Original article
Discovery of novel2,6-disubstituted pyridazinone derivatives
as acetylcholinestera inhibitors
Weiqiang Xing a,1,Yan Fu b,1,Zhangxing Shi a,Dong Lu a,Haiyan Zhang b,*,Youhong Hu a,*
a State Key Laboratory of Drug Rearch,Department of Medicinal Chemistry,Shanghai Institute of Materia Medica,Chine Academy of Sciences,
555Zu Chong Zhi Road,Shanghai201203,China
b State Key Laboratory of Drug Rearch,Department of Neuropharmacology,Shanghai Institute of Materia Medica,Chine Academy of Sciences,
555Zu Chong Zhi Road,Shanghai201203,China
a r t i c l e i n f o
Article history:
Received3November2012 Received in revid form
24January2013
Accepted27January2013 Available online8February2013
Keywords:
Pyridazinone Acetylcholinestera Alzheimer’s dia
Docking study a b s t r a c t
2,6-Disubstituted pyridazinone4was identified by HTS as a novel acetylcholinestera(AChE)inhibitor. Under SAR development,compound17e stood out as displaying high AChE inhibitory activity and AChE/ butyrylcholinestera(BuChE)lectivity in vitro.Docking studies revealed that17e might interact with the catalytic active site(CAS)and the peripheral anionic site(PAS)simultaneously.Bad on this novel binding information,6-ortho-tolylamino and N-ethyl-N-isopropylacetamide substituted piperidine were disclod as new PAS and CAS binders.
Ó2013Elvier Masson SAS.All rights rerved.
1.Introduction
Alzheimer’s dia(AD)is a complex neurodegenerative dis-order of the central nervous system.It is estimated that nearly36 million people worldwide are now suffering from AD,and the figure would be incread to about66million by2030if no breakthroughs were made.Acetylcholinestera(AChE),a rine protea,is responsible for acetylcholine hydrolysis and plays a fundamental role in impul transmission by terminating the action of the neurotransmitter acetylcholine at the cholinergic synaps and neuromuscular junction[1].Among the various approaches for treating AD,inhibition of AChE is still prevailing in treating or alleviating the symptoms of AD.Tacrine(1,Fig.1),a nonlective AChE/butyrylcholinestera(BuChE)inhibitor,was thefirst drug approved by FDA in1993.Other lective inhibitors, such as donepezil(2,Fig.1),also reached the market quentially.In recent years,novel AChE inhibitors are continuingly discovered from natural resources or by synthetic approaches,bearing such as berberine[2e6],coumarin[7,8],benzofuran[9],b-carboline[10],quinoline[11e13],benzophenone[14,15],triazin[16],ferulic acid [17]and naphthyridine[18]frameworks as the primary pharma-cophoric scaffolds.
The most interesting structural character of AChE protein is the prence of a deep narrow gorge,at the bottom of which a triad lies in the catalytic anionic site(CAS).Besides,the peripheral anionic site(
PAS)lies at the entrance of the gorge as a regulatory site.Bad on thefindings,Pang et al.first reported a tacrine dimer3(Fig.1) as a bivalent AChE inhibitor which interacts with CAS and PAS simultaneously[19].This compound displayed much higher AChE inhibitory activity and specificity over BuChE than the parent compound1.Through this strategy,the cond generation AChE inhibitors are developed by utilizing one or two known scaffolds of AChE inhibitor and most of them posss elevated potency and improved AChE/BuChE lectivity profile[20e28].
Through an in vitro HTS campaign with diver compounds libraries inhou,pyridazinone derivative4(Fig.1)stood out as a potential AChE inhibitor with an IC50of 1.66m M.To our best knowledge,only a few compounds bearing this scaffold were reported with a liner pyridazine-donepezil hybrid5(Fig.1)in the literature[29e31],which behaved as a dual site binding inhibitor. By optimizing the3,6-substitutions on the pyridazine ring,high AChE affinity of the compound was achieved.However,this type of molecules displayed low AChE/BuChE lectivity,which might lead
*Corresponding authors.
E-mail address:hzhang@mail.shcnc.ac(H.Zhang),yhhu@mail.shcnc.ac (Y.Hu).
1Authors equally contributed to this
work.Contents lists available at SciVer ScienceDirect
European Journal of Medicinal Chemistry jo urnal homepag e:www.elvie
0223-5234/$e e front matterÓ2013Elvier Masson SAS.All rights rerved.
dx.doi/10.1016/j.ejmech.2013.01.056
European Journal of Medicinal Chemistry63(2013)95e103
to undesirable peripheral side effects.As the compound 4bears different substituents on pyridazinone ring from 5a /5b and does not share any structural similarity with donepezil,we realized that the SAR of 4might not parallel with that of 5a /5b or donepezil,thus leading this de novo pyridazinone molecule to be worth of opti-mization for finding diver AChE inhibitors with high potency and AChE/BuChE lectivity.2.Results and discussion 2.1.Chemistry
The general synthetic route to build the focud AChE-targeted inhibitor library is illustrated in Scheme 1.Compound 9could be obtained by a quential process involving alkylation of corre-sponding pyridazinone (6)[32e 34]and followed by reduction of the nitro group under Fe/HOAc in re fluxing EtOH.The final products were obtained by 9condensing the substituted piperidines in the prence of triphosgene.2.2.SAR study and docking study
Structural inspections of the compounds in our HTS library revealed that the terminal 4-diethyl amide piperidine in 4may be esntial for activity.Firstly,we commenced with our work by changing the substituents on the pyridazinone ring (Table 1).Replacing the phenyl in 4by a single hydrogen (10)caud a slight loss in activity,which indicated that the phenyl might function as a hydrophobic pharmacophore.As the AChE binding pocket is a narrow hydrophobic gorge,a small hydrophobic methyl group was added to the phenyl (11a e c )for modulating the binding af finity.
Compound 11c ,a meta -methyl substituted pattern,displayed a slight enhancement in activity with an IC 50of 0.746m M.Besides,other meta -halo substituted analogs (11d e e )remained biological activity at enzymatic level.When the conjugated system in 4was extended by replacing the phenyl with naphthyl (12a e b ),or by adding a cyano group on meta position (12c ),the potency of com-pounds was evidently enhanced.The most promising compound 12b displayed a potent activity with an IC 50of 0.188m M,which was about 9folds more potent than the no-conjugation extended compound 4.
In order to get a comprehensive understanding of the SAR,docking study was performed.Crystal complex (code:1EVE)of donepezil and AChE protein was downloaded from Protein Data Bank,in which the positively charged piperidine nitrogen in donepezil achieves a caution-p interaction with Ph
e330and the terminal benzyl ring involves in p e p stacking with aromatic Trp84.Docking results showed that 12b may function as a bi-functional AChE inhibitor with novel binding conformation,which the naph-thyl occupies in the PAS pocket and the 4-diethyl amide piperidine is located at the CAS near the bottom of the gorge.The carbonyl oxygen on pyridazinone ring possibly takes part in hydrogen bonding interaction with Phe288and the 2-benzyl on pyridazinone involves in p e p stacking with Tyr334(Fig.2).No direct hydrogen bond interaction was obrved in the urea motif of 12b ,however,just as donepezil complex,a water bridged hydrogen bonding might occur in biological
circumstances.
Fig.1.Reprentative AChE inhibitors.
Scheme 1.Synthesis of analogues a .牛津小学英语6b教案
Table 1
AChE inhibitory activity of 6-aryl substituted pyridazinones.
excellency
No.R IC 50(m M)No.R IC 50(m M)
4
1.66
11d
0.623
10H 4.90
11e 0.648
11a
2.61
12a 0.462
11b
2.01
12b 0.188
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As shown in Fig.3,docking results revealed that the naphthyl for PAS binder in 12b not only acts as a hydrophobic group involving in the van der Waals interactions with the protein,but also functions as a p e p interaction element,which mainly works by the extended aromatic ring B.Considering the hydrophobic effects induced by the phenyl A in 12b and the methyl in 11c ,a novel 6-aniline pyr-idazinone scaffold was designed,in which the ring B is remained as a p e p interaction motif and ring A is broken by a single nitrogen linker.In this new scaffold,p e p stacking ef ficiency of ring B may also be elevated due to the incread rotation freedom.
Bad on above analys,6-substituted aniline pyridazinones were synthesized.As shown in Table 2,when the naphthyl (hydrophobic-p motif)in 12b was replaced by a single aniline (nitrogen-p motif,13),the AChE inhibitory activity of 13decread drastically (about 17folds).However,the activity of 14was restored when the aniline was changed to more hydrophobic naphthalen-1-amine,which indicated that the hydrophobic substitutions on the aniline ring played a great part in binding af finity.Subquently,other analogs with different substituted anilines were synthesized (15a e f ).The activity of compounds was decread in the prence of para substitutions (15c ,f ).The ortho substitutions signi ficantly incread the binding af finity probably due to a similar hydro-phobic effect
induced by ring A in 12b .In addition,a prevailing ‘Magic Methyl ’effect emerged in the pyridazinone compounds [35],a single 2-methyl aniline derivative (15a )showed the prom-inent inhibitory potency with an IC 50of 0.049m M.It was about 34and 4-fold more potent than 4and 12b ,respectively.Further enlarging the steric hindrance to dimethyl,ethyl or isopropyl (16a e c )decread the activity.
Docking studies showed that 15a displays a similar binding mode as 12b (Fig.4A).Overlapping compounds 15a ,4and 12b (Fig.4B)revealed that changing the phenyl in 4with naphthyl (12b )or adding a conjugated group (12c )made it possible for p e p stacking with Trp279,which led to an increa in activity.In the ca of 15a ,the 2-methyl aniline does act as a bi-functional group:the aromatic phenyl stacks with Trp279while the terminal methyl fit within the PAS hydrophobic pocket perfectly.
After the modi fication of the active compounds for PAS binding site,we optimized the CAS binding motifs to synthesize compounds 17a e f (Table 3).Cyclic analog 17a with the terminal morpholine motif lost the activity totally.Among the acyclic amide ries (17b e f ),the inhibitory activity of the compounds strongly paralleled with the hydrophobic effect of the substitutions.Among them,the N -ethyl-N -isopropyl amide derivative 17e showed slightly more potent than 15a .This phenomenon is consistent with the hydro-phobic environment of the CAS binding pocket.2.3.Selectivity pro file for A
ChE/BuChE
Moreover,the most potent and reprentative compounds were lected for evaluation of AChE/BuChE lectivity pro file.As shown in Table 4,compared with the reported 3-amino-6-aryl pyridazine ries [29,30],2,6-disubstituted pyridazin-3-one ries displayed about 200-fold or higher AChE/BuChE lectivity.For this scaffold,AChE inhibitory activity of the compounds could be enhanced by modifying PAS binding motif without affecting the BuChE af finity by comparing 4with 12b ,or 13with 15a .In the catalytic binding pocket,increasing the hydrophobic groups of the amide (17d e e )lowered the IC 50s for AChE accompanying with slight increasing for the binding af finities of BuChE,However,the lectivity pro files were still
maintained.
Fig. 2.Model of compound 12a bound to AChE.The naphthyl and 4-substituted piperidine interact with PAS and CAS
respectively.
Fig.3.Designing strategy for aniline ries.
Table 2
AChE inhibitory activity of 6-aniline substituted pyridazinones.
No.Ar IC 50(m M)No.Ar IC 50(m M)
13 3.28
15d 0.119
140.126
15e 0.135
高二物理公式15a 0.049
15f 4.84
15b 1.42
16a 0.607
15c 19.8
16b 0.188
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3.Conclusion
In this report,a ries of novel 2,6-disubstituted pyridazinone derivatives for AChE inhibition was optimized.Under the SAR development,high AChE af finity of the compounds was achieved by optimizing different substituents on the pyridazinone ring,without sacri ficing the AChE/BuChE lectivity pro file.
Docking study revealed that the pyridazinones might behave as dual binding site inhibitors with novel binding conformation.Utilizing this information,a delicate SAR study was performed at PAS binding site and 6-ortho-tolylamino substitution was opti-mized for involving in both p e p stacking and “Magic Methyl ”hydrophobic interaction.Moreover,rather than the prevailing N -benzyl piperidine reported in donepezil,a novel hydrophobic 4-substituted piperidine motif for catalytic pocket binder was dis-clod,which also provided a new platform for designing AChE inhibitor.Further biological evaluations of the pyridazinone r-ies are ongoing.
4.Experimental ction 4.1.General information
Reagents were puri fied prior to u unless otherwi stated.Column chromatography was carried out on silica gel (200e大学明天网
300mesh).1H NMR and 13C NMR spectral data were recorded in DMSO-d 6,CD 3OD or acetone-d 6on Varian Mercury 500,400or 300NMR spectrometer and Chemical shifts (d )were reported in parts per million (ppm),and the signals were described as brs (broad singlet),d (doublet),dd (doublet of doublet),m (multiple),q (quarter),s (singlet),and t (triplet).Coupling constants (J values)were given in Hz.Low-resolution mass spectra (ESI)was obtained using Agilent HPLC-MS (1260-6120B)and high-resolution mass spectra (ESI)were obtained using Waters Q-Tof Ultima apparatus.4.2.General procedures
itemizeTo a solution of pyridazinone derivative [32e 34](0.5mmol)in DMF (10mL)was added 1-(chloromethyl)3-nitrobenzene (0.52mmol)and Cs 2CO 3(0.55mmol),the resulting reaction mix-ture was stirred at 40e 50 C until no starting materials was detected by TLC (about 3h).The solvent was removed under reduced pressure and the residue was dissolved in EtOAc (30mL),washed with brine (3Â10mL).The organic layer was dried over anhydrous Na 2SO 4and concentrated in vacuo.The crude product was dissolved in 95%ethanol (50mL)containing 10mmol acetic acid.Iron powder (2mmol)was added and the resulting
mixture
Fig. 4.(A)Model of compound 15a bound to AChE.The ortho -tolylamino and 4-substituted piperidine interact with PAS and CAS,respectively.(B)Overlap of 4(magenta),12b (yellow),and 15a (green).The naphthyl in 12b and ortho-methyl amine in 15a function as p e p stacking element and involve in PAS van der Waals interaction.乐知英语
Table 3
SAR study at catalytic binding site.
No.R IC 50(m M)
17a
NA
17b 7.60
17c 0.473
17d 0.046
15a 0.050
17e 0.028
17f 0.116
Table 4
Selectivity pro file of reprentative compounds for AChE versus BuChE.No.
IC 50(m M)Ratio of IC 50(BuChE/AChE)
AChE
BuChE 4 1.66>40>2412b 0.188>40>21213 3.28>40>1215a 0.049>40>81617c 0.473>40>8417d 0.04610.021717e
0.028
5.50
196
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was stirred for5h.After cooled to room temperature,the reaction mixture wasfiltered through celite and thefilter cake was washed with95%ethanol(3Â15mL).The combined ethanol layers were evaporated in vacuo and the residue was re-dissolved in ethyl acetate(30mL).The organic layer was washed with brine (3Â10mL)and2M NaOH(10mL)quentially.The organic layer was dried over anhydrous Na2SO4,evaporated in vacuo to afford the crude2-aminobenzyl-6-substituted-pyridazin-3(2H)-ones,which were ud without further purification.
To a stirred solution of2-aminobenzyl-6-substituted-pyridazin-3(2H)-one and triphosgene(1mmol)in dry dichloromethane (5mL)was added triethylamine(2mmol)under nitrogen atmos-phere.A solution of the corresponding piperidine(1mmol)in dichloromethane(5mL)was added5e10min later and the mixture was stirred at room temperature overnight.The reaction mixture was diluted with dichloromethane(15mL)and washed with water (3Â20mL).The organic phas were parated,dried over anhy-drous Na2SO4and concentrated in vacuo.The residue was purified by column chromatography to afford the products.
4.2.1.N4,N4-Diethyl-N1-(3-((6-oxo-3-phenylpyridazin-1(6H)-yl) methyl)phenyl)piperidine-1,4-dicarboxamide(4)
Yield:81%:1H NMR(300MHz,acetone-d6)d8.03e7.87(m,4H), 7.61e7.38(m,5H),7.19(t,J¼7.9Hz,1H),7.04(d,J¼7.7Hz,1H),6.99 (d,J¼9.9Hz,1H),5.32(s,2H),4.30e4.15(m,2H),3.47e3.37(m,2H), 3.32(d,J¼6.8Hz,2H),2.99e2.74(m,3H),1.68(s,4H),1.18(t, J¼7.0Hz,3H),1.03(t,J¼6.9Hz,3H);13C NMR(125MHz,MeOD-d4) d174.67,160.25,156.20,145.43,139.91,136.53,134.31,131.02,129.41, 129.22,128.51,128.35,125.74,122.58,120.41,120.20,55.33(e CH2e Ph e),43.31(e NHCON(CH2CH2)2CHCO e),41.84(e CON(CH2CH3)2),
40.29(e CON(CH2CH3)2),38.32(e NHCON(CH2CH2)2CHCO e),28.39
(e NHCON(CH2CH2)2CHCO e),13.85(e CON(CH2CH3)2),11.90(e CON(CH2CH3)2);HRMS(ESI):m/z[MþH]þ488;HRMS(ESI)calcd for C28H33N5O3Na[MþNa]þ510.2481,found510.2492.
4.2.2.N4,N4-Diethyl-N1-(3-((6-oxopyridazin-1(6H)-yl)methyl) phenyl)piperidine-1,4-dicarboxamide(10)
Yield:58%:1H NMR(300MHz,acetone-d6)d8.06(s,1H),7.82 (dd,J¼3.6,1.4Hz,1H),7.53(d,J¼8.2Hz,1H),7.47(s,1H),7.33(dd, J¼9.5,3.8Hz,1H),7.16(t,J¼7.9Hz,1H),
6.94(d,J¼7.7Hz,1H), 6.87(dd,J¼9.5,1.6Hz,1H),5.21(s,2H),4.29e4.15(m,2H),3.43 (q,J¼7.1Hz,2H),3.32(q,J¼7.0Hz,2H),2.98e2.77(m,3H),1.73e 1.59(m,4H),1.18(t,J¼7.1Hz,3H),1.03(t,J¼7.0Hz,3H);13C NMR(125MHz,MeOD-d4)d174.73,161.04,156.17,139.92,137.38, 136.56,132.36,129.08,128.25,122.32,120.22,120.04,54.88(e CH2e Ph e),43.29(e NHCON(CH2CH2)2CHCO e),41.81(e CON(CH2CH3)2), 40.25(e CON(CH2CH3)2),38.28(e NHCON(CH2CH2)2CHCO e),
28.39(e NHCON(CH2CH2)2CHCO e),13.73(e CON(CH2CH3)2),11.78
(e CON(CH2CH3)2);LCMS(ESI)m/z412[MþH]þ;HRMS(ESI)calcd for C22H30N5O3[MþH]þ412.2349,found412.2331.
4.2.3.Diethyl-N1-(3-((6-oxo-3-(o-tolyl)pyridazin-1(6H)-yl)methyl) phenyl)piperidine-1,4-dicarboxamide(11a)
Yield:69%:1H NMR(300MHz,acetone-d6)d8.01(s,1H),7.53(t, J¼8.3Hz,3H),7.41(d,J¼7.1Hz,1H),7.35e7.23(m,3H),7.17(t, J¼7.7Hz,1H),7.03e6.92(m,2H),5.27(s,2H),4.31e4.16(m,2H), 3.42(q,J¼7.0Hz,2H),3.34e3.26(m,2H),2.
87(ddd,J¼21.2,14.9, 7.1Hz,3H),2.32(s,3H),1.75e1.61(m,4H),1.17(t,J¼7.1Hz,3H),1.02 (t,J¼7.0Hz,3H);13C NMR(125MHz,MeOD-d4)d174.72,159.99, 156.17,147.71,139.97,136.68,135.94,134.90,134.25,130.60,128.91, 128.80,128.71,128.29,125.72,122.49,120.40,120.06,55.02(e CH2e Ph e),43.30(e NHCON(CH2CH2)2CHCO e),41.80(e CON(CH2CH3)2),
40.25(e CON(CH2CH3)2),38.29(e NHCON(CH2CH2)2CHCO e),28.39
(e NHCON(CH2CH2)2CHCO e),19.20(e NH e Ph e CH3),13.73(e CON(CH2CH3)2),11.79(e CON(CH2CH3)2);LCMS(ESI)m/z[MþH]þ502;HRMS(ESI)calcd for C29H35N5O3Na[MþNa]þ524.2638,found 524.2657.
4.2.4.N4,N4-Diethyl-N1-(3-((6-oxo-3-(p-tolyl)pyridazin-1(6H)-yl) methyl)phenyl)piperidine-1,4-dicarboxamide(11b)
Yield:61%:1H NMR(300MHz,acetone-d6)d8.02(s,1H),7.90(d, J¼9.7Hz,1H),7.78(d,J¼8.3Hz,2H),7.53(d,J¼10.8Hz,2H),7.27 (d,J¼8.0Hz,2H),7.17(t,J¼7.8Hz,1H),7.02(d,J¼7.4Hz,1H),6.95 (d,J¼9.7Hz,1H),5.29(s,2H),4.30e4.15(m,2H),3.42(q,J¼7.2Hz, 2H),3.31(q,J¼7.0Hz,2H),2.96e2.73(m,
3H),2.35(s,3H),1.71e1.58 (m,4H),1.17(t,J¼7.1Hz,3H),1.02(t,J¼7.0Hz,3H);13C NMR (126MHz,MeOD-d4)d174.72,160.21,156.21,145.44,139.94,139.53, 136.66,131.53,130.93,129.26,129.11,128.29,125.62,122.50,120.31, 120.13,55.18(e CH2e Ph e),43.28(e NHCON(CH2CH2)2CHCO e), 41.79(e CON(CH2CH3)2),40.24(e CON(CH2CH3)2),38.28(e NHCON (CH2CH2)2CHCO e),28.37(e NHCON(CH2CH2)2CHCO e),19.80(e NH e Ph e CH3),13.71(e CON(CH2CH3)2),11.77(e CON(CH2CH3)2); LCMS(ESI)m/z[MþH]þ502;HRMS(ESI)calcd for C29H35N5O3Na [MþNa]þ524.2638,found524.2622.
4.2.
5.N4,N4-Diethyl-N1-(3-((6-oxo-3-(m-tolyl)pyridazin-1(6H)-yl) methyl)phenyl)piperidine-1,4-dicarboxamide(11c)
Yield:73%:1H NMR(300MHz,acetone-d6)d8.08(s,1H),7.88(d, J¼9.8Hz,1H),7.72(s,1H),7.65(d,J¼7.8Hz,1H),7.61(s,1H),7.51 (d,J¼8.8Hz,1H),7.32(t,J¼7.7Hz,1H),7.22(d,J¼7.6Hz,1H),7.17 (t,J¼7.9Hz,1H),7.01(d,J¼7.5Hz,1H),6.94(d,J¼9.7Hz,1H),5.29 (s,2H),4.28e4.15(m,2H),3.47e3.28(m,4H),3.00e2.71(m,3H), 2.37(s,3H),1.75e1.55(m,4H),1.22e1.10(t,
J¼7.0Hz,3H),1.01(t, J¼7.0Hz,3H);13C NMR(125MHz,MeOD-d4)d174.66,160.15, 156.08,145.40,140.02,138.35,136.63,134.19,131.01,129.91,129.23, 128.40,128.33,126.24,122.88,122.43,120.22,120.00,55.24(e CH2e Ph e),43.28(e NHCON(CH2CH2)2CHCO e),41.80(e CON(CH2CH3)2), 40.24(e CON(CH2CH3)2),38.25(e NHCON(CH2CH2)2CHCO e),
28.39(e NHCON(CH2CH2)2CHCO e),20.13(e NH e Ph e CH3e),13.79
(e CON(CH2CH3)2),11.86(e CON(CH2CH3)2);LCMS(ESI)m/z [MþH]þ502;HRMS(ESI)calcd for C29H35N5O3Na[MþNa]þ524.2638,found524.2637.
4.2.6.N1-(3-((3-(3-Bromophenyl)-6-oxopyridazin-1(6H)-yl) methyl)phenyl)-N4,N4-diethylpiperidine-1,4-dicarboxamide(11d) Yield:86%:1H NMR(300MHz,acetone-d6)d8.08(s,1H),8.03e 7.93(m,2H),7.90(d,J¼8.0Hz,1H),7.60(s,2H),7.51(d,J¼8.1Hz, 1H),7.42(t,J¼7.7Hz,1H),7.18(t,J¼7.7Hz,1H),7.00(t,J¼9.3Hz, 2H),
5.31(s,2H),4.28e4.15(m,2H),3.42(d,J¼7.0Hz,2H),3.31(d, J¼7.2Hz,2H),3.01e2.76(m,3H),1.67(s,4H),1.17(t,J¼
6.8Hz,3H), 1.02(t,J¼6.9Hz,3H);13C NMR(125MHz,MeOD-d4)d174.72, 160.14,156.16,143.77,139.99,136.47,132.03,130.76,130.25,129.50, 128.54,128.33,124.49,122.54,120.30,120.12,55.28(e CH2e Ph e),
43.28(e NHCON(CH2CH2)2CHCO e),41.80(e CON(CH2CH3)2),40.25
(e CON(CH2CH3)2),38.27(e NHCON(CH2CH2)2CHCO e),28.38(e NHCON(CH2CH2)2CHCO e),13.72(e CON(CH2CH3)2),11.78(e CON(CH2CH3)2);LCMS(ESI)m/z[MþH]þ566;HRMS(ESI)calcd for C28H32N5O3NaBr[MþNa]þ588.1586,found588.1569.
4.2.7.N1-(3-((3-(3-Chlorophenyl)-6-oxopyridazin-1(6H)-yl) methyl)phenyl)-N4,N4-diethylpiperidine-1,4-dicarboxamide(11e) Yield:92%:1H NMR(300MHz,acetone-d6)d8.06(s,1H),7.96(d, J¼9.8Hz,2H),7.85(d,J¼7.1Hz,1H),7.63(s,1H),7.55e7.43(m,3H), 7.18(t,J¼7.8Hz,1H),7.02(t,J¼8.1Hz,2H),
5.32(s,2H),4.30e4.19 (m,2H),3.50e3.25(m,4H),3.01e2.75(m,3H),1.78e1.60(m,4H), 1.18(dd,J¼8.3,5.8Hz,3H),1.03(t,J¼7.0Hz,3H);13C NMR (125MHz,MeOD-d4)d174.72,160.16,15
6.18,143.90,139.98,136.47, 136.29,134.54,130.79,130.03,129.50,129.05,128.32,125.61, 124.07,122.
55,120.33,120.13,55.28(e CH2e Ph e),43.27(e NHCON(CH2CH2)2CHCO e),41.79(e CON(CH2CH3)2),40.24(e
W.Xing et al./European Journal of Medicinal Chemistry63(2013)95e10399
CON(CH2CH3)2),38.27(e NHCON(CH2CH2)2CHCO e),28.37(e NHCON(CH2CH2)2CHCO e),13.71(e CON(CH2CH3)2),11.77(e CON(CH2CH3)2);LCMS(ESI)m/z[MþH]þ522;HRMS(ESI)calcd for C28H32N5O3NaCl[MþNa]þ544.2091,found544.2084.
4.2.8.N4,N4-Diethyl-N1-(3-((3-(naphthalen-2-yl)-6-oxopyridazin-
1(6H)-yl)methyl)phenyl)piperidine-1,4-dicarboxamide(12a) Yield:88%:1H NMR(300MHz,acetone-d6)d8.39(s,1H),8.25e 7.70(m,6H),7.68(s,1H),7.53(d,J¼6.3Hz,3H),7.19(t,J¼7.8Hz, 1H),7.06(d,J¼7.6Hz,1H),6.98(d,J¼9.8Hz,1H),5.33(s,2H), 4.30e4.15(m,2H),3.44e3.29(m,4H),2.98e2.73(m,3H),1.68(m, 4H),1.23e1.09(t,J¼7.0Hz,3H),1.02(t,J¼7.0Hz,3H);13C NMR (125MHz,MeOD-d4)d174.69,160.16,156.18,145.07,140.00,136.63, 133.66,133.14,131.52,130.89,129.24,128.33,128.22,128.20,127.23, 126.61,126.21,125.33,122.82,122.57,120.33,120.09,55.24(e CH2e Ph e),43.27(e NHCON(CH2CH2)
2CHCO e),41.78(e CON(CH2CH3)2), 40.23(e CON(CH2CH3)2),38.25(e NHCON(CH2CH2)2CHCO e),
28.36(e NHCON(CH2CH2)2CHCO e),13.70(e CON(CH2CH3)2),11.77
(e CON(CH2CH3)2);LCMS(ESI)m/z[MþH]þ538;HRMS(ESI)calcd for C32H35N5O3Na[MþNa]þ560.2638,found560.2643.
4.2.9.N4,N4-Diethyl-N1-(3-((3-(naphthalen-1-yl)-6-oxopyridazin-
1(6H)-yl)methyl)phenyl)piperidine-1,4-dicarboxamide(12b) Yield:76%:1H NMR(300MHz,acetone-d6)d8.19e7.87(m,4H), 7.72e7.46(m,7H),7.22(t,J¼7.8Hz,1H),7.03(q,J¼8.7Hz,2H),5.34 (s,2H),4.30e4.16(m,2H),3.47e3.27(m,4H),3.15e2.25(m,3H), 1.80e1.50(m,4H),1.23e1.12(t,J¼7.0Hz,3H),1.03(t,J¼7.0Hz,3H); 13C NMR(100MHz,MeOD-d
4)d174.74,160.15,156.21, 147.00,140.08,136.73,134.90,133.93,132.83,130.49,129.56,129.22, 128.46,128.20,127.13,126.62,125.93,124.87,124.70,122.86,120.70, 120.26,55.04(e CH2e Ph e),43.35(e NHCON(CH2CH2)2CHCO e), 41.88(e CON(CH2CH3)2),40.34(e CON(CH2CH3)2),38.36(e NHCON(CH2CH2)2CHCO e),28.46(e NHCON(CH2CH2)2CHCO e), 13.8
6(e CON(CH2CH3)2),11.91(e CON(CH2CH3)2);LCMS(ESI)m/z [MþH]þ538;HRMS(ESI)calcd for C32H35N5O3Na[MþNa]þ560.2638,found560.2631.
4.2.10.N1-(3-((3-(3-Cyanophenyl)-6-oxopyridazin-1(6H)-yl) methyl)phenyl)-N4,N4-diethyl piperidine-1,4-dicarboxamide(12c) Yield:79%:1H NMR(300MHz,acetone-d6)d8.31(s,1H),8.23(d, J¼7.8Hz,1H),8.05(s,1H),8.01(s,1H),7.82(d,J¼7.6Hz,1H),7.70e 7.65(m,2H),7.48(d,J¼7.9Hz,1H),7.19(t,J¼7.8Hz,1H),7.05(d, J¼7.7Hz,1H),7.00(d,J¼9.8Hz,1H),4.30e4.17(m,2H),3.43e3.25 (m,4H),3.00e2.80(m,3H),1.70e1.60(m,4H),1.12(t,J¼7.0Hz,3H), 1.03(t,J¼7.0Hz,3H);13C NMR(125MHz,MeOD-d4)d174.71,160.04, 156.10,143.14,140.06,136.39,13
5.65,132.42,130.56,130.07,129.64, 129.27,128.36,122.58,120.29,120.01,117.87,112.71,55.26(e CH2e Ph e),43.29(e NHCON(CH2CH2)2CHCO e),41.80(e CON(CH2CH3)2),
40.25(e CON(CH2CH3)2),38.27(e NHCON(CH2CH2)2CHCO e),28.39
(e NHCON(CH2CH2)2CHCO e),13.74(e CON(CH2CH3)2),11.80(e CON(CH2CH3)2);LCMS(ESI)m/z[MþH]þ513;HRMS(ESI)calcd for C29H32N6O3Na[MþNa]þ535.2434,found535.2439.
儿童暑假夏令营4.2.11.N4,N4-Diethyl-N1-(3-((6-oxo-3-(phenylamino)pyridazin-
1(6H)-yl)methyl)phenyl)piperidine-1,4-dicarboxamide(13) Yield:64%:1H NMR(300MHz,CD3OD-d4)d7.47(d,J¼8.2Hz, 2H),7.41(s,1H),7.33(d,J¼8.7Hz,1H),7.25e7.07(m,4H),7.08(d, J¼7.6Hz,1H),6.92(t,J¼7.8Hz,2H),5.20(s,2H),4.30e4.15(m,2H), 3.50e3.30(m,4H),3.05e2.72(m,3H),1.80e1.64(m,4H),1.23(t, J¼7.0Hz,3H),1.10(t,J¼7.1Hz,3H);13C NMR(125MHz,MeOD-d4)d 174.74,158.48,156.24,145.41,140.36,139.85,137.09,130.15,128.22, 128.17,122.72,121.03,120.72,120.05,117.66,101.92,54.02(e CH2e Ph e),43.28(e NHCON(CH2CH2)2CHCO e),41.79(e CON(CH2CH3)2),
40.24(e CON(CH2CH3)2),38.30(e NHCON(CH2CH2)2CHCO e),28.38
(e NHCON(CH2CH2)2CHCO e),13.69(e CON(CH2CH3)2),11.75(e CON(CH2CH3)2);LCMS(ESI)m/z[MþH]þ503;HRMS(ESI)calcd for C28H34N6O3Na[MþNa]þ525.2590,found525.2618.
4.2.12.N4,N4-Diethyl-N1-(3-((3-(naphthalen-1-ylamino)-6-oxopyridazin-1(6H)-yl)methyl)phenyl)piperidine-1,4-dicarboxamide(14)
Yield:81%:1H NMR(300MHz,acetone-d6)d8.23(d,J¼8.8Hz, 1H),8.16(s,1H),8.03(s,1H),7.99e7.91(m,1H),7.88(d,J¼4.7Hz, 1H),7.63e7.38(m,7H),7.18(t,J¼7.8Hz,1H),6.99(d,J¼7.8Hz,1H), 6.87(d,J¼9.8Hz,1H),5.10(s,2H),4.30e4.15(m,2H),3.48e3.39 (m,2H),3.39e3.25(m,2H),3.00e2.18(m,3H),1.79e1.62(m,4H), 1.19(t,J¼6.8Hz,3H),1.04(t,J¼7.1Hz,3H);13C NMR(125MHz, MeOD-d4)d174.84,158.86,156.34,146.75,139.88,137.14,135.47, 134.45,130.31,128.29,128.10,128.03,127.27,125.56,125.40,125.33, 123.40,122.83,121.69,120.85,120.16,117.56,54.11(e CH2e Ph e),
43.39(e NHCON(CH2CH2)2CHCO e),41.91(e CON(CH2CH3)2),40.36
(e CON(CH2CH3)2),38.41(e NHCON(CH2CH2)2CHCO e),28.51(e NHCON(CH2CH2)2CHCO e),13.82(e CON(CH2CH3)2),11.89(e CON(CH2CH3)2);LCMS(ESI)m/z[MþH]þ553;HRMS(ESI)calcd for C32H36N6O3Na[MþNa]þ575.2747,found575.2739.
4.2.13.N4,N4-Diethyl-N1-(3-((6-oxo-3-(o-tolylamino)pyridazin-
1(6H)-yl)methyl)phenyl)piperidine-1,4-dicarboxamide(15a) Yield:80%:1H NMR(300MHz,DMSO-d6)d
8.51(s,1H),7.93(s, 1H),7.60(d,J¼9.0Hz,1H),7.38(m,2H),7.31e7.05(m,3H),6.96e 6.79(m,3H),4.20e4.12(m2H),3.40e3.24(m,4H),2.94e2.69(m, 3H),2.16(s,3H),1.67e1.44(m,4H),1.13(t,J¼7.0Hz,3H),0.99(t, J¼7.0Hz,3H);13C NMR(125MHz,MeOD-d4)d174.73,158.67, 156.21,146.39,139.77,137.96,137.05,130.17,130.02,129.93,128.15, 127.87,125.84,123.29,122.65,121.85,120.75,120.04,53.87(e CH2e Ph e),43.28(e NHCON(CH2CH2)2CHCO e),41.80(e CON(CH2CH3)2),
40.25(e CON(CH2CH3)2),38.30(e NHCON(CH2CH2)2CHCO e),28.40
(e NHCON(CH2CH2)2CHCO e),16.77(e NH e Ph e CH3),13.71(e CON(CH2CH3)2),11.77(e CON(CH2CH3)2);LCMS(ESI)m/z[MþH]þ517;HRMS(ESI)calcd for C29H36N6O3Na[MþNa]þ539.2747,found 539.2742.
4.2.14.N4,N4-Diethyl-N1-(3-((6-oxo-3-(m-tolylamino)pyridazin-
altar>englishtown1(6H)-yl)methyl)phenyl)piperidine-1,4-dicarboxamide(15b) Yield:78%:1H NMR(300MHz,acetone-d6)d8.15(s,1H),8.01(s, 1H),7.56(s,2H),7.40(s,1H),7.34(d,J¼7.9Hz,1H),7.26e7.10(m,3H), 7.07(d,J¼7.5Hz,1H),6.83(d,J¼9.8Hz,1H),6.74(d,J¼6.8Hz,1H), 5.15(s,2H),4.30e4.20(m,2H),3.44(t,J
¼7.0Hz,2H),3.34(q, J¼7.0Hz,2H),3.00e2.92(m,3H),2.28(s,3H),1.75e1.64(m,4H),1.20 (d,J¼7.0Hz,3H),1.05(t,J¼7.0Hz,3H);13C NMR(125MHz,MeOD-d4)d174.66,158.38,156.14,145.39,140.29,139.92,138.00, 137.23,129.97,128.30,128.19,128.14,122.79,121.82,120.80,120.09, 118.22,114.84,53.96(e CH2e Ph e),43.28(e NHCON(CH2CH2)2 CHCO e),41.81(e CON(CH2CH3)2),40.25(e CON(CH2CH3)2),38.26
(e NHCON(CH2CH2)2CHCO e),28.41(e NHCON(CH2CH2)2CH CO e),
20.41(e NH e Ph e CH3),13.80(e CON(CH2CH3)2),11.87(e CON(CH2CH3)2);LCMS(ESI)m/z[MþH]þ517;HRMS(ESI)calcd for C29H36N6O3Na[MþNa]þ593.2747,found593.2744.九月英文缩写
4.2.1
5.N4,N4-Diethyl-N1-(3-((6-oxo-3-(p-tolylamino)pyridazin-
1(6H)-yl)methyl)phenyl)piperidine-1,4-dicarboxamide(15c) Yield:82%:1H NMR(300MHz,DMSO-d6)d8.88(s,1H),8.49(s, 1H),7.45(s,1H),7.40e7.32(m,3H),7.20e7.13(m,2H),7.02(d, J¼8.5Hz,2H),6.88
e6.78(m,2H),4.15e4.05(m,2H),3.30e3.19(m, 4H),2.90e2.70(m,3H),2.19(s,3H),1.65e1.40(m,4H),1.11(t, J¼7.1Hz,3H),0.97(t,J¼7.0Hz,3H);13C NMR(100MHz,MeOD-d4) d174.79,158.51,156.32,145.63,139.90,137.90,137.17,130.55,130.06, 128.74,128.28,128.22,122.79,120.78,120.11,117.87,54.12(e CH2e Ph e),43.36(e NHCON(CH2CH2)2CHCO e),41.86(e CON(CH2CH3)2), 40.31(e CON(CH2CH3)2),38.36(e NHCON(CH2CH2)2CHCO e),28.46
W.Xing et al./European Journal of Medicinal Chemistry63(2013)95e103 100
