Antimalarial Activity of 9a-N Substituted 15-Membered Azalides with

Dec 12, 2011 - Novel classes of antimalarial drugs are needed due to emerging drug resistance. Azithromycin, the first macrolide investigated for mala...
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Antimalarial Activity of 9a-N Substituted 15-Membered Azalides with Improved in Vitro and in Vivo Activity over Azithromycin Mihaela Perić,*,†,∥ Andrea Fajdetić,†,○ Renata Rupčić,†,○ Sulejman Alihodžić,†,○ Dinko Ž iher,†,○ Mirjana Bukvić Krajačić,†,○ Kirsten S. Smith,‡,# Zrinka Ivezić-Schönfeld,†,● Jasna Padovan,†,○ Goran Landek,†,○ Dubravko Jelić,†,○ Antun Hutinec,†,○ Milan Mesić,†,○ Arba Ager,§ William Y. Ellis,‡ Wilbur K. Milhous,‡,⊥ Colin Ohrt,‡ and Radan Spaventi†,○ †

GlaxoSmithKline Research Centre Zagreb Ltd., Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, Washington, DC 20910, United States § Department of Microbiology and Immunology, University of Miami, Miami, Florida 33177, United States # U.S. Army Medical Materiel Development Activity, 1430 Veterans Drive, Fort Detrick, Maryland 21702, United States ‡

S Supporting Information *

ABSTRACT: Novel classes of antimalarial drugs are needed due to emerging drug resistance. Azithromycin, the first macrolide investigated for malaria treatment and prophylaxis, failed as a single agent and thus novel analogues were envisaged as the next generation with improved activity. We synthesized 42 new 9a-N substituted 15-membered azalides with amide and amine functionalities via simple and inexpensive chemical procedures using easily available building blocks. These compounds exhibited marked advances over azithromycin in vitro in terms of potency against Plasmodium falciparum (over 100-fold) and high selectivity for the parasite and were characterized by moderate oral bioavailability in vivo. Two amines and one amide derivative showed improved in vivo potency in comparison to azithromycin when tested in a mouse efficacy model. Results obtained for compound 6u, including improved in vitro potency, good pharmacokinetic parameters, and in vivo efficacy higher than azithromycin and comparable to chloroquine, warrant its further development for malaria treatment and prophylaxis.



INTRODUCTION Malaria continues to be one of the most widespread infectious diseases of our time. This disease is caused by infection with one or more protozoan parasites of the genus Plasmodium in humans. Plasmodium falciparum is responsible for the most serious form of the disease that could be complicated by coma and death if left untreated. The malaria infection is estimated to affect 250 million people worldwide leading to almost 1 million fatal outcomes each year.1 Sub-Saharan Africa is the most distressed region where the vast majority of deaths occur in children under five years of age and in pregnant women. Even though during the past century a large number of drugs for the treatment of malaria have been developed, their utility in curing malaria is declining due to the selection of single-drug or © 2011 American Chemical Society

multidrug resistant parasites. This further fosters the need for the discovery of new antimalarial drugs.2 Azithromycin is a semisynthetic macrolide antibiotic that has become a blockbuster drug due to its wide spectrum of activity and superior pharmacokinetic and safety properties. Antimalarial potential of azithromycin was discovered by Walter Reed Army Institute of Research.3−6 It was demonstrated that azithromycin is a slow acting antimalarial7,8 that exerts its activity by inhibiting protein synthesis on the prokaryote-like ribosome in Plasmodium organelle, the apicoplast.9 Currently, azithromycin is being extensively evaluated in clinical trials to assess its potential use and combination partner in different Received: November 29, 2011 Published: December 12, 2011 1389

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Scheme 1. Synthesis of Novel 15-Membered Azalidesa

a Reagents and conditions: i, 3 or 4, arylhalide, DMSO, 100 °C, 14 h; ii, 3−5, aldehyde, TEA, NaBH4, MeOH, rt, 24 h; iii, 3−5, carboxylic acid, HOBT, EDCxHCl, TEA, DCM, rt, 24 h; iv, 3 (to compound 14), CH2CHCOR4, CHCl3, reflux, 48 h; v, LiOH, H2O, THF, rt, 3 h; vi, 15, (alkyl)aryl amine, HOBT, EDCxHCl, TEA, DCM, rt, 24 h; vii, H2, 10% Pd/C, EtOH, 4 bar, rt.

malaria-related indications.10−19 As no evidence for the superiority or equivalence to other antimalarials has been confirmed so far, the future of azithromycin for the treatment of malaria is uncertain.20 Antimalarial drug discovery today is focusing efforts on creating new molecules that are more potent and safer than currently available drugs and at the same time affordable to the target underprivileged population.2 These drugs should be dosed orally and infrequently to ensure better patient compliance. A very important feature of any potential new class is the activity against strains resistant to other antimalarial drug classes.7 Because of azithromycin-specific mode of

action,21 oral bioavailability, long half-life, and good safety record proven in children and pregnant women, we propose more potent analogues from this class could offer properties needed for malaria treatment and prophylaxis. We recently reported extensive synthesis of new 9asubstituted 15-membered azalide compound libraries comprising of (thio)urea derivatives, investigated their in vitro and in vivo antimalarial activity,22 and analyzed the structure−activity relationship.22,23 This research led to the discovery of novel urea and thiourea derivatives of 15-membered azalides with promising in vitro antimalarial activity. Here we report additional work undertaken in this area to avoid initial 1390

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Chart 1. Structures of the Substituents (R) Used in This Study

8−10 were obtained by reductive alkylation of amines 3−5 with appropriate aldehydes in the presence of triethylamine (TEA) and sodium borohydride. Amides 11−13 were prepared by acylation of corresponding amines 3−5 with various acids in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 1-hydroxybenzotriazole (HOBT) in dichloromethane at room temperature. On the other hand, amides 16 were obtained by esterification of carboxylic acid 15 with various amines using the above-described conditions for the preparation of amides 11−13. Starting carboxylic acid 15 was prepared by the Michael-type addition of amine 2 with methyl or ethyl acrylate followed by ester hydrolysis of 14 under basic conditions.29

difficulties with in vivo efficacy. A set of 42 novel 9a-N substituted amide and amine azalide compounds were synthesized, their in vitro antimalarial activity was determined, and structure−activity relationship (SAR) was established. Furthermore, five compounds were selected for further profiling by investigating their pharmacokinetic properties and in vivo antimalarial efficacy in an adapted Plasmodium berghei mouse model.



CHEMISTRY Identification of promising lead molecules as starting points for new antimalarial programs represents a persistent challenge for scientists within the field.24 Because a preliminary library of 9aN substituted urea and thiourea azalide derivatives has set forth compounds with improved in vitro antimalarial activity over azithromycin,22,23 we expanded research in this field. Here we describe the discovery of novel classes of 9a-N substituted azalides that were designed to additionally explore the chemical space around 9a-N substituted 15-membered azalide scaffolds. In the view of the stringent requirement for novel antimalarials to enable low cost therapy for uncomplicated malaria and intermittent preventive treatment (less than one US dollar per treatment),2 we designed novel 9a-N substituted derivatives as low cost compounds by using inexpensive starting materials and short synthetic procedures. Structural features that guided the design of novel macrolides included (1) replacement of the ureido/thioureido group with an amido and amino functionality and (2) retaining the aryl or heteroaryl group for improving antimalarial activity.25 Here we report the synthesis of 42 novel compounds belonging to four new series of 15-membered azalides (Scheme 1). Three series were completed starting from corresponding primary amine intermediates 3−5, the synthesis and characterization of which was described previously.26−28 Amine compounds 6 and 7 were prepared by the substitution of primary amines 3 and 4 with aryl and heteroaryl halides at elevated temperature. The chlorine atom from the quinoline moiety was removed by catalytic hydrogenation of compounds 6u and 7u yielding compounds 6t and 7t. Amine compounds



RESULTS AND DISCUSSION

In Vitro Activity. The in vitro activity of 42 amides and amines of 15-membered azalides was evaluated against P. falciparum strain TM91C235, a multidrug resistant clone from Southeast Asia and HepG2 hepatocellular carcinoma cell line. The results presented as IC50 values are shown in Table 1 for compounds from two amine classes and in Table 2 for two different amide scaffolds. Results from antimalarial in vitro screening indicated a high percentage of compounds (>90%) with substantially improved activity over azithromycin (up to >100-fold for the most potent compounds) and a majority of compounds (>60%) with IC50 at least 10-fold enhanced. As already observed with (thio)urea derivatives,25 classes of compounds described here also exhibit higher in vitro potencies against resistant strains than against the sensitive strain (data not shown). The presented library discloses compounds from an early lead-optimization phase where the influence of major structural features on the antimalarial activity was investigated, primarily the removal of cladinose and desosamine sugars, type of linker at the 9a-N position and the nature of the aromatic substituent R (Scheme 1). As sugar substiutents, L-cladinose and Ddesosomine, present in azithromycin have been shown to contribute substantially to its antibacterial activity,30 we investigated the significance of sugar unit(s) on antimalarial 1391

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Table 1. In Vitro Activity of 9a-N Substituted Amine Derivatives 6−10 Determined against P. falciparum TM91C235 Strain and HepG2 Hepatocellular Carcinoma Cell Line

a

des = D-desosamine. bclad = L-cladinose, ND = not determined.

The influence of the linker at the 9a-N position and the nature of the aromatic moiety on antimalarial activity were analyzed in more detail by examining the amine (6 and 8) and amide (11 and 16) compounds containing both sugars. Generally, phenyl derivatives (8a, 11b, and 16b, IC50 of 104, 233, and 261, respectively) were significantly more active in comparison to other tested monocyclic aromatic derivatives, that is, five-membered heteroaromatic oxazole derivative 11p (IC50 of 878) and six-membered heteroaromatic pyridine derivatives (8m, 8n, and 8o, IC50 of 1478, 391, and 568, respectively). On the other hand, phenyl derivatives (8f, 11i, 11h, and 16h) were less active than their naphthyl analoques

activity by comparing activities of paired analogues with the same 9a-N substituent and different sugar content (11p, 13p; 8o, 9o; 8n, 9n; 8t, 9t; 11h, 12h; 8r, 9r, 10r; 10l, 11l, 12l; 8s, 9s, 10s; 8f, 9f; 6t, 7t; 6u, 7u). All aglycones (10r, 10s, and 13p) were 3−4-fold less active in comparison to their decladinosyl analogues (9r and 9s) and 24−37-fold less active than analogues with both sugars (8r, 8s, and 11p). Furthermore, the activity of decladinosyl derivatives was lower than the activity of corresponding analogues with both sugars (i.e., IC50 of 8o < 9o; 11h < 12h; 6t < 7t) with one exception (IC50 of 6u > 7u). 1392

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Table 2. In Vitro Activity of 9a-N Substituted Amide Derivatives 11−13 and 16 Determined against P. falciparum TM91C235 Strain and HepG2 Hepatocellular Carcinoma Cell Line

a

des = D-desosamine. bclad = L-cladinose, ND = not determined.

(8a, 11b, and 16b). Quinolyl derivatives (i.e., 6t, 6u, 8r, and 8s) showed excellent antimalarial activity, suggesting that steric bulk and heteroatom in bicyclic aromatic system might have a positive impact on the antimalarial potency. Introduction of a chlorine atom on the quinoline moiety retained superior antimalarial activity irrespective of the presence of cladinose (6u and 7u). However, antimalarial activity significantly decreased when the methoxy group was positioned on C-6 of the quinoline ring (compound 6v, IC50 of 854) in contrast to the positive influence of electron donating groups on naphthyl ring derivatives (8j and 11l). SAR observations regarding linker influence were expanded comparing our earlier published 9a-N substituted 15-membered azalides having urea and thiourea moieties in the linker22,23 with amide and amine analogues reported here. The most populated analogue sets with naphthyl and phenyl aromatic moieties (Table 3) reveal a significant effect of the linker used on antimalarial activity. The activity increased among classes as follows: urea > amide (16b, 16h, 11b, 11h) > amine (8a, 8f). Occasionally, compounds with increased antimalarial potency also showed a slight increase in cytotoxicity (Tables 1 and 2). However, IC50 values determined in HepG2 cell line were relatively high (85% of compounds had IC50 ≥ 20 μM) generating favorable selectivity index (ratio between HepG2

Table 3. Structure−Activity Relationship of Amide (16b, 16h, 11b, 11h) and Amine (8a, 8f) Linked Derivatives Compared with Urea Derivatives,31 All Containing Analogous Aromate Substitutents (IC50 Determined against P. falciparum TM91C235 Strain)

IC50 and P. falciparum IC50 above 100) in almost all cases where calculations were possible. The overall selectivity index for novel 9a-N substituted amides (11−13 and 16) and amines (6−10) was superior to the selectivity index of previous classes 1393

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Table 4. Pharmacokinetic Parameters Estimated in Blood after Intravenous (IV) and Oral Gavage (PO) Administration to CD1 Mice (5 mg/kg IV and 25 mg/kg PO)a CLs (mL/min/kg) azithromycin 6u 6t 8t 11c 16k

16 4.0 7.7 10.2 23.3 10.5

± ± ± ± ± ±

5.3 0.2 1.5 0.3 3.3 3.3

Vss (L/kg) 8.8 2.9 4.9 4.9 5.0 7.8

± ± ± ± ± ±

3.6 1.3 1.5 1.1 1.3 2.1

T1/2 (h)

oral F (%)

fu

CLu (mL/min/kg)

± ± ± ± ± ±

72 20.4 1.7 19.5 12.7 23.6

0.69 0.42 0.39 0.57 0.43 0.13

23.2 9.5 20 18 54 81

8.3 17.8 14.4 8.7 6.9 16.5

1.1 9.4 1.1 1.6 0.7 3.5

a CL = blood clearance, Vss = steady-state volume of distribution, T1/2 = half life, F = bioavailability, fu = fraction unbound in mouse plasma, CLu = CLs/fu, assuming Cbl/Cpl = 1, a = n = 1, b = n = 2.

Chart 2. Structures of the Azalide Compounds Used for in Vivo Study in the Mouse Model

consisting of urea and thiourea derivatives22,23 due to an overall marked improvement in antimalarial activity accompanied by decrease in cytotoxicity. Preliminary Pharmacokinetics and in Vivo Antimalarial Activity. The in vivo pharmacokinetic profiles (Table 4) were investigated in the mouse for five compounds (Chart 2). Two amides 16k and 11c and three amines 6u, 6t, 8t were selected based on their favorable in vitro profile (IC50 against P. falciparum < 200 nM and HepG2 IC50 > 30 μM) and high solubility when prepared as acetic salts (≥10 mg/kg). In vitro metabolic stability studies in liver microsomes (mouse, rat, and human) indicated good stability across species, resulting in low intrinsic clearance (CLi < 0.6 mL/min·g liver), with the exception of compound 16k which showed a low to moderate intrinsic CLi (1.8 mL/min·g liver) in human liver microsomes. Plasma protein binding (PPB) in the mouse suggested these compounds have low PPB ranging from 43 to 61%, with the exception of compound 16k which was 87% protein bound. In line with the in vitro mouse microsomal stability results, all compounds had a very low blood CL (100 fold in the P. falciparum in vitro assay that translated in the in vivo efficacy after IV dosing by curing mice more efficiently than azithromycin and comparable to chloroquine. Detailed pharmacology, safety, and mode of action studies are additionally needed to reveal the true potential of this class, and compound 6u in particular, for its clinical use in the treatment and prophylaxis of malaria.



EXPERIMENTAL SECTION

Chemistry. All commercial reagents (Merck, Sigma-Aldrich) were used as provided unless otherwise indicated, and all solvents are of the highest purity unless otherwise noted. The purity of final compounds was assessed by the analytical LC-MS method and found to be ≥95% unless otherwise stated. The LC-MS analyses were performed using Waters Acquity UPLC instrument equipped with diode-array detector and MS detector, Waters SQD, using the following method: column, Waters Acquity UPLC BEH C18, 2.1 × 50 mm, 1.7 μm particles; mobile phase A, 0.1% HCOOH in water, mobile phase B, 0.1% HCOOH in CH3CN, isocratic 5% B in 1.5 min, then gradient 5−80% B in 7.25 min followed by 1.25 min at 90% B. Mass spectra were obtained on a Waters Micromass ZQmass spectrometer for ES+-MS. Electrospray positive ion mass spectra were acquired using a Micromass Q-Tof2 hybrid quadrupole time-of-flight mass spectrometer, equipped with a Z-spray interface, over a mass range of 100−2000 Da, with a scan time of 1.5 s and an interscan delay of 0.1 s in a continuum mode. Reserpine was used as the external mass calibrant lock mass ([M + H]+ = 609.2812 Da). The elemental composition was calculated using a MassLynx v4.1 for the [M + H]+ and the mass error quoted within ±5 ppm range. NMR spectra were recorded on a Bruker Avance DRX500 or Bruker Avance DPX300 spectrometer in CDCl3 or DMSO and chemical shifts are reported in ppm using TMS as an internal standard. In synthetic procedures, column chromatography was carried out over Merck Kieselgel 60 (230−400 mesh) or on SPE cartrige with average size silica 50 μm. Thin layer chromatography was performed on 0.24 mm silica gel plates Merck TLC 60F254. The eluent used was indicated and solvent ratios refer to volume. In general, organic solutions were dried with anhydrous Na2SO4 or K2CO3, evaporation and concentration were carried out under reduced pressure below 40 °C, unless otherwise noted. All final compounds were isolated as amorphous solid. 9a-[3-(7-Chloro-quinolin-4-ylamino)propyl]-9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A (6u). 4,7-Dichloroquinoline (3.0 g, 15.5 mmol) was added to a solution of intermediate 3 (4.09 g, 5.05 mmol) in DMSO (12 mL). The reaction mixture was stirred at 100 °C for 14 h and then the mixture of H2O (100 mL) and EtOAc (100 mL) was added. The solvents were separated and the EtOAc layer was washed with brine (50 mL), dried over K2CO3, and evaporated in a vacuum. The residue was precipitated from EtOAchexane. The precipitated solid was filtered and purified by column chromatography on silica gel using solvent system DCM/MeOH/ NH4OH = 90:9:0.5. Work up of the chromatography fraction and precipitation from EtOAc/hexane yielded compound 6u (1.2 g, Y = 25%); 1H NMR (500 MHz, DMSO-d6) δ 8.39 (d, J = 5.19 Hz, 1H), 1396

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7.32 Hz, 3H), 1.07 (d, J = 5.80 Hz, 3H), 1.05 - 1.07 (m, 1H), 1.00 (s, 3H), 0.97 - 1.00 (m, 6H), 0.83 (d, J = 6.71 Hz, 3H), 0.79 (t, J = 7.32 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 9.31, 9.31, 10.90, 14.92, 18.13, 18.39, 20.89, 21.28, 21.36, 22.09, 26.96, 27.24, 27.72, 29.77, 34.74, 39.43, 40.23, 40.23, 40.23, 40.27, 44.16, 48.74, 49.75, 60.49, 62.86, 64.58, 64.76, 67.03, 70.60, 72.65, 73.38, 74.30, 75.23, 76.32, 77.29, 77.81, 82.53, 94.90, 101.91, 139.10, 152.28, 176.23; MS (ESI) m/z calcd for C45H80N7O12 (M + H+) 910.5865; found 910.5859. 9a-[3-(Quinolin-4-ylamino)propyl]-9-deoxo-9-dihydro-9aaza-9a-homoerythromycin A (6t). To a solution of compound 6u (50 mg, 0.05 mmol) in EtOH (15 mL), 10% Pd/C (30 mg) was added and the reaction mixture was hydrogenated in Parr apparatus at 4 bar of hydrogen pressure for 24 h. The catalyst was filtrated off and solvent evaporated under reduced pressure. Product was purified by column chromatography (SPE column 5 g, eluent: DCM/MeOH/NH4OH = 90:9:0.5) and then precipitated from EtOAc:hexane yielding compound 6t (41 mg, Y = 89%); 1H NMR (500 MHz, DMSO-d6) δ 8.38 (d, J = 5.19 Hz, 1H), 8.21 (d, J = 7.63 Hz, 1H), 7.76 (d, J = 8.24 Hz, 1H), 7.59 (td, J = 1.22, 7.63 Hz, 1H), 7.32−7.45 (m, 1H), 7.13 (t, J = 5.19 Hz, 1H), 6.44 (d, J = 5.49 Hz, 1H), 4.91 (dd, J = 2.75, 10.07 Hz, 1H), 4.79 (d, J = 4.88 Hz, 1H), 4.43 (d, J = 7.02 Hz, 1H), 4.29 (s, 1H), 4.23 (d, J = 6.71 Hz, 1H), 4.04−4.10 (m, 1H), 4.04 (dd, J = 2.00, 6.00 Hz, 1H), 4.00 (d, J = 7.30 Hz, 1H), 3.62−3.70 (m, J = 2.00, 6.20, 6.20, 6.20, 11.00 Hz, 1H), 3.54 (d, J = 7.30 Hz, 1H), 3.52 (d, J = 7.50 Hz, 1H), 3.23−3.27 (m, 2H), 3.22 (s, 3H), 3.04 (dd, J = 7.48, 9.92 Hz, 1H), 2.94−3.01 (m, 1H), 2.91 (dd, J = 5.65, 8.70 Hz, 1H), 2.76 (q, J = 7.02 Hz, 1H), 2.71 (dq, J = 1.00, 7.50 Hz, 1H), 2.62−2.66 (m, 1H), 2.60 (dd, J = 3.50, 13.20 Hz, 1H), 2.43 (ddd, J = 3.80, 10.30, 12.00 Hz, 1H), 2.27 (d, J = 14.65 Hz, 1H), 2.22 (s, 6H), 2.10 (dd, J = 9.92, 12.66 Hz, 1H), 1.87 - 2.00 (m, 3H), 1.72 - 1.84 (m, 2H), 1.59 (ddd, J = 1.50, 3.00, 11.00 Hz, 1H), 1.50 (dd, J = 4.88, 14.65 Hz, 1H), 1.45−1.51 (m, 1H), 1.33−1.42 (m, 2H), 1.19 (s, 3H), 1.15 (d, J = 6.10 Hz, 3H), 1.13 (s, 3H), 1.11 (d, J = 7.02 Hz, 3H), 1.08−1.11 (m, 1H), 1.07 (d, J = 5.80 Hz, 3H), 1.01 (d, J = 6.50 Hz, 3H), 1.01 (s, 3H), 0.99 (d, J = 7.63 Hz, 3H), 0.84 (d, J = 6.71 Hz, 3H), 0.80 (t, J = 7.32 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 9.25, 9.33, 10.90, 14.84, 18.09, 18.38, 20.89, 21.25, 21.34, 22.30, 25.65, 27.10, 27.84, 29.86, 34.72, 40.23, 40.23, 40.45, 40.90, 44.16, 48.57, 48.72, 59.58, 62.62, 64.56, 64.79, 66.99, 70.52, 72.66, 73.43, 74.27, 75.03, 76.37, 77.28, 77.82, 82.57, 94.88, 98.04, 101.93, 118.76, 121.65, 123.58, 128.55, 128.79, 148.11, 149.88, 150.47, 176.30; MS (ESI) m/z calcd for C49H83N4O12 (M + H+) 919.6008; found 919.5990. 9a-[3-(Quinolin-4-ylamino)propyl]-3-O-decladinosyl-9deoxo-9-dihydro-9a-aza-9a-homoerythromycin A (7t). According to the procedure described for compound 6t starting from the 7t (0.31 g, 0.39 mmol) product 7u (0.22 g, Y = 75%) was obtained; 1H NMR (500 MHz, DMSO-d6) δ 8.38 (d, J = 5.19 Hz, 1H), 8.23 (d, J = 8.54 Hz, 1H), 7.76 (d, J = 8.24 Hz, 1H), 7.51−7.65 (m, 1H), 7.35− 7.44 (m, 1H), 7.03 (t, J = 4.88 Hz, 1H), 6.42 (d, J = 5.49 Hz, 1H), 5.03 (dd, J = 1.00, 12.00 Hz, 1H), 5.00 (d, J = 5.80 Hz, 1H), 4.42 (d, J = 7.32 Hz, 1H), 4.29 (s, 1H), 4.25−4.29 (m, 1H), 3.53 (d, J = 5.80 Hz, 1H), 3.46−3.50 (m, 1H), 3.45 (s, 1H), 3.35−3.43 (m, 1H), 3.17−3.30 (m, 2H), 3.09 (t, J = 8.70 Hz, 1H), 2.85−2.99 (m, 1H), 2.76 (br. s., 1H), 2.65−2.72 (m, 1H), 2.53−2.62 (m, 1H), 2.47−2.50 (m, 1H), 2.45 (ddd, J = 4.00, 10.00, 12.00 Hz, 1H), 2.21 (s, 6H), 2.00−2.08 (m, 1H), 1.93−1.98 (m, 1H), 1.87−1.93 (m, 1H), 1.75−1.86 (m, 3H), 1.67−1.76 (m, 1H), 1.53−1.62 (m, 1H), 1.35−1.47 (m, 1H), 1.14 (d, J = 6.71 Hz, 3H), 1.09 (s, 3H), 1.08−1.13 (m, 2H), 1.09 (d, J = 6.10 Hz, 3H), 1.01 (s, 3H), 0.98 (d, J = 6.71 Hz, 3H), 0.88 (d, J = 7.50 Hz, 3H), 0.87 (d, J = 6.30 Hz, 3H), 0.78 (t, J = 7.48 Hz, 3H); 13C NMR (125 MHz, DMSO-d6) δ 7.96, 8.26, 10.60, 15.91, 17.74, 20.94, 21.25, 21.49, 26.52, 27.02, 29.11, 30.16, 36.37, 39.27, 40.30, 40.30, 40.98, 50.79, 58.19, 60.39, 64.41, 68.27, 70.17, 73.32, 74.34, 75.99, 76.17, 76.42, 89.86, 98.03, 103.37, 118.76, 121.70, 123.52, 128.50, 128.86, 148.18, 149.89, 150.55, 175.31; MS (ESI) m/z calcd for C41H69N4O9 (M + H+) 761.5065; found 761.5052. General Procedure for Reductive Alkylation. Appropriate aldehyde (1 equiv), TEA (0.3 equiv), and macrolide intermediate 3−5 (1.2 equiv) were added to degasses MeOH solution and the reaction mixture was stirred under N2 at room temperature for 1 h. NaBH4 (2

equiv) was than added and the reaction mixture was stirred overnight at room temperature. The MeOH was evaporated and the residue extracted between DCM and water yielding crude products which were purified on SPE column eluating gradiently starting from 100% DCM and ending with mixture of DCM/MeOH/NH4OH = 90:9:0.5 yielding compounds 8−10. 9a-{3-[(Quinolin-4-ylmethyl)amino]propyl}-9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A (8t). White powder (Y = 40%); 1H NMR (500 MHz, DMSO-d6) δ 8.83 (d, J = 4.27 Hz, 1H), 8.21 (d, J = 8.24 Hz, 1H), 8.02 (d, J = 8.54 Hz, 1H), 7.74 (t, J = 7.48 Hz, 1H), 7.60 (t, J = 7.48 Hz, 1H), 7.55 (d, J = 4.27 Hz, 1H), 4.93 (d, J = 10.38 Hz, 1H), 4.80 (d, J = 4.88 Hz, 1H), 4.43 (d, J = 7.32 Hz, 1H), 4.23 (d, J = 7.32 Hz, 1H), 4.21 (s, 2H), 4.12 (s, 1H), 4.04 (dq, J = 6.30, 9.60 Hz, 1H), 3.96 - 3.99 (m, 1H), 3.99 (d, J = 5.80 Hz, 1H), 3.66 (dq, J = 5.65, 10.53 Hz, 1H), 3.53 (s, 1H), 3.50 (d, J = 6.41 Hz, 1H), 3.21 (s, 3H), 3.03 (ddd, J = 1.00, 7.32, 10.00 Hz, 1H), 2.95−3.02 (m, 1H), 2.82 (t, J = 7.78 Hz, 1H), 2.63−2.76 (m, 3H), 2.53−2.59 (m, 2H), 2.37−2.46 (m, 2H), 2.24 (d, J = 14.00 Hz, 1H), 2.20 (s, 6H), 2.13 (dd, J = 9.16, 12.82 Hz, 1H), 1.86−1.97 (m, 2H), 1.73−1.82 (m, 1H), 1.62−1.72 (m, 1H), 1.54−1.60 (m, 2H), 1.45−1.53 (m, 1H), 1.30−1.44 (m, 3H), 1.17 (s, 3H), 1.08−1.13 (m, 9H), 1.06 (d, J = 6.10 Hz, 3H), 0.96−1.01 (m, 9H), 0.95−0.98 (m, 1H), 0.88 (d, J = 6.71 Hz, 3H), 0.78 (t, J = 7.32 Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ 176.59, 150.49, 147.93, 146.47, 129.77, 129.25, 127.05, 126.51, 124.25, 120.07, 102.19, 95.32, 82.97, 78.34, 77.62, 76.89, 75.53, 74.53, 73.86, 72.99, 70.96, 67.39, 65.14, 64.87, 63.54, 59.48, 49.23, 49.09, 48.90, 47.46, 44.55, 40.64, 40.64, 40.64, 40.52, 35.05, 30.29, 28.58, 27.68, 26.96, 22.87, 21.73, 21.49, 21.27, 18.76, 18.22, 15.37, 11.23, 9.74, 8.93; MS (ESI) m/z calcd for C50H85N4O12 (M+H+) 933.6164; found 933.6162. 9a-{3-[(3-Phenylpropanoyl)amino]propyl}-9a-aza-9-deoxo9-dihydro-9a-homoerythromycin A (11c). To a solution of 3phenylpropanoic acid (0.28 g, 1.89 mmol) in dry DCM (100.0 mL), TEA (2.63 mL, 18.9 mmol), HOBT (0.512 g, 3.79 mmol), intermediate 3 (1.5 g, 1.89 mmol) and EDC × HCl (1.45 g, 7.58 mmol) were added. The reaction mixture was stirred at room temperature overnight, and then H2O (50 mL) was added and extracted with DCM (3 × 50 mL). Combined organic layers were washed with brine (50 mL), dried over Na2SO4, and evaporated in vacuum. The residue was purified by column chromatography on silicagel using solvent system DCM/MeOH/NH4OH = 90:7:0.5 to yield compound 11c (1.29 g, 74%). 1 H NMR (500 MHz, DMSO-d6) δ 0.79 (t, J = 7.3 Hz, 3 H), 0.86 (d, J = 6.7 Hz, 3 H), 0.98 (m, 6 H), 1.00 (s, 3 H), 1.07 (d, J = 5.8 Hz, 3 H), 1.10 (d, J = 7.3 Hz, 4 H), 1.13 (s, 3 H), 1.15 (d, J = 6.1 Hz, 3 H), 1.20 (s, 3 H), 1.36 (m, 2 H), 1.43 (m, 1 H), 1.45 (m, 1 H), 1.50 (dd, J = 14.8, 4.4 Hz, 1H), 1.60 (m, 2 H), 1.77 (m, J = 13.0, 7.3, 7.3, 7.3, 2.0 Hz, 1 H), 1.88 (br. s.,1 H), 1.94 (dq, J = 7.6, 6.0 Hz, 1 H), 2.06 (t, J = 11.4 Hz, 1 H), 2.22 (s, 6 H), 2.27 (d, J = 15.0 Hz, 1 H), 2.34 (t, J = 7.8 Hz, 2 H), 2.44 (m, 2 H), 2.54 (m, 1 H), 2.71 (m, 2 H), 2.80 (t, J = 7.8 Hz, 3 H), 2.91 (t, J = 8.4 Hz, 1 H), 2.98 (m, 2 H), 3.04 (m, 1 H), 3.22 (s, 3 H), 3.50 (m, 2 H), 3.66 (dq, J = 10.0, 5.9 Hz, 1 H), 3.96 (d, J = 6.1 Hz, 1 H), 4.01 (d, J = 4.9 Hz, 1 H), 4.06 (dq, J = 9.6, 6.3 Hz, 1 H), 4.23 (d, J = 7.3 Hz, 1 H), 4.28 (s, 1 H), 4.42 (d, J = 7.0 Hz, 1 H), 4.79 (d, J = 4.3 Hz, 1 H), 4.88 (d, J = 10.1, Hz, 1 H), 7.17 (m, 3 H), 7.25 (m, 2 H), 7.80 (t, J = 5.2 Hz, 1 H); 13C NMR (101 MHz, DMSO-d6) δ 9.60, 9.63, 11.25, 15.23, 18.42, 18.75, 21.24, 21.59, 21.70, 22.62, 27.35, 27.58, 28.17, 30.33, 31.39, 35.05, 37.32, 40.61, 40.81, 44.49, 48.81, 49.08, 60.13, 63.10, 64.87, 65.11, 67.30, 70.90, 72.99, 73.72, 74.58, 75.32, 76.64, 77.61, 78.13, 82.86, 95.20, 102.27, 126.06, 128.39, 128.47, 141.65, 171.30, 176.61; MS (ESI) m/z calcd for C49H86N3O13 (M + H+) 924.6161; found 924.6139. 9a-{3-[(Naphtalen-1-yl-acetyl)amino]propyl}-3-O-decladinosyl-9a-aza-9-deoxo-9-dihydro-9a-homoerythromycin A (12h). PS-Carbodiimide resin (PS-CDI, loading: 1.2 mmol/g) (105.2 mg, 0.126 mmol) was added to a dry reaction vessel. The naphtalen-1-ylacetic acid (186.2 mg, 0.095 mmol) dissolved in dry DCM (1.5 mL), was added to the dry resin. The mixture was stirred at room temperature for 1 h upon which intermediate 4 (50 mg, 0.063 mmol) dissolved in dry DCM (0.8 mL) was added. The reaction mixture was 1397

dx.doi.org/10.1021/jm201615t | J. Med. Chem. 2012, 55, 1389−1401

Journal of Medicinal Chemistry

Article

13C NMR (101 MHz, DMSO-d6) δ ppm: 9.41, 10.98, 14.93, 18.19, 18.45, 20.96, 21.35, 21.40, 22.08, 26.53, 27.77, 30.07, 32.37, 34.79, 40.24, 40.38, 44.18, 47.09, 48.79, 59.96, 64.59, 64.88, 67.02, 70.56, 72.72, 73.78, 74.31, 76.42, 77.33, 77.89, 82.60, 94.97, 101.90, 174.20, 176.30. 9a-{1-[(1S)-1-(1-Naphthalenyl)ethyl]amino)propanoyl}-9deoxo-9-dihydro-9a-aza-9a-homoerythromycin A (16k). TEA (62.4 μL, 0.45 mmol), HOBT (12.2 mg, 0.09 mmol), (1S)-1-(1naphthalenyl)ethanamine (8.5 mg, 0.05 mmol), and EDC × HCl (34.5 mg, 0.18 mmol) were added to a solution of intermediate 3 (36.3 mg, 0.045 mmol) in dry DCM (3.0 mL). The reaction mixture was stirred at room temperature overnight. Solvent was evaporated under reduced pressure giving 98.2 mg of yellowish crude product which was purified on preparative LC-MS (XTerra Prep RP18 column, 5 μm, 19 × 100 mm) using gradient system for elution: (0.1% HCOOH in H2O/ CH3CN) in which HCOOH: CH3CN was changed from 95:5 to 60:40 to give the title compound (14.9 mg, yield 35%). 1 H NMR (400 MHz, DMSO-d6) δ 0.78 (t, J = 7.34 Hz, 3 H), 0.85 (d, J = 6.70 Hz, 3 H), 0.93−1.02 (m, 9 H), 1.04−1.16 (m, 13 H), 1.18 (s, 3 H), 1.30−1.43 (m, 3 H), 1.47 (d, J = 6.82 Hz, 3 H), 1.51 (d, J = 4.39 Hz, 1 H), 1.59 (m, 1 H), 1.69−1.81 (m, 1 H), 1.83−1.98 (m, 2 H), 2.00−2.12 (m, 1 H), 2.23 (s, 6 H), 2.28 (m, 1 H), 2.36−2.46 (m, 2 H), 2.55 (m, 1 H), 2.64−2.78 (m, 3 H), 2.86−2.94 (m, 1 H), 3.01− 3.07 (m, 2 H), 3.08−3.17 (m, 1 H), 3.21 (s, 3 H), 3.45−3.53 (m, 2 H), 3.63−3.72 (m, 1 H), 3.95−4.03 (m, 1 H), 4.03−4.10 (m, 2 H), 4.23 (dd, J = 8.09, 3.01 Hz, 1 H), 4.27 (br. s., 1 H), 4.42 (d, J = 7.17 Hz, 1 H), 4.78 (d, J = 4.05 Hz, 1 H), 4.85 - 4.93 (m, 1 H), 5.62−5.72 (m, 1 H), 7.43−7.58 (m, 4 H), 7.81 (d, J = 8.09 Hz, 1 H), 7.92 (d, J = 8.44 Hz, 1 H), 8.08 (d, J = 8.21 Hz, 1 H), 8.44 (d, J = 7.98 Hz, 1 H); 13C NMR (101 MHz, DMSO-d6) δ 9.44, 9.78, 10.99, 14.87, 18.16, 18.43, 20.96, 21.33, 21.40, 21.49, 22.28, 27.72, 29.99, 33.40, 34.77, 40.29, 43.83, 44.11, 48.79, 64.61, 64.87, 67.04, 70.54, 72.73, 73.45, 74.28, 74.96, 76.44, 77.32, 77.82, 82.60, 94.91, 101.93, 122.27, 123.13, 125.40, 125.53, 126.06, 127.17, 128.58, 130.38, 133.35, 140.23, 170.38, 176.28; MS (ESI) m/z calcd for C52H86N3O13 (M + H+) 960.6161; found 960.6176. Biology. Compound Susceptibility Testing. The in vitro antimalarial activity of novel 15-membered azalide analogues was determined using the tritiated hypoxanthine incorporation assay of Desjardins et al.,38 as modified by Milhous et al.39 with the exception of exposing the parasite to the drug for 48 h.7 The P. falciparum clone used was TM91C235,40 a strain from Southeast Asia that shows high level resistance to mefloquine and a number of other antimalarials. Compounds and control antimalarials (chloroquine, mefloquine, and azithromycin) were diluted 2-fold over 11 different concentration and IC50's were determined using a nonlinear logistic dose response program. The presented IC50's represent averaged values in cases where multiple results were generated and relative errors were usually