HK1235775B - Pyrrolidine-2,5-dione derivatives, pharmaceutical compositions and methods for use as ido1 inhibitors - Google Patents

Description

Pyrrolidine-2,5-dione derivatives, pharmaceutical compositions, and methods for use as IDO1 inhibitors

Field of the Invention

The present invention relates to pyrrolidine-2,5-dione derivatives, including pharmaceutically acceptable enantiomers, salts, solvates and prodrugs thereof. The compounds of the present invention are inhibitors of IDO1 (indoleamine 2,3-dioxygenase-1) and are useful as therapeutic compounds, in particular for the treatment and/or prevention of cancer.

Background of the Invention

Indoleamine 2,3-dioxygenase 1 (IDO1) is an intracellular monomeric, heme-containing enzyme that catalyzes the first and rate-limiting step in the catabolism of L-tryptophan (Trp) along the kynurenine pathway, resulting in the production of N-formylkynurenine. 95% of Trp is metabolized via this kynurenine pathway. The kynurenine pathway (KYN) triggers the production of neuroactive and immunomodulatory metabolites collectively known as kynurenine and provides a precursor for supplemental dietary niacin, which is used for the biosynthesis of NAD+ and NADP+.

By locally depleting tryptophan and increasing kynurenine, IDO1, expressed by antigen-presenting cells (APCs), such as dendritic cells (plasmacytoid DCs in tumor-draining lymph nodes), can significantly affect T cell proliferation and survival and activate regulatory T cells, thereby reducing proinflammatory responses. IDO1 may thus provide "immune privilege" to tissues undergoing chronic inflammation, such as infectious and allergic diseases, transplantation, and cancer. Because this tolerogenic response is expected to play a role in various pathophysiological conditions, tryptophan metabolism and kynurenine production by IDO1 may represent a key interface between the immune and nervous systems. IDO1 expression is upregulated by proinflammatory cytokines and can be detected in various tissues, including the placenta, spleen, thymus, lung, digestive tract, and central nervous system (reviewed in Munn et al., Trends Immunol, 2013, 34, 137-43).

IDO1 has become a promising molecular target for new therapeutic agents for the treatment of cancer and other diseases characterized by reduced local Trp levels and/or imbalanced levels of cytotoxic metabolites produced by the kynurenine pathway (reviewed in Munn et al., Trends Immunol, 2013, 34, 137-43). In fact, inhibition of IDO1 activity as a therapeutic strategy has been tested in preclinical models of many diseases using the most widely used IDO1 inhibitor, the tryptophan analog L-1-methyltryptophan (L-1MT). Treatment with L-1MT, alone or in combination with other agents, attenuated disease severity in animal models of arthritis, ischemia-reperfusion injury, endotoxic shock, human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection, airway inflammation, and cancer (Uyttenhove et al., Nat Med, 2003, 9, 10, 1269-1274; Holmgaard et al., J Exp Med, 2013, 210, 7, 1389-1402). Regarding cancer, IDO1 induction was observed in vivo during allogeneic tumor rejection, indicating a possible role for this enzyme in the tumor rejection process (Uyttenhove et al., Nat Med, 2003, 9, 10, 1269-1274; Holmgaard et al., J Exp Med, 2013, 210, 7, 1389-1402). Cervical cancer cells (or HeLa cells) co-cultured with peripheral blood lymphocytes (PBLs) acquire an immunosuppressive phenotype by upregulating IDO1 activity. It is believed that the reduction in PBL proliferation following treatment with interleukin-2 (IL2) is due to the release of IDO1 by tumor cells in response to interferon (IFN)-g (γ) secretion by PBLs. IDO1 activity in tumor cells can thus be used to impair anti-tumor responses, a process in which IFNg plays a major role. Additional evidence for a tumor immune resistance mechanism based on tryptophan degradation by IDO1 comes from the finding that a large proportion of human tumors constitutively express IDO1 and that IDO1 expression by immunogenic mouse tumor cells prevents their rejection (reviewed in Munn et al., Front Biosci, 2012, 4, 734-45; Godin-Ethier et al., Clin Cancer Res 2011, 17, 6985-6991; Johnson et al., Immunol Invest 2012, 41, 6-7, 765-797). This effect is accompanied by a lack of recruitment of specific T cells to the tumor site and can be partially rescued by systemic treatment of mice with an IDO1 inhibitor without overt toxicity (Holmgaard et al., J Exp Med, 2013, 210, 7, 1389-1402).

IDO1 expression has been confirmed by immunohistochemistry in a wide range of cancer patients. Altered tryptophan to kynurenine ratios in IDO1 mRNA, protein, or blood have been detected in patients with malignant melanoma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, prostate cancer, cervical cancer, brain cancer, endometrial cancer, and ovarian cancer, among others. The presence of IDO1 is an independent predictor of worse clinical outcome in several malignancies (reviewed in Munn et al., Front Biosci, 2012, 4, 734-45).

Although great interest has been generated in the potential of IDO1 inhibitors as pharmaceutical agents, initial inhibitors were identified by modifying Trp rather than by discovering molecules with novel structural backbones. In the early 2000s, the best IDO1 inhibitors primarily included competitive Trp derivatives (e.g., L-1-MT) and non-competitive carbolines, which exhibited affinities in the micromolar range. Since 2006, several potent nanomolar IDO1 inhibitors with novel structural backbones have been discovered by high-throughput screening, computational screening, or natural product isolation and optimization of core pharmacophores in structures. Many of these IDO1 inhibitors have low micromolar activity or limited pharmacokinetics. Two IDO1 inhibitors are currently being tested in Phase I/II clinical trials for the treatment of recurrent or refractory solid tumors (reviewed in Dolušić et al., Expert Opin Ther Pat. 2013, 23, 1367-81).

At the same time, the importance of awakening and consolidating tumor immune surveillance is now widely accepted as a key aspect of anticancer therapy (Motz et al., Immunity, 2013, 39, 1, 61-73). Immunoscoping of infiltrating T cell subsets is being developed as a biomarker and allows for the determination of patient responsiveness to treatment (Galon et al., J Transl Med, 2012, 10, 1). Therefore, the search for new and potent IDO1 inhibitors remains of great interest.

Therefore, there is a need for new IDO1 inhibitors with improved potency for cancer treatment and/or prevention.

SUMMARY OF THE INVENTION

The compounds, compositions, and methods herein help to fill the current need for IDO1 inhibitors that can be administered to any patient diagnosed with cancer or any individual at risk of developing cancer.

In one aspect, a pharmaceutical composition or medicament is provided, comprising a compound of Formula Ia:

or a pharmaceutically acceptable enantiomer, salt, solvate or prodrug thereof, wherein:

X ^a represents -NH- or -CQ ² =CQ ³ -;

Q ² and Q ³ each independently represent H or a C1 to C6 alkyl group, preferably, Q ² and Q ³ each independently represent H or a methyl group, more preferably, Q ² and Q ³ represent H;

R ¹ and R ² each independently represent H, halogen, cyano, C1 to C6 alkyl or C1 to C6 alkoxy. Preferably, R ¹ and R ² each independently represent H or halogen.

In another aspect, a pharmaceutical composition is provided, comprising a compound of Formula Ia:

or a pharmaceutically acceptable enantiomer, salt, solvate or prodrug thereof, wherein:

X ^a represents -NH- or -CQ ² =CQ ³ -;

Q ² and Q ³ each independently represent H or a C1 to C6 alkyl group, preferably, Q ² and Q ³ each independently represent H or a methyl group, more preferably, Q ² and Q ³ represent H;

R ¹ and R ² each independently represent H, halogen, cyano, C1 to C6 alkyl or C1 to C6 alkoxy, preferably, R ¹ and R ² each independently represent H or halogen;

and at least one pharmaceutically acceptable carrier.

Also provided are compounds of formula Ia:

or a pharmaceutically acceptable enantiomer, salt, solvate or prodrug thereof, wherein:

X ^a represents -NH- or -CQ ² =CQ ³ -;

Q ² and Q ³ each independently represent H or a C1 to C6 alkyl group, preferably, Q ² and Q ³ each independently represent H or a methyl group, more preferably, Q ² and Q ³ represent H;

R ¹ and R ² each independently represent H, halogen, cyano, C1 to C6 alkyl or C1 to C6 alkoxy. Preferably, R ¹ and R ² each independently represent H or halogen.

In one embodiment, the compounds of Formula I and/or Formula Ia have deuterium atoms substituted for hydrogen atoms therein, i.e., are optionally deuterated. In one embodiment, the compounds of Formula I are deuterated at a chiral carbon and can be used to prepare deuterated compounds of Formula I' and/or Formula I". The compounds described herein, including those of Formula I, Formula Ia, Formula Ib, Formula I', and Formula I", and their deuterated counterparts, can be used to treat and/or prevent cancer and endometriosis and/or as IDO1 inhibitors.

Also provided are compounds of formula Ia':

or a pharmaceutically acceptable enantiomer, salt, solvate or prodrug thereof, wherein:

X ^a represents -NH- or -CQ ² =CQ ³ -;

Q ² and Q ³ each independently represent H or a C1 to C6 alkyl group, preferably, Q ² and Q ³ each independently represent H or a methyl group, more preferably, Q ² and Q ³ represent H;

R ¹ and R ² each independently represent H, halogen, cyano, C1 to C6 alkyl or C1 to C6 alkoxy. Preferably, R ¹ and R ² each independently represent H or halogen.

In one embodiment, the compounds of Formula I and/or Ia are deuterated at the chiral center, such as in the structure of Formula Ia':

or a pharmaceutically acceptable enantiomer, salt, solvate or prodrug thereof, wherein:

X ^a represents -NH- or -CQ ² =CQ ³ -;

Q ² and Q ³ each independently represent H or a C1 to C6 alkyl group, preferably, Q ² and Q ³ each independently represent H or a methyl group, more preferably, Q ² and Q ³ represent H;

R ¹ and R ² each independently represent H, halogen, cyano, C1 to C6 alkyl or C1 to C6 alkoxy, preferably, R ¹ and R ² each independently represent H or halogen. In one embodiment, the racemic compounds of Formula I and/or Ia can be deuterated using the techniques described herein and/or those known to those skilled in the art. Such compounds can be used in medicaments or pharmaceutical compositions and/or for producing deuterated R-enantiomers and/or deuterated S-enantiomers. Such deuterated enantiomers themselves can be used in medicaments or pharmaceutical compositions as described herein.

In addition, compounds of formula I', formula I'' or mixtures thereof are provided:

and pharmaceutically acceptable salts, solvates and precursors thereof, wherein

X represents -NQ ¹ - or -CQ ² =CQ ³ -;

Q ¹ , Q ² and Q ³ each independently represent H or C1 to C6 alkyl, preferably, Q ¹ is H, and Q ² and Q ³ each independently represent H or methyl, more preferably, Q ¹ , Q ² and Q ³ each represent H;

R ¹ and R ² each independently represent H, halogen, cyano, C1 to C6 alkyl or C1 to C6 alkoxy. Preferably, R ¹ and R ² each independently represent H or halogen.

In another embodiment, Q ¹ is H and X represents -NH- or -CQ ² =CQ ³ -;

Q ² and Q ³ each independently represent H or a C1 to C6 alkyl group, preferably, Q ² and Q ³ each independently represent H or a methyl group, more preferably, Q ² and Q ³ each represent H;

R ¹ and R ² each independently represent H, halogen, cyano, C1 to C6 alkyl or C1 to C6 alkoxy. Preferably, R ¹ and R ² each independently represent H or halogen.

In another embodiment, a composition comprising a compound of Formula I' and/or Formula I" is provided. The composition may contain a racemic compound. Alternatively, the composition may contain a mixture of compounds of Formula I' and Formula I" produced separately. Such a compound may contain a 1:1 ratio of Formula I' to Formula I", as present in a racemate, or the R-enantiomer may be present in an amount greater than 50%. In another alternative, the composition may contain greater than 50% of the S-enantiomer. Optionally, one or both of the racemate or enantiomers may be deuterated, for example at a chiral carbon.

The present invention further discloses a compound of formula Ib:

and pharmaceutically acceptable enantiomers, salts, solvates and prodrugs thereof, wherein:

X represents -NQ ¹ - or -CQ ² =CQ ³ -;

Q ¹ , Q ² and Q ³ each independently represent H or an alkyl group, preferably, Q ¹ is H, Q ² and Q ³ each independently represent H or a methyl group, more preferably, Q ¹ , Q ² and Q ³ represent H;

R ^1b and R ^2b each independently represent H, halogen, cyano, alkyl or alkoxy, preferably, R ^1b and R ^2b each independently represent H or halogen;

The condition is

When X represents -NQ ¹ -, then R ^1b and R ^2b are not both H, and R ^1b and R ^2b are not both F; in one embodiment, Q 1 is H.

When X represents -CQ ² =CQ ³ -, then R ^1b and R ^2b are not both H.

In another embodiment, when Q1 is H, X represents -NH- or -CQ ² =CQ ³ -.

Q ² and Q ³ each independently represent H or an alkyl group, Q ² and Q ³ each independently represent H or a methyl group, more preferably, Q ² and Q ³ represent H;

R ^1b and R ^2b each independently represent H, halogen, cyano, alkyl or alkoxy, preferably, R ^1b and R ^2b each independently represent H or halogen;

The condition is

When X represents -NH-, then R ^1b and R ^2b are not both H, and when X represents -NH-, then R ^1b and R ^2b are not both F; when X represents -CQ ² =CQ ³ -, then R ^1b and R ^2b are not both H.

According to one embodiment, the compound of formula I' is selected from:

(a) (-)-(R)-3-(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(b) (-)-(R)-3-(1H-indol-3-yl)pyrrolidine-2,5-dione;

(c) (-)-(R)-3-(5-chloro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(d) (R)-3-(6-chloro-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione; or

(e) (R) -3- (6-bromo-5-fluoro-1H-indol-3-yl) pyrrolidine-2,5-dione or a pharmaceutically acceptable salt or solvate thereof. In another embodiment, the compound of formula II' is selected from:

(a'')(S)-3-(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(b'')(S)-3-(1H-indol-3-yl)pyrrolidine-2,5-dione;

(c'')(S)-3-(5-chloro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(d'')(S)-3-(6-chloro-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione; or

(e'')(S)-3-(6-bromo-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione or a pharmaceutically acceptable salt or solvate thereof. In yet another embodiment, the compound is:

3-(6-chloro-5-fluoro- 1H -indol-3-yl)pyrrolidine-2,5-dione;

( R )-3-(6-chloro-5-fluoro- 1 H -indol-3-yl)pyrrolidine-2,5-dione;

3-(6-bromo-5-fluoro- 1H -indol-3-yl)pyrrolidine-2,5-dione;

( R )-3-(6-bromo-5-fluoro- 1H -indol-3-yl)pyrrolidine-2,5-dione,

3-(Naphthalen-1-yl)pyrrolidine-2,5-dione;

3-(6-Fluoronaphthalen-1-yl)pyrrolidine-2,5-dione;

3-(7-Fluoronaphthalen-1-yl)pyrrolidine-2,5-dione;

3-(6-chloronaphthalen-1-yl)pyrrolidine-2,5-dione; or

3-(7-Chloronaphthalen-1-yl)pyrrolidine-2,5-dione

or a pharmaceutically acceptable salt or solvate thereof, or a deuterated form thereof.

The present invention also discloses a method for preparing a compound of formula I', I'' or Ib, comprising reacting maleimide with a compound of formula (i) or (ib):

(i) (ib)

wherein X, R ¹ and R ² are as defined in formula I′ or I″ and R ^1b and R ^2b are as defined in formula Ib, and optionally the enantiomers are separated.

In another aspect, a compound having the structure of Formula II' is provided:

,

or a pharmaceutically acceptable salt or solvate thereof. In one embodiment, the compound is a free base, i.e., not in salt form or solvate form. Also provided is a pharmaceutical composition comprising a compound of formula II', alone or optionally in combination with a compound having the structure of formula II":

or a mixture or admixture of a pharmaceutically acceptable salt thereof.

Other aspects and advantages of the present invention will become apparent from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing the effect of increasing amounts of Compound 2 of the present invention on T cell proliferation (as measured by thymidine incorporation) in a SKOV-3 - PBMC co-culture assay.

Figure 2 is a graph showing circulating kynurenine concentrations in the blood of healthy mice following treatment with Compound 2 of the present invention or with vehicle.

Figure 3 is a graph showing different studies of tumor growth of 4T1 tumors in a mouse breast cancer model after treatment with Compound 1 or vehicle. Figure 3 shows tumor growth of 4T1 tumors after treatment with Compound 1 twice daily at a dose of 100 mg/kg. The upper line represents the vehicle and the lower line represents Compound 1.

Figure 4 is a graph showing tumor growth of PanCO2 tumors in mice after treatment with test compounds. The upper line represents vehicle and the lower line represents compound 1.

Figure 5 is a graph showing kynurenine concentrations within 4T1 tumors in mice following treatment with Compound 2 of the present invention or with vehicle.

Figure 6 is a graph showing kynurenine concentrations within CT26 tumors in mice following treatment with Compound 2 of the present invention or with vehicle.

Figure 7 is a graph showing tumor growth of CT26 tumors in Balb/c mice using Test Compound 1 at 200 mg/kg bid (open circles), 600 mg/kg bid (closed triangles) compared to vehicle (squares).

Detailed Description of the Invention

Compound

Compounds of formula I:

and pharmaceutically acceptable enantiomers, salts, solvates and prodrugs thereof, wherein:

X represents -NQ ¹ - or -CQ ² =CQ ³ -;

Q ¹ , Q ² and Q ³ each independently represent H or an alkyl group, preferably, Q ¹ , Q ² and Q ³ each independently represent H or a methyl group, more preferably, Q ¹ , Q ² and Q ³ represent H;

R ¹ and R ² each independently represent H, halogen, cyano, alkyl or alkoxy. Preferably, R ¹ and R ² each independently represent H or halogen.

In another embodiment, Q ¹ is H, X represents -NH- or -CQ ² =CQ ³ -;

Q ² and Q ³ each independently represent H or alkyl, preferably, Q ² and Q ³ each independently represent H or methyl, more preferably, Q ² and Q ³ represent H; R ¹ and R ² each independently represent H, halogen, cyano, alkyl or alkoxy, preferably, R ¹ and R ² each independently represent H or halogen.

Also provided herein are compounds of Formula I and pharmaceutically acceptable enantiomers, salts, solvates, and prodrugs thereof, which have at least one deuterium atom substituted for a hydrogen atom. In one embodiment, a compound of Formula I or any subformula thereof provided herein, wherein the subformula comprises Ia, Ib, I', I, II, II', II", at a chiral center, as exemplified below in the structure of Formula Ia':

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

Formulas I, Ia, and Ib are not drawn with reference to stereochemistry and thus each encompasses racemic compounds and individual stereoisomers, i.e., R- and/or S-stereoisomers. In one embodiment, these stereoisomers may have the structures of Formula I' (R-stereoisomer) and Formula II' (S-enantiomer).

Exemplary compounds of Formula I are shown in the Tables and Examples herein and include:

3-(5-Fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(-)-(R)-3-(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(+)-(S)-3-(5-Fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(1H-indol-3-yl)pyrrolidine-2,5-dione;

(-)-(R)-3-(1H-indol-3-yl)pyrrolidine-2,5-dione;

(+-)-(S)-3-(1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(5-chloro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(-)-(R)-3-(5-chloro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(+)-(S)-3-(5-chloro-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(6-chloro-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(R)-3-(6-chloro-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(S)-3-(6-chloro-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(6-bromo-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(R)-3-(6-bromo-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(S)-3-(6-bromo-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(5-Bromo-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(5-methyl-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(5-methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(2,5-Dioxopyrrolidin-3-yl)-1H-indole-5-carbonitrile;

3-(5,6-difluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(5-Fluoro-6-methyl-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(6-Fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(6-chloro-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(6-bromo-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(6-methyl-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(6-methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(2,5-Dioxopyrrolidin-3-yl)-1H-indole-6-carbonitrile;

3-(Naphthalen-1-yl)pyrrolidine-2,5-dione;

3-(6-Fluoronaphthalen-1-yl)pyrrolidine-2,5-dione;

3-(7-Fluoronaphthalen-1-yl)pyrrolidine-2,5-dione;

3-(6-chloronaphthalen-1-yl)pyrrolidine-2,5-dione; and

3-(7-Chloronaphthalen-1-yl)pyrrolidine-2,5-dione.

Optionally, these compounds of formula I may be deuterated, for example, at a chiral center. An exemplary deuterated compound (3- ² H)-3-(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione is provided in the Examples below. Other deuterated compounds may include, for example

(-)-(R)-(3- ² H)-3-(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(+)-(S)-(3- ² H)-3-(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(3- ² H)-3-(1H-indol-3-yl)pyrrolidine-2,5-dione;

(-)-(R)-(3- ² H)-3-(1H-indol-3-yl)pyrrolidine-2,5-dione;

(+-)-(S)-(3- ² H)-3-(1H-indol-3-yl)pyrrolidine-2,5-dione;

(3- ² H)-3-(5-chloro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(-)-(R)-(3- ² H)-3-(5-chloro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(+)-(S)-(3- ² H)-3-(5-chloro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(3- ² H)-3-(6-chloro-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(R)-(3- ² H)-3-(6-chloro-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(S)-(3- ² H)-3-(6-chloro-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(3- ² H)-3-(6-bromo-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(R)-3-(6-bromo-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(S)-(3- ² H)-3-(6-bromo-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(3- ² H)-3-(5-bromo-1H-indol-3-yl)pyrrolidine-2,5-dione;

(3- ² H)-3-(5-methyl-1H-indol-3-yl)pyrrolidine-2,5-dione;

(3- ² H)-3-(5-methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(2,5-Dioxopyrrolidin-3-yl)-1H-indole-5-carbonitrile;

(3- ² H)-3-(5,6-difluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(3- ² H)-3-(5-fluoro-6-methyl-1H-indol-3-yl)pyrrolidine-2,5-dione;

(3- ² H)-3-(6-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(3- ² H)-3-(6-chloro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(3- ² H)-3-(6-bromo-1H-indol-3-yl)pyrrolidine-2,5-dione;

(3- ² H)-3-(6-methyl-1H-indol-3-yl)pyrrolidine-2,5-dione;

(3- ² H)-3-(6-methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione;

(3- ² H)-3-(2,5-dioxopyrrolidin-3-yl)-1H-indole-6-carbonitrile;

(3- ² H)-3-(naphthalen-1-yl)pyrrolidine-2,5-dione;

(3- ² H)-3-(6-fluoronaphthalen-1-yl)pyrrolidine-2,5-dione;

(3- ² H)-3-(7-fluoronaphthalen-1-yl)pyrrolidine-2,5-dione;

(3- ² H)-3-(6-chloronaphthalen-1-yl)pyrrolidine-2,5-dione; and

(3- ² H)-3-(7-chloronaphthalen-1-yl)pyrrolidine-2,5-dione.

In one embodiment, preferred compounds of formula I are those of formula I' or I":

and pharmaceutically acceptable salts, solvates, and prodrugs thereof, wherein X, R ¹ and R ² are as defined in Formula I.

As described herein, a racemic compound of Formula I may contain about 50% of a compound of Formula I' and about 50% of a compound of Formula I'' based on a molar ratio of one isomer (about 48 mol% to about 52 mol% or about a 1:1 ratio). In another embodiment, a composition, medicament, or method of treatment may involve combining approximately equimolar ratios (about 48% to 52%) of compounds of Formula I' and Formula I'' produced separately. In another embodiment, a medicament or pharmaceutical composition may contain a mixture of individual compounds of Formula I' and Formula I'' in different ratios. In one embodiment, the pharmaceutical composition contains an excess (greater than 50%) of the R-enantiomer (Formula I'). Suitable molar ratios of R/S may be about 1.5:1, 2:1, 3:1, 4:1, 5:1, 10:1, or more. In another embodiment, the pharmaceutical composition may contain an excess of the S-enantiomer (Formula I'') and the provided R/S ratio is reversed. Other suitable amounts of R/S may be selected. For example, the R-enantiomer can be present in an amount of at least about 55% to 100% or at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, about 95%, about 98% or 100%. In other embodiments, the S-enantiomer can be present in a higher percentage, for example, in an amount of at least about 55% to 100% or at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, about 95%, about 98% or 100%. All ratios between all of these exemplary embodiments and greater than and less than thereof and still within the present invention are included. (The term "ratio" used herein (above and below) always refers to a molar ratio). The composition can contain a mixture of a racemate and a separate compound of Formula I' and/or Formula I'' in free alkali and/or salt form.

Optionally, one or both of the racemates or enantiomers may be deuterated. Such deuterated compounds may be in the form of salts. For example, deuterated stereoisomers may be characterized by the following structures:

.

wherein X (or ^Xa ), ^R1 and ^R2 are as defined above in Formulas I and Ia. Without wishing to be bound by theory, it has been described in the literature that one enantiomer (isomer or stereoisomer) can often be converted into a racemate and/or the other enantiomer in plasma. It is believed that the deuteration at the chiral center of these compounds slows down the conversion of individual stereoisomers to racemates and/or the other stereoisomer in plasma.

In one embodiment, preferred compounds of formula I are those of formula Ia:

and pharmaceutically acceptable enantiomers, salts, solvates and prodrugs thereof, wherein:

X ^a represents -NH- or -CQ ² =CQ ³ -;

Q ² and Q ³ each independently represent H or an alkyl group, preferably, Q ² and Q ³ each independently represent H or a methyl group, more preferably, Q ² and Q ³ represent H;

R ¹ and R ² each independently represent H, halogen, cyano, alkyl or alkoxy. Preferably, R ¹ and R ² each independently represent H or halogen.

In one embodiment, preferred compounds of formula I are those of formula Ib:

and pharmaceutically acceptable enantiomers, salts, solvates and prodrugs thereof, wherein:

X represents -NQ ¹ - or -CQ ² =CQ ³ -;

Q ¹ , Q ² and Q ³ each independently represent H or alkyl; optionally, the alkyl group is a C1 to C6 alkyl group, preferably, Q ¹ , Q ² and Q ³ each independently represent H or methyl, more preferably, Q ¹ , Q ² and Q ³ represent H;

R ^1b and R ^2b each independently represent H, halogen, cyano, alkyl or alkoxy; optionally, alkyl is C1 to C6 alkyl and alkoxy is C1 to C6 alkoxy, preferably R ^1b and R ^2b each independently represent H or halogen;

The condition is

When X represents -NQ ¹ -, then R ^1b and R ^2b are not both H, and R ^1b and R ^2b are not both F;

When X represents -CQ ² =CQ ³ -, then R ^1b and R ^2b are not both H.

In another embodiment, Q ¹ is H and X represents -NH ¹ - or -CQ ² =CQ ³ -;

Q ² and Q ³ each independently represent H or alkyl; optionally, the alkyl group is a C1 to C6 alkyl group, preferably, Q ² and Q ³ each independently represent H or methyl, more preferably, Q ² and Q ³ represent H;

R ^1b and R ^2b each independently represent H, halogen, cyano, alkyl or alkoxy; optionally, alkyl is C1 to C6 alkyl and alkoxy is C1 to C6 alkoxy, preferably R ^1b and R ^2b each independently represent H or halogen;

The condition is

When X represents -NH-, then R ^1b and R ^2b are not both H, and R ^1b and R ^2b are not both F;

When X represents -CQ ² =CQ ³ -, then R ^1b and R ^2b are not both H.

Particularly preferred compounds of formula I according to the invention are those shown in Table 1 hereinafter.

Table 1

Cpd n° structure Chemical name 1 1a 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 twenty one twenty two twenty three 3-(Naphthalen-1-yl)pyrrolidine-2,5-dione twenty four 3-(6-Fluoronaphthalen-1-yl)pyrrolidine-2,5-dione 25 3-(7-Fluoronaphthalen-1-yl)pyrrolidine-2,5-dione 26 3-(6-Chloronaphthalen-1-yl)pyrrolidine-2,5-dione 27 3-(7-Chloronaphthalen-1-yl)pyrrolidine-2,5-dione

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

In Table 1, the term "Cpd" means compound.

The compounds in Table 1 were named using ^{ChemBioDraw®} Ultra version 12.0 (PerkinElmer).

According to a preferred embodiment, particularly preferred compounds of formula I of the present invention are compounds numbered 1, 1a, 2, 4, 6, 7, 8, 9, 10, 14, 16, 22, 24, 25, 26, 27 in Table 1.

Compounds of Formula I and its subformulas contain asymmetric centers and thus exist as different stereoisomers. Therefore, the present invention encompasses all possible stereoisomers and includes not only racemic compounds but also individual enantiomers and non-racemic mixtures thereof. When a compound is desired to be in the form of a single enantiomer, the compound can be obtained by stereospecific synthesis, by resolution of the final product or any convenient intermediate, or by chiral chromatographic methods, each of which is known in the art. Resolution of the final product, intermediate, or starting material can be carried out by any suitable method known in the art.

The compounds of this invention can be in the form of pharmaceutically acceptable salts. The pharmaceutically acceptable salts of formula I compound include alkali salts, which form non-toxic salts, including salts of, for example, aluminum, calcium, choline, magnesium, potassium, sodium, zinc, and tetramethylammonium hydroxide. Although less desirable, other alkalis can be selected, including, for example, ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, N, N'-dibenzylethylenediamine, chloroprocaine (chloroprocaine), diethanolamine, procaine (procaine), N-benzylphenethylamine, diethylamine, piperazine, tris (hydroxymethyl) aminomethane, benzathine, diethylamine, diethanolamine, glycine, lysine, meglumine, ethanolamine, tromethamine, 2-(diethylamino) ethanol, ethanolamine, morpholine, and 4-(2-hydroxyethyl) morpholine. A half salt of an alkali can also be formed, such as a half calcium salt.

Pharmaceutically acceptable salts of compounds of formula I may be prepared by one or more of these methods:

(i) reacting a compound of formula I and its subformulae with a desired base;

(ii) removing an acid- or base-labile protecting group from a suitable precursor of a compound of formula I (or a subformula thereof) or performing a ring opening of a suitable cyclic precursor, such as a lactone or lactam, using the desired acid; or

(iii) converting one salt of a compound of formula I (or a subformula thereof) into another salt by reaction with a suitable acid or with the aid of a suitable ion exchange column.

All of these reactions are usually carried out in solution. The salt can be precipitated from the solution and collected by filtration or can be recovered by evaporating the solvent. The degree of ionization of the salt can vary from completely ionized to almost non-ionized.

The compounds of the present invention can be administered in the form of pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" is intended to include all acceptable salts, such as those that can be used as dosage forms for modifying solubility or hydrolysis characteristics or that can be used in sustained-release or prodrug formulations. Depending on the specific functionality of the compounds of the present invention, pharmaceutically acceptable salts of the compounds of the present invention include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as tetramethylammonium hydroxide.

These salts can be prepared by standard procedures, for example, by reacting the free acid with a suitable organic or inorganic base. In the presence of a basic group such as an amino group, acid salts, i.e., hydrochlorides, hydrobromides, acetates, pamoates, etc., can be used as dosage forms.

Additionally, where an alcohol group is present, pharmaceutically acceptable esters may be employed, such as acetate, maleate, pivaloyloxymethyl, and the like, and those esters known in the art to modify solubility or hydrolysis characteristics for use as sustained release or prodrug formulations.

All references to compounds of formula I include references to their enantiomers, salts, solvates, polymorphs, multicomponent complexes and liquid crystals.

The compounds of the present invention include compounds of formula I as defined above, including all polymorphs and crystal habits thereof, prodrugs and isomers (including optical, geometric and tautomers) thereof and isotopically labeled compounds of formula I.

In addition, with respect to the salts of the compounds of the present invention, although pharmaceutically acceptable salts are generally preferred, it should be noted that the present invention in its broadest sense also encompasses non-pharmaceutically acceptable salts, which can be used, for example, to separate and/or purify the compounds of the present invention. For example, salts formed using optically active acids or bases can be used to form diastereomeric salts, which can facilitate the separation of optically active isomers of the compounds of Formula I above.

As used herein, the term "free base" refers to the non-salt form of the compound of Formula I.

Unless otherwise specified, references herein to Formula I include subformulae thereof, such as Formulas Ia, Ib, Ia', I', I'', II, II', and II''.

The present invention also generally encompasses all pharmaceutically acceptable prodrugs and prodrugs of the compounds of Formula I.

Preparation method

The compounds of formula I can be prepared in various ways using reactions known to those skilled in the art.

The present invention further relates to the preparation of compounds of formula I:

and pharmaceutically acceptable enantiomers, salts, solvates and prodrugs thereof, wherein X, R ¹ and R ² are as defined in Formula I;

The first method comprises:

The compound of formula (i):

Wherein X, ^R1 and ^R2 are as defined in Formula I

Reaction with maleimide to provide a compound of formula I;

and optionally separating the enantiomers of formula I' and I".

According to one embodiment, the method can be carried out in the presence of a suitable solvent, preferably in acetic acid or acetonitrile, such as, but not limited to, acetic acid, acetonitrile, DMSO, dichloroethane, DMF, water or a mixture thereof. According to one embodiment, the method can be carried out in the presence or absence of a suitable catalyst, such as, but not limited to, a protic acid, such as, but not limited to, acetic acid, hydrochloric acid or sulfuric acid; or a Lewis acid, such as, but not limited to, zinc chloride, zinc acetate, zinc trifluoromethanesulfonate, aluminum chloride, cobalt chloride, cobalt acetate or ferric chloride.

According to one embodiment, the method can be carried out at a temperature of 20°C to about 200°C, preferably at a temperature of 60°C to 200°C or about 150°C to about 200°C, with or without microwave irradiation, for a period of 10 minutes to several hours, for example 10 minutes to 48 h.

According to one embodiment, the optional separation of the enantiomers of formula I' and I" starting from the corresponding compound of formula I can be achieved by chiral HPLC, for example but not limited to using Chiralpak® AS-H, Chiralcel® OJ-H or Chiralpak® IC columns, using a suitable solvent mixture as eluent, for example but not limited to supercritical _CO2 , ethanol, methanol, hexane.

According to one embodiment, the optional separation of the enantiomers of formula I' and I'' starting from the corresponding compound of formula I can be achieved by resolution using an optically pure acid, such as but not limited to camphorsulfonic acid or tartaric acid, or using an optically pure base, such as but not limited to brucine, depending on the nature of the compound of formula I.

The present invention further relates to a second process for preparing compounds of formula I:

and pharmaceutically acceptable enantiomers, salts, solvates and prodrugs thereof, wherein X, R ¹ and R ² are as defined in Formula I;

It comprises making a compound of formula (ii):

wherein X, R ¹ and R ² are as defined in Formula I; and

Z ¹ and Z ² represent H or an alkyl group, wherein Z ¹ and Z ² may form a ring;

Reaction with maleimide to provide a compound of formula I;

and optionally separating the enantiomers of formula I' and I".

According to one embodiment, the process can be carried out with or without the use of a catalyst such as, but not limited to, [RhOH(cod)] ₂ .

According to one embodiment, the process may be carried out in the presence of a base such as, but not limited to, trimethylamine (TEA), diethylisopropylamine (DIEA), sodium hydroxide (NaOH), potassium hydroxide (KOH), potassium phosphate (K ₃ PO ₄ ), potassium carbonate (K ₂ CO ₃ ), sodium carbonate (Na ₂ CO ₃ ), preferably TEA or DIEA.

According to one embodiment, the process may be carried out in the presence of a suitable solvent such as, but not limited to, dioxane, tetrahydrofuran (THF), dimethylformamide (DMF), water or mixtures thereof, preferably in dioxane or THF.

According to one embodiment, the method may be performed at a temperature of 20°C to about 150°C with or without microwave irradiation for a period of 10 minutes to several hours, for example 10 minutes to 24 h.

According to one embodiment, the optional separation of the enantiomers of formula I' and I" starting from the corresponding compound of formula I can be achieved by chiral HPLC, for example but not limited to using Chiralpak® AS-H, Chiralcel® OJ-H or Chiralpak® IC columns, using a mixture of suitable solvents as eluents, for example but not limited to supercritical _CO2 , ethanol, methanol, hexane.

According to one embodiment, the optional separation of the enantiomers of formula I' and I'' starting from the corresponding compound of formula I can be achieved by resolution using an optically pure acid, such as but not limited to camphorsulfonic acid or tartaric acid, or using an optically pure base, such as but not limited to brucine, depending on the nature of the compound of formula I.

The present invention further relates to a third method for preparing a compound of formula I:

and pharmaceutically acceptable enantiomers, salts, solvates and prodrugs thereof, wherein X, R ¹ and R ² are as defined in Formula I;

It includes:

(a) reacting the compound of formula (iii)

wherein X, R ¹ and R ² are as defined in Formula I;

To obtain the compound of formula (iv)

wherein X, R ¹ and R ² are as defined in Formula I;

(b) reacting the compound of formula (iv) with maleic anhydride to obtain the compound of formula (v)

wherein X, R ¹ and R ² are as defined in Formula I;

and

(c) reacting the compound of formula (iv) with urea to obtain a compound of formula I

(d) optionally separating the enantiomers of formula I' and I".

According to one embodiment, step (a) may be carried out in the presence of a nitrite, such as but not limited to NaNO ₂ , KNO ₂ , tert-butyl nitrite or isoamyl nitrite.

According to one embodiment, step (a) may be carried out in the presence of a suitable acid, such as but not limited to HBF ₄ .

According to one embodiment, step (a) may be carried out in the presence of a suitable solvent, such as but not limited to water.

According to one embodiment, step (a) may be performed at a temperature of -20°C to about 20°C, preferably at 0°C.

According to one embodiment, step (a) may be performed for a period of 10 minutes to several hours, for example 10 minutes to 24 h.

According to one embodiment, step (b) may be carried out in the presence of a suitable catalyst, such as but not limited to TiCl ₃ .

According to one embodiment, step (b) may be performed in the presence of a suitable base, such as but not limited to NaOH or KOH.

According to one embodiment, step (b) may be carried out in the presence of a suitable solvent, such as but not limited to acetone, methyl ethyl ketone.

According to one embodiment, step (b) may be performed at a temperature of -20°C to about 20°C, preferably at 0°C.

According to one embodiment, step (b) may be performed for a period of 10 minutes to several hours, for example 10 minutes to 24 h.

According to one embodiment, step (c) may be carried out in the absence or presence of a suitable solvent at a temperature of 100°C to about 200°C, preferably at 180°C.

According to one embodiment, step (c) may be performed for a period of 10 minutes to several hours, for example 10 minutes to 24 h.

According to one embodiment, the optional separation of the enantiomers of formula I' and I" starting from the corresponding compound of formula I can be achieved by chiral HPLC, for example but not limited to using Chiralpak® AS-H, Chiralcel® OJ-H or Chiralpak® IC columns, using a mixture of suitable solvents as eluents, for example but not limited to supercritical _CO2 , ethanol, methanol, hexane.

According to one embodiment, the optional separation of the enantiomers of formula I' and I'' starting from the corresponding compound of formula I can be achieved by resolution using an optically pure acid, such as but not limited to camphorsulfonic acid or tartaric acid, or using an optically pure base, such as but not limited to brucine, depending on the nature of the compound of formula I.

In general, the synthetic route for any individual compound of Formula I will depend on the specific substituents on each molecule and the current availability of the desired intermediates; such factors will again be understood by those skilled in the art.

According to another general procedure, a compound of formula I may be converted into an alternative compound of formula I using appropriate interconversion techniques well known to those skilled in the art.

Additionally, compounds of formula I and related formulae can be obtained by liberating the compound of formula I from a functional derivative thereof by treatment with a solvolytic or hydrogenolytic agent.

Preferred starting materials for solvolysis or hydrogenolysis are those that conform to formula I and related formulae, but contain corresponding protected amino and/or hydroxyl groups instead of one or more free amino and/or hydroxyl groups, preferably those carrying amino-protecting groups instead of H atoms bonded to N atoms, especially those carrying R*-N groups, wherein R* represents an amino-protecting group, instead of an HN group and/or those carrying hydroxy-protecting groups instead of the H atom of a hydroxyl group, for example those that conform to formula I, but carry -COOR** groups, wherein R** represents a hydroxy-protecting group, instead of a -COOH group.

There may also be a plurality of identical or different protected amino and/or hydroxyl groups in the molecule of the starting material. If the protecting groups present are different from one another, they can in many cases be cleaved selectively.

The term "amino-protecting group" is a generally known term and relates to a group that is suitable for protecting (blocking) an amino group from chemical reactions but is easily removable after the desired chemical reaction is carried out elsewhere in the molecule. Typical examples of such groups are, in particular, unsubstituted or substituted acyl, aryl, aralkoxymethyl or aralkyl groups. Since the amino-protecting group is removed after the desired reaction (or reaction sequence), its type and size are also unimportant; however, preference is given to those having 1 to 20, in particular 1 to 8, carbon atoms. The term "acyl" is to be understood in the broadest sense in conjunction with the process according to the invention. It encompasses acyl groups derived from aliphatic, araliphatic, aromatic or heterocyclic carboxylic acids or sulfonic acids, and in particular alkoxycarbonyl, aryloxycarbonyl and especially aralkoxycarbonyl groups. Examples of such acyl groups are alkanoyl groups such as acetyl, propionyl and butyryl; aralkanoyl groups such as phenylacetyl; aralkanoyl groups such as benzoyl and tolyl; aryloxyalkanoyl groups such as POA; alkoxycarbonyl groups such as methoxycarbonyl, ethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, BOC (tert-butoxycarbonyl) and 2-iodoethoxycarbonyl; aralkyloxycarbonyl groups such as CBZ ("benzyloxycarbonyl"), 4-methoxybenzyloxycarbonyl and FMOC; and arylsulfonyl groups such as Mtr. Preferred amino protecting groups are BOC and Mtr, and further CBZ, Fmoc, benzyl and acetyl.

Likewise, the term "hydroxy-protecting group" is a commonly known term and relates to a group that is suitable for protecting a hydroxy group from chemical reactions but can be easily removed after the desired chemical reaction has been carried out elsewhere in the molecule. Typical of such groups are the above-mentioned unsubstituted or substituted aryl, aralkyl or acyl groups, and additionally alkyl groups. The nature and size of the hydroxy-protecting group are not critical, since it is removed again after the desired chemical reaction or reaction sequence; groups having 1 to 20, in particular 1 to 10, carbon atoms are preferred. Examples of hydroxy-protecting groups are especially benzyl, 4-methoxybenzyl, p-nitrobenzoyl, p-toluenesulfonyl, tert-butyl and acetyl, with benzyl and tert-butyl being particularly preferred.

Depending on the protecting group used, the compounds of formula I and related formulae are released from their functional derivatives, such as strong mineral acids, such as hydrochloric acid, perchloric acid or sulfuric acid; strong organic carboxylic acids, such as trichloroacetic acid, TFA, or sulfonic acids, such as benzene- or p-toluenesulfonic acid. Other inert solvents may be present, but are not always required. Suitable inert solvents are preferably organic solvents, such as carboxylic acids, such as acetic acid; ethers, such as THF or dioxane; amides, such as DMF; halogenated hydrocarbons, such as dichloromethane; and also alcohols, such as methanol, ethanol or isopropanol; and water. In addition, mixtures of the above solvents are suitable. Trifluoroacetic acid (TFA) is preferably used in excess without adding another solvent, and perchloric acid is preferably used in the form of a mixture of acetic acid and 70% perchloric acid in a ratio of 9:1. The reaction temperature for cleavage is advantageously from about 0°C to about 50°C, preferably from 15°C to 30°C (room temperature).

BOC, OtBu, and Mtr groups can be cleaved, for example, preferably using TFA in dichloromethane or using approximately 3N to 5N HCl in dioxane at 15-30°C, and the FMOC group can be cleaved using approximately 5% to 50% solutions of dimethylamine, diethylamine, or piperidine in DMF at 15-30°C.

Protective groups that can be removed by hydrogenolysis (e.g., CBZ, benzyl, or amidino groups released from their oxadiazole derivatives) can be cleaved, for example, by treatment with hydrogen in the presence of a catalyst (e.g., a noble metal catalyst, such as palladium, which is advantageously located on a support such as carbon). Suitable solvents herein are those mentioned above, especially, for example, alcohols such as methanol or ethanol, or amides such as DMF. Hydrogenolysis is typically performed at a temperature of about 0° C. to 100° C. and a pressure of about 1 bar to 200 bar, preferably at 20-30° C. and 1-10 bar. For example, CBZ groups are very successfully hydrogenolyzed on 5% to 10% Pd/C in methanol or using ammonium formate (instead of hydrogen) on Pd/C in methanol/DMF at 20-30° C.

Examples of suitable inert solvents are hydrocarbons, for example hexane, petroleum ether, benzene, toluene or xylene; chlorinated hydrocarbons, for example trichloroethylene, 1,2-dichloroethane, tetrachloromethane, trifluoromethylbenzene, chloroform or dichloromethane; alcohols, for example methanol, ethanol, isopropanol, n-propanol, n-butanol or tert-butanol; ethers, for example diethyl ether, diisopropyl ether, tetrahydrofuran (THF) or dioxane; glycol ethers, for example ethylene glycol monomethyl or monoethyl ether or ethylene glycol dimethyl ether (diglyme); ketones, for example acetone or butanone; amides, for example acetamide, dimethylacetamide, N-methylpyrrolidone (NMP) or dimethylformamide (DMF); nitriles, for example acetonitrile; sulfoxides, for example dimethyl sulfoxide (DMSO); carbon disulfide; carboxylic acids, for example formic acid or acetic acid; nitro compounds, for example nitromethane or nitrobenzene; esters, for example ethyl acetate, or mixtures of the solvents mentioned.

_Esters can be hydrolyzed, for example, using HCl, _H2SO4 or using LiOH, NaOH or KOH in water, water/THF, water/THF/ethanol or water/dioxane at temperatures between 0°C and 100°C.

Furthermore, free amino groups can be acylated in a customary manner using acyl chlorides or anhydrides or alkylates using unsubstituted or substituted alkyl halides, advantageously in an inert solvent, for example dichloromethane or THF, and/or in the presence of a base, for example triethylamine or pyridine, at temperatures between -60°C and +30°C.

For all protection and deprotection methods, see Philip J. Kocienski, in “Protecting Groups”, Georg Thieme Verlag Stuttgart, New York, 1994, and Theodora W. Greene and Peter G. M. Wuts, in “Protective Groups in Organic Synthesis”, Wiley Interscience, 3rd edition, 1999.

The reaction schemes described in the Examples section are illustrative only and should not be construed as limiting the invention in any way.

use

The present invention further relates to a medicament comprising as active ingredient at least one compound according to the invention or a pharmaceutically acceptable enantiomer, salt, solvate or prodrug thereof or a deuterated form thereof.

In the present invention, the expression "compound of the present invention" encompasses compounds of Formula I and its subformulae or pharmaceutically acceptable enantiomers, salts, solvates and prodrugs or deuterated forms thereof. Examples are identified in Table 1 and in the Examples. Exemplary compounds include:

3-(5-Fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(-)-(R)-3-(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(+)-(S)-3-(5-Fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(1H-indol-3-yl)pyrrolidine-2,5-dione;

(-)-(R)-3-(1H-indol-3-yl)pyrrolidine-2,5-dione;

(+-)-(S)-3-(1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(5-chloro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(-)-(R)-3-(5-chloro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(+)-(S)-3-(5-chloro-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(6-chloro-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(R)-3-(6-chloro-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(S)-3-(6-chloro-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(6-bromo-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(R)-3-(6-bromo-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

(S)-3-(6-bromo-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(5-Bromo-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(5-methyl-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(5-methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(2,5-Dioxopyrrolidin-3-yl)-1H-indole-5-carbonitrile;

3-(5,6-difluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(5-Fluoro-6-methyl-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(6-Fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(6-chloro-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(6-bromo-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(6-methyl-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(6-methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione;

3-(2,5-Dioxopyrrolidin-3-yl)-1H-indole-6-carbonitrile;

3-(Naphthalen-1-yl)pyrrolidine-2,5-dione;

3-(6-Fluoronaphthalen-1-yl)pyrrolidine-2,5-dione;

3-(7-Fluoronaphthalen-1-yl)pyrrolidine-2,5-dione;

3-(6-chloronaphthalen-1-yl)pyrrolidine-2,5-dione; and

3-(7-Chloronaphthalen-1-yl)pyrrolidine-2,5-dione.

Optionally, the compounds of formula I may be deuterated, for example at a chiral center. An exemplary deuterated compound is (3- ² H)-3-(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione.

In one embodiment, the compound has the structure of Formula II: or a pharmaceutically acceptable salt thereof. The compound may be a racemate, wherein each stereoisomer is present in an amount of about 50 mol% (48% to 52%). Alternatively or additionally, a single enantiomer of the compound is used in a pharmaceutical composition. In one embodiment, the enantiomer is characterized by the structure of Formula II':

,

It exists in the form of a free base (non-salt). Optionally, the compound exists in the form of a pharmaceutically acceptable salt or solvate thereof. In another embodiment, the (S)-enantiomer is additionally or alternatively present in the composition. This enantiomer is characterized by the structure of Formula II'':

It is in free base form or, optionally, in salt form. The pharmaceutical composition may contain a mixture of compounds of Formula II' and Formula II". Various ratios of the two compounds may be selected. For example, the ratio may be about 1:1, or the compound of Formula II' may be present at greater than 50%, greater than 95%, greater than 90%, or about 95% to 100%. Similarly, in other compositions, the compound of Formula II' may be present at greater than 50%. The discussion of suitable ratios and molar percentages of enantiomers relative to compounds of Formula I and its subformulas previously described in the specification is hereby incorporated by reference.

The present invention also provides pharmaceutical compositions comprising a compound of the invention or a pharmaceutically acceptable enantiomer, salt, solvate, or prodrug thereof and at least one pharmaceutically acceptable carrier, diluent, excipient, and/or adjuvant. A carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and, in the case of a pharmaceutically acceptable carrier, not deleterious to the recipient thereof in the amounts employed in the pharmaceutical.

According to one embodiment, the present invention also encompasses pharmaceutical compositions containing, in addition to the compounds of the present invention or pharmaceutically acceptable enantiomers, salts, solvates and prodrugs thereof as active ingredients, other therapeutic agents and/or active ingredients.

By way of non-limiting example, the compounds of the invention may be formulated into pharmaceutical preparations in a form suitable for oral administration, parenteral administration (e.g., by intravenous, intramuscular or subcutaneous injection or intravenous infusion), topical administration (including ophthalmic administration), administration by inhalation, skin patch, implant, suppository, etc. Such suitable administration forms - which may be solid, semi-solid or liquid, depending on the mode of administration - as well as the methods and carriers, diluents and excipients used in their preparation will be clear to those skilled in the art; reference is made to the latest edition of Remington's Pharmaceutical Sciences.

Some preferred, but non-limiting examples of such formulations include tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, creams, lotions, soft and hard gelatin capsules, suppositories, drops, sterile injectable solutions and sterile packaged powders (which are normally reconstituted before use) for administration in bolus form and/or for continuous administration, which may be formulated using carriers, excipients and diluents suitable for such formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, polyethylene glycol, cellulose, (sterile) water, methylcellulose, methyl and propyl hydroxybenzoates, talc, magnesium stearate, edible, vegetable and mineral oils, or suitable mixtures thereof. The preparation may optionally contain other substances commonly used in pharmaceutical preparations, such as lubricants, wetting agents, emulsifiers and suspending agents, dispersants, disintegrants, extenders, fillers, preservatives, sweeteners, flavorings, flow regulators, release agents, etc. The composition may also be formulated so as to provide quick, sustained or delayed release of the active compound contained therein.

The pharmaceutical preparations of the present invention are preferably in unit dosage form and may be conveniently packaged in, for example, boxes, blisters, vials, bottles, sachets, ampoules or any other suitable single-dose or multi-dose holders or containers (which may be appropriately labeled); optionally with one or more inserts containing product information and/or instructions for use.

Depending on the condition to be prevented or treated and the route of administration, the active compounds of the invention can be administered in a single daily dose, in one or more divided daily doses or essentially continuously, for example using instillation.

The present invention also relates to the use of a compound of the present invention, or a pharmaceutically acceptable enantiomer, salt, solvate, or prodrug thereof, in the treatment and/or prevention of cancer and endometriosis. In one embodiment, the present invention relates to the use of a compound of the present invention, or a pharmaceutically acceptable enantiomer, salt, solvate, or prodrug thereof, in the treatment and/or prevention of cancer. In another embodiment, the present invention relates to the use of a compound of the present invention, or a pharmaceutically acceptable enantiomer, salt, solvate, or prodrug thereof, in the treatment and/or prevention of endometriosis.

In one embodiment, the compounds of the present invention, or a pharmaceutically acceptable enantiomer, salt, solvate, or prodrug thereof, are used to treat and/or prevent cancer and endometriosis. According to one embodiment, the compounds of the present invention, or a pharmaceutically acceptable enantiomer, salt, solvate, and prodrug thereof, are used to treat and/or prevent cancer. According to another embodiment, the compounds of the present invention, or a pharmaceutically acceptable enantiomer, salt, solvate, and prodrug thereof, are used to treat and/or prevent endometriosis.

The present invention further relates to a method for treating or preventing cancer and endometriosis, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable enantiomer, salt, solvate, or prodrug thereof. In one embodiment, the present invention relates to a method for treating or preventing cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable enantiomer, salt, solvate, or prodrug thereof. In another embodiment, the present invention relates to a method for treating or preventing endometriosis, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable enantiomer, salt, solvate, or prodrug thereof.

In one embodiment, the compounds of the invention, or pharmaceutically acceptable enantiomers, salts, solvates or prodrugs thereof, are used to increase immune recognition and destruction of cancer cells.

The compounds of the present invention are therefore useful as medicaments, inter alia, for the prevention and/or treatment of cancer.

The present invention further provides use of the compound of the present invention or a pharmaceutically acceptable enantiomer, salt, solvate or prodrug thereof for the preparation of a medicament for treating and/or preventing cancer.

Various cancers are known in the art. Cancers can be metastatic or non-metastatic. Cancers can be familial or spontaneous. In some embodiments, the cancer is selected from leukemia and multiple myeloma. In one embodiment, the cancer is leukemia. In one embodiment, the cancer is multiple myeloma.

Other cancers that can be treated using the methods of the present invention include, for example, benign and malignant solid tumors and benign and malignant non-solid tumors. In one embodiment, the cancer is a benign solid tumor. In one embodiment, the cancer is a malignant solid tumor. In one embodiment, the cancer is a benign non-solid tumor. In one embodiment, the cancer is a malignant non-solid tumor.

Examples of solid tumors include, but are not limited to, bile duct cancer, brain cancer (including glioblastoma and medulloblastoma), breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, intraepithelial neoplasia (including Bowen's disease and Paget's disease), liver cancer, lung cancer, neuroblastoma, oral cancer (including squamous cell carcinoma), ovarian cancer (including those derived from epithelial cells, stromal cells, germ cells and mesenchymal cells), pancreatic cancer, prostate cancer, rectal cancer, kidney cancer (including adenocarcinoma and Wilms tumor), sarcomas (including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma), skin cancer (including melanoma, Kaposi's sarcoma, sarcoma), basal cell carcinoma, and squamous cell carcinoma), testicular cancer, including germ cell tumors (seminomas and nonseminomas such as teratomas and choriocarcinomas), stromal tumors, germ cell tumors, and thyroid cancer (including adenocarcinoma and medullary carcinoma).

In one embodiment, the cancer is bile duct cancer. In one embodiment, the cancer is brain cancer, including glioblastoma and medulloblastoma. In one embodiment, the cancer is breast cancer. In one embodiment, the cancer is cervical cancer. In one embodiment, the cancer is choriocarcinoma. In one embodiment, the cancer is colon cancer. In one embodiment, the cancer is endometrial cancer. In one embodiment, the cancer is esophageal cancer. In one embodiment, the cancer is gastric cancer. In one embodiment, the cancer is intraepithelial neoplasia, including Bowen's disease and Paget's disease. In one embodiment, the cancer is liver cancer. In one embodiment, the cancer is lung cancer. In one embodiment, the cancer is neuroblastoma. In one embodiment, the cancer is oral cancer, including squamous cell carcinoma. In one embodiment, the cancer is ovarian cancer, including those derived from epithelial cells, stromal cells, germ cells, and mesenchymal cells. In one embodiment, the cancer is pancreatic cancer. In one embodiment, the cancer is prostate cancer. In one embodiment, the cancer is rectal cancer. In one embodiment, the cancer is kidney cancer, including adenocarcinoma and Wilms' tumor. In one embodiment, the cancer is a sarcoma, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma. In one embodiment, the cancer is skin cancer, including melanoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell carcinoma. In one embodiment, the cancer is testicular cancer, including germinal tissue tumors (seminomas and non-seminomas, such as teratomas and choriocarcinomas). In one embodiment, the cancer is a stromal tumor. In one embodiment, the cancer is a germ cell tumor. In one embodiment, the cancer is thyroid cancer, including thyroid adenocarcinoma and medullary carcinoma.

Examples of non-solid tumors include, but are not limited to, hematological tumors. As used herein, hematological tumors is a term of art that includes lymphoid disorders, myeloid disorders, and AIDS-related leukemias.

Lymphoid disorders include, but are not limited to, acute lymphocytic leukemia and chronic lymphoproliferative disorders (e.g., lymphomas, myelomas, and chronic lymphoid leukemia). Lymphomas include, for example, Hodgkin's disease, non-Hodgkin's lymphomas, and lymphocytic lymphomas. Chronic lymphoid leukemias include, for example, T-cell chronic lymphoid leukemia and B-cell chronic lymphoid leukemia.

In one embodiment, the lymphoid disorder is acute lymphocytic leukemia. In one embodiment, the lymphoid disorder is a chronic lymphoproliferative disorder (e.g., lymphoma, myeloma, and chronic lymphoid leukemia). In one embodiment, the lymphoma is Hodgkin's disease. In one embodiment, the lymphoma is non-Hodgkin's lymphoma. In one embodiment, the lymphoma is a lymphocytic lymphoma. In one embodiment, the chronic lymphoid leukemia is T-cell chronic lymphoid leukemia. In one embodiment, the chronic lymphoid leukemia is B-cell chronic lymphoid leukemia.

The present invention also provides a method of delaying the onset of cancer in an individual, comprising administering to the individual in need thereof a pharmaceutically effective amount of a compound of the present invention or a pharmaceutically acceptable enantiomer, salt, solvate, or prodrug thereof.

The present invention further relates to the use of the compounds of the present invention or pharmaceutically acceptable enantiomers, salts, solvates and prodrugs thereof as IDO1 inhibitors.

Therefore, in a particularly preferred embodiment, the present invention relates to the use of compounds of formula I and subformulae, in particular those in Table 1 above, or pharmaceutically acceptable enantiomers, salts, solvates and prodrugs thereof, as IDO1 inhibitors.

Therefore, in another aspect, the present invention relates to the use of these compounds or their enantiomers, salts, solvates and prodrugs for the synthesis of IDO1 inhibitors.

According to another feature of the invention, there is provided a method of modulating IDO1 activity in a subject in need of such treatment comprising administering to the subject an effective amount of a compound of the invention or a pharmaceutically acceptable enantiomer, salt, solvate or prodrug thereof.

According to another feature of the present invention, there is provided a use of a compound of the present invention or a pharmaceutically acceptable enantiomer, salt, solvate or prodrug thereof for the preparation of a medicament for modulating IDO1 activity in a subject in need of such treatment, the use comprising administering to the subject an effective amount of a compound of the present invention or a pharmaceutically acceptable enantiomer, salt, solvate or prodrug thereof.

definition

In the present invention, the following terms have the following meanings:

In the case where a group is substituted, such group may be substituted by one or more substituents and preferably one, two or three substituents. Substituents may be selected from, but are not limited to, halogen, hydroxy, oxo, nitro, amide, carboxyl, amino, cyano, haloalkoxy and haloalkyl.

The term "halogen" or "halo" means fluorine (F), chlorine (Cl), bromine (Br) or iodine (I). Preferred halogens are fluorine and chlorine.

The term "alkyl" by itself or as part of another substituent refers to a hydrocarbon radical of the formula _{CnH2n +1} , where n is a number greater than or equal to 1. Typically, the alkyl radicals of the present invention comprise 1 to 6 carbon atoms (C1, C2, C3, C4, C5, or C6 carbons, inclusive), preferably 1 to 4 carbon atoms, and more preferably 1 to 3 carbon atoms. Alkyl radicals may be straight or branched and may be substituted as indicated herein. Suitable alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl, pentyl and its isomers (e.g., n-pentyl, isopentyl), and hexyl and its isomers (e.g., n-hexyl, isohexyl). Optionally, the alkyl radical may be substituted with 1, 2, or 3 substituents. The substituents may be hydroxy, amino-, halogen, or C1-C3 alkyl. In one embodiment, the halogen substituent is F or Br. In another embodiment, the alkyl substituent is methyl. The term "alkoxy" refers to any radical O-alkyl.

The term "amino" refers to a _-NH2 group or any group thereof derived by replacing one or two hydrogen atoms with an organic aliphatic or aromatic group. Preferably, the group derived from _-NH2 is an "alkylamino" group, i.e., an N-alkyl group, including monoalkylamino and dialkylamino groups. According to a specific embodiment, the term "amino" refers to _NH2 , NHMe or _NMe2 .

The term "amino protecting group" refers to a protecting group for an amine functional group. According to a preferred embodiment, the amino protecting group is selected from the group comprising arylsulfonyl, tert-butoxycarbonyl, methoxymethyl, p-methoxybenzyl or benzyl.

The term "solvate" is used herein to describe compounds of the present invention that contain stoichiometric or substoichiometric amounts of one or more pharmaceutically acceptable solvent molecules, such as ethanol. When the solvent is water, the term "hydrate" is used.

The compounds of the present invention include compounds of formula I as defined above, including all polymorphs and crystal habits thereof, prodrugs thereof and isotopically labeled compounds of formula I.

The present invention also generally encompasses all pharmaceutically acceptable prodrugs and prodrugs of the compounds of Formula I.

The term "prodrug" as used herein refers to a pharmacologically acceptable derivative of a compound of Formula I, such as an amide, whose in vivo biotransformation product generates a biologically active drug. Prodrugs are generally characterized by increased bioavailability and ease of metabolism in vivo to the biologically active compound.

As used herein, the term "prodrug" means any compound that is modified to form a drug substance, wherein the modification may occur inside or outside the body and before or after the prodrug reaches the area of the body where drug administration is indicated.

The term "subject" refers to a mammal, preferably a human. In one embodiment, the subject may be a "patient," i.e., a warm-blooded animal, more preferably a human, who is awaiting or receiving medical care or was/is/will be the target of a medical procedure, or is being monitored for the development of a disease.

The term "human" refers to humans of both sexes and at any stage of development (ie, newborn, infant, toddler, adolescent, adult).

As used herein, the terms "treat," "treating," and "treatment" are meant to include alleviating, diminishing, or abolishing a condition or disease and/or its attendant symptoms.

As used herein, the terms "prevent," "preventing," and "prevention" refer to methods of: delaying or arresting the onset of a condition or disease and/or its attendant symptoms; protecting a subject from a condition or disease; or reducing a subject's risk of developing a condition or disease.

As used herein, the term "therapeutically effective amount" (or more simply "effective amount") means an amount of an active agent or active ingredient sufficient to achieve the desired therapeutic or prophylactic effect in a subject to which it is administered.

The term "administration" or variations thereof (eg, "administering") means providing an active agent or ingredient, alone or as part of a pharmaceutically acceptable composition, to a subject in which the condition, symptom, or disease is to be treated or prevented.

"Pharmaceutically acceptable" means that the ingredients of the pharmaceutical composition are compatible with each other and not deleterious to the subject to which they are administered.

The term "inhibitor" refers to a natural or synthetic compound that has a biological effect of inhibiting, significantly reducing, or downregulating the expression of a gene and/or protein, or a biological effect of inhibiting or significantly reducing the biological activity of a protein. Thus, an "IDO1 inhibitor" refers to a compound that has a biological effect of inhibiting, significantly reducing, or downregulating the expression of a gene encoding IDO1 and/or the expression of IDO1 and/or the biological activity of IDO1.

"D" and "d" both refer to deuterium. "dx.y" refers to substitution with x to y deuterium atoms. "Stereoisomers" refers to both enantiomers and diastereomers. A group is "substituted" with a substituent when one or more hydrogen atoms of the group are replaced with a corresponding number of substituent atoms (if the substituent is an atom) or groups (if the substituent is a group). For example, "substituted with deuterium" means replacing one or more hydrogen atoms with a corresponding number of deuterium atoms.

The words “comprise,” “comprises,” and “comprising” are to be interpreted in an inclusive manner rather than in an exclusive manner. The words “consist,” “consisting,” and variations thereof are to be interpreted in an exclusive manner rather than in an inclusive manner.

As used herein, unless otherwise specified, the term "about" means a variation of 10% from a given reference value.

Example

The present invention will be better understood with reference to the following examples. These examples are intended to represent specific embodiments of the present invention and are not intended to limit the scope of the invention.

I. Chemical Examples

MS data provided in the Examples described below were obtained as follows: Mass Spectrometry: LC/MS Agilent 6110 (ESI) or Waters Acquity SQD (ESI).

The NMR data provided in the Examples described below were obtained as follows: Bruker Ultrashield™ 400 PLUS and Bruker Fourier 300 MHz using TMS as the internal standard.

Microwave chemistry was performed on a single mode microwave reactor, Initiator Microwave System EU from Biotage.

Unless otherwise reported, preparative HPLC purifications were performed using a mass-directed automated purification Fractionlynx from Waters equipped with an Xbridge ^™ Prep C18 OBD column 19×150 mm 5 μm. All HPLC purifications were performed using gradients of CH ₃ CN/H ₂ O/NH ₄ HCO ₃ (5 mM), CH ₃ CN/H ₂ O/TFA (0.1%) or CH ₃ CN/H ₂ O/NH ₃ H ₂ O (0.1%).

Compound 1: 3-(5-Fluoro- 1H -indol-3-yl)pyrrolidine-2,5-dione

A. Path A

A mixture of 5-fluoro- 1H -indole (300 mg; 2.22 mmol) and maleimide (646 mg; 6.65 mmol) in AcOH (2 mL) was stirred at 170°C in a microwave reaction for 2 h. The reaction mixture was concentrated in _vacuo . The residue was neutralized to pH 7-8 with saturated aqueous NaHCO₃ and extracted with EtOAc ( ₁₀ mL x ₃ ). The combined organic layers were dried over anhydrous _Na₂SO₄ , filtered, concentrated, and purified by preparative HPLC to provide 180 mg (35%) of the title compound as _a yellow solid. LC-MS for _{C₁₂H₆FN₂O₂} ^- _H- [MH]: Calcd: 231.1; Found: 231.0. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ [ppm]: 11.30 (brs, 1H), 11.14 (s, 1H), 7.41 (d, J = 2.5 Hz, 1H), 7.36 (dd, J = 9.0, 4.6 Hz, 1H), 7.20 (dd, J = 10.1, 2.5 Hz, 1H), 6.94 (ddd, J = 9.2, 9.0, 2.5 Hz, 1H), 4.33 (dd, J = 9.5, 5.5 Hz, 1H), 3.17 (dd, J = 18.0,9.5 Hz, 1H), 2.79 (dd, J = 18.0, 5.5 Hz, 1H).

Path B:

Alternatively, a mixture of 5-fluoroindole (5.00 g, 5.00 g, 35.5 mmol, 96 mass %, 1.00) and maleimide (1.5 equivalents, 5.17 g, 53.3 mmol, 1.50) was charged to a 50 mL vessel, and then acetonitrile (3 L/kg, 15.0 mL, 11.7 g, 286 mmol, 100 mass %) and zinc chloride (1.05 equivalents, 5.08 g, 37.3 mmol, 100 mass %) were added. The reaction was heated to 85° C. over 10 minutes and then maintained at 85° C. for 24 hours. While still at 85° C., water (6 L/kg, 30.0 mL, 30.0 g, 1670 mmol, 100 mass %) was slowly added while maintaining the temperature above 80° C. A yellow solid precipitated. The reaction mixture was cooled to 50° C. over 1 hour, then stirred at 50° C. for 2 hours, and then cooled to 10° C. over 1 hour. The reaction was stirred at 10° C. for 1 hour. The solid was filtered off, and the filter cake was washed twice with 5 ml of 1:1 ACN/water to provide the isolated compound (6.85 g, 6.85 g, 29.5 mmol, 83.1% yield).

For purification, the resulting isolated compound (6.85 g, 6.85 g, 29.5 mmol, 100% by mass) was loaded into a vessel, followed by the addition of tetrahydrofuran (6 L/kg, 41.1 mL, 36.4 g, 505 mmol, 100% by mass). This mixture was heated to 66°C to form a homogeneous solution. Heptane (4 L/kg, 27.4 mL, 18.7 g, 187 mmol, 100% by mass) was slowly added at 66°C; after 5 volumes, a solid began to precipitate. The mixture was cooled to 25°C over 3 hours, then filtered and washed with heptane, and then dried in a high vacuum oven overnight. Isolated compound: (4.93 g, 4.93 g, 21.2 mmol, 100% by mass, 72.0% yield).

This isolated compound 2 (1.00 g, 4.3 mmol, 100 mass %) was loaded into a 50 ml vessel and tetrahydrofuran (6 L/kg, 6 mL, 100 mass %) and heptane (6 L/kg, 6 mL, 100 mass %) were added. The slurry was stirred at 25° C. for 48 hours. The solid was filtered and dried in a high vacuum oven overnight. Isolated compound: (0.89 g, 0.89 g, 3.83 mmol, 100 mass %, 89.00% yield).

Compound 1a: (3- ² H)-3-(5-fluoro-1 H -indol-3-yl)pyrrolidine-2,5-dione

To a solution of 3-(5-fluoro-1H-indol-3-yl)-pyrrolidine-2,5-dione (compound 1, 200 mg, 0.87 mmol) in _D₂O (3 mL) was added _{K₂CO₃ (} 300 mg, 2.2 mmol). The reaction was stirred at 40°C overnight. The mixture _was extracted with EtOAc. The organic layer was dried, filtered, concentrated, and purified by preparative HPLC to afford the title compound as a yellow solid (20 mg, 10%). LC-MS for _{C₁₂H₈DFN₂O₂} ^- _H- [MH _] ^- : Calcd: 232.1; Found: 232.1. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ [ppm]: 11.28 (s, 1H), 11.15 (s, 1H), 7.41 (d, J = 2.1 Hz, 1H), 7.36 (dd, J = 8.7, 4.5 Hz, 1H), 7.20 (dd, J = 10.2, 2.4 Hz, 1H), 6.97-6.90 (m, 1H), 3.19-3.13 (m,1H), 2.80-2.74 (m, 1H).

Compound 2: (-)-( R )-3-(5-fluoro- 1H -indol-3-yl)pyrrolidine-2,5-dione

150 mg of compound 1 was separated by chiral preparative HPLC to obtain 50 mg of the title compound as a yellow solid. Preparative chiral HPLC: Chiralpak® AS-H 250 mm × 20 mm 5 μm; mobile phase: _CO₂ /IPA = 60/40; flow rate: 50 mL/min, 214 nm, ambient temperature. Analytical chiral HPLC: Chiralpak® IC 250 mm × _4.6 mm ₅ μm; mobile phase: hexane/EtOH = 70/30; flow rate: 1.0 mL/min, 230 nm, ambient temperature; retention time: 6.25 min. P1: 96.3% ee [α] ²⁵⁴ _D = -75.4 (c = 0.0014, MeOH). LC-MS: calculated for _{C₁₂H₆FN₂O₂} + _H ⁺ [M+H] ⁺ : 233.1; found: 233.1. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ [ppm]: 11.30 (brs, 1H), 11.14(s, 1H), 7.41(d, J = 2.5 Hz, 1H), 7.36 (dd, J = 9.0, 4.6 Hz, 1H), 7.20 (dd, J = 10.1, 2.5 Hz, 1H), 6.94 (ddd, J = 9.2, 9.0, 2.5 Hz, 1H), 4.33 (dd, J = 9.5,5.5 Hz, 1H), 3.17 (dd, J = 18.0, 9.5 Hz, 1H), 2.79 (dd, J = 18.0, 5.5 Hz, 1H).

Compound 2a: (+)-( S )-3-(5-fluoro- 1H -indol-3-yl)pyrrolidine-2,5-dione

Compound 2a was isolated as the second eluting enantiomer from the chiral separation. Chiral HPLC: Retention time: 6.96 min. 98.5% ee [α] ²⁵⁴ _D = 70 (c = 0.0014, MeOH).

Compound 3: 3-( 1H -indol-3-yl)pyrrolidine-2,5-dione

Following the general procedure as outlined for compound 1, starting from 1H -indole (2.00 g; 17.1 mmol) and maleimide (4.96 g; 51.1 mmol), 2.50 g (68%) of _the title compound were obtained _{as a} yellow solid after purification by silica gel chromatography (petroleum ether/EtOAc = 1/1). LC-MS for _C12H10FN2O2 +H ⁺ [M+H] ⁺ : calculated value: 215.1; found value: 215.1. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ [ppm]: 11.29 (s, 1H), 11.02 (s, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.32 (d, J = 2.4 Hz, 1H),7.12-7.07 (m, 1H), 7.02 – 6.97 (m, 1H), 4.33 (dd, J = 9.5, 5.3 Hz, 1H), 3.18(dd, J = 18.0, 9.5 Hz, 1H), 2.76 (dd, J = 18.0, 5.3 Hz, 1H).

Compound 4: (-)-( R )-3-( 1H -indol-3-yl)pyrrolidine-2,5-dione

250 mg of compound 3 was separated by chiral preparative HPLC to obtain 100 mg of the title compound as a yellow solid. Preparative chiral HPLC: Chiralcel OJ-H 250 mm × 4.6 mm 5 μm; mobile phase: _CO₂ /MeOH = 60/40; flow rate: 50 mL/min, 230 nm, ambient temperature. Analytical chiral HPLC: Chiralcel IC 250 mm × _4.6 mm ₅ μm; mobile phase: hexane/EtOH = 70/30; flow rate: 1.0 mL/min, 230 nm, ambient temperature; retention time: 7.632 min. P1: 99.7% ee [α] ²⁵⁴ _D = -64.6 (c = 0.01, MeOH). LC-MS: Calcd for _{C₁₂H₁₀FN₂O₂} + _H ⁺ [M+H] ⁺ : 215.1; Found: 215.1. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ [ppm]: 11.29 (s, 1H), 11.02 (s,1H), 7.42 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.32 (d, J = 2.4 Hz,1H), 7.12-7.07 (m, 1H), 7.02 – 6.97 (m, 1H), 4.33 (dd, J = 9.5, 5.3 Hz, 1H), 3.18 (dd, J = 18.0, 9.5 Hz, 1H), 2.76 (dd, J = 18.0, 5.3 Hz, 1H).

Compound 4a: (+)-( S )-3-( 1H -indol-3-yl)pyrrolidine-2,5-dione

Compound 4a was isolated as the second eluting enantiomer from the chiral separation. Chiral HPLC: Retention time: 9.028 min. 99.6% ee [α] ²⁵⁴ _D = 64.5 (c = 0.01, MeOH).

Compound 5: 3-(5-chloro- 1H -indol-3-yl)pyrrolidine-2,5-dione

Following the general procedure outlined for compound 1, starting from 5-chloro- 1H -indole (2.00 g; 13.2 mmol) and maleimide (3.84 g; 39.6 mmol), 160 mg (4.9%) of the title compound were obtained as a yellow solid after purification by silica gel chromatography (petroleum ether/EtOAc = 3/1). LC-MS for C ₁₂ H ₉ ClN ₂ O ₂ —H ^— [MH] ^— : calculated value: 247.0; found value: 247.0. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ [ppm]: 11.30 (br s, 1H), 11.25 (br s, 1H), 7.49 (d, J = 2.0 Hz, 1H), 7.42 (d, J = 2.0 Hz, 1H), 7.39 (d, J =8.6 Hz, 1H), 7.10 (dd, J = 8.6, 2.0 Hz, 1H), 4.36 (dd, J = 9.5, 5.5 Hz, 1H), 3.17 (dd, J = 18.0, 9.5 Hz, 1H), 2.80 (dd, J = 18.0, 5.5 Hz, 1H).

Compound 6: (-)-( R )-3-(5-chloro- 1H -indol-3-yl)pyrrolidine-2,5-dione

120 mg of compound 5 was separated by chiral preparative HPLC to obtain 25 mg of the title compound. Preparative chiral HPLC: Chiralpak® IC 250 mm × 20 mm 5 μm; mobile phase: hexane/EtOH = 70/30; flow rate: 15 mL/min, 214 nm, ambient temperature. Analytical chiral HPLC: Chiralpak® IC 250 mm × 4.6 mm 5 μm; mobile phase: hexane/EtOH = 70/30; flow rate: 1.0 mL/min, 230 nm, ambient temperature; retention time: 6.073 min. P1: 99.5% ee [α] ²⁵⁴ _D = -69.0 (c = 0.0042, MeOH). LC-MS for _{C 12} H ₉ ClN ₂ O ₂ +H ⁺ [M+H] ^+: Calcd: 249.0; Found: 249.1. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ [ppm]: 11.29 (br s, 1H), 11.25 (br s,1H), 7.49 (d, J = 2.0 Hz, 1H), 7.42 (d, J = 2.4 Hz, 1H), 7.39 (d, J = 8.6 Hz,1H), 7.10 (dd, J = 8.6, 2.0 Hz, 1H), 4.36 (dd, J = 9.5, 5.5 Hz, 1H), 3.17 (dd, J = 18.0, 9.5 Hz, 1H), 2.80 (dd, J = 18.0, 5.5 Hz, 1H).

Compound 6a: (+)-( S )-3-(5-chloro- 1H -indol-3-yl)pyrrolidine-2,5-dione

Compound 6a was isolated as the second eluting enantiomer from the chiral separation. Chiral HPLC: Retention time: 6.868 min. P1: 99.6% ee [α] ²⁵⁴ _D = 67.4 (c = 0.0038, MeOH).

Compound 7: 3-(6-chloro-5-fluoro- 1H -indol-3-yl)pyrrolidine-2,5-dione

Following the general procedure as outlined for compound 1, starting from 6-chloro-5-fluoro- 1H -indole (300 mg; 1.77 mmol) and maleimide (513 mg; 5.28 mmol), 110 mg (23%) of the title compound were obtained _{as a yellow solid after purification by preparative HPLC. LC-MS for C12H8ClFN2O2 - H-} ^[ _MH ] ^- : calculated: 265.1; found: 265.0. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ [ppm]: 11.30 (br s, 1H), 11.27 (br s, 1H), 7.54 (d, J = 6.4 Hz, 1H), 7.47 (s, 1H), 7.46 (d, J = 10.2 Hz, 1H), 4.35 (dd, J = 9.4, 5.8 Hz, 1H), 3.16 (dd, J = 18.0, 9.4 Hz, 1H), 2.81 (dd, J = 18.0, 5.8 Hz, 1H).

Compound 8: ( R )-3-(6-chloro-5-fluoro- 1H -indol-3-yl)pyrrolidine-2,5-dione

70 mg of compound 7 was separated by chiral preparative HPLC to obtain 25 mg of the title compound. Preparative chiral HPLC: Chiralpak® AS-H 250 mm × 20 mm 5 μm; mobile phase: _CO₂ /IPA = 60/40; flow rate: 50 mL/min, 220 nm, ambient temperature. Analytical chiral HPLC: Chiralpak® IA 250 mm × 4.6 mm 5 μm; mobile phase: _CO₂ /IPA/DEA = 70/30/0.2; flow rate: 1.0 mL/min, 230 nm, ambient temperature; retention time: 3.72 min _. P1: >99.5% ee LC-MS: Calcd: 265.1 for _{C₁₂H₈ClFN₂O₂ - H-} ^[ MH] ^- ; Found: 265.1. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ [ppm]: 11.30 (br s, 1H), 11.27 (br s, 1H), 7.54 (d, J = 6.4 Hz, 1H), 7.47 (s, 1H), 7.46 (d, J = 10.2 Hz, 1H), 4.35 (dd, J = 9.4, 5.8 Hz, 1H), 3.16 (dd, J = 18.0, 9.4 Hz, 1H), 2.81 (dd, J = 18.0, 5.8 Hz, 1H).

Compound 8a: ( S )-3-(6-chloro-5-fluoro- 1H -indol-3-yl)pyrrolidine-2,5-dione

Compound 8a was isolated as the second eluting enantiomer from the chiral separation. Chiral HPLC: Retention time: 5.48 min. 99.6% ee

Compound 9: 3-(6-bromo-5-fluoro- 1H -indol-3-yl)pyrrolidine-2,5-dione

Following the general procedure as outlined for compound 1, starting from 6-bromo-5-fluoro- 1H -indole (213 mg; 1.00 mmol) and maleimide (388 mg; 4.00 mmol), 70 mg (23%) of the title compound were obtained as _{a yellow solid after purification by preparative HPLC. LC-MS for C12H8BrFN2O2 -} ^H- _[ MH _] ^- : calculated: 309.0; found: 308.9. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ [ppm]: 11.31 (s, 1H), 11.27 (s, 1H), 7.66 (d, J =6.0 Hz, 1H), 7.48 (d, J = 1.7 Hz, 1H), 7.44 (d, J = 9.8 Hz, 1H), 4.36 (dd, J = 9.2, 5.6 Hz, 1H), 3.17 (dd, J = 18.0, 9.2 Hz, 1H), 2.82 (dd, J = 18.0, 5.6Hz, 1H).

Compound 10: ( R )-3-(6-bromo-5-fluoro- 1H -indol-3-yl)pyrrolidine-2,5-dione

60 mg of compound 9 was separated by chiral preparative HPLC to obtain 22 mg of the title compound. Preparative chiral HPLC: Chiralpak® AD-H 250 mm × 20 mm 5 μm; mobile phase: _CO₂ /MeOH = 60/40; flow rate: 50 mL/min, 214 nm, ambient temperature. Analytical chiral HPLC: Chiralpak® ID 250 mm × 4.6 mm 5 μm; mobile phase: _CO₂ /MeOH = 60/40; flow rate: 3.0 mL/min, 230 nm, ambient temperature; retention time: 2.14 min _{. P1: >99.5% ee. LC-MS: Calcd: 309.0 for C₁₂H₈BrFN₂O₂ -} H- _[ ^MH _] ^- ; Found: 308.8. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ [ppm]: 11.31 (s, 1H), 11.27 (s, 1H), 7.66 (d, J = 6.0 Hz, 1H), 7.48 (d, J = 1.7 Hz, 1H), 7.44 (d, J = 9.8 Hz, 1H), 4.36 (dd, J = 9.2, 5.6 Hz, 1H), 3.17 (dd, J = 18.0, 9.2 Hz, 1H), 2.82 (dd, J = 18.0, 5.6 Hz, 1H).

Compound 10a: ( S )-3-(6-bromo-5-fluoro- 1H -indol-3-yl)pyrrolidine-2,5-dione

Compound 10a was isolated as the second eluting enantiomer from the chiral separation. Chiral HPLC: Retention time: 4.20 min. 98.9% ee

Compound 11: 3-(5-bromo- 1H -indol-3-yl)pyrrolidine-2,5-dione

Following the general procedure as outlined for compound 1, starting from 5-bromo- 1H -indole (500 mg; 2.56 mmol) and maleimide (666 mg; 6.86 mmol), 160 mg (21%) of _the title compound were obtained as _a yellow solid after purification by preparative HPLC. LC-MS for _C12H9BrN2O2 +H ⁺ [M+H] ⁺ : calculated: 293.0; found: _293.0 . ¹ H NMR (400 MHz, DMSO- d ₆ ) δ [ppm]: 11.29 (s, 1H), 11.26 (s, 1H), 7.64 (d, J = 1.8Hz, 1H), 7.40 (d, J = 2.4 Hz, 1H), 7.35 (d, J = 8.6 Hz, 1H), 7.21 (dd, J =8.6, 1.8 Hz, 1H), 4.36 (dd, J = 9.5, 5.5 Hz, 1H), 3.17 (dd, J = 18.0, 9.5 Hz, 1H), 2.80 (dd, J = 18.0, 5.5 Hz, 1H).

Compound 12: 3-(5-methyl- 1H -indol-3-yl)pyrrolidine-2,5-dione

Following the general procedure as outlined for compound 1, starting from 5-methyl- 1H -indole (300 mg; 2.29 mmol) and maleimide (670 mg; 6.87 mmol), 200 mg (38%) of the title compound were obtained as _a yellow solid after _{recrystallization in MeOH. LC-MS for C13H12N2O2 +} H ⁺ [M+H] ^+: Calcd: 229.1; Found: _229.1 . ¹ H NMR (400MHz, DMSO- d ₆ ) δ [ppm]: 11.27 (s, 1H), 10.88 (s, 1H), 7.26 (dd, J = 8.3, 2.0Hz), 7.25 (d, J = 2.0 Hz, 1H), 7.19 (s, 1H), 6.92 (d, J = 8.3 Hz, 1H), 4.29 (dd, J = 9.5, 5.3 Hz, 1H), 3.16 (dd, J = 18.0, 9.5 Hz, 1H), 2.74 (dd, J =18.0, 5.3 Hz, 1H), 2.36 (s, 3H).

Compound 13: 3-(5-methoxy- 1H -indol-3-yl)pyrrolidine-2,5-dione

Following the general procedure as outlined for compound 1, starting from 5-methoxy- 1H -indole (200 mg; 1.36 mmol) and maleimide (407 mg; 4.19 mmol), 170 mg (51%) _{of the title compound were obtained as a yellow solid after purification by preparative HPLC. LC-MS for C13H12N2O3 + H +} ^[ M+H] ⁺ : calculated: 245.1; found: 245.1. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ [ppm]: 11.25 (brs, 1H), 10.86 (s, 1H), 7.27 (d, J = 2.2 Hz 1H), 7.26 (d, J = 8.6 Hz, 1H), 6.91 (d, J = 2.2 Hz, 1H), 6.76 (dd, J = 8.6, 2.2 Hz, 1H), 4.30 (dd, J = 9.6, 5.3 Hz, 1H), 3.74 (s, 3H), 3.18 (dd, J = 17.9, 9.6 Hz, 1H), 2.75 (dd, J = 17.9, 5.3 Hz, 1H).

Compound 14: 3-(2,5-dioxopyrrolidin-3-yl) -1H -indole-5-carbonitrile

A mixture of 3-(5-bromo- 1H -indol-3-yl)pyrrolidine-2,5-dione (compound 11; 500 mg; 1.71 mmol) and CuCN (231 mg; 2.58 mmol) in NMP (3 mL) was stirred at 200° C. in a microwave reactor for 1.5 h. The reaction mixture was purified by preparative HPLC to provide 110 mg (27%) of the title compound as a green solid. LC-MS for C ₁₃ H ₉ N ₃ O ₂ +H ⁺ [M+H] ^+: Calcd: 240.1; Found: 240.1. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ[ppm]: 11.63 (brs, 1H), 8.04 (s, 1H), 7.57 (d, J = 1.8 Hz, 1H), 7.54 (d, J =8.8 Hz, 1H), 7.45 (dd, J = 8.6, 1.8 Hz, 1H), 4.44 (dd, J = 9.5, 5.8 Hz, 1H), 3.18 (dd, J = 17.8, 9.5 Hz, 1H), 2.87 (dd, J = 17.8, 5.8 Hz, 1H).

Compound 15: 3-(5,6-difluoro- 1H -indol-3-yl)pyrrolidine-2,5-dione

Following the general procedure as outlined for compound 1, starting from 5,6-difluoro- 1H -indole (200 mg; 1.31 mmol) and maleimide (380 mg; 3.91 mmol), 15 mg (5%) _{of the title compound were obtained as a yellow solid after purification by preparative HPLC. LC-MS for C12H8F2N2O2 + H + [} ^M +H] ^+: calcd: 251.1; found: 251.0. ¹ HNMR (300 MHz, DMSO- d ₆ ) δ [ppm]: 11.27 (brs, 1H), 11.21 (brs, 1H), 7.45 (dd, J = 11.5, 8.0 Hz, 1H), 7.41 (d, J = 1.8 Hz, 1H), 7.37 (dd, J = 11.2, 7.0 Hz, 1H), 7.48-7.34 (m, 3H), 4.34 (dd, J = 9.3, 5.6 Hz, 1H), 3.16 (dd, J = 18.0, 9.3 Hz, 1H), 2.80 (dd, J = 18.0, 5.6 Hz, 1H).

Compound 16: 3-(5-Fluoro-6-methyl- 1H -indol-3-yl)pyrrolidine-2,5-dione

Following the general procedure as outlined for compound 1, starting from 5-fluoro-6-methyl- 1H -indole (1.00 g; 6.70 mmol) and maleimide (2.10 g; 21.6 mmol), 42 mg (0.2%) of the title compound were obtained _{as a} yellow solid after purification by preparative HPLC. LC-MS for _C13H11FN2O2 +H ⁺ [M+H] ⁺ : calculated: 247.1; found: _247.1 . ¹ HNMR (300 MHz, DMSO- d ₆ ) δ [ppm]: 11.28 (s, 1H), 10.99 (s, 1H), 7.31 (d, J =2.5 Hz, 1H), 7.22 (d, J = 6.4 Hz, 1H), 7.13 (d, J = 10.8 Hz, 1H), 4.29 (dd, J = 9.4, 5.4 Hz, 1H), 3.16 (dd, J = 18.0, 9.4 Hz, 1H), 2.76 (dd, J = 18.0, 5.4Hz, 1H), 2.30 (d, , J = 1.6 Hz, 3H).

Compound 17: 3-(6-Fluoro- 1H -indol-3-yl)pyrrolidine-2,5-dione

Following the general procedure outlined for compound 1, starting from 6-fluoro- 1H -indole (4.00 g; 29.6 mmol) and maleimide (8.80 g; 90.7 mmol), 3.0 g (44%) of the title compound was obtained as an orange solid after purification by silica gel chromatography (petroleum ether/EtOAc = 3/1-2/3). LC-MS for _{C 12} H ₉ FN ₂ O ₂ —H ^— [MH] ^— : calculated value: 231.1; found value: 231.1. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ [ppm]: 11.10 (s, 1H), 7.43 (dd, J = 8.7, 5.4 Hz, 1H), 7.33 (d, J = 2.0 Hz, 1H), 7.14 (dd, J = 10.1, 2.3 Hz, 1H), 6.87 (td, J = 9.8, 8.7, 2.3 Hz, 1H), 4.34 (dd, J = 9.5, 5.4 Hz, 1H), 3.17 (dd, J = 18.0, 9.5 Hz, 1H), 2.77 (dd, J = 18.0, 5.4 Hz, 1H).

Compound 18: 3-(6-chloro- 1H -indol-3-yl)pyrrolidine-2,5-dione

Following the general procedure as outlined for compound 1, starting from 6-chloro- 1H -indole (0.50 g; 3.3 mmol) and maleimide (0.96 g; 9.9 mmol), 100 mg (12%) of the title compound was obtained as a yellow solid after purification by silica gel chromatography (petroleum ether/EtOAc = 5/1). LC-MS for C ₁₂ H ₉ ClN ₂ O ₂ —H ^— [MH] ^— : calculated value: 247.0; found value: 247.0. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ [ppm]: 11.27 (brs, 1H), 11.17 (s, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 1.8 Hz, 1H), 7.38 (d, J = 2.4 Hz, 1H), 7.03 (dd, J = 8.4, 1.8 Hz, 1H), 4.34 (dd, J = 9.5, 5.5 Hz, 1H), 3.17 (dd, J =18.0, 9.5 Hz, 1H), 2.77 (dd, J = 18.0, 5.5 Hz, 1H).

Compound 19: 3-(6-Bromo- 1H -indol-3-yl)pyrrolidine-2,5-dione

Following the general procedure as outlined for compound 1, starting from 6-bromo- 1H -indole (2.00 g; 10.2 mmol) and maleimide (2.96 g; 30.5 mmol), 1.5 g (50%) of the title compound was obtained as _a yellow solid after purification by preparative _HPLC . LC-MS for _C12H9BrN2O2 +H ⁺ [M+H] ⁺ : calculated: 293.0; found: _293.0 . ¹ H NMR (300 MHz, DMSO- d ₆ ) δ [ppm]: 11.30 (brs, 1H), 11.18 (s, 1H), 7.56 (d, J = 1.6Hz, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.37 (d, J = 2.4 Hz, 1H), 7.14 (dd, J =8.5, 1.7 Hz, 1H), 4.34 (dd, J = 9.5, 5.4 Hz, 1H), 3.17 (dd, J = 18.0, 9.5 Hz, 1H), 2.77 (dd, J = 18.0, 5.4 Hz, 1H).

Compound 20: 3-(6-methyl- 1H -indol-3-yl)pyrrolidine-2,5-dione

Following the general procedure as outlined for compound 1, starting from 6-methyl- 1H -indole (0.20 g; 1.52 mmol) and maleimide (0.44 g; 4.53 mmol), 0.22 g (63%) of _the title compound was obtained as a yellow solid after purification by preparative HPLC. LC-MS for _C13H12N2O2 ^- _H- [MH _] ^- : calculated: 227.1; found: 227.1. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ [ppm]: 10.85 (brs, 2H), 11.18 (s, 1H), 7.28 (d, J = 8.0Hz, 1H), 7.20 (d, J = 2.3 Hz, 1H), 7.16 (s, 1H), 6.83 (d, J = 8.0 Hz, 1H), 4.28 (dd, J = 9.5, 5.3 Hz, 1H), 3.17 (dd, J = 18.0, 9.5 Hz, 1H), 2.73 (dd, J = 18.0, 5.3 Hz, 1H), 2.38 (s, 3H).

Compound 21: 3-(6-methoxy- 1H -indol-3-yl)pyrrolidine-2,5-dione

Following the general procedure as outlined for compound 1, starting from 6-methoxy- 1H -indole (0.20 g; 1.36 mmol) and maleimide (0.40 g; 4.12 mmol), 80 mg (24%) of _the title compound were obtained as a yellow solid after purification by preparative HPLC. LC-MS for _C13H12N2O3 ^- _H- [MH _] ^- : calculated: 243.1; found: 243.1. ¹ HNMR (400 MHz, DMSO- d ₆ ) δ [ppm]: 11.26 (s, 1H), 10.81 (s, 1H), 7.29 (d, J =8.7 Hz, 1H), 7.16 (d, J = 2.2 Hz, 1H), 6.86 (d, J = 2.2 Hz, 1H), 6.66 (dd, J = 8.7, 2.2 Hz, 1H), 4.27 (dd, J = 9.5, 5.2 Hz, 1H), 3.75 (s, 3H), 3.16 (dd, J = 18.0, 9.5 Hz, 1H), 2.73 (dd, J = 18.0, 5.2 Hz, 1H).

Compound 22: 3-(2,5-dioxopyrrolidin-3-yl) -1H -indole-6-carbonitrile

Following the general procedure as outlined for compound 14, starting from 3-(6-bromo- 1H -indol-3-yl)pyrrolidine-2,5-dione (compound 19; 0.20 g; 0.68 mmol) and CuCN (90 mg; _1.00 mmol), 14 mg (8.6%) _{of the title compound were obtained as a yellow solid after purification by preparative HPLC. LC-MS for C13H9N3O2 + H} ⁺ [M+H] ⁺ : calculated: 240.1; found: 240.1. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ [ppm]: 11.63 (brs, 1H), 11.32(s, 1H), 7.88 (s, 1H), 7.68 – 7.62 (m, 2H), 7.35 (dd, J = 9.5, 5.6 Hz, 1H), 4.42 (dd, J = 17.8, 9.5 Hz, 1H), 3.18 (dd, J = 18.0, 9.9 Hz, 1H), 2.82 (dd, J = 17.8, 5.6 Hz, 1H).

Compound 23: 3-(Naphthalen-1-yl)pyrrolidine-2,5-dione

To a solution of 1-naphthaleneboronic acid (0.27 g; 1.57 mmol) in 1,4-dioxane (9 mL) and water (1.4 mL) was added _Et₃N (0.10 g; 0.99 mmol), [RhOH(cod)] _₂ (23 mg; 0.05 mmol), and maleimide (100 mg; 1.03 mmol). The dark brown mixture was heated at ₅₀ °C for 2.5 h, cooled to room temperature, and concentrated in vacuo. The residue was diluted with _H₂O (10 mL) and extracted with DCM (20 mL x 3). The combined organic layers were dried _over anhydrous _Na₂SO₄ , filtered, concentrated, and purified by preparative HPLC to afford 136 mg (59%) of the title compound as a white solid. LC-MS for _{C₁₄H₁₁NO₂} ^- _H- [MH] ^- : Calcd: 224.1; Found: 224.1. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ [ppm]:11.50 (s, 1H), 8.02-7.95 (m, 2H), 7.89 (d, J = 9.1 Hz, 1H), 7.63 – 7.53 (m,2H), 7.53 – 7.46 (m, 1H), 7.41 (d, J = 7.1 Hz, 1H), 4.96 (dd, J = 9.6, 5.7Hz, 1H), 3.32 (dd, J = 18.0, 9.6 Hz, 1H), 2.71 (dd, J = 18.0, 5.7 Hz, 1H).

Compound 24: 3-(6-Fluoronaphthalen-1-yl)pyrrolidine-2,5-dione

Step 1: 6-Fluoronaphthalene-1-diazonium tetrafluoroborate

To a solution of 6-fluoronaphthalen-1-amine (500 mg; 3.10 mmol) and _HBF4 (40%; 2 mL; 12.6 mmol) in _H2O (2 mL) at 0°C was added a cold solution of _NaNO2 (214 mg; 3.10 mmol) in _H2O (0.5 mL) dropwise. The reaction was stirred at room temperature for 1 h. The precipitate was collected by filtration, washed with EtOH (5 mL), _Et2O (5 mL), and dried under vacuum to afford 0.40 g (50%) of the title compound as an off-white solid, which was used directly in the next step without further purification.

Step 2: 2-(6-Fluoronaphthalen-1-yl)succinic acid

Maleic anhydride (150 mg; 1.54 mmol) was added to an aqueous NaOH solution (4 M; 0.70 mL; 2.8 mmol). The resulting solution was added to _an aqueous TiCl solution (15%; 3.2 g; 3.11 mmol) at 0-5°C, followed by acetone (2 mL). The cooling bath was removed and 6-fluoronaphthalene-1-diazonium tetrafluoroborate (Step 1: 400 mg; 1.54 mmol) was slowly added over 0.7 h. The suspension was stirred at room temperature for 1.5 h, concentrated to remove the acetone, and extracted with _Et2O (10 mL x 3). The aqueous layer was acidified to pH~1 with HCl (1 M) and extracted with EtOAc (10 mL x 3). The combined organic layers were dried over anhydrous _Na2SO4 , filtered, and concentrated to provide 190 mg (47%) _of the title compound as a brown solid, which was used directly in the next step without further purification. LC-MS calculated _{for C14H11FO4+NH4 + [} ^M + _{NH4 ]} ⁺ : 280.1; found: 280.0.

Step 3:

A mixture of 2-(6-fluoronaphthalen-1-yl)succinic acid (190 mg; 0.72 mmol) and urea (170 mg; 2.83 mmol) was stirred at 180°C for 1 h. The reaction mixture was purified by silica gel chromatography (petroleum ether/EtOAc = 1/1) to give a yellow solid, which was further purified by preparative HPLC to provide 63 mg ( ₃₆ %) of the title compound as _{a white solid. LC-MS for C14H10FNO2 +} H ⁺ [M+H] ⁺ : Calcd: 244.1; Found: 243.9. ¹ H NMR (300 MHz, DMSO- d ₆ ) δ [ppm]:8.08 (dd, J = 9.3, 5.6 Hz, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.76 (dd, J = 10.2,2.7 Hz, 1H), 7.56 – 7.46 (m, 2H), 7.38 (d, J = 6.6 Hz, 1H), 4.95 (dd, J =9.4, 5.6 Hz, 1H), 3.30 (dd, J = 18.0, 9.4 Hz, 1H), 2.71 (dd, J = 18.0, 5.6Hz, 1H).

Compound 25: 3-(7-Fluoronaphthalen-1-yl)pyrrolidine-2,5-dione

Step 1: 7-Fluoronaphthalene-1-diazonium tetrafluoroborate

Following the general procedure as outlined for compound 24 - Step 1, starting from 7-fluoronaphthalen-1-amine (300 mg; 1.86 mmol), _HBF4 (40%; 1.5 mL; 9.4 mmol), _H2O (5 mL), _NaNO2 (260 mg; 3.77 mmol) in _H2O (4 mL) _, 300 mg (62%) of the title compound was obtained as an off-white solid, which was used directly in the next step without further purification. LC-MS for _C10H6FN2 ⁺ [M _] ⁺ : Calcd: 173.1; Found: 173.0.

Step 2: 2-(7-Fluoronaphthalen-1-yl)succinic acid

Following the general procedure as outlined for compound 24 - Step 2, starting from maleic anhydride (110 mg; 1.12 mmol), aqueous NaOH (4 M; 0.7 mL; 2.8 mmol), aqueous TiCl ( ₁₅ %; 2.36 g; 2.32 mmol), acetone (2 mL) and 7-fluoronaphthalene-1-diazonium tetrafluoroborate (Step 1: 300 mg; 1.15 mmol), 200 mg (66%) of the title compound were obtained as a brown solid, which was used directly in the next step without further purification.

Step 3:

Following the general procedure as outlined for compound 24 - Step 3, starting from 2-(7-fluoronaphthalen-1-yl)succinic acid (Step 2; 200 mg; 0.76 mmol) and urea (180 mg; 3.00 mmol), 3.3 mg (1.8%) of the title compound was obtained as a white solid after purification by silica gel chromatography (petroleum ether/EtOAc = 1/1) and preparative HPLC. LC-MS for C ₁₄ H ₁₀ FNO ₂ —H ^— [MH] ^— : calculated value: 242.1; found value: 242.0. ¹ H NMR (300 MHz, MeOH- d ₄ ) δ [ppm]:7.99 (dd, J = 9.0, 5.9 Hz, 1H), 7.88 (dd, J = 6.8, 2.0 Hz, 1H), 7.70 (d, J =11.1, 2.0 Hz, 2H), 7.50-7.42 (m, 1H), 7.41 – 7.32 (m, 1H), 4.88 (dd, J = 9.5,5.1 Hz, 1H), 3.43 (dd, J = 18.2, 9.5 Hz, 1H), 2.72 (dd, J = 18.2, 5.1 Hz, 1H).

Compound 26: 3-(6-chloronaphthalen-1-yl)pyrrolidine-2,5-dione

Step 1: 6-Chloronaphthalene-1-diazonium tetrafluoroborate

Following the general procedure as outlined for compound 24 - Step 1, starting from a solution of 6-chloronaphthalen-1-amine (1.00 g; 5.63 mmol), _HBF4 (40%; 4 mL; 25.2 mmol), _H2O (4 _mL ), and _NaNO2 (390 mg; 5.65 mmol) in _H2O (1 mL), 1.50 g (96% _{) of the title compound was obtained as a purple solid, which was used directly in the next step without further purification. LC-MS for C10H6ClN2 +} ^[ M] ⁺ : Calcd: 189.0; Found: 188.9.

Step 2: 2-(6-chloronaphthalen-1-yl)succinic acid

Following the general procedure as outlined for compound 24 - Step 2, starting from maleic anhydride (216 mg; 2.20 mmol), aqueous NaOH (4 M; 6.0 mL; 24 mmol), aqueous TiCl ( ₁₅ %; 4.5 g; 4.4 mmol), acetone (2 mL) and 6-chloronaphthalene-1-diazonium tetrafluoroborate (Step 1: 600 mg; 2.17 mmol), 600 mg (99%) of the title compound were obtained as a black solid, which was used directly in the next step without further purification.

Step 3:

Following the general procedure as outlined for compound 24 - Step 3, starting from 2-(6-chloronaphthalen-1-yl)succinic acid (Step 2; 600 mg; 2.15 mmol) and urea (600 mg; 9.99 mmol), 10 mg (2%) of the title compound was obtained as a yellow solid after purification by silica gel chromatography (petroleum ether/EtOAc = 2/1) and preparative HPLC. LC-MS for C ₁₄ H ₁₀ ClNO ₂ —H ^— [MH] ^— : calculated value: 258.0; found value: 257.9. ¹ H NMR (400 MHz, MeOH- d ₄ ) δ [ppm]: 8.02(d, J = 9.0 Hz, 1H), 7.96 (d, J = 1.8 Hz, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.57– 7.49 (m, 2H), 7.42 (d, J = 7.3 Hz, 1H), 4.96 (dd, J = 9.8, 5.3 Hz, 1H), 3.44 (dd, J = 18.3, 9.8 Hz, 1H), 2.77 (dd, J = 18.2, 5.3 Hz, 1H).

Compound 27: 3-(7-chloronaphthalen-1-yl)pyrrolidine-2,5-dione

Step 1: 7-Chloronaphthalene-1-diazonium tetrafluoroborate

Following the general procedure as outlined for compound 24 - Step 1, starting from 7-chloronaphthalen-1-amine (0.45 g; 2.53 mmol), _HBF4 (40%; 2.5 mL; 15.7 mmol), _H2O (2 mL) and _NaNO2 (190 mg; 2.75 mmol) in _H2O (4 mL) _, 400 mg (57%) of the title compound was obtained as an _{off-white solid, which was used directly in the next step without further purification. LC-MS for C10H6ClN2 +} ^[ M] ⁺ : Calcd: 189.0; Found: 188.9.

Step 2: 2-(7-chloronaphthalen-1-yl)succinic acid

Following the general procedure as outlined for compound 24 - Step 2, starting from maleic anhydride (213 mg; 2.17 mmol), aqueous NaOH solution (4 M; 0.7 mL; 2.8 mmol), _aqueous TiCl solution (15%; 4.46 g; 4.3 mmol), acetone (2 mL) and 7-chloronaphthalene-1-diazonium tetrafluoroborate (Step 1: 600 mg; 2.17 mmol), 500 mg (83%) of the title compound was obtained as a brown solid, which was used directly in the next step without further purification.

Step 3:

Following the general procedure as outlined for compound 24 - Step 3, starting from 2-(7-chloronaphthalen-1-yl)succinic acid (Step 2; 500 mg; 1.79 mmol) and urea (430 mg; 7.16 mmol), 2.5 mg (0.5%) of the title compound was obtained as a white solid after purification by silica gel chromatography (petroleum ether/EtOAc = 1/1) and preparative HPLC. LC-MS for C ₁₄ H ₁₀ ClNO ₂ +H ⁺ [M+H] ⁺ : calculated value: 260.0; found value: 260.0. ¹ H NMR (300 MHz, MeOH- d ₄ ) δ [ppm]:8.08 (s, 1H), 7.95 (d, J = 8.7 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.59 – 7.53(m, 3H), 4.94 (dd, J = 9.6, 5.4 Hz, 1H), 3.44 (dd, J = 18.3, 9.6 Hz, 1H), 2.75 (dd, J = 18.3, 5.4 Hz, 1H).

II. Biological Examples

II.1. Assay for IDO1 enzyme activity determination

The compounds of the present invention inhibit the enzymatic activity of human IDO1.

To measure human IDO1 enzyme activity, the reaction mixture contained (final concentration) potassium phosphate buffer (50 mM, pH 6.5), ascorbic acid (10 mM), methylene blue (5 μM), and human recombinant IDO1 enzyme (prepared as described in Rohrig et al., J Med Chem, 2012, 55, 5270-5290; final concentration 5 μg/mL) without or with the indicated concentrations of the compounds of the invention (total volume 112.5 μL). The reaction was initiated by adding 37.5 μL of L-Trp (final concentration 100 μM) at room temperature. The reaction was carried out at room temperature for 15 minutes and terminated by adding 30 μL of 30% (w/v) trichloroacetic acid.

To convert N -formylkynurenine to kynurenine, the reaction mixture was incubated at 65°C for 30 min. Then, 120 μL of 2.5% (w/v) 4-(dimethylamino)-benzaldehyde in acetic acid was added, and the mixture was incubated at room temperature for 5 min. Kynurenine concentration was determined by measuring absorbance at 480 nm. A standard curve was constructed using pure kynurenine. IDO1 activity was measured using 10 consecutive concentrations of the compounds of the invention as described above. Data were fitted using Prism software (GraphPad Software, Inc.).

The biological activities of representative compounds are summarized in the table below:

Compound 1 0.15 1a 0.21 2 0.12 2a >50 3 3.0 4 1.8 4a >50 5 2.1 6 2.2 6a >50 7 0.49 9 0.29 10 0.62 10a 8.0 11 0.37 12 53 13 53 14 12 15 1.8 16 46 17 3.4 18 2.1 19 0.42 20 54 twenty two 1.7 twenty three 18 twenty four 1.7 25 4.6

In one embodiment, it is generally desirable to select compounds with an IC50 below 5 μM for further study.

II.2.A Cell-based assay for IDO activity determination: hIDO1 P815 cells

The compounds of the present invention inhibit the activity of human IDO in hIDO1 P815 cells [(ATCC® TIB-64™), Mus musculus mastocytoma cells, available from American Type Culture Collection (ATCC), Manassas VA].

The assay was performed in 96-well flat-bottom plates seeded with P815 cells overexpressing hIDO1 (prepared as described in Rohrig et al., J Med Chem, 2012, 55, 5270-5290) at a concentration of 2 × 10 ⁵ cells/well in a final volume of 200 μL. To determine IDO1 activity, cells were incubated in IMDM (Invitrogen) supplemented with 2% FBS and 2% penicillin/streptomycin for 24 hours at 37°C, 5% CO ₂ in the presence of various concentrations of the compounds of the invention.

The plate was then centrifuged at 1000 rpm for 5 min, and 100 μL of supernatant was collected in a conical plate, 30 uL of TCA 30% was added, and the supernatant was further centrifuged at 3000 x g for 10 minutes. 100 μL of supernatant was collected in a flat-bottomed plate and incubated in 100 μL of 2% (w/v) 4-(dimethylamino)-benzaldehyde in acetic acid at room temperature for 5 min. Kynurenine concentration was determined by measuring absorbance at 480 nm. A standard curve was drawn using pure kynurenine. IDO1 activity was measured using 10 different concentrations of the compounds of the invention as described above. Data were fitted using Prism software (GraphPad Software, Inc.).

The biological activities of representative compounds are summarized in the table below:

Compound 1 0.094 2 0.009 2a 0.45 3 0.92 4 0.24 4a 3.30 5 0.59 15 0.26 18 0.50

In one embodiment, it is generally desirable to select compounds with an IC50 below 5 μM for further study.

II.2.B Cell-based assay for IDO1 activity determination: HeLa cells

The compounds of the present invention inhibit the activity of human IDO1 in HeLa cells [human adenocarcinoma cells, ® CCL-2™].

The assay was performed in 96-well flat-bottom plates seeded with the human cervical cancer HeLa cell line and stimulated with IFNγ.

For adhesion, HeLa cells (at a concentration of 5 × 10 ³ cells/well) were incubated overnight at 37°C, 5% CO ₂ in EMEM (Lonza) supplemented with 10% FBS, 2% penicillin/streptomycin, and 2 mM Ultraglutamin in a final volume of 200 μL.

To stimulate IDO1 expression, cells were then incubated for _{two days} at 37°C, 5% CO2 in EMEM (Lonza) supplemented with 2% FBS, 2% penicillin/streptomycin, 2 mM Ultraglutamine, and 100 ng/mL IFNγ (R&D).

To determine IDO1 activity, the culture medium was removed and the cells were incubated for one day at 37°C, 5% _CO2 in EMEM (Lonza) supplemented with 2% FBS and 2% penicillin/streptomycin in the presence of various concentrations of the compounds of the invention. 100 μL of supernatant was then collected in a conical plate, 30 μL of 30% TCA was added, and the cells were centrifuged at 3000 x g for 10 minutes. 100 μL of supernatant was collected in a flat-bottomed plate and incubated in 100 μL of 2% (w/v) 4-(dimethylamino)-benzaldehyde in acetic acid at room temperature for 5 minutes. Kynurenine concentration was determined by measuring absorbance at 480 nm. A standard curve was drawn using pure kynurenine. Data were fitted using Prism software (GraphPad Software, Inc.).

The biological activities of representative compounds are summarized in the table below:

Compound 1 1.0 2 0.77 6 3.4 8 3.6 9 7.0 11 5.9

In one embodiment, it is generally desirable to select compounds with an IC50 below 5 μM for further study.

II.2.C Assay for the determination of IDO1 activity in human blood: whole blood packed leukocytes

The compounds of the present invention inhibit the activity of human IDO1 in a human whole blood assay (whole blood concentrated leukocytes).

Human whole blood leukocyte concentrates are obtained as a by-product in the preparation of red blood cell and platelet concentrates from whole blood donors (as described in van der Meer et al., Vox Sang , 1999, 76 (2), 90-99).

The assay was performed in 96-well flat-bottom plates containing undiluted human whole blood packed leukocytes (containing 2% penicillin/streptomycin) stimulated for 18 hours with lipopolysaccharide (LPS) (12.5 µg/mL) and recombinant human interferon-gamma (rhIFNg) (50 ng/mL) to induce the conversion of tryptophan to kynurenine. Plasma was collected after centrifugation and plasma kynurenine levels were determined by LC-MS/MS (HPLC column Unison™ UK-Phenyl, 75 × 4.6, 3 µm, flow rate: 0.8 mL/min, 4-minute gradient from water + 0.2% acetic acid to methanol + 0.1% formic acid, retention time: 2.7 min; API 4000™ MS-MS system from AB Sciex, ESI+ mode, precursor ion: 209.2, product ion: 94.1).

To determine the effect of IDO1 inhibition on kynurenine production, compounds of the present invention were co-incubated at various concentrations and data were fitted using Prism software (GraphPad Software, Inc.).

The biological activities of representative compounds are summarized in the following table (the results are an average of the results using blood from several different donors):

Compound Number of individual blood donors 1 3.36±0.51 13 2 3.26±0.71 15

II.2.D Cell-based assay for IDO1-dependent T cell proliferation: SKOV-3 PBMC co-culture

The compounds of the present invention restored T cell proliferation in a SKOV-3 PBMC co-culture assay.

The assay was performed in 96-well flat-bottom plates seeded with the human ovarian adenocarcinoma SKOV-3 cell line [SKOV-3; SKOV3] (ATCC® HTB-77™) and co-cultured with human peripheral blood mononuclear cells (PBMCs) stimulated with CD3/CD28 beads and rhIL-2.

For adhesion, irradiated SKOV-3 cells (150 × ¹⁰ cells/well) were incubated overnight at 37°C, 5% CO₂ in _Iscove 's Modified Dulbecco's Medium (IMDM) (Lonza) supplemented with 50% FBS, 2% penicillin/streptomycin, and 2 mM Ultraglutamin in a final volume of 150 μL. Isolated PBMCs (stimulated with CD3/CD28 beads and rhIL-2 (30 U/mL)) were added at a 1:1 ratio. After 24 hours of co-culture, ^3H -thymidine (1 μCi/10 μL) was added to assess proliferation following overnight incubation in the presence of 50% serum (TopCount counter, Perkin Elmer).

To determine the effect of IDO1 inhibition on the restoration of T cell proliferation, compounds of the invention were co-incubated at various concentrations.

Compound 2 exhibited an _EC50 of 0.074 μM in this assay (mean of three independent experiments). Figure 1 shows the effect of increasing concentrations of Compound 2 on thymidine incorporation.

II.3. In vivo suppression of blood kynurenine levels in healthy mice

The compounds of the present invention reduce the amount of kynurenine in the blood of healthy mice.

Briefly, mice were treated orally and by gavage (dosage 5 mL/kg, 10 mice/group) with different doses of a compound of the invention suspended in 0.5% hydroxypropyl methylcellulose (HPMC) K4M/0.25% Tween 20 or with a vehicle control (0.5% HPMC K4M/0.25% Tween 20). Two hours later, blood was harvested, plasma was prepared, and the amount of kynurenine present was determined by LC-MS-MS (HPLC column Unison UK-Phenyl, 75 × 4.6, 3 μm, flow rate: 0.8 mL/min, 4-minute gradient from water + 0.2% acetic acid to methanol + 0.1% formic acid, retention time: 2.7 min; API4000™ MS-MS system from AB Sciex, ESI+ mode, parent ion: 209.2, daughter ion: 94.1).

Compound 1 inhibited circulating kynurenine by 41% (p<0.0001) at 100 mg/kg and by 59% (p<0.0001) at 200 mg/kg: see table below.

vehicle Compound 1 100 mg/kg Compound 1 200 mg/kg Plasma kynurenine concentration (mean ± standard error of the mean) 187.6±17.8 ng/mL 111.1±27.0 ng/mL 77.7±9.2 ng/mL

Compound 2 inhibited circulating kynurenine by 39% (p<0.0001) at 10 mg/kg, 55% (p<0.0001) at 30 mg/kg and 68% (p<0.0001) at 100 mg/kg: see table below and Figure 2.

vehicle Compound 2 10 mg/kg Compound 2 30 mg/kg Compound 2 100 mg/kg Plasma kynurenine concentration (mean ± standard error of the mean) 201±15.7 ng/mL 122±3.5 ng/mL 91.0±4.4 ng/mL 64.0±3.8 ng/mL

Example II.4: In vivo efficacy studies in the 4T1 breast cancer syngeneic model

The in vivo efficacy study of the compounds of this invention was implemented in the 4T1 homologous tumor model of Balb/c mice implanted in the mammary gland orthotopically. 100,000 4T1 breast cancer cells (ATCC® CRL-2539™) were orthotopically implanted into the mammary glands of 7-week-old Balb/c mice (Day 0). When the tumor mean value reached 60 mm ³ (Day 7 to Day 11), animals were randomly assigned to different treatment groups based on tumor size. The compounds of this invention were orally administered twice a day (approximately at 9 am and 5 pm) from the random assignment day. The compound was suspended in Methocel™ cellulose ether vehicle and ultrasonically treated, then orally administered to the animals using a gavage needle. Treatment was given every day until the end of the study. The weight changes of all experimental animals were monitored twice a week. Tumor volume was measured twice a week by a caliper device and calculated using the following formula: tumor volume=0.5 × (length × width ² ). The study was terminated before tumor volume reached 2000 mm. TGI (tumor growth inhibition %) was determined to be 100. The table below and Figure 3A show that Compound 1 inhibits 4T1 tumor growth in vivo.

deal with TGI (tumor growth inhibition) Methocel 736.4 0% Compound 1 100 mg/kg, twice a day 443.7 43.4%

Example II.5: In vivo efficacy studies using the PancO2 pancreatic cancer syngeneic model

The in vivo efficacy study of the compounds of the present invention was conducted in a PancO2 homologous tumor model of subcutaneously implanted C57/Bl6 mice. 5 million PancO2 pancreatic cancer cells were subcutaneously implanted into 7-week-old C57/Bl6 mice (Day 0). When the tumor average reached 60 mm ³ (Days 10 to 12), the animals were randomly assigned to different treatment groups based on tumor size. The compound was orally administered twice a day (approximately at 9 am and 5 pm) starting from the randomized day. The compound was suspended in Methocel vehicle and sonicated, then orally administered to the animals using a gavage needle. Treatment was given daily until the end of the study. Weight changes in all experimental animals were monitored weekly. Tumor volume was measured weekly using a caliper device and calculated using the following formula: tumor volume = 0.5 × (length × width ² ). The study was terminated before the tumor volume reached 2000 mm. TGI (tumor growth inhibition %) was determined as. The following table and Figure 4 show that compound 1 inhibited PancO2 tumor growth in vivo .

deal with TGI (tumor growth inhibition) Methocel 598.2 0% Compound 1 200 mg/kg, twice a day 457.0 26.2%

In a separate study conducted under the same conditions, compound 2 (100 mg/kg, twice daily) was studied. Methocel vehicle or 100 mg/kg compound 2 was orally administered twice a day (8 hours apart) starting from the randomized day. Compound 2 was resuspended in Methocel vehicle and sonicated, then orally administered to the animals using a gavage needle. Treatment was given daily until the end of the study. Tumor volume was measured weekly using a caliper device and calculated using the following formula: tumor volume = 0.5 × (length × width ² ). Mice were considered dead when tumor size reached 400 mm ^3. The table below shows that compound 2 inhibited PancO2 tumor growth in vivo . SEM refers to standard error of measurement.

deal with TGI +/- SEM (tumor growth inhibition) Methocel® 677.6 +/- 39.2 0% Compound 2 - 100 mg/kg twice daily 586.6 +/- 48.4 16.8% +/- 8.2

Example II.6: In vivo efficacy study of inhibition of tryptophan degradation in 4T1 tumor tissue

The compounds of this invention can reduce the kynurenine concentration in mouse tumors, such as the 4T1 homologous tumors of Balb/c mice orthotopically implanted in the mammary gland. 100,000 4T1 breast cancer cells were orthotopically implanted into the mammary glands of 7-week-old Balb/c mice (day 0). When the tumor mean value reached 60 mm ³ (day 6), animals were randomly assigned to different treatment groups (n=10/group) based on tumor size. From day 6 to day 26, animals were treated with Methocel vehicle until the tumor reached 1500 mm ³ to 2000 mm ³ size. Compound 1 was suspended in Methocel vehicle and ultrasonicated, then orally administered to animals using a gavage needle. On the 26th and 27th days, Methocel vehicle or 200 mg/kg compound 1 was orally administered twice a day (approximately at 9 am and 5 pm). The next morning, treatment was given and mice were killed 4 hours after compound 1 was administered. Tumors were taken out, weighed, and frozen on dry ice. Tumor kynurenine concentrations were analyzed by LC/MS-MS. Compound 1 reduced kynurenine concentrations by 47% (p<0.0001): see the table below and Figure 5.

deal with Kynurenine concentration (ng/g tumor) Mean ± SEM Methocel 787.5 ± 46.2 Compound 1, 200 mg/kg 417.2 ± 55.7

Example II.7: In vivo efficacy study of inhibition of tryptophan degradation in CT26 tumor tissue

A. The compounds of the present invention can reduce the concentration of kynurenine in mouse tumors.

In the present study, CT26 homologous tumors were subcutaneously implanted into Balb-c mice. More particularly, 500,000 (500,000) CT26 colon cancer cells [CT26.WT, available from ATCC® CRL-2628™] were subcutaneously implanted into 7-week-old Balb/c mice (day 0). When the tumor mean value reached 150 mm ³ (day 11), animals were randomly assigned to different treatment groups (n=10/group) based on tumor size. Compound 1 was suspended in Methocel™ (methylcellulose) vehicle and ultrasonicated and then orally administered to animals using a gavage needle. After the tumor reached a size of 1500 mm ³ to 2000 mm ³ , mice were orally administered Methocel vehicle or compound 1 at 200 mg/kg twice a day (approximately at 9 am and 5 pm) for 2 days. The next morning, treatment was given and mice were sacrificed 2h after compound 1 was administered. Tumors were removed, weighed, and frozen on dry ice. Tumors were analyzed for kynurenine concentration by LC/MS-MS.

Compound 1 reduced kynurenine concentration by 59% (p<0.0001): see the table below and Figure 6.

deal with Kynurenine concentration (ng/g tumor) Mean ± SEM Methocel 2124 ± 272 Compound 1, 200 mg/kg 876 ± 68

B. Compound 1 inhibits tumor growth in vivo.

In a separate study, the anti-tumor efficacy of IDO-1 inhibition was tested in the CT26 colon syngeneic mouse tumor model using a series of different treatment regimens. The model was essentially as described above, except that 1 × 10 ⁶ cells in phosphate-buffered saline (PBS) were subcutaneously implanted into the flank of 8-week-old Balb/c female mice (10 per group) on day 0. Mice were randomly assigned to treatment groups (100 mg/kg twice daily, 200 mg/kg twice daily, or 600 mg/kg twice daily) based on tumor size on day 9 from the start of treatment. The results are shown in the table below and in Figure 7.

Group Dosage mg/kg schedule TGI%(D15) TGI%(D17) TGI%(D20) N vehicle - Twice a day - - - 10 Compound 1 100 Twice a day 29 33 20 10 Compound 1 200 Twice a day 38 41 34 10 Compound 1 600 Twice a day 36 51 38 10

At the highest dose of 600 mg/kg twice daily, there was significant tumor growth inhibition (TGI) of up to 51%. At the lower doses of 100 mg/kg and 200 mg/kg twice daily, the TGI based on the mean of the tumor measurement groups was slightly lower and thus indicated dose proportionality.

All disclosures cited in this specification and priority applications, including U.S. Provisional Patent Application No. 61/996,976, filed May 15, 2014, are incorporated herein by reference. Although the present invention has been described with reference to specific embodiments, it should be understood that modifications may be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims (31)

1. A pharmaceutical composition comprising a compound of formula I', a compound of formula I'", or a mixture thereof: Or its pharmaceutically acceptable enantiomer or salt, wherein: X represents -NH- or -CQ ² = CQ ³ -; ^Q2 and ^Q3 each independently represent H or C1 to C6 alkyl groups; ^R1 and ^R2 each independently represent H, halogen, cyano, C1 to C6 alkyl or C1 to C6 alkoxy; And at least one pharmaceutically acceptable carrier. 2. The pharmaceutical composition according to claim 1, wherein ^R1 and ^R2 each independently represent H or a halogen. 3. The pharmaceutical composition according to claim 1, wherein the composition comprises a racemic mixture comprising equimolar amounts of compound I' and compound I''. 4. The pharmaceutical composition according to claim 1 or claim 2, wherein the composition comprises different molar amounts of compound I' and compound I''. 5. The pharmaceutical composition of claim 4, wherein the composition comprises a mixture of a compound of formula I' and a compound of formula I'", wherein the composition comprises more than 50% of the compound of formula I'. 6. The pharmaceutical composition of claim 5, wherein the composition comprises at least 95% to 100% of the compound of formula I'. 7. The pharmaceutical composition according to claim 1, wherein the compound of formula I' is selected from: (a)(-)-(R)-3-(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione; (b)(-)-(R)-3-(1H-indol-3-yl)pyrrolidine-2,5-dione; (c)(-)-(R)-3-(5-chloro-1H-indol-3-yl)pyrrolidine-2,5-dione; (d)(R)-3-(6-chloro-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione; and (e)(R)-3-(6-bromo-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione, Or a pharmaceutically acceptable salt of any one of (a) to (e). 8. The pharmaceutical composition of claim 1, comprising a racemic mixture of compounds of formula I' and I'", wherein the racemate is selected from: (i) 3-(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione; (ii) 3-(1H-indol-3-yl)pyrrolidine-2,5-dione; (iii) 3-(5-chloro-1H-indol-3-yl)pyrrolidine-2,5-dione; (iv) 3-(6-chloro-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione; (v)3-(6-bromo-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione; (vi) 3-(5-bromo-1H-indol-3-yl)pyrrolidine-2,5-dione; (vii)3-(5-methyl-1H-indol-3-yl)pyrrolidine-2,5-dione; (viii) 3-(5-methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione; (ix)3-(2,5-dioxopyrrolidone-3-yl)-1H-indole-5-carboxynitrile; (x)3-(5,6-difluoro-1H-indol-3-yl)pyrrolidine-2,5-dione; (xi)3-(5-fluoro-6-methyl-1H-indol-3-yl)pyrrolidine-2,5-dione; (xii)3-(6-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione; (xiii)3-(6-chloro-1H-indol-3-yl)pyrrolidine-2,5-dione; (xiv)3-(6-bromo-1H-indol-3-yl)pyrrolidine-2,5-dione; (xv)3-(6-methyl-1H-indol-3-yl)pyrrolidine-2,5-dione; (xvi)3-(6-methoxy-1H-indol-3-yl)pyrrolidine-2,5-dione; (xvii)3-(2,5-dioxopyrrolidone-3-yl)-1H-indole-6-carboxynitrile; (xviii)3-(naphth-1-yl)pyrrolidine-2,5-dione; (xix)3-(6-fluoronaphth-1-yl)pyrrolidine-2,5-dione; (xx)3-(7-fluoronaphth-1-yl)pyrrolidine-2,5-dione; (xxii)3-(6-chloronaphth-1-yl)pyrrolidine-2,5-dione; or (xxiii)3-(7-chloronaphth-1-yl)pyrrolidine-2,5-dione. 9. The pharmaceutical composition according to claim 1, wherein the compound of formula I” is selected from: (a”)(S)-3-(5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione; (b”)(S)-3-(1H-indol-3-yl)pyrrolidine-2,5-dione; (c”)(S)-3-(5-chloro-1H-indol-3-yl)pyrrolidine-2,5-dione; (d”)(S)-3-(6-chloro-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione; and (e”)(S)-3-(6-bromo-5-fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione, or a pharmaceutically acceptable salt thereof. 10. The pharmaceutical composition according to any one of claims 1-9, wherein the compound of formula I' and/or the compound of formula I” is a free base. 11. The pharmaceutical composition according to any one of claims 1-9, wherein the compound of formula I' and/or the compound of formula I” is a salt. 12. The pharmaceutical composition according to claim 11, wherein the salt is selected from aluminum, calcium, choline, potassium, sodium and zinc salts. 13. A drug comprising a compound of formula I', a compound of formula I'", or a mixture thereof: Or its pharmaceutically acceptable salt, wherein: X represents -NH- or -CQ ² = CQ ³ -; ^Q2 and ^Q3 each independently represent H or C1 to C6 alkyl groups; ^R1 and ^R2 each independently represent H, halogen, cyano, C1 to C6 alkyl or C1 to C6 alkoxy, wherein the compound is optionally deuterated. 14. The medicament according to claim 13, wherein ^R1 and ^R2 each independently represent H or halogen. 15. Compounds of formula I', compounds of formula I”, or mixtures thereof: Or its pharmaceutically acceptable salt, wherein: X represents -NH- or -CQ ² = CQ ³ -; ^Q2 and ^Q3 each independently represent H or C1 to C6 alkyl groups; ^R1 and ^R2 each independently represent H, halogen, cyano, C1 to C6 alkyl, or C1 to C6 alkoxy; wherein the compound is optionally deuterated. Use in the preparation of medicines for the treatment and/or prevention of cancer or endometriosis. 16. The use according to claim 15, wherein ^R1 and ^R2 each independently represent H or a halogen. 17. The use according to claim 15 or claim 16, wherein the cancer is selected from malignant melanoma, acute myeloid leukemia, pancreatic cancer, colorectal cancer, prostate cancer, cervical cancer, brain cancer, endometrial cancer, and ovarian cancer. 18. Compounds of formula I', compounds of formula I”, or mixtures thereof: Or its pharmaceutically acceptable salt, wherein: X represents -NH- or -CQ ² = CQ ³ -; ^Q2 and ^Q3 each independently represent H or C1 to C6 alkyl groups; ^R1 and ^R2 each independently represent H, halogen, cyano, C1 to C6 alkyl or C1 to C6 alkoxy, wherein the compound is optionally deuterated; Its use in the preparation of IDO1 inhibitors. 19. As per the use of claim 18, wherein ^R1 and ^R2 each independently represent H or halogen. 20. Compounds having the structure of formula II': The use of its pharmaceutically acceptable salts in the preparation of IDO1 inhibitors. 21. The use according to claim 20, wherein the compound is a free base. 22. Compounds having the structure of formula II”: The use of its pharmaceutically acceptable salts in the preparation of IDO1 inhibitors. 23. The compound of claim 22, wherein the compound is a free base. 24. A pharmaceutical composition comprising a compound of formula II': Or its pharmaceutically acceptable salts and pharmaceutically acceptable carriers. 25. A pharmaceutical composition comprising a compound of formula II": Or its pharmaceutically acceptable salts and pharmaceutically acceptable carriers. 26. A pharmaceutical composition comprising a mixture of compounds of formula II' and formula II': Or its pharmaceutically acceptable salt. 27. The pharmaceutical composition of claim 26, wherein the compound of formula II' and the compound of formula II'' are present in a 1:1 molar ratio. 28. The pharmaceutical composition of claim 26, wherein the compound of formula II' is present in an amount of at least 75 mol%. 29. The pharmaceutical composition of claim 26, wherein the compound of formula II' is present in an amount of at least 90 mol%. 30. A pharmaceutical composition comprising a compound with the following structure: Or its pharmaceutically acceptable salt, or its deuterated form, and a pharmaceutically acceptable carrier. 31. The composition according to claim 30, wherein the compound of formula II is in the form of a free base.