-C(CH3)=CH-CH3 、-CH2-CH(CH3)-CH=CH-CH3、-CH(CH3)-CH2-CH=CH-CH3、-CH2-CH=CH-CH(CH3)2 、 -CH2-CH=C(CH3)-C2H5 、-CH2-C(CH3)=CH-C2H5 、 -CH(CH3)-CH=CH-C2H5 、-CH=CH-CH2-CH(CH3)2、-CH=CH-CH(CH3)-C2H5、-CH=C(CH3)-C3H7、-C(CH3)=CH-C3H7、-CH2-CH(CH3)-C(CH3)=CH2、-C[C(CH3)3]=CH2、-CH(CH3)-CH2-C(CH3)=CH2、-CH(CH3)-CH(CH3)-CH=CH2、-CH=CH-C2H4-CH=CH2 、 -C(CH3)2-CH2-CH=CH2 、-CH2-C(CH3)=C(CH3)2 、 -CH(CH3)-CH=C(CH3)2 、-C(CH3)2-CH=CH-CH3 、 -CH=CH-CH2-CH=CH-CH3 、-CH(CH3)-C(CH3)=CH-CH3 、 -CH=C(CH3)-CH(CH3)2 、-C(CH3)=CH-CH(CH3)2、-C(CH3)=C(CH3)-C2H5、-CH=CH-C(CH3)3、-C(CH3)2-C(CH3)=CH2 、 -CH(C2H5)-C(CH3)=CH2 、-C(CH3)(C2H5)-CH=CH2 、 -CH(CH3)-C(C2H5)=CH2 、-CH2-C(C3H7)=CH2、-CH2-C(C2H5)=CH-CH3、-CH(C2H5)-CH=CH-CH3、-C(C4H9)=CH2、-C(C3H7)=CH-CH3、-C(C2H5)=CH-C2H5、-C(C2H5)=C(CH3)2、-C[CH(CH3)(C2H5)]=CH2、-C[CH2-CH(CH3)2]=CH2、-C2H4-CH=CH-CH=CH2、-CH2-CH=CH-CH2-CH=CH2、-C3H6-C≡C-CH3、-CH2-CH=CH-CH=CH-CH3、-CH=CH-CH=CH-C2H5、-CH(CH3)-CH2-C≡CH、-CH(CH3)-C≡C-CH3、-C2H4-CH(CH3)-C≡CH、-CH=CH-CH=C(CH3)2、-CH2-CH(CH3)-CH2-C≡CH、-CH=CH-C(CH3)=CH-CH3、-CH=C(CH3)-CH=CH-CH3、-CH2-CH(CH3)-C≡CH、-C(CH3)=CH-CH=CH-CH3、-C≡CH、-C≡C-CH3、-CH2-C≡CH、-C2H4-C≡CH、-CH2-C≡C-CH3、-C≡C-C2H5、-C3H6-C≡CH、-C2H4-C≡C-CH3、-CH2-C≡C-C2H5, -C≡C-C3H7, -CH(CH3)-C≡CH, -C4H8-C≡CH, -C2H4-C≡C-C2H5, -CH2 -C≡C-C3H7, -C≡C-C4H9, -C≡C-CH2-CH(CH3)2, -CH(CH3)-C2H4-C≡CH, -CH2-CH(CH3) -C≡C-CH3, -C(CH3)(C2H5)-C≡CH, -CH(CH3)-CH2-C≡C-CH3, -CH(CH3)-C≡C-C2H5, -C H2-C≡C-CH(CH3)2, -C≡C-CH(CH3)-C2H5, -CH2-C≡CC≡C-CH3, -CH(C2H5)-C≡C-CH3, - C(CH3)2-C≡C-CH3, -CH(C2H5)-CH2-C≡CH, -CH2-CH(C2H5)-C≡CH, -C(CH3)2-CH2-C≡ CH, -CH2-C(CH3)2-C≡CH, -CH(CH3)-CH(CH3)-C≡CH, -CH(C3H7)-C≡CH, -CH2-CH(C≡C H)2, -C≡CC≡CH, -CH2-C≡CC≡CH, -C≡CC≡C-CH3, -CH(C≡CH)2, -C2H4-C≡CC≡CH, -CH2-C ≡C-CH2-C≡CH, -C≡C-C2H4-C≡CH, -C≡CC(CH3)3, -C≡C-CH2-C≡C-CH3, -C≡CC≡C-C2H5,

[0026] or

[0027] R 5 and R 6 or R 6 and R 7 Together with the two carbon atoms of the phenyl ring to which they are attached, they can form a 4- to 8-membered ring system, which is optionally substituted by one or more substituents selected from R 10 、R 11 、R 12 and R 13 ;

[0028] or an enantiomer, a diastereomer, a tautomer, a mixture of enantiomers, a mixture of diastereomers, a mixture of tautomers, a hydrate, a solvate, or a pharmaceutically acceptable salt of the above compounds.

[0029] Preferably, the present invention relates to compounds of formula (I):

[0030] in

[0031] A stands for

[0032] B stands for -OR 3 、-O-CH2-R 3 、-O-CH2-CH2-R 3 、-O-CH2-CH2-CH2-R 3 ; R 1 represent

[0033] R 2a and R 2b Independently represents -H or C 1-3 alkyl, and the C 1-3 The alkyl group is optionally substituted with 1 to 6 fluorine atoms or -OH;

[0034] R 3 represent

[0035] R 4 Represents -H, halogen, C 1-4 Alkyl, -OC 1-4 Alkyl, C 3-4 Cycloalkyl, -OC 3-4 Cycloalkyl, and the C 1-4 Alkyl, -OC 1-4 Alkyl, -OC 3-4 Cycloalkyl and C 3-4 The cycloalkyl group may be optionally substituted with 1 to 6 fluorine atoms;

[0036] R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 and R 13 represents independently of each other -H, -cyclo-C3H5, -cyclo-C4H7, -cyclo-C5H9, -cyclo-C6H 11 、-cyclo-C7H 13, -cycloC3H5O, -OH, -OCH3, -OC2H5, -OC3H7, -O-cyclo-C3H5, -OCH(CH3)2, -OC(CH3)3, -OC4H9, -OPh, -OCH2-Ph, -OCPh3, -CH2-OCH3, -C2H4-OCH3, -C3H6-OCH3, -CH2-OC2H5, -C2H4-OC2H5, -C3H6-OC2H5, -CH2-OC3H7, -C2H4-OC3H7, -C3H6-OC3H7, -CH2-O-cyclo-C3H5, -C2H4-O-cyclo-C3H5, -C3H6-O-cyclo-C3H5, -CH2-OCH(CH 3)2, -C2H4-OCH(CH3)2, -C3H6-OCH(CH3)2, -CH2-OC(CH3)3, -C2H4-OC(CH3)3, -C3H6-OC(CH3)3, -CH2-OC4H9, -C2H4-OC4H9, -C3H6-OC4H9, -CH2-OPh, - C2H4-OPh, -C3H6-OPh, -CH2-OCH2-Ph, -C2H4-OCH2-Ph, -C3H6-OCH2-Ph, -SH, -SCH3, -SC2H5, -SC3H7, -S-cyclo-C3H5, -SCH(CH3)2, -SC(CH3)3, -F, -Cl, -Br , -I, -CN, -COCH3, -COC2H5, -COC3H7, -CO-cyclo-C3H5, -COCH(CH3)2, -COC(CH3)3, -COOH, -COOCH3, -COOC2H5, -COOC3H7, -COO-cyclo-C3H5, -COOCH(CH3)2, -COOC(CH3)3, -OOC-CH3, -OOC-C2H5, -OOC-C3H7, -OOC-cyclo-C3H5, -OOC-CH(CH3)2, -OOC-C(CH3)3, -CONH2, -CONHCH3, -CONHC2H5, -CONHC3H7, -CONH-cyclo-C3H 5. -CONH[CH(CH3)2], -CONH[C(CH3)3], -CON(CH3)2, -CON(C2H5)2, -CON(C3H7)2, -CON(ring-C3H5)2, -CON[CH(CH3)2]2, -CON[C(CH3)3]2, -NHCOCH3, -NH COC2H5, -NHCOC3H7, -NHCO-ring-C3H5, -NHCO-CH(CH3)2, -NHCO-C(CH3)3, -NHCO-OCH3, -NHCO-OC2H5, -NHCO-OC3H7, -NHCO-O-ring-C3H5, -NHCO-OCH(CH3)2,-NHCO-OC(CH3)3, -NH2, -NHCH3, -NHC2H5, -NHC3H7, -NH-cyclo-C3H5, -NHCH(CH3)2, -NHC(CH3)3, -N(CH3)2, -N(C2H5)2, -N(C3H7)2, -N(cyclo-C3H5)2, -N[CH(CH3)2]2, -N[C(CH3)3]2, -SOCH3, -SOC2H5, -SOC3H7, -SO-cyclo-C3H5, -SOCH(CH3)2, -SOC(CH3)3, -SO2CH3, -SO2C2H5, -SO2C3H7, -SO2-cyclo-C3H5, -SO2CH(CH3)2, -SO2C(CH3)3, -SO3H, -SO3CH3, -SO3C2H5, -SO3C3H7, -SO3-cyclo-C3H5, -SO3CH(CH3)2, -SO3C(CH3)3, -SO2NH2, -SO2NHCH3, -SO2NHC2H5, -SO2NHC3H7, -SO2NH-cyclo-C3H5, -SO2NHCH(CH3)2, -SO2NHC(CH3)3, -SO2N(CH3)2, -SO2N(C2H5)2, -SO2N(C3H7)2, -SO2N(cyclo-C3H5)2, -SO2N[CH(CH3)2]2, -SO2N[C(CH3)3]2, -O-S(=O)CH3, -O-S(=O)C2H5, -O-S(=O)C3H7, -O-S(=O)-cyclo-C3H5, -O-S(=O)CH(CH3)2, -O-S(=O)C(CH3)3, -S(=O)(=NH)CH3, -S(=O)(=NH)C2H5, -S(=O)(=NH)C3H7, -S(=O)(=NH)-cyclo-C3H5, -S(=O)(=NH)CH(CH3)2, -S(=O)(=NH)C(CH3)3, -NH-SO2-CH3, -NH-SO2-C2H5, -NH-SO2-C3H7, -NH-SO2-cyclo-C3H5, -NH-SO2-CH(CH3)2, -NH-SO2-C(CH3)3, -O-SO2-CH3, -O-SO2-C2H5, -O-SO2-C3H7, -O-SO2-cyclo-C3H5, -O-SO2-CH(CH3)2, -O-SO2-C(CH3)3, -OCH2F, -OCHF2, -OCF3, -CH2-OCF3, -C2H4-OCF3, -C3H6-OCF3, -CH2-OCHF2, -C2H4-OCHF2, -C3H6-OCHF2, -OC2F5, -CH2-OC2F5, -C2H4-OC2F5, -C3H6-OC2F5, -O-COOCH3-O-COOC2H5, -O-COOC3H7, -O-COO-cyclo-C3H5, -O-COOCH(CH3)2, -O-COOC(CH3)3, -NH-CO-NH2, -NH-CO-NHCH3, -NH-CO-NHC2H5, -NH-CO-NHC3H7, -NH-C(=NH)-NH2, -NH-CO-N(C3H7)2, -NH-CO-NH[CH(CH3)2], -NH-CO-NH[C(CH3)3], -NH-CO-N(CH3)2, -NH-CO-N(C2H5)2, -NH-CO-NH-cyclo-C3H5, -NH-CO-N(cyclo-C3H5)2, -NH-CO-N[CH(CH3)2]2, -NH-C(=NH)-NHCH3, -NH-C(=NH)-NHC2H5, -NH-C(=NH)-NHC3H7, -O-CO-NH-cyclo-C3H5, -NH-C(=NH)-NH-cyclo-C3H5, -NH-C(=NH)-NH[CH(CH3)2], -O-CO-NH[CH(CH3)2], -NH-C(=NH)-NH[C(CH3)3], -NH-C(=NH)-N(CH3)2, -NH-C(=NH)-N(C2H5)2, -NH-C(=NH)-N(C3H7)2, -NH-C(=NH)-N(cyclo-C3H5)2, -O-CO-NHC3H7, -NH-C(=NH)-N[CH(CH3)2]2, -NH-C(=NH)-N[C(CH3)3]2, -O-CO-NH2, -O-CO-NHCH3, -O-CO-NHC2H5, -O-CO-NH[C(CH3)3], -O-CO-N(CH3)2, -O-CO-N(C2H5)2, -O-CO-N(C3H7)2, -O-CO-N(cyclo-C3H5)2, -O-CO-N[CH(CH3)2]2, -O-CO-N[C(CH3)3]2, -O-CO-OCH3, -O-CO-OC2H5, -O-CO-OC3H7, -O-CO-O-cyclo-C3H5, -O-CO-OCH(CH3)2, -O-CO-OC(CH3)3, -CH2F, -CHF2, -CF3, -CH2-CH2F, -CH2-CHF2, -CH2-CF3, cyclo-C8H, 15 , -Ph, -CH2-Ph, -CH2-CH2-Ph, -CH=CH-Ph, -CPh3, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9, -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H 11、-CH(CH3)-C3H7、-CH2-CH(CH3)-C2H5、-CH(CH3)-CH(CH3)2、-C(CH3)2-C2H5、-CH2-C(CH3)3、-CH(C2H5)2、-C2H4-CH(CH3)2、-C6H 13 、-C7H 15 、-C8H 17、-C3H6-CH(CH3)2、-C2H4-CH(CH3)-C2H5、-CH(CH3)-C4H9、-CH2-CH(CH3)-C3H7 、 -CH(CH3)-CH2-CH(CH3)2 、-CH(CH3)-CH(CH3)-C2H5、 -CH2-CH(CH3)-CH(CH3)2 、-CH2-C(CH3)2-C2H5、-C(CH3)2-C3H7、-C(CH3)2-CH(CH3)2、-C2H4-C(CH3)3、-CH(CH3)-C(CH3)3、-CH=CH2、-CH2-CH=CH2、-C(CH3)=CH2、-CH=CH-CH3、-C2H4-CH=CH2、-CH2-CH=CH-CH3、-CH=CH-C2H5、-CH2-C(CH3)=CH2、-CH(CH3)-CH=CH、-CH=C(CH3)2、-C(CH3)=CH-CH3、-CH=CH-CH=CH2、-C3H6-CH=CH2、-C2H4-CH=CH-CH3、-CH2-CH=CH-C2H5、-CH=CH-C3H7、-CH=CH-CH=CH-CH3、-C2H4-C(CH3)=CH2、-CH2-CH(CH3)-CH=CH2、-CH(CH3)-CH2-CH=CH2、-CH2-CH=C(CH3)2、-CH2-C(CH3)=CH-CH3、-CH(CH3)-CH=CH-CH3、-CH=CH-CH(CH3)2、-CH=C(CH3)-C2H5、-C(CH3)=CH-C2H5、-C(CH3)=C(CH3)2、-C(CH3)2-CH=CH2、-CH(CH3)-C(CH3)=CH2、-C4H8-CH=CH2、-C3H6-CH=CH-CH3、-C2H4-CH=CH-C2H5、-CH2-CH=CH-C3H7、-CH=CH-C4H9、-C3H6-C(CH3)=CH2 、 -C2H4-CH(CH3)-CH=CH2 、-CH2-CH(CH3)-CH2-CH=CH2 、 -C2H4-CH=C(CH3)2 、-CH(CH3)-C2H4-CH=CH2 、 -C2H4-C(CH3)=CH-CH3 、-CH2-CH(CH3)-CH=CH-CH3、-CH(CH3)-CH2-CH=CH-CH3、-CH2-CH=CH-CH(CH3)2 、 -CH2-CH=C(CH3)-C2H5 、-CH2-C(CH3)=CH-C2H5 、 -CH(CH3)-CH=CH-C2H5 、-CH=CH-CH2-CH(CH3)2、-CH=CH-CH(CH3)-C2H5、-CH=C(CH3)-C3H7、-C(CH3)=CH-C3H7、-CH2-CH(CH3)-C(CH3)=CH2、-C[C(CH3)3]=CH2、-CH(CH3)-CH2-C(CH3)=CH2、-CH(CH3)-CH(CH3)-CH=CH2、-CH=CH-C2H4-CH=CH2 、 -C(CH3)2-CH2-CH=CH2 、-CH2-C(CH3)=C(CH3)2 、 -CH(CH3)-CH=C(CH3)2 、-C(CH3)2-CH=CH-CH3 、 -CH=CH-CH2-CH=CH-CH3 、-CH(CH3)-C(CH3)=CH-CH3 、 -CH=C(CH3)-CH(CH3)2 、-C(CH3)=CH-CH(CH3)2、-C(CH3)=C(CH3)-C2H5、-CH=CH-C(CH3)3、-C(CH3)2-C(CH3)=CH2 、 -CH(C2H5)-C(CH3)=CH2 、-C(CH3)(C2H5)-CH=CH2 、 -CH(CH3)-C(C2H5)=CH2 、-CH2-C(C3H7)=CH2、-CH2-C(C2H5)=CH-CH3、-CH(C2H5)-CH=CH-CH3、-C(C4H9)=CH2、-C(C3H7)=CH-CH3、-C(C2H5)=CH-C2H5、-C(C2H5)=C(CH3)2、-C[CH(CH3)(C2H5)]=CH2、-C[CH2-CH(CH3)2]=CH2、-C2H4-CH=CH-CH=CH2、-CH2-CH=CH-CH2-CH=CH2、-C3H6-C≡C-CH3、-CH2-CH=CH-CH=CH-CH3、-CH=CH-CH=CH-C2H5、-CH(CH3)-CH2-C≡CH、-CH(CH3)-C≡C-CH3、-C2H4-CH(CH3)-C≡CH、-CH=CH-CH=C(CH3)2、-CH2-CH(CH3)-CH2-C≡CH、-CH=CH-C(CH3)=CH-CH3、-CH=C(CH3)-CH=CH-CH3、-CH2-CH(CH3)-C≡CH、-C(CH3)=CH-CH=CH-CH3、-C≡CH、-C≡C-CH3、-CH2-C≡CH、-C2H4-C≡CH、-CH2-C≡C-CH3、-C≡C-C2H5、-C3H6-C≡CH、-C2H4-C≡C-CH3、-CH2-C≡C-C2H5, -C≡C-C3H7, -CH(CH3)-C≡CH, -C4H8-C≡CH, -C2H4-C≡C-C2H5, -CH2 -C≡C-C3H7, -C≡C-C4H9, -C≡C-CH2-CH(CH3)2, -CH(CH3)-C2H4-C≡CH, -CH2-CH(CH3) -C≡C-CH3, -C(CH3)(C2H5)-C≡CH, -CH(CH3)-CH2-C≡C-CH3, -CH(CH3)-C≡C-C2H5, -C H2-C≡C-CH(CH3)2, -C≡C-CH(CH3)-C2H5, -CH2-C≡CC≡C-CH3, -CH(C2H5)-C≡C-CH3, - C(CH3)2-C≡C-CH3, -CH(C2H5)-CH2-C≡CH, -CH2-CH(C2H5)-C≡CH, -C(CH3)2-CH2-C≡ CH, -CH2-C(CH3)2-C≡CH, -CH(CH3)-CH(CH3)-C≡CH, -CH(C3H7)-C≡CH, -CH2-CH(C≡C H)2, -C≡CC≡CH, -CH2-C≡CC≡CH, -C≡CC≡C-CH3, -CH(C≡CH)2, -C2H4-C≡CC≡CH, -CH2-C ≡C-CH2-C≡CH, -C≡C-C2H4-C≡CH, -C≡CC(CH3)3, -C≡C-CH2-C≡C-CH3, -C≡CC≡C-C2H5,

[0037] or

[0038] R 5 and R 6 or R 6 and R 7 Together with the two carbon atoms of the phenyl ring to which they are attached, they can form a 4- to 8-membered ring system, which is optionally substituted by one or more substituents selected from R 10 、R 11 、R 12 and R 13 ;

[0039] or an enantiomer, a diastereomer, a tautomer, a mixture of enantiomers, a mixture of diastereomers, a mixture of tautomers, a hydrate, a solvate, or a pharmaceutically acceptable salt of the above compounds.

[0040] These compounds are suitable for enhancing the levels of cytokines secreted by stimulated immune cells and thereby increasing the local activity of immune cells in the vicinity of the stimulated immune cells.These findings make the compounds of the present invention useful for treating and / or preventing neoplastic and / or infectious diseases.

[0041] The term "pharmaceutically acceptable salt" refers to a salt of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound. The compounds of the present invention can form salts with organic or inorganic acids or bases. Examples of suitable acids for the formation of such acid addition salts are hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid, p-aminosalicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, maleic acid, sulfonic acid, phosphonic acid, perchloric acid, nitric acid, formic acid, propionic acid, gluconic acid, lactic acid, tartaric acid, hydroxymaleic acid, pyruvic acid, phenylacetic acid, benzoic acid, p-aminobenzoic acid, p-hydroxybenzoic acid, methanesulfonic acid, ethanesulfonic acid, nitrous acid, hydroxyethanesulfonic acid, ethylenesulfonic acid, p-toluenesulfonic acid, naphthylsulfonic acid, p-aminobenzenesulfonic acid, camphorsulfonic acid, china acid, mandelic acid, o-methylmandelic acid, hydrogen-benzenesulfonic acid, picric acid, adipic acid, D-o-tolyltartaric acid, hydroxymalonic acid, (o-, m-, p-)-toluic acid, naphthylaminesulfonic acid, trifluoroacetic acid, and other inorganic or organic acids well known to those skilled in the art. The salts are prepared by contacting the free base form of a compound of formula (I) with a sufficient amount of the desired acid to produce the salt in a conventional manner well known to those skilled in the art.

[0042] In the case where the compounds of the present invention carry acidic groups, they may also form salts with inorganic or organic bases. Examples of suitable inorganic or organic bases are, for example, NaOH, KOH, NH4OH, tetraalkylammonium hydroxides, lysine or arginine, etc. Salts may be prepared in a conventional manner using methods well known in the art, for example by treating a solution of a compound of formula (I) with an acid solution selected from the above group.

[0043] The term "4- to 8-membered ring system" as used herein refers to a 4- to 8-membered aromatic ring or a 4- to 8-membered heteroaromatic ring, a 4- to 8-membered carbocyclyl group or a 4- to 8-membered heterocyclyl group. 5 and R 6 or R 6 and R 7 The 4- to 8-membered ring system is formed and in the 4- to 8-membered ring system, two carbon atoms of the phenyl ring are included, on which R 5 and R 6 or R 6 and R 7 was replaced.

[0044] Preferably, the 4- to 8-membered aromatic ring represents phenyl and naphthyl, wherein these phenyl and naphthyl residues may be substituted by 1 to 4 substituents selected from R 10 to R 13 However, it is obvious to the skilled person that the term "may be substituted" means that the substituent R 10 to R 13 One of them replaces a hydrogen atom.

[0045] Preferably, the "4- to 8-membered heteroaromatic ring" comprises at least one heteroatom such as O, S, SO, SO2, N, NO and a double bond, wherein these monounsaturated 4-membered heterocyclic residues may be substituted by 1 to 4 substituents selected from R 10 to R 13 It is obvious to the skilled person that the term "may be substituted" means that the substituent R 10 to R 13 One of them replaces a hydrogen atom.

[0046] Preferably, the "4- to 8-membered heterocyclyl group" includes at least one heteroatom such as O, S, SO, SO2 and N and optionally one carbonyl (CO) bond, one or more double bonds, wherein the 4- to 8-membered heterocyclyl group may be substituted by 1 to 4 substituents selected from R 10 to R 13 It is obvious to the skilled person that the term "may be substituted" means that the substituent R 10 to R 13 One of them replaces a hydrogen atom.

[0047] Preferably, the "4 to 8 membered carbocyclyl group" may optionally include one or more double bonds and may be substituted by 1 to 4 substituents selected from R 10 to R 13 It is obvious to the skilled person that the term "may be substituted" means that the substituent R 10 to R 13 One of them replaces a hydrogen atom.

[0048] Preferably, in formula (I), R 5 and R 6 or R 6 and R 7 The following 4 to 6 membered ring system can be formed, wherein the 4 to 6 membered ring system can be optionally substituted with 1 to 4 substituents selected from R 10 to R 13 :

[0049]

[0050] Therefore, in formula (I), R 3 Preferably represents the following bicyclic ring, wherein R5 and R 6 or R 6 and R 7 To form a 4 to 6 membered ring system fused to a phenyl ring, wherein the 4 to 6 membered ring system may be optionally substituted with 1 to 4 substituents selected from R 10 to R 13 .

[0051]

[0052] More preferably, in formula (I), R 5 and R 6 or R 6 and R 7 The following 4 to 6 membered ring system can be formed, wherein the 4 to 6 membered ring system can be optionally substituted with 1 to 4 substituents selected from R 10 to R 13 :

[0053]

[0054] Therefore, in formula (I), R 3 Preferably represents the following bicyclic ring, wherein R 5 and R 6 or R 6 and R 7 To form a 4 to 6 membered ring system fused to a phenyl ring, wherein the 4 to 6 membered ring system may be optionally substituted with 1 to 4 substituents selected from R 10 to R 13 :

[0055]

[0056] Still more preferably, in formula (I), R 5 and R 6 or R 6 and R 7 The following 6-membered ring system can be formed, wherein the 6-membered ring system can be optionally substituted with 1 to 4 substituents selected from R 10 to R 13 :

[0057]

[0058] More preferably, the present invention relates to compounds of formula (I):

[0059]

[0060] in

[0061] A stands for

[0062] B is -OR 3 、-O-CHR 3 R 3* 、-O-CH2-CH2-R 3 or -O-CH2-CH2-CH2-R 3 ; R 1 represent

[0063] R 2a and R 2b Each independently represents -H, -F, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CH2OCH3, -CH2CH2OCH3, -CF2-CH3, -CHF-CHF2, -CHF-CF3, -CF2-CF3; wherein R 2a Not -H;

[0064] R 3 represent or preferably

[0065] R 3* represents -H, -F, -CH3, -C2H5, -C3H7, -CH(CH3)2, -CH2F, -CHF2, -CF3, -CH2-CF3;

[0066] R 4 and R 4* represents independently of one another -H, -F, -Cl, -Br, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -OCH3, -OC2H5, -OC3H7, -OCH(CH3)2, -OCH2F, -OCHF2, -OCF3, -CH2-CF3, -CHF-CH2F, -CHF-CHF2, -CHF-CF3, -CF2-CH3, -CF2-CH2F, -CF2-CHF2, -CF2-CF3,

[0067] R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 and R 13represents independently of each other -H, -cyclo-C3H5, -cyclo-C4H7, -cyclo-C5H9, -cyclo-C6H 11 、-cyclo-C7H 13, -cycloC3H5O, -OH, -OCH3, -OC2H5, -OC3H7, -O-cyclo-C3H5, -OCH(CH3)2, -OC(CH3)3, -OC4H9, -OPh, -OCH2-Ph, -OCPh3, -CH2-OCH3, -C2H4-OCH3, -C3H6-OCH3, -CH2-OC2H5, -C2H4-OC2H5, -C3H6-OC2H5, -CH2-OC3H7, -C2H4-OC3H7, -C3H6-OC3H7, -CH2-O-cyclo-C3H5, -C2H4-O-cyclo-C3H5, -C3H6-O-cyclo-C3H5, -CH2-OCH(CH 3)2, -C2H4-OCH(CH3)2, -C3H6-OCH(CH3)2, -CH2-OC(CH3)3, -C2H4-OC(CH3)3, -C3H6-OC(CH3)3, -CH2-OC4H9, -C2H4-OC4H9, -C3H6-OC4H9, -CH2-OPh, - C2H4-OPh, -C3H6-OPh, -CH2-OCH2-Ph, -C2H4-OCH2-Ph, -C3H6-OCH2-Ph, -SH, -SCH3, -SC2H5, -SC3H7, -S-cyclo-C3H5, -SCH(CH3)2, -SC(CH3)3, -F, -Cl, -Br , -I, -CN, -COCH3, -COC2H5, -COC3H7, -CO-cyclo-C3H5, -COCH(CH3)2, -COC(CH3)3, -COOH, -COOCH3, -COOC2H5, -COOC3H7, -COO-cyclo-C3H5, -COOCH(CH3)2, -COOC(CH3)3, -OOC-CH3, -OOC-C2H5, -OOC-C3H7, -OOC-cyclo-C3H5, -OOC-CH(CH3)2, -OOC-C(CH3)3, -CONH2, -CONHCH3, -CONHC2H5, -CONHC3H7, -CONH-cyclo-C3H 5. -CONH[CH(CH3)2], -CONH[C(CH3)3], -CON(CH3)2, -CON(C2H5)2, -CON(C3H7)2, -CON(ring-C3H5)2, -CON[CH(CH3)2]2, -CON[C(CH3)3]2, -NHCOCH3, -NH COC2H5, -NHCOC3H7, -NHCO-ring-C3H5, -NHCO-CH(CH3)2, -NHCO-C(CH3)3, -NHCO-OCH3, -NHCO-OC2H5, -NHCO-OC3H7, -NHCO-O-ring-C3H5, -NHCO-OCH(CH3)2,-NHCO-OC(CH3)3, -NH2, -NHCH3, -NHC2H5, -NHC3H7, -NH-cyclo-C3H5, -NHCH(CH3)2, -NHC(CH3)3, -N(CH3)2, -N(C2H5)2, -N(C3H7)2, -N(cyclo-C3H5)2, -N[CH(CH3)2]2, -N[C(CH3)3]2, -SOCH3, -SOC2H5, -SOC3H7, -SO-cyclo-C3H5, -SOCH(CH3)2, -SOC(CH3)3, -SO2CH3, -SO2C2H5, -SO2C3H7, -SO2-cyclo-C3H5, -SO2CH(CH3)2, -SO2C(CH3)3, -SO3H, -SO3CH3, -SO3C2H5, -SO3C3H7, -SO3-cyclo-C3H5, -SO3CH(CH3)2, -SO3C(CH3)3, -SO2NH2, -SO2NHCH3, -SO2NHC2H5, -SO2NHC3H7, -SO2NH-cyclo-C3H5, -SO2NHCH(CH3)2, -SO2NHC(CH3)3, -SO2N(CH3)2, -SO2N(C2H5)2, -SO2N(C3H7)2, -SO2N(cyclo-C3H5)2, -SO2N[CH(CH3)2]2, -SO2N[C(CH3)3]2, -O-S(=O)CH3, -O-S(=O)C2H5, -O-S(=O)C3H7, -O-S(=O)-cyclo-C3H5, -O-S(=O)CH(CH3)2, -O-S(=O)C(CH3)3, -S(=O)(=NH)CH3, -S(=O)(=NH)C2H5, -S(=O)(=NH)C3H7, -S(=O)(=NH)-cyclo-C3H5, -S(=O)(=NH)CH(CH3)2, -S(=O)(=NH)C(CH3)3, -NH-SO2-CH3, -NH-SO2-C2H5, -NH-SO2-C3H7, -NH-SO2-cyclo-C3H5, -NH-SO2-CH(CH3)2, -NH-SO2-C(CH3)3, -O-SO2-CH3, -O-SO2-C2H5, -O-SO2-C3H7, -O-SO2-cyclo-C3H5, -O-SO2-CH(CH3)2, -O-SO2-C(CH3)3, -OCH2F, -OCHF2, -OCF3, -CH2-OCF3, -C2H4-OCF3, -C3H6-OCF3, -CH2-OCHF2, -C2H4-OCHF2, -C3H6-OCHF2, -OC2F5, -CH2-OC2F5, -C2H4-OC2F5, -C3H6-OC2F5, -CH2F-CHF2, -CF3, -CH2-CH2F, -CH2-CHF2, -CH2-CF3, cyclo-C8H, 15 , -Ph, -CH2-Ph, -CH2-CH2-Ph, -CH=CH-Ph, -CPh3, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9, -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H 11 , -CH=CH2, -CH2-CH=CH2, -C(CH3)=CH2, -CH=CH-CH3, -C≡CH, -C≡C-CH3, -CH2-C≡CH,

[0068] Preferably R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 and R 13 represents independently of each other -H, -cyclo-C3H5, -cyclo-C4H7, -cyclo-C5H9, -cyclo-C6H 11 、-cyclo-C7H 13, -cycloC3H5O, -OH, -OCH3, -OC2H5, -OC3H7, -O-cyclo-C3H5, -OCH(CH3)2, -OC(CH3)3, -OC4H9, -OPh, -OCH2-Ph, -OCPh3, -CH2-OCH3, -C2H4-OCH3, -C3H6-OCH3, -CH2-OC2H5, -C2H4-OC2H5, -C3H6-OC2H5, -CH2-OC3H7, -C2H4-OC3H7, -C3H6-OC3H7, -CH2-O-cyclo-C3H5, -C2H4-O-cyclo-C3H5, -C3H6-O-cyclo-C3H5, -CH2-OCH(CH 3)2, -C2H4-OCH(CH3)2, -C3H6-OCH(CH3)2, -CH2-OC(CH3)3, -C2H4-OC(CH3)3, -C3H6-OC(CH3)3, -CH2-OC4H9, -C2H4-OC4H9, -C3H6-OC4H9, -CH2-OPh, -C2H4-OPh, -C3H6-OPh, -CH2-OCH2-Ph, -C2H4-OCH2-Ph, -C3H6-OCH2-Ph, -F, -Cl, -Br, -I, -CN, -COCH3, -COC2H5, -COC3H7, -CO-ring-C3H5, -COCH(CH3)2, -COC(CH3)3, -COOH, -COOCH3, -COOC2H5, -COOC3H7, -COO-ring-C3H5, -COOCH(CH3)2, -COOC(CH3)3, -OOC-CH3, -OOC-C2H5, -OOC-C3H7, -OOC-ring-C3H5, -OO C-CH(CH3)2, -OOC-C(CH3)3, -CONH2, -CONHCH3, -CONHC2H5, -CONHC3H7, -CONH-ring-C3H5, -CONH[CH(CH3)2], -CONH[C(CH3)3], -CON(CH3)2, -CON(C2H5) 2, -CON(C3H7)2, -CON(cyclo-C3H5)2, -CON[CH(CH3)2]2, -CON[C(CH3)3]2, -NHCOCH3, -NHCOC2H5, -NHCOC3H7, -NHCO-cyclo-C3H5, -NHCO-CH(CH3)2, -NHCO-C(CH3)3, -NHCO-OCH3, -NHCO-OC2H5, -NHCO-OC3H7, -NHCO-O-cyclo-C3H5, -NHCO-OCH(CH3)2, -NHCO-OC(CH3)3, -NH2, -NHCH3, -NHC2H5, -NHC3H7, -NH-cyclo-C3H5,-NHCH(CH3)2, -NHC(CH3)3, -N(CH3)2, -N(C2H5)2, -N(C3H7)2, -N(cyclo-C3H5)2, -N[CH(CH3)2]2, -N[C(CH3)3]2, -SOCH3, -SOC2H5, -SOC3H7, -SO-cyclo-C3H5, -SOCH(CH3)2, -SOC(CH3)3, -SO2CH3, -SO2C2H5, -SO2C3H7, -SO2-cyclo-C3H5, -SO2CH(CH3)2, -SO2C(CH3)3, -SO3H, -SO3CH3, -SO3C2H5, -SO3C3H7, -SO3-cyclo-C3H5, -SO3CH(CH3)2, -SO3C(CH3)3, -SO2NH2, -SO2NHCH3, -SO2NHC2H5, -SO2NHC3H7, -SO2NH-cyclo-C3H5, -SO2NHCH(CH3)2, -SO2NHC(CH3)3, -SO2N(CH3)2, -SO2N(C2H5)2, -SO2N(C3H7)2, -SO2N(cyclo-C3H5)2, -SO2N[CH(CH3)2]2, -SO2N[C(CH3)3]2, -O-S(=O)CH3, -O-S(=O)C2H5, -O-S(=O)C3H7, -O-S(=O)-cyclo-C3H5, -O-S(=O)CH(CH3)2, -O-S(=O)C(CH3)3, -O-SO2-CH3, -O-SO2-C2H5, -O-SO2-C3H7, -O-SO2-cyclo-C3H5, -O-SO2-CH(CH3)2, -O-SO2-C(CH3)3, -OCH2F, -OCHF2, -OCF3, -CH2-OCF3, -C2H4-OCF3, -C3H6-OCF3, -CH2-OCHF2, -C2H4-OCHF2, -C3H6-OCHF2, -OC2F5, -CH2-OC2F5, -C2H4-OC2F5, -C3H6-OC2F5, -CH2F, -CHF2, -CF3, -CH2-CH2F, -CH2-CHF2, -CH2-CF3, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9, -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H, 11 , -CH=CH2, -CH2-CH=CH2, -C(CH3)=CH2, -CH=CH-CH3, -C≡CH, -C≡C-CH3, -CH2-C≡CH,

[0069]

[0070] or

[0071] R 5 and R 6 or R 6 and R 7 Together with the two carbon atoms of the phenyl ring to which they are attached, they can form a 4- to 8-membered ring system, which is optionally substituted by one or more substituents selected from R 10 、R 11 、R 12 and R 13 ;

[0072] or an enantiomer, a diastereomer, a tautomer, a mixture of enantiomers, a mixture of diastereomers, a mixture of tautomers, a hydrate, a solvate, or a pharmaceutically acceptable salt of the above compounds.

[0073] In all formulae disclosed herein, B preferably represents -OR 3 、-O-CHR 3 R 3* or -O-CH2-CH2-R 3 .

[0074] Furthermore, it was found that compounds of formula (I) (wherein B represents -OR 3 ) are able to cross the blood-brain barrier well. Therefore, for indications where the ability to cross the blood-brain barrier is important, those compounds of formula (I) are particularly preferred, wherein B represents -OR 3 .

[0075] Thus, as disclosed herein, a group of compounds of formula (I) is claimed wherein B represents only -OR 3 , in order to claim protection for the group of compounds that can readily cross the blood-brain barrier.

[0076] Therefore, the remaining compounds of formula (I) are claimed, wherein B represents -O-CHR 3 R 3* or -O-CH2-CH2-R 3 or -O-CH2-CH2-CH2-R 3 .

[0077] In addition, the inventors found that R 1 The residues are particularly important for inhibitory activity. 1 The residue must have a hydroxyl group (-OH) and must also have at least one substituent R different from hydrogen (-H). 2a and an optional second substituent R 2b .

[0078] For all formulae disclosed herein, the substituent R 2a and R 2b The definition is as follows:

[0079] R 2a and R 2b Each independently represents -H, -F, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CH2OCH3, -CH2CH2OCH3, -CF2-CH3, -CHF-CHF2, -CHF-CF3, -CF2-CF3; wherein R 2a Not -H;

[0080] Or in other words:

[0081] R 2a represents -F, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CH2OCH3, -CH2CH2OCH3, -CF2-CH3, -CHF-CHF2, -CHF-CF3, -CF2-CF3; and

[0082] R 2b Represents -H, -F, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CH2OCH3, -CH2CH2OCH3, -CF2-CH3, -CHF-CHF2, -CHF-CF3, -CF2-CF3.

[0083] Therefore, in R 2a Under conditions other than hydrogen (-H), it is preferred that

[0084] R 2a and R 2b represent, independently of one another, -H, -F, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CH2OCH3, -CF2-CH3, -CHF-CHF2, -CHF-CF3, -CF2-CF3;

[0085] More preferably, R 2a and R 2brepresent, independently of one another, -H, -F, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CH2OCH3, -CF2-CH3, -CHF-CHF2;

[0086] More preferably, R 2a and R 2b represent, independently of one another, -H, -F, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CF2-CH3, -CHF-CHF2, -CHF-CF3, -CF2-CF3;

[0087] More preferably, R 2a and R 2b represent, independently of one another, -H, -F, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CF2-CH3, -CHF-CHF2, -CHF-CF3, -CF2-CF3;

[0088] More preferably, R 2a and R 2b represent, independently of one another, -H, -F, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CH2OCH3, -CF2-CH3, -CHF-CHF2;

[0089] More preferably, R 2a and R 2b represent, independently of one another, -H, -F, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CF2-CH3, -CHF-CHF2;

[0090] More preferably, R 2a and R 2b represent, independently of one another, -H, -F, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CF2-CH3, -CHF-CHF2;

[0091] More preferably, R 2a and R 2brepresent, independently of one another, -H, -F, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CF2-CH3, -CHF-CHF2;

[0092] More preferably, R 2a and R 2b represents independently of one another -H, -F, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH (always in R 2a different from hydrogen).

[0093] Also preferred are compounds wherein R 2a and R 2b Both represent -CH3 or -C2H5, and more preferably -CH3.

[0094] Furthermore, preference is also given to compounds in which R 2b Represents -H and R 2aRepresentative -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CH2OCH3, -CF2-CH3, -CHF-CHF2, -CHF-CF3, -CF2-CF3; and more preferably -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CF2-CH3, -CHF-CHF2, -CHF-CF3, -CF 2-CF3; and more preferably -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CF2-CH3, -CHF-CHF2, -CHF-CF3, -CF2-CF3; and more preferably -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CH2OCH3, -CF2-CH3, -CHF-CHF2; and more preferably -CH3, -C2H5 , -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CH2OCH3, -CF2-CH3, -CHF-CHF2; and more preferably -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CF2-CH3, -CHF-CHF2; and more preferably -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, - CH2OH, -CF2-CH3, -CHF-CHF2; and more preferably -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH; and more preferably -CH3, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH; and more preferably -CH3, -CH2F, -CHF2, -CF3, -CHF-CH2F, -CH2OH; and more preferably -CH3, -CH2F, -CHF2, -CF3, -CHF-CH2F, -CH2OH; and more preferably -CH3, -CH2F, -CHF2, -CF3, -CH2OH.

[0095] R 2b preferably represents -H or -CH3; and more preferably if R 2a represents -CH3, then R 2b represents -CH3, and if R 2a Different from -CH3, then R 2b Stands for -H.

[0096] More preferably, in formula (I), R 3 represent

[0097]

[0098] and

[0099] More preferably, R 3 represent

[0100]

[0101] And more preferably R 3 represent

[0102]

[0103] and

[0104] More preferably, R 3 represent

[0105]

[0106] Furthermore, in all formulae disclosed herein, it is preferred that R 10 、R 11 、R 12 and R 13 Represents hydrogen (-H).

[0107] Also preferred are compounds of formula (I) wherein

[0108] R 2a represents -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CH2OCH3; and

[0109] R 2b represents -H or -CH3; and preferably if R 2a represents -CH3, then R 2b represents -CH3, and if R 2a Different from -CH3, then R 2b Stands for -H.

[0110] B stands for -OR 3 or -O-CHR 3 R 3* ;

[0111] R 3 represent

[0112]

[0113] R 3* represents -H, -F, -CH3, -C2H5, -C3H7, -CH(CH3)2, -CH2F, -CHF2, -CF3, -CH2-CF3;

[0114] R 4 and R 4* represents independently of one another -H, -F, -Cl, -Br, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -OCH3, -OC2H5, -OC3H7, -OCH(CH3)2, -OCH2F, -OCHF2, -OCF3, -CH2-CF3, -CHF-CH2F, -CHF-CHF2, -CHF-CF3, -CF2-CH3, -CF2-CH2F, -CF2-CHF2, -CF2-CF3,

[0115] And A, R 1 、R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 and R 13 has the same meaning as defined in claim 1 or as defined herein.

[0116] In all formulae disclosed herein, and in particular in residue A, the substituent R 4Preferably represents -H, -F, -Cl, -Br, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -OCH3, -OC2H5, -OCH(CH3)2, -OCH2F, -OCHF2, -OCF3, -CH2-CF3, -CHF-CH2F, -CHF-CHF2, -CHF-CF3, -CF2-CH3, -CF2-CH2F, -CF2-CHF2 or -CF2-CF3; more preferably -H, -F, -Cl, -Br, -CH2F, -CHF2, -CF3, -OCH3, -OC2H5, -OCH(CH3)2, -OCH2F, -OC HF2, -OCF3, -CH2-CF3, -CHF-CH2F, -CHF-CHF2, -CHF-CF3, -CF2-CH3, -CF2-CH2F, -CF2-CHF2 or -CF2-CF3; more preferably -H, -F, -Cl, -CH2F, -CHF2, -CF3, -OCH3, -OC2H5, -OCH2F, -OCHF2, -OCF3, -CH2-CF3, -CHF-CH2F, -CHF-CHF2, -CHF-CF3, -CF2-CH3, -CF2-CH2F, -CF2-CHF2 or -CF2-CF3; more preferably -H, -F, -C l, -CHF2, -CF3, -OCH3, -OC2H5, -OCHF2, -OCF3, -CH2-CF3, -CHF-CH2F, -CHF-CHF2, -CHF-CF3, -CF2-CH3, -CF2-CH2F, -CF2-CHF2 or -CF2-CF3; more preferably -H, -F, -Cl, -CHF2, -CF3, -OCH3, -OC2H5, -OCHF2, -OCF3, -CHF-CHF2, -CHF-CF3, -CF2-CH3, -CF2-CH2F, -CF2-CHF2 or -CF2-CF3; more preferably -H, -F, -Cl, - CHF2, -CF3, -OCH3, -OC2H5, -OCHF2, -OCF3, -CHF-CHF2, -CHF-CF3, -CF2-CHF2 or -CF2-CF3; more preferably -H, -F, -Cl, -CHF2, -CF3, -OCH3, -OC2H5, -OCHF2, -OCF3, -CF2-CHF2 or -CF2-CF3; more preferably -H, -F, -Cl, -CHF2, -CF3, -OCH3, -OC2H5, -OCHF2 or -OCF3; more preferably -H, -F, -Cl, -CHF2, -CF3, -OCH3, -OCHF2 or -OCF3.

[0117] With R 4 The preferred combination of definitions of the substituent R4* Preferably represents -H, -F, -Cl, -CH2F, -CHF2, -CF3, -OCHF2 or -OCF3, more preferably R 4* Preferably represents -H, -F, -Cl, -CHF2, -CF3, -OCHF2 or -OCF3, more preferably -H, -F, -Cl, -CHF2, -OCHF2 or -OCF3, more preferably -H, -F, -Cl, -OCHF2 or -OCF3, more preferably -H, -F, -Cl or -OCF3, more preferably -H, -F or -OCF3, more preferably -H or -F, more preferably -H.

[0118] In all formulae disclosed herein, the substituent R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 and R 13 or substituent R 5 、R 6 、R 7 、R 8 and R 9 Preferably, independently of one another, represents -H, -cyclo-C3H5, -cyclo-C4H7, -cyclo-C5H9, -cyclo-C6H 11, -OH, -OCH3, -OC2H5, -OC3H7, -O-cyclo-C3H5, -OCH(CH3)2, -OC(CH3)3, -OC4H9, -CH2-OCH3, -C2H4-OCH3, -CH2-OC2H5, -CH2-O-cyclo-C3H5, -CH2-OCH(CH3)2, -F, -Cl, -Br, -I, -CN, -COCH3, -COC2H5, -COC3H7, -CO-cyclo-C3H5, -COCH(CH3)2, -COC(CH3)3, -COOH, -COOCH3, -COOC2H5, -COOC3H7, -COO-cyclo-C3H5, -CO OCH(CH3)2, -COOC(CH3)3, -OOC-CH3, -OOC-C2H5, -OOC-C3H7, -OOC-ring-C3H5, -OOC-CH(CH3)2, -OOC-C(CH3)3, -CONH2, -CONHCH3, -CONHC2H5, -CONHC3 H7, -CONH-ring-C3H5, -CONH[CH(CH3)2], -CONH[C(CH3)3], -CON(CH3)2, -CON(C2H5)2, -CON(C3H7)2, -CON(ring-C3H5)2, -CON[CH(CH3)2]2, -CON[C(CH3)3 ]2, -NHCOCH3, -NHCOC2H5, -NHCOC3H7, -NHCO-cyclo-C3H5, -NHCO-CH(CH3)2, -NHCO-C(CH3)3, -NH2, -NHCH3, -NHC2H5, -NHC3H7, -NH-cyclo-C3H5, -NHCH(CH3)2, -NHC(CH3)3, -N(CH3)2, -N(C2H5)2, -SOCH3, -SOC2H5, -SOC3H7, -SO-cyclo-C3H5, -SOCH(CH3)2, -SOC(CH3)3, -SO2CH3, -SO2C2H5, -SO2C3H7, -SO2-cyclo-C3 H5, -SO2CH(CH3)2, -SO3H, -SO3CH3, -SO3C2H5, -SO3C3H7, -SO3-ring-C3H5, -SO3CH(CH3)2, -SO2NH2, -SO2NHCH3, -SO2NHC2H5, -SO2NHC3H7, -SO2NH-ring-C 3H5, -SO2NHCH(CH3)2, -SO2NHC(CH3)3, -SO2N(CH3)2, -SO2N(C2H5)2, -OS(=O)CH3, -OS(=O)C2H5, -OS(=O)C3H7, -OS(=O)-ring-C3H5, -OS(=O)CH(CH3)2,-O-SO2-CH3, -O-SO2-C2H5, -O-SO2-C3H7, -O-SO2-cyclo-C3H5, -O-SO2-CH(CH3)2, -OCH2 F. -OCHF2, -OCF3, -CH2-OCF3, -C2H4-OCF3, -CH2-OCHF2, -C2H4-OCHF2, -C3H6-OCHF2 , -OC2F5, -CH2-OC2F5, -CH2F, -CHF2, -CF3, -CH2-CH2F, -CH2-CHF2, -CH2-CF3, -CH3, -C2H5, -C3H7, -CH(CH3)2, -C4H9, -CH2-CH(CH3)2, -CH(CH3)-C2H5, -C(CH3)3, -C5H, 11 , -CH=CH2, -CH2-CH=CH2, -C(CH3)=CH2, -CH=CH-CH3, -C≡CH, -C≡C-CH3, -CH2-C≡CH;

[0119] or

[0120] R 5 and R 6 or R 6 and R 7 Together with the two carbon atoms of the phenyl ring to which they are attached, they may form a 6-membered aromatic or 6-membered N-heteroaromatic ring system, which is optionally substituted by one or more substituents selected from R 10 、R 11 、R 12 and R 13 ; and the ring system is preferably unsubstituted (R 10 =R 11 =R 12 =R 13 =-H).

[0121] More preferably, in all formulae disclosed herein, the substituent R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 and R 13 or substituent R 5 、R 6 、R 7 、R 8 and R 9 Preferably, independently of one another, represents -H, -cyclo-C3H5, -cyclo-C4H7, -cyclo-C5H9, -cyclo-C6H 11, -OH, -OCH3, -OC2H5, -OC3H7, -O-cyclo-C3H5, -OCH(CH3)2, -OC(CH3)3, -OC4H9, -CH2-OCH3, -C2H4-OCH3, -CH2-OC2H5, -CH2-O-cyclo-C3H5, -CH2-OCH(CH3)2, -F, -Cl, -Br, -I, -CN, -COCH3, -COC2H5, -COC3H7, -CO-cyclo-C3H5, -COCH(CH3)2, -CONH2, -CONHCH3, -CONHC2H5, -CONHC3H7, -CONH-cyclo-C3H5, -CON(CH3)2, -CON(C2H5)2, -NHCOCH3, -NHCOC2H5, -NHCOC3H7, -NHCO-cyclo-C3H5, -NHCO-CH(CH3)2, -NH2, -NHCH3, -NHC2H5, -NHC3H7, -NH-cyclo-C3H5, -NHCH(CH3)2, -NHC(CH3)3, -N(CH3)2, -N(C2H5)2, -SOCH3, -SOC2H5, -SOC3H7, -SO-cyclo-C3H5, -SOCH(CH3)2, -SO2CH3, -SO2C2H5, -SO2C3H7, -SO2-cyclo-C3H5, -SO2CH(CH3)2, -SO3H, -SO3CH3, -SO3C2H5, -SO3C3H7, -SO3-cyclo-C3H5, -SO3CH(CH3)2, -SO2NH2, -SO2NHCH3, -SO2NHC2H5, -SO2NHC3H7, -SO2NH-cyclo-C3H5, -SO2N(CH3)2, -SO2N(C2H5)2, -O-S(=O)CH3, -O-S(=O)C2H5, -O-S(=O)C3H7, -O-S(=O)-cyclo-C3H5, -O-S(=O)CH(CH3)2, -O-SO2-CH3, -O-SO2-C2H5, -O-SO2-C3H7, -O-SO2-cyclo-C3H5, -O-SO2-CH(CH3)2, -OCH2F, -OCHF2, -OCF3, -CH2-OCF3, -C2H4-OCF3, -CH2-OCHF2, -C2H4-OCHF2, -C3H6-OCHF2, -OC2F5, -CH2-OC2F5, -CH2F, -CHF2, -CF3, -CH2-CH2F, -CH2-CHF2, -CH2-CF3, -CH3, -C2H5, -C3H7, -CH(CH3)2;

[0122] or

[0123] R 5and R 6 or R 6 and R 7 Together with the two carbon atoms of the phenyl ring to which they are attached, they may form a 6-membered aromatic or 6-membered N-heteroaromatic ring system containing 1 or 2 nitrogen atoms, said ring system being optionally substituted by one or more substituents selected from R 10 、R 11 、R 12 and R 13 ; and the ring system is preferably unsubstituted (R 10 =R 11 =R 12 =R 13 =-H).

[0124] More preferably, in all formulae disclosed herein, the substituent R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 and R 13 or substituent R 5 、R 6 、R 7 、R 8 and R 9Preferably, independently of one another, -H, -cyclo-C3H5, -OH, -OCH3, -OC2H5, -OC3H7, -O-cyclo-C3H5, -OCH(CH3)2, -CH2-OCH3, -CH2-OC2H5, -F, -Cl, -Br, -CN, -COCH3, -COC2H5, -CONH2, -CONHCH3, -CONHC2H5, -CON(CH3 )2, -SOCH3, -SOC2H5, -SOC3H7, -SO-cyclo-C3H5, -SOCH(CH3)2, -SO2CH3, -SO2C2H5, -SO2C3H7, - SO2-ring-C3H5, -SO2CH(CH3)2, -SO3H, -SO3CH3, -SO3C2H5, -SO3C3H7, -SO3-ring-C3H5, -SO3CH(C H3)2, -OS(=O)CH3, -OS(=O)C2H5, -OS(=O)C3H7, -OS(=O)-ring-C3H5, -OS(=O)CH(CH3)2, -O-S O2-CH3, -O-SO2-C2H5, -O-SO2-C3H7, -O-SO2-ring-C3H5, -O-SO2-CH(CH3)2, -OCH2F, -OCHF2, - OCF3, -CH2-OCF3, -C2H4-OCF3, -CH2-OCHF2, -C2H4-OCHF2, -C3H6-OCHF2, -OC2F5, -CH2-OC2 F5, -CH2F, -CHF2, -CF3, -CH2-CH2F, -CH2-CHF2, -CH2-CF3, -CH3, -C2H5, -C3H7, -CH(CH3)2;

[0125] or

[0126] R 5 and R 6 or R 6 and R 7 Together with the two carbon atoms of the phenyl ring to which they are attached, they can form a 6-membered aromatic or 6-membered N-heteroaromatic ring system containing one nitrogen atom, said ring system being optionally substituted by one or more substituents selected from R 10 、R 11 、R 12 and R 13 ; and the ring system is preferably unsubstituted (R 10 =R 11 =R 12 =R 13 =-H).

[0127] Preferably R 3 represent in

[0128] Substituent R 5 -R 7 has the meaning as disclosed above in the first two pages, and more preferably R 5 -R 7 Each of them is independently selected from -H, -OCH3, -OC2H5, -OC3H7, -O-cyclo-C3H5, -OCH(CH3)2, -F, -Cl, -Br, -CN, -SO2CH3, -SO2C2H5, -OCH2F, -OCHF2, -OCF3, -CH2-OCF3, -CH2-OCHF2, -OC2F5, -CH2F, -CHF2, -CF3, -CH2-CHF2, -CH2-CF3, -CH3, -C2H5, -C3H7, -CH(CH3)2; more preferably selected from -H, -OCH3, -OC2H5, - F, -Cl, -CN, -SO2CH3, -SO2C2H5, -OCH2F, -OCHF2, -OCF3, -CH2F, -CHF2, -CF3, -CH3, -C2H5; more preferably selected from -H, -OCH3, -OC2H5, -F, -Cl, -CN, -SO2CH3, -SO2C2H5, -OCH2F, -OCHF2, -OCF3, -CH2F, -CHF2, -CF3, -CH3; more preferably selected from -H, -OCH3, -F, -Cl, -OCHF2, -OCF3, -CHF2, -CF3, -CH3.

[0129] Furthermore, it is preferred that the substituent R 5 -R 7 Only one of represents hydrogen and the other two are different from hydrogen. More preferably, R 5 Different from hydrogen and R 6 Represents hydrogen or R 7 Represents hydrogen or R 6 and R 7 represents hydrogen. Therefore, the residue R 3 Para-substitution is preferred.

[0130] In all formulae disclosed herein, the substituent R 3 Preferably represents

[0131]

[0132] In certain embodiments, the present invention relates to compounds of formula (I):

[0133]

[0134] in

[0135] A stands for

[0136] B is -OR 3 、-O-CHR 3 R 3* 、-O-CH2-CH2-R 3 or -O-CH2-CH2-CH2-R 3 ; R 1 represent

[0137] R 2a and R 2b Each independently represents -H, -F, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CH2OCH3, -CH2CH2OCH3, -CF2-CH3, -CHF-CHF2, -CHF-CF3, -CF2-CF3; wherein R 2a Not -H;

[0138] R 3 represent

[0139]

[0140] R 3* represents -H, -F, -CH3, -C2H5, -C3H7, -CH(CH3)2, -CH2F, -CHF2, -CF3, -CH2-CF3;

[0141] R 4 and R 4* represents independently of one another -H, -F, -Cl, -Br, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -OCH3, -OC2H5, -OCH(CH3)2, -OCH2F, -OCHF2, -OCF3, -CH2-CF3, -CHF-CH2F, -CHF-CHF2, -CHF-CF3, -CF2-CH3, -CF2-CH2F, -CF2-CHF2, -CF2-CF3,

[0142] Preferably, R 4 Represents -H, -F, -Cl, -CH3, -CH2F, -CHF2, -CF3, -OCH3, -OCH(CH3)2, -OCHF2, -OCF3, -CF2-CHF2, and more preferably -H, -F, -Cl, -CH3, -CH2F, -CHF2, -CF3, -OCH3, -OCHF2, -OCF3,

[0143] Preferably, R 4* represents -H, -F, -Cl, -CH2F, -CHF2, -CF3, -OCHF2 or -OCF3, more preferably, R 4* Represents -H or -F;

[0144] And R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 and R 13 has the same meaning as defined herein, and R 10 、R 11 、R 12 and R 13 Preferably represents hydrogen.

[0145] Preferably, the present invention relates to compounds of formula (I),

[0146] in

[0147] A stands for

[0148] B is -OR 3 、-O-CHR 3 R 3* 、-O-CH2-CH2-R 3 or -O-CH2-CH2-CH2-R 3 ; and preferably B represents -OR 3 or -O-CHR 3 R 3* ;

[0149] R 1 represent

[0150] R 2a represents -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CH2OCH3; and

[0151] R 2b represents -H or -CH3; and preferably if R 2a represents -CH3, then R 2b represents -CH3, and if R2a Different from -CH3, then R 2b Stands for -H.

[0152] R 3 represent

[0153]

[0154] Preferably, R 3 represent

[0155]

[0156] R 3* represents -H, -F, -CH3, -C2H5, -C3H7, -CH(CH3)2, -CH2F, -CHF2, -CF3, -CH2-CF3;

[0157] R 4 and R 4* represents independently of one another -H, -F, -Cl, -Br, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -OCH3, -OC2H5, -OCH(CH3)2, -OCH2F, -OCHF2, -OCF3, -CH2-CF3, -CHF-CH2F, -CHF-CHF2, -CHF-CF3, -CF2-CH3, -CF2-CH2F, -CF2-CHF2, -CF2-CF3,

[0158] Preferably, R 4 Represents -H, -F, -Cl, -CH3, -CH2F, -CHF2, -CF3, -OCH3, -OCH(CH3)2, -OCHF2, -OCF3, -CF2-CHF2,

[0159] Preferably, R 4* represents -H, -F, -Cl, -CH2F, -CHF2, -CF3, -OCHF2 or -OCF3, more preferably, R 4* Represents -H or -F;

[0160] And R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 and R 13 has the same meaning as defined herein, and R 10 、R11 、R 12 and R 13 Preferably represents hydrogen.

[0161] Preferably, in the compounds of formula (I) as defined herein, R 1 represent

[0162]

[0163] More preferably, R 1 represent

[0164]

[0165] In certain embodiments, the present invention relates to compounds of formula (Ia) or (Ib):

[0166]

[0167] in

[0168] n is 0, 1, 2 or 3; preferably n is 0, 1 or 2, more preferably n is 0 or 1;

[0169] R 1 、R 2a 、R 2b 、R 4 、R 5 、R 6 、R 7 、R 8 and R 9 has the same meaning as defined above.

[0170] Preferred are compounds of formula (Ia),

[0171]

[0172] in

[0173] n is 0, 1 or 2, more preferably n is 0 or 1;

[0174] R 1 represent

[0175]

[0176] R 4Represents -H, -F, -Cl, -CH3, -CH2F, -CHF2, -CF3, -OCH3, -OCH(CH3)2, -OCH2F, -OCHF2, -OCF3, -C H2-CF3, -CHF-CH2F, -CHF-CHF2, -CHF-CF3, -CF2-CH3, -CF2-CH2F, -CF2-CHF2, -CF2-CF3,

[0177] Preferably, R 4 Represents -H, -F, -Cl, -CH3, -CH2F, -CHF2, -CF3, -OCH3, -OCH(CH3)2, -OCHF2, -OCF3, -CF2-CHF2, More preferably, R 4 represents -H, -F, -Cl, -CH2F, -CHF2, -CF3, -OCHF2, -OCF3, -CF2-CHF2, more preferably, R 4 represents -H, -F, -CH2F, -CHF2, -CF3, -OCHF2, -OCF3, -CF2-CHF2;

[0178] And R 5 to R 8 has the same meaning as defined herein.

[0179] More preferably, in formula (I), (Ia) or (Ib),

[0180] R 1 represent

[0181] More preferably, R 1 represent

[0182] In certain embodiments, the present invention relates to compounds of any one of the following formulae (II-1) to (II-5), (III-1) to (III-6), (IV-1) to (IV-8), (V-1) to (V-5), (VI-1) to (VI-2), and (VII-1) to (VII-3):

[0183]

[0184]

[0185]

[0186] where R 1 、R 3* 、R 4 、R 4*、R 5 、R 6 、R 7 、R 8 、R 10 、R 11 、R 12 and R 13 has the same meaning as defined in the same meaning as defined above.

[0187] In certain embodiments, the present invention relates to a compound of any one of the following formulae (II-1) to (II-5), (III-1) to (III-6), (IV-1) to (IV-6), (V-1) to (V-3):

[0188]

[0189]

[0190] where R 1 、R 4 、R 5 、R 6 、R 7 、R 8 、R 10 、R 11 、R 12 and R 13 has the same meaning as defined above.

[0191] Preferably, in the compounds of any one of the following formulae (II-1) to (II-5), (III-1) to (III-6), (IV-1) to (IV-6), (V-1) to (V-3), (II-1) to (II-5), (III-1) to (III-6), (IV-1) to (IV-8), (V-1) to (V-5), (VI-1) to (VI-2) and (VII-1) to (VII-3):

[0192] R 1 represent

[0193]

[0194] Preferably, R 1 represent

[0195]

[0196] More preferably, R 1 represent More preferably, R 1 represent

[0197] Preferably, in the compounds of any one of the following formulae (II-1) to (II-5), (III-1) to (III-6), (IV-1) to (IV-8), (V-1) to (V-5), (VI-1) to (VI-2) and (VII-1) to (VII-3),

[0198] R 4 Represents -H, -F, -Cl, -Br, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -OCH3, -OC2H5, -OCH(CH3)2, -OCH2F, -OCHF2, -OCF3, -CH2-CF3, -CHF-CH2F, -CHF-CHF2, -CHF-CF3, -CF2-CH3, -CF2-CH2F, -CF2-CHF2, -CF2-CF3,

[0199] Preferably, R 4 Represents -H, -F, -Cl, -CH3, -CH2F, -CHF2, -CF3, -OCH3, -OC2H5, -OCH(CH3)2, -OCH2F, -OCHF2, -OCF3 , -CH2-CF3, -CHF-CH2F, -CHF-CHF2, -CHF-CF3, -CF2-CH3, -CF2-CH2F, -CF2-CHF2, -CF2-CF3,

[0200] More preferably, R 4 Represents -H, -F, -Cl, -CH3, -CH2F, -CHF2, -CF3, -OCH3, -OCH(CH3)2, -OCHF2, -OCF3, -CF2-CHF2, And more preferably -H, -F, -Cl, -CH3, -CH2F, -CHF2, -CF3, -OCH3, -OCH(CH3)2, -OCHF2, -OCF3, -CF2-CHF2.

[0201] More preferably, R 4 represents -H, -F, -Cl, -CH3, -CH2F, -CHF2, -CF3, -OCH3, -OCHF2, -OCF3 or -CF2-CHF2; and more preferably -H, -F, -Cl, -CHF2, -CF3, -OCH3, -OCHF2, -OCF3; and most preferably R 4 Yes -OCF3.

[0202] In a preferred embodiment, the present invention relates to compounds of formula (I),

[0203]

[0204] in

[0205] A stands for

[0206] B stands for -OR 3 or -O-CH2-R 3 ;

[0207] R 1 represent

[0208] Preferably R 1 represent More preferably, R 1 represent

[0209] R 3 represent

[0210]

[0211] R 4 represents -H, -F, -Cl, -CH3, -CF3, -OCH3, -OCHF2 or -OCF3; and

[0212] R 5 、R 6 、R 7 and R 8 independently represent -H, -F, -Cl, -CN, -CH3, -CHF2, -CF3, -OCHF2, -OCF3 or -SO2CH3;

[0213] or an enantiomer, a diastereomer, a tautomer, a mixture of enantiomers, a mixture of diastereomers, a mixture of tautomers, a hydrate, a solvate, or a pharmaceutically acceptable salt of the above compounds.

[0214] More preferred are compounds of any one of the following formulae (III-1) to (III-2), (III-4) to (III-6), (IV-1) to (IV-2) and (IV-4) to (IV-6):

[0215]

[0216]

[0217] in

[0218] R 1represent Preferably

[0219] R 1 represent

[0220] More preferably, R 1 represent

[0221] R 4 Represents -F, -Cl, -Br, -CH3, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, -OCH3, -OCH2F, -OCHF2, -OCF3, -OCH2CH2F, -OCH2CF3 or -OCF2CF3;

[0222] Preferably, R 4 represents -H, -F, -Cl, -CH3, -CF3, -OCH3, -OCHF2 or -OCF3; and

[0223] R 5 、R 6 、R 7 and R 8 independently represent -H, -F, -Cl, -CN, -CH3, -CHF2, -CF3, -OCHF2, -OCF3 or -SO2CH3;

[0224] or an enantiomer, a diastereomer, a tautomer, a mixture of enantiomers, a mixture of diastereomers, a mixture of tautomers, a hydrate, a solvate, or a pharmaceutically acceptable salt of the above compounds.

[0225] In certain embodiments, the present invention relates to compounds of any one of the following formulae (I), (II-1) to (II-5), (III-1) to (III-6), (IV-1) to (IV-8), (V-1) to (V-5), (VI-1) to (VI-2), and (VII-1) to (VII-3): wherein R 3 represent

[0226]

[0227] Preferably, R 3 represent

[0228]

[0229]

[0230] More preferably, R 3represent

[0231]

[0232] More preferably, R 3 represent Best

[0233] In a preferred embodiment, the present invention relates to compounds of formula (I),

[0234]

[0235] in

[0236] A stands for

[0237] B stands for -OR 3 、-O-CH2-R 3 or -O-CHR 3 R 3* ;

[0238] R 1 represent

[0239] R 3 represent

[0240]

[0241] R 3* represents -H, -CH3, -C2H5, -C3H7, -CH(CH3)2, -CH2F, -CHF2, -CF3 or -CH2-CF3;

[0242] R 4 and R 4* represents independently of one another -H, -F, -Cl, -CH3, -CH2F, -CHF2, -CF3, -OCH3, -OCH(CH3)2, -OCH2F, -OCHF2, -OCF3, -CH2-CF3, -CHF-CH2F, -CHF-CHF2, -CHF-CF3, -CF2-CH3, -CF2-CH2F, -CF2-CHF2, -CF2-CF3,

[0243] Preferably, R 4 Represents -H, -F, -Cl, -CH3, -CH2F, -CHF2, -CF3, -OCH3, -OCH(CH3)2, -OCHF2, -OCF3, -CF2-CHF2, More preferably, R 4 represents -H, -F, -Cl, -CH2F, -CHF2, -CF3, -OCHF2, -OCF3, -CF2-CHF2, more preferably, R 4 represents -H, -F, -CH2F, -CHF2, -CF3, -OCHF2, -OCF3, -CF2-CHF2;

[0244] Preferably, R 4* represents -H, -F, -Cl, -CH2F, -CHF2, -CF3, -OCHF2 or -OCF3, more preferably, R 4* Represents -H or -F;

[0245] and

[0246] R 5 、R 6 、R 7 and R 8 independently represent -H, -F, -Cl, -CN, -CH3, -CHF2, -CF3, -OCH3, -OCHF2, -OCF3 or -SO2CH3;

[0247] or an enantiomer, a diastereomer, a tautomer, a mixture of enantiomers, a mixture of diastereomers, a mixture of tautomers, a hydrate, a solvate, or a pharmaceutically acceptable salt of the above compounds.

[0248] In a preferred embodiment, the present invention relates to compounds of formula (I),

[0249]

[0250] in

[0251] A stands for

[0252] B stands for -OR 3 or -O-CH2-R 3 ;

[0253] R 1 represent

[0254]

[0255] Preferably R 1 represent More preferably, R 1 represent R 3 represent

[0256]

[0257] Preferably, R 3 represent

[0258]

[0259]

[0260] R 4 represents -H, -F, -Cl, -CH3, -CF3, -OCH3, -OCHF2 or -OCF3;

[0261] or an enantiomer, a diastereomer, a tautomer, a mixture of enantiomers, a mixture of diastereomers, a mixture of tautomers, a hydrate, a solvate, or a pharmaceutically acceptable salt of the above compounds.

[0262] Particularly preferred compounds according to the present invention include those presented in Table 1.

[0263] Table 1

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301] or an enantiomer, a diastereomer, a tautomer, a mixture of enantiomers, a mixture of diastereomers, a mixture of tautomers, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof.

[0302] The most preferred compounds according to the present invention include compounds 142, 156, 248 and 329 or enantiomers, diastereomers, tautomers, mixtures of enantiomers, mixtures of diastereomers, mixtures of tautomers, hydrates, solvates, pharmaceutically acceptable salts thereof.

[0303]

[0304] Synthesis of compounds

[0305] The compound of formula (I) can be prepared with reference to the method shown in the following reaction scheme. As described below, by selecting suitable reagents and performing appropriate substitutions, the compound of formula (I) can be generated. Those of ordinary skill in the art can easily select solvent, temperature, pressure and other reaction conditions. The raw materials are commercially available or can be easily prepared by those of ordinary skill in the art. Those of ordinary skill in the art can apply other synthetic routes to prepare the compound of formula (I) according to methods similar to those disclosed in the literature.

[0306] Solution 1

[0307]

[0308] The present invention also relates to a process for producing a compound of formula (Ib),

[0309]

[0310] The method comprises the following steps:

[0311] Step A) Providing compound (I-1*)

[0312]

[0313] Step B1)

[0314] B1a) CC coupling reaction between compound (I-1*) and (A1*) in the presence of Pd catalyst and base

[0315]

[0316] To obtain compound (I-4*);

[0317] B1b) Removal of the protecting group P from compound (I-4*) 1

[0318]

[0319] To obtain compound (Ib);

[0320]

[0321] in

[0322] X represents a leaving group, preferably a halogen;

[0323] P 1 represents a hydroxyl protecting group;

[0324] R' represents hydrogen or C 1-3 Alkyl; or two R' together form a pinacol moiety;

[0325] n、R 2a 、R 2b 、R 4 and R 5 to R 9 has the same meaning as defined in formula (Ib).

[0326] Alternatively, a method for producing a compound of formula (Ib), comprising the steps of:

[0327] Step A) Providing compound (I-1*)

[0328]

[0329] Step B2)

[0330] B2a) CC coupling reaction between compound (I-1*) and (A2*) in the presence of a Pd catalyst and a base

[0331]

[0332] To obtain compound (I-2*)

[0333]

[0334] B2b) performing a coupling reaction between compounds (I-2*) and (B*)

[0335]

[0336] To obtain compound (Ib)

[0337]

[0338] in

[0339] X represents a leaving group, preferably a halogen;

[0340] P 1 represents a hydroxyl protecting group;

[0341] R' represents hydrogen or C 1-3Alkyl; or two R' together form a pinacol moiety;

[0342] n、R 2a 、R 2b 、R 4 and R 5 to R 9 has the same meaning as defined in formula (Ib).

[0343] Alternatively, a method for producing a compound of formula (Ib), comprising the steps of:

[0344] Step A2) provides compound (I-3*)

[0345]

[0346] Step B3)

[0347] B2a) Execution of the reaction of compound (I-3*) and (R 1 *) coupling reaction between

[0348]

[0349] To obtain compound (Ib)

[0350]

[0351] in

[0352] X represents a leaving group, preferably a halogen;

[0353] n、R 2a 、R 2b 、R 4 and R 5 to R 9 has the same meaning as defined in formula (Ib).

[0354] Carried out in the presence of a palladium catalyst and a base

[0355] In step B1a), the brominated compound (I-1*) is reacted with compound A1* as a boronic acid derivative by Suzuki coupling.

[0356]

[0357] Alternatively, in step B2a), the brominated compound (I-1*) is reacted with compound A2* as a boronic acid derivative by Suzuki coupling reaction.

[0358]

[0359] Where R' represents hydrogen or C 1-3Alkyl; or two R' together form a pinacol moiety;

[0360] Preferably, the boronic acid derivative (A1*) or (A2*) can be boronic acid (R′=-H) or an ester of boronic acid, such as its isopropyl ester (R′=-CH(CH3)2), pinacol moiety (R′-R′=-C(CH3)2-C(CH3)2-).

[0361] The palladium catalyst is Pd(0) or Pd(II) catalyst. The Pd(0) catalyst can be tetrakis(triphenylphosphine)palladium(0)[Pd(PPh3)4], tris(dibenzylideneacetone)dipalladium(0)[Pd2(dba)3]. The Pd(II) catalyst can be dichlorobis(triphenylphosphine)-palladium(II)[Pd(PPh3)2Cl2], palladium(II) acetate and triphenylphosphine or more preferably [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(Pd(dppf)Cl2). The reaction is preferably carried out in a solvent such as dioxane, DMF, DME, THF or a mixture of isopropanol and water and in the presence of a base such as aqueous sodium bicarbonate solution or K3PO4.

[0362] P 1 is a hydroxy protecting group and preferably a silicon-based protecting group selected from trimethylsilyl (TMS), triethylsilyl (TES), isopropyldimethylsilyl (IPMDS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), [2-(trimethylsilyl)ethoxy]methyl (SEM), 2-(trimethylsilyl)ethoxycarbonate (Teoc), more preferably tert-butyldimethylsilyl (TBS) or tert-butyldiphenylsilyl (TBDPS).

[0363] Medical uses

[0364] Surprisingly, the compounds of the present invention were found to effectively activate immune cells, particularly CD4+ and / or CD8+ cells as shown in Table B-1, and natural killer (NK) cells in the context of T cell receptor (TCR) and NK cell receptor signaling, respectively. This allows for highly selective TCR or NK cell receptor-mediated activation of the immune system against mutated tissues or tissues infected by foreign pathogens. Thus, by applying the present invention in clinical settings, highly effective treatments can be achieved by minimizing the risk of serious side effects. Due to the possibility of administering subnanomolar concentrations of EC 50This is further demonstrated by the discovery of highly effective compounds. Therefore, the present invention opens up a broad administration window to strike a balance between effective treatment and toxic off-target effects, thereby achieving sustained tolerability over multiple treatment courses.

[0365] As noted above, the present invention also relates to the use of the compounds of the present invention in a pharmaceutical setting. Here, the pharmaceutical setting is to be understood in the broadest sense as any method for improving a patient's health and / or well-being. The terms "pharmaceutical" and "pharmaceutical" are to be understood interchangeably.

[0366] Another aspect of the present invention relates to a pharmaceutical composition comprising at least one compound of the present invention and at least one pharmaceutically acceptable carrier.

[0367] Preferably, the pharmaceutical composition comprises at least one compound of the present invention and at least one pharmaceutically acceptable carrier, excipient and / or diluent. More preferably, the pharmaceutical composition further comprises at least one stimulator for activating immune cells.

[0368] In this respect also concerning pharmaceutical compositions, the definitions detailed above apply mutatis mutandis.

[0369] The pharmaceutically acceptable carrier according to the present invention can be any pharmaceutically acceptable additive, therefore, any additive that is nontoxic to the patient. Exemplarily, the pharmaceutically acceptable carrier can comprise a solvent, such as, for example, water, dimethyl sulfoxide (DMSO), ethanol, vegetable oil, paraffin oil or a combination thereof. In addition, the carrier can contain one or more detergents, one or more foaming agents (for example, sodium lauryl sulfate (SLS) / sodium dodecyl sulfate (SDS)), one or more coloring agents (for example, TiO , food coloring), one or more vitamins, one or more salts (for example, sodium, potassium, calcium, zinc salts), one or more wetting agents (for example, sorbitol, glycerol, mannitol, propylene glycol, polydextrose), one or more enzymes, one or more preservatives (for example, benzoic acid, methyl paraben), one or more tissue improvers (texturingagent) (for example, carboxymethyl cellulose (CMC), polyethylene glycol (PEG), sorbitol), one or more emulsifiers, one or more fillers, one or more polishing agents (glacing agent), one or more separating agents, one or more antioxidants, one or more herbal and plant extracts, one or more stabilizers, one or more polymers (e.g., hydroxypropyl methacrylamide (HPMA), polyethyleneimine (PEI), carboxymethylcellulose (CMC), polyethylene glycol (PEG)), one or more uptake mediators (e.g., polyethyleneimine (PEI), dimethyl sulfoxide (DMSO), cell penetrating peptides (CPP), protein transduction domains (PTD), antimicrobial peptides, etc.), one or more antibodies, one or more sweeteners (e.g., sucrose, potassium acesulfame, sodium saccharin, steviol glycosides), one or more counterstain dyes (e.g., fluorescein, fluorescein derivatives, Cy dyes, AlexaFluor dyes, S dyes, rhodamine, quantum dots, etc.), one or more homeopathic ingredients, one or more taste substances and / or one or more flavors.

[0370] Suitable diluents are substances that generally constitute the major portion of a composition or dosage form. Suitable diluents include sugars such as lactose, sucrose, mannitol, and sorbitol, starches derived from wheat, corn, rice, and potatoes, and celluloses such as microcrystalline cellulose. The amount of diluent in the composition can be from about 5 to about 95 weight %, preferably from about 25 to about 75 weight %, and more preferably from about 30 to about 60 weight % of the total composition.

[0371] Suitable excipients are binders, disintegrants, lubricants, glidants and / or colorants.

[0372] The term disintegrant refers to a material added to a composition to support the decomposition (disintegration) and release of the pharmaceutically active ingredient of the drug. Suitable disintegrants include starch, "cold water soluble" modified starches such as sodium starch glycolate, natural and synthetic gums such as locust bean gum, karaya gum, guar gum, tragacanth gum and agar, cellulose derivatives such as methylcellulose and sodium carboxymethylcellulose, microcrystalline cellulose and cross-linked microcrystalline cellulose such as croscarmellose sodium, alginates such as alginic acid and sodium alginate, clays such as bentonite and effervescent mixtures. The amount of disintegrant in the composition can be from about 2 to about 20% by weight of the composition, more preferably from about 5 to 10% by weight.

[0373] Adhesive is that powder particles are bonded or " glued " together and by forming particle it has the material of viscosity, therefore is used as " adhesive " in preparation.Adhesive can increase existing bonding strength in diluent or filler.Suitable adhesive comprises sugar such as sucrose, the starch derived from wheat, corn, rice and potato, natural gum such as gum arabic, gelatin and tragacanth, seaweed derivative such as alginic acid, sodium alginate and calcium ammonium alginate (calcium ammonium alginate), cellulose material such as methylcellulose, sodium carboxymethylcellulose and hydroxypropyl methylcellulose, polyvinylpyrrolidone and inorganic compound such as magnesium aluminum silicate.The amount of adhesive in composition can be about 2 to about 20 weight % of composition, preferably about 3 to about 10 weight %, and more preferably about 3 to about 6 weight %.

[0374] Lubricant refers to a substance that is added to the dosage form so that the tablet particles, etc., are released from the mold after being compressed by reducing friction or wear. Suitable lubricants include metal stearates such as magnesium stearate, calcium stearate or potassium stearate, stearic acid, high melting point waxes and other water-soluble lubricants such as sodium chloride, sodium benzoate, sodium acetate, sodium oleate, polyethylene glycol and D, L-leucine. Lubricants are usually added in the last step before compression because they must be present on the particle surface. The amount of lubricant in the composition can be about 0.2 to about 5 weight % of the composition, preferably about 0.5 to about 2 weight % of the composition, and more preferably about 0.3 to about 1.5 weight %.

[0375] Glidants are materials that prevent the components of a pharmaceutical composition from clumping and improve the flow characteristics of the granules, thereby making the flow smooth and uniform. Suitable glidants include silicon dioxide and talc. The amount of glidant in the composition may be from about 0.1 to about 5 weight percent, preferably from about 0.5 to about 2 weight percent, of the final composition.

[0376] Coloring agent is the excipient that makes composition or dosage form coloring.Such excipient can comprise the food grade dye that is adsorbed on suitable adsorbent (such as clay or aluminum oxide).The amount of coloring agent can be about 0.1 to about 5 weight % of composition, preferably about 0.1 to about 1 weight %.

[0377] The pharmaceutical composition of the present invention comprises at least one compound of the present invention. Optionally, the pharmaceutical composition may also comprise more than one compound of the present invention, such as a combination of two, three, four, five or even more compounds of the present invention.

[0378] Optionally, the pharmaceutical composition may also include one or more other pharmaceutically active agents, such as, for example, one or more other stimulants for activating immune cells, which may be other pharmaceutically active ingredients in the pharmaceutical composition except the compound of the present invention. Examples of such other stimulants for activating immune cells are provided below.

[0379] The compounds of the present invention, their pharmaceutically acceptable salts and pharmaceutical compositions can be used as medicines.

[0380] Therefore, another aspect of the present invention relates to the use of a compound or pharmaceutical composition according to the present invention as a medicament.

[0381] Insofar as this aspect relates to use as a medicine, the definitions detailed above apply mutatis mutandis.

[0382] In the context of the present invention, the terms "medicament", "therapeutic", "medicine", "drug", "therapeutic agent", "pharmaceutic", "pharmaceutical agent", "prophylactic agent" and the like are to be understood in the broadest sense as any kind of compound suitable for use in a medical context, i.e., for the treatment and / or prevention of a pathological condition.

[0383] The compound or pharmaceutical composition comprising the compound can be administered to the patient by any means known in the art, such as, for example, orally, by injection, nasally, transdermally / percutaneously, etc. Administration can be local administration (e.g., intratumoral, intranodal (i.e., into lymph nodes), intrathecal, intracerebroventricular (icv), topically or intravitreal) or systemic administration (e.g., intravenous (iv), intraarterial (ia), intraperitoneal (ip), intramuscular (im), subcutaneous (subcutaneous), oral, nasal). Preferably, administration is oral, intravenous, subcutaneous, intratumoral or intranodal administration, in particular oral or intravenous administration.

[0384] Administration can be a single administration ((acute) single administration) or can be repeated administration, such as, for example, repeated pulse dose administration or long-term administration. Repeated administration can, for example, be administration twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, more than ten times or even permanent administration. Between two administrations, there can be a time interval of less than one hour, one hour or more, six hours or more, twelve hours or more, twenty-four hours or more. Administration can be once a day, twice a day, three times a day, four times a day, every other day, every three days, once a week, every two weeks, once a month, twice a year or once a year. Those skilled in the art can determine a clinically feasible administration regimen based on balancing efficacy and toxicity.

[0385] Preferably, the medicament is suitable for treating or preventing pathological conditions associated with an inadequate immune response. In other words, the present invention also relates to a medicament suitable for enhancing an immune response. As used in the context of immune response activity, the terms "activate," "enhance," "strengthen," "increase," "trigger," "stimulate," etc., may be understood interchangeably in the broadest sense to mean providing an increased immune response activity.

[0386] In the context of the present invention, the enhancement of the immune response is preferably a local enhancement of the immune response, ie an enhancement of the immune response in the vicinity of the antigen recognized by the immune cells responsible for the respective immune response.

[0387] More specifically, antigen-stimulated, TCR-connected immune cells stimulated by the compounds of the present invention exhibit significantly increased secretion of several cytokines (such as, for example, IL-2, IFN-γ and / or TNF-α), as well as increased proliferation and cytotoxicity expressed by increased expression of cytotoxic factors (e.g., granzyme B), whereas corresponding unstimulated immune cells do not exhibit this. This results in local secretion of cytokines and effective antigen-specific cytotoxicity near the vegetation and / or infectious pathogen, thereby improving the local immune response to the vegetation and / or infectious pathogen. More preferably, the compounds of the present invention provide therapeutic or preventive interventions that increase the local effect efficiency of anti-tumor or antiviral T cells, B cells, and NK cells. However, in the absence of tumor or pathogen-associated antigens, the adverse effects of systemic cytokine levels can be widely avoided. When antigens are exemplarily located on the surface of neoplastic cells (e.g., cancer cells) and / or antigen-presenting cells (e.g., mature dendritic cells), the activity of immune cells (particularly T cells) contacted with such antigens may increase. It has been found that after administration of the compounds of the present invention, stimulated immune cells, particularly activated T cells in contact with their cognate antigens (e.g., tumor and / or pathogen antigens), show increased local activation of the immune system in the tumor microenvironment and draining lymph nodes. Therefore, the immune response enhanced near the vegetation can become a driving force for the enhanced physiological immune response supporting the cytotoxicity of the vegetation. This can further trigger antigen diffusion and neoantigen presentation achieved by APCs, thereby inducing a wider range of T cell-specific immune profiles. When the antigen is exemplarily located on the surface of virally infected cells (e.g., human papilloma (HPV) or hepatitis C infected cells), the activity of the immune cells in contact with such antigens may increase.

[0388] It is noteworthy that, compared with the strategy based on vaccination (for example, tumor vaccination), about its activity, the compound of the present invention does not necessarily require that immune cells have been in contact with specific tumor antigens, and the stimulation of cells can also be achieved by other means, for example, stimulating TCR / CD3 pathways and / or costimulatory pathways such as CD28. In the context of the present invention, the enhanced immune response is preferably characterized by an increase in the secretion of at least one cytokine, more preferably characterized by an increase in the secretion of at least one cytokine selected from IL-2, IFN-γ, TNF-α, IL-1 and IL-6, and even more preferably characterized by an increase in the secretion of at least one cytokine selected from IL-2, IFN-γ and TNF-α. Particularly preferably, the enhanced immune response is preferably characterized by an increase in the secretion of at least two cytokines, such as, particularly preferred IL-2 and IFN-γ, IL-2 and TNF-α or IFN-γ and TNF-α. Also highly preferred is an increase in at least three cytokines, such as IL-2, IFN-γ and TNF-α. Additionally or alternatively, expression of other markers associated with immune activity may also be increased, such as, for example, CD40 ligand (CD40L, also known as CD154), granzyme / perforin, CD69, CD25 and / or CD71. Preferably, such a marker is CD40L.

[0389] As mentioned above, known tumor infiltrating lymphocytes (TIL), particularly IL-2 and IFN-γ for the enhancement of T cells for neoplastic and / or infectious antigens produce and are associated with the immunity for neoplastic and / or infectious lesions that enhance.TNF-α also has such an effect.IL-2 can directly activate CD8 cells and natural killer (NK) cells. Therefore, its release may be useful to neoplastic and / or infectious lesions, but it may also have a general effect on promoting T cell survival. Therefore, its release during antigen presenting cells (APC) stimulation of T cells (for example, in lymph nodes) can also enhance immune response.

[0390] Granzyme / perforin is considered an effector molecule and therefore a marker for direct killing of neoplastic cells (particularly tumor cells) and may be released specifically proximal to or even within the neoplasm. Similarly, IFN-γ and TNF-α may also activate immune cells (such as, for example, NK cells and myeloid cells), but may also directly upregulate apoptotic pathways in neoplastic cells.

[0391] IL-6 is a pleiotropic cytokine that is particularly known for supporting B cell survival, and therefore its release in lymph nodes may also support B cell survival.

[0392] The release of IL-1 and IL-6 may enhance the development of T helper cells (such as Th17 cells), which are known to play an important role in immunity against neoplastic and infectious diseases. Therefore, the presence of elevated IL-6 and IL-1 levels in neoplastic and / or infectious lesions and lymph nodes may have a beneficial effect on the immune response.

[0393] CD25, CD69, CD71 and CD40L are well-known surface markers of T cell activation and are known to show the effect of compounds on the activation level of individual T cells. CD69 is particularly an early marker of T cell activation. CD25 is an IL-2 receptor, and high (higher) expression generally supports (more) rapid expansion of activated T cells. CD71 is a receptor for transferrin and generally supports T cells in providing Fc for proliferation. CD40L is a receptor on T helper cells that is known to support APC and B cell activation, survival and proliferation.

[0394] Specifically, the increase in IL-2, IFN-γ and / or TNF-α secretion and / or CD40L expression is also exemplified in the Examples section below. All of these markers are well-known factors in the anti-neoplastic immune response, thus demonstrating the anti-neoplastic (particularly, anti-tumor) activity of the compounds of the present invention.

[0395] This increase in cytokine secretion by immune cells can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold, compared to the corresponding cytokine secreted by immune cells exposed to the same stimulant and cultured under comparable conditions but not exposed to the compounds of the invention. Those skilled in the art will note that the rate of increase generally also depends on the amount of the compounds of the invention acting on the corresponding immune cells in a dose-dependent manner. Therefore, in many cases, it will also depend on the dose of the compounds of the invention administered to the patient in a dose-dependent manner. Within an appropriate dosage range, higher doses will generally also result in a greater increase. Those skilled in the art will also appreciate that dose-dependency is also related to the patient's weight, the patient's fat and body water content, the patient's individual metabolic rate for inactivating and / or eliminating the compound, the patient's individual immune status, and the like. Therefore, those skilled in the art can adjust the dosage accordingly.

[0396] Additionally or alternatively, the proliferation rate of immune cells (such as T cells, NK cells, B cells and / or monocytes) can also be increased. For example, the proliferation of CD4+ and / or CD8+ cells can be increased. Additionally or alternatively, the maintenance (i.e., survival rate, activity time or survival time) of immune cells (such as, for example, T cells, NK cells, B cells and / or monocytes (for example, CD4+ and / or CD8+ cells)) can also be increased.

[0397] Notably, the compounds of the present invention can also increase T cell reactivity to tumor antigens presented by MHC I to CD8 T cells or by MHC II to CD4 T cells, regardless of tumor type and regardless of tumor antigen. In addition, the compounds of the present invention can increase the immune activity of the patient's NK cells to help destroy tumor cells that have reduced MHC-I-mediated tumor antigen display. In addition, B cells and other immune cells can further enhance the strong antigen-specific T cell response, and these cells may also be targeted by the compounds of the present invention. Therefore, the compounds of the present invention can also eliminate immune neglect against neoplastic and / or infectious pathogens, so that the patient's specific tumor antigens are recognized as non-self, and thus the patient's own immune system can be reactivated to attack those tumor cells present in the patient's body, regardless of the corresponding type of neoplastic and / or infectious disease.

[0398] In view of the above, in another aspect, the present invention relates to a compound of the present invention, a pharmaceutically acceptable salt thereof or a pharmaceutical composition for use in treating or preventing neoplastic and / or infectious diseases.

[0399] Disclosed herein is a method of treating or preventing a neoplastic and / or infectious disease in a patient, comprising administering to the patient an amount of a compound or pharmaceutical composition of the present invention sufficient to treat or prevent the neoplastic and / or infectious disease in the patient.

[0400] In this respect also, as regards such medical uses and methods of treatment or prevention, respectively, the definitions set out in detail above (in particular in the context of compounds, pharmaceutical compositions and their use as medicaments) apply mutatis mutandis.

[0401] As used throughout the present invention, the term "patient" can be understood in the broadest sense as any subject or individual to be prevented or treated with the aid of the compounds or pharmaceutical compositions of the present invention, particularly those having or at risk of developing a neoplastic and / or infectious disease, whether or not clinical symptoms are present. The patient can be any animal, including humans. Preferably, the patient is a mammal (e.g., a human, mouse, rat, cow, pig, dog, cat, horse, donkey, goat, etc.), most preferably a human.

[0402] In the context of the present invention, the term "disease" is understood in its broadest sense to mean any pathological condition, whether or not clinical symptoms are present. Thus, a disease may be associated with a phenotype or may be latent. Preferably, a disease is a pathological condition that is accompanied by one or more clinical symptoms.

[0403] In the context of the present invention, a disease can be a chronic disease and / or an acute disease. Preferably, it is a chronic disease. A chronic disease has persistent or otherwise long-term effects. In the context of the present invention, chronic diseases also include diseases with a relapsing course, i.e., recurrent diseases with repeated relapses interspersed with periods of remission. Therefore, as used herein, a chronic disease can be understood in the broadest sense to mean any disease (with or without clinical symptoms) that lasts for at least 1 week, at least 1 month, at least 3 months, at least 6 months, at least 1 year, or even several years. When the patient is a human, a chronic disease is generally understood to last for at least 1 month or preferably at least 3 months. This understanding also applies to the present invention. In this context, it is understood that, for example, a neoplastic disease typically, but not necessarily, grows for months or even years before the first clinical symptoms appear. Nevertheless, a neoplastic disease is present from the time the first neoplastic cells appear, usually without any clinical symptoms. Therefore, a recognized neoplastic disease itself is usually, but not necessarily, a chronic disease. Similarly, when an infectious disease such as human immunodeficiency virus (HIV) infection begins to cause clinical symptoms and is first diagnosed, it is usually a chronic disease.

[0404] As used herein, neoplastic disease can be understood in the broadest sense as any tissue resulting from uncontrolled cell growth. In many cases, the vegetation at least causes a large mass of tissue, which is optionally innervated by blood vessels. It may or may not comprise the formation of one or more metastases / metastatic lesions. Neoplastic disease of the present invention can be any vegetation classified by the International Statistical Classification of Diseases and Related Health Problems 10th Revision (ICD-10) category C00-D48 of the International Statistical Classification of Diseases and Related Health Problems 10th Revision of the present invention.

[0405] Exemplarily, a neoplastic disease according to the present invention may be the presence of one or more malignant neoplasms (tumors) (ICD-10 categories C00-C97), may be the presence of one or more in situ neoplasms (ICD-10 categories D00-D09), may be the presence of one or more benign neoplasms (ICD-10 categories D10-D36), or may be the presence of one or more neoplasms of indeterminate or unknown behavior (ICD-10 categories D37-D48). Preferably, a neoplastic disease according to the present invention indicates the presence of one or more malignant neoplasms, i.e., is a malignant tumor (ICD-10 categories C00-C97).

[0406] In a more preferred embodiment, the neoplastic disease is cancer.

[0407] Cancer can be understood in the broadest sense as any malignant neoplastic disease, i.e., the presence of one or more malignant neoplasms in a patient. Cancer can be a solid or hematological malignancy. Preferably, cancer can be treated by at least one immunotherapy (including, for example, therapeutic antibodies directed against tumor antigens and / or experimental protocols such as, for example, cancer vaccination).

[0408] Subtypes of cancer can be classified in different ways, such as by where in the body the main or only tumor mass is found, or by the tissue of origin from which the tumor arose.

[0409] Illustratively, such malignant neoplasms according to the present invention may be located on or in the lips, oral cavity and throat (ICD-10 categories C00-C14), on or in the digestive organs (ICD-10 categories C15-C26), on or in the respiratory system and intrathoracic organs (ICD-10 categories C30-C39), on or in bones and articular cartilage (ICD-10 categories C40-C41), on or in the skin (ICD-10 categories C43-C44), on or in connective and soft tissues (ICD-10 categories C45-C49), on or in the breast and female genital organs (ICD-10 categories C50-C58), on or in the male genital organs (ICD-10 categories C51-C59), on or in the thyroid gland (ICD-10 categories C60-C61), on or in the thyroid gland (ICD-10 categories C62-C63), on or in the thyroid gland (ICD-10 categories C64-C65), on or in the thyroid gland (ICD-10 categories C66-C67), on or in the thyroid gland (ICD-10 categories C68-C69), on or in the thyroid gland (ICD-10 categories C69-C70), on or in the thyroid gland (ICD-10 categories C71-C72), on or in the thyroid gland (ICD-10 categories C73-C74), on or in the thyroid gland (ICD-10 categories C75-C76), on or in the thyroid gland (ICD-10 categories C77-C78), on or in the thyroid gland (ICD-10 categories C79-C80), on or in the thyroid gland (ICD-10 categories C81-C82), on or in the thyroid gland (ICD-10 categories C83-C84), on or in the thyroid gland (ICD-1 Neoplasms may be secondary and poorly defined (ICD-10 categories C76-C80), described as or presumed to be primary, lymphoid, hematopoietic, and related tissue neoplasms (ICD-10 categories C81-C96), or may be independent (primary) multisite neoplasms (ICD-10 category C97).

[0410] Illustratively, the cancer in the context of the present invention may be selected from carcinomas (i.e. cancers originating from epithelial cells; for example, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma and small cell carcinoma), sarcomas (i.e. cancers originating from connective tissue; for example, Askin's tumor, botryoid sarcoma, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma and soft tissue sarcoma), hematological cancers such as lymphoma, leukemia or myeloma. Hematological cancers contemplated herein include, but are not limited to, lymphomas and leukemias (i.e., cancers that arise from hematopoietic (blood-forming) cells; e.g., mature B-cell neoplasms, mature T-cell and natural killer (NK) cell neoplasms, Hodgkin lymphoma, immunodeficiency-associated lymphoproliferative disorders, lymphocytic leukemias, myeloid leukemias), germ cell tumors (i.e., cancers that arise from pluripotent cells of the sex organs; e.g., germ cell tumors (including dysgerminomas and seminomas), dysgerminomas, seminomas), blastomas (i.e., cancers that arise from immature "precursor" cells or embryonic tissue; e.g., hepatoblastoma, medulloblastoma, Wilms' tumor, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma, glioblastoma), and melanoma and its precursors (i.e., Cancers that arise from melanocytes; for example, lentigo maligna, superficial spreading melanoma, acral lentiginous melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma, soft tissue melanoma), as well as non-melanoma skin cancers (i.e., non-melanoma cancers that arise from the skin, for example, basal cell carcinoma, squamous cell carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, Kaposi's sarcoma, keratoacanthoma, spindle cell tumor, sebaceous gland carcinoma, microcystic adnexal carcinoma, Paget's disease of the breast, atypical fibroxanthoma, leiomyosarcoma, angiosarcoma) and gliomas (i.e., cancers that arise from cells of the brain or spinal column, for example, ependymoma, astrocytoma, oligodendroglioma, brain stem glioma, optic nerve glioma, mixed glioma).

[0411] In certain embodiments of the invention herein, the cancer is a non-hematological cancer such as a sarcoma, carcinoma, or melanoma. Preferably, the non-hematological cancer can be the formation of one or more solid tumors, such as, for example, selected from melanoma, neuroblastoma, lung cancer, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, epithelial squamous cell carcinoma, and breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, testicular cancer, colon cancer, colorectal cancer, hepatocellular carcinoma, bladder cancer, stomach cancer, esophageal cancer, pancreatic cancer, gastric cancer, liver cancer, mesothelioma, thyroid cancer, adrenal cancer, brain cancer, and head and neck cancer.

[0412] More preferably, the compound of the present invention or the pharmaceutical composition of the present invention can be used for preventing and / or treating cancer, wherein the cancer is interstitial fibrosis, prostate cancer, colon cancer, melanoma, lung cancer, rectal cancer, breast cancer, multiple myeloma, gastrointestinal cancer, non-small cell lung cancer (NSCLC).

[0413] Alternatively, the cancer may be the formation of one or more hematopoietic tumors, such as, for example, selected from multiple myeloma, non-Hodgkin's lymphoma, AML (acute myeloid leukemia, DLBCL (diffuse large B-cell lymphoma) and B-CLL (B-cell chronic lymphocytic lymphoma).

[0414] In an alternative preferred embodiment, the disease may be an infectious disease.

[0415] As used in the context of the present invention, the term "infectious disease" can be understood in the broadest sense as any pathological condition caused by one or more biological factors that are foreign to the body and can stimulate an immune response in the patient's body. An infectious disease may be with or without an inflammatory response (inflammation). Preferably, the infectious disease in the context of the present invention is accompanied by an inflammatory response. Exemplarily, this immune response in the patient's body can be caused in more detail by the following factors: the biological factor itself (for example, by the presence of its surface antigen), an antigen from the biological factor provided on major histocompatibility complex I or II (MHC I or MHC II), the proliferation of the biological factor, the reaction of the host tissue to such a biological factor, a compound produced or caused by such a biological factor (for example, a toxin, a chemical semiochemical, a cytokine, etc.), or an antigen formed from a hapten of such a biological factor. Such a biological factor may be a non-vital factor or a vital factor. Illustratively, the infectious disease can be caused by a biological agent selected from viruses, viroids, prions, microorganisms (such as bacteria), nematodes (such as roundworms and pinworms), arthropods (e.g., ticks, mites, fleas and lice), fungi, ringworms and tapeworms. Preferably, the infectious disease according to the present invention is caused by a virus or bacteria, specifically a viral infection.

[0416] The viral infection in the context of the present invention can be an infection of any virus. The viral infection can be an acute viral infection or a chronic viral infection. Preferably, it is a chronic viral infection. Non-limiting examples of clinically important viral families and species in the context of the present invention include adenovirus, herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpes virus type 8, human papillomavirus, BK virus, JC virus, smallpox, hepatitis B virus, human bocavirus, parvovirus B19, human astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C virus, virus, yellow fever virus, dengue virus, West Nile virus, rubella virus, hepatitis E virus, human immunodeficiency virus, influenza virus, Guanarito virus, Junin virus, Lassa virus, Machupo virus, Sabiá virus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus, Nipah virus, rabies virus, hepatitis D virus, rotavirus, orbivirus, colonovirus, and Banna virus.

[0417] In the context of the present invention, those viral infections associated with downregulation of the immune response are of particular interest, such as, for example, asymptomatic or symptomatic human immunodeficiency virus (HIV) infection (Acquired Immune Deficiency Syndrome (AIDS)).

[0418] In addition, viral infections associated with neoplasia, such as, for example, herpes simplex virus type 1 or type 2 (HSV), are also particularly interesting. Here, the compounds or pharmaceutical compositions of the present invention may simultaneously have pharmaceutical activity against viral infections and the neoplasms caused by the viral infections.

[0419] Furthermore, viral infections that have a long incubation period and can thus escape attack by the immune system (such as, for example, HSV 1 or HSV 2) are of particular interest.

[0420] Optionally, but not necessarily, infectious diseases, particularly chronic infectious diseases (e.g., chronic viral infections), may be associated with inflammation. In this context, inflammation can be characterized by an increase in NF-κB activity, C-reactive protein (CRP) levels, interferon-γ (IFN-γ) levels, interleukin 1 (IL-1) levels, and / or interleukin 8 (IL-8) levels.

[0421] In the context of treating or preventing neoplastic and / or infectious diseases, the compounds of the present invention can be used as the sole pharmaceutically active agent, or can be used in combination with one or more other pharmaceutically active agents. Exemplarily, such other pharmaceutically active agents can be stimulators for activating immune cells, can be antiproliferative agents (e.g., anticancer agents such as chemotherapy, antimetabolites, hormones, antibodies (Ab)), antiviral agents and / or antibiotics.

[0422] Preferably, such other pharmaceutically active agents are biological compounds, such as therapeutic monoclonal antibodies that have been shown to be effective in treating neoplasms. Exemplarily, such therapeutic monoclonal antibodies are directed against PD-1 molecules, PD-L1 molecules or another ligand of PD-1 molecules, CTLA-4 molecules, TIM3 molecules, LAG3 molecules, VISTA molecules or BTLA-4 molecules.

[0423] The utility of such combination therapies will depend on the pharmacokinetic and pharmacodynamic properties of the selected compounds and agents used in such combination therapies (adjuvant therapies).

[0424] Optionally, another medicament can be used in conjunction with, before or after one or more compounds of the present invention. As used herein, concomitant administration can be used in a single composition (e.g., combined in a pharmaceutical composition of the present invention) or in two separate compositions, which can also optionally be used by the same or different routes of administration (e.g., by injection, oral, nasal, transdermal, etc.). As used herein, when a compound of the present invention is used before or after, there can be a time interval of less than one hour, one hour or more, three hours or more, six hours or more, twelve hours or more, 24 hours or more, two days or more, or one week or more between the administration of the compound and the other medicament.

[0425] As mentioned above in the context of the compounds of the present invention, one or more other medicaments can also be administered once (single administration) or can be repeated administration, such as, for example, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, more than ten times or even permanently. Between two administrations, there can be a time interval of less than one hour, one hour or more, six hours or more, twelve hours or more, twenty-four hours or more. Administration can be once a day, twice a day, three times a day, four times a day, once every other day, once every three days, once a week, once every two weeks, once a month, twice a year or once a year.

[0426] In a preferred embodiment, the compounds or pharmaceutical compositions of the present invention are administered in combination with one or more other stimulators of activated immune cells.

[0427] The immunocyte used in the context of the present invention can be any immunocyte known in the art.Immune cell can be a cell of the adaptive immune system (for example, T cell or B cell), a cell of the innate immune system (natural killer (NK) cell, macrophage, monocyte) and / or a cell under two groups (for example, dendritic cell (DC); Antigen presenting cell (APC)) and / or a cell known to have phagocytic activity, such as basophil, neutrophil or eosinophilic granulocyte.

[0428] In a preferred embodiment, the immune cells are peripheral blood mononuclear cells (PBMC).

[0429] In a preferred embodiment, the immune cells are selected from those having T cell antigen receptors (TCRs) and CD4 and / or CD8 co-receptors on their cell surfaces. Those immune cells having CD4 co-receptors on their cell surfaces can be selected from T helper cells (Th cells), macrophages, and dendritic cells (DCs). Those immune cells having CD8 co-receptors on their cell surfaces can be selected from cytotoxic T cells, natural killer (NK) cells, cortical thymocytes, and dendritic cells (DCs).

[0430] In another preferred embodiment, the immune cells are selected from the group consisting of T cells, NK cells, monocytes and B cells.

[0431] In a particularly preferred embodiment, the immune cells are T cells and / or NK cells.

[0432] As is well known, T cells and NK cells have special effects on neoplasia (particularly anti-tumor immunity) and infectious diseases. NK cells are preferably lymphocytes that can be stimulated by Fc receptors (FcR). In particular, in the context of antibody-dependent cellular cytotoxicity (ADCC), this may have some benefits in the context of the present invention (e.g., as a single application or when co-administering therapeutic antibodies against neoplastic and / or infectious antigens).

[0433] It is well known that B cells play an important role in the humoral response to infectious pathogens. It is also known in the art that B cells can also play a role in generating humoral immunity against neoplastic cells.

[0434] Monocytes can be understood as the temporary phenotype of myeloid cells present in a large number of PBMCs. In addition, myeloid cells include dendritic cells (DC). Macrophages may have a positive and inhibitory effect on immunity, particularly anti-neoplastic therapy. Particularly interesting macrophages include M1-type macrophages. Myeloid cells can also be included in myeloid-derived suppressor cells (MDSC) that have a negative impact on anti-neoplastic immune response. Myeloid cells can be stimulated by FC receptors (FcR). Especially in the context of antibody-dependent cellular cytotoxicity (ADCC), this may have some benefits in the context of the present invention (for example, as a separate application or when co-administered for therapeutic antibodies against neoplastic and / or infectious antigens). It can also enhance the humoral immunity against tumor antigens that naturally occur in patients.

[0435] Activation of immune cells may be understood in the broadest sense as an increase in the immune activity of such cells and / or an increase in the cell proliferation of such cells.

[0436] In a more preferred embodiment, the other stimulatory agents for activated immune cells are selected from one or more antigens of the neoplasms and / or infectious pathogens to be treated, one or more TCR or CD3 agonists, one or more CD28 agonists, one or more agonists for other co-stimulatory T cell surface receptors (such as CD40L, CD69, OX40, GITR, CD137, CD27 and / or HVEM), and combinations of two or more thereof.

[0437] A TCR / CD3 agonist can be any agent that triggers CD3. It can be a peptide or non-peptide agonist that binds to the extracellular side of the TCR / CD3 with or without MHC-dependent antigen presentation, an agonist that binds to the intracellular side of the TCR / CD3, or an agent that activates an intracellular signaling pathway triggered by TCR / CD3 engagement. Preferably, the CD3 agonist can be an anti-CD3 antibody, a peptide antigen presented by MHC I or MHC II, an anti-CD3 antibody fragment, or an anti-CD3 antibody mimetic. Highly preferably, the CD3 agonist is a tumor antigen presented by MHC I or MHC II.

[0438] The CD28 agonist can be any agent that triggers CD28. It can be an agonist that binds to the extracellular side of CD28, it can be an agonist that binds to the intracellular side of CD28, or it can be an agent that activates the intracellular signal transduction pathway triggered by CD28. Preferably, the CD28 agonist can be an anti-CD28 antibody, an anti-CD28 antibody fragment, an anti-CD28 antibody mimetic, or a protein containing a natural ligand of CD28 (such as B7.1 or B7.2). Highly preferably, the CD28 agonist is an (agonistic) anti-CD28 antibody or an Ig fusion protein containing a natural ligand of CD28 (such as B7.1 or B7.2).

[0439] Antibodies in the context of the present invention can be monoclonal or polyclonal antibodies of any species or origin. They can bind to any epitope contained in a polypeptide with a corresponding cognate antigen (e.g., CD3 or CD28, respectively), including post-translational modifications thereof. Cognate antigens can, for example, be linear epitopes, structural epitopes, primary epitopes, and / or secondary epitopes. Antibodies can be of natural origin, genetically engineered, and / or synthetic origin.

[0440] Antibody fragments can be understood in the broadest sense as any fragment of an antibody that still has binding affinity for its target polypeptide. For example, an antibody fragment can be an antigen-binding fragment (Fab fragment), a truncated antibody containing one or two complementarity determining regions (CDRs), or an antibody variable fragment (Fv). Antibody fragments can be of natural origin, genetically derived, and / or synthetic origin.

[0441] Antibody mimics can be understood in the broadest sense as such organic compounds: it is similar to an antibody, can specifically bind to an antigen, and it generally has a molecular mass in the range of about 3kDa to about 25kDa. Antibody mimics can be, for example, Affibody (Affibody) molecules (Affibodies), Affilins, Affitins, Anticalins, Avimers, DARPins, Fynomers, Kunitz domain peptides, single domain antibodies (for example, VHH antibodies or VNAR antibodies), monomeric antibodies, diabodies, triabodies, flexible antibodies (flexibodies) and tandabs. Antibody mimics can be of natural origin, genetic technology origin and / or synthetic origin.

[0442] Peptide antigens can be understood in the broadest sense as organic compounds that specifically bind to MHC I or MHC II molecules and that are generally composed of 8-30 amino acids and preferably 9-25 amino acids. The peptides can be of natural, genetically engineered and / or synthetic origin.

[0443] Preferably, the additional stimulatory agent for activating immune cells is a combination of one or more CD3 agonists and one or more CD28 agonists. Particularly preferably, the additional stimulatory agent for activating immune cells is a combination of at least one (agonistic) anti-CD3 antibody and at least one (agonistic) anti-CD28 antibody. As will be apparent from the examples shown below, stimulation of immune cells by contacting these with (agonistic) anti-CD3 antibodies and / or (agonistic) anti-CD28 antibodies mechanistically mimics T cells, regardless of the specific antigen recognized by the individual TCRs. Stimulation with (agonistic) anti-CD3 antibodies and (agonistic) anti-CD28 antibodies can very well mimic the activation of T cells in patients.

[0444] In addition or alternatively, immune cells can also be triggered by antigens of vegetation and / or infectious pathogens (for example, by inoculating one or more antigens to the patient). So, antigen is considered as stimulant. The antigen of vegetation and / or infectious pathogen can be, for example, a vaccine comprising one or more antigens of vegetation and / or infectious pathogens, such as, for example, a vaccine based on polypeptide, a polynucleotide vaccine, an oligosaccharide vaccine or a vaccine based on the vegetation fragment of the same type or the infectious pathogen fragment of the same type. Those skilled in the art know that multiple methods for providing such vaccines are provided. Several antitumor and antiviral vaccines are also commercially available.

[0445] Additionally or alternatively, immune cells can also be triggered by antigen presenting cells (APCs) loaded with antigens. The APCs loaded with antigens are then considered as other stimulants. In this context, antigens are also antigens of the aforementioned vegetation and / or infectious pathogens.

[0446] Additionally or alternatively, one or more other stimulatory agents can be selected from checkpoint blockade therapeutics (particularly T cell surface receptor binding agents, such as, for example, those that bind to one or more of the following: CTLA4, PD-1, PDL-1, TIM3, LAG3, BTLA, VISTA and / or its ligands (e.g., anti-CTLA4, anti-PD-1 and / or anti-PDL-1 antibodies)), cytokines (e.g., IL-2, IL-15 and / or IL-7), APC activators (e.g., CD40 agonists), adoptive cellular agents (particularly adoptive T cells (e.g., chimeric immunoreceptor T cell therapy, such as, for example, CAR T cell therapy), dendritic cell therapy (e.g., sipuleucel-T) and / or natural killer cell therapy), enhancers of T cell function (e.g., lenalidomide and related agents), enhancers of natural killer cell function (e.g., anti-KIR antibodies), and therapeutic antibodies against tumor antigens.

[0447] Optionally, in particular when the patient suffers from a neoplastic disease, one or more chemotherapeutic agents, cytokines and / or other anti-tumor agents may be administered to the patient in addition to one or more compounds of the invention. Exemplarily, such chemotherapeutic agents, cytokines and anti-cancer agents may be selected from polyclonal or monoclonal antibodies (e.g., rituximab, trastuzumab, cetuximab, bevacizumab, basiliximab, daclizumab), antimetabolites (e.g., 5-fluorouracil, azathioprine, 6-mercaptopurine, mercaptopurine, pyrimidine, thioguanine, fludarabine, floxuridine, cytosine arabinoside (cytarabine), pemetrexed, raltitrexed, pralatrexate, methotrexate), alkylating agents (e.g., dichloromethane, cyclophosphamide, chlorambucil, ifosfamide), platinums (e.g., cisplatin, carboplatin, oxaliplatin), plant alkaloids and terpenoids (e.g., vinca alkaloids (vinca alkaloids), ... Vincristine, vinblastine, vinorelbine, vindesine), taxanes (e.g., paclitaxel), cyclophosphamide), topoisomerase inhibitors (e.g., camptothecins: irinotecan, topotecan, etoposide, etoposide phosphate, teniposide), melphalan, antineoplastic agents (e.g., doxorubicin (adriamycin), liposomal doxorubicin, epirubicin, bleomycin), dactinomycin, aminoglutethimide, amsacrine, anastrozole, purine and pyrimidine base antagonists, anthracycline antibiotics, aromatase inhibitors, asparaginase, antiestrogens, bexarotene, buserelin, busulfan, camptothecin derivatives, capecitabine, carmustine, cladribine, cytarabine, cytosine arabinoside, alkylating agents Growth inhibitors, dacarbazine, daunorubicin, docetaxel, epirubicin, estramustine, etoposide, exemestane, fludarabine, fluorouracil, folic acid antagonists, formestane, gemcitabine, glucocorticoids, goserelin, hormones and hormone antagonists and mesine, hydroxyurea, idarubicin, irinotecan, letrozole, leuprorelin, lomustine, mercaptopurine, miltefosine, mitomycin, mitotic inhibitors, mitoxantrone, nimustine, procarbazine, tamoxifen, temozolomide, teniposide, testolactone, thiotepa, topoisomerase inhibitors, treosulfan, tretinoin, triptorelin, trofosfamide, cytostatic antibiotics, everolimus, pimecrolimus, tacrolimus, acrolimus Spectinomycin, spiramycin, sirolimus (rapamycin), roxithromycin, ascomycin, bafilomycin, erythromycin, midecamycin, josamycin, concancamycin, clarithromycin, troleandomycin, polyphylline, tobramycin, mutamycin, dactinomycin, dactinomycin, phaeomycin, statins (e.g., cerivastatin, simvastatin, lovastatin, somatostatin, fluvastatin, nystatin, rosuvastatin, atorvastatin, pravastatin, pitavastatin, pentostatin), 4-hydroxycyclophosphamide, bendamustine, thymosin alpha-1, aclarubicin, fludarabine-5'-dihydrogen phosphate, hydroxyurea,Aldesleukin, pegaspargase, cepharanthin, epothilone A and B, azathioprine, mycophenolate mofetil, c-myc antisense, b-myc antisense, betulinic acid, camptothecin, melanocyte-stimulating hormone (α-MSH), activated protein C, IL-1β inhibitor, fumaric acid and its esters, dermicidin, calcipotriol, taclacitol, flavonoid, β-lapachone, podophyllotoxin, betulin, podophyllotoxin 2-ethylhydrazide, sagramostim, (rhu GM-CSF), pegylated interferon alfa-2b, lenograstim (r-HuG-CSF), filgrastim, polyethylene glycol, cephalosine, selectin (cytokine antagonist), CETP inhibitors, cadherins, cytokinin inhibitors, COX inhibitors (COX-2 or COX-3 inhibitors), angiopeptides, ciprofloxacin, fluroblastin, bFGF antagonist, probucol, prostaglandins, 1,11-dimethoxyeanthin- 6-one, 1-hydroxy-11-methoxycanthin-6-one, scopoletin, colchicine, NO donors, pentaerythritol tetranitrate, sydnonimines, S-nitroso derivatives, staurosporine, β-estradiol, α-estradiol, estriol, estrone, ethinylestradiol, fosestrol, medroxyprogesterone, estradiol cyclopentanepropionate, estradiol benzoate, tranilast, urophylla propionate, verapamil, cyclosporine A, paclitaxel and its derivatives such as 6-α-hydroxy Paclitaxel, baccatin, taxotere, mofebuzone, acemetacin, diclofenac, clonazolac, dapsone, o-carbamoyl-phenoxy-acetic acid, lidocaine, ketoprofen, mefenamic acid, piroxicam, meloxicam, chloroquine phosphate, penicillamine, hydroxychloroquine, auranofin, aurothiobutane disodium, oxacerol, celecoxib, beta-sitosterol, adenosylmethionine, myricaine, polidocanol, norvanillamide, levomenthol, benzocaine, aescin, elipticine, Calbiochem D-24851, colcemid, cytochalasin AE, indanocine, nocodazole, bacitracin, vitronectin receptor antagonist, azelastine, free nucleic acids, nucleic acids incorporated into viral transmitters, DNA and RNA fragments, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, antisense oligonucleotides, VEGF inhibitors, IGF-1, active agents from the group of antibiotics such as cefadroxil, cefazolin, cefaclor, cefoxitin, gentamicin, penicillin, dicloxacillin, oxacillin, sulfonamides, metronidazole, antithrombotic agents, argatroban, aspirin, abciximab, synthetic antithrombin, bivalirudin, comitidine, enoxaparin, GpIIb / IIIa platelet membrane receptor, antibodies to factor Xa inhibitors, heparin, hirudin, r-hirudin, PPACK, protamine, prourokinase, streptokinase, warfarin,Urokinase, vasodilators, dipyramidole, trapidil, nitroprusside, PDGF antagonists, triazolopyrimidines, seramin, ACE inhibitors, captopril, cilazapril, lisinopril, enalapril, losartan, thioproteinase inhibitors, prostacyclin, valperprost, interferon alpha, beta, and gamma, histamine antagonists, serotonin blockers, apoptosis inhibitors, apoptosis modulators, NF-kB or Bcl-xL antisense oligonucleotides, halofop Ketone, nifedipine, tocopherol, molsidomide, tea polyphenols, epicatechin gallate, epigallocatechin gallate, boswellic acid and its derivatives, leflunomide, anakinra, etanercept, sulfasalazine, tetracycline, triamcinolone, procainimide, retinoic acid, quinidine, disopyramide, flecainide, propafenone, sotalol, amiodarone, natural and synthetic steroids such as cyclohexine A, inonotus betulin, maquiroside A, mansonine, strebloside, hydrocortisone, betamethasone, dexamethasone, fenoprofen, ibuprofen, indomethacin, naproxen, phenylbutazone, acyclovir, ganciclovir, zidovudine, antifungals, clotrimazole, flucytosine, griseofulvin, ketoconazole, miconazole, terbinafine, chloroquine, mefloquine, quinine, natural terpenoids, hippocaesculin, barberry saponin-C21-angelate 14-dehydroagrostistachin, agroskerin, agrostistachin, 17-hydroxyagrostistachin, ovatodiolids, 4,7-oxoanisomelic acid, baccharinoids B1, B2, B3 and B7, turmeric saponins, bruceanol A, B and C, bruceantinoside C, yadanziosides N and P, isodeoxycurvifoliin, tomenphantopin A and B, cynoglossum A, B, C and D, ursolic acid, hyptatic acid A, zeorin, isorisaldehyde, maytenfoliol, effusantin A, excisanin A and B, longikaurin B, sculponeatin C, kamebaunin, leukamenin A and B, 13,18-dehydro-6-α-senecioyloxychaparrine, euphorbic acid A and B, regenilol, triptolide, magnetoside, schizonepetaside, aristolochic acid, anopterin, hydroxyanopterin, anthracene, protoanthracene, berberine,cheliburin chloride, ciprofloxacin, sinococuline, combrestatins A and B, cudraisoflavone A, curcumin, dihydroxanthin, nitidine chloride, 12-β-hydroxypregnene-3,20-dione ginkgolide, ginkgol, ginkgolic acid, helenalin, schizone, schizone-N-oxide, lasiocarpine, inonotus betulin, glycoside 1a, acanthoside A and B, larreatin, malloterin, mallotochromanol, isobutyrylmallotochromanol, marchantin A, maytansine, lycoridicin, margetine, liriodenine, bisparthenolidine, oxypsorospermin, aristolochic acid-All, periplocoside A, ghalakinoside, deoxypsorospermin, psychorubin, ricin A, sanguinarine, manwu wheat acid acid), methylsorbifolin, chromone of spathelia, stizophyllin, akagerine, dihydrousambaraensine, hydroxyusambarine, strychnine amylamine, strychnine theophylline, usambarine, usambarensine, daphnetin, lariciresinol, methoxylariciresinol, syringaresinol, umbelliferone, afromoson, acetylvismione B, desacetylvismione A, vismione A and B), radiation therapy (e.g., intensity modulated radiation therapy (IMRT), three-dimensional conformal radiation therapy (3DCRT), stereotactic body radiation therapy (SBRT), stereotactic radiosurgery (SRS), image-guided radiation therapy (IGRT), particle therapy (e.g., proton therapy), brachytherapy, radioisotope therapy (RIT) (e.g., with iodine-131, lutetium-177, strontium-89, and samarium (153Sm) lexidrone and / or yttrium-90)), anti-angiogenic therapy (e.g., , carboxyamidotriazole, TNP-470, CM101,, suramin, SU5416, thrombospondin, VEGFR antagonists, angiostatic steroids + heparin, cartilage-derived angiogenesis inhibitors, matrix metalloproteinase inhibitors, 2-methoxyestradiol, Tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, soluble VEGFR-1 and NRP-1, angiopoietin 2, angiostatins (e.g., TSP-1 and TSP-2 angiostatins), endostatin,Angiostatin, angiostatin, calreticulin, platelet factor-4, TIMP and CDAI, Meth-1 and Meth-2, CXCL10 prothrombin (kringle domain-2), antithrombin III fragment prolactin, VEGI, SPARC, osteopontin, mammary cord protein, proliferation protein-related protein, restin), kinase inhibitors (e.g., imatinib, imatinib mesylate, gefitinib, erlotinib, pazopanib, apatinib), proteasome inhibitors (e.g., bortezomib), PARP inhibitors (e.g., eneparib, olaparib), and combinations of two or more thereof.

[0448] Alternatively or additionally, if the patient suffers from neoplastic and / or infectious disease or is in the risk of suffering from neoplastic and / or infectious disease, one or more cytokines, hormones or their analogs (for example, selective estrogen receptor modulator tamoxifen, IL-2, IFN-α, IFN-β, IFN-γ, IL-4, IL-12, IL-18, platelet factor-4, TNF-α) can be further administered. These cytokines, hormones or their analogs can further trigger the patient's immune system. As previously mentioned, the high doses of many such medicaments may cause serious side effects. However, lower dosages can optionally be used to support treatment or prevention of the present invention.

[0449] Optionally, in particular when the patient suffers from a viral infection, in addition to one or more compounds of the present invention, one or more antiviral compounds may be administered to the patient. Such antiviral compounds may be exemplarily selected from entry or fusion inhibitors, nucleoside / nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, integrase inhibitors, and protease inhibitors.

[0450] Optionally, in particular when the patient suffers from a bacterial infection, in addition to one or more compounds of the present invention, one or more antibacterial antibiotics may be administered to the patient. Such antibacterial antibiotics may be exemplarily selected from antibiotics that target bacterial cell walls (e.g., penicillins and cephalosporins) or cell membranes (e.g., polymyxins), interfere with essential bacterial enzymes (e.g., rifamycins, intercalamycins, quinolones, and sulfonamides), and / or target polypeptide synthesis (e.g., macrolides, lincosamides, and tetracyclines).

[0451] Furthermore, the treatment or prevention according to the present invention may also be combined with other treatment modalities, such as, for example, radiation therapy (exemplarily based on x-ray radiation, ultraviolet (UV) radiation (e.g., UV-A, UV-B and / or UV-C radiation), alpha radiation, beta radiation, gamma radiation or cosmic radiation) and / or surgery.

[0452] As described above, it should be understood that the compounds of the present invention are particularly useful for treating and / or preventing neoplastic and / or infectious diseases in patients. However, the compounds of the present invention can be used not only for in vivo applications, but also for any type of ex vivo and / or in vitro applications. Exemplarily, they can also be used to activate immune cells in vivo, ex vivo, and in vitro.

[0453] Illustratively, the compounds of the present invention can be used to support any method of generating and / or expanding immune cells in vitro and / or in vitro, particularly but not necessarily for further use in adoptive cell therapy (ACT). For ACT, preferably antigen-specific T cells can be used. Such methods can also provide activated DCs, which can optionally be used in DC vaccination protocols.

[0454] In another aspect, the present invention relates to a method for producing activated immune cells in vitro or ex vivo, the method comprising the following steps:

[0455] (i) providing immune cells;

[0456] (ii) contacting the cells of step (i) with:

[0457] (a) at least one compound as defined in any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, and optionally

[0458] (b) one or more other stimulatory agents that activate said immune cells; and

[0459] (iii) culturing the cells of step (ii) under conditions suitable to maintain the viability of said cells.

[0460] In connection with such methods, the definitions detailed above also apply mutatis mutandis.

[0461] The method is performed ex vivo and / or in vitro, ie is an ex vivo and / or in vitro method. Therefore, in the context of this aspect relating to such a method, the immune cells are preferably activated outside the organism, in particular outside the patient.

[0462] Preferably, immune cells (e.g., T cells and / or natural killer cells) are mature immune cells. Such cells (particularly T cells) can be CD4+ and / or CD8+ cells. Immune cells can be obtained from any source suitable for this purpose. Alternatively or additionally, cells can also be B cells, such as, for example, CD19+B cells. Those skilled in the art know the various methods for obtaining such immune cells. Exemplarily, mature immune cells can be obtained from a blood sample (e.g., stored blood preservation products or fresh blood). Then, peripheral blood mononuclear cells (PBMCs) can be obtained, exemplarily from the buffy coat after centrifugation of the blood sample, and optionally further separated / purified, exemplarily, by labeling cell type-specific surface markers with fluorescently labeled antibodies, followed by fluorescence-activated cell sorting (flow cytometry), or by labeling cell type-specific surface markers with metal bead-labeled antibodies, followed by magnetic extraction of desired cells.

[0463] Alternatively, mature immune cells can also be obtained from cell culture. Methods for obtaining buffy coats and further separating and purifying cells are illustrated in the Examples section below. (Mature) immune cells are also commercially available.

[0464] Alternatively, immature immune cells or their precursors can be used and matured in an intermediate step by well-known methods of supplementing with corresponding cytokines and growth factors.

[0465] Subsequently, the immune cells are contacted with at least one compound of the present invention and optionally one or more other stimulants for activating immune cells. Those skilled in the art will immediately note that these compounds and agents can be added to the cells in any type of solution or culture medium suitable for the cells. Exemplarily, such a solution or culture medium may also be included in the composition defined in the context of the pharmaceutical composition above. Other stimulants for activating immune killer cells can be understood in the broadest sense as defined above. Optionally, other agents can be administered simultaneously with, before, or after one or more compounds of the present invention. Cells can be contacted with compounds and / or agents for less than 30 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours, at least 12 hours, at least 1 day or longer.

[0466] After or simultaneously with contacting the cells with the compound and optional reagent (step (ii)), the cells are cultured under conditions suitable for maintaining the viability of the cells (step (iii)). Thus, steps (ii) and (iii) may be performed as one step (simultaneously) or as two separate steps (subsequently) or partially overlapped in time. Typically, the cells are cultured in a suitable cell culture medium (e.g., X-Vivo 15) optimized to allow cell culture in the absence of FCS RPMI 1640 (optionally supplemented with fetal calf serum (FCS)) at 5% CO 2 and a temperature of 30° C.-39° C., preferably (approximately) 37° C. Preferably, the cells are cultured for at least 1 hour, at least 2 hours, at least 5 hours, at least 12 hours, at least 1 day, or at least 3 days.

[0467] As a result of the methods of the present invention, activated immune cells (eg, T cells and / or natural killer cells) can be obtained.

[0468] These activated immune cells can optionally be separated by any method known in the art (optional step (iv)). Optionally, as a further step (v), the activated immune cells can then be administered to patients in need thereof. Alternatively, the isolated activated immune cells obtained from step (iv) or the cells of step (iii) can also be stored and / or preserved (for example, dispersed in a culture medium containing DMSO and stored at -80°C). Alternatively, the isolated activated immune cells of step (iv) or the cells of step (iii) can be used for any other in vitro and / or in vivo purposes. Exemplarily, such activated immune cells can be used for further research purposes of activated immune cells (particularly activated T cells and / or natural killer cells).

[0469] Exemplarily, activated immune cells (particularly activated T cells and / or natural killer cells) can be used to produce cytokines secreted by the cells. Then, a further step (iv) is to culture the cells until the level of the desired cytokine secreted into the culture medium reaches the desired level, followed by separation and optionally the step (v) of purifying the desired cytokine. The separation of cytokines and optional purification can be carried out by any method known in the art, such as, for example, chromatographic methods. Optionally, such cytokines (for example, freezing, drying or freeze drying) can be stored and / or preserved subsequently.

[0470] Furthermore, the compounds of the present invention can be further used as research tools to study immune cell activation in more detail.

[0471] In another aspect, the present invention relates to the compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof, for use as a drug for preventing or treating neoplastic and / or infectious diseases, heart disease (wherein the heart disease is myocardial infarction), acute coronary syndrome, myocardial ischemia, ischemic cardiomyopathy, myocardial reperfusion injury, non-ischemic cardiomyopathy or acute or chronic heart failure.

[0472] Description of the drawings

[0473] Figure 1 Treatment schedule for in vivo efficacy study of Compound 142 with oral administration of the compound. Treatment schedule for efficacy study 1 of the POC study monitoring tumor volume and survival of orally treated mice. Arrows indicate oral administration of Compound 142.

[0474] Figure 2 Effect of Compound 142 on in vivo tumor growth rate. Mean tumor volume ± SEM of mice treated orally with Compound 142 on day 3: 30 mg / kg + daily: 10 mg / kg or day 3: 9 mg / kg + daily: 3 mg / kg. Statistically significant differences (p < 0.05) between treatment groups and vehicle control groups were calculated using two-way analysis of variance.

[0475] Figure 3A -B. M21 cell growth after co-culture of stimulated PBMCs M21 melanoma cells were incubated with isolated PBMCs and stimulated with anti-CD3 / 28 and compound 142 at 5 different concentrations.

[0476] Figure 4A B. M21 cell growth after co-culture with stimulated T cells. M21 melanoma cells were incubated with purified CD4+ and CD8+ T cells and stimulated with anti-CD3 / 28 and various concentrations of Compound 142. EC50s were calculated using GraphPad Prism 32 hours after addition of anti-CD3 / 28 and Compound 142.

[0477] Figure 5A -C. M21 cell growth after co-culture with stimulated T cells. M21 melanoma cells were incubated with purified CD4+ and CD8+ T cells and stimulated with anti-CD3 / 28 and various concentrations of compound 142, compound 156, or compound 119.

[0478] Figure 6 M21 cell growth after co-culture of unstimulated PBMCs and T cells M21 melanoma cells were incubated with unstimulated PBMCs or T cells and 10 μM compound 142.

[0479] Figure 7Treatment schedule for in vivo efficacy experiments with orally administered Compound 142. Treatment schedule for efficacy experiment 2 of a POC study monitoring tumor volume and survival in orally treated mice. Arrows indicate oral administration of Compound 142. Asterisks indicate treatment with 40 mg / kg Compound 142 every 48 hours, except on days 7 (30 mg / kg) and 9 (60 mg / kg).

[0480] Figure 8 Treatment schedule for in vivo efficacy study of Compound 142 with oral administration of the compound. Treatment schedule for efficacy study 3 of the POC study monitoring tumor volume and survival of orally treated mice. Arrows indicate oral administration of Compound 142.

[0481] Figure 9 Treatment schedule for in vivo efficacy experiments with orally administered Compound 142 or Compound 120. Treatment schedule for efficacy experiment 4 of a POC study monitoring tumor volume and survival in orally treated mice. Black arrows indicate daily oral administration of Compound 142 or Compound 120, while gray arrows depict oral Compound 142 treatment every three days.

[0482] Figure 10 Treatment schedule for in vivo efficacy studies of Compound 142, Compound 156, or Compound 119 with oral administration of the compounds. Treatment schedule for efficacy study 5, monitoring tumor volume and survival in orally treated mice. Arrows indicate daily administration of Compound 142, Compound 156, or Compound 119.

[0483] Figure 11 Treatment regimen for in vivo efficacy experiments with orally administered Compound 142, Compound 156, or Compound 119. Treatment regimen for efficacy experiment 6 of a POC study monitoring tumor volume and survival in orally treated mice. Arrows indicate daily oral administration of Compound 142, Compound 156, or Compound 119.

[0484] Figure 12 Effect of Compound 142 on in vivo tumor growth rate. Mean tumor volume ± SEM of mice treated orally with Compound 142 60 mg / kg D3 + 40 mg / kg once every two days* or vehicle. Asterisks indicate treatment with 40 mg / kg Compound 142 every 48 hours, except on Day 7 (30 mg / kg) and Day 9 (60 mg / kg). Statistically significant differences (p < 0.05) between treatment groups and vehicle control groups were calculated using two-way ANOVA.

[0485] Figure 13Effect of Compound 142 on in vivo tumor growth rate. Mean tumor volume ± SEM of mice treated orally with Compound 142 30 mg / kg D4 + 10 mg / kg once daily, 9 mg / kg D4 + 3 mg / kg once daily, or vehicle. Statistically significant differences (p < 0.05) between treatment groups and vehicle control groups were calculated using two-way ANOVA.

[0486] Figure 14 Effects of Compound 142 and Compound 120 on in vivo tumor growth rate. Mean tumor volume ± SEM of mice treated orally with Compound 142 30 mg / kg D4 + 10 mg / kg once daily or 30 mg / kg every three days, Compound 120 25 mg / kg once daily, or vehicle. Statistically significant differences (**p < 0.01) between treatment groups and vehicle control groups were calculated using two-way ANOVA.

[0487] Figure 15A , B and C1-C4. Effects of Compound 142, Compound 156 and Compound 119 on in vivo tumor growth rate and survival. A: Mean tumor volume ± SEM of mice treated orally with Compound 142 30 mg / kg D4 + 10 mg / kg once daily, Compound 156 20 mg / kg D4 + 10 mg / kg once daily, Compound 119 10 mg / kg once daily, or vehicle. Statistically significant differences between treatment groups and vehicle control groups were calculated using two-way ANOVA (**p < 0.01, ****p < 0.0001). B: Survival of mice treated orally with Compound 142 30 mg / kg D4 + 10 mg / kg once daily, Compound 156 20 mg / kg D4 + 10 mg / kg once daily, Compound 119 10 mg / kg once daily, or vehicle. C: Mean tumor volume ± SEM and tumor volume of each mouse receiving primary B16-SIY challenge or secondary B16-SIY repeat challenge.

[0488] Figure 16A B. Effects of Compound 142, Compound 156, and Compound 119 on In Vivo Tumor Growth Rate. A: Mean tumor volume ± SEM of mice treated orally with Compound 142, Compound 156, or Compound 119 7 to 21 days after B16-SIY inoculation. Statistically significant differences between treatment groups and vehicle control groups were calculated using two-way ANOVA (**p<0.01, ***p<0.001). B: Survival of mice treated orally with Compound 142, Compound 156, or Compound 119 7 to 21 days after B16-SIY inoculation.

[0489] Figure 17 Treatment schedule for in vivo efficacy studies of Compound 142, Compound 156, and Compound 119 with oral administration of compounds. Treatment schedule for efficacy studies of POC studies monitoring tumor volume and survival in orally treated mice. Arrows indicate oral drug treatment.

[0490] Figure 18A B. Effects of Compound 142, Compound 156, and Compound 119 on in vivo tumor growth rate. A: Mean tumor volume ± SEM of mice treated orally with Compound 142, Compound 156, or Compound 119 7 to 21 days after EO771 inoculation. Statistically significant differences (p < 0.01) between treatment groups and the vehicle control group were calculated using a two-way ANOVA. B: Survival of mice treated orally with Compound 142, Compound 156, or Compound 119 7 to 21 days after EO771 inoculation.

[0491] Figure 19 Treatment schedule for in vivo efficacy study of compound 142 with oral administration of compound. Treatment schedule for efficacy study of POC study monitoring tumor growth in orally treated mice. Arrows indicate oral drug treatment.

[0492] Figure 20 Effect of Compound 142 on Tumor Growth Rate in Vivo. Mean tumor volumes ± SEM of mice orally treated 7 to 28 days after GL261-LUC2-iRFP inoculation. Statistically significant differences (p<0.0001) between treatment groups and vehicle control groups were calculated using two-way ANOVA.

[0493] Figure 21A B. In vitro CD4+ or CD8+ T cell stimulation: Purified T cells were stimulated with anti-CD3, anti-CD3 / CD28, or CEFx viral peptides and compound 142 at 8 different concentrations.

[0494] Preparation Example

[0495] General information:

[0496] All reactions involving reagents or intermediates sensitive to air or moisture were carried out in flame-dried glassware under an argon atmosphere. Dry solvents (THF, toluene, MeOH, DMF, DCM) were used as commercial products. Data were recorded on a Bruker DRX400 (400 MHz). 1 H-NMR and 13C-NMR. Multiplicities are expressed as: br s (broadened singlet), s (singlet), d (doublet), t (triplet), q (quartet), quin (quintet), m (multiplet); and coupling constants (J) are given in Hertz (Hz). HPLC-electrospray mass spectra (HPLC ES-MS) were obtained using a Waters Acquity Performance Liquid Chromatography (UPLC) equipped with an SQ 3100 mass detection spectrometer. Column: Acquity UPLC BEH C18 1.7 μm, 2.1 x 50 mm. Flow rate: 0.5 ml / min. Eluents: A: HO containing 0.05% formic acid, and B: ACN containing 0.05% TFA. All chemicals and solvents were purchased from commercial sources such as Sigma-Aldrich, Fluka, TCI, Acros Organics, ABCR, Alfa Aesar, Enamine, VWR, Combi-Blocks, Apollo Scientific, Aquilla Pharmatech, Ark Pharm, D-L Chiral Chemicals, ChemBridge, Renno Tech, Accela, Key Organics, Pharmablock and ChemImpex. Unless otherwise noted, all commercially available compounds were used as received without further purification.

[0497] The abbreviations used in this specification and the following examples are:

[0498] mCPBA (m-chloroperbenzoic acid), CHX (cyclohexane), DAST (diethylaminosulfur trifluoride), DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), DCM (dichloromethane), DIPEA (N,N-diisopropylethylamine), DMF (dimethylformamide), DMSO (dimethyl sulfoxide), LCMS (liquid chromatography mass spectrometry), Ms (methylsulfonyl, methanesulfonyl), p-TSA (PTSA, p-toluenesulfonic acid), Pd (dppf )Cl2([1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride), SEM([2-(trimethylsilyl)ethoxy]methyl), TBDMS(tert-butyldimethylsilyl), TFA(trifluoroacetic acid), THF(tetrahydrofuran), TMAD(N,N,N',N'-tetramethylazodicarbonamide), TMB(1,3,5-trimethoxybenzene), TLC(thin layer chromatography), TPP(triphenylphosphine), Tos(toluenesulfonyl, p-toluenesulfonyl);

[0499] ACK (ammonium-chloride-potassium), CD (cluster of differentiation), CCR (CC motif receptor), DMEM (Dulbecco's modified Eagle's medium), FBS (fetal bovine serum), EDTA (ethylenediaminetetraacetic acid), FACS (fluorescence-activated cell sorting), HPbCD (2-hydroxypropyl-β-cyclodextrin), HPMC (hydroxypropyl-methylcellulose), PEG400 (polyethylene glycol), Pen-Strep (penicillin-streptomycin), PBMC (peripheral blood mononuclear cells), PBS (phosphate-buffered saline).

[0500] General information

[0501] General procedures and synthetic routes to the disclosed compounds

[0502] In the following sections, some general procedures are described that enable those skilled in the art to synthesize many of the key intermediates and final compounds disclosed in this patent. The synthetic schemes are not limited to the synthetic pathways and reactions outlined. The materials described herein can also be obtained by other conditions or reaction sequences disclosed in the literature.

[0503] Synthetic pathway

[0504] Path 1

[0505]

[0506] Path 2

[0507]

[0508] Path 3

[0509]

[0510] General Procedures

[0511] General Procedure A: Mitsunobu Reaction

[0512]

[0513] Phenol (1.0 eq.) and alcohol (1.5 eq.) were dissolved in dry THF (0.1 M). The solution was dried over molecular sieves. The molecular sieves were removed, cooled to 0°C, TPP (2.2 equivalents) was added and the reaction vessel was sealed. Stirred at 0°C for 30 min, then TMAD (2.6 equivalents) was added and stirred at 0°C for another 30 min. Heat to 55°C and stir overnight. After the reaction was complete, celite was added and the solvent was evaporated. Purification was performed by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient).

[0514] General Procedure B: Nucleophilic Substitution

[0515]

[0516] Phenol (1.0 equivalent), halide (1.5 equivalents) and K2CO3 (3.0 equivalents) are dissolved in dry acetonitrile (0.1M). The mixture is stirred at 60°C overnight in a sealed reaction vessel. After the reaction is complete, diatomaceous earth is added and volatiles are removed under reduced pressure. Purification is performed by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient). An alternative suitable base is Cs2CO3, and acetonitrile can be replaced by THF, DMF or acetone. Depending on the availability of the raw materials, the equivalents of phenol and halide can be reversed.

[0517] General Procedure C: Aryl-trimethoxyphenyliodonium salts

[0518]

[0519] The iodinated aromatic hydrocarbon (1.0 equivalent) was dissolved in dry acetonitrile (0.15 M). The solution was acidified with pTSA (1.1 equivalents). mCPBA (1.1 equivalents) was then added and stirred at 80° C. for 1-2 h. After the oxidation step was complete, 1,3,5-trimethoxybenzene (TMB) was added and stirred at 80° C. for another 30 min. After completion, celite was added and the volatiles were removed under reduced pressure. Purification was performed by normal phase column chromatography (silica gel, DCM / methanol gradient).

[0520] General Procedure D: O-Arylation with Aryl-Trimethoxyphenyliodonium Salts

[0521]

[0522] Phenol (1.2 equivalents) and K2CO3 (3.0 equivalents) were suspended in dry acetonitrile (0.25 M). The mixture was heated to 55° C. and the iodonium salt was added to the stirred solution. Stirring was continued at 55° C. overnight. Celite was added and volatiles were removed under reduced pressure. Purification was performed by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient). In order to expand the reaction range, phenol and K2CO3 can be replaced by aliphatic alcohols and NaH.

[0523] General Procedure E: Miyaura borylation

[0524]

[0525] Aryl halide (1.0 equiv), bisvaleryl diboron (1.2 equiv), Pd(dppf)Cl2*DCM (0.1 equiv), and KOAc (3.0 equiv) were dissolved in dry 1,4-dioxane (0.1 M). The reaction vessel was sealed and stirred at 90°C overnight. Upon completion, celite was added and the volatiles were removed under reduced pressure. Purification was performed by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient).

[0526] General Procedure F: Suzuki cross-linking

[0527]

[0528] 8-Bromoxanthine derivative (1.0 equiv), boronate (1.2 equiv), Pd(dppf)Cl2*DCM (0.1 equiv), and K3PO4 (3.0 equiv) were dissolved in a 4:1 mixture of 1,4-dioxane and water (0.1 M). The reaction vessel was sealed and stirred at 90°C overnight. After the reaction was complete, celite was added and the volatiles were removed under reduced pressure. Purification was performed by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate / methanol gradient). The boronate can be displaced by boronic acid.

[0529] General Procedure G: TBDMS Deprotection of Alcohols

[0530]

[0531] The TBDMS-protected alcohol (1.0 equivalent) was dissolved in THF (0.1 M). Concentrated hydrochloric acid (15 equivalents) was added and stirred at room temperature overnight. After the deprotection was completed, volatiles were removed under reduced pressure. The residue was dissolved in DMSO and purified by reverse phase HPLC (C18 column, water (0.1% TFA) and ACN (0.1% TFA) gradient). The desired fraction was lyophilized to produce the final compound.

[0532] General Procedure H: SEM Protection

[0533]

[0534] The xanthine derivative (1.0 equivalent) was dissolved in dry THF (0.2 M) and DIPEA (6.0 equivalents). SEM-Cl (4.0 equivalents) was added and continued to stir at room temperature overnight. If the reaction was incomplete, more DIPEA and SEM-Cl were added. After completion, saturated NaHCO solution was added, the mixture was concentrated under reduced pressure, diatomaceous earth was added and volatiles were removed under reduced pressure. Purification was performed by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient).

[0535] General Procedure I: TBDMS and SEM-Deprotection

[0536]

[0537] The compound (1.0 equivalent) of SEM and TBDMS protection is dissolved among the THF (0.1M).Add concentrated hydrochloric acid (15.0 equivalent) and stir at room temperature overnight.Under reduced pressure, remove volatile matter and add a solution of ammonia in methanol (25.0 equivalent, 7M).Stir at room temperature for 30min.Again under reduced pressure, remove volatile matter and resistates is dissolved among the DMSO and pass through reversed-phase HPLC (C18 post, water (0.1%TFA) / acetonitrile (0.1%TFA) gradient) purifying.Required fraction low pressure lyophilization is to produce final compound.

[0538] General Procedure J: Deoxofluorination of Alcohols

[0539]

[0540] The alcohol (1.0 equivalent) was dissolved in dry DCM (0.1 M) and cooled to 0°C. Deoxofluor (1.1 M equivalent) was added and the reaction vessel was sealed. Stirred at 20°C for 2 hours. After the reaction was complete, celite was added and the solvent was evaporated. Purification can be achieved by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient). An alternative suitable reagent is DAST.

[0541] General Procedure K: Deoxofluorination of Aldehydes and Ketones

[0542]

[0543] The aldehyde or ketone (1.0 equivalent) is dissolved in dry DCM (0.1 M) and cooled to 0°C. Deoxofluor (2.1 equivalents) is added and the reaction vessel is sealed. Stir at room temperature. After the reaction is complete, celite is added and the volatiles are evaporated. Purify by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient). DAST can be used instead of Deoxofluor.

[0544] General Procedure L: Dess-Martin Oxidation

[0545]

[0546] The alcohol (1.0 equivalent) was dissolved in dry DCM (0.1 M). Dess-Martin periodinane (2.0 equivalents) was added and the reaction vessel was sealed. Stirred at 60°C. After the reaction was complete, celite was added and the volatiles were evaporated. Purification was performed by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient).

[0547] General Procedure M: Nucleophilic Aromatic Substitution

[0548]

[0549] Aryl fluoride (1.0 equivalent), phenol (1.5 equivalents) and K2CO3 (3.0 equivalents) are dissolved in dry DMSO (0.1M). The mixture is stirred at 110°C for 3h in a sealed reaction vessel. After the reaction is complete, diatomaceous earth is added and volatiles are removed under reduced pressure. Purification is performed by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient). An alternative suitable base is Cs2CO3. Depending on the availability of the raw materials, the equivalents of phenol and halide can be reversed. The reaction can be performed under microwave irradiation instead of normal heating.

[0550] General Procedure N: Nucleophilic Aromatic Substitution (Pyridine)

[0551]

[0552] Aryl fluoride (1.0 equivalent), phenol (1.5 equivalents) and K2CO3 (3.0 equivalents) are dissolved in dry DMF (0.1M). The mixture is stirred at 110°C in a sealed reaction vessel. After the reaction is complete, diatomaceous earth is added and volatiles are removed under reduced pressure. Purification is performed by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient). Alternative suitable bases are Cs2CO3 or K3PO4. Depending on the availability of the raw materials, the equivalents of phenol and halide can be reversed. Alcohol can also be used instead of phenol. The reaction can be performed under microwave irradiation instead of normal heating.

[0553] General Procedure O: Overlapping (telescoped) SNAr / Suzuki Reactions

[0554]

[0555] Aryl fluoride (1.5 equiv), alcohol (1.5 equiv), and K3PO4 (4.0 equiv) were dissolved in dry 1,4-dioxane (0.1 M). The mixture was stirred in a sealed reaction vessel at 100°C. After the first step, (hetero)aryl bromide (1.0 equiv), Pd(dppf)Cl2*DCM (0.1 equiv), and water were added to give a 4:1 ratio of 1,4-dioxane:water. The mixture was stirred at 100°C. After the second reaction, celite was added and the volatiles were removed under reduced pressure. Purification was performed by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient). Depending on the availability of the starting materials, the equivalents of the boronic acid and halide components could be reversed. Alcohols could also be used instead of phenol.

[0556] General Procedure P: N-3 Alkylation of 7-Methylxanthine Derivatives

[0557]

[0558] A 7-methylxanthine derivative (1.0 equivalent), MgO (1.0 equivalent) and TBAB (1.0 equivalent) were dissolved in dry DMSO (0.1 M) and heated to 100° C. Epoxide (1.1 equivalents) was added and the reaction vessel was sealed. Stirring was continued at 100° C. After the reaction was complete, celite was added and the volatiles were evaporated. Purification was performed by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate methanol gradient).

[0559] General Procedure Q: TBDMS Deprotection

[0560]

[0561] The xanthine derivative (1.0 equivalent) and CsF (5.0 equivalent) of TBDMS protection are dissolved in dry EtOH / DMSO5 / 1 (0.1M). The reaction vessel is sealed and stirred at 60°C. After the reaction is completed, diatomaceous earth is added and volatiles are evaporated. Purification is performed by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate / methanol gradient). The reaction can be performed under microwave radiation instead of normal heating. Example

[0562] Preparation of 8-bromo-3-(2-hydroxypropyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione (1)

[0563]

[0564] To a stirred solution of 8-bromo-7-methyl-3,7-dihydro-1H-purine-2,6-dione (5 g, 20.49 mmol) in DMF (50 mL) at room temperature was added 1-bromo-2-propanol (4.24 g, 30.73 mmol) and DBU (3.1 g, 20.49 mmol). The resulting reaction mixture was stirred at 90 ° C for 16 h. After the reaction was completed, the reaction mass was cooled to room temperature. The reaction mixture was concentrated under reduced pressure to obtain a crude reaction mass. The substance was diluted with DCM (100 mL) and the precipitated solid was collected. The crude material was dried under vacuum to provide compound 1 (3.66 g, 59%) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ: 11.16 (brs, 1H), 4.80 (d, J = 5.2 Hz, 1H), 4.06-4.00 (m, 1H), 3.88-3.78 (m, 4H), 3.69 (dd, J = 13.4, 5.6 Hz, 1H), 1.05 (d, J = 6.0 Hz, 3H). LCMS (ESI+): Found for C9H11BrN4O3: 303.1 [M+H] + , the calculated value is 302.00.

[0565] Preparation of (R)-8-bromo-3-(3,3-difluoro-2-hydroxypropyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione (3b)

[0566]

[0567] To a mixture of 8-bromo-7-methyl-3,7-dihydro-1H-purine-2,6-dione (3.13 g, 12.8 mmol), MgO (429 mg, 10.6 mmol) and tetrabutylammonium bromide (3.77 g, 11.7 mmol) in 60 ml of DMSO at 60 ° C was added 1.0 g (10.6 mmol) of (R)-2-(difluoromethyl)oxirane. The reaction was stirred overnight and filtered on celite. The filtrate was concentrated under reduced pressure and purified by reverse phase column chromatography (RP18, water / acetonitrile gradient) to produce (R)-8-bromo-3-(3,3-difluoro-2-hydroxypropyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione 3b.

[0568] LCMS (ESI+): found for C9H9BrF2N4O3: 339 / 341 (1:1) [M+H] + , the calculated value is 338.0 / 340.0.

[0569] Preparation of 5-(difluoromethyl)-2,2-dimethyl-1,3-dioxane 69

[0570]

[0571] To a stirred solution of 2,2-dimethyl-1,3-dioxane-5-carbaldehyde (300 mg, 2.08 mmol) in DCM (6 mL) was added DAST (0.55 mL, 4.16 mmol) at room temperature, and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated. The crude product was purified by column chromatography, eluting the compound with 10% EtOAc in petroleum ether. The fractions containing the compound were combined and evaporated to give 69 (0.2 g, 58%) as a brown liquid. 1H NMR (400 MHz, CDCl3) δ ppm: 6.09 (td, J = 56.0, 7.2 Hz, 1H), 4.04-4.08 (m, 2H), 3.99-3.90 (m, 2H), 1.89-1.98 (m, 1H), 1.46 (s, 3H), 1.40 (s, 3H).

[0572] Preparation of 2-(difluoromethyl)propane-1,3-diol 70

[0573]

[0574] To a stirred solution of compound 69 (1.3 g, 7.82 mmol, 1.0 equiv) in MeOH:H₂O (3:126 mL) was added p-TSA (1.61 g 9.38 mmol, 1.2 equiv) at 0°C, and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was evaporated to remove excess volatiles, diluted in EtOAc, and washed with water. The separated organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure to provide compound 70 (400 mg, 41%) as a pale yellow syrup. 1H NMR (400 MHz, CDCl₃) δ ppm: 6.09 (td, J = 56.0, 5.2 Hz, 1H), 4.02-3.95 (m, 4H), 2.05-2.24 (m, 2H).

[0575] Preparation of 3,3-difluoro-2-(hydroxymethyl)propyl methanesulfonate 111

[0576]

[0577] To a stirred solution of compound 70 (200 mg, 1.58 mmol, 1.0 equiv) in DCM (4 mL) at 0°C were added triethylamine (0.22 mL, 1.58 mmol, 1.0 equiv) and methanesulfonyl chloride (0.12 mL, 1.58 mmol, 1.0 equiv). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was evaporated under reduced pressure to remove excess volatiles to provide compound 111 (120 mg, 37%). The crude compound was used in the next step as a pale yellow syrup without purification. 1H NMR (400 MHz, CDCl3) δ ppm: 6.12-5.82 (m, 1H), 4.49-4.39 (m, 3H), 3.92-3.89 (m, 1H), 3.06-3.08 (m, 3H), 2.73-2.44 (m, 1H).

[0578] Preparation of 8-bromo-3-(3,3-difluoro-2-(hydroxymethyl)propyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione 71

[0579]

[0580] To a stirred solution of compound 111 (100 mg, 0.49 mmol, 1.0 equiv) in DMF (1 mL) was added 8-bromo-7-methyl-3,7-dihydro-1H-purine-2,6-dione (179 mg, 0.73 mmol, 1.5 equiv) and DBU (73 mg, 0.49 mmol, 1.0 equiv). The reaction mixture was stirred at 90 ° C for 16 h. The reaction mixture was allowed to cool to room temperature. It was diluted with water and extracted with EtOAc (2 x 10 mL). The organic layer was washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. Using 3% methanol in dichloromethane as the mobile phase, the crude product was purified by column chromatography to obtain compound 71 (100 mg, 58%) as a colorless solid. 1H NMR (400 MHz, CDCl3) δ ppm: 11.31 (s, 1H), 6.11 (td, J = 56.0, 4 Hz, 1H), 4.84 (t, J = 5.2 Hz, 1H), 4.13 (dd, J = 14 Hz, 1H), 3.98 (dd, J = 14 Hz, 1H), 3.82 (s, 3H), 3.52 (t, J = 5.2 Hz, 2H). LCMS (ESI+): Found for C10H11BrF2N4O3: 355.3 [M+H] + , the calculated value is 354.0.

[0581] Preparation of 8-bromo-7-methyl-3-(3,3,3-trifluoro-2-(hydroxymethyl)propyl)-3,7-dihydro-1H-purine-2,6-dione 71a

[0582] 2-((8-Bromo-7-methyl-2,6-dioxo-1,2,6,7-tetrahydro-3H-purin-3-yl)methyl)-3,3,3-trifluoropropionic acid

[0583]

[0584] To a stirred solution of 8-bromo-7-methyl-3,7-dihydro-1H-purine-2,6-dione (2.5 g, 10.20 mmol, 1.0 equiv) and 2-(trifluoromethyl)acrylic acid (1.43 g, 10.20 mmol, 1.0 equiv) in DMF (50 ml) was added DBU (1.5 ml, 10.20 mmol, 1.0 equiv) in an airtight microwave vial under argon at room temperature. The reaction mixture was then stirred at 80 ° C for 30 min under microwave irradiation until LC-MS indicated complete consumption of the starting material. All volatiles were evaporated under reduced pressure to give a crude material (2.8 g), which was used in the next step without further purification. LC-MS: found for C10H8BrF3N4O4 is 386.06 [M+H] +, calculated to be 383.97.

[0585] Methyl 2-((8-bromo-7-methyl-2,6-dioxo-1,2,6,7-tetrahydro-3H-purin-3-yl)methyl)-3,3,3-trifluoropropionate

[0586]

[0587] To a stirred solution of 2-((8-bromo-7-methyl-2,6-dioxo-1,2,6,7-tetrahydro-3H-purin-3-yl)methyl)-3,3,3-trifluoropropanoic acid (2.8 g, 7.27 mmol, 1.0 eq) in MeOH (28 ml) at 0° C. under argon atmosphere was added sulfuric acid (143 mg, 1.45 mmol, 0.2 eq). The reaction mixture was then stirred at 70° C. for 16 hours until the starting material was completely consumed. All volatile solvents were evaporated under reduced pressure to give the crude product, which was purified by column chromatography (230-400 mesh silica gel) using 58% ethyl acetate in petroleum ether as eluent. The compound-containing fractions were concentrated and dried to afford methyl 2-((8-bromo-7-methyl-2,6-dioxo-1,2,6,7-tetrahydro-3H-purin-3-yl)methyl)-3,3,3-trifluoropropanoate as a colorless solid. LC-MS: Found for C11H10BrF3N4O4: 399.12 [M+H]+, Calcd: 397.98.

[0588] 8-Bromo-7-methyl-3-(3,3,3-trifluoro-2-(hydroxymethyl)propyl)-3,7-dihydro-1H-purine-2,6-dione 71a

[0589]

[0590] To a stirred solution of methyl 2-((8-bromo-7-methyl-2,6-dioxo-1,2,6,7-tetrahydro-3H-purin-3-yl)methyl)-3,3,3-trifluoropropanoate (900 mg, 2.25 mmol, 1.0 eq) in THF (20 ml) at 0° C. under argon atmosphere was added LiBH 4 (2M in THF) (2.3 ml, 4.5 mmol, 2 eq). The reaction mixture was then stirred at room temperature for 5 h. The progress of the reaction was monitored by LC-MS, which indicated complete consumption of the starting material. The reaction mixture was quenched with ice-cold water (10 mL) and washed with ethyl acetate (5×10 % HCl). The crude product was purified by column chromatography (230-400 mesh silica gel) using 75% ethyl acetate in petroleum ether as eluent. The fractions containing the compound were concentrated and dried to provide 8-bromo-7-methyl-3-(3,3,3-trifluoro-2-(hydroxymethyl)propyl)-3,7-dihydro-1H-purine-2,6-dione as a colorless solid. LC-MS: found 371.19 [M+H]+ for C10H10BrF3N4O3, calculated 369.99.

[0591] For example, the building blocks disclosed in Table A-1 can be prepared analogously to the methods described for derivatives 1, 3b, 71 or 71a.

[0592] Table A-1:

[0593]

[0594]

[0595] Preparation of 8-bromo-3-(2-((tert-butyldimethylsilyl)oxy)propyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione (8)

[0596]

[0597] To a stirred solution of compound 1 (3.5 g, 11.55 mmol) in DMF (35 mL) at room temperature was added imidazole (5.2 g, 34.7 mmol) and TBDMSCl (2.36 g, 34.7 mmole). The resulting reaction mixture was stirred at room temperature for 16 h. After the reaction was completed (monitored by LC-MS), the reaction mass was cooled to room temperature. The reaction mixture was evaporated under reduced pressure. The crude material was purified by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient) to provide 8 (4.22 g, 87.5%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ: 11.28 (s, 1H), 4.22-4.19 (m, 1H), 3.95-3.90 (m, 1H), 3.81 (s, 3H), 3.70 (dd, J = 13.2, 4.0 Hz, 1H), 1.12 (d, J = 6 Hz, 3H), 0.70 (s, 9H), -0.05 (s, 3H), -0.25 (s, 3H). LCMS (ESI+): Found for C15H25BrN4O3Si: 417.3 [M+H] + , the calculated value is 416.1.

[0598] Preparation of 8-bromo-3-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluoropropyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione 13

[0599]

[0600] To a stirred solution of compound 71 (100 mg, 0.28 mmol, 1.0 eq) in dry DMF (2 mL) was added imidazole (39 mg, 0.57 mmol, 2.0 eq) and TBDMS-Cl (86 mg, 0.57 mmol, 2.0 eq). The reaction mixture was then stirred at room temperature for 16 h. The reaction mixture was diluted with water and extracted with EtOAc (2 x 10 mL). The organic layer was washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by column chromatography using 3% methanol in dichloromethane to obtain compound 13 (75 mg, 58%) as a colorless solid. LCMS (ESI+): found for C16H25BrF2N4O3Si is 467.3 [M+H] +, calculated value is 466.01. 1H NMR (400 MHz, DMSO-d6) δppm: 11.30 (s, 1H), 6.11 (td, J = 56.0, 4.4 Hz, 1H), 4.13-4.04 (m, 2H), 3.81 (s, 3H), 3.72 (d, J = 4.8 Hz, 2H), 2.67-2.51 (m, 1H), 0.81 (s, 9H), -0.010 (s, 3H), -0.018 (s, 3H).

[0601] For example, the structural units in Table A-2 below can be synthesized as described for compound 8 or 13.

[0602] Table A-2:

[0603]

[0604]

[0605] Preparation of 8-(4-(benzyloxy)-3-methoxyphenyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione 15

[0606]

[0607] Under a nitrogen atmosphere, 500 mg of 8-bromo-7-methyl-3,7-dihydro-1H-purine-2,6-dione, 790 mg of (4-(benzyloxy)-3-methoxyphenyl)boronic acid, 170 mg of Pd(dppf)Cl2*DCM and 423 mg of K2CO3 were suspended in 20 ml of 1,4-dioxane / water (1:1) and heated to 100°C until the reaction was complete. After cooling to room temperature, 15 ml of water was added to precipitate the product, which was collected by filtration. The crude product was then triturated with 30 ml of acetonitrile overnight. The solid was collected, washed and dried to produce 680 mg of compound 15. LCMS (ESI-): found for C20H18N4O4 was 377 [MH] - , the calculated value is 378.13.

[0608] Preparation of 8-(4-(Benzyloxy)-3-fluorophenyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione 15a

[0609]

[0610] Anhydrous sodium carbonate (216 mg, 2.04 mmol, 2.0 equiv) was added to a solution of 8-bromo-7-methyl-3,7-dihydro-1H-purine-2,6-dione (250 mg, 1.02 mmol, 1.0 equiv) and (4-(benzyloxy)-3-fluorophenyl)boronic acid (276 mg, 1.12 mmol, 1.1 equiv) in a solvent mixture of 1,4-dioxane (6 ml) and H2O (3 ml) at room temperature under an argon atmosphere. The entire reaction mixture was degassed with argon for 15 minutes, and then [1,1′-bis(diphenylphosphino)ferrocene]dichloro-palladium(II) complex with dichloromethane (41.7 mg, 0.051 mmol, 0.05 equiv) was added. The reaction was then stirred at 100°C under microwave irradiation for 1 hour. H2O (20 ml) was added and extracted with EtOAc (3 x 150 ml). The combined organic extracts were washed with brine (1 x 30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to provide a crude product. The resulting crude product was purified by CHCl 50%-60% ACN in H2O. 18 Purification by reverse phase column chromatography on silica gel afforded 8-(4-(benzyloxy)-3-fluorophenyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione (142 mg) as a colorless solid. LC-MS: Found for C19H15FN4O3: 367.14 [M+H] + , the calculated value is 366.1.

[0611] Preparation of 7-methyl-8-(3-(trifluoromethoxy)-4-(4-(trifluoromethoxy)phenoxy)-phenyl)-3,7-dihydro-1H-purine-2,6-dione 15b

[0612]

[0613] 7-Methyl-8-(3-(trifluoromethoxy)-4-(4-(trifluoromethoxy)phenoxy)phenyl)-3,7-dihydro-1H-purine-2,6-dione was prepared similarly to 8-(4-(benzyloxy)-3-fluorophenyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione. LC-MS: Found for C20H12F6N4O5 503.2 [M+H]+, Calcd 502.1.

[0614] Preparation of 8-(4-(benzyloxy)-3-methoxyphenyl)-3-(2-hydroxypropyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione 16

[0615]

[0616] Under nitrogen atmosphere, 150 mg of compound 15, 0.051 ml of 1-bromo-2-propanol, and 46 mg of Na2CO3 were suspended in 3.9 ml of DMF and stirred at 50°C for 72 h. After cooling to room temperature, some water was added to precipitate the product, which was collected, washed, and dried. Pure compound 16 was obtained after purification by preparative TLC (DCM / MeOH, 15:1). LCMS (ESI+): found for C23H24N4O5: 437 [M+H] + , the calculated value is 436.17.

[0617] Preparation of 8-(4-(benzyloxy)-3-fluorophenyl)-3-(3,4-difluoro-2-hydroxybutyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione 203

[0618] 4,4-Dimethyl-3,5,8-trioxabicyclo[5.1.0]octane

[0619]

[0620] To a stirred solution of 2,2-dimethyl-4,7-dihydro-1,3-dioxepin (10 g, 78.02 mmol, 1.0 equiv) in DCM (250 ml) was added m-CPBA (26.84 g, 156.04 mmol, 2.0 equiv) and NaHCO (13.1 g, 156.04 mmol, 2.0 equiv) at 0°C under an inert atmosphere. The mixture was then stirred at room temperature for 16 h. The resulting solid was filtered through filter paper and washed with excess DCM (3 x 100 mL). The combined filtrate was washed with saturated NaHCO (2 x 100 mL), brine (2 x 50 mL), dried over anhydrous NaSO, filtered and concentrated to afford the crude material, which was purified by silica gel (100-200 mesh) column chromatography using 15-20% EtOAc in petroleum ether to yield 4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane (7.5 g) as a colorless liquid. 1 H NMR (400MHz, CDCl3) δppm: 4.03 (qd, J = 14.4Hz, 1.2Hz, 4H), 3.21-3.20 (m, 4H), 1.37 (s, 3H), 1.32 (s, 3H).

[0621] 2-(2,2-Dimethyl-1,3-dioxolan-4-yl)-2-fluoroethane-1-ol

[0622]

[0623] Under an argon stream, triethylamine trihydrofluoride (97%) (83.86 g, 520.2 mmol, 3.0 equiv) was added to a Teflon bottle containing 4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane (25 g, 173.4 mmol, 1.0 equiv) at room temperature. The bottle was then tightly sealed and stirred at 65°C for 24 h. The reaction mass was cooled to room temperature and poured into ice-cold saturated NaHCO3 solution (300 ml) and stirred for 30 minutes before extraction with EtOAc (3 x 300 ml). The combined organic layers were washed with saturated NaHCO3 solution (1 x 100 mL), brine (1 x 100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to provide the crude product. It was purified by silica gel (230-400 mesh) column chromatography using 35% EtOAc in petroleum ether as eluent to yield 2-(2,2-dimethyl-1,3-dioxolan-4-yl)-2-fluoroethane-1-ol (7.2 g) as a colorless liquid. 1 H NMR (400MHz, CDCl3) δppm: 4.65-4.45(m,1H), 4.36-4.27(m,1H), 4.13-4.08(m,1H), 3.95-3.82(m,3H), 2.04(br s,1H), 1.44(s,3H), 1.38(s,3H).

[0624] 4-(1,2-difluoroethyl)-2,2-dimethyl-1,3-dioxolane

[0625]

[0626] DAST (8.84 mL, 67 mmol, 2.5 eq) was added to a stirred solution of 2-(2,2-dimethyl-1,3-dioxolan-4-yl)-2-fluoroethane-1-ol (4.4 g, 26.8 mmol, 1.0 eq) in toluene (60 ml) at room temperature under an argon atmosphere. After 5 minutes, pyridine (6.42 mL, 80.4 mmol, 3.0 eq) was added to the reaction at room temperature and then stirred at room temperature for 8 h. The reaction was quenched with saturated aqueous NaHCO solution (50 mL) and extracted with EtOAc (2 x 100 ml). The combined organic layers were washed with saturated NaHCO (1 x 50 mL), brine (1 x 50 mL), dried over anhydrous NaSO, filtered and concentrated under reduced pressure to afford 4-(1,2-difluoroethyl)-2,2-dimethyl-1,3-dioxolane (0.8 g) as a light yellow liquid. The crude product obtained was used in the next step without further purification. 1H NMR (400MHz, CDCl3) δppm: 4.75-4.53 (m, 3H), 4.32-4.22 (m, 1H), 4.13-4.08 (m, 1H), 3.95 (dd, J = 8.4Hz, 6.4Hz, 1H), 1.43 (s, 3H), 1.37 (s, 3H).

[0627] 3,4-Difluorobutane-1,2-diol

[0628]

[0629] Under an argon atmosphere, 4-(1,2-difluoroethyl)-2,2-dimethyl-1,3-dioxolane (0.800 g, 4.81 mmol, 1.0 equivalent) was dissolved in methanol (10 ml) and cooled to ℃. Then, p-toluenesulfonic acid monohydrate (91.58 mg, 0.481 mmol, 0.1 equivalent) was added and the reaction was stirred at room temperature for 5 h. All volatiles were removed under reduced pressure. The resulting residue was dissolved in EtOAc (100 ml) and the organic layer was washed with saturated NaHCO (3 x 20 ml) and brine (2 x 20 ml). The organic layer was dried over anhydrous NaSO, filtered and concentrated under reduced pressure to provide a crude product. The crude product obtained was purified by silica gel (230-400 mesh) column chromatography using 40% EtOAc in petroleum ether as eluent to afford 3,4-difluorobutane-1,2-diol (170 mg) as a colorless liquid. 1 H NMR (400MHz, CDCl3) δppm: 4.79-4.67 (m, 3H), 4.01-3.92 (m, 1H), 3.84-3.75 (m, 1H), 2.45 (d, J = 5.2Hz, 1H), 1.94 (t, J = 5.6Hz, 3H).

[0630] 3,4-Difluoro-2-hydroxybutyl trifluoromethanesulfonate

[0631]

[0632] To a stirred solution of 3,4-difluorobutane-1,2-diol (20 mg, 0.159 mmol, 1 equivalent) in dry DCM (2 ml) was added 99% triethylamine (24.1 mg, 0.238 mmol, 1.5 equivalents) followed by 98% trifluoromethanesulfonic anhydride (53.697 mg, 0.190 mmol, 1.2 equivalents). The addition of all reagents was completed at 0 ° C and under an argon atmosphere. The reaction was then stirred at room temperature for 3 h. The progress of the reaction was monitored by TLC, which showed complete exhaustion of the starting material (SM) (50% EtOAc solution in petroleum ether, R f=0.55, H2SO4). The reaction was quenched by adding ice-cold water (5 mL) and extracted with DCM (2 x 30 ml). The combined organic extracts were washed with 1N NaHCO3 (1 x 5 mL), brine (1 x 10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to produce the crude product. The resulting crude product was immediately used in the next step.

[0633] 8-(4-(Benzyloxy)-3-fluorophenyl)-3-(3,4-difluoro-2-hydroxybutyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione 203

[0634]

[0635] Cesium carbonate (56.1 mg, 0.172 mmol, 2.0 equiv) was added to a solution of 8-(4-(benzyloxy)phenyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione (30 mg, 0.086 mmol, 1 equiv) in dry DMF (2 mL) at room temperature under an argon atmosphere and stirred for 30 minutes under an argon atmosphere. 3,4-difluoro-2-hydroxybutyl trifluoromethanesulfonate (28.90 mg, 0.112 mmol, 1.3 equiv) was then added to the reaction flask and the reaction was stirred at 60 ° C for 30 h. The progress of the reaction was monitored by LC-MS. H2O (10 ml) was added and extracted with EtOAc (2 x 100 ml). The combined organic extracts were washed with ice-cold water (3 x 20 mL), brine (3 x 20 mL), dried over anhydrous Na2SO4, filtered through cotton and concentrated to provide a crude product. The crude material was first purified by reverse phase (C 18 Column chromatography) to yield, after lyophilization, 8-(4-(benzyloxy)-3-fluorophenyl)-3-(3,4-difluoro-2-hydroxybutyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione as an off-white solid. 1 H NMR (400 MHz, DMSO-d6) δ ppm: 11.11 (s, 1H), 7.71 (dd, J = 12.4 Hz, 2.0 Hz, 1H), 7.60-7.58 (m, 1H), 7.50-7.34 (m 6H), 5.47 (br s, 1H), 5.28 (s, 2H), 4.84-4.58 (m, 3H), 4.23-4.12 (m, 2H), 4.05-4.00 (m, 1H), 3.98 (s, 3H). LC-MS: found for C23H21F3N4O4: 475.3 [M+H] + , the calculated value is 474.15.

[0636] Preparation of 7-methyl-3-(4,4,4-trifluoro-2-hydroxybutyl)-8-(3-(trifluoromethoxy)-4-(4-(trifluoromethoxy)phenoxy)phenyl)-3,7-dihydro-1H-purine-2,6-dione 216

[0637]

[0638] 30 mg of 7-methyl-8-(3-(trifluoromethoxy)-4-(4-(trifluoromethoxy)phenoxy)phenyl)-3,7-dihydro-1H-purine-2,6-dione 15b (1.0 equiv), 2.6 mg of MgO (1.0 equiv), and 21.2 mg of TBAB (1.0 equiv) were dissolved in 1 mL of dry DMSO (0.1 M) and heated to 100° C. 6.4 μL of 2-(2,2,2-trifluoroethyl)oxirane (1.1 equiv) was added and the reaction vessel was sealed. The mixture was stirred at 100° C. After the reaction was complete, celite was added and the volatiles were evaporated. Purification by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate / methanol gradient) gave 7-methyl-3-(4,4,4-trifluoro-2-hydroxybutyl)-8-(3-(trifluoromethoxy)-4-(4-(trifluoromethoxy)phenoxy)phenyl)-3,7-dihydro-1H-purine-2,6-dione 216. LCMS (ESI+): found for C24H17F9N4O6: 629.5 [M+H] + , the calculated value is 628.1.

[0639] For example, the compounds in Table A-3 below were synthesized as illustrated for compound 16, 203, or 216.

[0640] Table A-3:

[0641]

[0642] Preparation of (4-(trifluoromethoxy)phenyl)(2,4,6-trimethoxyphenyl)iodonium 4-methylbenzenesulfonate 104

[0643]

[0644] 20.0 g of 1-iodo-4-(trifluoromethoxy)benzene was dissolved in 250 ml of dry acetonitrile. 13.15 g of pTSA and 26.0 g of mCPBA were added and the reaction was stirred at 80° C. for 2 h. After the oxidation was complete, 12.8 g of 1,3,5-trimethoxybenzoate was added and stirring was continued at 80° C. for another 30 min. The mixture was concentrated under reduced pressure and the residue was adsorbed on celite. Volatiles were removed under reduced pressure. Purification was performed by normal phase column chromatography (silica gel, DCM / methanol gradient). LCMS (ESI+): C16H15F3IO4 + The measured value is 454.8[M] + , the calculated value is 455.00.

[0645] Preparation of (2-(trifluoromethoxy)phenyl)(2,4,6-trimethoxyphenyl)iodonium 4-methylbenzenesulfonate 108

[0646]

[0647] 150 mg of 1-iodo-2-(trifluoromethoxy)benzene was dissolved in 3 ml of dry acetonitrile. 117 mg of pTSA and 167 mg of mCPBA were added and the reaction was stirred at 55 ° C for 1 hour. After the oxidation was completed, 96 mg of 1,3,5-trimethoxybenzene was added and stirring was continued at 55 ° C for another 30 minutes. The mixture was adsorbed on celite. The volatiles were removed under reduced pressure. Purification was carried out by normal phase column chromatography (silica gel, DCM / methanol gradient). LCMS (ESI+): C16H15F3IO4 + The measured value is 454.6[M] + , the calculated value is 455.0.

[0648] The compounds in Table A-4 below were synthesized as compounds 104, 108 or as described in the literature. Other iodonium salts are also commercially available.

[0649] Table A-4:

[0650]

[0651]

[0652]

[0653] Preparation of 1-bromo-4-(4-(trifluoromethoxy)phenoxy)benzene 105

[0654]

[0655] 8.29 g of 4-bromophenol, 20.0 g of iodonium tosylate 104, and 17.65 g of KCO were heated to 55°C overnight in 250 ml of acetonitrile. Some of the solvent was removed under reduced pressure, and the residue was adsorbed onto celite. Compound 105 was purified by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient). GCMS (EI): m / z found for C13H8BrF3O2: 334.2 [M] +˙ , the calculated value is 333.96.

[0656] Preparation of 4,4,5,5-tetramethyl-2-(4-(4-(trifluoromethoxy)phenoxy)phenyl)-1,3,2-dioxaborolane 106

[0657]

[0658] 10 g of bromide 105, 11.43 g of bisvaleryldiborane, 1.22 g of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium in dichloromethane, and 11.91 g of potassium acetate were mixed in 200 ml of 1,4-dioxane. The reaction was stirred at 100°C until completion. Some of the solvent was removed under reduced pressure and the residue was adsorbed on celite. Purification was performed by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient). LCMS (ESI+): found for C19H20BF3O4: 381.2 [M+H] + , the calculated value is 380.14.

[0659] Preparation of 4-bromo-2-(difluoromethoxy)-1-((4-methylbenzyl)oxy)benzene 130

[0660]

[0661] Under nitrogen atmosphere, 52 mg of p-tolylmethanol and 53 mg of KOtBu were added to 2 ml of ice-cold toluene. The mixture was allowed to warm to room temperature. 388 mg of iodonium salt 129 was added and stirring was continued at room temperature for 1.5 h. The reaction mixture was adsorbed on diatomaceous earth and purified by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient). LCMS (ESI+): the measured value for C15H13BrF2O2 was 343.4 [M+H] + , the calculated value is 342.01.

[0662] Preparation of 2-(3-(difluoromethoxy)-4-((4-methylbenzyl)oxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 131

[0663]

[0664] 85 mg of bromide 130, 94 mg of bisvaleryldiborane, 42 mg of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium in dichloromethane and 49 mg of potassium acetate were mixed in 1.5 mL of 1,4-dioxane. The reaction was stirred at 85°C until completion and then directly adsorbed onto celite. Purification was performed by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient). LCMS (ESI+): found for C21H25BF2O4 was 391.3 [M+H] + , the calculated value is 390.18.

[0665] Preparation of 2-(4-((4-(difluoromethyl)benzyl)oxy)-3-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 17

[0666]

[0667] 500 mg of 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol, 603 mg (1.3 equivalents) of 1-(bromomethyl)-4-(difluoromethyl)benzene and 138 mg of KCO were heated to 60° C. in 10 ml of dry acetonitrile and stirred until the reaction was complete. The mixture was adsorbed on celite, dried and purified by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient) to give compound 17. LCMS (ESI+): Found for CHOBF0: 379 [M+H] + , the calculated value is 378.16.

[0668] Preparation of 2-(4-(Benzyloxy)-3-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 20

[0669]

[0670] 324 mg of benzyl alcohol and 500 mg of 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol in 5 ml of THF were mixed with The mixture was dried over molecular sieves and then transferred to a solution of 1.15 g of triphenylphosphine in 5 ml of THF at 0° C. under nitrogen. 860 mg of TMAD was added. After 30 min, the reaction mixture was heated to 55° C. until the reaction was complete. The mixture was adsorbed onto celite, dried, and purified by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient) to give compound 20. LCMS (ESI+): found for C₂OH₂SBO₄: 341 [M+H] + , the calculated value is 340.18.

[0671] Preparation of 1-(benzyloxy)-4-bromo-2-(trifluoromethoxy)benzene 23

[0672]

[0673] Benzyl bromide (4.16 g), 4-bromo-2-(trifluoromethoxy)phenol (2.5 g) and Cs2CO3 (9.5 g) were mixed in 30 ml of THF and stirred at 60°C until the reaction was complete. The mixture was adsorbed on celite, dried and purified by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient) to give compound 23. LCMS (ESI+): Found for C14H10BrF3O2: 348.6 [M+H] + , the calculated value is 347.98.

[0674] Preparation of 2-(4-(benzyloxy)-3-(trifluoromethoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 24

[0675]

[0676] 3.5 g of the bromo derivative 23, 0.411 g of Pd(dppf)Cl2*DCM, 4.0 g of potassium acetate, and 3.84 g of divaleryldiboron were heated to 100°C in 30 ml of 1,4-dioxane until the reaction was complete. Most of the solvent was evaporated, and the residue was adsorbed on celite and purified by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient) to provide pure compound 24. LCMS (ESI+): Found for C20H22BF3O4: 395 [M+H] + , the calculated value is 394.16.

[0677] Preparation of 4,4,5,5-tetramethyl-2-(3-(trifluoromethoxy)-4-(4-(trifluoromethoxy)phenoxy)phenyl)-1,3,2-dioxaborolane 337c

[0678]

[0679] 11.0 g of the bromo derivative 333f, 2.16 g of Pd(dppf)Cl2*DCM, 10.4 g of potassium acetate, and 10.1 g of bis(pentanoyl)diboron) in 100 ml of 1,4-dioxane were heated to 90°C until the reaction was complete. Most of the solvent was evaporated, and the residue was adsorbed onto celite and purified by normal phase column chromatography (330 g of silica gel, cyclohexane / ethyl acetate gradient) to provide pure boronate 337c. LCMS (ESI+): Found for C20H19BF6O5: 465.3 [M+H]+, Calcd: 464.12.

[0680] 1-(4-Chlorophenyl)-2,2-difluoroethane-1-ol 331a

[0681]

[0682] 219 mg 4-chlorobenzaldehyde (1.0 eq) was dissolved in 12 ml dry DMF (0.1 M) and 236 mg CsF (1.0 eq) was added. The mixture was heated to 40°C and 400 μL TMS-C was added. F2 H (2.0 equiv) was added and the reaction vessel was sealed. Stirred at 40°C for 4 h. After the addition was complete, 3.1 mL of 1 M TBAF solution (2.0 equiv) was added. After the reaction was complete, celite was added and the volatiles were evaporated. Purification was performed by reverse phase column chromatography (C18, acetonitrile / water) to yield 1-(4-chlorophenyl)-2,2-difluoroethane-1-ol. GCMS (EI): Found for C8H7ClF2O: 192.2 [M] ·+ , the calculated value is 192.0.

[0683] 1-(3,4-Dichlorophenyl)-2,2-difluoroethane-1-ol 331b

[0684]

[0685] 1-(3,4-Dichlorophenyl)-2,2-difluoroethane-1-ol was prepared as described for 1-(4-chlorophenyl)-2,2-difluoroethane-1-ol 331a. GCMS (EI): found for C8H6Cl2F2O 226.2 [M]+, calculated 226.0.

[0686] 1-(4-Chloro-3-fluorophenyl)-2,2-difluoroethane-1-ol 331c

[0687]

[0688] 1-(4-Chloro-3-fluorophenyl)-2,2-difluoroethane-1-ol was prepared as described for 1-(4-chlorophenyl)-2,2-difluoroethane-1-ol 331a. GCMS (EI): found for C8H6ClF3O 210.2 [M]+, calculated 210.0.

[0689] 1-(3-Chloro-4-methylphenyl)-2,2-difluoroethane-1-ol 331d

[0690]

[0691] 1-(3-Chloro-4-methylphenyl)-2,2-difluoroethane-1-ol was prepared as described for 1-(4-chlorophenyl)-2,2-difluoroethane-1-ol 331a. GCMS (EI): found for C9H9ClF2O 206.2 [M]+, calculated 206.0.

[0692] 1-(2-(Benzyloxy)-5-bromophenyl)-2,2-difluoroethane-1-ol 332a

[0693]

[0694] 1500 mg of 2-(benzyloxy)-5-bromobenzaldehyde (1.0 equivalent) was dissolved in 20 ml of dry DMF (0.1 M) and 782 mg of CsF (1.1 equivalent) was added. The mixture was heated to 40° C., 1.66 ml of TMS-CF2H (2.5 equivalents) was added to the reaction and the reaction vessel was sealed. The mixture was stirred at 40° C. for 4 h. After the addition was complete, 10.3 ml of 1 M TBAF solution (2.0 equivalents) was added. After the reaction was complete, celite was added and the solvent was evaporated. 1-(2-(benzyloxy)-5-bromophenyl)-2,2-difluoroethane-1-ol was purified by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate / methanol gradient). GCMS (EI): Found for C15H10BrF5O3 was 342.4 [M] ·+ , the calculated value is 342.0.

[0695] 1-(5-Bromo-2-(4-(trifluoromethoxy)phenoxy)phenyl)-2,2-difluoroethane-1-ol 333a

[0696]

[0697] Prepared similarly to that described for 1-(2-(benzyloxy)-5-bromophenyl)-2,2-difluoroethane-1-ol from 5-bromo-2-(4-(trifluoromethoxy)phenoxy)benzaldehyde. GCMS (EI): found for C15H13BrF2O2: 412.2 [M] ·+ , the calculated value is 412.0.

[0698] 1-(2-(Benzyloxy)-5-bromophenyl)-2,2-difluoroethane-1-one 332b

[0699]

[0700] 500 mg of 1-(2-(benzyloxy)-5-bromophenyl)-2,2-difluoroethane-1-ol (1.0 equivalent) was dissolved in 4 ml of dry DCM (0.1 M). 1235 mg of Dess-Martin periodinane (2.0 equivalent) was added and the reaction vessel was sealed. Stirring was continued at 60° C. for 6 h. After the reaction was complete, celite was added and the volatiles were evaporated. Purification by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient) gave 1-(2-(benzyloxy)-5-bromophenyl)-2,2-difluoroethane-1-one. GCMS (EI): Found for C15H11BrF2O2: 340.0 [M] ·+ , the calculated value is 340.0.

[0701] 1-(5-Bromo-2-(4-(trifluoromethoxy)phenoxy)phenyl)-2,2-difluoroethane-1-one 333b

[0702]

[0703] Prepared similarly to that described for 1-(2-(benzyloxy)-5-bromophenyl)-2,2-difluoroethane-1-one from 1-(5-bromo-2-(4-(trifluoromethoxy)phenoxy)phenyl)-2,2-difluoroethane-1-ol. GCMS (EI): Found for C15H8BrF5O3: 410.0 [M] ·+ , the calculated value is 410.0.

[0704] 1-(Benzyloxy)-4-bromo-2-(1,1,2,2-tetrafluoroethyl)benzene 332c

[0705]

[0706] 150 mg of 1-(2-(benzyloxy)-5-bromophenyl)-2,2-difluoroethane-1-one (1.0 equivalent) was dissolved in 5 ml of dry DCM (0.1 M) and cooled to 0°C. 322 μl of Deoxofluor (50 wt% solution in toluene, 2.1 equivalents) was added and the reaction vessel was sealed. Stirring was continued at 20°C for 2 h. After the reaction was complete, celite was added and the volatiles were evaporated. Purification by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient) gave 1-(benzyloxy)-4-bromo-2-(1,1,2,2-tetrafluoroethyl)benzene. GCMS (EI): Found for C15H11BrF4O: 361.9 [M] ·+ , the calculated value is 362.0.

[0707] 1-(Benzyloxy)-4-bromo-2-(difluoromethyl)benzene 332d

[0708]

[0709] Prepared similarly to that described for 1-(benzyloxy)-4-bromo-2-(1,1,2,2-tetrafluoroethyl)benzene from 2-(benzyloxy)-5-bromobenzaldehyde. GCMS (EI): found for C14H11BrF2O: 312.0 [M] ·+ , the calculated value is 312.0.

[0710] 4-Bromo-2-(difluoromethyl)-1-(4-(trifluoromethoxy)phenoxy)benzene 333d

[0711]

[0712] Prepared similarly to that described for 1-(benzyloxy)-4-bromo-2-(1,1,2,2-tetrafluoroethyl)benzene from 5-bromo-2-(4-(trifluoromethoxy)phenoxy)benzaldehyde. GCMS (EI): found for C14H8BrF5O2: 382.2 [M] ·+ , the calculated value is 382.0.

[0713] 4-Bromo-2-(1,1,2,2-tetrafluoroethyl)-1-(4-(trifluoromethoxy)phenoxy)benzene 333e

[0714]

[0715] Prepared similarly to that described for 1-(benzyloxy)-4-bromo-2-(1,1,2,2-tetrafluoroethyl)benzene from 1-(5-bromo-2-(4-(trifluoromethoxy)phenoxy)phenyl)-2,2-difluoroethane-1-one. GCMS (EI): found for C15H8BrF7O2: 432.4 [M] ·+ , the calculated value is 432.0.

[0716] 1-(Benzyloxy)-4-bromo-2-(fluoromethyl)-benzene 332e

[0717]

[0718] 250 mg of (2-(benzyloxy)-5-bromophenyl)methanol (1.0 eq) was dissolved in 5 ml of dry DCM (0.1 M) and cooled to 0°C. 314 μl of Deoxofluor (50 wt% solution in toluene, 1.1 eq) was added and the reaction vessel was sealed. Stirring was continued at 20°C for 2 h. After the reaction was complete, celite was added and the solvent was evaporated. Purification by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient) gave 1-(benzyloxy)-4-bromo-2-(fluoromethyl)-benzene. GCMS (EI): Found for C14H12BrFO: 294.0 [M] ·+ , the calculated value is 294.0.

[0719] 4-Bromo-2-(trifluoromethoxy)-1-(4-(trifluoromethoxy)phenoxy)benzene 333f

[0720]

[0721] 5 g of 4-bromo-1-fluoro-2-(trifluoromethoxy)benzene (1.0 eq), 4.1 ml of 4-(trifluoromethoxy)-phenol (1.5 eq) and 5.3 g of KCO (3.0 eq) were dissolved in 20 ml of dry DMSO (0.1 M). The mixture was stirred at 110° C. for 3 h in a sealed reaction vessel. After the reaction was complete, celite was added and the volatiles were removed under reduced pressure. Purification by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient) gave 4-bromo-2-(trifluoromethoxy)-1-(4-(trifluoromethoxy)phenoxy)benzene. GCMS (EI): found for C₁₄H₁₃BrF₆O₃: 416.3 [M] ·+ , the calculated value is 415.9.

[0722] 5-Bromo-3-isopropoxy-2-(4-(trifluoromethoxy)phenoxy)pyridine 334a

[0723]

[0724] 200 mg of 5-bromo-2-fluoro-3-isopropoxypyridine (1.0 equiv), 259 uL of 4-(trifluoromethoxy)phenol (1.5 equiv) and 402 mg of KCO (3.0 equiv) were dissolved in 2 mL of dry DMF (0.1 M). The mixture was stirred at 110° C. for 3 h in a sealed reaction vessel. After the reaction was complete, celite was added and the volatiles were removed under reduced pressure. Purification by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient) gave 5-bromo-3-isopropoxy-2-(4-(trifluoromethoxy)phenoxy)pyridine. LCMS (ESI+): found for C15H13BrF3NO3 was 392.1 [M+H] + , the calculated value is 391.0.

[0725] 5-Bromo-3-methyl-2-(4-(trifluoromethoxy)phenoxy)pyridine 334b

[0726]

[0727] Prepared similarly to that described for 5-bromo-3-isopropoxy-2-(4-(trifluoromethoxy)phenoxy)pyridine from 5-bromo-2-fluoro-3-methylpyridine. LCMS (ESI+): found for C13H9BrF3NO2: 348.0 [M+H] +, the calculated value is 347.0.

[0728] 5-Bromo-2-(4-(trifluoromethoxy)phenoxy)pyridine 334c

[0729]

[0730] Prepared similarly to that described for 5-bromo-3-isopropoxy-2-(4-(trifluoromethoxy)phenoxy)pyridine from 5-bromo-2-fluoropyridine. LCMS (ESI+): found for C12H7BrF3NO2 334.1 [M+H] + , the calculated value is 333.0.

[0731] 5-Bromo-3-(difluoromethyl)-2-(4-(trifluoromethoxy)phenoxy)pyridine 334d

[0732]

[0733] Prepared similarly to that described for 5-bromo-3-isopropoxy-2-(4-(trifluoromethoxy)phenoxy)pyridine from 5-bromo-3-(difluoromethyl)-2-fluoropyridine. LCMS (ESI+): found for C13H7BrF5NO2: 383.0 [M+H] + , the calculated value is 383.0.

[0734] 5-Bromo-3-methoxy-2-(4-(trifluoromethoxy)phenoxy)pyridine 334e

[0735]

[0736] Prepared similarly to that described for 5-bromo-3-isopropoxy-2-(4-(trifluoromethoxy)phenoxy)pyridine from 5-bromo-2-fluoro-3-methoxypyridine. LCMS (ESI+): found for C13H9BrF3NO3: 363.0 [M+H] + , the calculated value is 363.0.

[0737] 5-Bromo-3-chloro-2-((4-methylbenzyl)oxy)pyridine 334f

[0738]

[0739] 200 mg of 5-bromo-3-chloro-2-fluoropyridine (1.0 equivalent), 151 mg of p-tolylmethanol (1.3 equivalents) and 438 mg of K CO (2.0 equivalents) were dissolved in 6 ml of dry DMSO (0.15 M). The reaction vessel was sealed and stirred at 120° C. for 10 min under microwave irradiation. Purification was performed by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient) to produce 5-bromo-3-chloro-2-((4-methylbenzyl)oxy)pyridine. LCMS (ESI+): the found value for C H BrClNO was 312.5 [M+H] + , the calculated value is 311.0.

[0740] Analogously to 5-bromo-3-chloro-2-((4-methylbenzyl)oxy)pyridine 334f, other 5-bromo-2-alkoxypyridine derivatives were synthesized, such as 5-bromo-3-chloro-2-(1-phenylethoxy)pyridine (LCMS (ESI+): found for C13H11BrClNO312.1 [M+H] + , calculated value is 311.0) and 5-bromo-3-chloro-2-((4-(difluoromethyl)benzyl)oxy)pyridine (LCMS (ESI+): found for C14H13BrClNO348.0 [M+H] + , calculated value is 347.0).

[0741] Preparation of 2-(4-(Benzyloxy)-3-cyclobutyloxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 337i

[0742]

[0743] 150 mg of 2-(benzyloxy)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (1.0 eq.) and 47 μl of cyclobutanol (1.5 eq.) were dissolved in 5 ml of dry THF (0.1 M). The solution was dried over molecular sieves. The molecular sieves were removed, cooled to 0°C, 241 mg of TPP (2.2 equivalents) was added and the reaction vessel was sealed. Stirred at 0°C for 30 min, then 187 mg of TMAD (2.6 equivalents) was added and stirred at 0°C for another 30 min. Heat to 55°C and stir overnight. After the reaction was complete, celite was added and the solvent was evaporated. Purification by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient) gave 2-(4-(benzyloxy)-3-cyclobutyloxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane. LCMS (ESI+): found for C23H29BO4 was 531.4 [M+H] + , the calculated value is 530.2.

[0744] The building blocks in Table A-5 below were synthesized, for example, as illustrated for compounds 17, 20, 24, 106, 131, 337c, 337i, and others.

[0745] Table A-5:

[0746]

[0747]

[0748]

[0749]

[0750]

[0751]

[0752]

[0753]

[0754]

[0755]

[0756]

[0757]

[0758] Preparation of 8-(4-((4-(difluoromethyl)benzyl)oxy)-3-fluorophenyl)-7-methyl-3-(3,3,3-trifluoro-2-hydroxypropyl)-3,7-dihydro-1H-purine-2,6-dione 18

[0759]

[0760] To 730 mg of bromide 4, 70 mg of boronate ester 17, 100 mg of K3PO4 and 26.6 mg of Pd(dppf)Cl2 was added 3 ml of 1,4-dioxane / water (5:1) under nitrogen, and the mixture was heated to 100°C until the starting material was consumed. The mixture was adsorbed on celite, dried and purified by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient) to give compound 18. LCMS (ESI+): Found for C23H18F6N4O4 is 529.3 [M+H] + , the calculated value is 528.12.

[0761] Preparation of 8-(4-(benzyloxy)-3-methoxyphenyl)-3-(3,3-difluoro-2-hydroxypropyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione 68

[0762]

[0763] Under nitrogen, 3 ml of 1,4-dioxane / water (5:1) was added to 50 mg of bromide 10, 66 mg of boronate ester 20, 70 mg of K 3 PO 4 and 18.7 mg of Pd (dppf) Cl 2, and the mixture was heated to 100 ° C until the starting material was exhausted. The mixture was adsorbed on celite, dried and purified by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient) to obtain a protected intermediate. The protecting group was removed by dissolving the intermediate in 1.5 ml of THF / 200 μl of a 2M aqueous hydrochloric acid solution and stirring the mixture at 60 ° C overnight. The mixture was adsorbed on celite and purified by reverse phase column chromatography (RP18, water / acetonitrile gradient) to produce compound 68. LCMS (ESI +): the found value of C 23 H 22 F 2 N 4 O 5 was 473.3 [M + H] + , the calculated value is 472.16.

[0764] Preparation of 8-(4-(benzyloxy)-3-(trifluoromethoxy)phenyl)-3-(2-hydroxypropyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione 120

[0765]

[0766] 150 mg of 8-(4-(benzyloxy)-3-(trifluoromethoxy)phenyl)-3-(2-((tert-butyldimethylsilyl)oxy)-propyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione (1.0 equiv) and 192 mg of CsF (5.0 equiv) were dissolved in 5 ml of dry EtOH / DMSO 9 / 1 (0.1 M). The reaction vessel was sealed and stirred at 80° C. overnight. After the reaction was complete, celite was added and the volatiles were evaporated. Purification by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate / methanol gradient) gave 8-(4-(benzyloxy)-3-(trifluoromethoxy)phenyl)-3-(2-hydroxypropyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione 120. LCMS (ESI+): found for C23H21F3N4O5: 491.2 [M+H] + , the calculated value is 490.1.

[0767] Preparation of 8-(5-chloro-6-((4-chloro-3-fluorobenzyl)oxy)pyridin-3-yl)-7-methyl-3-(3,3,3-trifluoro-2-hydroxypropyl)-3,7-dihydro-1H-purine-2,6-dione 299

[0768]

[0769] 150 mg of 3-chloro-2-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (1.5 equivalents), (4-chloro-3-fluorophenyl)methanol (1.5 equivalents), and 90 mg of K3PO4 (4.0 equivalents) were dissolved in 3 ml of dry 1,4-dioxane (0.1 M). The mixture was stirred at 100°C for 5 hours in a sealed reaction vessel. After the initial reaction was complete, 35 mg of 8-bromo-7-methyl-3-(3,3,3-trifluoro-2-hydroxypropyl)-3,7-dihydro-1H-purine-2,6-dione (1.0 equivalents), 5.9 mg of Pd(dppf)Cl2*DCM (0.05 equivalents), and 750 μL of water were added. The mixture was stirred at 100°C for 2 hours. After the second reaction was complete, celite was added and the volatiles were removed under reduced pressure. Purification by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient) yielded 8-(5-chloro-6-((4-chloro-3-fluorobenzyl)oxy)pyridin-3-yl)-7-methyl-3-(3,3,3-trifluoro-2-hydroxypropyl)-3,7-dihydro-1H-purine-2,6-dione. LCMS (ESI+): Found for C21H15Cl2F4N5O4: 548.4 [M+H]+, Calcd: 547.0.

[0770] The compounds in Table A-6 below were synthesized as illustrated for compounds 18, 68, 120, 299 or other compounds using 8-bromoxanthine derivatives (e.g., disclosed in Table A-1 or A-2) and boronic acids or esters (e.g., commercially available or disclosed in Table A-5 or prepared analogously to procedures disclosed herein or in the literature).

[0771] Table A-6:

[0772]

[0773]

[0774]

[0775]

[0776]

[0777]

[0778]

[0779]

[0780]

[0781]

[0782] Preparation of 8-bromo-3-(2-((tert-butyldimethylsilyl)oxy)propyl)-7-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-3,7-dihydro-1H-purine-2,6-dione 42

[0783] 2.55 ml of SEMCl was added and the reaction mixture was stirred overnight. Additional SEMCl (0.5 ml) and DIPEA (1 ml) were added and the reaction was stirred again at room temperature overnight. Ethyl acetate was added and the organic phase was washed with saturated NaHCO solution, dried over MgSO and volatiles were removed under reduced pressure. The crude product was sufficiently pure for use in the next reaction without further purification. LCMS (ESI+): found for C21H39BrN4O4Si2 was 547.3 [M+H] + , the calculated value is 546.17.

[0784] Preparation of 3-(2-((tert-butyldimethylsilyl)oxy)propyl)-8-(3-fluoro-4-hydroxyphenyl)-7-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-3,7-dihydro-1H-purine-2,6-dione 43

[0785]

[0786] Under an inert atmosphere, 2.5 g of bromide 42, 1.3 g of 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol, 0.39 g of Pd(dppf)Cl2*DCM, and 2.91 g of K3PO4 in 30 ml of 1,4-dioxane / water (4:1) were stirred at 90°C overnight. The mixture was concentrated under reduced pressure and adsorbed onto celite. Purification was performed by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient) to yield phenol 43. LCMS (ESI+): found for C27H43FN4O5Si2 was 579.4 [M+H] + , the calculated value is 578.28.

[0787] Preparation of 8-(4-(benzyloxy)-3-fluorophenyl)-3-(2-hydroxypropyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione 44

[0788]

[0789] 30 mg of phenol 43 and 12 mg of benzyl alcohol were dissolved in 1.5 ml of THF and Molecular sieve drying.Then under nitrogen atmosphere at 0 ℃, this solution is transferred to 30mg triphenylphosphine in 0.5ml THF.After 30min, TMAD is added and after another 30min at 0 ℃, the mixture is heated to 55 ℃ and spent the night.The mixture is adsorbed on diatomaceous earth and purified by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate / methanol gradient) to produce a protected intermediate.In order to remove two protecting groups, the crude product is dissolved in 750 μl THF and 750 μl concentrated hydrochloric acid aqueous solution is added.After stirring at 30 ℃, volatiles are removed, and the crude product is dissolved in 2ml ammonia (0.5M solution in dry 1,4-dioxane) at room temperature.After 5min, volatiles are removed again, the crude product is dissolved in some DMSO, and directly purified by reverse phase HPLC (C18 column, water (0.1% TFA) / acetonitrile (0.1% TFA) gradient).The desired fraction is lyophilized to produce title compound 44. LCMS (ESI+): found for C22H21FN4O4: 425.3 [M+H] + , the calculated value is 424.15.

[0790] Preparation of 8-(4-(4-chlorophenoxy)-3-fluorophenyl)-3-(2-hydroxypropyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione 197

[0791]

[0792] 130mg phenol 43, 218mg iodonium salt 159 and 62mg K2CO3 were stirred in 4ml acetonitrile at 55°C until phenol was fully converted. The mixture was adsorbed on diatomaceous earth and purified by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient). The protected intermediate was dissolved in 3ml DCM and 500 μl TFA and stirred at 40°C overnight. Volatiles were removed and the crude product was dissolved in 3ml ammonia (7M solution in methanol) at room temperature. Volatiles were removed again after 5min, the crude product was dissolved in some DMSO and directly purified by reverse phase HPLC (C18 column, water (0.1% TFA) / acetonitrile (0.1% TFA) gradient). The desired fraction was lyophilized to produce title compound 197. LCMS (ESI+): the measured value for C21H18ClFN4O4 was 445.4[M+H] + , the calculated value is 444.1.

[0793] Preparation of 3-(2-((tert-butyldimethylsilyl)oxy)propyl)-8-(4-hydroxy-3-(trifluoromethyl)phenyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione 154

[0794]

[0795] Under an inert atmosphere, 100 mg of bromide 8, 104 mg of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(trifluoromethyl)phenol, 30.4 mg of Pd(dppf)2Cl*DCM, and 153 mg of K3PO4 were mixed with 2.5 ml of 1,4-dioxane / water (4:1) and stirred at 90°C overnight. The mixture was concentrated under reduced pressure and adsorbed onto celite. Purification was performed by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient) to give phenol 154. LCMS (ESI+): found for C22H29F3N4O4Si was 499.2 [M+H] + , the calculated value is 498.19.

[0796] Preparation of 3-(2-hydroxypropyl)-7-methyl-8-(3-(trifluoromethoxy)-4-(4-(trifluoromethoxy)phenoxy)phenyl)-3,7-dihydro-1H-purine-2,6-dione 155

[0797]

[0798] 100 mg of phenol 154, 138 mg of iodonium salt 104 and 111 mg of K CO were stirred in 4 ml of acetonitrile at 55° C. until the phenol was fully converted. The mixture was adsorbed on diatomaceous earth and purified by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate / methanol gradient). The protected intermediate was dissolved in 2 ml of THF and 500 μl of concentrated hydrochloric acid and stirred at 28° C. overnight. Purification was carried out by reverse phase HPLC (C18 column, water (0.1% TFA) / acetonitrile (0.1% TFA) gradient). The desired fraction was lyophilized to produce the title compound 155. LCMS (ESI+): the found value for C H F N O was 545.2 [M+H] + , the calculated value is 544.12.

[0799] Preparation of 3-(2-((tert-butyldimethylsilyl)oxy)propyl)-8-(4-hydroxy-3-(trifluoromethyl)phenyl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione 157

[0800]

[0801] Under an inert atmosphere, 100 mg of bromide 8, 104 mg of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(trifluoromethoxy)phenol, 30.4 mg of Pd(dppf)Cl2*DCM, and 153 mg of K3PO4 were mixed with 2.5 ml of 1,4-dioxane / water (4:1) and stirred at 90°C overnight. The mixture was concentrated under reduced pressure and adsorbed onto celite. Purification was performed by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate gradient) to give phenol 157. LCMS (ESI+): found for C22H29F3N4OOSi was 515.5 [M+H] + , the calculated value is 514.19.

[0802] Preparation of 3-(2-hydroxypropyl)-7-methyl-8-(3-(trifluoromethoxy)-4-(4-(trifluoromethoxy)phenoxy)phenyl)-3,7-dihydro-1H-purine-2,6-dione 156

[0803]

[0804] 100 mg of phenol 157, 138 mg of iodonium salt 104 and 111 mg of K CO were stirred in 4 ml of acetonitrile at 55° C. until the phenol was fully converted. The mixture was adsorbed on diatomaceous earth and purified by normal phase column chromatography (silica gel, cyclohexane / ethyl acetate / methanol gradient). The protected intermediate was dissolved in 2 ml of THF and 500 μl of concentrated hydrochloric acid and stirred at 28° C. overnight. Purification was carried out by reverse phase HPLC (C18 column, water (0.1% TFA) / acetonitrile (0.1% TFA) gradient). The desired fraction was lyophilized to produce the title compound 156. LCMS (ESI+): the found value for C H F N O was 561.1 [M+H] + , the calculated value is 560.11.

[0805] The compounds in Table A-7 below were synthesized as illustrated for compounds 44, 155, 156, 197 or other compounds.

[0806] Table A-7:

[0807]

[0808]

[0809]

[0810]

[0811] Biological Examples

[0812] 1. Example B-1: Immunostimulation assay using CD4+ and CD8+ T cells

[0813] In Example B-1, the principle of selecting effective compounds based on structure activity relationship (SAR) using human anti-CD3 / 28 stimulated T cells is shown. The enhanced activation of stimulated T cells was measured by measuring CD69 expression by flow cytometry.

[0814] 1.1 Materials used for bioassays

[0815] product Distributor Catalog Number LymphoPrep StemCell 07851 CD4 microbeads Miltenyi 130-045-101 CD8 microbeads Miltenyi 130-045-201 Dynabeads Human T-Activator CD3 / 28 <![CDATA[Gibco TM ]]> 11131D

[0816] 1.2 Procedure / Description

[0817] 1.2.1 Collection of PBMCs from buffy coat

[0818] Leukocyte-rich buffy coats were ordered from the Austrian Red Cross, diluted with PBS to a total volume of 480 ml, 30 ml transferred to a 50 ml falcon, and 11 ml lymphoprep added. The cell suspension was centrifuged at 2200 rpm for 20 min and the layer containing PBMCs was transferred, washed three times with PBS, and counted.

[0819] 1.2.2 Isolation of CD4+ and CD8+ T cells

[0820] Put 10 7 PBMCs were magnetically labeled with CD4 and CD8 microbeads according to the manufacturer's instructions and isolated using an autoMACS device. For dose-response experiments using purified CD4+CD8+ T cells, 80,000 cells were seeded into each 96-well flat-bottom cell culture dish.

[0821] 1.2.3 Serial Dilution of Compounds of the Invention

[0822] The 10 mM stock of the compound of the invention was further diluted with DMSO and equal amounts were transferred to 96-well plates containing T cells. The final concentrations tested were: 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM, 0.003 μM and 0.001 μM.

[0823] 1.2.4 Stimulation Protocol for EC50 Test

[0824] For stimulation assays, 80,000 purified CD4+ and CD8+ T cells were seeded into each 96-well plate and incubated with 1 μg / ml anti-CD3, 1 μg / ml anti-CD28, and a compound of the invention ranging from 0.001 μM to 30 μM. DMSO-only wells served as controls to determine minimal T cell activation after stimulation, and CD3 / 28-coupled Dynabeads were used to determine maximal T cell activation. Cells were cultured for 16 hours in a humidified incubator at 37°C and 5% CO2.

[0825] 1.2.5 Determination of EC50 values of compounds of the present invention by surface FACS

[0826] T cells were stained for surface antigens (anti-CD4 and anti-CD8) and cell activation markers (anti-CD25 and anti-CD69). In addition, live cells were stained using a fixable viability dye. The staining was performed for 15 minutes and cells were analyzed using a Fortessa flow cytometer and FlowJo software. GraphPad EC was calculated using a variable slope model (agonist versus response - variable slope). 50 .

[0827] The assay results are summarized in Table B-1.

[0828] Table B-1 shows the immunostimulation assay data. Immunostimulation of CD4+ and CD8+ T cells is indicated as EC 50 [nM] ("-" = not measured). Compounds with activity designated "A" provide EC 50 ≤100 nM; compounds with activity designated "B" provided 100 nM <EC 50 ≤500 nM; compounds with activity designated "C" provide 500 nM <EC 50 ≤1000 nM; compounds with activity designated "D" provide 1000 nM <EC 50 ≤5000 nM; compounds with activity designated "E" provide EC 50 >5000nM.

[0829]

[0830]

[0831]

[0832]

[0833]

[0834] 2 Example B-2: In vitro killing efficacy

[0835] Compounds 119, 142, 156, 120, 288, 248, 240, 238, 213, and 207 were tested for their potential to enhance PBMC- or T cell-mediated killing of allogeneic M21 melanoma cells using an Xcelligence-based approach.

[0836] 2.1 Materials used for bioassays

[0837] product Distributor Catalog Number LymphoPrep StemCell 07851 CD4 microbeads Miltenyi 130-045-101 CD8 microbeads Miltenyi 130-045-201 Dynabeads Human T-Activator CD3 / 28 <![CDATA[Gibco TM ]]> 11131D RPMI 10% FCS PenStrep

[0838] 2.2 Procedure / Description

[0839] 2.2.1 Inoculation of M21 melanoma cells

[0840] E-plates were coated with 10 μg / ml fibronectin at 37°C for 1 hour. Subconfluent M21 melanoma cells were trypsinized, counted, and 5,000 cells were seeded into each fibronectin-coated 96-well plate in RPMI containing 10% FCS and 1% PenStrep. M21 cells were grown in a humidified incubator at 37°C and 5% CO2 and co-cultured with purified PBMCs or T cells 20-24 hours after seeding.

[0841] 2.2.2 Collection of PBMCs from buffy coat

[0842] Leukocyte-rich buffy coats were ordered from the Austrian Red Cross, diluted to a total volume of 480 ml with PBS, 30 ml transferred to a 50 ml falcon, and 11 ml lymphoprep added. The cell suspension was centrifuged at 2200 rpm for 20 min and the PBMC layer was transferred, washed three times with PBS, and counted. For dose-response experiments using PBMC co-cultures, 25,000 cells were seeded into each 96-well E-plate containing M21 (PBMC:M21 ratio of 5:1).

[0843] 2.2.3 Isolation of CD4+ and CD8+ T cells

[0844] Put 10 7 PBMCs were magnetically labeled with CD4 and CD8 microbeads according to the manufacturer's instructions and isolated using an autoMACS device. For dose response experiments using purified T cell co-cultures, 25,000 cells were seeded into each 96-well E-plate containing M21 (T cells:M21 was 5:1).

[0845] 2.2.4 Serial Dilution of Compounds of the Invention (LMW Compounds)

[0846] 10mM stock of the compound of the invention was further diluted with DMSO and equal amounts were transferred to 96-well plates containing M21 and PBMC or T cells. The final concentrations tested were: 10μM, 3μM, 1μM, 0.3μM and 0.1μM or 10μM, 3μM, 1μM, 0.3μM and 0.1μM, 0.03μM, 0.01μM or 0.003μM.

[0847] 2.2.5 Xcelligence in vitro killing assay

[0848] The E-plates containing M21 cell layers and isolated PBMC or T cells were incubated with 1 μg / ml anti-CD3, 1 μg / ml anti-CD28, and LMW compounds ranging from 0.1 μM to 10 μM. DMSO-only and M21-only (without effector cells) wells and wells containing unstimulated effector cells without anti-CD3 / 28 served as controls. The E-plates were incubated in a moisturizing incubator at 37°C and 5% CO2 for 40 hours, and the M21 growth was monitored every 15 min using an Xcelligence device.

[0849] 2.2.6 Determination of EC50 values of the compounds of the present invention using Xcelligene

[0850] Xcelligence data were analyzed using GraphPad Prism and EC50s were calculated using a variable slope model (agonist versus response - variable slope).

[0851] 2.3 Results

[0852] 2.3.1 In vitro killing assay using stimulated PBMCs

[0853] Stimulated PBMCs from two different donors were used to assess M21 melanoma cell killing over 40 hours in the presence of five different concentrations of Compound 142. PBMCs from both donors demonstrated potent M21 killing following anti-CD3 / CD28 stimulation and ± Compound 142 addition, but without dose-dependency following increasing doses of Compound 142 ( Figure 3A and 3B ).

[0854] Figure 3A and 3B Depicted is the growth of M21 cells following co-culture with stimulated PBMCs. M21 melanoma cells were incubated with isolated PBMCs and stimulated with anti-CD3 / 28 and compound 142 at five different concentrations.

[0855] 2.3.2 In vitro killing assay using stimulated CD4+ and CD8+ T cells

[0856] Stimulated CD4+ and CD8+ T cells from one donor (Donor 2) were co-cultured with M21 melanoma cells after addition of Compound 142, and cell growth was monitored for 40 hours. EC50s were calculated using GraphPad Prism 32 hours after addition of anti-CD3 / 28 and Compound 142. Purified T cells exhibited dose-dependent M21 killing after anti-CD3 / CD28 stimulation and increasing concentrations of Compound 142, with an EC50 of 628 nM ( Figure 4A and4B ).

[0857] Figure 4A and 4B Depicted is the growth of M21 cells after co-culture with stimulated T cells. M21 melanoma cells were incubated with purified CD4+ and CD8+ T cells and stimulated with anti-CD3 / 28 and varying concentrations of compound 142. EC50s were calculated using GraphPad Prism 32 hours after addition of anti-CD3 / 28 and compound 142.

[0858] Stimulated CD4+ and CD8+ T cells from another donor were co-cultured with M21 melanoma cells after addition of various concentrations of compound 142 (donor 3), compound 156 (donor 1), or compound 119 (donor 1), and cell growth was monitored for 40 hours. Purified T cells showed dose-dependent M21 killing ( Figure 5A -C).

[0859] Figure 5A -C depicts M21 cell growth after co-culture with stimulated T cells. M21 melanoma cells were incubated with purified CD4+ and CD8+ T cells and stimulated with anti-CD3 / 28 and various concentrations of Compound 142, Compound 156, or Compound 119.

[0860] Stimulated CD4+ and CD8+ T cells from the same donors as above were co-cultured with M21 melanoma cells after addition of Compound 142, Compound 120, Compound 156, Compound 288, Compound 119, Compound 248, Compound 240, Compound 238, Compound 213 (active R enantiomer of Compound 238) or Compound 207 (active R enantiomer of Compound 240), and cell growth was monitored for 40 hours. Purified T cells exhibited dose-dependent M21 killing ( Figure 5D EC values were calculated 32 h after compound addition using GraphPad Prism. 50 .

[0861] Figure 5D-M depicts M21 cell growth after co-culture with stimulated T cells. M21 melanoma cells were incubated with purified CD4+ and CD8+ T cells and stimulated with anti-CD3 / 28 and various concentrations of Compound 142, Compound 120, Compound 156, Compound 288, Compound 119, Compound 248, Compound 240, Compound 238, Compound 213, or Compound 207.

[0862] 2.3.3 In vitro killing assay using unstimulated PBMCs and T cells

[0863] The killing efficiency of unstimulated PBMCs and CD4+ and CD8+ T cells from one donor (Donor 2) co-cultured with M21 melanoma cells was evaluated in the presence of 10 μM Compound 142. Unstimulated PBMCs cultured with M21 cells showed effective killing after addition of Compound 142 compared to the DMSO control, while on the other hand, co-culture of purified T cells with M21 cells did not show enhanced killing over 40 hours ( Figure 6 ).

[0864] Figure 6 Depicted is the growth of M21 cells following co-culture of unstimulated PBMCs and T cells. M21 melanoma cells were incubated with unstimulated PBMCs or T cells and 10 μM Compound 142.

[0865] 3 Example B-3: Efficacy against B16-SIY melanoma

[0866] Compounds 142, 156, 120, 119, 288, 248, 240, 238, 213, 207, 260, 262, and 329 showed preclinical efficacy in the murine B16-SIY melanoma model.

[0867] Individual compounds of the invention were selected based on their potency, ADME, and PK characteristics and tested in immunocompetent C57BL / 6J mice implanted with B16-SIY melanoma cells.

[0868] 3.1 Materials used for bioassays

[0869] product Distributor Catalog Number DMEM, low glucose, pyruvate, HEPES <![CDATA[Gibco TM ]]> 12320032 Heat-inactivated FBS <![CDATA[Gibco TM ]]> 10500064 Penicillin-streptomycin (10.000 U / ml) <![CDATA[Gibco TM ]]> 15140122 L-glutamine (200 mM) <![CDATA[Gibco TM ]]> 25030024 Trypsin-EDTA <![CDATA[Gibco TM ]]> 25300054 PBS <![CDATA[Gibco TM ]]> 14190-144 DMSO Sigma / Merck D2650 PEG400 Sigma / Merck 8.07485 HPbCD (2-Hydroxypropyl-β-cyclodextrin) Sigma / Merck 332607 HPMC (Hydroxypropyl Methylcellulose) Sigma / Merck 09963 ACK lysis buffer <![CDATA[Gibco TM ]]> A1049201 Antibodies for flow cytometry Biolegend Viability dye <![CDATA[Invitrogen TM ]]> 65-0865-14 <![CDATA[LEGENDplex TM Mouse Proinflammatory Chemokine Panel (13-plex)]]> Biolegend 740451 <![CDATA[LEGENDplex TM Mouse Th17 Set (7-Plex) and Filter Plate V03]]> Biolegend 741047

[0870] 3.2 Procedure / Description

[0871] 3.2.1 Animals and Ethics

[0872] Eight-week-old C57BL / 6 mice were purchased from Charles River Medical University. All animal experiments were performed in accordance with the institutional guidelines of the Austrian Institute of Molecular Pathology and approved by the Austrian Ministry of Science in accordance with the European Community Animal Care Rules. 3 Or when the humane endpoint was reached, the mice were sacrificed.

[0873] 3.2.2 B16-SIY cell culture

[0874] B16-SIY melanoma cells were cultured in DMEM containing 10% FCS, 1% PenStrep, and 1% glutamine and grown in a humidified incubator at 37°C and 5% CO2. For tumor inoculation, subconfluent B16-SIY cells were trypsinized, washed, counted, and resuspended in PBS or 50% PBS and 50% Matrigel to a concentration of 1×10 7 The final concentration was 10 B16-SIY cells / ml.

[0875] 3.2.3 Tumor inoculation and compound treatment

[0876] 3.2.3.1 Efficacy Experiment 1: Compound 142

[0877] Thirty C57BL / 6 mice were intradermally inoculated with 1x10 6 B16-SIY cells were implanted and tumor-bearing mice were randomly assigned to three different groups 3 days after implantation: Compound 142 30 mg / kg 3 days after B16-SIY inoculation and then 10 mg / kg daily (10 mice), Compound 142 9 mg / kg 3 days after B16-SIY inoculation and then 3 mg / kg daily (10 mice), or vehicle control (10 mice). Compound 142 was diluted in 10% DMSO, 20% PEG400, 21% HPbCD, 0.35% HPMC and 48.65% H2O and orally treated with two different treatment regimens: d3: 30 mg / kg + once daily: 10 mg / kg or d3: 9 mg / kg + once daily: 3 mg / kg ( Figure 1 10% DMSO, 20% PEG400, 21% HPbCD, 0.35% HPMC, and 48.65% H2O served as vehicle controls. Tumors were measured daily and mice were sacrificed 21 days after B16-SIY inoculation ( Figure 1 ).

[0878] 3.2.3.2 Efficacy Experiment 2: Compound 142

[0879] Thirty C57BL / 6 mice were intradermally inoculated with 1x10 6B16-SIY cells were transplanted and tumor-bearing mice were randomly assigned to three different groups 3 days after transplantation: Compound 142 treatment (10 mice, 60 mg / kg D3 + 40 mg / kg every 2 days; 40 mg / kg every 48 hours, except for 7 days (30 mg / kg) and 9 days (60 mg / kg) after inoculation), vehicle control (10 mice, 100 μl once a day) or remained untreated (10 mice). Compound 142 was diluted in 10% DMSO, 20% PEG400, 21% HPbCD, 0.35% HPMC and 48.65% H2O and orally treated with different treatment regimens ( Figure 7 Mice receiving compound 142 were treated with 60 mg / kg 3 days after B16-SIY inoculation and then 40 mg / kg of compound 142 every 48 hours (60 mg / kg D3 + 40 mg / kg every 2 days*), except for 7 days (30 mg / kg) and 9 days (60 mg / kg) after inoculation. Tumors were measured daily and mice were sacrificed 19 days after B16-SIY inoculation.

[0880] Figure 7 The treatment schedule for the POC study monitoring tumor volume and survival of mice for Compound 142 in an in vivo efficacy experiment of orally administered compounds is shown. The arrows indicate oral drug treatment with Compound 142.

[0881] Asterisks indicate 40 mg / kg Compound 142 treatment every 48 hours, except on days 7 (30 mg / kg) and 9 (60 mg / kg).

[0882] 3.2.3.3 Efficacy Experiment 3: Compound 142

[0883] 35 C57BL / 6J mice were intradermally inoculated with 1x10 6 B16-SIY cells were implanted and tumor-bearing mice were randomly assigned to three different groups 3 days after implantation: Compound 142 30 mg / kg 4 days after B16-SIY inoculation and then 10 mg / kg daily (10 mice, 30 mg / kg D4 + 10 mg / kg once a day), Compound 142 9 mg / kg 4 days after B16-SIY inoculation and then 3 mg / kg daily (10 mice, 9 mg / kg D4 + 3 mg / kg once a day), or vehicle control (10 mice, 100 μl once a day). Compound 142 was diluted in 10% DMSO, 20% PEG400, 21% HPbCD, 0.35% HPMC and 48.65% H2O and orally treated with two different treatment regimens ( Figure 810% DMSO, 20% PEG400, 21% HPbCD, 0.35% HPMC, and 48.65% H2O served as vehicle controls. Tumors were measured daily and mice were sacrificed 21 days after B16-SIY inoculation.

[0884] Figure 8 The treatment schedule for the POC study monitoring tumor volume and survival of mice for Compound 142 in an in vivo efficacy experiment of orally administered compounds is shown. The arrow indicates oral Compound 142 administration.

[0885] 3.2.3.4 Efficacy Experiment 4: Compound 142 or 120

[0886] 40 C57BL / 6J mice were intradermally inoculated with 1x10 6 B16-SIY cells were randomly assigned to four different groups 3 days after transplantation: Compound 142 treatment (10 mice, 30 mg / kg D4 + 10 mg / kg once a day), Compound 142 treatment (10 mice, 30 mg / kg every three days), Compound 120 (10 mice, 25 mg / kg once a day), and vehicle control (10 mice, 100 μl once a day). Compound 142 and Compound 120 were diluted in 10% DMSO, 20% PEG400, 21% HPbCD, 0.35% HPMC and 48.65% H2O and orally treated with mice ( Figure 9 10% DMSO, 20% PEG400, 21% HPbCD, 0.35% HPMC, and 48.65% H2O served as vehicle controls. Tumors were measured daily and mice were sacrificed 20 days after B16-SIY inoculation.

[0887] Figure 9 The treatment schedule for a POC study monitoring tumor volume and survival in mice for Compound 142 or Compound 120 in an in vivo efficacy experiment of orally administered compounds is shown. Black arrows indicate daily oral administration of Compound 142 or Compound 120, and gray arrows depict oral Compound 142 treatment every three days.

[0888] 3.2.3.5 Efficacy Test 5: Compound 142, 156 or 119

[0889] 40 C57BL / 6J mice were intradermally inoculated with 1x10 6B16-SIY cells were implanted and the tumor-bearing mice were randomly assigned to four different groups 3 days after implantation: compound 142 treatment (10 mice, 30 mg / kg D4 + 10 mg / kg once a day), compound 156 (10 mice, 20 mg / kg D4 + 10 mg / kg once a day), compound 119 (10 mice, 10 mg / kg once a day), and vehicle control (10 mice, 100 μl once a day). Compounds 142, 155, and 119 were diluted in 10% DMSO, 20% PEG400, 21% HPbCD, 0.35% HPMC, and 48.65% H2O and orally treated with mice ( Figure 10 10% DMSO, 20% PEG400, 21% HPbCD, 0.35% HPMC, and 48.65% H2O served as vehicle controls. Tumors were measured daily and mice were treated from 4 to 21 days after B16-SIY inoculation, followed by a 40-day observation period without treatment. 3 Mice were sacrificed upon reaching a humane endpoint. Mice that survived beyond day 61 were repeat-challenged on day 62 with an additional intradermal inoculation of B16-SIY cells as described above.

[0890] Figure 10 The treatment schedule for the POC study monitoring tumor volume and survival of mice in an in vivo efficacy experiment for orally administered compounds is shown. Arrows indicate daily oral administration of compounds 142, 156, or 119.

[0891] 3.2.3.6 Efficacy Experiment 6: Compounds 142, 156 and 119

[0892] 55 C57BL / 6J mice were intradermally inoculated with 1x10 6 B16-SIY cells were implanted and the tumor-bearing mice were randomly assigned to five different groups 6 days after transplantation, and then administered daily on day 7: compound 142 treatment (10 mice, 30 mg / kg D7+10 mg / kg once a day), compound 156 treatment (10 mice, 20 mg / kg D7+10 mg / kg once a day), compound 156 treatment (10 mice, 6 mg / kg D7+3 mg / kg once a day), compound 119 (10 mice, 10 mg / kg once a day), and vehicle control (10 mice, 100 μl once a day). Compounds 142, 156, and 119 were diluted in 10% DMSO, 20% PEG400, 21% HPbCD, 0.35% HPMC, and 48.65% H2O and orally treated with mice ( Figure 1110% DMSO, 20% PEG400, 21% HPbCD, 0.35% HPMC, and 48.65% H2O served as vehicle controls. Tumors were measured daily and were removed when the tumor volume was ≥1000 mm 3 Mice were sacrificed upon reaching a humane endpoint. Mice that survived beyond day 65 were repeat-challenged on day 66 with an additional intradermal B16-SIY cell inoculation as described above and terminated on day 82.

[0893] Figure 11 The treatment schedule for the POC study monitoring tumor volume and survival of mice in an in vivo efficacy experiment for orally administered compounds is shown. Arrows indicate daily oral administration of compounds 142, 156, or 119.

[0894] 3.2.3.7 Efficacy Experiment 7: Compound 156 and Anti-PD1

[0895] 41 C57BL / 6J mice were intradermally inoculated with 1x10 6 B16-SIY cells were transplanted and tumor-bearing mice were randomly assigned to 4 different groups (10 mice / group) 3 days after transplantation. Group 1 received compound 156 (30 mg / kg D3 + 10 mg / kg once a day) and rat IgG2a isotype control antibody (BioXcell, clone 2A3; 10 mg / kg twice a week), group 2 received compound 156 (30 mg / kg D3 + 10 mg / kg once a day) and anti-PD1 antibody (BioXcell, clone RMP1-14; 10 mg / kg twice a week), group 3 received vehicle control (100 μl once a day) and rat IgG2a isotype control antibody (BioXcell, clone 2A3; 10 mg / kg twice a week), and group 4 received vehicle control (100 μl once a day) and anti-PD1 antibody (BioXcell, clone RMP1-14; 10 mg / kg twice a week). Compound 156 was diluted in 10% DMSO, 20% PEG400, 21% HPbCD, 0.35% HPMC and 48.65% H2O and orally treated in mice ( Figure 23 10% DMSO, 20% PEG400, and 21% HPbCD, 0.35% HPMC, and 48.65% H2O served as vehicle controls. Anti-PD1 and isotype control antibodies were diluted in PBS and administered intraperitoneally. Tumors were measured three times a week and mice were treated from 3 days to 19 days after B16-SIY inoculation. On day 19 or when tumor volume ≥ 2000 mm 3 Or the mice were sacrificed when the humane endpoint was reached.

[0896] Figure 23The treatment schedule for compound 156, isotype control, and anti-PD1 in vivo efficacy experiments with oral administration of compound and intraperitoneal antibody is shown. Black arrows indicate daily oral administration of compound 156, and gray arrows depict twice-weekly intraperitoneal antibody treatment.

[0897] 3.2.3.8 Efficacy Experiment 8: Compounds 156, 238, 213, 207 and 248

[0898] 125 C57BL / 6J mice were intradermally inoculated with 1x10 6 B16-SIY cells were injected into the mice and the tumor-bearing mice were randomly assigned to 11 different groups 3 days after transplantation: compound 156 (10 mice, 10 mg / kg once a day), 2 groups of compound 248 (20 mice, 20 mg / kg D4 + 10 mg / kg once a day), compound 238 (10 mice, 20 mg / kg D4 + 10 mg / kg once a day), compound 207 (10 mice, 20 mg / kg D4 + 10 mg / kg once a day), compound 213 (10 mice, 20 mg / kg D4 + 10 mg / kg once a day), compound 248 (10 mice, 20 mg / kg once a day), compound 248 (10 mice, 6 mg / kg D4 + 3 mg / kg once a day), compound 248 (10 mice, 2 mg / kg D4 + 1 mg / kg once a day) and two groups of vehicle controls (20 mice, 100 μl once a day). Compounds 156, 843, 207 and 213 were diluted in 10% DMSO, 20% PEG400, 21% HPbCD, 0.35% HPMC and 48.65% HO. Compound 248 was diluted in 10% DMSO, 20% PEG400, 21% HPbCD, 0.35% HPMC and 48.65% HO or 20% diethylene glycol monoethyl ether, 20% TPGS, 0.6% HPMC and 59.4% HO. 10% DMSO, 20% PEG400 and 21% HPbCD, 0.35% HPMC and 48.65% H2O or 20% diethylene glycol monoethyl ether, 20% TPGS, 0.6% HPMC and 59.4% H2O served as vehicle controls. Tumors were measured three times a week and mice were treated from 4 to 21 days after B16-SIY inoculation, followed by a 40-day observation period without treatment ( Figure 25 ). When the tumor volume ≥1000mm 3 Mice were sacrificed upon reaching a humane endpoint. Mice that survived beyond day 61 were repeat-challenged on day 62 with an additional intradermal B16-SIY cell vaccination as described above and terminated on a specific day (experiment ongoing).

[0899] Figure 25 The treatment schedule for compounds 156, 238, 213, 207, and 248 for the in vivo efficacy experiment of Experiment 8, where tumor volume and survival of mice were monitored for orally administered compounds, is shown. Arrows indicate oral drug treatment.

[0900] 3.2.3.9 Efficacy Experiment 9: Compounds 156, 329, 213 and 262

[0901] C57BL / 6J mice were intradermally inoculated with 1x10 6 B16-SIY cells were injected into the mice and the tumor-bearing mice were randomly assigned to 10 different groups 3 days after transplantation: compound 156 (8 mice, 10 mg / kg once a day), compound 329 (8 mice, 10 mg / kg once a day), compound 213 (8 mice, 10 mg / kg once a day), compound 213 (8 mice, 25 mg / kg once a day), compound 213 (8 mice, 60 mg / kg once a day), compound 262 (8 mice, 10 mg / kg once a day), compound 329 (7 mice, 20 mg / kg once a day), and vehicle control (8 mice, 100 μl once a day). Compound 156, 329, 213 or 262 were diluted in 20% diethylene glycol monoethyl ether, 20% TPGS, 0.6% HPMC and 59.4% H2O. 20% diethylene glycol monoethyl ether, 20% TPGS, 0.6% HPMC and 59.4% H2O served as vehicle control. Tumors were measured 3 times per week and mice were treated from 4 to 21 days after B16-SIY inoculation, followed by a no-treatment observation period ( Figure 27 When the tumor volume is ≥1000 mm 3 Or the mice were sacrificed when the humane endpoint was reached.

[0902] Figure 27 The treatment schedule for in vivo efficacy experiments of compounds 156, 329, 213, and 262 where the mice were monitored for tumor volume and survival rate with orally administered compounds is shown (data not shown - experiments in progress). Arrows indicate oral drug treatments.

[0903] 3.3 Results

[0904] 3.3.1 Efficacy Experiment 1: Compound 142

[0905] C57BL / 6 mice were intradermally inoculated with B16-SIY cells and treated orally with 30 mg / kg of compound 142 3 days after B16-SIY injection, followed by 10 mg / kg daily (d3: 30 mg / kg + once daily: 10 mg / kg), or received 9 mg / kg of compound 142 3 days after B16-SIY inoculation, followed by 3 mg / kg daily (d3: 9 mg / kg + once daily: 3 mg / kg). Vehicle-treated mice were used as controls. Compared to vehicle controls, the tumor volume of mice treated with both compound 142 regimens was significantly reduced within 21 days ( Figure 2 ).

[0906] 3.3.2 Efficacy Experiment 2: Compound 142

[0907] C57BL / 6J mice were intradermally inoculated with B16-SIY cells and treated orally with 60 mg / kg of compound 142 3 days after B16-SIY injection, and then administered 40 mg / kg of compound 142 every 48 hours (60 mg / kg D3 + 40 mg / kg every 2 days; 40 mg / kg every 48 hours, except for 7 days (30 mg / kg) and 9 days (60 mg / kg) after inoculation). Mice that received vehicle (100 μl once a day) or remained untreated served as controls. Compared to all other treatment groups, mice administered compound 142 showed a significant reduction in tumor size over 19 days ( Figure 12 ).

[0908] Figure 12 The effect of compound 142 on tumor growth rate in vivo is shown. Mean tumor volume ± SEM of mice treated orally with compound 142 60 mg / kg D3 + 40 mg / kg once every 2 days* or vehicle. Asterisks indicate 40 mg / kg compound 142 treatment every 48 hours, except on day 7 (30 mg / kg) and day 9 (60 mg / kg). Statistically significant differences (p < 0.05) were calculated between the treatment group and the vehicle control group using two-way analysis of variance.

[0909] 3.3.3 Efficacy Experiment 3: Compound 142

[0910] C57BL / 6J mice were intradermally inoculated with B16-SIY cells and treated orally with 30 mg / kg of compound 142 4 days after B16-SIY injection, followed by 10 mg / kg daily (30 mg / kg D4 + 10 mg / kg once daily), or received 9 mg / kg of compound 142 4 days after B16-SIY inoculation, followed by 3 mg / kg daily (9 mg / kg D4 + 3 mg / kg once daily). Vehicle-treated mice (100 μl once daily) were used as controls. Compared to vehicle controls, tumor volume in mice treated with both compound 142 regimens was significantly reduced over 21 days ( Figure 13 ).

[0911] Figure 13 The effect on tumor growth rate in vivo is shown. Mean tumor volume ± SEM of mice treated orally with Compound 142 30 mg / kg D4 + 10 mg / kg once daily, 9 mg / kg D4 + 3 mg / kg once daily, or vehicle. Statistically significant differences (p < 0.05) between treatment groups and vehicle control group were calculated using two-way ANOVA.

[0912] 3.3.4 Efficacy Experiment 4: Compound 142 or 120

[0913] C57BL / 6J mice were inoculated intradermally with B16-SIY cells and treated orally with 30 mg / kg compound 142 4 days after B16-SIY injection, followed by 10 mg / kg (30 mg / kg D4+10 mg / kg once a day) or 30 mg / kg compound 142 every day for 4 days after B16-SIY inoculation, followed by 30 mg / kg every three days (30 mg / kg every three days). Compound 120 was administered daily from 4 days after B16-SIY inoculation (25 mg / kg once a day), and vehicle-treated mice (100 μl once a day) served as controls. Compared to vehicle controls, the tumor volume in mice was significantly reduced in 20 days after treatment with the compound 142 regimen. Compared to the vehicle-treated control group, the application of compound 120 did not affect tumor growth ( Figure 14 ).

[0914] Figure 14 The effects of compounds 142 and 120 on tumor growth rates in vivo are shown. Mean tumor volumes ± SEM of mice treated orally with compound 142 30 mg / kg D4 + 10 mg / kg once daily or 30 mg / kg every three days, compound 120 25 mg / kg once daily, or vehicle. Statistically significant differences (**p<0.01) between treatment groups and vehicle control groups were calculated using two-way ANOVA.

[0915] 3.3.5 Efficacy Experiment 5: Compound 142, 156 or 119

[0916] C57BL / 6J mice were intradermally inoculated with B16-SIY cells and orally treated with 30 mg / kg compound 142 4 days after B16-SIY injection, followed by 10 mg / kg daily (30 mg / kg D4 + 10 mg / kg once a day) until 21 days, or 20 mg / kg compound 156 was administered 4 days after B16-SIY inoculation, followed by 10 mg / kg daily (20 mg / kg D4 + 10 mg / kg once a day) until 21 days. Compound 119 was administered daily starting 4 days after B16-SIY inoculation (10 mg / kg once a day) until 21 days, and vehicle-treated mice (100 μl once a day) served as controls. Mice were then observed for 40 days without treatment. Compared to vehicle controls, mice treated with compounds 142, 156, and 119 showed a significant reduction in tumor volume and prolonged survival over 61 days ( Figure 15A -C). Three mice that received compound 156 between days 4 and 21 survived beyond day 61 and were repeated with additional intradermal B16-SIY cell inoculations on day 62. Three previously untreated mice without primary tumors were intradermally inoculated with B16-SIY cells and will serve as controls.

[0917] Figure 15A -C shows the effects of compounds 142, 156 and 119 on tumor growth rate and survival in vivo. A: Mean tumor volume ± SEM of mice treated with compound 142 30 mg / kg D4 + 10 mg / kg once a day, compound 156 20 mg / kg D4 + 10 mg / kg once a day, compound 119 10 mg / kg once a day or oral administration of vehicle. Statistically significant differences (**p < 0.01, ****p < 0.0001) were calculated between the treatment group and the vehicle control group using two-way ANOVA. B: Survival of mice treated with compound 142 30 mg / kg D4 + 10 mg / kg once a day, compound 156 20 mg / kg D4 + 10 mg / kg once a day, compound 119 10 mg / kg once a day or oral administration of vehicle. C: Mean tumor volume ± SEM and tumor volume of each mouse with initial B16-SIY challenge or secondary B16-SIY repeated challenge.

[0918] 3.3.6 Efficacy Experiment 6: Compound 142, 156 or 119

[0919] B16-SIY cells were inoculated into the dermis of C57BL / 6J mice and treated orally with 30mg / kg compound 142 7 days after B16-SIY injection, followed by 10mg / kg (30mg / kg D7+10mg / kg once a day) every day until 21 days. 20mg / kg compound 156 was applied 7 days after B16-SIY inoculation, followed by 10mg / kg treatment (20mg / kg D7+10mg / kg once a day) every day until 21 days, or 6mg / kg compound 156 was applied 7 days after B16-SIY inoculation, followed by 3mg / kg (6mg / kg D7+3mg / kg once a day) every day until 21 days. Compound 119 was applied every day from 7 days after B16-SIY inoculation until 21 days, and vehicle-treated mice (100 μl once a day) served as controls. Mice were subsequently observed for 44 days without treatment. Compared to vehicle control, mice treated with compound 156 and compound 119 showed significant reduction in tumor volume and prolonged survival in 65 days (Figure 16). Between 7 days and 21 days, 1 mouse receiving compound 156 20mg / kg D7+10mg / kg once a day and 1 mouse receiving 6mg / kg D7+3mg / kg once a day survived more than the 65th day and were repeatedly challenged with another intradermal B16-SIY cell inoculation on the 66th day. Three previously untreated age-matched mice without primary tumors were inoculated with B16-SIY cells in the dermis and will serve as controls. The mice of the repeated attack did not show primary or secondary B16-SIY tumor growth in 16 days compared to the control and terminated on the 82nd day.

[0920] Figure 16A A and B depict the effects of compounds 142, 156, and 119 on tumor growth rate in vivo. A: Mean tumor volume ± SEM of mice receiving oral treatment with compound 142, compound 156, or compound 119 7 to 21 days after B16-SIY inoculation. Statistically significant differences (**p<0.01, ***p<0.001) were calculated between the treatment group and the vehicle control group using two-way ANOVA. B: Survival of mice receiving oral treatment with compound 142, compound 156, or compound 119 7 to 21 days after B16-SIY inoculation.

[0921] 3.3.7 Efficacy Experiment 7: Compound 156 and Anti-PD1

[0922] C57BL / 6J mice were intradermally inoculated with B16-SIY cells and treated orally with 30 mg / kg of compound 156 3 days after B16-SIY inoculation, followed by daily administration of 10 mg / kg (30 mg / kg D3 + 10 mg / kg once a day) until day 19, and additional intraperitoneal injections of anti-PD1 antibody (10 mg / kg twice a week) or rat IgG2a isotype control antibody (10 mg / kg twice a week) twice a week starting 4 days after tumor cell inoculation until day 19. Vehicle-treated mice (100 μL / mouse once a day) that were additionally injected with anti-PD1 antibody (10 mg / kg twice a week) or rat IgG2a isotype control antibody (10 mg / kg twice a week) twice a week starting 4 days after tumor cell inoculation until day 19 served as controls. Compared to the vehicle and isotype control administration groups, mice treated with vehicle and anti-PD1, compound 156 and isotype control, and compound 156 and anti-PD1 showed significant reductions in tumor volume over 19 days. The combination of compound 156 and anti-PD1 showed the best response with respect to tumor growth control and tumor weight ( Figure 24A -B).

[0923] Figure 24A and B depict the effects of compound 156 and anti-PD-1 antibody monotherapy and combination therapy on in vivo tumor growth rate. A: Mean tumor volume ± SEM of mice treated orally with a combination of compound 156 or vehicle and anti-PD-1 or rat IgG2a isotype control antibody administered intraperitoneally 3 to 19 days after B16-SIY inoculation. Treatment groups were administered as follows: (grey) vehicle [once daily] orally + isotype [10 mg / kg twice weekly] intraperitoneally; (yellow) vehicle [once daily] orally + anti-PD-1 [10 mg / kg twice weekly] intraperitoneally; (purple) compound 156 [30 mg / kg D3 + 10 mg / kg once daily] orally + isotype [10 mg / kg twice weekly] intraperitoneally; (turquoise) compound 156 [30 mg / kg D3 + 10 mg / kg once daily] orally + anti-PD-1 [10 mg / kg twice weekly] intraperitoneally. Statistically significant differences between treatment groups and vehicle control group were calculated using two-way ANOVA (*p<0.5, ****p<0.0001).B: Tumor weights of individual mice receiving oral and intraperitoneal treatments as in A.

[0924] 3.3.8 Efficacy Experiment 8: Compounds 156, 238, 213, 207 and 248

[0925] C57BL / 6J mice were intradermally inoculated with B16-SIY cells and treated orally 4 days after B16-SIY inoculation with 10 mg / kg of compound 156 once daily (10 mg / kg once daily) or 20 mg / kg of compound 248, compound 238, compound 207 (R enantiomer of compound 240), or compound 213 (R enantiomer of compound 238), followed by daily administration of 10 mg / kg (20 mg / kg D4 + 10 mg / kg once daily) until day 21. Compound 248 was also evaluated using the following dosing regimens: 20 mg / kg once daily, 6 mg / kg D4 + 3 mg / kg once daily, 2 mg / kg D4 + 1 mg / kg once daily, and 20 mg / kg D4 + 10 mg / kg once daily treatment regimens compared in two different formulations. Mice treated with the vehicle for 4 days after B16-SIY inoculation until 21 days (100 μL / mouse once a day) served as controls. Mice were then observed for 40 days without treatment. Compared to the vehicle control, mice treated with compound 156, compound 248, compound 238, compound 207, and compound 213 showed significant reduction in tumor volume and prolonged survival over 61 days ( Figure 26A -D).

[0926] Between days 4 and 21, 4 mice receiving compound 156, 6 mice receiving compound 248 20 mg / kg once daily, 6 mice receiving compound 248 20 mg / kg D4 + 10 mg / kg once daily, 3 mice receiving compound 248 6 mg / kg D4 + 3 mg / kg once daily, 5 mice receiving compound 238, 1 mouse receiving compound 207, 2 mice receiving compound 213, and 2 mice receiving compound 248 20 mg / kg D4 + 10 mg / kg once daily in different formulations survived beyond day 61 and were repeated with additional intradermal B16-SIY cell inoculations on day 62. Five previously untreated mice without primary tumors were intradermally inoculated with B16-SIY cells and will serve as controls (experiments in progress).

[0927] Figure 26AFigures 1 through 2 depict the effects of compounds 156, 238, 213, 207, and 248 on in vivo tumor growth rate and survival. A: Mean tumor volume ± SEM of mice treated orally with compound 156, compound 248, or vehicle 4 to 21 days after B16-SIY inoculation. B: Survival of mice treated orally in A. C: Mean tumor volume ± SEM of mice treated orally with compound 156, 238, 213, 207, and 248 or vehicle 4 to 21 days after B16-SIY inoculation. D: Survival of mice treated orally in C.

[0928] 3.3.9 Efficacy Experiment 9: Compounds 156, 329, 213 and 262

[0929] C57BL / 6J mice were intradermally inoculated with B16-SIY cells and treated orally with 10 mg / kg of compound 156, compound 329 (R enantiomer of compound 248), compound 213 (R enantiomer of compound 238), and compound 262. In addition, compound 213 was evaluated using the following dosage regimens: 25 mg / kg once daily and 60 mg / kg once daily, and compound 329 was also administered at 20 mg / kg once daily from day 4 to day 21 after inoculation. Vehicle-treated mice (100 μL / mouse) served as controls. Mice were subsequently observed without treatment (experiment ongoing).

[0930] All compounds and treatment regimens resulted in significant reductions in tumor volume and prolonged survival over several days compared to vehicle control ( Figure 28 , experiments in progress).

[0931] Figure 28 Depicted are the effects of compounds 156, 329, 213, and 262 on in vivo tumor growth rate. Mean tumor volumes ± SEM of mice receiving oral treatment with compounds 156, 329, 213, and 262 or vehicle 4 to 18 days after B16-SIY inoculation.

[0932] 4. Example B-4: Efficacy against EO771 breast cancer

[0933] Compounds 156 and 119 were selected based on their potency, ADME, and PK properties and tested in immunocompetent C57BL / 6J mice engrafted with EO771 breast cancer cells. The compound formulation and dose used in the proof-of-concept (POC) study were based on PK studies performed at the Lead Discovery Center (LDC), Dortmund.

[0934] Compounds 156 and 119 showed preclinical efficacy in the murine EO771 breast cancer model.

[0935] 4.1 Materials used for bioassays

[0936]

[0937] 4.2 Procedure / Description

[0938] 4.2.1 Animals and Ethics

[0939] Eight-week-old C57BL / 6J mice were purchased from Charles River Medical University. All animal experiments were performed in accordance with the institutional guidelines of the Institute of Molecular Pathology, Austria, and were approved by the Austrian Ministry of Science according to the European Community Animal Care Rules.

[0940] 4.2.2 EO771 cell culture

[0941] EO771 breast cancer cells were cultured in DMEM containing 10% FCS, 1% PenStrep, 1% sodium pyruvate, 1% non-essential amino acid solution, and 1% L-glutamine and grown in a humidified incubator at 37°C and 5% CO2. For tumor inoculation, subconfluent EO771 cells were trypsinized, washed, counted, and resuspended in 50% PBS and 50% Matrigel to a size of 20 x 10 6 The final concentration of EO771 cells / ml.

[0942] 4.2.3 Efficacy test

[0943] 4.2.3.1 Tumor inoculation and compound treatment

[0944] Efficacy of the Treatment Regimen Using the POC Study Experiment 1 monitored tumor volume and survival in mice treated with the orally administered drug.

[0945] 46 C57BL / 6J mice were inoculated in the fourth mammary fat pad with 0.5x10 6EO771 cells were implanted and tumor-bearing mice were randomly assigned to four different groups 6 days after implantation: Compound 142 treatment (10 mice, 30 mg / kg D7 + 10 mg / kg once daily), Compound 156 (10 mice, 20 mg / kg D7 + 10 mg / kg once daily), Compound 119 (10 mice, 10 mg / kg once daily), and vehicle control (10 mice, once daily). Compounds 142, 156, and 119 were diluted in 10% DMSO, 20% PEG400, 21% HPbCD, 0.35% HPMC, and 48.65% HO and orally treated from day 7 to day 21. 100 μL of 10% DMSO, 20% PEG400, 21% HPbCD, 0.35% HPMC, and 48.65% HO served as a vehicle control. Tumors were measured daily and when the tumor volume was ≥700 mm 3 Or the mice were sacrificed when the humane endpoint was reached.

[0946] Figure 17 Depicted is the treatment regimen for compounds 142, 156, and 119 in an in vivo efficacy experiment of orally administered compounds. Arrows indicate oral drug treatment.

[0947] 4.3 Results

[0948] C57BL / 6J mice bearing EO771 tumors received 30 mg / kg of compound 142 7 days after EO771 injection, followed by daily administration of 10 mg / kg (30 mg / kg D7 + 10 mg / kg once a day) until day 21, or 20 mg / kg of compound 156 7 days after EO771 inoculation, followed by daily administration of 10 mg / kg (20 mg / kg D7 + 10 mg / kg once a day) until day 21. Compound 119 (10 mg / kg once a day) was administered daily starting 7 days after EO771 inoculation until day 21, and vehicle-treated mice (100 μL / mouse once a day) served as controls. When the tumor volume was ≥700 mm 3Mice were sacrificed or when a humane endpoint was reached. Mice were then observed for 40 days without treatment. Administration of compound 156 resulted in complete tumor rejection in 8 of the 10 mice, and mice treated with compound 119 showed a reduced tumor growth rate compared to the vehicle control group after the end of treatment. Mice treated with compound 156 and compound 119 showed prolonged survival compared to the vehicle control group after the end of treatment. Compared to the vehicle control group, administration of compound 142 did not affect tumor growth or survival. Between days 7 and 21, 3 mice receiving compound 142, 8 mice receiving compound 156, 4 mice receiving compound 119, and 2 untreated mice survived beyond day 61 and were repeatedly challenged with additional EO771 cell inoculations on day 62. 8 previously untreated age-matched mice without primary tumors were inoculated with EO771 cells and served as controls. Compared to controls, repeat-challenged mice showed no primary or secondary EO771 tumor growth over 20 days and were terminated at day 82 ( FIG. 18 ).

[0949] Figure 18A A and B depict the effects of compounds 142, 156, or 119 on tumor growth rate in vivo. A: Mean tumor volume ± SEM of mice treated orally with compounds 142, 156, or 119 7 to 21 days after EO771 inoculation. Statistically significant differences (p < 0.01) between treatment groups and vehicle control were calculated using two-way ANOVA. B: Survival of mice treated orally with compounds 142, 156, or 119 7 to 21 days after EO771 inoculation.

[0950] 5 Example B-5: Efficacy against GL261-LUC2-iRFP glioma

[0951] Compound 142 showed preclinical efficacy in the murine GL261-LUC2-iRFP glioma model.

[0952] 5.1 Materials used for bioassays

[0953]

[0954]

[0955] 5.2 Procedure / Description

[0956] 5.2.1 Animals and Ethics

[0957] Eight-week-old C57BL / 6J mice were purchased from Charles River Medical University. All animal experiments were performed in accordance with the institutional guidelines of the Institute of Molecular Pathology, Austria, and were approved by the Austrian Ministry of Science according to the European Community Animal Care Rules.

[0958] 5.2.2 GL261-LUC2-iRFP cell culture

[0959] GL261-LUC2-iRFP glioma cells were cultured in DMEM containing 10% FCS, 1% PenStrep, and 1% L-glutamine and grown in a humidified incubator at 37°C and 5% CO2. For tumor inoculation, subconfluent GL261-LUC2-iRFP cells were trypsinized, washed, counted, and resuspended in PBS to a volume of 50 × 10 6 The final concentration of GL261-LUC2-iRFP cells / ml.

[0960] 5.2.3 Efficacy test

[0961] 5.2.3.1 Tumor inoculation and compound treatment

[0962] Twenty C57BL / 6J mice were inoculated in the left hemisphere with 0.1x10 6 GL261-LUC2-iRFP cells were transplanted and the tumor-bearing mice were randomly assigned to two different groups 7 days after transplantation: compound 142 treatment (10 mice, 30 mg / kg D7+10 mg / kg once a day), and vehicle control (10 mice, once a day). Compound 142 was diluted in 10% DMSO, 20% PEG400, 21% HPbCD, 0.35% HPMC and 48.65% H2O and treated mice orally from day 7 to day 28. 100 μL 10% DMSO, 20% PEG400 and 21% HPbCD, 0.35% HPMC and 48.65% H2O served as vehicle control. The 4th, 10th, 14th, 17th, 21st, 24th, 28th, and 31st days after GL261-LUC2-iRFP inoculation were used. Tumor growth was monitored using the Spectrum in vivo imaging system. Mice were sacrificed 31 days after GL261-LUC2-iRFP inoculation or when a humane endpoint was reached.

[0963] Figure 19 Depicted is the treatment regimen of Compound 142 in an in vivo efficacy experiment of orally administered compound. Arrows indicate oral drug treatment.

[0964] 5.3 Results

[0965] C57BL / 6J mice bearing GL261-LUC2-iRFP tumors received 30 mg / kg of compound 142 7 days after GL261-LUC2-iRFP injection, followed by daily administration of 10 mg / kg (30 mg / kg D7 + 10 mg / kg once daily) until day 28, with vehicle-treated mice (100 μl / mouse once daily) serving as controls. Compound 142-treated mice showed reduced tumor volume compared to vehicle controls at day 31 ( Figure 20 ).

[0966] Figure 20 The effect of compound 142 on tumor growth rate in vivo is depicted. Mean tumor volume ± SEM of mice receiving oral treatment 7 to 28 days after GL261-LUC2-iRFP inoculation. Statistically significant differences (p < 0.0001) between treatment groups and vehicle control group were calculated using two-way ANOVA.

[0967] 6 Example B-6: T cell stimulation efficacy against viral antigens in vitro

[0968] Compound 142 was tested for its potential to enhance PBMC or T cell-mediated immunity against a viral peptide cocktail (CEFX) by measuring CD69 expression by flow cytometry.

[0969] 6.1 Materials used for bioassays

[0970] product Distributor Catalog Number LymphoPrep StemCell 07851 CD4 microbeads Miltenyi 130-045-101 CD8 microbeads Miltenyi 130-045-201 CEFX Ultra SuperStim Pool JPT PM-CEFX-2

[0971] 6.2 Procedure / Description

[0972] 6.2.1 Collection of PBMCs from buffy coat

[0973] Leukocyte-rich buffy coats were ordered from the Austrian Red Cross, diluted with PBS to a total volume of 480 ml, 30 ml transferred to a 50 ml falcon, and 11 ml lymphoprep added. The cell suspension was centrifuged at 2200 rpm for 20 min and the layer containing PBMCs was transferred, washed three times with PBS, and counted.

[0974] 6.2.2 Isolation of CD4+ and CD8+ T cells

[0975] Put 10 7 PBMCs were magnetically labeled with CD4 and CD8 microbeads according to the manufacturer's instructions and isolated using an autoMACS device.

[0976] 6.2.3 Serial Dilution of LMW Compounds

[0977] 10 mM LMW compound stocks were further diluted with DMSO and equal amounts were transferred to 96-well plates containing T cells. Final concentrations tested were: 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM and 0.01 μM.

[0978] 6.2.4 Stimulation Protocol for EC50 Test

[0979] For stimulation assays, 80,000 purified CD4+ and CD8+ T cells were seeded into each 96-well plate and incubated with 1 μg / ml anti-CD3, 1 μg / ml anti-CD3 and 1 μg / ml anti-CD28 or 0.5 μg / mL CEFx viral peptides and LMW compounds ranging from 0.01 μM to 30 μM. DMSO-only wells and wells without anti-CD3, anti-CD28 or CEFx served as controls. Stimulated and unstimulated cells were cultured in a moisturizing incubator at 37°C and 5% CO2 for up to 3 days.

[0980] 6.2.5 Determination of EC50 values of LMW compounds by surface FACS

[0981] T cells were stained for surface antigens (anti-CD4 and anti-CD8) and cell activation markers (anti-CD25 and anti-CD69). In addition, live cells were stained using a fixable viability dye. The staining was performed for 15 minutes and cells were analyzed using a Fortessa flow cytometer and FlowJo software. EC was calculated using GraphPad Prism and a variable slope model (agonist versus response - variable slope). 50 .

[0982] 6.3 Results

[0983] 6.3.1 In vitro CD4+ and CD8+ T cell stimulation assays

[0984] Purified CD4+ and CD8+ T cells were incubated with anti-CD3, anti-CD3 / CD28, or CEFx viral peptides and compound 142, and T cell activation was monitored 24 and 72 hours after stimulation. Purified T cells showed dose-dependent increases in the expression of CD25 and CD69 after stimulation with anti-CD3, anti-CD3 / CD28, or CEFx and increasing concentrations of compound 142.

[0985] Compound 142 enhanced virus-specific T cell activation in cells from healthy individuals (EC50 CD4+: 807 nm, EC50 CD8+: 504 nm). Compound 142 had no antigen-specific independent effect on T cell activation.

[0986] Figure 21AA and B depict in vitro CD4+ and CD8+ T cell stimulation, respectively. Purified T cells were stimulated with anti-CD3, anti-CD3 / CD28, or CEFx viral peptides and eight different concentrations of compound 142.

[0987] 7 Example B-7: In vitro phenotyping in human tissue and disease context

[0988] Example B-7 demonstrates the characterization of compound 142 in the Eurofins BioMAP colorectal cancer (CRC) panel of human primary cell-based systems. These systems are designed to mimic complex human tissue and disease biology to evaluate the effects of small molecules in the immunosuppressed tumor microenvironment (TME) and cell-based model systems of human vasculature, skin, lung, and inflammatory tissue. By quantitatively measuring the activity of biomarkers in this extensive panel and comparing the activity of known bioactive agents in the BioMAP reference database, the safety, efficacy, and function of the agents under investigation can be predicted.

[0989] Compound 142 induces biomarker patterns of activated lymphocytes in in vitro cultures that simulate immune responses in human tissues and diseases. Compound 142 triggers inflammatory biomarker signatures in human in vitro cell cultures that simulate immune responses in human tissues and diseases, especially suppressive tumor microenvironments. BioMAP assays were used to measure biomarker expression in cancer cell lines, primary immune, and tissue cell co-cultures treated with compound 142 compared to vehicle controls. Compound 142 was active, with 17 annotated reads, and had no cytotoxicity at the concentrations tested (10 μM and 3.3 μM). Compound 142 affected inflammation-related activities (increased sTNFα, IP-10, MCP-1), matrix remodeling activities (reduced collagen I, collagen III), angiogenesis-related activities (reduced uPA, sVEGF), and immune-related activities (reduced sIL-10, sIL-17A; increased sIFNγ, sIL-2, sIL-6).

[0990] 8 Example B-8: EC using prior art compounds 50 Experimental comparison

[0991] Example B-8 relates to a comparison of the compounds of the present invention with prior art xanthine derivatives. Compared to the compounds of the present invention (i.e., compound 119), prior art xanthine derivatives (Examples 103 and 104 of patent application WO 2000 / 09507 A1) showed no or minimal T cell activation in vitro.

[0992] Compound 119 and two xanthine derivatives from patent application WO 2000 / 09507A1 were tested for their potential to enhance CD3 / 28-stimulated T cell activation. Flow cytometry was used to measure CD69 expression, thereby measuring the activation of stimulated human CD4+ and CD8+ T cells. Compound 119 induced increased T cell activation at concentrations below 1 μM, while xanthine derivatives Examples 103 and 104 from patent application WO 2000 / 09507A1 showed either no or marginal T cell activation at 10 μM.

[0993] 1.1 Materials used for bioassays

[0994] product Distributor Catalog Number LymphoPrep StemCell 07851 CD4 microbeads Miltenyi 130-045-101 CD8 microbeads Miltenyi 130-045-201 Dynabeads Human T-Activator CD3 / 28 <![CDATA[Gibco TM ]]> 11131D

[0995] 1.2 Procedure / Description

[0996] 1.2.1 Collection of PBMCs from buffy coat

[0997] Leukocyte-rich buffy coats were ordered from the Austrian Red Cross, diluted with PBS to a total volume of 480 ml, 30 ml transferred to a 50 ml falcon, and 11 ml lymphoprep added. The cell suspension was centrifuged at 2200 rpm for 20 min and the layer containing PBMCs was transferred, washed three times with PBS, and counted.

[0998] 1.2.2 Isolation of CD4+ and CD8+ T cells

[0999] Put 10 7 PBMCs were magnetically labeled with CD4 and CD8 microbeads according to the manufacturer's instructions and isolated using an autoMACS device. For dose-response experiments using purified CD4+CD8+ T cells, 80,000 cells were seeded into each 96-well flat-bottom cell culture dish.

[1000] 1.2.3 Serial Dilution of LMW Compounds

[1001] 10 mM LMW compound stocks were further diluted with DMSO and equal amounts were transferred to 96-well plates containing T cells. Final concentrations tested were: 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM, 0.003 μM, and 0.001 μM.

[1002] 1.2.4EC 50 Trial stimulation regimen

[1003] For stimulation assays, 80,000 purified CD4+ and CD8+ T cells were seeded into each 96-well plate and incubated with 1 μg / ml anti-CD3, 1 μg / ml anti-CD28, and LMW compounds ranging from 0.001 μM to 30 μM. DMSO-only wells served as controls to determine minimal T cell activation after stimulation, and CD3 / 28-coupled Dynabeads were used to determine maximal T cell activation. Cells were cultured for 16 hours in a humidified incubator at 37°C and 5% CO2.

[1004] 1.2.5 Determination of EC of LMW compounds by surface FACS 50 value

[1005] T cells were stained for surface antigens (anti-CD4 and anti-CD8) and cell activation markers (anti-CD69). In addition, live cells were stained using a fixable viability dye. The staining was performed for 15 minutes and cells were analyzed using a Fortessa flow cytometer and FlowJo software. EC was calculated using GraphPad Prism and a variable slope model (agonist versus response - variable slope). 50 .

[1006] 1.3 Results

[1007] 1.3.1EC 50 Sure

[1008] Stimulated CD4+ and CD8+ T cells from two donors were incubated with increasing concentrations of compound 142, Examples 103 and 104 of patent application WO2000 / 09507A1, and T cell activation was measured 16 hours after stimulation. Compound 142 showed a dose-dependent increase in human T cell activation after anti-CD3 / CD28 stimulation, EC 50 The xanthine derivatives Example 103 and Example 104 of patent application WO2000 / 09507A1 showed limited T cell activation at 10 μM and had no effect on the EC of CD4+ T cells. 50 Greater than 1 μM (donor 1) ( Figure 22A -B, Table B-2).

[1009] Table B-2: EC 50 Values of CD4+ and CD8+ human T cells from donor 1

[1010]

[1011]

[1012] CD4+ and CD8+ T cells from another donor (Donor 2) showed no T cell activation or a dose-dependent decrease in T cell activation following Examples 103 and 104 of patent application WO 2000 / 09507 A1 ( Figure 22C -D, Table B-3).

[1013] Table B-3: EC 50 Values of CD4+ and CD8+ human T cells from donor 2

[1014]

Claims

1. Compounds of formula (I) in A stands for B is -OR 3 、-O-CHR 3 R 3* 、-O-CH2-CH2-R 3 or -O-CH2-CH2-CH2-R 3 ; R 1 represent R 2a and R 2b Each independently represents -H, -F, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CH2OCH3, -CH2CH2OCH3, -CF2-CH3, -CHF-CHF2, -CHF-CF3, -CF2-CF3; wherein R 2a Not -H; R 3 represent R 3* represents -H, -F, -CH3, -C2H5, -C3H7, -CH(CH3)2, -CH2F, -CHF2, -CF3, -CH2-CF3; R 4 and R 4* represents independently of one another -H, -F, -Cl, -Br, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -OCH3, -OC2H5, -OC3H7, -OCH(CH3)2, -OCH2F, -OCHF2, -OCF3, -CH2-CF3, -CHF-CH2F, -CHF-CHF2, -CHF-CF3, -CF2-CH3, -CF2-CH2F, -CF2-CHF2, -CF2-CF3, R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 and R 13 represents independently of each other -H, -cyclo-C3H5, -cyclo-C4H7, -cyclo-C5H9, -cyclo-C6H 11 、-cyclo-C7H 13 , -cycloC3H5O, -OH, -OCH3, -OC2H5, -OC3H7, -O-cyclo-C3H5, -OCH(CH3)2, -OC(CH3)3, -OC4H9, -OPh, -OCH2-Ph, -OCPh3, -CH2-OCH3, -C2H4-OCH3, -C3H6-OCH3, -CH2-OC2H5, -C2H4-OC2H5, -C3H6-OC2H5, -CH2-OC3H7, -C2H4-OC3H7, -C3H6-OC3H7, -CH2-O-cyclo-C3H5, -C2H4-O-cyclo-C3H5, -C3H6-O-cyclo-C3H5, -CH2-OCH(CH 3)2, -C2H4-OCH(CH3)2, -C3H6-OCH(CH3)2, -CH2-OC(CH3)3, -C2H4-OC(CH3)3, -C3H6-OC(CH3)3, -CH2-OC4H9, -C2H4-OC4H9, -C3H6-OC4H9, -CH2-OPh, - C2H4-OPh, -C3H6-OPh, -CH2-OCH2-Ph, -C2H4-OCH2-Ph, -C3H6-OCH2-Ph, -SH, -SCH3, -SC2H5, -SC3H7, -S-cyclo-C3H5, -SCH(CH3)2, -SC(CH3)3, -F, -Cl, -Br , -I, -CN, -COCH3, -COC2H5, -COC3H7, -CO-cyclo-C3H5, -COCH(CH3)2, -COC(CH3)3, -COOH, -COOCH3, -COOC2H5, -COOC3H7, -COO-cyclo-C3H5, -COOCH(CH3)2, -COOC(CH3)3, -OOC-CH3, -OOC-C2H5, -OOC-C3H7, -OOC-cyclo-C3H5, -OOC-CH(CH3)2, -OOC-C(CH3)3, -CONH2, -CONHCH3, -CONHC2H5, -CONHC3H7, -CONH-cyclo-C3H 5. -CONH[CH(CH3)2], -CONH[C(CH3)3], -CON(CH3)2, -CON(C2H5)2, -CON(C3H7)2, -CON(ring-C3H5)2, -CON[CH(CH3)2]2, -CON[C(CH3)3]2, -NHCOCH3, -NH COC2H5, -NHCOC3H7, -NHCO-ring-C3H5, -NHCO-CH(CH3)2, -NHCO-C(CH3)3, -NHCO-OCH3, -NHCO-OC2H5, -NHCO-OC3H7, -NHCO-O-ring-C3H5, -NHCO-OCH(CH3)2,-NHCO-OC(CH3)3, -NH2, -NHCH3, -NHC2H5, -NHC3H7, -NH-cyclo-C3H5, -NHCH(CH3)2, -NHC(CH3)3, -N(CH3)2, -N(C2H5)2, -N(C3H7)2, -N(cyclo-C3H5)2, -N[CH(CH3)2]2, -N[C(CH3)3]2, -SOCH3, -SOC2H5, -SOC3H7, -SO-cyclo-C3H5, -SOCH(CH3)2, -SOC(CH3)3, -SO2CH3, -SO2C2H5, -SO2C3H7, -SO2-cyclo-C3H5, -SO2CH(CH3)2, -SO2C(CH3)3, -SO3H, -SO3CH3, -SO3C2H5, -SO3C3H7, -SO3-cyclo-C3H5, -SO3CH(CH3)2, -SO3C(CH3)3, -SO2NH2, -SO2NHCH3, -SO2NHC2H5, -SO2NHC3H7, -SO2NH-cyclo-C3H5, -SO2NHCH(CH3)2, -SO2NHC(CH3)3, -SO2N(CH3)2, -SO2N(C2H5)2, -SO2N(C3H7)2, -SO2N(cyclo-C3H5)2, -SO2N[CH(CH3)2]2, -SO2N[C(CH3)3]2, -O-S(=O)CH3, -O-S(=O)C2H5, -O-S(=O)C3H7, -O-S(=O)-cyclo-C3H5, -O-S(=O)CH(CH3)2, -O-S(=O)C(CH3)3, -S(=O)(=NH)CH3, -S(=O)(=NH)C2H5, -S(=O)(=NH)C3H7, -S(=O)(=NH)-cyclo-C3H5, -S(=O)(=NH)CH(CH3)2, -S(=O)(=NH)C(CH3)3, -NH-SO2-CH3, -NH-SO2-C2H5, -NH-SO2-C3H7, -NH-SO2-cyclo-C3H5, -NH-SO2-CH(CH3)2, -NH-SO2-C(CH3)3, -O-SO2-CH3, -O-SO2-C2H5, -O-SO2-C3H7, -O-SO2-cyclo-C3H5, -O-SO2-CH(CH3)2, -O-SO2-C(CH3)3, -OCH2F, -OCHF2, -OCF3, -CH2-OCF3, -C2H4-OCF3, -C3H6-OCF3, -CH2-OCHF2, -C2H4-OCHF2, -C3H6-OCHF2, -OC2F5, -CH2-OC2F5, -C2H4-OC2F5, -C3H6-OC2F5, -O-COOCH3-O-COOC2H5, -O-COOC3H7, -O-COO-cyclo-C3H5, -O-COOCH(CH3)2, -O-COOC(CH3)3, -NH-CO-NH2, -NH-CO-NHCH3, -NH-CO-NHC2H5, -NH-CO-NHC3H7, -NH-C(=NH)-NH2, -NH-CO-N(C3H7)2, -NH-CO-NH[CH(CH3)2], -NH-CO-NH[C(CH3)3], -NH-CO-N(CH3)2, -NH-CO-N(C2H5)2, -NH-CO-NH-cyclo-C3H5, -NH-CO-N(cyclo-C3H5)2, -NH-CO-N[CH(CH3)2]2, -NH-C(=NH)-NHCH3, -NH-C(=NH)-NHC2H5, -NH-C(=NH)-NHC3H7, -O-CO-NH-cyclo-C3H5, -NH-C(=NH)-NH-cyclo-C3H5, -NH-C(=NH)-NH[CH(CH3)2], -O-CO-NH[CH(CH3)2], -NH-C(=NH)-NH[C(CH3)3] -NH-C(=NH)-N(CH3)2, -NH-C(=NH)-N(C2H5)2, -NH-C(=NH)-N(C3H7)2, -NH-C(=NH)-N(cyclo-C3H5)2, -O-CO-NHC3H7, -NH-C(=NH)-N[CH(CH3)2]2, -NH-C (=NH)-N[C(CH3)3]2, -O-CO-NH2, -O-CO-NHCH3, -O-CO-NHC2H5, -O-CO-NH[C(CH3)3], -O-CO-N(CH3)2, -O-CO-N(C2H5)2, -O-CO-N(C3H7)2, -O-CO-N(cyclic -C3H5)2、-O-CO-N[CH(CH3)2]2、-O-CO-N[C(CH3)3]2、-O-CO-OCH3、-O-CO-OC2H5、-O-CO-OC3H7、-O-CO-O-C3H5、-O-CO-OCH(CH3)2、-O-CO-OC(CH3)3、-CH2F、-CHF2、-CF3、-CH2-CH2F、-CH2-CHF2、-CH2-CF3、-C8H 15 、-Ph、-CH2-Ph、-CH2-CH2-Ph、-CH=CH-Ph、-CPh3、-CH3、-C2H5、-C3H7 、-CH(CH3)2、-C4H9、-CH2-CH(CH3)2、-CH(CH3)-C2H5、-C(CH3)3、-C5H 11 、-CH(CH3)-C3H7、-CH2-CH(CH3)-C2H5、-CH(CH3)-CH(CH3)2、-C(CH3)2-C2H5、-CH2-C(CH3)3、-CH(C2H5)2、-C2H4-CH(CH3)2、-C6H 13 、-C7H 15 、-C8H 17 、-C3H6-CH(CH3)2、-C2H4-CH(CH3)-C2H5、-CH(CH3)-C4H9、-CH2-CH(CH3)-C3H7、-CH(CH3)-CH2-CH(CH3)2、-CH(CH3)-CH(CH3)-C2H5、-CH2-CH(CH3)-CH(CH3)2、-CH2-C(CH3)2-C2H5、-C(CH3)2-C3H7、-C(CH3)2-CH(CH3)2、-C2H4-C(CH3)3、-CH(CH3)-C(CH3)3、-CH=CH2、-CH2-CH=CH2、-C(CH3)=CH2、-CH=CH-CH3、-C2H4-CH=CH2、-CH2-CH=CH-CH3、-CH=CH-C2H5、-CH2-C(CH3)=CH2、-CH(CH3)-CH=CH、-CH=C(CH3)2、-C(CH3)=CH-CH3、-CH=CH-CH=CH2、-C3H6-CH=CH2、-C2H4-CH=CH-CH3、-CH2-CH=CH-C2H5、-CH=CH-C3H7、-CH=CH-CH=CH-CH3、-C2H4-C(CH3)=CH2、-CH2-CH(CH3)-CH=CH2、-CH(CH3)-CH2-CH=CH2、-CH2-CH=C(CH3)2、-CH2-C(CH3)=CH-CH3、-CH(CH3)-CH=CH-CH3、-CH=CH-CH(CH3)2、-CH=C(CH3)-C2H5、-C(CH3)=CH-C2H5、-C(CH3)=C(CH3)2、-C(CH3)2-CH=CH2、-CH(CH3)-C(CH3)=CH2、-C4H8-CH=CH2、-C3H6-CH=CH-CH3、-C2H4-CH=CH-C2H5、-CH2-CH=CH-C3H7、-CH=CH-C4H9、-C3H6-C(CH3)=CH2、-C2H4-CH(CH3)-CH=CH2、 -CH2-CH(CH3)-CH2-CH=CH2、-C2H4-CH=C(CH3)2、-CH(CH3)-C2H4-CH=CH2、-C2H4-C(CH3)=CH-CH3、-CH2-CH(CH3)-CH=CH-CH3、-CH(CH3)-CH2-CH=CH-CH3、-CH2-CH=CH-CH(CH3)2、-CH2-CH=C(CH3)-C2H5、-CH2-C(CH3)=CH-C2H5、-CH(CH3)-CH=CH-C2H5、-CH=CH-CH2-CH(CH3)2、-CH=CH-CH(CH3)-C2H5、-CH=C(CH3)-C3H7、-C(CH3)=CH-C3H7、-CH2-CH(CH3)-C(CH3)=CH2、-C[C(CH3)3]=CH2、-CH(CH3)-CH2-C(CH3)=CH2、-CH(CH3)-CH(CH3)-CH=CH2、-CH=CH-C2H4-CH=CH2、-C(CH3)2-CH2-CH=CH2、-CH2-C(CH3)=C(CH3)2、-CH(CH3)-CH=C(CH3)2、-C(CH3)2-CH=CH-CH3、-CH=CH-CH2-CH=CH-CH3、-CH(CH3)-C(CH3)=CH-CH3、-CH=C(CH3)-CH(CH3)2、-C(CH3)=CH-CH(CH3)2、-C(CH3)=C(CH3)-C2H5、-CH=CH-C(CH3)3、-C(CH3)2-C(CH3)=CH2、-CH(C2H5)-C(CH3)=CH2、-C(CH3)(C2H5)-CH=CH2、-CH(CH3)-C(C2H5)=CH2、-CH2-C(C3H7)=CH2、-CH2-C(C2H5)=CH-CH3、-CH(C2H5)-CH=CH-CH3、-C(C4H9)=CH2、-C(C3H7)=CH-CH3、-C(C2H5)=CH-C2H5、-C(C2H5)=C(CH3)2、-C[CH(CH3)(C2H5)]=CH2、-C[CH2-CH(CH3)2]=CH2、-C2H4-CH=CH-CH=CH2、-CH2-CH=CH-CH2-CH=CH2、-C3H6-C≡C-CH3、-CH2-CH=CH-CH=CH-CH3、-CH=CH-CH=CH-C2H5、-CH(CH3)-CH2-C≡CH、-CH(CH3)-C≡C-CH3、-C2H4-CH(CH3)-C≡CH、-CH=CH-CH=C(CH3)2、-CH2-CH(CH3)-CH2-C≡CH, -CH=CH-C(CH3)=CH-CH3, -CH=C(CH3)-CH=CH-CH3, -CH2-CH(CH3)-C≡CH, -C(CH3)=CH-CH=CH-CH3, -C≡CH, -C≡C-CH3, -CH2-C≡CH, -C2H4-C≡CH, -CH2-C≡C-CH3, -C≡C-C2H5, -C3H6-C≡CH, -C2H4-C≡C-CH3, -CH2-C≡C-C2H5, -C≡C-C3H7, -CH(CH3)-C≡CH, -C4H8-C≡CH, -C2H4-C≡C-C2H5, -CH2-C≡C-C3H7, -C≡C-C4H9, -C≡C-CH2-CH(CH3)2, -CH(CH3)-C2H4-C≡CH, -CH2-CH(CH3)-C≡C-CH3, -C(CH3)(C2H5)-C≡CH, -CH(CH3)-CH2-C≡C-CH3, -CH(CH3)-C≡C-C2H5, -CH2-C≡C-CH(CH3)2, -C≡C-CH(CH3)-C2H5, -CH2-C≡C-C≡C-CH3, -CH(C2H5)-C≡C-CH3, -C(CH3)2-C≡C-CH3, -CH(C2H5)-CH2-C≡CH, -CH2-CH(C2H5)-C≡CH, -C(CH3)2-CH2-C≡CH, -CH2-C(CH3)2-C≡CH, -CH(CH3)-CH(CH3)-C≡CH, -CH(C3H7)-C≡CH, -CH2-CH(C≡CH)2, -C≡C-C≡CH, -CH2-C≡C-C≡CH, -C≡C-C≡C-CH3, -CH(C≡CH)2, -C2H4-C≡C-C≡CH, -CH2-C≡C-CH2-C≡CH, -C≡C-C2H4-C≡CH, -C≡C-C(CH3)3, -C≡C-CH2-C≡C-CH3, -C≡C-C≡C-C2H5 or R 5 and R 6 or R 6 and R 7 Together with the two carbon atoms of the phenyl ring to which they are attached, they can form a 4- to 8-membered ring system, which is optionally substituted by one or more substituents selected from R 10 、R 11 、R 12 and R 13 ; or an enantiomer, a diastereomer, a tautomer, a mixture of enantiomers, a mixture of diastereomers, a mixture of tautomers, a hydrate, a solvate, or a pharmaceutically acceptable salt of the above compounds.

2. The compound according to claim 1, wherein R 3 represent And R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 and R 13 Has the same meaning as defined in claim 1.

3. The compound according to claim 1 or 2, wherein R 2a represents -CH3, -C2H5, -CH2F, -CHF2, -CF3, -CH2-CF3, -CHF-CH2F, -CH2OH, -CH2CH2OH, -CH2OCH3; and R 2b Represents -H or -CH3; B stands for -OR 3 or -O-CHR 3 R 3* ; R 3 represent R 3* represents -H, -F, -CH3, -C2H5, -C3H7, -CH(CH3)2, -CH2F, -CHF2, -CF3, -CH2-CF3; R 4 and R 4* represents independently of one another -H, -F, -Cl, -Br, -CH3, -C2H5, -CH2F, -CHF2, -CF3, -OCH3, -OC2H5, -OC3H7, -OCH(CH3)2, -OCH2F, -OCHF2, -OCF3, -CH2-CF3, -CHF-CH2F, -CHF-CHF2, -CHF-CF3, -CF2-CH3, -CF2-CH2F, -CF2-CHF2, -CF2-CF3, And R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 and R 13 Has the same meaning as defined in claim 1.

4. The compound according to any one of claims 1 to 2, wherein B represents -O-CHR 3 R 3* 、-O-CH2-CH2-R 3 or -O-CH2-CH2-CH2-R 3 ;and R 3 and R 3* Has the same meaning as defined in claim 1.

5. The compound according to any one of claims 1 to 3, wherein B represents -OR 3 ; and R 3 Has the same meaning as defined in claim 1.

6. The compound according to any one of claims 1 to 5, wherein the compound has any one of the following formulae (II-1) to (II-5), (III-1) to (III-6), (IV-1) to (IV-8), (V-1) to (V-5), (VI-1) to (VI-2), and (VII-1) to (VII-3): where R 1 、R 3* 、R 4 、R 4* 、R 5 、R 6 、R 7 、R 8 、R 10 、R 11 、R 12 and R 13 has the same meaning as defined in any one of claims 1-5.

7. The compound according to any one of claims 1 to 6, wherein in A stands for B stands for -OR 3 、-O-CH2-R 3 or -O-CHR 3 R 3* ; R 1 represent R 3 represent R 3* represents -H, -F, -CH3, -C2H5, -C3H7, -CH(CH3)2, -CH2F, -CHF2, -CF3, -CH2-CF3; R 4 and R 4* represents independently of one another -H, -F, -Cl, -CH3, -CH2F, -CHF2, -CF3, -OCH3, -OC2H5, -OC3H7, -OCH(CH3)2, -OCH2F, -OCHF2, -OCF3, -CH2-CF3, -CHF-CH2F, -CHF-CHF2, -CHF-CF3, -CF2-CH3, -CF2-CH2F, -CF2-CHF2, -CF2-CF3, and R 5 、R 6 、R 7 and R 8 independently of one another represent -H, -F, -Cl, -CN, -CH3, -CHF2, -CF3, -OCH3, -OCHF2, -OCF3 or -SO2CH3.

8. The compound according to any one of claims 1 to 7, wherein R 3 represent 9. The compound according to claim 1, wherein the compound is selected from: or an enantiomer, a diastereomer, a tautomer, a mixture of enantiomers, a mixture of diastereomers, a mixture of tautomers, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof.

10. A pharmaceutical composition comprising as an active ingredient at least one compound according to any one of claims 1 to 9 and at least one pharmaceutically acceptable carrier, excipient and / or diluent. The pharmaceutical composition according to claim 10 , further comprising at least one stimulator for activating immune cells.

12. A compound according to any one of claims 1 to 9, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 9 or 10 for use as a medicament.

13. The compound according to any one of claims 1 to 9, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 9 or 10 for use in preventing or treating neoplastic and / or infectious diseases.

14. The compound, pharmaceutically acceptable salt thereof or pharmaceutical composition for use according to claim 13, wherein the compound, pharmaceutically acceptable salt thereof or pharmaceutical composition is administered in combination with one or more other stimulators for activating immune cells.

15. A method for producing activated immune cells in vitro or ex vivo, the method comprising the following steps: (i) providing immune cells; (ii) contacting the cells of step (i) with: (a) at least one compound as defined in any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, and optionally (b) one or more other stimulatory agents that activate the immune cells; and (iii) culturing the cells of step (ii) under conditions suitable to maintain the viability of said cells.

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