DESC:Field of the invention:
This invention relates to small molecules of Formula (I) which are biphenyl pyridine rigid compounds with polycyclic heterocycles therapeutically useful as immune modulators. These compounds disrupt the PD-1/PD-L1 checkpoint pathway, and preferably also one or more related pathways. The one more related checkpoint pathway could be, for instance, the VISTA pathway or any other pathway. These compounds are useful in treating, preventing, or ameliorating diseases such as cancer or infections.

(I)
Background of the Invention:
Immune checkpoints (ICs) have pivotal roles in regulating immune responses. The inhibitory ICs in the tumor microenvironment (TME) have been implicated in the immune evasion of tumoral cells. Therefore, identifying and targeting these inhibitory ICs might be critical for eliminating tumoral cells. ICs, which can be expressed on immune cells and tumour cells, can regulate immune responses. These molecules are essential in maintaining the body’s homeostasis; however, the expression of inhibitory ICs, e.g., programmed cell death ligand 1 (PD-L1), can shield tumoral cells from anti-tumoral immune responses. Thus, suppressing these inhibitory ICs has gained special attention in treating various cancers.
PD-L1 is a 40 kDa immune checkpoint protein encoded in humans by the CD274 gene. Upon binding to its receptor PD-1, which is expressed on activated B cells, T cells, and myeloid cells, PD-L1 initiates signalling pathways that lead to downregulation of T cell proliferation and activation, facilitating tumour cell escape from T cell- mediated immune surveillance, thereby contributing to cancer severity and progression. PD-L1 expression has been shown on a wide variety of solid tumours (e.g., breast, lung, colon, ovarian, melanoma, bladder, liver, salivary, stomach, gliomas, thyroid, thymic epithelial, head, and neck (Brown J A et al., 2003. J. Immunol. 170:1257-66; Dong H et al. 2002. Nat. Med. 8:793-800; Hamanishi J, et al. 2007. Proc. Natl. Acad. Sci. USA 104:3360-65; Strome S E et al. 2003. Cancer Res. 63:6501-5; Inman B A et al. 2007. Cancer 109:1499-505; Konishi J et al. 2004. din. Cancer Res. 10:5094-100; Nakanishi J et al._2007. Cancer lmmunol. Immunother_56:1173-82; which are incorporated herein by reference), and the protein has arisen as an attractive target for the development of anti-cancer therapeutics. PD-L1 expression is further involved in the evasion of immune responses involved in infectious diseases (e.g., chronic viral infections including HBV and HIV).
As such, PD-L1 also serves as a therapeutic target for the treatment of a variety of infectious diseases. Therapeutic efficacy of PD-L1 antagonists (and of PD-1 antagonists) has been validated in clinical trials. Immune checkpoint inhibitors, such as monoclonal antibodies targeting programmed death 1 (PD-1) and programmed death ligand-1 (PD-L1), have achieved enormous success in the treatment of several cancers. However, monoclonal antibodies are expensive to produce, have poor tumour penetration, and may induce autoimmune side effects, all of which limit their application. Still, there is a need for more potent, better and/or selective immune modulators of PD-1 pathway.
To date, six FDA-approved therapeutic antibodies targeting PD-1/PD-L1, including Nivolumab, Pembrolizumab, Avelumab, Atezolizumab, Cemiplimab and Durvalumab, have achieved significant clinical results in the indications and long-lasting relief.
Despite the unprecedented success of monoclonal antibodies, their inherent disadvantages include high manufacturing costs, low tissue penetration, insufficient oral availability, and immunogenicity. In addition to lower manufacturing costs, higher stability, and better tissue and tumour penetration, small-molecule inhibitors can provide better therapeutic indices compared to monoclonal drugs, not only based on optimal drug efficacy. The parameters are more flexible for clinical and oral administration and a reasonable half-life can be maintained to avoid systemic immunogenicity. Therefore, small molecule inhibitors can be administered alone or in combination with therapeutic antibodies to provide promising alternative therapeutic strategies to address drug resistance and low clinical responses.
So far, there are no FDA-approved small-molecule inhibitors of PD-L1, although several patents and publications have disclosed a series of small-molecule inhibitors targeting the PD-1/PD-L1 pathway. To meet the demands of the domestic market, more alternative compounds with novel frameworks are urgently needed for future clinical applications.
VISTA (V-domain immunoglobulin suppressor of T cell activation) is a well-established immune regulatory receptor. VISTA, also referred to as PD-1 homolog (PD-1H), differentiation of embryonic stem cells 1 (Dies1), DD1a, Gi24, SISP1, B7-H5, and C10orf54, is a novel inhibitory IC. VISTA can be substantially overexpressed in the tumour-infiltrating immune cells of various cancers, e.g., melanoma, gastric cancer, prostate cancer, colorectal cancer, and acute myeloid leukaemia. Besides, recent findings have shown that VISTA can also be expressed on tumoral cells, e.g., hepatocellular, endometrial cancer, ovarian cancer, gastric cancer, and non-small cell lung cancer. (Kakavand H, Jackett LA, Menzies AM, Gide TN, Carlino MS, Saw RPM, et al. Modern Pathol an Off J U States Can Acad Pathol Inc (2017) 30(12):1666–76 ; Kuklinski LF, Yan S, Li Z, Fisher JL, Cheng C, Noelle RJ, et al. Cancer Immunol Immunother CII (2018) 67(7):1113–21; Gao J, Ward JF, Pettaway CA, Shi LZ, Subudhi SK, Vence LM, et al. Nat Med (2017) 23(5):551–5; Xie S, Huang J, Qiao Q, Zang W, Hong S, Tan H, et al. Cancer Immunol Immunother CII (2018) 67(11):1685–94; Wang L, Jia B, Claxton DF, Ehmann WC, Rybka WB, Mineishi S, et al.. Oncoimmunology (2018) 7(9):e1469594; Zhang M, Pang H-J, Zhao W, Li Y-F, Yan L-X, Dong Z-Y, et al.. BMC Cancer (2018) 18(1):511; Liao H, Zhu H, Liu S, Wang H. Oncol Lett (2018) 16(3):3465–72; Böger C, Behrens H-M, Krüger S, Röcken C. Oncoimmunology (2017) 6(4): e1293215. 51. Villarroel-Espindola F, Yu X, Datar I, Mani N, Sanmamed M, Velcheti V, et al. Res an Off J Am Assoc Cancer Res (2018) 24(7):1562–73, all of which are incorporated by reference.)
As a novel inhibitory IC, VISTA can be overexpressed on myeloid lineages, lymphoid lineages, tumour-infiltrating immune cells, and tumour cells; thus, it can substantially inhibit anti-tumoral immune responses. Since VISTA expression on tumour cells can lead to tumour development and its signalling pathway is different from the signalling pathways of other inhibitory ICs such as PD-L1, dual blockade of tumoral VISTA/PD-L1 can lead to a synergic inhibitory effect on tumour development. The combined blockade of inhibitory ICs, e.g., VISTA/PD-1 blockade, can increase the response rates of affected patients to cancer therapies.
So far, there are no FDA-approved small-molecule inhibitors targeting the PD-1/PD-L1 and VISTA pathway. CA-170 is the only known agent in the clinical phase that can directly target PD-L1 and VISTA.
In conclusion, it is crucial to develop novel and dual target PD-1/PD-L1 and VISTA inhibitors for clinical immunotherapy such as cancer drugs.

Summary of invention:
The present invention provides biphenyl pyridine rigid compounds with a terminal polycyclic heterocycle, such as a seven-membered or eight-membered bicyclic spiro heterocycle. These compounds can inhibit the programmed cell death Ligand 1 (PD-1/PD-L1) signalling pathway and / or the VISTA pathway. These small molecule antagonists of PD-1/PD-L1/VISTA interactions show potent anti-tumour activity in vitro and in vivo. The compounds have been shown to have efficacy comparable to that of monoclonal antibodies. Without wishing to be bound by theory, it is speculated that the compounds may act, at least in part, by relieving PD-1/PD-L1 -induced T cell exhaustion.

The present invention provides a compound represented by Formula I:

(I)
wherein:
R1, R2 and R4 are each independently selected from hydrogen, halogen, hydroxy, cyano, C1-6 alkyl, C1-6 alkoxy, cycloalkyl containing up to 6 carbon atoms, alkenyl, alkynyl, and -NR’R5;
R3 is selected from hydrogen, halogen, hydroxy, cyano, C1-6 alkyl, C3-6 cycloalkyl, alkenyl, alkynyl, NR’R5, and -OR6;
R’ is selected from H and C1-6 alkyl;
R5 is selected from hydrogen, C1-6 alkyl, cycloalkyl containing up to 6 carbon atoms, alkenyl, and alkynyl;
R6 is selected from C1-6 alkyl and -L2-A;
L2 is absent or is C1-6 alkylene;
A is selected from aryl and 5- to 14-membered heteroaryl, which may be optionally substituted with one or two substituents each independently selected from cyano, halogen, hydroxy, and amino; and
L1 is optionally substituted polycyclic heterocyclyl;
or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof.

Compounds of formula (I) include stereoisomers such as geometric isomers of compounds of formula (I) as drawn above.

The invention also provides a method of treating, preventing or ameliorating disease, the method comprising administering a compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined herein, or a pharmaceutical composition, to a subject. Similarly, the invention provides a compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined herein, or a pharmaceutical composition, for use in a method of treating, preventing or ameliorating disease as defined herein. Further, the invention also provides the use of a compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined herein, or a pharmaceutical composition, in the manufacture of a medicament for use in a method of treating, preventing or ameliorating disease as defined herein.

The invention also provides an in vitro method, such as an assay method, which comprises using a compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined herein to:
(i) inhibit the PD-1/PD-L1 checkpoint pathway;
(ii) inhibit the activity of VISTA;
(iii) reverse T-cell exhaustion;
(iv) increase T-cell function;
(v) increase CD8+ T-cell population;
(vi) decrease expression of PD-1;
(vii) decrease expression of TIM3;
(viii) increase expression of CD107a;
(ix) increase expression of Granzyme-B; and/or
(x) IFNg restoration
(xi) Increasing T cell mediated cytotoxicity to tumour cells

Brief Description of the figures
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced in the drawing.
Fig 1(a) Binding interactions and pose of NAT-24 in PD-L1 dimer (PDB 5N2F) interface. Fig 1(b) Binding interactions and pose of NAT-25 in VISTA ECD (PDB 6MVL).
Fig 2 is a flow chart illustrating the virtual screening for putative PD-1/PD-L1 inhibitors and species capable of binding to VISTA, described in the computational example below. It is a schematic presentation of the various stages of the Structure-based virtual screening and workflow employed for the identification of PD-1/PD-L1 and VISTA small-molecule inhibitors, using Schrödinger software.
Fig 3 (a) Microscale thermophoresis (MST) dose-response curve of four compounds described herein, binding to His-tagged human PD-L1. (b) Melting curves and shifts of melting temperature (Tm) of hPD-L1 in the presence of those four compounds.
Fig 4(a) IC50 values of Cpd-1, Cpd-2, Cpd-3 and Cpd-4 in HTRF assay represented as bar graphs. (b) IC50 values of Cpd-1, Cpd-2, Cpd-3 and Cpd-4 in HTRF-dimerization assay represented as bar graphs.
Fig 5(a) EC50 values of Cpd-1, Cpd-2, Cpd-3 and Cpd-4 in NFAT reported bioassay represented as bar graphs. (b) IC50 values of Cpd-1, Cpd-2, Cpd-3 and Cpd-4 in VISTA/VSIG3 assay represented as bar graphs.
Fig. 6 includes results of the experiments demonstrated in Example 54, using compounds 1-4 (referred to as Cp 1, Cp 2, Cp 3 and Cp 4 respectively). Figure 6(a)-(d) refer to a CHO-PDL1 cell line with no treatment and treatment with the compounds, followed by cell surface PD-L1 detected by flow cytometry at 72 hours (fig. 6(a)); 48 hours (fig. 6(b)); 17 hours (fig. 6(c)) and 2 hours (fig. 6(d)). Figure 6(e) refers to a MDAMB231 cell line with no treatment and treatment with compounds 1 and 3 followed by cell surface PD-L1 detected by flow cytometry at 72 hours. Figure 6(f) refers to a U251 cell line with no treatment and treatment with compounds 1 and 3 followed by cell surface PD-L1 detected by flow cytometry at 17 hours. Figure 6(g) represents a HCC827 cell line with no treatment and treatment with compounds 1 and 3, followed by cell surface PD-L1 detected by flow cytometry at 72 hours.
Fig.7 shows the results of an experiment described in Example 55 below, wherein apoptosis of cells was measured in an Annexin V assay after coculturing CD8 T cells with a variety of cell lines and compounds 1 and 3 of the invention. Figure 7(a) shows the results using MDAMB231 cells. Figure 7(b) shows the results using U251 cells. Figure 7(c) shows the results using HCC827 cells.
Fig.8 shows the results of an experiment described in Example 55 below, wherein the effect of compounds 1, 2 and 3 according to the invention on exhausted T cells was measured. Figure 8(a) shows the level of CD8+, and exhaustion markers PD1 and TIM3, in exhausted T cells. Figures 8(b) to (d) show the level of those markers after treatment with compounds 1, 2 and 3 respectively. The CD8 cell count increases, while exhaustion markers PD1 and TIM3 decrease. Figure 8(e) shows the level of exhaustion marker PD1, and of IFNG, in a co-culture of exhausted T cells with MDAMB231. Figures 8(f) and 8(g) shows the level of those markers in such cultures when treated with compound 1 and compound 3 of the invention. The expression of exhaustion marker PD1 is decreased while expression of IFNG is restored (increases).

Detailed Description of the invention:

Development of the invention

As discussed above, small-molecule inhibitors may represent desirable alternatives to monoclonal antibody (mAb) drugs in therapies which target immune checkpoint receptors. Small molecules can in theory offer advantages over mAb drugs. Small molecules can provide increased oral bioavailability, bio-efficiency, and short half-life activity, which is particularly important for autoimmune or adverse immune events. Small molecules can also offer a greater diffusion rate within the tumor microenvironment (TME), and the possibility of avoiding the macrophage-mediated resistance observed in anti-PD-1 therapy.
Small molecules are technically difficult to identify and assess. Together with a challenging design, the limited structural elucidation of the targets has been a challenge in the development of PD-1/PD-L1 small-molecule inhibitors. Before 2015, no human PD-1/PD-L1 X-ray structure was resolved, and the murine form does not allow the assessment of the extent of plasticity or interactions established with PD-L1. In recent years, several human PD-1 and PD-L1 X-ray structures have been resolved and expose the structural differences in murine and human forms within the binding modes between proteins, as well as the plasticity in the complex formation.
The design of inhibitors directly targeting the PD-1/PD-L1 interaction interface has been limited by the large, hydrophobic, and flat interface between proteins without deep binding pockets. Recently, different X-ray structures of PD-L1 with a class of small-molecule inhibitors have been resolved. Bristol Myers Squibb (BMS) compounds were the first non-peptide-based compounds able to inhibit PD-1/PD-L1 interaction, however, they are reported to have poor drug-like properties. In general, these inhibitors bind to PD-L1 leading to a deep cylindrical, hydrophobic pocket created by the interface of two monomers [Zak, K. M. et al. 2016, supra].
The present inventors have sought to develop new inhibitors capable of disrupting PD-1/PD-L1 binding. The inventors have employed in silico analysis described in the computational example below and identified a novel skeleton structure capable of targeting the PD-L1 dimer interface. The novel skeleton structure is also capable of targeting VISTA.
As described in the Examples section that follows, the inventors then followed a trans-disciplinary approach to discover novel small molecules that can modulate PD-1/PD-L1 interaction. The in silico analyses were combined with in vitro, ex vivo and experimental studies to assess the ability of novel compounds to modulate PD-1/PD-L1 interaction and enhance T-cell function.
In particular, the inventors recognised that the types of assays previously developed to validate the effect of PD-1/PD-L1 small-molecule inhibitors are highly limited. These are biochemical assays employed for hit validation and/or engineered cell-based assays. The present inventors decided to evaluate compounds activity using innovative cell-based assays. Initially, studies were focused on PD-1/PD-L1 inhibition, and for that, different cell lines were selected. Two types of cancer cells, breast cancer, and brain cell lines were thus selected to perform the in vitro studies looking at the impact of the hit compounds on PD-L1/PD-1 interaction and internalization experiments based on flow cytometry were established with various concentration of the compounds at various time-intervals.
The inventors also employed in vitro cancer models using different cancer cells and peripheral blood mononuclear cells (PBMC) / T cells isolated from healthy volunteers.
These studies led to the identification of novel small molecules able to promote T-cell activation by targeting the PD-1/PD-L1 co-inhibitory interactions. These novel small molecules were shown to have efficacy comparable to that of approved monoclonal antibodies.
These newly identified small molecules were shown by experiment to have a variety of useful effects, suggesting significant medical potential (for instance in cancer immunotherapy). These effects include:
• reversal of T cell exhaustion
• restoration of IFNg, CD107a and Granzyme-B upregulation
• Increasing level of CD8+ T cells, for instance in co-culture with cancer cells
• High apoptotic activity, for instance in a coculture of CD8+ Tcells with MDAMB231/U251/HCC827 cells.
• Inhibition of binding of PD-1 to PD-L1, for instance in MDAMB231, HCC827 and U251 cell lines thus enhancing the T-cell activation
• Inhibition of VISTA
• Downregulating expression of exhaustion marker PD-1 on T cells
• Decreasing expression of exhaustion marker TIM3
• Increasing T cell mediated cytotoxicity to tumour cells

These effects were in some cases observed to an even greater extent than was demonstrated by an approved monoclonal antibody.

Definitions

As used herein, “C1-6 alkyl” refers to a straight-chain or branched-chain saturated hydrocarbon group, wherein “C1-6” means that the moiety has 1-6 carbon atoms, that is, the group contains 1, 2, 3, 4, 5 or 6 carbon atoms. Typically a C1-6 alkyl group or moiety is a C1-3 alkyl group or moiety. A C1-3 alkyl group or moiety is a linear or branched alkyl group or moiety containing from 1 to 3 carbon atoms. Examples of C1-6 alkyl groups and moieties include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl and 3-methyl-butyl. Examples of C1-3 alkyl groups and moieties include methyl, ethyl, n-propyl, and i-propyl. Methyl is preferred. For the avoidance of doubt, where two alkyl moieties are present in a group, the alkyl moieties may be the same or different.
As used herein, “alkenyl” refers to a straight-chain or branched-chain unsaturated hydrocarbon group, comprising at least one carbon-carbon double bond. Typically, an alkenyl group is a C2-6 alkenyl group, having 2-6 carbon atoms. Examples of alkenyl groups include ethenyl (also called vinyl) and propenyl groups.
As used herein, “alkynyl” refers to a straight-chain or branched-chain unsaturated hydrocarbon group, comprising at least one carbon-carbon triple bond. Typically, an alkynyl group is a C2-6 alkynyl group, having 2-6 carbon atoms. Examples of alkynyl groups include ethynyl and propynyl groups.
As used herein, “C1-6 alkylene” refers to a linear or branched alkylene group or moiety. Examples include methylene, ethylene and n-propylene groups and moieties.
As used herein, “C1-6 alkoxy” refers to an alkyl ether group. A C1-6 alkoxy group is a moiety of formula C1-6 alkyl-O-, where C1-6alkyl is as defined above. Exemplary C1-6 alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy. Typically, a C1-6 alkoxy group is a C1-3 alkoxy group of formula C1-3alkyl-O-, where C1-3 alkyl is as defined above. Methoxy is preferred.
As used herein, “cycloalkyl” refers to a non-aromatic saturated hydrocarbon ring. A cycloalkyl moiety typically contains from 3 to 8 carbon atoms. Additionally, a cycloalkyl moiety is typically monocyclic. Preferably, a cycloalkyl is a C3-6 cycloalkyl, containing 3 to 6 carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
As used herein, “cycloalkenyl” refers to a non-aromatic unsaturated hydrocarbon ring comprising at least one carbon-carbon double bond. A cycloalkenyl moiety typically contains from 3 to 8 carbon atoms. Additionally, a cycloalkenyl moiety is typically monocyclic. Preferably, a cycloalkenyl is a C3-6cycloalkenyl, containing 3 to 6 carbon atoms. Examples include cyclopropenyl, cyclobutenyl, cyclopentenyl and cyclohexenyl.
As used herein, a heterocyclyl group or moiety is a non-aromatic, saturated or unsaturated carbocyclic ring in which one or more, for example 1, 2 or 3, of the carbon atoms are replaced with a heteroatom selected from N, O, and S. Preferably, each such heteroatom is selected from N and O. Where the heteroatom is S, the sulphur atom may be substituted by O, and may for instance be in the form of an -S(O)- or -S(O)2- moiety. A heterocyclyl moiety typically is typically 3- to 12-membered (that is, 3 to 12 atoms form the ring or rings). Preferably, a heterocyclyl moiety is 7- to 10-membered. The most preferred heterocyclyl moieties herein are 7- or 8-membered. If the heterocyclyl group or moiety is unsaturated, it may comprise one or more double bonds. Typically, if the heterocyclyl group or moiety is unsaturated, it has only one double bond, which is usually a carbon-carbon double bond. Preferably, the heterocyclyl is saturated and does not comprise any unsaturated bonds within the ring.
A heterocyclyl group or moiety may be monocyclic or polycyclic. Where the heterocyclyl moiety is polycyclic, it typically contains two, or three rings; preferably a polycyclic heterocyclyl contains two rings and may be referred to as a “bicyclic heterocyclyl”.
A polycyclic heterocyclyl may be a spiroheterocyclyl group or moiety. “Spiroheterocyclyl” as used herein refers to polycylic heterocyclyl that shares a carbon atom (called a spiro atom) between two monocyclic rings. The spiroheterocyclyl may be a monospiroheterocyclyl, a bispiroheterocyclyl or a polyspiroheterocyclyl according to the number of spiro atoms (that is, atoms shared between the rings) present. Preferably the spiroheterocyclyl is a bispiroheterocyclyl, containing two rings. Exemplary spiroheterocyclyl groups include
, , and .
A polycyclic heterocyclyl may be a fused heterocyclyl. As used herein, “fused heterocyclyl” refers to a polycyclic heterocyclyl in which each ring shares an adjacent pair of carbon atoms with another ring or rings in the polycyclic heterocyclyl system. Where a fused heterocyclyl is unsaturated, one or more (preferably 1,2,3 or 4) of the rings may contain one or more (preferably 1,2 or 3) double bonds. Typically the fused heterocyclyl is unsaturated. Depending on the number of rings present, the fused heterocyclyl may for instance be bicyclic, tricyclic, or tetracyclic. Preferably the fused heterocyclyl is bicyclic. Exemplary fused heterocyclic groups include:
and .
As used herein, an “aryl” group or moiety refers to a monocyclic or polycyclic aromatic carbocycle. The aryl group is typically a C6-14 aryl group comprising from 6 to 14 carbon atoms. Preferably, the aryl group is a C6-10 aryl group comprising from 6 to 10 carbon atoms. Typically the aryl group is phenyl or naphthyl. Phenyl is preferred.
As used herein, “heteroaryl” refers to a monocyclic or polycyclic 5- to 14-membered aromatic ring, typically a 5- to 10-membered ring and preferably a 5- or 6-membered ring, containing at least one heteroatom selected from N, O and S. Usually the heteroaryl contains 1, 2 or 3 heteroatoms. Preferably the heteroaryl is monocyclic. Examples include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, furanyl, thienyl, pyrazolidinyl, pyrrolyl, oxadiazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl thiadiazolyl, imidazolyl, pyridazolyl and pyrazolyl groups.
As used herein, a halogen refers to fluorine, chlorine, bromine, or iodine and is preferably chlorine or fluorine.
As used herein, a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or p-toluenesulphonic acid. Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases such as alkyl amines, aralkyl amines and heterocyclic amines.
As used herein, a “subject” is typically a human or an animal. Preferably a subject is a mammal. More preferably, the subject is a human.

Compounds of formula (I)

Provided herein is a compound represented by Formula I:

(I)
wherein:
R1, R2 and R4 are each independently selected from hydrogen, halogen, hydroxy, cyano, C1-6 alkyl, C1-6 alkoxy, cycloalkyl containing up to 6 carbon atoms, alkenyl, alkynyl, and -NR’R5;
R3 is selected from hydrogen, halogen, hydroxy, cyano, C1-6alkyl, C3-6cycloalkyl, alkenyl, alkynyl, NR’R5, and -OR6;
R’ is selected from H and C1-6 alkyl;
R5 is selected from hydrogen, C1-6 alkyl, cycloalkyl containing up to 6 carbon atoms, alkenyl, and alkynyl;
R6 is selected from C1-6 alkyl and -L2-A;
L2 is absent or is C1-6 alkylene;
A is selected from aryl and 5- to 14-membered heteroaryl, which may be optionally substituted with one or two substituents each independently selected from cyano, halogen, hydroxy, and amino; and
L1 is optionally substituted polycyclic heterocyclyl;
or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof.
A cycloalkyl containing up to 6 carbon atoms is typically C3-6 cycloalkyl.
Compounds of formula (I) may be optionally substituted at the positions indicated (for instance at L1). By “optionally substituted” is meant that a hydrogen atom may or may not be replaced with a substituent other than hydrogen. Where a substituent is undefined, it is typically selected from halogen, hydroxy, cyano, C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkyl and -NR’2, where R’ is selected from H and C1-6 alkyl. Where the number of substituents is undefined, it is typically selected from one, two or three unless the context provides otherwise.
Preferably, R’ is H (herein, H and hydrogen are used interchangeably).
Preferably, R5 is selected from hydrogen and C1-6 alkyl, and more preferably from hydrogen and C1-3 alkyl. For instance, R5 may be H, methyl or ethyl.
In an aspect, R1, R2 and R4 are each independently selected from hydrogen, halogen, cyano, C1-6 alkyl, C1-6 alkoxy, cycloalkyl containing up to 6 carbon atoms, alkenyl, alkynyl, and -NHR5;
R3 is selected from hydrogen, halogen, cyano, C1-6alkyl, cycloalkyl containing up to 6 carbon atoms, alkenyl, alkynyl, NHR5, and -OR6;
-OR6 is either C1-6 alkoxy or -L2-A;
L2 is absent or is methylene; and
A is aryl which is optionally substituted with a cyano group.
Typically, R1 is selected from halogen, cyano, C1-6alkyl, and C1-6 alkoxy. Preferably R1 is selected from halogen, cyano, C1-3 alkyl, and C1-3 alkoxy. Most preferably, R1 is halogen. Thus, preferably, R1 may be selected from chlorine, fluorine, cyano, methyl, ethyl, i-propyl, n-propyl, methoxy, ethoxy, i-propoxy, and n-propoxy; for instance from chlorine, cyano, methyl and methoxy; particularly chlorine.
Typically, R2 is selected from hydrogen and C1-6 alkyl. Preferably R2 is selected from hydrogen and C1-3 alkyl. Most preferably R2 is hydrogen. Thus, preferably, R2 may be selected from hydrogen, methyl, ethyl, i-propyl, and n-propyl; particularly hydrogen.
Typically, R4 is selected from hydrogen, halogen, cyano, C1-6alkyl, and C1-6 alkoxy. Preferably, R4 is selected from hydrogen, halogen, C1-3 alkyl, and C1-3 alkoxy. Most preferably, R4 is selected from hydrogen and C1-3 alkyl. Thus, preferably, R4 may be selected from hydrogen, chlorine, fluorine, methyl, ethyl, i-propyl, n-propyl, methoxy, ethoxy, i-propoxy, and n-propoxy; for instance from hydrogen, chlorine, fluorine, methyl and methoxy; particularly hydrogen or methyl.
Typically, R3 is selected from hydrogen, halogen, C1-6 alkyl, and -OR6. Preferably, R3 is selected from hydrogen, halogen, C1-3 alkyl, and -OR6. Most preferably, R3 is -OR6. Thus, preferably, R3 may be selected from hydrogen, chlorine, fluorine, methoxy, ethoxy, i-propyl, n-propyl, methoxy, ethoxy, i-propoxy, n-propoxy and -OR6.
In an aspect, R6 may be C1-6 alkyl. Preferably, R6 may be C1-3 alkyl. Thus, preferably, R6 may be methyl, ethyl, i-propyl or n-propyl; particularly methyl.
In another aspect, R6 is L2-A and A is C6-10 aryl optionally substituted with one or two substituents each independently selected from cyano and halogen. In this aspect, it is preferred that A is a C6 aryl group. It is also preferred that the one or two substituents, where present, are each cyano. It is also preferred that A carries 0 or 1 substituents. In this aspect, it is most preferred that A is a C6 aryl substituted with one cyano group.
In this aspect, L2 is absent or is a C1-6 alkylene moiety. Preferably, L2 is absent or is methylene. Most preferably, L2 is methylene.
It is more preferred that R6 is C1-6 alkyl, as discussed above, rather than -L2-A.
Accordingly, R6 is selected from C1-6alkyl and L2-A where L2 is absent or is a C1-6alkylene moiety and A is C6-10aryl optionally substituted with one or two substituents each independently selected from cyano and halogen. Typically, R6 is selected from C1-3 alkyl and L2-A wherein L2 is absent or is methylene and A is C6 aryl substituted with 0 or 1 cyano groups. For instance, R6 may be selected from methyl, ethyl, i-propyl, n-propyl, phenyl, benzyl, cyanophenyl and cyanobenzyl. Preferably, R6 is selected from methyl, ethyl, i-propyl, n-propyl, and cyanobenzyl. Most preferably, R6 is C1-3 alkyl, and in particular methyl.

The compound of formula (I) carries an L1 group which is an optionally substituted polycyclic heterocyclyl. Typically, L1 is of formula:

wherein
n is 0, 1, 2, 3, 4 or 5;
a wavy bond line indicates a covalent bond;
two of RA, RB, RC and RD join together to form a 3- to 8-membered cycloalkyl, cycloalkenyl, or heterocyclyl ring which is optionally substituted by one, two or three substituents each independently selected from halogen, hydroxy, cyano, C1-6 alkyl, and C1-6 alkoxy; and either
(i) the other two of RA, RB, RC and RD are each independently selected from hydrogen, halogen, hydroxy, cyano, C1-6 alkyl, and C1-6 alkoxy, or
(ii) the other two of RA, RB, RC and RD are absent and the carbon atoms between the remaining two of RA, RB, RC and RD are joined by a double bond.
In aspect (i), the polycyclic heterocyclyl is a spiroheterocyclyl. In aspect (ii), the polycyclic heterocyclyl is a fused heterocyclyl. Typically, L1 is a seven- to ten-membered spiroheterocyclyl or fused heterocyclyl.
Preferably, n is 1, 2 or 3. Most preferably, n is 1 or 2.
It is also preferred that the two of RA, RB, RC and RD which do not join together to form a ring are each independently selected from hydrogen and C1-3alkyl. Most preferably, the two of RA, RB, RC and RD which do not join together to form a ring are hydrogen.
It is preferred that the two of RA, RB, RC and RD which join together to form a 3- to 8-membered ring form cycloalkyl or heterocyclyl ring. Where a heterocyclyl ring is thus formed, it is preferred that the heterocyclyl ring comprises a single heteroatom, most preferably oxygen.
The ring formed by RA, RB, RB and RD may or may not be substituted. Typically it is not substituted. However, where it is substituted it is preferred that it is substituted by one substituent selected from halogen, hydroxy, and C1-3 alkoxy. Thus, preferred substituents include chlorine, fluorine, hydroxy, methoxy, ethoxy, i-propoxy and n-propoxy. Most preferred is hydroxy.
Examples of L1 include: , , , , , , , , , , , , , , , , , , , , , and .
Preferably L1 is selected from , , , , and .
Most preferably L1 is selected from and .

Thus, typically:
R1 is selected from halogen, cyano, C1-6alkyl, and C1-6 alkoxy;
R2 is selected from hydrogen and C1-6 alkyl;
R4 is selected from hydrogen, halogen, cyano, C1-6alkyl, and C1-6 alkoxy;
R3 is selected from hydrogen, halogen, C1-6 alkyl, and -OR6;
R6 is selected from C1-3 alkyl and L2-A wherein L2 is absent or is methylene and A is C6 aryl substituted with 0 or 1 cyano groups; and
L1 is of formula:

wherein
n is 0, 1, 2, 3, 4 or 5;
a wavy bond line indicates a covalent bond;
two of RA, RB, RC and RD join together to form a 3- to 8-membered cycloalkyl, cycloalkenyl, or heterocyclyl ring which is optionally substituted by one, two or three substituents each independently selected from halogen, hydroxy, cyano, C1-6 alkyl, and C1-6 alkoxy; and either
(i) the other two of RA, RB, RC and RD are each independently selected from hydrogen, halogen, hydroxy, cyano, C1-6 alkyl, and C1-6 alkoxy, or
(ii) the other two of RA, RB, RC and RD are absent and the carbon atoms between the remaining two of RA, RB, RC and RD are joined by a double bond.
In a preferred aspect:
R1 is selected from halogen, cyano, C1-3 alkyl, and C1-3 alkoxy;
R2 is selected from hydrogen and C1-3 alkyl;
R4 is selected from hydrogen, halogen, C1-3 alkyl, and C1-3 alkoxy;
R3 is selected from hydrogen, halogen, C1-3 alkyl, and -OR6;
R6 is selected from C1-3 alkyl, and cyanobenzyl; and
L1 is of formula:

wherein n is 1, 2 or 3;
two of RA, RB, RC and RD join together to form a 3- to 8-membered cycloalkyl or heterocyclyl ring which is optionally substituted by one substituent selected from halogen, hydroxy, and C1-3 alkoxy; and
the other two of RA, RB, RC and RD are each independently selected from hydrogen and C1-6 alkyl.

In a highly preferred aspect:
R1 is halogen;
R2 is hydrogen;
R4 is selected from hydrogen and C1-3 alkyl;
R3 is -OR6;
R6 is C1-3 alkyl, and in particular methyl; and
L1 is of formula:

wherein n is 1 or 2;
two of RA, RB, RC and RD join together to form a 3- to 8-membered cycloalkyl ring or a heterocyclyl ring which cycloalkyl or heterocyclyl ring which comprises a single heteroatom, which is oxygen; and which 3- to 8-membered ring is optionally substituted by one hydroxy substituent; and
the other two of RA, RB, RC and RD are each hydrogen.

Compounds of formula (I) containing one or more chiral center may be used in
enantiomerically or diastereoisomerically pure form, or in the form of a mixture of isomers. For the avoidance of doubt, the compounds of the invention may be used in any tautomeric form. Further, for the avoidance of doubt, compounds of formula (I) may take the form of any stereoisomer, such as any geometric isomer, of a structure as shown herein.
Compounds of the invention may be provided as a pharmaceutically acceptable salt. Compounds of the invention may be provided in the form of a hydrate, solvate or prodrug. A hydrate is a particular form of solvate wherein the solvent molecule(s) associated with the compound is/are water molecules. Preferably, a compound of the invention is a compound of formula (I) or a pharmaceutically acceptable salt thereof.

Compounds of formula (I’)

The compounds of the invention may be compounds of formula (I’); geometric isomers thereof or pharmaceutically acceptable compounds thereof; or salts, hydrates, solvates or prodrugs thereof.
Compounds of formula (I’) are compounds of formula (I) wherein:
R1, R2, R3 and R4 is selected from hydrogen, halogen, C1-C6 alkoxy, C1-C6 alkyl, C1-C6 cycloalkyl, alkenyl, alkynyl, NHR4, C1-C3 alkyl and Cyano.
R3 may also be selected from substituted or unsubstituted aryloxy.
The "alkyl" in this context refers to a straight-chain or branched-chain alkyl group, wherein the C1-C6 group means that the moiety has 1-6 carbon atoms, that is, the group contains 1, 2, 3, 4, 5 or 6 carbon atoms.
The "alkoxy" in this context refers to an alkyl ether alkyl group, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy, etc.
The "halogen" in this context refers to fluorine, chlorine, bromine, or iodine.
The L1 –is polycyclic heterocyclyl which includes spiro and fused heterocyclyls. “Spiroheterocyclyl” refers to polycylic heterocyclyl that shares a carbon atom (called a spiro atom) between the monocyclic rings, wherein one or more (Preferably 1,2, or 3) of the ring atoms are heteroatoms selected from nitrogen, oxygen, or sulphur. The spiroheterocyclyl may be a monospiroheterocyclyl, a bicyspiroheterocyclyl or a polyspiroheterocyclyl according to the number of spiro atoms shared between the rings. Spiroheterocyclyl includes, but not limited to:

“Fused heterocyclyl” refers to polycyclic heterocyclyl in which each ring shares an adjacent pair of carbon atoms with other rings in the system, wherein one or more (preferably 1,2,3 or 4) of the rings may contain one or more (preferably 1,2 or 3) double bonds, but none of the rings have a fully conjugated-electron system, wherein one or more (preferably 1,2, or 3) of the ring atoms are heteroatoms selected from nitrogen or oxygen and the remaining ring atoms are carbon atoms. Depending on the number of rings, it may be bicyclic, tricyclic, tetracyclic or polycyclic, fused heterocyclyl includes, but not limited to:

Specific compounds of the invention

Compounds of formula (I) can, for example, be prepared according to the reaction schemes outlined in the “Synthesis examples” below. 81 compounds according to the invention were synthesised. These compounds are listed in Table 1, below, along with NMR and mass spectrometry analysis data. Each compound is assigned a “compound ID” number from NAT-1 to NAT-81. In an aspect, the compound of formula (I) may be a compound as listed in Table 1, or a hydrate, prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof.

Table-1: representative examples of the disclosure
Com-pound ID Structural formula Formula weight Compound IUPAC name/ NMR and mass

NAT-1
458.55 3-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methoxy-3-pyridyl] methyl]-3-azabicyclo [3.2.0] heptan-6-ol
1H NMR (DMSO-d6, 400 MHz): d 7.97 (d, J = 7.6 Hz, 1H), 7.38-7.36 (m, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.21-7.19 (m, 1H), 7.13 (d, J = 8.0 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.84-6.80 (m, 2H), 4.65 (brs, 1H), 4.28 (s, 4H), 4.13 (brs, 1H), 3.90 (s, 3H), 3.74-3.60 (m, 2H), 2.86 (d, J = 5.6 Hz, 1H), 2.75 (d, J = 8.0 Hz, 1H), 2.43-2.47 (m, 2H), 2.19 (s, 3H), 2.06-2.03 (m, 1H), 1.81-1.78 (m, 1H). m/z: 459.37 (M+H) +

NAT-2
559.65 3-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-3-[(6-hydroxy-3-aza-bicyclo[3.2.0]heptan-3-yl) methyl]-2-pyridyl]oxymethyl] benzonitrile
1H NMR (400 MHz, DMSO-d6): 8.01 (d, J = 7.6 Hz, 1H), 7.92 (s, 1H), 7.85-7.76 (m, 2H), 7.62-7.58 (m, 1H), 7.30-7.27 (m, 2H), 7.21-7.15 (m, 2H), 6.92 (d, J = 8.4 Hz, 1H), 6.83-6.78 (m, 2H), 5.46 (s, 2H), 4.65 (m, 1H), 4.28 (s, 4H), 4.13-4.11 (m, 1H), 3.84-3.67 (m, 2H), 2.87-2.75 (m, 2H), 2.43-2.47 (m, 2H), 2.21-2.17 (m, 1H), 2.06 (s, 3H), 1.81-1.78 (m, 1H). m/z: 560.44 (M+H) +

NAT-3 444.52 6-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methoxy-3-pyridyl] methyl]-2-oxa-6-azaspiro [3.3] heptane.
1H NMR (CDCl3, 400 MHz): d 7.55 (d, J = 7.6 Hz, 1H), 7.38-7.36 (m, 1H), 7.29-7.22 (m, 2H), 7.0 (d, J = 7.6 Hz, 1H), 6.92-6.88 (m, 2H), 6.84-6.82 (m, 1H), 4.77 (s, 4H), 4.30 (s, 4H), 3.96 (s, 3H), 3.57 (s, 2H), 3.49 (s, 4H), 2.25 (s, 3H). m/z: 445.3 (M+H) +
NAT-4
458.55 2-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methoxy-3-pyridyl] methyl]-6-oxa-2-azaspiro [3.4] octane
1H NMR (CDCl3, 400 MHz): d 7.71 (d, J = 7.6 Hz, 1H), 7.38-7.36 (m, 1H), 7.30-7.24 (m, 2H), 7.05 (d, J = 7.2 Hz, 1H), 6.92-6.88 (m, 2H), 6.84-6.82 (m, 1H), 4.30 (s, 4H), 4.0-3.97 (m, 5H), 3.89 (s, 2H), 3.83-3.73 (m, 6H), 2.25 (s, 3H), 2.21 (t, J = 7.2 Hz, 2H). m/z: 459.3 (M+H) +
NAT-5
570.64 2-[6-[(3-cyanophenyl) methoxy]-5-(6-oxa-2-azaspiro [3.4] octan-2-ylmethyl)-2-pyridyl]-6-(2,3-dihydro-1,4-benzodioxin-6-yl) benzonitrile
1H NMR (DMSO-d6, 400 MHz): d 7.94 (s, 1H), 7.85-7.74 (m, 4H), 7.71-7.67 (m, 1H), 7.63-7.57 (m, 2H), 7.50 (d, J = 8.0 Hz, 1H), 7.11 (s, 1H), 7.07 (d, J = 8.0 Hz, 1H), 7.0 (d, J = 8.0 Hz, 1H), 5.57 (s, 2H), 4.31 (s, 4H), 3.73 (s, 2H), 3.66-3.63 (m, 4H), 3.26 (s, 4H), 2.04 (t, J = 8.0 Hz, 2H). m/z: 571.6 (M+H) +
NAT-6
448.16 6-[[2-chloro-6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-3-pyridyl] methyl]-2-oxa-6-azaspiro [3.3] heptane
1H NMR (CDCl3, 400 MHz): d 7.77 (d, J = 8.0 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.32 – 7.29 (m, 1H), 7.27 – 7.25 (m, 2H), 6.90 (d, J = 8.0 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 6.82 – 6.79 (dd, J = 2.0 Hz 8.0 Hz, 1H), 4.75 (s, 4H), 4.30 (s, 4H), 3.70 (s, 2H), 3.52 (s, 4H), 2.18 (s, 3H). m/z: 449.4 (M+H) +
NAT-7
462.1 2-[[2-chloro-6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-3-pyridyl] methyl]-6-oxa-2-azaspiro [3.4] octane
1H NMR (CDCl3, 400 MHz,): d 7.82 (d, J = 7.6 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.32 – 7.24 (m, 3H), 6.90 (d, J = 8.4 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 6.80 (dd, J = 2.0 Hz 8.4 Hz, 1H), 4.30 (s, 4H), 3.89 (s, 2H), 3.81 (t, J = 6.8 Hz, 2H), 3.77 (s, 2H), 3.38 (s, 4H), 2.19 (s, 3H), 2.15 (t, J = 6.8 Hz, 2H). m/z: 463.1 (M+H)+
NAT-8
428.52 6-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methyl-3-pyridyl] methyl]-2-oxa-6-azaspiro [3.3] heptane
1H NMR (CDCl3, 400 MHz): d 7.60 (d, J = 7.6 Hz, 1H), 7.33 – 7.29 (m, 2H), 7.26 – 7.21 (m, 2H), 6.91 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 1.6 Hz, 1H), 6.83 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 4.79 (s, 4H), 4.32 (s, 4H), 3.59 (s, 2H), 3.47 (s, 4H), 2.57 (s, 3H), 2.17 (s, 3H). m/z: 429.6 (M+H) +
NAT-9
442.23 2-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methyl-3-pyridyl] methyl]-6-oxa-2-azaspiro [3.4] octane
1H NMR (400 MHz, CDCl3): d 7.63 (d, J = 7.6 Hz, 1H), 7.31 – 7.27 (m, 2H), 7.24 – 7.19 (m, 2H), 6.89 (d, J = 8.4 Hz, 1H), 6.86 (d, J = 2.0 Hz, 1H), 6.81 (dd, J = 2.0 Hz, 6.4 Hz, 1H), 4.30 (s, 4H), 3.87 (s, 2H), 3.80 (t, J = 6.8 Hz, 2H), 3.64 (s, 2H), 3.31 (s, 4H), 2.57 (s, 3H), 2.16 (s, 3H), 2.13 (t, J = 7.2 Hz, 2H).
m/z: 443.3 (M+H)+
NAT-10
472.24 2-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methoxy-4-methyl-3-pyridyl]methyl]-6-oxa-2-azaspiro[3.4] octane
1H NMR (400 MHz, CDCl3): d 7.36 (dd, J = 1.6 Hz, 5.6 Hz, 1H), 7.24 – 7.21 (m, 2H), 6.92-6.88 (m, 2H), 6.85-6.82 (m, 2H), 4.31 (s, 4H), 3.94 (s, 3H), 3.82 (s, 2H), 3.75 (t, J =7.2 Hz, 2H), 3.67 (s, 2H), 3.31 (brs, 4H), 2.40 (s, 3H), 2.25 (s, 3H), 2.08 (t, J = 7.2 Hz, 2H).
m/z: 473.3 (M+H)+
NAT-11 458.22 6-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methoxy-4-methyl-3-pyridyl] methyl]-2-oxa-6-azaspiro [3.3] heptane
1H NMR (400 MHz, CDCl3): d 7.37 (dd, J = 1.6 Hz, 5.6 Hz, 1H), 7.26 – 7.25 (m, 2H), 6.94 –6.90 (m, 2H), 6.86 – 6.83 (m, 2H), 4.76 (s, 4H), 4.32 (s, 4H), 3.96 (s, 3H), 3.63 (brs, 2H), 3.50 (brs, 4H), 2.40 (s, 3H), 2.27 (s, 3H).m/z: 459.2 (M+H)+
NAT-12
476.21 2-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-5-fluoro-2-methoxy-3-pyridyl] methyl]-6-oxa-2-azaspiro [3.4] octane
1H NMR (400 MHz, CDCl3): d 7.56 (d, J = 10.8 Hz, 1H), 7.27 – 7.26 (m, 2H), 7.1 (t, J = 4.8 Hz, 1H), 6.93 – 6.88 (m, 2H), 6.84 – 6.82 (m, 1H), 4.31 (s, 4H), 3.97 (s, 3H), 3.7 – 3.73 (m, 4H), 3.36 (d, J = 14.0 Hz, 1H), 3.23 (d, J = 13.6 Hz, 1H), 3.1 (d, J = 8.0 Hz, 4H), 2.03 (t, J = 7.2 Hz, 2H), 1.97 (s,3H). m/z: 477.3 (M+H)+
NAT-13
462.20 6-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-5-fluoro-2-methoxy-3-pyridyl] methyl]-2-oxa-6-azaspiro [3.3] heptane
1H NMR (400 MHz, DMSO-d6): d 7.64 (d, J = 11.2 Hz, 1H), 7.29 – 7.26 (m, 1H), 7.23 – 7.21 (m, 1H), 7.13 – 7.10 (m, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.84 – 6.79 (m, 2H), 4.54 (s, 4H), 4.28 (s, 4H), 3.87 (s, 3H), 3.2 (brs, 6H), 1.88 (s, 3H).
m/z: 463.2 (M+H)+
NAT-14
469.20 2-(2,3-dihydro-1,4-benzodioxin-6-yl)-6-[6-methoxy-5-(6-oxa-2-azaspiro [3.4] octan-2-ylmethyl)-2-pyridyl] benzonitrile
1H NMR (400 MHz, DMSO-d6): d 7.79-7.76 (m, 3H), 7.58-7.55 (m, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.10 (d, J = 2.4 Hz, 1H), 7.06 (dd, J = 8.0 Hz, 2.4 Hz, 1H), 7.0 (d, J = 8.0 Hz, 1H), 4.31 (s, 4H), 3.98 (s, 3H), 3.72 (s, 2H), 3.64 (t, J = 8.0 Hz, 2H), 3.58 (s, 2H), 3.24 (s, 4H), 2.03 (t, J = 8.0 Hz, 2H). m/z: 470.2 (M+H)+
NAT-15
455.18 2-(2,3-dihydro-1,4-benzodioxin-6-yl)-6-[6-methoxy-5-(2-oxa-6-azaspiro [3.3] heptan-6-ylmethyl)-2-pyridyl] benzonitrile
1H NMR (400 MHz, DMSO-d6): d 7.79-7.78 (m, 2H), 7.74 (d, J = 8.0 Hz, 1H), 7.58-7.55 (m, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.10 (d, J = 2.0 Hz, 1H), 7.06 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.0 (d, J = 8.0 Hz, 1H), 4.62 (s, 4H), 4.30 (s, 4H), 3.97 (s, 3H), 3.52 (s, 2H), 3.38 (s, 4H). m/z: 456.1 [M+H ]+
NAT-16
483.22 2-(2,3-dihydro-1,4-benzodioxin-6-yl)-6- [6-methoxy-4-methyl-5-(6-oxa-2-azaspiro[3.4]octan-2-ylmethyl)-2-pyridyl]benzonitrile
1H NMR (400 MHz, DMSO-d6): d 7.79-7.78 (m, 2H), 7.56 (t, J = 4.8 Hz, 1H), 7.30 (s, 1H), 7.10 (d, J = 2.0 Hz, 1H), 7.05 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.0 (d, J = 8.0 Hz, 1H), 4.30 (s, 4H), 3.96 (s, 3H), 3.67-3.59 (m, 6H), 3.19 (s, 4H), 2.42 (s, 3H), 1.97 (t, J = 4.8 Hz, 2H). m/z 484.2[M+H]+
NAT-17
469.20 2-(2,3-dihydro-1,4-benzo dioxin-6-yl)-6-[6-methoxy-4-methyl-5-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)-2-pyridyl]benzonitrile
1H NMR (400 MHz, DMSO-d6): d 7.79 (d, J = 4.0 Hz, 2H), 7.56 (t, J = 4.0 Hz, 1H), 7.29 (s, 1H), 7.09 (s, 1H), 7.05 (d, J = 8.0 Hz, 1H), 7.0 (d, J = 8.0 Hz, 1H), 4.58 (s, 4H), 4.31 (s, 4H), 3.95 (s, 3H), 3.55 (s, 2H), 3.33 (s, 4H), 2.39 (s, 3H): m/z: 470.2 [M+H]+
NAT-18
473.15 2- [6-chloro-5-(6-oxa-2-aza spiro [3.4] octan-2-ylmethyl)-2-pyridyl]-6-(2,3-dihydro-1,4-benzodioxin-6-yl) benzonitrile
1H NMR (400 MHz, DMSO-d6): d 8.02 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.13 (d, J = 2.4 Hz, 1H), 7.08 (dd, J = 8.0 Hz, 2.4 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 4.31 (s, 4H), 3.74 (s, 4H), 3.65 (t, J = 8.0 Hz, 2H), 3.30 (s, 4H), 2.05 (t, J = 8.0, 2H). m/z: 474.2 [M+H]+
NAT-19
459.13 2- [6-chloro-5-(2-oxa-6-aza spiro [3.3] heptan-6-ylmethyl)-2-pyridyl]-6-(2,3-dihydro-1,4-benzodioxin-6-yl) benzonitrile
1H NMR: (CDCl3, 400 MHz): d 7.68-7.64 (m, 2H), 7.58 (d, J = 8.0 Hz, 1H), 7.35 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.20 (t, J = 8.0 Hz, 1H), 7.14 (d, J = 2.0 Hz, 1H), 7.02 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H), 4.75 (s, 4H), 4.29 (s, 4H), 3.57 (s, 2H), 3.47 (s, 4H); m/z: 460.1 [M+H]+ and 462.1 [M+2H]+
NAT-20
487.19 2-(2,3-dihydro-1,4-benzodioxin-6-yl)-6-[3-fluoro-6-methoxy-5-(6-oxa-2-azaspiro[3.4]octan-2-ylmethyl)-2-pyridyl]benzonitrile
1H NMR (400 MHz, DMSO-d6): d 7.80-7.77 (m, 2H), 7.62-7.59 (m, 2H), 7.12 (d, J = 2.0 Hz, 1H), 7.07 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 4.30 (s, 4H), 3.93 (s, 3H), 3.62-3.57 (m, 4H), 3.41 (s, 2H), 3.05 (s, 4H), 1.93 (t, J = 8.0 Hz, 2H). m/z: 488.2 [M+H]+
NAT-21
473.18 2-(2,3-dihydro-1,4-benzodioxin-6-yl)-6-[3-fluoro-6-methoxy-5-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)-2-pyridyl]benzonitrile
1H NMR: (CDCl3, 400 MHz): d 7.51 (d, J = 12.0 Hz, 1H), 7.35 (t, J = 4.8 Hz, 1H), 7.26 (d, J = 4.8 Hz, 2H), 7.11 (d, J = 2.0 Hz, 1H), 7.03 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 6.93 (d, J = 8.0 Hz, 1H), 4.71 (s, 4H), 4.30 (s, 4H), 3.51 (s, 2H), 3.28 (s, 4H), 3.23 (s, 3H). m/z: 474.1[M+H]+, 475.1[M+2H]+
NAT-22
478.17 2-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl)phenyl]-2-methoxy-3-pyridyl] methyl]-6-oxa-2-azaspiro [3.4] octane
1H NMR (400 MHz, DMSO-d6): d 7.71 (d, J = 7.2 Hz, 1H), 7.53 – 7.51 (m, 1H), 7.45 (t, J = 8.8 Hz, 1H), 7.40 – 7.37 (m, 1H), 7.21 (d, J = 7.6 Hz, 1H), 6.95 – 6.88 (m, 3H), 4.30 (s, 4H), 3.88 (s, 3H), 3.73 (s, 2H), 3.64 (t, J = 6.8 Hz, 2H), 3.57 (brs, 2H), 3.24 (brs, 4H), 2.03 (t, J = 6.8 Hz, 2H). m/z: 479.1 (M+H)+ and 481.1(M+2H)+
NAT-23
464.15 6-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl)phenyl]-2-methoxy-3-pyridyl]methyl]-2-oxa-6-azaspiro[3.3] heptane
1H NMR (400 MHz, CDCl3): d 7.57 (d, J = 7.6 Hz, 1H), 7.52-7.49 (m, 1H), 7.37 – 7.30 (m, 2H), 7.19 (d, J = 7.6 Hz, 1H), 7.0 (s, 1H), 6.93 (t, J = 10.0 Hz, 2H), 4.77 (s, 4H), 4.31 (s, 4H), 3.98 (s, 3H), 3.58 (s, 2H), 3.49 (s, 4H).
m/z: 465.0 (M+H)+ and 467.0
NAT-24
492.18 2-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl) phenyl]-2-methoxy-4-methyl-3-pyridyl]methyl]-6-oxa-2-aza spiro[3.4] octane
1H NMR (400 MHz, CDCl3): d 7.49 (dd, J = 2.0 Hz, 5.2 Hz, 1H), 7.36 – 7.29 (m, 2H), 7.04 (s, 1H), 7.0 – 6.98 (m, 1H), 6.94 – 6.93 (m, 2H), 4.31 (s, 4H), 3.96 (s, 3H), 3.82 (s, 2H), 3.76 (t, J = 7.2 Hz, 2H), 3.69 (brs, 2H), 3.31 (brs, 4H), 2.41 (s, 3H), 2.08 (t, J = 7.2 Hz, 2H).
m/z: 493.1 (M+H)+ and 495.1
NAT-25
478.17 6-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl)phenyl]-2-methoxy-4-methyl-3-pyridyl]methyl]-2-oxa-6-aza spiro[3.3] heptane
1H NMR (400 MHz, CDCl3): d 7.49 (dd, J = 2.0 Hz, 5.2 Hz, 1H), 7.36 – 7.29 (m, 2H), 7.03 (s, 1H), 7.01 – 6.98 (m, 1H), 6.93 – 6.92 (m, 2H), 4.74 (s, 4H), 4.31 (s, 4H), 3.96 (s, 3H), 3.60 (s, 2H), 3.46 (s, 4H), 2.37 (s, 3H). m/z: 479.1(M+H)+ and 481.1
NAT-26
496.16 2-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl)phenyl]-5-fluoro-2-methoxy-3-pyridyl]methyl]-6-oxa-2-azaspiro[3.4] octane
1H NMR (400 MHz, CDCl3): d 7.58 (d, J = 10.8 Hz, 1H), 7.39 – 7.34 (m, 2H), 7.23 – 7.21 (m, 1H), 7.03 – 7.02 (m, 1H), 6.98 – 6.92 (m, 2H), 4.31 (s, 4H), 4.0 (s, 3H), 3.77 – 3.73 (m, 4H), 3.43 – 3.27 (m, 2H), 3.12 (brs 4H), 2.04 (t, J = 6.8 Hz, 2H). m/z: 497.2 (M+H)+ and 499.2
NAT-27
482.14 6-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl)phenyl]-5-fluoro-2-methoxy-3-pyridyl]methyl]-2-oxa-6-azaspiro[3.3] heptane
1H NMR (400 MHz, CDCl3): d 7.52 (d, J = 11.2 Hz, 1H), 7.39 – 7.33 (m, 2H), 7.21 – 7.20 (m, 1H), 7.02 – 7.01 (m, 1H), 6.98 – 6.92 (m, 2H),4.69 (s, 4H), 4.31 (s, 4H), 4.0 (s, 3H), 3.35 – 3.19 (m, 6H). m/z: 483.2 (M+H)+ and 485.1
NAT-28
474.22 2-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methoxy-phenyl]-2-methoxy-3-pyridyl]methyl]-6-oxa-2-azaspiro[3.4] octane
1H NMR: (CDCl3, 400 MHz): d 7.79 (dd, J = 7.6 Hz, 2.0 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.32 (dd, J = 7.6 Hz, 2.0 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 7.13 (d, J = 2.0 Hz, 1H), 7.09 (dd, J = 7.6 Hz, 2.0 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 4.30 (s, 4H), 4.02 (s, 3H), 3.87 (s, 2H), 3.79 (t, J = 7.2 Hz, 2H), 3.65 (s, 2H), 3.35 (s, 4H), 3.34 (s, 3H), 2.13 (t, J = 7.2 Hz, 2H). m/z: 475.2 (M+H)+
NAT-29
460.20 6-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methoxy-phenyl]-2-methoxy-3-pyridyl] methyl]-2-oxa-6-azaspiro [3.3] heptane
1H NMR: (CDCl3, 400 MHz): ? 7.79 (dd, J = 8.0 Hz, 1.6 Hz, 1H), 7.54 (d, J = 1.6 Hz, 2H), 7.32 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.22 (t, J = 8.0 Hz, 1H), 7.13 (d, J = 2.0 Hz, 1H), 7.08 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 4.76 (s, 4H), 4.30 (s, 4H), 4.01 (s, 3H), 3.62 (s, 2H), 3.54 (s, 4H), 3.34 (s, 3H). m/z: 461.2 [M+H]+.
NAT-30
478.17 2-[[2-chloro-6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methoxy-phenyl]-3-pyridyl]methyl]-6-oxa-2-azaspiro [3.4]octane
1H NMR: (CDCl3, 400 MHz): d 7.67-7.61 (m, 2H), 7.58 (d, J = 7.6 Hz, 1H), 7.34 (dd, J = 7.6 Hz, 2.0 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 7.13 (d, J = 2.0 Hz, 1H), 7.10 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 4.29 (s, 4H), 3.90 (s, 2H), 3.81 (t, J = 6.8 Hz, 2H), 3.63 (s, 2H), 3.29 (s, 4H), 3.25 (s, 3H), 2.11 (t, J = 6.8 Hz, 2H). m/z: 479.2 [M+H]+ and 481.2.
NAT-31
464.15 6-[[2-chloro-6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methoxy-phenyl]-3-pyridyl]methyl]-2-oxa-6-azaspiro [3.3] heptane
1H NMR: (CDCl3, 400 MHz): d 7.66-7.62 (m, 2H), 7.49 (d, J = 8.0 Hz, 1H), 7.33 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.21 (t, J = 8.0 Hz, 1H), 7.19 (d, J = 2.0 Hz, 1H), 7.04 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 4.77 (s, 4H), 4.30 (s, 4H), 3.59 (s, 2H), 3.46 (s, 4H), 3.24 (s, 3H). m/z: 465.1 [M+H]+ and 467.1.
NAT-32
458.22 2-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methoxy-phenyl]-2-methyl-3-pyridyl]methyl]-6-oxa-2-azaspiro[3.4] octane
1H NMR: (CDCl3, 400 MHz): d 7.66-7.64 (m, 2H), 7.60 (d, J = 7.6 Hz, 1H), 7.32 (dd, J = 7.6 Hz, 2.0 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 7.08 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 4.30 (s, 4H), 3.87 (s, 2H), 3.79 (t, J = 6.8 Hz, 2H), 3.64 (s, 2H), 3.30 (s, 4H), 3.27 (s, 3H), 2.59 (s, 3H), 2.12 (t, J = 6.8 Hz, 2H). m/z: 459.2 [M+H]+
NAT-33
444.20 6-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methoxy-phenyl]-2-methyl-3-pyridyl]methyl]-2-oxa-6-azaspiro[3.3] heptane
1H NMR: (CDCl3, 400 MHz): d 7.66-7.63 (m, 2H), 7.55 (d, J = 8.0 Hz, 1H), 7.32 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.22 (t, J = 8.0 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 7.08 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 6.91 (d, J = 8.0 Hz, 1H), 4.78 (s, 4H), 4.30 (s, 4H), 3.58 (s, 2H), 3.45 (s, 4H), 3.27 (s, 3H), 2.58 (s, 3H). m/z: 445.3 [M+H]+
NAT-34
488.23 2-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methoxy-phenyl]-2-methoxy-4-methyl-3-pyridyl]methyl]-6-oxa-2-aza spiro [3.4] octane
1H NMR: (CDCl3, 400 MHz): d 7.77 (dd, J = 7.6 Hz, 2.0 Hz, 1H), 7.38 (s, 1H), 7.31 (dd, J = 7.6 Hz, 2.0 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 7.08 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 4.30 (s, 4H), 3.99 (s, 3H), 3.81 (s, 2H), 3.75 (t, J = 7.2 Hz, 2H), 3.66 (s, 2H), 3.36 (s, 3H), 3.29 (s, 4H), 2.39 (s, 3H), 2.07 (t, J = 7.2 Hz, 2H). m/z: 489.3 [M+H]+
NAT-35
474.22 6-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methoxy-phenyl]-2-methoxy-4-methyl-3-pyridyl] methyl]-2-oxa-6-aza spiro [3.3] heptane
1H NMR: (CDCl3, 400 MHz): d 7.77 (dd, J = 8.0 Hz, 1.6 Hz, 1H), 7.40 (s, 1H), 7.32 (dd, J = 8.0 Hz, 1.6 Hz, 1H), 7.22 (t, J = 8.0 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 7.08 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 4.73 (s, 4H), 4.31 (s, 4H), 4.00 (s, 3H), 3.69 (s, 2H), 3.58 (s, 4H), 3.35 (s, 3H), 2.37 (s, 3H). m/z: 475.3 [M+H]+
NAT-36
492.21 2-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methoxy-phenyl]-5-fluoro-2-methoxy-3-pyridyl]methyl]-6-oxa-2-aza spiro [3.4]octane
1H NMR: (CDCl3, 400 MHz) d 7.59 (d, J = 11.2 Hz, 1H), 7.39-7.37 (m, 1H), 7.23 (s, 1H), 7.22 (d, J = 2.0 Hz, 1H), 7.13 (d, J = 2.0 Hz, 1H), 7.07 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 4.31 (s, 4H), 3.99 (s, 3H), 3.77 (s, 2H), 3.74 (t, J = 6.8 Hz, 2H), 3.61 (s, 2H), 3.21 (s, 3H), 3.13 (s, 4H), 2.04 (t, J = 6.8 Hz, 2H). m/z: 493.2 [M+H]+
NAT-37
478.19 6-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methoxy-phenyl]-5-fluoro-2-methoxy-3-pyridyl]methyl]-2-oxa-6-aza spiro [3.3] heptane
1H NMR: (CDCl3, 400 MHz): d 7.53 (d, J = 12.0 Hz, 1H), 7.38 (t, J = 4.8 Hz, 1H), 7.21 (d, J = 4.8 Hz, 2H), 7.12 (d, J = 2.0 Hz, 1H), 7.07 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 4.69 (s, 4H), 4.31 (s, 4H), 3.99 (s, 3H), 3.55 (s, 2H), 3.27 (s, 4H), 3.20 (s, 3H). m/z: 479.1 [M+H]+
NAT-38
439.19 2-(2,3-dihydro-1,4-benzodioxin-6-yl)-6-[6-methyl-5-(2-oxa-6-azaspiro[3.3] heptan-6-ylmethyl)-2-pyridyl] benzonitrile
1H NMR: (CDCl3, 400 MHz): d 7.68-7.62 (m, 3H), 7.55 (d, J = 8.0 Hz, 1H), 7.45 (d, J = 8.0 Hz, 2.0 Hz, 1H), 7.09-7.06 (m, 2H), 6.97 (d, J = 8.0 Hz, 1H), 4.78 (s, 4H), 4.31 (s, 4H), 3.59 (s, 2H), 3.45 (s, 4H), 2.60 (s, 3H). m/z: 440.2 [M+H]+
NAT-39
453.21 2-(2,3-dihydro-1,4-benzo dioxin-6-yl)-6-[6-methyl-5-(6-oxa-2-azaspiro[3.4] octan-2-ylmethyl)-2-pyridyl]benzo nitrile
1H NMR: (DMSO-d6, 400 MHz) d 7.72 (d, J = 8.0 Hz, 1H), 7.68-7.62 (m, 2H), 7.54 (d, J = 8.0 Hz, 1H), 7.45 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.09-7.06 (m, 2H), 6.96 (d, J = 8.0 Hz, 1H), 4.30 (s, 4H), 3.87 (s, 2H), 3.79 (t, J = 6.8 Hz, 2H), 3.65 (s, 2H), 3.30 (s, 4H), 2.61 (s, 3H), 2.13 (t, J = 6.8 Hz, 2H). m/z: 454.2 [M+H]+
NAT-40
482.12 2-[[2-chloro-6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl)phenyl]-3-pyridyl]methyl]-6-oxa-2-azaspiro [3.4]octane
1H NMR (400 MHz, CDCl3): d 7.84 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.50 – 7.47 (m, 1H), 7.38 – 7.33 (m, 2H), 6.97 – 6.91 (m, 3H), 4.30 (s, 4H), 3.89 (s, 2H), 3.82 – 3.77 (m, 4H), 3.38 (s, 4H), 2.14 (t, J = 7.2 Hz, 2H). m/z: 483.1 (M+H)+ and 485.1
NAT-41
468.1 6-[[2-chloro-6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl)phenyl]-3-pyridyl]methyl]-2-oxa-6-azaspiro [3.3] heptane
1H NMR (400 MHz, CDCl3): d 7.78 (d, J = 7.6 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.50 – 7.47 (m, 1H), 7.36 – 7.35 (m, 2H), 6.97 (brs, 1H), 6.92 (brs, 2H), 4.80 (s, 4H), 4.31 (s, 4H), 3.71 (s, 2H), 3.52 (s, 4H).
m/z: 469.35 (M+H)+ and 471.0
NAT-42
448.16 6-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl)phenyl]-2-methyl-3-pyridyl]methyl]-2-oxa-6-azaspiro[3.3] heptane
1H NMR (400 MHz, CDCl3): d 7.60 (d, J = 7.6 Hz, 1H), 7.46 – 7.43 (m, 1H), 7.40 – 7.30 (m, 3H), 6.97– 6.92 (m, 3H), 4.78 (s, 4H), 4.30 (s, 4H), 3.58 (s, 2H), 3.45 (s, 4H), 2.57 (s, 3H). m/z: 449.1 (M+H)+ and 451.1
NAT-43
462.17 2-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl)phenyl]-2-methyl-3-pyridyl]methyl]-6-oxa-2-azaspiro[3.4] octane
1H NMR (400 MHz, CDCl3): d 7.64 (d, J = 7.6 Hz, 1H), 7.46 – 7.44 (m, 1H), 7.40 – 7.30 (m, 3H), 6.98 – 6.92 (m, 3H), 4.30 (s, 4H), 3.87 (s, 2H), 3.80 (t, J = 6.8 Hz, 2H), 3.65 (s, 2H), 3.31 (s, 4H), 2.59(s, 3H), 2.13 (t, J = 6.8 Hz, 2H).
m/z: 463.1 (M+H)+ and 465.1
NAT-44
453.21 2-(2,3-dihydro-1,4-benzodioxin-6-yl)-6-[4-methyl-5-(6-oxa-2-azaspiro[3.4] octan-2-ylmethyl)-2-pyridyl] benzo nitrile
1H NMR (400 MHz, DMSO-d6): d 8.50 (s, 1H), 7.79 (t, J = 8.0 Hz, 1H), 7.74-7.70 (m, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.57 (dd, J = 8.0 Hz, 1.2 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 7.07 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.0 (d, J = 8.0 Hz, 1H), 4.31 (s, 4H), 3.72 (s, 2H), 3.72-3.62 (m, 6H), 3.23-3.19 (m, 4H), 2.39 (s, 3H), 2.03 (t, J = 6.8 Hz, 2H). m/z: 454.2 (M+H)+
NAT-45
453.21 2-(2,3-dihydro-1,4-benzodioxin-6-yl)-6-[3-methyl-5-(6-oxa-2-azaspiro[3.4] octan-2-ylmethyl)-2-pyridyl]benzo nitrile
1H NMR (400 MHz, DMSO-d6): d 8.39 (s, 1H), 7.79 (t, J = 8.0 Hz, 1H), 7.71 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.11 (d, J = 2.0 Hz, 1H), 7.07 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.0 (d, J = 8.0 Hz, 1H), 4.30 (s, 4H), 3.71 (s, 2H), 3.66-3.62 (m, 4H), 3.20 (s, 4H), 2.20 (s, 3H), 2.02 (t, J = 8.0 Hz, 2H). m/z: 454.2 (M+H)+
NAT-46
443.16 2-(2,3-dihydro-1,4-benzodioxin-6-yl)-6-
[4-fluoro-5-(2-oxa-6-azaspiro[3.3]
heptan-6-ylmethyl)-2-pyridyl]benzonitrile
1H NMR (400 MHz, DMSO-d6): d 8.52 (d, J = 4.8 Hz, 1H), 7.84 (t, J = 8.0 Hz, 1H), 7.66 (d, J = 8.0 Hz, 2H), 7.54 (t, J = 4.8 Hz, 1H), 7.11 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 4.62 (s, 4H), 4.31 (s, 4H), 3.69 (s, 2H), 3.42 (s, 4H). m/z: 444.2 (M+H)+
NAT-47
443.16 2-(2,3-dihydro-1,4-benzodioxin-6-yl)-6-[3-fluoro-5-(2-oxa-6-azaspiro [3.3] heptan-6-ylmethyl)-2-pyridyl] benzonitrile
1H NMR (400 MHz, DMSO-d6): d 8.48 (s, 1H), 7.84 (t, J = 8.0 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.68-7.65 (m, 2H), 7.12 (d, J = 2.0 Hz, 1H), 7.07 (dd, J = 2.0 Hz, 2.4 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 4.63 (s, 4H), 4.31 (s, 4H), 3.66 (s, 2H), 3.39 (s, 4H).
m/z: 444.2 (M+H)+
NAT-48
457.18 2-(2,3-dihydro-1,4-benzo dioxin-6-yl)-6-[3-fluoro-5-(6-oxa-2-azaspiro [3.4]octan-2-ylmethyl)-2-pyridyl] benzonitrile
1H NMR (400 MHz, DMSO-d6): d 8.53 (d, J = 8.0 Hz, 1H), 7.85 (t, J = 8.0 Hz, 1H), 7.67 (d, J = 8.0 Hz, 2H), 7.58 (t, J = 8.0 Hz,1H), 7.11 (d, J = 2.0 Hz, 1H), 7.07 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 4.31 (s, 4H), 3.74 (s, 4H), 3.64 (t, J = 8.0 Hz, 2H), 3.27 (s, 4H), 2.02 (t, J = 8.0 Hz, 2H). m/z: 458.2 (M+H)+
NAT-49
473.15 2-[3-chloro-5-(6-oxa-2-azaspiro[3.4] octan-2-yl methyl)-2-pyridyl]-6-(2,3-dihydro-1,4-benzodioxin-6-yl) benzonitrile
1H NMR (400 MHz, DMSO-d6): d 8.57 (s, 1H), 8.0 (s, 1H), 7.83 (t, J = 8.0 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.11-7.0 (m, 3H), 4.31 (s, 1H), 3.72 (s, 4H), 3.64 (t, J = 8.0 Hz, 2H), 3.22 (s, 4H), 2.03 (t, J = 8.0 Hz, 2H). m/z: 474.2 (M+H)+
NAT-50
459.13 2-[3-chloro-5-(2-oxa-6-aza spiro[3.3] heptan-6-yl methyl)-2-pyridyl]-6-(2,3-dihydro-1,4-benzodioxin-6-yl) benzonitrile
1H NMR (400 MHz, DMSO-d6): d 8.55 (s, 1H), 7.98 (s, 1H), 7.83 (t, J = 8.0 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.11 (d, J = 2.0 Hz, 1H), 7.07 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 4.62 (s, 4H), 4.31 (s, 4H), 3.64 (s, 2H), 3.38 (s, 4H), 3.31 (s, 2H).
m/z: 460.1 (M+H)+ and 462.1
NAT-51
480.16 2-[[2-chloro-6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-5-fluoro-3-pyridyl]methyl]-6-oxa-2-azaspiro[3.4]octane
1H-NMR (400 MHz, CDCl3): d 7.65 (d, J = 9.2 Hz, 1H), 7.31 – 7.28 (m, 3H), 6.90 (d, J = 8.4 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 6.82 – 6.79 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 4.29 (s, 4H), 3.89 (s, 2H), 3.81 (t, J = 7.2 Hz, 2H), 3.74 (s, 2H), 3.39 (s, 4H), 2.15 (t, J = 7.2 Hz, 2H), 2.10 (s, 3H).
m/z: 481.1 (M+H)+ and 483.1
NAT-52
466.15 6-[[2-chloro-6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-5-fluoro-3-pyridyl]methyl]-2-oxa-6-azaspiro[3.3]heptane
1H-NMR (400 MHz, CDCl3): d 7.59 (d, J = 9.2 Hz, 1H), 7.31 – 7.28 (m, 3H), 6.90 (d, J = 8.0 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 6.82 – 6.79 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 4.80 (s, 4H), 4.30 (s, 4H), 3.68 (s, 2H), 3.54 (s, 4H), 2.10 (s, 3H). m/z: 467.1 (M+H)+ and 469.2
NAT-53
476.19 2-[[2-chloro-6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-4-methyl-3-pyridyl]methyl]-6-oxa-2-azaspiro[3.4]octane
1H-NMR (400 MHz, CDCl3): d 7.30 – 7.22 (m, 3H), 7.16 (s, 1H), 6.89 (d, J = 8.4 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 6.81 – 6.78 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 4.30 (s, 4H), 3.83 (s, 2H), 3.82 (s, 2H), 3.76 (t, J = 7.2 Hz, 2H), 3.35 (s, 4H),2.45 (s, 3H), 2.19 (s, 3H), 2.09 – 2.04 (m, 2H). m/z: 477.1 (M+H)+ and 479.2
NAT-54
462.17 6-[[2-chloro-6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-4-methyl-3-pyridyl]methyl]-2-oxa-6-azaspiro[3.3]heptane
1H-NMR (400 MHz, CDCl3): d 7.28 – 7.27 (m, 3H), 7.16 (s, 1H), 6.90 (d, J = 8.0 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 6.81 – 6.78 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 4.75 (s, 4H), 4.30 (s, 4H), 3.77 (s, 2H), 3.52 (s, 4H), 2.47 (s, 3H), 2.18 (s, 3H), m/z: 463.1 (M+H)+ and 465.2
NAT-55
467.56 2-[5-(3,3a,4,5,6,6a-hexahydro-1H-cyclo penta[c]pyrrol-2-ylmethyl)-6-methoxy-2-pyridyl]-6-(2,3-dihydro-1,4-benzo dioxin-6-yl)benzo nitrile
1H-NMR (400 MHz, CDCl3): d 7.89 (d, J = 7.6 Hz, 1H), 7.72 – 7.69 (dd, J = 1.2 Hz, 8.0 Hz, 1H), 7.63 (t, J = 8.0 Hz, 1H), 7.45 – 7.43 (dd, J = 1.2 Hz, 7.6 Hz, 1H), 7.31 (d, J = 7.2 Hz, 1H), 7.10 – 7.07 (m, 2H), 6.97 (d, J = 8.0 Hz, 1H), 4.31 (s, 4H), 4.07 (s, 3H), 3.58 (s, 2H), 2.81 (t, J = 7.6 Hz, 2H), 2.62 (brs, 2H), 2.22 – 2.19 (m, 2H), 1.67 – 1.45 (m, 6H).
m/z: 468.2 (M+H)+
NAT-56
497.58 2-(2,3-dihydro-1,4-benzo dioxin-6-yl)-6-[6-methoxy-5-(2-oxa-8-azaspiro[4.5] decan-8-ylmethyl)-2-pyridyl] benzo nitrile
1H-NMR (400 MHz, CDCl3): d 7.79 (d, J = 7.6 Hz, 1H), 7.72 – 7.69 (dd, J = 1.6 Hz, 8.0 Hz, 1H), 7.63 (t, J = 8.0 Hz, 1H), 7.46 – 7.44 (dd, J = 1.2 Hz, 7.6 Hz, 1H), 7.32 (d, J = 7.6 Hz, 1H), 7.10 – 7.07 (m, 2H), 6.98 – 6.96 (dd, J = 0.4 Hz, 8.0 Hz, 1H), 4.32 (, 4H), 4.08 (s, 3H), 3.86 (t, J = 7.2 Hz, 2H), 3.57 (s, 2H), 3.55 (s, 2H), 2.50 (brs, 4H), 1.75 (t, J = 7.2 Hz, 2H), 1.66 – 1.63 (m, 4H).
m/z: 498.3 (M+H)+
NAT-57
509.01 8-[[2-chloro-6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-5-fluoro-3-pyridyl]methyl]-2-oxa-8-azaspiro[4.5]decane
1H-NMR (400 MHz, CDCl3): d 7.75 (d, J = 9.2 Hz, 1H), 7.31 – 7.28 (m, 3H), 6.90 (d, J = 8.2 Hz, 1H), 6.86 (s, 1H), 6.82 – 6.79 (dd, J = 1.6 Hz, 8.0 Hz, 1H), 4.30 (s, 4H), 3.89 (t, J = 7.2 Hz, 2H), 3.62 (s, 2H), 3.58 (s, 2H), 2.56 – 2.53 (m, 4H), 2.12 (s, 3H), 1.78 (t, J = 7.2 Hz, 2H), 1.69 (brs, 4H).
m/z: 509.2 (M+H)+ and 511.3
NAT-58
486.60 8-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methoxy-3-pyridyl]methyl]-2-oxa-8-azaspiro[4.5] decane
1H-NMR (400 MHz, CDCl3): d 7.70 (d, J = 7.2Hz, 1H), 7.40 – 7.38 (dd, J = 1.2 Hz, 7.2 Hz, 1H), 7.29 – 7.22 (m, 2H), 7.00 (d, J = 7.6 Hz, 1H), 6.92 – 6.89 (m, 2H), 6.85 – 6.82 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 4.31 (s, 4H), 3.97 (s, 3H), 3.85 (t, J = 7.2 Hz, 2H), 3.56 (s, 2H), 3.54 (s, 2H), 2.51 – 2.49 (m, 4H), 2.27 (s, 3H), 1.75 (t, J = 7.2 Hz, 2H), 1.66 – 1.63 (m, 4H). m/z: 487.2 (M+H)+
NAT-59
456.58 2-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methoxy-3-pyridyl]methyl]-3,3a,4,5,6,6a-hexahydro-1H-cyclopenta[c]pyrrole
1H-NMR (400 MHz, CDCl3): d 7.70 (d, J = 7.6 Hz, 1H), 7.40 – 7.38 (dd, J = 1.6 Hz, 7.6 Hz, 1H), 7.29 – 7.22 (m, 2H), 7.00 (d, J = 7.2 Hz, 1H), 6.92 – 6.89 (m, 2H), 6.85 – 6.82 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 4.31 (s, 4H), 3.97 (s, 3H), 3.58 (s, 2H), 2.85 (t, J = 8.0 Hz, 2H), 2.62 (brs, 2H), 2.27 (s, 3H), 2.19 – 2.15 (m, 2H), 1.69 – 1.61 (m, 4H), 1.51 – 1.45 (m, 2H). m/z: 457.2 (M+H)+
NAT-60
507.02 8-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl)phenyl]-2-methoxy-3-pyridyl]methyl]-2-oxa-8-azaspiro[4.5] decane
1H-NMR (400 MHz, CDCl3): d 7.72 (d, J = 7.6 Hz, 1H), 7.54 – 7.52 (dd, J =2.0 Hz, 7.2 Hz, 1H), 7.37 – 7.30 (m, 2H), 7.22 – 7.20 (d, J = 7.2 Hz, 1H), 6.99 (d, J = 1.2 Hz, 1H), 6.94 – 6.93 (m, 2H), 4.31 (s, 4H), 3.99 (s, 3H), 3.85 (t, J = 7.2 Hz, 2H), 3.56 (s, 2H), 3.54 (s, 2H), 2.51 – 2.49 (m, 4H), 1.75 (t, J = 7.2 Hz, 2H), 1.66 – 1.63 (m, 4H). m/z: 507.1 (M)+ and 509.2
NAT-61
476.99 2-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl)phenyl]-2-methoxy-3-pyridyl]methyl]-3,3a,4,5,6,6a-hexahydro-1H-cyclopenta[c]pyrrole
1H-NMR (400 MHz, CDCl3): d 7.72 (d, J = 7.6 Hz, 1H), 7.54 – 7.52 (dd, J = 2.0 Hz, 7.2 Hz, 1H), 7.37 – 7.29 (m, 2H), 7.20 (d, J = 7.6 Hz, 1H), 6.99 – 6.91 (m, 3H), 4.30 (s, 4H), 3.99 (s, 3H), 3.58 (s, 2H), 2.83 (t, J = 7.6 Hz, 2H), 2.62 (brs, 2H), 2.20 – 2.17 (m, 2H), 1.71 – 1.63 (m, 4H), 1.51 – 1.45 (m, 2H). m/z: 477.3 (M+H)+ and 479.2
NAT-62
521.05 8-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl)phenyl]-2-methoxy-4-methyl-3-pyridyl]methyl]-2-oxa-8-azaspiro[4.5]decane
1H NMR: (CDCl3, 400 MHz) d7.52 (d, J = 7.6 Hz, 2.0 Hz, 1H), 7.36-7.29 (m, 2H), 7.04 (s, 1H), 6.99 (d, J = 2.0 Hz, 1H), 6.94-6.93 (m, 2H), 4.31 (s, 4H), 3.95 (s, 3H), 3.84 (t, J = 7.2 Hz, 2H), 3.55 (s, 2H), 3.52 (s, 2H), 2.45 (s, 4H), 2.43 (s, 3H), 1.73 (t, J = 7.2 Hz, 2H), 1.57 (s, 4H). m/z: 521.1 (M)+ and 523.2
NAT-63
491.02 2-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl)phenyl]-2-methoxy-4-methyl-3-pyridyl]methyl]-3,3a,4,5,6,6a-hexahydro-1H-cyclopenta[c]pyrrole
1H NMR: (CDCl3, 400 MHz) d 7.51 (d, J = 7.2 Hz, 2.0 Hz, 1H), 7.35-7.28 (m, 2H), 7.02 (s, 1H), 6.99 (d, J = 2.0 Hz, 1H), 6.94-6.92 (m, 2H), 4.30 (s, 4H), 3.95 (s, 3H), 3.59 (s, 2H), 2.74-2.53 (m, 4H), 2.42 (s, 3H), 2.20-2.17 (m, 2H), 1.68-1.25 (m, 6H). m/z: 491.2 (M)+ and 493.2
NAT-64
439.51 2-(2,3-dihydro-1,4-benzodioxin-6-yl)-6-[3-methyl-5-(2-oxa-6-azaspiro[3.3]hept an-6-ylmethyl)-2-pyridyl] benzonitrile
1H NMR (400 MHz, DMSO-d6): d 8.37 (s, 1H), 7.79 (t, J = 8.0 Hz, 1H), 7.66 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.11 (d, J = 2.0 Hz, 1H), 7.07 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.0 (d, J = 8.0 Hz, 1H), 4.62 (s,4H), 4.30 (s, 4H), 3.57 (s, 2H), 3.35 (s, 4H), 2.20 (s, 3H). m/z: 440.2 (M+H)+
NAT-65
503.98 2-[6-chloro-4-methoxy-5-(6-oxa-2-azaspiro[3.4]octan-2-ylmethyl)-2-pyridyl]-6-(2,3-dihydro-1,4-benzo dioxin-6-yl)benzonitrile
1H NMR (400 MHz, DMSO-d6): d 7.85-7.78 (m, 2H), 7.64 (d, J = 8.0 Hz, 1H), 7.60 (s, 1H), 7.14 (d, J = 2.4 Hz, 1H), 7.09 (dd, J = 8.0 Hz, 2.4 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 4.31 (s, 4H), 3.98 (s, 3H), 3.69 (s, 4H), 3.61 (t, J = 8.0 Hz, 2H), 3.27-3.26 (m, 4H), 1.98 (s, 2H). m/z: 504.2 (M+H)+ and 506.2
NAT-66
439.19 2-(2,3-dihydro-1,4-benzodioxin-6-yl)-6-[4-methyl-5-(2-oxa-6-azaspiro[3.3] hept an-6-ylmethyl)-2-pyridyl] benzo nitrile
1H NMR (400 MHz, DMSO-d6): d 8.37 (s, 1H), 7.79 (t, J = 8.0 Hz, 1H), 7.66 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.11 (d, J = 2.0 Hz, 1H), 7.07 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.0 (d, J = 8.0 Hz, 1H), 4.62 (s,4H), 4.30 (s, 4H), 3.57 (s, 2H), 3.35 (s, 4H), 2.20 (s, 3H).
m/z: 440.2 (M+H)+
NAT-67
481.59 2-(2,3-dihydro-1,4-benzodioxin-6-yl)-6-[6-methyl-5-(2-oxa-8-azaspiro[4.5] decan-8-ylmethyl)-2-pyridyl] benzonitrile
1H NMR (400 MHz, DMSO-d6): d 7.81-7.75 (m, 3H), 7.64 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 7.07 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.0 (d, J = 8.0 Hz, 1H), 4.31 (s, 4H), 3.72 (t, J = 8.0 Hz, 2H), 3.51 (s, 2H), 3.43 (s, 2H), 2.57 (s, 3H), 2.40 (s, 4H), 1.68 (t, J = 8.0 Hz, 2H), 1.54-1.51 (m, 4H). m/z: 482.2 (M+H)+
NAT-68
451.56 2-[5-(3,3a,4,5,6,6a-hexahydro-1H-cyclopenta[c]pyrrol-2-ylmethyl)-6-methyl-2-pyridyl]-6-(2,3-dihydro-1,4-benzo dioxin-6-yl)benzonitrile
1H NMR (400 MHz, DMSO-d6): d 7.81-7.74 (m, 3H), 7.64 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 7.07 (dd, J = 8.0 Hz, 2.4 Hz, 1H), 7.0 (d, J = 8.0 Hz, 1H), 4.31 (s, 4H), 3.56 (s, 2H), 2.56 (s, 5H), 2.24 (t, J = 7.6 Hz, 2H), 1.65-1.62 (m, 3H), 1.43-1.30 (m, 3H). m/z: 491.2 (M+H)+ and 493.2
NAT-69
471.98 2-[5-(3,3a,4,5,6,6a-hexahydro-1H-cyclopenta[c]pyrrol-2-ylmethyl)-3-chloro-2-pyridyl]-6-(2,3-dihydro-1,4-benzodioxin-6-yl)benzonitrile
1H NMR (400 MHz, DMSO-d6): d 8.59 (s, 1H), 8.02 (s, 1H), 7.84 (t, J = 8.0 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.11 (d, J = 2.0 Hz, 1H), 7.07 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 4.31 (s, 4H), 3.64 (s, 2H), 2.63 (t, J = 8.0 Hz, 2H), 2.19-2.16 (m, 2H), 1.65-1.60 (m, 3H), 1.39-1.37 (m, 3H). m/z: 472.3 (M+H)+ and 474.3
NAT-70
462.17 2-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl)phenyl]-4-methyl-3-pyridyl]methyl]-6-oxa-2-azaspiro[3.4] octane
1H NMR: (CDCl3, 400 MHz): d 8.50 (s, 1H), 7.44 (dd, J = 6.8 Hz, 2.4 Hz, 1H), 7.38-7.31 (m, 3H), 6.98 (s, 1H), 6.92 (d, J = 2.0 Hz, 2H), 4.30 (s, 4H), 3.85 (s, 2H), 3.78 (t, J = 7.2 Hz, 2H), 3.64 (s, 2H), 3.27 (s, 4H), 2.40 (s, 3H), 2.10 (t, J = 7.2 Hz, 2H). m/z: 463.2 (M+H)+ and 465.2
NAT-71
460.19 2-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl)phenyl]-2-methyl-3-pyridyl]methyl]-3,3a,4,5,6,6a-hexahydro-1H-cyclopenta[c]pyrrole
1H NMR: (CDCl3, 400 MHz): d 7.66 (d, J = 8.0 Hz, 1H), 7.46 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.38-7.29 (m, 3H), 6.97 (s, 1H), 6.91 (d, J = 2.0 Hz, 2H), 4.29 (s, 4H), 3.55 (s, 2H), 2.67-2.58 (m, 7H), 2.26 (d, J = 6.0 Hz, 2H), 1.68-1.41 (m, 6H). m/z: 461.2 (M+H)+ and 463.2
NAT-72 472.24 2-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methoxy-phenyl]-4-methoxy-3-pyridyl]methyl]-3,3a,4,5,6,6a-hexa hydro-1H-cyclopenta[c] pyrrole
1H NMR: (CDCl3, 400 MHz): d 8.52 (s, 1H), 7.70 (dd, J = 7.6 Hz, 2.0 Hz, 1H), 7.42 (s, 1H), 7.33 (dd, J = 7.6 Hz, 2.4 Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.14 (d, J = 2.0 Hz, 1H), 7.09 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 4.30 (s, 4H), 3.89 (s, 3H), 3.61 (s, 2H), 3.30 (s, 3H), 2.87-2.60 (m, 4H), 2.16-2.13 (m, 2H), 1.62-1.44 (m, 6H).
m/z: 473.3 (M+H)+
NAT-73
458.22 2-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-4-methoxy-3-pyridyl]methyl]-6-oxa-2-azaspiro [3.4] octane
1H-NMR (400 MHz, CDCl3): d 8.43 (s, 1H), 7.32 – 7.24 (m, 3H), 6.91 – 6.87 (m, 3H), 6.83 – 6.80 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 4.30 (s, 4H), 3.89 (s, 3H), 3.85 (s, 2H), 3.78 (t, J = 7.2 Hz, 2H), 3.67 (s, 2H), 3.37 – 3.33 (m, 4H), 2.17 (s, 3H), 2.13 (t, J = 7.2 Hz, 2H).
m/z: 459.4 (M+H)+
NAT-74
444.2 6-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-4-methoxy-3-pyridyl]methyl]-2-oxa-6-azaspiro[3.3] heptane
1H-NMR (400 MHz, CDCl3): d 8.38 (s, 1H), 7.32 – 7.23 (m, 3H), 6.92 – 6.87 (m, 2H), 6.87 (d, J = 1.6 Hz, 1H), 6.83 – 6.81 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 4.77 (s, 4H), 4.31 (s, 4H), 3.89 (s, 3H), 3.60 (s, 2H), 3.49 (s, 4H), 2.17 (s, 3H).
m/z: 445.3 (M+H)+
NAT-75
440.57 2-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-4-methyl-3-pyridyl]methyl]-3,3a,4,5,6,6a-hexahydro-1H-cyclopenta[c]pyrrole
1H-NMR (400 MHz, CDCl3): d 8.47 (s, 1H), 7.32 – 7.30 (dd, J = 2.0 Hz, 7.6 Hz, 1H), 7.28 – 7.19 (m, 3H), 6.91 – 6.87 (m, 2H), 6.83 – 6.81 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 4.30 (s, 4H), 3.55 (s, 2H), 2.64 – 2.56 (m, 4H), 2.43 (s, 3H), 2.26 – 2.24 (m, 2H), 2.18 (s, 3H), 1.68 – 1.62 (m, 4H), 1.46 – 1.39 (m, 2H).
m/z: 441.3 (M+H)+
NAT-76
502 2-[6-chloro-5-(2-oxa-8-azaspiro[4.5] decan-8-ylmethyl)-2-pyridyl]-6-(2,3-dihydro-1,4-benzodioxin-6-yl) benzonitrile
1H NMR (400 MHz, DMSO-d6): d 8.0 (d, J = 8.0 Hz, 1H), 7.76-7.73 (m, 2H), 7.67 (t, J = 8.0 Hz, 1H), 7.49 (dd, J = 8.0 Hz, 1.2 Hz, 1H), 7.09-7.05 (m, 2H), 6.97 (d, J = 8.0 Hz, 1H), 4.31 (s, 4H), 3.86 (t, J = 8.0 Hz, 2H), 3.65 (s, 2H), 3.58 (s, 2 H), 2.52 (s, 4H), 1.76 (t, J = 8.0 Hz, 2H), 1.70-1.65 (m, 4H).
m/z: 502.2 (M)+ and 504.2
NAT-77
502 2-[5-(3,3a,4,5,6,6a-hexahydro-1H-cyclopenta[c]pyrrol-2-ylmethyl)-6-chloro-4-methoxy-2-pyridyl]-6-(2,3-dihydro-1,4-benzodioxin-6-yl)benzo nitrile
1H NMR (400 MHz, CDCl3): d 7.81 (dd, J = 8.0 Hz, 1.2 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 7.49 (dd, J = 8.0 Hz, 1.2 Hz, 1H), 7.35 (s, 1H), 7.08-7.05 (m, 2H), 6.97 (d, J = 8.0 Hz, 1H), 4.31 (s, 4H), 3.97 (s, 3H), 3.78 (s, 2H), 2.93 (t, J = 8.0 Hz, 2H), 2.57 (s, 2H), 2.18-2.15 (m, 2H), 1.60-1.47 (m, 6H).
m/z: 502.3 (M)+ and 504.2
NAT-78 499.39 6-[[4-chloro-6-[2-chloro-3-(2,3-dihydro-1,4-benzo dioxin-6-yl)phenyl]-2-methoxy-3-pyridyl]methyl]-2-oxa-6-azaspiro[3.3]Heptane
1H NMR (400 MHz, DMSO-d6): d 7.55 (dd, J = 8.0 Hz, 1.6 Hz, 1H), 7.47 (t, J = 8.0 Hz, 1H), 7.42 (dd, J = 8.0 Hz, 1.6 Hz, 1H), 7.35 (s, 1H), 6.95-6.93 (m, 2H), 6.90 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 4.58 (s, 4H), 4.28 (s, 4H), 3.90 (s, 3H), 3.62 (s, 2H), 3.40 (s, 4H).
m/z: 499.3 (M)+ and 501.3
NAT-79 513.41 2-[[4-chloro-6-[2-chloro-3-(2,3-dihydro-1,4-benzo dioxin-6-yl)phenyl]-2-methoxy-3-pyridyl]methyl]-6-oxa-2-azaspiro[3.4]octane
1H NMR (400 MHz, DMSO-d6): d 7.55 (dd, J = 8.0 Hz, 1.6 Hz, 1H), 7.47 (t, J = 8.0 Hz, 1H), 7.42 (dd, J = 8.0 Hz, 1.6 Hz, 1H), 7.35 (s, 1H), 6.95-6.88 (m, 3H), 4.28 (s, 4H), 3.91 (s, 3H), 3.68-3.59 (m, 6H), 3.25 (s, 4H), 1.97 (t, J = 6.8 Hz, 2H).
m/z: 513.2 (M)+ and 515.2
NAT-80
541.47 8-[[4-chloro-6-[2-chloro-3-(2,3-dihydro-1,4-benzo dioxin-6-yl)phenyl]-2-methoxy-3-pyridyl]methyl]-2-oxa-8-aza spiro[4.5]decane
1H NMR (400 MHz, DMSO-d6): d 7.57 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.47 (t, J = 8.0 Hz, 1H), 7.43 (dd, J = 8.0 Hz, 1.6 Hz, 1H), 7.37 (s, 1H), 6.95-6.93 (m, 2H), 6.90 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 4.29 (s, 4H), 3.91 (s, 3H), 3.70 (t, J = 7.2 Hz, 2H), 3.56 (s, 2H), 3.40 (s, 2H), 2.43 (s, 4H), 1.65 (t, J = 7.2 Hz, 2H), 1.48-1.46 (m, 4H).
m/z: 541.2 (M)+ and 543.2
NAT-81
511.44 2-[[4-chloro-6-[2-chloro-3-(2,3-dihydro-1,4-benzo dioxin-6-yl)phenyl]-2-methoxy-3-pyridyl]methyl]-3,3a,4,5,6,6a-hexahydro-1H-cyclopenta [c]pyrrole
1H NMR (400 MHz, DMSO-d6): d 7.56 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.47 (t, J = 8.0 Hz, 1H), 7.42 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.35 (s, 1H), 6.95-6.93 (m, 2H), 6.90 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 4.29 (s, 4H), 3.90 (s, 3H), 3.65 (s, 2H), 2.72-2.68 (m, 2H), 2.66 (s, 4H), 2.17-2.13 (m, 2H), 1.57-1.40 (m, 6H).
m/z: 511.2 (M)+ and 513.2

In particular, the compound may be selected from:
, , , , , , , , , , , ,
, , , , , , , , , , , ,

, or ,
or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof.

Methods of treatment
The compounds of the present invention are therapeutically useful. Accordingly, the invention provides a method of therapy which comprises administering to a subject a compound of formula (I) as defined herein, or a prodrug, hydrate, solvate or pharmaceutically acceptable salt thereof. Similarly, the invention provides a compound of formula (I) as defined herein, or a prodrug, hydrate, solvate or pharmaceutically acceptable salt thereof, for use in treating the human or animal body.
In particular, the compounds of the invention are capable of interfering with PD-1/PD-L1 interaction, and consequently may be useful in treating conditions associated with PD-1 and/or PD-L1 function. Similarly, the compounds of the invention are capable of enhancing T-cell function, and so may be useful in treating disorders where enhanced T-cell function is desirable. Exemplary disorders of this type include cancer, neurodegenerative diseases and infectious diseases.
The compounds may additionally interfere with an additional checkpoint pathway, such as the VISTA checkpoint pathway. Accordingly, the compounds of the invention may be useful in interfering with an interaction between (i) PD-1 and PD-L1 and/or (ii) VISTA and VSIG-3. Preferably the compounds of the invention are useful in interfering with the PD-1/PD-L1 pathway and the VISTA/VSIG-3 pathway. By “interfering with” or “disrupting” is typically meant that the compound of the invention binds to one of the components of the pathway. For instance, the compound of the invention may bind to PD-1, PD-L1, VISTA or VSIG-3.
For example, the compounds of the invention may be useful in treating a disorder associated with overexpression of PD-L1, such as a cancer associated with overexpression of PD-L1. Such cancers are known to the skilled person and particularly include brain cancer, non-small cell lung cancer, Triple negative breast cancer, bladder cancer, gall bladder cancer, liver cancer, and thyroid cancer.
Similarly, the compounds of the invention may be useful in treating a disorder associated with overexpression of VISTA, such as a cancer associated with overexpression of VISTA.

Accordingly, the invention provides a method of treating, preventing and/or ameliorating disease, the method comprising administering a compound of formula (I) as defined herein, or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition to a subject.
The method typically comprises inhibiting the PD-1/PD-L1 checkpoint pathway.
Alternatively or additionally, the method may comprise inhibiting the activity of VISTA. For instance, the method may comprise interfering in the interaction between VISTA and VSIG-3.
The compounds of the invention have been shown to reverse T-cell exhaustion. For instance, they have been shown to have efficacy in one or more of: increasing T-cell function, increasing CD8+ T-cell population; decreasing expression of exhaustion markers PD-L1 and TIM3, increasing expression of CD107a, increasing expression of Granzyme B, and increasing expression of IFNG. Accordingly, the above method may comprise one or more of:
(i) inhibit the PD-1/PD-L1 checkpoint pathway;
(ii) inhibit the activity of VISTA;
(iii) reverse T-cell exhaustion;
(iv) increase T-cell function;
(v) decrease expression of PD-1 in exhausted T cells;
(vi) decrease expression of TIM3 exhausted T cells;
(vii) increase CD8+ T-cell population;
(viii) increase expression of CD107a;
(ix) increase expression of Granzyme-B; and/or
(x) IFNg restoration.
(xi) Increasing T cell mediated cytotoxicity to tumour cells

In practice, the method of therapy outlined above is typically a method of treating, ameliorating or preventing a disorder selected from a proliferative disorder, cancer, a neurodegenerative disorder, an autoimmune disorder or an infectious disorder.
The compounds of the invention have activity in interfering with the PD-1/PD-L1 pathway. Accordingly, it is preferred that the method is a method of treating, ameliorating or preventing a disorder associated with overexpression of PD-L1. Particularly preferably, the disorder is a cancer associated with overexpression of PD-L1. Exemplary cancers include brain cancer, breast cancer, lung cancer, renal cancer, bladder cancer, thyroid cancer, liver cancer, and gall bladder cancer. Most preferably the cancer is selected from breast cancer, brain cancer, thyroid cancer and lung cancer.
Alternatively, the disorder may be an infectious disorder such as, for instance, any of HIV, Septic shock, and Hepatitis A, B, C or D.
The subject may have or be at risk of developing a disorder as described above. For instance, the subject may have or be at risk of developing a proliferative disorder, cancer, a neurodegenerative disorder, or an infectious disorder.

The compounds of the invention have efficacy as discussed herein when taken alone. However, it would also be possible to utilize these compounds in a method of co-therapy with another therapeutic agent. Accordingly, in an aspect, the method described herein additionally comprises administering to the subject a further therapeutic agent.
The another therapeutic agent may be, for instance, another small molecule or a monoclonal antibody. Preferably, the another therapeutic agent may be an inhibitor of the PD-1/PD-L1 pathway, and/or an inhibitor of the VISTA pathway (such as an inhibitor of the interaction between VISTA and VSIG-3).
In a preferred embodiment, the further therapeutic agent is selected from Nivolumab, Pembrolizumab, Avelumab, Atezolizumab, Cemiplimab and Durvalumab. These are monoclonal antibodies currently approved for the treatment of disorders by interfering with the PD-1/PD-L1 pathway. For instance, the method may be a method of treating, ameliorating and/or preventing a disorder by administering to a subject (i) a compound of formula (I) as defined herein, or a prodrug, hydrate, solvate or pharmaceutically acceptable salt thereof, and (ii) at least one of Nivolumab, Pembrolizumab, Avelumab, Atezolizumab, Cemiplimab and Durvalumab.
The two or more aforementioned therapeutic agents may be administered simultaneously or sequentially.

The invention provides a compound of formula (I), or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined herein, or a pharmaceutical composition, for use in any of the above-identified methods. For instance, the invention provides a compound of formula (I), or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined herein, or a pharmaceutical composition, for use in a method of treating, ameliorating or preventing a disorder associated with overexpression of PD-L1. Particularly preferably, the disorder is a cancer associated with overexpression of PD-L1 selected from brain cancer, breast cancer, lung cancer, renal cancer, bladder cancer, thyroid cancer, liver cancer, and gall bladder cancer.
The invention also provides another therapeutic agent, for use in a method of co-therapy with the compound of formula (I) as defined herein. For instance, the invention provides:
Nivolumab, for use in a method of therapy comprising administering to a subject (i) a compound of formula (I), or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined herein, and (ii) Nivolumab. Typically the method is a method of treating, ameliorating or preventing a disorder associated with overexpression of PD-L1. Particularly preferably, the disorder is a cancer associated with overexpression of PD-L1 selected from brain cancer, breast cancer, lung cancer, renal cancer, bladder cancer, thyroid cancer, liver cancer, and gall bladder cancer.
Similarly, the invention provides Pembrolizumab, for use in a method of therapy comprising administering to a subject (i) a compound of formula (I), or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined herein, and (ii) Pembrolizumab.
Similarly, the invention provides Avelumab, for use in a method of therapy comprising administering to a subject (i) a compound of formula (I), or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined herein, and (ii) Avelumab.
Similarly, the invention provides Atezolizumab, for use in a method of therapy comprising administering to a subject (i) a compound of formula (I), or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined herein, and (ii) Atezolizumab.
Similarly, the invention provides Cemiplimab, for use in a method of therapy comprising administering to a subject (i) a compound of formula (I), or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined herein, and (ii) Cemiplimab.
Similarly, the invention provides Durvalumab, for use in a method of therapy comprising administering to a subject (i) a compound of formula (I), or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined herein, and (ii) Durvalumab.

The invention also provides the use of a compound of formula (I), or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the manufacture of a medicament for use in a method as described above.

In vitro methods

The above-discussed therapeutic applications typically concern in vivo methods of using the compounds described herein. However, the compounds of formula (I), and prodrugs, hydrates, solvates and pharmaceutically acceptable salts thereof, are also useful in in vitro methods.
For instance, these compounds are particularly useful in assays concerning PD-1/PD-L1 binding. Flow cytometry assays are particularly preferred.

Accordingly, the invention also provides an in vitro method which comprises using a compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined herein to:
(i) inhibit the PD-1/PD-L1 checkpoint pathway;
(ii) inhibit the activity of VISTA;
(iii) reverse T-cell exhaustion;
(iv) increase T-cell function;
(v) decrease expression of PD-1 in exhausted T cells;
(vi) decrease expression of TIM3 exhausted T cells;
(vii) increase CD8+ T-cell population;
(viii) increase expression of CD107a;
(ix) increase expression of Granzyme-B; and/or
(x) IFNg restoration.
(xi) Increasing T cell mediated cytotoxicity to tumour cells

EXAMPLES

The invention will now be described with reference to synthetic examples, a computational example, and activity examples. It is to be understood that the invention is not necessarily limited in its application to the details set forth in the following examples. The skilled person will appreciate that the invention can be practiced or carried out in other ways. Modifications of the present examples which fall within the meaning and range of equivalency of the claims are therefore intended to be embraced within them.

Materials and methods
Cell Lines, Growth Conditions and Reagents:
MDA-MB-231, HCC827, U251 were all originally obtained from ATCC and maintained as per the instructions.
CHO-PDL1, CHO-VISTA, CHO-PD1 cell line was purchased from Abeomics INC-USA and maintained according to the manufacturer's instructions.
All cell lines were routinely screened for mycoplasma contamination.
PD-1/PD-L1 binding assay kit was purchased from Cisbio Assays.
Human IFN-gamma GenLISA™ ELISA 96 wells KIT was purchased from Krishgen Biosystems, India)
Paraformaldehyde 4% (v/v), PD-L1 recombinant human protein and Invitrogen SYPRO® orange (5000× solution) was purchased from ThermoFisher Scientific.
MTT® reagent, bovine serum albumin (BSA) and Ficoll® Paque Plus was purchased from Sigma-Aldrich.
BMS202 was purchased from Selleckchem (Houston, USA).
Anti-CD3 and anti-CD28 monoclonal antibodies were acquired from Invitrogen
Recombinant human IFN-? and recombinant human IL-2 were purchased from Peprotech. Fluorochrome-labeled antibodies for flow cytometry were purchased from Invitrogen, Thermofischer.
Computational example:
In Silico identification of PD-1/PD-L1 and VISTA dual Small-Molecule Inhibitors
Inventors have employed in-silico analysis starts from hPD-L1 IgV domain (5N2F) and based on strategy of virtual screening, free energy of the binding Molecular mechanics with generalized Born and surface area solvation (MM/GBSA), Molecular dynamics (MD) simulations using Schrödinger software, discovers active small molecules targeting PD-L1as shown in Fig. 1A.
Due to non-availability of VISTA crystal co-ordinates with small molecule inhibitor; inventors started the study from the defining of binding site based on mutation data and literature at Extra cellular domain (ECD) of VISTA (6MVL) followed by virtual screening, free energy of the binding Molecular mechanics with generalized Born and surface area solvation (MM/GBSA) and Molecular dynamics (MD) simulations using Schrödinger software, discovers a further active small molecule as shown in Fig 1B.
A collection of approximately 5000 novel structures were proposed and screened using molecular docking into the PD-L1 dimer interface. Structure-based virtual screening for the identification of PD-1/PD-L1 and VISTA small-molecule inhibitors was performed as shown in FIG. 2. beginning with pre-filtering the compounds on the basis of Lipinski’s rule of 5, followed by the screening in silico using the XP scoring function of GLIDE module of Schrodinger software, followed by the free energy binding using PRIME, molecular dynamics using DESMOND. Shortlisted the molecules after visual inspection and ADME filtering of the top-ranked compounds within the binding pocket.
Only the compounds that presented favourable binding conformations and the interaction with key binding site amino acid residues were retained for further studies

Synthetic examples

All compounds of the invention can be synthesized according to the method of route 1, below, in which the final step is reductive amination reaction of the corresponding starting material. The method of route 1 employs a substituted benzaldehyde and appropriate amine.
Following are the synthetic schemes for the compounds of inventions:

NAT-1 (example of route 1)

Example-1: Preparation of 6-chloro-2-methoxy-pyridine-3-carbaldehyde (Intermediate-3)

Stage-1: Preparation of 6-chloro-2-methoxy-pyridine-3-carboxylic acid (Intermediate-1)

Dissolved 2,6-dichloropyridine-3-carboxylic acid (40.0 g, 0.208 mole) in tetrahydrofuran (160 mL) and methanol (80 mL) at room temperature. Charged lithium hydroxide monohydrate (26.18 g, 0.624 mole) to the reaction mixture at room temperature for 20 h. The completeness of the reaction confirmed by TLC. The solvent completed distilled under reduced pressure. The crude mass was diluted with water and acidified (pH 4-5) with 2N hydrochloric acid. Filtered the solid and dried in tray dried at 55°C to afford 39.0 g of compound INT-1. 1H-NMR (DMSO-d6, 400 MHz): d 13.24 (brs, 1H), 8.15 (d, J = 8.0 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H), 3.93 (s, 3H).

Stage-2: Preparation of (6-chloro-2-methoxy-3-pyridyl) methanol (Intermediate-2)

Dissolved compound INT-I (38.0 g, 0.2025 mole) in tetrahydrofuran (380.0 mL) at room temperature under nitrogen atm. Charged Lithium aluminum hydride 2.4M in tetrahydrofuran solution (101.2 mL, 0.243 mole) at 0-5 °C to the reaction mixture. The reaction mixture was stirred for 7.30 hours at room temperature. The completeness of the reaction confirmed by TLC. The reaction mixture cooled to 0 °C and quenched with aqueous ammonium chloride. The reaction mixture raised to room temperature and filtered. Charged ethyl acetate to the filtrate, separated the organic layer and distilled under reduced pressure to afford 29.4 g of compound INT-2. 1H-NMR (CDCl3, 400 MHz): d 7.55 (d, J = 7.2 Hz, 1H), 6.91 (d, J = 7.6 Hz, 1H), 4.63 (s, 2H), 3.99 (s, 3H), 2.13 (brs, 1H).

Stage-3: Preparation of 6-chloro-2-methoxy-pyridine-3-carbaldehyde (Intermediate-3)

Charged compound INT-2 (15.0 g, 0.0866) in dichloromethane (300.0 mL) at room temperature. Charged dess martin periodinane (43.9 g, 0.1036) at room temperature and stirred for 4 hours. The completeness of the reaction confirmed by TLC. The reaction mixture was quenched with 1N sodium hydroxide, extracted in Dichloromethane. The organic layer distilled under reduced pressure to give crude compound. Which was purified by silica gel column chromatography (2-5% ethyl acetate in hexane) to afford 13.3 g of Compound INT-3. 1H-NMR (CDCl3, 400 MHz): d 10.30 (d, J = 0.8 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.03 (d, J1 = 0.4 Hz J2 = 7.6 Hz, 1H), 4.09 (s, 3H).

Example-2: Preparation of 6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methoxy-pyridine-3-carbaldehyde (Intermediate-7)

Stage-1: Preparation of 2-(2,3-dihydro-1,4-benzodioxin-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Intermediate-4)

Charged 6-bromo-2,3-dihydro-1,4-benzodioxine (100.0 g, 0.4650 mole) and Bis(pinacolato)diboron (141.7 g, 0.5580 mole) was dissolved in 1,4-doxane (1000.0 mL) at room temperature. The reaction mixture de-gasified for 10 min at room temperature. Charged potassium acetate (136.9 g, 1.395 mole) followed by [1,1'-Bis (diphenylphosphino) ferrocene] dichloropalladium(II) DCM complex (18.97 g, 0.023 mole). De-gasified the reaction mixture for 10 min at room temperature. The reaction mixture stirred at 100°C for 6 hours. The completeness of the reaction confirmed by TLC. The reaction mixture cooled to room temperature and filtered through cilite bed. The filtrate diluted with water and extracted with ethyl acetate. The organic layer distilled under reduced pressure. The crude purified by silica gel column chromatography (0-5% ethyl acetate in hexane) to afford 124 g of Compound INT-4. 1H-NMR (CDCl3, 400 MHz): d 7.32 – 7.28 (m, 2H), 6.85 (d, J = 8.0 Hz, 1H), 4.27 – 4.23 (m, 4H), 1.32 (s, 9H).
Stage-2: Preparation of 6-(3-bromo-2-methyl-phenyl)-2,3-dihydro-1,4-benzodioxine (Intermediate-5)

Charged compound INT-4 (18.0 g, 0.0686 mole) and 1,3-dibromo-2-methyl- benzene (18.87 g, 0.0755 mole) into Tetrahydrofuran (360mL) at room temperature. The reaction mixture was de-gasified for 10 minutes at room temperature. Charged potassium carbonate (28.40 g, 0.2058 mole), DM water (50.0 mL) followed by [1,1'-Bis (diphenylphosphino) ferrocene] dichloropalladium(II) DCM complex (2.8 g, 0.00343 mole). De-gasified the reaction mixture for 10 min at room temperature. The reaction mixture was stirred at 62°C for 10 hours. The completeness of the reaction was confirmed by TLC. The reaction mixture was brought to room temperature and filtered through celite bed. The filtrate was diluted with DM water and extracted with ethyl acetate. The organic layer was distilled under reduced pressure and the crude was purified by silica gel column chromatography (10% ethyl acetate in hexane) to afford 12.8 g of Compound INT-5. 1H-NMR (DMSO-d6, 400 MHz): d 7.59 – 7.57 (dd, J1 = 2.4 Hz J2 = 7.2 Hz, 1H), 7.19 – 7.13 (m, 2H), 6.91 (d, J = 8.4 Hz, 1H), 6.81 (d, J = 2.0 Hz, 1H), 6.77 – 6.75 (dd, J1 = 2.0 Hz J2 = 8.0 Hz, 1H), 4.28 (s, 4H), 2.26 (s, 3H).

Stage-3: Preparation of 2-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl] -4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Intermediate-6)

Charged Compound INT-5 (12.8 g, 0.0419 mole) and Bis(pinacolato)diboron (12.78 g, 0.0503 mole) into 1,4-doxane (200 mL) at room temperature. The reaction mixture was de-gasified for 10 minutes at room temperature. Charged potassium acetate (12.33 g, 0.1257 mole) followed by [1,1'-Bis (diphenylphosphino) ferrocene] dichloropalladium(II) DCM complex (1.71 g, 0.00209 mole). De-gasified the reaction mixture for 10 min at room temperature. The reaction mixture was stirred at 100°C for 3 hours. The completeness of the reaction was confirmed by TLC. The reaction mixture was brought to room temperature and filtered through celite bed. The filtrate was diluted with DM water and extracted with ethyl acetate. The organic layer was distilled under reduced pressure and the crude was purified by silica gel column chromatography (0-5% ethyl acetate in hexane) to afford 14.0 g of Compound INT-6. 1H-NMR (CDCl3, 400 MHz): d 7.74 – 7.71 (dd, J1 = 2.0 Hz J2 = 7.6 Hz, 1H), 7.26 – 7.24 (m, 1H), 7.19(t, J = 7.2 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H), 6.80 (d, J = 2.0 Hz, 1H), 6.76 – 6.73 (dd, J1 = 2.0 Hz J2 = 8.0 Hz, 1H), 4.29 (s, 4H), 2.43 (s, 3H), 1.35 (s, 9H).

Stage-4: Preparation of 6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methoxy-pyridine-3-carbaldehyde (Intermediate-7)

Charged compound INT-6 (1.0 g, 0.0028 mole) and compound INT-3 (0.53 g, 0.0031 mole) into 1,4-dioxane (20 mL) at room temperature. The reaction mixture was de-gasified for 10 minutes at room temperature. Charged potassium carbonate (1.17 g, 0.0085 mole), DM water (6.0 mL) followed by [1,1'-Bis (diphenylphosphino) ferrocene] dichloropalladium(II) DCM complex (0.11 g, 0.00014 mole). De-gasified the reaction mixture for 10 minutes at room temperature. The reaction mixture was stirred at 95 °C for 8 h. The completeness of the reaction was confirmed by TLC. The reaction mixture was brought to room temperature and filtered through celite bed. The filtrate was diluted with DM water and extracted with ethyl acetate. The organic layer was distilled under reduced pressure and the crude was purified by silica gel column chromatography (15% ethyl acetate in hexane) to afford 0.71 g of Compound INT-7. 1H-NMR (CDCl3, 400 MHz): d 10.41 (d, J = 0.8 Hz, 1H), 8.18 (d, J = 7.6 Hz, 1H), 7.42 – 7.40 (m, 1H), 7.32 – 7.30 (m, 2H), 7.18 – 7.16 (dd, J1 = 0.8 Hz J2 = 7.6 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 2.0 Hz, 1H), 6.84 – 6.82 (dd, J1 = 2.0 Hz J2 = 8.4 Hz, 1H), 4.31 (s, 4H), 4.10 (s, 3H), 2.28 (s, 3H). m/z: 362.31 (M+H) +

Example-3: Preparation of 3-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methoxy-3-pyridyl] methyl]-3-azabicyclo [3.2.0]heptan-6-ol (NAT-1)

Dissolved compound INT-7 (0.15 g, 0.00041 mole) and compound 3-azabicyclo [3.2.0] heptan-6-ol.HBr salt (0.161 g, 0.00083) in dichloromethane (10.0mL) at room temperature. Charged acetic acid (0.1 mL) to the reaction mixture and stirred for 4 hours at 80-85°C. Reaction mass was brought to RT, charged Sodium triacetoxyborohydride (0.263 g, 0.0012 mole) and stirred for 3-4 hours at 80-85°C. The completeness of the reaction was confirmed by TLC. The reaction mixture was diluted with DM water (15mL), extracted with dichloromethane (20mL). The organic layer was washed with 10% sodium bicarbonate solution (15mL) followed by DM water (15mL). The organic layer was separated and distilled under reduced pressure to afford crude compound. The crude compound was purified by silica gel column chromatography (5-10% methanol in MDC) to afford 30mg of NAT-1 as solid. 1H NMR (DMSO-d6, 400 MHz): d 7.97 (d, J = 7.6 Hz, 1H), 7.38-7.36 (m, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.21-7.19 (m, 1H), 7.13 (d, J = 8.0 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.84-6.80 (m, 2H), 4.65 (brs, 1H), 4.28 (s, 4H), 4.13 (brs, 1H), 3.90 (s, 3H), 3.74-3.60 (m, 2H), 2.86 (d, J = 5.6 Hz, 1H), 2.75 (d, J = 8.0 Hz, 1H), 2.43-2.47 (m, 2H), 2.19 (s, 3H), 2.06-2.03 (m, 1H), 1.81-1.78 (m, 1H). m/z: 459.37 (M+H) +

NAT-2

Intermediate preparation
Example-4: Preparation of 3-[(6-chloro-3-formyl-2-pyridyl)oxymethyl] benzonitrile (Intermediate-11)

Stage-1: Preparation of 6-chloro-2-[(3-cyanophenyl)methoxy]pyridine-3-carboxylic acid (Intermediate-8)

Charged tetrahydrofuran (96.0mL) and sodium hydride (3.3g, 0.1375) into round bottom flask at room temperature. Cooled the reaction mass temperature to 15-20°C. Dissolved 3-(hydroxymethyl)benzonitrile (9.15g, 0.0687) in tetrahydrofuran (96.0mL), charged to reaction mixture and stirred for 15-20 min at 15-20°C. Dissolved 2,6-dichloro-4-methyl-pyridine-3-carboxylic acid (12.0 g, 0.2426 mole) in tetrahydrofuran (96 mL) and charged to reaction mixture and stirred for 15-20 min at 15-20°C. The mass temperature was raised to room temperature and stirred for 3-hrs. The completeness of the reaction was confirmed by TLC. Quenched the reaction mass with 1N hydrochloric acid (120.0mL) and extracted with ethyl acetate twice (120mL x 2). The organic layer was washed with 5% sodium bicarbonate and acidified the organic layer with 50% acetic acid solution. White solid formation was observed and stirred for 30 min. Filtered the solid and dried in hot air oven at 45°C for 4hrs to give 12.2 g of compound INT-8. 1H NMR (DMSO-d6, 400 MHz): d 13.24 (s, 1H), 8.21 (d, J = 8.0 Hz, 1H), 7.99 (s, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 5.48 (s, 2H).

Stage-2: Preparation of methyl 6-chloro-2-[(3-cyanophenyl) methoxy]pyridine-3-carboxylate (Intermediate-9)

Charged INT-8 (12.0g, 0.0415 mole) in dimethylformamide (120.0mL) into round bottom flask at room temperature. Charged potassium carbonate (17.2g, 0.1247 mole) to the reaction at 25-30°C. Stirred the mass for 5-10 min. Added slowly methyl iodide (12.9 mL, 0.2078) to the reaction mass at 25-30°C and stirred for 3-4hrs. Filtered the reaction and charged the reaction mass into DM water (240.0mL) slowly. Solid formation was observed. Filtered the solid and washed the solid with DM water (200.0mL). Dried the compound in hot air oven at 60°C for 4hrs to give 12.2 g of compound INT-9. 1H NMR (DMSO-d6, 400 MHz): d 8.26 (d, J = 8.0 Hz, 1H), 7.98 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.64 (t, J = 8.0 Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H), 5.50 (s, 2H), 3.84 (s, 3H).

Stage-3: Preparation of 3-[[6-chloro-3-(hydroxymethyl)-2-pyridyl] oxymethyl] benzonitrile (Intermediate-10)

Charged INT-9 (12.2g, 0.0403 mole) in tetrahydrofuran (1220.0mL) and methanol (122.0mL) into round bottom flask at 25-30°C. Charged slowly sodium borohydride (6.1g, 0.1612 mole) to the reaction mass. Stirred the reaction mass for 16-17 hrs. Distilled off the solvent from the reaction mass, charged DM water (120.0mL) and stirred for 10min. Extracted with ethyl acetate (120mL) and distilled off organic solvent to give 11.1g of compound INT-10. 1H NMR (DMSO-d6, 400 MHz): d 7.93 (s, 1H), 7.83-7.78 (m, 3H), 7.61 (t, J = 8.0 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 5.40 (s, 2H), 5.31 (t, J = 8.0 Hz, 1H), 4.50 (d, J = 8.0 Hz, 2H).

Stage-4: Preparation of 3-[(6-chloro-3-formyl-2-pyridyl) oxymethyl] benzonitrile
(Intermediate-11)

Charged compound INT-10 (11 g, 0.40 mole) in dichloromethane (165.0 mL) at 25-30°C. Charged dess martin periodinane (20.3 g, 0.048 mole) at 0-5°C and stirred for 4 hours. The completeness of the reaction was confirmed by TLC. Quenched the reaction mixture with 1N sodium hydroxide, extracted in dichloromethane (165mL). Distilled the organic layer under reduced pressure to give 10.2 g of Compound INT-11. 1H NMR (DMSO-d6, 400 MHz): d 10.26 (s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.05 (s, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.63 (t, J = 8.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 5.55 (s, 2H).

Example-5: Preparation of 3-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-3-formyl-2-pyridyl] oxymethyl] benzonitrile (Intermediate-12)

Dissolved compound INT-11 (0.4 g, 0.0145 mole) and compound INT-6 (0.51 g, 0.0145 mole) in 1,4-dioxane (80.0 mL) at 25-30°C. De-gasify the reaction mixture for 10 min at 25-30°C. Charged potassium carbonate (0.604 g, 0.044 mole), DM water (4.0 mL) followed by [1,1'-Bis (diphenylphosphino) ferrocene]dichloropalladium(II) DCM complex (0.3 g, 0.00036 mole). De-gasified the reaction mixture for 10 min at 25-30°C. Stirred the reaction mixture at 95 °C for 8 hours. The completeness of the reaction was confirmed by TLC. The reaction mixture was brought to room temperature and filtered through cilite bed. The filtrate was diluted with DM water and extracted with ethyl acetate. The organic layer was distilled under reduced pressure. The crude was purified by column chromatography using silica gel 100-200mesh to afford 0.32 g of Compound INT-12. 1H NMR (DMSO-d6, 400 MHz): d 10.40 (s, 1H), 8.23 (d, J = 7.6 Hz, 1H), 8.04 (s, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.81-7.79 (m, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.40-7.32 (m, 3H), 7.28-7.26 (m, 1H), 6.93 (d, J = 8.0 Hz, 1H), 6.94-6.80 (m, 2H), 5.61 (s, 2H), 4.29 (s, 4H), 2.1 (s, 3H). m/z: 463.35 (M+H) +

Example-6: Preparation of 3-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-3-formyl-2-pyridyl] oxymethyl] benzonitrile (NAT-2)

Dissolved compound INT-12 (0.15 g, 0.00032 mole) and compound 3-azabicyclo[3.2.0]heptan-6-ol.HBr salt (0.125 g, 0.00064 mole) in dichloromethane (10 mL) at 25-30°C. Charged acetic acid (0.1 mL) to the reaction mixture and stirred for 4 hours at 80-85°C. Reaction mass temperature was brought to 25-30°C, charged Sodium triacetoxyborohydride (0.206 g, 0.00097 mole) and stirred for 3-4 hours at 80-85°C. The completeness of the reaction was confirmed by TLC. Diluted the reaction mixture with DM water (150.0mL), extracted with dichloromethane (150.0mL). Washed the organic layer with 10% sodium bicarbonate (150mL) followed by DM water. Separated the organic layer and distil under reduced pressure to afford crude compound. The crude compound was purified by column chromatography using silica gel 100-200 mesh (5-10% methanol in MDC) to afford 30 mg of NAT-2 as solid. 1H NMR (400 MHz, DMSO-d6): 8.01 (d, J = 7.6 Hz, 1H), 7.92 (s, 1H), 7.85-7.76 (m, 2H), 7.62-7.58 (m, 1H), 7.30-7.27 (m, 2H), 7.21-7.15 (m, 2H), 6.92 (d, J = 8.4 Hz, 1H), 6.83-6.78 (m, 2H), 5.46 (s, 2H), 4.65 (m, 1H), 4.28 (s, 4H), 4.13-4.11 (m, 1H), 3.84-3.67 (m, 2H), 2.87-2.75 (m, 2H), 2.43-2.47 (m, 2H), 2.21-2.17 (m, 1H), 2.06 (s, 3H), 1.81-1.78 (m, 1H). m/z: 560.44 (M+H) +
NAT-3

Intermediate preparation
Example-7: Preparation of 6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methoxy-pyridine-3-carbaldehyde (Intermediate-7)

Refer NAT-1 for intermediate-7 preparation.
Example-8: Preparation of 6-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methoxy-3-pyridyl] methyl]-2-oxa-6-azaspiro [3.3]heptane (NAT-3)

Dissolved compound INT-7 (2.75 g, 0.0076 mole) and compound 2-oxa-6-azaspirov [3.3] heptane (0.83 g, 0.00837 mole) in dimethylformamide (30.0mL) at 25-30°C. Charged acetic acid (2.7 mL) to the reaction mixture and stirred for 16 hours at 25-30°C. Charged Sodium triacetoxyborohydride (4.83 g, 0.0228 mole) at 25-30°C and stir for 3-4 hours. Confirmed the completeness of the reaction by TLC. Diluted the reaction mixture with DM water (275mL) and extracted with dichloromethane (275mL). Washed the organic layer with 10% sodium bicarbonate (275mL) and followed by DM water. Separated the organic layer and distilled under reduced pressure to afford crude compound. Purified the crude compound by column chromatography using silica gel 100-200 mesh (5-10% methanol in MDC) to afford 1.6 g of NAT-3 as solid. 1H NMR (CDCl3, 400 MHz): d 7.55 (d, J = 7.6 Hz, 1H), 7.38-7.36 (m, 1H), 7.29-7.22 (m, 2H), 7.0 (d, J = 7.6 Hz, 1H), 6.92-6.88 (m, 2H), 6.84-6.82 (m, 1H), 4.77 (s, 4H), 4.30 (s, 4H), 3.96 (s, 3H), 3.57 (s, 2H), 3.49 (s, 4H), 2.25 (s, 3H). m/z: 445.3 (M+H) +

NAT-4

Intermediate preparation
Example-9: Preparation of 6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methoxy-pyridine-3-carbaldehyde (Intermediate-7)

Refer NAT-1 for intermediate-7 preparation.
Example-10: Preparation of 2-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methoxy-3-pyridyl] methyl]-6-oxa-2-azaspiro [3.4] octane (NAT-4)

Dissolved compound INT-7 (2.0 g, 0.00553 mole) and compound 6-oxa-2-azaspiro[3.4]octane di trifluoroacetate (2.45 g, 0.0071 mole) in dimethylformamide (20mL) at 25-30°C. Charged N,N-diisopropyl ethylamine (3.3 mL, 0.0193 mole) to the reaction mixture and stir for 16 hours at 25-30°C. Charge Sodium triacetoxyborohydride (2.34 g, 0.011 mole) at 25-30°C and stirred for 3-4 hours. The completeness of the reaction was confirmed by TLC. Diluted the reaction mixture with DM water (200mL) and extracted with dichloromethane (200mL). Washed the organic layer with 10% sodium bicarbonate followed by DM water. Separated The organic layer and distilled under reduced pressure to afford crude compound. Purified The crude compound by column chromatography using silica gel 100-200 mesh (5-10% methanol in MDC) to afford 1.0 g of NAT-4 as solid. 1H NMR (CDCl3, 400 MHz): d 7.71 (d, J = 7.6 Hz, 1H), 7.38-7.36 (m, 1H), 7.30-7.24 (m, 2H), 7.05 (d, J = 7.2 Hz, 1H), 6.92-6.88 (m, 2H), 6.84-6.82 (m, 1H), 4.30 (s, 4H), 4.0-3.97 (m, 5H), 3.89 (s, 2H), 3.83-3.73 (m, 6H), 2.25 (s, 3H), 2.21 (t, J = 7.2 Hz, 2H). m/z: 459.3 (M+H) +

NAT-5

Intermediate preparation
Example-11: Preparation of 3-[(6-chloro-3-formyl-2-pyridyl) oxymethyl] benzonitrile (Intermediate-11)

Refer NAT-2 for intermediate-11 preparation.
Example-12: Preparation of 2-(2,3-dihydro-1,4-benzodioxin-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Intermediate-4)

Refer NAT-1 for intermediate-4 preparation.
Example-13: Preparation of 2-chloro-6-(2,3-dihydro-1,4-benzodioxin-6-yl) benzonitrile (Intermediate-13)

Dissolved INT-4 (60.0 g, 0.2289 mole) and 2,6-dichlorobenzonitrile (59.0 g, 0.3433 mole) in 1,4-dioxane (900.0 mL) at 25-30°C. De-gasified the reaction mixture for 10 min at 25-30°C. Charged potassium carbonate (94.9 g, 0.686 mole), DM water (180 mL) followed by [1,1'-Bis (diphenylphosphino) ferrocene] dichloropalladium (II) DCM complex (9.34 g, 0.114 mole). De-gasified the reaction mixture for 10 min at 25-30°C. Stirred the reaction mixture at 90-95°C for 10 hours. Confirmed completeness of the reaction by TLC. Reaction mixture was brought to 25-30°C and filtered through celite bed. Diluted the filtrate with DM water (600 mL) and extract with ethyl acetate (600mL x 2). Distilled the organic layer under reduced pressure. Purified the crude by column chromatography using silica gel100-200 mesh (10% ethyl acetate in hexane) to afford 51.2 g of Compound INT-13. IHNMR: (400 MHz, CDCl3): d 7.54-7.50 (t, J=8.0 Hz, 1H), 7.47-7.44 (m, 1H), 7.35 (d, J=7.6 Hz, 1H), 7.06-7.02 (m, 2H), 6.96 (d, J=8.4 Hz, 1H), 4.31 (s, 4H).
Example-14: Preparation of 2-(2,3-dihydro-1,4-benzodioxin-6-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) benzonitrile (Intermediate-14)

Dissolved INT-13 (12.8 g, 0.0736 mole) and bis(pinacolato)diboron (22.4 g, 0.0883 mole) in 1,4-doxane (200 mL) at 25-30°C. De-gasified the reaction mixture for 10 min at 25-30°C. Charge potassium acetate (21.6 g, 0.2208 mole) followed by [1,1'-Bis (diphenylphosphino) ferrocene] dichloropalladium (II) DCM complex (4.2 g, 0.0051 mole). De-gasified the reaction mixture for 10 min at 25-30°C. Stirred the reaction mixture at 90-95°C for 16 hours. Confirmed the completeness of the reaction by TLC. The reaction mass was brought to 25-30°C and filtered through celite bed. Diluted the filtrate with DM water (200mL) and extracted with ethyl acetate (200mL x 2). Distilled the organic layer under reduced pressure. Purified the crude by column chromatography using silica gel 100-200 mesh (0-5% ethyl acetate in hexane) to afford 15.1 g of Compound INT-14. 1HNMR: (DMSO-d6, 400 MHz): d 7.76 (d, J=8.0 Hz, 1H), 7.71 (t, J=7.6 Hz, 1H), 7.62 (d, J=7.6 Hz, 1H), 7.04 (s, 1H), 6.99 (m, 2H), 4.3 (s, 4H), 1.3 (s, 12H).

Example-15: Preparation of 2-[6-[(3-cyanophenyl) methoxy]-5-formyl-2-pyridyl]-6-(2,3-dihydro-1,4-benzodioxin-6-yl) benzonitrile (Intermediate-15)

Dissolved INT-11 (4.0 g, 0.0146 mole) and compound 14 (5.6 g, 0.0154 mole) in 1,4-dioxane (80mL) at 25-30°C. De-gasified the reaction mixture for 10 min at 25-30°C. Chargde potassium carbonate (6.07 g, 0.044 mole), DM water (40.0 mL) followed by [1,1'-Bis (diphenylphosphino) ferrocene] dichloropalladium(II) DCM complex (0.23 g, 0.00029 mole). De-gasified the reaction mixture for 10 minutes at room temperature. Stirred the reaction mass at 95 °C for 8 hours. Confirmed the completeness of the reaction by TLC. The reaction mass was brought to room temperature and filtered through celite bed. Diluted the filtrate with DM water and extracted with ethyl acetate. Distilled the organic layer under reduced pressure. Purified the crude by column chromatography using silica gel 100-200 mesh (15% ethyl acetate in hexane) to afford 4.7 g of Compound INT-15. 1H NMR (DMSO-d6, 400 MHz): d 10.38 (s, 1H), 8.32 (d, J = 8.0 Hz, 1H), 8.04 (s, 1H), 7.90-7.80 (m, 4H), 7.72 (d, J = 8.0 Hz, 1H), 7.68-7.66 (m, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.14 (s, 1H), 7.09 (d, J = 8.0 Hz, 1H) 7.02 (d, J = 8.0 Hz, 1H), 5.72 (s, 2H), 4.31 (s, 4H).

Example-16: Preparation of 2-[6-[(3-cyanophenyl) methoxy]-5-(6-oxa-2-azaspiro [3.4]octan-2-ylmethyl)-2-pyridyl]-6-(2,3-dihydro-1,4-benzodioxin-6-yl) benzonitrile (NAT-5)

Dissolved INT-15 (0.2 g, 0.00042 mole) and compound 6-oxa-2-azaspiro[3.4]octane di trifluoroacetate (0.214 g, 0.00063 mole) in ethyl acetate (4 mL) at 25-30°C. Charged N,N-diisopropyl ethylamine (0.26 mL, 0.00152 mole) to the reaction mixture and stirred for 16 hours at 25-30°C. Charged Sodium triacetoxyborohydride (0.134 g, 0.00063 mole) at 25-30°C and stirred for 3-4 hours. Confirmed the completeness of the reaction by TLC. Diluted the reaction mixture with DM water (10mL). Separated organic layer and washed with 10% sodium bicarbonate followed by DM water. Separated organic layer and distilled under reduced pressure to afford crude compound. Purify the crude compound by column chromatography using silica gel 100-200 mesh (5-10% methanol in MDC) to afford 20 mg of NAT-5 as solid. 1H NMR (DMSO-d6, 400 MHz): d 7.94 (s, 1H), 7.85-7.74 (m, 4H), 7.71-7.67 (m, 1H), 7.63-7.57 (m, 2H), 7.50 (d, J = 8.0 Hz, 1H), 7.11 (s, 1H), 7.07 (d, J = 8.0 Hz, 1H), 7.0 (d, J = 8.0 Hz, 1H), 5.57 (s, 2H), 4.31 (s, 4H), 3.73 (s, 2H), 3.66-3.63 (m, 4H), 3.26 (s, 4H), 2.04 (t, J = 8.0 Hz, 2H). m/z: 571.6 (M+H) +

NAT-6

Intermediate preparation
Example-17: Preparation of 2-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Intermediate-6)

Refer NAT-1 for intermediate-6 preparation.
Example-18: Preparation of 2-chloro-6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl] pyridine-3-carboxylic acid (Intermediate-16)

Dissolved 2,6-dichloropyridine-3-carboxylic acid (15.0g, 0.0781 mole) and INT-6 (27.5 g, 0.0781 mole) in a mixture of 1,4-dioxane (150mL) and ethanol (150mL) at 25-30°C. De-gasified the reaction mixture for 10 min at 25-30°C. Charged potassium carbonate (37.7 g, 0.2733 mole), DM water (150.0 mL) followed by [1,1'-Bis (diphenylphosphino) ferrocene]dichloropalladium (II) DCM complex (2.74 g, 0.0039 mole). De-gasified the reaction mixture for 10 min at 25-30°C. Stirred the reaction mixture for 7 hours at 95 °C. Confirmed the completeness of the reaction by TLC. Distilled the reaction mass under reduced pressure to give crude. Diluted the crude with DM water (150.0mL) and extracted with ethyl acetate (150mL). Acidified the aqueous layer with 4N hydrochloric acid and extracted with ethyl acetate (150mL x 2). Distilled the organic layer under reduced pressure to give 32.0 g of Compound INT-16. 1H NMR (DMSO-d6, 400 MHz): d 13.79 (brs, 1H), 8.30 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.39 – 7.33 (m, 2H), 7.29 – 7.27 (dd, J = 2.4 Hz 7.2 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.85 – 6.80 (m, 2H), 4.28 (s, 4H), 2.15 (s, 3H).
Example-19: Preparation of [2-chloro-6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-3-pyridyl] methanol (Intermediate-17)

Dissolved INT-16 (32.0g, 0.0838 mole) in tetrahydrofuran (320.0 mL) at 25-30°C and cooled to 0-10°C. Charged BH3.DMS complex (2M in THF) solution to the reaction mass slowly. After completion the mass temperature was raised to 65-70°C and maintained for 8hrs. Confirmed the completeness of the reaction by TLC. Cooled the reaction mass to 0-10°C, quenched with saturated ammonium chloride solution (320.0mL) and extracted with ethyl acetate (320mL). Separated organic solvent and distilled under reduced pressure. Purified the crude compound by column chromatography using silica gel 100-200 mesh (ethyl acetate/hexane) to afford 27.9 g of Compound INT-17. 1H NMR (DMSO-d6, 400 MHz): d 8.02 (d, J = 7.6 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.31 (d, J = 4.4 Hz, 2H), 7.25 (m, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.84 – 6.79 (m, 2H), 5.59 (t, J = 5.6 Hz, 1H), 4.60 (d, J = 5.6 Hz, 2H), 4.28 (s, 4H), 2.12 (s, 3H).

Example-20: Preparation of 2-chloro-3-(chloromethyl)-6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl] pyridine (Intermediate-18)

Dissolved INT-17 (2.0g, 0.00543 mole) in dichloromethane (30.0 mL) at 25-30°C. Charged thionyl chloride (0.75mL, 0.0108 mole) drop wise to the reaction mass at 25-30°C. Maintained the reaction mass for 6 hours at 25-30°C. Confirmed the completeness of the reaction confirmed by TLC. Quenched the reaction with ice cold water (20 mL) and extracted with ethyl acetate (30 mL). Separated organic solvent and distilled under reduced pressure to give 1.8 g of Compound INT-18. 1H NMR (CDCl3, 400 MHz): d 7.91 (d, J = 8.0 Hz, 1H), 7.41 (d, J = 7.6 Hz, 1H), 7.34-7.31 (m, 1H), 7.29-7.27 (m, 2H), 6.91 (d, J = 8.4 Hz, 1H), 6.86 – 6.85 (m, 1H), 6.82-6.79 (m, 1H), 4.75 (s, 2H), 4.30 (s, 4H), 2.20 (s, 3H).

Example-21: Preparation of 6-[[2-chloro-6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-3-pyridyl] methyl]-2-oxa-6-azaspiro [3.3] heptane (NAT-6)

Dissolved INT-18 (0.9g, 0.00232 mole) in acetonitrile (10.0 mL) at 25-30°C. Charged 2-oxa-6-azaspiro [3.3] heptane (0.34g, 0.00349 mole) followed by N, N-diisopropylamine (0.8 mL, 0.00464 mole) at 25-30°C. Maintained the reaction for 5 hours at 85-90°C. Confirmed the completeness of the reaction by TLC. Distilled the solvent under reduced pressure to get crude compound. Dilutee The crude compound with DM water (10mL) and extracted with ethyl acetate (10.0mLx2). Distilled the organic solvent under reduced pressure to give crude compound. Purified the crude compound by column chromatography using silica gel 100-200 mesh (ethyl acetate/hexane) to afford 0.7 g of Compound NAT-6. 1H NMR (CDCl3, 400 MHz): d 7.77 (d, J = 8.0 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.32 – 7.29 (m, 1H), 7.27 – 7.25 (m, 2H), 6.90 (d, J = 8.0 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 6.82 – 6.79 (dd, J = 2.0 Hz 8.0 Hz, 1H), 4.75 (s, 4H), 4.30 (s, 4H), 3.70 (s, 2H), 3.52 (s, 4H), 2.18 (s, 3H). m/z: 449.4 (M+H) +

NAT-7

Example-22: Preparation of 2-chloro-3-(chloromethyl)-6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl] pyridine (Intermediate-18)

Refer NAT-6 for intermediate-18 preparation.
Example-23: Preparation of 2-[[2-chloro-6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-3-pyridyl] methyl]-6-oxa-2-azaspiro [3.4] octane (NAT-7)

Dissolved INT-18 (0.9g, 0.00232 mole) in acetonitrile (10.0 mL) at 25-30°C. Charged 6-oxa-2-azaspiro [3.4] octane ditrifluoroacetate (1.19g, 0.00349 mole) followed by N, N-diisopropylamine (1.21 mL, 0.00696 mole) at 25-30°C. Maintained the reaction at 85-90°C for 5 hours. Confirmed The completeness of the reaction by TLC. Distilled the solvent under reduced pressure to give crude. Diluted the crude compound with DM water (10.0mL) and extracted with ethyl acetate (10.0mLx2). Distilled the organic solvent under reduced pressure to give crude compound. Purified the crude compound by column chromatography using silica gel 100-200 mesh (ethyl acetate/hexane) to afford 0.7 g of Compound NAT-7. 1H NMR (CDCl3, 400 MHz,): d 7.82 (d, J = 7.6 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.32 – 7.24 (m, 3H), 6.90 (d, J = 8.4 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 6.80 (dd, J = 2.0 Hz 8.4 Hz, 1H), 4.30 (s, 4H), 3.89 (s, 2H), 3.81 (t, J = 6.8 Hz, 2H), 3.77 (s, 2H), 3.38 (s, 4H), 2.19 (s, 3H), 2.15 (t, J = 6.8 Hz, 2H). m/z: 463.1
(M+H)+

NAT-8

Intermediate preparation
Example-24: Preparation of 2-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Intermediate-6)

Refer NAT-1 for intermediate-6 preparation.
Example-25: Preparation of (6-chloro-2-methyl-3-pyridyl) methanol (Intermediate-19)

Dissolved 6-chloro-2-methyl-pyridine-3-carboxylic acid (5.0 g, 0.02914 mole) in tetrahydrofuran (100 mL) at 25-30°C under nitrogen atmosphere. Charged BH3.DMS complex 2.0M in tetrahydrofuran solution (17.4 mL, 0.0349 mole) at 0-5 °C to the reaction mixture. Stirred the reaction mixture for 10hours at 60-65°C. Confirmed the completeness of the reaction by TLC. Cooled the reaction mixture to 0-5°C and quenched with 2N aqueous hydrochloric acid. Raised the reaction mass temperature to 25-30°C and extracted with ethyl acetate (100mL). The organic layer was washed with DM water (100mL). Distilled the ethyl acetate under reduced pressure to afford 3.7 g of compound INT-19. 1H NMR (CDCl3, 400 MHz): d 7.75 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 4.5 (s, 4H), 2.38 (s, 3H).

Example-26: Preparation of [6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methyl-3-pyridyl] methanol (Intermediate-20)

Dissolved INT-19 (0.5 g, 0.00317 mole) and compound INT-6 (1.45 g, 0.0412 mole) in 1,4-dioxane (40 mL) at 25-30°C. Degasify the reaction mixture for 10 min at 25-30°C. Charged potassium carbonate (1.31 g, 0.00951 mole), DM water (2.0 mL) followed by [1,1'-Bis (diphenylphosphino)ferrocene]dichloropalladium (II) DCM complex (0.051 g, 0.000063 mole). De-gasified the reaction mixture for 10 min at 25-30°C. Stirred the reaction mixture at 90-95°C for 7 hours. Confirmed the completeness of the reaction by TLC. Reaction mixture was brought to at 20-25°C and filtered through celite bed. Diluted the filtrate with DM water and extracted with ethyl acetate. Distilled the organic layer under reduced pressure. Purified the crude by column chromatography (15% ethyl acetate in hexane) using silica gel 60-120 mesh to afford 0.74 g of Compound INT-20. 1H NMR (CDCl3, 400 MHz): d 7.78 (d, J = 7.6 Hz, 1H), 7.33 – 7.28 (m, 3H), 7.20 – 7.18 (m, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.83-6.78 (m, 2H), 5.27 (t, J = 5.2 Hz, 1H), 4.57 (d, J = 5.6 Hz, 2H), 4.28 (s, 4H), 2.47 (s, 3H), 2.10 (s, 3H). m/z: 348.1 (M+H) +

Example-27: Preparation of 3-(chloromethyl)-6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methyl-pyridine (Intermediate-21)

Dissolved INT-20 (0.74g, 0.00213 mole) in dichloromethane (15mL) at 25-30°C. Charged thionyl chloride (0.29mL, 0.00426 mole) drop wise to the reaction mass at 25-30°C. Maintained the reaction for 4 hours at 25-30°C. Confirmed the completeness of the reaction confirmed by TLC. Quenched the reaction with 10% sodium carbonate solution (10.0mL). Distilled the organic solvent under reduced pressure to yield 0.64 g of Compound INT-21. 1H NMR (CDCl3, 400 MHz): d 7.73 (d, J = 8.0 Hz, 1H), 7.33 – 7.28 (m, 4H), 6.94-6.90 (m, 2H), 6.85-6.83 (m, 1H), 6.83-6.78 (m, 1H), 4.70 (s, 2H), 4.32 (s, 4H), 2.74 (s, 3H), 2.20 (s, 3H). m/z: 366.3 (M+H) +

Example-28: Preparation of 6-[[6-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methyl-phenyl]-2-methyl-3-pyridyl]methyl]-2-oxa-6-azaspiro[3.3] heptane (NAT-8)

Dissolved INT-21 (0.9g, 0.00090 mole) in acetonitrile (10mL) at 25-30°C. Charged 2-oxa-6-azaspiro [3.3] heptane (0.071g, 0.00099 mole) followed by triethylamine (0.25 mL, 0.00180 mole) at 75-80°C for 4 hours. Confirmed the completeness of the reaction by TLC. Distilled the solvent under reduced pressure to give crude. Diluted Crude compound with DM water (10mL) and extracted with ethyl acetate (10mL x 2). Distilled organic solvent under reduced pressure to give crude compound. Purified crude compound by column chromatography using silica gel 100-200 mesh (ethyl acetate/hexane) to afford 0.24 g of Compound NAT-8. 1H NMR (CDCl3, 400 MHz): d 7.60 (d, J = 7.6 Hz, 1H), 7.33 – 7.29 (m, 2H), 7.26 – 7.21 (m, 2H), 6.91 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 1.6 Hz, 1H), 6.83 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 4.79 (s, 4H), 4.32 (s, 4H), 3.59 (s, 2H), 3.47 (s, 4H), 2.57 (s, 3H), 2.17 (s, 3H). m/z: 429.6 (M+H) +

NAT-22 and NAT-23

Example-29: Preparation of 6-chloro-2-methoxy-pyridine-3-carboxylic acid (Intermediate-1)
Refer NAT-1 for intermediate-1 preparation.
Example-30: Preparation of (6-chloro-2-methoxy-3-pyridyl)methanol (Intermediate-2)
Refer NAT-1 for intermediate-2 preparation.
Example-31: Preparation of 2-(2,3-dihydro-1,4-benzodioxin-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Intermediate-4)
Refer NAT-1 for intermediate-4 preparation.
Intermediate preparation of 2-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl) phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Intermediate-23)

Example-32: preparation of 6-(3-bromo-2-chloro-phenyl)-2,3-dihydro-1,4-benzodioxine (Intermediate-22)

Charged compound INT-4 (128.9 g, 0.4650 mole) and 1,3-dibromo-2-chloro-benzene (125.7 g, 0.4650 mole) into 1,4-dioxane (1290 mL) at room temperature. The reaction mixture was de-gasified for 10 minutes at room temperature. Charged potassium carbonate (192.6 g, 1.395 mole), DM water (200.0 mL) followed by [1,1'-Bis (diphenylphosphino) ferrocene] dichloropalladium(II) DCM complex (9.49 g, 0.011625 mole). De-gasified the reaction mixture for 10 min at room temperature. The reaction mixture was stirred at 80-85°C for 10 hours. The completeness of the reaction was confirmed by TLC. The reaction mixture was brought to room temperature and filtered through celite bed. The filtrate was diluted with DM water and extracted with ethyl acetate. The organic layer was distilled under reduced pressure and the crude was purified by silica gel column chromatography (10% ethyl acetate in hexane) to afford 100 g of Compound INT-22. 1H NMR (400 MHz, CDCl3): d 7.77 – 7.74 (dd, J = 1.6 Hz, 7.6 Hz, 1H), 7.37 – 7.35 (dd, J = 1.6 Hz, 7.6 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 2.0 Hz, 1H), 6.86 – 6.84 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 4.28 (s, 4H).
Example-33: preparation of 2-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl) phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Intermediate-23)
Charged Compound INT-22 (100 g, 0.191 mole) and Bis(pinacolato)diboron (94.0 g, 0.229 mole) into 1,4-doxane (1000 mL) at room temperature. The reaction mixture was de-gasified for 10 minutes at room temperature. Charged potassium acetate (90.8 g, 0.5741 mole) followed by [1,1'-Bis (diphenylphosphino) ferrocene] dichloropalladium(II) DCM complex (6.3 g, 0.0077 mole). De-gasified the reaction mixture for 10 min at room temperature. The reaction mixture was stirred at 100°C for 7 hours. The completeness of the reaction was confirmed by TLC. The reaction mixture was brought to room temperature and filtered through celite bed. The filtrate was diluted with DM water and extracted with ethyl acetate. The organic layer was distilled under reduced pressure and the crude was purified by silica gel column chromatography (5-15% ethyl acetate in hexane) to afford 65 g of Compound INT-23.
1H NMR (400 MHz, CDCl3): d 7.59 – 7.57 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 7.34 – 7.32 (dd, J = 2.0 Hz, 7.6 Hz, 1H), 7.27 – 7.23 (m, 1H), 6.92 (d, J = 1.6 Hz, 2H), 6.89 – 6.88 (m, 1H), 4.29 (s, 4H), 1.38 (s, 12H).

Example-34 preparation of [6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl) phenyl]-2-methoxy-3-pyridyl]methanol (Intermediate-24)

Dissolved INT-2 (23 g, 0.1324 mole) and compound INT-23 (59.2 g, 0.1589 mole) in 1,4-dioxane (276 mL) at 25-30°C. Degasify the reaction mixture for 10 min at 25-30°C. Charged potassium carbonate (54.8 g, 0.3972 mole), DM water (69 mL) followed by [1,1'-Bis (diphenylphosphino) ferrocene] dichloropalladium (II) DCM complex (2.7 g, 0.0033 mole). De-gasified the reaction mixture for 10 min at 25-30°C. Stirred the reaction mixture at 90-95°C for 7 hours. Confirmed the completeness of the reaction by TLC. Reaction mixture was brought to at 20-25°C and filtered through celite bed. Diluted the filtrate with DM water and extracted with ethyl acetate. Distilled the organic layer under reduced pressure. Purified the crude by column chromatography (15% ethyl acetate in hexane) using silica gel 60-120 mesh to afford 45.0 g of Compound INT-24. 1H NMR (400 MHz, DMSO-d6): d 7.82 - 7.80 (d, J = 7.6Hz, 1H), 7.53 – 7.51 (dd, J = 2.8 Hz, 7.6 Hz, 1H), 7.48 – 7.44 (t, J = 7.6 Hz, 1H), 7.40 – 7.38 (dd, J = 2.0 Hz, 7.6 Hz, 1H), 7.24 (d, J = 7.6 Hz, 1H), 6.95 – 6.93 (m, 2H), 6.91 – 6.88 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 5.23 (t, J = 5.6 Hz, 1H), 4.51 (d, J = 5.6 Hz, 2H), 4.29 (s, 4H), 3.89 (s, 3H). m/z: 384.2 (M+), 386.2 (M+2H) +.

Example-35 preparation of 6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl) phenyl]-3-(chloromethyl)-2-methoxy-pyridine (Intermediate-25)

Dissolved INT-24 (24.0g, 0.0703 mole) in dichloromethane (540 mL) at 25-30°C. Charged thionyl chloride (16.72 g, 0.1406 mole) drop wise to the reaction mass at 10-15°C. Maintained the reaction for 4 hours at 20-25°C. Confirmed the completeness of the reaction confirmed by TLC. Quenched the reaction with 10% sodium carbonate solution (10.0mL). Distilled the organic solvent under reduced pressure to yield 21.0 g of Compound INT-25. 1H NMR (400 MHz, CDCl3): d 7.74 – 7.72 (d, J = 7.6 Hz, 1H), 7.53 – 7.50 (dd, J = 2.8 Hz, 7.2 Hz, 1H), 7.38 – 7.33 (m, 2H), 7.26 – 7.22 (m, 1H), 7.00 – 6.99 (m, 1H), 6.94 – 6.93 (m, 2H), 4.66 (s, 2H), 4.31 (s, 4H), 4.04 (s, 3H).
m/z: 402.1 (M+), 404.1 (M+2H)+, 406.1 (M+4H)+.

Example-36: Preparation of 2-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl) phenyl]-2-methoxy-3-pyridyl]methyl]-6-oxa-2-azaspiro[3.4]octane (NAT-22)

Dissolved INT-25 (16.8g, 0.04176 mole) in acetonitrile (168.0 mL) at 25-30°C. Charged 6-oxa-2-azaspiro [3.4] octane ditrifluoroacetate (21.3g, 0.0626 mole) or INT-32 (7.07g, 0.0625 mole) followed by N, N-diisopropylamine (26.9 g, 0.2088 mole) at 25-30°C. Maintained the reaction at 65-70°C for 5 hours. Confirmed The completeness of the reaction by TLC. Distilled the solvent under reduced pressure to give crude. Diluted the crude compound with DM water (168.0mL) and extracted with ethyl acetate (168.0 mL). Distilled the organic solvent under reduced pressure to give crude compound. Purified the crude compound by column chromatography using silica gel 100-200 mesh (ethyl acetate/hexane) to afford 11.0 g of Compound NAT-22. 1H NMR (400 MHz, DMSO-d6): d 7.71 (d, J = 7.2 Hz, 1H), 7.53 – 7.51 (m, 1H), 7.45 (t, J = 8.8 Hz, 1H), 7.40 – 7.37 (m, 1H), 7.21 (d, J = 7.6 Hz, 1H), 6.95 – 6.88 (m, 3H), 4.30 (s, 4H), 3.88 (s, 3H), 3.73 (s, 2H), 3.64 (t, J = 6.8 Hz, 2H), 3.57 (brs, 2H), 3.24 (brs, 4H), 2.03 (t, J = 6.8 Hz, 2H). m/z: 479.1 (M+H)+ and 481.1(M+2H)+

Example-37: Preparation of 6-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl) phenyl]-2-methoxy-3-pyridyl]methyl]-2-oxa-6-azaspiro[3.3]heptane (NAT-23)

Dissolved INT-25 (15.0g, 0.04176 mole) in acetonitrile (150.0 mL) at 25-30°C. Charged 2-oxa-6-azaspiro [3.3] heptane (7.39g, 0.0745 mole) followed by N, N-diisopropylamine (9.7 g, 0.0745 mole) at 25-30°C. Maintained the reaction at 65-70°C for 5 hours. Confirmed The completeness of the reaction by TLC. Distilled the solvent under reduced pressure to give crude. Diluted the crude compound with DM water (168.0mL) and extracted with ethyl acetate (150.0 mL). Distilled the organic solvent under reduced pressure to give crude compound. Purified the crude compound by column chromatography using silica gel 100-200 mesh (ethyl acetate/hexane) to afford 11.5 g of Compound NAT-23. 1H NMR (400 MHz, CDCl3): d 7.57 (d, J = 7.6 Hz, 1H), 7.52-7.49 (m, 1H), 7.37 – 7.30 (m, 2H), 7.19 (d, J = 7.6 Hz, 1H), 7.0 (s, 1H), 6.93 (t, J = 10.0 Hz, 2H), 4.77 (s, 4H), 4.31 (s, 4H), 3.98 (s, 3H), 3.58 (s, 2H), 3.49 (s, 4H).
m/z: 465.0 (M+H) + and 467.0 (M+2H) +

NAT-24 and NAT-25

Example-38: Preparation of Intermediate (6-chloro-2-methoxy-4-methyl-3-pyridyl)methanol (INT-27)

Step-1: Preparation of 6-chloro-2-methoxy-4-methyl-pyridine-3-carboxylic acid (INT-26)

Dissolved 60% NaH (52.38 1.3104 mole) in tetrahydrofuran (1080 mL) at 25-30°C under nitrogen atmosphere. Cool the reaction mass to 5-10°C. Dissolved 2,6-dichloro-4-methyl-pyridine-3-carboxylic acid (108 g, 0.5241mole) in tetrahydrofuran (540 mL) and added slowly to the reaction mass at 5-10°C. Maintained the reaction mixture at 60-65°C for 4h. The completeness of the reaction confirmed by TLC. DM water charged slowly to the reaction mass. The crude mass was diluted with water and acidified (pH 1-2) with 2N hydrochloric acid at 5-10°C. Filtered the solid and dried in tray dried at 75°C to afford 95.0 g of compound INT-26. 1H-NMR (DMSO-d6, 400 MHz): d 13.44 (s, 1H), 7.09 (s, 1H), 3.87 (s, 3H), 2.27 (s, 3H). m/z: 202.1 [M+H] + and 204.1 [M+2H] +
Step-2: Preparation of Intermediate (6-chloro-2-methoxy-4-methyl-3-pyridyl) methanol (INT-27)

Dissolved INT-26 (94.0 g, 0.4662 mole) in tetrahydrofuran (940 mL) at 25-30°C under nitrogen atmosphere. Charged BH3.DMS complex 2.0M in tetrahydrofuran solution (466.25 mL, 0.9325 mole) at 0-5 °C to the reaction mixture. Stirred the reaction mixture for 4hours at 60-65°C. Confirmed the completeness of the reaction by TLC. Cooled the reaction mixture to 0-5°C and quenched with 10% ammonium chloride solution. Raised the reaction mass temperature to 25-30°C. and extracted with ethyl acetate (100mL). The organic layer was washed with DM water (100mL). Filtered the solid and dried in tray dried at 70°C to afford 86 g of compound INT-27. 1H NMR (DMSO-d6, 400 MHz): d 6.96 (s, 1H), 4.44 (s, 2H), 3.84 (s, 3H), 2.34 (s, 3H). m/z: 188.1 [M+H]+ and 190.1 [M+2H] +
Example-39: preparation of 2-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl) phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Intermediate-23)

Refer NAT-22 for intermediate-23 preparation.
Example-40: preparation of [6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl) phenyl]-2-methoxy-5-methyl-3-pyridyl]methanol (Intermediate-28)

Dissolved INT-23 (86 g, 0.2307 mole) and compound INT-27 (34.63 g, 0.1846 mole) in 1,4-dioxane (860 mL) at 25-30°C. Degasify the reaction mixture for 10 min at 25-30°C. Charged potassium carbonate (95.33 g, 0.6923 mole), DM water (172 mL) followed by [1,1'-Bis (diphenylphosphino) ferrocene] dichloropalladium (II) DCM complex (4.71 g, 0.00576 mole). De-gasified the reaction mixture for 10 min at 25-30°C. Stirred the reaction mixture at 90-95°C for 6 hours. Confirmed the completeness of the reaction by TLC. Reaction mixture was brought to at 20-25°C and filtered through celite bed. Diluted the filtrate with DM water and extracted with ethyl acetate. Distilled the organic layer under reduced pressure. Purified the crude by column chromatography (15% ethyl acetate in hexane) using silica gel 60-120 mesh to afford 58.8 g of Compound INT-28. 1H NMR (DMSO-d6, 400 MHz): d 7.49-7.43 (m, 2H), 7.38 (dd, J = 2.0 Hz, 1.6 Hz, 1H), 7.06 (s, 1H), 6.95-6.92 (m, 2H), 6.89 (dd, J = 2.0 Hz, 1.6 Hz, 1H), 4.80 (t, J = 5.2 Hz, 1H ), 4.54 (d, J = 5.2 Hz, 2H ), 4.28 (s, 4H), 3.92 (s, 3H), 2.40 (s, 3H). m/z: 398.2 (M+H) + and 400.2 (M+2H) +

Example-41: preparation of 2-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl) phenyl]-5-(chloromethyl)-6-methoxy-3-methyl-pyridine (Intermediate-29)

Dissolved INT-28 (15.5g, 0.0389 mole) in dichloromethane (155 mL) at 25-30°C. Charged thionyl chloride (11.30 mL, 0.1558 mole) drop wise to the reaction mass at 10-15°C. Maintained the reaction for 4 hours at 20-25°C. Confirmed the completeness of the reaction confirmed by TLC. Quenched the reaction with 10% sodium carbonate solution (10.0mL). Distilled the organic solvent under reduced pressure to yield 14.8 g of Compound INT-29. 1H NMR (DMSO-d6, 400 MHz): d 7.49 (dd, J = 2.4 Hz, 2.4 Hz, 1H), 7.37-7.31 (m, 2H), 7.06 (s, 1H), 6.99-6.98 (m, 2H), 6.93 (dd, J = 2.4 Hz, 2.4 Hz, 1H), 4.74 (s, 2H), 4.31 (s, 4H), 4.02 (s, 3H), 2.45 (s, 3H). m/z: 416 (M+), 418.2 (M+2H)+, 420.2 (M+4H)+.

Example-42: Preparation of 2-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl) phenyl]-2-methoxy-5-methyl-3-pyridyl]methyl]-6-oxa-2-azaspiro[3.4]octane (NAT-24)

Dissolved INT-29 (14.8g, 0.0355 mole) in acetonitrile (148.0 mL) at 25-30°C. Charged 6-oxa-2-azaspiro [3.4] octane ditrifluoroacetate (7.04g, 0.0711 mole) or INT-32 (6.02g, 0.0532 mole) followed by N, N-diisopropylamine (24.72 mL, 0.1422 mole) at 25-30°C. Maintained the reaction at 70-75°C for 5 hours. Confirmed The completeness of the reaction by TLC. Distilled the solvent under reduced pressure to give crude. Diluted the crude compound with DM water (148.0mL) and extracted with ethyl acetate (148.0 mL). Distilled the organic solvent under reduced pressure to give crude compound. Purified the crude compound by column chromatography using silica gel 100-200 mesh (ethyl acetate/hexane) to afford 10.7 g of Compound NAT-24. 1H NMR (400 MHz, CDCl3): d 7.49 (dd, J = 2.0 Hz, 5.2 Hz, 1H), 7.36 – 7.29 (m, 2H), 7.04 (s, 1H), 7.0 – 6.98 (m, 1H), 6.94 – 6.93 (m, 2H), 4.31 (s, 4H), 3.96 (s, 3H), 3.82 (s, 2H), 3.76 (t, J = 7.2 Hz, 2H), 3.69 (brs, 2H), 3.31 (brs, 4H), 2.41 (s, 3H), 2.08 (t, J = 7.2 Hz, 2H). m/z: 493.1 (M+H) + and 495.1 (M+2H) +

Example-43: Preparation of 6-[[6-[2-chloro-3-(2,3-dihydro-1,4-benzodioxin-6-yl) phenyl]-2-methoxy-5-methyl-3-pyridyl]methyl]-2-oxa-6-azaspiro[3.3]heptane (NAT-25)

Dissolved INT-29 (10.6g, 0.02546 mole) in acetonitrile (106.0 mL) at 25-30°C. Charged 2-oxa-6-azaspiro [3.3] heptane (13.03g, 0.03819 mole) followed by N, N-diisopropylamine (22.19 g, 0.1273 mole) at 25-30°C. Maintained the reaction at 65-70°C for 5 hours. Confirmed The completeness of the reaction by TLC. Distilled the solvent under reduced pressure to give crude. Diluted the crude compound with DM water (106.0mL) and extracted with ethyl acetate (106.0 mL). Distilled the organic solvent under reduced pressure to give crude compound. Purified the crude compound by column chromatography using silica gel 100-200 mesh (ethyl acetate/hexane) to afford 7.2 g of Compound NAT-25. 1H NMR (400 MHz, CDCl3): d 7.49 (dd, J = 2.0 Hz, 5.2 Hz, 1H), 7.36 – 7.29 (m, 2H), 7.03 (s, 1H), 7.01 – 6.98 (m, 1H), 6.93 – 6.92 (m, 2H), 4.74 (s, 4H), 4.31 (s, 4H), 3.96 (s, 3H), 3.60 (s, 2H), 3.46 (s, 4H), 2.37 (s, 3H).
m/z: 479.1 (M+H) + and 481.1 (M+2H) +
Salts formation of NAT-24 is described below.

Example-44: Preparation of Intermediate 6-oxa-2-azaspiro [3.4] octane INT-32
Preparation of Intermediate 6-oxa-2-azaspiro [3.4] octane INT-32

Example-45: Preparation of Intermediate 2-benzyl-6-oxa-2-azaspiro[3.4]octan-3-one INT-30
Step-I: Dissolved Tetrahydrofuran-3-carboxylic acid (100g, 0.861 mole) in dichloromethane (1000 mL) at 25-30°C. Charged pyridine (1.38 mL, 0.017 mole) at 25-30°C. Charged thionyl chloride (65.0 mL, 0.895 mole) drop wise to the reaction mass at 25-30°C about 20-30 min. Maintained the reaction for 4 hours at 25-30°C.
Step-2: Dissolved 1,3,5 Tri benzyl 1,3,5 triazinane (101.59g, 0.284 mole) in dichloromethane (500 mL) at 25-30°C. Charged BF3 etherate (271g, 0.861 mole) at 25-30°C about 10-15min. Maintained the reaction for 2 hours at 25-30°C. Charged triethylamine (503.8 mL, 3.615 mol) to step-I reaction mass at 25-30°C about 30-40 min and maintained for 30min.Charged Step-II reaction mass into step-I reaction mass at -67.5±2.5 °C about 15-20 min and maintained for 20min. raised the reaction mass temperature to 2.5±2.5 °C and maintained for 20min. Charged DM water (500 mL) at 2.5±2.5 °C and raise the temperature to 25-30°C.Striierd the reaction mass for 15-16hrs at 25-30°C. Separated the dichloromethane layer and washed with 2M KHSO4 solution (500 mL) followed by sat.NaHCO3 aqueous solution (500 mL) and DM water (500 mL). Dichloromethane layer distilled at 45-50°C to give 122.3g of INT-30 1H NMR (400 MHz, CDCl3): d 7.38-7.21 (m, 5H), 4.45-4.34 (m, 2H), 4.03-3.95 (m, 2H), 3.92-3.83 (m, 2H), 3.25-3.18 (m, 2H), 2.46-2.38 (m, 1H), 2.14-2.07 (m, 1H). m/z: 218.3 (M+H) +

Example-46: Preparation of Intermediate 2-benzyl-6-oxa-2-azaspiro[3.4]octane Hydrochloride salt INT-31
Charged Tetrahydrofuran (900.0mL) and aluminum chloride (88.3g, 0.662 mole) at -22.5±2.5°C under nitrogen atm about 20-25min. Charged LiAlH4 (2.0 M in THF) solution (331 mL, 0.662 mole) to the reaction mass at -22.5±2.5°C under nitrogen atm about 30-40min. Maintained the reaction mass for 1hr at 25-30°C. Charged INT-30 (120g, 0.552 mole) dissolved in Tetrahydrofuran (900 mL) into the reaction mass at -10±2.5°C about 30-40min. Maintained the reaction mass for 2hrs at 0±5°C. Confirmed The completeness of the reaction by TLC. Charged ethyl acetate (180 mL) into the reaction mass at -5±5°C about 15–20 minutes. Maintained the reaction mass for 12hrs at -5±5°C. Charged ethanolamine (240 mL, 1.104 mole) into the reaction mass at -5±5°C about 25–30min. Maintained the reaction mass for 14hrs at 27.5±2.5°C. Filtered the reaction mass and distilled the tetrahydrofuran at 47.5±2.5°C. Charged DM water (444 mL) and dichloromethane (1200 mL) at 27.5±2.5°C, stirred for 15min. Separated the dichloromethane layer and distilled at 47.5±2.5°C to give crude compound (Purity by GC 87.0%)
The crude compound dissolved in ethyl acetate (1370 mL) at 27.5±2.5°C. Charged IPA. HCl (274mL) at 12.5±2.5°C. Maintained the reaction mass temperature at 27.5±2.5°C about 40min. Filtered 2-benzyl-6-oxa-2-azaspiro[3.4]octane hydrochloride salt and washed the salt with with n-heptane (685mL) and isolated as hydrochloride salt INI-31 (Purity by HPLC 99.0%)
The 2-benzyl-6-oxa-2-azaspiro[3.4]octane hydrochloride salt and charged ethyl acetate (1370 mL), basified with 2N NaOH solution about 11-12 pH at 12.5±2.5°C. Separated the organic layer and distilled at 47.5±2.5°C to give 44.4g of INT-31 base (Purity by HPLC 99.0%). 1H NMR (400 MHz, DMSO-d6): d 7.31-7.20 (m, 5H), 3.68 (s, 2H), 3.62 (t, J = 8.0 Hz, 2H), 3.52 (s, 2H), 3.13-3.09 (m, 4H), 1.98 (t, J = 8.0 Hz, 2H). m/z: 204.1 (M+H) +

Example-47: Preparation of Intermediate 6-oxa-2-azaspiro [3.4] octane INT-32
Dissolved INT-31 (144g, 0.708 mol) and ethyl alcohol (2880 mL) in autoclave at 27.5±2.5 °C. Charged 10% Pd/C (72g) into the reaction mass at 27.5±2.5°C under nitrogen atm. Maintained the reaction mass for 24hrs under hydrogen atm (70psi) at 27.5±2.5°C. Confirmed The completeness of the reaction by TLC. The reaction mass filtered using Celite bed and washed with ethyl alcohol (720 mL) under N2 atm. The filtrate was distilled at 42.5±2.5°C to give 68g of INT-32. 1H NMR (400 MHz, DMSO-d6): d 3.69 (s, 2H), 3.62 (t, J = 8.0 Hz, 2H), 3.40 (s, 4H), 2.71 (s,1H), 1.99 (t, J = 8.0 Hz, 2H).

Activity examples

By way of initial summary, methods for testing the ability of compounds of present invention to compete for the binding of PD-1 / PD-L1 were as follows:
Biophysical methods Microscale Thermophoresis (MST), Differential scanning fluorimetry (DSF) were used to determine these compounds affinity towards the protein of interest.
A series of in vitro assays were conducted to establish the immunomodulation of the compounds – these included non-cell-based assays, and Homogeneous time-resolved fluorescence (HTRF) technique for PD1/PDL1 axis disruption and identification of dimerization.
Cell based assays were also performed, involving (PBMCs)/ T cells/ Exhausted Tcells/CD8+Tcells. These assays were executed using Flow cytometry for determination of CD8+T-cell population, IFNg secretion, internalization, and apoptosis. Thus, the immunomodulation properties of these compounds were established.
In addition, in some cases an HTRF -dimerization assay was conducted to establish PDL1-dimerization.

Example-48:
Determination of binding affinity of the compounds by MicroScale Thermophoresis (MST)
Experimental Materials:
Dye Kit: MonolithTM Series Protein Labeling Kit RED-tris-NTA 2nd Generation and RED-NHS 2nd Generation; Capillary: MO-K022 Monolith NT.115 Capillaries; Buffer: pH 7.4, PBST; Proteins: Human hPD-L1 / B7-H1 (19-134) Protein, His Tag (MALS verified) Human PD-L1 (19-134), His Tag is expressed from human 293 cells (HEK293). It contains AA Phe 19 - Tyr 134, Predicted N-terminus: Phe 19; VISTA/B7-H5 Protein, Human, Recombinant (His Tag), A DNA sequence encoding the human Gi24 (AAH20568.1) (Met1-Ala194) was expressed with a polyhistidine tag at the C-terminus; Human B7-1 / CD80 Protein, His Tag (MALS verified), Human B7-1, His Tag is expressed from human 293 cells (HEK293). It contains AA Val 35 - Asn 242. Predicted N-terminus: Val 35 B71-H5228; MO. Affinity Analysis v2.2.4 software.
Experimental model: Monolith NT.115
Experimental steps (binding affinity of the compounds with human PD-L1 / human VISTA / human B7-1)
The binding affinity between human PD-L1 / human VISTA / human B7-1 and synthesized compounds of the present invention was determined by MicroScale Thermophoresis (MST). The proteins were labelled according to the instructions of the Monolith RED-Tris-NTA second-generation protein labelling kit / RED-NHS second generation protein labelling kit (Nano Temper Technologies). Diluted the compounds into a solution with a concentration ranging from 10.0 µM to 305.0 pM (10 µM, diluted 16 times, each time with two-fold dilution, and the final concentration was 305.0 pM). At room temperature, mixed the compound solution and the labelled protein evenly at a ratio of 1:1, incubated in the dark for 10 minutes, and used a glass capillary to absorb the mixture for detection. Used MO.Affinity Analysis v2.2.4 software to process the data and obtained the binding affinity value between the compound and proteins.
The obtained Kd values for hPD-L1 and compounds are shown in Table 2. The ranges are as follows: A: ? 50 nM; B: 50 nM - 100 nM; C: 100 nM - 200 nM: D: 200 nM - 600 nM; E: ? 600 nM
Table.2. Binding affinity of compounds with hPD-L1 protein.
S.No Compound ID MST Kd S.No Compound ID MST Kd S.No Compound ID MST Kd
1 NAT-1 A 34 NAT-34 D 67 NAT-67 E
2 NAT-2 C 35 NAT-35 D 68 NAT-68 D
3 NAT-3 A 36 NAT-36 C 69 NAT-69 E
4 NAT-4 A 37 NAT-37 E 70 NAT-70 D
5 NAT-5 E 38 NAT-38 D 71 NAT-71 E
6 NAT-6 A 39 NAT-39 D 72 NAT-72 E
7 NAT-7 A 40 NAT-40 A 73 NAT-73 C
8 NAT-8 E 41 NAT-41 B 74 NAT-74 B
9 NAT-9 B 42 NAT-42 B 75 NAT-75 E
10 NAT-10 A 43 NAT-43 A 76 NAT-76 D
11 NAT-11 A 44 NAT-44 A 77 NAT-77 D
12 NAT-12 A 45 NAT-45 B 78 NAT-78 C
13 NAT-13 B 46 NAT-46 A 79 NAT-79 C
14 NAT-14 A 47 NAT-47 D 80 NAT-80 E
15 NAT-15 A 48 NAT-48 E 81 NAT-81 D
16 NAT-16 C 49 NAT-49 E
17 NAT-17 D 50 NAT-50 E
18 NAT-18 C 51 NAT-51 E
19 NAT-19 C 52 NAT-52 E
20 NAT-20 E 53 NAT-53 D
21 NAT-21 C 54 NAT-54 D
22 NAT-22 A 55 NAT-55 A
23 NAT-23 A 56 NAT-56 C
24 NAT-24 A 57 NAT-57 E
25 NAT-25 A 58 NAT-58 D
26 NAT-26 A 59 NAT-59 A
27 NAT-27 A 60 NAT-60 B
28 NAT-28 D 61 NAT-61 B
29 NAT-29 C 62 NAT-62 A
30 NAT-30 C 63 NAT-63 A
31 NAT-31 C 64 NAT-64 E
32 NAT-32 D 65 NAT-65 D
33 NAT-33 C 66 NAT-66 D

Exemplary data for four compounds of the invention, each falling into category A in the table above, is shown in Figure 3. These compounds are referred to as compound 1, compound 2, compound 3 and compound 4 hereafter.

The obtained Kd values for hVISTA protein and compounds are shown in Table 3. The ranges are as follows: A: ? 50 nM; B: 50 nM - 200 nM; C: 200 nM - 1000 nM.
Table.3. Binding affinity of compounds with hVISTA protein.
S.No Compound ID MST Kd
1 NAT-3 B
2 NAT-4 A
3 NAT-6 B
4 NAT-7 A
5 NAT-22 B
6 NAT-23 B
7 NAT-24 C
8 NAT-25 B
9 NAT-57 C
10 NAT-75 Not Detected

The obtained Kd values for Human B7-1/CD80 protein are shown in Table 4. The ranges are as follows: A ? 50 nM; B: 50 nM - 200 nM; C: 200 nM - 1000 nM: D: 1.0 µM -2.0 µM

Table.4. Binding affinity of compounds with human B7-1 / CD80 protein.
S.No Compound ID MST Kd S.No Compound ID MST Kd
1 NAT-3 B 14 NAT-23 B
2 NAT-4 A 15 NAT-24 A
3 NAT-6 C 16 NAT-25 A
4 NAT-7 A 17 NAT-26 A
5 NAT-9 B 18 NAT-27 A
6 NAT-10 B 19 NAT-40 A
7 NAT-11 C 20 NAT-41 C
8 NAT-12 A 21 NAT-42 B
9 NAT-13 A 22 NAT-43 B
10 NAT-14 B 23 NAT-44 B
11 NAT-15 A 24 NAT-45 A
12 NAT-16 A 25 NAT-46 B
13 NAT-22 B

Example-49
Determination of Thermal shift (?Tm) by Differential Scanning Fluorimetry (DSF) - Effect of the Compounds on Thermal Stability
Experimental Materials:
Kit: Protein Thermal Shift™ Dye Kit (Applied Biosystems (Thermo Fisher Scientific) # 4461146), Protein Thermal Shift Dye, Protein Thermal Shift Buffer; MicroAmp™ Optical 8-Tube Strip with Attached Optical Caps, 0.2 mL (Applied Biosystems (Thermo Fisher Scientific) Cat# A30588); Protein: Human hPD-L1 / B7-H1 (19-134) Protein, His Tag (MALS verified) Human PD-L1 (19-134), His Tag is expressed from human 293 cells (HEK293). It contains AA Phe 19 - Tyr 134, Predicted N-terminus: Phe 19
Experimental model: QuantStudio™ 3 Real-Time PCR System (software version 1.4.0) Applied Biosystems (Applied Biosystems (Thermo Fisher Scientific))
Experimental steps
The compounds were dissolved in DMSO with a concentration of 10 mM. Lyophilized human protein hPD-L1 protein was dissolved in distilled water at a concentration of 40 µM. hPD-L1 was used in a final concentration of 10 µM, and the compounds dissolved in DMSO were added to the protein sample to the final concentration of 50 µM. The protein incubated with DMSO alone was included as a control. 1000X protein thermal shift dye was diluted with protein thermal shift buffer to prepare 8X protein thermal shift dye and used immediately while protecting from light to reduce photo bleaching. Added assay buffer (PBS), protein and compounds into the well. Sealed the PCR plate, then spin the PCR plate 1000 rpm for 1 minute. After an incubation period (5 min at room temperature) for protein equilibration with compound, protein thermal shift dye was mixed with the protein. Place the PCR plate into the PCR instrument and run the temperature at 25 oC for 2 min, then scan to 95 oC at 0.05 oC/s and heat at 95 oC for 2 min. The thermal denaturation assay was performed in a total volume of 20 µL. All samples were run in triplicates.

Table -5 Summary table of Tm and ?Tm for NAT-compounds/ human PD-L1 protein
S.No Compound ID Concentration Tm value ?Tm (Tm-To)
hPD-L1 Protein 10 µM 52.83 ± 0.3 ? (To) -NA-
1 NAT-3 50 µM 58.00 ± 0.54 ? 5.17 ?
2 Compound 1 50 µM 62.04 ± 0.99 ? 9.21 ?
3 Compound 2 50 µM 60.99 ± 0.35 ? 8.16 ?
4 Compound 3 50 µM 60.40 ± 0.13 ? 7.57 ?
5 Compound 4 50 µM 60.83 ± 0.44 ? 8.0 ?

Example-50: To determine the IC50 values of the compounds by in-vitro Homogeneous time resolved fluorimetry (HTRF) PD1/PDL-1 binding assay.
Experimental materials
PD1 / PD-L1 binding kit (CisBio # 64PD1PEG); Assay Plate: 384 well white opaque proxiplate (Perkin Elmer # 6008280); Centrifuge tubes; Serological Pipettes; Easy pet.
Experimental protocol
Conducted as per the procedure given in the PD1 / PD-L1 binding kit.
Results: The obtained IC50 values for HTRF assay are shown in Table- 6.
The ranges are as follows: A: ? 50 nM; B: 50 nM ? IC50 ? 250 nM; C: 250 nM ? IC50 ? 600 nM; D: ? 600 nM.

Table-6: IC50 values of the compounds in HTRF assay
S.No Compound
ID HTRF IC50 S.No Compound HTRF IC50 S.No Compound
ID HTRF IC50
1 NAT-1 B 36 NAT-36 D 71 NAT-71 D
2 NAT-2 C 37 NAT-37 D 72 NAT-72 D
3 NAT-3 A 38 NAT-38 C 73 NAT-73 B
4 NAT-4 B 39 NAT-39 C 74 NAT-74 B
5 NAT-5 D 40 NAT-40 B 75 NAT-75 D
6 NAT-6 C 41 NAT-41 B 76 NAT-76 D
7 NAT-7 C 42 NAT-42 B 77 NAT-77 C
8 NAT-8 D 43 NAT-43 B 78 NAT-78 C
9 NAT-9 D 44 NAT-44 C 79 NAT-79 D
10 NAT-10 A 45 NAT-45 D 80 NAT-80 D
11 NAT-11 A 46 NAT-46 D 81 NAT-81 C
12 NAT-12 D 47 NAT-47 C
13 NAT-13 D 48 NAT-48 D
14 NAT-14 B 49 NAT-49 D
15 NAT-15 A 50 NAT-50 D
16 NAT-16 A 51 NAT-51 D
17 NAT-17 B 52 NAT-52 D
18 NAT-18 B 53 NAT-53 C
19 NAT-19 C 54 NAT-54 C
20 NAT-20 D 55 NAT-55 A
21 NAT-21 C 56 NAT-56 B
22 NAT-22 A 57 NAT-57 D
23 NAT-23 A 58 NAT-58 C
24 NAT-24 A 59 NAT-59 A
25 NAT-25 A 60 NAT-60 B
26 NAT-26 C 61 NAT-61 B
27 NAT-27 B 62 NAT-62 A
28 NAT-28 C 63 NAT-63 A
29 NAT-29 B 64 NAT-64 D
30 NAT-30 C 65 NAT-65 C
31 NAT-31 C 66 NAT-66 C
32 NAT-32 C 67 NAT-67 D
33 NAT-33 D 68 NAT-68 C
34 NAT-34 C 69 NAT-69 D
35 NAT-35 C 70 NAT-70 C

Example-51: To determine the IC50 values of test compounds by in-vitro HTRF dimerization PDL1/PDL-1 binding assay.
Experimental materials
PDL1 FC Tag Protein; PDL1 HIS Tag Protein; Allophycocyanin; XL665; Assay Plate: 384 well white opaque Proxiplate (Perkin Elmer # 6008280); Centrifuge tubes; Serological Pipettes; Easy pet.
Experimental protocol:
Added 2 ul compound (10X) in 2 % DMSO in assay plate. Compound dilutions were prepared in Europium detection buffer, provided in the KIT. Added 4 ul of diluted Tag1- PDL-1 HIS Tag in all the wells except buffer wells. Dilute Tag1-PDL-1 HIS Tag stock (40uM) to 200nM with Europium detection buffer(40nM/well). Added 4 ul of diluted Tag-2-PDL-1 FC Tag in all the wells except buffer and negative control wells. Dilute Tag2-PD-L1 FC Tag stock (1923nM) to 60nM with Europium detectionbuffer(12nM/well). Added Europium detection buffer, 2 ul in positive control wells, 6 ul in negative control wells and 20 ul in buffer control wells. Add 10 µL of pre-mixed Anti-Tag1 XL665 antibody and Anti-Tag2 Allophycocyanin reagent to all the wells except buffer wells. Diluted Anti-Tag1 XL665 antibody 625nM to 12nM (3nM/well) and Anti-Tag2 Allophycocyanin reagent stock 2960nM to 230nM (58nM/well) with Europium detection buffer. Sealed the plate and incubate for 1 hour at room temperature. Post incubation, removed the plate sealer and read on an HTRF® compatible reader.
Table-7: IC50 values demonstrated by the lead compounds in HTRF-dimerization assay
S.No. Compound ID IC50 (nM)
1 Compound 1 74
2 Compound 2 79
3 Compound 3 94
4 Compound 4 20

Example-52: Determination of EC50 values of the compounds in intercellular PD1/PDL-1 blockade bioassay (NFAT)
Experimental materials
PD-1/PD-L1 Blockade Bioassay kit (Promega # J1250); Assay Plate: 96 well white clear bottom (Corning 3610); Centrifuge tubes; Serological Pipettes; Easy pet; Microplate reader
Experimental protocol: Conducted as per the procedure given in the screening assay kit.
Table-8: EC50 values demonstrated by the lead compounds in NFAT assay
S.No. Compound ID EC50 (uM)
1 Compound 1 0.121
2 Compound 2 0.955
3 Compound 3 0.671
4 Compound 4 0.664
Example-53: To determine the binding activity of the compounds in VISTA/ VSIG3 biotinylated inhibition assay.

Experimental materials:

VSIG-3: VISTA [Biotinylated] Inhibitor Screening Assay Kit ; Catalog #79782(BPS Bioscience); PBS (Phosphate buffered saline); Luminometer or microplate reader capable of reading chemiluminescence; Adjustable micropipettor and sterile tips

Experimental procedure: Conducted as per the procedure given in the screening assay kit.
Table-9: IC50 values demonstrated by the lead compounds in VISTA/VSIG3 assay.
S. No Compound ID IC50 (uM)
VISTA/VSIG3
1 Compound 1 0.078
2 Compound 2 0.048
3 Compound 3 0.795
4 Compound 4 0.034

Experiment-54: PDL1 internalization- The selected cell lines were incubated with the selected compounds to assess compound-binding affinities, and consequently internalization. Results are shown in Figure 4.

Experimental Protocol-1:
CHO-PDL1(high PDL1 expressing) Cancer Cells (0.1×106 cells) were plated in 6-well plate and incubated for 24h. Next day treated with test compounds (5uM) tested for PDL1 inhibition, DMSO (background), and Atezolizumab (I uM, positive control) for 17 hours. Non-confluent cell cultures are trypsinized into single-cell suspension, washed with PBS and centrifuged. Cells are subsequently stained with PDL1-fluorescent proxy (1 µg/100 µL) for 30 min at 4°C, washed twice and re-suspended in fluorescence-activated cell sorting (FACS) buffer. Cells were analysed using Flow cytometer.
Internalization efficiency (%) = [(fluorescence intensity of the control group-fluorescence intensity of the experimental group)/fluorescence intensity of the control group] × 100%

Table-10: Internalization demonstrated by the lead compounds at different incubation time points with CHO-PDL1 cells (PDL1 expressing) cells.

Compound ID Number of hours
72h 48h 17h
Compound 1 (Internalization %) 44.10 41.01 30.50%
Compound 2 (Internalization %) 32.77 23.69 21.70%
Compound 3Internalization %) 45.77 62.50 41.1%
Compound 4 Internalization %) 6.60 Not done Not done
Atezolizumab
Internalization % 85.90 88.43 85.19
As shown herein, the highest reduction on PD-L1 accessibility upon treatment with the different compounds was observed for the most prolonged incubation time (72 hours) except for compound 3, and therefore the subsequent experiments were performed by incubating the compounds for 72 hours.
Experimental Protocol-2:

CHO-PDL1 were digested with trypsin. Cells were resuspended in a complete medium and placed in a 15 mL centrifuge tube. The cells were centrifuged at 1000 rpm for 5 mins and resuspended in an FACS staining buffer (1x PBS with 2% FBS) at a concentration of 2 × 107 cells/mL. The cell suspension was dispensed into a 1.5 mL EP tube at 50 µL/tube (1 × 106 cells). Compound 3 (5 uM) and the cell suspensions diluted with an FACS staining buffer were combined to a final volume of 100 µL so that the concentration of compound 3 in the combined liquid was within the appropriate range ( 5uM) pipetted to mix, and then incubated on ice for 60 mins. 1 mL of an FACS staining buffer was used to wash the cells, which were then centrifuged at 300 × g for 5 mins at 4 ?C to discard unbound compound. After adding 200 µL of the FACS staining buffer to each tube, the tube was incubated at 37 ?C for 2h to observe compound internalization bound to the cell surface. At each time point, another sample tube incubated at 4 ?C was set as a negative control on the basis that the antibody internalization ability under this condition was very weak. To terminate internalization, 1 mL of the ice-cold FACS staining buffer was added to each tube. The tubes were then immersed in an ice bath and centrifuged at 300 g at 4 ?C for 5 mins. Then, 100 µL of the secondary antibody solution containing 5 µL of the anti-PDL1 was added to each sample. They were incubated at 4 ?C for 30 mins in the dark. The cells were washed with 1 mL of the FACS staining buffer and centrifuged at 300 × g for 5 min at 4 ?C to discard unbound secondary antibodies. The cells were washed twice, then resuspended in a 300 µL ice-cold FACS staining buffer. Flow cytometry was used to detect the fluorescence intensity. The degree of internalization of cell surface-bound NAT-24 was determined by the percentage of decrease in the mean fluorescence intensity (MFI) of samples incubated at 37 ?C compared to the control samples incubated at 4 ?C. The following formula was used to calculate the internalization efficiency of each antibody in the cells: internalization efficiency (%) = [(fluorescence intensity of the control group-fluorescence intensity of the experimental group)/fluorescence intensity of the control group] × 100%

Table-11: Internalization demonstrated by the lead compounds after incubation with CHO-PDL1(PDL1 expressing) cells for 2h.
S. No Compound ID Internalization after 2h
%
1 Compound 3 21.30%
2 Atezolizumab 78.55%

Experimental Protocol-3:

MDAMB231 (high PDL1 expressing) Cancer Cells (0.1×106 cells) were plated in 6-well plate and incubated for 24h. Next day treated with test compounds (3uM) tested for PDL1 inhibition, DMSO (background), and Atezolizumab (1 uM, positive control) for 17 hours. Non-confluent cell cultures were trypsinized into single-cell suspension, washed with PBS and centrifuged. Cells were subsequently stained with PDL1-fluorescent proxy (1 µg/100 µL) for 30 min at 4°C, washed twice and re-suspended in fluorescence-activated cell sorting (FACS) buffer. Cells were analysed using Flow cytometer.
Internalization efficiency (%) = [(fluorescence intensity of the control group-fluorescence intensity of the experimental group)/fluorescence intensity of the control group] × 100%

Table-12: Internalization demonstrated by the lead compounds after incubation with MDAMB231 cells at different time points.

Compound ID Number of hours
72h 17h
Compound 1 (Internalization %) 37.00 11.57
Compound 2 (Internalization %) 16.80 Not done
Compound 3 Internalization %) 49.8 18.78
Compound 4 Internalization %) 8.50 Not done
Atezolizumab
Internalization % 93.80 95.05

Experimental Protocol-3:
U251 (PD-L1 expressing) Cancer Cells (0.1×106 cells) were plated in 6-well plate and incubated for 24h. Next day treated with test compounds (10uM) tested for PD-L1 inhibition, DMSO (background), and Atezolizumab (positive control) for 17 hours. Non-confluent cell cultures are trypsinized into single-cell suspension, washed with PBS and centrifuged. Cells were subsequently stained with PD-L1-fluorescent proxy (1 µg/100 µL) for 30 min at 4°C, washed twice and re-suspended in fluorescence-activated cell sorting (FACS) buffer. Cells were analysed using Flow cytometer.
The PD-L1 internalization in cells will be measured by a decrease in MFI relative to background.
Internalization efficiency (%) = [(fluorescence intensity of the control group-fluorescence intensity of the experimental group)/fluorescence intensity of the control group] × 100%

Table-13: Internalization demonstrated by the lead compounds after incubation with U251 cells at different time points.
Compound ID Number of hours
72h 17h
Compound 1 (Internalization %) 41.38% 24.91
Compound 2 (Internalization %) Not done Not done
Compound 3 Internalization %) 74.94 44.91
Compound 4 Internalization %) Not done Not done
Atezolizumab
Internalization % 89.16 94.19

Experimental Protocol-4:
8505C (Thyroid cancer) PDL1 expressing) Cancer Cells (0.1×106 cells) were plated in 6-well plate and incubated for 24h. Next day treated with test compounds (10uM) tested for PDL1 inhibition, DMSO (background), and Atezolizumab (positive control) for 17 hours. Non-confluent cell cultures were trypsinized into single-cell suspension, washed with PBS and centrifuged. Cells were subsequently stained with PDL1-fluorescent proxy (1 µg/100 µL) for 30 min at 4°C, washed twice and re-suspended in fluorescence-activated cell sorting (FACS) buffer. Cells were analysed using Flow cytometer.
The PD-L1 internalization in cells was be measured by a decrease in MFI relative to background.
Internalization efficiency (%) = [(fluorescence intensity of the control group-fluorescence intensity of the experimental group)/fluorescence intensity of the control group] × 100%

Table-14: Internalization demonstrated by the lead compounds after incubation with 8505Ccells at 17 hours
Compound ID Number of hours
17h
Compound 1 (Internalization %) No internalization
Compound 2 (Internalization %) 18.99%
Compound 3Internalization %) No internalization
Compound 4 Internalization %) 25.43%

Experimental Protocol-4:
HCC827 (Thyroid cancer) PDL1 expressing) Cancer Cells (0.1×106 cells) were plated in 6-well plate and incubated for 24h. Next day treated with test compounds (5uM) tested for PDL1 inhibition, DMSO (background), and Atezolizumab (positive control) for 17 hours. Non-confluent cell cultures were trypsinized into single-cell suspension, washed with PBS and centrifuged. Cells were subsequently stained with PDL1-fluorescent proxy (1 µg/100 µL) for 30 min at 4°C, washed twice and re-suspended in fluorescence-activated cell sorting (FACS) buffer. Cells were analysed using Flow cytometer.
The PD-L1 internalization in cells was be measured by a decrease in MFI relative to background.
Internalization efficiency (%) = [(fluorescence intensity of the control group-fluorescence intensity of the experimental group)/fluorescence intensity of the control group] × 100%

Table-15: Internalization demonstrated by the lead compounds after incubation with HCC827 cells at 72h.
S. No Compound ID Internalization after 72h
%
1 Compound 1 36.07%
2 Compound 3 60.80%

Experiment-55: Reversal of T-cell Exhaustion by the lead compounds demonstrated by PD1 down regulation thereby IFNg restoration
a. Experimental protocol for T-cell Exhaustion
• Start with 1-1.5 × 106 purified T cells/ml in culture medium in a suitable tissue culture plate or tissue culture flask.
• Add Dyna beads Human T-Activator CD3/CD28 (Thermo) at a bead-to-cell ratio of 1:1.
• Add 30 U/ml rIL-2.
• Incubate in a humidified CO2 incubator at 37°C, according to your specific experimental requirements.
• Examine cultures daily, noting cell size and shape. Cell shrinking and reduced proliferation rate is typically observed in exhausted cell cultures.
• Every 48 h, cells were counted and restimulated with a fresh batch of CD3/CD28 Dynabeads.
• After two or three stimulations, the Dynabeads were removed by LS column through Magnetic separation.
• The Exhausted T cells were Counted and frozen for further experiments.

a. Experimental protocol-1 -treatment of compounds with Exhausted T cells in the absence of cancer cells (determination of PD1, TIM3 and CD8 by flow cytometer)
* Exhausted T cells (105 cells) were plated and activated with (CD3+CD28) and incubated with compounds (10uM) for 72h, sup was subjected to FACS analysis to check Tcell Exhaustion reversal. Early-stage Exhaustion marker PD1 and late stage Exhaustion marker TIM3 expression of Ex T cells got reduced and CD8 population got increased by treating the compounds with Exhausted T cells in the absence of cancer cells

Table-16
MARKERS Exhausted T cells EX T cells+ Compound 1 EX T cells+ Compound 2 EX T cells+ Compound 3
CD8+ (%) 29.758 68.137 69.579 70.122
PD1 (%) 77.590 69.249 72.620 64.946
TIM3 (%) 53.270 36.964 41.268 28.424

b. Experimental protocol 2 for the treatment of compounds with Exhausted T cells cocultured with MDAMB231 cells (determination of IFNg restoration and PD1 expression by flow cytometer)
Exhausted T-cells were plated and cocultured with MDA-MB-231 cancer cells in the presence of test compounds (3, 1, 0.3uM) for 72h. Atezolizumab is used a positive control (0.3uM) T cells and cancer cells were utilized at a ratio of 3:1. The supernatant containing T cells was collected, The level of IFNg restored by the activated T cells and PD1 expression in the supernatant was detected by Flow cytometer. Highest IFNg restoration and downregulation of PD1 expression is noticed at 0.3uM concentration of test compound.
Table-17:

Compound ID IFNg restoration and PD1 down regulation
IFNg (%) PD1 (%)
Exhausted T cells 6.78 37.426
Coculture 40.174 18.035
Compound 1
46.533 7.299
Compound 3 54.071 6.889
Atezolizumab 52.252 12.412

Experiment-55: T cell mediated cytotoxicity to tumour cells-
Apoptosis assay (Annexin V -flow cytometry based)
Exemplary results are shown in Figure 5 and Figure 6.
Experimental protocol for the isolation of human Peripheral blood mononuclear cells (PBMCs) from whole blood of the heathy volunteer
1. Process Whole blood cells from healthy donor on a density gradient, like Ficoll Hypaque to enrich for mononuclear cells.
2. Recover the "buffy coat" containing the mononuclear cells and wash the cells two times with excess DPBS to remove any residual separation media. This can be done by spinning the cells for 10 minutes at 200 x g.
3. After the second wash step, disrupt the cell pellet by “racking” the tube, resuspend the cells in H-Lyse Buffer from R&D Systems®’ Human Erythrocyte Lysing Kit (Catalog # WL1000) that has been diluted to 1X strength with sterile distilled water. Quickly vortex the tube (10 mL of 1X H Lyse solution per 250 million cells is recommended).
4. Incubate the cells for 10 minutes at room temperature and then fill the tube with 1X Wash Buffer from the Lysing kit. Note: The Wash Buffer must also be diluted with sterile water to 1X strength prior to use.
5. Spin the cells for 10 minutes at 200 x g and then resuspend the cells in a small volume of 1X MagCellect Buffer.
6. Perform a cell count and then adjust the cell concentration to 1 x 108 cells per mL with cold 1X MagCellect Buffer.
Experimental protocol for Human CD8 T Cells Isolation (KIT:Cat no:MAGH112)
1. Transfer 1 x 108 cells (1 mL volume) into a 15 mL Centrifuge Tube. Add 100 µL of MagCellect Human CD8+ T Cell Biotinylated Antibody Cocktail. Gently mix the cell-antibody suspension, avoiding bubble formation, and incubate at 2-8 °C in a refrigerator for 15 minutes.
2. Add 200 µL of Streptavidin Ferrofluid to the cell suspension, mix gently and incubate at 2-8 °C in a refrigerator for 15 minutes.
3. At the end of the incubation period bring the volume of the reaction in the tube to 2 mL by adding 0.7 mL of 1X MagCellect Buffer. Mix gently to ensure that all reactants in the tube are in suspension.
4. Proceed to magnetic separation.
5. Place column in the magnetic field of a suitable MACS Separator
6. Prepare column by rinsing with 3 mL of buffer.
7. Apply cell suspension (Max 109) onto the column. Collect flow-through containing unlabelled cells, representing the enriched CD8 T cells.
8. Wash column with 3 mL of buffer. Collect unlabelled cells that pass through, representing the enriched CD8 T cells, and combine with the flow-through from step 7.
9. Spin down the flow-through at 300g for 10 min.
10. Count the cells and freeze them in 90% FBS and 10 % DMSO in LN2
Experimental protocol 1-apoptosis assay by treating the lead compounds with MDAMB231 and CD8+ T cells (Annexin V assay).
• CD8+ T cells isolated from PBMCs were plated with MDAMB231 cancer cells at a 1:1 ratio (3 x 105) in 96-well plates.
• T cells were stimulated with anti-CD3 and anti-CD28 antibodies. Cells were cocultured at 37_ for 20 hours in the presence of the compounds (3 uM) and mAB(1 uM)
• Cells were washed in FACS buffer and then resuspended in Annexin binding buffer containing FITC-conjugated Annexin V. Mix and incubate for 10min at RT. Wash cells in binding buffer and resuspend in binding buffer and add Propidium Iodide (PI).
• Performed flow cytometer analysis. Compound-1 and Compound-3 displayed more apoptotic feature when CD8+ T cells were cocultured with MDAMB231 cells.
Table-18
Compound ID ANNEXINE V+ PI- (%) ANNEXINE V+ PI+ (%)
Apoptotic Late stage Apoptotic
Coculture 25.955 55.096
Compound 1
23.720 61.060
Compound 3 23.300 63.120
Atezolizumab 19.384 42.493
Experimental protocol -2-Apoptosis assay by treating the lead compounds with U251and CD8+ T cells (Annexin V assay).
• CD8+ T cells isolated from PBMCs were plated with U251 cancer cells at a 1:1 ratio (3 x 105) in 96-well plates.
• T cells were stimulated with anti-CD3 and anti-CD28 antibodies. Cells were cocultured at 37_ for 20 hours in the presence of the compounds (10 uM) and mAB(1 uM)
• Cells were washed in FACS buffer and then resuspended in Annexin binding buffer containing FITC-conjugated Annexin V. Mix and incubate for 10min at RT. Wash cells in binding buffer and resuspend in binding buffer and add Propidium Iodide (PI).
• Performed flow cytometer analysis. Compound-1 and Compound-3 displayed more apoptotic feature when CD8+ T cells were cocultured with U251 cells.
Table 19
Compound ID ANNEXINE V+ PI- (%) ANNEXINE V+ PI+ (%)
Apoptotic Late stage Apoptotic
Coculture 38.154 55.501
Compound 1
45.910 50.882
Compound 3 37.896 59.561
Atezolizumab 38.306 45.148
Experimental protocol -3-Apoptosis assay by treating the lead compounds with HCC827and CD8+ T cells (Annexin V assay).
• CD8+ T cells isolated from PBMCs were plated with HCC827cancer cells at a 1:1 ratio (3 x 105) in 96-well plates.
• T cells were stimulated with anti-CD3 and anti-CD28 antibodies. Cells were cocultured at 37_ for 20 hours in the presence of the compounds ( uM) and mAB(1 uM)
• Cells were washed in FACS buffer and then resuspended in Annexin binding buffer containing FITC-conjugated Annexin V. Mix and incubate for 10min at RT. Wash cells in binding buffer and resuspend in binding buffer and add Propidium Iodide (PI).
• Performed flow cytometer analysis. Compound-1 and Compound-3 displayed more apoptotic feature when CD8+ T cells were cocultured with HCC827 cells.
Table 20
Compound ID ANNEXINE V+ PI+ (%) ANNEXINE V+ PI- (%)
Late stage Apoptotic Apoptotic
Coculture 64.830 24.670
Compound 1
70.040 23.120
Compound 3 70.510 21.960
Atezolizumab 72.410 20.530

Experiment-56: Activation of the T-cells by coculturing with MDAMB231/U251 cells and treating with the test compounds demonstrated by the IFNg restoration and increase in T cell markers.
Experimental Protocol -1
PBMCs isolated three healthy donors were used in this experiment. One day before co-culture, PBMCs were resuspended in T cell media and cultured overnight at 37°C. Prior to co-culture MDAMB231 cells were treated overnight with 200 ng. mL-1 human recombinant interferon IFN-?. PBMCs were seeded at a density of 0.1×106 cells/well and stimulated with CD3, CD28, IL2. Co-culture of PBMCs was performed with MDAMB231 cells at 2:1 cells in the presence of test compounds (3 uM) and Mab for 72 hours. The cells were then stained with fluorochrome-labelled antibodies and analysed by flow cytometer.

Table-21

CD8+T-CELL ACTIVATION MARKERS (AVERAGE OF THREE DONORS)

Markers STIM
PBMCs STIM PBMCs + MDAMB231
Coculture Coculture +
Compound 1
Coculture
Compound 3
Coculture +Atezolizumab
(1uM)
CD8+ GATING
CD8+ (%) 38.469 41.769 44.576 43.796 43.599
CD69+ (%) 66.471 87.685 86.242 89.102 88.275
CD107A (%) 37.210 54.859 65.00 68.097 58.811
IFNg (%) 29.832 30.02 30.986 32.405 28.633
Gran-B 88.797 95.764 97.335 96.585 96.536

Experimental protocol 2:

U251(1.5 x 105)- in 6-well plate- 24h treatment with 500IU/ml of IFNg cocultured (2:1) with Activated T cells and compounds (10uM) for 72h, sup was subjected to Flow cytometry analysis to check CD8+ activation markers.
Table-22
Compound ID T cell activation markers
CD8+ IFNg (%) Granzyme-B (%)
Coculture 68.165 86.544 68.997
Compound 1
83.898 92.929 81.241
Compound 3 79.838 87.663 82.148
Atezolizumab
71.00 87.148 77.289
Above experiment is demonstrated in one specimen donor T cells.
Experiment-57:
ADME and pharmacokinetics of the lead compounds

Experimental protocol 1
Determination of Permeability of lead compounds in MDR1-MDCK monolayer-
MDR1 transfected MDCK monolayers cultured for 5 days were used for the in vitro transport studies and were obtained from Netherland Cancer Institute. Cells were split every other day at a split ratio of 1:3~1:5 and grown in Dulbecco's Modified Eagle Medium supplemented with 10% FBS in the presence of antibiotics (1% Pen-Strip). For transport studies, cells were seeded onto polycarbonate Transwell filter membranes (Millipore) at a density of 500,000 cells/well. After 24 h post seeding, changed medium and cultured for another 4 days before transport experiments. For transport studies, donor solutions were prepared by diluting the stock solutions of test compounds in transport medium (HBSS buffer with 10mM HEPES, pH 7.4). Receiver solutions were the same HBSS buffer with 10mM HEPES, pH 7.4. The transport of test compounds (5 µM) was measured in duplicate in two directions [apical to basolateral (A?B) and basolateral to apical (B?A)]. The permeability coefficient for membrane transport of test compounds was determined using the following equation:

Papp (cm/sec) = (Vr/C0) (1/S) (dC/dt)
(Papp = apparent permeability, Vr = volume of medium in the receiver chamber, C0 = PAR of the test drug in the receiver chamber, S = surface area of monolayer, dC/dt = drug PAR in the receiver chamber with time).; Area of 24-well = 0.7 cm2 ; Peak area ratio = Analyte peak area/IS peak area
Efflux ratio was defined as:
Efflux ratio =(???????? ??-??)/(???????? ??-??)
Table-23: Permeability of lead compounds in MDR1-MDCK layer

Compound ID Permeability @ 5 uM (MDR1-MDCK) incubated for 60 min- B-A/A-B
Efflux ratio
(A-B) 106 cm/Sec (B-A) 106 cm/Sec
Compound 1 Salt 4.32 6.90 1.60
Compound 2 Salt 4.23 7.88 1.86
Compound 3 Salt
5.95 9.71 1.63
Compound 4 Salt
5.87 9.01 1.54
Digoxin 0.65 23.2 35.90
Propranolol 43.2 44.8 1.04
Conclusions-All the lead compounds are not PgP substrates and moderately permeable.
Experimental protocol 2
Time dependent Inhibition (TDI) of CYP 3A4 by the lead compounds in Human liver microsomes
Stock preparation:
Standard working solution preparation:
• Working stock preparation: 10 mM of test compounds and 100mM of mefipristone were prepared using DMSO.
• The test compounds and reference standard, mifepristone were tested at 1 µM and 10 µM final concentration.
• The final protein concentration was maintained at 0.1 mg/ mL.
Buffer preparation: 66.7 mM potassium phosphate buffer (pH 7.4) was used as assay buffer and was prepared by mixing appropriate volumes of 1 M K2HPO4 and 1 M KH2PO4 stock buffers. Experiment was performed as per below condition.
Table 24
CYP450 isoform Activity Final midazolam (substrate conc. In µM) Microsomal protein conc. (mg/mL) Final mifepristone (Inhibitor conc. in µM)
3A4 1’-Hydroxylation 2.5 0.1 10

Methodology (n=2):
Preparation of incubation mixture
1. Thawed the microsomes by keeping on ice. 2. Prepared microsomal suspension in potassium phosphate buffer, to yield the required final protein concentration. 3. Mixed the contents gently by inverting and equilibrated at 37 ± 1°C for 5 min.
Experimental procedure
Primary-incubation steps: 1. Added 898.8 µL of the above incubation mixture into three polypropylene tubes labeled “Test”, “Test control” and “Positive control”; 2. Spiked 1.12 µL of the "Test" and “Positive” compound from 10mM and 100mM respectively for +NADPH and -NADPH incubations; 3. Similarly spiked 1.12 µL of DMSO to the tube labeled “Test control” ; 4. Added 100 µL of 10 mM NADPH to +NADPH incubations and 100 µL of buffer to -NADPH incubations. 5. Mixed the incubation mixture and incubated for 1, 5, 10, 20, 30 and 40 mins.
Secondary-incubation steps:
6. At respective time points, two aliquots (2 x 20 µL) from each of the above primary incubation mixtures were transfered into secondary incubation containing Midazolam (2.5µM); 7. Further, added 20µL of NADPH to all the tubes and incubated in shaking water bath at 37 ± 1°C for 10 minutes.; 8. After completion of incubation time, stop the reactions by adding 300 µL of quenching solution. ; 9. Vortexed the tubes gently and centrifuged at 4000 rpm for 20 minutes at 4°C.; 10. Collect the supernatant and submited the samples to the LC-MS/MS system. 11. Metabolite formation for each probe substrate estimated employing LC-MS/MS system.
Data Analysis:
Percent remaining: [Mean peak area ratio at each timepoint / Mean peak area ratio at 0 min] x 100
Bioanalysis: Bioanalysis was done on LC-MS/MS as per the following conditions.

Mass spectrometer : LCMS-8045 Triple Quad
HPLC : Shimadzu Nxera 30 AC
Chromatographic conditions
Mobile phase A 0.1 % formic acid in Milli-Q water
Mobile phase B Acetontirile
Flow rate (mL/min) 0.80
Run time (min) 3.50
Injection volume (µL) 5.00
Sampling 200 µL + 200 µL quenching solutions
Internal standard Loperamide
HPLC column Atlantis dC18 (4.6×50 mm, 3.1 µm)
Gradient Program
Time (min) Mobile phase A (%) Mobile phase B (%)
0.01 95 5
1.40 5 95
2.80 5 95
3.00 95 5
3.50 Controller Stop

Mass spectrometry conditions
S.No. Compound Name Ionization Mode RT (min) MRM (Q1>Q3)
1 OH-Midazolam Positive 1.66 342.20 / 324.00
2 Loperamide Positive 1.80 477.30 / 266.25

Table-25

Compound ID % inhibition (without NADPH)
0 min 1 min 5 min 10 min 20 min 30 min 40 min
Mifepristone 64.0 65.8 65.1 63.5 67.9 71.2 69.5
Compound 1 NI NI NI NI NI NI NI
Compound 2 NI NI NI NI NI NI NI
Compound 3 NI NI NI NI NI NI NI
Compound 4 NI NI NI NI NI NI NI

Table 26
Compound ID % inhibition (with NADPH)
0 min 1 min 5 min 10 min 20 min 30 min 40 min
Mifepristone 65.1 69.7 83.6 85.4 88.9 89.9 92.6
Compound 1 NI NI NI 5.57 NI NI NI
Compound 2 NI NI NI NI NI NI NI
Compound 3 NI NI NI 2.91 NI NI NI
Compound 4 NI NI NI 1.06 NI NI NI
NI- NO INHIBITION
Conclusion- CYP3A4 is the main human enzyme responsible for phase I metabolism of drugs. Inhibition of CYP3A4 could lead to drug toxicity, drug-drug interactions, and other adverse effects, and is usually undesired. All the compounds are having minimum liability for Drug-Drug interactions.

Experimental protocol 3

Plasma protein binding of the lead compounds in mouse and human plasma
Procedure:
To evaluate the ability of compound to bind the plasma proteins, the most common approach of plasma protein binding using equilibrium dialysis was used. Compound was tested at a final concentration of 3 µM in mouse and human plasma. An aliquot of 150 µL plasma containing compound was added in first half (plasma side) of the well of 96-well micro-equilibrium dialysis device. An aliquot of 150 µL of 100 mM sodium phosphate buffer pH 7.4 was added in the second half (buffer side) of the well of 96-well HT equilibrium dialysis device. The plate containing plasma and buffer was equilibrated at 37 ± 1 °C for 4.5 h, with constant shaking on an orbital shaker. Samples were collected from respective halves after the completion of incubation time. The proteins were precipitated using organic solvents. The samples were subjected to centrifugation and the supernatants were analysed analysis on LC-MS/MS.
Conclusion:
All the compounds had a high binding in mouse and human plasma with fraction bound greater than 99.50%. The stability and recovery of all the compounds in plasma was good. In vitro High protein plasma binding has minimum liability for the compounds targeting brain.

Experimental protocol -4
In Vitro Assessment of PXR Activation Potential of lead compounds in Plated Cultures of DPX2 Cells-
Study Design
1. Preparation for Cell Seeding
1) Prepared the culture medium for DPX2 cells using DPX2 culturing medium supplemented with 10% FBS.
2) Cultivated DPX2 cells in T-75 flasks in a cell culture incubator set at 37°C, 5% CO2, 95% relative humidity. Allowed cells to reach 80-90% confluence before detaching and splitting.
3) Rinsed cultivated cells in T-75 flasks with 5 mL PBS. Aspirate off, add 1.5 mL trypsin, and incubated at 37 °C for approximately 5 minutes or until the cells detach and float. Inactivated trypsin by adding excess serum containing medium.
4) Removed cell suspension to a conical tube and pellet cells by centrifugation at 150 ´ g for 5 minutes. Resuspended cells in seeding medium at a density of 3.2´105 cells/mL. Transferred 25 µL to each well of 384-well cell culture plate. Placed plate(s) in incubator and incubate at 37°C for 24 hours.
2. Incubation with Test Compounds
1) Prepared stock solutions of test compounds and inducers in DMSO. The final concentrations for positive control rifampicin is 10 µM. The final work concentration for test compounds were as requested. Final concentration of DMSO in the treatment group should be 0.1%.
2) Removed the plate(s) from the incubator and directly add 25 nL of the negative control, inducers, or test article solutions, each in triplicate. Returned plate(s) to the incubator for 24 hours.
3) Check cell morphology and monolayer integrity prior to initiating the experiment with the substrates to ensure that the monolayers are of acceptable quality for the study.
3. Determination of Quantitation of PXR Activation
1) After 48 of treatment, the cultures were ready for the determination of quantitation of PXR activation.
2) Allowed the CellTiter-Fluor™ Cell Viability Assay kit and One-Glo Luciferase Assay System to reach room temperature. Transferred the GF-AFC Substrate (10 µL) into the Assay Buffer container (10 ml) to form 2× reagent, then diluted to 1× reagent by adding 10 mL PBS. Transferred the ONE-Glo Luciferase Transfer Assay Substrate into the ONE-Glo Luciferase Assay Buffer.
3) Removed the plate(s) from the incubator. Aspirated the dosing medium from each well. Poured contents of the tube containing 1× CellTiter-Fluor™ Cell Viability Assay reagent into a sterile media trough. Using a multichannel pipette, gently add 25 µL of the reagent into each well. Incubate the plate for 30 minutes at 37 °C.
4) Removed the 384-well plate from the incubator, briefly allowed to cool to ambient temperature. Measured the fluorescence of individual wells with a microplate reader in fluorescence mode at 400 nm excitation and 505 nm emission.
5) Poured ONE-Glo Assay reagent into a media trough, add 25 µL of the reagent into each well. Carefully agitated the plate to mix the reagents contained in the well. After 5 minutes, readed the luminescence of individual wells using a luminometer.

4. Data Analysis
All calculations were carried out using Microsoft Excel.
The normalized luciferase activity is determined by RLU/RFU, where the RLU means the relative luminescence unites for each of the three replicates for each test compounds at each dosage, and RFU means the relative fluorescence unites for each of the three replicates for each test compounds at each dosage. The RLU and RFU for vehicle was the average of the three replicates for vehicle sample respectively.
The fold-activation mRNA level was determined by the equation:

The percent of control (%) is determined by the equation:
Percent of control (%)= (Fold of activation compound/Fold of activation positive control)*100
The viability (%) is determined by the equation:
Viability (%)= (RFU compound/RFU DMSOl)*100

Table-27

NO. Compound ID Conc. (µM) Fold Activation Percent of Control (%) Cell Viability (%) Comment
Control Rifampicin 10 15.5 100 98.7 Inducer
1 Compound 1 salt 10 1.88 12.1 76.6 Not an Inducer
2 Compound 3 salt 10 1.22 7.84 73.1 Not an inducer
3 Compound 4 salt 10 1.33 10.0 91.4 Not an Inducer

Conclusions: All the lead compounds are not CYP3A4 inducers
Experiment-58;
Experimental protocol:
To assess the cardiac risk of the lead compounds through hERG assay
Table-28

Screening of the compounds at 10 points (30 uM to 1.53 nM)- (IC50 Values-uM)
Compound 1 Compound 2 Compound 3 Compound 4 E4031
20.00 12.30 13.70 15.20 15.9 nM

Conclusion- Lead compounds are reasonably safe and are not Cardiotoxic as per hERG-assay. Safety margin (IC50>10uM); E-4031 is an experimental class III antiarrhythmic drug that blocks potassium channels of the hERG type.
,CLAIMS:WE CLAIM:

1. A compound represented by Formula I:

(I)
wherein:
R1, R2 and R4 are each independently selected from hydrogen, halogen, hydroxy, cyano, C1-6 alkyl, C1-6 alkoxy, cycloalkyl containing up to 6 carbon atoms, alkenyl, alkynyl, and -NR’R5;
R3 is selected from hydrogen, halogen, hydroxy, cyano, C1-6 alkyl, C3-6cycloalkyl, alkenyl, alkynyl, NR’R5, and -OR6;
R’ is selected from H and C1-6 alkyl;
R5 is selected from hydrogen, C1-6alkyl, cycloalkyl containing up to 6 carbon atoms, alkenyl, and alkynyl;
R6 is selected from C1-6alkyl and -L2-A;
L2 is absent or is C1-6 alkylene;
A is selected from aryl and 5- to 14-membered heteroaryl, which may be optionally substituted with one or two substituents each independently selected from cyano, halogen, hydroxy, and amino; and
L1 is optionally substituted polycyclic heterocyclyl;
or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof.

2. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to claim 1, wherein:
R1, R2 and R4 are each independently selected from hydrogen, halogen, cyano, C1-6 alkyl, C1-6 alkoxy, cycloalkyl containing up to 6 carbon atoms, alkenyl, alkynyl, and -NHR5;
R3 is selected from hydrogen, halogen, cyano, C1-6 alkyl, cycloalkyl containing up to 6 carbon atoms, alkenyl, alkynyl, NHR5, and -OR6;
-OR6 is either C1-6 alkoxy or -L2-A;
L2 is absent or is methylene; and
A is aryl which is optionally substituted with a cyano group.

3. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to claim 1 or claim 2, wherein R1 is selected from halogen, cyano, C1-6 alkyl, and C1-6 alkoxy; preferably from halogen, cyano, C1-3 alkyl, and C1-3 alkoxy; and more preferably halogen.

4. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to any of claims 1 to 3, wherein R2 is selected from hydrogen and C1-6 alkyl, preferably hydrogen.

5. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to any of claims 1 to 4, wherein R3 is selected from hydrogen, halogen, C1-6 alkyl, and -OR6; preferably from hydrogen, halogen, C1-3 alkyl, and -OR6.

6. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to any of claims 1 to 5, wherein R4 is selected from hydrogen, halogen, cyano, C1-6 alkyl, and C1-6 alkoxy; preferably from hydrogen, halogen, C1-3 alkyl, and C1-3 alkoxy; and more preferably from hydrogen and C1-3 alkyl.

7. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to any of claims 1 to 6, wherein R6 is C1-6 alkyl.

8. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to any of claims 1 to 6, wherein R6 is L2-A and A is C6-10 aryl optionally substituted with one or two substituents each independently selected from cyano and halogen; preferably wherein A is C6 aryl optionally substituted with a cyano group.

9. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to any of claims 1 to 6 or 8 wherein L2 is methylene.

10. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to any of claims 1 to 9, wherein L1 is of formula:

wherein
n is 0, 1, 2, 3, 4 or 5;
a wavy bond line indicates a covalent bond;
two of RA, RB, RC and RD join together to form a 3- to 8-membered cycloalkyl, cycloalkenyl, or heterocyclyl ring which is optionally substituted by one, two or three substituents each independently selected from halogen, hydroxy, cyano, C1-6 alkyl, and C1-6alkoxy; and either
(i) the other two of RA, RB, RC and RD are each independently selected from hydrogen, halogen, hydroxy, cyano, C1-6 alkyl, and C1-6alkoxy, or
(ii) the other two of RA, RB, RC and RD are absent and the carbon atoms between the remaining two of RA, RB, RC and RD are joined by a double bond.

11. The compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt according to any of claims 1 to 10, wherein L1 is selected from:
, , , , , , , , , , , , , , , , , , , , , and ;
preferably from , , , , and ;
and more preferably from and .
12. The compound according to any of claims 1 to 11, which is selected from:
, , , , , , , , , ,
, ,
, , , , , , , , , , , ,
, or ,
or a prodrug, hydrate, solvate, or pharmaceutically acceptable salt thereof.
13. A method of treating, preventing or ameliorating disease, the method comprising administering a compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined in any of claims 1 to 12 to a subject.

14. The method according to claim 13, wherein the method comprises inhibiting the PD-1/PD-L1 checkpoint pathway.

15. The method according to claim 13 or claim 14, wherein the method comprises inhibiting the activity of VISTA.

16. The method according to any of claims 13 to 15, wherein the method comprises one or more of:
(i) inhibit the PD-1/PD-L1 checkpoint pathway;
(ii) inhibit the activity of VISTA;
(iii) reverse T-cell exhaustion;
(iv) increase T-cell function;
(v) decrease expression of PD-1 in exhausted T cells;
(vi) decrease expression of TIM3 exhausted T cells;
(vii) increase CD8+ T-cell population;
(viii) increase expression of CD107a;
(ix) increase expression of Granzyme-B; and/or
(x) IFNg restoration.
(xi) Increasing T cell mediated cytotoxicity to tumour cells

17. The method according to any of claims 13 to 16, wherein the method is a method of treating, ameliorating and/or preventing a disorder selected from a proliferative disorder, cancer, a neurodegenerative disorder, or an infectious disorder.

18. The method according to any of claims 13 to 17, wherein the method is a method of treating, ameliorating and/or preventing a disorder associated with overexpression of PD-L1, preferably wherein the disorder is a cancer associated with overexpression of PD-L1.

19. The method according to any of claims 13 to 18, wherein the method is a method of treating, ameliorating and/or preventing cancer selected from brain cancer, breast cancer, lung cancer, renal cancer, bladder cancer, thyroid cancer, liver cancer, and gall bladder cancer; preferably breast cancer, brain cancer, thyroid cancer and lung cancer.

20. The method according to any of claims 13 to 19, wherein the method additionally comprises administering to the subject a further therapeutic agent.

21. The method according to claim 20, wherein the further therapeutic agent is selected from Nivolumab, Pembrolizumab, Avelumab, Atezolizumab, Cemiplimab and Durvalumab.

22. A compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined in any of claims 1 to 12, for use in a method as defined in any of claims 13 to 21.

23. Use of a compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined in any of claims 1 to 12, in the manufacture of a medicament for use in a method as defined in any of claims 13 to 21.

24. An in vitro method which comprises using a compound, prodrug, hydrate, solvate, or pharmaceutically acceptable salt as defined in any of claims 1 to 12 to:
(i) inhibit the PD-1/PD-L1 checkpoint pathway;
(ii) inhibit the activity of VISTA;
(iii) reverse T-cell exhaustion;
(iv) increase T-cell function;
(v) decrease expression of PD-1 in exhausted T cells;
(vi) decrease expression of TIM3 exhausted T cells;
(vii) increase CD8+ T-cell population;
(viii) increase expression of CD107a;
(ix) increase expression of Granzyme-B; and/or
(x) IFNg restoration.
(xi) Increasing T cell mediated cytotoxicity to tumour cells