discovery and in vivo evaluation of ( s )- n -(1-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9 h...

Discovery and in Vivo Evaluation of (S)‑N‑(1-(7-Fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)‑9H‑purin-6-amine (AMG319) and RelatedPI3Kδ Inhibitors for Inflammation and Autoimmune DiseaseTimothy D. Cushing,*,† Xiaolin Hao,† Youngsook Shin,† Kristin Andrews,⊥ Matthew Brown,†

Mario Cardozo,† Yi Chen,† Jason Duquette,† Ben Fisher,† Felix Gonzalez-Lopez de Turiso,† Xiao He,†

Kirk R. Henne,‡ Yi-Ling Hu,∥ Randall Hungate,⊥ Michael G. Johnson,† Ron C. Kelly,§ Brian Lucas,†

John D. McCarter,⊥ Lawrence R. McGee,† Julio C. Medina,† Tisha San Miguel,⊥ Deanna Mohn,∥

Vatee Pattaropong,† Liping H. Pettus,⊥ Andreas Reichelt,⊥ Robert M. Rzasa,⊥ Jennifer Seganish,†

Andrew S. Tasker,⊥ Robert C. Wahl,⊥ Sharon Wannberg,∥ Douglas A. Whittington,# John Whoriskey,∥

Gang Yu,∥ Leeanne Zalameda,⊥ Dawei Zhang,⊥ and Daniela P. Metz∥

†Department of Therapeutic Discovery, ‡Department of Pharmacokinetics and Drug Metabolism, and §Department of PharmaceuticsResearch and Development, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, California 94080, United States∥Department of Inflammation Research, and ⊥Department of Therapeutic Discovery, Amgen Inc., One Amgen Center Drive,Thousand Oaks, California 91320, United States#Department of Therapeutic Discovery, Amgen Inc., 360 Binney Street, Cambridge, Massachusetts 02142, United States

*S Supporting Information

ABSTRACT: The development and optimization of a series of quinolinyl-purines as potent and selective PI3Kδ kinase inhibitors with excellentphysicochemical properties are described. This medicinal chemistry effort ledto the identification of 1 (AMG319), a compound with an IC50 of 16 nM in ahuman whole blood assay (HWB), excellent selectivity over a large panel ofprotein kinases, and a high level of in vivo efficacy as measured by two rodentdisease models of inflammation.

■ INTRODUCTION

Phosphoinositides (PIs) play key roles in multiple cell functionsin their capacity as second messengers, including cell survival,signal transduction, and control of membrane trafficking andtransport.1,2 Dysfunctional regulation of the various phosphoi-nositides has been implicated in a variety of diseases includingcancer, autoimmune disorders, and inflammation, to name afew. Phosphoinositides are lipids consisting of two bilayer fattyacids attached via a glycerol phosphate linkage to a cytosolic 1-inositol. Lipid kinases known as PI3Ks utilize ATP tophosphorylate the 3-OH of the inositol ring moiety, convertingthe PI phosphatidylinositol 4,5-bisphosphate (PIP2) tophosphatidylinositol 3,4,5-trisphosphate (PIP3).3 PIP3 func-tions as an anchoring site for protein kinases containing thehighly conserved pleckstrin homology (PH) domain.4 Thesekinases in turn are further activated (phosphorylated ordephosphorylated), regulating a great number of importantcellular functions. Arguably, the most important PIP3 bindingprotein kinase, the serine/threonine kinases AKT (or PKB),

controls much of the growth, survival, and proliferation of thecell through further downstream proteins such as mTOR,GSK3β, Foxo3a, p70S6K, and NF-κB. Calcium mobilizationand gene transcription are also governed by PH-domaincontaining tyrosine kinases of the Tec family; BTK, ETK, andITK.5 Thus, phosphoinositide metabolizing enzymes such asPI3Ks are attractive targets to modulate the functions of thesesecond messengers.Of the several classes of PI3Ks, the most studied is class 1,

which is further delineated into two subclasses (class 1A andclass 1B). Class 1A (PI3Kα, -β, and -δ) are downstream ofreceptor tyrosine kinases (RTKs), while the class 1B (PI3Kγ) isdownstream of G-protein-coupled receptors (GPCRs). Thesefour homologous kinases exist as heterodimers of regulatory

Special Issue: New Frontiers in Kinases

Received: October 20, 2014

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and catalytic subunits. The regulatory subunit contains domainsthat allow for anchoring to cell surface receptors and otherregulatory proteins, and the catalytic subunit (p110α, -β, -γ, -δ)contains the ATP binding domain. It is this catalytic subunitand its ATP binding site that are the focus of small moleculeinhibitor development. By the competitive inhibition of ATPbinding, the phosphorylation of PIP2 is blocked and PIP3 isnot formed. The important regulatory proteins such as AKT areprevented from anchoring to the cell membrane, thus inhibitingtheir function.While PI3Kα and PI3Kβ are ubiquitously expressed, PI3Kγ

and PI3Kδ are found in leukocytes with PI3Kδ nearly confinedto spleen, thymus, and peripheral blood leukocytes.6 Althoughthe dysregulation of PI3Kα and PI3Kβ is implicated in theetiology of solid tumors, the dysregulation of PI3Kγ and PI3Kδhas been implicated in diseases of the innate and adaptiveimmune system such as rheumatoid arthritis (RA), systemiclupus erythematosus (SLE), and hematological malignancies.Our interest in developing a small molecule inhibitor targetingPI3Kδ stems from its more restrictive expression pattern, thedata accumulated from genetically modified mice and twosuccessful biologics targeting B cells. These biologics, themonoclonal antibodies rituximab and belimumab, have provenefficacious against RA and SLE, respectively. Because PI3Kδ hasa singular role in B cell maturation and function,7 its targetingshould mimic the effects of the two biologic drugs. In additionto B cell modulation, PI3Kδ plays a partial role in T-cellactivation8 and neutrophil trafficking9 and also contributes toactivation of other leukocytes that have been implicated inautoimmune disease, such as macrophages, dendritic cells, andNK T-cells.10

New and surprising insights into the role of PI3Kδ inleukocyte function stem from two recent publicationsdescribing a dominant gain-of-function PI3Kδ mutation inseveral unrelated European families with an unusual combina-tion of lymphoproliferation and immunodeficiency thatpredisposes them to lung and ear infections.11 Theimmunodeficiency associated with this mutation is somewhatsurprising, and the mechanism is currently not understood. Itwas speculated that the hyperactive p110δ-AKT-mTORpathway promotes aerobic glycolysis, limiting the functionand survival of lymphocytes and driving them to premature

senescence, thus impairing proper immune responses topathogens.12 Chronic inflammation is not unique to auto-immune disease but is also associated with long-term exposureto stress or physical insult to the body and can lead tocontinuous signaling of the PI3Kδ pathway in respondingleukocytes. For example, in chronic obstructive pulmonarydisease (COPD), constant exposure to cigarette smoke isassociated with high levels of PI3Kδ and phospho-AKT in thelung.13 Like individuals with the gain-of-function PI3Kδmutations, COPD patients are plagued by recurrent bacterialrespiratory infections, including H. inf luenza and S. peumoniae,suggesting that a similar immunodeficiency may be associatedwith persistent PI3Kδ signaling in COPD as is seen with thegenetic gain-of function mutation. The leukocyte defect in thepatients with the gain-of-function mutation can be amelioratedby treatment with rapamycin, an inhibitor of the mTORsignaling pathway downstream of PI3Kδ. It is tempting tospeculate that inhibition of PI3Kδ may be a viable treatmentoption not just for complex autoimmune disorders such as SLEand RA, where PI3Kδ likely drives B-cell and other leukocytesubset activities that culminate in pathology,14−16 but also forchronic nonautoimmune conditions, such as COPD, wherehigh levels of PI3Kδ and phospho-AKT are prominent but maydrive immune deficiencies and hence persistent infections.In this report we discuss the SAR development and in vivo

properties of a series of compounds that selectively target thePI3Kδ isoform in the context of autoimmune and inflammatorydisorders. This effort culminated in the discovery ofpurinylquinoline 1 (AMG319). Although this compound wasdeemed exceptional compared to the other analogs in thisseries, we determined that it would be more advantageous todevelop this compound in an oncology setting. 1 is currentlyundergoing phase 1 clinical trials targeting hematologicalmalignancies.17−20

■ RESULTS AND DISCUSSION

Genesis of Compounds. Knight et al. published alandmark paper21 describing an interesting class of compoundsexisting in a propeller-shaped configuration exemplified by 2(PIK-39) that was profoundly more potent (>50-fold) towardthe PI3Kδ isoform relative to the other class 1 PI3Ks. Thisselectivity between the highly homologous isoforms was

Figure 1. SAR development of the purinylquinoline series, which led to compound 1. By utilization of known literature compounds and insightsgained from two internal kinase programs, compounds 4 and 5 were generated, which in turn led to analog 6. Shown are IC50 values against PI3Kδ(enzyme, Alphascreen assay; for a description see Experimental Section).

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attributed to the unusual overall shape of the molecules.Typical flat kinase inhibitors such as 3 (LY294002)22 bind inthe ATP-binding pocket with no significant difference betweenthe PI3K isoforms. In contrast the quinazolinone 2 forces a keymethionine (Met804 PI3Kγ, conserved in all isoforms) toreorient, accommodating the large quinazolinone moiety. Thisligand-induced methionine reorientation induces a shift of the

peptide backbone that propagates through the upper loop(amino acids 803−811 in PI3Kγ), leading to a conformationalchange in the ATP-binding region enhancing isoform selectivityin favor of the PI3Kδ isoform. It was this theory of binding andselectivity that we set out to exploit.Molecular modeling indicated that an overlap between the

known PI3K inhibitors 2, 3, and a cinnoline template (a key

Table 1. Initial SAR around the Linker between Purine and Quinoline Bicycle

aAlphascreen assay. bIn vitro anti-IgM/CD40L-induced B cell proliferation (as measured by thymidine incorporation) assay. cRat liver microsomal(RLM)/human liver microsomal (HLM) % turnover (TO) (the percentage of compound that is metabolized in 30 min at 37 °C). dPhosphatebuffered saline, pH 7.4, Symyx benchtop system, equilibrated 42−78 h. eRat clearance dosed iv, 0.5 mg/kg, 100% DMSO. fRat % oral bioavailabilitydosed po 2.0 mg/kg, 1% Tween 80, 1% methyl cellulose, 98% water.

Table 2. Comparison of (S)-Methyl Analogs to Des-Methyl Analogs

aAbility of compound to inhibit anti-IgM induced AKT phosphorylation (Ser473) in mouse B cells; phospho-AKT (pAKT) expression on B220+gated B cells was determined by flow cytometry in mouse splenocytes. bCompound pretreated HWB was stimulated with anti-IgD to induce CD-69expression on B cells (6 h) and was evaluated by flow cytometry. cThe intrinsic unbound human whole blood (HWBint,u) potency was derived bymultiplying the PPB fraction unbound by the total HWB potency ( f u × HWB). dCYP: cytochrome p450 competitive assay, probe substrates(midazolam (3A4), 5 μM) (bufuralol (2D6), 8 μM) with compound (3.0 μM) reported as % inhibition (LC/MS). ePassive permeability in LLC-PK1 cells, apparent permeability (Papp) measured by mass balance (LC/MS). fTested at 1.0 μM concentration in rat and human plasma, proteinbinding measured by ultracentrifugation and LC/MS. gIntrinsic unbound hepatic clearance equals the hepatic (total) clearance divided by thefraction unbound (CLint,u = CLH/f u).

hThis compound has a high extraction ratio such that the clearance depends exclusively on hepatic blood flow.iThese drugs have intermediate extraction ratios (0.3 < 0.7) so that the numbers in parentheses could be dependent on liver blood flow, unbounddrug fraction, and the intrinsic metabolic clearance. jDose po 2.0 mg/kg, 1% Tween 80, 1% methylcellulose, 98% water.


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functional component of a p38 program)23 could provide anovel structural starting point (A) with a twist similar to thepropeller shape found in 2 (Figure 1). By combining theseprecursor structures and a gem-dimethylindoline found usefulin a NIK (nuclear factor κB inducing kinase) program,24 weobtained 4, a compound with modest activity in a PI3Kδenzyme assay. However, after replacement of the cinnoline witha quinoline moiety, compounds were found that dramaticallyimproved the potency, such as 525 (0.30 μM) (Figure 1).Further SAR development of this indoline series as

exemplified by 4 and 5 led to very potent, selective, andbioavailable analogs.25 On the basis of these results, it wasenvisioned that the quinoline moiety could provide a significant

advantage as a replacement of the quinazolinone found in 2.Compound 6 had similar enzyme potency to 2, although withsignificantly weaker cell potency.

SAR of Compounds. Although 6 was a reasonable startingpoint, the thioether moiety was a potential metabolic liability.The ether-linked analog 7 was significantly more potent in thePI3Kδ enzyme and cell assays (Table 1). To determine theoptimal linkage, several related analogs, 8, 9, and 10, weresynthesized. The methylene linked 10 had very weak PI3Kδpotency, but the ether-linked 8 and amino-linked 9 were similarin PI3Kδ potency to 7. Additionally, 8 was 34-fold selective forPI3Kδ over the PI3Kγ isoform, while the N-linked 9 was only7-fold selective. These analogs exhibited improved potency and

Table 3. SAR: Potency and Selectivity Data for Analogsa

aFor a description of the assays see Tables 1 and 2. bThe intrinsic unbound human whole blood (HWBint,u) potency was derived by multiplying thePPB fraction unbound by the total HWB potency ( f u × HWB). f u values are found in Table 4.


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selectivity but were poorly soluble and had high turnover inmicrosomes and consequently displayed poor pharmacokineticsin the rat. The observed high clearance was hypothesized to bedue to action of aldehyde oxidase (AO) or xanthine oxidase(XO) on the purine ring.26 The similarly potent and selectivepyrrolopyrimidine 11 was an attempt to blunt this activity, but11 still suffered high microsomal clearance, much like 8 and 9(Table 2). In spite of the low solubility and high metabolism ofthe ether-linked analogs 7, 8, and 11, we were interested in

these compounds because of their higher selectivity; however, itbecame apparent that the compounds are chemically unstable,as the fragment 9H-purin-6-ol (from 7, 8) was identified insolutions after several days at neutral pH. This fragmentationcould occur via an SN2 hydrolysis or an SN1 ionization or both.Analog 12 was synthesized with a methyl group α to the ethermoiety in an attempt to decrease the rate of putative SN2hydrolysis. However, steric hindrance to minimize the SN2mechanism might actually enhance an SN1 mechanism, leading

Table 4. SAR: Physicochemical Properties and PK of Analogs in Table 3

aPermeability; for a description of this and other assays see Tables 1 and 2. bDose iv, 0.5 mg/kg, 100% DMSO. cDose po, 2.0 mg/kg, 1% Tween 80,1% methylcellulose, 98% water. dIntrinsic unbound hepatic clearance equals the hepatic (total) clearance divided by the fraction unbound (CLint,u =CLH/f u). This value is an indication of how the unbound exposure increased with decreasing CLint,u (AUCu = dose/CLint,u.).

eThe equation CLint,u =CLH/f u holds only for compounds with low extraction ratios (<0.3), where f u(CLint, u) ≪ Q (liver blood flow). These drugs have intermediateextraction ratios (0.3 < 0.7) so that the numbers in parentheses could be dependent on liver blood flow, unbound drug fraction, and the intrinsicmetabolic clearance.


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to no net improvement, which was the outcome with 12.Analog 12 exhibited no improvement in stability over the des-methyl analogs but the enzyme potency of 12 toward PI3Kδincreased 5-fold over the des-methyl analog 11. This potencyenhancement was not unexpected, as the methyl moiety wasadvantageous in similar propeller shaped molecules27 becauseof a stereospecific ligand−protein interaction.28 This isdemonstrated by a comparison of the PI3Kδ enzyme potencyof the S-methyl isomer (12, IC50 = 7.1 nM) and R-methylisomer (13, IC50 = 2.6 μM). This S-methyl isomer stereo-selectivity is a property of the entire series (data not shown),perhaps due to a steric clash between the R-methyl group andthe protein. Analog 12 also had significantly improved cellularpotency including a 60-fold positive shift in the B cellproliferation assay (as measured by 3H thymidine incorpo-ration; for details see Experimental Section) compared to 11.Because the metabolic instability of the ether-linked com-pounds was still quite high, our focus shifted to the amino-linked analog 9, which was less potent but chemically stable.The identification of metabolites of 9 treated with rat livermicrosomes implied that the metabolic instability of 9 and 12was partially due to oxidative metabolism of the aromaticmethyl groups. This adverse metabolism led to the synthesis ofdichloro 14. Analog 14 was found to be more stable in theRLM assay than 9 and 12. The major metabolites of 9 werefound in the quinoline and toluyl moieties, but the majormetabolites of 14 were confined to the purine moiety. Thelower metabolism of 14 translated to a slightly improved ratclearance of 1.6 L h−1 kg−1. Additionally, 14 had a rat oralbioavailability of 43% compared to 9% for 9. This is interesting,as both 9 and 14 have low solubility but high permeability(LLC-PK1 parental cell line, Papp of 47 × 10−6 and 41 × 10−6

cm/s, respectively). Thus, the higher oral bioavailability of 14compared to 9 implied that the oral bioavailability was afunction of metabolic stability (Table 2).Analog 14 was the first compound tested in the in vivo

models in spite of the high intrinsic rat clearance and highCYP2D6 inhibition. Additionally, the main driver of the invivo/in vitro correlation, the human whole blood (HWB) assay(anti-IgD induced CD-69 on B cells in 90% human wholeblood), was still suboptimal (HWBint,u = 8.4 nM). Thenoticeable improvement in potency in the enzyme and B cellproliferation assay with 12 compared to 11 led to the synthesisof the (S)-methyl containing 15, the direct analog of 14. Analog15 had the expected increase in potency in both enzyme andcellular assays over 14, including the critical HWB assay wherethe increase in potency was >9-fold (HWBint,u of 0.85 vs 8.4nM). The cellular potency improvement was noticed in otherα-methyl analogs as well. For example, 16 compared veryfavorably to 17, with the B cell proliferation cellular activityimproved over 11-fold and the HWB intrinsic unboundpotency increased by a factor of 5. The intrinsic rat iv clearancefor these analogs 12, 14, 15, 16, and 17 remained high despitethe α-methyl group’s contribution to improvements in otherproperties such as potency.Much of the improvement in enzyme and cellular potency is

likely because the methyl group limits the free rotation aroundthe bond at the 3-position of the quinoline, rigidifying thestructure in a low energy state closer to the bindingconformation. Molecular modeling calculations indicate thatthis difference is about 1 kcal/mol, equivalent to a 10-fold shiftin potency, which is generally consistent with what is observed.With the superiority of the S-methyl group established, we

embarked on further SAR development to improve theseanalogs. An intensive effort to explore the substitution patternaround the 2-phenyl group and the quinoline core structure wasundertaken (Table 3 and Table 4). Substituting the chlorine on15 for fluorine gave 18 providing nearly a 2-fold improvementin the HWB intrinsic unbound potency and a 4-foldimprovement in the rat iv intrinsic clearance. The quinoxaline19, an analog of 16, provided a very similar overall profile withalmost identical cellular and HWB potency. The biochemicalselectivity profile and PK were very similar, but 19 had a lowerlevel of solubility. Because of the improved HWB with fluorinesubstitution on the pendent phenyl ring, it was hypothesizedthat a similar improvement would be actualized by replacing the8-chloroquinoline. The 8-fluoroquinoline 20, a direct analog of16, had the same high bioavailability (F = 100%), similar (high)intrinsic rat iv clearance, but high intrinsic unbound HWBpotency. The cellular potency (B cell proliferation and pAKT)was also similar. Additional fluorine containing analogs 21 and22 displayed a similar intrinsic unbound HWB potency andhigh intrinsic rat clearance, in the case of 22. The related 7-fluoroquinoline analog 23 displayed no advantage over 20. Theintrinsic clearance and HWB activity were very similar, and theoral bioavailability was worse (41% vs 100%). Replacing the 3-fluorophenyl of 23 with 2-methylsulfonylphenyl provided 24, acompound with weaker (17-fold) intrinsic unbound HWBpotency but with a large increase in PBS solubility (62 vs >200μg/mL). The rPPB fraction unbound ( f u) was significantlyhigher, 0.30 (as was hPPB f u), leading to the lower rat ivintrinsic clearance (3.7 L h−1 kg−1) and potentially leading tothe lower oral bioavailability.29

The solubility of the compounds remained problematic. Inthe early analogs 8, 9, and 11 there appeared to be acorrespondence between low solubility and poor bioavailability.A slight improvement in solubility as in 14, 16, and 17 providedan improvement in oral bioavailability but could still beattributed to the high permeability found in these compounds.For example, 19 had poor solubility, high permeability, but highoral bioavailability. In general, the overall permeability of theseries of compounds was quite high and most small structuralchanges had little effect. For example, the (S)-methyl analog 16and des-methyl 17 had identical Papp values of 37 × 10−6 cm/s.The increase in oral bioavailability of 16 compared to 17 couldbe attributed to the increased rigidity of 16 relative to 17. Largeincreases in the PBS solubility led to decreased activity in a bilesalt export pump (BSEP) assay, thought to be a keytoxicological assay for the assessment of compounds. Analog23 had BSEP activity of 19 μM, while the soluble analog 24 wastested at >133 μM. There was also a trend for increases insolubility providing positive effect on CYP inhibition. There-fore, our focus remained on further increasing the solubility inspite of the increased rPPB (and hPPB) unbound fraction,which was interesting30 but initially at least to the detriment ofthe rat PK.31 The goal was to provide a compound that wouldbe highly tractable in a clinical setting.The high solubility of 24, while an intriguing result, was not

replicated with 25, an analog of 16. 3-Methylsulfonyl 25 hadlower solubility than 24, and the oral bioavailability (36%) waslower than 16. Additionally, 25 was less potent in the B cellproliferation assay and was less potent in the HWB assay(HWBint,u) compared to 16 or even 24, although 24 and 25displayed an acceptable permeability.The 3-pyridyl-2-quinoline analog 26 had significantly higher

solubility in PBS buffer compared to 7-fluorophenyl 20 but


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suffered high CYP3A4 inhibition (92%) and higher rat ivclearance. The CYP3A4 inhibition was reduced by placing amethyl group ortho to the 3-pyridyl nitrogen of 26, but thisanalog, 27 still possessed high rat iv clearance (CL = 2.9 L h−1

kg−1). 27 also had very high solubility, similar to 26, and waspotent in the HWB assay (HWBint,u = 5 nM). The high rat ivclearance was modulated by the addition of a fluoro group tothe pyridine ring providing 28, which lowered the rat ivclearance (CL = 0.78 L h−1 kg−1) substantially. The CYP3A4inhibition also decreased in the 2-pyridyl 29, but this analogexhibited higher rat iv clearance. Similarly, 2-pyridyl 30, ananalog of 16, did not have the CYP3A4 inhibition of the 3-pyridyl analogs, but it did have higher CYP2D6 inhibition. Inspite of this, 30 had a reasonable overall profile, including HWBpotency (HWBint,u = 2.8 nM), solubility, and permeability. Thecompound had good oral exposure (F = 87%) but stillunacceptable high rat iv clearance (CLint,u = 37 L h−1 kg−1).The pyridyl 31, an analog of quinoxaline 19 and 30, had lowerrat iv clearance (CLint,u = 8.5 L h−1 kg−1) than both of these andhigher % F than 14. 8-Chloroquinoxaline 32, an analog of 31,had similar properties but with significantly weaker HWBpotency. Although the electron withdrawing 8-cyano sub-stituted quinoline 33 had a lower rat iv clearance than thecorresponding 8-fluoro 29, the intrinsic unbound clearance wassimilar. A comparison between 33 and 30 is revealing. 33 hadsimilar intrinsic unbound HWB potency, 2.5 nM vs 2.8 nM,respectively, but 33 had a significantly higher unbound fractionin rat plasma (0.14 vs 0.027). The in vivo rat iv clearance wassimilar, but the intrinsic clearance of 33 is considerably less withCLint,u values of 7.0 and 37 L h−1 kg−1 for 33 and 30,respectively, indicating a level of protection toward metabolismprovided by the cyano group, although 33 had a lower rat oralbioavailability than 30 (26% vs 87%). The SAR indicated thatthe 2-pyridyl substituted quinoline was nearly optimal,providing greater solubility, lower CYP inhibition, and generallylower intrinsic unbound clearance without sacrificing intrinsicunbound HWB potency. However, the question of where andof what type of electron withdrawing group in the quinolinering was best remained open. Clearly, the necessity of anelectron withdrawing group in the quinoline resulting in adecrease of the in vivo rat iv clearance was demonstrated withcompounds like 33. In general, the difference in SAR between achlorine substituent and a fluorine substituent on the quinolinecore is subtle. Consider the difference in solubility between 16,20, and 23. The fluoroquinolines are slightly less lipophilic andmore soluble than the chloroquinoline. It is only when the 2-pyridyl moiety is combined with the fluoroquinoline core or thechloroquinoline core that the differences in PK start tomanifest. For example, a larger difference in CLint,u values isdisplayed by the 2-pyridyl analogs 29 (8.1 L h−1 kg−1) and 30(37 L h−1 kg−1) than the related pair 16 (43 L h−1 kg−1) and 20(54 L h−1 kg−1). The other 7-fluoro substituted 34, 35, and 1had very high solubility, permeability, and low CYP inhibition.All three compounds are potent in the two cellular and HWBassays, and the rat iv intrinsic unbound clearance was also verylow in 1 and 35, lower than found in 34. The rat oralbioavailability was also higher for 1 than 35. The 7-fluoroquinoline 1 had lower rat iv intrinsic unbound clearancethan 8-fluoroquinoline 29 and 8-chloroquinoline 30 andwithout the CYP2D6 inhibition signal found in 30. 1 hadminimal CYP3A4/2D6 inhibition and did not inhibit CYPs(1A2, 2C8, 2C9, 2C19, 2E1, all >20 μM). There was no timedependent inhibition (TDI) against CYPs 3A4, 2D6, 1A2, and

2C9 nor CYP induction (3A4, 2D6, 1A2, 2B6) as measured inhepatocytes.32 1 was clean in a hERG binding assay (>25 μM),and an Ames micronucleus test proved negative. 1 had minimaleffects in a BSEP assay up to >200 μM. Additionally, 1 wasclean in a large side effect profiling panel (CEREP) and had noactivity in a large panel (Kinomescan) of 359 unique proteinkinases tested at 10 μM drug concentration (Figure 2).

In Vivo Pharmacology. An in vivo model (phosphor-ylation of AKT) was employed as a way to correlate the in vitropAKT cellular data with in vivo pAKT levels. Similar to the invitro studies, membrane-bound immunoglobulin (such as IgMor IgD) serves as a functional B cell receptor through which thecells can be activated via cross-linkage of the receptor withsoluble antibody to the receptor, which leads to increasedphosphorylation of AKT within the cells; in vitro thisphosphorylation can be completely blocked with functionalPI3Kδ-selective inhibitors. For this purpose, in these in vivostudies, mice were injected with anti-IgM to stimulatecirculating B cells. A B-cell transgenic mouse, line 3751,33

was used as these mice are engineered to only express surface-bound IgM and are devoid of circulating IgM. The lack ofcirculating IgM is critical, as circulating IgM can form immunecomplexes with the injected antibody that could lead toimmune-complex-mediated toxicities. Animals were dosed withcompound 15 min prior to iv delivery of FITC-labeled anti-IgMto stimulate B cells. After 30 min the animals were sacrificedand the blood and spleen analyzed for B cells activation bymeasuring pAKT via flow cytometry (Figure 3a,b). When thepAKT vs unbound blood concentrations of the compound wasplotted, an IC50 value was generated (Figure 3c). This was donefor several compounds, and there was a striking correlationbetween the in vivo generated IC50 values and the in vitro IC50values (Table 5). This experiment was also used as a gauge forthe multidose functional, 10-day protein−antigen (KLH-keyhole limpet hemocyanin) challenge model in rats (Table6). These studies were performed to determine the correlation

Figure 2. Kinase dendogram of 1. Red balls on the protein kinasefamily “tree” mark the level of binding. Binding assay results are shownas Kd values (nM) against 359 separate protein kinases, tested at 10μM compound concentration. As can be seen from the figure, 1 wasinactive under these criteria. All kinases tested at >35 POC and 80%were >70 POC. Kinase selectivity experiment and figure were suppliedby DiscoveRx, formerly AMBIT Inc.


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between biochemical coverage (i.e., pAKT) with functionalactivity in vivo. This model allowed assessment of compoundimpact on T-cell-dependent primary B cell responses bymeasuring the ability of the compounds to reduce KLH-specificIgM and IgG production. Doses were chosen that would start

to provide coverage at trough over the calculated in vivo pAKTIC50 values. Some of these doses were envisioned to provide acomplete knockdown of the KLH-specific Ig response.34 Thekey compound 1 achieved this coverage at the 3 mg/kg level,which also covered the HWB (CD-69) IC90 at trough for a full24 h period, perhaps a more relevant marker of how thecompound would behave clinically. The lower doses 0.1, 0.3,and 1 mg/kg covered trough concentrations between the HWBIC50 and IC90 and evinced partial efficacy. Similarly, the plasmaconcentration of 1 covered the IC90 at the 1 mg/kg dose of themouse anti-IgM pAKT in vitro assay (Figure 4).Comparisons of inhibitory activity in HWB to establish

potency are confounded by differences in PPB betweencompounds. PPB corrected HWB IC50 values (HWBint,u)reflect intrinsic activity normalized for unbound compound inthe assay matrix. Compound 1 was among the more activemolecules in blood (HWBint,u = 2.6 nM), and it also exhibitedmeaningful in vivo efficacy when tested in the in vivo pAKTand KLH antigen-challenge pharmacology models. Not uniqueto 1 was the observation that molecules in the series possessedrelatively low PPB in rat and human (Table 4), which likelyenabled good ex vivo HWB and in vivo activity. Though lowPPB could be a factor leading to high hepatic clearance (CLH)in vivo, compounds with sufficiently low intrinsic clearance(CLint,u) are able to maintain efficacious exposures at lower

Figure 3. Vehicle, benchmark compound 2-(((2-amino-9H-purin-6-yl)amino)methyl)-5-methyl-3-(o-tolyl)quinazolin-4(3H)-one (D-073),28,35 and1 were dosed orally to the transgenic (IgMm) mice. Fifteen minutes later they were injected (iv) with anti-IgM/FITC. Blood (a) and spleen tissue(b) were collected after 30 min, stained, and analyzed for phospho-AKT (p-AKT) levels by flow cytometry. The cells were gated on B220+ (B cells)and FITC positive cells analyzed. Mann−Whitney U test was used to evaluate differences between groups. The asterisk (∗) denotes statisticalsignificance (p < 0.05) when compared to the control group. (c) Unbound concentrations were plotted against % remaining pAKT activity (100 − %pAKT inhibition) measured in peripheral blood B cells for each corresponding dose. Total plasma concentrations of 1 from two separateexperiments (a) were converted to estimated unbound concentrations using mouse plasma protein binding data ( fu = 0.0442). The estimated in vivounbound drug concentration pAKT IC50 was 1.9 nM and was in reasonable agreement with the pAKT murine splenocyte B-cell-based in vitrounbound drug concentration IC50 of 1.1 nM [calculated from 1.5 nM total drug IC50 × 0.711f u,media. Quantitation of 1 in mouse plasma wasperformed using a sensitive and selective LC−MS/MS method. MS analysis was carried out using electrospray ionization (ESI) in the positive mode,with detection of analyte and internal standard N-((1R)-1-(3-(4-(ethyloxy)phenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl)ethyl)-N-(3-pyridinylmethyl)-2-(4((trifluoromethyl)oxy)phenyl)acetamide by multiple reaction monitoring (MRM). The lower limit of quantitation (LLOQ) inthe assay was 0.3 ng/mL.

Table 5. In Vitro−in Vivo pAKT IC50 Correlations forTested Analogs

calculated, unbound drug concentration IC50 (nM)a

analog in vitro pAKT in vivo pAKT

14 3.0 2.016 3.0 2.019 0.68 0.7322 1.7 2.223 0.68 1.324 2.4 4.530 2.4 2.933 4.3 5.534 3.4 5.01 1.1 1.9

aIn vitro−in vivo pAKT IC50 correlations for selected analogs.Analyses done in a similar way as in Figure 3c. All values corrected forPPB (in vivo) and serum protein binding (in vitro). R2 correlation is0.62


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overall doses. Analog 1 exhibits the lowest rat CLint,u (2.7 L h−1

kg−1) in the series (Table 4), which translated to the lowestED50 values for IgM and IgG reduction among compounds ofsimilar potency tested in the KLH antigen-challenge model.These observations were key in the selection of 1 as a clinicalcandidate.Structure and Selectivity. The hypothesis that substitut-

ing a quinoline would be a viable structural isostere of thequinazolinone in 2 proved to be correct and was supported bythe data and cocrystal structures (Figure 5). Figure 5a showsthe propeller-shaped 1 bound in the ATP binding pocket ofPI3Kγ. The 7-fluoroquinoline lies in the inducible specificitypocket between Trp812 and Met804, and the pyridine projectsout of the ATP-binding pocket toward a site referred to ashydrophobic region II.36 Figure 5b shows the inhibitor withselected residue differences between the class I PI3K isoforms,and several key intramolecular hydrogen bonds are indicated asdashed lines, including the energetically important hingeVal882-purine contact. Figure 5c shows the overlay of PI3Kγ+ 1 with the published cocrystal structure of 2 (yellow) boundto PI3Kδ (PDB code 2WXF).36 This overlay illustrates thesimilar binding conformation of the two structural types andhighlights the similarity of the two ATP binding pockets.Structural and computational analyses suggest that the maindifference in the selectivity of the propeller-shaped kinaseinhibitors among the various PI3K isoforms is due todifferences in the flexibility of the P-loop.36,37 This loop(analogous to the P-loop found in protein kinases) comprisesresidues 752−758 in PI3Kδ and is more accommodating of theperpendicular quinoline and quinazolinone of 1 and 2,respectively, than are similar loops in other isoforms.The significant conformational similarity between 1 and 2 is

replicated with other analogs in this series. The selectivitydifferences observed between the analogs are subtle and havemuch to do with a slight shifting of the inhibitor in the bindingsite due to small structural changes. For example, the ether-linked 8 was 5-fold more selective for PI3Kδ selective overPI3Kγ than the amino-linked 9, but structural studies andmodeling show a very close overlap. Generally, although PI3Kδ

potency improved, there was little difference in selectivity witha methyl substituent in the amino or ether linkage, as acomparison of the pairs 11 vs 12, 14 vs 15, and 17 vs 16 shows(Table 2). The largest selectivity gain involved PI3Kα/δ where1-ethanamine 15 exhibited a 4-fold improvement in selectivityover methanamine 14. However, for the ether-linked pair 11and 12 the selectivity difference of PI3Kα/δ was reversed. Theethoxy-linked 12 was 4-fold less selective compared to themethoxy-linked 11. The final pair was most similar tomethylamine 17 having a 3-fold PI3Kγ/δ selectivity advantagecompared to 1-ethylamine 16. There was only a smallselectivity trend when substituting the quinoline core for aquinoxaline. The PI3Kβ/δ selectivity of quinoxaline 31 was 6-fold greater than the PI3Kβ/δ selectivity of quinoline 30, andthere was a 50-fold difference in favor of 31 comparing PI3Kγ/δ selectivity. For the structurally similar pair 16 and 19 theselectivity differential was less pronounced. Quinoxaline 19 was5-fold more PI3Kγ/δ selective than quinoline 16 and only 4-fold more PI3Kβ/δ selective. However, this trend wascontradicted with quinoline 1 and quinoxaline 34, which hadnearly equivalent selectivity on average. With respect to thesubstituted phenyl (or pyridyl) group, e.g., the third arm of thepropeller, there appeared to be a small preference for 2-substituted phenyl groups compared to 3-substitution, as theselectivity difference between 16 and 18 bears out. 18 is nearly7-fold more selective for PI3Kδ versus PI3Kγ. The X-raycocrystal structure of 16 indicates that the 3-fluoro substituentmakes a positive (σ-hole) contact with the carbonyl of Ala885in PI3Kγ while the 2-fluoro substituent is unable to make thiscontact in the same orientation (pointing toward Trp812).38

The three other isoforms have a serine instead of alanine at thisposition. This analysis is complicated in that the barrier towardphenyl ring rotation remains small, even though the final lowenergy form adopts a 90° configuration relative to the quinolinemoiety (Figure 6a). The 2-chlorophenyl 15 also proved muchweaker in potency toward PI3Kα, which has almost 10-foldgreater selectivity than 3-fluoro 16. A fluorine substituted ateither the 7- or 8-position of the quinoline exhibits a larger levelof selectivity than the 8-chloroquinolines, as a comparison of 8-

Table 6. % Inhibition of the KLH Induced Inflammatory Response with Selected Analogs

% KLH inhibitiona with dose q.d. of

analog day 10 30 mg/kg 25 mg/kg 10 mg/kg 3 mg/kg 2.5 mg/kg 1 mg/kg 0.3 mg/kg 0.1 mg/kg ED50b

1 IgM 88 ± 5 80 ± 13 67 ± 15 3 ± 23* 0.25IgG 95 ± 1 83 ± 7 48 ± 14 32 ± 11* 0.35

1c IgM 100d 99d 99d 90 ± 4 0.17IgG 100d 99d 96 ± 2 54 ± 23* 0.28

14 IgM 96 ± 3 89 ± 4 −0.9 ± 45* 6.8IgG 70 ± 12 64 ± 10 14 ± 10* 7.9

16 IgM 100d 99d 92 ± 2 77 ± 5 0.20IgG 100d 100d 84 ± 7 27 ± 20* 0.56

19 IgM 96 ± 2 94 ± 2 91 ± 3 58 ± 24* 0.26IgG 100d 95 ± 2 59 ± 18 42 ± 18* 0.35

22 IgM 99 ± 1 96 ± 2 80 ± 5 46 ± 7* 1.2IgG 100 ± 22 100 ± 20 73 ± 20 20 ± 11* 2.1

30 IgM 89 ± 5 87 ± 5 62 ± 10 65 ± 11 0.51IgG 76 ± 10 71 ± 9 19 ± 12* 26 ± 14* 2.0

34 IgM 83 ± 7 84 ± 4 61 ± 9 29 ± 7* 0.8IgG 96 ± 5 81 ± 9 42 ± 18 32 ± 13* 1.4

aPercent inhibition of the IgM and IgG response of several compounds and the corresponding doses in the KLH model; all data points have p < 0.05compared to vehicle control except for those marked with an asterisk (∗). bED50 (mg/kg) was calculated from these data. cExperiment with 1repeated. All experiments performed as described in Figure 4. dCalculated % SEM is <1.


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fluoro- 20 (PI3Kγ/δ = 24), 7-fluoro- 23 (PI3Kγ/δ = 18), and 8-chloro- 16 (PI3Kγ/δ = 3) or 7-fluoro- 1 (PI3Kγ/δ = 48), 8-fluoro- 29 (PI3Kγ/δ = 57), and 8-chloro- 30 (PI3Kγ/δ = 2)

shows. This selectivity differential was similar for the other twoisoforms. Structurally, it was hypothesized that while the purinering is exactly superimposed, the 7- and 8-fluoroquinolines are

Figure 4. Vehicle or 1 in 2% HPMC, 1% Pluronic F68, 10% Captisol, pH 2.0, was administered (0.1, 0.3, 1, 3 mg/kg) q.d. po for 10 days in femaleLewis rats (N = 8/dose group). At 2 h after the first dosing, 200 μL of PBS containing 60 μg of KLH was administered to each rat intravenously. Tendays after the KLH priming, rats were euthanized and blood was taken by cardiac puncture for the measurement of KLH specific IgG (a) and IgM(b) by ELISA. The y-axis is represented as a mean serum dilution factor. Error bars represent the standard error of the mean (SEM) of eight rats. (c)After administration of 1, plasma was harvested to assess exposures in each dose group. Unbound drug concentrations (mean ± SD) were measuredby LC−MS/MS and plotted relative to HWBint,u CD-69 (IC50 = 2.0 nM; IC90 = 15 nM) and in vitro mouse anti IgM pAKTunbound (IC50 = 1.1 nM;IC90 = 9.2 nM) estimates, represented as dashed lines. The percent values are the attenuation of KLH-specific IgG responses from (a).

Figure 5. (a) Cocrystal structure of p110γ + 1 at 2.7 Å resolution. The orientation of the pyridine ring could not be determined from the electrondensity and was assigned arbitrarily. Dashed lines indicate hydrogen bonds; the red sphere is an ordered water molecule. (b) Amino acid differencescluster on the P-loop (top) and at the mouth of the active site. Not all differences are shown. (c) Overlay of 1 (green) bound to PI3Kγ (blue) and 2(yellow) bound to PI3Kδ (beige). The P-loop in PI3Kγ is colored pink. Residue labels correspond to PI3Kγ.


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slightly puckered out of the plane relative to their 8-chloroquinoline analogs. This rotational difference wasattributed to a larger steric demand placed on the chloroquino-line, enhancing the isoform selectivity. Additional selectivitygains over PI3Kγ were found on going to the 2-pyridylquino-line analogs 1 and 29, which were thought to sit lower in thebinding pocket than the substituted phenyl or pyridine analogs,such as 23, 20, or 27 (Figure 6b,c). The protein kinaseselectivity found with 1 (Figure 2) can generally be attributedto the structural differences between the PI3K lipid kinases and

protein kinases. The PI3Ks have a large “mouth” perpendicularto the ATP binding region not typically found in proteinkinases. Additionally the adenine binding pocket is larger inlipid kinases than found in most protein kinases.

Chemistry. 2-Chloroquinoline-3-carbaldehydes 36a39 and36b40 were converted to various substituted 2-phenylquinolines38a−d via a Suzuki coupling and the aldehydes 38b−d directlyreduced to primary alcohols (40b−d). Alcohol 40a wasaccessed most smoothly by the NaBH4 reduction of 36afollowed by a Suzuki coupling of the resulting 37. The carbon

Figure 6. (a) Cocrystal structure of p110γ + 16 at 2.30 Å resolution. The 3-fluoro substituent points toward the carbonyl of Ala885 enhancingpotency. (b) Crystal structure of p110γ + 27 at 2.40 Å resolution. (c) Overlay of 1 (green) onto p110γ + 27 (magenta). 1 sits lower in the pocketcompared to 27, enhancing selectivity over PI3Kγ.

Scheme 1a

aReagents and conditions: (i) NaBH4 (1.5 equiv), THF (0.5−0.9 M), or MeOH (0.1 M), 0 °C, 0.5−4 h; (ii) boronic acid (1.1 or 1.3 equiv),Pd(PPh3)4 (0.05 equiv), Na2CO3 (5.0 equiv), CH3CN−H2O, 100 °C; (iii) o-tolylboronic acid (1.5 equiv), PdCl2(PPh3)4 (0.05 equiv), Na2CO3 (4.0equiv), DME, EtOH, H2O, 150 °C, microwave, 60%; (iv) MeMgBr (2.0 equiv), THF (0.2 or 0.3 equiv), 0 °C; (v) NaH (4.0 equiv), DMSO (0.4 M),DABCO (4.0 equiv), chiral purification as needed; (vi) 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (2.0 equiv), DABCO (2.0 equiv), NaH (2 equiv),DMSO (2.4 mL), rt, 4 h, chiral HPLC; (vii) SOCl2 (5.0 equiv), CHCl3 (0.25 M); (viii) CBr4 (1.5 equiv), PPh3 (1.5 equiv), DCM (0.17 M), 0 °C,0.5 h; (ix) NaN3 (3.0 equiv), DMSO (0.25 M); (x) Pd−C, H2, MeOH, rt; (xi) (bromomethyl)triphenylphosphonium bromide (1.2 equiv),potassium tert-butoxide (6.8 equiv), −78 to 40 °C, 5 h, 41%; (xii) NaOH (0.7 mL, 4.0 equiv, 2 M), THF (0.7 mL), 6-mercaptopurine monohydrate(1.5 equiv), 4 h, reflux, 60%; (xiii) 6-chloropurine (1.2 equiv) or 6-brompurine (1.2 equiv), DIEA (2.0 equiv), EtOH (0.05 M) or n-BuOH, reflux;(xiv) 6-iodopurine (3.0 equiv), DMF (0.1 M), Et3N (2.90 mL), CuI (0.4 equiv), PdCl2(PPh3)2 (0.2 equiv), 50 °C, 3 h, 41%; (xv) EtOH/MeOH(1:1, 2 mL), 10% Pd/C (0.05 equiv), 34%.


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linked 10 was prepared from 38a via a Corey−Fuchsprocedure41 providing 39a followed by a Sonogashira cross-coupling and reduction of the resulting alkyne 39b. Aldehydes38a and 38b were treated with a Grignard reagent to supply thesecondary alcohols 40e and 40f, respectively. Conversion of thealcohols 40b−d to the primary halides 41a−c was performedwith SOCl2 or carbon tetrabromide and triphenyphosphine.Chlorides 41a and 41b were displaced with azide to provide42a and 42b followed by hydrogenation with Pd/C to providethe amines 43a and 43b, which were coupled with 6-chloropurine to provide the analogs 14 and 17. Alternatively,the ether linked analogs 7, 8, and 11 were prepared directlyfrom intermediates 40a and 40d by coupling with either the 6-chloropurine or 3-chloropyrrolopyrimidine and sodium hydridewith DABCO in DMSO. In a similar way 12 and 13 wereprepared from 40e followed by SFC chiral purification. Thethioether 6 was prepared via the thiolate of 6-mercaptopurineand 41c (Scheme 1).This overall sequence was also employed to provide

enantiomerically pure 15. The α-methyl 40f was subjectedsequentially in one pot to thionyl chloride, sodium azide, andhydrogenation to produce 43c, which was treated with 6-chloropurine and after chiral HPLC provided 15. Because ofthe inherent difficulties in purification of racemic compoundson a larger scale, we needed to find a more satisfactory methodof chiral synthesis. This was explored in the synthesis of 9.When a Mitsunobu reaction was employed with phthalimide,40a produced 44 followed by cleavage to amine 45, which wasthen coupled with chloropurine to provide 9 (Scheme 2).In the subsequent chiral synthesis the Misunobu reaction was

utilized to install the amino group after a stereospecificreduction of a ketone. The commercial 2-chloro-7-fluoroquino-line-3-carbaldehydes 47a and 47b (prepared in one step fromN-(3,5-difluorophenyl)acetamide) were treated with methyl-magnesium bromide to provide secondary alcohols 49a and49b. Alternatively, the secondary alcohols 49c and 49d couldbe prepared in one step from the commercially available 2,8-dichloroquinoline (48a) or 2-chloro-8-fluoroquinoline (48b)condensed with acetaldehyde and LDA. These secondaryalcohols were then oxidized to ketones 50a−d followed by thekey chiral reduction involving (+) DIP-Cl in THF to supply51a−d in high enantiomeric excess. These chiral alcohols were

inverted, protected, and transformed to the amino containingphthalimides 52a−d via a Mitsunobu reaction. The 2-chloroquinoline intermediates 52a−d were treated to Suzukicoupling conditions. Standard conditions involving boronic acidand tetrakistriphenylphosphine)palladium(0) with sodiumcarbonate in acetonitrile−water caused a ring opening of thephthalimide in conjunction with the coupling. These benzoicacid intermediates could be isolated, characterized (53a−c),and converted to the free amines 54a and 54c with refluxingconcentrated HCl in ethanol and for 54b, by the addition ofhydrazine monohydrate. Alternatively, the ring openedintermediate (uncharacterized) of 53d was converted in situback to the phthalimide 53d with hot ethanolic HCl. Toprevent the hydrolysis, the Suzuki reaction was modified toavoid water by employing 1,1′-bis(diphenylphosphino)-ferrocenepalladium(II) dichloride−dichloromethane complexand dry DMF. Under these conditions the fluorophenylph-thalimides 53e and 53f were isolated. For the synthesis of thepyridyl compounds Stille couplings of 52a−d were employedproviding 2-pyridylquinolines 55a, 55b, 55d, and 55e. A Stillecoupling was also used to convert the 8-chloroquinoline moietyof 55b to the cyanoquinoline 55c. Additionally, for theconversion of 52c to 55f, a copper(I) mediated Suzukicoupling was utilized, which in turn could be converted to55g via a palladium catalyzed cross-coupling involvingtrimethylaluminum. Subsequently the isolated phthalimides53d−f, 55a−e, and 55g were converted to the amines 54d−fand 56a−f with hydrazine. These amines in turn wereconverted to the target molecules 16, 18, 20, 22, 23, 25, 2829, 30, 33, 35, and 1 with chloro- or bromopurines as describedpreviously (Scheme 3).We modified this general procedure in several instances

(Scheme 4). For example, instead of relying on thephthalimide, the amine could be accessed by way of a reductionof an azide, which was easily prepared from 51d by aMitsunobu reaction with sodium azide to give 57. This in turnwas subjected to either a Stille or Suzuki coupling providing58a and 58b. The azides at this stage were easily reduced in theusual way with triphenylphosphine providing 59a and 59bfollowed by the final SNAr reaction to give analogs 26 and 21.Experimentally, low yields resulted when a thioetherphthalimide adduct was oxidized to the sulfone. This issue

Scheme 2a

aReagents and conditions: (i) SOCl2 (5 equiv), CHCl3 (0.2 M); (ii) NaN3 (2 equiv), DMSO (0.28 M), 4 h; (iii) 10% Pd/C, H2, MeOH (0.09 M),70%, three steps; (iv) 6-chloropurine, DIEA, n-BuOH, 115 °C; (v) chiral HPLC; (vi) phthalimide (2.0 equiv), PPh3 (1.7 equiv), ADDP (1.7 equiv),THF (0.2 M), rt, 35 h, 43%; (vii) NH2NH2−H2O (5.0 equiv), EtOH (0.08 M), reflux, 2 h, 97%; (viii) 6-chloropurine (2.0 equiv), Et3N (2.0 equiv),DMF (0.3 M), rt to 90 °C, 19 h, 40%.


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was surmounted by converting 52a to a tert-butyloxycarbonylprotected amine 60, which was subjected to the Suzukicoupling providing 2-(methylthio)phenylquinoline 61. Thisanalog could be easily oxidized to the sulfone 62 followed bythe standard SNAr reaction to yield analog 24. Compound 65was prepared in several steps from aldehyde 36b as outlinedpreviously in Scheme 1. Racemic 65 was then converted to 66by displacement of the chloride in 65 with phthalimidepotassium salt. After deprotection to the amine 67, SNArdisplacement and chiral HPLC purification provided 27. The

alternative series analog 4 was prepared by treatment of 1-(2-amino-5-chlorophenyl)propan-1-one with sodium nitrite underacidic conditions to providing the cyclized cinnoline alcohol 68.This was converted to chloride 69 with POCl3 and subjected toa hot solution of 4-(3,3-dimethylindolin-6-yl)morpholine in 1-pentanol to provide the product (Scheme 4).The three quinoxoline analogs 31, 32, and 34 were prepared

in several steps (Scheme 5). Starting with the condensation ofethyl 2-oxobutanoate and 2-amino, 3-chloroaniline in PPAsupplied 8-chloroethylquinoxalinone (70a) together with a

Scheme 3a

aReagents and conditions: (i) POCl3 (7.0 equiv), DMF (2.5 equiv); (ii) MeMgBr (2.0 equiv), THF; (iii) LDA, THF; CH3CHO, −78 °C; (iv)MnO2 (10.0 equiv), Tol (0.17 M), reflux, 2 h; (v) (+) DIP-Cl (2.2 equiv), THF (0.45 M), −47 °C; (vi) phthalimide (1.2 equiv), DIAD (1.2 equiv),PPh3 (1.2 equiv), 0 °C to rt; (vii) boronic acid (1.1−1.8 equiv), Pd(PPh3)4 (0.05−0.15 equiv), Na2CO3 (5.0 equiv), MeCN, water, 80−90 °C; (viii)2-tri-n-butylstannylpyridine, PPh3, 1,4-dioxane, 100 °C; (ix) 2-chloro-5-fluoropyridin-3-ylboronic acid (1.2 equiv), Pd(PPh3)4 (0.5 equiv), CsF (3.0equiv), CuI (0.2 equiv), 1,2-ethanediol, dimethyl ether (1.0 equiv), microwave, 100 °C, 1 h, 40%; (x) K2CO3 (0.4 equiv (for 53f)), or Na2CO3 (5.2equiv, (for 53e)), Pd(dppf)Cl2 (0.1 equiv), dry DMF (0.15 M), phenylboronic acid (2.0 equiv), 100 °C, 2.5 h; (xi) Pd(CF3CO2)2, Xphos, tributyltincyanide, NMP, dicyclohexylamine, 160 °C, 3.25 h, 67.5%; (xii) AlMe3 (5.0 equiv), dioxane (0.02 M), Pd(PPh3)4 (0.2 equiv), reflux, 4 h, 75%; (xiii)hydrazine or hydrazine hydrate (1.5−20 equiv), EtOH (0.1 M), 50−90 °C, 0.5−4 h; (xiv) conc HCl (10−20 equiv), EtOH (0.2−0.5 M), reflux, 4 hto 6 days and (54b) hydrazine monohydrate (10 equiv); (xv) conc HCl, sealed flask, 24 h, 76.5% of 56f; (xvi) 6-chloropurine (1.0−1.1 equiv) or 6-bromopurine (1.2 equiv), DIEA (1.0−3.0 equiv), n-BuOH (0.1−1.0 M), 100−118 °C.


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minor amount of the 5-chloro regioisomer. 7-Fluoroquinox-alinone 70b was prepared by DDQ oxidation of thecommercially available 3-ethyl, 7-fluoro-3,4-dihydroquinoxali-none.42 Bromo compounds 71a and 71b were subsequentlyprepared by treatment of 70a and 70b with 1,3-dibromo-5,5-dimethylimidazolidine-2,4-dione, benzoyl peroxide in CCl4.These intermediates were then subjected to POCl3 treatmentto supply chloroquinolines 72a and 72b, which were thentreated with potassium phthalimide to give 73a and 73b. Thesynthesis of the 3,8-dichloroquinoxaline 73c was done in aslightly different manner. 3-Chlorobenzene-1,2-diamine wascondensed and cyclized with methyl 3-(1,3-dioxoisoindolin-2-yl)-2-oxobutanoate43 to produce the phthalimide 74, which wasconverted to the chloroquinoxaline 73c as before. A Stille

coupling installed the 2-pyridyl group supplying 75a−cfollowed by a deprotection of the phthalimide, leaving theamino intermediates 76a−c. The amine 76d was obtained bychiral HPLC purification of 76c. These amines were thenconverted to the final products 31, 32, and 34 by treatmentwith DIEA and chloropurine followed by a final chiralpurification using SFC.Finally, the synthesis of 19 was done in a very similar way to

the other quinoxalines (Scheme 6). 2-Aminobutanic acid washeated with 1-chloro-3-fluoro-2-nitrobenzene to provide 78,which was reductively cyclized to deliver the dihydroquinoxa-line 79. The oxidation of 79 with DDQ supplied 70a,44 whichwas converted to the 2-chloroquinoxaline 80. At this point, the3-fluorophenyl group was installed with a Suzuki coupling to

Scheme 4a

aaReagents and conditions: (i) PPh3, DIAD, DPPA, THF, 0 °C to rt; (ii) boronic acid, Pd(PPh3)4, Na2CO3, MeCN, water, 80−100 °C; (iii) PPh3,THF, water, 60 °C; (iv) DIEA, n-BuOH, 6-bromopurine or 6-chloropurine, 100−130 °C; (v) NH2NH2 (10 equiv), EtOH, 90 °C, 0.5 h, then Boc2O(1.1 equiv), Et3N (1.0 equiv), reflux, 76%; (vi) OsO4 (0.13 equiv), NMO (3.0 equiv), acetone (0.79 M), 100%; (vii) TFA, DCM, rt, 1 h; (viii)MeMgBr (2.0 equiv), THF, 0 °C, 98%; (ix) SOCl2 (5.0 equiv), CHCl3 (0. 3M), rt, 3 h; (x) potassium phthalimide (2.5 equiv), DMF (0.37 M), 1.5h, 100 °C; (xi) NH2NH2 (10 equiv), EtOH (0.1 M), reflux, 1.5 h, 60%, three steps; (xii) chiral HPLC; (xiii) NaNO2 (1.1 equiv), HCl (5.0 N), 92%;(xiv) POCl3, (15.1 equiv), 90 °C, 3 h, 78%; (xv) 4-(3,3-dimethylindolin-6-yl)morpholine, 1-pentanol, 140 °C, 51%.


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give 81, which was then brominated to provide 82. Thesecondary bromide in 82 was displaced with azide to give 83,which was reduced with trimethylphosphine to give the racemicamine 84, which was treated to the standard SNAr conditions,and following a chiral purification 19 was obtained. Althoughthis was a lengthy sequence, it provided the desired product in

high yield and allowed for reasonable scaling up, even with thefinal purification (Scheme 6).

■ CONCLUSIONIn this report we review the discovery and development of 1focusing on inflammation and autoimmunity (1 is currently

Scheme 5a

aReagents and conditions: (i) ethyl 2-oxobutanoate (1.0 equiv), PPA (1.0 equiv), 100−160 °C, 2.7 h, 60%; (ii) 1,4-dioxane (0.2 M), DDQ (1.05equiv), rt, 2.5 h, 95%; (iii) 1, 3-dibromo-5,5-dimethylimidazolidine-2,4-dione (0.7 equiv), benzoyl peroxide (0.1 equiv), CCl4 (1.0 equiv), 20 h, 80°C to reflux, 80−100%; (iv) POCl3 (10−13 equiv), 1.5−2 h, 100−110 °C; (v) potassium phthalimide (1.0 equiv), DMF (0.3 M), 1−3 h, rt, 70−76%; (vi) 2-tri-n-butylstannylpyridine (1.3−2.0 equiv), Pd(PPh3)4 (0.1 equiv), 1,4-dioxane or toluene, 110 °C, 19−29 h; (vii) methyl, 3-(1,3-dioxoisoindolin-2-yl)-2-oxobutanoate (1.0 equiv), EtOAc (0.4 M), rt, 3 h, ∼3.5:1 mixture of regioisomers; (viii) hydrazine hydrate (5−10 equiv),EtOH, 95 °C, 1−1.5 h; (ix) chiral purification SFC or chiral HPLC; (x) DIEA (3.0 equiv), n-BuOH (0.1 or 0.28 M), 6-chloropurine (1.0 equiv) or 6-bromopurine (1.0 equiv), 100−110 °C, 5−24 h.

Scheme 6a

aReagents and conditions: (i) 2-aminobutanic acid (1.0 equiv), K2CO3 (1.0 equiv), DMSO, 80 °C, 18 h, 76%; (ii) SnCl2−2H2O (5.0 equiv), HCl,EtOH, 90 °C, 2.5 h, 94%; (iii) DDQ (1.0 equiv), 1,4-dioxane, rt, 2 h, 97%; (iv) POCl3 (10 equiv), 100 °C, 2.0 h, 90%; (v) (3-fluorophenyl)boronicacid (1.0 equiv), Pd(PPh3)4 (0.05 equiv), Na2CO3 (5.0 equiv), CH3CN−H2O (3:1, 0.1 M), 8 h, 89%; (vi) 1,3-dibromo-5,5-dimethylimidazolidine-2,4-dione (0.55 equiv), benzoyl peroxide (0.1 equiv), CCl4 (0.2 M), reflux, 17 h, 92% ; (vii) NaN3 (2 equiv), DMSO (0.3 M), rt, 2 h, 100%; (viii)PMe3 (1.0 equiv, 1 M), THF−H2O (4:1, 0.17 M), rt, 1.7 h, 87.8%; (ix) 6-chloropurine (1.0 equiv), DIEA (3.0 equiv), 1-BuOH (1.0 M), 100 °C, 22h, 48%; (x) chiral separation using SFC, 37%.


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undergoing clinical trials for oncology). 1 has a high degree ofpotency toward PI3Kδ and selectivity over the other PI3Kisoforms. PI3Kδ is known to be a key player in theinflammatory response; therefore, small molecule inhibitors ofPI3Kδ would be very useful in diseases of dysfunctionalimmunity such as RA and SLE. Although many analogsreported here such as 14 and 16 had reasonable profiles in bothin vitro assays and in vivo rodent models of disease, these werenot our only considerations. Mindful of potential pitfalls thatcould occur in the clinic and beyond, we focused on thephysicochemical properties of these analogs as well as theirpotency and selectivity. PI3K isoform selectivity gains werefound by pinpointing electron withdrawing groups on eitherthe 2-aryl group or the quinoline core, with the 7-fluoroquino-line being the most advantageous. In large part, the high kinaseselectivity of 1 was due to the unusual shape of the molecule,which limits its ability to bind to the ATP binding site ofprotein kinases. Because permeability was consistently high forthis series, we focused on improving solubility. This focusyielded compounds that were substantially improved in theiroverall in vitro profiles, such as increased HWB potency anddecreasing CYP inhibition. A consequence of this was lowerplasma protein binding. Initially, this led to compounds withhigher clearance, but structural changes in the inhibitorsprovided compounds with much lower intrinsic clearance. Thishad the effect of increasing the unbound drug concentration,which led to higher efficacy in the rat KLH model. Keydevelopments along this path include the discovery of the 2-methylsulfonylphenyl moiety as found in 24, a group whichdramatically increased the solubility. Additionally, the 2-pyridine substituted quinoline moiety, as in 1, was an improvedsoluble isostere of the methylsulfonylphenyl group, furtherenhancing the compound profile including lowering of the rativ clearance. Although the pAKT mouse model was used onlyto benchmark compounds and many compounds performedsimilarly, it was the close correlation between the in vitro and invivo assays that allowed a smooth selection of doses for the 10-day KLH IgG-IgM response rat model. It was the performancein this model that led to the selection of compounds such as 1for further preclinical evaluation. Finally, we will be reportingon additional structurally related, potent, and selective PI3Kδinhibitors for inflammation and more details pertaining to 1including details related to its use in an oncology clinical settingin due course.

■ EXPERIMENTAL SECTIONGeneral Biology. Recombinant Expression of PI3Ks. Full

length p110 subunits of PI3kα, -β, and -δ, N-terminally labeled withpolyHis tag were coexpressed with p85 with baculovirus expressionvectors in Sf9 insect cells. P110/p85 heterodimers were purified bysequential Ni-NTA, Q-HP, and Superdex-100 chromatography.Purified α, β, and δ isoforms were stored at −20 °C in 20 mM Tris,pH 8, 0.2 M NaCl, 50% glycerol, 5 mM DTT, 2 mM Na cholate.Truncated PI3Kγ, residues 114−1102, N-terminally labeled withpolyHis tag, was expressed using baculovirus in Hi5 insect cells. The γisoform was purified by sequential Ni-NTA, Superdex-200, Q-HPchromatography, and stored frozen at −80 °C in NaH2PO4, pH 8, 0.2M NaCl, 1% ethylene glycol, 2 mM β-mercaptoethanol.In Vitro Assays. PI3K Enzyme Assays. A PI3K Alphascreen assay

(PerkinElmer, Waltham, MA) was used to measure the activity of apanel of four phosphoinositide 3-kinases: PI3Kα, PI3Kβ, PI3Kγ, andPI3Kδ. Enzyme reaction buffer was prepared using sterile water(Baxter, Deerfield, IL) and 50 mM Tris-HCl, pH 7, 14 mM MgCl2, 2mM sodium cholate, and 100 mM NaCl. 2 mM DTT was added freshon the day of the experiment. The Alphascreen buffer was made using

sterile water and 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.10%Tween 20, and 30 mM EDTA. Then 1 mM DTT was added fresh onthe day of the experiment. Compound source plates used for this assaywere 384-well Greiner clear polypropylene plates containing testcompounds at 5 mM and diluted 1:2 over 22 concentrations. Columns23 and 24 contained only DMSO, as these wells comprised thepositive and negative controls, respectively. Source plates werereplicated by transferring 0.5 μL per well into 384-well Optiplates(PerkinElmer, Waltham, MA). Each PI3K isoform was diluted inenzyme reaction buffer to 2× working stocks. PI3Kα was diluted to 1.6nM, PI3Kβ was diluted to 0.8 nM, PI3Kγ was diluted to 15 nM, andPI3Kδ was diluted to 1.6 nM. PI(4,5)P2 (Echelon Biosciences, SaltLake City, UT) was diluted to 10 μM, and ATP was diluted to 20 μM.This 2× stock was used in the assays for PI3Kα and PI3Kβ. For assayof PI3Kγ and PI3Kδ, PI(4,5)P2 was diluted to 10 μM and ATP wasdiluted to 8 μM to prepare a similar 2× working stock. Alphascreenreaction solutions were made using beads from the anti-GSTAlphascreen kit (PerkinElmer, Waltham, MA). Two 4× workingstocks of the Alphascreen reagents were made in Alphascreen reactionbuffer. In one stock, biotinylated-IP4 (Echelon Biosciences, Salt LakeCity, UT) was diluted to 40 nM and streptavadin-donor beads werediluted to 80 μg/mL. In the second stock, PIP3-binding protein(Echelon Biosciences, Salt Lake City, UT) was diluted to 40 nM andanti-GST-acceptor beads were diluted to 80 μg/mL. As a negativecontrol, a reference inhibitor at a concentration ≫Ki (40 μM) wasincluded in column 24 as a negative (100% inhibition) control. Using a384-well Multidrop (Titertek, Huntsville, AL), 10 μL/well of 2×enzyme stock was added to columns 1−24 of the assay plates for eachisoform. An amount of 10 μL/well of the appropriate substrate 2×stock (containing 20 μM ATP for the PI3Kα and -β assays andcontaining 8 μM ATP for the PI3Kγ and -δ assays) was then added tocolumns 1−24 of all plates. Plates were then incubated at roomtemperature for 20 min. In the dark, 10 μL/well of the donor beadsolution was added to columns 1−24 of the plates to quench theenzyme reaction. The plates were incubated at room temperature for30 min. Still in the dark, 10 μL/well of the acceptor bead solution wasadded to columns 1−24 of the plates. The plates were then incubatedin the dark for 1.5 h. The plates were read on an Envision multimodeplate reader (PerkinElmer, Waltham, MA) using a 680 nm excitationfilter and a 520−620 nm emission filter.

Human B Cell Proliferation Assay. B cells were purified from humanperipheral blood mononuclear cells (PBMCs) by negative selection.Approximately 3 × 104 purified B cells per well were seeded into a 96-well plate. Compounds were dissolved in DMSO at a concentration of10 mM, and a 10-point, 3-fold serial dilution of the compound wascarried out in DMSO. Then 0.5 μL of compound was added to eachwell in duplicates so that the final DMSO concentration was 0.25%and the highest compound concentration was 10 μM. Afterpreincubating for 30 min, B cells were treated with 2 μg/mL ofanti-human IgM antibody plus 300 ng/mL human CD40L or 5 ng/mLhuman IL-4 plus 200 ng/mL of CD40L as a counterscreen to evaluatethe off-target effects (data not shown). The plates were incubated at 37°C and 5% CO2 for 72 h, then pulsed with 0.5 μCi per well 3Hthymidine for 18 h, and B cells were collected to count theincorporation of 3H thymidine.

Murine B Cell in Vitro Anti-IgM Induced pAKT Assay. B cells wereharvested from BALB/c mouse spleens as approved by the AmgenInstitutional Animal Care and Use Committee. The 1 ×106

splenocytes were plated into 96-deep well plates in dilution medium(PBS + 0.5% BSA) and rested for 30 min. A 96-well compounddilution plate was prepared starting at 2.5 mM with a 10-point 1:3serial dilution in 100% DMSO, with lanes 6 and 12 containing DMSOonly to provide stimulation and no stimulation control values. Afurther 10× compound dilution plate was prepared starting at 25 μMby transferring 2 μL of diluted compounds into 198 μL of dilutionmedia in a 96-well U bottom plate. Ten microliters of dilutedcompound was transferred to the resting splenocytes for a final startingconcentration of 2.5 μM, mixed, and incubated 45 min at roomtemperature. To stimulate the cells, 10 μL of antimurine IgM (20 μg/mL final concentration in dilution medium) was added to columns 1−


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11. No anti-IgM was added to column 12 containing the nostimulation control wells. Cells were mixed and incubated 15 min atroom temperature. The stimulation was ended by the addition of 37°C prewarmed 1× lyse/fix solution (BD Biosciences). The plate wascovered, shaken, and incubated at 37 °C for 10 min for RBC lysis andcell fixation. The cells were centrifuged, and the supernatant wasremoved. To permeabilize the cells, 600 μL of 90% ice cold methanolwas added per well while vigorously shaking. The plate was sealed andstored at −20 °C until further FACS analysis. For flow cytometryanalysis, the plates were centrifuged and washed twice to removeresidual methanol and then stained with 50 μL of the followingantibodies: CD45R/B220, Pacific Blue, 1:100 dilution; anti-phospho-AKT (Ser473) (193H12) rabbit mAb, 1:100 dilution; Zap70(pY319)/Syk (pY352), PE, 10 μL/well; ERK1/2 (pT202/pY204),FITC, 10 μL/well. The samples were stained for 1 h at roomtemperature and then washed with 1.8 mL of wash buffer (2% FBS inPBS). To detect rabbit anti-mouse pAKT, 50 μL of anti-rabbit AlexaFluor-647 secondary (1:500) in dilution medium was added per well.The plate was incubated 15 min at room temperature and then washedtwice with wash buffer. An amount of 200 μL of cells was transferredto a 96-well U bottom tissue culture plate and analyzed on a BectonDickenson LSRII flow cytometer. Then 5−10000 CD45R/B220+ Bcells were analyzed for their mean fluorescent activity (MFI) of pAKT.pSyk and pERK1/2 MFI levels were also measured on the B cells ascontrols for on target activity of the compounds. To derive IC50 values,percent of control (POC) values for pAKT, pSyk, and pERK1/2 wereplotted against the compound concentration (log μM) in GraphPadPrism, version 5.0.In Vivo Studies. pAKT Mouse in Vivo Studies. IgM membrane only

homozygous transgenic mice (6- to 12-week-old female) were orallydosed with compound or vehicle control (n = 5 per group). At 15 minafter treatment, mice were tail iv injected with 50 μg of endotoxin-freeFITC-labeled μ chain specific anti-IgM (Jackson Immunoresearch) orPBS only control. Blood and spleen tissue were collected after 30 minof stimulation for drug concentration and B cell pAKT analysis viaflow cytometry. Briefly, blood and dispersed splenocytes were fixedwith BD Phosflow lyse/fix buffer (Becton Dickinson), pelleted, andpermeabilized with cold 90% MeOH. Cells were then stained withpAKT (Cell Signaling Technology) and Alexa-647 secondary(Invitrogen) and B220-Pacific Blue (Becton Dickinson) for FACSanalysis. Stimulated B220+/anti-IgM FITC+ B cells were analyzed forpAKT levels with B220+/FITC− B cells from anti-IgM untreated miceserving as a control. Mice were maintained and experiments wereperformed at Amgen Inc., Thousand Oaks, CA, under a protocolapproved by the Institutional Animal Care and Use Committee(IACUC).KLH Rat in Vivo Studies. Female Lewis rats (N = 8/dose group)

were dosed po with test compounds or vehicle (2% HPMC, 1%Pluronic F68, 10% Captisol, pH 2.0) once a day for 10 days at variousdoses. Two hours after the first dosing, 200 μL of PBS containing 60μg of KLH was administered to each rat intravenously. Ten days afterthe KLH priming, rats were euthanized and blood was taken by cardiacpuncture for the measurement of KLH specific IgG and IgM byELISA.Crystallography. Human p110γ(144−1102) was expressed,

purified, and crystallized, and inhibitor complexes were preparedaccording to published procedures.45,46 Diffraction data for p110γ + 1were collected on an FR-E rotating anode X-ray source equipped withan RAXIS IV++ detector. Diffraction data for p110γ + 16 and p110γ +27 were collected at the Advanced Photon Source, beamline 31-ID,using λ = 0.9793 Å and a MAR 165 mm CCD detector. Data wereprocessed using the HKL software suite,47 and the structures wererefined using REFMAC starting from previously solved models ofPI3Kγ.48 Model building was performed with COOT.49

General Chemistry. All solvents and chemicals used were reagentgrade unless otherwise noted. Anhydrous solvents were purchasedfrom Aldrich and used without further purification. Analytical thinlayer chromatography (TLC) and column (flash) chromatographywere performed on Merck silica gel 60 (230−400 mesh) utilizingCombi-Flash or Biotage instrumentation. Microwave reactions were

performed with a CEM Discover benchtop apparatus. Concentrationrefers to solvent removal, which was conducted with a Buchi typerotary evaporator. Residual solvent was removed using a vacuummanifold (1 Torr). Reported yields are isolated yields. All finalcompounds were purified to ≥95% purity as determined by an Agilent1100 series HPLC instrument with UV detection at 220 nm using thefollowing method: Zorbax SB-C8 column (3.5 μm, 150 mm × 4.6 mmi.d.), eluting with binary solvent systems A and B using a 5−95% B(0−15 min) gradient elution [A, H2O with 0.1% TFA; B, CH3CN with0.1% TFA]; flow rate 1.5 mL/min. Preparative reversed-phase highpressure liquid chromatography (RP-HPLC) was performed using anAgilent 1100 series HPLC and Phenomenex Gemini C18 column (5μm, 100 mm × 30 mm i.d.), eluting with binary solvent systems A andB using a gradient elution [A, H2O with 0.1% TFA; B, CH3CN with0.1% TFA] with UV detection at 220 nm. Low resolution electrosprayionization (ESI) mass spectrometry analysis was conducted on anAgilent 1100 series LC/MSD electrospray mass spectrometer with UVdetection at 254 nm. Mass spectrometry results are reported as theratio of mass over charge, followed by the relative abundance of eachion in parentheses. Compounds were analyzed in the positive ESImode with acetonitrile/water with 0.1% formic acid. NMR spectrawere recorded on a Varian Gemini 400 MHz or Bruker Avance 500 or600 MHz or NMR spectrometer. Chemical shifts (δ) are reported inparts per million (ppm) relative to residual undeuterated solvent orTMS as internal reference. Spin−spin coupling constants (J) arereported in hertz (Hz) and obtained from the splitting patterns, whichare indicated as follows: s = singlet; d = doublet; t = triplet; q =quartet; qn = quintet; dd = doublet of doublet; dt = doublet of triplets;m = multiplet; br = broad peak. Enantiomeric excess determinationswere done with chiral HPLC on Chiralpak AD-H or OD-H, OH-H,AS-H, and IA columns, eluting with isopropanol in hexane. Preparativechiral HPLC was done with Chiracel OD-H columns (0.46 mm × 250mm, 5 mm) with isopropanol in hexane as eluent. Larger scalequantitative separations were done with supercritical fluid chromatog-raphy (SFC). An analytical vial of approximately 1 mg/mL (inmethanol) was prepared from the sample material. Analytical SFCscreening was performed on multiple chiral phases. The gradientmethod and columns used in the screen are below. Chiral columns:AD-H, AS-H, OD-H, OJ-H, IA, IB, IC, Lux2 (150 mm × 4.6 mm, 5μm). Mobile phase: A, liquid CO2; B, methanol (0.2% DEA). CO2 andmethanol are mixed according to the gradient below, and mobile phaseis held to an elevated (100 bar), constant outlet pressure at the outletat all times during gradient. Oven/column temp: 40 °C. Flow rate: 5.0mL/min total. Outlet pressure: 100 bar. Gradient, percent B, time(min): 5.00, 0.00; 60.00, 4.50; 60.00, 6.00; 5.00, 6.50; 5.00, 7.00. Onthe basis of the separation observed, an isocratic method wasdeveloped with the best column to project conditions on preparative(quantitative) scale and for QC analysis following prep. Typicalconditions: OD-H + IC 250 mm × 30 mm columns in-series using 36g/min IPA (0.2% DEA) + 54 g/min CO2 on Thar 350 SFC. Outletpressure = 125 bar. Temp = 18 °C. Wavelength = 327 nm. Used 2.0mL injections of 5.0 g/750 mL (9:1 IPA/ACN) sample solution.

4-(1-(6-Chloro-3-methylcinnolin-4-yl)-3,3-dimethylindolin-6-yl)morpholine (4). A solution of 4-(3,3-dimethylindolin-6-yl)-morpholine (232 mg, 1.00 mmol, 0.75 equiv) and 69 (320 mg, 1.5mmol, 1.0 equiv) in 1-pentanol (10 mL, 0.15 M) was heated to 140 °Covernight. The reaction mixture was cooled to rt and poured intowater and extracted with DCM (2 × 50 mL). The organic layer waswashed with saturated NaHCO3, brine and dried over MgSO4. Thesolvent was removed under reduced pressure and the crude residuepurified by chromatography, eluting with 0−100% EtOAc/hexane.Yield 51% (208 mg, 0.51 mmol). 1H NMR (500 MHz, [D6]DMSO) δppm 8.44 (1 H, d, J = 9.0 Hz), 7.84 (1 H, dd, J = 9.0, 2.2 Hz), 7.50 (1H, d, J = 2.0 Hz), 6.89 (1 H, s), 6.41 (1 H, s), 5.88 (1 H, s), 2.71 (3 H,s), 1.24 (6 H, s). Mass spectrum (ESI) m/e = 409.1 (M + 1). HRMS(ESI) m/z calculated for C23H25ClN4O + H+ [M + H]: 409.1795.Found: 409.1795.

3-((9H-Purin-6-ylthio)methyl)-2-(2-methoxyphenyl)-8-meth-ylquinoline (6). To a stirred biphasic mixture of NaOH (2 M, 0.7mL, 1.40 mmol) and 41c (122 mg, 0.356 mmol) in THF (0.7 mL) was


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added 6-mercaptopurine monohydrate (91 mg, 0.54 mmol). After 4 h,refluxing for the last 3 h, the mixture was cooled to rt, diluted withH2O (10 mL), brought to pH 6 by the addition of 2 N HCl, saturatedwith NaCl, and extracted with THF. The combined organic layerswere dried (MgSO4) and concentrated. The resulting light yellow solidwas recrystallized from THF/MeOH to provide 6. White powder;yield 60% (89 mg, 0.214 mmol). 1H NMR (400 MHz, [D8]THF) δppm 2.70 (3 H, s), 3.79−3.84 (3 H, m), 4.71 (2 H, s), 7.02−7.11 (2 H,m), 7.32−7.50 (5 H, m), 7.62 (1 H, d, J = 8.02 Hz), 8.09 (1 H, s), 8.37(1 H, s) 8.55 (1 H, s). Mass spectrum (ESI) m/e = 414.0 (M + 1).HRMS (ESI) m/z calculated for C23H19N5OS + H+ [M + H]:414.1389. Found: 414.1385.General Procedure for the Synthesis of 7 and 8. A mixture of

6-chloropurine (2.0 equiv) or 4-chloropyrrolo[2.3-d]pyrimidine (75mg, 0.49 mmol) and DABCO (4.0 equiv) in DMSO (1 mL) wasstirred at rt for 4 h and was then added via cannula to a mixture of 40a(94 mg, 0.36 mmol) or 40d (70 mg, 0.25 mmol) and sodium hydride,60% dispersion in mineral oil (4.0 equiv) in DMSO (0.4 M) that hadbeen stirred at rt for 15 min prior to the addition. The final mixturewas stirred at rt for 3.5 h, neutralized by the addition of glacial aceticacid, diluted with brine, and extracted with EtOAc. The combinedorganic layers were dried over MgSO4 and concentrated underreduced pressure.3-((9H-Purin-6-yloxy)methyl)-2-(2-methoxyphenyl)-8-meth-

ylquinoline (7). The residue was purified by column chromatog-raphy, eluting with 0−2% DCM/MeOH to provide 7. White solid;yield 84% (0.083 g, 0.21 mmol). 1H NMR (400 MHz, [D6]DMSO) δppm 13.41 (1 H, s), 8.51 (1H, s), 8.39 (1 H, s), 7.87 (1 H, d, J = 7.8Hz), 7.65 (1 H, d, J = 7.0 Hz), 7.49−7.56 (1 H, m), 7.34−7.46 (2 H,m), 7.12 (1 H, d, J = 8.2 Hz), 7.03 (1 H, t, J = 7.2 Hz), 5.58 (2 H, s),3.72 (3 H, s), 2.69 (3 H, s). Mass spectrum (ESI) m/e = 398.2 (M +1). HRMS (ESI) m/z calculated for C23H19N5O2 + H+ [M + H]:398.1617. Found: 398.1609.3-(((9H-Purin-6-yl)oxy)methyl)-8-methyl-2-(o-tolyl)quinoline

(8). The resulting yellow oil was dissolved in DCM, evaporated ontosilica gel (deactivated with 2 M NH3 in MeOH), and purified by flashchromatography, eluting with 5% MeOH/DCM. White solid; yield64% (136 mg, 0.228 mmol). 1H NMR (400 MHz, CDCl3) δ ppm 8.63(1 H, s), 8.40 (1 H, s), 8.34 (1 H, s), 7.84 (1 H, d, J = 8.02 Hz), 7.65(1 H, d, J = 6.85 Hz), 7.53 (1 H, dd, J = 8.02 Hz), 7.36 (1 H, d, J =7.24 Hz), 7.29 (1 H, d, J = 7.88 Hz), 7.17−7.26 (1 H, m), 5.60 (2 H,s), 4.89 (4 H, s), 3.32−3.38 (8 H, m), 2.77 (3 H, s), 2.17 (3 H, s), 1.31(1 H, s). Mass spectrum (ESI) m/e = 282 (M + 1). HRMS (ESI) m/zcalculated for C23H19N5O + H+ [M + H]: 382.1668. Found: 382.1672.N-((8-Methyl-2-o-tolylquinolin-3-yl)methyl)-9H-purin-6-

amine (9). A mixture of 45 (50 mg, 0.19 mmol), 6-chloropurine (59mg, 0.38 mmol), and triethylamine (0.053 mL, 0.38 mmol) in DMF(0.6 mL) was stirred at rt for 1 h, then 60 °C for 12 h and 90 °C for 6h. The crude mixture was evaporated onto silica gel and purified byflash chromatography (Biotage Si 25+M, 5−10% MeOH/DCM) toprovide 64 mg of a colorless glass. The compound was further purifiedby reversed-phase HPLC (Gilson, H2O/MeCN/THF) to provide 9.White solid; yield 40% (29 mg, 0.076 mmol). 1H NMR (400 MHz,[D4]methanol) δ ppm 8.29 (1 H, br s), 8.12 (1 H, br s), 8.07 (1 H, brs), 7.70 (1 H, d, J = 7.63 Hz), 7.56 (1 H, d, J = 6.06 Hz), 7.38−7.49 (1H, m), 7.33 (2 H, d, J = 6.85 Hz), 7.18−7.30 (4 H, m), 4.73 (2 H, brs), 2.72 (3 H, br s). Mass spectrum (ESI) m/e = 381.2 (M + 1).HRMS (ESI) m/z calculated for C23H20N6 + H+ [M + H]: 381.1828.Found: 381.182.3-(2-(7H-Purin-6-yl)ethyl)-8-methyl-2-(o-tolyl)quinoline (10).

To a solution of 39b (0.069g, 0.14 mmol) in EtOH/MeOH (1:1, 2mL) was added Pd/C (0.05 equiv). The resulting mixture wasevacuated/backfilled with N2 (3×) and with hydrogen (3×). Themixture was stirred under a hydrogen balloon overnight. At this time itwas filtered through a Celite pad. Solvent was removed under reducedpressure and the crude residue was purified by flash chromatography,eluting with 0−10% MeOH/DCM to afford (10). Yield 34% (0.018g,0.047 mmol). 1H NMR (400 MHz, [D6]DMSO) δ ppm 13.38 (1 H,s), 8.70 (1 H, br s), 8.46 (1 H, br s), 8.29 (1 H, br s), 7.73 (1 H, br s),7.40−7.60 (2 H, m), 7.15−7.38 (4 H, m), 3.12 (2 H, br s), 2.61 (3 H,

s), 2.00 (3 H, s), 1.24 (1 H, br s), 0.85 (1 H, br s). Mass spectrum(ESI) m/e = 380 (M + 1). HRMS (ESI) m/z calculated for C24H21N5+ H+ [M + H]: 380.1875. Found: 380.1879.

3-(((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)oxy)methyl)-8-methyl-2-(o-tolyl)quinoline (11). A mixture of 4-chloropyrrolo[2.3-d]-pyrimidine (75 mg, 0.49 mmol) and DABCO (109 mg, 0.973mmol) in dry DMSO (1 mL) was stirred at rt for 4 h and was thenadded via cannula to a mixture of 40a (70 mg, 0.27 mmol) and sodiumhydride, 60% dispersion in mineral oil (39 mg, 0.98 mmol, 3.7 equiv)in DMSO (0.5 M) that had been stirred at rt for 15 min prior to theaddition. The final mixture was stirred at rt for 3.5 h, neutralized by theaddition of glacial acetic acid, diluted with brine, and extracted withEtOAc. The combined organic layers were dried over MgSO4 andconcentrated under reduced pressure. The residue was purified bycolumn chromatography, eluting with 0−2% DCM/MeOH to provide(11). White solid; yield 69% (70 mg, 0.18 mmol). 1H NMR([D6]DMSO) δ 12.0 (1 H, s), 8.60 (1 H, s), 8.26 (1 H, s), 7.90 (1 H,d, J = 7.9 Hz), 7.66 (1 H, d, J = 7.0 Hz), 7.54 (1 H, t, J = 7.4 Hz),7.22−7.37 (5 H, m), 6.50 (1 H, d, J = 2.1 Hz), 5.45 (2 H, s), 2.68 (3H, s), 2.11 (3 H, s). Mass spectrum (ESI) m/e = 381 (M + 1). HRMS(ESI) m/z calculated for C24H20N4O + H+ [M + H]: 381.1715.Found: 381.1713.

(S)-3-(1-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)oxy)ethyl)-8-meth-yl-2-(o-tolyl)quinoline (12) and (R)-3-(1-((7H-Pyrrolo[2,3-d]-pyrimidin-4-yl)oxy)ethyl)-8-methyl-2-(o-tolyl)quinoline (13).4-Chloro-7H-pyrrolo[2,3-d]pyrimidine (0.31 g, 2.0 mmol) andDABCO (0.45 g, 4.0 mmol) were stirred in DMSO (1.4 mL) at rtfor 4 h. 40a (0.28 g, 1.0 mmol) was stirred in 1.0 mL of DMSO, andsodium hydride, 60% dispersion in mineral oil (2 equiv) was added.After 10 min, the pyrrolopyrimidine−DABCO solution was added tothe quinolinyl alcohol, and the mixture was stirred overnight.Chromatography, eluting with 0−100% EtOAc in hexane followedby chiral separation using IA column (5% isopropanol in hexane), gavethe pure isomers

(S)-3-(1-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)oxy)ethyl)-8-meth-yl-2-(o-tolyl)quinoline (12). Yield 10% (34 mg, 0.10 mmol). 1HNMR (400 MHz, [D6]DMSO) δ ppm 11.99 (1 H, br s), 8.59 (1 H, brs), 8.25 (1 H, br s), 8.08 (1 H, br s), 7.88 (1 H, d, J = 7.04 Hz, 2 H),7.61 (1 H, d, J = 6.7 Hz), 7.49 (1 H, t, J = 7.04 Hz), 7.31−7.36 (3 H,m), 6.55 (2 H, br s), 2.65 (3 H, s.), 2.11 (3 H, s), 1.69 (3 H, br s).Mass spectrum (ESI) m/e = 395.2 (M + 1). HRMS (ESI) m/zcalculated for C25H22N4O + H+ [M + H]: 395.1872. Found: 395.1881.

(R)-3-(1-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)oxy)ethyl)-8-methyl-2-(o-tolyl)quinoline (13). Yield 11% (40 mg, 0.11 mmol).1H NMR (400 MHz, [D6]DMSO) δ ppm 11.98 (1 H, br s), 8.59 (1 H,br s), 8.25 (1 H, br s), 8.08 (1 H, br s), 7.86 (2 H, d, J = 7.04 Hz), 7.61(1 H, d, J = 6.7 Hz), 7.49 (1 H, t, J = 7.04 Hz), 7.31−7.36 (3 H, m)6.55 (2 H, br s), 2.64 (3 H, s), 2.11 (3 H, s), 1.68 (3 H, br s). Massspectrum (ESI) m/e = 395.2 (M + 1). HRMS (ESI) m/z calculated forC25H22N4O + H+ [M + H]: 395.1872. Found: 395.1873.

General Procedure for the Synthesis of 38a−d and 63. To astirred mixture of 2-chloro-8-methylquinoline-3-carbaldehyde (36a)(1.00 equiv) or 2,8-dichloroquinoline 3-carbaldehyde (36b) (1.00equiv) and Pd(PPh3)4 (0.05 equiv), and Na2CO3 (5.0 equiv, 2 Maqueous) in CH3CN−water (3:1, 0.1 M) was added o-tolyboronic acid(1.5 g, 11.0 mmol, 1.1 equiv) or 2-chlorophenylboronic acid (4.20 g,30.9 mmol, 1.1 equiv) or 3-fluorophenylboronic acid (1.6 g, 12 mmol,1.3 equiv) or 2-methoxyphenylboronic acid (0.167 g, 1.10 mmol, 1.1equiv) or 2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-pyridine (1.01 g, 4.87 mmol, 1.1 equiv), and the mixture was heatedat 100 °C under N2 for several hours. The mixture was partitionedbetween EtOAc and H2O, the organic layer was separated, and theaqueous layer was extracted with EtOAc. The organic layer was driedover Na2SO4, filtered, and concentrated. Column chromatography(silica gel) with 0−25% gradient of EtOAc in hexane provided 2-arylquinoline-3-carbaldehydes.

8-Methyl-2-(2-methylphenyl)quinoline-3-carbaldehyde(38a). Starting amount of 6 (2.1 g, 10 mmol); white solid; yield 90%(2.35g, 8.99 mmol). 1H NMR (400 MHz, CDCl3) δ ppm 9.96 (1 H,s), 8.83 (1 H, s), 7.88 (1 H, d, J = 7.8 Hz), 7.74 (1 H, d, J = 6.3 Hz),


dx.doi.org/10.1021/jm501624r | J. Med. Chem. XXXX, XXX, XXX−XXXR

7.55 (1 H, t, J = 7.8 Hz), 7.36−7.46 (4 H, m), 2.84 (3 H, s), δ 2.30 (3H, s). Mass spectrum (ESI) m/e = 262 (M + 1).8-Chloro-2-(2-chlorophenyl)quinoline-3-carbaldehyde

(38b). Starting amount of 36b (6.35 g, 28.1 mmol); yellow solid; yield74%, (6.3 g, 21 mmol). 1H NMR (400 MHz, [D6]DMSO) δ ppm10.25 (1 H, s), 8.93 (1 H, s), 8.14 (1 H, d, J = 8.6 Hz), 8.03 (1 H, d, J= 9.0 Hz), 7.55−7.64 (1 H, t, J = 8.0 Hz). Mass spectrum (ESI) m/e =302.0 and 304.0 (M + 1).8-Chloro-2-(3-fluorophenyl)quinoline-3-carbaldehyde (38c).

Starting amount of 36b (2.0 g, 8.8 mmol); yield 71% (1.8 g, 6.3mmol). Mass spectrum (ESI) m/e = 286 (M + 1).2-(2-Methoxyphenyl)-8-methylquinoline-3-carbaldehyde

(38d). Starting amount of 36a (0.206 g, 1.00 mmol); white solid; yield90% (0.250g, 0.900 mmol). 1H NMR (500 MHz, [D6]DMSO) δ ppm9.86 (1 H, s), 8.88 (1 H, s), 8.13 (1 H, d, J = 8.1 Hz), 7.85 (1 H, d, J =7.1 Hz), 7.56−7.74 (3 H, m), 7.22−7.30 (2 H, m), 3.78 (3 H, s), 2.80(3 H, s). Mass spectrum (ESI) m/e = 278 (M + 1).8-Chloro-2-(2-methylpyridin-3-yl)quinoline-3-carbaldehyde

(63). Starting amount of 36b (1.00 g, 4.24 mmol); off-white solid;yield 86% (1.25g, 3.82 mmol). 1H NMR (400 MHz, [D6]DMSO) δppm 9.96 (1 H, s), 9.16 (1 H, s), 8.62 (1 H, dd, J = 4.9, 1.8 Hz), 8.31(1 H, dd, J = 8.2, 1.2 Hz), 8.18 (1 H, dd, J = 7.4, 1.2 Hz), 7.80 (1 H,dd, J = 7.6, 1.8 Hz), 7.76 (1 H, dd, J = 8.2, 7.4 Hz), 7.40 (1 H, dd, J =7.4, 4.7 Hz), 2.35 (3 H, s). Mass spectrum (ESI) m/e = 283 (M + H).3-Ethynyl-8-methyl-2-(o-tolyl)quinoline (39a). To

(bromomethyl)triphenylphosphonium bromide (1.00 g, 2.30 mmol)in THF (12 mL) at −78 °C was added portionwise potassium tert-butoxide (4.8 equiv). The reaction mixture was stirred at −78 °C, and38a (0.500 g, 1.92 mmol) was added. The resulting mixture was stirredat −78 °C for 4 h. At this time, more potassium tert-butoxide (2 equiv)was added. The reaction mixture was allowed to warm to rt and heatedto 40 °C for 1 h. Water was added and the mixture diluted with DCMand washed with water, brine and dried over MgSO4. Solvent wasremoved and the crude was purified by flash chromatography, elutingwith 0−50% EtOAc/hexane to give 39a. Yield 41% (207 mg, 0.787mmol). Mass spectrum (ESI) m/e = 258 (M + 1).3-((7H-Purin-6-yl)ethynyl)-8-methyl-2-(o-tolyl)quinoline

(39b). To a solution of 6-iodo-7H-purine (586 mg, 2.34 mmol) and39a (204 mg, 0.790 mmol) in DMF (0.1 M) was added TEA (2.9mL). The resulting mixture was degassed followed by the addition ofCuI (0.4 equiv) and PdCl2(PPh3)2 (0.2 equiv). The reaction mixturewas heated to 50 °C for 3 h. The reaction mixture was cooled to rt.Solvent was removed under reduced pressure and the crude residuewas purified by flash chromatography, eluting with 0−10% MeOH/DCM to afford 39b. Yield 41% (123 mg, 0.324 mmol). Mass spectrum(ESI) m/e = 376 (M + 1).(8-Methyl-2-o-tolylquinolin-3-yl)methanol (40a). A mixture of

37 (310 mg, 1.49 mmol), o-tolylboronic acid (304 mg, 2.24 mmol),PdCl2(PPh3)2 (52.4 mg, 0.0746 mmol), and Na2CO3 (0.329 mg, 5.97mmol) in (7:3:2 DME/H2O/EtOH) (7.2 mL) was heated at 150 °Cin a microwave reactor. After 15 min the mixture was partitionedbetween EtOAc (25 mL) and H2O (25 mL), the layers were separated,and the aqueous layer was extracted with EtOAc (2 × 25 mL). Thecombined organic layers were dried (MgSO4) and concentrated. Theresulting black oil was dissolved in DCM, evaporated onto silica gel,and purified by flash chromatography (Biotage Si 25+M, 10−20%EtOAc/hexane) to provide (40a) White solid; yield 60% (237 mg,0.898 mmol). 1H NMR (400 MHz, CDCl3) δ ppm 8.28 (1 H, s), 7.71(1 H, d, J = 8.0 Hz), 7.55 (1 H, d, J = 8.0 Hz), 7.44 (1 H, dd, J = 8.0Hz), 7.37−7.26 (5 H, m), 4.92 (2 H, d, J = 4.0 Hz), 2.77 (3 H, s), 2.13(3 H, s), 1.80 (1 H, br s). Mass spectrum (ESI) m/e = 264.4 (M + 1).General Procedure for the Synthesis of 37 and 40b−d. To a

solution of 36a (1.05 g, 5.11 mmol) or 38a (1.28 g, 4.90 mmol) or38b (1.18 g, 3.91 mmol) or 38c (2.0 g, 7.0 mmol) or 38d (0.800 g,2.88 mmol) in THF (0.5 M) or MeOH (0.1 M, 37 synthesis) at 0 °Cwas added NaBH4 (1.5 equiv) and stirred for 2 h (or 0.5 h, 37synthesis). Water was added and the aqueous layer extracted withEtOAc. The organic layer was dried over Na2SO4, filtered, andconcentrated. The residue was purified by column chromatography on

silica gel using 50% of EtOAc in hexane to provide crude product(unless otherwise noted).

(2-Chloro-8-methylquinolin-3-yl)methanol (37). The MeOHwas removed under reduced pressure, and the aqueous layer wasextracted with EtOAc (3 × 50 mL). The combined organic layers weredried (MgSO4) and concentrated to provide 37. White solid; yield99% (1.05 g, 5.06 mmol). The crude product was sufficiently pure tobe used in the next step without further purification. 1H NMR (400MHz, CDCl3) δ ppm 8.23 (1 H, s), 7.68 (1 H, d, J = 8.0 Hz), 7.56 (1H, d, J = 8.0 Hz), 7.47−7.43 (1 H, dd, J = 8.0, 8.0 Hz), 4.92 (2 H, d, J= 4.0 Hz), 2.77 (3 H, s), 2.13 (1 H, br s). Mass spectrum (ESI) m/e =208.0 (M + 1).

2-(2-Chlorophenyl)-8-chloroquinolin-3-yl)methanol (40b).Yellow solid; yield 57%, (0.675 g, 2.22 mmol). 1H NMR (400 MHz,[D6]DMSO) δ ppm 8.56 (1 H, s), 8.10 (1 H, dd, J = 8.2, 1.2 Hz), 7.94(1 H, dd, J = 7.6, 1.4 Hz), 7.63 (2 H, t, J = 7.8 Hz), 7.44−7.59 (3 H,m), 5.54 (1 H, t, J = 5.3 Hz). Mass spectrum (ESI) m/e = 304.0 and306.1 (M + 1).

(8-Chloro-2-(3-fluorophenyl)quinolin-3-yl)methanol (40c).Yield 70% (1.4 g, 4.9 mmol). Mass spectrum (ESI) m/e = 288.1 (M+ 1).

(2-(2-Methoxyphenyl)-8-methylquinolin-3-yl)methanol(40d). White solid; yield 99% (0.80 g, 2.8 mmol). 1H NMR (400MHz, [D6]DMSO) δ ppm 8.41 (1 H, s), 7.92 (1 H, d, J = 7.8 Hz),7.64 (1 H, d, J = 7.0 Hz), 7.50−7.59 (2 H, m), 7.33 (1 H, dd, J = 7.4,1.6 Hz), 7.22 (1 H, d, J = 7.8 Hz), 7.16 (1 H, t, J = 7.2 Hz), 5.37 (1 H,t, J = 5.5 Hz), 3.79 (3 H, s), 2.72 (3 H, s). Mass spectrum (ESI) m/e =280.1 (M + 1).

General Procedure for the Synthesis of 40e, 40f, 49a, 49b,64. To a mixture of 38a (0.36 g, 1.4 mmol) or 38b (500 mg, 1.7mmol) or 63 (1.07 g, 3.80 mmol) or 47a (334 g, 1.60 mol) or 47b(2.3 g, 10 mmol) in THF (0.2−0.3 M) at 0 °C was added dropwise asolution of a methylmagnesium iodide or bromide (2 equiv, 3 M), andthe mixture was stirred overnight before adding saturated NH4Clsolution. The mixture was extracted with EtOAc, and the organic layerwas dried (Na2SO4) and concentrated. The residue was purified bycolumn chromatography on silica gel (eluent, 1:1 EtOAc/hexane) toprovide 1-(2-phenylquinolin-3-yl) alcohols.

1-(8-Methyl-2-(o-tolyl)quinolin-3-yl)ethanol (40e). Yield 97%(0.37 g, 1.4 mmol). 1H NMR (400 MHz, CDCl3) δ 8.34 (1 H, s), 7.66(1 H, d, J = 8.2 Hz), 7.48 (1 H, d, J = 6.7 Hz), 7.39 (1 H, t, J = 7.8Hz), 7.19−7.27 (4 H, m), 2.69 (3 H, s), 2.08 (3 H, s), 1.30 (3 H, m).Mass spectrum (ESI) m/e = 278 (M + 1).

1-(8-Chloro-2-(2-chlorophenyl)quinolin-3-yl)ethanol (40f).White solid; yield 87% (460 mg, 1.45 mmol). Mass spectrum (ESI)m/e = 318.0 (M + 1).

1-(2-Chloro-7-fluoroquinolin-3-yl)ethanol (49a). Yield 68%(244 g, 1.08 mmol). Mass spectrum (ESI) m/e = 226 (M + 1).

1-(2-Chloro-5,7-difluoroquinolin-3-yl)ethanol (49b). Pale yel-low solid; yield 71% (1.75 g, 7.17 mmol). 1H NMR (400 MHz,CDCl3) δ ppm 8.63 (1 H, s), 7.50 (1 H, d, J = 9.49 Hz), 7.06−7.13 (1H, m), 5.38 (1 H, qd, J = 6.13, 3.91 Hz), 2.19 (1 H, d, J = 3.91 Hz),1.58 (3 H, s). Mass spectrum (ESI) m/e = 244 (M + 1).

1-(8-Chloro-2-(2-methylpyridin-3-yl)quinolin-3-yl)ethanol(64). Orange oil; yield 98% (1.12 g, 3.72 mmol). 1H NMR (400 MHz,[D6]DMSO) δ ppm 8.68 (1 H, s), 8.59 1 H, dd, J = 4.9, 1.8 Hz), 8.10(l H, dd, J = 8.2, 1.2 Hz), 7.94 (l H, dd, J = 7.6, 1.4 Hz), 7.74 (l H, dd,J = 7.8, 1.6 Hz), 7.58−7.65 (l H, m), 7.39 (l H, dd, J = 7.6, 4.9 Hz),5.47 (l H, d, J = 4.3 Hz), 4.64 (l H, br s), 2.25 (3 H, s), 1.20 (3 H, d, J= 7.4 Hz). Mass spectrum (ESI) m/e = 299 (M + 1).

General Procedure for the Synthesis of 41a and 41b. To astirred solution of 40b (0.675 g, 2.22 mmol) or 40c (150 mg, 0.52mmol) in CHCl3 (0.25 M) was added SOCl2 (5 equiv) at rt for 2 h.The mixture was concentrated, and the residue was partitionedbetween EtOAc and saturated aqueous NaHCO3 solution. The organiclayer was separated, washed with water and brine, dried over Na2SO4,filtered, and concentrated. The crude product was purified by columnchromatography on a Redi-Sep column using 0−100% gradient ofEtOAc in hexane to provide 3-(chloromethyl)-2-arylquinolines.


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8-Chloro-3-(chloromethyl)-2-(2-chlorophenyl)quinoline(41a). Yellow foam; yield 96% (685 mg, 2.13 mmol). 1H NMR (400MHz, [D6]DMSO) δ ppm 8.53 (1 H, s), 7.88 (1 H, dd, J = 8.2, 1.2Hz), 7.81 (1 H, dd, J = 7.4, 1.2 Hz), 7.48 (1 H, d, J = 7.4 Hz), 7.42−7.46 (1 H, m), 7.27−7.41 (3 H, m), 4.63 (1 H, d, J = 9.8 Hz), 4.33−4.45 (1 H, m). Mass spectrum (ESI) m/e = 322.0 and 324.0 (M + 1)8-Chloro-3-(chloromethyl)-2-(3-fluorophenyl)quinoline

(41b). Yield 91% (160 mg, 0.477 mmol). Mass spectrum (ESI) m/e =306 (M + 1).3-(Bromomethyl)-2-(2-methoxyphenyl)-8-methylquinoline

(41c). Solid CBr4 (1.5 equiv) was added to a mixture of 40d (313 mg,1.12 mmol) and triphenylphosphine (1.5 equiv) in DCM (0.17 M) at0 °C, and the mixture was stirred at 0 °C for 0.5 h. The crude mixturewas concentrated under reduced pressure, evaporated onto silica gel,and purified by flash chromatography, eluting with 0−10% EtOAc/hexane to provide 41c. White solid; yield 91% (347 mg, 1.02 mmol) asa white solid. 1H NMR (400 MHz, CDCl3) δ ppm 8.26 (1 H, s), 7.68(1 H, d, J = 8.0 Hz), 7.55 (1 H, d, J = 8.0 Hz), 7.49−7.41 (3 H, m),7.14 (1 H, td, J = 1.0, 8.0 Hz), 7.01 (1 H, d, J = 8.0 Hz), 4.56 (2 H, brs), 3.77 (1 H, s), 2.78 (3 H, s). Mass spectrum (ESI) m/e = 342 (M +1).General Procedure for the Synthesis of 42a and 42b. To a

solution of 41a (220 mg, 0.68 mmol) or 41b (187 mg, 0.611 mmol) inDMSO (0.25 M) was added NaN3 (3 equiv) at rt, and the mixture wasstirred for 4 h. The mixture was diluted with water, extracted withEtOAc, and the organic layer was washed with water, dried overNa2SO4, filtered, and concentrated under reduced pressure.3-(Azidomethyl)-8-chloro-2-(2-chlorophenyl)quinoline

(42a). Yield 80% (224 mg, 0.547 mmol). Mass spectrum (ESI) m/e =329 (M + 1).3-(Azidomethyl)-8-chloro-2-(3-fluorophenyl)quinoline

(42b). Yield 100% (191 mg, 0.611 mmol). Mass spectrum (ESI) m/e= 313 (M + 1).General Synthesis of 43a and 43b. To a N2 purged solution of

42a (180 mg, 0.547 mmol) or 42b (191 mg, 0.611 mmol) in MeOH(2 mL) was added Pd/C (cat.), placed under a H2 balloon overnight.After concentration, the crude mixture was applied to a Redi-Sepcolumn using 0−100% DCM/(89:9:1 DCM/MeOH/NH4OH) toprovide the product.(8-Chloro-2-(2-chlorophenyl)quinolin-3-yl)methanamine

(43a). Light yellow solid; yield 81% (134 mg, 0.442 mmol). 1H NMR(400 MHz, [D6]DMSO) δ ppm 8.62 (1 H, s), 8.05 (1 H, dd, J = 8.6,1.2 Hz), 8.00 (1 H, dd, J = 7.4, 1.2 Hz), 7.63−7.73 (2 H, m), 7.47−7.62 (3 H, m), 3.90 (1 H, s), 3.75 (1 H, s). Mass spectrum (ESI) m/e= 303.1 and 305.0 (M + 1).(8-Chloro-2-(3-fluorophenyl)quinolin-3-yl)methanamine

(43b). Yield 51% (90 mg, 0.31 mmol). Mass spectrum (ESI) m/e =287.0 (M + 1).1-(8-Chloro-2-(2-chlorophenyl)quinolin-3-yl)ethanamine

(43c). A stirred solution of 40f (460 mg, 1.44 mmol) in CHCl3 (7mL) was treated with SOCl2 (0.5 mL, 5 equiv) dropwise. After 2 h themixture was concentrated to dryness. The residue was dissolved inDMSO (5 mL) and treated with NaN3 (0.19 g, 2.92 mmol, 2.0 equiv)at rt. After 4 h the reaction mixture was partitioned between EtOAc(20 mL) and water (15 mL), and the water layer was extracted withEtOAc (5 mL), washed with water, brine, dried over Na2SO4, andconcentrated to give a foam. The foam was dissolved in MeOH (15mL) and treated with Pd−C (10%, 40 mg) and the reaction stirredunder a H2 balloon for 2 h. The mixture was filtered and concentrated.Chromatography on silica gel (DCM/MeOH, 20/1) gave 43c. Offwhite foam; yield 70% (320 mg, 1.01 mmol). It was slighty impurewith starting material and was taken on without further purification.1H NMR (400 MHz, CDCl3) δ ppm 8.59 (1 H, s), 8.47 (1 H, s),7.75−7.84 (3 H, m), 7.40−7.55 (5 H, m), 4.09−4.29 (1 H, m), 1.20 (3H, d, J = 6.7 Hz). Mass spectrum (ESI) m/e = 317.0 (M + 1).2-((8-Methyl-2-o-tolylquinolin-3-yl)methyl)isoindoline-1,3-

dione (44). To a stirred mixture of 40a (258 mg, 0.980 mmol),phthalimide (216 mg, 1.47 mmol), PPh3 (321 mg, 1.22 mmol) in THF(5 mL) at rt was added solid ADDP (309 mg, 1.22 mmol). After 24 hanother 0.5 equiv of phthalimide, ADDP, and PPh3 was added and themixture was stirred at rt. After 5 h, the mixture was concentrated,

evaporated onto silica gel, and purified by flash chromatography(Biotage Si 40+M, 15−25% EtOAc/hexanes) to provide 44. White oil;yield 43% (165 mg, 0.420 mmol). 1H NMR (400 MHz, CDCl3) δppm 7.96 (1 H, s), 7.85 (2 H, dd, J = 4.0, 8.0 Hz), 7.74 (2 H, dd, J =4.0, 8.0 Hz), 7.59 (1 H, d, J = 8.0 Hz), 7.54 (1 H, d, J = 8.0 Hz), 7.40(1 H, t, J = 8.0 Hz), 7.37−7.29 (4 H, m), 4.79 (1 H, d, J = 16.0 Hz),2.78 (3 H, s), 2.22 (3 H, s). Mass spectrum (ESI) m/e = 393.2 (M +1).

(8-Methyl-2-o-tolylquinolin-3-yl)methanamine (45). A mix-ture of 44 (155 mg, 0.395 mmol) and hydrazine monohydrate (0.120mL, 1.98 mmol) in EtOH (5 mL) was heated under reflux for 2 h. Themixture was cooled to rt, concentrated, and partitioned betweensaturated aqueous NaHCO3 (25 mL) and EtOAc (25 mL). Theaqueous layer was extracted with EtOAc (2 × 25 mL). The organiclayer was dried (MgSO4) and concentrated. The resulting yellow oilwas dissolved in DCM, evaporated onto silica gel, and purified by flashchromatography (Biotage Si 25+M, 10−20% EtOAc/hexane) toprovide 45. Cloudy oil; yield 97% (101 mg, 383 mmol). 1H NMR(400 MHz, CDCl3) δ ppm 8.21 (1 H, s), 7.70 (1 H, d, J = 8.0 Hz),7.54 (1 H, d, J = 8.0 Hz), 7.43 (1 H, dd, J = 8.0, Hz), 7.36−7.27 (5 H,m), 3.8 (2 H, s), 2.77 (3 H, s), 2.13 (1 H, br s). Mass spectrum (ESI)m/e = 263.2 (M + 1).

2-Chloro-5,7-difluoroquinoline-3-carbaldehyde (47b). To astirred solution of POCl3 (9.2 mL, 7.0 equiv) was added dropwiseDMF (2.8 mL, 2.5 equiv) at 0 °C to rt for 30 min. To the resultingsemisolid was added N-(2,3-difluorophenyl)acetamide (2.45 g, 140mmol) in one portion, and the resulting mixture heated at 75 °Covernight. After cooling to rt, the reaction mixture was pouredcarefully into ice−water (300g). The white solid was filtered, washedwith water, NaHCO3, water and dried in the air to give 47b. Whitesolid; yield 67% (2.78 g, 9.38 mmol). 1H NMR (500 MHz, CDCl3) δppm 10.57 (1 H, s), 9.00 (1 H, d, J = 0.73 Hz), 7.58 (1 H, d, J = 9.16Hz), 7.18 (1 H, t, J = 8.81 Hz). Mass spectrum (ESI) m/e = 228 (M +1). Mass spectrum (ESI) m/e = 210 (M + 1).

1-(2,8-Dichloroquinolin-3-yl)ethanol (49c). A freshly preparedsolution of LDA (1.1 equiv) in THF (100 mL) was kept in 0 °C for 30min and cooled to −78 °C before the addition of a solution of 48a(8.4 g, 42.4 mmol) in THF (44 mL) dropwise. After 45 min,acetaldehyde (3.6 mL, 1.5 equiv) was added dropwise. After 30 min,saturated NH4Cl was added and partitioned between EtOAc (150 mL)and water (100 mL). The organic layer was washed with water, brine,dried over Na2SO4, and concentrated. After flash chromatography(silica gel) (DCM/hexane, 3/2) of the residue hexane (80 mL) wasadded to the colorless oil and the solution was left overnight. Filtrationgave 49c. White solid; yield 56% (5.8 g, 24 mmol). 1H NMR (400MHz, CDCl3) δ ppm 8.43 (1 H, s), 7.84 (1 H, d, J = 8.0 Hz), 7.79 (1H, d, J = 8.0 Hz), 7.50 (1 H, t, J = 8.0 Hz), 5.40 (1 H, q, J = 8.0 Hz),1.63 (3 H, d, J = 8.0 Hz). Mass spectrum (ESI) m/e = 242 (M + 1).

2 1-(2-Chloro-8-fluoroquinolin-3-yl)ethanol (49d). A solutionof 48b (24.3 g, 108 mmol) in THF (200 mL) was placed into ajacketed dropping funnel on top of a flask cooled to −78 °C,containing freshly prepared LDA (1.9 equiv) in THF (80 mL). Thesolution was added dropwise maintaining temperature below −70 °C.After the addition, the mixture was allowed to stir for 5 min and thenthe entire reaction mixture was added dropwise (via cannula) to athree-necked flask with cold acetaldehyde (22.6 mL, 400 mmol) in 600mL of THF at −78 °C. The temperature was maintained below −70°C. The mixture was allowed to stir for an additional 25 min. At thistime, NH4Cl (200 mL, 50% saturated) and EtOAc (500 mL) wereadded. The layers were separated, and the aqueous layer was extractedwith EtOAc (100 mL). The organic layers were washed with saturatedNaCl solution (200 mL), dried over MgSO4, filtered, andconcentrated. The resulting oil was deposited on 100 g of silica geland passed through 200 g silica plug using hexane/EtOAc 8:2 (6 L).(Note: Only the middle 2 L was collected first, and last 2 L was setaside.) Crystallization was from 9:1 hexanes/EtOAc. The mixture wascooled to rt and stirred overnight. It was filtered and washed with 9:1hexanes/EtOAc, air-dried, then vacuum-dried to give 49d. Whitepowder; yield 54% (10.9 g, 48.3 mmol). 1H NMR (400 MHz,(CDCl3) δ ppm 8.43 (1 H, br s), 7.64 (1 H, td, J = 7.8,5.1 Hz), 7.41 (1


dx.doi.org/10.1021/jm501624r | J. Med. Chem. XXXX, XXX, XXX−XXXT

H, ddd, J = 10.2, 7.4, 1.2 Hz), 5.39 (1 H, qdd, J = 6.3, 3.9, 0.8 Hz),2.22 (1 H, d, J = 3.9 Hz), 1.62 (3 H, d, J = 6.3 Hz). Mass spectrum(ESI) m/e = 226.0 (M + 1).General Procedure for 50a−d. A mixture of 49a (7.7 g, 34

mmol) or 49b (1.75 g, 7.18 mmol) or 49c (5.00 g, 207 mmol) or 49d(6.20 g, 27.5 mmol) and MnO2 (10 equiv) in toluene (0.17 M) washeated to reflux for 2 h. Filtration followed with removal of solventgave the product.(R)-1-(2-Chloro-7-fluoroquinolin-3-yl)ethanol (50a). Off-white

solid; yield 81% (6.2 g, 28 mmol). Mass spectrum (ESI) m/e = 224 (M+ 1).1-(2-Chloro-5,7-difluoroquinolin-3-yl)ethanone (50b). Pale

yellow solid; yield 85% (1.47 g, 6.12 mmol). Mass spectrum (ESI)m/e = 242 (M + 1).1-(2,8-Dichloroquinolin-3-yl)ethanone (50c). White solid;

yield 91% (4.50 g, 18.8 mmol). Mass spectrum (ESI) m/e = 239.9(M + 1).1-(2-Chloro-8-fluoroquinolin-3-yl)ethanone (50d). White

solid; yield 72% (4.43 g, 19.8 mmol). 1H NMR (400 MHz,(CDCl3) δ ppm 8.40 (1 H, d, J = 7.6 Hz), 7.71 (1 H, br d, J = 8.2Hz), 7.56 (1 H, td, J =7.8, 5.1 Hz), 7.54 (1 H, ddd, J = 8, 7.8, 1:6 Hz).Mass spectrum (ESI) m/e = 224 (M + 1).General Procedure for 51a−d. To a stirred solution of (+) DIP-

Cl (2.2 equiv) in anhydrous THF (0.3 M) cooled to −47 °C (dry ice,acetonitrile bath) (or −78 °C for 51c synthesis) was added 50a (62.0g, 277 mmol) or 50b (34.7 g, 144 mmol) or 50c (26.2 g, 109 mmol)or 50d (63.4 g, 283 mmol) as a solution in THF (0.45 M) dropwise.The mixture was allowed to warm to 10 °C for 5 h or rt overnight. Atthis time, acetone (1.1 M) and 10% Na2CO3 (1.1 M) were added at 0°C and allowed to stir for 1 h at rt. EtOAc (0.22 M) was added, andthe layers were separated. The organic phase was washed with a 50%saturated NaHCO3 solution, brine, dried over MgSO4, filtered, andconcentrated to provide product.(R)-1-(2-Chloro-7-fluoroquinolin-3-yl)ethanol (51a). The re-

sulting yellow residue was treated with hexane (100 mL) and water(165 mL), and the mixture was stirred at rt overnight. The resultingwhite solid was filtered, dissolved in ethyl acetate (400 mL), washedwith brine and water, and then dried over MgSO4 and evaporated invacuo to give 51a. White solid; yield 72% (45.0 g, 199 mmol). ChiralHPLC on AD column (isopropanol in hexane, 10%) showed >99% ee.Mass spectrum (ESI) m/e = 226 (M + 1).(R)-1-(2-Chloro-5,7-difluoroquinolin-3-yl)ethanol (51b). The

dried solids were treated with hexane (300 mL) and water (200 mL),stirred for 20 min, filtered, and washed with water and hexane to afford51b. White solid; yield 91% (31.7 g, 130 mmol). Chiral HPLC on ADcolumn (isopropanol in hexane, 10%) showed >99% ee . Massspectrum (ESI) m/e = 244 (M + 1).(R)-1-(2,8-Dichloroquinolin-3-yl)ethanol (51c). The dried

solids were treated with hexane (300 mL) and water (200 mL) andstirred for 20 min, filtered, and washed with water and hexane. ChiralHPLC on IA column (isopropanol in hexane, 10%) showed a ratio of20:1 for two enantiomers. Recrystallization from a mixture of EtOAc(70 mL) and hexane (250 mL) provided 51c. White needles; yield90% (23.7 g, 97.9 mmol). Chiral HPLC AD-H column (isopropanol inhexane, 10%) showed >99 ee. 1H NMR (400 MHz, CDCl3) δ ppm8.43 (1 H, s), 7.84 (1 H, d, J = 8.0 Hz), 7.79 (1 H, d, J = 8.0 Hz), 7.50(1 H, t, J = 8.0 Hz), 5.40 (1 H, q, J = 8.0 Hz), 1.63 (3 H, d, J = 8.0Hz). Mass spectrum (ESI) m/e = 242 (M + 1).(R)-1-(2-Chloro-8-fluoroquinolin-3-yl)ethanol (51d). The

dried solids were treated with hexanes (1.5 L) and H2O (1 L) andstirred at rt for 3 h before a white solid was filtered and washed withwater and hexane to give 51d. White solid; yield 83% (53.3 g, 236mmol). Chiral HPLC AD-H column (isopropanol in hexane, 10%)showed >99 ee. 1H NMR (400 MHz, (CDCl3) δ ppm 8.43 (1 H, br s),7.64 (1 H, br d, J = 8.2 Hz), 7.50 (1 H, td, J = 7.8, 4.7 Hz), 7.41 (1 H,ddd, J = 10.2, 7.8, 1.2 Hz), 5.40 (1 H, qd, J = 5.9, 0.8 Hz), 2.22 (1 H,br s), 1.62 (3 H, d, J = 6.3 Hz). Mass spectrum (ESI) m/e = 226 (M +1).General Procedure for 52a−d. To a solution of 51a (3.03 g, 13.4

mmol) or 51b (1.17 g, 4.80 mmol) or 51c (22.2 g, 91.7 mmol) or 51d

(8.15g, 36.1 mmol) in THF (0.2 M) were added PPh3 (1.2 equiv),phthalimide (1.2 equiv), and DIAD (1.2 equiv) dropwise at 0 °C to rt.The reaction mixture was stirred 4 h to overnight. The mixture waspartioned between EtOAc or Et2O and water. The organic layer wasseparated, washed with water, brine, dried over Na2SO4, andconcentrated. The residue was purified by column chromatographyon silica gel, eluting with 0−25% EtOAc/hexane to give the product.

(S)-2-(1-(2-Chloro-7-fluoroquinolin-3-yl)ethyl)isoindoline-1,3-dione (52a). White foam; yield 80% (3.80 g, 10.7 mmol). Massspectrum (ESI) m/e = 355 (M + 1).

(S )-2-(1-(2-Chloro-5,7-difluoroquinolin-3-yl)ethyl)-isoindoline-1,3-dione (52b). Yield 80% (1.08 g, 3.84 mmol). 1HNMR (500 MHz, CDCl3) δ ppm 8.76 (1 H, s), 7.80−7.84 (2 H, m),7.73 (2 H, dd, J = 5.5, 3.1 Hz), 7.45−7.49 (1 H, m), 7.11 (1 H, td, J =9.3, 2.2 Hz), 5.94 (1 H, q, J = 7.3 Hz), 1.98 (3 H, d, J = 7.1 Hz). Massspectrum (ESI) m/e = 373 (M + 1).

(S)-2-(1-(2,8-Dichloroquinolin-3-yl)ethyl)isoindoline-1,3-dione (52c). Chromatography on silica gel, eluting with 1:1 DCM/hexane. White foam; yield 88% (29.8 g, 80.3 mmol). 1H NMR (400MHz, CDCl3) δ ppm 8.49 (l H, s), 7.77−7.73 (4 H, m), 7.66−7.63 (2H, m), 7.43 (1 H, t, J = 8.0 Hz), 5.89 (1 H, q, J = 8.0 Hz), 1.91 (3 H, d,J = 8.0 Hz). Mass spectrum (ESI) m/e = 372 (M + l).

(S)-2-(1-(2-Chloro-8-fluoroquinolin-3-yl)ethyl)isoindoline-1,3-dione (52d). Chromatography using 100% dichloromethane;yield 82% (10.5 g, 29.6 mmol). 1H NMR (500 MHz, CDCl3) δ ppm8.59 (1 H, s), 7.82 (2 H, dd, J = 5.5, 3.1 Hz), 7.69−7.75 (3 H, m), 7.53(1 H, td, J = 8.1, 4.9 Hz), 7.43 (1 H, ddd, J = 10.1, 7.8, 1.1 Hz), 5.97 (1H, q, J = 7.1 Hz), 1.99 (3 H, d, J = 7.1 Hz). Mass spectrum (ESI) m/e= 355.2 (M + 1).

(S)-2-(1-(2,8-Dichloroquinolin-3-yl)ethyl)isoindoline-1,3-dione (53d). A stirred suspension of 52c (250 mg, 0.673 mmol), 3-(methylsulfonyl)phenylboronic acid (162 mg, 0.810 mmol, 1.2 equiv),Na2CO3 (214 mg, 2.01 mmol, 3.0 equiv), Pd(Ph3)4 (35 mg, 0.034mmol, 0.05 equiv) in MeCN (0.1 M) and H2O (0.3 M) was heated to90 °C overnight. The reaction mixture was cooled to rt and partitionedbetween EtOAc and water. The organic layer was isolated andextracted with NaOH (1.0 N) The combined water layers werewashed with DCM or EtOAc/hexane and neutralized to pH 3 withHCl (3.0 N). The resulting milky mixture was extracted with DCM.Combined extracts were washed with brine, dried over Na2SO4, andconcentrated. The product was dissolved in EtOH (0.1 M) and 4 NHCl/dioxane to give a 25:1 EtOH/4 N HCl dioxane solution. Thereaction mixture was heated to 90 °C overnight. After cooling to rt, thereaction mixture was concentrated to dryness to provide 53d, usedwithout characterization. White solid; yield, 62% (205 mg, 0.418mmol).

General Procedure for 53e and 53f. To a stirred mixture of 52d(9.30 g, 26.2 mmol (for 53f)) or 52d (3.02 g, 8.51 mmol, 53esynthesis), K2CO3 (14.49 g, 10.49 mmol, 0.4 equiv (for 53f)), orNa2CO3 (4.71 g, 44.4 mmol, 53e synthesis), Pd(dppf)2Cl2 (0.1 equiv),in dry DMF (0.15 M), purged with N2 was added phenylboronic acid(2.0 equiv), and the mixture was heated to 100 °C. After 2.5 h themixture was diluted with EtOAc, washed with water, brine, dried overMgSO4, filtered, and concentrated to give a crude product.

2-((S)-1-(8-Fluoro-2-(3-fluorophenyl)quinolin-3-yl)ethyl)-isoindoline-1,3-dione (53e). Recrystallization was from EtOAc/hexanes. Yield 70% (2.45 g, 5.91 mmol). 1H NMR (400 MHz, CDCl3)δ ppm 8.63 (1 H, s), 7.66−7.75 (5 H, m), 7.53 (1 H, td, J = 7.9, 4.9Hz), 7.42 (1 H, ddd, J = 10.4, 7.8, 1.4 Hz), 7.35 (1 H, td, J = 7.9, 5.7Hz), 7.21 (1 H, dt, J = 7.7, 1.2 Hz), 7.04−7.14 (2 H, m), 5.80 (1 H, q,J = 7.0 Hz), 1.90 (3 H, d, J = 7.0 Hz). Mass spectrum (ESI) m/e =415.0 (M + l).

2-((S)-1-(8-Fluoro-2-(3, 5-difluorophenyl)quinolin-3-yl)-ethyl)isoindoline-1,3-dione (53f). Purification: silica gel plug(400g), 30% EtOAc in hexanes, then DCM. Concentration andrecrystallization with EtOAc (100 mL) and hexanes (400 mL) gave avoluminous white precipiate, which was filtered and washed with 200mL of 9:1 hexanes/EtOAc(200 mL), isolating 7 g of white solid. It wasdissolved in chloroform. Silica gel plug (15 g) and chloroform (150mL) were used. Concentration and recrystallization were from


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EtOAc/hexanes. White solid; yield 66% (4.80 g, 11.1 mmol). 1H NMR(400 MHz, CDCl3) δ ppm 8.66 (1 H, s), 7.68−7.76 (5 H, m), 7.55 (1H, td, J = 8.0, 4.7 Hz), 7.44 (1 H, ddd, J = 10.3, 7.7, 1.2 Hz), 6.94 (2H, dt, J = 5.8, 2.0 Hz), 6.83 (1 H, tt, J = 9.0, 2.3 Hz), 5.78 (1 H, q, J =7.0 Hz), 1.92 (3 H, d, J = 7.0 Hz). Mass spectrum (ESI) m/e = 432.9(M + l).General Procedure for 53a−c. A stirred mixture of 52c (20.4 g,

55.1 mmol, 53a synthesis) or 52c (19.0 g, 51.2 mmol, 53b synthesis)or 52a (19.0 g, 53.6 mmol, 53c synthesis), the appropriate boronicacid (1.1−1.6 equiv), Pd(PPh3)4 (0.05−0.1 equiv), and Na2CO3 (5.0equiv) in acetonitrile−water (3:1) (0.1 M) was heated to 100 °C. After1−4 days, the mixture was cooled to rt, concentrated, and partitionedbetween DCM and water. The water layer (pH 10−11) was washedwith DCM to remove byproducts. The organic and acidic aqueouslayers were separated. The aqueous layer was treated with 2 N HCl(0.2 M in substrate) and extracted with DCM. The organic layer waswashed with water brine, dried over Na2SO4, filtered, and concentrated2-(((S)-1-(8-Chloro-2-(3-fluorophenyl)quinolin-3-yl)ethyl)-

carbamoyl)benzoic Acid (53a). yield, ∼quant (25.9 g, 55.1 mmol).1H NMR (400 MHz, [D6]DMSO) δ ppm 12.87 (1 H, s), 8.99 (1 H, d,J = 7.0 Hz), 8.61 (1 H, s), 7.92−8.03 (2 H, m), 7.78 (1 H, dd, J = 7.8,1.2 Hz), 7.47−7.67 (6 H, m), 7.35−7.45 (2 H, m), 5.26−5.38 (1 H,m), 1.34 (3 H, d, J = 6.7 Hz). Mass spectrum (ESI) m/e = 448.9 (M +1).2-((1S)-1-(8-Chloro-2-(2-fluorophenyl)quinolin-3-yl)-

ethylcarbamoyl)benzoic Acid (53b). Brown solid; yield 91% (20.9g, 46.5 mmol). 1H NMR (400 MHz, [D6]DMSO) δ ppm 12.86 (1 H,br. s), 8.92 (1 H, br s), 8.62 (1 H, s), 8.02 (1 H, dd, J = 8.4, 1.2 Hz),7.96 (1 H, dd, J = 7.5, 1.3 Hz), 7.75(1 H, dd, J = 7.5, 1.1 Hz), 7.53−7.67 (4 H, m), 2.03 (2 H, s), 7.46−7.52 (1 H, m), 7.36−7.45 (3 H, m),5.09 (1 H, quin, J = 6.9 Hz), 1.36 (3 H, d, J = 6.8 Hz). Mass spectrum(ESI) m/e = 449.0 (M + 1).2-((1S)-1-(7-Fluoro-2-(3-fluorophenyl)quinolin-3-yl)-

ethylcarbamoyl)benzoic Acid (53c). Pale yellow solid; yield,∼quant (23.4 g, 54 mmol). Mass spectrum (ESI) m/e = 432.9 (M + l).General Procedure for 54a−c. To a stirred suspension of 53a

(24.7 g, 55.1 mmol) or 53b (20.9 g, 46.5 mmol) or 53c (23.4 g, 54.1mmol) in EtOH (0.2−0.5 M) was added conc HCl (10−20 equiv) andheated to reflux. After 4 h to 6 days, the mixture was cooled to rt andthe ethanol removed under vacuum.(1S ) -1- (8-Chloro-2-(3-fluorophenyl)quinol in-3-yl ) -

ethanamine (54a). The mixture was refluxed for 6 days. To theresidual acidic aqueous mixture was added ice−water (200 mL) at 0°C with stirring. The mixture was treated with 10 N NaOH (∼50 mL)to pH ≈ 10 and extracted with DCM. The organic layer was washedwith brine, dried over Na2SO4, filtered, and concentrated to give abrown syrup which was purified. Column chromatography: 330 g ofRedi-Sep column using 0−20% DCM/(89:9:1 DCM/MeOH/NH4OH) over 50 min and then 20% DCM/(89:9:1 DCM/MeOH/NH4OH) for 50 min as eluent to give 54a. Light yellow syrup; yield84% (13.8 g, 46.1 mmol). 1H NMR (400 MHz, [D6]DMSO) δ ppm8.74 (1 H, s), 8.01 (1 H, dd, J = 8.2, 1.2 Hz), 7.92 (1 H, dd, J = 7.4, 1.2Hz), 7.54−7.63 (2 H, m), 7.43−7.52 (2 H, m), 7.32−7.39 (1 H, m),4.25 (1 H, q, J = 6.7 Hz), 2.03 (2 H, s), 1.21 (3 H, d, J = 6.7 Hz). Massspectrum (ESI) m/e = 300.9 (M + 1).(1S ) -1- (8-Chloro-2-(2-fluorophenyl)quinol in-3-yl ) -

ethanamine (54b). The mixture was refluxed for 4 days. The mixturewas poured into ice−water. The aqueous acidic mixture (pH ≈ 1.5)was extracted with DCM and concentrated to give a brown syrup. Theremaining aqueous mixture was then treated with saturated aqueousNaHCO3 solution (200 mL) and 10 N NaOH (10 mL) to give pH ≈11 and extracted with DCM. The organic layer was washed with water,brine, dried over Na2SO4, filtered, and concentrated to give 54b.Yellow syrup; yield 5.3% (0.74 g, 2.5 mmol). The brown syrup wasdissolved in DCM (200 mL) and washed with saturated aqueousNaHCO3 solution and extracted with DCM. The combined organiclayers were washed with water, brine, dried over Na2SO4, filtered, andconcentrated under reduced pressure to give a mixture of (major)desired product and the (minor) ethyl ester as a dark red syrupy solid,which was suspended in EtOH (230 mL) and treated with hydrazine

monohydrate (22.5 mL, 465 mmol). This mixture was refluxed for 1.5h, cooled to rt, and the resulting precipitate was filtered and washedwith EtOAc. The filtrate was concentrated under reduced pressure,redissolved in EtOAc (200 mL), and washed with water (200 mL).The organic layer was dried over MgSO4, filtered, and concentratedunder reduced pressure to give an orange syrup, which was purified bycolumn chromatography on a 330 g Redi-Sep column using 0−50%DCM/(89:9:1 DCM/MeOH/NH4OH) over 25 min and then 50%isocratic of DCM/(89:9:1 DCM/MeOH/NH4OH) for 25 min aseluent to give 54b yellow syrup. Combined yield 86% (12.0 g, 39.8mmol). 1H NMR (400 MHz, [D6]DMSO) δ ppm 8.74 (1 H, s), 8.03(1 H, dd, J = 8.2, 1.2 Hz), 7.93 (1 H, dd, J = 7.5, 1.3 Hz), 7.50−7.66 (3H, m), 7.34−7.43 (2 H, m), 4.03 (1 H, q, J = 6.4 Hz), 1.98 (2 H, br s),1.16 (3 H, d, J = 5.5 Hz). Mass spectrum (ESI) m/e = 301.0 (M + l).

(1S ) -1- (7-Fluoro-2-(3-fluorophenyl)quinol in-3-yl ) -ethanamine (54c). The mixture was refluxed for 4 h. The residuewas treated with hydrazine hydrate (27.0 mL, 536 mmol) in EtOH(200 mL) at 70 °C for 3 h. The resultant mixture was concentrated,filtered, and rinsed with DCM−MeOH (9:1). The combined filtrateswere concentrated. Purification of the residue by flash chromatographyover silica gel, with gradient elution, 0−10% MeOH in DCM with0.1% aqueous NH4OH, gave 54c. Yield 78% (11.95 g, 42.03 mmol).1H NMR (400 MHz, [D6]DMSO) δ ppm 8.70 (1 H, s), 8.12 (1 H, dd,J = 9.0, 6.5 Hz), 7.73 (1 H, dd, J = 10.6, 2.7 Hz), 7.51−7.67 (3 H, m),7.39−7.48 (2 H, m), 7.29−7.37 (1 H, m), 4.21 (1 H, q, J = 6.5 Hz),1.20 (3 H, d, J = 6.5 Hz). Mass spectrum (ESI) m/e = 285.1 (M + 1).

General Procedure for 55a, 55b, 55d, and 55e. A mixture of52a (85.6 g, 241 mmol) or 52b (100 mg, 0.268 mmol) or 52c (13.6 g,36.6 mmol) or 52d (0.803 g, 2.26 mmol) and Pd(PPh3)4 (0.05 equiv)or (0.1 equiv, for 55a synthesis) and 2-(tributylstannyl)pyridine (1.2equiv) or (1.0 equiv, for 55b synthesis) in dioxane (0.12 M) or toluene(0.13 M, for 55b synthesis) was heated to 100−120 °C under N2 for 1day, 4 days (55b synthesis), or 3 days (55e synthesis) and thenallowed to cool to rt. Removal of solvent followed by colunmchromatography on silica gel (gradient elution EtOAc/hexane)provided the product.

(S)-2-(1-(8-Fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-isoindoline-1,3-dione (55a). Chromatography, 40 g CombiFlash,10% EtOAc/hexane to 100% EtOAC, gave 55a. Yellow foam; yield83% (0.750 g, 1.89 mmol,). 1H NMR (400 MHz, [D6]DMSO) δ ppm8.78 (1 H, s), 8.51 (1 H, d, J = 3.9 Hz), 7.95−8.01 (1 H, m), 7.73−7.81 (3 H, m), 7.59−7.71 (5 H, m), 7.37 (1 H, dd, J = 7.0, 5.5 Hz),6.22 (1 H, q, J = 6.9 Hz), 1.82 (3 H, d, J = 7.0 Hz). Mass spectrum(ESI) m/e = 398.0 (M + l).

2-((S)-1-(8-Chloro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-isoindoline-1,3-dione (55b). The sample was dissolved in EtOAc,washed with saturated NaCl solution, dried with sodium sulfate,filtered, and concentrated. Chromatography, 330 g silica gel columnwith 0−70% EtOAc/hexane, gave 55b. Yellow orange oil; yield 73%(11 g, 27 mmol). 1H NMR (400 MHz, CDCl3) δ ppm 8.68 (1 H, s),8.65−8.67 (1 H, m), 7.93 (1 H, dt, J = 7.8, 1.0 Hz), 7.85 (2 H, ddd, J =10.5, 7.9, 1.2 Hz), 7.74 (1 H, td, J = 7.7, 1.8 Hz), 7.62−7.70 (4 H, m),7.46−7.52 (1 H, m), 7.30 (1 H, ddd, J = 7.6, 4.9, 1.2 Hz), 6.58 (1 H, q,J = 7.0 Hz), 2.01 (3 H, d, J = 7.0 Hz). Mass spectrum (ESI) m/e =414.0 (M + 1).

3-((S)-1-(1,3-Dioxoisoindolin-2-yl)ethyl)-2-(pyridin-2-yl)-quinoline-8-carbonitrile (55c). To a flask were charged 55b (3.00g, 7.25 mmol), Pd(CF3CO2)2 (121 mg, 0.362 mmol), 2-dicyclohex-ylphosphino-2′,4′,6′-triisopropylbiphenyl (Xphos) (346 mg, 0.725mmol), and tributyltin cyanide (2.29 g, 7.25 mmol). NMP (4 mL)and dicyclohexylamine (10 mL) were added, and the reaction washeated to 160 °C (in a hot oil bath) under N2. After 45 min additionalamounts of Pd(CF3CO2)2 (0.05 equiv), Xphos (0.1 equiv), anddicyclohexylamine (5 mL) were added. Additional amounts ofPd(CF3CO2)2 (0.05 equiv), Xphos (0.1 equiv), and dicyclohexylamine(5 mL) were added every 30 min for 1.5 h (3×). After an additional 1h, the mixture was cooled to rt and partitioned between EtOAc andsaturated NaHCO3. The organic layer was washed with brine, driedwith MgSO4, filtered, and evaporated in vacuo. Column chromatog-raphy (hexanes/EtOAc, 1:0 to 1:2) gave 55c. Yield 68% (1.98 g, 4.90


dx.doi.org/10.1021/jm501624r | J. Med. Chem. XXXX, XXX, XXX−XXXV

mmol). 1H NMR (400 MHz, CDCl3) δ ppm 8.74 (1 H, s), 8.68 (1 H,dq, J = 4.9, 0.8 Hz), 8.17 (1 H, dd, J = 8.2, 1.2 Hz), 8.13 (1 H, dd, J =7.2, 1.4 Hz), 8.01−8.04 (1 H, m), 7.78 (1 H, td, J = 7.7, 1.8 Hz), 7.61−7.70 (5 H, m), 7.34 (1 H, ddd, J = 7.5, 4.8, 1.4 Hz), 6.63−6.70 (1 H,m), 2.02 (3 H, d, J = 7.4 Hz). Mass spectrum (ESI) m/e = 405.1 (M +1).(S)-2-(1-(5,7-Difluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-

isoindoline-1,3-dione (55d). Off white solid; yield 76% (85.0 mg,0.205 mmol). 1H NMR (400 MHz, CDCl3) δ ppm 8.88 (1 H, s),8.59−8.70 (1 H, m), 7.61−7.75 (5 H, m), 7.58 (1 H, d, J = 9.78 Hz),7.31 (2 H, ddd, J = 7.43, 4.70, 1.17 Hz), 7.11 (1 H, td, J = 9.39, 2.35Hz), 6.33 (1 H, q, J = 7.04 Hz), 1.98 (3 H, d, J = 7.04 Hz). Massspectrum (ESI) m/e = 416 (M + 1).(S)-2-(1-(7-Difluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-

isoindoline-1,3-dione (55e). The reaction mixture was cooled to rtand decanted. The final 200 mL solution was filtered to remove Pdresidue. The combined solvents were concentrated to 300 mL, filtered,washed (300 mL, 1:1 EtOAc/hexane), and dried to give a tan solid(82.1 g). The original mother liquor was concentrated to 100 mL andtreated with (200 mL, 1:1 EtOAc/hexane) to precipitate an additional2.2 g. The solids were combined yield 88% (84.3 g 212 mmol). 1HNMR (500 MHz, CDCl3) δ ppm 8.69 (1 H, s), 8.65−8.67 (1 H, m),7.94 (1 H, dd, J = 8.9, 6.0 Hz), 7.71−7.76 (2 H, m), 7.63−7.69 (5 H,m), 7.36−7.41 (1 H, m), 7.32 (1 H, ddd, J = 7.6, 4.9, 1.2 Hz), 6.31 (1H, q, J = 7.1 Hz), 1.98 (3 H, d, J = 7.3 Hz). Mass spectrum (ESI) m/e= 398.2 (M + 1).2-((S)-1-(8-Chloro-2-(2-chloro-5-fluoropyridin-3-yl)quinolin-

3-yl)ethyl)isoindoline-1,3-dione (55f). A mixture of 52c (280 mg,0.754 mmol), 2-chloro-5-fluoropyridin-3-ylboronic acid (159 mg,0.907 mmol), Pd(PPh3)4 (436 mg, 0.38 mmol), cesium fluoride(344 mg, 2.26 mmol), and copper(I) iodide (29 mg, 0.15 mmol) in1,2-ethanediol, dimethyl ether (3.0 mL, 0.75 mmol) was subjected tomicrowave at 100 °C for 1 h and cooled to rt. The mixture was filteredand rinsed with EtOAc. The filtrates were collected and concentrated.Purification of the residue by flash chromatography over silica gel,eluting with 0−100% EtOAc in hexane, gave 55f. Yield 40% (140 mg,0.300 mmol). 1H NMR (500 MHz, [D6]DMSO) δ ppm 9.02 (1 H, s),8.96 (1 H, s), 8.59 (1 H, d, J = 2.9 Hz), 8.51 (1 H, d, J = 2.9 Hz), 8.31(1 H, dd, J = 8.4, 3.1 Hz), 8.24 (1 H, ddd, J = 8.3, 2.9, 1.2 Hz), 8.00−8.06 (2 H, m), 7.79 (6 H, ddd, J = 5.4, 3.1, 2.0 Hz), 7.65−7.75 (7 H,m), 5.49−5.56 (1 H, m), 5.40 (1 H, q, J = 7.3 Hz), 1.91 (3 H, d, J =7.1 Hz), 1.85 (3 H, d, J = 6.8 Hz) (note that there is a mixture of tworotational isomers, ratio 1:1.4). Mass spectrum (ESI) m/e = 466.0 (M+ 1).2-((S)-1-(8-Chloro-2-(5-fluoro-2-methylpyridin-3-yl)quinolin-

3-yl)ethyl)isoindoline-1,3-dione (55g). A mixture of 55f (140 mg,0.300 mmol), dioxane (15 mL, 0.02 M), trimethylaluminum (0.5 mL,1.50 mmol), and Pd(PPh3)4 (69 mg, 0.060 mmol) was refluxed for 4 hunder N2 and then cooled to rt. The reaction mixture was acidifiedwith 2 N HCl and the solvent evaporated. The residue was dilutedwith water, treated with NaOH and the mixture extracted with EtOAc.The combined extracts were washed with water, brine, dried, andconcentrated. Purification of the residue by flash chromatography oversilica gel, eluting with 0−100% EtOAc in hexane, gave 55g. Yield 75%(100 mg, 0.225 mmol). 1H NMR (500 MHz, CDCl3) δ ppm 8.70 (1H, d, J = 7.1 Hz), 8.44 (1 H, d, J = 17.1 Hz), 7.85−7.94 (2 H, m),7.66−7.77 (4 H, m), 7.55 (1 H, t, J = 7.9 Hz), 7.38 (1/2 H, d, J = 7.1Hz), 7.14 (1/2 H, d, J = 6.1 Hz), 5.41−5.63 (1 H, m), 1.85−1.96 (3 H,m), 1.56 (3 H, s). Mass spectrum (ESI) m/e = 445.9 (M + 1).(1S)-1-(8-Chloro-2-(5-fluoro-2-methylpyridin-3-yl)quinolin-

3-yl)ethanamine (56f). A mixture of 55g (100 mg, 0.224 mmol) andconc HCl in a sealed flask was heated to reflux for 24 h. The solventwas removed and the residue purified by flash chromatography oversilica gel, gradient elution, 0−10% MeOH in DCM with 0.1% aqueousNH4OH, gave 56f. Yield 76% (54.2 mg, 0.172 mmol). 1H NMR (500MHz, [D4]MeOH) δ ppm 8.68 (1 H, s), 8.51 (1 H, d, J = 2.7 Hz),7.98 (1 H, dd, J = 8.4, 1.1 Hz), 7.91 (1 H, dd, J = 7.5, 1.3 Hz), 7.64−7.82 (1 H, m), 7.56−7.63 (1 H, m), 5.50 (2 H, s), 3.88−4.15 (1 H, m),2.32 (3 H, s), 1.33 (3 H, d, J = 6.6 Hz). Mass spectrum (ESI) m/e =317 (M + 1).

General Procedure for 54d−f and 56a−e. A stirred solution of53d (205 mg, 0.418 mmol) or 53e (2.45 g, 5.91 mmol) or 53f (8.50 g,19.7 mmol) or 55a (270 mg, 0.679 mmol) or 55b (16.8 g, 40.6 mmol)or 55c (7.50 g, 18.5 mmol) or 55d (83 mg, 0.20 mmol) or 55e (43.8 g,110 mmol) in EtOH (0.1 M) was treated with NH2NH2 (10 equiv) orhydrazine, monohydrate (20 equiv, 56a synthesis) (25 equiv, 54esynthesis) (1.5 equiv, 56c synthesis) (10 equiv, 56e synthesis)dropwise at rt. The mixture was then heated to 50−90 °C for 0.5−4 hand cooled to rt [unless noted below]. The reaction mixture wasfiltered and the filter cake washed with EtOAc. The organic layer wasconcentrated, partially dissolved in EtOAc, washed with water and theaqueous layer was extracted with EtOAc. The combined organic layerswere washed with water, brine, dried over Na2SO4, and concentratedto give the product.

(1S)-1-(8-Chloro-2-(3-(methylsulfonyl)phenyl)quinolin-3-yl)-ethanamine (54d). Yellow oil. Mass spectrum (ESI) m/e = 361.2 (M+ 1). The crude material was used immediately for 25.

(1S ) -1- (8-Fluoro-2-(3-fluorophenyl)quinol in-3-yl ) -ethanamine (54e). Yield 80% (1.34 g, 4.71 mmol). 1H NMR (400MHz, (CDCl3) δ ppm 8.51 (1 H, d, J = 1.6 Hz), 7.65 (1 H, br d, J =8.21 Hz), 7.52−7.42 (2 H, series of m), 7.39 (1 H, ddd, J = 10.5, 7.4,1.2 Hz), 7.34 (1 H, dt, J = 7.5, 1.2 Hz), 7.30 (1 H, ddd, J = 9.4, 2.7, 1.6Hz), 7.16 (1 H, tdd, J = 8.6, 2.4, 0.8 Hz), 4.48 (1 H, q, J = 6.2 Hz),1.37 (d, J = 6.7 Hz, 3 H). Mass spectrum (ESI) m/e = 285 (M + 1).

(S)-1-(2-(3,5-Difluorophenyl)-8-fluoroquinolin-3-yl)-ethanamine (54f). Yellow oil; yield 100% (5.9 g, 19.5 mmol). 1HNMR (400 MHz, CDCl3) δ ppm 1.37 (3 H, d, J = 6.65 Hz), 1.55 (2H, br s), 4.47 (1 H, q, J = 6.26 Hz), 6.92 (1 H, tt, J = 8.80, 2.35 Hz),7.10−7.17 (2 H, m), 7.38−7.44 (1 H, m), 7.51 (1 H, td, J = 7.92, 4.89Hz), 7.68 (1 H, d, J = 8.22 Hz), 8.53 (1 H, d, J = 1.56 Hz).

(1S)-1-(8-Fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethanamine(56a). The crude oil was used without purification. Yield 70% (0.127g, 0.475 mmol). Mass spectrum (ESI) m/e = 430.1 (M + 1).

(1S)-1-(8-Chloro-2-(pyridin-2-yl)quinolin-3-yl)ethanamine(56b). Tan oil; yield 100% (11.6 g, 40.9 mmol). The crude materialwas heated to 90 °C/2 mmHg to remove a colorless liquid byproduct.1H NMR (500 MHz, CDCl3) δ ppm 8.69 (1 H, dt, J = 4.9, 0.9 Hz),8.46 (1 H, s), 8.18 (1 H, d, J = 7.8 Hz), 7.92 (1 H, td, J = 7.7, 1.7 Hz),7.78−7.83 (2 H, m), 7.47 (1 H, t, J = 7.8 Hz), 7.38 (1 H, ddd, J = 7.6,4.9, 1.0 Hz), 4.84 (1 H, q, J = 6.8 Hz), 1.45 (3 H, d, J = 6.6 Hz). Massspectrum (ESI) m/e = 284.0 (M + 1).

3-((S)-1-Aminoethyl)-2-(pyridin-2-yl)quinoline-8-carbonitrile(56c). After heating to 85 °C for 4 h, the mixture was cooled to rt andtreated with DCM (200 mL) and NaHCO3 (saturated aqueoussolution). The separated aqueous layer was extracted with DCM, andthe combined organic layers were dried, filtered, and evaporated invacuo. The resulting residue was taken up in EtOAc (100 mL) andwater (100 mL) and acified to pH 2 with 1.0 M HCl. The aqueouslayer was separated and the pH adjusted (pH 14) with 4.0 M NaOH.The aqueous layer was then extracted with DCM, dried, filtered, andevaporated in vacuo. Yield 97% (4.93 g, 17.9 mmol). 1H NMR (400MHz, CDCl3) δ ppm 1.41 (3 H, d, J = 6.65 Hz), 2.06 (2 H, br s), 4.89(1 H, q, J = 6.65 Hz), 7.35 (1 H, ddd, J = 7.43, 4.69, 1.17 Hz), 7.55 (1H, dd, J = 8.22, 7.43 Hz), 7.86 (1 H, td, J = 7.73, 1.76 Hz), 8.02−8.07(2 H, m), 8.19−8.23 (1 H, m), 8.49 (1 H, s), 8.63−8.66 (1 H, m).Mass spectrum (ESI) m/e = 275.2 (M + 1).

(S)-1-(5,7-Difluoro-2-(pyridin-2-yl)quinolin-3-yl)ethanamine(56d). White solid; yield 100% (57 mg, 0.20 mmol). Mass spectrum(ESI) m/e = 286 (M + 1).

(1S)-1-(7-Fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethanamine(56e). The filtrate was concentrated in vacuo, and then it wasredissolved in EtOAc (1.5 L) and washed with water (500 mL). Theaqueous layer was extracted with EtOAc (1 × 300 mL). The combinedorganic layers were reduced to a volume of 800 mL and then treatedwith water (800 mL) and acidified to pH 2 with aqueous HCl (4.0 M).The aqueous layer was washed with DCM (300 mL) and the pHadjusted (pH 14) with NaOH (4.0 M) and extracted with EtOAc (2 ×500 mL). The combined organic layers were dried (MgSO4), filtered,and evaporated in vacuo. Yield 93% (27.5 g, 103 mmol). 1H NMR(400 MHz, CDCl3) δ ppm 8.69−8.73 (1 H, m), 8.44 (1 H, s), 7.85−7.95 (3 H, m), 7.76 (1 H, dd, J = 10.2, 2.3 Hz), 7.33−7.42 (2 H, m),


dx.doi.org/10.1021/jm501624r | J. Med. Chem. XXXX, XXX, XXX−XXXW

4.63 (1 H, q, J = 6.7 Hz), 1.43 (3 H, d, J = 6.7 Hz). Mass spectrum(ESI) m/e = 268.2 (M + 1).General Procedure for 1, 15, 16, 17, 18, 19, 22, 23, 25, 29,

30, 31, 33, 34, 35. A mixture of 54d (1.51 g, 4.18 mmol) or 56b(11.0 g, 38.8 mmol) or 54f (5.9 g, 19.5 mmol) or 54a (530 mg, 1.76mmol) or 54b (12.0 g, 39.8 mmol) or 56a (123 mg, 0.460 mmol) or54c (12.0 g, 42 mmol) or 56c (4.93 g, 18.0 mmol) or 56d (57 mg,0.20 mmol) or 56e (35.1 g, 131 mmol) or 84 (41.1 g, 136 mmol) or43c (165 mg, 0.520 mmol) or 76b (206 mg, 0.724 mmol) or 76d(11.8 g, 44.1 mmol) or 43b (30.0 mg, 0.105 mmol) and 6-chloropurine (1.0−1.2 equiv) and DIEA (1.0−3.0 equiv) in n-BuOH(0.1−1.0 M) or EtOH (0.05 M (17) was heated to 100−118 °C. Afterheating overnight, the mixture was concentrated to provide a residue.The residue was partitioned between EtOAc and water. The waterlayer was extracted with EtOAc or DCM. The combined organic layerswere washed with water, brine, dried with MgSO4 and concentrated togive a crude product. Chromatography was with silica gel, 0−100%DCM/(89:9:1 DCM/MeOH/NH4OH) [unless otherwise noted].(S)-N-(1-(7-Fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-

purin-6-amine (1). DIEA (1.2 equiv) and 6-chloropurine (1 equiv)were heated to 110 °C, 24 h. The crude yield, 57 g, was divided intoapproximately 8 g portions and each portion purified on a 330 g Redi-Sep column by using a 20−60% DCM/(89:9:1 DCM/MeOH/NH4OH) over 30 min, isocratic at 60% for 30 min. Combined purifiedfractions provided a white solid, which was crushed into powder in amortar and pestle and dried at 120 °C under high vacuum (300mTorr) in a Kugelrohr apparatus. White powder; yield 69% (35 g, 62mmol). 1H NMR (400 MHz, [D6]DMSO) δ ppm 12.76 (1 H, br s),8.69 (1 H, br s), 8.63 (1 H, s), 8.21 (1 H, br s), 7.96−8.12 (4 H, m),7.93 (1 H, s), 7.76 (1 H, dd, J = 10.4, 2.5 Hz), 7.45−7.57 (2 H, m),6.00 (1 H, d, J = 1.2 Hz), 1.61 (3 H, d, J = 6.7 Hz). Mass spectrum(ESI) m/e = 386.0 (M + 1).(S)-N-(1-(8-Chlorophenyl)quinolin-3-yl)ethyl)-9H-purin-6-

amine (15). 6-Chloropurine (1.1 equiv), DIEA (1.5 equiv), n-BuOH(0.17 M), and 115 °C were used. Purification with reverse-phaseHPLC provided the racemic product (105 mg). Chiral HPLC wasused (Chiralcel OD-H column, 0.46 mm × 250 mm, 5 mm)(isopropanol in hexane, 50%). The sample was then dissolved in DCM(1 mL) and put into two vials on chiral separation (15 min method)(retention time of 4.9 min) to give 15. White solid; yield 9% (20 mg,0.046 mmol). 1H NMR (400 MHz, CDCl3) δ ppm 8.47 (1 H, br s),8.36 (1 H, br s), 8.22 (1 H, br s), 7.94 (1 H, br s), 7.68−7.87 (3 H,m), 7.41−7.54 (2 H, m), 7.16−7.30 (2 H, m.), 5.42−5.73 (1 H, m),3.98−4.10 (1 H, m), 1.22 (3 H, d, J = 5.9 Hz). Mass spectrum (ESI)m/e = 435.1 (M + 1) (retention time of 6.7 min). (R)-N-(1-(8-Chlorophenyl)quinolin-3-yl)ethyl)-9H-purin-6-amine white solid;yield 10% (22 mg, 0.051 mmol). 1H NMR (400 MHz, CDCl3) δppm 8.45 (1 H, s), 8.36 (1 H, s), 8.21 (1 H, s), 7.92 (1 H, s), 7.69−7.83 (3 H, m), 7.39−7.50 (1 H, m), 7.34−7.16 (2 H, m), 5.43−5.71 (1H, m), 4.04 (1 H, dt, J = 12.3, 6.0 Hz), 1.22 (3 H, d, J = 6.3 Hz). Massspectrum (ESI) m/e = 435.1 (M + 1).N-((S)-1-(8-Chloro-2-(3-fluorophenyl)quinolin-3-yl)ethyl)-9H-

purin-6-amine (16). DIEA (3 equiv), 6-chloropurine (1.5 equiv),and 100 °C were used. White solid; yield 65% (480 mg, 1.15 mmol).1H NMR (400 MHz, CD3OD) δ ppm 8.43 (1 H, s), 8.05 (1 H, s),7.98 (1 H, s), 7.73−7.80 (2 H, m), 7.48−7.53 (1 H, m), 7.44−7.49 (1H, m), 7.35−7.44 (2 H, m), 7.04−7.11 (1 H, m), 1.44−1.47 (d, 3 H).Mass spectrum (ESI) m/e = 419 (M + 1). HRMS (ESI) m/zcalculated for C22H16ClFN6 + H+ [M + H]: 419.1187. Found:419.1179.N-((8-Chloro-2-(3-fluorophenyl)quinolin-3-yl)methyl)-9H-

purin-6-amine (17). 6-Chloropurine (1.2 equiv) was used.Chromatography, EtOAc/hexane (0−50%), gave a yield of 47%(20.0 mg, 0.0494 mmol). 1H NMR (500 MHz, [D6]DMSO) δ ppm8.41 (1 H, s), 8.31 (1 H, s), 8.13 (2 H, s), 7.95 (2 H, dd, J = 19.07,7.83 Hz), 7.61−7.55 (6 H, m), 7.38 (1 H, m), 4.88 (2 H, br s). Massspectrum (ESI) m/e = 405.1 (M + 1). HRMS (ESI) m/z calculated forC21H14ClFN6 + H+ [M + H]: 405.1031. Found: 405.1023.N-((S)-1-(8-Chloro-2-(2-fluorophenyl)quinolin-3-yl)ethyl)-9H-

purin-6-amine (18). DIEA (3 equiv), 6-chloropurine (1.0 equiv), n-

BuOH (1.0 equiv), and 100 °C were used. The residue was purified bycolumn chromatography with Redi-Sep column using 0−35% gradientof 89:9:1 DCM/MeOH/NH4OH in DCM over 14 min and then 35%isocratic 89:9:1 DCM/MeOH/NH4OH in DCM for 25 min as eluentto give a light yellow solid, which was suspended in DCM and filteredto give a white solid. Yield 44% (7.42g, 17.7 mmol). 1H NMR (400MHz, [D6]DMSO) δ ppm 12.86 (1 H, s), 8.67 (1 H, s), 8.20 (1 H, s),8.09 (1 H, s), 7.95−8.03 (2 H, m), 7.93 (1 H, dd, J = 7.6, 1.0 Hz), 7.691 H, s), 7.58 (1 H, t, J = 7.8 Hz), 7.46−7.55 (1 H, m), 7.25−7.39 (2 H,m), 5.38 (1 H, s), 1.55 (3 H, d, J = 7.0 Hz). Mass spectrum (ESI) m/e= 419.0 (M + 1). HRMS (ESI) m/z calculated for C22H16ClFN6 + H+

[M + H]: 419.1187. Found: 419.1190.N-((S)-1-(5-Chloro-3-(3-fluorophenyl)quinoxalin-2-yl)ethyl)-

9H-purin-6-amine (19). The reaction involved 6-chloropurine (1.00equiv), n-BuOH (1.0 M), DIEA (3.0 equiv), 100 °C, 22 h. Forpurification, the residue (brown syrupy solid) was suspended in water(500 mL), resulting in a precipitate, which was collected by filtrationand washed with water (500 mL) to give a yellow solid. This wassuspended in EtOAc−hexane (1:1, 400 mL), filtered, washed withEtOAc−hexane (1:1, 500 mL), and dried to give the crude product(43.0 g). The organic filtrate still contained product. The yellow solid(43.0 g) was purified by flash column chromatography using 20−100%gradient of 89:9:1 DCM/MeOH/NH4OH in DCM as eluent to give alight yellow solid, which was suspended in DCM (150 mL), filtered,washed with DCM (150 mL), dried under vacuum to give the pureracemic product. Off-white solid; yield 48% (27.2 g, 64.8 mmol). 1HNMR (400 MHz, [D6]DMSO) δ ppm 12.90 (1 H, br s), 7.89−8.20 (5H, m), 7.82 (1 H, t, J = 8.0 Hz), 7.61−7.74 (2 H, m), 7.48−7.60 (1 H,m), 7.26−7.38 (1 H, m), 5.72 (1 H, br s), 1.55 (3 H, d, J = 5.5 Hz).Mass spectrum (ESI) m/e = 419.8 (M + 1). All chiral HPLC(Chiralcel OD-H column, 0.46 mm × 250 mm, 5 mm) using 5%isocratic of isopropanol in hexane as eluent: two peaks at 9.503 and12.045 min at 254 nm; using 3% isocratic of isopropanol in hexane aseluent, two peaks at 15.141 and 19.733 min at 254 nm. Chiralseparation: OD-H + IC 250 mm × 30 mm columns in series using 36g/min IPA (0.2% DEA) + 54 g/min CO2 on Thar 350 SFC. Outletpressure = 125 bar; temp = 18C; wavelength = 327 nm. Used 2.0 mLinjections of 5.0 g/750 mL (9:1 IPA/ACN) sample solution, i.e., 6.7mg/mL (13.3 mg/injection). Cycle time = 3.2 min; run time = 8.0min. First peak on OD-H column: 99% pure. Chromatography (toremove <1% dipurine byproduct) with 330 g Redi-Sep column using0−20% DCM/(89:9:1 DCM/MeOH/NH4OH) over 50 min, then20% DCM/(89:9:1 DCM/MeOH/NH4OH) for 50 min, then 20−30% DCM/(89:9:1 DCM/MeOH/NH4OH) over 100 min as eluentto give the desired product 19. Yield 37% (9.93 g, 23.6 mmol). 1HNMR (400 MHz, [D6]DMSO) δ ppm 12.90 (1 H, br s), 7.87−8.29 (5H, m), 7.82 (1 H, t, J = 8.0 Hz), 7.61−7.74 (2 H, m), 7.48−7.61 (1 H,m), 7.24−7.39 (1 H, m), 5.72 (1 H, br s), 1.55 (3 H, d, J = 5.9 Hz).Mass spectrum (ESI) m/e = 420.1 (M + 1). Enantiomeric excessanalysis: >99% ee. HRMS (ESI) m/z calculated for C21H15ClFN7 + H+

[M + H]: 420.1140. Found: 420.1132.(S)-N-(1-(2-(3,5-Difluorophenyl)-8-fluoroquinolin-3-yl)ethyl)-

9H-purin-6-amine (22).White solid; yield 72% (5.9 g, 14 mmol). 1HNMR (400 MHz, CDCl3) δ ppm 8.34−8.37 (2 H, m), 8.01 (1 H, s),7.56 (1 H, d, J = 7.8 Hz), 7.42−7.52 (3 H, m), 7.34−7.41 (1 H, m),6.90 (1 H, tt, J = 9.0, 2.3 Hz), 6.60 (1 H, br s), 5.79 (1 H, br s), 1.55 (3H, d, J = 6.7 Hz). Mass spectrum (ESI) m/e = 421.1(M + 1). HRMS(ESI) m/z calculated for C22H15F3N6 + H+ [M + H]: 421.1389.Found: 421.1378.

N-((S)-1-(7-Fluoro-2-(3-fluorophenyl)quinolin-3-yl)ethyl)-9H-purin-6-amine (23). After chromatography, the product was washedin DCM−hexane (1:1). White solid; yield 52% (8.8 g, 22 mmol). 1HNMR (500 MHz, [D4]MeOH) δ ppm 8.57 (1 H, s), 8.14 (1 H, s),8.08 (1 H, br s), 8.01 (1 H, dd, J = 9.0, 6.1 Hz), 7.66 (1 H, dd, J = 10.3,2.4 Hz), 7.47−7.56 (3 H, m), 7.44 (1 H, td, J = 8.7, 2.6 Hz), 7.18 (1H, td, J = 8.5, 2.1 Hz), 5.60−5.77 (1 H, br., s), 1.57 (3 H, d, J = 6.8Hz). Mass spectrum (ESI) m/e = 402.9 (M + l). HRMS (ESI) m/zcalculated for C22H16F2N6 + H+ [M + H]: 403.1483. Found: 403.1482.

N-((S)-1-(7-Fluoro-2-(2-(methylsulfonyl)phenyl)quinolin-3-yl)ethyl)-9H-purin-6-amine (24). To a solution of 62 (15 g, 34


dx.doi.org/10.1021/jm501624r | J. Med. Chem. XXXX, XXX, XXX−XXXX

mmol) in DCM (50 mL, 0.67 M) was added TFA (50 mL, 653 mmol,19 equiv) at rt. After 1 h, the reaction mixture was concentrated andneutralized with saturated NaHCO3 (pH 7). The resulting mixture wasextracted with EtOAc (3 × 100 mL). The organic layer was washedwith NaHCO3, brine, dried over Na2SO4, and concentrated to give awhite foam. This material was dissolved in n-BuOH (100 mL) andtreated with 6-chloropurine (5.48 g, 1.05 equiv) and DIEA (7.05 mL,1.2 equiv) at 130 °C overnight. The mixture was concentrated todryness dissolved in EtOAc, washed with water, brine, and dried overMgSO4. Chromatography, silica gel (20:1:0.01 DCM/MeOH/NH4OH), gave a white solid; yield 51% (8.0 g, 17 mmol). 1H NMR(400 Hz, [D4]MeOH) δ ppm 9.35 (l H, s), 8.53−8.47 (m, 2 H), 8.25(l H, s), 7.98−7.76 (6 H, m), 5.85 (s, br, 0.4 H), 5.58−5.56 (m, 0.6H), 3.19 (3 H, s), 1.88−1.81 (3 H, m). 1H NMR (500 MHz, CDCl3) δppm 8.94 (1 H, s), 8.23 (1 H, dd, J = 7.9, 1.1 Hz), 8.02 (1 H, dd, J =9.0, 5.9 Hz), 7.79−7.83 (1 H, m), 7.74−7.78 (1 H, m), 7.65 (1 H, dd, J= 9.7, 2.6 Hz), 7.45 (1 H, td, J = 8.7, 2.7 Hz), 7.37 (1 H, dd, J = 7.3,1.2 Hz), 4.08−4.17 (1 H, m), 1.65 (3 H, d, J = 6.8 Hz). Mass spectrum(ESI) m/e = 463.0 (M + l). HRMS (ESI) m/z calculated forC23H19FN6O2S + H+ [M + H]: 463.1352. Found: 463.1343.(S)-N-(1-(8-chloro-2-(3-(methylsulfonyl)phenyl)quinolin-3-

yl)ethyl)-9H-purin-6-amine (25). The reaction mixture was purifiedby reverse phase HPLC (MeCN/H2O/0.1%TFA, 0−50%). Whitepowder; yield 22%, (440 mg, 0.919 mmol) 1H NMR (500 MHz,[D4]MeOH) δ ppm 8.66 (1 H, s), 8.35−8.40 (2 H, m), 8.31 (1 H, brs), 7.90−8.06 (4 H, m), 7.76 (1 H, br s), 7.60 (1 H, t, J = 7.9 Hz), 5.72(1 H, br s), 3.14 (3 H, s), 1.71 (3 H, d, J = 6.8 Hz). Mass spectrum(ESI) m/e = 479.0 (M + l). HRMS (ESI) m/z calculated forC23H19ClN6O2S + H+ [M + H]: 479.1057. Found: 479.1052.N-((S)-1-(8-Fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-

purin-6-amine (29). Yield 38% (68.0 mg, 0.176 mmol). 1H NMR(500 MHz, [D4]MeOH) δ ppm 8.66 (1 H, d, J = 4.4 Hz), 8.62 (1 H,d, J = 1.0 Hz), 8.04−8.08 (2 H, m), 7.89−7.97 (2 H, m), 7.77 (1 H, d,J = 8.3 Hz), 7.57 (1 H, td, J = 8.1, 4.9 Hz), 7.42−7.50 (2 H, m), 5.98(1 H, br s), 1.69 (3 H, d, J = 6.8 Hz). Mass spectrum (ESI) m/e =386.2 (M + l).N-((S)-1-(8-Chloro-2-(pyridin-2-yl)quinolin-3-yl) ethyl)-9H-

purin-6-amine (30). After chromatography, the resulting whitefoam was treated with hot hexane and filtered to give a white powder;yield 96% (15.7 g, 37.2 mmol) 1H NMR (400 Hz, [D6]DMSO) δ ppm12.66 (l H, br s.), 8.54 (l H, s), 8.50 (l H, s), 8.12 (l H, s), 7.92−7.84(2 H, m), 7.77−7.74 (3 H, m), 7.39 (1 H, t, J = 8.0 Hz), 7.34 (1 H, t, J= 8.0 Hz), 5.91 (l H, s), 1.48 (3 H, d, J = 4.0 Hz). Mass spectrum(ESI) m/e = 402 (M + l). HRMS (ESI) m/z calculated forC21H16ClN7 + H+ [M + H]: 402.1234. Found: 402.1229.N-((S)-1-(5-Chloro-3-(pyridin-2-yl)quinoxalin-2-yl)ethyl)-9H-

purin-6-amine (31) and N-(1-(5-Chloro-3-(pyridin-2-yl)-quinoxalin-2-yl)ethyl)-9H-purin-6-amine (77b). The reactioninvolved 6-chloropurine (1.0 equiv), DIEA (3.0 equiv), n-BuOH(0.1 M), 100 °C, 24 h. For purification, the residue was dissolved inDCM−MeOH (9:1, 50 mL), washed with water (30 mL), brine (30mL), dried over Na2SO4, filtered, and concentrated. Chromatography,40 g of Redi-Sep column using 0−100% gradient of 89:9:1 DCM/MeOH/NH4OH and DCM over 14 min and then 100% isocratic89:9:1 DCM/MeOH/NH4OH for 14 min, gave a light yellow solid,which was suspended in MeOH and filtered to give 77b. Off-whitesolid; yield 63% (0.183 g, 0.455 mmol). 1H NMR (400 MHz,[D6]DMSO) δ ppm 12.87 (1 H, br s), 8.75 (1 H, br s), 8.00−8.18 (5H, m), 7.96 (1 H, s), 7.90 (1 H, br s), 7.84 (1 H, dd, J = 8.4, 7.6 Hz),7.55 (1 H, br s), 6.24 (1 H, br s), 1.71 (3 H, d, J = 5.1 Hz). Massspectrum (ESI) m/e = 402.9 (M + 1). Analytical chiral HPLCparameters. Chiralpak AD-H column: 0.46 mm × 250 mm, 5 mm(isopropanol in hexane, 10%), first peak at 13.431 min and secondpeak at 22.605 min at 254 nm. Chiralcel OD-H column: 0.46 mm ×250 mm, 5 mm, using 10% isocratic of isopropanol in hexane as eluent,first peak at 7.275 min and second peak at 9.237 min at 254 nm. Forchiral separation, the racemic mixture (152.26 mg) was separatedusing SFC dissolved in 43.3 mL of ethanol, and 0.75 mL injections ofthis solution onto the SFC system at conditions below resulted inisolation of two major peaks. Column: 150 mm × 30 mm OD-H (5

μm). Mobile phase: A, liquid CO2, 52.5 g/min,; B, methanol (0.2%DEA), 22.5 mL/min. Mobile phase temp: ambient. Outlet pressure:100 bar. Wavelength: 220 nm. Peak 1 (first peak at 4.82 min): 31. Tansolid; 37% (562 mg, 0.267 mmol). 1H NMR (400 MHz, [D6]DMSO)δ ppm 12.36−13.26 (1 H, m), 8.76 (1 H, br s), 8.00−8.17 (5 H, m),7.96 (1 H, s), 7.90 (1 H, br s), 7.80−7.87 (1 H, m), 7.55 (1 H, br s),6.24 (1 H, br s), 1.71 (3 H, d, J = 5.9 Hz). Mass spectrum (ESI) m/e =402.9 (M + 1). Enantiomeric excess analysis: >99% ee, first-elutingenantiomer on OH-H column. HRMS (ESI) m/z calculated forC20H15ClN8 + H+ [M + H]: 403.1186. Found: 403.1179. Peak 2(second peak at 5.18 min on OD-H column): N-((R)-1-(5-chloro-3-(pyridin-2-yl)quinoxalin-2-yl)ethyl)-9H-purin-6-amine, tan solid; yield33% (50.9 mg, 0.241 mmol). 1H NMR (400 MHz, [D6]DMSO) δppm 12.46 (1 H, br s), 8.75 (1 H, br s), 8.00−8.17 (5 H, m), 7.96 (1H, s), 7.90 (1 H, d, J = 7.4 Hz), 7.80−7.87 (1 H, m), 7.50−7.60 (1 H,m), 6.24 (1 H, br s), 1.71 (3 H, d, J = 6.7 Hz). Mass spectrum (ESI)m/e = 402.9 (M + 1). Enantiomeric excess analysis: >99% ee, second-eluting enantiomer on OH-H column.

Note that the stereochemistry of the two isomers was arbitrarilyassigned based on the comparison of retention time on chiral HPLC ofsimilar analogs (first peak as S-isomer and second peak as R-isomer),confirmed by crystal structures of PI3Kγ protein and 27.

(S)-3-(1-((9H-Purin-6-yl)amino)ethyl)-2-(pyridin-2-yl)-quinoline-8-carbonitrile (33). Yield 68% (4.80 g, 12.2 mmol). (S)Chiral separation by SFC: dissolved in 423 mL of methanol, and 5.0mL injections of this solution onto the SFC system at conditionsbelow resulted in isolation of two peaks, one major and one minor.Column: 250 mm × 30 mm AS-H (5 μm). Mobile phase: A, liquidCO2, 99 g/min; B, methanol (0.2% DEA), 37 mL/min. Mobile phasetemp: 24 °C. Outlet pressure: 140 bar. Wavelength: 265 nm.Enantiomeric excess determination: 98.2% ee. 1H NMR (400 MHz,[D4]MeOH) δ ppm 1.75 (3 H, d, J = 7.04 Hz), 6.22 (1 H, br s), 7.46−7.53 (1 H, m), 7.69−7.76 (1 H, m), 7.95−8.03 (1 H, m), 8.07 (2 H, s),8.16 (1 H, d, J = 7.83 Hz), 8.26 (2 H, ddd, J = 12.52, 7.83, 1.17 Hz),8.69 (1 H, s), 8.72 (1 H, d, J = 5.09 Hz). Mass spectrum (ESI) m/e =393.0 (M + l). HRMS (ESI) m/z calculated for C22H16N8 + H+ [M +H]: 393.1576. Found: 393.1565.

(S)-N-(1-(6-Fluoro-3-(pyridin-2-yl)quinoxalin-2-yl)ethyl)-9H-purin-6-amine (34). The reaction involved 6-chloropurine (1.0equiv), DIEA (3.0 equiv), n-BuOH (0.1 M), 110 °C, 22 h. Forpurification, the residue was dissolved in DCM (1 L). The solutionwas washed with water (2 × 500 mL). The organic layer was driedover Na2SO4, filtered, and concentrated to give a brown solid. Thebrown solid was suspended in DCM (100 mL), sonicated, filtered, andwashed with DCM (100 mL) to give an off-white solid.Chromatography: 330 g Redi-Sep column using 0%−50% DCM/(89:9:1 DCM/MeOH/NH4OH) over 25 min and then 50% DCM/(89:9:1 DCM/MeOH/NH4OH) for 25 min as eluent to give 34 and71. White solid; yield 33% (5.68 g, 14.7 mmol). 1H NMR (500 MHz,[D6]DMSO) δ ppm 12.79 (1 H, br s), 8.74 (1 H, br s), 8.19 (1 H, dd,J = 9.3, 5.9 Hz), 7.99−8.14 (3 H, m), 7.96 (1 H, s), 7.93 (1 H, dd, J =9.5, 2.7 Hz), 7.83−7.89 (1 H, m), 7.80 (1 H, td, J = 8.9, 2.8 Hz), 7.54(1 H, br s), 6.16 (1 H, br s), 1.68 (3 H, d, J = 6.6 Hz). Mass spectrum(ESI) m/e = 387 (M + 1). HRMS (ESI) m/z calculated for C20H15FN8+ H+ [M + H]: 387.1482. Found: 387.148.

(S)-N-(1-(5,7-Difluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-purin-6-amine (35). The reaction involved 6-chlorpurine (1.0equiv), DIEA (1.2 equiv), n-BuOH (0.1 M), 110 °C. Purificationwas by reverse HPLC on C18 (MeCN/H2O/TFA, 0−50%). Themajor peak was collected and treated with HCl (3 N) andconcentrated to give a white powder; yield 74% (60 mg, 0.15mmol). 1H NMR (400 MHz, [D4]MeOH) δ ppm 1.87 (3 H, d, J =6.26 Hz), 5.77 (1 H, br s), 7.38−7.52 (1 H, m), 7.70 (1 H, d, J = 9.39Hz), 8.18 (1 H, t, J = 6.46 Hz), 8.49 (2 H, br s) 8.60 (1 H, d, J = 7.04Hz), 8.74 (1 H, t, J = 7.43 Hz), 8.99 (1 H, s), 9.11 (d, J = 5.09 Hz, 1H). Mass spectrum (ESI) m/e = 404.0 (M + 1). HRMS (ESI) m/zcalculated for C21H15F2N7 + H+ [M + H]: 404.1435. Found: 404.1431.

General Procedure for the Synthesis of 14, 20, 21, 26, 27,28, and 32. To a round bottomed flask was charged 43a (7.20 g, 23.8mmol) or 54e (22 mg, 0.077 mmol) or 56f (54 mg, 0.17 mmol) or


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76a (314 mg, 1.10 mmol) or 59a (60 mg, 0.22 mmol) or (59b (66 mg,0.22 mmol) or 67 (0.68 g, 2.3 mmol), DIEA (2.0−3.0 equiv), 6-bromopurine (1.2 equiv), and n-BuOH (0.1−1.0 M). The mixture washeated to 100−118 °C for 14−24 h, cooled to rt, and the solvent wasremoved in vacuo to provide a crude product. Purification of theresidue was by flash chromatography over silica gel, gradient elution,0−10% MeOH/DCM with 0.1% aqueous NH4OH [unless otherwisenoted].N-((8-Chloro-2-(2-chlorophenyl)quinolin-3-yl)methyl)-9H-

purin-6-amine (14). Crude product: orange syrup. Columnchromatography: (silica gel) 50% of 89:9:1 DCM/MeOH/NH4OHin DCM. Yield: light yellow solid suspended in EtOH (150 mL). Themixture was refluxed for 30 min, cooled to rt. A precipitate formed andwas collected by filtration, washed with cold EtOH (100 mL). Off-white solid; yield 80% (7.78 g, 19.0 mmol). 1H NMR (400 MHz,[D6]DMSO) δ ppm 12.94 (1 H, s), 8.36 (1 H, s), 8.05−8.29 (3 H, m),7.99 (1 H, dd, J = 8.4, 1.0 Hz), 7.93 (1 H, dd, J = 7.4, 1.2 Hz), 7.44−7.68 (5 H, m), 4.50−4.82 (2 H, m). Mass Specrum (ESI) major peakof m/z 421.1 [M + H]+. Elemental analysis calculated (%) forC21H16Cl4N6 (di-HCl salt): C 51.04, H 3.26, N 17.01. Found: C 51.74,H 3.77, N 16.79. HRMS (ESI) m/z calculated for C21H14Cl2N6 + H+

[M + H]: 421.0735. Found: 421.0725.N-((S)-1-(8-Fluoro-2-(3-fluorophenyl)quinolin-3-yl)ethyl)-7H-

purin-6-amine (20). n-BuOH (0.1 M) yield 58% (18 mg, 0.045mmol). 1H NMR (400 MHz, CDCl3) δ ppm 8.33 (2 H, s), 7.97 (1 H,s), 7.65−7.61 (2 H, m), 7.52 (1 H, d, J = 8.2 Hz), 7.47−7.39 (2 H, m),7.35 (1 H, ddd, J = 10.6,7.8, 1.6 Hz), 6.76 (1 H, br s), 5.80 (1 H, br s),1.51 (3 H, d, J = 7.0 Hz). Mass spectrum (ESI) m/e = 403 (M + 1).HRMS (ESI) m/z calculated for C22H16F2N6 + H+ [M + H]:403.1483. Found: 403.1487.N-((S)-1-(2-(2,5-Difluorophenyl)-8-fluoroquinolin-3-yl)ethyl)-

7H-purin-6-amine (21). Purified by reverse phase chromatography(5−30% gradient). Yield 59% (54 mg, 0.13 mmol). 1H NMR (400MHz, CDCl3) δ ppm 8.37 (1 H, s), 8.24 (1 H, s), 7.94 (1 H, s), 7.60(1 H, d, J = 8.2 Hz), 7.48 (2 H, td, J = 8.0, 5.1 Hz), 7.35−7.43 (2 H,m), 7.09 (2 H, d, J = 3.9 Hz), 6.40 (1 H, br s), 5.54 (1 H, d, J = 9.8Hz), 1.68 (3 H, d, J = 7.0 Hz). Mass spectrum (ESI) m/e = 420.9 (M+ 1).N-((S)-1-(8-Fluoro-2-(pyridin-3-yl)quinolin-3-yl)ethyl)-7H-

purin-6-amine (26). DIEA (3.0 equiv) was used. Purified by reversephase chromatography (5−30% gradient). Yield 23% (20 mg, 0.053mmol). 1H NMR (400 MHz, CDCl3) δ ppm 9.15 (1 H, s), 8.71 (1 H,dd, J = 4.7, 1.6 Hz), 8.34−8.38 (1 H, m), 8.26−8.30 (1 H, m), 8.22 (1H, d, J = 7.8 Hz), 7.97 (1 H, s), 7.54 (1 H, d, J = 7.4 Hz), 7.32−7.48 (4H, m), 6.67 (1 H, br s), 5.75 (1 H, br s), 1.53 (3 H, d, J = 6.7 Hz).Mass spectrum (ESI) m/e = 385.9 (M + 1).N-((S)-1-(8-Chloro-2-(2-methylpyridin-3-yl)quinolin-3-yl)-

ethyl)-9H-purin-6-amine (27). The reaction involved 6-bromopur-ine (1.1 equiv), DIEA (2.7 equiv), n-BuOH (2.5 M), reflux, 18 h.Chromatography, Redi-Sep column using 0−50% gradient of 89:9:1DCM/MeOH/NH4OH in DCM, gave a yellow solid. The yellow solidwas suspended in MeOH and filtered to give N-(1-(8-chloro-2-(2-methylpyridin-3-yl)quinolin-3-yl)ethyl)-9H-purin-6-amine as an off-white solid, which was dissolved in MeOH−CH2Cl2 (1:4, 5 mL),filtered, and separated on a Chiralpak IA column using 20% isocratic ofisopropanol in hexane for 40 min as eluent to give the pure isomer as awhite solid; yield 55% (0.867g, 1.15 mmol). 1H NMR (400 MHz,[D6]DMSO) δ ppm 12.90 1 H, s), 8.59 (2 H, d, J = 61.6 Hz), 7.70−8.37 (6 H,m), 7.58 (1 H, t, J = 7.8 Hz), 7.32 (1 H, s), 5.34 (1 H, br s),2.32 (3 H, s), 1.53 (3 H, br s). Mass spectrum (ESI) m/e = 416 (M +1). HRMS (ESI) m/z calculated for C22H18ClN7 + H+ [M + H]:416.1390. Found: 416.1389.N-((S)-1-(8-Chloro-2-(5-fluoro-2-methylpyridin-3-yl)-

quinolin-3-yl)ethyl)-9H-purin-6-amine (28). n-BuOH (1.0 M);yield 37% (27 mg, 0.063 mmol). 1H NMR (500 MHz, [D6]DMSO) δppm 12.85 (1 H, br s), 8.70 (1 H, br s), 8.49 (1 H, br s), 8.11 (2 H, brs), 8.02 (1 H, d, J = 8.1 Hz), 7.96 (1 H, dd, J = 7.6, 1.2 Hz), 7.85−7.93(1 H, m), 7.62 (1 H, t, J = 7.9 Hz), 5.77 (3 H, s), 5.37 (1 H, br s), 4.10(1 H, q, J = 5.4 Hz), 2.27 (3 H, br s). Mass spectrum (ESI) m/e =433.9 (M + 1)

N-((S)-1-(8-Chloro-3-(pyridin-2-yl)quinoxalin-2-yl)ethyl)-9H-purin-6-amine (32) and N-(1-(8-Chloro-3-(pyridin-2-yl)-quinoxalin-2-yl)ethyl)-9H-purin-6-amine (77a). The reactioninvolved n-BuOH (0.28 M), DIEA (3.0 equiv), and 6-bromopurine(1 equiv), 110 °C, 5 h. Chromatography provided 77a. Tan solid; yield77% (343 mg, 0.851 mmol). 1H NMR (400 MHz, [D6]DMSO) δ ppm12.87 (1 H, br s), 8.74 (1 H, br s), 7.96−8.20 (6 H, m), 7.85 (2 H, t, J= 8.0 Hz), 7.53 (1 H, br s), 6.33 (1 H, br s), 1.67 (3 H, d, J = 6.3 Hz).Mass spectrum (ESI) m/e = 403.1 (M + 1). The racemic tan solid(343 mg) was purified by Lotus separation with Chiralcel OJ-H (2 cm× 25 cm) 08-9805, 25% methanol/CO2, 100 bar, 80 mL/min, to give32. Yield 37% (143 mg, 0.315 mmol). 99% ee (Chiracel OJ-H (25 cm× 0.46 cm), 25% methanol (0.1% DEA)/CO2, 100 bar, 3 mL/min, tR= 2.59 min). 1H NMR (400 MHz, dichloromethane-d2) δ ppm 8.85(d, J = 4.30 Hz, 1 H), 8.28 (1 H, s), 8.20 (1 H, d, J = 7.83 Hz), 8.07 (1H, dd, J = 8.41, 1.37 Hz), 7.98 (1 H, td, J = 7.63, 1.56 Hz), 7.93 (1 H,br s), 7.91 (1 H, dd, J = 7.83, 1.17 Hz), 7.71 (1 H, dd, J = 8.41, 7.63Hz), 7.53 (1 H, br s), 7.50 (1 H, dd, J = 7.83, 4.69 Hz), 6.68 (1 H, brs), 1.70 (3 H, d, J = 6.26 Hz), 1.54 (1 H, br s). Mass spectrum (ESI)m/e = 403.1 (M + 1). HRMS (ESI) m/z calculated for C20H15ClN8 +H+ [M + H]: 403.1186. Found: 403.1180.

(S)-3-(1-Azidoethyl)-2-chloro-8-fluoroquinoline (57). Triphe-nylphosphine (1.81 g, 6.9 mmol, 1.2 equiv) was dissolved in anhydrousTHF (30 mL) and cooled to 0 °C. To this solution was added DIAD(1.36 mL, 6.9 mmol). The mixture was stirred for 30 min at 0 °C, and(R)-1-(2-chloro-8-fluoroquinolin-3-yl)ethanol 51d (1.31 g, 5.81mmol) in THF (30 mL) was added, followed by diphenylphosphorylazide (1.37 mL, 6.3 mmol). The mixture was allowed to warm to rtand stirred overnight. The crude material was purified by flashchromatography 0−3% EtOAc/hexane to give 57. Yield 76% (1.10 g,4.43 mmol). 1H NMR (400 MHz, (CDCl3) δ ppm 8.30 (1 H, d, J =1.2 Hz), 7.67 (1 H, d, J = 8.2 Hz), 7.54 (1 H, td, J = 7.8, 4.7 Hz), 7.45(1 H, ddd, J = 10.2, 7.8, 1.2 Hz) 5.22 (1 H, q, J = 6.7 Hz), 1.68 (3 H, d,J = 6.7 Hz). Mass spectrum (ESI) m/e = 250.9 (M + 1).

General Procedure for the Synthesis of 58a and 58b. To around bottomed flask, purged with N2 were added 57 (100 mg, 0.399mmol, 1.0 equiv), Pd(PPh3)4 (0.05 equiv), Na2CO3 (2.0 equiv),pyridin-3-ylboronic acid (1.5 equiv), or 2,5-difluorophenylboronic acid(1.5 equiv) in 3:1 MeCN/H2O, 0.1 M. This was heated to 80 °C for1−1.5 h. The mixture was cooled to rt and the solvent removed. Theresidue was treated with EtOAc (20 mL) and washed with a saturatedsolution of NaHCO3 (5 mL) and NaCl (5 mL), dried over MgSO4,filtered, and concentrated. Chromatography involved 50:50 hexanes/EtOAc and 12 g Redi-Sep column.

3-((S)-1-Azidoethyl)-8-fluoro-2-(pyridin-3-yl)quinoline (58a).Yield 64% (75 mg, 0.26 mmol). 1H NMR (400 MHz, CDCl3) δ ppm8.86 (1 H, d, J = 2.3 Hz), 8.76 (1 H, dd, J = 5.1, 1.6 Hz), 8.41 (1 H, d,J = 1.6 Hz), 7.97 (1 H, dt, J = 7.8, 2.0 Hz), 7.73 (1 H, d, J = 8.2 Hz),7.57 (1 H, td, J = 7.9, 4.9 Hz), 7.46−7.51 (2 H, m), 4.89 (1 H, q, J =6.7 Hz), 1.58−1.60 (1 H, m), 1.59 (3 H, d, J = 7.0 Hz). Mass spectrum(ESI) m/e = 294.0 (M + 1).

3-((S)-1-Azidoethyl)-2-(2,5-difluorophenyl)-8-fluoroquino-line (58b). Chromatography (5% EtOAc in hexanes, 23 g silica, then5% ether in hexanes, 10 g silica) gave 58b. Yield 64% (84 mg, 0.26mmol). 1H NMR (400 MHz, CDCl3) δ ppm 8.40 (1 H, d, J = 1.2 Hz),7.73 (1 H, d, J = 8.2 Hz), 7.58 (1 H, td, J = 7.9, 4.9 Hz), 7.47 (1 H,ddd, J = 10.3, 7.7, 1.2 Hz), 7.23−7.26 (1 H, m), 7.16−7.21 (2 H, m),4.81 (1 H, q, J = 6.3 Hz), 1.56 (3 H, s). Mass spectrum (ESI) m/e =328.9 (M + 1).

General Procedure for the Synthesis of 59a and 59b. Thereaction involved dissolving 58a (72 mg, 0.24 mmol) or 58b (80 mg,0.24 mmol), PPh3 (1.2 equiv), H2O (20 equiv) in THF (0.1 M). Themixture was heated to 60 °C overnight. Solvent was removed. Dilutionwas with ether (6 mL), and extraction was with HCl (1 N). Theaqueous layer was adjusted to pH 12 with NaOH and extracted withether (20 mL). The organic layer was dried ove MgSO4, filtered, andconcentrated.

(1S)-1-(8-Fluoro-2-(pyridin-3-yl)quinolin-3-yl)ethanamine(59a). Yield 100% (66 mg, 0.24 mmol). 1H NMR (400 MHz, CDCl3)δ ppm 8.85 (1 H, dd, J = 2.3, 0.8 Hz), 8.71 (1 H, dd, J = 4.7, 1.6 Hz),


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8.56 (1 H, d, J = 1.6 Hz), 7.96 (1 H, dt, J = 7.8, 2.0 Hz), 7.68 (1 H, d, J= 7.4 Hz), 7.37−7.58 (5 H, m), 4.44−4.51 (1 H, m), 1.37 (3 H, d, J =6.7 Hz). Mass spectrum (ESI) m/e = 268.0 (M + 1).(1S)-1-(2-(2,5-Difluorophenyl)-8-fluoroquinolin-3-yl)-

ethanamine (59b). Yield 90% (66 mg, 0.22 mmol). 1H NMR (400MHz, CDCl3) δ ppm 8.52 (1 H, s), 7.63−7.71 (2 H, m), 7.43−7.57 (2H, m), 7.39 (1 H, ddd, J = 10.5, 7.7, 1.0 Hz), 7.19−7.26 (1 H, m), 7.13(2 H, td, J = 6.2, 1.8 Hz), 4.29 (1 H, q, J = 6.3 Hz), 1.32 (3 H, br s).Mass spectrum (ESI) m/e = 303.0 (M + 1).S-tert-Butyl (1-(2-Chloro-7-fluoroquinolin-3-yl)ethyl)-

carbamate (60). To a rt solution of 52a (1.0 g, 2.8 mmol) inEtOH (10 mL) was added dropwise NH2NH2 (10 equiv, 0.88 mL),and the solution heated to 90 °C for 0.5 h. After cooling to rt, thereaction mixture was concentrated and partitioned between EtOAc(20 mL) and H2O (5 mL). The organic layer was separated, washedwith water, saturated NaCl, dried over Na2SO4, and concentrated togive a colorless oil, which was dissolved in THF (10 mL) and treatedwith Boc2O (1.1 equiv, 0.68 g) and Et3N (1.0 equiv, 0.39 mL) andrefluxed. After cooling to rt, the reaction mixture was concentrated andpurified by column chromatography on silica gel (EtOAc/hexane, 1:9).White solid; yield 76% (0.70 g, 2.2 mmol). 1H NMR (400 MHz,CDCl3) δ ppm 8.12 (1 H, s), 7.82 (1 H, dd, J = 9.0, 5.9 Hz), 7.66 (1H, dd, J = 9.8, 2.7 Hz), 7.36 (1 H, td, J = 8.6, 2.3 Hz), 5.13−5.24 (1 H,m), 5.07 (1 H, br s), 1.56 (9 H, s), 1.43 (3 H, br s). Mass spectrum(ESI) m/e = 325.1 (M + 1).(S)-tert-Butyl (1-(7-Fluoro-2-(2-(methylthio)phenyl)quinolin-

3-yl)ethyl)carbamate (61). A mixture of 60 (382 mg, 1.2 mmol), 2-(methylthio)phenylboronic acid (257 mg, 1.3 equiv), Na2CO3 (623mg, 5.0 equiv), Pd(PPh3)4 (93 mg, 0.081 mmol), MeCN (9 mL), andwater (3 mL) was heated to 85 °C under N2 overnight. After coolingto rt, the reaction was partitioned between EtOAc (10 mL) and water(5 mL). The organic layer was separated, washed, dried (MgSO4, andconcentrated. The residue was purified by column chromatography onsilica gel to give a white solid; yield 95% (460 mg, 1.12 mmol). Massspectrum (ESI) m/e = 413.2 (M + 1).(S)-tert-Butyl (1-(7-Fluoro-2-(2-(methylsulfonyl)phenyl)-

quinolin-3-yl)ethyl)carbamate (62). Under an N2 atmosphere, 61(19.5 g, 47.3 mmol) was dissolved in acetone (600 mL) and water(150 mL). NMO (17.0 g, 142 mmol, 3.0 equiv) was added followed byOsO4 (601 mg, 2.36 mmol 0.05 equiv). The mixture was allowed tostir at rt for 4 h. The mixture was treated with an additional amount ofOsO4 (1.0 g, 3.93 mmol, 0.083 equiv) and stirred overnight. Thereaction mixture was treated with saturated Na2S2O3 solution (15 mL)and the acetone partially removed by concentration. The remainingsolution was diluted with EtOAc and washed with saturated Na2S2O3

solution and saturated NaCl. The organic layer was dried (Na2SO4),filtered, and concentrated in vacuo. The crude residue was purified byflash chromatography, eluting with 0−50% EtOAc in hexanes toprovide a white foam. Yield 100% (21 g, 47 mmol). 1H NMR (500MHz, CDCl3) δ ppm 8.22−8.27 (2 H, m), 7.91 (1 H, ddd, J = 18.8,8.9, 6.0 Hz), 7.74−7.83 (1 H, m), 7.59−7.72 (2 H, m), 7.35−7.49 (2H, m), 5.46 (1 H, br s), 4.94 (1 H, br s), 4.76−4.87 (1 H, m), 3.27−3.36 (3 H, m), 1.63 (1.5 H, d, J = 6.6 Hz), 1.42 (4.5 H, br s), 1.34 (1.5H, d, J = 6.6 Hz), 1.26 (4.5 H, s) (mixture of two atropoisomers ratio1.1:1). Mass spectrum (ESI) m/e = 445.1 (M + 1).8-Chloro-2-(2-methylpyridin-3-yl)quinoline-3-carbaldehyde

(63). A mixture of 36b (1.00 g, 4.42 mmol), Pd(PPh3)4 (0.26 g, 0.22mmol), and Na2CO3 (2.34 g, 22.1 mmol) in CH3CN−H2O (90 mL,3:1) was stirred at 100 °C. After 3 h, the mixture was cooled to rt andpartitioned between EtOAc (150 mL) and water (150 mL). Theorganic layer was washed with brine (2 × 100 mL), dried over Na2SO4,filtered, and concentrated under reduced pressure to give a yellowsolid. Column chromatography, eluting with 0−100% gradient ofEtOAc/hexane, gave an off-white solid; yield 86% (1.08g, 3.82 mmol).1H NMR (400 MHz, [D6]DMSO) δ ppm 9.96 (1 H, s), 9.16 (1 H, s),8.62 (1 H, dd, J = 4.9, 1.8 Hz), 8.31 (1 H, dd, J = 8.2, 1.2 Hz), 8.18 (1H, dd, J = 7.4, 1.2 Hz), 7.80 (1 H, dd, J = 7.6, 1.8 Hz), 7.76 (1 H, dd, J= 8.2, 7.4 Hz), 7.40 (1 H, dd, J = 7.4, 4.7 Hz), 2.35 (3 H, s). Massspectrum (ESI) m/e = 283 (M + H).

1-(8-Chloro-2-(2-methylpyridin-3-yl)quinolin-3-yl)ethanol(64). To a stirred heterogeneous mixture of 63 (1.07 g, 3.80 mmol) inTHF (14.6 mL, 3.80 mmol) was added MeMgBr (1.90 mL, 5.70mmol, 3 M in diethyl ether) dropwise at 0 °C, and the mixture wasallowed to warm to rt. After 2 h saturated NH4Cl (50 mL) was addedand the mixture extracted with EtOAc (2 × 50 mL). The organic layerwas washed with water (50 mL), brine (50 mL), dried over Na2SO4,filtered, and concentrated to give an orange syrup (1.44 g). Colunmchromatography, eluting with 0−100% gradient of EtOAc in hexane,gave an orange solid; yield 98% (1.12g, 3.73 mmol). 1H NMR (400MHz, [D6]DMSO) δ ppm 8.68 (1 H, s), 8.59 (1 H, dd, J = 4.9, 1.8Hz), 8.10 (1 H, dd, J = 8.2, 1.2 Hz), 7.94 (1 H, dd, J = 7.6, 1.4 Hz),7.74 (1 H, dd, J = 7.8, 1.6 Hz), 7.58−7.65 (1 H, m), 7.39 (1 H, dd, J =7.6, 4.9 Hz), 5.47 (1 H, d, J = 4.3 Hz), 4.64 (1 H, br s), 2.25 (3 H, s),1.20 (3 H, d, J = 7.4 Hz). Mass spectrum (ESI) m/e = 299 (M + 1).

8-Chloro-3-(1-chloroethyl)-2-(2-methylpyridin-3-yl)-quinoline Hydrochloride (65). To a rt stirred solution of 64 (1.11 g,3.71 mmol) in chloroform (12.37 mL, 0.3 M) was treated with thionylchloride (1.35 mL, 18.6 mmol) dropwise. After 3 h, the mixture wasconcentrated under reduced pressure and coevaporated three timeswith DCM to give an off-white syrupy solid (1.31 g, 3.71 mmol). Thecrude product was carried on to the next step without purification. 1HNMR (400 MHz, [D6]DMSO) δ ppm 9.04 (1 H, s), 8.94 (1 H, dd, J =5.7, 1.4 Hz), 8.56 (1 H, d, J = 7.4 Hz), 8.19 (1 H, dd, J = 8.2, 1.2 Hz),8.07 (1 H, dd, J = 7.4, 1.2 Hz), 8.02 (1 H, dd, J = 7.6, 5.7 Hz), 7.71−7.77 1 H, m), 5.25 (1 H, d, J = 6.3 Hz), 2.52 (3 H, s), 1.92 (3 H, d, J =6.7 Hz). Mass spectrum (ESI) m/e = 317 (M + 1).

2-(1-(S-Chloro-2-(2-methylpyridin-3-yl)quinolin-3-yl)ethyl)-isoindoline-l,3-dione (66). To a stirred solution of 65 (1.31 g, 3.71mmol) in DMF (10 mL, 0.37 M) at 100 °C was added potassiumphthalimide (1.72 g, 9.28 mmol). After 1.5 h, the mixture wasconcentrated and triturated with water (50 mL). The resulting solidwas filtered, washed with NaOH (2 N, 50 mL), water (500 mL), andair-dried to give a tan solid; yield 100% (1.58 g, 3.70 mmol). Theproduct was carried on without purification for the next step. Massspectrum (ESI) m/e = 428 (M + 1).

1-(8-Chloro-2-(2-methylpyridin-3-yl)quinolin-3-yl)-ethanamine (67). To a stirred suspension of 66 (1.58 g, 3.70 mmol)in EtOH (37 mL, 0.1 M) was added hydrazine, anhydrous (1.16 mL,37 mmol, 10 equiv), and the resulting mixture was refluxed. After 1.5h, the mixture was cooled to rt. The byproduct was filtered off, washedwith MeOH (100 mL) and the filtrate concentrated under reducedpressure to give a yellow solid. Chromatography, RediSep colunmusing 0−100% (89:9:1 MeOH/NH4OH)/DCM, gave a yellow syrup;yield 60% from 64 (1.1 g, 2.2 mmol). 1H NMR (400 MHz,[D6]DMSO) δ ppm 8.75 (1 H, s), 8.58 (l H, dd, J = 4.9, 1.8 Hz), 8.04(1 H, dd, J = 8.2, 1.2 Hz), 7.92 (1 H, dd, J = 7.4, 1.2 Hz), 7.77 (1 H, s),7.60 (1 H, dd, J = 8.2, 7.4 Hz), 7.34−7.44 (1 H, m), 4.09 (1 H, d, J =4.7 Hz), 2.25 (3 H, s), 2.05 (2 H, br s), 1.13 (3 H, d, J = 6.7 Hz). Massspectrum (ESI) m/e = 298 (M + 1).

6-Chloro-3-methylcinnolin-4-ol (68). A cooled (0 °C) suspen-sion of 1-(2-amino-5-chlorophenyl)propan-1-one (4.88 g, 26.6 mmol)in HCl (35 mL, 5 N) was added to a solution of sodium nitrite (2.02 g,29.2 mmol) in 10 mL of water dropwise. After the addition, themixture was stirred at rt for 15 min. The clear yellow solution was thenheated at 85 °C for 2 h, and a large amount of solid precipitated. Thereaction was cooled to rt, filtered, and the solid was washed with water,Et2O and dried on the vacuum pump. Off white amorphous solid;yield 92% (4.73 g, 24.3 mmol). 1H NMR (400 MHz, [D6]DMSO) δppm 7.97 (1 H, d, J = 2.3 Hz), 7.77 (1 H, dd, J = 9.1, 2.4 Hz), 7.60 (1H, d, J = 9.0 Hz), 2.27 (3 H, s). Mass spectrum (ESI) m/e = 195 (M +1).

4,6-Dichloro-3-methylcinnoline (69). A stirred mixture of 68(1.38 g, 7.09 mmol) and phosphorus oxychloride (10.0 mL, 107mmol, 15.1 equiv) was heated at 90 °C for 3 h. The reaction wascooled to rt, and the excess POCl3 was removed in vacuo. The residuewas dissolved in DCM and washed with saturated NaHCO3. Theorganic layer was dried, filtered, and concentrated to dryness to give ablack amorphous solid. The material was dissolved in DCM, washedwith H2O, and dried in vacuo to provide a light-yellow amorphous


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solid; yield 78% (1.18g, 5.54 mmol). 1H NMR (400 MHz, CDCl3) δppm 8.49 (1 H, d, J = 9.0 Hz), 8.17 (1 H, d, J = 2.2 Hz), 7.77 (1 H, dd,J = 9.0, 2.2 Hz), 3.06 (3 H, s). Mass spectrum (ESI) m/e = 215.0 (M +1).8-Chloro-3-ethylquinoxalin-2(1H)-one (70a). A 3 L round-

bottom flask fitted with an overhead stirrer was charged with 3-chlorobenzene-1,2-diamine (54.8 g, 384 mmol) and polyphosphoricacid (500.0 mL, 384.2 mmol). The resulting black viscous solution washeated to 80 °C. To the viscous mixture at 80 °C was added ethyl 2-oxobutanoate (50.0 g, 384 mmol), and the mixture was stirred at 100−160 °C. After 2 h 40 min, the mixture was cooled to 0 °C, thoroughlymixed with water (1.0 L), and cautiously neutralized with NaOH (1.35L, 10 N) to pH ≈ 7.0. The resulting precipitate was collected byfiltration, washed with water (1.50 L), and dried to give a mixture oftwo regiosiomers as a wet red solid. This (72.43 g) was suspended inEtOH (750 mL) and refluxed. After 1 h, the mixture was allowed tocool to rt over 15 h. The solid was collected by filtration and washedwith EtOH (500 mL) to give the desired product in 94% purity (6% 5-chloro-3-ethylquinoxalin-2(1H)-one). Violet solid, yield 60% (48.4 g,232 mmol) 94% purity as judged by HPLC (6% 5-chloro-3-ethylquinoxalin-2(1H)-one). 1H NMR (400 MHz, [D6]DMSO) δppm 11.85 (1 H, s), 7.71 (1 H, dd, J = 7.8, 1.2 Hz), 7.61 (1 H, dd, J =7.8, 1.2 Hz), 7.28 (1 H, t, J = 8.0 Hz), 2.83 (2 H, q, J = 7.2 Hz), 1.22(3 H, t, J = 7.2 Hz). Mass spectrum (ESI) m/e = 208.9 (M + 1).3-Ethyl-7-fluoroquinoxalin-2(1H)-one (70b). To a round-

bottom flask fitted with an overhead stirrer charged with 3-ethyl-7-fluoro-3,4-dihydroquinoxalin-2(1H)-one (60.61 g, 312.1 mmol) and1,4-dioxane (1.35 L) was added DDQ (75.9 g, 327.7 mmol), and themixture was stirred at rt. After 2.5 h, the mixture was concentratedunder reduced pressure to give a brown residue. This was suspendedin saturated NaHCO3 (2 L), stirred for 30 min, filtered and the solidwashed with saturated NaHCO3 (1 L) to give a light green solid. Thiswas resuspended in saturated NaHCO3 (500 mL), well mixed, filtered,and washed with saturated NaHCO3 (500 mL) to give an off-whitesolid. The off-white solid was suspended in water (500 mL), wellmixed, filtered, washed with water (1 L), air-dried overnight, and driedunder high vacuum at 22 °C for 2 h to give the product. Off-whitesolid yield 95% (56.87 g, 295.9 mmol). 1H NMR (400 MHz,[D6]DMSO) δ ppm 12.36 (1 H, br s), 7.76 (1 H, dd, J = 9.0, 5.9 Hz),7.11 (1 H, td, J = 8.8, 2.7 Hz), 7.00 (1 H, dd, J = 9.6, 2.7 Hz), 2.78 (2H, q, J = 7.4 Hz), 1.20 (3 H, t, J = 7.4 Hz). Mass spectrum (ESI) m/e= 193 (M + 1).General Procedure for the Synthesis of 71a and 71b. To a

stirred suspension of 70a (1.32 g, 6.33 mmol) or 70b (20.6 g, 107mmol) and 1,3-dibromo-5,5-dimethylhydantoin (0.7 equiv) in carbontetrachloride (1 equiv) was added benzoyl peroxide (0.1 equiv), andthe mixture was heated at reflux. After 20 h, the mixture was cooled tort, and saturated NaHCO3 (0.1 M) was added. A precipitate wascollected by filtration and washed with water to give a solid. The twolayers from the filtrate were separated. The organic layer was driedover Na2SO4, filtered, combined with the solid, and concentratedunder reduced pressure to give the product.3-(1-Bromoethyl)-8-chloroquinoxalin-2(1H)-one (71a). Yel-

low solid; yield 100% (1.96 g, 6.33 mmol). 1H NMR (400 MHz,[D6]DMSO) δ ppm 12.22 (1 H, br s), 7.79 (1 H, d, J = 7.8 Hz), 7.72(1 H, d, J = 8.2 Hz), 7.34 (1 H, t, J = 7.8 Hz), 5.66 (1 H, q, J = 6.9Hz), 2.00 (3 H, d, J = 6.7 Hz). Mass spectrum (ESI) m/e = 286.7 [M +H (79Br)]+ and 288.7 [M + H (81Br)]+

3-(1-Bromoethyl)-7-fluoroquinoxalin-2(1H)-one (71b). Tansolid; yield 80% (23.19 g, 85.54 mmol). 1H NMR (400 MHz,[D6]DMSO) δ ppm 12.68 (1 H, br s), 7.85 (1 H, dd, J = 9.0, 5.9 Hz),7.19 (1 H, td, J = 8.8, 2.7 Hz), 7.05 (1 H, dd, J = 9.8, 2.7 Hz), 5.62 (1H, q, J = 6.7 Hz), 1.99 (3 H, d, J = 6.7 Hz. Mass spectrum (ESI) m/e =271 (M + 1).2-(1-(8-Chloro-3-hydroxyquinoxalin-2-yl)ethyl)isoindoline-

1,3-dione (74). To a stirred solution of methyl 3-(1,3-dioxoisoindo-lin-2-yl)-2-oxobutanoate (1.13 g, 4.33 mmol) in EtOAc (9.85 mL, 4.33mmol) was added 3-chlorobenzene-1,2-diamine (0.618 g, 4.33 mmol)at rt. After 3 h the resulting precipitate was filtered to give a ∼3.5:1mixture of regioisomers, with the desired regiomer 2-(1-(5-chloro-3-

hydroxyquinoxalin-2-yl)ethyl)isoindoline-1,3-dione 74 being domi-nant. Yield 69%, (1.53g, 3.00 mmol). 1H NMR (400 MHz,[D6]DMSO) δ ppm 12.57 (0.62 H, br s), 11.96 (0.16 H, br s),7.84−7.91 (4 H, m), 7.66−7.78 (0.47 H, m), 7.48−7.54 (0.75 H, m),7.42−7.46 (0.7 H, m), 7.32 (0.25 H, t, J = 8.0 Hz), 7.27 (0.68 H, dd, J= 8.0, 1.4 Hz), 5.54−5.63 (1.0 H, m), 1.80 (2.18 H, d, J = 7.0 Hz),1.75 (0.62 H, d, J = 7.0 Hz). Mass spectrum (ESI) m/e = 353.8 (M +1). (Stuctural assignment of 74 was verified with pure 73c by 1H−15NHMBC NMR.)

General Procedure for the Synthesis of 72a and 72b. Aheterogeneous mixture of 71b (58.86 g, 217.1 mmol) or 71a (1.84 g,6.41 mmol) and POCl3 (10 equiv) was stirred at 100−110 °C. After 2h, the mixture was cooled to rt and poured into ice with stirring andcarefully neutralized with NH4OH and water. The resulting precipitatewas collected by filtration, rinsed with water, and dried under highvacuum.

2-(1-Bromoethyl)-3,5-dichloroquinoxaline (72a). Brown solid;yield 88% (1.68 g, 5.51 mmol) including 3,5-dichloro-2-(1-chloroethyl)quinoxaline. 1H NMR (400 MHz, [D6]DMSO) δ ppm8.10−8.17 (2 H, m), 7.88−7.95 (1 H, m), 5.85 (1 H, q, J = 6.7 Hz),2.16 (3 H, d, J = 6.7 Hz). Mass spectrum (ESI) m/e = 306.7 (M + 1).

2-(1-Bromoethyl)-3-chloro-6-fluoroquinoxaline (72b). Brownsolid; yield 93% (58.31 g, 201.5 mmol) including trace of 3-chloro-2-(1-chloroethyl)-6-fluoroquinoxaline. Mass spectrum (ESI) m/e = 288(M + H (79Br)). The brown solid was carried on crude withoutpurification for the next step.

General Procedure for 73a and 73b. To a stirred rt solution of72b (57.17 g, 197.5 mmol) or 72a (22.4 g, 73.2 mmol) in DMF (0.3M) was added potassium phthalimide (1.0 equiv). After 1−3 h, waterwas added.

2-(1-(3,5-Dichloroquinoxalin-2-yl)ethyl)isoindoline-1,3-dione (73a). Precipitate was filtered, washed with water, and dried.Orange solid; yield 71% (19.2 g, 51.6 mmol). 1H NMR (500 MHz,[D6]DMSO) δ ppm 8.16 (1 H, dd, J = 8.3, 1.2 Hz), 8.12 (1 H, dd, J =7.7, 1.3 Hz), 7.92 (1 H, dd, J = 8.4, 7.7 Hz), 7.87 (4 H, s), 5.86−5.92(1 H, m), 1.87 (3 H, d, J = 6.8 Hz). Mass spectrum (ESI) m/e = 371.8(M + 1).

2-(1-(3-Chloro-6-fluoroquinoxalin-2-yl)ethyl)isoindoline-1,3-dione (73b). The mixture was extracted with DCM (3 × 500 mL).The organic layer was washed with brine (1 L), dried over MgSO4,filtered, and concentrated to give a red liquid. This was filtered thougha plug of silica (5.5 in. diameter × 5 in. height) and eluted with 20−100% EtOAc/hexane to give the desired product. Pink solid; yield 76%(53.4 g, 150 mmol). 1H NMR (400 MHz, [D6]DMSO) δ ppm 8.25 (1H, dd, J = 9.4, 5.9 Hz), 7.81−7.95 (6 H, m), 5.86 (1 H, q, J = 6.8 Hz),1.86 (3 H, d, J = 7.0 Hz). Mass spectrum (ESI) m/e = 356 (M + 1).

2-(1-(3,8-Dichloroquinoxalin-2-yl)ethyl)isoindoline-1,3-dione (73c). A 3.5:1 mixture of 74 and 2-(1-(8-chloro-3-hydroxyquinoxalin-2-yl)ethyl)isoindoline-1,3-dione (4.50 g, 12.7mmol) was stirred in POCl3 (16.0 mL, 172 mmol) at 110 °C for1.5 h. The mixture was cooled to rt and poured into ice with stirring.NH4OH was added with additional ice until the mixture was slightlybasic. The resulting mixture was extracted with DCM, and thecombined organic layers were dried over MgSO4, filtered, andevaporated. Column chromatography (2×), 330 g Redi-Sep column,8% EtOAc for 10 min, then gradient to 25% EtOAc in hexane, gave ayield of 49%, (2.30 g, 6.19 mmol). 1H NMR (400 MHz, [D6]DMSO)δ ppm 8.09−8.12 (1 H, m), 8.05 (1 H, dd, J = 8.6, 1.2 Hz), 7.90−7.93(1 H, m), 7.87−7.89 (4 H, m), 5.89 (1 H, q, J = 7.0 Hz), 1.92 (3 H, d,J = 6.7 Hz). Mass spectrum (ESI) m/e = 371.8 (M + 1). (Stucturalassignment was verified by 1H−15N HMBC NMR.) 73a yield, 9%(0.405g, 1.09 mmol) regioisomer impurity from starting material.

General Procedure for the Synthesis of 75a−c. A stirredsolution of 73a (0.526 g, 1.41 mmol), or 73b (50.14 g, 140.9 mmol),or 73c (500 mg, 1.34 mmol), 2-tri-n-butylstannylpyridine (1.3−2.0equiv), and Pd(PPh3)4 (0.1 equiv) in 1,4-dioxane (0.12 M) was heatedto 110 °C. After 19−29 h, the mixture was cooled to rt andconcentrated to give a residue.

2-(1-(8-Chloro-3-(pyridin-2-yl)quinoxalin-2-yl)ethyl)-isoindoline-1,3-dione (75a). Solvent: toluene (0.27 M). Chroma-


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tography: 40 g silica gel column with 0−40% EtOAc/hexane. Whitesolid, yield 82% (457 mg, 1.10 mmol). 1H NMR (400 MHz,dichloromethane-d2) δ ppm 1.85 (3 H, d, J = 7.04 Hz) 6.77 (1 H, q, J= 7.04 Hz) 7.26−7.33 (1 H, m) 7.65−7.77 (6 H, m) 7.85 (1 H, d, J =6.26 Hz) 7.94 (1 H, d, J = 7.83 Hz) 8.04 (1 H, dd, J = 8.41, 1.37 Hz)8.61−8.70 (1 H, m). Mass spectrum (ESI) m/e = 415.0 (M + 1).2-(1-(5-Chloro-3-(pyridin-2-yl)quinoxalin-2-yl)ethyl)-

isoindoline-1,3-dione (75b). Chromatography: 40 g Redi-Sepcolumn, 0−100% gradient EtOAc/hexane, 14 min, 100% isocraticEtOAc. Brown solid; yield 70%, (0.411 g, 0.991 mmol). 1H NMR (400MHz, [D6]DMSO) δ ppm 8.51 (1 H, dt, J = 4.8, 1.3 Hz), 8.10 (2 H,ddd, J = 7.9, 6.6, 1.2 Hz), 7.87−7.92 (1 H, m), 7.75−7.80 (4 H, m),7.68 (2 H, dd, J = 5.5, 2.7 Hz), 7.36 (1 H, ddd, J = 6.0, 4.8, 2.9 Hz),6.45−6.52 (1 H, m), 1.78 (3 H, d, J = 7.0 Hz). Mass spectrum (ESI)m/e = 414.9 (M + 1).2-(1-(6-Fluoro-3-(pyridin-2-yl)quinoxalin-2-yl)ethyl)-

isoindoline-1,3-dione (75c). To the residue was added DCM (200mL). The mixture was heated under reflux for 20 min and then cooledto 0 °C with stirring. The solid was collected by filtration and washedwith EtOAc−hexane (1:5, 500 mL) to give the desired product as adark brown solid. This was dissolved in DCM/MeOH (9:1, 400 mL),filtered through a plug of silica gel, and rinsed with DCM/MeOH (9:1,600 mL). The filtrate was concentrated under reduced pressure to givea brown solid, which was suspended in EtOAc−hexane (1:9, 400 mL)and heated under reflux for 40 min. The mixture was cooled to rt,filtered, washed with EtOAc−hexane (1:9, 600 mL), and dried to give75c. Tan solid; yield 85% (47.76 g, 119.9 mmol). 1H NMR (400 MHz,[D6]DMSO) δ ppm 8.47−8.53 (1 H, m), 8.20 (1 H, dd, J = 9.4, 5.9Hz), 7.94 (1 H, dd, J = 9.4, 2.7 Hz), 7.85 (1 H, td, J = 8.9, 2.9 Hz),7.64−7.80 (6 H, m), 7.30−7.37 (1 H, m), 6.42 (1 H, q, J = 6.9 Hz),1.76 (3 H, d, J = 7.0 Hz). Mass spectrum (ESI) m/e = 399 (M + 1).General Procedure for the Synthesis of 76a−c. To a stirred

slurry of 75a (457 mg, 1.10 mmol) or 75b (0.405 g, 0.977 mmol) or75c (47.26 g, 118.6 mmol) in EtOH (0.1−0.5-M) was addedhydrazine monohydrate (5−10 equiv). The mixture was heated toreflux for 1−1.5 h.1-(8-Chloro-3-(pyridin-2-yl)quinoxalin-2-yl)ethanamine

(76a). The reaction mixture was filtered while hot, and the filter cakewas rinsed with methanol. Filtrate was concentrated to dryness andpartitioned between EtOAc and saturated NaHCO3. The aqueouslayer was extracted again with EtOAc. The organic layers werecombined and dried with Na2SO4 and filtered to give 76a. Yellow oil;yield, quant (322 mg, 1.13 mmol). 1H NMR (400 MHz, [D6]DMSO)δ ppm** 1.39 (3 H, d, J = 6.65 Hz), 2.21 (2 H, br s), 4.74 (1 H, q, J =6.65 Hz), 7.60 (1 H, ddd, J = 6.75, 4.79, 2.15 Hz), 7.84 (1 H, t, J =7.82 Hz), 8.04−8.15 (4 H, m), 8.77 (1 H, d, J = 4.69 Hz). Massspectrum (ESI) m/e = 285.1 (M + 1).1-(5-Chloro-3-(pyridin-2-yl)quinoxalin-2-yl)ethanamine

(76b). The mixture was concentrated under reduced pressure.Column chromatography, 40 g Redi-Sep column using 0−100%DCM/(89:9:1 DCM/MeOH/NH4OH) over 14 min and then 100%isocratic 89:9:1 DCM/MeOH/NH4OH for 3 min, gave a yellow solid;yield 78% (0.216 g, 0.760 mmol). 1H NMR (400 MHz, [D6]DMSO) δppm 8.77 (1 H, ddd, J = 4.9, 1.4, 1.2 Hz), 8.07−8.14 (3 H, m), 8.03 (1H, dd, J = 7.8, 1.2 Hz), 7.83−7.90 (1 H, m), 7.57−7.64 (1 H, m), 4.76(1 H, q, J = 6.4 Hz), 2.49 (2 H, br s), 1.37 (3 H, d, J = 6.7 Hz). Massspectrum (ESI) m/e = 285.0 (M + 1).1-(6-Fluoro-3-(pyridin-2-yl)quinoxalin-2-yl)ethanamine

(76c) and (S)-1-(6-Fluoro-3-(pyridin-2-yl)quinoxalin-2-yl)-ethanamine (76d). The precipitate was broken up with a spatula,filtered, washed with EtOAc (3 × 250 mL portions). The filtrate wasconcentrated under reduced pressure. The residue was redissolved inEtOAc (600 mL) and water (300 mL). The organic layer wasseparated, and the aqueous layer was back-extracted with EtOAc (3 ×100 mL). The EtOAc layer was dried over MgSO4, filtered, andconcentrated to give 76c. Brown solid; yield 97% (31.0 g, 115.3mmol). 1H NMR (400 MHz, [D6]DMSO) δ ppm 8.75 (1 H, dq, J =4.7, 0.9 Hz), 8.20 (1 H, dd, J = 9.0, 5.9 Hz), 7.98−8.11 (2 H, m), 7.91(1 H, dd, J = 9.4, 2.7 Hz), 7.78−7.86 (1 H, m), 7.56−7.61 (1 H, m),4.66 (1 H, q, J = 6.7 Hz), 2.08 (2 H, br s), 1.35 (3 H, d, J = 6.7 Hz).

LC−MS (ESI) m/e = 269 (M + 1). HPLC: a peak at 4.991 min,97.59% pure at 254 nm, 104104-8-1-HPLC. Analytical chiral HPLC:Chiralpak AD-H column, 0.46 mm × 250 mm, 5 mm) (isopropanol inhexane, 10%), two peaks at 9.350 min and at 10.678 min at 254 nm.Quantitative chiral purification (SFC): dissolved in 800 mL ofmethanol (plus few drops TFA for solubility). The 1.3 mL injectionsof this solution onto the SFC system at conditions below resulted inisolation of two major peaks. Column: 250 mm × 30 mm AS-H (5μm). Mobile phase: A, liquid CO2, 54 g/min; B, methanol (0.2%DEA), 36 mL/min. Mobile phase, temp: 22 °C. Outlet pressure: 117bar. Wavelength: 327 nm. After chiral separation, each fraction wascoevaporated two times with toluene and ethanol to removediethylamine which was contained in methanol as eluent onpurification. 76d: brown syrupy solid; yield 41% (12.67 g, 47.2mmol). Chiral HPLC (Chiralpak AD-H column, 0.46 mm × 250 mm,5 mm) (isopropanol in hexane, 10%): a peak at 9.135 min (first-eluting enantiomer) at 254 nm. Enantiomeric excess analysis: 98.86%ee, second-eluting enantiomer on AS-H column. 1H NMR (400 MHz,[D6]DMSO) δ ppm 8.73−8.78 (1 H, dq, J = 4.8, 1.2, 0.9 Hz), 8.20 (1H, dd, J = 9.4, 5.9 Hz), 7.99−8.10 (2 H, m), 7.91 (1 H, dd, J = 9.4, 2.7Hz), 7.82 (1 H, td, J = 8.9, 2.9 Hz), 7.58 (1 H, ddd, J = 7.4, 4.9, 1.4Hz), 4.66 (1 H, q, J = 6.4 Hz), 2.07 (2 H, br s), 1.35 (3 H, d, J = 6.7Hz). Mass spectrum (ESI) m/e = 269 (M + 1).

2-((3-Chloro-2-nitrophenyl)amino)butanoic Acid (78). Astirred mixture of 1-chloro-3-fluoro-2-nitrobenzene (37.7 g, 215mmol), 2-aminobutanoic acid (22.0 g, 215 mmol), and K2CO3 (30.0g, 215 mmol) in DMSO (80.0 mL) was heated to 80 °C. After 18 hthe mixture was poured into water (300 mL) and extracted with Et2O.The aqueous layer was acidified to generate a solid, which wasrecovered by filtration. The solid was recrystallized from toluene (110mL) to afford fine yellow needles. Yield 76% (56 g, 162 mmol). 1HNMR (400 MHz, [D6]DMSO) δ ppm 13.02 (1 H, br s), 7.35 (1 H, t, J= 8.2 Hz), 6.82−6.90 (2 H, m), 6.25 (1 H, d, J = 7.8 Hz), 4.14 (1 H,td, J = 7.2, 5.5 Hz), 1.74−1.92 (2 H, m), 0.88 (3 H, t, J = 7.2 Hz).Mass spectrum (ESI) m/e = 259.0 (M + 1).

8-Chloro-3-ethyl-3,4-dihydroquinoxalin-2(1H)-one (79). To astirred solution of 72 (10.0 g, 38.7 mmol) in EtOH (99.1 mL, 38.7mmol) and HCl (64.4 mL, 193 mmol, 3 N) was added SnCl2·2H2O(44.0 g, 193 mmol). The mixture was heated under reflux. After 2.5 h,the mixture was cooled to rt, concentrated to remove excess EtOH,which precipitated a yellow solid. The mixture was then cautiouslytreated with an excess of KOH (300 mL, 10 M), water (300 mL) andextracted with DCM (4 × 200 mL). The organic layer was washedwith water (3 × 300 mL), dried over MgSO4, filtered, andconcentrated to give 79. Light yellow syrup; yield 94% (7.65 g, 36.3mmol). 1H NMR (400 MHz, [D6]DMSO) δ ppm 9.70 (1 H, s), 6.75−6.81 (1 H, m), 6.66−6.73 (2 H, m), 6.36 (1 H, d, J = 1.2 Hz), 3.69 (1H, ddd, J = 6.7, 5.2, 1.8 Hz), 1.53−1.71 (2 H, m), 0.93 (3 H, t, J = 7.4Hz). Mass spectrum (ESI) m/e = 211.0 (M + 1).

8-Chloro-3-ethylquinoxalin-2(1H)-one (70a). To a rt stirredsolution of 79 (1.10 g, 5.23 mmol) in 1,4-dioxane (52.3 mL) wasadded DDQ (1.19 g, 5.23 mmol). After 2 h, the mixture wasconcentrated and the residue was dissolved in DCM (200 mL). Thesolution was washed with saturated NaHCO3 (2 × 100 mL), water (2× 100 mL), brine (100 mL), dried over Na2SO4, filtered, andconcentrated to give 74. Light yellow solid; yield 97% (1.06 g, 5.08mmol). 1H NMR (400 MHz, [D6]DMSO) δ ppm 11.87 (1 H, br s),7.71 (1 H, dd, J = 8.2, 0.8 Hz), 7.62 (1 H, d, J = 7.8 Hz), 7.28 (1 H, t, J= 8.0 Hz), 2.83 (2 H, q, J = 7.2 Hz), 1.23 (3 H, t, J = 7.4 Hz). Massspectrum (ESI) m/e = 208.9 (M + 1).

3,5-Dichloro-2-ethyl-4a,8a-dihydroquinoxaline (80). A stirredsuspension of 70a (20.0 g, 96 mmol) in POCl3 (147 g, 959 mmol) washeated at 100 °C. After 2 h, the reaction was cooled to rt andconcentrated. The resulting black residue was slowly treated with ice(∼40 mL) followed by water (30 mL). The mixture was treated withNH4OH (∼35 mL) until pH 12. The resulting black precipitate wasfiltered and washed with water and dried under vacuum overnight togive 80. Yield 90% (21.8 g, 86 mmol). 1H NMR (400 MHz, CDCl3) δppm 7.99 (1 H, dd, J = 8.4, 1.2 Hz), 7.82 (1 H, dd, J = 7.5, 1.1 Hz),


dx.doi.org/10.1021/jm501624r | J. Med. Chem. XXXX, XXX, XXX−XXXAC

7.64−7.69 (1 H, m), 3.20 (2 H, q, J = 7.4 Hz), 1.45 (3 H, t, J = 7.4Hz). Mass spectrum (ESI) m/e = 227.0 (M + 1).5-Chloro-2-ethyl-3-(3-fluorophenyl)quinoxaline (81). A 5 L

three-neck round-bottom flask with an overhead stirrer was chargedwith 80 (90.0 g, 396 mmol), 3-fluorophenylboronic acid (55.4 g, 396mmol), Pd(PPh3)4 (22.9 g, 19.8 mmol), and Na2CO3 (210 g, 198mmol) in acetonitrile−water (3:1) (4000 mL) and was heated at 85°C (internal temperature, 79.2 °C). After 8 h, the mixture was cooledto rt. The product crystallized as an orange solid overnight. Theorganic layer was decanted and concentrated to give a wet orangesolid. The wet orange solid was suspended in water (500 mL), stirredat rt for 30 min, filtered, washed with water (1 L), and dried undervacuum to give the desired product (77.6 g). The bottom aqueouslayer containing the orange crystalline solid was filtered and washedwith water (1.5 L) to give (45.4 g) both samples that contained a smallamount of triphenylphosphine oxide. The two samples were combinedand recrystallized from hot isopropanol (1 L), providing 81. Yield 88%(100.6 g, 351 mmol). 1H NMR (400 MHz, [D6]DMSO) δ ppm 8.08(1 H, dd, J = 8.6, 1.2 Hz), 7.97−8.02 (1 H, m), 7.80−7.86 (1 H, m),7.53−7.66 (3 H, m), 7.41 (1 H, dddd, J = 9.2, 8.0, 2.7, 1.2 Hz), 3.04 (2H, q, J = 7.4 Hz), 1.24 (3 H, t, J = 7.4 Hz). Mass spectrum (ESI) m/e= 286.9 (M + 1).2-(1-Bromoethyl)-5-chloro-3-(3-fluorophenyl)quinoxaline

(82). To a 5 L three-neck round-bottom flask fitted with an overheadstirrer was charged 81 (80.3 g, 280 mmol) and 1,3-dibromo-5,5-dimethylhydantoin (44.0 g, 154 mmol) in CCl4 (1.50 L, 0.2 M) wasadded benzoyl peroxide (9.045 g, 28.01 mmol) with heating at reflux.After 17 h, the mixture was allowed to cool and saturated aqueousNaHCO3 (100 mL) was added. The mixture was concentrated underreduced pressure to give a brown solid, which was suspended insaturated NaHCO3 (1 L). The resulting precipitate was collected byfiltration, washed with water (2 L), and dried under vacuum to give asolid (140.8 g), which was dissolved in DCM (2 L), dried overNa2SO4, filtered, and concentrated under reduced pressure to give abrown solid (104.3 g), which was again dissolved in DCM (500 mL),filtered through a silica gel pad, and washed with 30% of EtOAc inhexane (2 L) to provide 82. Yellow solid; yield 92% (94.3 g, 258mmol). 1H NMR (400 MHz, [D6]DMSO) δ ppm 8.14−8.18 (1 H,m), 8.09−8.13 (1 H, m), 7.88−7.95 (1 H, m), 7.59−7.70 (3 H, m),7.42−7.50 (1 H, m), 5.64 (1 H, q, J = 6.6 Hz), 2.15 (3 H, d, J = 6.7Hz). Mass spectrum (ESI) m/e = 364.8 [M + H (79Br)]+ and 366.7 [M+ H (81Br)]+.2-(1-Azidoethyl)-5-chloro-3-(3-fluorophenyl)quinoxaline

(83). To a solution of 82 (94.3 g, 258 mmol) in DMSO (860 mL, 258mmol) was added NaN3 (33.6 g, 516 mmol), and the mixture wasstirred at rt. After 2 h, water (1 L) was added to the mixture cooledwith an ice bath. The resulting precipitate was collected by filtration,washed with water (1 L), and dried under vacuum overnight to give83. Dark brown solid; yield 100%. (90.2 g, 258 mmol). 1H NMR (400MHz, [D6]DMSO) δ ppm 8.17 (1 H, dd, J = 8.4, 1.4 Hz), 8.11 (1 H,dd, J = 7.8, 1.2 Hz), 7.88−7.94 (1 H, m), 7.55−7.69 (3 H, m), 7.41−7.48 (1 H, m), 4.89 (1 H, q, J = 6.7 Hz), 1.65 (3 H, d, J = 6.7 Hz).Mass spectrum (ESI) m/e = 327.9 (M + 1).1-(5-Chloro-3-(3-fluorophenyl)quinoxalin-2-yl)ethanamine

(84). To a stirred solution of 83 (84.6 g, 258. mmol) in THF−H2O(4:1, 1500 mL) at 0 °C was added PMe3 (387 mL, 387 mmol, 1.0 M/THF). After 1 h 40 min, the mixture was poured into NaOH (500 mL,2 N) with cooling. The mixture was extracted with EtOAc (3 × 500mL). The organic layer was washed with brine (2 × 1 L), dried overMgSO4, and concentrated to give a red oil. Chromatography, 30%DCM/(89:9:1 DCM/MeOH/NH4OH), gave 79. Red oil; yield 88%(77.9 g, 226 mmol). 1H NMR (400 MHz, [D6]DMSO) δ ppm 8.10 (1H, dd, J = 8.2, 1.2 Hz), 8.00−8.05 (1 H, m), 7.85 (1 H, dd, J = 8.4, 7.6Hz), 7.57−7.68 (3 H, m), 7.39−7.46 (1 H, m), 4.32 (1 H, q, J = 6.7Hz), 2.06 (2 H, br s), 1.31 (3 H, d, J = 6.7 Hz). Mass spectrum (ESI)m/e = 301.9 (M + 1).

■ ASSOCIATED CONTENT*S Supporting InformationComparison of analogs 1 and 2 and control compound D-073;additional synthesis procedures; and crystallographic datacollection and refinement statistics. This material is availablefree of charge via the Internet at http://pubs.acs.org.Accession CodesAtomic coordinates and structure factors for the cocrystalstructures of PI3Kγ with compounds 1, 16, and 27 have beendeposited in the PDB with accession codes 4WWN, 4WWO,and 4WWP, respectively.

■ AUTHOR INFORMATIONCorresponding Author*Phone: 650-243-8265. E-mail: [email protected]; E-mail:[email protected] authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe are indebted to the following people: For expert NMRelucidation we thank David Chow and Smriti Khera. Foranalytical or preparative scale chiral and chiral SFC separations,thanks go to Manuel Ventura and Brent Murphy. Forprocurement and large scale synthesis of certain advancedintermediates thanks go to Eric Wohlheiter Yong-Jae Kim,Oliver Thiel, Rob Milburn, Johann Chan, and MikeAchmatowicz. For off target cellular assay deployment andexpertise not discussed in this report thanks go to Sandy Ross,David Fong, Kent Miner, and Kathy Chen. For in vitro drugmetabolism and disposition experiments thanks go to SilviaWong and Thuy Tran. For in vivo rodent pharmacokineticstudy conduct thanks go to Stacy Fide and Craig Uyeda. Foruseful discussions in the course of this investigation thanks goto Robert J. Zamboni, and for fruitful discussion on themanuscript thanks go to Margaret Chu-Moyer. Finally, forsome assistance in the preparation of this manuscript thanks goto Minna Bui.

■ ABBREVIATIONS USEDADDP, 1,1′-(azodicarbonyl)dipiperidine; AKT, protein kinaseB; AO, aldehyde oxidase; BSEP, bile salt export pump; BTK,Bruton’s tyrosine kinase; CD, cluster of differentiation; CYP,cytochrome P450; DIAD, diisopropyl azodicarboxylate; DIEA,N,N-diisopropylethylamine; (+) DIP-Cl, (+)-B-chlorodiisopi-nocampheylborane; DPPA, diphenylphosphoryl azide; ELISA,enzyme-linked immunosorbent assay; ETK, cryptic autophos-phorylating protein tyrosine kinase; FITC, fluorescein iso-thiocyanate; FOXO3a, forkhead box O3; GPCR, G-protein-coupled receptor; GSK3β, glycogen synthase kinase 3; HepG2,hepatocellular carcinoma; HLM, human liver microsomal;HWB, human whole blood; ITK, IL2-inducible T-cell kinase;KLH, keyhole limpet hemycin; LLC-PK1, Lilly Laboratoriescell porcine kidney; mTOR, mammalian target of rapamycin;NIK, nuclear factor κB inducing kinase; NF-κB, nuclear factorκ-light-chain-enhancer of activated B cells; MAPK, mitogen-activated protein kinase; PBS, phosphate buffer saline; PI,phosphoinositide; PIP2, phosphatidylinositol 4,5-bisphosphate;PIP3, phosphatidylinositol 3,4,5-trisphosphate; PH, pleckstrinhomology; PKB, protein kinase B; POC, percent of control;P70S6, serine/threonine kinase target substrate is S6 ribosomalprotein; RA, rheumatoid arthritis; RLM, rat liver microsomal;


dx.doi.org/10.1021/jm501624r | J. Med. Chem. XXXX, XXX, XXX−XXXAD

http://pubs.acs.org

mailto:[email protected]

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RTK, receptor tyrosine kinase; SLE, systemic lupus eryth-ematosus; XO, xanthine oxidase; SEM, standard error of themean; Xphos, 2-dicyclohexylphosphino-2,4,6-triisopropyl-1,1-biphenyl

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dx.doi.org/10.1021/jm501624r | J. Med. Chem. XXXX, XXX, XXX−XXXAF

discovery and in vivo evaluation of ( s )- n -(1-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9 h...

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