Buchwald reaction as the key step for the synthesis of metabolically more stable analogs of amodiaquine

European Journal of Medicinal Chemistry 46 (2011) 3052e3057

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Original article

Buchwald reaction as the key step for the synthesis of metabolically more stable analogs of amodiaquine Nicolas Le Fur a, b, c, Guillaume Hochart a, b, c, Paul-Emmanuel Larchanché a, b, c, Patricia Melnyk a, b, c, * a

Univ Lille Nord de France, F-59000 Lille, France UDSL, EA 4481, UFR Pharmacie, F-59006 Lille, France c UMR CNRS 8161, F-59000 Lille, France b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 February 2011 Received in revised form 14 April 2011 Accepted 15 April 2011 Available online 21 April 2011

Amodiaquine is one of the most active anti-malarial 4-aminoquinoline but its metabolization is believed to generate hepatotoxic derivatives. Previously, we described new analogs of amodiaquine and amopyroquine, in which hydroxyl group was replaced by various amino groups and identified highly potent compounds with lower toxicity. We describe here the synthesis of new analogs that have been modified on their 40 - and 50 -positions in order to reduce their metabolization. A new synthetic strategy was developed using Buchwald coupling reaction as the key step. Ó 2011 Elsevier Masson SAS. All rights reserved.

Keywords: Drugs Heterocycles Quinolines Palladium Substitution

1. Introduction In spite of the recent decline of the pathology, malaria still remains a major health problem with about 243 million cases worldwide in 2008 and 860 000 deaths in more than 100 countries [1,2]. 85e90% of cases and deaths were in the African Region and this constitutes one of the main obstacles to socio-economic development in sub-Saharian Africa and other tropical regions in the world. Chloroquine (CQ, Fig. 1) was a mainstream drug in the fight against Plasmodium falciparum, but its efficacy was restricted by the emergence of resistant parasites. Quinoline anti-malarials concentrate in the parasite food vacuole and are thought to exert their activity by preventing effective formation of hemozoin by interacting to heme through pep stacking of their planar aromatic structures, resulting in heme-mediated toxicity towards the parasite [3]. The lack of an enzyme drug target for quinoline antimalarials is probably a chief reason why resistance development to these drugs is relatively slow. Amodiaquine (AQ, Fig. 1), another

* Corresponding author. EA 4481 GRIIOT, UDSL, UFR Pharmacie, 3 rue du Pr Laguesse, BP83, F-59006 Lille cedex, France. Fax: þ33 (0) 3 20 95 90 09. E-mail address: [email protected] (P. Melnyk). 0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2011.04.047

4-aminoquinoline, proved to be effective against CQ-resistant strains [4,5]. But in the 1980s, cases of agranulocytosis, neutropenia and hepatisis were reported associated with AQ prophylaxis and its prophylactic use was then stopped [6]. AQ toxicity has been explained by the presence of its 4-hydroxyanilino moiety, which is believed to undergo extensive metabolization to its quinoneimine variant and nucleophilic attack on its 50 -position (Fig. 2) [7,8]. Formation of this reactive species in vivo and subsequent binding to cellular proteins and lipids could affect cellular functions either directly or by immunological response [9,10]. This bio-activation was found to be accompanied by the expression of a drug-related antigen on the cell surface, suggesting a type II hypersensitivity reaction and causing the myelotoxicity of AQ [11,12]. Nevertheless, AQ is commercialised in combination with an artemisinin derivative as CoarsucamÒ speciality for curative treatment. For years, our laboratory has been involved in the design and synthesis of AQ-analogs in order to improve the anti-malarial activity while preventing metabolization [13e15]. Furthermore, as AQanalogs obtained by the replacement of the N-diethylamino function of the side chain with a pyrrolidine cycle or a N-tert-butyl group were proved to be metabolically less labile [16,17], we completed our study with the development of parallel amopyroquine-analogs series. Amopyroquine (ApQ, Fig. 1), a structural analog of AQ in

N. Le Fur et al. / European Journal of Medicinal Chemistry 46 (2011) 3052e3057

5' 4' OH N

HN Cl

HN 4

N

Cl

Chloroquine (CQ)

3'

5'

4' NR

2

NR2

HN Cl

N

Amodiaquine (AQ) : NR2 = NEt2 Amopyroquine (ApQ) : NR2 = pyrrolidine

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3'

NR'2

HN

N

Cl

1 NR2 = N-methylpiperazino, NR'2 = pyrrolidino 2 NR2 = morpholino, NR'2 = pyrrolidino

NR2 N

N

3 NR2 = N-methylpiperazino 4 NR2 = morpholino

Fig. 1. Structure of some aminoquinoline anti-malarials.

Fig. 3. Structure of some potent amodiaquine analogs and target compounds.

which the diethylamino group in the side chain is replaced with a pyrrolidine cycle was shown to be more active than both CQ and AQ against 11 CQ-resistant isolates of P. falciparum [18,19]. We recently developed a series of analogs where 40 -hydroxyl group was replaced by various amino substituents (Fig. 3) [20]. The substitution of 40 -hydroxy by N-methylpiperazino (compound 1) or morpholino group (compound 2) provided low nanomolar activity upon K1 chloroquino-resistant strain and good cytotoxicity. The most potent morpholino analog 2 was selected for in vivo experiments. This compound presented good in vivo anti-malarial activity, very close to that of the reference compound AQ, with a >99.9% reduction of parasitaemia observed at day 4 and day 11. In order to avoid potential metabolization in 50 -position, we decided to modify our best compounds 1 and 2 by preparing their 50 -methylated analogs 3 and 4 (Fig. 3). In our previous work, 40 -amino analogs were synthesized thanks to an SNAr reaction of activated fluoro derivatives [16] and 40 -alkyl or aryl analogs were successfully obtained [21] using SuzukieMiyaura cross-coupling reaction [22], as in the case of tebuquine [23]. In the present work, both 3-methyl-4-fluoronitro and 3-methyl4-bromonitro aryl precursor were commercially available and we planned to test both synthetic approaches to yield our target compounds 3 and 4 (Fig. 4): Buchwald cross-coupling reaction [24] with bromo derivative or SNAr reaction with fluoro compound. As previously described by our group, Mannich amino side chain could be introduced through Tcherniak-Einhorn reaction [21].

defluorobenzyl amine 7 (33%) were isolated. The synthesis of target compounds from fluoro starting material was then given up. A first attempt of Buchwald coupling reaction was realized on phtalimido compound 5b using different conditions: Pd(OAc)2 or Pd2(dba)3 as palladium source, P(o-tolyl)3 as ligand, tBuONa or Cs2CO3 as base, toluene or dioxane as solvent and diverse reaction conditions from room temperature to 90  C. All our efforts failed, compound 5b remained unchanged. We decided then to build the amino side chain before the coupling reaction. As expected [21], hydrazinolysis of compound 5b with hydrazine hydrate provided benzylamine 6b with good yield. Benzylamine 6b was reacted with dibromobutane to yield compound 9. Unfortunately all attempts to realize Buchwald coupling from benzylamine 6b as a substrate failed. We then decided to protect the amino side chain by preparing Boc derivative 10. This compound was then subjected to diverse experimental conditions. Optimized yields of 68% for compound 11 and 71% for 12 were obtained with Pd2(dba)3 as palladium source, racemic BINAP as ligand, Cs2CO3 as base, dioxane as solvent, 90  C for about 20 h. BINAP was the only ligand allowing us to obtain the expected compounds. These conditions were unsuccessful when pyrrolidino intermediate 9 was used as the substrate. The end of the synthesis was classically performed with an acidic deprotection of Boc group, reduction of nitro group and regioselective nucleophilic aromatic substitution of the chlorine atom in position 4 of the 4,7-dichloroquinoline by aniline compounds 17 and 18 (Scheme 2) to afford derivatives 3 and 4 in good yields. The yield of the last step was considerably improved by the use of one equivalent of HCl, enhancing therefore C4 electrophilicity of the quinoline nucleus. Target compounds were finally obtained after an eight step procedure with global yields of 19% (3) and 18% (4).

2. Results and discussion In order to obtain key intermediates 11 and 12 (Scheme 1), the 30 -amino side chain was introduced via a super-electrophilic Tcherniak-Einhorn amidomethylation of the commercially available 4-fluoro-3-methylnitro-benzene and 4-bromo-3-methylnitrobenzene with N-hydroxymethylphtalimide in trifluoromethanesulfonic acid according to Olah et al. procedure [25]. As the substrates were strongly deactivated, the use of a super acid as catalyst and solvent was necessary. When deprotection of phtalimido group was engaged for fluoro compound 5a, amino derivative 6a could not be obtained. Within a complex mixture, indazole 8 (31%) and

3. Conclusion Despite recent progress, malaria still remains a major health problem. Aminoquinoline derivatives are historically interesting compounds to treat this pathology but some of them, such as amodiaquine, can present metabolization issues leading to hepatotoxic effects. We have described here a new synthesis of amodiaquine analogs in order to prevent their metabolization. Target

Nucleophilic attack

Buchwald or SNAr reaction

5'

OH HN

NEt2

N

P450 [O]

Cl

N

Cl

N

O

N

NEt2 Binding to cellular proteins HEPATOTOXICITY

Amodiaquine (AQ)

Fig. 2. Oxidation of AQ to the toxic quinoneimine electrophilic metabolite mediated by cytochrome P450.

HN

N

X

N O2N

X

N

Y O2N Y = F, Br

Cl

N 3 X = N-Me 4X=O

Tcherniak-Einhorn reaction

Fig. 4. Retrosynthetic strategy.

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N. Le Fur et al. / European Journal of Medicinal Chemistry 46 (2011) 3052e3057

H N N

F O2N

O2 N NH2

b

NH2

6a

O2 N

7

8

Y=F

Y

Y

a

O2N

O2 N O

Y = F, Br

Br

b Y = Br O2N

N

c

5a Y = F 5b Y = Br

O2 N

NH2

O

Br

d

6b

N

e O2N

NHBoc 10

X

NHBoc 11 X = N-Me 12 X = O

Br O2N 9

N

Scheme 1. Synthesis of key intermediates 11 and 12. Reagents: (a) PhtCH2OH, TfOH, 0 C to r.t. (70-94%); (b) hydrazine hydrate, CH3CN, reflux (75%); (c) Br(CH2)4Br, K2CO3, CH3CN, reflux (67%); (d) Boc2O, THF, r.t (90%); (e) N-methylpiperazine or morpholine, Pd2dba3, (þ/) BINAP, Cs2CO3, 1,4-dioxanne, 90 C (68-71%).

4.2. Synthesis of key intermediates 11 and 12

compounds 3 and 4 were efficiently obtained thanks to Buchwald coupling reaction and Tcherniak-Einhorn amidomethylation. Further work will evaluate the impact of this methyl group for metabolic stability and anti-malarial activity.

4.2.1. 2-[(2-Fluoro-3-methyl-5-nitrophenyl)methyl]-2,3-dihydro1H-isoindole-1,3-dione 5a N-(Hydroxymethyl)phtalimide (7.09 g, 40.0 mmol) was dissolved in 25 mL of triflic acid at 0  C. The mixture was stirred for 20 min and then 2-fluoro-5-nitrotoluene (6.2 g, 1eq) was added. This solution was allowed to warm to rt and was stirred overnight. The mixture was poured slowly into 500 mL of ice-cold water. The aqueous layer was extracted twice with CH2Cl2. The combined organic layers were washed with 200 mL of water, dried over Na2SO4, filtered and evaporated to yield expected compound 5a as a white powder (8.6 g, 70% yield). 1H NMR (300 MHz, CDCl3) d 8.06 (s, 1H, H-4), 8.04 (s, 1H, H-6), 7.90 (m, 2H, H-Ar), 7.71 (m, 2H, H-Ar), 4.97 (s, 2H, CH2), 2.38 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3) d 167.9 (C), 164.6 (C), 161.1 (C), 134.7 (CH), 132.1 (C), 127.2 (d, C, JCF ¼ 19.9 Hz), 126.8 (d, CH, JC-F ¼ 6.8 Hz), 124.7 (d, CH, JC-F ¼ 18.3 Hz), 124.0 (CH), 123.4 (d, CH, JC-F ¼ 5.4 Hz), 35.4 (CH2), 15.0 (CH3); LC/MS tR ¼ 4.8 min; PHPLC > 99%.

4. Experimental section 4.1. General procedures All reactions were monitored by thin-layer chromatography carried out on 0.2 mm E. Merck silica gel plates (60F-254) using UV light as a visualizing agent. 1H and 13C NMR spectra were obtained using a Bruker 300 MHz spectrometer, chemical shifts (d) were expressed in ppm relative to TMS used as an internal standard. Purity and identity of intermediate compounds were checked by LC/MS, using a Waters Alliance 2695 system (X-Terra column, ionization mass spectrometer). The following eluent systems were used: A (H2O/TFA, 100:0.01) and B (CH3CN/H2O/TFA, 80:20:0.01). Retention times (tR) were obtained, at flow rate of 0.3 mL/min, using the following conditions: a gradient run from 100% eluent A to 100% eluent B over 8 min, then 100% eluent B for 90 s. The purity of final compounds was verified by two types of high pressure liquid chromatography (HPLC) columns: C18 Deltapak (C18 N) and C4 Interchrom UP5WC4-25QS (C4) on a Shimadzu system equipped with a UV detector set at 254 nm, same eluents as for LCMS method, at flow rate of 1 mL/min, with a 40 min method: a gradient run from 100% eluent A during 1 min, then to 100% eluent B over the next 30 min. For some compounds mass spectra were recorded on a MALDI-TOF Voyager-DE-STR (Applied Biosystems) apparatus. Reagents were obtained from Acros, Aldrich, Lancaster, Novabiochem and Avocado. The following abbreviations were used: AcOEt (ethyl acetate), rt (room temperature), Quino (quinoline).

N O2N

X

NHBoc 11 X = N-Me 12X = O

N

a O2N

NH2

13 X = N-Me 14 X = O

X

4.2.2. 2-[(2-Bromo-3-methyl-5-nitrophenyl)methyl]-2,3-dihydro1H-isoindole-1,3-dione 5b N-(Hydroxymethyl)phtalimide (2.36 g, 13.3 mmol) was dissolved in 20 mL of triflic acid at 0  C. The mixture was stirred for 20 min and then 2-bromo-5-nitrotoluene (2.88 g, 1eq) was added. This solution was allowed to warm to rt and was stirred for 18 h. The mixture was poured slowly into 300 mL of ice-cold water. The aqueous layer was extracted with CH2Cl2. The combined organic layers were washed with 100 mL of water, dried over Na2SO4, filtered and evaporated to yield expected compound 5b as a white solid (4.71 g, 94% yield). 1H NMR (300 MHz, CDCl3) d 7.97 (d, 1H, J ¼ 2.6 Hz, H-4), 7.84 (m, 2H, H-Ar), 7.71 (m, 3H, H-Ar, H-6), 4.95 (s, 2H, CH2), 2.48 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3) d 167.7 (C), 146.7 (C), 140.9 (C), 137.3 (C), 134.5 (CH), 132.3 (C), 131.7 (C), 124.1

N

b O2N 15 X = N-Me 16 X = O

N

X

N

c H2N 17 X = N-Me 18 X = O

X

N

d HN

N Cl

X

N

N 3 X = N-Me 4X=O

Scheme 2. Synthesis of target compounds 3 and 4. Reagents: (a) HCl, MeOH, r.t. (quant.); (b) Br(CH2)4Br K2CO3, CH3CN, reflux (53-58%); (c) HCOONH4, Pd/C (10%), EtOH, r.t. (99%); (d) 4,7-dichloroquinoline, HCl (1M, aq.), CH3CN, reflux (84-71%).

N. Le Fur et al. / European Journal of Medicinal Chemistry 46 (2011) 3052e3057

(CH), 123.8 (CH), 119.9 (CH), 42.3 (CH2), 23.8 (CH3); LC/MS tR ¼ 9.9 min; PHPLC ¼ 99%. 4.2.3. (2-Bromo-3-methyl-5-nitrophenyl)methanamine 6b 4.4 mL of hydrazine hydrate was added to 2-[(2-bromo-3methyl-5-nitrophenyl)methyl]-2,3-dihydro-1H-isoindole-1,3dione 5b (4.4 g, 11.7 mmol) in 220 mL of acetonitrile. The mixture was stirred under reflux overnight and then was allowed to cool to rt. The phtalhydrazide side product was removed by filtration and washed with 100 mL portions of acetonitrile. The combined acetonitrile filtrates were evaporated and the residue was partitioned between water and AcOEt. The organic layer was extracted with two 100 mL portions of aqueous HCl 1 M. The combined acidic aqueous layers were basified to pH ¼ 10 with solid potassium hydroxide and then extracted with AcOEt. The organic layer was dried over Na2SO4 filtered and evaporated to yield expected compound 6b as an orange powder (2.15 g, 75% yield). 1H NMR (300 MHz, CDCl3) d 8.10 (d, 1H, J ¼ 2.8 Hz, H-4), 7.94 (d, 1H, J ¼ 2.6 Hz, H-6), 3.96 (s, 2H), 2.47 (s, 3H); 13C NMR (75 MHz, CDCl3) d 146.8 (C), 144.4(C), 140.4(C), 132.9(C), 123.4 (CH), 120.6 (CH), 47.0 (CH2), 23.8 (CH3). LC/MS tR ¼ 5.4 min; PHPLC > 99%; m/z (ESI) 244.9e246.9 [M þ H]þ. 4.2.4. (3-Methyl-5-nitrophenyl)methanamine 7 Brown oil (1.5 g, 33% yield). 1H NMR (300 MHz, CDCl3) d 8.00 (s, 1H, H-6), 7.91 (s, 1H, H-4), 7.48 (s, 1H, H-2), 3.96 (s, 2H, CH2), 2.45 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3) d 148.7 (C), 145.1 (C), 140.2 (C), 134.7 (CH), 122.6 (CH), 119.5 (CH), 45.9 (CH2), 21.5 (CH3). LC/MS tR ¼ 4.9 min; PHPLC ¼ 95%; m/z (ESI) 167.1 [M þ H]þ. 4.2.5. 7-Methyl-5-nitro-1H-indazole 8 Brown solid (1.5 g, 31% yield). 1H NMR (300 MHz, CD3OD) d 8.63 (s, 1H, H-4), 8.31 (s, 1H, H-3), 8.04 (s, 1H, H-6), 2.65 (s, 3H, CH3); 13C NMR (75 MHz, CD3OD) d 144.9 (C), 144.7 (C), 139.1 (CH), 124.1 (C), 122.7 (CH), 118.2 (CH), 18.0 (CH3). LC/MS tR ¼ 7.1 min; PHPLC ¼ 95%; m/z (ESI) 177.9 [M þ H]þ. 4.2.6. 1-(2-Bromo-3-methyl-5-nitro-benzyl)-pyrrolidine 9 A mixture of (2-bromo-3-methyl-5-nitrophenyl)methanamine 6b (735 mg, 3 mmol), 1,4-dibromobutane (0.54 mL, 1.5eq) and K2CO3 (2.07 g, 5eq) in 125 mL of acetonitrile was refluxed for 48 h. After filtration and evaporation of the filtrate, the residue was purified by flash chromatography (CH2Cl2/MeOH/NH4OH//9/1/0.1) to yield expected compound 9 as a yellow powder (601 mg, 67% yield). 1H NMR (300 MHz, CDCl3) d 8.21 (d, 1H, J ¼ 2.8 Hz, H-4), 7.98 (d, 1H, J ¼ 2.8 Hz, H-6), 3.79 (s, 2H, CH2), 2.6 (m, 4H, 2NeCH2), 2.46 (s, 3H, CH3), 1.78 (m, 4H, 2CH2); 13C NMR (75 MHz, CDCl3) d 147.0 (C), 141.7 (C), 140.2 (C), 133.5 (C), 123.3 (CH), 122.2 (CH), 60.2 (CH2), 54.5 (CH2), 24.0 (CH3), 23.9 (CH2). LC/MS tR ¼ 5.9 min; PHPLC ¼ 99%; m/z (ESI) 298.3e300.3 [M þ H]þ. 4.2.7. tert-Butyl N-[(2-bromo-3-methyl-5-nitrophenyl)methyl] carbamate 10 Di-tert-butyl dicarbonate (1.51 g, 6.90 mmol) was added to (2bromo-3-methyl-5-nitrophenyl) methanamine 6b (1.54 g, 1.1 eq) in 60 mL of THF. The mixture was stirred overnight at rt. After evaporation of the solvent, the residue was thoroughly washed with pentane and filtered off to yield expected compound 10 as offwhite powder (1.93 g, 90% yield). 1H NMR (300 MHz, CDCl3) d 7.95 (s, 2H, H-4, H-6), 5.12 (br, 1H, NH), 4.37 (d, 2H, J ¼ 6.3 Hz, NeCH2), 2.45 (s, 3H, CH3), 1.41 (s, 9H, 3 CH3); 13C NMR (75 MHz, CDCl3) d 155.7 (C), 146.8 (C), 140.5 (C), 140.4 (C), 132.5 (C), 123.7 (CH), 120.4 (CH), 45.2 (CH2), 28.3(CH3), 23.7 (CH3). LC/MS tR ¼ 9.5 min; PHPLC > 99%; m/z (ESI) 369.0e371.0 [M þ Na]þ; 330.0e331.9 [M þ H-Me]þ; 244.9e247.0 [M þ H-Boc]þ.

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4.2.8. tert-Butyl N-{[3-methyl-2-(4-methylpiperazino)-5nitrophenyl]methyl}carbamate 11 In a oven-dried flask and under a nitrogen atmosphere, were placed tert-butyl N-[(2-bromo-3-methyl-5-nitrophenyl)methyl] carbamate 10 (150 mg, 0.43 mmol), (þ/) BINAP (14 mg, 0.05 eq), Cs2CO3 (196 mg, 1.4 eq), Pd2dba3 (10 mg, 0.05 eq) and 3 mL of dry 1,4-dioxane. N-Methylpiperazine (58 mL, 1.2 eq) was added and the mixture was stirred at 90  C for 16 h. The solution was filtered through a celite pad and evaporated. The residue was purified by flash chromatography on silica gel (CH2Cl2/MeOH//9/1 with 1% triethylamine) to yield expected compound 11 as a pale brown powder (106 mg, 68% yield). 1H NMR (300 MHz, CDCl3) d 7.91 (s, 1H, H-4), 7.82 (s, 1H, H-6), 5.04 (br, 1H, NH), 4.34 (d, 2H, J ¼ 6.1 Hz, NeCH2), 3.3e2.9 (m, 4H, 2NeCH2), 2.6e2.3 (m, 7H, 2NeCH2, CH3), 1.40 (s, 9H, 3CH3); 13C NMR (75 MHz, CDCl3) d 156.0 (C), 153.5 (C), 144.6 (C), 139.1 (C), 137.9 (C), 125.6 (CH), 120.5 (CH), 55.8 (CH2), 49.6 (CH2), 46.4 (CH3), 41.5 (CH2), 28.3 (CH3), 20.1 (CH3). LC/MS tR ¼ 5.4 min; PHPLC > 99%; m/z (ESI) 365.2 [M þ H]þ. 4.2.9. tert-Butyl N-{[3-methyl-2-morpholino-5-nitrophenyl] methyl}carbamate 12 In a oven-dried flask and under a nitrogen atmosphere, were placed tert-butyl N-[(2-bromo-3-methyl-5-nitrophenyl)methyl] carbamate 10 (500 mg,1.45 mmol), (þ/) BINAP (45 mg, 0.07 mmol), Cs2CO3 (661 mg, 2.03 mmol), Pd2dba3 (33 mg, 0.07 eq) and 10 mL of dry 1,4-dioxane. Morpholine (152 mL, 1.74 mmol) was added and the mixture was stirred at 90  C for 24 h. The solution was filtered through a celite pad and evaporated. The residue was purified by flash chromatography on silica gel (CH2Cl2/AcOEt//9/1) to yield expected compound 12 as a pale brown powder (360 mg, 71% yield). 1H NMR (300 MHz, CDCl3) d 8.00 (d, 1H, J ¼ 2.1 Hz, H-4), 7.92 (d, 1H, J ¼ 2.7 Hz, H-6), 5.05 (brs, 1H, NH), 4.46 (d, 2H, J ¼ 6.0 Hz, NeCH2), 3.9e3.8 (m, 4H, 2OeCH2), 3.5e2.7 (m, 4H, 2NeCH2), 2.45 (s, 3H, CH3), 1.46 (s, 9H, 3 CH3); 13C NMR (75 MHz, CDCl3) d 156.5 (C), 153.5 (C), 145.4 (C), 139.8 (C), 138.6 (C), 126.3 (CH), 121.3 (CH), 68.3 (CH2), 50.6 (CH2), 42.1 (CH2), 29.0 (CH3), 20.7 (CH3). LC/MS tR ¼ 9.0 min; PHPLC ¼ 99%; m/z (ESI) 352.2 [M þ H]þ. 4.3. Synthesis of target compounds 3 and 4 4.3.1. [3-Methyl-2-(4-methylpiperazino)-5-nitrophenyl] methanamine 13 1.2 mL of acetyl chloride was slowly added to 10 mL of MeOH at 0  C. The solution was stirred for 30 min at rt and then tert-butyl N-{[3-methyl-2-(4-methylpiperazino)-5-nitrophenyl]methyl}carbamate 11 (300 mg, 0.82 mmol) dissolved in 5 mL of MeOH was added. The reaction mixture was stirred for 2 h and then evaporated. The residue was dissolved in 15 mL of water and washed with diethyl ether. The aqueous layer was made alkaline (pH ¼ 10) with an aqueous NaOH 1 M and extracted with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered and evaporated to yield expected compound 13 as a pale yellow oil (216 mg, quantitative yield). 1H NMR (300 MHz, CDCl3) d 8.05 (d, J ¼ 2.9 Hz, 1H, H-4), 7.85 (d, J ¼ 2.8 Hz,1H, H-6), 3.89 (s, 2H, NeCH2), 3.4e2.8 (m, 4H, 2NeCH2), 2.7e2.3 (m, 4H, 2NeCH2), 2.37 (s, 3H, NeCH3), 2.32 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3) d 153.9 (C), 144.6 (C), 143.1 (C), 137.9 (C), 125.2 (CH), 121.3 (CH), 55.9 (CH2), 49.8 (CH2), 46.4 (CH3), 43.4 (CH2), 20.1 (CH3). LC/MS tR ¼ 4.4 min; PHPLC ¼ 99%; m/z (ESI) 265.2 [M þ H]þ. 4.3.2. (3-Methyl-2-morpholino-5-nitrophenyl)methanamine hydrochloride 14 0.81 mL of acetyl chloride was slowly added to 5 mL of MeOH at 0  C. The solution was stirred for 30 min at rt and then tertbutyl N-{[3-methyl-2-morpholino-5-nitrophenyl]methyl}carbamate 12 (200 mg, 0.57 mmol) dissolved in 5 mL of MeOH was added. The

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reaction mixture was stirred for 3 h and then evaporated to yield expected compound 14 as an orange powder (162 mg, 99% yield). 1H NMR (300 MHz,) d 8.16 (d, J ¼ 3.0 Hz, 1H, H-4), 7.98 (d, J ¼ 2.7 Hz, 1H, H-6), 3.9e3.7 (m, 6H, 2OeCH2, CH2), 3.2e3.1 (m, 4H, 2NeCH2), 2.6e2.5 (m, 4H, 2NeCH2), 2.48 (s, 3H, CH3), 1.9e1.8 (m, 4H, 2CH2); 13C NMR (75 MHz, CD3OD) d 153.8 (C), 145.1 (C), 139.4 (C), 133.9 (C), 127.3 (CH), 121.4 (CH), 67.4 (CH2), 50.2 (CH2), 39.4 (CH2), 19.1 (CH3). MALDITOF m/z ¼ 306.3 [M þ H]þ. HPLC (C4) tR ¼ 5.4 min; PHPLC > 99%; HPLC (C18) tR ¼ 13.3 min; PHPLC > 99%. 4.3.3. 1-Methyl-4-[2-methyl-4-nitro-6-(pyrrolidinomethyl)phenyl] piperazine 15 A mixture of [3-methyl-2-(4-methylpiperazino)-5-nitrophenyl] methanamine 13 (188 mg, 0.71 mmol), 1,4-dibromobutane (93 mL, 1.1 eq) and K2CO3 (490 mg, 5 eq) in 15 mL of acetonitrile was refluxed for 48 h. After filtration and evaporation of the filtrate, the residue was purified by flash chromatography (CH2Cl2/MeOH/ NH4OH//9/1/0.1) to yield expected compound 15 as a pale yellow oil (120 mg, 53% yield). 1H NMR (300 MHz, CDCl3) d 8.02 (d, J ¼ 2.9 Hz, 1H, H-4), 7.76 (d, J ¼ 2.8 Hz, 1H, H-6), 3.58 (s, 2H, CH2), 3.1e3.0 (m, 4H, 2NeCH2), 2.5e2.3 (m, 8H, 4NeCH2), 2.28 (s, 3H, NeCH3), 2.25 (s, 3H, CH3), 1.7e1.6 (m, 4H, 2CH2); 13C NMR (75 MHz, CDCl3) d 154.5 (C), 143.8 (C), 139.0 (C), 137.3 (C), 125.0 (CH), 123.5 (CH), 57.0 (CH2), 55.8 (CH2), 54.0 (CH2), 49.7 (CH2), 46.7 (CH3), 23.5 (CH2), 20.3 (CH3). LC/MS tR ¼ 4.2 min; PHPLC ¼ 95%; m/z (ESI) 319.3 [M þ H]þ. 4.3.4. 4-[2-Methyl-4-nitro-6-(pyrrolidinomethyl)phenyl] morpholine 16 A mixture of (3-methyl-2-morpholino-5-nitrophenyl)methanamine hydrochloride 14 (160 mg, 0.56 mmol), 1,4-dibromobutane (80 mL, 0.67 mmol) and K2CO3 (542 mg, 3.92 mmol) in 10 mL of acetonitrile was heated at 60  C for 48 h. After filtration and evaporation of the filtrate, the residue was purified by flash chromatography (CH2Cl2/MeOH/NH4OH//9/1/0.1) to yield expected compound 16 as a pale yellow oil (100 mg, 58% yield). 1H NMR (300 MHz, CD3OD) d 8.16 (d, J ¼ 3.0 Hz, 1H, H-4), 7.98 (d, J ¼ 2.7 Hz, 1H, H-6), 3.9e3.7 (m, 6H, CH2, 2OeCH2), 3.3e3.2 (m, 4H, 2NeCH2), 2.6e2.5 (m, 4H, 2NeCH2), 2.48 (s, 3H, CH3), 1.90e1.80 (m, 4H, 2CH2); 13C NMR (75 MHz, CD3OD) d 154.5 (C), 144.6 (C), 139.2 (C), 138.4 (C), 125.0 (CH), 123.3 (CH), 67.8 (CH2), 57.0 (CH2), 54.1 (CH2), 50.4 (CH2), 23.4 (CH2), 19.3 (CH3). LC/MS tR ¼ 6.2 min; PHPLC ¼ 99%; m/z (ESI) 306.3 [M þ H]þ. 4.3.5. 3-Methyl-4-(4-methylpiperazino)-5-(pyrrolidinomethyl) aniline 17 1-Methyl-4-[2-methyl-4-nitro-6-(pyrrolidinomethyl)phenyl] piperazine 15 (90 mg, 0.28 mmol) was hydrogenated using ammonium formate (107 mg, 6 eq) and Pd/C (10% Pd, 15 mg, 0.05 eq) in 2 mL of EtOH. The mixture was stirred overnight at rt and then filtered through a celite pad. The filtrate was evaporated and the residue was dissolved in CH2Cl2 and washed with a saturated aqueous solution of Na2CO3. The aqueous layer was extracted with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered and evaporated to yield expected compound 17 as a pale yellow oil (84 mg, quantitative yield). 1H NMR (300 MHz, CDCl3) d 6.65 (d, J ¼ 2.8 Hz, 1H, H-4), 6.30 (d, J ¼ 2.8 Hz,1H, H-6), 3.64 (s, 2H, CH2), 3.45 (brs, 2H, NH2), 3.2e2.9 (m, 4H, 2NeCH2), 2.5e2.3 (m, 8H, 4NeCH2), 2.28 (s, 3H, NeCH3), 2.20 (s, 3H, CH3), 1.77e1.67 (m, 4H, 2CH2); 13C NMR (75 MHz, CDCl3) d 144.1 (C), 141.0 (C), 139.6 (C), 138.5 (C), 117.1 (CH), 114.3 (CH), 56.5 (CH2), 56.0 (CH2), 54.3 (CH2), 50.3 (CH2), 46.7 (CH3), 23.7 (CH2), 19.9 (CH3). LC/MS m/z (ESI) 289.3 [M þ H]þ. 4.3.6. 3-Methyl-4-morpholino-5-(pyrrolidinomethyl)aniline 18 4-[2-Methyl-4-nitro-6-(pyrrolidinomethyl)phenyl] morpholine 16 (62 mg, 0.20 mmol) was hydrogenated using ammonium formate (128 mg, 2.00 mmol) and Pd/C (10% Pd, 21 mg, 0.02 mmol)

in 2 mL of EtOH. The mixture was stirred overnight at rt and then filtrated through a celite pad. The filtrate was evaporated and the residue was dissolved in CH2Cl2 and washed with a saturated aqueous solution of Na2CO3. The aqueous layer was extracted with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered and evaporated to yield expected compound 18 as a pale yellow oil (55 mg, 99% yield). 1H NMR (300 MHz, CD3OD) d 6.63 (d, J ¼ 3 Hz, 1H, H-4), 6.45 (d, J ¼ 3 Hz, 1H, H-6), 3.8e3.7 (m, 8H, 2OeCH2, CH2, NH2), 3.3e3.2 (m, 2H, NeCH2), 2.9e2.8 (m, 2H, NeCH2), 2.5e2.6 (m, 4H, 2NeCH2), 2.28 (s, 3H, CH3), 1.8e1.7 (m, 4H, 2CH2); 13C NMR (75 MHz, CD3OD) d 144.8 (C), 139.2 (C), 137.9 (2C), 117.6 (CH), 114.8 (CH), 67.9 (2CH2), 56.0 (CH2), 54.0 (2CH2), 50.8 (2CH2), 22.9 (2CH2), 18.3 (CH3). LC/MS tR ¼ 4.6 min; PHPLC ¼ 95%; m/ z (ESI) 276.3 [M þ H]þ. 4.3.7. 7-Chloro-N-[3-methyl-4-(4-methylpiperazino)-5(pyrrolidinomethyl)phenyl]quinolin-4-amine 3 3-Methyl-4-(4-methylpiperazino)-5-(pyrrolidinomethyl)aniline 17 (70 mg, 0.24 mmol) and 4,7-dichloroquinoline (50 mg, 1eq) were refluxed overnight in 5 mL of acetonitrile with 1.25 mL of HCl 1 M. The reaction mixture was then evaporated and purified by flash chromatography (CH2Cl2/MeOH/NH4OH 9:1:0.1) to yield expected compound 3 as a white powder (92 mg, 84% yield). 1H NMR (300 MHz, CDCl3) d 8.55 (d, J ¼ 5.3 Hz, 1H, QuinoH-2), 8.03 (d, J ¼ 2.1 Hz, 1H, QuinoH-8), 7.83 (d, J ¼ 9.0 Hz, 1H, QuinoH-5), 7.45 (dd, J ¼ 2.1 and 9.0 Hz, 1H, QuinoH-6), 7.27 (m, 1H, H-4), 7.02 (d, J ¼ 2.7 Hz, 1H, H-6), 6.96 (d, J ¼ 5.3 Hz, 1H, QuinoH-3), 6.57 (brs, 1H, NH), 3.76 (s, 2H, CH2), 3.3e3.1 (m, 4H, 2NeCH2), 2.7e2.4 (m, 8H, 4NeCH2), 2.40 (s, 3H, NeCH3), 2.38 (s, 3H, CH3), 1.8e1.7 (m, 4H, 2CH2). 13C NMR (300 MHz, MeOH-d4) d 151.2 (CH), 150.2 (C), 148.5 (C), 144.6 (C), 138.8 (C), 137.7 (C), 135.2 (C), 126.6 (C), 125.3 (CH), 123.5 (CH), 122.7 (CH), 118.8 (C), 101.4 (CH), 56.15 (CH2), 55.65 (CH2), 54.07 (CH2), 49.38 (CH2), 45.34 (CH2), 22.96 (CH2), 18.95 (CH3). LC/MS tR ¼ 5.5 min; PHPLC > 99%; m/z (ESI) 450.1 [M þ H]þ. HPLC (C4) tR ¼ 7.1 min; PHPLC ¼ 97%; HPLC (C18) tR ¼ 15.9 min; PHPLC > 99%. 4.3.8. 7-Chloro-N-[3-methyl-4-morpholino-5-(pyrrolidinomethyl) phenyl]quinolin-4-amine 4 3-Methyl-4-morpholino-5-(pyrrolidinomethyl)aniline 18 (46 mg, 0.17 mmol) and 4,7-dichloroquinoline (40 mg, 0.20 mmol) were refluxed overnight in 5 mL of acetonitrile with 0.51 mL of HCl 1 M. The reaction mixture was then evaporated and purified by flash chromatography (CH2Cl2/MeOH/NH4OH//95/5/0 to 90/10/1) to yield expected compound 4 as a white powder (53 mg, 71% yield). 1H NMR (300 MHz, CDCl3) d 8.43 (d, J ¼ 5.5 Hz, 1H, QuinoH-2), 8.24 (d, J ¼ 9.0 Hz, 1H, QuinoH-5), 7.90 (d, J ¼ 1.9 Hz, 1H, QuinoH-8), 7.55 (d, J ¼ 2.1 Hz, 1H, H-4), 7.30 (dd, J ¼ 1.9 and 9.0 Hz, 1H, QuinoH-6), 7.15 (d, J ¼ 2.0 Hz, 1H, H-6), 6.89 (d, J ¼ 5.5 Hz, 1H, QuinoH-3), 4.15 (s, 1H, NH), 3.89 (s, 2H, CH2), 3.69 (t, J ¼ 9.2 Hz, 2H, OeCH2), 3.38 (t, J ¼ 9.4 Hz, 2H, OeCH2), 3.1e3.0 (m, 4H, 2NeCH2), 2.8e2.7 (m, 4H, 2NeCH2), 2.37 (s, 3H, CH3), 1.9e1.8 (m, 4H, 2CH2); 13C NMR (75 MHz, CDCl3) d 151.0 (CH), 148.8 (C), 148.7 (C), 144.3 (C), 138.8 (C), 137.9 (C), 135.8 (C),134.4 (C),127.6 (CH),126.1 (CH),125.8 (CH),123.6 (CH),122.6 (CH), 118.5 (C), 102.5 (CH), 68.2 (CH2), 55.1 (CH2), 54.3 (CH2), 50.8 (CH2), 23.7 (CH2), 20.5 (CH3). LC/MS tR ¼ 6.2 min; PHPLC > 99%; m/z (ESI) 437.3 [M þ H]þ; HPLC (C4) tR ¼ 7.9 min; PHPLC > 99%; HPLC (C18) tR ¼ 15.3 min; PHPLC > 99%. Acknowledgments We express our thanks to Gérard Montagne, Emmanuelle Boll and Hervé Drobecq for analytical and spectroscopic analysis and Robin Segard for his contribution in organic synthesis. The authors thank Dr Laurence Agouridas for fruitful discussion and proofreading of the manuscript. This work was supported by Université de Lille II.

N. Le Fur et al. / European Journal of Medicinal Chemistry 46 (2011) 3052e3057

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