Patent Application
20200239420
Compounds, methods and pharmaceutical compositions for the prevention and/or treatment of HIV; including the prevention of the progression of AIDS and general immunosuppression, by administering certain indoleamine 2,3-dioxygenase compounds in therapeutically effective amounts are disclosed. Methods for preparing such compounds and methods of using the compounds and pharmaceutical compositions thereof are also disclosed.
Indoleamine-2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that catalyzes the oxidation of the indole ring of tryptophan to produce N-formyl kynurenine, which is rapidly and constitutively converted to kynurenine (Kyn) and a series of downstream metabolites. IDO1 is the rate limiting step of this kynurenine pathway of tryptophan metabolism and expression of IDO1 is inducible in the context of inflammation. Stimuli that induce IDO1 include viral or bacterial products, or inflammatory cytokines associated with infection, tumors, or sterile tissue damage. Kyn and several downstream metabolites are immunosuppressive: Kyn is antiproliferative and proapoptotic to T cells and NK cells (Munn, Shafizadeh et al. 1999, Frumento, Rotondo et al. 2002) while metabolites such as 3-hydroxy anthranilic acid (3-HAA) or the 3-HAA oxidative dimerization product cinnabarinic acid (CA) inhibit phagocyte function (Sekkai, Guittet et al. 1997), and induce the differentiation of immunosuppressive regulatory T cells (Treg) while inhibiting the differentiation of gut-protective IL-17 or IL-22 -producing CD4+ T cells (Th17 and Th22)(Favre, Mold et al. 2010). IDO1 induction, among other mechanisms, is likely important in limiting immunopathology during active immune responses, in promoting the resolution of immune responses, and in promoting fetal tolerance. However, in chronic settings, such as cancer, or chronic viral or bacterial infection, IDO1 activity prevents clearance of tumor or pathogen and if activity is systemic, IDO1 activity may result in systemic immune dysfunction (Boasso and Shearer 2008, Li, Huang et al. 2012). In addition to these immunomodulatory effects, metabolites of IDO1 such as Kyn and quinolinic acid are also known to be neurotoxic and are observed to be elevated in several conditions of neurological dysfunction and depression. As such, IDO1 is a therapeutic target for inhibition in a broad array of indications, such as to promote tumor clearance, enable clearance of intractable viral or bacterial infections, decrease systemic immune dysfunction manifest as persistent inflammation during HIV infection or immunosuppression during sepsis, and prevent or reverse neurological conditions.
Despite the success of antiretroviral therapy (ART) in suppressing HIV replication and decreasing the incidence of AIDS-related conditions, HIV-infected patients on ART have a higher incidence of non-AIDS morbidities and mortality than their uninfected peers. These non-AIDS conditions include cancer, cardiovascular disease, osteoporosis, liver disease, kidney disease, frailty, and neurocognitive dysfunction (Deeks 2011). Several studies indicate that non-AIDS morbidity/mortality is associated with persistent inflammation, which remains elevated in HIV-infected patients on ART as compared to peers (Deeks 2011). As such, it is hypothesized that persistent inflammation and immune dysfunction despite virologic suppression with ART is a cause of these non-AIDS-defining events (NADEs).
HIV infects and kills CD4+ T cells, with particular preference for cells like those CD4+ T cells that reside in the lymphoid tissues of the mucosal surfaces (Mattapallil, Douek et al. 2005). The loss of these cells combined with the inflammatory response to infection result in a perturbed relationship between the host and all pathogens, including HIV itself, but extending to pre-existing or acquired viral infections, fungal infections, and resident bacteria in the skin and mucosal surfaces. This dysfunctional host:pathogen relationship results in the over-reaction of the host to what would typically be minor problems as well as permitting the outgrowth of pathogens among the microbiota. The dysfunctional host:pathogen interaction therefore results in increased inflammation, which in turn leads to deeper dysfunction, driving a vicious cycle. As inflammation is thought to drive non-AIDS morbidity/mortality, the mechanisms governing the altered host:pathogen interaction are therapeutic targets.
IDO1 expression and activity are increased during untreated and treated HIV infection as well as in primate models of SIV infection (Boasso, Vaccari et al. 2007, Favre, Lederer et al. 2009, Byakwaga, Boum et al. 2014, Hunt, Sinclair et al. 2014, Tenorio, Zheng et al. 2014). IDO1 activity, as indicated by the ratio of plasma levels of enzyme substrate and product (Kyn/Tryp or K:T ratio), is associated with other markers of inflammation and is one of the strongest predictors of non-AIDS morbidity/mortality (Byakwaga, Boum et al. 2014, Hunt, Sinclair et al. 2014, Tenorio, Zheng et al. 2014). In addition, features consistent with the expected impact of increased IDO1 activity on the immune system are major features of HIV and SIV induced immune dysfunction, such as decreased T cell proliferative response to antigen and imbalance of Treg:Th17 in systemic and intestinal compartments (Favre, Lederer et al. 2009, Favre, Mold et al. 2010). As such, we and others hypothesize that IDO1 plays a role in driving the vicious cycle of immune dysfunction and inflammation associated with non-AIDS morbidity/mortality. Thus, we propose that inhibiting IDO1 will reduce inflammation and decrease the risk of NADEs in ART-suppressed HIV-infected persons.
As described above, inflammation associated with treated chronic HIV infection is a likely driver of multiple end organ diseases [Deeks 2011]. However, these end organ diseases are not unique to HIV infection and are in fact the common diseases of aging that occur at earlier ages in the HIV-infected population. In the uninfected general population inflammation of unknown etiology is a major correlate of morbidity and mortality [Pinti, 2016 #88]. Indeed, many of the markers of inflammation are shared, such as IL-6 and CRP. If, as hypothesized above, IDO1 contributes to persistent inflammation in the HIV-infected population by inducing immune dysfunction in the GI tract or systemic tissues, then IDO1 may also contribute to inflammation and therefore end organ diseases in the broader population. These inflammation-associated end organ diseases are exemplified by cardiovascular diseases, metabolic syndrome, liver disease (NAFLD, NASH), kidney disease, osteoporosis, and neurocognitive impairment. Indeed, the IDO1 pathway has links in the literature to liver disease (Vivoli abstracts at Italian Assoc. for the Study of the Liver Conference 2015], diabetes [Baban, 2010 #89], chronic kidney disease [Schefold, 2009 #90], cardiovascular disease [Mangge, 2014 #92; Mangge, 2014 #91], as well as general aging and all cause mortality [Pertovaara, 2006 #93]. As such, inhibition of IDO1 may have application in decreasing inflammation in the general population to decrease the incidence of specific end organ diseases associated with inflammation and aging.
IDO expression can be detected in a number of human cancers (for example; melanoma, pancreatic, ovarian, AML, CRC, prostate and endometrial) and correlates with poor prognosis (Munn 2011). Multiple immunosuppressive roles have been ascribed to the action of IDO, including the induction of Treg differentiation and hyper-activation, suppression of Teff immune response, and decreased DC function, all of which impair immune recognition and promote tumor growth (Munn 2011). IDO expression in human brain tumors is correlated with reduced survival. Orthotropic and transgenic glioma mouse models demonstrate a correlation between reduced IDO expression and reduced Treg infiltration and an increased long term survival (Wainwright, Balyasnikova et al. 2012). In human melanoma a high proportion of tumors (33 of 36 cases) displayed elevated IDO suggesting an important role in establishing an immunosuppressive tumor microenvironment (TME) characterized by the expansion, activation and recruitment of MDSCs in a Treg-dependent manner (Holmgaard, Zamarin et al. 2015). Additionally, host IDO expressing immune cells have been identified in the draining lymph nodes and in the tumors themselves (Mellor and Munn 2004). Hence, both tumor and host-derived IDO are believed to contribute to the immune suppressed state of the TME.
The inhibition of IDO was one of the first small molecule drug strategies proposed for re-establishment of an immunogenic response to cancer (Mellor and Munn 2004). The d-enantiomer of 1-methyl tryptophan (D-1MTor indoximod) was the first IDO inhibitor to enter clinical trials. While this compound clearly does inhibit the activity of IDO, it is a very weak inhibitor of the isolated enzyme and the in vivo mechanism(s) of action for this compound are still being elucidated. Investigators at Incyte optimized a hit compound obtained from a screening process into a potent and selective inhibitor with sufficient oral exposure to demonstrate a delay in tumor growth in a mouse melanoma model (Yue, Douty et al. 2009). Further development of this series led to INCB204360 which is a highly selective for inhibition of IDO-1 over IDO-2 and TDO in cell lines transiently transfected with either human or mouse enzymes (Liu, Shin et al. 2010). Similar potency was seen for cell lines and primary human tumors which endogenously express IDO1 (IC50s˜3-20 nM). When tested in co-culture of DCs and naïve CD4+CD25− T cells, INCB204360 blocked the conversion of these T cells into CD4+FoxP3+ Tregs. Finally, when tested in a syngeneic model (PAN02 pancreatic cells) in immunocompetent mice, orally dosed INCB204360 provided a significant dose-dependent inhibition of tumor growth, but was without effect against the same tumor implanted in immune-deficient mice. Additional studies by the same investigators have shown a correlation of the inhibition of IDO1 with the suppression of systemic kynurenine levels and inhibition of tumor growth in an additional syngeneic tumor model in immunocompetent mice. Based upon these preclinical studies, INCB24360 entered clinical trials for the treatment of metastatic melanoma (Beatty, O'Dwyer et al. 2013).
In light of the importance of the catabolism of tryptophan in the maintenance of immune suppression, it is not surprising that overexpression of a second tryptophan metabolizing enzyme, TDO2, by multiple solid tumors (for example, bladder and liver carcinomas, melanomas) has also been detected. A survey of 104 human cell lines revealed 20/104 with TDO expression, 17/104 with IDO1 and 16/104 expressing both (Pilotte, Larrieu et al. 2012). Similar to the inhibition of IDO1, the selective inhibition of TDO2 is effective in reversing immune resistance in tumors overexpressing TDO2 (Pilotte, Larrieu et al. 2012). These results support TDO2 inhibition and/or dual TDO2/IDO1 inhibition as a viable therapeutic strategy to improve immune function.
Multiple pre-clinical studies have demonstrated significant, even synergistic, value in combining IDO-1 inhibitors in combination with T cell checkpoint modulating mAbs to CTLA-4, PD-1, and GITR. In each case, both efficacy and related PD aspects of improved immune activity/function were observed in these studies across a variety of murine models (Balachandran, Cavnar et al. 2011, Holmgaard, Zamarin et al. 2013, M. Mautino 2014, Wainwright, Chang et al. 2014). The Incyte IDO1 inhibitor (INCB204360, epacadostat) has been clinically tested in combination with a CTLA4 blocker (ipilimumab), but it is unclear that an effective dose was achieved due to dose-limited adverse events seen with the combination. In contrast recently released data for an on-going trial combining epacadostat with Merck's PD-1 mAb (pembrolizumab) demonstrated improved tolerability of the combination allowing for higher doses of the IDO1 inhibitor. There have been several clinical responses across various tumor types which is encouraging. However, it is not yet known if this combination is an improvement over the single agent activity of pembrolizumab (Gangadhar, Hamid et al. 2015). Similarly, Roche/Genentech are advancing NGL919/GDC-0919 in combination with both mAbs for PD-L1 (MPDL3280A, Atezo) and OX-40 following the recent completion of a phase 1a safety and PK/PD study in patients with advanced tumors.
IDO1 activity generates kynurenine pathway metabolites such as Kyn and 3-HAA that impair at least T cell, NK cell, and macrophage activity (Munn, Shafizadeh et al. 1999, Frumento, Rotondo et al. 2002) (Sekkai, Guittet et al. 1997, Favre, Mold et al. 2010). Kyn levels or the Kyn/Tryp ratio are elevated in the setting of chronic HIV infection (Byakwaga, Boum et al. 2014, Hunt, Sinclair et al. 2014, Tenorio, Zheng et al. 2014), HBV infection (Chen, Li et al. 2009), HCV infection (Larrea, Riezu-Boj et al. 2007, Asghar, Ashiq et al. 2015), and TB infection(Suzuki, Suda et al. 2012) and are associated with antigen-specific T cell dysfunction (Boasso, Herbeuval et al. 2007, Boasso, Hardy et al. 2008, Loughman and Hunstad 2012, Ito, Ando et al. 2014, Lepiller, Soulier et al. 2015). As such, it is thought that in these cases of chronic infection, IDO1-mediated inhibition of the pathogen-specific T cell response plays a role in the persistence of infection, and that inhibition of IDO1 may have a benefit in promoting clearance and resolution of infection.
IDO1 expression and activity are observed to be elevated during sepsis and the degree of Kyn or Kyn/Tryp elevation corresponded to increased disease severity, including mortality (Tattevin, Monnier et al. 2010, Darcy, Davis et al. 2011). In animal models, blockade of IDO1 or IDO1 genetic knockouts protected mice from lethal doses of LPS or from mortality in the cecal ligation/puncture model (Jung, Lee et al. 2009, Hoshi, Osawa et al. 2014). Sepsis is characterized by an immunosuppressive phase in severe cases (Hotchkiss, Monneret et al. 2013), potentially indicating a role for IDO1 as a mediator of immune dysfunction, and indicating that pharmacologic inhibition of IDO1 may provide a clinical benefit in sepsis.
In addition to immunologic settings, IDO1 activity is also linked to disease in neurological settings (reviewed in Lovelace Neuropharmacology 2016(Lovelace, Varney et al. 2016)). Kynurenine pathway metabolites such as 3-hydroxykynurenine and quinolinic acid are neurotoxic, but are balanced by alternative metabolites kynurenic acid or picolinic acid, which are neuroprotective. Neurodegenerative and psychiatric disorders in which kynurenine pathway metabolites have been demonstrated to be associated with disease include multiple sclerosis, motor neuron disorders such as amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, Alzheimer's disease, major depressive disorder, schizophrenia, anorexia (Lovelace, Varney et al. 2016). Animal models of neurological disease have shown some impact of weak IDO1 inhibitors such as 1-methyltryptophan on disease, indicating that IDO1 inhibition may provide clinical benefit in prevention or treatment of neurological and psychiatric disorders.
It would therefore be an advance in the art to discover IDO inhibitors that effective the balance of the aforementioned properties as a disease modifying therapy in chronic HIV infections to decrease the incidence of non-AIDS morbidity/mortality; and/or a disease modifying therapy to prevent mortality in sepsis; and/or an immunotherapy to enhance the immune response to HIV, HBV, HCV and other chronic viral infections, chronic bacterial infections, chronic fungal infections, and to tumors; and/or for the treatment of depression or other neurological/neuropsychiatric disorders.
Briefly, in one aspect, the present invention discloses compounds of Formula I
or a pharmaceutically acceptable salt thereof wherein:
each n is independently 2, 1, or 0 (i.e. is absent);
Q1 is —C(O)NH—, NHC(O)—, or a 5-9 membered heterocycle wherein said heterocycle contains 1-3 hetero atoms independently selected from O, S, and N, and wherein said heterocycle may optionally be substituted with 1-4 substituents independently selected from halogen, OH, C1-3alkyl, OC1-3alkyl, C1-3fluoroalkyl, CN, and NH2;
Ar1 is C5-9aryl, or 5-9 membered heteroaryl, wherein aryl and heteroaryl include bicycles and heteroaryl contains 1-3 hetero atoms independently selected from O, S, and N, and wherein Ar1 may optionally be substituted with 1-4 substituents independently selected from halogen, OH, C1-3alkyl, OC1-3alkyl, C1-3fluoroalkyl, CN, and NH2; and
Ar2 is C5-9aryl, or 5-9 membered heteroaryl, wherein heteroaryl contains 1-3 hetero atoms independently selected from O, S, and N, and wherein Ar1 may optionally be substituted with 1-4 substituents independently selected from halogen, OH, C1-3alkyl, OC1-3alkyl, C1-3fluoroalkyl, CN, and NH2.
In another aspect, the present invention discloses a method for treating diseases or conditions that would benefit from inhibition of IDO. Examples include, is inflammation associated with HIV infection; chronic viral infections involving hepatitis B virus or hepatitis C virus; cancer; or sepsis.
In another aspect, the present invention discloses pharmaceutical compositions comprising a compound of Formula I or a pharmaceutically acceptable salt thereof.
In another aspect, the present invention provides a compound of Formula I or a pharmaceutically acceptable salt thereof for use in therapy.
In another aspect, the present invention provides a compound of Formula I or a pharmaceutically acceptable salt thereof for use in treating diseases or condition that would benefit from inhibition of IDO.
In another aspect, the present invention provides use of a compound of Formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in treating diseases or conditions that would benefit from inhibition of IDO.
In another aspect, the present invention discloses a method for treating a viral infection in a patient mediated at least in part by a virus in the retrovirus family of viruses, comprising administering to said patient a composition comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof. In some embodiments, the viral infection is mediated by the HIV virus.
In another aspect, a particular embodiment of the present invention provides a method of treating a subject infected with HIV comprising administering to the subject a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.
In yet another aspect, a particular embodiment of the present invention provides a method of inhibiting progression of HIV infection in a subject at risk for infection with HIV comprising administering to the subject a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof. Those and other embodiments are further described in the text that follows.
Preferably Ar1 is quinoline, isoquinoline, quinazoline, quinoxaline, indole, azaindole, benzodiazole, phenyl, pyridyl, diazole, or pyrimidine, and wherein Ar1 may optionally be substituted with a substituent selected from halogen, OH, C1-3alkyl, OC1-3alkyl, C1-3fluoroalkyl, CN, and NH2. More preferably Ar1 is quinoline, isoquinoline, or indole, and may optionally be substituted with a substituent selected from halogen, OH, C1-3alkyl, OC1-3alkyl, C1-3fluoroalkyl, CN, and NH2. Most preferably Ar1 is quinoline optionally substituted with a halogen.
Preferably Ar3 is phenyl or thiophene, optionally substituted with a halogen.
Preferred pharmaceutical compositions include unit dosage forms. Preferred unit dosage forms include tablets.
In particular, it is expected that the compounds and composition of this invention will be useful for prevention and/or treatment of HIV; including the prevention of the progression of AIDS and general immunosuppression. It is expected that in many cases such prevention and/or treatment will involve treating with the compounds of this invention in combination with at least one other drug thought to be useful for such prevention and/or treatment. For example, the IDO inhibitors of this invention may be used in combination with other immune therapies such as immune checkpoints (PD1, CTLA4, ICOS, etc.) and possibly in combination with growth factors or cytokine therapies (IL21, IL-7, etc.).
In is common practice in treatment of HIV to employ more than one effective agent. Therefore, in accordance with another embodiment of the present invention, there is provided a method for preventing or treating a viral infection in a mammal mediated at least in part by a virus in the retrovirus family of viruses which method comprises administering to a mammal, that has been diagnosed with said viral infection or is at risk of developing said viral infection, a compound as defined in Formula I, wherein said virus is an HIV virus and further comprising administration of a therapeutically effective amount of one or more agents active against an HIV virus, wherein said agent active against the HIV virus is selected from the group consisting of Nucleotide reverse transcriptase inhibitors; Non-nucleotide reverse transcriptase inhibitors; Protease inhibitors; Entry, attachment and fusion inhibitors; Integrase inhibitors; Maturation inhibitors; CXCR4 inhibitors; and CCR5 inhibitors. Examples of such additional agents are Dolutegravir, Bictegravir, and Cabotegravir.
“Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium, and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, and oxalate. Suitable salts include those described in P. Heinrich Stahl, Camille G. Wermuth (Eds.), Handbook of Pharmaceutical Salts Properties, Selection, and Use; 2002.
The present invention also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or ACN are preferred.
The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
In one embodiment, the pharmaceutical formulation containing a compound of Formula I or a salt thereof is a formulation adapted for oral or parenteral administration. In another embodiment, the formulation is a long-acting parenteral formulation. In a further embodiment, the formulation is a nano-particle formulation.
The present invention is directed to compounds, compositions and pharmaceutical compositions that have utility as novel treatments for immunosuppresion. While not wanting to be bound by any particular theory, it is thought that the present compounds are able to inhibit the enzyme that catalyzes the oxidative pyrrole ring cleavage reaction of I-Trp to N-formylkynurenine utilizing molecular oxygen or reactive oxygen species.
Therefore, in another embodiment of the present invention, there is provided a method for the prevention and/or treatment of HIV; including the prevention of the progression of AIDS and general immunosuppression.
The following examples serve to more fully describe the manner of making and using the above-described invention. It is understood that these examples in no way serve to limit the true scope of the invention, but rather are presented for illustrative purposes. In the examples and the synthetic schemes below, the following abbreviations have the following meanings. If an abbreviation is not defined, it has its generally accepted meaning.
1H NMR spectra were recorded on a Bruker Ascend 400 spectrometer or a Varian 400 spectrometer. Chemical shifts are expressed in parts per million (ppm, δ units). Coupling constants are in units of hertz (Hz). Splitting patterns describe apparent multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), m (multiplet), br (broad).
The analytical low-resolution mass spectra (MS) were recorded on Waters ACQUITY UPLC with SQ Detectors using a Waters BEH C18, 2.1×50 mm, 1.7 μm using a gradient elution method.
Solvent A: 0.1% formic acid (FA) in water;
Solvent B: 0.1% FA in acetonitrile;
At 0° C., to a suspension of NaH (60% in oil) (6.92 g, 288 mmol) in anhydrous THF (650 mL) under nitrogen with vigorous stirring was added the triethyl phosphonoacetate (52.5 g, 288 mmol.) dropwise. After stirred at 0° C. for 30 min, 1,4-cyclohexanedione monoethylene ketal (41 g, 260 mmol) in THF (150 mL) was added dropwise. The resulting mixture was allowed to warm up to room temperature and stirred overnight. The reaction mixture was poured into saturated aq. NH4Cl and extracted with EtOAc. The organics were washed sequentially with water and brine, and dried over Na2SO4. Filtration and concentration in vacuum gave a crude product, which was purified by flash chromatography (silica gel, 0-30% EtOAc in PE) to afford the title compound (56 g, 95% yield). (ESI) m/z calcd for C12H18O4: 226.12. Found: 227.33 (M+1)+.
At 0° C., to a suspension of NaH (60% in oil) (2.82 g, 70.4 mmol) in anhydrous DMF (120 mL) under nitrogen with vigorous stirring was added the trimethyl sulfoxonium iodide (15.5 g, 70.4 mmol.) portion wise. After stirred at 0° C. for 30 min, ethyl 2-(1,4-dioxaspiro[4.5]decan-8-ylidene)acetate (8.76 g, 38.7 mmol) in DMF (30 mL) was added dropwise. The resulting mixture was allowed to warm up to room temperature and stirred for another 3 h. The reaction mixture was poured into saturated aq. NH4Cl and extracted with EtOAc. The organics were washed sequentially with water and brine, and dried over Na2SO4. Filtration and concentration in vacuum gave a crude product, which was purified by flash chromatography (silica gel, 10-30% EtOAc in PE) to afford the title compound (5.2 g, 56% yield). (ESI) m/z calcd for C13H20O4: 240.14. Found: 241.56 (M+1)+.
To a solution of ethyl 7,10-dioxadispiro[2.2.46.23]dodecane-1-carboxylate (5.0 g, 20.8 mmol) in acetone (50 mL), was added 6 N HCl (20 mL, 120 mmol) dropwise. After the reaction mixture was stirred at room temperature overnight, water and EtOAc were added and the layers were separated. The organics were washed sequentially with water and brine, and dried over Na2SO4. Filtration and concentration in vacuum gave a crude product, which was purified by flash chromatography (silica gel, 0-20% EtOAc in PE) to afford the title compound (2.6 g, 64% yield). (ESI) m/z calcd for C11H16O3: 196.11. Found: 197.26 (M+1)+.
At −78° C., to a solution of ethyl 6-oxospiro[2.5]octane-1-carboxylate (2.57 g, 13.11 mmol) in THF, was added LiHMDS (13.77 mL, 13. mmol) dropwise during 30 min and the reaction mixture was stirred at the same temperature for another 1 h. Then a solution of N-phenyl bis-(trifluromethanesulfonamide) (4.92 g, 13.77 mmol) in THF (20 mL) was added to the reaction mixture dropwise. After stirred at room temperature overnight, the reaction mixture was quenched with aq. NH4Cl and the resulting mixture was extracted with EtOAc. The combined organic layers were washed sequentially with water and brine, and dried over Na2SO4. Filtration and concentration in vacuum gave a crude product, which was purified by flash chromatography (silica gel, 0-20% EtOAc in PE) to afford the title compound (2.36 g, 55% yield). (ESI) m/z calcd for C12H15F3O5S: 328.06. Found: 329.42 (M+1)+.
A suspension of ethyl 6-(((trifluoromethyl)sulfonyl)oxy)spiro[2.5]oct-5-ene-1-carboxylate (2.26 g, 6.89 mmol), quinolin-4-ylboronic acid (1.79 g, 10.34 mmol), Pd(PPh3)4 (796 mg, 0.689 mmol), Na2CO3 (1.83 g, 11.23 mmol), KBr (902 mg, 7.58 mmol) in dioxane (20 mL) and water (2 mL) was stirred at 100° C. for 14 hours under nitrogen atmosphere. After the reaction mixture was cooled to room temperature, this was partitioned between water and EtOAc and the layers were separated. The organics were washed sequentially with water and brine, and dried over Na2SO4. Filtration and concentration in vacuum gave a crude product, which was purified by flash chromatography to afford the title compound (1.01 g, 48% yield). (ESI) m/z calcd for C20H21NO2: 307.16. Found: 308.26 (M+1)+.
A mixture of ethyl 6-(quinolin-4-yl)spiro[2.5]oct-5-ene-1-carboxylate (500 mg, 1.63 mmol) and 10% Pd/C (0.2 g) in MeOH (10 mL) was stirred at room temperature under H2 atmosphere (15 psi) overnight. The resulting mixture was filtered through a pad of Celite and the filtrate was concentrated under reduced pressure to give the crude product which was purified by flash chromatography (silica gel, 0-30% EtOAc in PE) to afford the title compound (0.4 g, 79% yield) as a brown oil. (ESI) m/z calcd for C20H23NO2: 309.41. Found: 310.68 (M+1)+.
To a solution of 6-(quinolin-4-yl)spiro[2.5]octane-1-carboxylate (0.20 g, 0.65 mmol) in MeOH (6 mL) was added 1N NaOH aq. (2.6 mL, 2.6 mmol). After stirred at room temperature overnight, the resulting mixture was neutralized with 1N HCl and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated to The combined organic layers were dried over Na2SO4. Solvent was removed under vacuum and the residue was purified by Prep. TLC (5% MeOH in DCM) to afford the title compound. cis-isomer (80 mg, 44% yield): 1H NMR (400 MHz, MeOD) δ 8.77 (d, J=4.7 Hz, 1H), 8.28 (d, J=8.4 Hz, 1H), 8.20 (s, 1H), 8.03 (d, J=8.3 Hz, 1H), 7.80-7.73 (m, 1H), 7.69-7.62 (m, 1H), 7.45 (d, J=4.7 Hz, 1H), 3.62-3.51 (m, 1H), 2.23 (td, J=13.2, 3.5 Hz, 1H), 2.08-1.91 (m, 4H), 1.90-1.81 (m, 1H), 1.64-1.50 (m, 2H), 1.22-1.12 (m, 2H), 1.04-0.97 (m, 1H). LCMS (ESI) m/z calcd for C18H19NO2: 281.14. Found: 282.30 (M+1)+. trans-isomer (30 mg, 16% yield): 1H NMR (400 MHz, MeOD) δ 8.77 (d, J=4.3 Hz, 1H), 8.43 (s, 1H), 8.26 (d, J=8.5 Hz, 1H), 8.03 (d, J=8.4 Hz, 1H), 7.76 (t, J=7.6 Hz, 1H), 7.65 (t, J=7.7 Hz, 1H), 7.48 (d, J=4.6 Hz, 1H), 3.60-3.49 (m, J=11.6 Hz, 1H), 2.31-2.20 (m, J=13.0, 11.4 Hz, 1H), 2.10-1.96 (m, J=21.7, 13.7 Hz, 2H), 1.94-1.73 (m, 4H), 1.66-1.58 (m, J=7.5, 5.6 Hz, 1H), 1.17-1.08 (m, J=16.0, 10.5 Hz, 2H), 1.00-0.93 (m, J=7.7, 4.0 Hz, 1H). LCMS (ESI) m/z calcd for C18H19NO2: 281.14. Found: 282.34 (M+1)+.
To a stirred solution of cis-6-(quinolin-4-yl)spiro[2.5]octane-1-carboxylic acid (80 mg, 0.285 mmol) and 4-chloroaniline (44 mg, 0.342 mmol) in DCM (5 mL) was added DIPEA (56 mg, 0.427 mmol) followed by HATU (162 mg, 0.427 mmol). After stirred at r.t. overnight, the reaction mixture was quenched with brine and the resulting mixture was extracted with DCM (×3). The combined organic layers were dried over Na2SO4. Solvent was removed under vacuum and the residue was purified by Prep. HPLC to afford the title compound (81 mg, 73% yield). 1H NMR (400 MHz, CDCl3) δ 8.87 (d, J=4.8 Hz, 1H), 8.70 (s, 1H), 8.24 (d, J=8.4 Hz, 1H), 8.12 (d, J=8.5 Hz, 1H), 7.79 (t, J=7.3 Hz, 1H), 7.66 (t, J=7.6 Hz, 1H), 7.58 (d, J=8.6 Hz, 2H), 7.33 (d, J=4.9 Hz, 1H), 7.24 (d, J=8.7 Hz, 2H), 3.41 (t, J=11.8 Hz, 1H), 2.22-2.14 (m, 1H), 2.13-2.05 (m, 1H), 1.98-1.85 (m, 2H), 1.84-1.77 (m, 1H), 1.57-1.45 (m, 2H), 1.41-1.31 (m, 2H), 1.00-0.91 (m, 2H). LCMS (ESI) m/z calcd for C24H23ClN2O: 390.15. Found: 391.24/393.15 (M/M+2)+.
To a stirred solution of trans-6-(quinolin-4-yl)spiro[2.5]octane-1-carboxylic acid (30 mg, 0.107 mmol) and 4-chloroaniline (16 mg, 0.128 mmol) in DCM (2 mL) was added DIPEA (21 mg, 0.159 mmol) followed by HATU (60 mg, 0.159 mmol). After stirred at r.t. overnight, the reaction mixture was quenched with brine and the resulting mixture was extracted with DCM (×3). The combined organic layers were dried over Na2SO4. Solvent was removed under vacuum and the residue was purified by Prep. HPLC to afford the title compound (21 mg, 50% yield). 1H NMR (400 MHz, CDCl3) δ 8.87 (d, J=4.5 Hz, 1H), 8.12 (dd, J=19.7, 8.4 Hz, 2H), 7.71 (t, J=7.2 Hz, 1H), 7.57 (t, J=7.2 Hz, 1H), 7.54-7.42 (m, 3H), 7.34 (d, J=4.6 Hz, 1H), 7.32-7.27 (m, 2H), 3.47-3.39 (m, 1H), 2.26-2.19 (m, 1H), 2.12-2.00 (m, 3H), 1.84-1.75 (m, 3H), 1.58-1.54 (m, 1H), 1.35-1.32 (m, 1H), 1.18-1.13 (m, 1H), 0.97-0.92 (m, 1H). LCMS (ESI) m/z calcd for C24H23ClN2O: 390.15. Found: 391.24/393.15 (M/M+2)+.
At 0° C., A solution of 1.0 M potassium tert.-butoxide in THF (499 mL, 499 mmol) was slowly added to a mixture of methyltriphenylphosphonium bromide (178 g, 499 mmol) in THF (450 mL) at 0° C. After stirring for 1 h, 1,4-dioxa-spiro[4.5]decan-8-one (26 g, 166 mmol) was added. The resulting mixture was allowed to warm up to room temperature and stirred for 3 h. The reaction mixture was poured into saturated aq. NH4Cl and extracted with EtOAc. The organics were washed sequentially with water and brine, and dried over Na2SO4. Filtration and concentration in vacuum gave a crude product, which was purified by flash chromatography (silica gel, 0-30% EtOAc in PE) to afford the title compound (18 g, 70% yield). (ESI) m/z calcd for C9H14O2: 154.10. Found: 155.16 (M+1)+.
To a suspension of 8-methylene-1,4-dioxaspiro[4.5]decane (10 g, 64.8 mmol) and zinc-copper (12.7 g, 194 mmol) in Et2O (100 mL) was added trichloroacetyl chloride (8.66 mL, 77.8 mmol) drop wise at room temperature. After stirring at room temperature for 1 h, MeOH (30 mL) was added, then the mixture was cooled to −5° C. and zinc dust (12.7 g, 194 mmol) was added in portion over the period of 1 h at −5° C. The reaction mixture was allowed to warm to room temperature and celite was added. The resulting thick mass was filtered over celite pad and the cake was washed with excess ethyl acetate. The combined solution was washed with brine and dried over Na2SO4. Filtration and concentration in vacuum gave a crude product, which was purified by flash chromatography (silica gel, 0-30% EtOAc in PE) to afford the title compound (4.4 g, 34% yield). (ESI) m/z calcd for C11H16O3: 196.11. Found: 197.18 (M+1)+.
To a solution of 8,11-dioxadispiro[3.2.47.24]tridecan-2-one (1.4 g, 7.14 mmol) in MeOH (396 mL) was added dibenzylamine (2.8 g, 14.3 mmol) under nitrogen atmosphere and the resulting solution was stirred for 2 hours at room temperature. Then NaBH3CN (1.79 g, 28.5 mmol) was added portion wise and the reaction mixture was stirred at room temperature overnight. The resulting mixture was quenched with H2O, extracted with EtOAc. The combined organic layer was washed sequentially with water and brine, and dried over Na2SO4. Filtration and concentration in vacuum gave a crude product, which was purified by flash chromatography (silica gel, 0-30% EtOAc in PE) to afford the title compound (2.4 g, 89% yield). (ESI) m/z calcd for C25H31NO2: 377.24. Found: 378.41 (M+1)+.
To a solution of N,N-dibenzyl-8,11-dioxadispiro[3.2.47.24]tridecan-2-amine (1.05 g, 2.8 mmol) in acetone (10 mL), was added 12 N HCl (2 mL, 24 mmol) dropwise. After the reaction mixture was stirred for 2 h, water and EtOAc were added and the layers were separated. The organics were washed sequentially with water and brine, and dried over Na2SO4. Filtration and concentration in vacuum afforded the title compound (0.50 g, 91% yield). (ESI) m/z calcd for C23H27NO: 333.21. Found: 334.37 (M+1)+.
At −78° C., to a solution of N,N-dibenzyl-8,11-dioxadispiro[3.2.47.24]tridecan-2-amine (600 mg, 1.80 mmol) and N-phenyl bis-(trifluromethanesulfonamide) (964 mg, 2.70 mmol) in THF, was added LiHMDS (2.7 mL, 2.70 mmol) dropwise during 30 min and the reaction mixture was allowed to warm up to room temperature and stirred overnight. The reaction mixture was quenched with aq. NH4Cl and the resulting mixture was extracted with EtOAc. The combined organic layers were washed sequentially with water and brine, and dried over Na2SO4. Filtration and concentration in vacuum gave a crude product, which was purified by flash chromatography (silica gel, 0-10% EtOAc in PE) to afford the title compound (728 mg, 87% yield). (ESI) m/z calcd for C24H26F3NO3S: 465.16. Found: 466.24 (M+1)+.
A suspension of 2-(dibenzylamino)spiro[3.5]non-6-en-7-yl trifluoromethanesulfonate (728 mg, 1.56 mmol), quinolin-4-ylboronic acid (386 mg, 2.35 mmol), Pd(PPh3)4 (180 mg, 0.156 mmol), NaBr (177 mg, 1.72 mmol) in dioxane (10 mL) and water (2 mL) was stirred under nitrogen atmosphere at 100° C. for 14 hours. After the reaction mixture was cooled to room temperature, this was partitioned between water and EtOAc and the layers were separated. The organics were washed sequentially with water and brine, and dried over Na2SO4. Filtration and concentration in vacuum gave a crude product, which was purified by flash chromatography to afford the title compound (580 mg, 83% yield). (ESI) m/z calcd for C32H32N2: 444.26. Found: 445.33 (M+1)+.
A suspension of N,N-dibenzyl-7-(quinolin-4-yl)spiro[3.5]non-6-en-2-amine (580 mg, 1.30 mmol) and 10% Pd/C (290 mg) in EtOAc (10 mL) was stirred at room temperature under H2 atmosphere (15 psi) overnight. The resulting mixture was filtered through a pad of Celite and the filtrate was concentrated under reduced pressure to give the crude product which was purified by flash chromatography (silica gel, 0-50% EtOAc in PE) to afford the title compound (200 mg, 57% yield) as a brown oil. (ESI) m/z calcd for C18H26N2: 270.21. Found: 271.39 (M+1)+.
To a stirred solution of 7-(1,2,3,4-tetrahydroquinolin-4-yl)spiro[3.5]nonan-2-amine (100 mg, 0.369 mmol) and 4-chlorobenzoic acid (86.8mg, 0.555 mmol) in DCM (2 mL) was added DIPEA (143 mg, 1.107 mmol) followed by HATU (211 mg, 0.555 mmol). After stirred at Et. for 2 h, the reaction mixture was quenched with brine and the resulting mixture was extracted with DCM (×3). The combined organic layers were dried over Na2SO4. Solvent was removed under vacuum and the residue was purified by column chromatography on silica gel (0˜50% EA in PE) to afford the title compound (43 mg, 29% yield). LCMS (ESI) m/z calcd for C25H29ClN2O: 408.20. Found: 409.43/411.41 (M/M+2)+.
To a stirred solution of 4-chloro-N-(7-(1,2,3,4-tetrahydroquinolin-4-yl)spiro[3.5]nonan-2-yl)benzamide (43 mg, 0.105 mmol) in DME (2 mL) was added 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione (21 mg, 0.084 mmol) and the resulting mixture was stirred at 80° C. by microwave for 1.5 h. The reaction mixture was partitioned between ethyl acetate and water. The organic layer was separated and washed Na2CO3 and brine, dried over Na2SO4. Solvent was removed under vacuum and the residue was purified by Prep. HPLC to afford the title compound (12 mg, 28% yield). 1H NMR (400 MHz, DMSO) δ 8.82 (d, J=4.5 Hz, 1H), 8.70 (d, J=7.4 Hz, 1H), 8.22 (d, J=8.3 Hz, 1H), 8.02 (d, J=8.2 Hz, 1H), 7.89 (d, J=8.5 Hz, 2H), 7.75 (t, J=7.6 Hz, 1H), 7.63 (t, J=7.6 Hz, 1H), 7.54 (d, J=8.5 Hz, 2H), 7.41 (d, J=4.6 Hz, 1H), 4.46-4.38 (m, 1H), 3.38-3.32 (m, 1H), 2.40-2.34 (m, 1H), 2.17-2.10 (m, 1H), 1.98-1.90 (m, 2H), 1.88-1.62 (m, 7H), 1.58-1.50 (m, 1H). LCMS (ESI) m/z calcd for C25H25ClN2O: 404.17. Found: 405.38/407.35 (M/M+2)+.
To a stirred solution of 7-(1,2,3,4-tetrahydroquinolin-4-yl)spiro[3.5]nonan-2-amine (25 mg, 0.094 mmol) and 5-chlorothiophene-2-carboxylic acid (15 mg, 0.094 mmol) in DCM (2 mL) was added DIPEA (36 mg, 0.28 mmol) followed by HATU (36 mg, 0.094 mmol). After stirred at Et. for 2 h, the reaction mixture was quenched with brine and the resulting mixture was extracted with DCM (×3). The combined organic layers were dried over Na2SO4. Solvent was removed under vacuum and the residue was purified by column chromatography on silica gel (0˜50% EA in PE) to afford the title compound (23 mg, 59% yield). LCMS (ESI) m/z calcd for C23H27ClN2OS: 414.15. Found: 415.36/417.32 (M/M+2)+.
To a stirred solution of 5-chloro-N-(7-(1,2,3,4-tetrahydroquinolin-4-yl)spiro[3.5]nonan-2-yl)thiophene-2-carboxamide (23 mg, 0.055 mmol) in DME (2 mL) was added 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione (11 mg, 0.044 mmol) and the resulting mixture was stirred at 80° C. by microwave for 1.5 h. The reaction mixture was partitioned between ethyl acetate and water. The organic layer was separated and washed Na2CO3 and brine, dried over Na2SO4. Solvent was removed under vacuum and the residue was purified by Prep. HPLC to afford the title compound (12 mg, 53% yield). 1H NMR (400 MHz, DMSO) δ 8.82 (d, J=4.5 Hz, 1H), 8.70 (d, J=7.4 Hz, 1H), 8.22 (d, J=8.4 Hz, 1H), 8.02 (d, J=7.8 Hz, 1H), 7.75 (t, J=7.1 Hz, 1H), 7.68 (d, J=4.0 Hz, 1H), 7.63 (t, J=7.1 Hz, 1H), 7.40 (d, J=4.5 Hz, 1H), 7.19 (d, J=4.0 Hz, 1H), 4.36 (dd, J=16.1, 8.2 Hz, 1H), 3.39-3.35 (m, 1H), 2.38-2.33 (m, 1H), 2.16-2.10 (m, 1H), 1.96-1.88 (m, 2H), 1.86-1.74 (m, 4H), 1.72-1.52 (m, 4H). LCMS (ESI) m/z calcd for C23H23ClN2OS: 410.12. Found: 411.30/413.25 (M/M+2)+.
At 0° C., to a suspension of NaH (153 mg, 3.82 mmol, 60% in oil) in anhydrous THF (3 mL) under nitrogen with vigorous stirring was added the triethyl phosphonoacetate (972 mg, 4.34 mmol) dropwise. After stirred at 0° C. for 30 min, 8,11-dioxadispiro[3.2.47.24]tridecan-2-one (550 mg, 2.55 mmol) in THF (2 mL) was added dropwise. The resulting mixture was allowed to warm up to room temperature and stirred overnight. The reaction mixture was poured into saturated aq. NH4Cl and extracted with EtOAc. The organics were washed sequentially with water and brine, and dried over Na2SO4. Filtration and concentration in vacuum gave a crude product, which was purified by flash chromatography (silica gel, 0-30% EtOAc in PE) to afford the title compound (600 mg, 88% yield). LCMS (ESI) m/z calcd for C15H22O4: 266.15. Found: 267.27 (M+1)+.
A mixture of ethyl 2-(8,11-dioxadispiro[3.2.47.24]tridecan-2-ylidene)acetate (600 mg, 2.25 mmol) and 10% Pd/C (300 mg) in EtOH (10 mL) was stirred at room temperature under H2 atmosphere (15 psi) overnight. The resulting mixture was filtered through a pad of Celite and the filtrate was concentrated under reduced pressure to afford the title compound (0.59 g, 86% yield), which was used in the following step without purification. LCMS (ESI) m/z calcd for C15H24O4: 268.17. Found: 269.43 (M+1)+.
To a solution of ethyl 2-(8,11-dioxadispiro[3.2.47.24]tridecan-2-yl)acetate (0.59 g, 2.20 mmol) in acetone (10 mL), was added 1 N HCl (11 mL, 11.0 mmol) dropwise. After the reaction mixture was stirred at room temperature overnight, water and EtOAc were added and the layers were separated. The organics were washed sequentially with water and brine, and dried over Na2SO4. Filtration and concentration in vacuum gave a crude product, which was purified by flash chromatography (silica gel, 0-30% EtOAc in PE) to afford the title compound (468 mg, 95% yield). LCMS (ESI) m/z calcd for C13H20O3: 224.14. Found: 225.28 (M+1)+.
At −78° C., to a solution of N,N-dibenzyl-8,11-dioxadispiro[3.2.47.24]tridecan-2-amine (340 mg, 1.52 mmol) and N-phenyl bis-(trifluromethanesulfonamide) (704 mg, 1.97 mmol) in THF (8 mL), was added LiHMDS (1.97 mL, 1.97 mmol) dropwise during 30 min and the reaction mixture was allowed to warm up to room temperature and stirred overnight. The reaction mixture was quenched with aq. NH4Cl and the resulting mixture was extracted with EtOAc. The combined organic layers were washed sequentially with water and brine, and dried over Na2SO4. Filtration and concentration in vacuum gave a crude product, which was purified by flash chromatography (silica gel, 0-10% EtOAc in PE) to afford the title compound (220 mg, 41% yield). LCMS (ESI) m/z calcd for C14H19F3O5S: 356.09. Found: 357.40 (M+1)+.
A suspension of ethyl 2-(7-(((trifluoromethyl)sulfonyl)oxy)spiro[3.5]non-6-en-2-yl)acetate (220 mg, 0.62 mmol), quinolin-4-ylboronic acid (115 mg, 0.70 mmol), Pd(PPh3)4 (54 mg, 0.047 mmol), NaBr (69 mg, 0.67 mmol) and Na2CO3 (148 mg, 1.40 mmol) in dioxane (4.0 mL) and water (1.0 mL) was stirred under nitrogen atmosphere at 100° C. for 14 hours. After the reaction mixture was cooled to room temperature, this was partitioned between water and EtOAc and the layers were separated. The organics were washed sequentially with water and brine, and dried over Na2SO4. Filtration and concentration in vacuum gave a crude product, which was purified by flash chromatography to afford the title compound (71 mg, 34% yield). LCMS (ESI) m/z calcd for C22H25NO2: 335.19. Found: 336.35 (M+1)+.
A mixture of ethyl 2-(7-(quinolin-4-yl)spiro[3.5]non-6-en-2-yl)acetate (71 mg, 0.21 mmol) and 10% Pd/C (40 mg) in EtOH (3.0 mL) was stirred at room temperature under H2 atmosphere (15 psi) overnight. The resulting mixture was filtered through a pad of Celite and the filtrate was concentrated under reduced pressure to give the crude product which was purified by flash chromatography (silica gel, 0-50% EtOAc in PE) to afford the title compound (63 mg, 89% yield) as a brown oil. LCMS (ESI) m/z calcd for C22H27NO2: 337.20. Found: 338.27 (M+1)+.
To a solution of ethyl 2-(7-(quinolin-4-yl)spiro[3.5]nonan-2-yl)acetate (63 mg, 0.19 mmol) in MeOH (2 mL) was added 1N NaOH aq. (0.5 mL). After stirred at room temperature for 2h, the resulting mixture was neutralized with 1N HCl and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated to give the title compound (41 mg, 70% yield) as a pale solid, which was used in the following step without further purification. LCMS (ESI) m/z calcd for C20H23NO2: 309.17. Found: 310.20 (M+1)+.
To a stirred solution of 2-(7-(quinolin-4-yl)spiro[3.5]nonan-2-yl)acetic acid (45 mg, 0.145 mmol) and 4-chloroaniline (18.5 mg, 0.145 mmol) in DCM (2 mL) was added DIPEA (75 uL, 0.435 mmol) followed by HATU (55 mg, 0.145 mmol). After stirred at r.t. overnight, the reaction mixture was quenched with brine and the resulting mixture was extracted with DCM (×3). The combined organic layers were dried over Na2SO4. Solvent was removed under vacuum and the residue was purified by Prep. HPLC to afford the title compound (20 mg, 33% yield). 1H NMR (400 MHz, CDCl3) δ 9.99 (s, 1H), 8.80 (d, J=4.5 Hz, 1H), 8.21 (d, J=8.3 Hz, 1H), 8.01 (d, J=8.3 Hz, 1H), 7.76-7.71 (m, 1H), 7.66-7.57 (m, 3H), 7.38 (d, J=4.6 Hz, 1H), 7.34 (d, J=8.9 Hz, 2H), 2.68-2.61 (m, 1H), 2.48-2.42 (m, 3H), 2.16-2.10 (m, 1H), 1.99-1.89 (m, 2H), 1.80-1.69 (m, 3H), 1.66-1.47 (m, 6H). LCMS (ESI) m/z calcd for C26H27ClN2O: 418.18. Found: 419.32/421.27 (M/M+2)+.
To a solution of 8, 11-dioxadispiro[3.2.47.24]tridecan-2-one (4 g, 20.4 mmol) in MeOH (100 mL) was added NaBH4 (1.54 g, 40.8 mmol) portion wise. After stirred at room temperature for 30 min, the mixture was partitioned between ethyl acetate and aq. The layers were separated and the organic layer was washed with brine and dried over Na2SO4. Filtration and concentration in vacuum gave a crude product, which was purified by flash chromatography (silica gel, 0-30% EtOAc in PE) to afford the title compound (3.6 g, 34% yield). LCMS (ESI) m/z calcd for C11H18O3: 198.13. Found: 199.21(M+1)+.
At 0° C., to a solution of 8,11-dioxadispiro[3.2.47.24]tridecan-2-ol (3.6 g, 18.2 mmol) and TEA (7.6 mL, 54.5 mmol) in DCM (40 mL) was added MsCl (2.8 mL, 36.4 mmol) drop wise. After stirred at room temperature for 1 hour, the reaction mixture was partitioned between DCM and water. The layers were separated and the organic layer was washed with brine, dried over Na2SO4, concentrated in vacuum to give a crude product, which was used in the following step without purification (4.0 g, 80% yield). LCMS (ESI) m/z calcd for C12H20O5S: 276.10. Found: 277.36 (M+1)+.
A suspension of 8,11-dioxadispiro[3.2.47.24]tridecan-2-yl methanesulfonate (1.56 g, 5.64 mmol), KCN (551 mg, 8.46 mmol), 18-C-6 (1.49 g, 5.64 mmol) and NaI (845 mg, 5.64 mmol) in DMSO (15 mL) was stirred at 130° C. for 2 hours. After cooled to room temperature, the reaction mixture was partitioned between ethyl acetate and water. The layers were separated and the organic layer was washed with brine, dried over Na2SO4, concentrated in vacuum to give a crude product, which was purified by flash chromatography (silica gel, 0-30% EtOAc in PE) to afford the title compound (500 mg, 43% yield). LCMS (ESI) m/z calcd for C12H17NO2: 207.13. Found: 208.32(M+1)+.
To a solution of 8,11-dioxadispiro[3.2.47.24]tridecane-2-carbonitrile (500 mg, 2.41 mmol) in EtOH (2.4 mL) was added 1N LiOH (2.4 mL, 2.41 mmol) and the mixture was stirred at 90° C. for 2 hours. After cooled to room temperature, the reaction mixture was acidified with 1N HCl to pH 3-4 and partitioned between ethyl acetate and water. The layers were separated and the organic layer was washed with brine, dried over Na2SO4, concentrated in vacuum. The resulting crude product was dissolved in THF (5 mL) and treated with 2 N aq. HCl (4 mL). After stirred at room temperature for 4 hours, the reaction mixture was concentrated and the crude product was purified by flash chromatography (silica gel, 0-50% EtOAc in PE) to afford the title compound (360 mg, 69% yield). LCMS (ESI) m/z calcd for C10H14O3: 182.09. Found: 183.30(M+1)+.
To a suspension of 7-oxospiro[3.5]nonane-2-carboxylic acid (380 mg, 2.08 mmol), K2CO3 (862 mg, 6.24 mmol) in acetone (4 mL) was added methyl iodide (1.48 g, 10.4 mmol). After stirred at room temperature overnight, the reaction mixture was filtered through celite and the filtrate was concentrated under reduced press to give a crude product, which was purified by column chromatography (silica gel, 0-30% EtOAc in PE) to afford the title compound (360 mg, 88% yield). LCMS (ESI) m/z calcd for C11H16O3: 196.11. Found: 197.28(M+1)+.
Preparation of methyl 7-(((trifluoromethyl)sulfonyl)oxy)spiro[3.5]non-6-ene-2-carboxylate
At -78° C., to a solution of methyl 7-oxospiro[3.5]nonane-2-carboxylate (234 mg, 1.19 mmol) and N-phenyl bis-(trifluromethanesulfonamide) (639 mg, 1.79 mmol) in THF, was added 1M LiHMDS (1.79 mL, 1.79 mmol) drop wise over 30 min. The mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was quenched with aq. NH4C1 and the resulting mixture was extracted with EtOAc. The combined organic layers were washed sequentially with water and brine, and dried over Na2SO4. Filtration and concentration in vacuum gave a crude product, which was purified by flash chromatography (silica gel, 0-10% EtOAc in PE) to afford the title compound (340 mg, 87% yield). LCMS (ESI) m/z calcd for C12H15F3O5S: 328.06. Found: 329.27(M+1)+.
A suspension of methyl 7-(((trifluoromethyl)sulfonyl)oxy)spiro[3.5]non-6-ene-2-carboxylate (340 mg, 1.03 mmol), (6-fluoroquinolin-4-yl)boronic acid (339 mg, 1.24 mmol), Pd(PPh3)4 (239 mg, 0.21 mmol), KBr (112 mg, 1.03 mmol) in dioxane (4 mL) and water (1 mL) was stirred at 100° C. under nitrogen atmosphere for 14 hours. After cooled to room temperature, the reaction mixture was partitioned between water and EtOAc and the layers were separated. The layers were separated and the organic layer was washed with brine, dried over Na2SO4, concentrated in vacuum to give a crude product, which was purified by flash chromatography to afford the title compound (68 mg, 20% yield). LCMS (ESI) m/z calcd for C20H20FNO2: 325.15. Found: 326.37 (M+1)+.
A suspension of methyl 7-(6-fluoroquinolin-4-yl)spiro[3.5]non-6-ene-2-carboxylate (68 mg, 0.207 mmol) and 10% Pd/C (34 mg) in MeOH (2 mL) was stirred for 30 mins at room temperature under H2 atmosphere (15 psi). The resulting mixture was filtered through a pad of celite and the filtrate was concentrated under reduced pressure to give the crude product, which was purified by flash chromatography (silica gel, 0-50% EtOAc in PE) to afford the title compound (40 mg, 59% yield) as a colorless oil. LCMS (ESI) m/z calcd for C20H22FNO2: 327.16. Found: 328.34 (M+1)+.
To a solution of 7-(6-fluoroquinolin-4-yl)spiro[3.5]nonane-2-carboxylate (40 mg, 0.122 mmol) in MeOH (1mL) was added 1N aq. LiOH (0.3 mL). After stirred at room temperature overnight, the resulting mixture was neutralized with 1N HCl to pH 6-7 and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated to give the title compound (20 mg, 52% yield) as a pale solid, which was used in the following step without further purification. LCMS (ESI) m/z calcd for C19H20FNO2: 313.15. Found: 314.23 (M+1)+.
To a stirred solution of 7-(6-fluoroquinolin-4-yl)spiro[3.5]nonane-2-carboxylic acid (16 mg, 0.051 mmol) and 4-chloroaniline (7.8 mg, 0.061 mmol) in DCM (1 mL) was added DIPEA (19 uL, 0.153 mmol) followed by HATU (29 mg, 0.076 mmol). After stirred at r.t. overnight, the reaction mixture was quenched with brine and the resulting mixture was extracted with DCM (×3). The combined organic layers were dried over Na2SO4. Solvent was removed under vacuum and the residue was purified by Prep. HPLC to afford the title compound (3 mg, 14% yield). 1H NMR (400 MHz, CDCl3) δ 8.81 (d, J=4.2 Hz, 1H), 8.19-8.12 (m, 1H), 7.66 (dd, J=10.5, 2.6 Hz, 1H), 7.56-7.43 (m, 3H), 7.31-7.27 (m, 3H), 7.07 (s, 1H), 3.17-3.06 (m, 2H), 2.23-2.19 (m, 2H), 2.05 (dd, J=21.4, 9.3 Hz, 3H), 1.96-1.92 (m, 1H), 1.88-1.85 (m, 1H), 1.69-1.56 (m, 5H). LCMS (ESI) m/z calcd for C25H24CIFN2O: 422.16. Found: 423.40/425.37 (M/M+2)+.
Compounds of the present invention were tested via high-throughput cellular assays utilizing detection of kynurenine via mass spectrometry and cytotoxicity as end-points. For the mass spectrometry and cytotoxicity assays, human peripheral blood mononuclear cells (PBMC) (PB003F; AllCells®, Alameda, Calif.) were stimulated with human interferon-γ (IFN-γ) (Sigma-Aldrich Corporation, St. Louis, Mo.) and lipopolysaccharide from Salmonella minnesota (LPS) (Invivogen, San Diego, Calif.) to induce the expression of indoleamine 2, 3-dioxygenase (IDO1). Compounds with IDO1 inhibitory properties decreased the amount of kynurenine produced by the cells via the tryptophan catabolic pathway. Cellular toxicity due to the effect of compound treatment was measured using CellTiter-Glo® reagent (CTG) (Promega Corporation, Madison, Wis.), which is based on luminescent detection of ATP, an indicator of metabolically active cells.
In preparation for the assays, test compounds were serially diluted 3-fold in DMSO from a typical top concentration of 1 mM or 5 mM and plated at 0.5 μL in 384-well, polystyrene, clear bottom, tissue culture treated plates with lids (Greiner Bio-One, Kremsmünster, Austria) to generate 11-point dose response curves. Low control wells (0% kynurenine or 100% cytotoxicity) contained either 0.5 μL of DMSO in the presence of unstimulated (−IFN-γ/−LPS) PBMCs for the mass spectrometry assay or 0.5 μL of DMSO in the absence of cells for the cytotoxicity assay, and high control wells (100% kynurenine or 0% cytotoxicity) contained 0.5 μL of DMSO in the presence of stimulated (+IFN-γ/+LPS) PBMCs for both the mass spectrometry and cytotoxicity assays.
Frozen stocks of PBMCs were washed and recovered in RPMI 1640 medium (Thermo Fisher Scientific, Inc., Waltham, Mass.) supplemented with 10% v/v heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc., Waltham, Mass.), and 1× penicillin-streptomyci antibiotic solution (Thermo Fisher Scientific, Inc., Waltham, Mass.). The cells were diluted to 1,000,000 cells/mL in the supplemented RPMI 1640 medium. 50 μL of either the cell suspension, for the mass spectrometry assay, or medium alone, for the cytotoxicity assay, were added to the low control wells, on the previously prepared 384-well compound plates, resulting in 50,000 cells/well or 0 cells/well respectively. IFN-γ and LPS were added to the remaining cell suspension at final concentrations of 100 ng/ml and 50 ng/ml respectively, and 50 μL of the stimulated cells were added to all remaining wells on the 384-well compound plates. The plates, with lids, were then placed in a 37 oC, 5% CO2 humidified incubator for 2 days.
Following incubation, the 384-well plates were removed from the incubator and allowed to equilibrate to room temperature for 30 minutes. For the cytotoxicity assay, CellTiter-Glo® was prepared according to the manufacturer's instructions, and 40 μL were added to each plate well. After a twenty-minute incubation at room temperature, luminescence was read on an EnVision® Multilabel Reader (PerkinElmer Inc., Waltham, Mass.). For the mass spectrometry assay, 10 μL of supernatant from each well of the compound-treated plates were added to 40 μL of acetonitrile, containing 10 μM of an internal standard for normalization, in 384-well, polypropylene, V-bottom plates (Greiner Bio-One, Kremsmünster, Austria) to extract the organic analytes. Following centrifugation at 2000 rpm for 10 minutes, 10 μL from each well of the acetonitrile extraction plates were added to 90 μL of sterile, distilled H2O in 384-well, polypropylene, V-bottom plates for analysis of kynurenine and the internal standard on the RapidFire 300 (Agilent Technologies, Santa Clara, Calif.) and 4000 QTRAP MS (SCIEX, Framingham, Mass.). MS data were integrated using Agilent Technologies' RapidFire Integrator software, and data were normalized for analysis as a ratio of kynurenine to the internal standard.
The data for dose responses in the mass spectrometry assay were plotted as % IDO1 inhibition versus compound concentration following normalization using the formula 100−(100*((U−C2)/(C1−C2))), where U was the unknown value, C1 was the average of the high (100% kynurenine; 0% inhibition) control wells and C2 was the average of the low (0% kynurenine; 100% inhibition) control wells. The data for dose responses in the cytotoxicity assay were plotted as % cytotoxicity versus compound concentration following normalization using the formula 100−(100*((U−C2)/(C1−C2))), where U was the unknown value, C1 was the average of the high (0% cytotoxicity) control wells and C2 was the average of the low (100% cytotoxicity) control wells.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2018/058389 | 10/26/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62578704 | Oct 2017 | US |
