WO2016138988A1 - Piperazine inhibitors of bacterial gyrase and topoisomerase iv - Google Patents

Abstract

This invention relates to a piperazine derivative having the formula (I), wherein R' is selected from -H, -COOH and -CONH₂; Ft" is selected from -H, -COOH and -CONH₂ with the proviso that when R' is -COOH or -CONH₂ then R" is not -COOH or -CONH₂, and when R" is -COOH or -CONH₂ then R' is not -COOH or -CONH₂; A is selected from -(CH₂)₃-, -(CH₂)₂CH(OH)-, -CH₂-CH=CH- (cis and trans), (a); B is selected from -(CH₂)_n-, -(CH₂)_nO-, -(CH₂)_nS- and -(CH₂)_n-CH=CH- (cis and trans), (b); and wherein n = 1 -4; and Y is selected from C₁-C₇ alkyl (straight or branched), C₅-C₆ cycloalkyi optionally substituted with one or more halogens, thiophene or phenyl optionally substituted with one or more halogens or -C(hal)₃ wherein "hal" denotes a halogen, or a pharmaceutically acceptable salt thereof.

Description

Piperazine inhibitors of bacterial gyrase and topoisomerase IV Field of the invention

The present invention relates to a novel class of antibiotic compounds that are effective against methicillin-resistant Staphylococcus aureus (MRSA). The new class of antibiotics is constituted by a piperazine core that links to a fluoroquinoline and a hydrophobic moiety.

Background of the invention

Penicillin has been commercially available for half a century. However, the extensive use of penicillin has resulted in development in resistance among bacteria such as MRSA. The mortality rate for humans infected is 15-60% and in Europe 25,000 people die every year from infections caused by multidrug-resistant bacteria. MRSA strains are resistant to the β-lactam antibiotics (penicillins), but resistance to tet- racyclins, macrolides, lincosamides, aminoglycosides, trimethoprim, and in some cases also to fluoroquinolones is frequently observed. Vancomycin has for a long time been the drug of choice for MRSA infections, but this has now resulted in increasing vanco- mycin-resistance among MRSA.

Novexel and GSK have identified a new structural class of antibiotics, which consist of a quinolone and a partly aromatic heterocycle linked via a piperidine core. These antibiotics block the activity of bacterial topoisomerase and, accordingly, they are known as novel bacterial topoisomerase inhibitors (NBTIs). Novexel's lead compound NXL101 is a carboxy-piperidine based compound, whereas GSK progressed from the piperidine GSK2140944 compound (see below)

Structures of compounds NXL101 and GSK2140944 There are several advantages of topoisomerase inhibitors as antibiotics. Bacteria contain the two type IIA topoisomerases DNA gyrase (topoisomerase II) and topoisomerase IV. The active sites show a high degree of similarity, thus one antibiotic can poten- tially target those two distinct enzymes. Consequently, development of resistance would require mutations in both of the corresponding genes (gyrA for gyrase and parC for topoisomerase IV). Additionally, topoisomerase inhibitors might be effective against both Gram-positive and Gram-negative bacteria since the topoisomerase genes are highly conserved in both species. However, Gram-negative bacteria are typically harder to eradicate due to the outer membrane permeability barrier, multiple efflux pumps, as well as antibiotic- and target-modifying enzymes. Furthermore, DNA gyrase is not found in humans, and the human topoisomerases have distinct differences from the bacterial counterparts and are generally not affected by bacterial topoisomerase inhibitors.

During the phase I clinical trial of NXL101 , the compound was found to disturb the heart rhythm by prolongation of the QT interval. As this may lead to cardiac arrhythmia and sudden death, NXL101 was immediately discontinued. The typical cause of QT prolongation is blocking of the potassium channel in the heart encoded by the human ether-a-go-go related gene (hERG), commonly referred to as the hERG channel. The binding affinity for the hERG channel inhibitors is now always evaluated in pre-clinical studies of new drug candidates.

Thus, there is a medical need for new type of antibiotics that are effective towards pathogenic resistant bacterial strains and which have a safer profile than NXL101 regarding hERG.

Detailed description of the invention

The present invention provides a new class of antibiotics. The antibiotics target bacte- rial type II topoisomerase with a novel mode of action, which is distinct from the one of currently approved drugs. The antibiotics inhibit bacterial gyrase and topoisomerase IV and hamper DNA transcription and replication in living bacterial cells. Furthermore, this new class of antibiotics possesses a simple chemical structure, which facilitates its rapid preparation via chemical synthesis.

The present invention relates to a piperazine derivative having the formula (I)

Formula (I), wherein

Ft' is selected from -H, -COOH and -CONH₂;

Ft" is selected from -H, -COOH and -CONH₂ with the proviso that when Ft' is -COOH or -CONH2 then Ft" is not -COOH or -CONH₂, and when Ft" is -COOH or -CONH₂ then Ft' is not -COOH or -CONH₂;

A is selected from -(CH₂)₃-, -(CH₂)₂CH(OH)-, -CH₂-CH=CH- (cis and trans),

B is selected from -(CH₂)_n-, -(CH₂)_nO-, -(CH₂)_nS- and -(CH₂)_n-CH=CH- (cis and trans),

— _anc| wherein n = 1 -4; and

Y is selected from C1-C7 alkyl (straight or branched), C₅-C₆ cycloalkyi optionally substituted with one or more halogens, thiophene or phenyl optionally substituted with one or more halogens or -C(hal)₃ wherein "hal" denotes a halogen.

All cis-trans, R or S forms are encompassed in the above-mentioned formula (I) as well as all stereroisomers, enantiomers, racemic forms and mixtures thereof.

Compared with NXL101 the compounds of the present invention contain a piperazine core and not a piperidine core. The present inventors have found that changing the pi- peridine core with a piperazine core in general does not have negative effect on the antibiotic activity, but the linker A as well as the substituent R' or R" and the group B seem to have effect on reduction in hERG activity. Moreover, as seen from the examples herein, the fluor group in the quinoline moiety seems to be important for obtaining compounds with excellent antibiotic properties.

The R-configuration in the 2-position of the piperidine moiety seems to lead to more potent antibiotics, but compounds with S-configuration are still active and have suitable in vitro hERG characteristics. The R' group may be -H, -COOH or -CONH₂. From the ex- periments reported herein it is seen that changing R' from -COOH to -CONH₂ (all other structural elements being the same; see eg compound 53a and 54) does not affect the antibiotic activity, but had a 10-fold impact on hERG affinity. From the examples it is also seen that when R" is -COOH then both antibiotic activity is suitable and hERG affinity is acceptable. Accordingly, compounds are preferred, wherein R' is - COOH. Accordingly, preferred compounds have the following structure:

including the R- and the S-forms

With respect to the linker A suitable results have been obtained with all the linkers mentioned above. It seems as if the antibiotic activity decreases going from an alkanyl group to an alkenyl or an alkynyl group, but on the other hand the results with respect to hERG affinity are improved. Thus, all the linkers seem to either contribute to a potent antibacterial response or to a suitable hERG affinity, or both. Especially some of the linkers seem to contribute to excellent antibiotic and hERG effect, namely -CH₂-CH₂- CH₂- and -CH₂-CH₂-CHOH-:

Where FT, R", B and Y are as defined herein and FT" is H or OH.

All configurations are included in the above formula. As it appears from the examples herein a suitable configuration is:

Combining the information obtained from linker A as well as from R' and R" gives the following interesting structures:

wherein R'", B and Y are as defined above and the -COOH group in the 2- or 3-posi- tion of the piperazine ring can be in the R- or S-forms as described above. With respect the hydrophobic moiety -B-Y, the experiments reported herein show that Y groups like thiophene and phenyl are suitable optionally substituted with one or two halogens, especially fluorine, but also cycloalkanyl or straight or branched alkanyl groups are of interest (see eg compounds 58p and 58r):

wherein the phenyl-group may be substituted with one or two halogens:

especially one or two fluorine atoms. Other suitable Y groups are:

B is selected from -(CH₂)_n-, -(CH₂)_nO-, -(CH₂)_nS- and -(CH₂)_n-CH=CH- (cis and trans), — CH₂ — ^: _anc| wherein n = 1 -4. In the position closest to Y C, O and S lead to po- tent antibiotic compounds. It seems as if the compounds with O have a slightly better profile with respect to hERG and that disubstitution with F of the phenyl ring lead to compounds with good antibacterial effect and fine profile regarding hERG. Preferred linkers B are: -CH₂-CH₂-CH₂-, -0-CH₂-CH₂-, and -S-CH₂-CH₂-, where the -O- and the -S- part are coupled to Y and the -CH₂- part is coupled to the piperazine moiety. How- ever, as it appears from the results given herein suitable results are also obtained with any of the other mentioned B linkers.

In accordance with the above, the following structures are of interest:

wherein R', R", R'" are as defined above, X is C, O or S, and Y is H or halogen selected from F, Br, I, preferably F. As seen from the examples the phenyl ring may also be monosubstituted in the 3-position with a halogen.

Encompassed are also pharmaceutically acceptable salts of the compounds of the invention. Thus, when R' or R" is -COOH the suitable pharmaceutically acceptable salts include alkali salt, alkaline earth salts. Salts can also be obtained by reaction with am- monia or other types of amines. The hydrated version of the carboxylic acid is also acceptable. Suitable salts may be the sodium, potassium, lithium, magnesium, calcium, zink, aluminium salts. Other suitable salts include ammonium salt or salts of amine bases (ethanolamine, diethanolamine, trimethylamine, triethylamine, methylamine, propylamine, diisopropylamine, A/,/V-dimethylethanolamine, benzylamine, dicyclohexylamine, /V-benzyl-p-phenethylamine, Λ/,Λ/'-dibenzylethylenediamine, diphenylenediamine, ben- zhydrylamine, quinine, choline, arginine, lysine, leucine, dibenzylamine).

Pharmaceutically acceptable derivatives of the amine functions include the free base form or their acid addition or quaternary ammonium salts, for example their salts with mineral acids e.g. hydrochloric, hydrobromic, sulphuric nitric or phosphoric acids, or or- ganic acids, e.g. acetic, fumaric, succinic, maleic, citric, benzoic, p-toluenesulphonic, methanesulphonic, naphthalenesulphonic acid, camphorsulfonic or tartaric acids. Compounds may also be prepared as the /V-oxide.

The inventors have shown that the compounds of formula (I) have antibiotic activity, they act as topoisomerase inhibitors and they have suitable properties regarding hERG channel, i.e. they have sufficient low hERG inhibition.

Compounds of particular interest in the present context are included in Tables 1 -5. Based on structure-activity studies the following elements are important: OMe

Carbonyl not tolerated OH tolerated

OH and NH₂ are potent

3-Atom linker J OHand CO_∑H important for hERG

important for potency reduction

E-Alkene important

for hERG reduction

R-configuration important for potency

Mono fluorination of 2- or

3- position is tolerated, S, O and C are potent,

2,5-bisfluorination is potent O important for hERG reduction

Structure-activity relationships for piperazine-based antibiotic agents

As mentioned above, the piperazine derivatives of the present invention have been shown to act as MRSA antibiotics. Most of the compounds have been tested to identify the minimal inhibitory concentration against S. aureus; one β-lactam sensitive strain have been used, namely S. aureus MSSA (RV37) and one β-lactam resistant S. au- reus MRSA (CC398). Moreover, the compounds have been tested for inhibition of the bacterial enzymes gyrase and topoisomerase IV indicating that the compounds act as topoisomerase inhibitors. Finally, the compounds have been tested in an in vitro assay for hERG affinity. The results appear from the experimental section herein. The criteria to divide piperazine derivatives in groups of active versus non-active are based on results with the two conventional antibiotics ciprofloxaxin and vancomycin against S. aureus and E. faecalis.

The following tables show the MIC values of ciprofloxacin and vancomycin against S. aureus and E. faecalis according to the FDA. The data are divided according to the following criteria: susceptible, intermediate and resistant.

According to FDA standard vancomycin and ciprofloxacin powder should provide MIC values provided in the following table:

In the present context, the compounds of the invention are regarded as antibiotics if the MIC values for MSSA RV37 and/or MRSA CC398 are at the most 16 μg/mL. The most potent compounds are those having MIC values for MSSA RV37 and/or MRSA CC398 of at the most 1 μg/mL. The intermediate active compounds have MIC values from 1 to 10 μg/mL.

As seen from the examples herein compounds are provided, which have excellent antibiotic properties. Most of the exemplified and tested compounds have MIC values of 10 μg/mL or less and many of the compounds have MIC values of 5 μg/mL or less. Together with the excellent antibiotic properties, the compounds of the invention also seem to have suitable properties with respect to hERG inhibition.

With respect to hERG affinity it is generally assumed that the hERG affinity has to be less than 50% inhibition at 30 μΜ, i.e. the EC₅o has to be higher than 30 μΜ for replacement of the substrates (in the in vitro test kit) in the hERG channel protein. As seen from the tables herein, in some cases the hERG affinity is determined by % inhibition at 30 μΜ and in other cases by EC₅o for replacement of a substrate in a hERG test. To enable comparison of the two different assessments, the % inhibition at 30 μΜ will be represented by >30 μΜ or <30 μΜ in tables with a majority of compounds characterized by EC₅o, and EC50 will be represented as >50% inhibition or <50% inhibition in ta- bles with a majority of compounds characterized by % inhibition at 30 μΜ.

The tests performed are in vitro tests and only give a rough indication. The hERG channel inhibition assay is regarded as a highly sensitive measurement which will identify compounds exhibiting cardiotoxicity related to hERG inhibition in vivo. However, it is important to note that not all compounds which inhibit hERG activity in vitro will proceed to cause cardiotoxicity in vivo. The relevance of the in vitro data will depend on other factors such as the plasma concentrations reached in vivo. Moreover, some compounds may pass the in vitro test, but still cause cardiotoxicity in vivo. Accordingly, it is important to supplement the in vitro tests with other tests, preferably an in vivo test in a suitable test animal.

The hERG tests must be supplemented by other test such as manual or automated patch clamp assays, which are optional to use as preclinical cardiac risk indicators.

As mentioned above, the hERG test performed (as described in the experimental section) gives only an indication of the ability of the compound to inhibit the hERG channel in vitro. The inhibition should be as low as possible, but the test only gives indicative re- suits on which a selection may take place. In order to have a more conclusive evaluation of hERG inhibition a so-called patch-clamp test should be performed. In the present context, a compound of the invention is regarded as of interest, if the compound has excellent antibacterial effect. Preferably, the compound also have suitably low hERG inhibition. Preferably, when the compound is tested in the hERG test, the test re- suits in an EC50- value of 24 μΜ or more. However, a person skilled in the art will know that the values obtained from the hERG test are indicative and that further tests are necessary to ensure low cardiotoxicity of the compound in vivo.

In some cases, a compound is regarded as of interest with respect to a suitable hERG profile if the EC50 is higher than 30 μΜ, which corresponds to less than 50% inhibition at 30 μΜ, but as explained above, the in vitro results may differ markedly from in vivo results relating to cardiotoxicity.

The compounds are intended for use in the treatment of infections caused by pathogenic bacteria. Treatment includes a range of bacteria including Gram-positive and Gram-negative bacteria and mycobacteria. The infection may be on any part of the mammal body. Treatment of humans as well as household, farm and live-stock animals is within the scope of the present invention.

The compounds may be administered to a subject in need thereof in the form of a phar- maceutical composition comprising the compound together with one or more pharmaceutically acceptable excipients. The formulation may be designed to oral, parenteral or topical application. It may be in the form of a dosage form such as a solid dosage form and may contain an effective dose of the compound. A person skilled in the art will find guidance of how to formulate pharmaceutical composition in Remington's Pharmaceuti- cal Sciences (newest edition) and a person skilled in the art will know how a suitable dosage regime can be determined based on pharmacokinetic data and pre-clinical studies.

General description of methods for preparing compounds according to the invention In the following schemes are given details regarding the preparation of the compounds of the invention and intermediates:

Scheme 1 . Synthesis of quinoline building block 6

Scheme 3. Synthesis of quinoline-aldehyde building blocks 11 , 14, and 16

Scheme 4. Synthesis of /V-protected piperazine building blocks

Scheme 5. Synthesis of aldehyde building blocks

Scheme 6. S nthesis of compounds 53a, 53b and 54

Scheme 7. Synthesis of final compounds 58a-s, 59, 62a-e, 63a-f and 64a-e

Scheme 8. Synthesis of final compounds 68, 70, 72a-b, 73 and 74

Scheme 9. Solid-phase synthesis of final compounds 79a-b, 80 and 81

Legends to figures

Figure 1 . Gels showing inhibition of S. aureus gyrase supercoiling enzymatic activity. Ciprofloxacin tested in dosing range 0.05-50 μΜ and NXL101 and 58f tested in dosing range 0.05-10 μΜ. Figure 2. Gels showing inhibition of S. aureus topoisomerase IV relaxation enzymatic activity. Ciprofloxacin. NXL101 and 58f tested in concentrations 50-0.05 μΜ.

The invention is further illustrated in the following non-limiting examples. Materials and methods

Procedures and characterization All reagents and solvents were purchased from commercial suppliers and used without further purification. All solvents used were of HPLC-grade, which predominantly were used without further drying. Dry DMF was obtained from an Innovative Technology Pure Solv MD 7 Solvent Purification System. The glassware for dry reactions was dried over a Bunsen flame under vacuum before contact with solvent or reagents. Unless otherwise stated, reactions were run as open-system reactions, using only a loosely- fitted plastic plug in order to avoid contamination of the reaction mixture. Reaction products have been purified using flash column chromatography or preparative high- performance liquid chromatography (prep-HPLC). Reactions were routinely monitored using thin layer chromatography (TLC), ultra-performance liquid-chromatography mass-spectrometry (UPLC-MS) and/or high-performance liquid-chromatography with UV detection (HPLC-UV).

Analytical TLC was performed using Merck aluminium sheets covered with silica (C60). The plates were visualized using UV light and/or a KMn04 staining solution (3 g in water (300 mL), K2CO3 (20 g) and 5% NaOH (aq) (5 mL)) followed by heating.

Analytical HPLC-UV was accomplished on a Waters Alliance reversed phase (RP) HPLC system, employing a Waters 2695 Separations Module and a Waters 2998 Pho- todiode Array Detector. The column used was a Symmetry® C18 column (c 3.5 μηι,

4.6 x 75 mm; column temp: 25 °C; flow: 1 mL/min) with routine detection at 215 nm and 254 nm. Eluents A (0.1 % TFA in H₂0) and B (0.1 % TFA in MeCN) were used in a linear gradient (100% A to 100% B) in a total run time of 13 min. Analytical UPLC-MS (ESI) was performed on a S2 Waters ACQUITY RP-UPLC system equipped with a diode array detector using an ACQUITY UPLC BEH C18 column (d

1 .7 μηι, 2.1 x 50mm; column temp: 65 °C; flow: 0.6 mL/min), as well as a SQD ESI MS detector. Eluents A1 (0.1 % HCOOH in H₂0), A2 (0.1 % NH4COOCH3), B1 (0.1 %

HCOOH in MeCN) and B2 (0.1 % NH4COOCH3 in MeCN) were used in a linear gradient 5% B1 /B2 to 100% B1/B2 in a total run time of 2.6 min.

Analytical LC-HRMS (ESI) analysis was performed on an Agilent 1 100 RP-LC system equipped with a diode array detector using a Phenomenex Luna C18 column (c 3 μηι, 2.1 x 50 mm; column temp: 40 °C; flow: 0.4 mL/min). Eluents A (0.1 % HCOOH in H₂0) and B (0.1 % HCOOH in MeCN) were used in a linear gradient (20% B to 100% B) in a total run time of 15 min. The LC system was coupled to a Micromass LCT orthogonal time-of-flight mass spectrometer equipped with a Lock Mass probe operation in positive electrospray mode.

Flash column chromatography was achieved using a glass column packed with Merck Geduran® 60 silica gel (40-63 μηι particles) as stationary phase, and liquid phase as specified in the individual experimental procedures.

Preparative RP-HPLC was carried out on a Waters Alliance RP-HPLC system consisting of a Waters 2545 Binary Gradient Module equipped with an xBridgeTM Prep BEH130 C18 column OBDTM (c 5 μηι, 19 x 100mm; column temp: 25 °C; flow: 20 mL/min), a Waters Photodiode Array Detector (detecting at 210-600 nm), a Waters UV Fraction Manager and a Waters 2767 Sample Manager. Elution was carried out in a linear reversed phase gradient, combining H₂0 and MeCN (buffered with 0.1 % HCOOH or NH4COOCH3). Freeze drying was accomplished via a Thermo Scientific Heto Pow- erDry® LL 1500 freeze dryer.

All purified compounds have been routinely characterized by ¹ H NMR, ¹³C NMR, IR, RP-UPLC-MS and RP-HPLC-UV, as well as melting point and optical rotation where applicable. Novel compounds were further characterized via HRMS. For diastereomeric mixtures, only ¹ H NMR was obtained, as well as HRMS for acid analogs.

NMR spectra were recorded on a Bruker Ascend spectrometer with a Prodigy cry- oprobe (operating at 400 MHz for ¹ H NMR and at 100 MHz for ¹³C NMR), and analyzed via the NMR software MestReNova (version 6.2.1 -7569) released by Mestrelab Re- search S.L. The chemical shifts (δ) are reported in parts per million (ppm) and the coupling constants (J) in Hz. The majority of the spectra have been recorded in CDCI₃, and the signals were adjusted relative to this position (δ 7.26 ppm for ¹ H NMR and δ 77.2 ppm for ¹³C NMR). For spectra recorded in DMSO-d₆, the signals were adjusted relative to the DMSO signal (δ 2.5 ppm for ¹ H NMR and δ 39.5 ppm for ¹³C NMR). In some of the spectra of acid analogs, a residue of formic acid can be seen (δ 8.3 ppm for ¹ H NMR and δ 165.5 ppm for ¹³C NMR), which originates from the purification using preparative HPLC.

IR analyses were performed on a Bruker Alpha FT-IR spectrometer. Melting points were measured using a Stuart SMP30 melting point apparatus, and specified as an in- terval of melting temperatures (°C), or as a point from where the compound decomposed (dec). Optical rotation was measured on a Perkin-Elmer 341 polarimeter (polar- imeter cell 1 .0 ml_, 100 mm), with a sodium source lamp (589.3 nm, 23 °C).

Compounds have been drawn and named through use of the visualization software ChemDraw Ultra 14.0 released by PerkinElmer Informatics. Molecular- and exact masses have been calculated via this program as well.

Biological methods

Gyrase Supercoiling Assay

The assay was performed using S. aureus Gyrase Supercoiling Assay Kit (Inspiralis, Norwich, UK, Cat No. SAS4001 ). The assay was carried out according to the instructions from the supplier. Supercoiled pBR322 plasmid DNA was incubated with gyrase in the assay buffer supplied by the manufacturer. Different concentrations of the compounds were tested. Reactions were carried out for 30 minutes at 37 °C and terminated by addition of stop buffer (STEP) and chloroform/isoamyl alcohol. Samples were vor- texed, centrifuged and run through a 1 % agarose gel for 2 h at 80 V. Gels were stained with ethidium bromide and visualized under UV light.

Gyrase Supercoilling High Throughput Plate Assay

The assay was performed using S. aureus Gyrase Supercoiling High/Medium-Throughput Assay Kit (Inspiralis, Norwich, UK, Cat No. SATRG01 ). Black streptavidin-coated 96-well microplates were rehydrated by using Wash buffer followed by immobilize bioti- nylated oligo (TF01 ) onto the wells¹. Excess of oligo was removed by using Wash buffer. Enzyme assay was carried out by using pN01 followed by addition of enzyme and compounds. The plate was incubated for 30 minutes at 37 °C followed by addition of TE buffer and then 30 minutes incubation at room temperature. Unbound plasmid was washed off with TE buffer. DNA stain was added and fluorescence was measured in plate reader (Excitation: 495 nm; Emission 537 nm). Topoisomerase IV High Throughput Plate Assay

The assay was performed using S. aureus Topoisomerase IV High / Medium-Throughput Assay Kit (Inspiralis, Norwich, UK, Cat No. SATRIV01 ). Black streptavidin-coated 96-well microplates were rehydrated by using Wash buffer followed by immobilize bioti- nylated oligo (TF01 ) onto the wells¹. Excess of oligo was removed by using Wash buffer. Enzyme assay was carried out by using pN01 followed by addition of enzyme and compounds. The plate is incubated for 30 minutes at 37°C followed by addition of TE buffer and then 30 minutes incubation at room temperature. Unbound plasmid was washed off with TE buffer. DNA stain was added and fluorescence was measured in plate reader (Excitation: 495 nm; Emission 537 nm). hERG inhibition

The hERG channel inhibition was measured by the Predictor™ hERG Fluorescence Polarization Assay test kit (catalog no. PV5365) from Invitrogen (Carlsbad, CA). The binding assay was carried out according to the kit instructions². The Predictor™ hERG kit from Invitrogen is a homogeneous fluorescent assay that uses a simple add-and- read format. The assay is based on the principle of fluorescence polarization where a red fluorescent tracer is displaced from the hERG channel by compounds that bind to the channel. Assay performance was validated by using established hERG channel blockers. MIC determination by broth microdilution

The MIC values of the different compounds were determined by standard broth micro- dilution assay in 96-well sterile microplates³. The following two bacterial strains were used, S. aureus MSSA (RV37), DSM4910, ATCC35556 and S. aureus MRSA (CC398, SSI). The bacteria were grown for 24 h. at 37°C in Mueller Hinton media supplemented with 1 % glucose when appropriate. The compounds were tested in 1 1 different concentrations ranging from 50 μg/mL to 0.05 μg/mL. The MIC endpoint was determined as the lowest concentration of antibiotic at which there was no visible growth.

References for biological protocols

1 Maxwell A, Burton NP and O'Hagan N High-throughput assays for DNA gyrase and other topoisomerases. Nucl. Acids Res. (2006) 34 (15): e104.

²lnvitrogen. Predictor™ hERG Fluorescence Polarization Assay test kits. Protocol part no. PV5365.pps. Rev. date: 28 October 2009

³ Andrews JM. J. Determination of minimum inhibitory concentrations. Antimicrob. Chemother. (2001 ) 48 (suppl 1 ): 5-16.

References for the synthetic schemes: ¹ Flagstad, T.; Petersen, M. T.; Hinnerfeldt, D. M.; Givskov, M.; Nielsen, T. E. Synthesis 2014, 46, 3263.

¹ Li, B.; Zhang, Z.; Mangano, M. Org. Process Res. Dev. 2008, 12, 1273-1275.

1 Baque, E.; Carry, J.-C; Evers, M.; Hubert, P.; Mallerom, J.-L.; Mignani, S.; Pantel, G.; Tabart, M.; Viviani, F., US 2002/1 1 1492 A1 , 2002.

¹ Bi, L; Zhang, Y.; Zhao, M.; Wang, C; Chan, P.; Tok, J. B.-H.; Peng, S. Bioorg. Med. Chem. 2005, 13, 5640-5646.

¹ Mieczkowski, A.; Kozminski, W.; Jurczak, J. Synthesis 2009, 2010, 221-232.

1 Ranganathan, S.; Ranganathan, D.; Singh, S. K., Tetrahedron Lett. 1987, 28, 2893- 2894.

¹ Anisimov, A. Moscow Univ. Chem. Bull. 1981 , 22, 508-509.

¹ Peng, H.; Cheng, Y.; Ni, N.; Li, M.; Choudhary, G.; Chou, H. T.; Lu, C.-D.; Tai, P. C; Wang, B. ChemMedChem 2009, 4, 1457-1468

¹ Wu, J. -J.; Cheng, J.-H.; Zhang, J.; Shen, L; Qian, X.-H.; Cao, S. Tetrahedron 2011 , 67, 285-288.

Experimental and Examples

2,4-Dichloro-3-fluoro-6-methoxyquinoline (2)." Dimethyl 2-fluoromalo- nate (1 ) (20.0 g, 134 mmol) and LiOH (6.89 g, 287 mmol) were dissolved

in a mixture of water (20 mL) and MeOH (300 mL) and the reaction mixture was stirred for 22 h at rt. The reaction mixture was concentrated in vacuo and the residue was suspended in water (30 mL) and MTBE (200 mL). The suspension was cooled to 0 °C and cone. HCI (30 mL) was slowly added. The mixture was transferred to a separatory funnel and the layers were separated. The aqueous phase was extracted with MTBE (3 x 100 mL) and the combined organic layers were dried over Na₂S04 and concentrated in vacuo to give 2-fluoromalonic acid as a white solid (15.4 g, 94%). ¹ H NMR (400 MHz, DMSO-d₆) δ 5.48 (d, JHF = 48.0 Hz, 1 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.9 (d, JCF = 23.7 Hz), 85.4 (d, JCF = 189.2 Hz); IR (neat) cm ¹ : 2945, 1707, 1407, 1268, 1 127, 901 , 693; m.p.: 134.8 - 139.2 °C. 2-Fluoromalonic acid (15.4 g, 126 mmol) was added portion wise to POCI₃ (91 .7 ml_, 989 mmol) under an argon atmosphere and the solution was stirred for 35 min at reflux. The solution was then cooled to 60 °C and p- anisidine (15.5 g, 126 mmol) was added portion wise whereupon the reaction mixture was stirred for 2 h at reflux. Evolved HCI gas was trapped with 1 M NaOH. Excess POCI₃ was removed by distillation and the residue was cooled to rt, poured over crushed ice, and the mixture was stirred overnight. 25% NH4OH (aq.) was then added until pH 10 (210 ml.) and the mixture was stirred for 1 h 30 min at rt. The mixture was filtered and the filter cake was washed with water and purified by flash column chroma- tography on silica gel (EtOAc:heptane (1 :19 to 1 :14), R_f = 0.4 in 1 :9 EtOAc:heptane) to give the title compound as an off-white solid (1 1.5 g, 37%). ¹H NMR (400 MHz, CDCI₃) δ 7.91 (d, J = 9.2 Hz, 1 H), 7.38 (dd, J = 9.2, 2.7 Hz, 1 H), 7.33 (d, J = 2.7 Hz, 1 H), 3.98 (s, 3H); ¹³C NMR (100 MHz, CDCI₃) δ 159.7, 148.5 (d, JCF = 261.2 Hz), 139.8 (d, JCF = 4.4 Hz), 137.0 (d, JCF = 21.9 Hz), 130.6 , 127.9, 126.0 (d, JCF = 16.1 Hz), 123.1 (d, JCF = 3.0 Hz), 101 .7 (d, J_CF = 5.3 Hz), 55.8; IR (neat) cm^-1: 1620, 1586, 1494, 1376, 1347, 1234, 826, 787; m.p.: 140.2 - 141.1 °C. All spectroscopic data were consistent with those in the literature.

4-Chloro-3-fluoro-6-methoxyquinolin-2-ol (3).' Dichloroquinoline 2 (30.4 g, 124 mmol) was dissolved in AcOH (400 ml.) and the reaction

mixture was stirred for 42 h at reflux. Then water (280 ml.) was added and the mixture was cooled to rt and filtered. The filter cake was washed with water and dried in vacuo to give the title compound as a white solid (27.4 g, 94%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.48 (br s, 1 H), 7.36 (d, J = 8.9 Hz, 1 H), 7.27 (dd, J = 8.9, 2.7 Hz, 1 H), 7.24 (d, J = 2.7 Hz, 1 H), 3.84 (s, 3H); ¹³C NMR (100 MHz, DMSO-de₆) δ 155.7, 154.1 (d, JCF = 26.0 Hz), 148.7 (d, JCF = 250.9 Hz), 129.5 (d, JCF = 2.5 Hz), 125.4 (d, JCF = 15.5 Hz), 120.4 (d, JCF = 3.0 Hz), 1 18.0, 1 17.7, 106.1 (d, JCF = 6.3 Hz), 56.1 ; IR (neat) cm^-1: 2812, 1650, 1610, 1577, 1497, 1455, 1424, 1292, 1 199; m.p.: 254.6 - 259.1 °C.

4-Chloro-3-fluoro-6-methoxyquinolin-2-yl trifluoromethanesul-

fonate (4).' Quinolone 3 (27.4 g, 120 mmol) was suspended in CH2CI2 (350 ml.) and EtsN (21.7 ml_, 156 mmol) and cooled to 0 °C under an argon atmosphere. Tf₂O (24.5 ml_, 145 mmol) was added drop wise over 1 h, whereupon the mix- ture was stirred for 1 h at 0 °C. Then additional Et₃N (2.20 ml_, 15.6 mmol) and Tf₂0 (2.4 ml_, 14.5 mmol) were added at 0 °C and the mixture was stirred for 15 min. The reaction mixture was quenched with water (150 ml.) and transferred to a separatory funnel where the layers were separated. The aqueous phase was extracted with CH2CI2 (2 x 230 ml.) and the combined organic layers were dried over MgS04, concentrated in vacuo, and purified by flash column chromatography on silica gel (EtOAc:heptane (1 :1 ), JcF= 0.61 ). The product was washed with heptane to give an off-white solid (36.7 g, 85%). ¹H NMR (400 MHz, CDCI₃) δ 7.94 (d, J = 9.2 Hz, 1 H), 7.44 (dd, J = 9.2, 2.7 Hz, 1 H), 7.38 (d, J = 2.7 Hz, 1 H), 4.00 (s, 3H); ¹³C NMR (100 MHz, CDCI₃) δ 160.5, 143.9 (d, JcF = 264.5 Hz), 140.8 (d, J_CF = 17.2 HZ), 137.2 (d, J_CF = 4.4 Hz), 131 .1 (d, JCF = 1.6 Hz), 129.0 (d, JCF = 1 .5 Hz), 128.1 (d, JCF = 14.2 Hz), 124.0 (d, JCF = 2.9 Hz), 1 18.7 (q, JCF = 320.6 Hz), 102.0 (d, JCF = 5.2 Hz), 56.1 ; IR (neat) cm^-1: 3092, 2948, 1621 , 1505, 1423, 1391 , 1359, 1205, 1 131 , 1 104, 922, 808; m.p.: 67.7 - 69.6 °C. 4-Chloro-3-fluoro-6-methoxyquinoline (5).'" A solution of triflate 4 (34.6

g, 96.2 mmol) and Pd(PPh₃)₄ (1 .83 g, 1 .59 mmol) in THF (480 ml.) was charged with a magnetic stirring bar and heated to 50-60 °C in flame dried flask under an argon atmosphere. Pyridine (77.5 ml_, 962 mmol) and EtsSiH (153 ml_, 140 mmol) were then added and the mixture was stirred for 2 h. The temperature was raised to 65 - 70 °C and the mixture was stirred for another 1 h 20 min. The reaction was then quenched with sat. NaHCO₃ (20 ml.) and concentrated in vacuo. EtOAc (100 ml.) and sat. NaHC03 (280 ml.) were added and the layers were separated. The organic layer was washed with sat. NH₄CI (3 x 185 ml_), dried over MgS0₄, concentrated in vacuo, and then purified by flash column chromatography (EtOAc:heptane (1 :9 to 1 :4), f?_f = 0.42 in 1 :4 EtOAc:heptane) to give the title compound as a white solid (12.3 g, 61 %). Fractions where product was contaminated with impurities were unsuccessfully purified by flash column chromatography to give the title compound as an off-white solid (8.7 g, 43%, purity by RP-UPLC-MS = 92%). The two crops of product were combined and used directly in the next step. ¹H NMR (400 MHz, DMSO-d₆) δ 8.84 (s, 1 H), 7.99 (d, J = 9.2 Hz, 1 H), 7.45 (dd, J = 9.2, 2.7 Hz, 1 H), 7.32 (d, J = 2.7 Hz, 1 H), 3.95 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 159.3, 152.3 (d, JCF = 255.9 Hz), 141 .2 (d, JCF = 2.9 Hz),

138.1 (d, JCF = 26.4 Hz), 131.4, 127.3, 122.8 (d, JCF = 15.1 Hz), 122.1 (d, JCF = 2.9 Hz),

101 .2 (d, JCF = 5.4 Hz), 55.8; I R (neat) cm^-1: 3016, 2966, 1621 , 1495, 1460, 1354, 1306, 1223, 1018, 910, 824, 788; m.p.: 92.5 - 94.4 °C. All spectroscopic data were consistent with those in the literature. 3-Fluoro-4-iodo-6-methoxyquinoline (6).'" Chloroquinoline 5 (20.3 g, 9²-^{2 mmo1}) ^was dissolved in CH₂CI₂ (160 mL) and treated with 1 M HCI

in ether (105 mL), whereupon the solvents were removed in vacuo to give a yellow solid. The salt was suspended in MeCN (480 mL) together with Nal (144 g, 959 mmol) and stirred for 22 h at reflux. A solution containing 5% Na2S203 (aq) and 10% K2CO3 (aq) (350 mL) was then added and the solution was stirred for 10 min. MeCN was removed in vacuo and the remaining aqueous phase was filtered. The filter cake was washed with CH2CI2 to give the title compound as a white solid (21.0 g, 72% over two steps). ¹H NMR (400 MHz, CDCI₃) δ 8.46 (s, 1 H), 7.97 (d, J = 9.1 Hz, 1 H), 7.33 (dd, J = 9.1 , 2.7 Hz, 1 H), 7.27 (d, J = 2.7 Hz, 1 H), 4.00 (s, 3H); ¹³C NMR (100

MHz, CDCI₃) δ 160.3, 157.6 (d, JCF = 253.9 Hz), 141 .4 (d, JCF = 2.2 Hz), 137.1 (d, JCF =

30.3 Hz), 132.4, 131 .7, 122.0 (d, JCF = 3.1 Hz), 109.2 (d, JCF = 5.1 Hz), 93.9 (d, JCF =

22.4 Hz), 55.9; IR (neat) cm^-1: 3012, 2959, 2827, 1616, 1496, 1463, 1400, 1348, 1304, 1223, 1 126, 1017, 904, 822, 789; m.p.: 172.2-174.2 °C. All spectroscopic data were consistent with those in the literature.

Scheme 2. Synthesis of aldehyde building block 8^iv

3,3-Dimethoxypropanal (8).^iv A 250 mL flask was charged with a magnetic

stirring bar, 1 , 1 ,3,3-tetramethoxypropane (40 g, 0.243 mol), 6% H₃P0₄ (20 mL) and the mixture was stirred for 5 days at rt. Then Et.20 (300 mL) was added and the mixture was cooled in an ice bath. CaC03 (20 g) was added and after 5 min stirring the mixture was filtered. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography on silica gel (Et20:pentane (3:7), Rf = 0.2) to give the title compound as a colorless liquid (8.57 g, 30%). ¹H NMR (300 MHz, CDCI₃) δ 9.32 (s, 1 H), 4.42 (t, J = 3.9 Hz, 1 H), 2.97-2.94 (m, 6H), 2.40-2.09 (m, 2H). All spectroscopic data were consistent with those in the literature.

Scheme 3. Synthesis of quinoline-aldehyde building blocks 11 , 14, and 16

1 -(3-Fluoro-6-methoxyquinolin-4-yl)-3,3-dimethoxypropan-1 -ol (10). A 50 mL flask was charged with a magnetic stirring bar, iodoquin-

oline 6 (0.500 g, 1.65 mmol), THF (8 mL) and the mixture was cooled to -78 °C. 2 M /-PrMgCI in THF (1.07 mL, 2.14 mmol) was then added and the mixture was stirred for 10 min, whereupon 3,3-dimethoxypropanal 8 (0.292 g, 2.47 mmol) was added and the external cooling was removed. After stirring for 10 min NH4CI (aq) was added and the mixture was concentrated to remove the volatiles. The mixture was then taken up in water, extracted with CH2CI2 (3 x 50 mL) and the combined organic layers were concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (EtOAc:heptane (1 :1 ), R_f = 0.2) to give the title compound as a colorless oil (195 mg, 40%). ¹H NMR (400 MHz, CDCI₃) δ 8.50 (d, J = 1 .9 Hz, 1 H), 7.93 (d, J = 9.2 Hz, 1 H), 7.76 (d, J = 2.7 Hz, 1 H), 7.27 (dd, J = 9.5, 2.4 Hz, 1 H), 5.70 (dd, J = 9.5, 3.7 Hz, 1 H), 4.63 (t, J = 5.4 Hz, 1 H), 3.91 (s, 3H), 3.73 (s, 1 H), 3.39 (s, 3H), 3.36 (s, 3H), 2.51 (ddd, J = 14.5, 9.4, 5.4 Hz, 1 H), 2.07 (ddd, J = 14.5, 5.4, 4.0 Hz, 1 H); ¹³C NMR (100 MHz, CDCI₃) δ 158.4, 154.0 (d, J_CF = 253.7 Hz), 142.1 (d, J_CF = 2.5 Hz), 138.4 (d, JCF = 30.1 Hz), 131 .5, 130.0 (d, J_CF = 8.3 Hz), 127.9 (d, J_CF = 2.9 Hz), 120.8 (d, J_CF = 2.5 Hz), 103.4 (d, JCF = 5.3 Hz), 103.2, 64.5 (d, JCF = 4.6 Hz), 55.6, 54.0, 53.4, 39.1 ; IR (neat) cm^-1: 2938, 2832, 1621 , 1508, 1467, 1427, 1354, 1227, 1 123, 1053; HRMS (ESI) calcd for C15H19FNO4 [M + H]⁺ 296.1293, found 296.1298. 3-(3-Fluoro-6-methoxyquinolin-4-yl)-3-hydroxypropanal (11 ). Acetal 10 (47 mg, 0.16 mmol) was dissolved in CH₂CI₂ (0.6 mL), cooled to 0 °C

and added TFA:H₂0 (1 :1 , 0.3 mL) and the mixture was stirred for 10 min at this temperature followed by 2 h at rt. CH₂CI₂ (25 mL) and NaHCOs (sat, aq) (25 mL) were added and the layers were separated. The aqueous phase was extracted with CH₂CI₂ (2 x 25 mL) and the combined organic layers were dried using Na₂S0₄ and concentrated in vacuo to give the title compound as a mixture of dimeric structures. Mixture used directly without further purification.

3-(3-Fluoro-6-methoxyquinolin-4-y/)prop-2-yn-1 -ol (12). A suspension of iodoquinoline 6 (10.0 g, 33.0 mmol) in THF (200 mL) and Et₃N (130

mL) was degassed with argon for 1 h. Pd(PPh3)₄ (1.14 g, 0.990 mmol) and Cul (0.315 g, 1 .65 mmol) were added and the reaction mixture was degassed with argon for 1 h. Propargyl alcohol (2.15 mL, 39.6 mmol) was added via syringe and the reaction mixture was degassed for 30 min before stirring for 5 days at rt. Water (150 mL) and CH₂CI₂ (200 mL) were then added and the phases were separated. The aqueous phase was extracted with CH₂CI₂ (3 x 200 mL) and the combined organic layers were dried over Na₂S0₄ and concentrated in vacuo. The remaining solids were washed with CH₂CI₂ to give the title compound as a white solid, which was collected as crop 1 (5.80 g). The filtrate was concentrated in vacuo and purified by flash column chromatography on silica gel (EtOAc:heptane (2:3), R_f = 0.2) to give the title compound as an additional crop of white solid (1 .70 g). Combined yield: 7.50 g, 98%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.81 (s, 1 H), 8.03 - 7.94 (m, 1 H), 7.50 - 7.40 (m, 2H), 5.65 (t, J = 6.1 Hz, 1 H), 4.54 (d, J = 6.1 Hz, 2H), 3.95 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 159.18, 156.3 (d, J_CF = 259.1 Hz), 140.7 (d, J_CF = 2.5 Hz), 138.0 (d, J_CF = 26.7 Hz), 131 .3, 129.1 , 121.5 (d, J_CF = 2.7 Hz), 1 1 1 .9 (d, J_CF = 12.7 Hz), 105.1 (d, J_CF = 4.5 Hz), 103.4 (d, JCF = 5.2 Hz), 73.0 (d, J_CF = 0.9 Hz), 55.7, 49.7; IR (neat) cm^-1 : 3322, 3089, 2831 , 2661 , 1620, 1596, 1503, 1470, 1222, 1027; m.p.: 1 18.8-1 19.8 °C. 3-(3-Fluoro-6-methoxyquinolin-4-yl)propan-1 -ol (13). Alkyne 12 (7-50 9. 32-4 mmol) and Pd/C (3.45 g, 3.24 mmol) were placed under

an argon atmosphere. MeOH (325 mL) was added via syringe and the flask was evacuated, filled with hydrogen, and the reaction mixture was stirred for 2 days at rt. Then additional Pd/C (0.843 g, 0.792 mmol), (0.570 g, 0.139 mmol) and (1.87 g, 0.456 mmol) was added after 2 days, 5 days, and 8 days, respectively. After 9 days the reaction mixture was filtered through celite, washed with EtOAc, and concentrated in vacuo. The residue was washed with ChbCI₃ and the white solid was collected as crop 1 (3.44 g). The filtrate was concentrated in vacuo and purified by flash column chromatography on silica gel (EtOAc:heptane (1 :1 ), Rf = 0.2) to give a second crop of product (1 .70 g) as a white solid. Combined yield: 5.14 g, 67%. ¹H NMR (400 MHz, CDCIs) δ 8.57 (d, JHF = 0.9 Hz, 1 H), 7.98 (d, J = 9.0 Hz, 1 H), 7.30 (dd, J = 9.0, 2.7 Hz, 1 H), 7.28 (d, J = 2.5 Hz, 1 H), 3.94 (s, 3H), 3.72 (t, J = 6.1 Hz, 2H), 3.20 - 3.09 (m, 2H), 2.21 - 1 .84 (m, 3H); ¹³C NMR (100 MHz, CDCI₃) δ 158.7, 155.1 (d, JCF = 251.2 Hz), 141 .6 (d, JCF = 2.3 Hz), 138.2 (d, J_CF = 29.4 Hz), 131.7, 129.8 (d, J_CF = 13.0 Hz), 129.4 (d, JCF = 3.9 Hz), 120.5 (d, JCF = 2.7 Hz), 102.0 (d, JCF = 5.5 Hz), 61.9, 55.7, 32.0, 20.5 (d, JCF = 3.3 Hz); I R (neat) cm^-1: 3219, 2941 , 2877, 2842, 1620, 151 1 , 1431 , 1324, 1216, 1031 , 828; m.p. = 1 16.2 - 1 17.5 °C.

General procedure I: Oxidation of alcohols to aldehydes with Dess- Martin perio- dinane 3-(3-Fluoro-6-methoxyquinolin-4-yl)propanal (14). 3-(3-Fluoro-6- methoxyquin-olin-4-yl)propan-1-ol (13) (135 mg, 0.574 mmol) and DMP

(292 mg, 0.689 mmol) were dissolved in CH2CI2 (3 ml.) and stirred at rt. After 2 h the reaction mixture was quenched with a 2:1 solution of 10% Na2S203 (aq) and sat. NaHCO₃ (aq) (3 ml.) and stirred for 30 min, whereupon the layers were separated. The aqueous phase was extracted with CH2CI2 (2 x 5 ml.) and the combined organic layers were washed with water containing a few drops of 2 M NaOH (aq) (10 ml_), dried over Na2S0₄, to afford the title compound as a white solid (131 mg, >95%), which was used in the next step without further purification. ¹H NMR (300 MHz, CDCI₃) δ 9.88 (s, 1 H), 8.59 (s, J = 1.1 Hz, 1 H), 8.01 (d J = 9.2 Hz, 1 H), 7.32 (dd, J = 9.2, 2.7 Hz, 1 H), 7.15 (d, J = 2.7 Hz, 1 H), 3.95 (s, 3H), 3.35 (dt, J = 7.7, 1.8 Hz, 2H), 2.87 (t, J = 7.7 Hz, 2H); HRMS (ESI) calcd for C13H13FNO2 [M + H]⁺ 234.0925, found 234.0931 .

(Z)-3-(3-Fluoro-6-methoxyquinolin-4-yl)prop-2-en-1 -ol (15). A 50 ml. flask was charged with a magnetic stirring bar, a septum, 3-(3-

fluoro-6-methoxyquinolin-4-yl)prop-2-yn-1 -ol (12) (0.500 mg, 2.16 mmol), EtOAc (25 ml.) and Pd/C (1 15 mg, 0.108 mmol) and cooled to 0 °C in an ice bath. An H2-atmosphere was introduced via a balloon and the mixture was stirred for 4 h. The mixture was then filtered through celite, which was washed with EtOAc. The fil- trate was concentrated in vacuo and the residue was purified by flash column chromatography on silica gel (EtOAc:heptane (2:3), R_f = 0.2) to give the title compound as a white solid (421 mg, 84%).¹H NMR (400 MHz, CDCI₃) δ 8.59 (d, J_HF = 0.7 Hz, 1H), 7.96 (d, J= 9.2 Hz, 1H), 7.29 (dd, J =9.2, 2.7 Hz, 1H), 7.05 (d, J= 2.7 Hz, 1H), 6.56 (dd, J= 11.6, 1.3 Hz, 1H), 6.38 (dt, J = 11.6, 6.5 Hz, 1H), 4.13 (dt, J = 6.5, 1.3 Hz, 2H), 3.90 (s, 3H), 2.26 (br s, 1H); ¹³C NMR (100 MHz, CDCI₃) δ 158.9, 153.2 (d, J_CF = 253.4 Hz), 141.3 (d, JC_F = 2.3 Hz), 138.2 (d, J_CF = 29.6 Hz), 137.7 (d, J J_CF = 1.0 Hz), 131.3, 128.7 (d, J_CF = 2.7 Hz), 124.9 (d, J_CF = 13.5 Hz), 121.2 (d, J_CF = 2.7 Hz), 118.8, 102.6 (d, JC_F = 5.5 Hz), 60.6 (d, J_CF = 4.6 Hz), 55.7; IR (neat) cm^-1: 3500-3300, 2937, 2834, 1620, 1507, 1426, 1358, 1227, 1026, 830; HRMS (ESI) calcd for C13H13FNO2 [M + H]⁺ 234.0930, found 234.0927; m.p.: 97 - 99 °C.

(Z)-3-(3-Fluoro-6-methoxyquinolin-4-yl)acrylaldehyde (16) Following general procedure / using (Z)-3-(3-fluoro-6-methoxyquinolin-4-

yl)prop-2-en-1-ol (15) (85 mg, 0.36 mmol) and DMP (170 mg, 0.40 mmol) the title compound was obtained as a white solid (82 mg, >95%, cis:trans (3:1)) after extraction.¹H NMR (400 MHz, CDCI₃) δ 9.88 (dd, J= 7.4, 1.4 Hz, 1H), 9.60 (dd, J = 8.1, 2.0 Hz, 0.3H), 8.69 (s, 1H), 8.68 (s, 0.3H), 8.03 (d, J= 9.2 Hz, 1H), 8.03 (d, J = 9.1 Hz, 0.3H), 7.85 (dd, J= 16.3, 1.4 Hz, 0.3H), 7.60 (dd, J = 11.5, 2.0 Hz, 1H), 7.25 (d, J= 2.8 Hz, 0.3H), 7.03 (ddd, J= 16.3, 7.4 Hz, J_HF= 0.7 Hz, 0.3H), 6.98 (d, J= 2.7 Hz, 1H), 6.55 (ddd, J = 11.5, 8.1, JHF = 1.5 Hz, 1H), 3.97 (s, 0.3H), 3.96 (s, 1H), 3.91 (s, 1H), 3.90 (s, 3H), cis:trans (3:1); ¹³C NMR (100 MHz, CDCI₃) δ 193.4 (0.3C), 191.6 (d, JC_F = 3.1 Hz, 1C), 159.7 (0.3C), 159.6 (1C), 154.37 (d, J_CF = 263.3 Hz, 0.3C), 153.1 (d, JCF = 257.1 Hz, 1C), 142.0 (d, J_CF = 2.8 Hz, 0.3C), 141.5 (d, J_CF = 2.6 Hz, 1C), 139.8 (1C), 138.5 (d, J_CF = 29.2 Hz, 0.3C), 138.2 (d, J_CF = 28.7 Hz, 1C), 137.3 (d, J_CF = 10.4 Hz, 0.3C), 136.4 (1C), 134.6 (1C), 132.2 (0.3C), 131.9 (1C), 128.2 (d, J_CF = 1.6 Hz, 1C), 127.4 (d, JC_F = 1.6 Hz, 0.3C), 122.3 (d, J_CF = 12.5 Hz, 0.3C), 121.8 (0.3C), 121.6 (d, JCF = 2.8 Hz, 1C), 121.2 (d, JCF = 2.8 Hz, 0.3C), 102.2 (d, JCF = 5.3 Hz, 1C), 101.6 (d, JCF = 5.3 Hz, 0.3C), 55.9 (0.3C), 55.8 (1C), cis:trans (3:1); HRMS (ESI) calcd for C13H11FNO2 [M + H]⁺ 232.1, found 232.1.

Scheme 4. Synthesis of A/-protected piperazine building blocks

(R)-1 -Benzyl 2-methyl piperazine-1 ,2-dicarboxylate (19). (f?)-Pipera- zine-2-carboxylic acid dihydrochloride 17a (4.94 g, 24.3 mmol) was dis¬

solved in 6 M NaOH (aq) (13 mL) and added a suspension of Cu(OAc)2 (2.2 g, 1 1.4 mmol) in H₂0 (9 mL) The mixture was stirred for 10 minutes. Then acetone (36 mL) was added followed by slow addition of CbzCI (4.52 mL, 31.6 mmol) and the mixture was stirred at rt for 2 h and then concentrated in vacuo. The residue was taken up in H₂0 (20 mL), followed by addition of K₂C0₃ (4.4 g, 31 .6 mmol) and EDTA (4.5 g, 1 1.4 mmol) and then stirred for 10 min. A solution of B0C2O (3.5 g, 31.6 mmol) in diox- ane (20 mL) was then added and the mixture was stirred overnight at rt. HCI (cone, aq) was then added until pH 4-5, the aqueous phase was separated and further extracted with EtOAc (3 x 100 mL). The combined the organic layers were concentrated in vacuo. The residue was taken up in 0.78 M HCI in MeOH (premixed by addition of AcCI (6.6 mL) to MeOH (30 mL) at 0 °C) and stirred for 4 h at reflux. The mixture was concentrated in vacuo and then added EtOAc (100 mL) followed by addition of saturated NaHC03 (aq) until neutral pH. The phases were separated and the aqueous phase was extracted with EtOAc (2 x 50 mL). The combined organic layers were dried over Na₂S0₄ and concentrated in vacuo. The residue was purified by flash column chroma- tography on silica gel (1 % EtsN in EtOAc, Rf = 0.2) to give the title compound as a colorless oil (4.86 g, 72%). ¹H NMR (400 MHz, CDCI₃) δ 7.46 - 7.18 (m, 5H), 5.17 (s, 2H), 4.77 and 4.66 (2 x d, J = 2:5 Hz, 1 H), 3.93 (m, 1 H), 3.77 and 3.71 (2 x s, 3H), 3.56 and 3.51 (2 x d, J = 12.7 Hz, 1 H), 3.25 and 3.13 (2 x td, J = 12.7, 3.2 Hz, 1 H), 3.05 - 2.86 (m, 2H), 2.81 - 2.64 (m, 1 H), 2.41 (br s, 1 H), flotamers; ¹³C NMR (100 MHz, CDCI₃) δ 171 .3 and 171.2, 156.5 and 156.0, 136.5, 128.6 (2C), 128.2, 128.0 (2C), 67.6 and 67.5, 55.2 and 54.7, 52.6, 47.1 and 46.9, 45.1 , 42.3 and 42.0, rotamers; IR (neat) cm^-1: 3344, 2952, 291 1 , 2862, 1739, 1699, 1430, 1357, 1228, 763, 699; HRMS (ESI) calcd for C14H19N2O4 [M + H]⁺ 279.1339, found 279.1342; = - 44.7 (c 1.0, CHCI₃).

(R)-1 -Benzyl-3-methyl-piperazine-1 ,3-dicarboxylate (21 ). A suspen¬

sion of Cu(OAc)2 (4.50 g, 24.8 mmol) in water (9.2 mL) was added to a solution of (f?)-piperazine-2-carboxylic acid dihydrochloride (17a) (10.0 g, 49.2 mmol) in aqueous NaOH (6 M, 25.5 mL, 153 mmol) and stirred for 40 min at rt. Acetone (85 mL) was then added and the mixture was cooled to 0 °C, followed by drop-wise addition of CbzCI (9.20 mL, 64.4 mmol). After stirring at rt for another 1 h 40 min, EDTA (18.4 g, 49.2 mmol), K2CO3 (6.80 g, 64.0 mmol), and water (40 mL) were added. After 20 min, B0C2O (14.0 g, 64.1 mmol) in acetone (40 mL) was added and the mixture was stirred for 5 days at rt. Additional K2CO3 (3.40 g, 24.6 mmol) and Boc₂0 (1.10 g, 4.90 mmol) were added and the reaction mixture was stirred for 2 days. The acetone was removed in vacuo and the mixture was acidified to pH 5-6 using cone. HCI (aq) and then ex- tracted with EtOAc (3 x 100 mL). The combined organic layers were washed with brine (100 mL), dried over Na2S0₄, and concentrated in vacuo to give a yellow oil. AcCI (16.5 mL, 231 mmol) was added drop-wise to MeOH (75 mL) at 0 °C and the solution was added to the yellow oil and the mixture was stirred for 3 h 20 min at reflux and then concentrated in vacuo. EtOAc (200 mL) and sat. NaHCCb (aq) (100 mL) were added, the layers separated, and the aqueous phase was extracted with EtOAc (2 x 100 mL). The combined organic layers were dried over Na2S0₄, concentrated in vacuo, and purified by flash column chromatography on silica gel (100% EtOAc to EtOAc:MeOH (19:1 ), Rf = 0.2 in 100% EtOAc) to give the title compound as a yellow oil (5.60 g, 41 %). ¹H NMR (400 MHz, DMSO-d₆) δ 7.45 - 7.25 (m, 5H), 5.36 - 4.85 (m, 2H), 3.80 (br s, 1 H), 3.63 (s, 3H), 3.58 - 3.38 (m, 2H), 3.20 - 3.02 (m, 1 H), 2.95 - 2.81 (m, 1 H), 2.77 (br s, 1 H), 2.57 (t, J = 8.9 Hz, 1 H), 2.54 - 2.46 (m, 1 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 171 .5, 154.4, 136.9, 128.4, 127.9, 127.5, 66.3, 56.0, 51 .7, 45.6, 43.8, 43.2; IR (neat) cm^-1: 3340, 2952, 1737, 1693, 1426, 1205, 1 153, 1 1 17, 754, 697.

HRMS (ESI) calcd for C14H19N2O4 [M + H]⁺ 279.1339, found 279.1340;

1.0, CHCI₃).

General procedure II: Synthesis of bis-protected piperazine-2-carboxylic acid

(R)-1 -(((9H-Fluoren-9-yl)methoxy)carbonyl)-4-(ieri-butoxycarbonyl)pi- perazine-2-carboxylic acid (23a). To a solution of (R )-piperazine-2-

boxylic acid 17a (2.96 g, 22.7 mmol) in 2 M NaOH (aq) (12 mL) a suspension of Cu(OAc)₂ (2.06 g, 1 1 .4 mmol) in H₂0 (9 mL) was added and the mixture was stirred for 10 minutes. Then Boc₂0 (6.44 g, 29.5 mmol) dissolved in acetone (33 mL) was added followed by stirring at rt for 1 .5 h. Then the mixture was filtered and the filter cake was rinsed with cold acetone:H₂0 (2:1 ). The solids were dried in vacuo to give the copper complex 22a as a light-blue solid (5.54 g, 93%). The copper complex 22a (5.46 g, 10.5 mmol) was suspended in H₂0 (50 mL), followed by the addition of EDTA (3.89 g, 10.5 mmol) and K₂C03 (3.76 g, 27.2 mmol) and stirred at rt for 10 minutes. Then FmocCI (5.41 g, 20.9 mmol) dissolved in dioxane (50 mL) was added and the reaction mixture was stirred for 30 min at rt. The mixture was then concentrated in vacuo to remove dioxane and the aqueous phase was extracted with EtOAc (10 mL). The mixture was acidified (pH < 4) with cone. HCI (aq) and extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with brine, dried over MgSCU and concentrated in vacuo to give the title compound as a white amorphous solid (8.96 g, 95%). ¹ H NMR (400 MHz, CDCI₃) δ 7.79 - 7.70 (m, 2H), 7.57 (dd, J = 6.9, 4.6 Hz, 1 H), 7.51 (t, J = 7.8 Hz, 1 H), 7.43 - 7.35 (m, 2H), 7.35 - 7.26 (m, 2H), 4.88 - 4.35 (m, 4H), 4.34 - 4.16 (m, 1 H), 4.15 - 3.78 (m, 2H), 3.41 - 2.97 (m, 2H), 2.82 (s, 1 H), 1 .44 (s, 9H); ¹³C NMR (100 MHz, CDCI₃) δ 156.5, 155.7, 154.5, 143.8, 141 .5, 127.9, 127.3, 125.1 , 124.9, 120.2, 81 .0, 68.2, 54.0, 47.3, 44.4, 41 .4, 40.9, 28.4; IR (neat) cm ¹ : 3012, 2971 , 2940, 1697, 1637; [a]¾³= + 8.5 (c 1 .0, CHCI₃). (S)-1-(((9H-Fluoren-9-yl)methoxy)carbonyl)-4-(ieri-butoxycarbonyl)pi- perazine-2-carboxylic acid (23b).^v Following general procedure II, us-

ing (S)-piperazine-2-carboxylic acid dihydrochloride 17b (4.95 g, 24.4 mmol), 2 M NaOH (aq) (49 mL), Cu(OAc)₂ (2.22 g, 12.2 mmol), Boc₂0 (6.92 g, 31 .7 mmol), EDTA (4.53 g, 12.2 mmol), K₂C0₃ (4.38 g, 31 .7 mmol) and FmocCI (6.31 g, 24.4 mmol), the title compound was isolated as a white solid via extraction (7.72 g, 70%). ¹ H NMR (400 MHz, CDCI₃) δ 7.72 - 7.63 (m, 2H), 7.50 (dd, J = 7.0, 4.8 Hz, 1 H), 7.43 (t, J = 7.8 Hz, 1 H), 7.36 - 7.27 (m, 2H), 7.27 - 7.19 (m, 1 H), 4.79 - 4.31 (m, 4H), 4.25 - 4.10 (m, 1 H), 4.01 - 3.70 (m, 2H), 3.33 - 3.08 (m, 1 H), 3.07 - 2.89 (m, 1 H), 2.86 - 2.67 (m, 1 H), 1 .37 (s, 9H); ¹³C NMR (100 MHz, CDCI₃) δ 156.5, 155.7, 154.5, 143.8, 141 .5, 127.9, 127.3, 125.1 , 124.9, 120.2, 81 .0, 68.2, 54.0, 47.3, 44.4, 41 .4, 40.9, 28.4; m.p.: 179 - 180 ^°C; IR (neat) cm ¹ : 3020, 2966, 2915, 1757, 1681 , 1662; MS (ESI) calcd for C₂₅H₂₉N₂0₆ [M + H]⁺ 543.20, found 453.37; - 9.2 (c 1 .0, CHCI₃). All spectro-

scopic data were consistent with those in the literature. Scheme 5. Synthesis of aldehyde building blocks

General procedure III: Alkylation of thiols with bro oethanof 2-(Thiophen-2-ylthio)ethanol (25).^vii Thiophene-2-thiol (24) (5.00 g, 43.3

mmol) and 2-bromoethanol (5.81 g, 46.8 mmol) were dissolved in 4 M NaOH (aq) (10 mL) and stirred at reflux. After 2 h the reaction was allowed to cooled to rt and CH₂CI₂ (75 mL) was added, the layers were separated and the aqueous phase was extracted with CH2CI2 (2 x 75 mL). The combined organic layers were dried over MgSC , concentrated in vacuo and purified by flash column chromatography on silica gel (EtOAc:heptane (1 :4), R_f = 0.1 ) to give the title compound as a red/brown oil (5.89 g, 85%). ¹ H NMR (400 MHz, CDCI₃) δ 7.36 (dd, J = 5.4, 1 .1 Hz, 1 H), 7.16 (dd, J = 3.5, 1 .1 Hz, 1 H), 6.98 (dd, J = 5.4, 3.5 Hz, 1 H), 3.74 (t, J = 5.9 Hz, 2H), 2.96 (t, J = 5.9 Hz, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 134.7, 132.8, 130.0, 127.8, 60.2, 41 .9; IR (neat) crrr 1 : 3332, 3100, 2922, 1402, 1337, 1283, 1216, 1043, 845, 695, 495, 471 ; HRMS (ESI) calcd for C₆H₉OS₂ [M + H]⁺ 161 .0089, found 161 .0090. All spectroscopic data were consistent with those in the literature. 2-(Thiophen-2-ylthio)acetaldehyde (26).^viii Following general procedure I,

using alcohol 25 (5.39 g, 33.6 mmol), DMP (15.7 g, 3.70 mmol), 2 M NaOH (aq) (400 mL), dried over Na₂S04, and concentrated in vacuo. The title compound was isolated as a red/brown oil (2.82 g, 53%) after purification by flash column chromatog- raphy on silica gel (EtOAc:heptane (1 :9), fl_f = 0.2). ¹ H NMR (400 MHz, DMSO-d₆) δ 9.51 (t, J = 2.8 Hz, 1 H), 7.64 (dd, J = 5.3, 1 .2 Hz, 1 H), 7.22 (dd, J = 3.6, 1 .3 Hz, 1 H), 7.04 (dd, J = 5.3, 3.6 Hz, 1 H), 3.71 (d, J = 2.8 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) 5 196.1 , 134.7, 131 .2, 131 .0, 128.2, 47.3. All spectroscopic data were consistent with those in the literature.

3-(2-Fluorophenyl)propanal (28). AcCI (2.42 mL, 33.9 mmol) was dis-

solved in ethanol (7.4 mL), whereupon 3-(2-fluorophenyl)propanoic acid (0.500 g, 2.97 mmol) was added and the reaction was stirred at reflux. After 30 min the solvent was removed in vacuo and the remaining crude was taken up in sat. Na₂C0₃ (aq) (15 mL) and CH₂CI₂ (30 mL). The layers were separated and the aqueous phase was extracted with CH₂CI₂ (2 x 30 mL) and the combined organic layers were dried over Na₂S04 and concentrated in vacuo. The crude ester was dissolved in dry toluene (30 mL) and cooled to -78 °C followed by the dropwise addition of 1 M DIBAL-H in toluene (3.57 mL, 3.57 mmol). After stirring for 3 h at -78 °C, 1 M HCI (aq) (20 mL, 20 mmol) was added and the reaction mixture was removed from the cooling bath and allowed to heat to room temperature until clear separation of the two phases (1 h). Then the phases were separated and the aqueous phase was extracted with CH₂CI₂ (2 x 40 mL), whereupon the organic layers were combined, dried over MgSCU, concentrated in vacuo, and purified by flash column chromatography on silica gel (EtOAc:heptane (1 :9), R\ = 0.25) to give the title compound as a white amorphous solid (0.375 g, 83%). ¹ H NMR (400 MHz, CDCI₃) δ 9.82 (s, 1 H), 7.24 - 7.14 (m, 2H), 7.04 (m, 2H), 2.98 (t, J = 7.5 Hz, 2H), 2.78 (t, J = 7.5 Hz, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 201 .4, 161 .2 (d, JCF = 245.0 Hz), 130.8 (d, JCF = 4.8 Hz), 128.3 (d, JCF = 8.1 Hz), 127.3 (d, JCF = 15.6 Hz), 124.3 (d, JCF = 3.6 Hz), 1 15.5 (d, JCF = 21 .9 Hz), 44.0 (d, JCF = 1 .3 Hz), 22.0 (d, JCF = 2.8 Hz).

(E)-3-(2,5-Difluorophenyl)acrylaldehyde (30). AcCI (10.8 mL, 151

mmol) was dissolved in ethanol (34 mL), whereupon (E)-3-(2,5-difluoro- phenyl)acrylic acid (2.50 g, 13.6 mmol) was added and the reaction was stirred at reflux. After 30 min the solvent was removed in vacuo and the remaining crude was taken up in sat. Na2C0₃ (aq) (25 mL) and CH₂CI₂ (50 mL). The layers were separated and the aqueous phase was extracted with CH₂CI₂ (4 x 50 mL) and the combined organic layers were dried over Na₂S04 and concentrated in vacuo. The crude ester was dissolved in dry toluene (150 mL) and cooled to -78 °C followed by the dropwise addition of 1 M DIBAL-H in toluene (16.3 mL, 16.3 mmol). After stirring for 2 h at -78 °C a mixture of the ester, aldehyde and alcohol was obtained and 1 M DIBAL-H in toluene (8.15 mL, 8.15 mmol) was added to reduce the ester and the aldehyde to the alcohol. After stirring for another 1 h at -78 °C 1 M HCI (aq) (40 mL, 40 mmol) was added and the reaction mixture was removed from the cooling bath and allowed to heat to room temperature until clear separation of the two phases (1 h). Then the phases were sepa- rated and the aqueous phase was extracted with CH₂CI₂ (3 x 100 mL), whereupon the organic layers were combined, dried over Na₂S04 and concentrated in vacuo. Following general procedure I, the remaining crude was oxidized by DMP (6.33 g, 14.9 mmol) to give the title compound as a white amorphous solid (1 .03 g, 45%) after purification by flash column chromatography on silica gel (EtOAc:heptane (1 :9), R_f = 0.2). ¹ H NMR (400 MHz, CDCI₃) δ 9.73 (d, J = 7.6 Hz, 1 H), 7.60 (dd, J = 16.2, 1 .0 Hz, 1 H), 7.34 - 7.22 (m, 1 H), 7.17 - 7.07 (m, 2H), 6.74 (dd, J = 16.2, 7.6 Hz, 1 H); ¹³C NMR (100 MHz, CDCI₃) δ 193.5, 158.9 (dd, JCF = 244.1 , 2.4 Hz), 157.3 (dd, JCF = 250.8, 2.4 Hz), 143.3 (dd, JCF = 3.2, 2.4 Hz), 131 .4 (d, JCF = 5.0 Hz), 123.5 (dd, JCF = 14.1 , 8.0 Hz), 1 19.5 (dd, JCF = 24.4, 9.0 Hz), 1 17.7 (dd, JCF = 24.9, 8.5 Hz), 1 14.6 (dd, JCF = 24.6, 2.9 Hz).

2-(phenylthio)ethanol (35). ^ix Following general procedure III, thiophenol

(446 μί, 4.54 mmol) was alkylated using 2-bromoethanol (0.386 μί, 5.45 mmol) and 4 M NaOH (aq) (1 .14 mL) to give the title compound as a colorless oil (436 mg, 62%) after purification by flash column chromatography on silica gel (EtOAc:hep- tane (1 :4), R_f = 0.2). ¹ H NMR (400 MHz, CDCI₃) δ 7.42 - 7.36 (m, 2H), 7.33 - 7.27 (m, 2H), 7.24 - 7.19 (m, 1 H), 3.74 (t, J = 6.0 Hz, 2H), 3.1 1 (t, J = 6.0 Hz, 2H), 2.17 (s, 1 H); ¹³C NMR (100 MHz, CDCI₃) δ 134.9, 130.3, 129.2, 126.8, 60.4, 37.4. All spectroscopic data were consistent with those in the literature.

2-((2-Fluorophenyl)thio)ethanol (36). Following general procedure III, 2-fluorobenzenethiol (0.517 g, 4.03 mmol) was alkylated using 2-bromo¬

ethanol (0.504 g, 4.03 mmol) and 4 M NaOH (aq) (1 mL) to give the title compound as a colorless oil (0.358 g, 51 %) after purification by flash column chromatography on sil- ica gel (EtOAc:heptane (1 :4), R_f = 0.2).¹ H NMR (400 MHz, CDCI₃) δ 7.45 (td, J = 7.8, 1 .7 Hz, 1 H), 7.30 - 7.22 (m, 1 H), 7.15 - 7.03 (m, 2H), 3.71 (t, J = 5.9 Hz, 2H), 3.08 (t, J = 5.9 Hz, 2H), 2.02 (br s, 1 H); ¹³C NMR (100 MHz, CDCI₃) δ 162.3 (d, J_CF = 245.6 Hz), 133.8 (d, JCF = 1 .4 Hz), 129.5 (d, JCF = 7.9 Hz), 124.8 (d, JCF = 3.8 Hz), 121 .4 (d, JCF = 17.8 Hz), 1 16.1 (d, JCF = 22.8 Hz), 60.5, 37.4 (d, JCF = 2.2 Hz); IR (neat) cm^-1 : 3361 , 2929, 2879, 1571 , 1473, 1446, 1260, 1220, 1071 , 821 , 752; HRMS (ESI) calcd for C₈H₈FS⁺ [M - OH]⁺ 155.0325, found 155.0319.

2-((2,6-Difluorophenyl)thio)ethanol (37). Following general procedure

III, 2,5-difluorobenzenethiol (95 mg, 0.65 mmol) was alkylated using 2- bromoethanol (81 mg, 0.65 mmol) and 4 M NaOH (aq) (0.16 mL) to give the title com- pound as a colorless oil (56 mg, 45%) after purification by flash column chromatography on silica gel (EtOAc:heptane (1 :4), R_f = 0.2). ¹ H NMR (400 MHz, CDCI₃) δ 7.12 (ddd, J = 8.6, 5.8, 3.1 Hz, 1 H), 7.02 (td, J = 8.9, 4.6 Hz, 1 H), 6.95 - 6.86 (m, 1 H), 3.75 (t, J = 5.9 Hz, 2H), 3.10 (t, J = 5.9 Hz, 2H), 2.05 (s, 1 H); ¹³C NMR (100 MHz, CDCI₃) δ 158.6 (dd, JCF = 244.9, 2.6 Hz), 157.8 (dd, JCF = 241 .0, 2.7 Hz), 123.9 (dd, JCF = 20.4, 8.3 Hz), 1 18.7 (dd, JCF = 25.1 , 2.1 Hz), 1 16.0 (ddd, J = 147.3, 24.7, 8.5 Hz), 60.5, 36.7 (d, J = 2.3 Hz); IR (neat) cm^-1: 3356, 2932, 2881 , 1614, 1583, 1480, 1245, 1 186, 1059, 757; HRMS (ESI) calcd for C₈H₇F₂S [M - OH] ⁺ 173.0231 , found 173.0233.

2-((2-(Trifluoromethyl)phenyl)thio)ethanol (38). Following general pro-

cedure III, 2-(trifluoromethyl)benzenethiol (0.500 g, 2.81 mmol) was alkyl- ated using 2-bromoethanol (239 μL, 3.37 mmol) and 4 M NaOH (aq) (0.70 mL) to give the title compound as a colorless oil (450 mg, 72%) after purification by flash column chromatography on silica gel (EtOAc:heptane (3:7), R_f = 0.2). ¹H NMR (400 MHz, CDCI₃) δ 7.66 (d, J = 7.9 Hz, 1 H), 7.58 (d, J = 7.9 Hz, 1 H), 7.48 (t, J = 7.6 Hz, 1 H), 7.32 (t, J = 7.6 Hz, 1 H), 3.75 (t, J = 5.9 Hz, 2H), 3.17 (t, J = 5.9 Hz, 2H), 2.07 (s, 1 H); ¹³C NMR (1000 MHz, CDC1₃) δ 134.8, 132.4, 132.3, 131 .0 (q, JCF = 29.9 Hz), 127.1 (q, J_CF = 5.6 Hz), 123.9 (q, JCF = 274 Hz), 126.7, 60.4, 38.2; IR (neat) cm^-1 : 3346, 2932, 2879, 1593, 1440, 1310, 1257, 1 169, 1 1 1 1 , 1032, 759; HRMS (ESI) calcd for C9H9F3S [M - OH]⁺ 205.0293, found 205.0291 .

2-Phenoxyacetaldehyde (48). The aldehyde was freshly prepared from 2- phenoxyethanol (45) using general procedure I.

2-(2-Fluorophenoxy)ethan-1 -ol (46). 2-Fluorophenol (43) (2.00 g, 17.8 mmol), ethylenecarbonate (3.14 g, 35.7 mmol) and K2CO3 (3.95 g, 28.6

mmol) were heated to reflux in toluene (90 mL). After stirring for 26 h, the reaction mixture was concentrated in vacuo and purified by flash column chromatography on silica gel (EtOAc:heptane (1 :1 ), R_f = 0.2) to give the title compound as a colorless oil (1.93 g, 69%). ¹H NMR (400 MHz, CDCI₃) δ 7.12 - 7.02 (m, 2H), 6.98 (td, J = 8.4, 1.7 Hz, 1 H), 6.95 - 6.88 (m, 1 H), 4.18 - 4.08 (m, 2H), 4.15 - 4.1 1 (m, 2H), 3.97 (d, J = 4.1 Hz, 2H), 2.54 (s, 1 H); ¹³C NMR (100 MHz, CDCI₃) δ 153.0 (d, J_CF = 245.3 Hz), 146.8 (d, J_CF = 10.6 Hz), 124.5 (d, J_CF = 3.9 Hz), 121 .8 (d, J_CF = 6.9 Hz), 1 16.4 (d, J_CF = 18.3 Hz), 1 15.5 (d, JCF = 1 .7 Hz), 71.0, 61.4.

2-(2-Fluorophenoxy)acetaldehyde (49). The aldehyde was freshly pre-

pared from 2-phenoxyethanol (46) using general procedure I.

2-(2,5-Difluorophenoxy)acetaldehyde (50). 2,5-Difluorophenol (44)

(3.00 g, 23.1 mmol), ethylenecarbonate (4.01 g, 46.1 mmol) and K2CO3 (5.1 g, 36.9 mmol) were heated to reflux in toluene (1 15 mL). After stirring for 26 h, the reaction mixture was concentrated in vacuo and purified by flash column chromatography on silica gel (EtOAc:heptane (1 :1 ), R_f = 0:2) to the intermediate alcohol 47 (2.59 g, 65%). Following general procedure I, the alcohol was oxidized using DMP (6.9 g, 14.9 mmol) to give the title compound as a white amorphous solid (2.30 g, 90%) after purification by flash column chromatography on silica gel (EtOAc:heptane (3:7), R_f = 0.2). The compound was not characterized due to rapid decomposition.

Scheme 6. Synthesis of compounds 53a, 53b and 54

(R)-Methyl 1 -(3-(3-fluoro-6-methoxyquinolin-4-yl)propyl)-4-(2- (thiophen-2-ylthio)ethyl)piperazine-2-carboxylate (52a). A 50 mL flask was charged with a magnetic stirring bar and MeOH and

cooled to 0 °C. Then AcCI (215 μL) was added and the mixture was stirred for 5 min. The bis-protected piperazine 23a (500 mg, 1 .10 mmol) was added and the mixture was stirred for 2 h at reflux, and then concentrated in vacuo. CH2CI2 was added to the mixture and then removed in vacuo (repeated 3 times). The HCI salt was then dissolved in DMF (5.5 mL) and added aldehyde 14 (0.210 mg, 1.33 mmol) followed by NaBH(OAc)3 (formed in situ by addition of AcOH to NaBH₄ (50 mg, 1.33 mmol)) and the mixture was stirred for 1 h at rt. The reaction mixture was then concentrated in vacuo and the residue was purified by flash column chromatography on silica gel (EtOAc:heptane (2:3), R_f = 0.2) to give the intermediate 51 a (165 mg, 29%). The intermediate product 51a (157 mg, 0.309 mmol) was dissolved in DMF and added piperidine (61 μL, 0.618 mmol) and the mixture was stirred for 20 min at rt. The solvent was removed in vacuo and the residue was purified by flash column chromatography (gradient from 0-10% MeOH in EtOAc, R_f = 0.1 in EtOAc:MeOH (19:1 )) to give the free amine (79 mg, 89%). The amine (79 mg, 0.27 mmol) was then dissolved in CH2CI2 (3 mL) and added aldehyde 14 (64 mg, 0.27 mmol) followed by NaBH(OAc)₃ (formed in situ by addition of AcOH to NaBH₄ (13 mg, 0.33 mmol)) and the mixture was stirred for 20 min at rt. The solvent was removed and the residue was purified by flash column chromatography on silica gel (EtOAc:heptane (1 1 :9), R_f = 0.2) to give the title compound as a colorless oil (1 10 mg, 79%). ¹H NMR (400 MHz, CDCI₃) δ 8.57 (s, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.33 - 7.28 (m, 2H), 7.17 (d, J = 2.6 Hz, 1 H), 7.10 (dd, J = 3.5, 0.9 Hz, 1 H), 6.95 (dd, J = 5.3, 3.6 Hz, 1 H), 3.94 (s, 3H), 3.66 (s, 3H), 3.34 - 3.26 (m, 1 H), 3.13 - 2.95 (m, 3H), 2.91 - 2.76 (m, 3H), 2.75 - 2.65 (m, 1 H), 2.65 - 2.36 (m, 7H), 1 .85 (q, J = 7.4 Hz, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 172.4, 158.6, 155.0 (d, JCF = 251 .4 Hz), 141.6 (d, JCF = 2.3 Hz), 138.3 (d, JCF = 29.4 Hz), 134.4, 133.8, 131 .8, 129.9 (d, JCF = 13.0 Hz), 129.4, 129.3 (d, JCF = 3.9 Hz), 127.6, 120. 2 (d, JCF = 2.5 Hz), 102.0 (d, JCF = 5.5 Hz), 63.4, 57.4, 55.7, 55.4, 55.2, 53.0, 51 .7, 48.6, 35.8, 26.6, 22.0 (d, JCF = 3.1 Hz); IR (neat) cm^-1 : 2947, 2813, 1739, 1620, 1509, 1466, 1229, 1031 , 831 ; HRMS (ESI) calcd for C25H31 FN3O3S2 [M + H]⁺ 504.1785, found 504.1786

+ 16.7 (c 1 .0, CHCI₃). (S)-Methyl 1 -(3-(3-fluoro-6-methoxyquinolin-4-yl)propyl)-4-(2- (thiophen-2-ylthio)ethyl)piperazine-2-carboxylate (52b). The compound was prepared according to 52a starting from 23b (500 mg, 1.10 mmol) to give the title compound as a colorless oil (134

mg, 24% over the 3 steps). ¹ H NMR (400 MHz, CDCI₃) δ 8.56 (s,

1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.35 - 7.27 (m, 2H), 7.17 (d, J = 2.5 Hz, 1 H), 7.09 (d, J = 3.4 Hz, 1 H), 6.95 (dd, J = 5.2, 3.6 Hz, 1 H), 3.94 (s, 3H), 3.66 (s, 3H), 3.34 - 3.27 (m, 1 H), 3.12 - 2.95 (m, 3H), 2.90 - 2.77 (m, 3H), 2.75 - 2.66 (m, 1 H), 2.64 - 2.36 (m, 7H), 1.86 (q, J = 7.4 Hz, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 172.3, 158.6, 155.0 (d, J_CF = 251 .4 Hz), 141.6 (d, JCF = 2.3 Hz), 138.3 (d, J = 29.4 Hz), 134.4, 133.8, 131 .8, 129.9 (d, JCF = 13.0 Hz), 129.4, 129.3 (d, JCF = 3.9 Hz), 127.6, 120.2 (d, JCF = 2.6 Hz), 102.0 (d, JCF = 5.4 Hz), 63.4, 57.4, 55.7, 55.4, 55.2, 52.9, 51 .7, 48.7, 35.8, 26.6, 22.0 (d, JCF = 3.0 Hz); IR (neat) cm^-1 : 3072, 2948, 2814, 1740, 1620, 1509, 1468, 1229, 1031 , 831 , 705; HRMS (ESI) calcd for C25H31FN3O3S2 [M + H]⁺ 504.1785, found 504.1784;

General procedure IV: Hydrolysis of methyl ester

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(2-(thio- phen-2-ylthio)ethyl)piperazine-2-carboxylic acid (53a). Methyl ester 52a (0.040 g, 0.080 mmol) was charged in a glass vial. 0.9 M LiOH (aq) (260 μL, 0.234 mmol) and THF (520 μL) were then

added. The vial was sealed with a lid, and placed on a heated shaker (65 °C) for 1 -2 days, whereupon the reaction was quenched with 0.9 M AcOH (aq) (260 μL, 0.234 mmol). The reaction mixture was then concentrated under a flow of pressurized air, and purified by preparative HPLC to give the title compound as an off- white solid (25 mg, 51 %). ¹H NMR (500 MHz, DMSO-d₆) δ 8.67 (s, 1 H), 7.94 (d, J = 9.1 Hz, 1 H), 7.60 (d, J = 5.3 Hz, 1 H), 7.38 (dd, J = 9.1 , 2.6 Hz, 1 H), 7.34 (d, J = 2.6 Hz, 1 H), 7.17 (d, J = 3.5 Hz, 1 H), 7.04 (dd, J = 5.3, 3.6 Hz, 1 H), 3.94 (s, 3H), 3.20 - 3.15 (m, 1 H), 3.13 - 2.96 (m, 3H), 2.93 - 2.81 (m, 3H), 2.66 - 2.58 (m, 2H), 2.54 - 2.30 (m, 6H), 1.86 - 1.72 (m, 2H). ¹³C NMR (100 MHz, CDCI₃) δ 170.1 , 158.8, 155.0 (d, JCF = 251 .4 Hz), 141.6 (d, J_CF = 2.3 Hz), 138.2 (d, J_CF = 29.4 Hz), 134.8, 132.8, 131 .8, 130.2, 129.6 (d, JCF = 12.8 Hz), 129.3 (d, J_CF = 3.8 Hz), 127.9, 120.4 (d, J_CF = 2.6 Hz), 102.0 (d, JCF = 5.3 Hz), 60.8, 56.1 , 55.9, 54.9, 54.4, 51.4, 47.9, 34.9, 25.8, 21 .9 (d, J_CF = 3.1 Hz); IR (neat) cm^-1: 3477 (br), 2998, 2953, 2828, 1722, 1620, 1509, 1468, 1230, 1028, 831 ; HRMS (ESI) calcd for C27H31FN3O4 [M + H]⁺ 480.2293, found 480.2302;

13.5 (c 1 .0, CHCI₃).

(S)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(2-(thio- phen-2-yl-thio)ethyl)piperazine-2-carboxylic acid (53b). Following general procedure IV, methyl ester 52b (0.045 g, 0.089 mmol), was hydrolyzed with aqueous LiOH to give the title com¬

pound as a colorless oil (28 mg, 57%) after purification by preparative HPLC. ¹H NMR (300 MHz, DMSO-d₆) δ 8.67 (s, 1 H), 7.95 (d, J = 9.0 Hz, 1 H), 7.61 (dd, J = 5.3, 1 .2 Hz, 1 H), 7.42 - 7.32 (m, 2H), 7.18 (dd, J = 3.5, 1.3 Hz, 1 H), 7.04 (dd, J = 5.3, 3.5 Hz, 1 H), 3.95 (s, 3H), 3.23 - 2.97 (m, 4H), 2.95 - 2.81 (m, 3H), 2.68 - 2.56 (m, 2H), 2.50 - 2.29 (m, 6H), 1.87 - 1.71 (s, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 170.6, 158.8, 155 (d, JCF = 251 .5, Hz), 141 .5 (d, JCF = 2.2 Hz), 138.1 (d, JCF = 29.4 Hz), 134.6, 133.0, 131 .7, 130.0, 129.4 (d, JCF = 12.9 Hz), 129.2 (d, JCF = 3.9 Hz), 127.8, 120.4 (d, JCF = 2.4 Hz), 101 .7 (d, JCF = 5.4 Hz), 61.8, 56.2, 55.8, 54.9, 54.4, 51.3, 48.2, 34.0, 25.5, 21.8 (d, J_CF = 3.0 Hz).

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(2-(thio- phen-2-ylthio)-ethyl)piperazine-2-carboxamide (54). Carboxylic acid 53a (10 mg, 0.02 mmol) was dissolved in DMF (200 μL) where after DIPEA (1 1 μL, 0.06 mmol) and HATU (9 mg, 0.02

mmol) were added and the mixture was stirred for 5 min at rt. Then N H4CI (2 mg, 0.03 mmol) was added and the mixture was stirred for 1 h at rt. The mixture was then purified by preparative HPLC to give the title compound as an orange oil (7 mg, 70%). ¹ H NMR (400 MHz, CDCI₃) δ 8.81 (d, J_HF = 2.1 Hz, 1 H), 8.23 (d, J = 9:3 Hz, 1 H), 7.49 (dd, J =9.3, 2.5 Hz, 1 H), 7.41 (dd, J =5.4, 1.2 Hz, 1 H), 7.28 (d, J = 2:5 Hz, 1 H), 7.17 (dd, J = 3.6, 1 .2 Hz, 1 H), 7.00 (dd, J = 5.4, 3.6 Hz, 2H), 6.85 (br s, 2H), 4.00 (s, 3H), 3.86 (d, J = 6.0 Hz, 1 H), 3.48 - 3.29 (s, 1 H), 3.32 - 2.97 (m, 1 1 H), 2.97 - 2.87 (m, 1 H), 2.84 - 2.75 (m, 1 H), 2.1 1 - 2.01 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 160.5, 154.8 (d, JCF = 248.6 Hz), 136.1 , 135.6, 133.9 (d, JCF = 34.5 Hz), 131 .1 , 130.8 (d, JCF = 8.8 Hz), 130.1 (d, JCF = 4.1 Hz), 128.3, 127.1 , 124.1 , 1 17.4, 1 14.5, 102.0, 56.2, 55.2 54.1 , 53.2, 50.8, 47.8, 41.7, 32.8, 25.0, 22.4.

Scheme 7. Synthesis of compounds 58a-s, 59, 62a-e, 63a-f and 64a-e

1 -Benzyl 3-methyl (R)-4-(3-(3-fluoro-6-methoxyquinolin-4-yl)pro- pyl)piperazin-e-1 ,3-dicarboxylate (55). A solution of piperazine 21 (4.50 g, 16.2 mmol) in CH₂CI₂ (100 mL) was added to aldehyde 14 fol

lowed by the addition of AcOH (93.0 μL, 1 .62 mmol) and NaBH(OAc)₃ (5.14 g, 24.3 mmol). The reaction mixture was stirred for 1 h 50 min at rt before quenching with sat. aqueous NaHCO₃ (120 mL). The aqueous phase was extracted with CH2CI2 (3 x 200 mL), the combined organic layers were dried over Na2S0₄, con- centrated in vacuo, and purified by flash chromatography on silica gel (EtOAc:heptane (9:1 1 ), R_f = 0.20) to give the title compound as a yellow oil (6.80 g, 85%). ¹ H NMR (400 MHz, DMSO-d₆) δ 8.67 (s, 1 H), 7.95 (d, J = 9.1 Hz, 1 H), 7.43 - 7.23 (m, 7H), 5.06 (s, 2H), 3.94 (s, 3H), 3.90 - 3.76 (m, 1 H), 3.76 - 3.57 (m, 1 H), 3.54 - 3.45 (m, 3H), 3.41 - 3.37 (m, 1 H), 3.28 (dd, J = 13.2, 3.9 Hz, 1 H), 3.07 (t, J = 7.4 Hz, 3H), 2.94 (t, J = 9.2 Hz, 1 H), 2.73 - 2.65 (m, 1 H), 2.62 - 2.52 (m, 1 H), 2.48 - 2.38 (m, 1 H), 1.88 - 1.68 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 171.4, 158.4, 154.7 (d, JCF = 250.1 Hz), 154.3, 141 .2 (d, JCF = 2.1 Hz), 138.1 (d, JCF = 29.0 Hz), 137.0, 131 .5, 130.1 (d, JCF = 12.9 Hz), 129.0 (d, JCF = 3.9 Hz), 128.6, 128.0, 127.7, 120.6 (d, J_CF = 2.3 Hz), 102.6 (d, J_CF = 5.4 Hz), 66.5, 60.5, 55.8, 54.4, 51 .4, 46.4, 45.8, 43.5, 26.5, 21 .4 (d, J_CF = 2.6 Hz); IR (neat) cm-¹ : 3032, 3001 , 2949, 2860, 1737, 1699, 1620, 1508, 1465, 1427, 1281 , 1226, 1 1 18, 1027.

Methyl (R)-1 -(3-(3-fluoro-6-methoxyquinolin-4-yl)propyl) piperazine- 2-carbox-ylate (56). Compound 55 (6.80 g, 13.7 mmol) was dissolved in MeOH (80 mL) and placed under an argon atmosphere. Pd/C (1 .46 g,

1.37 mmol) and HC0₂NH₄ (3.46 g, 54.9 mmol) were added and the reaction mixture was stirred for 21 h at rt. The reaction mixture was filtered through celite, washed with MeOH (170 mL), and concentrated in vacuo. The residue was dissolved in CH2CI2 (100 mL) and sat. NaHC03 (100 mL) was added. The mixture was transferred to a separatory funnel and the layers were separated. The aqueous phase was extracted with CH2CI2 (3 x 200 mL) and the combined organic layers were dried over Na2S0₄ and then concentrated in vacuo and purified by flash column chromatography on silica gel (MeOH:Et₃N:CH₂CI₂ (1 :3:100), R_f = 0.25) to give the title compound as a yellow oil (3.60 g, 73%). ¹H NMR (400 MHz, CDCI₃) δ 8.58 (d, J_HF = 1.0 Hz, 1 H), 7.99 (d, J = 9.2 Hz, 1 H), 7.31 (dd, J = 9.2, 2.7 Hz, 1 H), 7.19 (d, J = 2.7 Hz, 1 H), 3.95 (s, 3H), 3.66 (s, 3H), 3.23 (t, J = 4.5 Hz, 1 H), 3.14 - 2.99 (m, 4H), 2.99 - 2.90 (m, 2H), 2.91 - 2.82 (m, 1 H), 2.76 - 2.64 (m, 1 H), 2.58 - 2.46 (m, 1 H), 2.41 - 2.31 (m, 1 H), 2.10 (br s, 1 H), 1.95 - 1.79 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 172.5, 158.6, 154.9 (d, J_CF = 251 .4 Hz), 141.5 (d, J_CF = 2.3 Hz), 138.2 (d, J_CF = 29.4 Hz), 131 .7, 129.8 (d, J_CF = 13.0 Hz), 129.2 (d, JCF = 3.9 Hz), 120.1 (d, J_CF = 2.6 Hz), 102.0 (d, J_CF = 5.4 Hz), 63.1 , 55.7, 55.6, 51.5, 49.2, 48.5, 45.7, 26.2, 21.9 (d, JCF = 3.1 Hz); IR (neat) cm^-1 : 3297, 2948, 2830, 1736, 1619, 1507, 1467, 1428, 1227, 1 130.

General procedure V: Reductive alkylation of amines

(R)-Methyl 1 -(3-(3-fluoro-6-methoxyquinolin-4-yl)propyl)-4- phenethylpip-erazine-2-carboxylate (57a). In a small vial, NaBH(OAc)₃ (freshly prepared by premixing AcOH (200 μL, 3.50

mmol) and NaBH₄ (8 mg, 0.24 mmol) for 5 min) was added to a solution of amine 56 (50 mg, 0.14 mmol) and 2-phenylacetaldehyde (31 μL, 0.28 mmol) in CH2CI2 (1.5 mL). The reaction mixture was stirred for 1 .5 h at rt, concentrated in vacuo, and purified by flash column chromatography on silica gel (EtOAc:heptane (1 :1 ), f?_f = 0.2) to give the title compound as a colorless oil (62 mg, >95%). ¹H NMR (400 MHz, CDCI₃) δ 8.55 (s, 1 H), 7.96 (d, J = 9.2 Hz, 1 H), 7.28 (dd, J = 9.2, 2.7 Hz, 1 H), 7.23 (d, J = 7.4 Hz, 2H), 7.191 - 7.13 (m, 4H), 3.92 (s, 3H), 3.64 (s, 3H), 3.36 - 3.27 (m, 1 H), 3.14 - 2.94 (m, 3H), 2.85 - 2.38 (m, 1 1 H), 1 .85 (p, J = 7.4 Hz, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 172.5, 158.6, 155.0 (d, JCF = 251 .4 Hz), 141.6 (d, JCF = 2.2 Hz), 140.2, 138.2 (d, JCF = 29.4 Hz), 131.8, 129.9 (d, JCF = 13.0 Hz), 129.3 (d, JCF = 3.9 Hz), 126.1 , 120.2 (d, JCF = 2.6 Hz), 102.0 (d, JCF = 5.4 Hz), 63.7, 60.1 , 55.6, 55.2, 53.2, 51.7, 48.9, 33.4, 26.5. 22.0 (d, JCF = 3.1 Hz); IR (neat) cm^-1 : 3064, 3027, 2949, 281 1 , 1741 , 1621 , 1509, 1467, 1321 , 1230, 1031 , 831 , 700; HRMS (ESI) calcd for

C27H33FN3O3 [M + H]⁺ 466.2500, found 466.2500; [a]¾³= + 13.6 (c 1 .0, CHCI₃).

(R)- Methyl 1 -(3-(3-fluoro-6-methoxyquinolin-4-yl)propyl)-4-

(4-phenyl-butyl)piperazine-2-carboxylate (57c). Following general procedure V, amine 56 (37 mg, 0.10 mmol) was re-

ductively alkylated using 4-phenylbutanal (23 mg, 0.15 mmol),

AcOH (5.9 μL, 0.10 mmol), and NaBH(OAc)₃ (43 mg, 0.20 mmol) in CH2CI2 (1 mL) to give the title compound as a colorless oil (26 mg, 51 %) after purification by flash column chromatography on silica gel (EtOAc:heptane (3.1 ), f?_f = 0.2). ¹H NMR (400 MHz, CDCI₃) δ 8.58 (d, J = 1.0 Hz, 1 H), 7.99 (d, J = 9.2 Hz, 1 H), 7.34 - 7.24 (m, 4H), 7.21 - 7.13 (m, 4H), 3.95 (s, 3H), 3.65 (s, 3H), 3.33 - 3.28 (m, 1 H), 3.14 - 2.96 (m, 3H), 2.88 - 2.76 (m, 1 H), 2.67 - 2.24 (m, 10H), 1.87 (p, J = 7.6 Hz, 2H), 1.68 - 1.54 (m, 2H), 1.54 - 1.43 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 172.6, 158.7, 155.0 (d, JCF = 251.4 Hz), 142.6, 141.7 (d, J_CF = 2.3 Hz), 138.3 (d, J_CF = 29.4 Hz), 131.8, 130.0 (d, J_CF = 13.0 Hz), 129.3 (d, JCF = 3.9 Hz), 128.5, 128.4, 125.8, 120.3 (d, J_CF = 2.6 Hz), 102.0 (d, J_CF = 5.4 Hz), 63.9, 58.1 , 55.7, 55.7, 55.3, 53.3, 51 .7, 49.0, 35.9, 29.3, 26.6, 26.4, 22.1 (d, J_CF = 3.1 Hz); IR (neat) cm-¹ : 2940, 2857, 281 1 , 1742, 1620, 1508, 1468, 1362, 1320, 1230, 1031 , 831 , 700; HRMS (ESI) calcd for C29H37FN3O3 [M + H]⁺ 494.2813 found 494.2814;

(R)-Methyl 1 -(3-(3-fluoro-6-methoxyquinolin-4-yl)propyl)-4- (3-(2-fluoro-phenyl)propyl)piperazine-2-carboxylate (57d). Following general procedure V, amine 56 (37 mg, 0.10 mmol)

was reductively alkylated using aldehyde 28 (23 mg, 0.15 mmol), AcOH (5.9 μL, 0.10 mmol), and NaBH(OAc)₃ (43 mg, 0.20 mmol) in CH₂CI₂ (1 mL) to give the title compound as a colorless oil (29 mg, 56%) after purification by flash column chromatography on silica gel (EtOAc:heptane (3:1 ), R_f = 0.25). ¹H NMR (400 MHz, CDCI₃) δ 8.58 (d, J = 1.0 Hz, 1 H), 7.99 (d, J = 9.2 Hz, 1 H), 7.31 (dd, J = 9.2, 2.7 Hz, 1 H), 7.21 - 7.1 1 (m, 3H), 7.07 - 6.94 (m, 2H), 3.95 (s, 3H), 3.67 (s, 3H), 3.36 - 3.27 (m, 1 H), 3.16 - 2.95 (m, 3H), 2.88 - 2.77 (m, 1 H), 2.76 - 2.26 (m, 10H), 1 .87 (p, J = 7.4 Hz, 2H), 1.76 (p, J = 7.5 Hz, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 172.6, 161 .3 (d, JCF = 244.6 Hz), 158.7, 155.0 (d, JCF = 251 .4 Hz), 141 .6, 138.3 (d, JCF = 29.4 Hz), 131.8, 130.8 (d, JCF = 5.2 Hz), 130.1 (d, JCF = 13.0 Hz), 129.3 (d, JCF = 3.9 Hz), 129.0 (d, JCF = 16.0 Hz), 127.6 (d, JCF = 8.1 Hz), 124.0 (d, JCF = 3.5 Hz), 120.3 (d, JCF = 2.6 Hz), 1 15.3 (d, JCF = 22.2 Hz), 102.0 (d, JCF = 5.5 Hz), 63.6, 57.4, 55.7, 55.5, 55.3, 53.3, 51.7, 48.9, 27.2, 26.8 (d, JCF = 2.4 Hz), 26.6, 22.1 (d, JCF = 3.0 Hz); IR (neat) cm^-1: 2949, 2813, 1741 , 1620, 1508, 1467, 1457, 1229, 1032, 831 , 757; HRMS (ESI) calcd for C28H34F2N3O3 [M + H]⁺ 498.2563 found 498.2555; = + 12.8 (c 1.0, CHCI₃).

(R)-Methyl 4-cinnamyl-1 -(3-(3-fluoro-6-methoxyquinolin-4- yl)propyl)-piperazine-2-carboxylate (57e). Following general procedure V, amine 56 (50 mg, 0.14 mmol) was reductively al¬

kylated using cinnamaldehyde (23 μί, 0.18 mmol) and

NaBH(OAc)₃ (freshly prepared by premixing AcOH (200 μL, 3.50 mmol) and NaBH₄ (8 mg, 0.24 mmol) for 5 min in CH2CI2 (1 .5 mL)) to give the title compound as a colorless oil (66 mg, >95%) after purification by flash column chromatography on silica gel (EtOAc:heptane (2:3), R_f = 0.2). ¹H NMR (400 MHz, CDCI₃) δ 8.57 (d, J = 0.6 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.35 (d, J = 7.2 Hz, 2H), 7.32 - 7.26 (m, 3H), 7.21 (t, J = 7.2 Hz, 1 H), 7.17 (d, J = 2.7 Hz, 1 H), 6.49 (d, J = 15.9 Hz, 1 H), 6.20 (dt, J = 15.9, 6.6 Hz, 1 H), 3.93 (s, 3H), 3.65 (s, 3H), 3.31 (t, J = 5.0 Hz, 1 H), 3.17 - 2.96 (m, 5H), 2.85 - 2.76 (m, 1 H), 2.72 - 2.64 (m, 2H), 2.59 - 2.48 (m, 3H), 2.47 - 2.39 (m, 1 H), 1 .87 (p, J = 7.4 Hz, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 172.5, 158.6, 155.0 (d, JCF = 251 .4 Hz), 141.6 (d, JCF = 2.3 Hz), 138.2 (d, JCF = 29.4 Hz), 136.9, 133.2, 131.8, 129.9 (d, JCF = 13.0 Hz), 129.2 (d, JCF = 3.9 Hz), 128.6, 127.6, 126.4, 126.2, 120.2 (d, JCF = 2.6 Hz), 102.0 (d, JCF = 5.4 Hz), 63.9, 60.8, 55.6, 55.6, 55.2, 53.1 , 51 .7, 49.1 , 26.5, 22.0 (d, JCF = 3.1 Hz); IR (neat) cm^-1: 3026, 2999, 2949, 2810, 2768, 1741 , 1621 , 1509, 1468, 1362, 1321 , 1230, 1032, 831 ; HRMS (ESI) calcd for C28H33FN3O3 [M + H]⁺ 478.2500, found 478.2502;

+ 8.0 (c 1.0, CHCI₃). (R ,E)-methyl 4-(3-(2,5-difluorophenyl)allyl)-1 -(3-(3-fluoro-6- methoxy-quinolin-4-yl)propyl)piperazine-2-carboxylate (57f). Following general procedure V, amine 56 (77 mg, 0.21

mmol) was reductively alkylated using aldehyde 30, AcOH (12 μL, 0.013 mmol), and NaBH(OAc)₃ (91 mg, 0.43 mmol) in CH₂CI₂ (2 mL) to give the title compound as a colorless oil (77 mg, 70%) after purification by flash column chromatography on silica gel (EtOAc:heptane (3:1 ), fl_f = 0.3). ¹H NMR (400 MHz, CDCI₃) δ 8.57 (d, J = 1.0 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.30 (dd, J = 9.2, 2.7 Hz, 1 H), 7.18 (d, J = 2.7 Hz, 1 H), 7.10 (ddd, J = 9.2, 5.8, 3.1 Hz, 1 H), 6.96 (td, J = 9.2, 4.6 Hz, 1 H), 6.91 - 6.82 (m, 1 H), 6.61 (d, J = 16.1 Hz, 1 H), 6.26 (dt, J = 16.1 , 6.5 Hz, 1 H), 3.94 (s, 3H), 3.66 (s, 3H), 3.35 - 3.29 (m, 1 H), 3.22 - 2.96 (m, 5H), 2.88 - 2.77 (m, 1 H), 2.76 - 2.60 (m, 2H), 2.60 - 2.49 (m, 3H), 2.48 - 2.39 (m, 1 H), 1.87 (p, J = 7.4 Hz, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 172.5, 158.9 (dd, J_CF = 241.6, 2.2 Hz), 158.6, 156.2 (dd, J_CF = 244.8, 2.2 Hz), 155.0 (d, J_CF = 251 .4 Hz), 141.7 (d, J_CF = 2.3 Hz), 138.3 (d, J_CF = 29.4 Hz), 131 .8, 130.3, 129.9 (d, J_CF = 13.0 HZ), 129.3 (d, J_CF = 3.9 Hz), 126.1 (dd, J_CF = 14.8, 8.0 Hz), 124.5, 120.2 (d, J_CF = 2.6 Hz), 1 16.8 (dd, J_CF = 25.2, 8.8 Hz), 1 15.2 (dd, JC_F = 24.4, 8.7 Hz), 1 13.2 (dd, J_CF = 24.5, 4.1 Hz), 102.0 (d, J_CF = 5.4 Hz), 63.7, 60.7, 55.7, 55.6, 55.2, 53.2, 51.7, 48.9, 26.6, 22.1 (d, JCF = 3.0 Hz); IR (neat) cm^-1: 2950, 2812, 1740, 1620, 1508, 1490, 1468, 1429, 1264, 1229, 1 193, 1 147, 1031 , 830; HRMS (ESI) calcd for C28H3i F₃N₃03 [M + H]⁺ 514.2312 found 514.2304; = + 1 1.3 (c 1.0,

CHCIs).

(R)-methyl 1 -(3-(3-fluoro-6-methoxyquinolin-4-yl)propyl)-4- (2-(phenyl-thio)ethyl)piperazine-2-carboxylate (57g). Following general procedure V, amine 56 (50 mg, 0.14 mmol) was re-

ductively alkylated using aldehyde 39 (23 mg, 0.15 mmol) and

NaBH(OAc)₃ (freshly prepared by premixing AcOH (200 μL, 3.50 mmol) and NaBH₄ (8 mg, 0.24 mmol) for 5 min) in CH2CI2 (1 .5 mL) to give the title compound as an orange oil (51 mg, 74%) after purification by flash column chromatography on silica gel

(EtOAc:heptane (3:2), R_f = 0.3). ¹H NMR (CDCI₃, 400 MHz) δ 8.59 (s, 1 H), 8.00 (d, J = 9.2 Hz, 1 H), 7.37 - 7.25 (m, 5H), 7.23 - 7.15 (m, 2H), 3.96 (s, 3H), 3.68 (s, 3H), 3.36 - 3.29 (m, 1 H), 3.13 - 3.05 (m, 3H), 3.02 (t, J = 7.5 Hz, 2H), 2.88 - 2.71 (m, 2H), 2.69 - 2.48 (m, 6H), 2.47 - 2.39 (m, 1 H), 1 .87 (p, J = 7.4 Hz, 2H); ¹³C NMR (CDCI₃, 100 MHz) δ 172.4, 158.6, 155.0 (d, JCF = 251 .4 Hz), 141.6 (d, JCF = 2.2 Hz), 138.3 (d, JCF = 29.4 Hz), 136.4, 131 .8, 129.9 (d, J = 12.9 Hz), 129.3, 129.2, 129.0, 126.1 , 120.2 (d, JCF = 2.6 Hz), 102.0 (d, JCF = 5.4 Hz), 63.5, 57.3, 55.7, 55.5, 55.2, 53.0, 51.7, 48.7, 30.9, 26.5, 22.0 (d, JCF = 3.0 Hz); IR (neat) cm^-1 : 2948, 2814, 1740, 1620, 1509, 1467, 1230, 1029, 831 ; HRMS (ESI) calcd for C27H33FN3O3S [M + H]⁺ 498.2221 , found 498.2218; [a]¾³= + 9.3 (c 1 .0, CHCI₃).

(R)-Methyl 1 -(3-(3-fluoro-6-methoxyquinolin-4-yl)propyl)-4- (2-((2-fluoro-phenyl)thio)ethyl)piperazine-2-carboxylate (57h). Following general procedure V, amine 56 (50 mg, 0.14

mmol) was reductively alkylated using aldehyde 40 (73 mg, 0.43 mmol) and NaBH(OAc)3 (freshly prepared by premixing AcOH (285 μL, 4.98 mmol) and NaBH₄ (24 mg, 0.62 mmol) for 5 min) in CH2CI2 (1.2 mL) to give the title compound as a yellow oil (27 mg, 38%) after purification by flash column chromatography on silica gel (EtOAc:heptane (1 :1 ), = 0.2). ¹H N MR (400 MHz, CDCI₃) δ 8.57 (s, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.36 (td, J = 7.5, 1 .6 Hz, 1 H), 7.30 (dd, J = 9.2, 2.6 Hz, 1 H), 7.23 - 7.16 (m, 2H), 7.10 - 7.01 (m, 2H), 3.94 (s, 3H), 3.67 (s, 3H), 3.30 (s, 1 H), 3.14 - 2.94 (m, 5H), 2.89 - 2.69 (m, 2H), 2.67 - 2.35 (m, 7H), 1.86 (p, J = 7.4 Hz, 2H); ¹³C NMR (100 MHz, CDCI₃) 1δ72.2, 161 .5 (d, JCF = 245.1 Hz), 158.5, 154.9 (d, JCF = 251.4 Hz), 141 .5, 138.1 (d, JCF = 29.4 Hz), 132.2 (d, JCF = 1.8 Hz), 131.7, 129.9, 129.2 (d, JCF = 3.8 Hz), 128. 5 (d, JCF = 7.8 Hz), 124.4 (d, JCF = 3.7 Hz), 123.0 (d, JCF = 17.4 Hz), 120.1 , 1 15.7 (d, JCF = 22.5 Hz), 101.9 (d, JCF = 5.4 Hz), 63.3, 57.2, 55.6, 55.3, 55.1 , 52.8, 51.6, 48.5, 30.6, 26.4, 21.9 (d, J_CF = 3.1 Hz); IR (neat) cm^-1: 2948, 2814, 1740, 1620, 1509, 14712, 1320, 1263, 1229, 1031 , 831 , 755; HRMS (ESI) calcd for

C27H32F2N3O3S [M + H]⁺ 516.2127, found 516.2122; + 12.6 (c 1 .0,CHCI₃).

(R)-Methyl 1 -(3-(3-fluoro-6-methoxyquinolin-4-yl)propyl)-4- (2-((2-(tri-fluoromethyl)phenyl)thio)ethyl)piperazine-2-car- boxylate (58i). Following general procedure V, amine 56 (37

mg, 0.10 mmol) was reductively alkylated using aldehyde 42 (70 mg, 0.17 mmol) and NaBH(OAc)3 (freshly prepared by premixing AcOH (200 μL, 3.50 mmol) and NaBH₄ (8 mg, 0.24 mmol) for 5 min) in CH2CI2 (1 .5 mL) to give the title compound as a yellow oil (46 mg, 59%) after purification by flash column chromatography on silica gel (EtOAc:heptane (3:2), R_f = 0.3). ¹ H NMR (400 MHz, CDCI₃) δ 8.50 (s, 1 H), 7.91 (d, J = 9.2 Hz, 1 H), 7.56 (d, J = 7.9 Hz, 1 H), 7.45 - 7.34 (m, 2H), 7.26 - 7.16 (m, 2H), 7.1 1 (d, J = 2.5 Hz, 1 H), 3.88 (s, 3H), 3.60 (s, 3H), 3.26 - 23.22 (m, 1 H), 3.06 - 2.90 (m, 5H), 2.81 - 2.65 (m, 2H), 2.61 - 2.32 (m, 7H), 1 .79 (p, J = 7.3, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 172.3, 158.7, 155.0 (d, JCF = 251 .4 Hz), 141 .6 (d, JCF = 2.2 Hz), 138.3 (d, JCF = 29.4 Hz), 136. 3, 132.1 , 131 .8, 131 .0, 129.9 (m), 129.3 (d, JCF = 3.9 Hz), 126.9 (q, J = 5.7 Hz), 126.0, 123.7 (q, J_CF = 274 Hz), 120.2 (d, J = 2.6 Hz), 102.1 (d, J = 5.4 Hz), 63.3, 56.9, 55.7, 55.5, 55.1 , 52.9, 51 .7, 48.6, 31 .7, 26.7, 22.0 (d, J = 3.1 Hz); IR (neat) cm ¹ : 2950, 2817, 1741 , 1620, 1509, 1469, 1313, 1230, 1 129, 1034, 831 , 764; HRMS (ESI) calcd for C28H32F4N3O3S [M + H]⁺ 566.2095, found 566.2091 ;

[a]¾³= + 7.6 (c 1 .0, CHCI₃).

(R)-Methyl 1 -(3-(3-f luoro-6-methoxyquinolin-4-yl)propyl)- 4-(2-((2-(tri-fluoromethyl)phenyl)thio)ethyl)piperazine-2- carboxylate (57j). Following general procedure V, amine 56

(37 mg, 0.10 mmol) was reductively alkylated using aldehyde 41 (89 mg, 0.21 mmol) and NaBH(OAc)₃ (freshly prepared by premixing AcOH (200 μL, 3.50 mmol) and NaBH₄ (8 mg, 0.24 mmol) for 5 min) in CH2CI2 (1 .5 mL) to give the title compound as a yellow oil (68 mg, 92%) after purification by flash column chromatography on silica gel (EtOAc:heptane (2:3), R_f = 0.2). ¹ H NMR (400 MHz, CDCI₃) δ 8.58 (s, 1 H), 8.00 (d, J = 9.2 Hz, 1 H), 7.32 (dd, J = 9.2, 2.5 Hz, 1 H), 7.19 (d, J = 2.4 Hz, 1 H), 7.08 - 6.95 (m, 2H), 6.90 - 6.82 (m, 1 H), 3.96 (s, 3H), 3.69 (s, 3H), 3.35 - 3.30 (m, 1 H), 3.14 - 2.96 (m, 5H), 2.89 - 2.73 (m, 2H), 2.67 - 2.39 (m, 7H), 1 .87 (p, J = 7.3 Hz, 2H); ¹³NMR (100 MHz, CDCI₃) δ 173.2, 158.6, 158.6 (dd, JCF = 244.3, 2.5 Hz) 157.2 (dd, JCF = 241 .4 Hz) 155.0 (dd, JCF = 251 .4, 2.5 Hz), 141 .6 (d, JCF = 2.2 Hz), 138.3 (d, JCF = 29.4 Hz), 131 .8, 129.9 (d, JCF = 13.0 Hz), 129.3 (d, JCF = 3.9 Hz), 125.6 (dd, JCF = 20.4, 8.3 Hz), 120.2 (d, JCF = 2.6 Hz), 1 17.3 (dd, JCF = 25.4, 2.4 Hz), 1 16.3 (dd, JCF = 25.2, 8.9 Hz), 1 14.1 (dd, JCF = 24.0, 8.1 Hz), 102.0 (d, JCF = 5.4 Hz), 63.3, 56.9, 55.7, 55.4, 55.1 , 52.9, 51 .7, 48.5, 30.3 (d, JCF = 2.3 Hz), 26.6, 22.0 (d, JCF = 3.0 Hz); IR (neat) crrr ¹ : 2949, 2815, 1739, 1612, 1509, 1481 , 1229, 1 186, 1031 , 831 , 757; HRMS (ESI) calcd for C27H31 F3N3O3S [M + H]⁺ 534.2033, found 534.2031 ; + 13.0 (c 1 .0, CHCI₃).

(R)-Methyl 1-(3-(3-fluoro-6-methoxyquinolin-4-yl)propyl)-4- (2-phenoxy-ethyl)piperazine-2-carboxylate (57k). Following general procedure V, amine 56 (50 mg, 0.14 mmol) was reduc¬

tively alkylated using aldehyde 48 (35 mg, 0.28 mmol) and NaBH(OAc)₃ (freshly prepared by premixing AcOH (400 μL, 7.00 mmol) and NaBH₄ (16 mg, 0.28 mmol) for 5 min) in CH₂CI₂ (1 .5 mL) to give the title compound as a colorless oil (46 mg, 69%) after purification by flash column chromatography on silica gel (EtOAc:heptane (1 :1 ), R_f = 0.2). ¹ H NMR (400 MHz, CDCI₃) δ 8.55 (s, 1 H), 7.96 (d, J = 9.2 Hz, 1 H), 7.28 (dd, J = 9.2, 2.9 Hz, 1 H), 7.23 (d, J = 8.0 Hz, 2H), 7.16 (d, J = 2.6 Hz, 1 H), 6.91 (t, J = 7.3 Hz, 1 H), 6.85 (d, J = 8.0 Hz, 2H), 4.03 (td, J = 5.7, 2.5 Hz, 2H), 3.91 (s, 3H), 3.61 (s, 3H), 3.31 (d, J = 5.7 Hz, 1 H), 3.12 - 2.93 (m, 3H), 2.86 - 2.69 (m, 5H), 2.58 (t, J = 4.6 Hz, 2H), 2.56 - 2.47 (m, 1 H), 2.42 (dt, J = 10.4, 5.0 Hz, 1 H), 1 .84 (p, J = 7.4 Hz, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 172.4, 158.7, 158.6, 155.0 (d, JCF = 251 .4 Hz), 141 .6 (d, JCF = 2.3 HZ), 138.2 (d, JCF = 29.4 Hz), 131 .8, 129.9 (d, J CF = 13.0 Hz), 129.5, 129.2 (d, JCF = 5.4 Hz), 120.9, 120.2 (d, JCF = 2.6 Hz), 1 14.6, 102.0 (d, JCF = 5.4 Hz), 65.8, 63.6, 56.9, 56.1 , 55.6, 55.2, 53.5, 51 .6, 48.8, 26.5, 22.0 (d, JCF = 3.1 Hz); IR (neat) cm ¹ : 2948, 2829, 1740, 1620, 1508, 1467, 1230, 1034, 831 , 755; HRMS (ESI) calcd for C27H33FN3O4 [M + H]⁺ 482.2450, found 482.2450;

+ 15.8 (c 1 .0, CHCI₃).

(R)-Methyl 4-(2-(2,5-dif luorophenoxy)ethyl)-1 -(3-(3-f luoro- 6-methoxy-quinolin-4-yl)propyl)- piperazine-2-carboxylate (57m). Following general procedure V, amine 56 (37 mg,

0.10 mmol) was reductively alkylated using aldehyde 50 (26 mg, 0.15 mmol) and NaBH(OAc)₃ (43 mg, 0.20 mmol) in CH₂CI₂ (1 mL) to give the title compound as a colorless oil (36 mg, 68%) after purification by flash column chromatography on silica gel (EtOAc:heptane (2:3), R_f = 0.2). ¹ H NMR (400 MHz, CDCI₃) δ 8.57 (s, 1 H), 7.98 (d, J = 8.6 Hz, 1 H), 7.30 (dd, J = 9.2, 2.6 Hz, 1 H), 7.18 (d, J = 2.6 Hz, 1 H), 6.99 (ddd, J = 10.6, 9.0, 5.3 Hz, 1 H), 6.67 (ddd, J = 9.7, 6.7, 2.9 Hz, 1 H), 6.62 - 6.51 (m, 1 H), 4.08 (t, J = 5.6 Hz, 2H), 3.94 (s, 3H), 3.64 (s, 3H), 3.33 (dd, J = 6.0, 3.5 Hz, 1 H), 3.16 - 2.96 (m, 3H), 2.92 - 2.77 (m, 4H), 2.77 - 2.70 (m, 1 H), 2.67 - 2.49 (m, 3H), 2.49 - 2.39 (m, 1 H), 1 .86 (p, J = 7.3 Hz, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 172.4, 158.8 (dd, JCF = 242.1 , 2.5 Hz), 158.7, 155.0 (d, JCF = 251 .4 Hz), 149.1 (dd, JCF = 241 .4, 3.3 Hz), 147.5 (dd, JCF = 12.6, 10.4 Hz), 141 .7 (d, JCF = 2.2 Hz), 138.3 (d, JCF = 29.4 Hz), 131 .8, 130.0 (d, JCF = 13.0 Hz), 129.3 (d, JCF = 3.9 Hz), 120.2 (d, JCF = 2.6 Hz), 1 16.3 (dd, JCF = 20.8, 10.2 Hz), 106.9 (dd, JCF = 23.8, 6.9 Hz), 103.1 (dd, JCF = 27.4, 1 .8 Hz), 102.0 (d, JCF = 5.4 Hz), 67.9, 63.5, 56.6, 56.2, 55.7, 55.2, 53.5, 51 .7, 48.7, 25.7, 22.0 (d, JCF = 3.1 Hz); IR (neat) cm ¹ : 2950, 2830, 1740, 1622, 151 1 , 1468, 1322, 1206, 1 156, 1031 , 832; + 7.0 (c 0.5, CHCI₃). (R)-Methyl 1 -(3-(3-fluoro-6-methoxyquinolin-4-yl)propyl)-4-hep- tylpiperazine-2-carboxylate (57o). Following general procedure

V, amine 56 (50 mg, 0.14 mmol) was reductively alkylated using

heptanal (27 μL , 0.15 mmol) and NaBH(OAc)3 (freshly prepared by premixing AcOH (200 μL, 3.50 mmol) and NaBH₄ (8 mg, 0.24 mmol) for 5 min) in CH2CI2 (1 .5 mL) to give the title compound as a colorless oil (68 mg, 92%) after purification by flash column chromatography on silica gel (EtOAc:heptane (2:3), R_f = 0.2). ¹H NMR (400 MHz, CDCI₃) δ 8.54 (d, J = 0.6 Hz, 1 H), 7.95 (d, J = 9.2 Hz, 1 H), 7.27 (dd, J = 9.2, 2.7 Hz, 1 H), 7.15 (d, J = 2.1 Hz, 1 H), 3.92 (s, 3H), 3.63 (s, 3H), 3.27 (t, J = 5.0 Hz, 1 H), 3.1 1 - 2.94 (m, 3H), 2.84 - 2.73 (m, 2H), 2.63 - 2.34 (m, 6H), 2.34 - 2.19 (m, 2H), 1.84 (p, J = 7.4 Hz, 2H), 1 .47 - 1 .35 (m, 2H), 1 .31 - 1 .18 (m, 8H), 0.84 (t, J = 6.8 Hz, 3H); ¹³C NMR (100 MHz, CDCI₃) δ 172.6, 158.6, 155.0 (d, JCF = 251 .5 Hz), 141.6 (d, JCF = 2.3 Hz), 138.2 (d, JCF = 29.4 Hz), 131 .8, 129.9 (d, JCF = 13.0 Hz), 129.2 (d, JCF = 3.9 Hz), 120.2 (d, JCF = 2.6 Hz), 101 .9 (d, JCF = 5.4 Hz), 63.9, 58.4, 55.6, 55.6, 55.3, 53.3, 51.6, 49.1 , 31.9, 29.3, 27.4, 26.8, 26.5, 22.7, 22.0 (d, JCF = 3.1 Hz), 14.15; IR (neat) cm^-1: 2930, 2856, 281 1 , 1743, 1621 , 1509, 1468, 1230, 1032, 831 ; HRMS (ESI) calcd for C26H39FN3O3 [M + H]⁺ 460.2970, found 460.2971 ; + 18.3 (c 1 .0, CHCI₃).

(R)-Methyl 4-(cyclohexylmethyl)-1 -(3-(3-fluoro-6-methoxy-quin- olin-4-yl)-propyl)piperazine-2-carboxylate (57r). Following general procedure V, amine 56 (37 mg, 0.10 mmol) was reductively

alkylated using cyclohexanecarbaldehyde (20 μL, 0.17 mmol) and NaBH(OAc)₃ (freshly prepared by premixing AcOH (200 μL, 3.50 mmol) and NaBH₄ (8 mg, 0.24 mmol) for 5 min) in CH2CI2 (1 .5 mL) to give the title compound as a colorless oil (42 mg, 66%) after purification by flash column chromatography on silica gel

(EtOAc:heptane (2:3), R_f = 0.2). ¹H NMR (CDCI₃, 400 MHz) δ 8.56 (d, J = 0.7 Hz, 1 H), 7.97 (d, J= 9.2 Hz, 1 H), 7.29 (dd, J = 9.2, 2.7 Hz, 1 H), 7.17 (d, J = 2.7 Hz, 1 H), 3.94 (s, 3H), 3.64 (s, 3H), 3.33 - 3.26 (m, 1 H), 3.12 - 2.94 (m, 3H), 2.86 - 2.76 (m, 1 H), 2.67 - 2.61 (s, 1 H), 2.60 - 2.47 (m, 2H) , 2.44 - 2.35 (m, 3H), 2.06 (dd, J = 7.1 , 1 .9 Hz, 2H), 1 .85 (p, J = 7.4 Hz, 2H), 1.75 - 1.59 (m, 5H), 1.48 - 1.36 (m, 1 H), 1.26 - 1.09 (m, 3H), 0.88 - 0.74 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 172.6, 158.6, 155.0 (d, JCF = 251.4 Hz), 141 .6 (d, JCF = 2.3 Hz), 138.3 (d, JCF = 29.4 Hz), 131.8, 130.0 (d, JCF = 13.0 Hz), 129.3 (d, JCF = 3,9 Hz), 120.3 (d, JCF = 2.6 Hz), 102.0 (d, JCF = 5.4 Hz), 65.1 , 63.7, 56.1 , 55.7, 55.3, 53.8, 51.5, 48.9, 35.1 , 31 .8, 31.7, 26.9, 26.6, 26.2, 26.2, 22.1 (d, JCF = 3.9 Hz); IR (neat) cm^-1 : 2922, 2849, 2807, 1743, 1621 , 1509, 1467, 1230, 1033, 831 ; HRMS (ESI) calcd for C26H37FN3O3 [M + H]⁺ 458.2813, found 458.281 1 ;

+ 1 1 .2 (c 1 .0, CHCIs).

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4- phenethylpiperazine- 2-carboxylic acid (58a). Following general procedure IV, methyl ester 57a (55mg, 0.12 mmol) was hy-

drolyzed to give the title compound as an amorphous solid after preparative HPLC (39 mg, 73%).¹H NMR (400 MHz, DMSO-d₆) δ 8.68 (d, J = 0.5 Hz, 1 H), 7.95 (d, J = 9.0 Hz, 1 H), 7.41 - 7.34 (m, 2H), 7.29 - 7.14 (m, 5H), 3.95 (s, 3H), 3.20 (dd, J = 5.6, 3.6 Hz, 1 H), 3.16 - 2.95 (m, 4H), 2.91 - 2.80 (m, 1 H), 2.77 - 2.52 (m, 7H), 2.47 - 2.34 (m, 2H), 1.88 - 1.71 (m, 2H); ¹³C NMR (100 MHz, DMSO-c/e) δ 171 .6, 158.2, 154.5 (d, JCF = 250.2 Hz), 141 .0 (d, JCF = 2.1 HZ), 140.1 , 137.9 (d, JCF = 28.9 Hz), 131 .3, 129.7 (d, JC_F = 13.2 Hz), 128.8 (d, JC_F = 4.0 Hz), 128.7, 128.2, 125.9, 120.5, 102.3 (d, JCF = 5.3 Hz), 62.8, 59.1 , 55.7, 55.0, 54.2, 51 .9, 47.9, 32.4, 25.9, 21.3 (d, JC_F = 2.4 Hz); IR (neat) cm^-1: 3054, 3029, 2960, 2943, 2924, 2820, 2773, 1620,

1513, 1451 , 1375, 1339, 1262, 1237, 1 178, 1 102, 1064, 1031 , 980, 864, 828, 792, 745, 698, 546, 510; HRMS (ESI) calcd for C26H31FN3O3 [M + H]⁺ 452.2344 found 452.2343;

= + 6.9 (c O.5, in CH₃OH).

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(3-phe- nylpropyl)piperazine-2-carboxylic acid (58b). Following general procedure IV, methyl ester 57b (58 mg, 0.12 mmol) was

hydrolyzed to give the title compound as an amorphous solid af- ter preparative HPLC (28 mg, 51 %). ¹ H NMR (400 MHz, CDCI₃) δ 8.50 (d, J_HF = 0.8 Hz, 1 H), 7.91 (d, J = 9.2 Hz, 1 H), 7.26 - 7.19 (m, 3H), 7.15 - 7.1 1 (m, 2H), 7.1 1 - 7.06 (m, 2H), 3.88 (s, 3H), 3.34 (t, J = 3.2 Hz, 1 H), 3.12 (d, J = 10.6 Hz, 1 H), 3.05 - 2.87 (m, 4H), 2.82 - 2.70 (m, 2H), 2.70 - 2.62 (m, 1 H), 2.61 - 2.46 (m, 5H), 2.40 - 2.30 (m, 1 H), 1 .88 - 1.78 (m, 4H); ¹³C NMR (100 MHz, CDCI₃) δ 171.3, 158.8, 155.0 (d, JCF = 251.4 Hz), 141 .5 (d, JC_F = 2.2 Hz), 140.7, 138.2 (d, J_CF = 29.5 Hz), 131.7, 129.9 (d, J_CF = 12.8 Hz), 129.4 (d, J_CF = 3.8 Hz), 128.7, 128.4, 126.4, 120.3 (d, J_CF = 2.5 Hz), 102.1 (d, J_CF = 5.4 Hz), 60.5, 56.6, 55.8, 54.6, 54.4, 51.6, 46.9, 33.2, 26.9, 26.0, 22.0 (d, J_CF = 3.0 Hz);MS (ESI) calcd for C27H33FN3O3 [M + H]⁺ 466.3 found 466.3. (R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(4-phe-

was hydrolyzed to give the title compound as an amorphous solid after preparative HPLC (16 mg, 65%). ¹H NMR (400 MHz, CDCI₃) δ 8.54 (d, J = 1.0 Hz, 1 H), 7.96 (d, J = 9.2 Hz, 1 H), 7.28 (dd, J = 9.2, 2.7 Hz, 1 H), 7.25 - 7.21 (m, 2H), 7.19 - 7.16 (m, 2H), 7.16 - 7.10 (m, 2H), 3.93 (s, 3H), 3.36 (t, J = 2.8 Hz, 1 H), 3.17 (d, J = 10.4 Hz, 1 H), 3.08 - 2.92 (m, 4H), 2.85 - 2.65 (m, 3H), 2.65 - 2.44 (m, 5H), 2.42 - 2.29 (m, 1 H), 2.06 - 1 .80 (m, 2H), 1 .73 - 1 .52 (m, 4H); ¹³C NMR (100 MHz, CDCI₃) δ 171 .4, 158.7, 155.0 (d, JCF = 251 .4 Hz), 141.7, 141.6, 138.2 (d, JCF = 29.5 Hz), 131 .8, 129.9 (d, JCF = 12.7 Hz), 129.4 (d, JCF = 3.9 Hz), 128.6, 128.5, 126.1 , 120.3, 102.1 (d, JCF = 5.4 Hz), 60.2, 57.1 , 55.8, 54.8, 54.4, 51 .6, 46.8, 35.5, 28.8, 26.2, 24.7, 22.0 (d, JCF = 3.1 Hz); IR (neat) cm^-1 : 3024, 2939, 2860, 2831 , 1716, 1509, 1468, 1454, 1362, 1322, 1231 , 1029, 750; HRMS (ESI) calcd for C28H35FN3O3 [M + H]⁺ 480.2657 found 480.2658; + 3.6 (c 1.0, CHCI₃).

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(3-(2-flu- orophenyl)-propyl)piperazine-2-carboxylic acid (58d). Following general procedure IV, methyl ester 57d (28 mg, 0.056

mmol) was hydrolyzed to give the title compound as an off-white amorphous solid after preparative HPLC (14 mg, 51 %). ¹H NMR (400 MHz, CDCI₃) δ 9.32 (br. s, 1 H), 8.59 (d, J = 0.9 Hz, 1 H), 8.00 (d, J = 9.2 Hz, 1 H), 7.33 (dd, J = 9.2, 2.6 Hz, 1 H), 7.24 - 7.14 (m, 3H), 7.1 1 - 6.98 (m, 2H), 3.97 (s, 3H), 3.45 (t, J = 3.2 Hz, 1 H), 3.21 (d, J = 9.6 Hz, 1 H), 3.16 - 3.00 (m, 4H), 2.87 (t, J = 7.0 Hz, 2H), 2.80 - 2.72 (m, 1 H), 2.73 - 2.60 (m, 5H), 2.55 - 2.44 (m, 1 H), 2.07 - 1 .85 (m, 4H); ¹³C NMR (100 MHz, CDCI₃) 6 171.2, 161.2 (d, JCF = 244.8 Hz), 158.8, 155.0 (d, JCF = 251 .4 Hz), 141.5 (d, JCF = 2.1 Hz), 138.1 (d, JCF = 29.5 Hz), 131.7, 130.7 (d, JCF = 4.9 Hz), 129.8 (d, JCF = 12.9 Hz), 129.4 (d, JCF = 3.8 Hz), 128.2 (d, JCF = 8.1 Hz), 127.6 (d, JCF = 15.9 Hz), 124.3 (d, JCF = 3.5 Hz), 120.4, 1 15.5 (d, JCF = 22.1 Hz), 102.1 (d, JCF = 5.4 Hz), 60.7, 56.6, 55.8, 54.7, 54.3, 51.6, 47.0, 26.7 (d, JCF = 2.2 Hz), 25.9, 25.7, 21.9 (d, JCF = 3.1 Hz); IR (neat) cm^-1: 2947, 2868, 2829, 1717, 1620, 1509, 1468, 1362, 1322, 1229, 1029, 831 , 757; HRMS (ESI) calcd for C27H32F2N3O3 [M + H]⁺ 484.2406 found

484.2400; = + 5.0 (c 1.0, CHCI₃).

(R)-4-Cinnamyl-1 -(3-(3-fluoro-6-methoxyquinolin-4-yl)pro- pyl)piperazin-e-2-carboxylic acid (58e). Following general procedure IV, methyl ester 57e (49 mg, 0.10 mmol) was hydro-

lyzed to give the title compound as an off-white amorphous solid after preparative HPLC (34 mg, 70%). ¹H NMR (400 MHz, CDCI₃) δ 9.49 (br s, 1 H), 8.56 (s, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.39 - 7.21 (m, 6H), 7.19 (d, J = 2.6 Hz, 1 H), 6.58 (d, J = 15.7 Hz, 1 H), 6.18 (dt, J = 15.7, 7.1 Hz, 1 H), 3.93 (s, 3H), 3.47 (t, J = 3.8 Hz, 1 H), 3.37 (d, J = 6.9 Hz, 2H), 3.27 - 3.14 (m, 2H), 3.10 - 2.82 (m, 5H), 2.82 - 2.67 (m, 2H), 2.59 (td, J = 9.1 , 2.7 Hz, 1 H), 2.07 - 1 .85 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 171 .4, 158.8, 155.0 (d, JCF = 251 .4 Hz), 141.4 (d, JCF = 2.2 Hz), 138.1 (d, JCF = 29.5 Hz), 137.0, 135.9, 131.7, 129.6 (d, JCF = 12.9 Hz), 129.3 (d, JCF = 3.7 Hz), 128.8, 128.5, 126.8, 121.1 , 120.4 (d, JCF = 2.4 Hz), 102.1 (d, JCF = 5.4 Hz), 61 .4, 59.6, 55.8, 54.7, 53.9, 51 .0, 47.1 , 25.7, 21.9 (d, JCF = 3.0 Hz); IR (neat) cm^-1: 3396, 2955, 2829, 1620, 1509, 1468, 1362, 1230, 1029, 750; HRMS (ESI) calcd for C27H31FN3O3 [M + H]⁺ 464.2344 found 464.2342; = + 14.9 (c 1.0, CHCI₃).

(R, E) -4-(3-(2,5-Difluorophenyl)allyl)-1 -(3-(3-fluoro-6-me- thoxyquinolin-4-yl)propyl)piperazine-2-carboxylic acid (58f). Following general procedure IV, methyl ester 57f (42

mg, 0.082 mmol) was hydrolyzed to give the title compound as an off-white amorphous solid after preparative HPLC (23 mg, 55%). ¹ H NMR (400 MHz, CDCI₃) δ 9.76 (br s, 1 H), 8.57 (d, J = 0.7 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.30 (dd, J = 9.2, 2.6 Hz, 1 H), 7.19 (d, J = 2.6 Hz, 1 H), 7.12 (ddd, J = 8.9, 5.7, 3.0 Hz, 1 H), 6.98 (td, J = 9.3, 4.5 Hz, 1 H), 6.90 (tt, J = 9.0, 3.7 Hz, 1 H), 6.68 (d, J = 16.0 Hz, 1 H), 6.26 (dt, J = 16.0, 6.9 Hz, 1 H), 3.94 (s, 3H), 3.48 (t, J = 3.7 Hz, 1 H), 3.36 (d, J = 6.8 Hz, 2H), 3.20 - 3.10 (m, 2H), 3.06 (t, J = 7.4 Hz, 2H), 3.02 - 2.82 (m, 3H), 2.74 (d, J = 12.2 Hz, 2H), 2.56 (t, J = 9.0 Hz, 1 H), 2.07 - 1 .91 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 171.0, 158.7 (dd, JCF = 242.4, 2.1 Hz), 158.7, 156.2 (dd, JCF = 245.8, 2.1 Hz), 154.9 (d, J = 251 .5 Hz), 141 .4 (d, JCF = 2.2 Hz), 138.0 (d, J = 29.5 Hz), 131.6, 129.5 (d, JCF = 12.8 Hz), 129.2 (d, JCF = 3.8 Hz), 127.6, 125.9, 125.0 (dd, JCF = 14.5, 7.8 Hz), 120.3 (d, JCF = 2.4 Hz), 1 16.9 (dd, JCF = 25.1 , 8.7 Hz), 1 16.0 (dd, JCF = 24.4, 8.8 Hz), 1 13.5 (dd, JCF = 24.6, 3.8 Hz), 101 .9 (d, J_CF = 5.3 Hz); I R (neat) cm^-1: 2946, 2830, 1718, 1621 , 1509, 1490, 1362, 1231 , 1030, 973, 830, 756; HRMS (ESI) calcd for C27H29F3N3O3 [M + H]⁺ 500.2156 found 500.2155; + 12.8 (c 1.O, CHCI₃).

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(2-(phe- nylthio)-ethyl)piperazine-2-carboxylic acid (58g). Following general procedure IV, methyl ester 57g (38 mg, 0.076 mmol)

was hydrolyzed to give the title compound as an off-white solid after preparative HPLC (27 mg, 72%). ¹H NMR (400 MHz, CDCI₃) δ 9.17 (br s, 1 H), 8.57 (d, J = 0.8 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.37 - 7.23 (m, 5H), 7.23 - 7.15 (m, 2H), 3.95 (s, 3H), 3.42 (t, J = 3.8 Hz, 1 H), 3.1 1 - 3.00 (m, 6H), 2.86 - 2.78 (m, 3H), 2.76 - 2.65 (m, 4H), 2.49 - 2.42 (m, 1 H), 2.09 - 1.86 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 170.4, 158.9, 155.0 (d, J_CF = 251 .4 Hz), 141.5 (d, J_CF = 2.3 Hz), 138.1 (d, J_CF = 29.5 Hz), 135.0, 131 .7, 130.2, 129.5 (d, JCF = 12.8 Hz), 129.3, 126.9, 120.4 (d, JCF = 2.5

Hz), 102.0 (d, JCF = 5.4 Hz), 61.4, 56.2, 55.8, 54.9, 54.3, 51 .3, 47.9, 30.4, 25.5, 21.9 (d, JCF = 3.1 Hz); IR (neat) cm^-1 : 3402, 2946, 1620, 1509, 1468, 1362, 1230, 742; HRMS (ESI) calcd for C26H31 FN3O3S [M + H]⁺ 484.2065 found 484.2061 ; = - 6.0 (c 1.0,

CHCIs).

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(2-((2- fluorophen-yl)thio)ethyl)piperazine-2-carboxylic acid (58h). Following general procedure IV, methyl ester 57h (30 mg,

0.057 mmol) was hydrolyzed to give the title compound as an off-white solid after preparative HPLC (17 mg, 59%). ¹H NMR (400 MHz, CDCI₃) δ 8.57 (d, J = 0.8 Hz, 1 H), 7.99 (d, J = 9.2 Hz, 1 H), 7.40 (td, J = 7.5, 1 .6 Hz, 1 H), 7.31 (dd, J = 9.2, 2.6 Hz, 1 H), 7.28 - 7.21 (m, 2H), 7.18 (d, J = 2.6 Hz, 1 H), 7.10 - 7.03 (m, 2H), 3.95 (s, 3H), 3.44 (t, J = 3.7 Hz, 1 H), 3.14 - 2.91 (m, 6H), 2.91 - 2.79 (m, 3H), 2.77 - 2.68 (m, 4H), 2.48 (dd, J = 14.6, 6.2 Hz, 1 H), 2.09 - 1 .87 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 170.2, 162.1 (d, JCF = 245.9 Hz), 158.9, 155.0 (d, JCF = 251 .4 Hz), 141 .4 (d, JCF = 2.3 Hz), 138.0 (d, J = 29.6 Hz), 133.6, 131.6, 129.6 (d, JCF = 8.0 Hz), 129.6 (d, JCF = 8.4 Hz), 129.3 (d, JCF = 3.7 Hz), 124.8 (d, JCF = 3.7 Hz), 121 .6, 120.5, 1 16.1 (d, JCF = 22.6 Hz), 102.0 (d, JCF = 5.5 Hz), 61.4, 56.3, 55.9, 54.9, 54.2, 51 .2, 47.9, 30.1 (d, JCF = 3.0 Hz), 25.5, 21 .9 (d, JCF = 3.2 Hz); IR (neat) cm^-1 : 3395, 2945, 2825, 1620, 1509, 1472, 1362, 1230, 756; HRMS (ESI) calcd for C26H30F2N3O3S [M + H]⁺ 502.1970 found 502.1968; = - 5.4 (c 0.50, CHCI₃).

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(2-((2- (trifluorome-thyl)phenyl)thio)ethyl)piperazine-2-carboxylic acid (58i). Following general procedure IV, methyl ester 57i

(40 mg, 0.071 mmol) was hydrolyzed to give the title compound as an off-white solid after preparative HPLC (27 mg, 70%). ¹ H NMR (400 MHz, CDCI₃) δ 9.00 (br s, 1 H), 8.57 (d, J = 0.8 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.64 (d, J = 7.8 Hz, 1 H), 7.55 - 7.41 (m, 2H), 7.37 - 7.27 (m, 2H), 7.17 (d, J = 2.7 Hz, 1 H), 3.94 (s, 3H), 3.44 (dd, J = 4.8, 3.5 Hz, 1 H), 3.15 - 2.99 (m, 5H), 2.99 - 2.88 (m, 2H), 2.88 - 2.78 (m, 2H), 2.78 - 2.67 (m, 4H), 2.52 (t, J = 8.6 Hz, 1 H), 2.09 - 1.90 (m, 2H); ¹³C NMR (100 MHz, CDCIs) δ 170.3, 158.9, 155.0 (d, JCF = 251.5 Hz), 141 .5 (d, JCF = 2.2 Hz), 138.0 (d, JCF = 29.5 Hz), 134.9, 132.4, 132.0, 131.7, 130.8 (q, J_CF = 30.1 Hz), 129.4, 129.2 (d, JCF = 3.7 Hz), 127.1 (q, J_CF = 5.6 Hz), 126.8, 123.8 (q, J_CF = 273.8 Hz), 120.5 (d, J_CF = 2.4 Hz), 102.0 (d, J_CF = 5.3 Hz), 62.1 , 56.1 , 55.8, 54.9, 54.1 , 51 .3, 48.1 , 31 .2, 25.3, 21.8 (d, JCF = 3.0 Hz); IR (neat) cm^-1: 3379, 2954, 2828, 1621 , 1509, 1469, 1313, 1231 , 1 129, 1034; HRMS (ESI) calcd for C27H30F4N3O3S [M + H]⁺ 552.1939 found 552.1937;

= + 2.4 (c 1.0, CHCI₃).

(R)-4-(2-((2,5-Difluorophenyl)thio)ethyl)-1 -(3-(3-fluoro-6- methoxy-quinolin-4-yl)propyl)piperazine-2-carboxylic acid

(58j). Following general procedure IV, methyl ester 57j (46

mg, 0.086 mmol) was hydrolyzed to give the title compound as an off-white solid after preparative HPLC (34 mg, 76%). ¹ H NMR (400 MHz, CDCI₃) δ 9.51 (br s, 1 H), 8.56 (d, J = 0.8 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.30 (dd, J = 9.2, 2.6 Hz, 1 H), 7.17 (d, J = 2.6 Hz, 1 H), 7.06 (ddd, J = 8.5, 5.7, 3.1 Hz, 1 H), 7.00 (ddd, J = 8.9, 4.6, 4.6 Hz, 1 H), 6.92 - 6.82 (m, 1 H), 3.95 (s, 3H), 3.44 (dd, J = 4.6, 3.5 Hz, 1 H), 3.14 - 2.98 (m, 6H), 2.93 (dd, J = 9.0, 3.9 Hz, 2H), 2.83 (td, J = 13.0, 6.9 Hz, 2H), 2.78 - 2.65 (m, 4H), 2.50 (t, J = 8.6 Hz, 1 H), 2.10 - 1 .87 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 170.3, 159.3 (dd, JCF = 97.6, 2.6 Hz), 158.9, 156.9 (d, JCF = 96.5 Hz), 155.0 (d, JCF = 251 .5 Hz), 141.46 (d, JCF = 2.2 Hz), 138.1 (d, JCF = 29.4 Hz), 131.7, 129.4 (d, JCF =

13.0 Hz), 129.3 (d, JCF = 3.8 Hz), 124.0 (dd, JCF = 20.3, 8.1 Hz), 120.4 (d, JCF = 2.5 Hz), 1 18.4 (d, JCF = 25.2 Hz), 1 16.7 (dd, JCF = 25.3, 8.9 Hz), 1 15.2 (dd, JCF = 24.0, 8.1 Hz), 102.0 (d, JCF = 5.4 Hz), 61.9, 56.2, 55.8, 54.9, 54.2, 51.2, 48.1 , 29.8, 25.4, 21.8 (d, JCF = 3.0 Hz); IR (neat) cm^-1: 3395, 2946, 2830, 1620, 1509, 1481 , 1230, 1 186, 757;

HRMS (ESI) calcd for C26H29F3N3O3S [M + H]⁺ 520.1876 found 520.1869.

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(2-phe- noxyethyl)-piperazine-2-carboxylic acid (58k). Following general procedure IV, methyl ester 57k (53 mg, 0.1 1 mmol) was

hydrolyzed to give the title compound as an off-white solid after preparative HPLC (38 mg, 74%). ¹ H NMR (400 MHz, CDCI₃) δ 9.43 (s, 1 H), 8.57 (d, J = 0.8 Hz, 1 H), 7.99 (d, J = 9.2 Hz, 1 H), 7.35 - 7.23 (m, 3H), 7.19 (d, J = 2.6 Hz, 1 H), 6.95 (t, J = 7.6 Hz, 1 H), 6.87 (d, J = 7.6 Hz, 2H), 4.12 (t, J = 5.0 Hz, 2H), 3.95 (s, 3H), 3.47 (dd, J = 3.8, 3.8 Hz, 1 H), 3.22 - 2.62 (m, 12H), 2.1 1 - 1.87 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 170.8, 158.9, 158.1 , 155.0 (d, J_CF = 251 .4 Hz), 141.4 (d, J_CF = 2.2 Hz), 138.0 (d, JC_F = 29.6 Hz), 131 .6, 129.7, 129.5, 129.3 (d, J_CF = 3.8 Hz), 121.5, 120.5 (d, J_CF = 2.5 Hz), 1 14.6, 102.0 (d, J_CF = 5.4 Hz), 64.1 , 61.5, 56.1 , 55.8, 54.8, 54.5, 51.7, 47.7, 25.5, 21.8 (d, JC_F = 3.1 Hz); IR (neat) cm^-1 : 3395, 2948, 2831 , 1620, 1509, 1467, 1231 , 1031 , 755; HRMS (ESI) calcd for C₂6H3₁ FN3O₄ [M + H]⁺ 468.2293 found 468.2291 ;

= + 9.9 (c 1.0, CHCIs).

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(2-(2- fluorophen-oxy)ethyl)piperazine-2-carboxylic acid (58I). Following general procedure V, amine 56 (32 mg, 0.089 mmol)

was reductively alkylated using fluorophenyl aldehyde 49 (41 mg, 0.27 mmol), and NaBH(OAc)₃ (94 mg, 0.44 mmol) in CH₂CI₂ (1 mL). The reaction mixture was concentrated in vacuo and the crude ester was hydrolyzed following general procedure IVto give the title compound as an off-white amorphous solid after preparative HPLC (2 mg, 4%). HRMS (ESI) calcd for C₂6H30F₂N3O₄ [M + H]⁺ 486.2199, found 486.2202.

(R)-4-(2-(2,5-Difluorophenoxy)ethyl)-1 -(3-(3-fluoro-6-meth- oxyquinolin-4-yl)propyl)piperazine-2-carboxylic acid (58m). Following general procedure IV, methyl ester 57m (35

mg, 0.068 mmol) was hydrolyzed to give the title compound as an off-white solid after preparative HPLC (20 mg, 59%). ¹ H NMR (400 MHz, CDCI₃) δ 8.91 (br. s, 1 H), 8.57 (d, J = 0.9 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.30 (dd, J = 9.2, 2.6 Hz, 1 H), 7.19 (d, J = 2.6 Hz, 1 H), 7.00 (ddd, J = 10.6, 9.0, 5.3 Hz, 1 H), 6.67 (ddd, J = 9.6, 6.7, 3.0 Hz, 1 H), 6.60 (ddt, J = 9.0, 7.8, 3.1 Hz, 1 H), 4.15 (t, J = 4.9 Hz, 3H), 3.95 (s, 3H), 3.46 (t, J = 3.6 Hz, 1 H), 3.21 - 3.10 (m, 1 H), 3.10 - 2.93 (m, 6H), 2.91 - 2.74 (m, 4H), 2.72 - 2.60 (m, 1 H), 2.1 1 - 1.89 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 170.4,

158.8, 158.6 (dd, JCF = 242.8, 2.5 Hz), 154.9 (d, JCF = 251.5 Hz), 149.0 (dd, JCF =

241 .9, 3.3 Hz), 146.7 (dd, J_CF = 12.6, 10.5 Hz), 141 .4 (d, J_CF = 2.2 Hz), 138.0 (d, J_CF = 29.5 Hz), 131 .6, 129.4 (d, J_CF = 12.6 Hz), 129.2 (d, J_CF = 3.8 Hz), 120.3 (d, J = 2.4 Hz), 1 16.5 (dd, J_CF = 20.7, 10.1 Hz), 107.6 (dd, J_CF = 23.7, 6.9 Hz), 103.2 (dd, J_CF = 27.4, 1 .6 Hz), 101.9 (d, JC_F = 5.4 Hz), 66.4, 61.3, 55.8, 55.7, 54.7, 54.5, 51.9, 47.8, 25.5, 21.7 (d, JCF = 3.1 Hz); IR (neat) cm^-1: 2947, 2927, 2843, 1621 , 151 1 , 1469, 1431 , 1362, 1324, 1264, 1230, 1206, 1 156, 1029, 833; + 7.0 (c 1.O, CHCI₃).

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-pentylpi- perazine-2-carboxylic acid (58n). Following general procedure

V, amine 56 (60.0 mg, 0.166 mmol) was reductively alkylated using

pentanal (41 mg, 0.27 mmol), and NaBH(OAc)₃ (53 mg, 0.25 mmol) in CH2CI2 (1.7 mL). The reaction mixture was concentrated in vacuo and the crude ester was hydrolyzed following general procedure IV \o give the title compound as an off-white amorphous solid after preparative HPLC (37 mg, 54%). ¹H NMR (400 MHz, CDCI₃) δ 8.56 (d, JHF = 0.7 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.30 (dd, J = 9.2, 2.6 Hz, 1 H), 7.21 (d, J = 2.6 Hz, 1 H), 3.96 (s, 3H), 3.43 - 3.36 (m, 1 H), 3.23 (d, J = 10.6 Hz, 1 H), 3.1 1 - 2.94 (m, 4H), 2.89 - 2.68 (m, 3H), 2.67 - 2.45 (m, 3H), 2.45 - 2.30 (m, 1 H), 2.06 - 1 .81 (m, 2H), 1 .64 - 1.49 (m, 2H), 1.38 - 1.20 (m, 4H), 0.89 (t, J = 6.9 Hz, 3H); ¹³C NMR (100 MHz, CDCI₃) 171δ.3, 158.6, 154.9 (d, J_CF = 251.3 Hz), 141 .5 (d, J_CF = 2.2 Hz), 138.1 (d, J_CF = 29.4 Hz), 131 .7, 129.8 (d, J_CF = 15.7 Hz), 129.3 (d, J_CF = 3.8 Hz), 120.1 , 102.0 (d, J_CF = 5.4 Hz), 60.0, 57.2, 55.7, 54.7, 54.4, 51.5, 46.7, 29.2, 26.1 , 24.8, 22.3, 21 .9 (d, J_CF = 3.0 Hz), 13.9; IR (neat) cm^-1: 3405, 2955, 2932, 2862, 1618, 1508, 1468, 1361 , 1228; HRMS (ESI) calcd for C23H33FN3O3 [M + H]⁺ 418.2501 , found 418.2490;

= +5.1 (c 1.0, in CHCI₃).

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-heptylpi- perazine-2-carboxylic acid (58o). Following general procedure

IV, methyl ester 57o (53 mg, 0.12 mmol) was hydrolyzed to give the

title compound as an off-white solid after preparative HPLC (27 mg, 53%). ¹H NMR (400 MHz, CDCI₃) δ 8.56 (d, J = 0.9 Hz, 1 H), 7.97 (d, J = 9.2 Hz, 1 H), 7.30 (dd, J = 9.2, 2.7 Hz, 1 H), 7.20 (d, J = 2.7 Hz, 1 H), 3.95 (s, 3H), 3.40 - 3.35 (m, 1 H), 3.30 - 3.26 (m, 1 H), 3.1 1 - 3.03 (m, 4H), 2.91 - 2.76 (m, 2H), 2.72 - 2.68 (m, 1 H), 2.65 - 2.49 (m, 3H), 2.43 - 2.37 (m, 1 H), 2.03 - 1.81 (m, 2H), 1.60 - 1.53 (m, 2H), 1.36 - 1.15 (m, 9H), 0.86 (t, J = 6.9 Hz, 3H); ¹³C NMR (100 MHz, CDCI₃) δ 171.8, 158.7, 155.0 (d, JCF = 251 .4 Hz), 141 .6 (d, JCF = 2.2 Hz), 138.2 (d, JCF = 29.5 Hz), 131 .8, 130.0 (d, JCF = 12.8 Hz), 129.4 (d, JCF = 3.8 Hz), 120.2 (d, JCF = 2.4 Hz), 102.2 (d, JCF = 5.3 Hz), 60.3, 57.4, 55.8, 54.8, 54.5, 51 .5, 46.6, 31 .7, 29.0, 27.1 , 26.3, 25.0, 22.6, 22.0 (d, JCF = 3.0 Hz), 14.2; IR (neat) cm^-1 : 3397, 2929, 2856, 1620, 1509, 1468, 1231 ; HRMS (ESI) calcd for C25H37FN3O3 [M + H]⁺ 446.2813 found 446.2816; = + 3.9 (c

1.0, CHCIs).

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-nonylpiper- azine-2-carboxylic acid (58p). Following general procedure V, amine 56 (60.0 mg, 0.17 mmol) was reductively alkylated using

nonal (43.0 μL, 0.25 mmol), and NaBH(OAc)₃ (53 mg, 0.25 mmol) in CH2CI2 (1.7 mL). The reaction mixture was concentrated in vacuo and the crude ester was hydrolyzed following general procedure IV \o give the title compound as an off-white amorphous solid after preparative HPLC (37.1 mg, 47%). ¹H NMR (400 MHz, CDCI₃) δ 8.57 (s, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.30 (dd, J = 9.2, 2.6 Hz, 1 H), 7.21 (d, J = 2.6 Hz, 2H), 2.88 - 2.77 (m, 1 H), 2.77 - 2.66 (m, 2H), 2.65 - 2.43 (m, 3H), 2.42 - 2.30 (m, 1 H), 2.09 - 1.79 (m, 2H), 1.65 - 1.47 (m, 2H), 1 .41 - 1 .15 (m, 12H), 0.87 (t, J = 6.8 Hz, 3H); ¹³C NMR (100 MHz, CDCI₃) δ 171.4, 158.7, 155.0 (d, JCF = 251 .3 Hz), 141.6 (d, JCF = 2.3 Hz), 138.3 (d, JCF = 29.5 Hz), 131 .8, 130.0 (d, J_CF = 12.8 Hz), 129.4 (d, J_CF = 4.0 Hz), 120.3 (d, J_CF = 2.5 Hz), 102.1 (d, J_CF = 5.5 Hz), 60.0, 57.3, 55.8, 54.8, 54.6, 51.7, 46.9, 32.0, 29.5, 29.4, 29.3, 27.2, 26.3, 25.3, 22.8, 22.0 (d, J_CF = 3.0 Hz), 14.2; IR (neat) cm^-1: 3418, 2923, 2852, 2815, 1619, 1509, 1468, 1321 , 1209; HRMS (ESI) calcd for C27H4i FN₃0₃ [M + H]⁺ 474.3127, found 474.3128; = + 4.1 (c 1.O, CHCI₃).

(R)-4-(Cyclopentylmethyl)-1 -(3-(3-fluoro-6-methoxyquinolin-4- yl)propyl)-piperazine-2-carboxylic acid (58q). Following general procedure V, amine 56 (60 mg, 0.17 mmol) was reductively alkyl¬

ated using cyclopentanecarbaldehyde (27 μί, 0.25 mmol), AcOH (1.0 μL, 0.017 mmol), and NaBH(OAc)₃ (53 mg, 0.25 mmol) in CH₂CI₂ (1 .7 mL). The reaction mixture was concentrated in vacuo and the crude ester was hydrolyzed following general procedure IVto give the title compound as an off-white amorphous solid after preparative HPLC (42 mg, 58%, over two steps). ¹H NMR (400 MHz, CDCI₃) δ 8.57 (d, JHF = 1.0 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.31 (dd, J = 9.2, 2.7 Hz, 1 H), 7.21 (d, J = 2.7 Hz, 1 H), 3.97 (s, 3H), 3.40 (t, J = 2.6 Hz, 1 H), 3.15 - 3.02 (m, 3H), 2.96 (d, J = 1 1.1 Hz, 1 H), 2.86 - 2.71 (m, 3H), 2.69 - 2.56 (m, 1 H), 2.52 - 2.40 (m, 3H), 2.41 - 2.28 (m, 1 H), 2.16 - 2.05 (m, 1 H), 2.05 - 1.87 (m, 2H), 1.86 - 1 .73 (m, 2H), 1 .68 - 1 .48 (m, 4H), 1 .22 - 1 .09 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 170.5, 158.8, 155.1 (d, JCF = 251.4 Hz), 141 .6 (d, JCF = 2.2 Hz), 138.2 (d, JCF = 29.4 Hz), 131.8, 129.9 (d, JCF = 12.7 Hz), 129.4 (d, JCF = 3.8 Hz), 120.4 (d, JCF = 2.5 Hz), 102.1 (d, JCF = 5.4 Hz), 63.1 , 59.8,

55.8, 54.9, 54.8, 52.3, 47.5, 36.3, 31.4, 31 .3, 26.1 , 25.3, 25.3, 22.0 (d, JCF = 3.2 Hz); IR (neat) cm^-1: 3406, 2948, 2866, 2828, 1619, 1508, 1454, 1322, 1229; HRMS (ESI) calcd for C24H33FN3O3 [M + H]⁺ 430.2501 , found 430.2503; = + 4.6 (c 1.0, in CHCI₃).

(R)-4-(cyclohexylmethyl)-1 -(3-(3-fluoro-6-methoxyquinolin-4- yl)propyl)pip-erazine-2-carboxylic acid (58r). Following general procedure V, amine 56 (60 mg, 0.17 mmol) was reductively alkyl¬

ated using cyclopentanecarbaldehyde (27 μL, 0.25 mmol), AcOH

(1.0 μL, 0.017 mmol), and NaBH(OAc)₃ (53 mg, 0.25 mmol) in CH2CI2 (1 .7 ml_). The reaction mixture was concentrated in vacuo and the crude ester was hydrolyzed following general procedure IV to give the title compound as an off-white amorphous solid after preparative HPLC (42 mg, 58%). Following general procedure IV, methyl ester 57r (32 mg, 0.069 mmol) was hydrolyzed to give the title compound as an off-white solid after preparative HPLC (24 mg, 77%, over two steps) ¹H NMR (400 MHz, CDCI₃) δ 9.58 (br s, 1 H), 8.57 (d, J = 1 .0 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.31 (dd, J = 9.2, 2.6 Hz, 1 H), 7.20 (d, J = 2.6 Hz, 1 H), 3.96 (s, 3H), 3.43 (dd, J = 3.2, 3.2 Hz, 1 H), 3.17 - 2.70 (m, 8H), 2.60 - 2.57 (m, 1 H), 2.47 - 2.40 (m, 2H), 2.34 (dd, J = 12.5, 7.2 Hz, 1 H), 2.10 - 1 .85 (m, 2H), 1 .81 - 1.63 (m, 5H), 1.61 - 1.52 (m, 1 H), 1.30 - 1.05 (m, 3H), 1.00 - 0.81 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 170.9, 158.9, 155.0 (d, JCF = 251 .3 Hz), 141 .4 (d, JCF = 2.3 Hz), 138.0 (d, JCF = 29.6 Hz), 131.6, 129.9 (d, JCF = 12.5 Hz), 129.4 (d, JCF = 3.8 Hz), 120.5 (d, JCF = 2.5 Hz), 102.0 (d, JCF = 5.4 Hz), 64.0, 60.4, 55.9, 54.9, 54.8, 52.2, 47.3, 34.0, 31.3, 26.3, 25.8, 25.7, 22.0 (d, JCF = 3.0 Hz); IR (neat) cm^-1: 3385, 2924, 2850, 1621 , 1509, 1468, 1231 ; HRMS (ESI) calcd for C25H35FN3O3 [M + H]⁺ 444.2657 found 444.2668; = + 2.4 (c 1.0, CHCI₃).

(R)-4-((4,4-Difluorocyclohexyl)methyl)-1 -(3-(3-fluoro-6-meth- oxyquinolin-4-yl)propyl)piperazine-2-carboxylic acid (58s). Following general procedure V, amine 56 (60.0 mg, 0.17 mmol)

was reductively alkylated using 4,4-difluorocyclo-hexanecarbalde- hyde (freshly prepared using general procedure /from (4,4-difluorocyclohexyl)metha- nol (35 μί, 0.33 mmol)), AcOH (1.0 μί, 0.017 mmol), and NaBH(OAc)₃ (88 mg, 0.41 mmol) in CH2CI2 (1.7 ml_). The reaction mixture was concentrated in vacuo and the crude ester was hydrolyzed following general procedure IV to give the title compound as a light brown solid after preparative HPLC (6 mg, 7%, over two steps). ¹H NMR (400 MHz, CDCI₃) δ 8.57 (d, JHF = 0.7 Hz, 1 H), 7.99 (d, J = 9.2 Hz, 1 H), 7.31 (dd, J = 9.2, 2.6 Hz, 1 H), 7.20 (d, J = 2.6 Hz, 1 H), 3.97 (s, 3H), 3.40 (t, J = 2.6 Hz, 1 H), 3.07 (t, J = 7.5 Hz, 2H), 3.00 - 2.82 (m, 2H), 2.82 - 2.55 (m, 4H), 2.45 (m, 1 H), 2.39 - 2.23 (m, 3H), 2.10 - 1 .59 (m, 9H), 1 .33 - 1.22 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 169.9, 158.8, 155.1 (d, JCF = 251.3 Hz), 141.6 (d, JCF = 2.3 Hz), 138.2 (d, JCF = 29.4 Hz), 131.9, 129.7 (d, JCF = 12.2 Hz), 129.4 (d, JCF = 3.7 Hz), 123.4 (dd, JCF = 242.7, 239.6 Hz), 120.4 (d, JCF = 2.5 Hz), 102.0 (d, J_CF = 5.4 Hz), 63.1 (d, J_CF = 2.2 Hz), 60.0, 55.8, 55.0, 54.9, 52.4, 47.8, 33.3 (dd, J_CF = 23.0, 3.6 Hz), 33.1 (dd, J_CF = 22.6, 3.2 Hz), 32.7, 27.50 (d, JCF = 9.7 Hz), 27.4 (d, J_CF = 9.7 Hz), 26.0, 22.0 (d, J_CF = 3.1 Hz); IR (neat) cm^-1 : 3397, 3071 , 2937, 2864, 2829, 1722, 1621 , 1509, 1468, 1372, 1231 , 1 1 14, 832, 756; HRMS (ESI) calcd for C25H33F3N3O3 [M + H]⁺ 480.2469, found 480.2461 ; = + 24.8

(R)-4-(3-(2,5-Difluorophenyl)propyl)-1 -(3-(3-fluoro-6-meth- oxyquinolin-4-yl)propyl)piperazine-2-carboxylic acid (59).

Methyl ester 57f (35 mg, 0.068 mmol) and 10% Pd/C (87 mg,

0.82 mmol) was dissolved in methanol under a hydrogen at- mosphere and stirred at rt for 1 h. The reaction mixture was then filtered through a pad of celite, concentrated in vacuo and redissolved in 0.3 M LiOH (aq) (THF:water (2:1 )) to a concentration of 0.1 M of the ester. After shaking in a closed reaction vial for 16 h at 65 °C, AcOH (3 equiv) was added, THF was removed under a flow of pressurized air, and the mixture was purified by preparative HPLC to give the title compound as an off- white amorphous solid (7.6 mg, 22% over two steps). ¹H NMR (400 MHz, CDCI₃) δ 8.56 (d, J = 0.9 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.31 (dd, J = 9.2, 2.6 Hz, 1 H), 7.19 (d, J = 2.6 Hz, 1 H), 7.00 - 6.91 (m, 1 H), 6.90 - 6.82 (m, 2H), 3.94 (s, 3H), 3.53 - 3.46 (m, 1 H), 3.35 - 3.19 (m, 2H), 3.13 - 2.87 (m, 5H), 2.83 - 2.49 (m, 7H), 1 .98 (q, J = 7.3 Hz, 2H), 1 .88 (q, J J = 7.4 Hz, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 170.9, 158.9, 158.7 (dd, JCF = 242.3, 2.2 Hz), 157.1 (dd, JCF = 240.4, 2.4 Hz), 155.0 (d, JCF = 251 .3 Hz), 141 .4, 138.0 (d, JCF = 29.5 Hz), 131.6, 130.0 (d, JCF = 1 1 .5 Hz), 129.4 (d, JCF = 3.9 Hz), 129.1 (d, JCF = 7.1 Hz), 120.5, 1 16.9 (dd, JCF = 23.8, 5.2 Hz), 1 16.5 (dd, JCF = 25.2, 8.7 Hz), 1 14.5 (dd, JCF = 23.9, 8.5 Hz), 102.1 (d, JCF = 5.3 Hz), 56.5, 55.8, 54.7, 54.0, 51.2, 46.7, 26.6, 25.6, 25.2, 21.8 (d, JCF = 3.0 Hz); IR (neat) cm^-1 : 2946, 2831 , 1673, 1621 , 1508, 1469, 1231 , 1207, 1 140, 1029, 831 , 753; HRMS (ESI) calcd for C27H31F3N3O3 [M + H]⁺ 502.2312, found 502.2306; = + 15.0 (c O.5, CHCI₃).

Methyl (R)-1 -(3-(3-fluoro-6-methoxyquinolin-4-yl)propyl)-4-(prop- 2-yn-1 -yl)-piperazine-2-carboxylate (60). Amine 56 (1.98 g, 5.48 mmol) was dissolved in DMF (32 mL), whereupon K2CO3 (1.14 g,

822 mmol) and propargyl bromide (0.650 mL, 5.48 mmol) were added and the reaction mixture was stirred for 21 h. Water (200 mL) and CH2CI2 (200 mL) were added and the layers were separated. The aqueous phase was extracted with EtOAc (2 x 200 mL) and the combined organic layers were dried over Na2S0₄, concentrated in vacuo, and purified by flash column chromatography (EtOAc:heptane (2:1 ), f?_f = 0.35) to give the title compound as an orange/brown amorphous solid (1.31 g, 60%). ¹H NMR (400 MHz, CDCI₃) δ 8.58 (d, J_HF = 1 .0 Hz, 1 H), 7.99 (d, J = 9.2 Hz, 1 H), 7.31 (dd, J = 9.2, 2.7 Hz, 1 H), 7.19 (d, J = 2.7 Hz, 1 H), 3.96 (s, 3H), 3.67 (s, 3H), 3.35 (t, J = 4.8 Hz, 1 H), 3.31 (d, J = 2.4 Hz, 2H), 3.18 - 2.96 (m, 3H), 2.88 - 2.72 (m, 3H), 2.59 (t, J = 5.0 Hz, 2H), 2.57 - 2.50 (m, 1 H), 2.50 - 2.42 (m, 1 H), 2.25 (t, J = 2.4 Hz, 1 H), 1 .87 (p, J = 7.4 Hz, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 172.5, 158.7, 155.1 (d, JCF = 251 .4 Hz), 141.7 (d, JCF = 2.3 Hz), 138.3 (d, JCF = 29.4 Hz), 131 .9, 130.0 (d, JCF = 13.0 Hz), 129.3 (d, JCF = 3.9 Hz), 120.3 (d, JCF = 2.6 Hz), 102.0 (d, JCF = 5.4 Hz), 78.2, 73.8, 63.4, 55.8, 55.2, 54.4, 51 .8, 51.5, 48.6, 46.9, 26.7, 22.1 (d, JCF = 3.1 Hz); IR (neat) cm^-1: 3296, 2949, 2829, 1737, 1620, 1508, 1467, 1320, 1227, 1 133; HRMS (ESI) calcd for C22H27F2N3O3 [M + H]⁺.2031 , found 400.2029; = + 17.1 (c 1.0, in CHCI₃).

General procedure VI: Sonogashira cross-coupling on alkyne 60

Methyl (R)-1 -(3-(3-fluoro-6-methoxyquinolin-4-yl)propyl)-4- (3-(3-fluoro-phenyl)prop-2-yn-1 -yl)piperazine-2-carboxylate (61a). Alkyne 60 (190 mg, 0.497 mmol) was dissolved in THF (3

mL) and EtsN (2 mL), whereupon 1-fluoro-3-iodobenzene (158 mg, 0.713 mmol) was added, and the mixture was degassed with argon for 40 min. Then Cul (5 mg, 0.02 mmol) and Pd(PPh₃)₄ (17 mg, 0.014 mmol) were added and the reaction mixture was degassed with argon for 30 min and stirred at rt under an argon atmosphere for 25 h. Water (10 mL) and CH2CI2 (15 mL) were added, the layers were separated, and the aqueous phase was extracted with CH2CI2 (3 x 15 mL). The combined organic layers were dried using Na2S0₄, concentrated in vacuo, and purified by flash column chromatography on silica gel (EtOAc:heptane (1 :1 ), R_f = 0.2) to give the title compound as a yellow oil (209 mg, 89%). ¹H NMR (400 MHz, CDCI₃) δ 8.65 (s, 1 H), 8.06 (d, J = 9.2 Hz, 1 H), 7.40 - 7.25 (m, 4H), 7.19 (d, J = 9.5 Hz, 1 H), 7.12 - 7.03 (m, 1 H), 4.01 (s, 3H), 3.76 (s, 3H), 3.60 (s, 2H), 3.49 - 3.43 (m, 1 H), 3.30 - 3.04 (m, 3H), 3.00 - 2.84 (m, 3H), 2.74 (t, J = 4.8 Hz, 2H), 2.69 - 2.50 (m, 2H), 1.96 (p, J = 7.4 Hz, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 172.4, 162.4 (d, JCF = 246.4 Hz), 158.6, 155.0 (d, JCF = 251.5 Hz), 141.6 (d, JCF = 2.2 Hz), 138.2 (d, JCF =29.4 Hz), 131.8, 129.9 (d, JCF = 8.7 Hz), 129.9 (d, JCF =13.0 Hz), 129.2 (d, JCF =3.8 Hz), 127.7 (d, JCF =3.0 Hz), 124.9 (d, JCF =9.5 Hz), 120.2 (d, J_CF =2.6 Hz), 1 18.6 (d, J_CF =22.7 Hz), 1 15.6 (d, J_CF =21.1 Hz), 102.0 (d, J_CF =5.4 Hz), 85.0, 84.6 (d, J_CF =3.3 Hz), 63.4, 55.6, 55.1 , 54.6, 51.8 (d, JCF =7.6 Hz), 48.7, 47.6, 26.6, 22.0 (d, J_CF =3.1 Hz).

Methyl (R)-1 -(3-(3-fluoro-6-methoxyquinolin-4-yl) propyl)- 4-(3-(p-tolyl)prop-2-yn-1 -yl)piperazine-2-carboxylate (61 b). Following general procedure VI, alkyne 60 (300 mg, 0.75

mmol) and 4-fluoro-iodobenzene (130 μL, 1 .1 mmol) were coupled using Cul (21 mg, 0.1 1 mmol) and Pd(PPh₃)₄ (87 mg, 0.075 mmol) in THF:Et₃N (3:2) (8 mL) to give the title compound as a yellow oil (286 mg, 76%) after purification by flash column chromatography on silica gel (EtOAc:heptane (1 :1 ), R_f = 0.2). ¹H NMR (400 MHz, CDCI₃) δ 8.57 (d, JHF = 0.8 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.43 - 7.36 (m, 2H), 7.30 (dd, J = 9.2, 2.7 Hz, 1 H), 7.18 (d, J = 2.7 Hz, 1 H), 7.01 - 6.95 (m, 2H), 3.93 (s, 3H), 3.67 (s, 3H), 3.51 (s, 2H), 3.37 (t, J = 4.8 Hz, 1 H), 3.20 - 2.95 (m, 3H), 2.89 - 2.76 (m, 3H), 2.70 - 2.61 (m, 2H), 2.61 - 2.43 (m, 2H), 1.94 - 1.83 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 172.4, 162.5 (d, JCF = 249.2 Hz), 158.7, 155.0 (d, JCF = 251 .4 Hz), 141 .7 (d, JCF = 2.3 Hz), 138.3 (d, JCF = 29.4 Hz), 133.7 (d, JCF = 8.3 Hz), 131.9, 129.9 (d, JCF = 13.0 Hz), 129.3 (d, JCF = 3.8 Hz), 120.3 (d, JCF = 2.6 Hz), 1 19.2 (d, JCF = 3.6 Hz), 1 15.7 (d, JCF = 22.0 Hz), 102.0 (d, J_CF = 5.5 Hz), 84.8, 83.6, 63.6, 55.7, 55.2, 54.7, 51.8, 51.8, 48.8, 47.7, 26.6, 22.1 (d, J_CF = 3.1 Hz); IR (neat) cm^-1: 3072, 2950, 2830, 1740, 1679, 1621 , 1507, 1468, 1229, 1 155, 834; HRMS (ESI) calcd for C28H30F2N3O3 [M + H]⁺ 494.2250, found 494.2250; = + 7.4 (c 1 .0, in CHCI₃).

Methyl (R)-4-(3-(3,4-difluorophenyl)prop-2-yn-1 -yl)-1 -(3-(3- fluoro-6-methoxyquinolin-4-yl)propyl)piperazine-2-carbox- ylate (61 c). Following general procedure VI, alkyne 60 (190

mg, 0.497 mmol) and 1 ,2-difluoro-4-iodobenzene (171 mg, 0.713 mmol) were coupled using Cul (5 mg, 0.02 mmol) and Pd(PPh₃)₄ (17 mg, 0.014 mmol in THF:Et₃N (3:2) (5 mL) to give the title compound as a yellow oil (214 mg, 88%) after purification by flash column chromatography on silica gel (EtOAc:heptane (1 :1 ), R_f = 0.2). ¹H NMR (400 MHz, CDCI₃) δ 8.57 (s, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.30 (dd, J = 9.2, 2.7 Hz, 1 H), 7.25 - 7.17 (m, 2H), 7.16 - 7.12 (m, 1 H), 7.1 1 - 7.03 (m, 1 H), 3.93 (s, 3H), 3.67 (s, 3H), 3.49 (s, 2H), 3.40 - 3.31 (m, 1 H), 3.19 - 2.96 (m, 3H), 2.91 - 2.74 (m, 3H), 2.64 (t, J = 4.9 Hz, 2H), 2.59 - 2.43 (m, 2H), 1.88 (p, J = 7.4 Hz, 2H); ¹³C NMR (100 MHz, CDCI₃) 1δ72.4, 158.7, 155.0 (d, JCF = 251 .4 Hz), 151.5 (dd, JCF = 59.4, 12.8 Hz), 149.1 (dd, JCF = 57.0, 12.8 Hz), 141.7 (d, JCF = 2.3 Hz), 138.3 (d, JCF = 29.4 Hz), 131 .9, 129.9 (d, J_CF =13.0 Hz), 129.3 (d, J_CF =3.9 Hz), 128.4 (dd, J_CF = 6.4, 3.6 Hz), 120.8 (d, JCF = 18.3 Hz), 120.2 (d, J_CF = 2.6 Hz), 1 19.9 (dd, J_CF = 7.7, 4.2 Hz), 1 17.5 (d, JCF = 18.5 Hz), 102.1 (d, J_CF = 5.4 Hz), 84.7 (d, J_CF = 1.7 Hz), 83.80 (dd, J_CF = 2.4, 1 .8 Hz), 63.5, 55.7, 55.2, 54.7, 51 .8, 51 .8, 48.8, 47.6, 26.6, 22.1 (d, J_CF = 3.1 Hz).

Methyl (R)-4-(3-(2,6-difluorophenyl)prop-2-yn-1 -yl)-1 -(3-(3- fluoro-6-methoxyquinolin-4-yl)propyl)piperazine-2-car- boxylate (61 d). Following general procedure VI, alkyne 60

(190 mg, 0.497 mmol) and 1 ,3-difluoro-2-iodobenzene (171 mg, 0.713 mmol) were coupled using Cul (5 mg, 0.02 mmol) and Pd(PPh₃)4 (28 mg, 0.024 mmol) in THF:EtsN (3:2) (5 mL) to give the title compound as a yellow oil (99 mg, 41 %) after purification by flash column chromatography on silica gel (EtOAc:heptane (1 :1 ), R_f = 0.2). ¹H NMR (400 MHz, CDCI₃) δ 8.56 (s, 1 H), 7.97 (d, J = 9.2 Hz, 1 H), 7.28 (dd, J = 9.2, 2.7 Hz, 1 H), 7.24 - 7.19 (m, 1 H), 7.17 (d, J = 2.7 Hz, 1 H), 6.87 (dd, J = 8.3, 7.1 Hz, 2H), 3.92 (s, 3H), 3.67 (s, 3H), 3.60 (d, J = 3.3 Hz, 2H), 3.42 - 3.37 (m, 1 H), 3.22 - 3.14 (m, 1 H), 3.12 - 2.99 (m, 2H), 2.98 - 2.89 (m, 1 H), 2.89 - 2.80 (m, 2H), 2.68 (t, J = 5.0 Hz, 2H), 2.63 - 2.47 (m, 2H), 1 .92 - 1 .77 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 172.4, 163.3 (dd, JCF = 253.3, 5.3 Hz), 158.7, 155.0 (d, JCF = 251.4 Hz), 141.6 (d, JCF = 2.2 Hz), 138.2 (d, JCF = 29.4 Hz), 131.7, 130.0 (d, J_CF = 13.0 Hz), 129.7 (t, J_CF = 9.9 Hz), 129.3 (d, JCF = 3.8 Hz), 120.4 (d, J_CF = 2.6 Hz), 1 1 1 .3 (d, J_CF = 5.6 Hz), 1 1 1 .1 (d, J_CF = 5.7 Hz), 102.1 (d, J_CF =1 9.8 Hz), 101 .9 (d, J_CF = 5.5 Hz), 94.4 (t, J_CF = 3.1 Hz), 72.8, 63.1 , 55.6, 55.0, 54.4, 51.7 (d, J_CF = 7.0 Hz), 48.5, 47.8, 26.7, 22.0 (d, J_CF = 3.1 Hz); IR (neat) cm^-1: 2951 , 2831 , 1738, 1620, 1508, 1468, 1321 , 1229, 1031 , 1003, 910, 830, 784, 732.

Methyl (R)-1 -(3-(3-fluoro-6-methoxyquinolin-4-yl)propyl)-4- (3-(2,4,6-trifluorophenyl)prop-2-yn-1 -yl)piperazine-2 -car- boxy I ate (61 e). Following general procedure VI, alkyne 60

(190 mg, 0.497 mmol) and 1 ,3,6-trifluoro-2-iodobenzene (245 mg, 0.951 mmol) were coupled using Cul (9 mg, 0.05 mmol) and Pd(PPhi3)₄ (44 mg, 0.038 mmol in THF:EtsN (3:2) (5 mL) to give the title compound as a yellow oil (160 mg, 63%) after purification by flash column chromatography on silica gel (EtOAc:hep- tane (1 :1 ), R_f = 0.2).¹H NMR (400 MHz, CDCI₃) δ 8.57 (d, J = 0.8 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.30 (dd, J = 9.2, 2.7 Hz, 1 H), 7.18 (d, J = 2.7 Hz, 1 H), 6.67 (dd, J = 8.6, 7.1 Hz, 2H), 3.94 (s, 3H), 3.68 (s, 3H), 3.59 (d, J = 3.1 Hz, 2H), 3.43 - 3.37 (m, 1 H), 3.22 - 3.13 (m, 1 H), 3.12 - 2.98 (m, 2H), 2.97 - 2.78 (m, 3H), 2.68 (t, J = 4.9 Hz, 2H), 2.63 - 2.48 (m, 2H), 1 .93 - 1.81 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 172.4, 163.6 (ddd, JCF = 254.2, 14.7, 8.0 Hz), 162.2 (dd, J_CF = 252.5, 14.8 Hz), 158.7, 155.1 (d, J_CF = 251 .4 Hz), 141.6 (d, JCF = 2.3 Hz), 138.3 (d, JCF = 29.4 Hz), 131 .8, 130.0 (d, J_CF = 13.0 Hz), 129.3 (d, JCF = 3.9 Hz), 120.3 (d, J_CF = 2.6 Hz), 101 .9 (d, J_CF = 5.4 Hz), 100.95 - 100.10 (m), 98.8 (td, JCF = 20.2, 4.8 Hz), 94.2 (dd, JCF = 5.4, 3.1 Hz), 71.8, 63.2, 55.7, 55.1 , 54.4, 51 .7, 51.7, 48.5, 47.8, 26.7, 22.0 (d, JCF = 3.1 Hz); IR (neat) cm^-1: 2951 , 2832, 1739, 1620, 1501 , 1443, 1322, 1229, 1 124, 1039, 999, 831 , 732.

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(3-(3-flu- orophen-yl)prop-2-yn-1 -yl)piperazine-2-carboxylic acid (62a). Following general procedure IV, methyl ester 61a (36

mg, 0.072 mmol) was hydrolyzed to give the title compound as an off-white amorphous solid after preparative HPLC (24 mg, 68%). ¹ H NMR (400 MHz, CDCIs) δ 8.57 (d, J = 0.8 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.34 - 7.23 (m, 2H), 7.23 - 7.15 (m, 2H), 7.15 - 7.09 (m, 1 H), 7.06 - 6.99 (m, 1 H), 3.95 (s, 3H), 3.65 - 3.41 (m, 3H), 3.14 - 2.95 (m, 4H), 2.93 - 2.76 (m, 5H), 2.76 - 2.66 (m, 1 H), 2.25 - 1.76 (m, 2H); ¹³C NMR (100 MHz, CDCIs) δ 170.2, 162.4 (d, JCF = 246.9 Hz), 158.9, 155.0 (d, JCF = 251 .5 Hz), 141 .5 (d, JCF = 2.3 Hz), 138.1 (d, J_CF = 29.4 Hz), 131.8, 130.2 (d, J_CF = 8.6 Hz), 129.4, 129.2, 127.9 (d, J_CF = 3.1 Hz), 124.2 (d, J_CF = 9.4 Hz), 120.4 (d, J_CF = 2.5 Hz), 1 18.8 (d, JCF = 22.8 Hz), 1 16.2 (d, JCF = 21 .1 Hz), 101 .9 (d, J_CF = 5.4 Hz), 85.9 (d, J_CF = 3.2 Hz), 82.8, 61.7, 55.8, 54.9, 53.1 , 50.5, 48.3, 46.9, 25.6, 21.8 (d, J_CF = 3.1 Hz); IR (neat) cm^-1: 2943, 2833, 1620, 1580, 1509, 1469, 1361 , 1231 , 1 150, 1028, 910, 787, 731 ; HRMS (ESI) calcd for C27H28F2N3O3 [M + H]⁺ 480.2093 found 480.2089.

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(3-(4- fluoro-phenyl)prop-2-yn-1 -yl)piperazine-2-carboxylic acid (62b). Following general procedure IV, methyl ester 61 b (63

mg, 0.13 mmol) was hydrolyzed to give the title compound as an off-white amorphous solid after preparative HPLC (32 mg, 53%). ¹ H NMR (400 MHz, CDCIs) δ 9.12 (br s, 1 H), 8.57 (d, J_HF = 0.7 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.46 - 7.36 (m, 2H), 7.30 (dd, J = 9.2, 2.6 Hz, 1 H), 7.15 (d, J = 2.6 Hz, 1 H), 7.07 - 6.93 (m, 2H), 3.93 (s, 3H), 3.67 - 3.35 (m, 3H), 3.25 - 3.15 (m, 1 H), 3.13 - 2.71 (m, 9H), 2.13 - 1.95 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 170.1 , 162.7 (d, JCF = 250.2 Hz), 158.9, 154.9 (d, JCF = 251.6 Hz), 141.3 (d, JC JCF F = 2.3 Hz), 137.9 (d, JCF = 29.5 Hz), 133.8 (d, JCF = 8.4 Hz), 131 .6, 129.0 (d, JC JCF F = 3.6 Hz), 128.9 (d, JCF = 12.3 Hz), 120.4 (d, JCF = 2.5 Hz), 1 18.3 (d, JCF = 3.5 Hz), 1 15.7 (d, J_CF = 22.1 Hz), 101 .7 (d, Jb_F = 5.4 Hz), 86.1 , 81.2, 62.8, 55.7, 54.9, 52.8, 49.7, 48.6, 46.8, 24.9, 21 .6 (d, J_CF = 3.0 Hz); IR (neat) cm ¹: 341 1 , 2946, 2831 , 1720, 1620, 1506, 1468, 1362, 1229, 1 155, 834, 754; HRMS (ESI) calcd for C27H28F2N3O3 [M + H]⁺ 480.2093, found 480.2090; = + 6.0 (c 1.0,

CHCIs).

(R)-4-(3-(3,4-Difluorophenyl)prop-2-yn-1 -yl)-1 -(3-(3-fluoro- 6-methoxy-quinolin-4-yl)propyl)piperazine-2-carboxylic acid (62c). Following general procedure IV, methyl ester 61 c

(30 mg, 0.059 mmol) was hydrolyzed to give the title com- pound as an off-white amorphous solid after preparative HPLC (27 mg, 92%). ¹H NMR (400 MHz, CDCI₃) δ 8.62 - 8.53 (m, 1 H), 7.99 (d, J = 9.2 Hz, 1 H), 7.31 (dd, J = 9.2, 2.7 Hz, 1 H), 7.25 - 7.15 (m, 3H), 7.14 - 7.06 (m, 1 H), 3.96 (s, 3H), 3.62 - 3.52 (m, 2H), 3.52 - 3.42 (m, 1 H), 3.14 - 2.97 (m, 3H), 2.96 - 2.59 (m, 7H), 2.13 - 1.87 (m, 2H); IR (neat) cm^-1 : 2944, 2834, 1672, 1620, 1513, 1469, 1231 , 732; HRMS (ESI) calcd for

C27H27F3N3O3 [M + H]⁺ 498.1999 found 498.1988.

(R)-4-(3-(2,6-Difluorophenyl)prop-2-yn-1 -yl)-1 -(3-(3-fluoro-6- methoxy-quinolin-4-yl)propyl)piperazine-2-carboxylic acid (62d). Following general procedure IV, methyl ester 61 d (25

mg, 0.049 mmol) was hydrolyzed to give the title compound as an off-white amorphous solid after preparative HPLC (12 mg, 50%). ¹ H NMR (400 MHz, CDCI₃) δ 8.58 (d, J = 0.9 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.33 - 7.27 (m, 2H), 7.20 (d, J = 2.7 Hz, 1 H), 6.96 - 6.87 (m, 2H), 3.96 (s, 3H), 3.71 (d, J = 1 .9 Hz, 2H), 3.50 (t, J = 2.9 Hz, 1 H), 3.07 (t, J = 7.7 Hz, 3H), 2.95 - 2.73 (m, 6H), 2.72 - 2.61 (m, 1 H), 2.1 1 - 1 .81 (m, 2H); IR (neat) (neat) cm^-1: 2952, 1674, 1621 , 1509, 1468, 1232, 1004; HRMS (ESI) calcd for C27H27F3N3O3 [M + H]⁺ 498.1999 found 498.1989.

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(3- (2,4,6-trifluoro-phenyl)prop-2-yn-1 -yl)piperazine-2-carbox- ylic acid (62e). Following general procedure IV, methyl ester

61e (40 mg, 0.076 mmol) was hydrolyzed to give the title compound as an off-white amorphous solid after preparative HPLC (13 mg, 34%). ¹H NMR (400 MHz, CDCI₃) δ 8.57 (d, J = 0.9 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.31 (dd, J = 9.2, 2.7 Hz, 1 H), 7.20 (d, J = 2.7 Hz, 1 H), 6.70 (dd, J = 8.6, 7.1 Hz, 2H), 3.96 (s, 3H), 3.68 (d, J = 2.6 Hz, 2H), 3.49 (t, J = 2.9 Hz, 1 H), 3.1 1 - 3.02 (m, 3H), 2.92 - 2.84 (m, 1 H), 2.84 - 2.70 (m, 5H), 2.70 - 2.59 (m, 1 H), 2.10 - 1.97 (m, 1 H), 1.96 - 1.83 (m, 1 H); IR (neat) (neat) cm^-1 : 3072, 2928, 2857, 1674, 1638, 1505, 1444, 1232, 1 126, 1040; HRMS (ESI) calcd for C27H26F4N3O3 [M + H]⁺ 516.1905 found 516.1902. General procedure VII: Hydrogenation of alkynes and subsequent hydrolysis of methyl ester

(R,Z )-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(3-(3- fluorophen-yl)allyl)piperazine-2-carboxylic acid (63a). Alkyne 61 a (37 mg, 0.068 mmol) and 10% Pd/C (1 mg, 0.001 mmol) was

dissolved in methanol under a hydrogen atmosphere and stirred at rt for 1 h. The reaction mixture was then filtered through a pad of celite, concentrated in vacuo and redissolved in 0.3 M LiOH (THF:water (2:1 )) to a concentration of 0.1 M of the ester. After shaking in a closed reaction vial for 16 h at 65 °C, AcOH (3 equiv) was added, THF was removed under a flow of pressurized air, and the mixture was purified by preparative HPLC to give the title compound as an off-white amorphous solid (14 mg, 39% over two steps). ¹H NMR (400 MHz, CDCI₃) δ 8.56 (d, J = 0.9 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.36 - 7.28 (m, 2H), 7.20 (d, J = 2.7 Hz, 1 H), 7.03 - 6.91 (m, 2H), 6.87 (dd, J = 9.8, 1.7 Hz, 1 H), 6.68 (d, J = 1 1.7 Hz, 1 H), 5.76 (dt, J = 12.0, 6.6 Hz, 1 H), 3.95 (s, 3H), 3.45 - 3.37 (m, 3H), 3.14 - 3.01 (m, 3H), 2.98 - 2.89 (m, 1 H), 2.86 - 2.72 (m, 3H), 2.72 - 2.62 (m, 1 H), 2.45 (dd, J = 1 1.5, 2.2 Hz, 1 H), 2.35 (td, J = 1 1.0, 4.2 Hz, 1 H), 2.07 - 1 .82 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 170.3, 162.7 (d, J_CF = 246.4 Hz), 158.6, 154.9 (d, JCF = 251 .4 Hz), 141 .4, 138.2, 138.1 (d, JCF = 29.4 Hz), 132.9, 131 .7, 130.0 (d, JCF = 8.5 Hz), 129.7 (d, JCF = 12.8 Hz), 129.3 (d, JCF = 3.7 Hz), 126.5, 124.5 (d, JCF = 2.9 Hz), 120.2, 1 15.5 (d, JCF = 21.6 Hz), 1 14.5 (d, JCF = 21 .1 Hz), 102.0 (d, JCF = 5.5 Hz), 60.1 , 55.7, 54.6, 54.6, 53.8, 51.8, 47.4, 25.9, 21 .8; IR (neat) cm^-1: 3382, 3012, 2944, 2832, 1674, 1620, 1580, 1509, 1469, 1231 , 732; HRMS (ESI) calcd for C27H30F2N3O3 [M + H]⁺ 482.2250 found 482.2244. (R ,Z)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(3-(4- fluo-rophen-yl)allyl)piperazine-2-carboxylic acid (63b). Fol- lowing general procedure VII, alkyne 61 b (109 mg, 0.220

mmol) was hydrogenated using 10% Pd/C (2 mg, 0.002 mmol) and hydrolyzed to give the title compound as an off-white amorphous solid after preparative HPLC (13 mg, 12%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.66 (s, 1 H), 7.94 (d, J = 9.1 Hz, 1 H), 7.41 - 7.28 (m, 4H), 7.21 -7.16 (m, 2H), 6.55 (d, J = 1 1 .9 Hz, 1 H), 5.68 (dt, J = 1 1 .8, 6.2 Hz, 1 H), 3.93 (s, 3H), 3.22 - 3.16 (m, 1 H), 3.13 (d, J = 5.9 Hz, 2H), 3.10 - 2.96 (m, 3H), 2.95 - 2.80 (m, 1 H), 2.70 - 2.56 (m, 2H), 2.48 - 2.42 (m, 2H), 2.32 (m, 1 H), 1.85 - 1.69 (m, 2H), 1.23 (s, 1 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 172.0, 161 .1 (d, JCF = 244.3 Hz), 158.2, 154.5 (d, JCF = 250.1 Hz), 141.0 (d, JCF = 2.1 Hz), 137.9 (d, JCF = 29.0 Hz), 133.0 (d, JCF = 3.2 Hz), 131.3, 130.8 (d, JCF = 8.1 Hz), 130.1 , 129.8 (d, JCF = 13.1 Hz), 129.2, 128.8 (d, JCF = 4.0 Hz), 120.5 (d, JCF = 2.3 Hz), 1 15.1 (d, JCF = 21.3 Hz), 102.4 (d, JCF = 5.3 Hz), 62.9, 55.6, 55.2, 55.1 , 54.2, 52.1 , 47.9, 26.0, 21.3 (d, JCF = 2.5 Hz); IR (neat) cm^-1 : 3380, 3012, 2928, 2830, 1721 , 1620, 1603, 1508, 1468, 1229, 1 157, 831 , 754; HRMS (ESI) calcd for C27H30F2N3O3 [M + H]⁺ 482.2250, found 482.2252; [a]¾° = -16.2 (c O.5, in CHCI₃).

(R ,Z)-4-(3-(3,4-difluorophenyl)allyl)-1 -(3-(3-fluoro-6-me- thoxyquinolin-4-yl)propyl)piperazine-2-carboxylic acid (63c). Following general procedure VII, alkyne 61 c (43 mg,

0.83 mmol) was hydrogenated using 10% Pd/C (1 mg, 0.001 mmol) and hydrolyzed to give the title compound as an off-white amorphous solid after preparative HPLC (21 mg, 51 %). ¹ H NMR (400 MHz, CDCI₃) δ 8.56 (d, J = 1.0 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.30 (dd, J = 9.2, 2.7 Hz, 1 H), 7.19 (d, J = 2.6 Hz, 1 H), 7.17 - 7.1 1 (m, 1 H), 7.03 - 6.96 (m, 1 H), 6.92 - 6.82 (m, 1 H), 6.61 (d, J = 1 1.7 Hz, 1 H), 5.82 - 5.68 (m, 1 H), 3.94 (s, 3H), 3.46 - 3.35 (m, 3H), 3.15 - 3.02 (m, 3H), 3.00 - 2.86 (m, 2H), 2.85 - 2.70 (m, 3H), 2.58 - 2.49 (m, 1 H), 2.45 - 2.35 (m, 1 H), 2.06 - 1.83 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 170.6, 158.7, 154.9 (d, JCF = 251 .4 Hz), 150.1 (dd, JCF = 248.7, 12.7 Hz), 149.7 (dd, JCF = 249.8, 12.5 Hz), 141 .4 (d, JCF = 2.2 Hz), 138.0 (d, JCF = 29.5 Hz), 133.0 (d, JCF = 5.8 Hz), 132.9 (d, JCF = 5.9 Hz), 132.1 , 131.6, 129.6 (d, JCF = 12.8 Hz), 129.2 (d, JCF = 3.8 Hz), 126.3, 125.0 (dd, JCF = 6.1 , 3.6 Hz), 120.2 (d, JCF = 2.5 Hz), 1 17.5 (t, JCF = 16.7 Hz), 102.0 (d, J_CF = 5.4 Hz), 60.7, 55.7, 54.6, 54.5, 53.8, 51.5, 47.4, 25.7, 21.8 (d, J_CF = 3.1 Hz); IR (neat) cm^-1 : 2943, 2833, 1673, 1620, 1513, 1429, 1276, 1231 , 732; HRMS (ESI) calcd for C27H29F3N3O3 [M + H]⁺ 500.2156 found 500.2143. (R ,Z)-4-(3-(2,6-Difluorophenyl)allyl)-1 -(3-(3-fluoro-6-methox- yquinolin-4-yl)propyl)piperazine-2-carboxylic acid (63d). Following general procedure VII, alkyne 61 d (25 mg, 0.049 mmol) was hydrogenated using 10% Pd/C (1 mg, 0.001 mmol) and hy-

drolyzed to give the title compound as an off-white amorphous solid after preparative HPLC (8 mg, 33%). ¹H NMR (400 MHz, CDCI₃) δ 8.57 (d, J = 1 .0 Hz, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.32 - 7.27 (m, 2H), 7.20 (d, J = 2.7 Hz, 1 H), 6.95 - 6.88 (m, 1 H), 6.45 (d, J = 1 1 .5 Hz, 1 H), 5.97 (dt, J = 1 1 .5, 6.7 Hz, 1 H), 3.95 (s, 3H), 3.40 (t, J = 2.8 Hz, 1 H), 3.17 - 3.12 (m, 2H), 3.09 - 3.00 (m, 3H), 2.92 - 2.86 (m, 1 H), 2.77 (td, J = 12.6, 5.9 Hz, 3H), 2.67 - 2.58 (m, 1 H), 2.41 (dd, J = 1 1 .6, 2.4 Hz, 1 H), 2.32 (td, J = 10.9, 5.1 Hz, 1 H), 2.07 - 1 .80 (m, 2H); IR (neat) cm^-1 : 2943, 1674, 1621 , 1509, 1465, 1232, 995, 906, 730; HRMS (ESI) calcd for C27H29F3N3O3 [M + H]⁺ 500.2156 found 500.2157.

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(3-(3- fluorophenyl)-propyl)piperazine-2-carboxylic acid (64a). Following general procedure VII, alkyne 61 a (32 mg, 0.066 mmol)

was hydrogenated using 10% Pd/C (7 mg, 0.007 mmol) and hy- drolyzed to give the title compound as an off-white amorphous solid after preparative HPLC (14 mg, 44%). ¹ H NMR (400 MHz, CDCI₃) δ 8.57 (d, J = 1.0 Hz, 1 H), 7.99 (d, J = 9.2 Hz, 1 H), 7.31 (dd, J = 9.2, 2.7 Hz, 1 H), 7.26 - 7.22 (m, 1 H), 7.22 - 7.19 (m, 1 H), 6.96 - 6.83 (m, 3H), 3.96 (s, 3H), 3.43 (t, J = 3.1 Hz, 1 H), 3.16 (d, J = 1 1 .3 Hz, 1 H), 3.07 (t, J = 7.2 Hz, 2H), 3.02 - 2.92 (m, 2H), 2.90 - 2.72 (m, 3H), 2.64 (t, J = 7.6 Hz, 2H), 2.61 - 2.53 (m, 3H), 2.48 - 2.36 (m, 1 H), 2.08 - 1 .80 (m, 4H); ¹³C NMR (100 MHz, CDCI₃) δ 171.0, 163.1 (d, JCF = 245.8 Hz), 158.8, 155.1 (d, JCF = 251 .3 Hz), 143.2 (d, JCF = 7.2 Hz), 141.4 (d, J_CF = 2.2 Hz), 138.1 (d, J_CF = 29.6 Hz), 131.6, 130.2 (d, J_CF = 8.4 Hz), 130.0 (d, J_CF = 12.9 Hz), 129.4 (d, J_CF = 3.9 Hz), 124.1 (d, J_CF = 2.8 Hz), 120.5, 1 15.3 (d, JCF = 20.9 Hz), 1 13.4 (d, JCF = 21 .0 Hz), 102.1 (d, JCF = 5.3 Hz), 60.3, 56.5, 55.9, 54.8, 54.3, 51.7, 47.1 , 32.9, 26.8, 25.9, 22.0; IR (neat) cm^-1: 2942, 2866, 1672, 1620, 1509, 1470, 1363, 1232; HRMS (ESI) calcd for C27H32F2N3O3 [M + H]⁺ 484.2406 found 484.2401 .

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(3-(4- fluorophen-yl)propyl)piperazine-2-carboxylic acid (64b). Following general procedure VII, alkyne 61 b (60 mg, 0.12

mmol) was hydrogenated using 10% Pd/C (7 mg, 0.006 mmol) and hydrolyzed to give the title compound as an off-white amorphous solid after preparative HPLC (31 mg, 53%). ¹H NMR (400 MHz, CDCI₃) δ 8.68 (br s, 1 H), 8.55 (s, 1 H), 7.97 (d, J = 9.2 Hz, 1 H), 7.29 (dd, J = 9.2, 2.6 Hz, 1 H), 7.18 (d, J = 2.6 Hz, 1 H), 7.12 - 7.05 (m, 2H), 6.99 - 6.90 (m, 2H), 3.93 (s, 3H), 3.43 (t, J = 3.5 Hz, 1 H), 3.20 (m, 2H), 3.09 - 2.98 (m, 3H), 2.98 - 2.79 (m, 2H), 2.76 - 2.54 (m, 6H), 2.55 - 2.45 (m, 1 H), 2.04 - 1.76 (m, 4H); ¹³C NMR (100 MHz, CDCI₃) δ 171.7, 161 .5 (d, J_CF = 244.1 Hz), 158.8, 155.0 (d, JCF = 251 .3 Hz), 141 .4 (d, J_CF = 2.2 Hz), 138.0 (d, J_CF = 29.5 Hz), 136.2 (d, JCF = 3.2 Hz), 131.6, 129.8 (d, JCF = 7.8 Hz), 129.7, 129.3 (d, J_CF = 3.8 Hz), 120.3 (d, JCF = 2.4 Hz), 1 15.4 (d, JCF = 21.1 Hz), 102.1 (d, J_CF = 5.4 Hz), 61 .0, 56.6, 55.8, 54.6, 54.2, 51.3, 46.6, 32.3, 26.7, 25.7, 21.9 (d, JCF = 2.9 Hz); IR (neat) cm^-1 : 3410, 3035, 2946, 2830, 1722, 1620, 1509, 1468, 1362, 1220, 1 156, 1029, 831 , 754; HRMS (ESI) calcd for C27H32F2N3O3 [M + H]⁺ 484.2406, found 484.2400; = +1 1.0 (c 1.0, in

(R)-4-(3-(3,4-difluorophenyl)propyl)-1 -(3-(3-fluoro-6-meth- oxyquinolin-4-yl)propyl)piperazine-2-carboxylic acid (64c). Following general procedure VII, alkyne 61 c (43 mg, 0.085

mmol) was hydrogenated using 10% Pd/C (9 mg, 0.008 mmol) and hydrolyzed to give the title compound as an off-white amorphous solid after prepar- ative HPLC (17 mg, 39%). ¹H NMR (400 MHz, CDCIs) δ 8.57 (d, J = 1 .0 Hz, 1 H), 7.99 (d, J = 9.2 Hz, 1 H), 7.31 (dd, J = 9.2, 2.6 Hz, 1 H), 7.20 (d, J = 2.7 Hz, 1 H), 7.06 (dt, J = 10.2, 8.3 Hz, 1 H), 6.96 (ddd, J = 1 1 .1 , 7.6, 2.1 Hz, 1 H), 6.89 - 6.83 (m, 1 H), 3.95 (s, 3H), 3.45 (t, J = 3.3 Hz, 1 H), 3.18 (d, J = 1 1 .0 Hz, 1 H), 3.1 1 - 2.95 (m, 4H), 2.87 (dd, J = 12.2, 5.1 Hz, 2H), 2.79 - 2.71 (m, 1 H), 2.66 - 2.54 (m, 5H), 2.51 - 2.41 (m, 1 H), 2.07 - 1 .76 (m, 4H); ¹³C NMR (100 MHz, CDCIs) δ 171.1 , 158.9, 155.1 (d, JCF = 251.2 Hz), 150.4 (dd, JCF = 248.1 , 12.7 Hz), 149.1 (dd, JCF = 246.4, 12.5 Hz), 141 .4 (d, JCF = 2.2 Hz), 138.0 (d, JCF = 29.7 Hz), 137.6 (d, JCF = 4.1 Hz), 137.6 (d, JCF = 4.1 Hz), 131.6, 129.9 (d, JCF = 12.9 Hz), 129.4 (d, JCF = 3.8 Hz), 124.3 (dd, JCF = 6.0, 3.5 Hz), 120.5 (d, JCF = 2.5 Hz), 1 17.3 (dd, JCF = 24.2, 16.9 Hz), 102.1 (d, JCF = 5.4 Hz), 60.6, 56.4, 55.9, 54.7, 54.2, 51 .6, 46.9, 32.3, 26.8, 25.8, 21 .9 (d, JCF = 3.1 Hz); IR (neat) cm^-1: 2944, 2868, 2834, 1672, 1620, 1516, 1432, 1283, 1231 ; HRMS (ESI) calcd for C27H31 F3N3O3 [M + H]⁺ 502.2312 found 502.2295. (R)-4-(3-(2,6-Difluorophenyl)propyl)-1 -(3-(3-fluoro-6-methox- yquinolin-4-yl)propyl)piperazine-2-carboxylic acid (64d). Following general procedure VII, alkyne 61 d (43 mg, 0.085 mmol)

was hydrogenated using 10% Pd/C (5 mg, 0.005 mmol) and hy- drolyzed to give the title compound as an off-white amorphous solid after preparative HPLC (4 mg, 15%). HRMS (ESI) calcd for C27H31 F3N3O3 calcd for [M + H]⁺ 502.2312 found 502.2304.

(R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-4-(3- (2,4,6-trifluor-ophenyl)propyl)piperazine-2-carboxylic acid (64e). Following general procedure VII, alkyne 61e (40

mg, 0.076 mmol) was hydrogenated using 10% Pd/C (16 mg, 0.015 mmol) and hydrolyzed to give the title compound as an off-white amorphous solid after preparative HPLC (6 mg, 14%). ¹H NMR (400 MHz, CDCI₃) δ 8.58 (d, J = 1.0 Hz, 1 H), 7.99 (d, J = 9.2 Hz, 1 H), 7.32 (dd, J = 9.2, 2.7 Hz, 1 H), 7.21 (d, J = 2.7 Hz, 1 H), 6.71 - 6.59 (m, 2H), 3.97 (s, 3H), 3.40 (t, J = 2.5 Hz, 1 H), 3.1 1 - 3.01 (m, 3H), 2.91 (d, J = 9.2 Hz, 1 H), 2.76 (ddd, J = 16.9, 13.2, 5.0 Hz, 3H), 2.62 (dt, J = 13.0, 7.6 Hz, 3H), 2.55 - 2.49 (m, 2H), 2.45 (dd, J = 1 1 .4, 2.3 Hz, 1 H), 2.34 (td, J = 1 1.3, 4.2 Hz, 1 H), 2.09 - 1 .96 (m, 1 H), 1.89 (ddd, J = 19.3, 13.0, 6.7 Hz, 1 H), 1 .80 (dt, J = 15.1 , 7.6 Hz, 2H); IR (neat) cm-¹ : 2943, 1672, 1621 , 1509, 1440, 1362, 1231 , 1 1 15, 998, 833; HRMS (ESI) calcd for C27H30F4N3O3 [M + H]⁺ 520.2218 found 520.2219.

Scheme 8. Synthesis of final compounds 68, 70, 72a-b, 73 and 74

Methyl (R)-4-(3-phenylpropyl)piperazine-2-carboxylate (66). Pi- p_{erazine 19} (2.83 g, 10.2 mmol) was dissolved in CH₂CI₂ (50 mL) un-

der stirring at rt, followed by addition of hydrocinnamaldehyde (1.61 mL, 12.2 mmol). After 15 min of stirring, cone. AcOH (58 μί, 1 .0 mmol) was added, and the solution was stirred for another 15 min. Then NaBH(OAc)3 (4.32 g, 20.4 mmol) was added, and the reaction subsequently ran to completion after 1.5 h. The organic phase was washed with 10% NaHC03 (aq) (25 mL), followed by separation, and extraction of the aqueous phase with CH2CI2 (50 mL). The combined organic phases were dried over Na2S0₄ and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (EtOAc:heptane (1 :3), R_f = 0.2), to give the intermediate amine as a colorless oil (2.46 g, 61 %). ¹H NMR (400 MHz, CDCI₃) δ 7.40 - 7.24 (m, 7H), 7.22 - 7.14 (m, 3H), 5.18 (s, 2H), 4.84 - 4.80 and 4.73 - 4.67 (2 x m, 1 H), 4.01 - 3.86 (m, 1 H), 3.77 and 3.72 (2 x s, 3H), 3.50 - 3.22 (m, 2H), 2.85 - 2.70 (m, 1 H), 2.69 - 2.53 (m, 2H), 2.43 - 2.26 (m, 2H), 2.23 - 2.01 (m, 2H), 1.84 - 1.73 (m, 2H), rotamers; ¹³C NMR (100 MHz, CDCI₃) δ 171.2 and 171.0 (1 C), 156.5 and 156.0 (1 C), 142.1 , 136.6, 128.6 (4C), 128.5 (2C), 128.2, 128.00, 127.97, 125.9, 67.6 and 67.5 (1 C), 57.0, 55.3 and 55.0 (1 C), 53.4, 52.7, 52.5, 41 .9 and 41 .7 (1 C), 33.2, 28.4, rotamers; IR (neat) cm^-1: 3027, 2948, 2813, 1747, 1701 , 1414, 1292, 1204, 1 1 10, 1040, 746, 697; HRMS (ESI) calcd for C23H29N2O4 [M + H]⁺ 397.2127, found 397.2126; = + 26.9 (c

1.0, CHCIs).

The intermediate amine (2.31 g, 5.83 mmol) was dissolved in MeOH (25 mL) under stirring at rt, whereupon NH4COOH (0.735 g, 1 1 .7 mmol) was added. After 25 min of stirring, 10% Pd/C (0.062 g, 0.058 mmol) suspended in MeOH (5 mL) was added to the solution. The reaction mixture was following stirred at rt under argon overnight. Addi- tional 10% Pd/C (0.620 g, 0.583 mmol) and NH4COOH (0.735 g, 1 1.7 mmol) were then added, whereupon the reaction ran to completion after 2 days. The reaction mixture was filtered through a pad of celite, followed by removal of MeOH in vacuo. The residue was suspended in water and extracted with EtOAc (2x50 mL). The combined organic phases were concentrated in vacuo and subjected to flash column chromatog- raphy on silica gel (CH₂CI₂:MeOH (95:5), f?_f = 0.2), to give the title compound 66 as a yellow oil (1 .05 g, 68%). ¹H NMR (400 MHz, CDCI₃) δ 7.40 - 7.33 (m, 2H), 7.30 - 7.23 (m, 3H), 3.82 (s, 3H), 3.67 (dd, J = 8.2, 3.2 Hz, 1 H), 3.18 - 3.1 1 (m, 1 H), 3.02 - 2.89 (m, 2H), 2.75 - 2.64 (m, 3H), 2.50 - 2.40 (m, 3H), 2.33 - 2.24 (m, 1 H), 2.18 (br s, 1 H), 1.95 - 1.84 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 172.7, 142.2, 128.5 (2C), 128.4 (2C), 125.8, 58.0, 57.3, 55.2, 53.7, 52.1 , 44.4, 33.6, 28.4; IR (neat) cm^-1 : 3352, 3025, 2946, 2808, 1737, 1657, 1452, 1434, 1201 , 1 143, 746, 699; HRMS (ESI) calcd for

C15H23N2O2 [M + H]⁺ 263.1760, found 263.1755; [a]¾° = - 6.8 (c 1.0, CHCI₃).

Methyl (2 R)-1 -(3-(3-fluoro-6-methoxyquinolin-4-yl)-3-hy- droxypropyl)-4-(3-phenylpropyl) piperazine-2-carboxylate (67). Freshly prepared aldehyde 11 (0.048 g, 0.16 mmol) and amine 66 (0.043 g, 0.16 mmol) were mixed and dissolved in

CH2CI2 (1.6 mL). Then NaBH(OAc)₃ (0.052 g, 0.24 mmol) was added, and the reaction mixture was stirred for 1 h at rt. Sat. NaHC03 (aq) (5 mL) was subsequently added, and the aqueous phase was extracted with CH2CI2 (3 x 10 mL). The combined organic phases were washed with brine (20 mL), dried over Na2S04 and concentrated in vacuo. The residue was subjected to flash column chromatography on silica gel (EtOAc:heptane (3:2), R_f = 0.2) to afford the title compound 67 as a colorless oil (diastereoisomeric mixture, 0.057 g, 71 % over two steps). ¹H NMR (400 MHz, CDCIs) δ 8.52 (s, 1 H), 7.98 - 7.91 (m, 2H), 7.30 - 7.23 (m, 3H), 7.19 - 7.13 (m, 3H), 5.79 (dd, J = 10.4, 2.6 Hz, 0.5H), 5.72 (dd, J = 10.0, 2.6 Hz, 0.5H), 3.90 (s, 3H), 3.75 (s, 1 .5H), 3.72 (s, 1 .5H), 3.50 - 3.33 (m, 1 .5H), 3.24 - 3.13 (m, 1 H), 3.06 - 2.96 (m, 0.5H), 2.88 - 2.23 (m, 12H), 1.83 - 1.66 (m, 3H), diastereoisomers (1 :1 ); MS (ESI) calcd for C28H35FN3O4 [M + H]⁺ 496.3, found 496.4. ¹ H NMR (400 MHz, CDCI₃) δ 8.52 (s, 1 H), 7.98 - 7.91 (m, 2H), 7.30 - 7.23 (m, 3H), 7.19 - 7.13 (m, 3H), 5.79 (dd, J = 10.4, 2.6 Hz, 0.5H), 5.72 (dd, J = 10.0, 2.6 Hz, 0.5H), 3.90 (s, 3H), 3.75 (s, 1 .5H), 3.72 (s, 1 .5H), 3.50 - 3.33 (m, 1.5H), 3.24 - 3.13 (m, 1 H), 3.06 - 2.96 (m, 0.5H), 2.88 - 2.23 (m, 12H), 1 .83 - 1 .66 (m, 3H), diastereoisomers (1 :1 ); MS (ESI) calcd for C28H35FN3O4 [M + H⁺] 496.3, found 496.4.

(2R)-1 -(3-(3-Fluoro-6-methoxyquinolin-4-yl)-3-hydroxypro- pyl)-4-(3-phenylpropyl)piperazine-2-carboxylic acid (68). Following general procedure IV, methyl ester 67 (56 mg,

0.1 1 mmol) was hydrolyzed to give the title compound as an off-white amorphous solid (1 :1 diastereomeric mixture) after preparative HPLC (43 mg, 78%). ¹ H NMR (400 MHz, CDCI₃) δ 8.51 (s, 0.5H), 8.49 (s, 0.5H), 7.94 (d, J = 9.2 Hz, 1 H), 7.91 - 7.85 (m, 0.5H), 7.82 - 7.77 (m, 0.5H), 7.29 - 7.21 (m, 3H), 7.20 - 7.09 (m, 3H), 5.83 (d, J = 8.2 Hz, 0.5H), 5.72 (d, J = 5.3 Hz, 0.5H), 3.85 (s, 1 .5H), 3.81 (s, 1 .5H), 3.63 - 3.49 (m, 1 .5H), 3.47 - 2.41 (m, 13H), 2.33 - 2.13 (m, 0.5H), 2.06 - 1 .79 (m, 3H), acid proton has exchanged, diastereoisomers (1 :1 ); HRMS (ESI) calcd for C27H33FN3O4 [M + H⁺] 482.2455, found 482.2457.

(2/7)-Methyl 4-(cyclohexylmethyl)-1 -(3-(3-fluoro-6-methoxyqui nolin-4-yl)-3-hydroxypropyl)piperazine-2-carboxylate (69). A solution of amine 19 (100 mg, 0.36 mmol), cyclohexanecarbalde-

hyde (52 mg, 0.47 mmol) and NaBH(OAc)₃ (1 14 mg, 0.54 mmol) in CH2CI2 (1 .8 mL) was stirred for 30 minutes at rt. The reaction mixture was then concentrated in vacuo, and purified by flash column chromatography on silica gel (EtOAc:hep- tane (7:3), R_f = 0.7) to give the Cbz-protected amine intermediate (96 mg, 71 %). The intermediate (59 mg, 0.16 mmol) was dissolved in MeOH followed by the addition of HCOONH4 (60 mg, 0.94 mmol) and Pd/C (9 mg, 0.009 mmol) and stirred for 10 min at rt. The mixture was then filtered through celite which was washed with MeOH and the filtrate was concentrated in vacuo. The residue was dissolved in EtOAc (40 mL) and washed with H2O (40 mL) and the phases were separated. The organic layer was dried over Na2S0₄ and concentrated in vacuo. The residue was taken up in CH2CI2 (1 mL) and stirred with aldehyde 11 (freshly prepared, 47 mg, 0.16 mmol) and NaBH(OAc)3 (50 mg, 0.24 mmol) for 15 min at rt. CH₂CI₂ (40 mL) and water (40 mL) was then added and the mixture transferred to a separatory funnel. The phases were separated and the aqueous phase was extracted with CH2CI2 (2 x 25 mL). The combined organic layers were dried over Na2S0₄ and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (EtOAc:heptane (1 :1 ), R_f = 0.2), to give the title compound as a colorless oil (40 mg, 38% over three steps). ¹H NMR (400 MHz, CDCI₃) δ 8.60 - 8.49 (m, 1 H), 8.01 - 7.93 (m, 2H), 7.28 (dd, J = 9.2, 2.7 Hz, 1 H), 5.81 (dd, J = 10.7, 2.7 Hz, 0.5H), 5.75 (dd, J = 10.2, 2.6 Hz, 0.5H), 3.92 (s, 1 .5H), 3.92 (s, 1 .5H), 3.77 (s, 1 .5H), 3.72 (s, 1.5H), 3.54 - 3.46 (m, 0.5H), 3.46 - 3.33 (m, 1 H), 3.33 - 3.21 (m, 0.5H), 3.22 - 3.13 (m, 1 H), 3.02 (td, J = 12.9, 3.3 Hz, 0,5H), 2.90 - 2.28 (m, 7H), 2.12 (d, J = 7.2 Hz, 2H), 1 .79 - 1 .59 (m, 6H), 1 .52 - 1.37 (m, 1 H), 1 .29 - 1.08 (m, 3H), 0.92 - 0.76 (m, 2H), mixture of two diastereomers.

(2R)-4-(Cyclohexylmethyl)-1 -(3-(3-fluoro-6-methoxyquinolin-4- yl)-3-hydroxypropyl)piperazine-2-carboxylic acid (70). Follo- wing general procedure IV, methyl ester 69 (31 mg, 0.065 mmol) was hydrolyzed to give the title compound as an off-white amor¬

phous solid (1 :1 diastereomeric mixture) after preparative HPLC (17 mg, 55%). ¹H NMR (400 MHz, CDCI₃) δ 8.51 (s, 1 H), 7.93 (d, J = 9.0 Hz, 1 H), 7.85 - 7.66 (m, 1 H), 7.24 (m, 1 H), 5.93 - 5.61 (m, 1 H), 3.87 (s, 1.5H), 3.86 (s, 1.5H), 3.60 - 3.48 (m, 1 H), 3.36 - 2.09 (m, 1 1 H), 2.07 - 1 .89 (m, 1 H), 1 .80 - 1 .41 (m, 6H), 1 .32 - 1 .05 (m, 3H), 1.02 - 0.73 (m, 2H), mixture of two diastereomers.

Methyl (2/7)-4-((E)-3-(2,5-difluorophenyl)allyl)-1 -(3-(3- fluoro-6-meth-oxyquinolin-4-yl)-3-hydroxypropyl)pipera zine-2-carboxylate (71 ). A solution of amine 19 (100 mg,

0.36 mmol), aldehyde 30 (52 mg, 0.47 mmol) and

NaBH(OAc)₃ (1 13 mg, 0.54 mmol) in CH2CI2 (1 .8 mL) was stirred for 30 minutes at rt. The reaction mixture was then concentrated in vacuo, and purified by flash column chromatography on silica gel (EtOAc:heptane (7:3), R_f = 0.2) to give the Cbz-protected amine intermediate (123 mg, 80%). The intermediate (68 mg, 0.14 mmol) was dissolved in MeOH followed by the addition of HCOONH₄ (20 mg, 0.31 mmol) and Pd/C (8 mg, 0.007 mmol) and stirred for 10 min at rt. The mixture was then filtered through celite which was washed with MeOH and the filtrate was concentrated in vacuo. The residue was dissolved in EtOAc (40 mL) and washed with H₂0 (40 mL) and the phases were separated. The organic layer was dried over Na2S0₄ and concentrated in vacuo. The residue was taken up in CH2CI2 (1 mL) and stirred with aldehyde 11 (freshly prepared, 41 mg, 0.14 mmol) and NaBH(OAc)₃ (44 mg, 0.21 mmol) for 15 min at rt. CH₂CI₂ (40 mL) and water (40 mL) was then added and the mixture transferred to a separatory funnel. The phases were separated and the aqueous phase was extracted with CH2CI2 (2 x 25 mL). The combined organic layers were dried over Na2S0₄ and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel

(EtOAc:heptane (3:1 ), R_f = 0.2), to give the title compound as a colorless oil (62 mg, 38% over three steps). ¹H NMR (400 MHz, CDCI₃) δ 8.55 (s, 1 H), 7.95 (d, J = 9.4 Hz, 1 H), 7.28 (dd, J = 9.2, 2.6 Hz, 1 H), 7.16 - 7.09 (m, 1 H), 7.00 (dd, J = 9.2, 4.6 Hz, 0.5H) 0.5H), 6.97 (dd, J = 9.4, 4.6 Hz, 0.5H), 6.92 - 6.85 (m, 1 H), 6.64 (d, J = 16.1 Hz, 1 H), 6.30 (t, J = 6.5 Hz, 0.5H), 6.26 (d, J = 6.6 Hz, 0.5H), 5.82 (dd, J = 10.6, 2.5 Hz, 0.5H), 5.75 (dd, J = 10.0, 2.4 Hz, 0.5H), 3.92 (s, 3H), 3.78 (s, 1.5H), 3.75 (s, 1.5H), 3.52 - 3.45 (m, 0.5H), 3.47. - 3.35 (m, 1 H), 3.26-3.13 (m, 3H), 3.10 - 2.96 (m, 0.5H), 2.9 - 2.25 (m, 7H), 1.85 - 1.66 (m, 1 H), mixture of two diastereomers.

(2 R-)4-((E)-3-(2,5-Difluorophenyl)allyl)-1 -(3-(3-fluoro-6- methoxy-quinolin-4-yl)-3-hydroxypropyl)piperazine-2- carboxylic acid (72a and 72b). Following general procedure IV, methyl ester 71 (44 mg, 0.083 mmol) was hydro-

lyzed to give two fractions of diastereomers 72a (dr 2:1 , 1 1 mg, 27%) and 72b (dr 1 :9, 9 mg, 21 %) after preparative HPLC. ¹H NMR (400 MHz, CDCI₃) δ 8.43 (s, 1 H), 7.85 (d, J = 9.2 Hz, 1 H), 7.65 (s, 1 H), 7.21 - 7.14 (m, 1 H), 7.09 - 6.99 (m, 1 H), 6.95 - 6.74 (m, 2H), 6.55 (d, J = 15.6 Hz, 1 H), 6.23 - 6.03 (m, 1 H), 5.71 (d, J = 7.6 Hz, 1 H), 3.75 (s, 3H), 3.62 - 3.52 (m, 1 H), 3.48-3.33 (m, 2H), 3.25 - 3.1 1 (m, 2H), 3.03 - 2.84 (m, 3H), 2.81 - 2.65 (m, 2H), 2.62 - 2.42 (m, 2H), 2.01 - 1 .85 (m, 1 H), mixture of two diastereomers.

Methyl (R)-4-(2-(thiophen-2-ylthio)ethyl)piperazine-2-carbox- ylate (65). Piperazine 19 (2.12 g, 7.62 mmol) and aldehyde 26 (1.45

g, 9.14 mmol) were dissolved in CH2CI2 (35 mL) together with acetic acid (43.5 μL, 0.762 mmol) and NaBH(OAc)₃ (3.23 g, 15.2 mmol). The mixture was stirred at rt for 1 h. The reaction was quenched with NaHC0₃ (25 mL) and water (50 mL). The aqueous phase was washed with CH₂CI₂ (2 x 50 mL) and the combined organic layers were dried over Na₂S04 and concentrated in vacuo. The compound was purified by flash chromatography on silica gel (1 :3 EtOAc:heptane, R_f = 0.2) to give the intermediate Cbz-protected amine as a brown oil (2.35 g, 73%). ¹ H NMR (400 MHz, CDCI₃) δ 7.41 - 7.28 (m, 6H), 7.14 - 7.07 (m, 1 H), 7.00 - 6.91 (m, 1 H), 5.22 - 5.06 (m, 2H), 4.89 - 4.59 (m, 1 H), 3.98 - 3.82 (m, 1 H), 3.74 (m, 3H), 3.47 - 3.1 1 (m, 2H), 2.86 (t, J = 7.3 Hz, 2H), 2.80 - 2.66 (m, 1 H), 2.66 - 2.49 (m, 2H), 2.32 - 2.19 (m, 1 H), 2.18 - 2.06 (m, 1 H); ¹³C NMR (100 MHz, CDCI₃) δ 170.7, 156.4, 155.9, 136.6, 133.9, 129.5, 128.6, 128.2, 128.0, 128.0, 127.7, 67.6, 57.2, 55.3, 54.9, 53.6, 52.5, 52.4, 41 .8, 35.8 (major rotamer).

The intermediate Cbz-protected amine (2.28 g, 5.42 mmol) was dissolved in diethyl ether (35 mL) and HBr (33% in acetic acid, 35 mL). After 2 h, water (100 mL) was added and the aqueous phase was washed with diethyl ether (3x 50 mL) to remove impurities. The aqueous phase was the basified with NaHC0₃ and extracted with diethyl ether (3 x 50 mL). The combined organic layers were dried over Na₂S04 and concentrated in vacuo to give the title product as a brown oil (0.679 g, 44%). ¹ H NMR (400 MHz, CDCI₃) δ 7.34 (dd, J = 5.4, 1 .2 Hz, 1 H), 7.12 (dd, J = 3.5, 1 .2 Hz, 1 H), 6.97 (dd, J = 5.3, 3.5 Hz, 1 H), 3.76 (s, 3H), 3.73 (dd, J = 7.9, 3.4 Hz, 1 H), 3.18 - 3.08 (m, 1 H), 2.99 - 2.86 (m, 4H), 2.70 - 2.61 (m, 3H), 2.59 - 2.50 (m, 1 H), 2.41 - 2.32 (m, 1 H); ¹³C NMR (100 MHz, CDCI₃) δ 176.6, 171 .3, 134.1 , 134.0, 129.6, 127.7, 57.7, 56.1 , 53.9, 52.6, 52.2, 43.1 , 35.4.

(R ,Z)-1 -(3-(3-fluoro-6-methoxyquinolin-4-yl)allyl)-4-(2-(thio- phen-2-ylthio)ethyl)piperazine-2-carboxylic acid (73). Amine 65 (0.081 g, 0.28 mmol) was suspended in CH₂CI₂ (710 μL) un¬

der stirring at rt. To this, NaBH(OAc)₃ (0.090 g, 0.43 mmol) was added, followed by addition of a suspension of aldehyde 16 (freshly prepared from alcohol 15 (0.121 g, 0.517 mmol)) in CH₂CI₂ (710 μί). The reaction mixture was left stirring overnight, whereupon water (5 mL) was added to the mixture, and the organic phase was washed with 10% NaHC0₃ (aq) (10 mL). The aqueous phase was then extracted with CH₂CI₂ (2 x 10 mL), followed by washing of the combined organic layers with brine (15 mL) and drying over Na₂S04. The concentrated residue was subjected to flash chromatography on silica gel (EtOAc:heptane (1 :1 ), Rf = 0.2) to give the intermediate ester, which was hydrolyzed using general procedure IVto give the title compound 73 as a white amorphous solid (0.019 g, 17% over two steps). ¹H NMR (400 MHz, CDCIs) δ 8.60 (s, 1 H), 7.98 (d, J = 9.2 Hz, 1 H), 7.34 - 7.27 (m, 2H), 7.09 (d, J = 3.5 Hz, 1 H), 7.00 (d, J = 2.5 Hz, 1 H), 6.93 (dd, J = 5.0, 3.5 Hz, 1 H), 6.69 (d, J = 1 1 .6 Hz, 1 H), 6.33 (dt, J = 1 1.6, 6.3 Hz, 1 H), 3.90 (s, 3H), 3.64 - 3.44 (m, 1 H), 3.39 - 3.24 (m, 2H), 3.09 - 2.90 (m, 1 H), 2.89 - 2.53 (m, 8H), 2.38 (m, 1 H), acid proton has exchanged; ¹³C NMR (100 MHz, CDCI₃) δ 170.3, 159.0, 152.9 (d, J_CF = 254.1 Hz), 141 .3 (d, JCF = 2.1 Hz), 138.3 (d, J_CF = 29.3 Hz), 134.5, 133.1 , 131.5, 123.0, 128.7 (d, J_CF = 2.6 Hz), 127.9, 127.8, 124.9, 122.6, 121.1 , 102.6 (d, JCF = 5.0 Hz), 62.7, 56.3, 55.8, 54.3, 54.2, 50.9, 48.1 , 34.8; IR (neat) cm^-1 : 3600-3100, 3071 , 2944, 2828, 1718, 1620, 1506, 1358, 1227, 831 , 752; HRMS (ESI) calcd for C24H27FN3O3S2 [M + H]⁺ 488.1478, found 488.1479; [a]¾° = + 16.0 (c 1.O, CHCI₃).

(R ,E)-1 -(3-(3-fluoro-6-methoxyquinolin-4-yl)allyl)-4-(2-(thio- phen-2-ylthio)-ethyl)piperazine-2-carboxylic acid (74). Amine 65 (0.081 g, 0.28 mmol) was suspended in CH₂CI₂ (710 μL), fol¬

lowed by addition of aldehyde 16 (freshly prepared from alcohol

15 (0.121 g, 0.517 mmol)) in CH2CI2 (710 μL). To this mixture, cone. AcOH (10 μL, 0.17 mmol) was added, and the reaction was left for stirring overnight at rt. The reaction was followed on HPLC-UV by taking out fractions of 10 μL and reducing it with

NaBH(OAc)3. As only one peak was seen on the HPLC, the reaction mixture was heated to reflux (40 °C) for 3 h. Then NaBH(OAc)₃ (0.090 g, 0.43 mmol) was added. Water (5.0 ml.) was added to the mixture, and the organic phase was washed with 10% NaHCCb (aq) (10 ml_). The aqueous phase was then extracted with CH2CI2 (2 x 10 ml_), followed by washing of the combined organic layers with brine (15 ml.) and drying over Na2S0₄. The concentrated residue was subjected to flash chromatography on silica gel (EtOAc:heptane (1 :1 ), R_f = 0.2), to give the intermediate ester, which was hydrolyzed using general procedure IVto give the title compound 74 as a beige amor- phous solid (0.017 g, 9% over two steps). ¹ H NMR (400 MHz, CDCI₃) δ 8.61 (d, JHF = 1.6 Hz, 1 H), 7.96 (d, J = 9.2 Hz, 1 H), 7.37 - 7.33 (m, 2H), 7.30 (dd, J = 9.2, 2.5 Hz, 1 H), 7.15 (d, J = 3.5 Hz, 1 H), 7.00 - 6.93 (m, 2H), 6.48 (dt, J = 16.1 , 6.7 Hz, 1 H), 3.95 (s, 3H), 3.69 - 3.52 (m, 2H), 3.10 - 2.88 (m, 6H), 2.84 - 2.68 (m, 3H), 2.63 - 2.49 (m, 1 H), acid proton has exchanged; ¹³C NMR (100 MHz, CDCI₃) δ 170.5, 159.0, 154.1 (d, J = 256.1 Hz), 141.6 (d, J = 2.4 Hz), 138.5 (d, J = 29.8 Hz), 135.9, 134.8, 132.8, 131.3, 130.2, 128.4 (d, J = 2.6 Hz), 127.9, 125.0 (d, J = 9.8 Hz), 123.8 (s), 121.1 (d, J = 2.1 Hz), 102.2 (d, J = 5.4 Hz), 60.2, 58.3, 56.2, 55.9, 54.7, 51 .7, 48.0, 34.8; IR (neat) cm ¹ : 3600-3200, 2941 , 2826, 1719, 1619, 1507, 1363, 1228, 1 150, 1028, 831 , 704; HRMS (ESI) calcd for C24H27FN3O3S2 [M + H]⁺ 488.1478, found 488.1477; = - 25.5 (c 1 .0,

CHCI₃).

Scheme 9. Solid-phase synthesis of final compounds 79a-b, 80 and 81

General protocols for solid-phase synthesis

All solid-phase synthesis reactions were performed in polyethylene syringes with a polypropylene filter. The syringes were attached to Teflon valves, which were connected with Teflon tubes allowing vacuum to be applied to the syringes. Before use, the amino-functionalized ChemMatrix resin (0.3 mmol/g) was washed with DMF (x 6), MeOH (x 6) and CH₂CI₂ (x 6) followed by lyophilization. Attachment of the 4-hydroxymethylbenzoic acid (HMBA) linker to the ChemMatrix resin was carried out by dissolving the acid (3 equiv) in DMF followed by addition of /V-ethyl- morpholine (NEM) (4 equiv) and TBTU (2.9 equiv). After 5 min the mixture was added to the resin and the mixture was occasionally stirred over 1 h. The resin was washed with DMF (x 6) and CH₂CI₂ (x 6) and lyophilized.

Attachment of the piperazine core to the HMBA linker was performed by mixing carbox- ylic acid 23a (3 equiv), MSNT (3 equiv) and /V-methylimidazole (Melm) (6 equiv) in dry CH2CI2. After 5 min, the mixture was added to the HMBA-functionalized resin and the mixture was occasionally stirred over 1 h. The resin was washed with dry DMF (x 1 ) and the procedure was repeated once, followed by washes with DMF (x 6) and CH₂CI₂ (x 6).

Boc-deprotection was performed by adding 50% TFA (CH₂CI₂) to the resin and the mix- ture was occasionally stirred over 1 h, followed by washes with CH₂CI₂ (x 6), 5% Et₃N (DMF) (x 2), DMF (x 6).

Fmoc-deprotection was performed by swelling the resin in 20% piperidine (DMF) for 2 min and then 5 min, followed by wash with DMF (x 6) and CH₂CI₂ (x 6).

Reductive alkylations were performed by swelling the resin in CH₂CI₂ or DMF followed by addition of aldehyde (3 - 5 equiv) and NaBH(OAc)₃ (5 equiv, either as powder or formed in situ by addition of AcOH to NaBH₄ in a separate flask). After 2 - 16 h the resin was washed with DMF (x 6) and CH₂CI₂ (x 6).

Sonogashira cross-couplings were performed by swelling the resin in CH₂CI₂:Et₃N (2:1 ) followed by addition of aromatic halide (5 equiv), Pd(PPh₃)4 (10 mol%), Cul (15 mol%) and the syringed was sealed. After shaking for 1 - 3 days the resin was washed with DMF (x 6), MeOH (x 6) and CH₂CI₂ (x 6).

Cleavage from the HMBA linker for UPLC-MS analysis was performed by treatment of 5 - 10 mg of resin with 0.1 M NaOH (aq) for 30 min, followed by neutralization with 0.1 M HCI (aq).

Cleavage to release NBTI analogs was performed by treatment of 200-300 mg resin with 0.1 M NaOH (aq) for 2-16 h followed by neutralization with 0.1 M HCI (aq). The resin was rinsed with MeCN (x 2). The filtrates were concentrated in vacuo and the residue was purified by preparative HPLC.

(2R)-1-(3-(3-fluoro-6-methoxyquinolin-4-yl)-3-hydroxyprop- yl)-4-(2-(thio-phen-2-ylthio)ethyl)piperazine-2-carboxylic acid (79a and 79b). General solid-phase synthesis protocols were followed to give the title compound as a mixture of diastereomers

(1:1). Crude purity according to UPLC-MS: 71%. The two diastereomers were then separated to give 79a and 79b.

79a: 9:1 dr.; ¹H N MR (400 MHz, CDCI₃) δ 8.49 (d, J= 1.2 Hz, 1H), 7.92 (d, J= 9.2 Hz, 1H), 7.77 (s, 1H), 7.32 (dd, J= 5.2, 0.7 Hz, 1H), 7.24 (dd, J =9.2, 2.5 Hz, 1H), 7.11 (d, J= 2.8 Hz, 1H), 6.94 (dd, J= 5.3, 3.6 Hz, 1H), 5.78 (d, J = 9.1 Hz, 1H), 4.78 (br. s, 2H), 3.81 (s, 3H), 3.57-3.16 (m, 3H), 3.05-2.60 (m, 9H), 2.33 (s, 1H), 1.97 (s, 1H); ¹³C NMR (100 MHz, CDCI₃) δ 171.6, 158.2, 153.8 (d, JCF = 253.9 Hz), 142.0, 138.4 (d, J = 30.1 Hz), 134.4, 133.2, 131.5, 130.4 (d, J = 7.7 Hz), 130.0, 128.1, 127.9, 120.4, 104.1,

65.2, 64.4, 56.5, 55.7, 54.5, 52.5, 50.9, 48.5, 34.9, 31.6; IR (neat) cm^-1; 3272 (br), 2953, 2829, 1621, 1509, 1466, 1354, 1230, 1145; HRMS (ESI) calcd for

C24H29FN3O4S2 [M + H]⁺ 506.1578, found 506.1574;

- 1.4 (c 1.0, CHCI₃).

79b: ¹H NMR (400 MHz, CDCI₃) δ 8.49 (s, 1 H), 7.91 (d, J = 9.2 Hz, 1 H), 7.71 (d, J = 2.3 Hz, 1 H), 7.31 (d, J = 5.3 Hz, 1 H), 7.22 (dd, J = 9.2, 2.3 Hz, 1 H), 7.09 (d, J = 3.4 Hz, 1H), 6.94 (dd, J = 5.3, 3.6 Hz, 1H), 5.77 (d, J = 7.2 Hz, 1H), 5.35 (br s, 2H), 3.78 (s, 3H), 3.67 - 3.38 (m, 3H), 3.05 - 2.89 (m, 2H), 2.86 - 2.51 (m, 9H), 2.07 - 1.93 (m, 1 H); ¹³C NMR (100 MHz, CDCI₃) δ 170.3, 158.4, 154.05 (d, JCF = 254.2 Hz), 142.0 (d, JCF = 2.1 Hz), 138.5 (d, JCF = 30.2 Hz), 134.3, 133.5, 131.6, 129.9, 129.8, 128.0 (d, JCF = 3.0 Hz), 127.8, 120.4, 103.8 (d, JCF = 4.6 Hz), 66.1, 65.6, 56.6, 55.7, 53.8, 53.8, 49.9, 48.9,

35.3, 30.9; IR (neat) cm^-1; 3203 (br), 2953, 2830, 1621, 1509, 1465, 1354, 1231, 1200, 1148; HRMS (ESI) calcd for C24H29FN3O4S2 [M + H]⁺ 506.1578, found

506.1573;[a]j3³=- 5.0 (d.O, CHCI₃).

(R)-4-(3-(3-Fluoro-6-methoxyquinolin-4-yl)propyl)-1-(2-(thio- phen-2-ylthio)-ethyl)piperazine-2-carboxylic acid (80). General solid-phase synthesis protocols were followed to give the title

compound. Crude purity according to UPLC-MS: >95%.¹H NMR

(400 MHz, CDCI₃) δ 8.58 (s, 1H), 8.00 (d, J= 9.2 Hz, 1H), 7.34-7.27 (m, 2H), 7.14 (d, J = 2.6 Hz, 1 H), 7.08 (dd, J = 3.5, 1.1 Hz, 1 H), 6.93 (dd, J = 5.3, 3.6 Hz, 1 H), 3.95 (s, 3H), 3.41 -3.37 (m, 1H), 3.30 (d, J= 11.3 Hz, 1H), 3.13-2.62 (m, 12H), 2.51 (td, J = 1 1.3, 3.6 Hz, 1 H), 2.06 - 1 .95 (m, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 171.8, 158.9, 154.9 (d, JCF = 251 .8, Hz), 141 .5 (d, JCF = 2.3 Hz), 138.0 (d, JCF = 29.2 Hz), 134.3, 133.5, 131.8, 129.2, 128.9 (d, JCF = 3.6 Hz), 128.4 (d, JCF = 12.8 Hz), 127.5, 120.4 (d, JCF = 2.5 Hz), 101.6 (d, J = 5.3 Hz), 70.6, 60.1 , 56.8, 55.8, 54.4, 54.0, 51.8, 46.5, 36.5, 24.7, 21.8 (d, J = 3.1 Hz); IR (neat) cm^-1 : 3402 (br), 3065, 2948, 2830, 1716, 1620, 1509, 1468, 1362, 1323, 1231 , 1 142; HRMS (ESI) calcd for C24H29FN3O3S2 [M + H]⁺ 490.1629, found 490.1527;

+ 1 1.7 (c 1.O, CHCI₃).

(R)-1 -(3-(3-fluoro-6-methoxyquinolin-4-yl)prop-2-yn-1 -yl)-4-(2- (thiophen-2-ylthio)ethyl)piperazine-2-carboxylic acid (81 ). General solid-phase synthesis protocols were followed to give the ti- t^le compound. Crude purity according to UPLC-MS: 62%. ¹H NMR

(400 MHz, CDCI₃) δ 8.62 (s, 1 H), 7.96 (d, J = 9.2 Hz, 1 H), 7.48 (d, J

= 2.7 Hz, 1 H), 7.36 (dd, J = 5.4, 0.9 Hz, 1 H), 7.32 (dd, J = 9.2, 2.8 Hz, 1 H), 7.16 (dd, J = 3.5, 0.9 Hz, 1 H), 6.97 (dd, J = 5.3, 3.6 Hz, 1 H), 4.05 - 3.94 (m, 1 H), 3.99 (s, 3H), 3.88 - 3.81 (m, 1 H), 3.72 (t, J = 3.2 Hz, 1 H), 3.16 - 2.90 (m, 6H), 2.84 - 2.70 (m, 3H), 2.65 - 2.55 (m, 1 H); ¹³C NMR (100 MHz, CDCI₃) δ 170.8, 159.7, 157.4 (d, J_CF = 260.5 Hz), 141 .2 (d, J = 2.7 Hz), 137.7 (d, J_CF = 27.0 Hz), 134.8, 132.8, 131 .3, 130.2, 130.1 , 127.9, 122.0 (d, JCF = 2.6 Hz), 1 13.0 (d, JCF = 12.8 Hz), 103.6 (d, JCF = 5.3 Hz), 99.1 , 75.5, 60.3, 56.2, 56.1 , 54.7, 51.8, 47.5, 45.7, 34.8; IR (neat) cm-¹; 3072, 2997, 2930, 2831 , 2359, 1720, 1672, 1619, 1504, 1226; HRMS (ESI) calcd for C24H29FN3O3S2 [M + H]⁺ 486.1316, found 486.1309; [a]¾³= - 3.4 (c 0.5, CHCI₃).

Table 1. Biological evaluation of piperazine-based antibiotic agents

88

PCT/EP2016/000357

81

Table 2. Biological evaluation of piperazine-based antibiotic agents

a Methicillin-susceptible S. aureus RV37. ^b Methicillin-resistant S. aureus CC398. ^c Determined as % inhibition of the hERG channel at 30 μΜ; number in parentheses indicates the established EC₅o for the hERG channel when available; ND = Not determined. ^d EC₅o for the hERG channel found to be >30 μΜ corresponding to <50% inhibition at 30 μΜ. ^e EC₅o for the hERG channel found to be <30 μΜ corresponding to >50% inhibition at 30 μΜ. ^f Determined against MRSA M2.

Table 6. Comparative data - compounds are prepared in accordance with the methods specified herein

Claims

Claims

1 . A piperazine derivative having the formula (I)

Formula (I) wherein

Ft' is selected from -H, -COOH and -CONH₂;

Ft" is selected from -H, -COOH and -CONH₂ with the proviso that when Ft' is -COOH or -CONH2 then Ft" is not -COOH or -CONH₂, and when Ft" is -COOH or -CONH₂ then Ft' is not -COOH or -CONH₂;

A is selected from -(CH₂)₃-, -(CH₂)₂CH(OH)-, -CH₂-CH=CH- (cis and trans),

B is selected from -(CH₂)_n-, -(CH₂)_nO-, -(CH₂)_nS- and -(CH₂)_n-CH=CH- (cis and trans),

Y is selected from C1-C7 alkyl (straight or branched), C₅-C₆ cycloalkyi optionally substituted with one or more halogens, thiophene or phenyl optionally substituted with one or more halogens or -C(hal)₃ wherein "hal" denotes a halogen,

or a pharmaceutically acceptable salt thereof.

2. A piperazine derivative according to claim 1 having one of the following formulas

Formula (II)

Formula (III)

Formula (IV)

wherein both cis and trans form are included, or

Formula (V), wherein R', R", B and Y are as defined in claim 1 ,

or a pharmaceutically acceptable salt thereof.

3. A compound according to claim 1 or 2, wherein Y-B- is selected from

Y-S-CH₂-CH₂-,

Y-CH₂-,

Y-CH2-CH2-,

Y-CH2-CH2-CH2-,

Y-CH=CH-CH₂-, and

Y-O-CH2-CH2- wherein Y is as defined in claim 1 ,

or a pharmaceutically acceptable salt thereof.

4. A compound according to any of the preceding claims, wherein Y- is selected from

wherein Ri and/or R₂ is H or halogen selected from F, Br and I

optionally substituted with one or two halogens selected from F, Br and I or Y-B- is a straight or branched alkyl group having from 4 to 9 carbon atoms, or a pharmaceutically acceptable salt thereof.

5. A compound according to any of the preceding claims having one of the following structures:

wherein R', R" are as defined above, R'" is H or OH, X is C, O or S, and Y is H or halo- gen selected from F, Br, I, preferably F, or a pharmaceutically acceptable salt thereof.

6. A compound according to any of the preceding claims selected from the compounds described in any of Tables 1 -5, or a pharmaceutically acceptable salt thereof.

7. A compound according to any of the preceding claims, wherein R' is -COOH and R" is H.

8. A compound according to any of the preceding claims for use in medicine.

9. A compound according to any of the preceding claims for use in the treatment of an infectious disease.

10. A compound for use according to claim 6, wherein the infectious disease is caused by a bacterium.

1 1 . A method for preparing a compound defined in any of claims 1 -5 essentially as described herein.