AU2020101052A4 - Indoleamine 2,3-dioxygenase inhibitors and their application in medicine - Google Patents

Abstract

The present invention relates to a compound represented by formula I, a pharmaceutical composition containing the compound of formula I, a method for inhibiting indoleamine 2,3-dioxygenase, and its use in medicine. R2 N ~ C yl(CR'b r-ArrNCY (I)

Description

Indoleamine 2,3-dioxygenase inhibitors and their application in medicine

This application claims benefit priority of Chinese Patent Application Serial

NO.201711498307.2 filed on November 2 9th, 2017, named as Indoleamine

2,3-dioxygenase inhibitors and their application in medicine and NO.

201810754253.6, filed on July 2 9th 2018, named as Indoleamine 2,3-dioxygenase

inhibitors and their application in medicine. Both of which are hereby

incorporated by reference in its entirety.

Field of the invention

The invention relates to a novel indoleamine 2,3-dioxygenase inhibitor with

inhibitory activity on tryptophan metabolism, and a pharmaceutical composition

containing it as an active ingredient.

Background of the invention

Tryptophan (TRP) is an a-amino acid used for protein biosynthesis. It

contains a-amino group, a-carboxylic acid group and side chain indole. It is

indispensable in human beings. The human body is not able to synthesize it, but

need to obtain it from the diet. Tryptophan is also a precursor to the synthetic

neurotransmitter serotonin and the hormone N-acetyl-5-methoxytryptamine

(melatonin). The heme-dependent enzyme indoleamine 2,3-dioxygenase (also

called IDO or IDO1) is a metabolic enzyme responsible for converting

tryptophan to N-formyl-kynurenine outside the liver; this is the first step in the

process of tryptophan metabolism, and also the rate-limiting step of the entire

process. N-formyl-kynurenine is a precursor of various biologically active

molecules kynurenine (or Kyn), and kynurenine has immunomodulatory

functions (Schwarcz et al, Nat Rev Neurosci. 2012; 13 ( 7): 465).

Indoleamine 2,3-dioxygenase (IDO) is widely expressed in solid tumors

(Uyttenhove et al, Nat Med. 2003; 10: 1269), and is also expressed in primary

cancer and metastatic cancer cells. IDO is induced by proinflammatory factors in

tumors, including type I and type II interferons produced by infiltrating

lymphocytes (Tnani and Bayard, Biochim Biophys Acta. 1999; 1451 (1): 59;

Mellor and Munn, Nat Rev Immunol 2004; 4 (10): 762; Munn, Front Biosci. 2012;

4: 734) and transforming growth factor-p (TGF-j) (Pallotta et al, Nat Immunol.

201 1; 12 (9): 870). In recent years, more and more evidence shows that IDO, as

an inducible enzyme, plays a major role in the regulation of immune cells.

Decreased tryptophan levels and increased kynurenine could suppress immune

effector cells and promote adaptive immune suppression by inducing and

maintaining regulatory T cells (Tregs; Munn, Front Biosci. 2012; 4: 734); the

concentration of tryptophan in T cells is also positively correlated in the immune

system. In the tumor immune microenvironment, activated or overexpressed IDO

leads to the exhaustion of tryptophan, which in turn leads to T cell death,

inactivation of the immune system, and ultimately to tumor immune tolerance

and immune escape. Existing studies have shown that the immune imbalance

caused by IDO is deeply involved in the formation and progression of tumors.

Therefore, IDO receptor has become an important target for tumor and other

immunotherapy. In addition to tumors, IDO is also associated with viral

infections, depression, organ transplant rejection, or autoimmune diseases

(Johnson and Munn, Immunol Invest 2012; 41 (6-7): 765). Therefore, drugs

targeting IDO are also of great value for the treatment of the above diseases. In

short, developing an active and selective IDO inhibitor that can effectively treat

diseases caused by harmful substances in the kynurenine pathway via regulating

kynurenine channels and maintaining the level of tryptophan in the body is

necessary as a monotherapy or combination therapies.

A large number of published preclinical data further confirmed the role of

IDO in the anti-tumor immunity. IDO inhibitors can be used to activate T cells,

thereby increasing the activation of T cells when they are suppressed by viruses

such as pregnancy, malignancy, or HIV. Forced IDO induction in cancer cells

proved to have a survival advantage (Uyttenhove et al, Nat Med. 2003; 10: 1269).

Another in vivo study shows that IDO inhibitors reduce the dependence on

lymphocytes by reducing kynurenine levels during tumor growth (Liu et al,

Blood. 2010; 115 (17): 3520). Preclinical studies have also shown that IDO

inhibitors have a synergistic effect if used in combination with other tumor drugs, such as radiotherapy, chemotherapy or vaccines. (Koblish et al, Mol Cancer Ther.

2010; 9 (2): 489, Hou et al, Cancer Res. 2007; 67 (2): 792; Sharma et al, Blood.

2009; 1 13 (24): 6102). The research on IDO inhibitors and anti-tumor drugs has made important

progress globally, such as INCB024360, NLG919 and BMS-986205 have all

entered the clinic. However, due to the limitation of the adversed effects,

INCB024360 makes the current clinical research dose (50mg bid, or 100mg bid)

about 30% of the optimal dose (300mg bid, 600mg bid), and therefore the clinical

activity is greatly limited; meanwhile, the pharacophore of INCB024360 is also a

toxicophore, which might explain that its derivatives still suffer from the similar

toxicity issue. The safety profiling of NLG919 is better in the expense ofworse

biological activity. BMS-986205 has just entered the clinic at present with

limitedclinical data revealed. We view the discovery of novel molecules based on

the structure of BMS-986520 a better chance to yield candidates with excellent

biological avicity and safety profiling, with the goal of curing tumors rather than

inhibiting them.

Detailed description of the invention

In one aspect, the present invention provides a compound represented by

formula I,

[R']m

/ /R

N Cy,-(CRaR b),-A'

(II)

in which ^^^ represents: orm-

A represent -C(O)-, -S(O) 2 - or -S(O)- ; in which, every R1 is respectively selected from hydrogen, halogen, hydroxyl, nitro, cyano, sulfonate, C 1 .6 alkyl, C 3 -6 cycloalkyl, C 2 -6 alkenyl,

C 2 -6alkynyl, CI.6 alkoxy, Ci-C6 haloalkyl , C-Chaloalkoxy, Ci-Chalocycloalkyl,

C 1 .6alkylthio, CI 6 alkylcarbonyl, C 1 .6 alkoxycarbonyl, di(CI 6 Alkyl) aminoC 2 -6 alkoxycarbonyl, amino, Ci-6 alkylamino, di(CI 6 alkyl) amino, carbamoyl,

Ci 6 alkylcarbamoyl, di (C1 .6 alkyl Group) carbamoyl, bis (C 1.alkyl)aminoC 2 -6 alkyl carbamoyl, sulfamoyl, CI.6 alkyl sulfamoyl, di (CI 6 alkyl) sulfamoyl, di(Ci 6 alkyl) amino C 26- alkylsulfamoyl, C1 6. alkylsulfonyl, C1 .6 alkylsulfinyl, di

(C 1 6 alkyl)phosphono, hydroxy CI 6 alkyl Group, hydroxycarbonyl C1-6 alkyl,

CI6 alkoxy CI.6 alkyl, C1.6 alkylsulfonylCI.6 alkyl, C 1 .6 alkylsulfinyl C 1 .6 alkyl, di(Ci.6 alkyl) phosphonoCI. 6 alkyl, hydroxy C 2 -6 alkoxy, C 1 .6 alkoxyC 2 -6 alkoxy, amino C1.6 alkyl, C 1 .6 alkylaminoC 1.6 alkyl, di(CI.6 alkyl)aminoCI 6 alkyl, di(Ci.6 alkyl) aminoacetyl, aminoC 2 -6 alkoxy,C1.6 alkylaminoC 2 -6 alkoxy,

di(CI.6 alkyl) amino C 2 -6 alkoxy, hydroxy C 2 -6 alkylamino, CI. 6alkoxyC 2 -alkylamino,aminoC 2- 6 alkylamino,C 1 .6 alkylaminoC 2-6 alkylamino,

di(C 1 6 alkyl)aminoC 2 -alkylamino; or adjacent RI Ring together to form a 3-8

membered ring, selectively, the ring contains 0, 1, 2, 3 heteroatoms.

Cy 1 is selected from the group consisting of a 5-15 membered bridged ring

group, a 5-15 membered spirocyclic group, a 5-15 membered bridged

heterocyclic group, or a 5-15 membered spiro heterocyclic group substituted with

an arbitrary substituent. The substituent is selected from halogen, hydroxyl,

C 1 .6alkyl, amino, halogenated C1.6 alkyl, mercapto, C 1 .6 alkylmercapto,

CI 6alkylamino, di(C1. 6 alkyl) amino, cyano; Ra, Rb, R2 are each independently selected from hydrogen, C1 -C 6 alkyl or

C 3 -6 cycloalkyl;

Cy 2 is C 5-C 1 0 aryl, C 5-C 10 heteroaryl, C 5-CIO cycloalkyl, C 5 -CIO heterocycloalkyl containing one or more substituents, the substituent is selected

from halogen, hydroxy, nitro, cyano, sulfonate, Ci-6 alkyl, C 2 -6 alkenyl,

C 2 -6alkynyl, C 3 -6 cycloalkyl, CI.6 alkoxy, C1 -C 6 haloalkyl, C1-Chaloalkoxy, C1 - 6 alkylthio, C 1 .6 alkylcarbonyl, C 1 .6 alkylthio, C1.6 alkylcarbonyl,

C 1.alkylcarbonyloxy,C 1 6alkoxycarbonyl,di(Ci. 6 alkyl)aminoC 26- alkoxycarbonyl,

amino, C 1 .6 alkylamino, di(Ci.6 alkyl)amino, carbamoyl, C 1 .6 alkylcarbamoyl,

di(Ci 6 alkyl) carbamoyl, di(C1. 6 alkyl) C 2 -6alkylcarbamoyl, sulfamoyl,

C 1.alkylsulfamoyl,di(Ci. 6 alkyl)sulfamoyl,di(C1 6 alkyl)aminoC 2 -6 alkylsulfamoyl, CI. 6alkylsulfonyl, C 1 .6 alkylsulfinyl, di(Ci.6 alkyl)phosphono, hydroxyCi.6 alkyl,

hydroxycarbonyl C 1.6 alkyl, C 1.6 alkoxyC 1 .6 alkyl, C 1.6 alkylsulfonylC 1.6 alkyl,

C 1.6alkylsulfinyl C1.6 alkyl, di(Ci 6 alkyl)phosphonoCi. 6 alkyl,hydroxyC 26- alkoxy,

C 1 .6 alkoxyC 2-6 alkoxy, aminoC 1.6 alkyl, C 1 .6 alkylaminoC 1.6 alkyl, di(C1. 6 alkyl)aminoC1-6 alkyl, di (CI.6 alkyl) aminoacetyl, aminoC 2 -6alkoxy,

C 1 . 6alkylaminoC 2 -6alkoxy, di(C1. 6 alkyl)aminoC 2 -6 alkoxy, hydroxyC 2 -6 alkylamino, C1.6 alkoxyC2-6alkylamino, aminoC 2 -6 alkylamino,

C 1 . 6alkylaminoC 2 - alkylamino, 6 di(C1. 6alkyl)aminoC 2 - 6alkylamino, -S(O)C 1 .6 alkyl;

or when two substituents are adjacent , Can form a 3-8 member ring, the 3-8

member ring can contain 0, 1, 2, 3 0, S, N atoms; m, n is 0, 1, 2, 3, 4.

The invention also provides a method for preparing a compound having the

structure of formula (X):

[R1m O [R1m oEt Rc Rd EtO' COOEt [R3]m EtO 0sl Og entylium salt 'OEt ~ N Base/Organic esolvent yin (Step I) 0 N /

RC Rd R2 R[R 11,1 I HN-Cy2 N-Cy 2 Grignard reagent 0

(x) Pathway T

In pathway I:

The base used in step (1) is selected from inorganic bases or organic bases,

including but not limited to: sodium hydride, calcium hydride, sodium amide,

sodium methoxide, sodium ethoxide, potassium hydroxide, sodium hydroxide,

lithium hydroxide, hydrogenation Lithium aluminum, tert-butyl lithium, tert-butyl potassium, potassium tert-butoxide, lithium diisopropylamide, barium

hydroxide, or any combination thereof;

The organic solvents used in step (1) include but are not limited to: 1,4-dioxane, N, N-dimethylformamide, dichloromethane, chloroform, DMSO, DMF, THF, acetone, methanol, Ethanol or any combination thereof; Wherein the ylium salt used in step (2) is selected from sulfur ylide or phosphorus ylide The Grignard reagent used in step (3) is selected from CH 3 MgCl, CH 3 MgBr,

C 2 H 5MgCl, C 2H 5MgBr, i-PrMgCl, i-PrMgBr, PhCH 2 MgCl, PhCH 2 MgBr or any combination thereof.

The invention also provides a method for preparing a compound having the structure of formula (XI):

0 1

[R']m 1 [F] [R] RC Rd

1_ _ 11s> ~ ylium salt N N-. Step) Ste base/organic solvent 0 Step( 2) N.

[R1 ]m Rc Cy-N [R1]m RcRd hydrolysis NH O N N'Cy2 step( 3) Step( 4) 0 N- N. (XI) Pathway II

In pathway II: Wherein the catalyst used in step (1) is selected from methyl titanate, ethyl titanate, n-propyl titanate, isopropyl titanate, butyl titanate or any combination thereof; The base used in step (2) is selected from inorganic bases or organic bases, including but not limited to: sodium hydride, calcium hydride, sodium amide, sodium methoxide, sodium ethoxide, potassium hydroxide, sodium hydroxide, lithium hydroxide, hydrogenation Lithium aluminum, tert-butyl lithium, tert-butyl potassium, potassium tert-butoxide, lithium diisopropylamide, barium hydroxide, or any combination thereof;

The organic solvents used in step (2) include but are not limited to:

1,4-dioxane, N, N-dimethylformamide, dichloromethane, chloroform, DMSO,

DMF, THF, acetone, methanol, Ethanol or any combination thereof;

Wherein the ylium salt used in step (2) is selected from sulfur ylide or

phosphorus ylide

The hydrolysis in step (3) is performed under acidic conditions, and the acid

is selected from but not limited to hydrochloric acid, sulfuric acid, hydrobromic

acid, oxalic acid, citric acid, formic acid, acetic acid or any combination thereof;

The organic solvents used in step (4) include but are not limited to:

1,4-dioxane, N, N-dimethylformamide, dichloromethane, chloroform, DMSO,

DMF, THF, acetone, methanol , Ethanol or any combination thereof.

The invention also provides a method for preparing a compound having the

structure of formula (XII):

[R 1] H2N-S'[P]m N [Fm Nm

Step( 1) 0 Br O N N Step(2

[ 1]m H

hydrolysis ' N H [Rlm 2 alkylation Step(3)Selective N Grignard reagent N0 Stp50 Step( 4)Stp5) N XII) Pathway III

In pathway III:

Wherein the catalyst used in step (1) is selected from methyl titanate, ethyl

titanate, n-propyl titanate, isopropyl titanate, butyl titanate or any combination

thereof;

Wherein the step (2) is performed under the action of a strong

non-nucleophilic base selected from but not limited to lithium diisopropylamide,

lithium diethylamide, isopropylcyclohexylamino Lithium, lithium

dicyclohexylamide, lithium 2,2,6,6-tetramethylpiperidino, lithium

hexamethyldisilazide;

The hydrolysis in step (3) is performed under acidic conditions, and the acid is selected from but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, oxalic acid, citric acid, formic acid, acetic acid or any combination thereof;

The Grignard reagent used in step (4) is selected from CH 3 MgCl, CH 3 MgBr,

C 2 H 5MgCl, C 2H 5MgBr, i-PrMgCl, i-PrMgBr, PhCH 2 MgCl, PhCH 2 MgBr or any combination thereof

Wherein when performing the alkylation reaction described in step (4), the

alkylation reaction reagent is selected from haloalkyl, the reaction is performed

under a Lewis acid as a catalyst, and the Lewis acid is preferably AlCl 3, FeC 2

, CuCl 2 .

The invention also provides a method for preparing a compound having the

structure of formula (XIII):

[R1 ]m 0 Rc d Rc d d~ BPh 3P Rd ~R RRl RCl dOxidation [RR]m Rd c~TS CN-/Ts

N Base/organic solvent NOrganicsolvent Base/ Step( 2) organic solvent Step( 1) Step( 3)

[R1R]q. R [M]n Rd 2 [R ]m RRd 2 RRdCRd Rd

[R]m Rd hydrolysis OH NHCy 2g N Step() N 0 Grignard reagentN CY step ( 5)XIII)

Pathway IV

In pathway IV,

The base used in step (1) and step (3) is selected from inorganic bases or

organic bases, including but not limited to: sodium hydride, calcium hydride,

sodium amide, sodium methoxide, sodium ethoxide, potassium hydroxide,

sodium hydroxide , Lithium hydroxide, lithium aluminum hydride, tert-butyl

lithium, tert-butyl potassium, potassium tert-butoxide, lithium diisopropylamide,

barium hydroxide, or any combination thereof;

The organic solvents used in step (1) to step (3) include but are not limited

to: 1,4-dioxane, N, N-dimethylformamide, dichloromethane, chloroform, DMSO,

DMF, THF, acetone, methanol, ethanol or any combination thereof;

The oxidant used in step (2) is selected from but not limited to m-chloroperoxybenzoic acid, Cr0 3, KMnO 4 , MnO 2 , NaCr 2 0 7, H10 4 , PbAc 4

, Os04, hydrogen peroxide or any combination thereof;

Wherein the hydrolysis reaction in step (4) is performed under acidic conditions, and the acid is selected from but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, oxalic acid, citric acid, formic acid, acetic acid or any combination thereof;

The Grignard reagent used in step (5) is selected from CH 3 MgCl, CH 3 MgBr,

C 2 H 5MgCl, C 2H 5MgBr, i-PrMgCl, i-PrMgBr, PhCH 2 MgCl, PhCH 2 MgBr or any combination thereof;

The invention also provides a method for preparing a compound having the formula (XIV): Ph9 H

[R]mPh []mN [R']m N OEtI Me 3Si N O OEt reduCtion OEt N- 0 0r Strong base N / 11 YN N, stp 1 N step( 2) N step (1) H Re 2 R*

[R ] N R [RN]R\ R 2 N selective alkylation [Ri] N HN0Cy2 Cy2 N Grignard reagent Nstep ( 4) Cy step( 3) N

XIV) Pathway V

In pathway V:

Wherein step (1) is carried out in the presence of alkali metal fluoride or alkaline earth metal fluoride, said alkali metal fluoride is selected from but not limited to LiF, NaF, KF, MgF 2 , CaF 2 ;

The reduction reaction in step (2) can be palladium-carbon catalytic hydrogenation reduction or Na / liquid ammonia reduction;

The Grignard reagent used in step (3) is selected from CH 3 MgCl, CH 3 MgBr,

C 2 H 5MgCl, C 2H 5MgBr, i-PrMgCl, i-PrMgBr, PhCH 2 MgCl, PhCH 2 MgBr or any

combination thereof;

Wherein when performing the alkylation reaction described in step (4), the

alkylation reaction reagent is selected from haloalkyl, the reaction is performed

under a Lewis acid as a catalyst, and the Lewis acid is preferably AlCl 3 , FeC 2

, CuCl 2 .

Examples

Here, when referring to a "compound" having a specific structural formula,

its stereoisomers, diastereomers, enantiomers, racemic mixtures, and isotopic

derivatives are also generally covered. It is well known to the technical persons

skilled in the art that salts, solvates, and hydrates of a compound are alternative

existing forms of the compound, and they can all be converted into the compound

under certain conditions, so when referring to a compound In general, it also

includes its pharmaceutically acceptable salts, and further includes its solvates

and hydrates. Similarly, when referring to a compound herein, it generally

includes its prodrugs, metabolites and nitrogen oxides.

The compound of the present invention may also be prepared in the form of a

pharmaceutically, acceptable salt formed with an inorganic or organic acid such as

hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, acetic

acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric

acid,fumaric acid, malic acid, mandelic acid, tartaric acid, citric acid, ascorbic acid,

palmitic acid, maleic acid, hydroxymaleic acid, benzoic acid, hydroxybenzoic acid,

phenylacetic acid, cinnamic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid and toluenesulfonic acid.

The pharmaceutically acceptable salt of the present invention may be prepared

by conventional methods, for example, by dissolving the compound in a

water-miscible organic solvent such as acetone, methanol, ethanol and acetonitrile,

adding thereto an excess amount of an organic acid or an aqueous solution of

inorganic acid, to induce precipitation of salts from the resulting mixture, removing the solvent and remaining free acid therefrom, and isolating the precipitated salts. Accordingly, the present invention provides a use of the inventive compound for the manufacture of a medicament for preventing or treating cancers, tumors, inflammatory diseases, autoimmune diseases, or immunologically mediated diseases. In addition, the present invention provides a pharmaceutical composition for preventing or treating cancers, tumors, inflammatory diseases, autoimmune diseases, or immunologically mediated diseases which comprises the inventive compound as an active ingredient. Synthesis The compounds of the present invention can be synthesized by known procedures with reference to the following description. All purchased solvents and reagents are used without treatment. All synthesized compounds can be analyzed and verified by, but not limited to, the following methods: LCMS (liquid chromatography mass spectrometry, liquid phase mass spectrometry) and NMR (nuclear magnetic resonance, nuclear magnetic resonance). Nuclear magnetic resonance (NMR) is measured by Bruker AVANCE-500 nuclear magnetic instrument. The deuterated solvent used in the measurement is deuterated dimethyl sulfoxide (d-DMSO), deuterated chloroform (CDCl3), tetramethylsilane (TMS) as Internal standard. The following abbreviations represent various types of splitting peaks: singlet (s), doublet (d), triplet (t), multiplet (m), broad (br). Thermo Fisher-MSQ Plus liquid-mass spectrometry was used for the determination of mass spectrometry (MS). General synthetic analysis and examples are described as follows: Example 1 N-(4-chlorophenyl)-6-(6-fluoroquinolin-4-yl)spiro[2.5]octane-I-carboxamide F H N

N /

Cl B2Pin 2 OBF F PhNf 2 - Pd(dPPf)C1 2

) 0 NaHMDS 2f~a KOAc, NaBr 0 -B 0 N 0( 0-1 TfOTB 0 4-dioxane 0'Pd(PPh3), K2C03 N O~~I MTBE fO 14-dixane2 3 N

1a lb Ic

F F F

Pd/C,H 2 04N HCI EtOTPHFCOONEt

PA N Aeoe N -t-BuONa, THIF N 0

Id le OEtt4-hlroniinI If C1

1 0F 1gt 1 IF H 11 - EtN >/S 0 4-chloroaniline

NaH, DMVSOn) i-PrMgCI, THF N 11

Ig I

StepI: To a solution of 1, 4-cyclohexanedione monoethylene acetal (10.0 g, 64.03

mmol) in tert-butyl methyl ether (250 mL) was added

N-phenyl-bis(trifluoromethanesulfonimide (22.9 g, 64.03 mmol). The mixture was

cooled to -78°C, and sodium bis(trimethylsilyl)amide (2.0 M in THF, 32 mL, 64.03

mmol) was added dropwise under N 2 . The mixture was stirred at -78°C for 1 h and

then warmed to r.t. for 16 h until TLC showed complete conversion of starting

material. The reaction was quenched by 3 mL KHSO 4 solution and filtered. The

filtrate was concentrated under reduced pressure. The residue was dissolved in 30 mL

MTBE, which was washed with 5% NaOH (45 mL x 3) and brine (50 mL). The

organic layer was dried over Na2 SO 4 , filtered and concentrated to give compound la

(34 g, yield: 93.6%) as red-orange oil. 'H NMR (500 MHz, CDC 3) 6 5.66 (J= 4.0 Hz,

1H), 4.01-3.96 (in, 4H), 2.56-2.52 (in, 2H), 2.42-2.40 (in, 2H), 1.90 (t, J= 6.5 Hz, 2H).

Step2: To a solution of compound la (13 g, 45.1 mmol) in dioxane (100 mL)

were added bis(pinacolato)diboron (14.9 g, 58.64 mmol), KOAc (13.3 g, 135.3

mmol) and Pd(dppf)C12 (1.65 g, 2.26 mmol). The reaction mixture was stirred at

100°C under N 2 for 16 h. The mixture was concentrated under reduced pressure.

The residue was suspended in EA and filtered over a pad of celite. The filtrate was concentrated and purified by column chromatography on silica gel to afford compound lb (7.6 g, yield: 63%) as a pale-yellow solid. 'H NMR (500 MHz,

CDC 3) 6 6.48-6.45 (m, 1H), 3.98 (s, 4H), 2.40-2.34 (m, 4H), 1.73 (t, J= 6.5 Hz, 2H),

1.25 (s, 12H).

Step 3: To a solution of compound lb (5.7 g, 21.48 mmol) in dioxane (60 mL)

and water (15 mL) were added 4-chloro-6-fluoroquinoline (3.0 g, 16.53 mmol),

potassium carbonate (6.8 g, 49.56 mmol), and tetrakis(triphenylphosphine)

palladium(0) (954 mg, 0.83mmol). The reaction mixture was stirred at100C under

N 2 for 16 h. The mixture was concentrated under reduced pressure. The residue

was diluted with H 2 0 and extracted with EtOAc. The organic layer was

concentrated. The residue was purified by column chromatography on silica gel to

afford compound I c(2.42 g, yield 51%) as pale-yellow oil. MS (ESI): mz 286.1

(M+H)f. 'H NMR (500 MHz, CDCl3) 6 8.81 (d, J= 4.5 Hz, 1H), 8.15 (dd, J= 9.0,5.5

Hz, 1H), 7.65 (dd, J= 10.0, 2.5 Hz, 1H), 7.49 (td, J= 9.0, 2.5 Hz, 1H), 7.26 (d, J=

4.5 Hz, 1H), 5.77 (t, J = 3.5 Hz, 1H), 4.08-40.6 (m, 4H), 2.65-2.60 (m, 2H),

2.56-2.53 (m, 2H), 2.00 (t, J= 6.5 Hz, 2H).

Step 4: To a solution of compound Il (2.42 g, 8.49 mmol) in isopropyl alcohol

(45 mL) was added 10% w/w palladium on carbon (300 mg). The mixture was

stirred at 55°C under hydrogen atmosphere for 16 h. The reaction was filtered over

a pad of celite. The filtrate was concentrated to give compound Id (2.04 g, yield:

84%) as slurry oil, which was used to the next step directly. MS (ESI): mz 288.1

(M+H)f.

Step 5: To a solution of compound Id (2.04 g, 7.11 mmol) in acetone (36 mL)

was added HCl (4N in water, 9 mL, 36 mmol) and the mixture was stirred at 45°C

for 16 h. The organic volatiles were removed and the remaining aqueous solution

was adjusted to pH=9 by 6N NaOH solution. The mixture was extracted with

EtOAc three times. The combined organic layers were washed with brine, dried

over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was

purified by column chromatography on silica gel to give compound le (1.17 g,

yield 67%) as a pale-yellow solid. MS (ESI): m/z 244.3 (M+H). 'H NMR (500

MHz, CDC 3) 68.85 (d, J= 4.5 Hz, 1H), 8.22 (dd, J= 9.0, 5.5 Hz, 1H), 7.74 (dd, J=

10.0, 2.5 Hz, 1H), 7.57-7.50 (m, 1H), 7.33 (d, J= 4.5 Hz, 1H), 3.74-3.66 (m, 1H),

2.72-2.58 (m, 4H), 2.41-2.34 (m, 2H), 2.11-2.00 (m, 2H).

Step 6: To a solution of triethyl phosphonoacetate (968 mg, 4.32 mmol) in dry

THF (16 mL) at 0°C, sodium tert-butoxide (415 mg, 4.32 mmol) was added. After

10 min, a solution of compound le (1 g, 4.12 mmol) in THF (4 mL) was added to

the above mixture and the resulting reaction mixture was stirred for 2 h. The

reaction was quenched by H 20, extracted with EtOAc three times. The combined

organic layers were washed with brine (20 mL), dried over Na 2 SO 4 , filtered and

concentrated under reduced pressure. The residue was purified by column

chromatography on silica gel to give compound If (1.18 g, yield: 92%) as a white

solid. MS (ESI): m/z 314.0 (M+H)f.'H NMR (500 MHz, CDC 3) 6 8.81 (d, J= 4.5

Hz, 1H), 8.17 (dd, J= 9.0,5.5 Hz, 1H), 7.72 (dd, J= 10.0, 2.5 Hz, 1H), 7.53-7.47 (m,

1H), 7.28 (d, J= 4.5 Hz, 1H), 5.75 (s, 1H), 4.19 (q, J= 7.0 Hz, 2H), 3.52-3.42 (m,

1H), 2.54-2.48 (m, 2H), 2.26-2.11 (m, 4H), 1.80-1.68 (m, 2H), 1.30 (t, J= 7.0 Hz, 3H).

Step 7: To a suspension of NaH (60% w/w in mineral oil, 383 mg, 9.57 mmol) in

DMSO (15 mL) was added trimethylsulfoxonium iodide (2.11 g, 9.57 mmol). After

the mixture was stirred at r.t. for 1.5 h, a solution of compound If (1.0 g, 3.19

mmol) in DMSO (5 mL) was added to the above mixture and the resulting reaction

mixture was stirred at r.t. for 16 h. The mixture was quenched by H 2 0 and extracted

with EtOAc. The organic layer was concentrated and the residue was purified by

column chromatography on silica gel to give compound ig (820 mg, yield: 78%)

as colorless oil. MS (ESI): m/z 328.1 (M+H).'H NMR (500 MHz, CDCl 3) 6 8.83 (d,

J = 4.5 Hz, 1H), 8.24 (dd, J= 9.0, 5.5 Hz, 1H), 7.71 (dd, J = 10.0, 2.5 Hz, 1H),

7.55-7.49 (m, 1H), 7.35 (d, J 4.5 Hz, 1H), 4.19 (q, J= 7.0 Hz, 2H), 3.32-3.24 (m,

1H), 2.17 (td, J= 13.0, 3.5 Hz, 1H), 2.07-1.90 (m, 4H), 1.87-1.78 (m, 1H), 1.58 (dd,

J= 8.0, 5.5 Hz, 1H), 1.46-1.37 (m, 1H), 1.30 (t, J= 7.0 Hz, 3H), 1.28-1.24 (m, 2H),

1.16-1.11 (m, 1H), 1.00 (dd, J= 8.0, 4.5 Hz, 1H).

Step 8: To a mixture of 4-chloroaniline (94 mg, 0.73 mmol) in dry THF (5 mL) at 0°C, isopropylmagnesium chloride solution (2.0 M in THF, 0.4 mL, 0.73 mmol) was added dropwise. After the mixture was stirred at r.t. for 5 min, a solution of compound ig (60 mg, 0.18mmol) in dry THF (2 mL) was added dropwise. The resulting mixture was stirred at r.t. for 16 h. The reaction was quenched by aq.

NH 4 Cl solution. The mixture was extracted with ethyl acetate three times and the

combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under

reduced pressure. The residue was purified by Prep-HPLC to give compound 1

(16.04 mg, yield: 21%) as a white solid. MS (ESI): m/z 408.9 (M+H).'H NMR

(500 MHz, d6 -DMSO) 6 10.37 (s, 1H), 8.81 (s,1H), 8.11-8.05 (m, 1H), 8.02 (d, J=

11.0 Hz, 1H), 7.69-7.63 (m, 3H), 7.38-7.31 (m, 3H), 3.48-3.40 (m, 1H), 2.20 (t, J=

12.0 Hz, 1H), 1.97-1.84 (m, 4H), 1.78 (d, J= 12.5 Hz, 1H), 1.72 (t, J= 6.5 Hz, 1H),

1.35-1.26 (m, 1H), 1.17-1.08 (m, 2H), 0.96-0.90 (m, 1H).

Example 2

N-(4-fluorophenyl)-6-(6-fluoroquinolin-4-yl)spiro[2.5]octane-I-carboxamide F H N

0F F N /

Compound 2 was prepared using the similar procedures as described for

compound 1 using 4-fluoro aniline to replace 4-chloro aniline. MS (ESI): mz 393.3

(M+H)f.'H NMR (500 MHz, d6 -DMSO) 6 10.28 (s, 1H), 8.81 (s, 1H), 8.10-8.05 (m,

1H), 8.03 (d, J= 11.0 Hz, 1H), 7.69-7.60 (m, 3H), 7.37 (s, 1H), 7.13 (t, J= 8.0 Hz,

2H), 3.49-3.40 (m, 1H), 2.20 (t, J= 12.0 Hz, 1H), 1.98-1.85 (m, 4H), 1.78 (d, J=

11.0 Hz, 1H), 1.72 (t, J = 6.5 Hz, 1H), 1.37-1.28 (m, 1H), 1.17-1.07 (m, 2H),

0.94-0.89 (m, 1H).

Example 3

N-(4-chlorobenzyl)-6-(6-fluoroquinolin-4-yl)spiro[2.5]octane-I-carboxamide

CI F NN F F F C

OEt NaOH HATU, DIPEA Cl

N EtOH0 I

Ig 3a 3

Step 1: To a solution of compound Ig (200 mg, 0.61 mmol) in ethanol (10 mL)

was added NaOH (2N in water, 4 mL, 8 mmol) and the mixture was stirred at

50°C for 2 h. The mixture was cooled to r.t. and adjusted to pH=1 by 4N HCl

solution. The mixture was extracted with EtOAc three times. The combined

organic layers were dried over Na 2 SO 4 , filtered and concentrated under reduced

pressure. The residue was purified by Prep-TLC to give compound 3a (150 mg,

yield 83%) as a white solid. MS (ESI): m/z 300.0 (M+H). 'H NMR (500 MHz,

d 6-DMSO) 6 12.02 (br, 1H), 8.83 (d, J= 4.5 Hz, 1H), 8.10 (dd, J= 9.0, 5.5 Hz, 1H),

8.03 (dd, J = 10.0, 2.5 Hz, 1H), 7.71-7.64 (m, 1H), 7.38 (d, J = 4.5 Hz, 1H), 3.48-3.41 (m, 1H), 2.21-2.13 (m, 1H), 2.01-1.80 (m, 4H), 1.75-1.65 (m, 1H), 1.51

(dd, J= 8.0, 5.5 Hz, 1H), 1.38-1.32 (m, 1H), 1.11-1.05 (m, 1H), 1.04-0.99 (m, 1H),

0.95 (dd, J= 7.5, 4.0 Hz, 1H).

Step 2: To a solution of compound 3a (40 mg, 0.13 mmol) in DMF (5 mL)

were added DIPEA (52 mg, 0.39 mmol) and HATU (61 mg, 0.16 mmol) and the

mixture was stirred at r.t. for 30 min. 4-chlorobenzylamine (57 mg, 0.39 mmol)

was added to the above mixture and the resulting reaction mixture was stirred at r.t.

for 2 h. The mixture was quenched by H 20(20 mL) and extracted with EtOAc three

times. The combined organic layers were dried over Na 2 SO 4 , filtered and

concentrated under reduced pressure. The residue was purified by Prep-HPLC to

give compound 3 (6.34 mg, yield 11%) as a white solid. MS (ESI): mz 423.4

(M+H)f.'H NMR (500 MHz, d6-DMSO) 6 8.81 (s, 1H), 8.68 (s, 1H), 8.12-8.07 (m,

1H), 8.00 (d, J= 11.0 Hz, 1H), 7.67 (t, J= 8.5 Hz,1H), 7.36-7.31 (m, 4H), 7.14 (s,

1H), 4.44 (dd, J= 15.0, 6.5 Hz, 1H), 4.18 (dd, J= 15.0 Hz, 5.0 Hz,1H), 3.42-3.34 (m,

1H), 2.13 (t, J= 12.5 Hz, 1H), 1.86-1.74 (m, 4H), 1.63 (d, J= 12.5 Hz,1H),1.54-1.48

(m, 1H), 1.25-1.15 (m, 1H), 1.08-0.96 (m, 2H), 0.82-0.76 (m, 1H).

Example 4

N-(4-chlorophenethyl)-6-(6-fluoroquinolin-4-yl)spiro[2.5]octane-1-carboxamide F H

0 C1 N /

Compound 4 was prepared using the similar procedures as described for

compound 3 using 4-chlorophenethylamine to replace 4-chlorobenzylamine. MS

(ESI): m/z 437.4 (M+H)+.H NMR (500 MHz, d 6-DMSO) 6 8.84 (d, J= 4.5 Hz, 1H),

8.19 (t, J= 5.5 Hz, 1H), 8.12-8.07 (m, 1H), 7.99 (d, J= 11.0 Hz, 111), 7.67 (t, J= 8.5

Hz, IH), 7.27 (d, J= 4.1 Hz,1H), 7.25-7.21 (m, 4H), 3.49-3.42 (m, 1H), 3.30-3.22

(m, 2H), 2.74 (t, J= 6.5 Hz, 2H), 2.11 (t, J= 12.5 Hz, 1H), 1.85-1.75 (m, 4H), 1.67 (d,

J = 12.5 Hz, 1H), 1.46-1.41 (m, 1H), 1.26-1.14 (m, 1H), 1.03-0.95 (m, 2H),

0.75-0.70 (m, 1H).

Example 5

6-(6-fluoroquinolin-4-yl)-N-(4-(trifluoromethyl)phenyl)spiro[2.5]octane-1-carboxami

de F H

NN CF 3 N /

F F H OH N T3P, Pyridine CF 3

N / N

3a 5

To a solution of compound 3a (40 mg, 0.13 mmol) in EA (5 mL) were

subsequently added pyridine (32 mg, 0.39 mmol) and

2,4,6-Tripropyl-1,3,5,2,4,6-Trioxatriphosphorinane-2,4,6-Trioxide(127img,0.33 mmol) and the mixture was stirred at r.t. for 10 min. 4-Aminobenzotrifluoride (65 mg, 0.39 mmol) was added to the above mixture and the resulting reaction mixture was stirred at r.t. for 16 h. The mixture was quenched by NaOH (2N in water, 2 mL) and diluted with H 2 0 (20 mL). The resulting mixture was extracted with EtOAc three times. The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by Prep-HPLC to give compound 5 (2.65 mg, yield 5%) as a white solid. MS (ESI): mz 437.4 (M+H)f.'H NMR (500 MHz, d-DMSO) 610.63 (s, 1H), 8.80 (d, J= 4.5 Hz, 1H), 8.10-8.05 (m, 1H), 8.03 (d, J= 11.0 Hz, 1H), 7.84 (d, J= 8.0 Hz, 2H), 7.69-7.63 (m, 3H), 7.36 (s, 1H), 3.48-3.41 (m, 1H), 2.25-2.17 (m, 1H), 1.99-1.85 (m, 4H),

1.80-1.74 (m, 2H), 1.35-1.25 (m, 1H), 1.21-1.09 (m, 2H), 0.99-0.95 (m, 1H).

Example 6 N-(4-cyanophenyl)-6-(6-fluoroquinolin-4-yl)spiro[2.5]octane-I-carboxamide F H N

C 0 CN N /

Compound 6 was prepared using the similar procedures as described for compound 5 using 4-aminobenzonitrile to replace 4-aminobenzotrifluoride. MS (ESI): m/z 400.4 (M+H)f.'H NMR (500 MHz, d6-DMSO) 6 10.70 (s, 1H), 8.80 (d, J = 4.5 Hz, 1H), 8.10-8.06 (m, 1H), 8.03 (d, J= 11.0 Hz, 1H), 7.82 (d, J= 8.5 Hz, 2H), 7.75 (d, J= 8.5 Hz, 2H), 7.66 (t, J= 9.0 Hz, 1H), 7.35 (d, J= 3.5 Hz, 1H), 3.48-3.40 (m, 1H), 2.25-2.17 (m, 1H), 1.99-1.83 (m, 4H), 1.81-1.74 (m, 2H), 1.33-1.23 (m,

1H), 1.21-1.10 (m, 2H), 1.02-0.96 (m, 1H).

Example 7 N-(6-chloropyridin-3-yl)-6-(6-fluoroquinolin-4-yl)spiro[2.5]octane-I-carboxamide F H N

N /

Compound 7 was prepared using the similar procedures as described for compound 5 using 5-amino-2-chloropyridine to replace 4-aminobenzotrifluoride. MS (ESI): m/z 410.4 (M+H).'H NMR (500 MHz, d6 -DMSO) 6 10.62 (s, 1H), 8.81 (s, 1H), 8.63 (s, 1H), 8.15-8.06 (m, 2H), 8.03 (d, J= 11.0 Hz, 1H), 7.66 (t, J= 8.5 Hz, 1H), 7.45 (d, J= 8.0 Hz, 1H), 7.38 (s, 1H), 3.48-3.41 (m, 1H), 2.25-2.16 (m, 1H), 1.99-1.82 (m, 4H), 1.82-1.72 (m, 2H), 1.35-1.26 (m, 1H), 1.20-1.09 (m, 2H),

1.00-0.95 (m, 1H).

Example 8 N-(5-chloropyridin-2-yl)-6-(6-fluoroquinolin-4-yl)spiro[2.5]octane-I-carboxamide F H

CI N /

Compound 8 was prepared using the similar procedures as described for compound 5 using 2-amino-5-chloropyridine to replace 4-aminobenzotrifluoride. MS (ESI): m/z 410.4 (M+H).'H NMR (500 MHz, d6 -DMSO) 6 11.04 (s, 1H), 8.80 (s, 1H), 8.38 (s, 1H), 8.13 (d, J= 8.5 Hz, 1H), 8.07 (t, J= 8.0 Hz, 1H), 8.02 (d, J= 11.0 Hz, 1H), 7.85 (d, J= 9.0 Hz, 1H), 7.66 (t, J= 8.0 Hz,1H), 7.34 (s,1H), 3.47-3.40 (m, 1H), 2.19 (t, J = 12.5 Hz, 1H), 2.02-1.84 (m, 5H), 1.78 (d, J = 11.0 Hz, 1H),

1.33-1.22 (m, 1H), 1.21-1.15 (m, 1H), 1.08 (d, J= 12.5 Hz, 1H), 0.98-0.92 (m, 1H).

Example 9 N-(4-chlorophenyl)-6-(6-fluoro-7-methylquinolin-4-yl)spiro[2.5]octane-I-carboxamid e F H Me N

CI N /

F F F H OEt Me OEt Me N LIDA, Mel 0 4-chloroaniline 0 C ~i-PrMgCI, THF 1g 9a 9

Step 1: To a mixture of diisopropylamine (123 mg, 1.22 mmol) in dry THF (5

mL) at -78°C, n-BuLi (2.5 M, 0.5 mL, 1.25 mmol) was added dropwise, followed

by a solution of compound Ig (200 mg, 0.61 mmol) in THF (2 mL). After the

mixture was stirred at -78°C for 1 h, a solution of CH3I (173 mg, 1.22 mmol) in

dry THF (2 mL) was added dropwise. The resulting mixture was stirred at -78°C

for 0.5 h and then warmed to r.t. for 16 h. The reaction was quenched by aq.

NH 4 Cl solution. The mixture was extracted with ethyl acetate three times and the

combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under

reduced pressure. The residue was purified by Prep-TLC to give compound 9a

(24 mg, yield: 12%) as colorless oil. MS (ESI): m/z 342.4 (M+H).

Step 2: To a mixture of 4-chloroaniline (36 mg, 0.28 mmol) in dry THF (2 mL)

at 0°C, isopropylmagnesium chloride solution (2.0 M in THF, 0.2 mL, 0.4 mmol)

was added dropwise. After the mixture was stirred at r.t. for 5 min, a solution of

compound 9a (24 mg, 0.07 mmol) in dry THF (1 mL) was added dropwise. The

resulting mixture was stirred at r.t. for 16 h. The reaction was quenched by aq.

NH 4 Cl solution. The mixture was extracted with ethyl acetate three times and the

combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under

reduced pressure. The residue was purified by Prep-HPLC to give compound 9

(12.01 mg, yield: 41%) as a white solid. MS (ESI): m/z 423.4 (M+H).'H NMR

(500 MHz, d6-DMSO) 6 10.37 (s, 1H), 8.75 (s, 1H), 7.99-7.91 (m, 2H), 7.80-7.56

(m, 3H), 7.34 (d, J= 8.0 Hz, 2H), 7.28 (s, 1H), 3.44-3.37 (m, 1H), 2.44 (s, 3H),

2.23-2.14 (m, 1H), 1.98-1.84 (m, 4H), 1.78-1.69 (m, 2H), 1.17-1.07 (m, 2H),

0.95-0.92 (m, 1H).

Example 10

(1S)-N-(4-chlorophenyl)-6-(6-fluoro-7-methylquinolin-4-yl)spiro[2.5]octane-I-carbox

amide

0 FNH Me F NNH H H

CI N

/ Compound 10

(1R)-N-(4-chlorophenyl)-6-(6-fluoro-7-methylquinolin-4-yl)spiro[2.5]octane-I-carbo

xamide

F ,H H Me 'N H CI N /!r

Compound 11

Compound 10 and Compound 11 were obtained by chiral column separation of

compound 9. Absolute stereochemistry arbitrarily assigned.

Compound 10: MS (ESI): m/z 423.4 (M+H). 'H NMR (500 MHz, d 6-DMSO) 6 10.39 (s, 1H), 8.75 (d, J= 4.5 Hz, 1H), 7.97 (d, J= 12.0 Hz, 1H),

7.94 (d, J= 8.0 Hz, 1H), 7.65 (d, J= 9.0 Hz, 2H), 7.34 (d, J= 9.0 Hz, 2H), 7.28

(d, J = 4.5 Hz, 1H), 3.44-3.38 (m, 1H), 2.44 (s, 3H), 2.24-2.14 (m, 1H),

1.97-1.82 (m, 4H), 1.80-1.74 (m, 1H), 1.73-1.69 (m, 1H), 1.32-1.24 (m, 1H),

1.17-1.12 (m, 1H), 1.10 (d,J= 13.0 Hz, 1H), 0.93 (dd,J= 7.5, 4.0 Hz, 1H).

Compound 93: MS (ESI): m/z 423.4 (M+H). 'H NMR (500 MHz, d 6-DMSO) 6 10.39 (s, 1H), 8.75 (d, J= 4.5 Hz, 1H), 7.97 (d, J= 12.0 Hz, 1H),

7.94 (d, J= 8.0 Hz, 1H), 7.65 (d, J= 9.0 Hz, 2H), 7.34 (d, J= 9.0 Hz, 2H), 7.28

(d, J = 4.5 Hz, 1H), 3.45-3.38 (m, 1H), 2.45 (s, 3H), 2.24-2.14 (m, 1H),

1.97-1.82 (m, 4H), 1.80-1.74 (m, 1H), 1.73-1.69 (m, 1H), 1.32-1.24 (m, 1H),

1.16-1.13 (m, 1H), 1.10 (d,J= 13.0 Hz, 1H), 0.93 (dd,J= 7.5, 4.0 Hz, 1H).

Biological evaluations: Example 1: Study on the activity of the compound of the present invention in Hela

cells

Hela cells were seeded in 96-well culture plates and incubated at 37C, 100% relative humidity, 5% CO2 incubator for 24 hours. The compound was dissolved in DMSO and diluted to an appropriate concentration, and then the compound was diluted 100-fold with DMEM medium containing interferon-y and 10% fetal bovine serum to the final concentration of effect. Aspirate the old medium from the 96-well plate and add 200 pL of each medium containing compound and interferon-y from the previous step. The content of tryptophan in the medium is 16 mg/L, and the concentration of interferon-y is 50 ng/mL. The cells were placed in a 37C, 100% relative humidity, and 5% CO2 incubator for 48 hours, then 140 pL of the cell culture supernatant was mixed with 15 pL of trichloroacetic acid, and placed at 52°C for 30 min, then centrifuged at room temperature to take the centrifugal supernatant. Mix with an equal volume of Ehrlich's reagent, measure the light absorption at 480 nm, and calculate theIC50 values. Compound NO. IC5 0

1 A

2 B 3 C 4 C 5 C 6 B

7 B 8 B

9 A 10 A

11 C

Note A: IC 50=0.1nM~IOnM B: IC5 0 = l0nM100nM C: IC5 0 >100nMo

Example 2: Study on the activity of the present invention in HEK293 cells highly expressing human IDO1 protein

HEK293-IDO cells with high expression of human IDO1 protein were

prepared by electrotransformation, and the cells were seeded in 96-well culture

plates, and cultured at 37C, 100% relative humidity, and 5% CO 2 incubator for

24 hours. Dissolve in DMSO and dilute to the appropriate concentration, then use

DMEM medium containing 10% fetal bovine serum to dilute the candidate

100-fold to the final concentration. Aspirate the old medium from the 96-well

plate and add 200 pL of the medium contained in the previous step to each well.

The content of tryptophan in the medium is 16 mg/L. The cells were placed in a

37C, 100% relative humidity, and 5% CO2 incubator for 24 hours. 140 pL of the

cell culture supernatant was mixed with 15 pL of trichloroacetic acid and placed

at 52°C for 30 min. Mix with an equal volume of Ehrlich's reagent, measure the

light absorption at 480 nm, and calculate the IC5 0 values.

Compound NO. IC 5 0 (HEK293 cell)

1 B

2 B

3 C 4 C

5 B

6 B

7 B

8 B

9 A

10 A

11 C

Note A: IC 50=0.1nM~IOnM B: IC5 0 = l0nM100nM C: IC5 0 >100nMo

Claims (1)

1. What is claimed is 1. The compound of any with the structure of:. NO. Compound structure NO. Compound structure F F

   H 2H

   0 0 F ci F

   NH 4 H \/IH N- _ N 0 F F

   NN ~ CF 3 N\/N

   00 F F

   7NN/H I 8 NH N

   F H F0 Me- Na 1 Me NH

   Me NN

   NI