Anti-Mycobacterial N-(2-Arylethyl)quinolin-3-amines Inspired by Marine Sponge-Derived Alkaloid

The synthesis and evaluation of simplified analogs of marine sponge-derived alkaloid 3-(phenethylamino)demethyl(oxy)aaptamine were performed to develop novel anti-mycobacterial substances. Ring truncation of the tricyclic benzo[de][1,6]-naphthyridine skeleton effectively weakened the cytotoxicity of the natural product, and the resulting AC-ring analog exhibited good anti-mycobacterial activity. A structure–activity relationship (SAR) study, synthesizing and evaluating some analogs, demonstrated the specificity and importance of the N-(2-arylethyl)quinolin-3-amine skeleton as a promising scaffold for anti-mycobacterial lead compounds.


Introduction
Tuberculosis (TB), a bacterial infection caused by Mycobacterium tuberculosis, remains a leading cause of mortality worldwide [1]. According to a World Health Organization report, there are an estimated 10 million new TB cases and 1.5 million deaths annually [2]. Considering the standard regimen, known as directly observed therapy short-course (DOTS), a minimum 6-month TB treatment course is requisite, mainly because most existing anti-TB drugs are effective against M. tuberculosis only during the active state. Therefore, new anti-mycobacterial lead compounds effective against M. tuberculosis are urgently needed to address both active and dormant states. Hypoxic conditions induce the dormant state of Mycobacterium sp., which has a drug susceptibility profile resembling that of latent M. tuberculosis infection, although the physiology of latent M. tuberculosis infection remains unclear [3][4][5].
Marine natural products have garnered considerable attention as rich and promising sources of drug candidates, especially in the field of anti-tubercular drug discovery [6,7]. Based on this background, we have previously established a screening system to isolate anti-dormant mycobacterial substances from marine organisms and marine-derived microorganisms through bioassay-guided separation [8,9]. In a recent study, we discovered 3-(phenethylamino)demethyl(oxy)aaptamine (PDOA, 1) as a promising anti-dormant mycobacterial substance derived from an Indonesian marine sponge of Aaptos sp. (Figure 1). Compound 1 showed potent antimicrobial activity against M. bovis BCG, with a minimum inhibitory concentration (MIC) value of 1.56 µM under both aerobic and hypoxic conditions (Table 1). Remarkably, compound 1 exhibited potent anti-mycobacterial activity against drug-sensitive M. tuberculosis H37Rv, as well as against extensively drug-resistant M. tuberculosis strains, with MIC values ranging between 1.5-6.0 µM [10].

Synthesis and Evaluation of Truncated Analogs of 1
Generally, natural products have complex chemical structures with various functional groups and exhibit diverse bioactivities by binding to multiple target molecules (proteins). Truncation of some moieties can extract the essential scaffold of the natural product to reduce the number of target proteins without losing specific bioactivity. In addition, a substantial amount of the truncated analog can be easily synthesized owing to its simple structure. Furthermore, downsizing the molecular weight of the compound might improve the absorption, distribution, metabolism, excretion and toxicity (ADMET) profile. Several successful examples of truncated natural product analogs have been reported [13][14][15]. Recently, we developed a simplified analog of cortistatin A, a complex marinederived anti-angiogenic steroidal alkaloid. The optimized analog, prepared using fewer than 10 steps, was found to exert potent and selective growth inhibitory activity against HUVECs, comparable with that of the natural product, and exhibited potent in vivo antitumor activity [16,17].
Therefore, we simplified the core structure of compound 1 to extract the essential scaffold. An initial structure-activity relationship (SAR) study of 1 and related naturallyoccurring congeners 2-4 revealed that the essential functionality of 1 for anti-mycobacterial activity could not be attributed to the tricyclic benzo[de] [1,6]-naphthyridine core structure but rather to the 2-phenethylamino side chain [10]. Considering the SAR, we planned to prepare mono-or bicyclic truncated analogs with 2-phenethylamino side chains and evaluate their anti-mycobacterial activity against M. bovis BCG. Figure 2 shows the structures of three bicyclic analogs: AB-ring analog 5, AC-ring analog 6, BC-ring analog 7, and monocyclic analog 8. These results imply that compound 1 might be a potential anti-TB drug exerting a novel mechanism of action. However, the scarcity of natural sources has hampered further evaluation. Although the total synthesis of 1 [10,11] can provide a sufficient amount of the compound, lead optimization of the tricyclic benzo[de] [1,6]-naphthyridine skeleton might be challenging. In addition, we found that 1 exhibited cytotoxicity against human umbilical vein endothelial cells (HUVECs) with an IC 50 value of 1.36 µM, which is comparable with the MIC against M. bovis BCG ( Table 1). Cytotoxicity of 1 against some tumor cells has also been reported [12]. To overcome these drawbacks, we engaged in the development of a truncated analog of 1 as a selective anti-TB drug. Herein, we present the synthesis and evaluation of various 3-substituted quinoline derivatives.

Synthesis and Evaluation of Truncated Analogs of 1
Generally, natural products have complex chemical structures with various functional groups and exhibit diverse bioactivities by binding to multiple target molecules (proteins). Truncation of some moieties can extract the essential scaffold of the natural product to reduce the number of target proteins without losing specific bioactivity. In addition, a substantial amount of the truncated analog can be easily synthesized owing to its simple structure. Furthermore, downsizing the molecular weight of the compound might improve the absorption, distribution, metabolism, excretion and toxicity (ADMET) profile. Several successful examples of truncated natural product analogs have been reported [13][14][15]. Recently, we developed a simplified analog of cortistatin A, a complex marine-derived anti-angiogenic steroidal alkaloid. The optimized analog, prepared using fewer than 10 steps, was found to exert potent and selective growth inhibitory activity against HUVECs, comparable with that of the natural product, and exhibited potent in vivo antitumor activity [16,17]. Therefore, we simplified the core structure of compound 1 to extract the essential scaffold. An initial structure-activity relationship (SAR) study of 1 and related naturallyoccurring congeners 2-4 revealed that the essential functionality of 1 for anti-mycobacterial activity could not be attributed to the tricyclic benzo[de] [1,6]-naphthyridine core structure but rather to the 2-phenethylamino side chain [10]. Considering the SAR, we planned to prepare mono-or bicyclic truncated analogs with 2-phenethylamino side chains and evaluate their anti-mycobacterial activity against M. bovis BCG. Figure 2 shows the structures of three bicyclic analogs: AB-ring analog 5, AC-ring analog 6, BC-ring analog 7, and monocyclic analog 8.
tumor activity [16,17]. Therefore, we simplified the core structure of compound 1 to extract the essential scaffold. An initial structure-activity relationship (SAR) study of 1 and related naturallyoccurring congeners 2-4 revealed that the essential functionality of 1 for anti-mycobacterial activity could not be attributed to the tricyclic benzo[de] [1,6]-naphthyridine core structure but rather to the 2-phenethylamino side chain [10]. Considering the SAR, we planned to prepare mono-or bicyclic truncated analogs with 2-phenethylamino side chains and evaluate their anti-mycobacterial activity against M. bovis BCG. Figure 2 shows the structures of three bicyclic analogs: AB-ring analog 5, AC-ring analog 6, BC-ring analog 7, and monocyclic analog 8.  First, analog 5 was synthesized, as shown in Scheme 1A. Condensation was performed between homoveratrylamine (9) and Cbz-glycine gave amide 10, which was further converted to dihydroisoquinoline 11 via Bischler-Napieralski cyclization. The following two-step oxidation/aromatization by O2 yielded isoquinoline 12, and subsequent treatment with 2-phenethyl bromide and NaH afforded the desired AB-ring analog 5 through alkylation and concomitant removal of the Cbz group. Second, AC-ring analog 6 was synthesized as follows (Scheme 1B). The Friedländer reaction [18] with two aldehydes, 13 and 14, and subsequent removal of the Boc group yielded quinolin-3-amine 16. Then, the copper-catalyzed cross-coupling reaction with 2-phenethylboronic acid provided the desired analog 6 [19]. In addition, BC-ring analog 7 was prepared via the C8-bromination of 1,6naphthyridine (17) and subsequent Buchwald-Hartwig amination with 2-phenethylamine (Scheme 1C). A similar amination reaction toward 3-bromopyridine (19) proceeded smoothly using a BrettPhos-ligated palladium catalyst [20] to provide monocyclic C-ring analog 8 [21] in good yield (Scheme 1D). Biological evaluation of the synthesized analogs revealed that quinoline analog 6, which mimics the AC ring of 1, exhibited good antibacterial activity against M. bovis BCG under aerobic conditions (Table 1, MIC = 6.25 µM). Conversely, analogs 5 (AB-ring mimic), 7 (BC-ring mimic), and 8 (C-ring mimic) exerted weak anti-mycobacterial activity. Interestingly, analog 6 showed diminished cytotoxicity against HUVECs (IC50 = 18 µM) when compared with analog 1, indicating that the truncation of the B-ring could remove the cytotoxic property of 1. Although analog 6 exhibited weak antibacterial activity against M. bovis BCG under hypoxic conditions (MIC = 50 µM), the initial SAR study revealed that the 3-substituted quinoline skeleton might be a minimal and promising scaffold for anti-mycobacterial drug lead.  Biological evaluation of the synthesized analogs revealed that quinoline analog 6, which mimics the AC ring of 1, exhibited good antibacterial activity against M. bovis BCG under aerobic conditions (Table 1, MIC = 6.25 µM). Conversely, analogs 5 (AB-ring mimic), 7 (BC-ring mimic), and 8 (C-ring mimic) exerted weak anti-mycobacterial activity. Interestingly, analog 6 showed diminished cytotoxicity against HUVECs (IC 50 = 18 µM) when compared with analog 1, indicating that the truncation of the B-ring could remove the cytotoxic property of 1. Although analog 6 exhibited weak antibacterial activity against M. bovis BCG under hypoxic conditions (MIC = 50 µM), the initial SAR study revealed that the 3-substituted quinoline skeleton might be a minimal and promising scaffold for anti-mycobacterial drug lead.

SAR Study of N-(2-Arylethyl)quinolin-3-amine Analog
Next, we prepared congeners of 6 to examine the SAR around the quinoline ring, as depicted in Scheme 2. p-Quinone-type analogs 25 and 32, mimicking the A-ring of 1, were obtained by oxidation of the corresponding quinolinols 24 and 31, respectively, using Fremy's salt [22]. Compound 23 was prepared from 3-bromoquinolin-5-ol (21) [23], with the side chain attached through Buchwald-Hartwig amination. The synthetic method for 30 was the same as that for 6 (Scheme 2B), starting from isovanillin (26). Thus, 26 was converted to 27 according to the literature [24], and the Friedländer reaction with aldehyde 14 afforded isoquinoline 28. Subsequent removal of the Boc group and a cross-coupling reaction with 2-phenethylboronic acid yielded 30. Selective cleavage of the 8-OCH 3 ether bond from 30 to 31 was achieved by treatment with 48% HBr aq and subsequent oxidation using Fremy's salt provided 32.

SAR Study of N-(2-arylethyl)quinolin-3-amine Analog
Next, we prepared congeners of 6 to examine the SAR around the quinoline ring, as depicted in Scheme 2. p-Quinone-type analogs 25 and 32, mimicking the A-ring of 1, were obtained by oxidation of the corresponding quinolinols 24 and 31, respectively, using Fremy's salt [22]. Compound 23 was prepared from 3-bromoquinolin-5-ol (21) [23], with the side chain attached through Buchwald-Hartwig amination. The synthetic method for 30 was the same as that for 6 (Scheme 2B), starting from isovanillin (26). Thus, 26 was converted to 27 according to the literature [24], and the Friedländer reaction with aldehyde 14 afforded isoquinoline 28. Subsequent removal of the Boc group and a cross-coupling reaction with 2-phenethylboronic acid yielded 30. Selective cleavage of the 8-OCH3 ether bond from 30 to 31 was achieved by treatment with 48% HBr aq and subsequent oxidation using Fremy's salt provided 32.
Analogs 25 and 32 exhibited weakened anti-mycobacterial activity and enhanced cytotoxicity, undoubtedly owing to the quinone structure (Table 1). In contrast, quinolonetype analog 35 exhibited no anti-mycobacterial or cytotoxic activity. These results indicated the uniqueness of the quinoline core structure in the scaffold, and the electron density of the aromatic ring might be pivotal for anti-mycobacterial activity.
We further explored the SAR of the side chains (Scheme 3). To explore the importance of the secondary amine moiety, phenacyl analog 36, N-alkyl analog 37/38, and ether analog 40 were prepared. Compound 36 was obtained through the acylation of quinolin-3-amine (16), and treatment of 6 or quinolin-3-ol (39) with the corresponding alkyl halide yielded 37, 38, and 40, respectively. Moreover, analogs 42-46 were synthesized to examine the appropriate structure of the alkyl chain. Notably, analogs 42-45 were obtained by Buchwald-Hartwig amination between 3-bromoquinoline (41) and the corresponding primary amines, and the alkynyl analog 46 was prepared through the alkylation of 16. indicated the uniqueness of the quinoline core structure in the scaffold, and the electron density of the aromatic ring might be pivotal for anti-mycobacterial activity. We further explored the SAR of the side chains (Scheme 3). To explore the importance of the secondary amine moiety, phenacyl analog 36, N-alkyl analog 37/38, and ether analog 40 were prepared. Compound 36 was obtained through the acylation of quinolin-3amine (16), and treatment of 6 or quinolin-3-ol (39) with the corresponding alkyl halide yielded 37, 38, and 40, respectively. Moreover, analogs 42-46 were synthesized to examine the appropriate structure of the alkyl chain. Notably, analogs 42-45 were obtained by Buchwald-Hartwig amination between 3-bromoquinoline (41) and the corresponding primary amines, and the alkynyl analog 46 was prepared through the alkylation of 16. Phenacyl amide analog 36, ether analog 40, and N-propargyl analog 38 exhibited significantly weakened anti-mycobacterial activity, whereas N-methyl analog 37 exhibited antibacterial activity comparable to that of 6 ( Table 1). These findings indicate that basic nitrogen at that position is essential for binding to the target molecule responsible for the anti-mycobacterial activity, and the steric hindrance around the nitrogen might interrupt binding. In addition, on comparing the anti-mycobacterial activities of analogs 42-46, we observed that the presence of an aromatic ring at the side chain terminal was indispensable, and the 2-naphthyl analog 44 exhibited the most potent antibacterial activity under hypoxic conditions (MIC 12.5 µM). Conversely, the markedly reduced anti-mycobacterial activity of 1-naphthyl analog 43 further confirmed the importance of the side chain, probably through precise structure recognition by the target molecule. Phenacyl amide analog 36, ether analog 40, and N-propargyl analog 38 exhibited significantly weakened anti-mycobacterial activity, whereas N-methyl analog 37 exhibited antibacterial activity comparable to that of 6 ( Table 1). These findings indicate that basic nitrogen at that position is essential for binding to the target molecule responsible for the anti-mycobacterial activity, and the steric hindrance around the nitrogen might interrupt binding. In addition, on comparing the anti-mycobacterial activities of analogs 42-46, we observed that the presence of an aromatic ring at the side chain terminal was indispensable, and the 2-naphthyl analog 44 exhibited the most potent antibacterial activity under hypoxic conditions (MIC 12.5 µM). Conversely, the markedly reduced anti-mycobacterial activity of 1-naphthyl analog 43 further confirmed the importance of the side chain, probably through precise structure recognition by the target molecule.
In summary, ring truncation of the marine-derived alkaloid PDOA (1) resulted in the development of N-(2-arylethyl)quinolin-3-amine as a promising scaffold for generating novel anti-mycobacterial substances. The SAR study revealed the specificity and importance of the side chain structure, and the 2-naphthyl analog 44 exhibited good antimycobacterial activity under aerobic and hypoxic conditions. Although it remains unclear whether the target molecule of the compound developed in the present study is the same as that of 1, further synthesis and evaluation of various analogs would lead to the development of potent and selective anti-mycobacterial drug candidates. Structural optimization for anti-TB activity/selectivity over cytotoxicity and mechanistic analysis will be undertaken in due course.

Antimicrobial Activity of the Compounds under Aerobic and Hypoxic Conditions
The minimum inhibitory concentrations (MICs) against M. bovis BCG Pasteur were determined using the established MTT method [27]. All of the testing samples were purified with reversed-phase HPLC, and the purity of >99% was confirmed by 1 H-NMR and HPLC. The samples were dissolved in DMSO, and the activity of the samples was evaluated by preparing samples in 2-fold dilution series from 200 µM (final concentration). The mid-log phase of M. bovis BCG (1 × 10 5 CFU/0.1 mL) was inoculated in a 96-well plate, and the serially diluted sample was added to the 96-well plate. In case of aerobic conditions, bacteria were incubated at 37 • C for 7 days. Alternatively, the hypoxic model was established based  [28]. The mycobacterial bacilli were grown in Middlebrook 7H9 broth at 37 • C under a nitrogen atmosphere containing 0.2% oxygen until the optical density at 600 nm reached 0.8. Subsequently, the bacilli were inoculated in a 96-well plate at the same density under aerobic conditions and incubated at 37 • C under a nitrogen atmosphere containing 0.2% oxygen for 14 days. After incubation, an aliquot (50 µL) of MTT solution (5.0 mg/mL) was added to each well and incubated at 37 • C for an additional 12 h under aerobic or hypoxic condition. The optical density at 560 nm was then measured to determine the MIC value. The reproducibility of the data was confirmed by three independent experiments.

Assay for Cytotoxicity of Compounds against HUVECs
HUVECs (5 × 10 5 cells/vial) was purchased from Kurabo Inc. and grown in HuMedia-EG2 medium with growth supplements (Kurabo Inc., Osaka, Japan). HUVECs in the culture medium was plated into each well of 96-well plates (2 × 10 3 cells/well/100 µL). After 24 h, the serially diluted compounds, which were dissolved in the medium containing no more than 0.5% EtOH, were added, and then the plates were incubated for an additional 72 h in a humidified atmosphere of 5% CO 2 at 37 • C. The cell proliferation was detected by WST-8 colorimetric reagent (Nacalai Tesque, Inc., Kyoto, Japan). The IC 50 value was determined by linear interpolation from the growth inhibition curve.
All the spectral data were identical to the reported ones [29].
All the spectral data were identical to the reported ones [29].
3.5.3. Benzyl (6,7-Dimethoxyisoquinoline-1-carbonyl)carbamate (12) A solution of 11 (10.7 mg, 0.030 mmol) in CHCl 3 (0.5 mL) was stirred for 3 days under air. Removal of the solvent from the mixture under reduced pressure gave a crude product, which was used for the next reaction without further purification. 1  Activated carbon (20.5 mg, 100 wt%) was added to a solution of the above product (20.0 mg, 0.054 mmol) in xylene (2.0 mL), and the whole mixture was stirred under an O 2 atmosphere at 120 • C for 10 h. After cooling to rt, the mixture was filtered through a Celite pad. Removal of the solvent from the filtrate under reduced pressure gave a crude product, which was purified with SiO 2 column chromatography (n-Hexane/AcOEt = 2:1) to give 12 (5.8 mg, 29%) as a yellow solid. 3.5.4. 6,7-Dimethoxy-N-phenethylisoquinoline-1-carboxamide (5) NaH (5.2 mg, 0.11 mmol) was added to a solution of 12 (5.0 mg, 0.014 mmol) in DMF (0.5 mL) at 0 • C and the whole mixture was stirred for 5 min. Phenethyl bromide (20 µL, 0.16 mmol) was added to the mixture and the whole mixture was stirred for 24 h at rt, 48 at 60 • C, and 9 h at 90 • C. After cooling to rt, H 2 O (1 mL) was added to the mixture and the whole mixture was extracted with AcOEt. Removal of the solvent from the organic phase under reduced pressure gave a crude product, which was purified with preparative TLC (n-Hexane/AcOEt = 2:1) to give 5 (3.3 mg, 72%) as a yellow solid. 1  3.5.5. tert-Butyl quinolin-3-ylcarbamate (15) 4 N NaOH aq. (49 µL, 0.20 mmol) was added dropwise to a solution of 2-aminobenzaldehyde (13, 28.8 mg, 0.18 mmol) and tert-butyl (2-oxoethyl)carbamate (14, 7.9 mg, 0.065 mmol) in MeOH (0.5 mL) and the whole mixture was stirred at rt for 18 h. Removal of the solvent from the mixture under reduced pressure gave a crude product, which was diluted with AcOEt and was then washed with H 2 O. Removal of the solvent from the AcOEt phase under reduced pressure gave a crude product, which was purified with preparative TLC (PTLC, CHCl 3 /MeOH = 60:1) to give 15 (10.4 mg, 52%) as a white solid.
All the spectral data were identical to the reported ones [30].
3.5.6. Quinolin-3-amine (16) TFA (120 µL) was added to a solution of 15 (6.4 mg, 0.026 mmol) in CH 2 Cl 2 (1.0 mL) at 0 • C and the whole mixture was stirred at rt for 24 h. Sat. NaHCO 3 aq. was added to the mixture and the whole mixture was extracted with CH 2 Cl 2 . Removal of the solvent from the organic phase under reduced pressure gave a crude product, which was purified with SiO 2 column chromatography (CH 2 Cl 2 /MeOH = 80:1, 1% Et 3 N) to give 16 (3.6 mg, 95%) All the spectral data were identical to the reported ones [31].

N-Phenethylquinolin-3-amine (6)
6 was prepared through the reported method [19]. All the spectral data were identical to the reported ones.
All the spectral data were identical to the reported ones [21].

3-Bromo-5-(methoxymethoxy)quinoline (22)
Chloromethyl methyl ether (84 µL, 1.10 mmol) and K 2 CO 3 (408 mg, 2.95 mmol) were added to a solution of 21 (225 mg, 1.00 mmol) in acetone (5 mL) and the whole mixture was stirred at rt for 2 h. H 2 O was added to the mixture and the whole mixture was extracted with AcOEt. Removal of the solvent from the AcOEt phase under reduced pressure gave a crude product containing 22, which was used for the next reaction without further purification.
3.5.14. 3-(Phenethylamino)quinolin-5-ol (24) Conc. HCl aq. (0.3 mL) was added to a solution of 23 (49.2 mg, 0.160 mmol) in MeOH (0.9 mL) and the whole mixture was stirred at rt for 3 h. The reaction mixture was neutralized with sat. NaHCO 3 aq. And the whole mixture was extracted with CHCl 3 containing 10% MeOH. Removal of the solvent from the organic phase under reduced pressure gave a crude product, which was purified with SiO 2 column chromatography (CHCl 3 /MeOH = 10:1) to give 24 (29.9 mg, 70% in 3 steps) as a yellow solid. 1 3.52 (t, J = 6.9 Hz, 2H), 2.99 (t, J = 6.9 Hz, 2H). 13 (8 mL) was added dropwise to the mixture. After stirring the whole mixture at rt for 1 h, acetone was removed from the mixture under reduced pressure, and the resulting aqueous phase was extracted with CH 2 Cl 2 . Removal of the solvent from the organic phase under reduced pressure gave a crude product, which was purified with PTLC (CHCl 3 /MeOH = 50:1) to give 25 (4.6 mg, 29%) as a red-purple solid. 1 (27) 27 was prepared from isovanillin (26) through the reported method [24]. All the spectral data were identical to the reported ones.
3.5.17. tert-Butyl (7,8-dimethoxyquinolin-3-yl)carbamate (28) 4 N NaOH aq. (124 µL, 2.4 mmol) was added dropwise to a solution of 27 (25.1 mg, 0.21 mmol) and 14 (149 mg, 0.93 mmol) in MeOH (1.0 mL), and the whole mixture was stirred at rt for 30 h. MeOH was removed from the mixture under reduced pressure, and the resulting aqueous phase was extracted with AcOEt. Removal of the solvent from the organic phase under reduced pressure gave a crude product, which was purified with SiO 2 column chromatography (n-hexane/AcOEt = 1:1) to give 28 (19.2 mg, 30%) as a tan oil. 1 (29) TFA (0.17 mL, 2.2 mmol) was added to a solution of 28 (34.8 mg, 0.11 mmol) in CH 2 Cl 2 (1.0 mL) at 0 • C, and the whole mixture was stirred at rt for 3 h. Sat. NaHCO 3 aq. was added to the mixture and the whole mixture was extracted with AcOEt. Removal of the solvent from the organic phase under reduced pressure gave a crude product, which was purified with PTLC (CHCl 3 /MeOH = 30:1) to give 29 (14.0 mg, 60%) as a tan oil. 1  Pyridine (6.3 µL, 0.078 mmol) and Cu(OAc) 2 (6.1 mg, 0.034 mmol) were added to a solution of 29 (5.3 mg, 0.026 mmol) in 1,4-dioxane (2.0 mL) and the whole mixture was stirred under reflux for 15 min. 2-Phenethylboronic acid (5.1 mg, 0.034 mmol) was added to the mixture and the whole mixture was further stirred under reflux for 14 h. After cooling to rt, H 2 O was added to the mixture and the whole mixture was extracted with AcOEt. Removal of the solvent from the organic phase under reduced pressure gave a crude product, which was purified with PTLC (CHCl 3 /MeOH = 30:1) to give 30 (3.0 mg, 38%) as a red-purple solid.

2-Phenyl-N-(quinolin-3-yl)acetamide (36)
A solution of phenacyl chloride (93 µL, 0.704 mmol) in CH 2 Cl 2 (2 mL) was added dropwise to a solution of 16 (68.4 mg, 0.474 mmol) and pyridine (402 µL, 4.99 mmol) in CH 2 Cl 2 (3 mL) and the whole mixture was stirred at rt for 7 h. Sat. NH 4 Cl aq. was added to the mixture and the whole mixture was extracted with CH 2 Cl 2 . Removal of the solvent from the organic phase under reduced pressure gave a crude product, which was purified with SiO 2 column chromatography (hexane/AcOEt = 1:1) to give 36 (82.5 mg, 66%) as a white solid. 1  A solution of 6 (25.0 mg, 0.101 mmol) in 2,2,2-trifluoroethanol (TFE, 0.25 mL) was added to a solution of HCHO aq. (18 µL, 0.500 mmol) in TFE (0.25 mL) and the whole mixture was stirred at rt for 5 min. NaBH 4 (7.6 mg, 0.201 mmol) was added to the mixture and the whole mixture was stirred at rt for 13 h. The reaction was quenched by the addition of H 2 O, and the whole mixture was extracted with AcOEt. Removal of the solvent from the organic phase under reduced pressure gave a crude product, which was purified with SiO 2 column chromatography (CHCl 3 /MeOH = 20:1) to give 37 (20.4 mg, 77%) as a pale yellow oil. 1  K 2 CO 3 (2.2 mg, 15.9 µmol) and propargyl bromide (52 µL, 0.480 mmol) were added to a solution of 6 (40.0 mg, 0.161 mmol) in acetone (2.4 mL) and the whole mixture was stirred at 60 • C for 32 h. After cooling to rt, H 2 O was added to the mixture and the whole mixture was extracted with AcOEt. Removal of the solvent from the organic phase under reduced pressure gave a crude product, which was purified with SiO 2 column chromatography (hexane/AcOEt = 2:1) to give 38 (7.0 mg, 15%) as a pale yellow oil. 1  NaH (60.0 mg, ca. 1.50 mmol) and 2-phenethyl bromide (205 µL, 1.52 mmol) were added to a solution of quinolin-3-ol (39) (149 mg, 1.03 mmol) in DMF (2 mL) and the whole mixture was stirred at rt for 18 h. Sat. NaHCO 3 aq. was added to the mixture and the whole mixture was extracted with AcOEt. Removal of the solvent from the organic phase under reduced pressure gave a crude product, which was purified with SiO 2 column chromatography (hexane/EtOAc = 1:1) to give 40 (108 mg, 42%) as a pale yellow oil. 1