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Investigation of a Novel Series of 2‑Hydroxyisoquinoline1,3(2H,4H)‑diones as Human Immunodeficiency Virus Type 1 Integrase Inhibitors Virginie Suchaud,†,‡ Fabrice Bailly,*,†,‡ Cedric Lion,†,‡ Christina Calmels,∥ Marie-Line Andréola,∥ Frauke Christ,§ Zeger Debyser,§ and Philippe Cotelle†,‡ †

Université Lille Nord de France, F-59000 Lille, France Equipe d'Accueil de Chimie Moléculaire et Formulation, Université Lille 1, F-59655 Villeneuve d’Ascq, France § Interdisciplinary Research Centrum KULAK and Center for Molecular Medicine, Katholieke Universiteit Leuven, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium ∥ Laboratoire de Microbiologie Fondamentale et Pathogénicité, UMR 5234 CNRS, Université Bordeaux Segalen, 146 Rue Léo Saignat, F-33076 Bordeaux, France ‡

ABSTRACT: We report herein further insight into the biological activities displayed by a series of 2-hydroxyisoquinoline1,3(2H,4H)-diones (HIDs). Substitution of the N-hydroxyimide two-metal binding pharmacophore at position 4 by carboxamido side chains was previously shown by us to be fruitful for this scaffold, since strong human immunodeficiency virus type 1 integrase (HIV-1 IN) inhibitors in the low nanomolar range associated with low micromolar anti-HIV activities were obtained. We investigated the influence of substitution at position 7 on biological activity. Introduction of electron-withdrawing functional groups such as the nitro moiety at position 7 led to a noticeable improvement of antiviral activity, down to low nanomolar antiHIV potencies, with advantageous therapeutic indexes going close to those of the clinically used raltegravir and retained potencies against a panel of IN mutants.

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INTRODUCTION It was recently reported that there were 34.2 million (31.8− 35.2 million) people living with human immunodeficiency virus type 1 (HIV-1) at the end of 2011, with 1.7 million (1.6−1.9 million) deaths related to acquired immunodeficiency syndrome (AIDS) and 2.5 million (2.2−2.8 million) new infections.1 Since the first use of zidovudine in the United States in 1987, extensive research led to a combinatorial treatment for HIV-1 infection. Standard highly active antiretroviral therapy (HAART) is based on potent drug cocktails belonging to several different groups such as nucleoside reverse transcriptase inhibitors (NRTIs), nucleotide reverse transcriptase inhibitors (NtRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), fusion inhibitors (FIs), coreceptor inhibitors (CRIs), and integrase inhibitors (INIs).2 Although improvements in HAART have resulted in major advances in longevity and quality of life for HIV-infected © 2014 American Chemical Society

patients and changed AIDS from a rapidly lethal disease into a chronic manageable condition, these treatment regimens are often associated with severe and long-term side effects. Moreover, in resource-limited environments, these treatments are almost unaffordable for the majority of patients and settings of clinical follow-up are less optimized. These accumulated factors led to treatment failures through the emergence of drugresistant strains. The retroviral infection can be only temporarily controlled but not eradicated due to the persistence of HIV-1 reservoirs. Thus, there is still a crucial need for innovative more potent, cheaper, and less toxic anti-HIV drugs, which could overcome the encountered problems, which are mainly drug resistance, toxicity, and pharmacokinetics profile. Targeting the HIV-1 integrase (IN) is a clinically validated approach for designing novel anti-HIV therapies. This viral Received: January 21, 2014 Published: May 3, 2014 4640

dx.doi.org/10.1021/jm500109z | J. Med. Chem. 2014, 57, 4640−4660

Journal of Medicinal Chemistry

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States.13 Nevertheless, novel classes of second-generation IN inhibitors are still needed. So far, the most innovative proposed strategies are illustrated by the design of small-molecule inhibitors of IN dimerization14 or of the interaction between lens epithelium-derived growth factor and IN.15−17 For our part, our team has been working for several years on the development of 2-hydroxyisoquinoline1,3(2H,4H)-diones as dual inhibitors of HIV-1 integrase and RT-associated ribonuclease H function (Figure 1) since this scaffold, referred to as N-hydroxyimide, was previously designed as a targeted active-site binding inhibitor with selectivity for two-metal ion enzymes.18 A pioneer series was constituted by compounds variously substituted at position 7 (Chart 1) with IC50 values against integrase ranging from 0.09 to 18.8 μM. Two hits were discovered with submicromolar IC50 values (R 7 = NHCOCH 2 Ph, IC 50 = 90 nM; R 7 = NHCOCH2(4-F)Ph, IC50 = 130 nM). Unfortunately these compounds exhibited high cellular toxicity, which limited their application as antiviral agents.19 Second, we presented the elaboration of a series of compounds substituted at position 4 by alkyl and arylalkyl chains (Chart 1) and evidenced two hits [R4 = C5H11, IC50 = 1.35 μM; R4 = (CH2)3Ph, IC50 = 2.60 μM] with still limiting high cellular toxicity.20 Then the crucial importance of a hydrophobic side chain bearing a mono- or polyhalogenated benzyl group in the pharmacophore of INSTIs was pointed out with the publication of prototype foamy virus (PFV) intasome crystal structures.21 This led us to substitute our scaffold at position 4 with aromatic carboxamido side chains, as is the case in FDA-approved raltegravir’s structure (Chart 1). Major advances were obtained with this novel series displaying strong low nanomolar IN inhibitory potencies, comparable to that of the clinically used raltegravir, and micromolar anti-HIV activities.22 A crystal structure of one selected hit compound, 101, bound to the wild-type prototype foamy virus intasome (MB76, Figure 1) revealed that the compact scaffold displaying all three Mg2+ chelating oxygen atoms from a single ring showed an overall binding mode similar to previous INSTIs.23 Compound 101 potently inhibited ST and 3′-P IN catalytic activities while it kept activity against a panel of raltegravir-resistant HIV-1 variants and did not induce any resistance selection in cell culture.23 All together, these data indicate the potential of this candidate IN inhibitor compared to previous INSTIs. The limited anti-HIV activities in the micromolar range were certainly related to low cell permeability and high protein binding and needed to be improved, and for this purpose, we considered further substitutions of the scaffold in a hit-to-lead optimization process. Herein we report the synthesis and biological

enzyme catalyzes two key reactions, strand transfer (ST) and 3′-processing (3′-P), to integrate the viral DNA into the host chromatin and establish irreversible infection. For this purpose it needs to be present as a tetramer and to interact with cellular cofactors.3 Raltegravir (Figure 1) resulted from intensive

Figure 1. Structures of FDA-approved raltegravir, elvitegravir, and dolutegravir and of our hit and lead compounds 101 and 80, pointing out the key components of the HIV-1 IN inhibitory pharmacophore: magnesium chelating moiety (red) and hydrophobic halogenobenzyl group (green).

studies on diketo acid derivatives and was the first anti-HIV drug approved by the Food and Drug Administration (FDA). It acts as a selective ST inhibitor by interacting with the magnesium cations of IN catalytic core.3,4 It requires a 400 mg twice daily dose and has already provoked the emergence of resistance associated with clear signature mutation pathways (N155H, Q148H/K/R, and Y143/C/R).5,6 Elvitegravir (Figure 1) was the second compound belonging to the same class of integrase strand transfer inhibitors (INSTIs) approved as a component of the elvitegravir Quad pill. This “4 in 1” tablet targets HIV-1 IN by elvitegravir boosted by the pharmacoenhancer cobicistat and HIV-1 RT by the two NRTIs emtricitabine and tenofovir disoproxil fumarate.7,8 Although this novel anti-HIV tablet will offer new perspective for patients failing existing antiviral regimes, cross-resistance with raltegravir rules out any treatment option for patients failing on raltegravir therapy.9,10 Dolutegravir (S/GSK1349572; Figure 1), although apparently superior to raltegravir, exhibits significant resistance overlap10−12 and has been recently approved in the United Chart 1. Synoptic View of Hit-to-Lead Optimization Processa

a

The process starts from the HDI scaffold and shows the four successive investigated series of compounds. 4641



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Scheme 1. Synthesis of Target Compounds V (79−100)a

a

Reagents and conditions: (i) 1.2 equiv of BnONH2, toluene, Dean−Stark apparatus, reflux for 4−15 h (20−50%); (ii) 10.0 equiv of KOH, MeOH/ H2O 1:2, 5 min−12 h, rt (51−92%); (iii) 5.0 equiv of R4NH2, toluene, Dean−Stark apparatus, reflux for 2−15 h (54−89%); (iv) BCl3, 5.0 or 6.0 equiv, CH2Cl2, 1 h, −78 °C, and then H2O, 0 °C (15−69%) or H2, 5% Pd/C, rt, 20 min−4 h (30−89%).

Scheme 2. Synthesis of Precursor 4a

a Reagents and conditions: (i) 4.0 equiv of SOCl2, MeOH, 0 °C, and then reflux for 24 h; (ii) 6.0 equiv of CH3I, 6.0 equiv of K2CO3, acetone, reflux for 12 h (43%); (iii) 1.3 equiv of LDA, THF, −78 °C, 30 min; (iv) CO2, 0 °C, 1 h, and then 3.0 M HCl; (v) 1.0 equiv of BOP, 5.0 equiv of Et(iPr)2N, 1.3 equiv of BnONH2, −20 °C for 1 h and then rt for 12 h (40%).

attempts to use the crude organic solution for the following coupling step with O-benzylhydroxylamine were also unsuccessful. This presumably reflects the instability of these acids even at low temperature. So we turned to alternative synthetic pathways based on the aromatic nucleophilic substitution of 2-bromobenzoic acids 5−8 (Scheme 3) or

comparative evaluation of a novel series of 2-hydroxyisoquinoline-1,3(2H,4H)-diones variously substituted at positions 4 and 7 (Chart 1). The hydrophobic carboxamido side chains interacting by π-stacking with retroviral DNA bases,21,23 revealed as a key component for noticeable anti-integrase and antiviral activities, were kept at position 4, and we investigated the effect of substitution of position 7 by small functions such as methoxy, nitro, amino, halide, trifluoromethyl, cyanide, and amide. We first turned our attention to this position since it is the most synthetically versatile one. Comparatively, a few compounds substituted at positions 6 and 8 were also studied.

Scheme 3. Synthesis of Precursors 9−16a

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CHEMISTRY Scheme 1 shows the synthetic route derived from our previously reported method,22 which was used for preparation of target 2-hydroxyisoquinoline-1,3(2H,4H)-diones. N-Benzyloxymalonamides II (4 and 29−42) were cyclized in mild alkaline conditions to afford the corresponding 2-benzyloxy-4methoxycarbonylisoquinoline-1,3(2H,4H)-dione ester precursors III (43−57). Esters 43−57 were converted into amides 58−78 by addition−elimination of various primary amines. Finally, the O-benzyl protecting group was removed by action of boron trichloride at −78 °C or hydrogenation at room temperature over 5% Pd/C. N-Benzyloxyamide intermediates II were obtained by two pathways. The first one was based upon a formerly reported route for the preparation of 4-alkoxycarbonylisoquinoline-1,3diones24 and was used only for the synthesis of 4 (Scheme 2). 4-Hydroxyhomophthalic acid 1,19 obtained in three steps from homophthalic acid, was successively esterified and etherified to give intermediate 2. Lithium diisopropyl amide-promoted deprotonation of the methylene group and reaction of the anion with carbon dioxide afforded acid 3, which was coupled with O-benzylhydroxylamine with activation with the BOP reagent to yield amide 4 (Scheme 2). This route was unsuccessful for other homophthalic derivatives. Problems were early encountered for the reaction of deprotonated 5-substituted methyl homophthalates with carbon dioxide. The carboxylic acids in β-position relative to the methyl ester could not be isolated in satisfactory yields, and

a

Reagents and conditions: (i) 0.08 equiv of CuBr, 2.4 or 4.8 equiv of NaH, methyl malonate, rt, and then Δ, 75 °C, 30 min−2 h 30 min (45−80%); (ii) 6.0 equiv of SOCl2, MeOH, 0 °C, and then reflux for 15 h (75−95%).

methyl 2-fluoro-5-nitrobenzoate 17 by methyl malonate (Scheme 4). Scheme 3 shows that ortho-halogenated benzoic acids 5−8 were converted to diesters 9−12 by the Hurtley reaction using copper bromide and sodium hydride.25,26 This copper-catalyzed direct arylation of β-dicarbonyl compounds was useful for the preparation of early precursors yielding final C-6- and C-8-substituted HIDs (81, 84, and 85). Unfortunately we could not extend the panel of C-6- and C-8-substituted HIDs due to limiting yields and poor reproducibility observed with this reaction. Compounds 9−12 upon reflux in a methanolic solution of thionyl chloride yielded triesters 13− 16. Alternatively, methyl 2-fluoro-5-nitrobenzoate 17 was displaced by methyl malonate under standard conditions of aromatic nucleophilic substitution to give the nitrated compound 18, which was then a convenient precursor for other functions (Scheme 4). Its reduction by catalytic hydrogenation on 5% Pd/C afforded the corresponding aniline 19. Diazotation by sodium nitrite in acid medium gave the diazonium salt, which was allowed to react with hydrogen 4642



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Scheme 4. Synthesis of Precursors 20−28a

Reagents and conditions: (i) 5.0 equiv of SOCl2, MeOH, 0 °C, and then reflux for 15 h (90%); (ii) 1.2 equiv of NaH, 1.0 equiv of CH2(CO2Me)2, THF, rt, and then reflux for 15 h (67%); (iii) H2, 5% Pd/C, MeOH, rt, 12 h (95%); (iv) 1.5 or 1.2 equiv of R5COCl, CH2Cl2 [or AcOEt, DMF, 2.5 equiv of Et(iPr)2N], rt, 4 h (R5 = -CH3, -CH2Ph, -Ph) or 12 h (33−96%); (v) (a) (X = F) 1.5 equiv of NaNO2, 2.0 equiv of HBF4, 6.0 M HCl, 0 °C, 1 h, and then Δ, 80 °C (42%); (b) (X = Cl, Br) 1.0 equiv of NaNO2, HCl(Br), 0 °C; (b) 1.0 equiv of CuCl(Br), HCl(Br), 0 °C, and then rt for 12 h (48−61%); (c) (X = CN) 1.0 equiv of NaNO2, HNO3, 0 °C, 20 min, and then 1.2 equiv of CuCN, 3.6 equiv of KCN, 65 °C, 30 min (51%).

a

Table 1 also shows the positive influence of the nitro function since all the 7-nitrated derivatives (80 and 96−100) were strongly active against HIV-1 integrase; 80, 96, 97, and 100 were even as potent as the clinically used INSTI raltegravir (IC50 = 10 nM). A glance at the data shows that the linker’s length between amide function and aromatic ring does not have a drastic influence on inhibitory property. Compounds 96 (R4 = NHCOPh) and 97 (R4 = NHCOCH2Ph) inhibited HIV-1 integrase in the same manner (IC50 = 10 nM), and there was only a slight difference between the potencies of 98 (R4 = NHCOpFPh, IC50 = 30 nM), 80 (R4 = NHCOpFCH2Ph, IC50 = 10 nM), and 99 (R4 = NHCOCH2CH2pFPh, IC50 = 60 nM). Except for 87, 88, and 94, which were characterized as tautomeric keto forms (Table 1), a common remarkable feature of the potent compounds (80, 81, 89, and 95−100) with IC50 values below 70 nM is that they were all characterized by NMR as pure enol forms in dimethyl sulfoxide (DMSO) solution (Table 1). This was also the case for 80 in phosphate buffer (pH 7). It seems that the strong electron-withdrawing effects of the nitro and cyano functions favor this tautomeric form of the N-hydroxyimide scaffold, which is able to directly interact with the magnesium ions of IN catalytic center. Indeed, we previously demonstrated that metal complexation is strictly dependent on the enolization abilities of the compounds.19 Regarding the tautomeric equilibrium, 83−87, substituted by halogen atoms, were obtained as pure keto forms (Table 1) or sometimes as mixtures of keto and enol forms with a large preference for the keto form (75%). All analogues with EDGs at C-7 (79, 82, and 90−94) were obtained as pure keto forms (Table 1). For most of the investigated molecules, the carboxamido side chain used was a p-fluorobenzyl group that has appeared recurrently since 2000 (L-731,988)27 and was later shown to invade a pocket at the protein−DNA interface that is natively occupied by the 3′-terminal base of viral DNA.21 In 95, we replaced this aromatic group by a linear hexyl chain to evaluate if a linear lipophilic side chain could give a similar fit into the hydrophobic pocket. This was the case since 95 (IC50 = 60 nM) was nearly as active as 80 (IC50 = 10 nM). When compared to the precedent series of investigated compounds,22 the

tetrafluoroborate, cuprous halides, and cyanide to afford the 5halogeno and 5-cyano derivatives 20−23 (Scheme 4). Reaction of aniline 19 with several acid chlorides also gave amides 24− 28, where R5 is an alkyl, (hetero)aryl, or alkyl(hetero)aryl group (Scheme 4). All the triester precursors 13−16 and 18− 28 were converted to the corresponding N-benzyloxymonomalonamide derivatives 29−42 by reflux with O-benzylhydroxylamine in toluene (Scheme 1). The first series of target compounds was dedicated to compounds bearing small groups such as methoxy (79), nitro (80), amino (82), halides (83, 86, 87), trifluoromethyl (88), cyano (89), and amides at position 7 (90−94) and a pfluorobenzylaminocarbonyl group at position 4 (Table 1). Due to promising biological activities of nitrated compound 80 (Table 1), a second series (95−100) incorporating various amide groups at position 4 while keeping the nitro function at position 7 was elaborated. These 2-hydroxyisoquinoline1,3(2H,4H)-diones were obtained either as keto forms (for 79, 82−88, and 90−94; Table 1) with traces of enol form or as enol forms (for the nitro and cyano derivatives 80, 81, 89, and 95−100; Table 1).

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RESULTS AND DISCUSSION Anti-integrase and Antiviral Properties: Initial SAR around 80. Table 1 shows the inhibitory properties of target compounds against HIV-1 integrase. Except for 85 (IC50 = 0.88 μM), all the series was active in the low submicromolar range (IC50 = 10−260 nM). There was no strict relationship between electronic effects of the substituent at position 7 and inhibitory properties. For example, 87 (R7 = Br, IC50 = 60 nM) and 89 (R7 = CN, IC50 = 70 nM), with electron-withdrawing groups (EWGs), and 91 (R7 = NHCOPh, IC50 = 80 nM) and 94 (R7 = NHCOCH2Thioph, IC50 = 60 nM), with electron-donating groups (EDGs), displayed IC50 values similar to our previously reported hit 101 (IC50 = 56 nM).22,23 Regarding the fluorinated (83−85) and nitrated (80, 81) series, there was no significant effect of the aromatic position of fluorine atom or nitro function on integrase inhibition. Low nanomolar integrase inhibition was obtained for 80 (R7 = NO2, IC50 = 10 nM), 81 (R6 = NO2, IC50 = 30 nM), and 88 (R7 = CF3, IC50 = 20 nM). 4643



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Table 1. Inhibition of HIV-1 IN and RNase H Activities, Antiviral Activity, and Cytotoxicity of Substituted 2Hydroxyisoquinoline-1,3(2H,4H)-diones 79−101 and Raltegravira

a

Concentration required to inhibit by 50% the in vitro overall integrase activity. bEffective concentration required to reduce HIV-1-induced cytopathic effect by 50% in MT-4 cells. cCytotoxic concentration required to reduce by 50% MT-4 cell viability. dTherapeutic index, defined by CC50/EC50 ratio. ePercentage inhibition of HIV-1 RNase H activity at 10 μM concentration. f Major tautomeric form present in DMSO solution.

(IC50 = 0.36 μM),22 with an IC50 value of 4.8 μM for the most active compound 80. Regarding antiviral activities, 79 (R7 = OMe, EC50 = 6.5 μM) and 82 (R7 = NH2, EC50 = 23.3 μM) substituted by EDGs were devoid of any noticeable antiviral effect. Amides 90−94 (except for 90) displayed important cytotoxicities, which hindered the

proportion of candidates reaching raltegravir’s potency was significantly increased. We also tested the series against HIV-1 RT-associated RNase H activity (Table 1), and 60−80% inhibition was obtained at 10 μM concentration for 80, 96, 97, and 100. These candidates, which are also the best ones against integrase, do not reach the formerly reported potency of 101 4644



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previous docking protocol,22 developed on the 3S3M crystallographic structure of dolutegravir in the PFV IN intasome active site28 by use of the CCDC Gold software33 and the CHEMPLP scoring function,34 was refined in order to introduce two additional factors that may influence the binding mode of our scaffold. First, ab initio minimization of our ligand followed by a conformational study of dihedral angle Φ clearly indicates that its most stable conformer displays an intramolecular hydrogen bond between the exocyclic amide hydrogen and the enol oxygen, thus forming a pseudo-6-membered ring (conformer A, Figure 2; data not shown). The energy of such an intra-

detection of any antiviral activity in cell culture. This phenomenon was observed before with pioneer 7-substituted compounds lacking the carboxamido side chain18 at position 4 and is confirmed herein. Addition of this side chain at position 4, which was revealed as critical for the anti-integrase properties of this scaffold, did not overcome the incapacitating cytotoxicities. In contrast, all compounds substituted by EWGs displayed noticeable antiviral activities. The halogenated derivatives (83, 86, and 87) displayed EC50 values in the micromolar range (0.73−2.99 μM). In the halogenated series, the antiviral activity decreasing from the fluorinated compound 83 (EC50 = 0.73 μM) to the brominated one 87 (EC50 = 2.99 μM) could be related to some extent to the halogen lipophilicity. Substitution of the scaffold by trifluoromethyl group, cyano function, and particularly nitro function was greatly beneficial. Except for 95, 99, and 100 acting in the micromolar range, low submicromolar anti-HIV activities were obtained for 80, 81, and 96−98, with EC50 values ranging from 110 to 270 nM. The micromolar antiviral potency of 100 (R4 = CONHCH2pOMePh, EC50 = 2.97 μM) showed the negative influence of replacement of the fluorine atom in 80 (R4 = CONHCH2pFPh, EC50 = 110 nM) by a methoxy group, as previously observed with our first series of carboxamides.22 The position of the nitro function on the benzenic ring of the scaffold did not impact the antiviral activity, with close EC50 values of 110 and 270 nM for the 7- and 6-substituted isomers 80 and 81, respectively. Compounds 96 (R4 = CONHPh) and 98 (R4 = CONHpFPh) bearing phenylcarboxamido side chains displayed EC50 values (around 0.15 μM) similar to those of their respective homologues 97 (R4 = CONHCH2Ph) and 80 (R4 = CONHCH2pFPh) with benzylcarboxamido side chains. In contrast, addition of another carbon between the amide function and the phenyl ring was a little unfavorable since 99, with a phenethyl side chain [R4 = CONH(CH2)2pFPh] inhibited HIV-1 replication in the micromolar range (EC50 = 0.95 μM). In comparison with our precedent hit compound 101 (EC50 = 2.34 μM),22,23 the antiviral activities of 80, 81, and 96−98 are at least 1 log unit lower and 80, with an EC50 value of 110 nM, emerged as a novel promising lead compound. As a whole, except for 88 (R4 = CF3) characterized as the keto form (Table 1), there is a good relationship between enolization ability of the compounds and their antiviral efficacy. Drug molecules with low submicromolar anti-HIV activities were characterized as 100% enol forms in solution (Table 1), and the tautomeric equilibrium has a great impact on the lipophilicity of the N-hydroxyimide moiety. Partition coefficients, log D, of 1.24 and 0.56 were determined for enol 80 and keto 101, respectively. A calculation (www.molinspiration.com) supports this discrepancy of lipophilicity between the tautomeric forms since log P values of 1.98 and 0.76 were calculated for the enol and keto forms of 80. From 101 to 80, the 2-fold increase of lipophilicity of the drugs can explain to some extent the increase of antiviral activity, since 80 is expected to have better cellular permeation than 101. Moreover, there was an advantageous window between antiviral efficacy and cellular toxicity (463−1100-fold) for the most potent compounds of the novel series. These therapeutic indexes not only represent progress in comparison with the precedent series (21−86fold)22 but also get close to the value of the clinically used raltegravir (1333-fold). Docking Study. In silico molecular docking calculations were performed in order to qualitatively evaluate the ability of lead compound 80 to fit in the IN catalytic pocket. Our

Figure 2. Proposed binding modes A and B of our 2-hydroxyisoquinoline-1,3(2H,4H)-diones depending on the ligand conformation.

molecular H-bond is difficult to quantitate in our case, as it requires discriminating between its effect and that of other steric and electrostatic factors within the ligand when dihedral angle Φ is changed, but typical values for such systems are estimated at 10−15 kJ·mol−1. Upon complexation with magnesium cations, two main conformers A and B were previously evidenced by our studies.22 Whereas conformer A retains it, conformer B requires the rupture of this intramolecular H-bond. Conformer B would also require significant internal torsion, as well as destabilizing close steric contacts between the carboxamide linkage and the aromatic hydrogen at position 5, in order to complex efficiently with the metal cofactors and occupy the cavity. Additionally, the previously reported crystal structure of 101 in complex with the PFV IN intasome (PDB 4IKF)23 identified binding mode A for this compound, in accordance with our previous docking predictions. This provides a strong array of presumptions in favor of conformer A: a significant energy barrier needs to be crossed indeed if binding mode B is to be attained. Because this intramolecular H-bond cannot be taken into account by the ChemPLP scoring function, we introduced a dynamic maximum distance constraint of 2.4 Å between these two atoms in the ligand during the docking runs, thus allowing flexibility while retaining the hydrogen bond. Second, in order to take into account the proximity of arginine 329 to the catalytic site, the flexibility of its side chain was also introduced in the method by performing the docking runs on a library of 34 Arg329 rotamers of the PFV IN intasome structure 3S3M. Enabling a modulation of this side chain’s conformation indeed gives the CCDC Gold Docking Suite’s genetic algorithm the opportunity to consider more diverse solutions, which may otherwise have been omitted due to steric clash between the ligand and the starting crystallized rotamer of Arg329. Parameters describing the relative weighted terms of the CHEMPLP fitness scoring function34 were also optimized in order to prevent aberrant binding modes and improve cluster size. 4645



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This optimized docking protocol yielded binding mode A as the best solution with very good clustering outcome, by far the best results with the ChemPLP scoring function and after rescoring with the ChemScore function. Although we have previously shown by NMR and molecular modeling that this scaffold complexes magnesium cations in the enolic form, the ionization state of the ligand is still uncertain in the catalytic site. However, docking the dianion or monoanion form led to the same poses (data not shown). As for 101, the obtained binding mode for 80 involves chelation of the two magnesium cofactors by oxygens at positions 1, 2, and 3 of our pharmacophore (Figure 3). The p-

no further insight into the mechanistic influence of the nitro moiety. It is not clear whether this improvement is due to additional H-bonds with Arg329 or to an increased magnesium chelation potency of the molecule due to the strong resonance electron-withdrawing effect of the nitro group or (most likely) a combination of both. Pharmacological Profile of 80. Given the potential of the novel lead compound 80, we investigated more deeply its pharmacological properties regarding its cellular mechanism of action and activities against viral subtypes and mutants. First of all, we performed time of addition experiments (TOA) to pinpoint the stage of the HIV life cycle that is inhibited by antiretroviral drugs. Compound 80 was tested together with a set of known HIV replication inhibitors. The compounds were added at different time points after infection of MT-4 cells with HIV-1 IIIB, and p24 antigen production in the supernatants was measured at 30 h postinfection. The loss of activity was delayed to 4−5, 10−11, and 25 h postinfection for RT inhibitor AZT, INSTI raltegravir, and protease inhibitor ritonavir, respectively (Figure 4). Compound 80 matched the profile observed for the INSTI raltegravir and our hit compound 101, attesting to its cellular inhibition of HIV-1 integrase (Figure 4).

Figure 3. Proposed binding mode of 80 as obtained by molecular docking in the PFV IN catalytic site (PDB 3S3M).Viral DNA is depicted in pink, PFV IN in blue, magnesium cations in green, and the ligand in gold. The pose involves (a) dual magnesium complexation through the three oxygens on the heterocyclic core, (b) π-stacking of the fluorobenzyl side chain with the invariant deoxycytosine C16, and (c) π-stacking of the central isoquinoline moiety with the invariant terminal 3′-deoxyadenosine A17.

Figure 4. Time of addition experiment. After infection of MT-4 cells with HIV-1 IIIB, inhibitors (50- and 100-fold EC50) were added at indicated time points spanning from 1 to 25 h postinfection. Virus replication was determined by p24 antigen determination in the supernatant at 30 h postinfection. Comparative profiles of RT inhibitor AZT, HDIs 101 and 80, INSTI raltegravir (Ral), and protease inhibitor ritonavir (Rit) are presented.

Considering the broad genetic diversity of HIV-1 and the variable prevalence of subtypes in different regions of the world, we then studied the anti-HIV activity of 80 against a spectrum of subtypes. The EC50 values of 80 against clades A, D, E, and F were 0.15, 0.49, 0.52, and 0.14 μM, respectively, showing that no significant variation in its anti-HIV activity was observed. As mentioned before, one of the most challenging problems in the search for novel marketable anti-integrase drugs is the crossresistance profile with current INSTIs. Clinical studies evidenced a rapid development of resistance to raltegravir, due to the low genetic barrier of HIV-1 IN. The most common key mutations are E92Q, Q148H, N155H, and G140S/Q148H. Table 2 shows the x-fold changes in EC50 values of 80 and 10123 tested against raltegravir-resistant strains. Like the previous hit 101, 80 retained completely its activity against E92Q and Q148H mutants. There was a loss of its activity with a 2- and 6-fold change in EC50 for the N155H mutant and the G140S/Q148H double mutant, respectively. But these decreases in antiviral activity appear negligible in comparison

fluorobenzyl moiety nicely occupies the hydrophobic pocket created between the conserved cytosine of viral DNA and Pro214 of PFV IN. The DNA terminal adenosine also stacks with the core of our scaffold, as it does for 101 and dolutegravir. It is not clear whether or not the nitro substituent at position 7 of our heterocyclic scaffold plays a significant role in enzymatic inhibition. The docked solution indicates the possibility for intermolecular hydrogen bonds between the nitro group and Arg329 (the distance between arginine hydrogens and nitro oxygens being measured respectively at 1.83 and 2.29 Å). Even though it has previously been reported in the literature that the H-bond acceptor character of nitro groups is only about half as strong as that of more commonly found carbonyl or alcohol moieties and that such interactions have less significant binding energies,29 the enzymatic activity of 80 (IC50 = 10 nM) is indeed increased by a 5-fold factor compared to our hit compound 101 (IC50 = 56 nM) and is also more active than cyano counterpart 89 (IC50 = 70 nM). At this point, we have 4646



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Table 2. Cross-Resistance (x-fold Change in EC50) of 10123 and 80 with Raltegravir virus strain E92Q Q148H N155H G140S/Q148H a

RALa

integrase 80

101 1 1 1 1

Table 4. Detailed Analysis of in Vitro Toxicity of 80 and Cerivastatin

1 1 2 6

1.0 μM

compd

30.0 μM

100.0 μM

IC50a (μM)

b

5 9 7 362

Cytotoxicity (Cell Number) −20.1 12.6 −7.4 ndc nd nd Intracellular Free Calciumd −1.3 18.8 18.3 nd nd nd Nuclear Sizeb 2.0 23.8 22.1 nd nd nd Membrane Permeabilityd 0 23.9 6.7 nd nd nd Mitochondrial Membrane Potentialb 8.4 −23.8 22.4 nd nd nd

80 cerivastatin 80 cerivastatin

Raltegravir.

with raltegravir, which displayed a 7- and 362-fold change in EC50 for the same mutants (Table 2). Preliminary ADMETox Evaluation of 80. The discrepancy between anti-integrase and antiviral potencies of 80 led us to perform a preliminary ADMETox evaluation. This drug candidate displayed good aqueous solubility of 200.0 μM and a partition coefficient (log D) of 1.24. At 10 μM concentration, a mean permeability coefficient Papp (Caco-2 cells, pH 6.5/7.4) below 0.1 × 10−6 cm/s was determined and high human plasma protein binding (mean of 99.9% protein bound; mean of 66.8% recovery) was observed, which certainly account for the lack of strong antiviral activity. P-glycoprotein (P-gp) is an important transporter protein found in cells throughout the body, such as those lining the intestine and blood−brain barrier. It is believed to play an important role in defining the extent of distribution of drug molecules and limiting their oral and brain exposures. Up to a concentration of 100.0 μM, 80 inhibited the P-gp efflux only at 7.6% extent (Table 3) while the reference compound, verapamil, reduced by 50% this efflux at a concentration of 8.0 μM. It was also remarkably stable in human liver microsomes, since the percentage remaining after 1 h exposure was 94% at 1.0 μM concentration. Cytochromes P450 are the principal enzymes for oxidative metabolism of drugs and other xenobiotics. Among the xenobiotic-metabolizing cytochromes P450, five formsCYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4appear to be most commonly responsible for the metabolism of drugs. Inhibition of cytochrome P450mediated metabolism is often the mechanism for drug−drug interactions. Table 3 shows that 80 at a concentration of 10 μM only slightly inhibited CYP1A2 and CYP2C19. On the contrary, there was a marked effect on CYP2C9 and CYP3A4 activities with 76.5% and 100% inhibition, respectively. In addition, an extended study on the toxicity of this compound was performed (Table 4). The effects on cell number, intracellular free calcium, nuclear size, membrane permeability, and mitochondrial potential were measured. Compound 80 did not display any severe toxicity in these tests, even at a concentration of 100 μM. In contrast, the reference compound, cerivastatin, displayed submicromolar IC50 values for each test.

80 cerivastatin 80 cerivastatin 80 cerivastatin

nd 0.96 nd 0.34 nd 0.13 nd 0.80 nd 0.60

a

Concentration required to inhibit a−e. bPercentage reduction relative to untreated control. cNot determined. dPercentage increase relative to untreated control.

Finally, 80 was tested in a hERG (human ether-a-go-go-related gene potassium channel 1) inhibition assay. Inhibition of this voltage-gated potassium ion channel, a transmembrane protein encoded by the hERG gene, is known to be undesirable due to the possibility of QT prolongation, which can lead to fatal cardiac arrhythmia. Compound 80 did not significantly inhibited the tail current with 2.6%, 5.1%, and 3.9% inhibition at concentrations of 0.1, 1.0, and 10 μM, respectively. Under the same conditions, the control compound E-4031 induced hERG-mediated cardiac toxicity with an IC50 value of 24 nM.

■

CONCLUSIONS The 2-hydroxyisoquinoline-1,3(2H,4H)-dione scaffold previously provided a hit compound 101 with strong anti-integrase potency but unsatisfying antiviral potency in the micromolar range. The lack of cross resistance with other INSTIs led us to further pharmacomodulate its structure by investigating the effect of 7-substitution. Interestingly, we found that the substitution by electron-withdrawing functions like cyano and, particularly, nitro led to enol compounds with strong HIV-1 IN inhibitory properties and improved low submicromolar antiHIV activities, which are at least 1 log unit lower than that of our previous hit compound 101. Compound 80, with an EC50 value of 110 nM and an advantageous window between antiviral efficacy and cellular toxicity (1100-fold), emerged as a novel promising lead compound. Like 101, this cellular

Table 3. Inhibition of Different Members of the Cytochrome P450 Mixed-Function Oxidase System and of P-gp Efflux by 80 and Reference Compounds compd 80 furafyline sulfaphenazole tranylcypromine quinidine ketoconazole

CYP1A2 a

1.4 1.4d nd nd nd nd

CYP2C9 a

76.5 nd 0.18d nd nd nd

CYP2C19 11.4 nd nd 3.1d nd nd

a

CYP2D6

CYP3A4

P-gp inhibitionc

b

106o nd nd nd nd 0.28d

−5.1, −3.2, 7.6

nd nd nd nd 0.017d nd

Percentage inhibition of control values at a 10.0 μM dose. bNot determined. cP-gp inhibition at 1.0, 30.0, and 100.0 μM in MDR1−MDCKII system. dIC50 values (micromolar).

a

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(5.0 mL) was added. After being stirred for 1 h at −20 °C and 12 h at room temperature, the mixture was washed with 2.0 M HCl, 1.0 M NaHCO3, and brine. The organic layer was dried over Na2SO4 and concentrated in vacuo. After column chromatography of the residue (eluent petroleum ether/AcOEt 70/30), 4 was obtained as a yellow oil (40%). 1H NMR (300 MHz, CDCl3): δ = 3.65 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 4.81 (s, 2H, OCH2), 5.13 (s, 1H, CH), 7.03 (dd, 1H, H4, 3JH4−H3 = 8.5 Hz, 4JH4−H6 = 2.6 Hz), 7.23 (s, 5H, HAr), 7.39 (d, 1H, H6, 4JH6−H4 = 2.6 Hz), 7.50 (d, 1H, H3, 3JH3−H4 = 8.5 Hz), 9.49 (s, 1H, NH). NMR 13C (75 MHz, CDCl3): δ = 52.1 (CH), 52.5 (OCH3), 52.7 (OCH3), 55.5 (OCH3), 77.9 (OCH2), 115.9 (CH), 118.4 (CH), 126.5 (CIV), 128.4 (2CH), 128.6 (CH), 129.3 (2CH), 129.9 (CIV), 133.0 (CH), 135.2 (CIV), 159.0 (C5), 165.7 (CO), 168.0 (CO), 169.6 (CO). ESI-MS: m/z = 388 (M + H)+. Synthesis of Precursors 13−16. 4-Fluoro-2-(1,3-dimethoxy1,3-dioxopropan-2-yl)benzoic Acid (9). A solution of 2-bromo-4fluorobenzoic acid (0.50 g, 2.3 mmol) and copper bromide (26 mg, 0.18 mmol, 0.08 equiv) in dimethyl malonate (5.0 mL) was degassed with argon for 10 min and put under argon atmosphere. Sodium hydride (60% in mineral oil, 225 mg, 5.6 mmol, 2.4 equiv) was added portionwise at room temperature over 10 min. Toluene (5 mL) was added and the mixture was heated at 75 °C for 2.5 h. After the mixture was cooled to room temperature, water (50 mL) was added and the mixture was extracted six times with ether (50 mL). The aqueous layer was acidified with 1.0 M HCl and extracted with ethyl acetate. The organic layer was dried over MgSO4 and concentrated in vacuum to give a sticky white residue, which was filtered and rinsed with toluene (minimal volume). Product 9 was obtained as a white solid (77%), mp 127−130 °C. 1H NMR (300 MHz, acetone-d6): δ = 3.61 (s, 6H, 2OCH3), 5.84 (s, 1H, CH), 7.21 (dd, 1H, H3, 3JH3−F = 10.1 Hz, 4 JH3−H5 = 2.6 Hz), 7.28 (ddd, 1H, H5, 3JH5−F = 8.7 Hz, 3JH5−H6 = 8.1 Hz, 4JH5−H3 = 2.7 Hz), 8.19 (dd, 1H, H6, 3JH6−H5 = 8.8 Hz, 4JH6−F = 6.6 Hz). 13C NMR (75 MHz, acetone-d6): δ = 53.0 (2OCH3), 54.9 (CH), 115.3 (d, CH, 2JC−F = 21.5 Hz), 117.9 (d, CH, 2JC−F = 23.7 Hz), 126.3 (d, C1, 4JC−F = 3.4 Hz), 134.7 (d, CH, 3JC−F = 9.4 Hz), 138.3 (d, C2, 3 JC−F = 8.9 Hz), 165.3 (C4, 1JC−F = 251.7 Hz), 167.6 (CO), 169.0 (2CO). ESI-MS: m/z = 271 (M + H)+. 6-Fluoro-2-(1,3-dimethoxy-1,3-dioxopropan-2-yl)benzoic Acid (10). Intermediate 10 was synthesized from 2-bromo-6-fluorobenzoic acid according to the protocol reported for synthesis of 9, using copper bromide (0.08 equiv) and sodium hydride (4.9 equiv): white solid (80%), mp 127−131 °C. 1H NMR (300 MHz, acetone-d6): δ = 3.73 (s, 6H, 2OCH3), 5.20 (s, 1H, CH), 7.30 (t, 1H, H5, 3JH5−F = 3JH5−H4 = 9.0 Hz), 7.34 (d, 1H, H3, 3JH3−H4 = 7.8 Hz), 7.59 (td, 1H, H4, 3JH4−H3 = 3JH4−H5 = 8.1 Hz, 4JC4−F = 5.7 Hz). 13C NMR (75 MHz, acetone-d6): δ = 52.6 (2OCH3), 54.4 (CH), 116.1 (d, C5, 2JC−F = 22.3 Hz), 122.2 (d, C1, 2JC−F = 16.5 Hz), 125.8 (d, C3, 4JC−F = 3.3 Hz), 132.3 (d, C4, 3 JC−F = 9.2 Hz), 134.2 (d, C2, 3JC−F = 2.7 Hz), 160.2 (C6, 1JC−F = 251.0 Hz), 165.5 (CO), 168.0 (2CO). ESI-MS: m/z = 271 (M + H)+. 4-Nitro-2-(1,3-dimethoxy-1,3-dioxopropan-2-yl)benzoic Acid (11). Intermediate 11 was synthesized from 2-bromo-4-nitrobenzoic acid according to the protocol reported for synthesis of 9: beige oil (45%). 1H NMR (300 MHz, acetone-d6): δ = 3.77 (s, 6H, 2OCH3), 5.93 (s, 1H, CH), 8.32−8.35 (m, 3H, HAr). 13C NMR (75 MHz, acetone-d6): δ = 52.9 (2OCH3), 54.4 (CH), 123.4 (CH), 125.4 (CH), 132.7 (CH), 136.1 (CIV), 136.6 (CIV), 150.0 (CIV, C4), 166.7 (CO), 168.2 (2CO). ESI-MS: m/z = 284 (M + H)+. 4-Trifluoromethyl-2-(1,3-dimethoxy-1,3-dioxopropan-2-yl)benzoic Acid (12). Intermediate 12 was synthesized from 2-bromo-4trifluoromethylbenzoic acid according to the protocol reported for synthesis of 9. However, after the work-up, a mixture of reactant and 12 was obtained and the coupled compound, which did not crystallize, was not isolated. The crude mixture was used for the next esterification step. Methyl 4-Fluoro-2-(1,3-dimethoxy-1,3-dioxopropan-2-yl)benzoate (13). Thionyl chloride (4.8 mL, 6.0 equiv) was added dropwise at 0 °C to a solution of 9 (3.16 g, 12.0 mmol) in methanol (50 mL). After 16 h of reflux, the mixture was concentrated in vacuum. The residue was taken up in ethyl acetate (100 mL) and washed with water (2 × 50 mL). The organic layer was dried over MgSO4 and

integrase inhibitor retained antiviral efficacies against raltegravir-resistant strains. On the whole, the preliminary ADMETox assessment of 80 evidenced a pharmacokinetic profile similar to that of 101. Weak permeability and high plasma protein binding certainly account for the lack of strong nanomolar antiviral activity. Without any severe toxicity, 80 displayed noticeable inhibitions of CYP2C9 and CYP3A4 activities. Further optimizing modulations of the scaffold will still be necessary to address these different issues and reach the required standard for a clinical candidate.

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EXPERIMENTAL SECTION

Chemistry: General Details. All reagents and solvents were purchased from Aldrich-Chimie (Saint-Quentin-Fallavier, France) of ACS reagent grade and were used as provided. Thin-layer chromatographic analyses were performed on plastic sheets precoated with silica gel 60F254 (Merck). SiO2, 40−63 mesh (Merck), or 30 μm HPSilprepacked SNAP columns (Biotage) were used for flash column chromatography. NMR spectra were obtained on an AC 300 Bruker spectrometer in the appropriate solvent with tetramethylsilane (TMS) as internal reference. Chemical shifts are reported in δ units (parts per million, ppm) and are assigned as singlet (s), doublet (d), doublet of doublets (dd), triplet (t), quartet (q), quintet (quin), sextuplet (sext), multiplet (m), and broad signals (br). Melting points were obtained on a Reichert Thermopan melting point apparatus, equipped with a microscope, and are uncorrected. Mass spectra (electrospray ionization, ESI) were recorded on a Micromass Quattro II spectrometer. High-resolution mass spectrometry (HRMS) measurements were made on an Apex Qe 9.4 T Bruker Daltonics spectrometer. Analytes dissolved in methanol (3 mM solutions) were diluted with water/methanol/formic acid solution (50/50/0.1 v/ v/v) to afford 3 μM solutions and infused into the mass spectrometer nano-ESI source in positive mode at a rate of 1 μL/min. Purity of the tested compounds was established by combustion analysis, confirming purity ≥95%. Elemental analyses (C, H, N) were performed by CNRS Laboratories (Vernaison, France); the analytical results were within ±0.4% of theoretical values. Synthesis of Precursor 4. Methyl 5-Methoxy-2-(2-methoxy-2oxoethyl)benzoate (2). Thionyl chloride (1.5 mL, 20.4 mmol) was added dropwise to a cooled solution of 2-carboxymethyl-5hydroxybenzoic acid19 1 (1.00 g, 5.1 mmol). After 24 h of reflux, the solvent was removed in vacuo. The crude residue was dissolved in dry acetone (50.0 mL), and potassium carbonate (4.20 g, 30.6 mmol) and methyl iodide (1.9 mL, 30.6 mmol) were added. After 12 h of reflux,volatiles were removed in vacuo. The residue was taken up in ethyl acetate and washed several times with 1.0 M NaOH and brine. The organic layer was dried over sodium sulfate and concentrated in vacuo to give an orange solid (43%), mp 212 °C. 1H NMR (300 MHz, DMSO-d6): δ = 3.48 (s, 3H, OCH3), 3.69 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 3.81 (s, 2H, CH2), 6.98 (dd, 1H, H4, 3JH4−H3= 8.4 Hz, 4JH4−H6 = 2.7 Hz), 7.15 (d, 1H, H3, 3JH3−H4 = 8.4 Hz), 7.36 (d, 1H, H6, 4JH6−H4 = 2.7 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 39.6 (CH2), 51.8 (OCH3), 52.0 (OCH3), 55.4 (OCH3), 115.8 (CH), 118.3 (CH), 128.0 (CIV), 130.4 (CIV), 133.3 (CH), 158.6 (C5), 167.2 (CO), 172.3 (CO). Methyl 2-{1-[(Benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2yl}-5-methoxybenzoate (4). A solution of freshly distilled diisopropylamine (0.77 mL, 5.5 mmol) in 10.0 mL of dry THF under an argon atmosphere was cooled to −78 °C, and 3.4 mL of 1.6 M n-butyllithium (5.5 mmol) was added. After 30 min of reaction at −78 °C, a solution of methyl 5-methoxy-2-(2-methoxy-2-oxoethyl)benzoate (2) (1.00 g, 4.2 mmol) in 5.0 mL of dry THF was added dropwise. After the solution was stirred for 30 min at −78 °C, the temperature was allowed to rise to 0 °C and the argon inlet was removed. Solid CO2 was added portionwise for 1 h. After acidification with 3.0 M HCl, the solution was extracted with ethyl acetate. BOP (2.00 g, 4.6 mmol) and diisopropylethylamine (3.5 mL, 21.0 mmol) were added at −20 °C to the precedent organic solution. After 20 min of stirring at −20 °C, a solution of O-benzylhydroxylamine (0.87 g, 5.5 mmol) in ethyl acetate 4648



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concentrated in vacuum to give a white solid (95%), mp 81−84 °C. 1H NMR (300 MHz, CDCl3): δ = 3.76 (s, 6H, 2OCH3), 3.84 (s, 3H, OCH3), 5.80 (s, 1H, CH), 7.07 (ddd, 1H, H5, 3JH5−F = 8.7 Hz, 3JH5−H6 = 7.7 Hz, 4JH5−H3 = 2.6 Hz), 7.13 (dd, 1H, H3, 3JH4−F = 9.7 Hz, 4JH3−H5 = 2.6 Hz), 8.03 (dd, 1H, H6, 3JH6−H5 = 8.8 Hz, 4JH6−F = 6.0 Hz); 13C NMR (75 MHz, CDCl3): δ = 52.4 (OCH3), 53.0 (2OCH3), 54.5 (CH), 115.3 (d, CH, 2JC−F = 21.3 Hz), 117.5 (d, CH, 2JC−F = 23.5 Hz), 125.4 (d, C1, 4JC−F = 3.2 Hz), 137.5 (d, CH, 3JC−F = 8.9 Hz), 138.3 (d, C2, 3JC−F = 8.9 Hz), 164.7 (d, C4, 1JC−F = 250.7 Hz), 166.4 (CO), 168.5 (2CO). ESI-MS: m/z = 285 (M + H)+. Methyl 6-Fluoro-2-(1,3-dimethoxy-1,3-dioxopropan-2-yl)benzoate (14). Intermediate 10 was treated with thionyl chloride according to the protocol reported for synthesis of 13. After the workup, NMR analysis of the crude product still showed the presence of benzoic acid. After column chromatography (eluent petroleum ether/ AcOEt 70/30), 14 was obtained as a white solid (75%), mp 75−78 °C. 1 H NMR (300 MHz, CDCl3): δ = 3.61 (s, 6H, 2OCH3), 3.76 (s, 3H, OCH3), 4.95 (s, 1H, CH), 6.98 (t, 1H, H5, 3JH5−F = 3JH5−H4 = 9.0 Hz), 7.14 (d, 1H, H3, 3JH3−H4 = 7.8 Hz), 7.31 (td, 1H, H4, 3JH4−H5 = 3 JH4−H3= 8.0 Hz, 4JH4−F = 5.8 Hz). 13C NMR (75 MHz, CDCl3): δ = 52.3 (OCH3), 52.6 (2OCH3), 54.4 (CH), 115.9 (d, C5, 2JC−F = 22.3 Hz), 120.9 (d, C1, 2JC−F = 15.7 Hz), 125.4 (d, C3, 4JC−F = 3.3 Hz), 132.0 (d, C4, 3JC−F = 9.2 Hz), 133.7 (d, C2, 3JC−F = 2.3 Hz), 160.2 (d, C6, 1JC−F = 252.0 Hz), 165.0 (CO), 167.8 (2CO). ESI-MS: m/z = 285 (M + H)+. Methyl 4-Nitro-2-(1,3-dimethoxy-1,3-dioxopropan-2-yl)benzoate (15). Intermediate 11was treated with thionyl chloride according to the protocol reported for synthesis of 13: beige solid (91%), mp 68− 72 °C. 1H NMR (300 MHz, CDCl3): δ = 3.70 (s, 6H, 2OCH3), 3.83 (s, 3H, OCH3), 5.63 (s, 1H, CH), 8.04 (d, 1H, H6, 3JH6−H5 = 8.6 Hz), 8.12 (dd, 1H, H5, 3JH5−H6 = 8.6 Hz, 4JH5−H3 = 2.1 Hz), 8.21 (d, 1H, H3, 4 JH3−H5 = 2.1 Hz). 13C NMR (75 MHz, CDCl3): δ = 52.8 (OCH3), 53.0 (2OCH3), 54.4 (CH), 122.8 (CH), 125.4 (CH), 132.0 (CH), 135.0 (CIV), 136.0 (CIV), 149.6 (CIV, C4), 165.7 (CO), 167.7 (2CO). ESI-MS: m/z = 312 (M + H)+. Methyl 4-Trifluoromethyl-2-(1,3-dimethoxy-1,3-dioxopropan-2yl)benzoate (16). Crude intermediate 12 was treated with thionyl chloride according to the protocol reported for synthesis of 13. Compound 16 was isolated as a yellow solid after column chromatography of the residue (eluent petroleum ether/EtOAc 80/ 20; 37% for two-step reaction from 2-bromo-4-trifluoromethylbenzoic acid), yellow oil. 1H NMR (300 MHz, CDCl3): δ = 3.77 (s, 6H, 2OCH3), 3.91 (s, 3H, OCH3), 5.82 (s, 1H, CH), 7.59 (d, 1H, H3, 3 JH3−H4 = 8.3 Hz), 7.78 (dd, 1H, H4, 3JH4−H3 = 8.3 Hz, 4JH4−H6 = 1.7 Hz), 8.27 (d, 1H, H6, 4JH6−H4 = 1.9 Hz). 13C NMR (75 MHz, CDCl3): δ = 52.37 (OMe), 52.66 (2OMe), 54.47 (CH), 122.56 (q, CF3, 1JC−F = 267.0 Hz), 127.80 (q, CH, 3JC−F = 3.8 Hz), 128.80 (q, CH, 3JC−F = 3.8 Hz), 130.16 (CIV), 130.41 (q, C5, 2JC−F = 33.0 Hz), 131.06 (C3), 138.24 (CIV), 165.92 (CO), 168.72 (2CO). ESI-MS: m/z = 335 (M + H)+. Synthesis of Precursors 20−28. Methyl 2-Fluoro-5-nitrobenzoate (17). Commercial 2-fluoro-5-nitrobenzoic acid (3.7 g, 20.0 mmol) was dissolved at 0 °C in MeOH (100 mL), and thionyl chloride (5.3 mL, 60.0 mmol) was added dropwise. The solution was heated under reflux for 24 h and concentrated in vacuo. The residue was dissolved in EtOAc and washed several times with 1.0 M NaOH. After the mixture was dried over Na2SO4, the solvent was evaporated in vacuo to yield 17 as a yellow oil, which crystallized upon standing as light yellow needles: yield 90%, mp 47−49 °C. 1H NMR (300 MHz, CDCl3): δ = 4.01 (s, 3H, OCH3), 7.35 (t, 1H, H3, 3JH3−H4 = 3JH3−F = 9.2 Hz), 8.44 (dt, 1H, H4, 3JH4−H3 = 9.2 Hz, 4JH4−F = 4JH4−H6 = 3.5 Hz), 8.87 (dd, 1H, H6, 4JH6−F = 5.9 Hz, 4JH6−H4 = 2.7 Hz). 13C NMR (75 MHz, CDCl3): δ = 47.7 (OCH3), 113.2 (d, C3, 2JC−F = 25.1 Hz), 114.5 (d, C1, 2JC−F = 12.0 Hz), 122.9 (d, C6, 3JC−F = 3.3 Hz), 124.3 (d, C4, 3JC−F = 10.9 Hz), 138.6 (C5), 157.4 (d, COOCH3, 3JC−F = 3.8 Hz), 159.8 (d, C2, 1JC−F = 269.0 Hz). Methyl 2-(1,3-Dimethoxy-1,3-dioxopropan-2-yl)-5-nitrobenzoate (18). An argon stream was passed over a suspension of sodium hydride (60% dispersion in mineral oil, 1.2 g, 25.1 mmol) in petroleum ether to discard mineral oil. Then sodium hydride was dispersed in THF

(30.0 mL), and a solution of methyl malonate (2.9 mL, 25.1 mmol) in THF (20.0 mL) was added dropwise under argon inert atmosphere. After 30 min of stirring at room temperature, a solution of 17 (5.0 g, 25.1 mmol) in THF (20.0 mL) was added dropwise, and the solution was heated under reflux for 15 h. The solvent was removed in vacuo. The residue was dissolved in EtOAc and washed several times with water. After column chromatography of the residue (eluent petroleum ether/EtOAc 80/20), product 18 was obtained as a yellow oil, which crystallized upon standing as light yellow needles: yield 67%, mp 63− 65 °C. 1H NMR (300 MHz, CDCl3): δ = 3.73 (s, 6H, 2OCH3), 3.90 (s, 3H, OCH3), 5.81 (s, 1H, CH), 7.61 (d, 1H, H3, 3JH3−H4 = 8.6 Hz), 8.31 (dd, 1H, H4, 3JH4−H3 = 8.6 Hz, 4JH4−H6 = 2.2 Hz), 8.80 (dd, 1H, H6, 4JH6−H4 = 2.2 Hz). 13C NMR (75 MHz, CDCl3): δ = 52.9 (2OCH3), 54.7 (OCH3), 59.7 (CH), 125.0 (CH), 126.8 (CH), 131.1 (CIV), 132.5 (CH), 140.3 (CIV), 146.9 (CIV, C5), 165.2 (CO), 167.4 (2CO). ESI-MS: m/z = 312 (M + H)+. Methyl 2-(1,3-Dimethoxy-1,3-dioxopropan-2-yl)-5-aminobenzoate (19). Compound 18 (4.0 g, 12.9 mmol) was dissolved in methanol (100 mL) and hydrogenated for 12 h at room temperature over Pd/C 5% (0.4 g). The catalyst was filtered and the solvent was removed in vacuo: pink solid, yield 95%, mp 89−90 °C. 1H NMR (300 MHz, CDCl3): δ = 3.79 (s, 6H, 2OCH3), 3.88 (s, 3H, OCH3), 5.63 (s, 1H, CH), 6.84 (d, 1H, H3, 3JH3−H4 = 8.3 Hz), 7.21 (dd, 1H, H4, 3 JH4−H3 = 8.3 Hz, 4JH4−H6 = 2.2 Hz), 7.32 (d, 1H, H6, 4JH6−H4 = 2.2 Hz).; 13C NMR (75 MHz, CDCl3): δ = 52.2 (2OCH3), 52.7 (OCH3), 54.1 (CH), 116.9 (CH), 118.7 (CH), 123.2 (CIV), 130.1 (CIV), 131.0 (CH), 146.6 (C5), 167.5 (CO), 169.6 (2CO). ESI-MS: m/z = 282 (M + H)+. Methyl 5-Fluoro-2-(1,3-Dimethoxy-1,3-dioxopropan-2-yl)benzoate (20). A solution of 19 (1.00 g, 3.6 mmol) in aqueous 6.0 M HCl (5.0 mL) was cooled at 0−5 °C. A solution of sodium nitrite (0.37 g, 5.4 mmol) in water (3.0 mL) was added dropwise while the temperature was maintained below 5 °C. After 10 min of stirring, a pink solution of the diazonium salt was obtained. Tetrafluoroboric acid (1.1 mL, 10.8 mmol) was added dropwise and the solution was stirred for 1 h at 5 °C. The formed precipitate was filtered and dried for 2 h at 50 °C in an oven. The residual powder was then heated at 80 °C for several minutes until the end of gas evolvement. The residue was dissolved in ethyl acetate and extracted several times with 1.0 M NaHCO3. The organic layer was dried over Na2SO4, and after removal of the volatiles in vacuo, the crude residue was purified by column chromatography with petroleum ether/ethyl acetate (70/30 v/v) as eluent to give a yellow oil (42%). 1H NMR (300 MHz, acetone-d6): δ = 3.61 (s, 6H, 2OCH3), 3.75 (s, 3H, OCH3), 5.54 (s, 1H, CH), 7.30 (td, 1H, H4, 3JH4−H3 = 3JH4−F = 8.3 Hz, 4JH4−H6 = 2.5 Hz), 7.36 (dd, 1H, H3, 3JH3−H4 = 8.3 Hz, 4JH3−F = 5.6 Hz), 7.58 (dd, 1H, H6, 3JH6−F = 9.7 Hz, 4JH6−H4 = 2.5 Hz). 13C NMR (75 MHz, acetone-d6): δ = 53.0 (OCH3), 53.1 (2OCH3), 55.0 (CH), 118.2 (d, CHAr, 2JC−F = 24.0 Hz), 120.2 (CH, 2JC−F = 21.3 Hz), 131.5 (CIV), 132.8 (CIV), 133.4 (C3, 3JC−F = 8.2 Hz), 162.6 (C5, 1JC−F = 246.0 Hz), 166.9 (CO), 169.3 (2CO); ESI-MS: m/z = 285 (M + H)+. Methyl 5-Chloro-2-(1,3-dimethoxy-1,3-dioxopropan-2-yl)benzoate (21). A solution of 19 (1.0 g, 3.6 mmol) in concentrate hydrochloric acid (8.0 mL) was cooled at 0−5 °C. A solution of sodium nitrite (0.25 g, 3.6 mmol) in water (3.0 mL) was added dropwise while the temperature was maintained below 5 °C. After 10 min of stirring, a pink solution of the diazonium salt was obtained. A cold solution of CuCl (0.5 g, 5.1 mmol) in concentrated HCl acid solution was added dropwise to the diazonium mixture while the temperature was maintained below 5 °C. After temperature rising and 12 h of stirring, water (10.0 mL) was added and the solution was extracted several times with ethyl acetate. Organic layers were dried over Na2SO4, and after removal of the volatiles in vacuo, the crude residue was purified by column chromatography with petroleum ether/ethyl acetate (70/30 v/v) as eluent to give a yellow oil (48%). 1 H NMR (300 MHz, CDCl3): δ = 3.80 (s, 6H, 2OCH3), 3.92 (s, 3H, OCH3), 5.76 (s, 1H, CH), 7.40 (d, 1H, H3, 3JH3−H4 = 8,3 Hz), 7.54 (dd, 1H, H4, 3JH4−H3 = 8.3 Hz, 4JH4−H6 = 1.7 Hz), 8.03 (d, 1H, H6, 4 JH6−H4 = 1.7 Hz). 13C NMR (75 MHz, acetone-d6): δ = 53.0 (OCH3), 53.2 (2OCH3), 54.7 (CH), 130.5 (CH), 131.9 (CIV), 132.8 (2CH), 4649



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133.2 (CIV), 133.5 (CIV), 166.1 (CO), 168.5 (2CO). ESI-MS: m/z = 301 and 303 (M + H)+. Methyl 5-Bromo-2-(1,3-dimethoxy-1,3-dioxopropan-2-yl)benzoate (22). The process followed in this step is the same as described for 21, except hydrobromic acid was used instead of hydrochloric acid, giving a white solid (61%). 1H NMR (300 MHz, CDCl3): δ = 3.80 (s, 6H, 2OCH3), 3.92 (s, 3H, OCH3), 5.74 (s, 1H, CH), 7.34 (d, 1H, H3, 3JH3−H4 = 8.3 Hz), 7.69 (dd, 1H, H4, 3JH4−H3 = 8.3 Hz, 4JH4−H6 = 1.5 Hz), 8.18 (d, 1H, H6, 4JH6−H4 = 1.5 Hz). 13C NMR (75 MHz, CDCl3): δ = 52.7 (OCH3), 53.0 (2OCH3), 54.2 (CH), 122.2 (C5), 131.0 (CIV), 131.9 (CH), 133.3 (CIV), 133.9 (CH), 135.5 (CH), 166.1 (CO), 168.5 (2CO); ESI-MS: m/z = 345 and 347 (M + H)+. Methyl 5-Cyano-2-(1,3-dimethoxy-1,3-dioxopropan-2-yl)benzoate (23). A solution of aniline 19 (2.07 g, 7.4 mmol) and sodium nitrite (0.5 g, 7.4 mmol) in 90% nitric acid (5.2 mL) and water (2.6 mL) was cooled at 0 °C and stirred for 20 min. It was added to icy water (50 mL), filtered, and kept in an ice bath. CuCN (0.80 g, 8.84 mmol) and KCN (1.73 g, 26.5 mmol) were dissolved in water (30 mL) and heated to 62 °C. The cold diazonium solution was added dropwise to the cyanide solution while the temperature was kept between 62 and 70 °C and the reaction mixture was always kept basic by addition of saturated aqueous NaHCO3. The stirred reaction mixture was maintained at 70 °C for another 30 min and then was allowed to cool to room temperature. The aqueous layer was extracted with EtOAc (3 × 70 mL), the combined organic extracts were dried over Na2SO4, and the solvent was evaporated in vacuo. Purification by flash chromatography with petroleum ether/ethyl acetate (70/30, % v/ v) as eluent gave 23 as yellow crystals (51%), mp 83 °C. 1H NMR (300 MHz, CDCl3): δ = 3.78 (s, 6H, 2OCH3), 3.93 (s, 3H, OCH3), 5.81 (s, 1H, CH), 7.59 (d, 1H, H3, 3JH3−H4 = 8,2 Hz), 7.82 (dd, 1H, H4, 3JH4−H3 = 8.2 Hz, 4JH4−H6 = 1.8 Hz), 8.32 (d, 1H, H6, 4JH6−H4 = 1.8 Hz). 13C NMR (75 MHz, CDCl3): δ = 52.5 (OCH3), 53.1 (2OCH3), 54.5 (CH), 112.5 (C5), 117.4 (CN), 130.6 (CIV), 131.5 (CH), 134.6 (CH), 135.3 (CH), 139.0 (CIV), 165.5 (CO), 167.9 (2CO). ESI-MS: m/z = 292 (M + H)+. Methyl 5-Acetamido-2-(1,3-dimethoxy-1,3-dioxopropan-2-yl)benzoate (24). Acetyl chloride (1.1 mL, 16.0 mmol) was added to a solution of 19 (3.0 g, 10.7 mmol) in dichloromethane (50 mL). After being stirred for 4 h at room temperature, the solution was washed with aqueous 1.0 M HCl. The organic phase was dried over Na2SO4. The solvent was removed under reduced pressure to give an oily residue, which was triturated in ether. After filtration, a white powder was obtained (96%), mp 81−83 °C.; 1H NMR (300 MHz, DMSO-d6): δ = 2.06 (s, 3H, CH3), 3.68 (s, 6H, 2OCH3), 3.80 (s, 3H, OCH3), 5.49 (s, 1H, CH), 7.26 (d, 1H, H3, 3JH3−H4 = 8.5 Hz), 7.80 (dd, 1H, H4, 3 JH4−H3 = 8.5 Hz, 4JH4−H6 = 1.9 Hz), 8.20 (d, 1H, H6, 4JH6−H4 = 1.9 Hz), 10.24 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 23.9 (CH3), 52.3 (OCH3), 52.6 (2OCH3), 54.3 (CH), 120.6 (CH), 122.6 (CH), 128.0 (CIV), 129.6 (CIV), 130.7 (CH), 139.1 (C5), 166.6 (CO), 168.4 (2CO), 168.7 (CO). ESI-MS: m/z = 324 (M + H)+. Methyl 5-Benzamido-2-(1,3-dimethoxy-1,3-dioxopropan-2-yl)benzoate (25). The process followed in this step is the same as described for 24 except benzoyl chloride was used (instead of acetyl chloride) to give a white powder (67%), mp 135−137 °C. 1H NMR (300 MHz, DMSO-d6): δ = 3.71 (s, 6H, 2OCH3), 3.87 (s, 3H, OCH3), 5.60 (s, 1H, CH), 7.35−7.37 (m, 1H, HAr), 7.58−7.61 (m, 3H, HAr), 8.02−8.06 (m, 3H, HAr), 8.49 (s, 1H, HAr), 10.59 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 52.3 (OCH3), 52.6 (2OCH3), 54.4 (CH), 122.0 (CH), 123.9 (CH), 127.7 (2CH), 128.4 (2CH), 128.7 (CIV), 129.6 (CIV), 130.7 (CH), 131.9 (CH), 134.4 (CIV), 139.1 (C5), 165.8 (CO), 166.7 (CO), 168.5 (2CO). ESI-MS: m/z = 386 (M + H)+. Methyl 2-(1,3-Dimethoxy-1,3-dioxopropan-2-yl)-5-picolinamidobenzoate (26). Thionyl chloride (1.1 mL, 15.7 mmol) was added dropwise to a cooled solution of 2-picolinic acid (1.05 g, 8.5 mmol) in a mixture of ethyl acetate (25 mL) and N,N-dimethylformamide (DMF; 5 mL). After 1 h of reflux, the volatiles were removed in vacuo and the crude acid chloride was dissolved in a mixture of ethyl acetate (25 mL) and DMF (5 mL). Diisopropylethylamine (3.5 mL, 21.4

mmol) and 19 (2.0 g, 7.1 mmol) were added. After 12 h of stirring at room temperature, the solution was washed several times with 1.0 M NaHCO3 and 1.0 M HCl. The organic phase was dried over Na2SO4 and concentrated in vacuo. Organic residues were triturated with ether, and insoluble materials were filtered to give a pink solid (81%), mp 153−155 °C. 1H NMR (300 MHz, CDCl3): δ = 3.81 (s, 6H, 2OCH3), 3.94 (s, 3H, OCH3), 5.77 (s, 1H, CH), 7.47 (d, 1H, H3, 3JH3−H4 = 8.5 Hz), 7.55 (ddd, 1H, HAr, 3JH−H = 7.7 Hz, 3JH−H = 4.8 Hz, 4JH−H = 1.2 Hz), 7.97 (td, 1H, HAr, 3JH−H = 7.7 Hz, 4JH−H = 1.6 Hz), 8.06 (dd, 1H, H4, 3JH4−H3 = 8.5 Hz, 4JH4−H6 = 2.4 Hz), 8.34 (d, 1H, H2′, 3JH2′−H3′ = 7.7 Hz), 8,45 (d, 1H, H6, 4JH6−H4 = 2.4 Hz), 8.66 (d, 1H, H5′, 3JH5′−H4′ = 4.7 Hz), 10.22 (s, 1H, NH). 13C NMR (75 MHz, CDCl3): δ = 52.9 (OCH3), 53.2 (2OCH3), 54.9 (CH), 122.7 (CH), 123.1 (CH), 124.7 (CH), 127.6 (CH), 129.5 (CIV), 130.2 (CIV), 131.1 (CH), 138.7 (CH), 138.8 (C5), 148.9 (CH), 150.0 (C1′), 163.4 (CO), 167.2 (CO), 168.9 (2CO). ESI-MS: m/z = 387 (M + H)+. Methyl 2-(1,3-Dimethoxy-1,3-dioxopropan-2-yl)-5(phenylacetamido)benzoate (27). The process followed in this step is the same as described for 24 except phenylacetyl chloride was used (instead of acetyl chloride) to give a white powder (76%), mp 126− 128 °C. 1H NMR (300 MHz, DMSO-d6): δ = 3.36 (s, 6H, 2OCH3), 3.68 (s, 3H, OCH3), 3.80 (s, 2H, OCH2), 5.51 (s, 1H, CH), 7.27 (d, 1H, H3, 3JH3−H4 = 8.2 Hz), 7.33 (s, 5H, HAr), 7.82 (dd, 1H, H4, 3JH4−H3 = 8.5 Hz, 4JH4−H6 = 1.9 Hz), 8.24 (d, 1H, H6, 4JH6−H4 = 1.9 Hz), 10.49 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 43.7 (CH2), 52.8 (OCH3), 53.1 (2OCH3), 54.8 (CH), 121.2 (CH), 123.2 (CH), 127.1 (CH), 128.8 (CIV), 128.8 (2CH), 129.6 (2CH), 130.2 (CIV), 131.2 (CH), 136.1 (CIV), 139.5 (C5), 167.1 (CO), 168.9 (2CO), 170.1 (CO). ESI-MS: m/z = 400 (M + H)+. Methyl 2-(1,3-Dimethoxy-1,3-dioxopropan-2-yl)-5-[2-(thiophen2-yl)acetamido]benzoate (28). The process followed in this step is the same as described for 24 except 2-thienylacetic acid was used instead of picolinic acid to give a white powder (33%), mp 135−137 °C. 1H NMR (300 MHz, CDCl3): δ = 3.78 (s, 6H, 2OCH3), 3.87 (s, 3H, OCH3), 3.95 (s, 2H, CH2), 5.68 (s, 1H, CH), 7.05−7.08 (m, 2H, Hthiophen), 7.32−7.34 (m, 2H, Hthiophen and H3), 7.70 (dd, 1H, H4, 3 JH4−H3 = 8.5 Hz, 4JH4−H6 = 1.9 Hz), 8.02 (d, 1H, H6, 4JH6−H4 = 1.9 Hz), 9.82 (s, 1H, NH). 13C NMR (75 MHz, CDCl3): δ = 38.2 (CH2), 52.5 (OCH3), 53.0 (2OCH3), 54.5 (CH), 122.2 (CH), 123.8 (CH), 125.7 (CH), 127.4 (CH), 127.5 (CH), 129.7 (CIV), 129.8 (CIV), 130.7 (CH), 135.5 (CIV), 137.8 (C5), 167.0 (CO), 168.5 (CO), 169.3 (2CO). ESI-MS: m/z = 406 (M + H)+. Synthesis of Malonamide Precursors 29−42. Methyl 2-{1[(Benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2-yl}-5-nitrobenzoate (29). A solution of intermediate 18 (0.31 g, 1.0 mmol) and Obenzylhydroxylamine (0.15 g, 1.2 mmol) in toluene (15.0 mL) was refluxed for 15 h in a Dean−Stark apparatus. After cooling, the solution was concentrated in vacuo. The residue was dissolved in EtOAc. The organic layer was washed with 2.0 M HCl and dried over Na2SO4. After concentration in vacuo and column chromatography of the residue (eluent petroleum ether/EtOAc 70/30), 29 was obtained as an orange oil, yield 50%. 1H NMR (300 MHz, CDCl3): δ = 3.65 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 4.83 (s, 2H, OCH2), 5.22 (s, 1H, CH), 7.26 (m, 5H, HAr), 7.75 (d, 1H, H3, 3JH3−H4 = 8.6 Hz), 8.32 (dd, 1H, H4, 3JH4−H3 = 8.6 Hz, 4JH4−H6 = 2.2 Hz), 8.75 (dd, 1H, H6, 3JH6−H5 = 8.6 Hz, 4JH6−H4 = 2.2 Hz), 9.62 (s, 1H, NH). 13C NMR (75 MHz, CDCl3): δ = 52.9 (CH), 53.2 (2OCH3), 78.8 (OCH2), 125.8 (CH), 126.8 (CH), 128.5 (2CH), 128.7 (CH), 129.3 (2CH), 130.2 (CIV), 133.7 (CH), 135.0 (CIV), 141.3 (CIV), 147.2 (CIV, C5), 164.1 (CO), 166.2 (CO), 168.4 (CO). ESI-MS: m/z = 403 (M + H)+. Methyl 2-{1-[(Benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2yl}-5-fluorobenzoate (30). White powder (30%), mp 118−119 °C. 1 H NMR (300 MHz, DMSO-d6): δ = 3.65 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 4.81 (s, 2H, OCH2), 5.15 (s, 1H, CH), 7.36 (m, 5H, HAr), 7.49−7.52 (m, 2H, HAr), 7.64−7.67 (m, 1H, HAr), 11.51 (s, 1H, NH). 13 C NMR (75 MHz, DMSO-d6): δ = 52.2 (CH), 52.9 (OCH3), 53.1 (OCH3), 77.3 (OCH2), 117.3 (d, CH, 2JC−F = 23.5 Hz), 119.8 (d, CH, 2 JC−F = 20.7 Hz), 128.8 (2CH), 128.9 (CH), 129.5 (2CH), 131.4 (d, C2, 4JC−F = 3.3 Hz), 131.9 (d, C1, 3JC−F = 7.1 Hz), 132.3 (d, C3, 3JC−F = 8.2 Hz), 136.1 (CIV), 161.4 (d, C5, 1JC−F = 244.4 Hz), 164.6 (CO), 4650



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166.3 (d, CO, 4JC−F = 2.7 Hz), 169.1 (CO). ESI-MS: m/z = 376 (M + H)+. Methyl 2-{1-[(Benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2yl}-5-chlorobenzoate (31). White powder (35%), mp 112−113 °C. 1 H NMR (300 MHz, acetone-d6): δ = 3.56 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 4.76 (s, 2H, OCH2), 5.26 (s, 1H, CH), 7.22 (m, 5H, HAr), 7.53 (m, 2H, H4 and H3), 7.78 (s, 1H, H6), 10.40 (s, 1H, NH). 13 C NMR (75 MHz, acetone-d6): δ = 52.0 (OCH3), 52.1 (OCH3), 52.2 (CH), 77.4 (OCH2), 128.3 (2CH), 128.4 (CH), 129.2 (2CH), 130.0 (CH), 131.5 (CIV), 132.0 (CH), 132.3 (CH), 133.2 (CIV), 134.0 (CIV), 135.9 (CIV), 164.5 (CO), 166.0 (CO), 168.5 (CO). ESI-MS: m/z = 392 and 394 (M + H)+. Methyl 2-{1-[(Benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2yl}-5-bromobenzoate (32). White powder (35%), mp 99−101 °C. 1 H NMR (300 MHz, CDCl3): δ = 3.64 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 4.81 (s, 2H, OCH2), 5.12 (s, 1H, CH), 7.24 (m, 5H, HAr), 7.45 (d, 1H, HAr, 3JH−H = 8.6 Hz), 7.62 (d, 1H, HAr, 3JH−H = 8.6 Hz), 8.02 (m, 1H, H6), 9.55 (s, 1H, NH). 13C NMR (75 MHz, CDCl3): δ = 52.3 (CH), 52.8 (OCH3), 53.0 (OCH3), 78.0 (OCH2), 122.1 (CIV), 128.5 (2CH), 128.7 (CH), 129.3 (3CH), 130.39 (CIV), 130.40 (CIV), 133.6 (CH), 135.0 (CIV), 135.6 (CH), 164.8 (CO), 167.0 (CO), 168.9 (CO). ESI-MS: m/z = 436 and 438 (M + H)+. Methyl 2-{1-[(Benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2yl}-5-trifluoromethylbenzoate (33). Brown oil (33%). 1H NMR (300 MHz, CDCl3): δ = 3.70 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 4.87 (s, 2H, CH2), 5.30 (s, 1H, CH), 7.29 (m, 5H, HAr), 7.75 (d, 1H, HAr, 3JH−H = 8.5 Hz), 7.79 (d, 1H, HAr, 3JH−H = 8.5 Hz), 8.22 (s, 1H, H6), 9.82 (s, 1H, NH). 13C NMR (75 MHz, CDCl3): δ = 52.8 (CH), 52.9 (OMe), 53.0 (OMe), 78.0 (OCH2), 123.5 (q, CF3, 1JC−F = 270.7 Hz), 127.80 (q, CH, 3JC−F = 3.8 Hz), 128.5 (2CH), 128.7 (CH), 129.0 (q, CH, 3JC−F = 3.8 Hz), 129.3 (2CH), 129.5 (CIV), 130.4 (q, C5, 2JC−F = 33.0 Hz), 132.9 (C3), 135.0 (CIV), 138.5 (CIV), 164.6 (CO), 166.9 (CO), 168.7 (CO). ESI-MS: m/z = 426 (M + H)+ Methyl 2-{1-[(Benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2yl}-5-cyanobenzoate (34). White powder (37%), mp 142−146 °C. 1 H NMR (300 MHz, acetone-d6): δ = 3.56 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 4.76 (s, 2H, OCH2), 5.34 (s, 1H, CH), 7.18−7.27 (m, 5H, HAr), 7.69 (d, 1H, H3, 3JH3−H4 = 8.2 Hz), 7.85 (dd, 1H, H4, 3JH4−H3 = 8.2 Hz, 4JH4−H6 = 1.8 Hz), 8.12 (d, 1H, H6, 4JH6−H4 = 1.8 Hz); 10.46 (s, 1H, NH). 13C NMR (75 MHz, acetone-d6): δ = 52.9 (OCH3), 53.0 (OCH3), 53.5 (CH), 78.2 (OCH2), 112.7 (C5), 118.2 (CN), 129.0 (2CH), 129.2 (CH), 129.90 (CH), 129.94 (2CH), 131.8 (CIV), 132.6 (CH), 134.7 (CH), 135.9 (CH), 136.5 (CIV), 140.6 (CIV), 164.8 (CO), 166.5 (CO), 168.9 (CO). ESI-MS: m/z = 383 (M + H)+. Methyl 5-Acetamido-2-{1-[(benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2-yl}benzoate (35). White powder (28%), mp 60−62 °C. 1 H NMR (300 MHz, DMSO-d6): δ = 2.06 (s, 3H, CH3), 3.63 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 4.81 (s, 2H, OCH2), 5.11 (s, 1H, CH), 7.36 (s, 6H, H3 and HAr), 7.73 (dd, 1H, H4, 3JH4−H3 = 8.5 Hz, 4JH4−H6 = 1.9 Hz), 8.20 (d, 1H, H6, 4JH6−H4 = 1.9 Hz), 10.20 (s, 1H, NH), 11.46 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 23.9 (CH3), 52.1 (OCH3), 52.24 (OCH3), 52.25 (CH), 76.8 (OCH2), 120.4 (CH), 122.5 (CH), 128.27 (2CH), 128.32 (CH), 128.9 (2CH), 129.0 (CIV), 129.6 (CIV), 129.9 (CH), 135.7 (CIV), 138.7 (C5), 164.4 (CO), 166.7 (CO), 168.7 (2CO). ESI-MS: m/z = 415 (M + H)+. Methyl 5-Benzamido-2-{1-[(benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2-yl}benzoate (36). Light yellow powder (23%), mp 85− 94 °C. 1H NMR (300 MHz, DMSO-d6): δ = 3.66 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 4.84 (s, 2H, OCH2), 5.17 (s, 1H, CH), 7.38 (m, 6H, HAr), 7.56 (m, 3H, HAr), 7.99 (m, 3H, HAr), 8.43 (s, 1H, H6), 10.52 (s, 1H, NH), 11.50 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 52.77 (2OCH3), 52.78 (CH), 77.3 (OCH2), 122.3 (CH), 124.4 (CH), 128.2 (2CH), 128.8 (2CH), 128.9 (3CH), 129.5 (2CH), 130.0 (CIV), 130.2 (CIV), 130.4 (CH), 132.3 (CH), 134.9 (CIV), 136.2 (CIV), 139.2 (C5), 164.9 (CO), 166.2 (CO), 167.2 (CO), 169.4 (CO). ESI-MS: m/ z = 491 (M + H)+. Methyl 2-{1-[(Benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2yl}-5-picolinamidobenzoate (37). White powder (20%), mp 207− 208 °C. 1H NMR (300 MHz, CDCl3): δ = 3.75 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.90 (s, 2H, OCH2), 5.27 (s, 1H, CH), 7.33 (s, 5H,

HAr), 7.60 (dd, 1H, HAr, 3JH−H = 6.1 Hz, 3JH−H = 6.7 Hz), 7.71 (d, 1H, HAr, 3JH−H = 7.9 Hz), 7.94−8.04 (m, 2H, HAr), 8.38 (d, 1H, HAr, 3JH−H = 7.9 Hz), 8.55 (s, 1H, HAr), 8.67 (d, 1H, H6, 4JH6−H4 = 4.4 Hz), 9.65 (s, 1H, NH), 10.33 (s, 1H, NH). 13C NMR (75 MHz, CDCl3): δ = 52.7 (CH), 52.8 (2OCH3), 77.3 (OCH2), 122.4 (CH), 123.0 (CHA), 124.7 (CH), 127.6 (CH), 128.78 (2CH), 128.84 (CH), 129.5 (2CH), 130.1 (CIV), 130.3 (CH), 130.5 (CIV), 136.2 (CIV), 138.4 (C5), 138.7 (CH), 149.0 (CH), 150.1 (C1′), 163.4 (CO), 164.9 (CO), 167.2 (CO), 169.4 (CO). ESI-MS: m/z = 478 (M + H)+. Methyl 2-{1-[(Benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2yl}-5-(2-phenylacetamido)benzoate (38). Light yellow powder (25%), mp 65−70 °C. 1H NMR (300 MHz, DMSO-d6): δ = 3.63 (s, 3H, OCH3), 3.66 (s, 2H, CH2), 3.79 (s, 3H, OCH3), 4.81 (s, 2H, OCH2), 5.13 (s, 1H, CH), 7.33−7.39 (m, 11H, H3 and 10HAr), 7.76 (dd, 1H, H4, 3JH4−H3 = 8.6 Hz, 4JH4−H6 = 2.3 Hz), 8.23 (d, 1H, H6, 4 JH6−H4 = 2.3 Hz), 10.46 (s, 1H, NH), 11.47 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 43.7 (CH2), 52.71 (CH), 52.73 (2OCH3), 77.3 (OCH2), 121.0 (CH), 123.1 (CH), 127.1 (CH), 128.7 (2CH), 128.8 (3CH), 129.4 (2CH), 129.6 (2CH), 129.8 (CIV), 130.1 (CIV), 130.5 (CH), 136.1 (CIV), 136.2 (CIV), 139.1 (C5), 164.9 (CO), 167.1 (CO), 169.4 (CO), 170.0 (CO). ESI-MS: m/z = 491 (M + H)+. Methyl 2-{1-[(Benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2yl}-5-[2-(thiophen-2-yl)acetamido]benzoate (39). Yellow powder (27%), mp 67−70 °C. 1H NMR (300 MHz, CDCl3): δ = 3.68 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.93 (s, 2H, CH2), 4.88 (s, 2H, OCH2), 5.12 (s, 1H, CH), 7.02−7.03 (m, 2H, HAr), 7.31 (m, 5H, HAr), 7.41−7.43 (m, 2H, HAr), 7.93 (s, 1H, HAr), 8.13 (s, 1H, HAr), 9.82 (s, 1H, NH), 10.25 (s, 1H, NH). 13C NMR (75 MHz, CDCl3): δ = 38.3 (CH2), 52.6 (CH), 52.7 (OCH3), 52.9 (OCH3), 78.1 (OCH2), 122.0 (CH), 123.8 (CH), 125.9 (CH), 127.5 (CH), 127.6 (CH), 128.6 (CH), 128.7 (CH), 129.2 (CIV), 129.3 (3CH), 130.1 (CIV), 132.5 (CH), 135.0 (CIV), 135.5 (CIV), 137.6 (CIV), 165.5 (CO), 167.8 (CO), 168.3 (CO), 169.5 (CO). ESI-MS: m/z = 497 (M + H)+. Methyl 2-{1-[(Benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2yl}-4-fluorobenzoate (40). Four hours of reflux; white solid (20%), mp 104−107 °C. 1H NMR (300 MHz, CDCl3): δ = 3.73 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 4.89 (s, 2H, OCH2), 5.33 (s, 1H, CH), 7.10 (td, 1H, H5, 3JH5−F = 3JH5−H6 = 8.7 Hz, 4JH5−H3 = 2.5 Hz), 7.41 (dd, 1H, H3, 3JH3−F = 9.6 Hz, 4JH3−H5 = 1.8 Hz), 8.00 (dd, 1H, H6, 3 JH6−H5 = 8.6 Hz, 4JH6−F = 6.0 Hz), 9.74 (br s, 1H, NH). 13C NMR (75 MHz, CDCl3): δ = 52.4 (OCH3), 52.6 (OCH3), 53.0 (CH), 78.0 (s, 2H, OCH2), 115.2 (d, CH, 2JC−F = 21.4 Hz), 119.1 (d, CH, 2JC−F = 23.3 Hz), 125.0 (d, C1, 4JC−F = 3.2 Hz), 128.5 (2CH), 128.6 (CH), 129.2 (2CH), 133.3 (d, C6, 3JC−F = 8.9 Hz), 135.0 (CIV), 137.8 (d, C2, 3 JC−F = 8.9 Hz), 164.9 (CO), 164.7 (C4, 1JC−F = 253.5 Hz), 167.2 (CO), 168.8 (CO). ESI-MS: m/z = 376 (M + H)+. Methyl 2-{1-[(Benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2yl}-6-fluorobenzoate (41). Six hours of reflux; white solid (38%), mp 156−160 °C. 1H NMR (300 MHz, DMSO-d6): δ = 3.68 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 4.73 (s, 1H, CH), 4.83 (s, 2H, OCH2), 7.31−7.37 (m, 7H, HAr), 7.60 (td, 1H, H4, 3JH4−H5 = 3JH4−H3= 8.0 Hz, 4 JH4−F = 5.8 Hz), 11.6 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 52.2 (OCH3), 52.7 (OCH3), 52.8 (CH), 77.0 (OCH2), 115.8 (d, C5, 2 JC−F = 21.8 Hz), 120.8 (d, C1, 2JC−F = 15.5 Hz), 125.6 (d, C3, 4JC−F = 3.0 Hz), 128.4 (2CH), 128.5 (CH), 129.1 (2CH), 132.6 (d, C4, 3JC−F = 9.2 Hz), 134.6 (d, C2, 3JC−F = 2.2 Hz), 135.7 (CIV), 159.5 (d, C6, 1 JC−F = 250.6 Hz), 163.4 (CO), 164.6 (CO), 168.2 (CO). ESI-MS: m/ z = 376 (M + H)+. Methyl 2-{1-[(Benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2yl}-4-nitrobenzoate (42). White solid (20%), mp 133−137 °C. 1H NMR (300 MHz, CDCl3): δ = 3.71 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 4.90 (s, 2H, OCH2), 5.20 (s, 1H, CH), 7.34 (m, 5H, HAr), 8.13 (d, 1H, H5, 3JH5−H6 = 8.6 Hz), 8.23 (d, 1H, H6, 3JH6−H5 = 8.6 Hz), 8.41 (s, 1H, H3), 9.90 (s, 1H, NH). 13C NMR (75 MHz, CDCl3): δ = 53.17 (CH), 53.20 (2OCH3), 78.2 (OCH2), 122.9 (CH), 127.1 (CH), 128.5 (2CH), 128.7 (CH), 129.3 (2CH), 131.9 (CH), 134.4 (CIV), 134.9 (CIV), 136.7 (CIV), 149.7 (CIV, C4), 164.2 (CO), 166.4 (CO), 168.6 (CO). ESI-MS: m/z = 403 (M + H)+. Cyclization of N-Benzyloxymalonamides 4 and 29−42. Methyl 2-(Benzyloxy)-7-methoxy-1,3-dioxo-1,2,3,4-tetrahydroiso4651



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temperature; white powder (90%), 100% enol form, mp 169−172 °C. H NMR (300 MHz, CDCl3): δ = 4.14 (s, 3H, OCH3), 5.28 (s, 2H, OCH2), 7.42−7.44 (m, 3H, HAr), 7.62−7.64 (m, 2H, HAr), 7.82 (dd, 1H, H6, 3JH6−H5 = 8.7 Hz, 4JH6−H8 = 1.4 Hz), 8.69 (s, 1H, H8), 16.05 (s, 1H, OH). 13C NMR (75 MHz, CDCl3): δ = 53.7 (OCH3), 79.4 (OCH2), 84.5 (C4), 108.0 (C7), 118.2 (CN), 125.4 (CH), 128.7 (2CH), 129.7 (CH), 130.2 (2CH), 133.17 (CIV), 133.28 (CH), 135.4 (CH), 136.4 (CIV), 157.4 (CO), 164.5 (CO), 173.2 (CO). ESI-MS: m/z = 351 (M + H)+. Methyl 7-Acetamido-2-(benzyloxy)-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylate (50). Yellow powder (70%), 100% keto form, mp 197−199 °C. 1H NMR (300 MHz, DMSO-d6): δ = 2.09 (s, 3H, CH3), 3.71 (s, 3H, OCH3), 5.05 (s, 2H, OCH2), 5.37 (s, 1H, H4), 7.36−7.42 (m, 4H, HAr), 7.54−7.57 (m, 2H, HAr), 7.89 (d, 1H, H3, 3 JH3−H4 = 8.6 Hz), 8.40 (d, 1H, H6, 4JH6−H4 = 1.9 Hz), 11.34 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 24.0 (CH3), 53.5 (OCH3), 54.0 (C4), 77.5 (OCH2), 117.7 (CH), 124.7 (CH), 125.0 (CIV), 126.3 (CIV), 127.9 (CH), 128.4 (2CH), 129.0 (CH), 129.5 (2CH), 134.2 (CIV), 139.8 (C5), 160.4 (CO), 163.0 (CO), 167.3 (CO), 168.8 (CO). ESI-MS: m/z = 383 (M + H)+. Methyl 7-Benzamido-2-(benzyloxy)-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylate (51). Orange powder (83%), 100% keto form, mp 185−187 °C. 1H NMR (300 MHz, DMSO-d6): δ = 3.86 (s, 3H, OCH3), 5.07 (s, 2H, OCH2), 5.42 (s, 1H, H4), 7.29−7.42 (m, 3H, HAr), 7.57−7.75 (m, 5H, HAr), 7.91−8.02 (m, 3H, HAr), 8.42 (d, 1H, HAr, 3JH−H = 8.0 Hz), 8.62 (d, 1H, H8, 4JH8−H7 = 2.5 Hz), 10.41 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 54.0 (OCH3), 54.5 (C4), 78.0 (OCH2), 119.6 (CH), 125.5 (CIV), 126.5 (CH), 127.5 (CIV), 128.2 (2CH), 128.4 (CH), 128.9 (2CH), 129.0 (2CH), 129.4 (CH), 130.0 (2CH), 132.4 (CH), 134.7 (CIV), 134.8 (CIV), 140.2 (C7), 160.9 (CO), 163.5 (CO), 166.3 (CO), 167.8 (CO). ESI-MS: m/ z = 445 (M + H)+. Methyl 2-(Benzyloxy)-1,3-dioxo-7-picolinamido-1,2,3,4-tetrahydroisoquinoline-4-carboxylate (52). Beige powder (51%), 100% keto form, mp 177−180 °C. 1H NMR (300 MHz, DMSO-d6): δ = 4.13 (s, 3H, OCH3), 5.30 (s, 2H, OCH2), 5.31 (s, 1H, H4), 7.42−7.43 (m, 4H, HAr), 7.56−7.65 (m, 3H, HAr), 7.98 (t, 1H, HAr, 3JH−H = 7.6 Hz), 8.36 (d, 1H, HAr, 3JH−H = 7.7 Hz), 8.41−8.49 (m, 2H, HAr), 8.68 (s, 1H, H8), 10.32 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 54.1 (OCH3), 54.5 (C4), 78.0 (OCH2), 119.9 (CH), 123.1 (CH), 125.5 (CIV), 126.9 (CH), 127.7 (CH), 127.8 (CIV), 128.3 (CH), 130.0 (2CH), 129.4 (CH), 130.1 (2CH), 134.7 (CIV), 138.7 (CH), 139.5 (CIV), 149.0 (CH), 150.0 (C1′), 160.8 (CO), 163.5 (CO), 163.6 (CO), 167.7 (CO). ESI-MS: m/z = 446 (M + H)+. Methyl 2-(Benzyloxy)-7-phenylacetamido-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylate (53). White powder (80%), 100% keto form, mp 187−196 °C. 1H NMR (300 MHz, DMSOd6): δ = 3.70 (s, 3H, OCH3), 3.99 (s, 2H, CH2), 5.05 (s, 2H, OCH2), 5.38 (s, 1H, H4), 7.26−7.35 (m, 8H, HAr), 7.54−7.56 (m, 3H, HAr), 7.92 (d, 1H, HAr, 3JH−H = 8.2 Hz), 8.42 (s, 1H, H8), 10.61 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 43.8 (CH2), 54.0 (OCH3), 54.5 (C4), 77.9 (OCH2), 118.4 (CH), 125.3 (CH), 125.6 (CIV), 127.1 (CH), 128.5 (CH), 128.8 (2CH), 128.9 (2CH), 129.6 (3CH), 130.0 (2CH), 134.7 (CIV), 136.1 (CIV), 136.3 (CIV), 140.1 (C5), 160.8 (CO), 163.5 (CO), 167.7 (CO), 170.1 (CO). ESI-MS: m/z = 459 (M + H)+. Methyl 2-(Benzyloxy)-1,3-dioxo-7-[2-(thiophen-2-yl)acetamido]1,2,3,4-tetrahydroisoquinoline-4-carboxylate (54). White powder (92%), 100% keto form, mp 179−181 °C. 1H NMR (300 MHz, DMSO-d6): δ = 3.71 (s, 3H, OCH3), 3.93 (s, 2H, CH2), 5.05 (s, 2H, OCH2), 5.39 (s, 1H, H4), 6.99−7.01 (m, 2H, HAr), 7.41 (m, 5H, HAr), 7.55−7.56 (m, 2H, HAr), 7.92 (d, 1H, HAr, 3JH−H = 7.7 Hz), 8.42 (s, 1H, H8), 10.63 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 38.0 (CH2), 54.0 (OCH3), 54.5 (C4), 78.0 (OCH2), 118.4 (CH), 125.4 (CH), 125.6 (CIV), 125.7 (CH), 127.0 (CH), 127.2 (CH), 127.3 (CIV), 128.6 (CH), 128.9 (2CH), 129.4 (CH), 130.0 (2CH), 134.7 (CIV), 137.1 (CIV), 140.0 (CIV), 160.8 (CO), 163.4 (CO), 167.7 (CO), 169.1 (CO). ESI-MS: m/z = 465 (M + H)+. Methyl 2-(Benzyloxy)-6-fluoro-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylate (55). Ten minutes of stirring at room

quinoline-4-carboxylate (43). Intermediate 4 (1.0 mmol) was dissolved in a solution of methanol (10.0 mL) and 2.0 M KOH (10.0 mL). After 12 h of stirring, the solution was acidified with 2.0 M HCl and extracted three times with dichloromethane (20.0 mL). The combined organic extracts were dried over Na2SO4 and concentrated in vacuo. The residue was triturated in ether to afford a yellow powder (61%), 100% keto form, mp 115−117 °C. 1H NMR (300 MHz, DMSO-d6): δ = 3.70 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 5.05 (s, 2H, OCH2), 5.38 (s, 1H, H4), 7.37−7.43 (m, 6H, HAr), 7.55−7.56 (m, 2H, HAr). 13C NMR (75 MHz, DMSO-d6): δ = 54.5 (C4), 55.6 (OCH3), 55.8 (OCH3), 78.3 (OCH2), 112.0 (CH), 118.1 (CH), 126.1 (CIV), 127.1 (2CH), 127.6 (CH), 128.9 (2CH), 130.8 (CH), 133.2 (CIV), 137.1 (CIV), 159.5 (CO), 161.0 (CO), 164.0 (CO), 169.9 (CO). ESIMS: m/z = 356 (M + H)+. Methyl 2-(Benzyloxy)-3-hydroxy-7-nitro-1-oxo-1,2-dihydroisoquinoline-4-carboxylate (44). Yellow solid (89%), mp 190−196 °C, 100% enol form. 1H NMR (300 MHz, CDCl3): δ = 4.17 (s, 3H, OCH3), 5.31 (s, 2H, OCH2), 7.45 (m, 3H, HAr), 7.63 (m, 2H, HAr), 8.43 (dd, 1H, H6, 3JH6−H5 = 9.4 Hz, 4JH6−H8 = 2.0 Hz), 8.60 (d, 1H, H5, 3 JH5−H6 = 9.4 Hz), 9.26 (d, 1H, H8, 4JH8−H4 = 1.9 Hz). 13C NMR (75 MHz, CDCl3): δ = 53.7 (OCH3), 79.4 (OCH2), 84.5 (CIV), 121.2 (CIV), 124.6 (CH), 125.6 (CH), 127.4 (CH), 128.7 (2CH), 129.6 (CH), 130.1 (2CH), 133.0 (CIV), 138.0 (CIV), 144.0 (CIV, C7), 157.5 (CO), 164.8 (CO), 173.1 (CO). ESI-MS: m/z = 371 (M + H)+. Methyl 2-(Benzyloxy)-7-fluoro-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylate (45). Yellow powder (77%), 100% keto form, mp 140−142 °C. 1H NMR (300 MHz, DMSO-d6): δ = 3.72 (s, 3H, OCH3), 5.05 (s, 2H, OCH2), 5.18 (s, 1H, H4), 7.42−7.43 (m, 3H, HAr), 7.55−7.67 (m, 4H, HAr), 7.86 (d, 1H, H8, 3JH8−F = 8.2 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 54.1 (CH3), 54.3 (C4), 78.0 (OCH2), 114.9 (d, CHAr, 2JC−F = 24.0 Hz), 122.3 (d, CHAr, 2JC−F = 22.4 Hz), 127.5 (CIV), 128.9 (2CH), 129.2 (CIV), 129.5 (CH), 130.0 (2CH), 130.7 (d, C5, 3JC−F = 8.2 Hz), 134.6 (CIV), 160.1 (CO), 162.3 (d, C7, 1 JC−F = 245.7 Hz), 163.2 (CO), 167.5 (CO). ESI-MS: m/z = 344 (M + H)+. Methyl 2-(Benzyloxy)-7-chloro-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylate (46). White powder (69%), 100% keto form, mp 161−163 °C. 1H NMR (300 MHz, DMSO-d6): δ = 3.72 (s, 3H, OCH3), 5.05 (s, 2H, OCH2), 5.48 (s, 1H, H4), 7.42 (m, 4H, HAr), 7.58 (m, 2H, HAr), 8.08 (s, 1H, H8), 8.43 (d, 1H, HAr, 3JH−H = 8.8 Hz). 13 C NMR (75 MHz, DMSO-d6): δ = 53.0 (OCH3), 54.2 (C4), 78.0 (OCH2), 126.6 (CH), 127.2 (CIV), 128.9 (3 CH), 129.5 (CH), 130.1 (2CH), 131.8 (CIV), 133.6 (CH), 134.7 (CIV), 134.7 (CIV), 161.3 (CO), 163.0 (CO), 167.3 (CO). ESI-MS: m/z = 360 and 362 (M + H)+. Methyl 2-(Benzyloxy)-7-bromo-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylate (47). White powder (77%), 100% keto form, mp 160−162 °C. 1H NMR (300 MHz, DMSO-d6): δ = 3.71 (s, 3H, OCH3), 5.05 (s, 2H, OCH2), 5.46 (s, 1H, H4), 7.42−7.59 (m, 5H, HAr), 7.76 (d, 1H, HAr, 3JH−H = 8.2 Hz), 7.94 (d, 1H, HAr, 3JH−H = 8.2 Hz), 8.20 (s, 1H, H8). 13C NMR (75 MHz, DMSO-d6): δ = 53.1 (OCH3), 54.5 (C4), 78.1 (OCH2), 122.2 (C7), 123.7 (CIV), 126.6 (CH), 128.9 (3 CH), 129.5 (CH), 129.6 (CH), 130.0 (CH), 133.4 (CIV), 134.6 (CIV), 136.3 (CH), 161.4 (CO), 163.0 (CO), 167.2 (CO). ESI-MS: m/z = 404 and 406 (M + H)+. Methyl 2-(Benzyloxy)-7-trifluoromethyl-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylate (48). Five minutes of stirring at room temperature; white powder (76%), 100% enol form, mp 160− 164 °C. 1H NMR (300 MHz, CDCl3): δ = 4.10 (s, 3H, OCH3), 5.25 (s, 2H, OCH2), 7.40−7.43 (m, 3H, HAr), 7.62 (dd, 2H, HAr, 3JH−H = 6.6 Hz, 4JH−H = 2.9 Hz), 7.79 (dd, 1H, H6, 3JH6−H5 = 8.9 Hz, 4JH6−H8 = 1.9 Hz), 8.50 (d, 1H, H5, 3JH5−H6 = 8.9 Hz), 8.63 (s, 1H, H8), 15.91 (s, 1H, OH). 13C NMR (75 MHz, CDCl3): δ = 53.5 (OCH3), 79.3 (OCH2), 84.2 (C4), 121.0 (CIV), 123.5 (q, CF3, 1JC−F = 234.7 Hz), 125.1 (CH), 125.8 (CH, 3JC−F = 4.1 Hz), 126.5 (q, C7, 2JC−F = 33.6 Hz), 128.7 (2CH), 129.5 (CH), 129.6 (CH, 3JC−F = 3.2 Hz), 133.2 (CIV), 135.6 (CIV), 158.0 (CO), 164.1 (CO), 173.3 (CO). ESI-MS: m/z = 394 (M + H)+. Methyl 2-(Benzyloxy)-7-cyano-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylate (49). Ten minutes of stirring at room

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60 precipitated and was isolated by filtration. After concentration in vacuo, the residue was triturated with ether and the precipitate was filtered and dried at room temperature to give an orange solid, yield 75%, mp 183−185 °C, 100% enol form. 1H NMR (300 MHz, DMSOd6): δ = 4.48 (d, 2H, CH2, 3JH−H = 5.6 Hz), 5.06 (s, 2H, OCH2), 7.15 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.8 Hz), 7.40 (m, 5H, HAr), 7.62 (m, 2H, HAr), 8.01 (dd, 1H, H6, 3JH6−H5 = 9.7 Hz, 4JH6−H8 = 2.3 Hz), 8.78 (d, 1H, H8, 4JH8−H6 = 2.3 Hz), 9.40 (d, 1H, H5, 3JH5−H6 = 9.7 Hz), 10.35 (t, 1H, NH, 3JH−H = 5.6 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 41.2 (CH2), 76.6 (OCH2), 90.2 (C4), 114.9 (d, C3′ and C5′, 2JC−F = 20.7 Hz), 115.8 (CIV), 123.6 (CH), 124.1 (CH), 124.6 (CH), 128.2 (2CH), 128.5 (CH), 129.21 (2CH), 129.22 (d, C2′ and C6′, 3JC−F = 7.6 Hz), 135.3 (CIV), 137.0 (d, C1′, 4JC−F = 3.3 Hz), 137.7 (CIV), 144.5 (C7), 159.2 (CO), 161.0 (d, C4′, 1JC−F = 240.0 Hz), 161.5 (CO), 167.8 (CO). ESI-MS: m/z = 464 (M + H)+. N-(4-Fluorobenzyl)-2-(benzyloxy)-7-fluoro-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (60). White powder (75%), 100% keto form, mp 215−217 °C. 1H NMR (300 MHz, DMSOd6): δ = 4.32 (d, 1H, CH2, 2JH−H = 15.6 Hz, 3JH−H = 5.3 Hz), 4.33 (d, 1H, CH2, 2JH−H = 15.6 Hz, 3JH−H = 5.3 Hz), 5.02 (d, 1H, OCH2, 2JH−H = 9.5 Hz), 5.03 (d, 1H, OCH2, 2JH−H = 9.5 Hz), 5.20 (s, 1H, H4), 7.18 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.5 Hz), 7.30 (dd, 2H, H2′ and H6′, 3JH−H = 8.2 Hz, 4JH−F = 5.7 Hz), 7.42−7.52 (m, 4 H, HAr), 7.56− 7.58 (m, 2H, HAr), 7.64 (t, 1H, H6, 3JH−H = 3JH−F = 8.5 Hz), 7.84 (d, 1H, HAr, 3JH−H = 8.5 Hz), 9.33 (t, 1H, NH, 3JH−H = 5.3 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 42.0 (CH2), 55.1 (C4), 77.4 (OCH2), 114.1 (d, CHAr, 2JC−F = 24.0 Hz), 115.2 (d, C3′ and C5′, 2JC−F = 21.3 Hz), 121.6 (d, CHAr, 2JC−F = 22.9 Hz), 127.3 (d, C8a, 3JC−F = 7.6 Hz), 128.4 (2CH), 128.9 (CH), 129,2 (d, C5, 3JC−F = 8.2 Hz), 129.3 (d, C2′ and C6′, 3JC−F = 8.2 Hz), 129.4 (2CH), 130.9 (d, C4a, 4JC−F = 2.7 Hz), 134.3 (CIV), 134.6 (d, C1′, 4JC−F = 2.7 Hz), 160.1 (d, C1, 4JC−F = 2.7 Hz), 161.3 (d, C7, 1JC−F = 241.1 Hz), 161.5 (C4′, 1JC−F = 244.9 Hz), 164.2 (CO), 165.9 (CO). ESI-MS: m/z = 437 (M + H)+. N-(4-Fluorobenzyl)-2-(benzyloxy)-7-chloro-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (61). White powder (81%), 100% keto form, mp 216−218 °C. 1H NMR (300 MHz, DMSOd6): δ = 4.31 (dd, 1H, CH2, 2JH−H = 15.4 Hz, 3JH−H = 5.0 Hz), 4.32 (dd, 1H, CH2, 2JH−H = 15.4 Hz, 3JH−H = 5.0 Hz), 5.01 (d, 1H, OCH2, 2 JH−H = 14.9 Hz), 5.02 (d, 1H, OCH2, 2JH−H = 14.9 Hz), 5.20 (s, 1H, H4), 7.18 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.3 Hz), 7.30 (dd, 2H, H2′ and H6′, 3JH−H = 7.6 Hz, 4JH−F = 4.5 Hz), 7.42−7.47 (m, 4H, HAr), 7.56−7.57 (m, 2H, HAr), 7.84 (d, 1H, HAr, 3JH−H = 8.2 Hz), 8.05 (s, 1H, H8), 9.33 (t, 1H, NH, 3JH−H = 5.0 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 42.0 (CH2), 55.3 (C4), 77.4 (OCH2), 115.2 (d, C3′ and C5′, 2JC−F = 21.3 Hz), 127.1 (CIV), 127.2 (CH), 128.4 (2CH), 128.8 (CH), 128.9 (CH), 129.2 (d, C2′ and C6′, 3JC−F = 8.2 Hz), 129.4 (2CH), 133.1 (CIV), 133.5 (CIV), 134.0 (CH), 134.3 (CIV), 134.5 (d, C1′, 4JC−F = 2.7 Hz), 160.0 (CO), 161.3 (d, C4′, 1JC−F = 241.6 Hz), 164.1 (CO), 165.7 (CO). ESI-MS: m/z = 453 and 455 (M + H)+. N-(4-Fluorobenzyl)-2-(benzyloxy)-7-bromo-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (62). White powder (77%), 100% keto form, mp 171−177 °C. 1H NMR (300 MHz, DMSOd6): δ = 4.33 (dd, 1H, CH2, 2JH−H = 15.4 Hz, 3JH−H = 5.4 Hz), 4.34 (dd, 1H, CH2, 2JH−H = 15.4 Hz, 3JH−H = 5.4 Hz), 5.02 (d, 1H, OCH2, 2 JH−H = 9.1 Hz), 5.03 (d, 1H, OCH2, 2JH−H = 9.1 Hz), 5.20 (s, 1H, H4), 7.18 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.6 Hz), 7.31 (dd, 2H, H2′ and H6′, 3JH−H = 8.0 Hz, 4JH−F = 5.7 Hz), 7.39−7.43 (m, 4 H, HAr), 7.56−7.58 (m, 2H, HAr), 7.95 (d, 1H, HAr, 3JH−H = 8.0 Hz), 8.18 (s, 1H, H8), 9.34 (t, 1H, NH, 3JH−H = 5.4 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 42.5 (CH2), 55.8 (C4), 77.9 (OCH2), 115.7 (d, C3′ and C5′, 2JC−F = 20.7 Hz), 121.8 (C7), 127.8 (CIV), 128.9 (2CH), 129.4 (CH), 129.5 (CH), 129.7 (d, C2′ and C6′, 3JC−F = 8.2 Hz), 129.9 (2CH), 130.6 (CH), 134.4 (CIV), 134.8 (CIV), 135.0 (d, C1′, 4JC−F = 3.3 Hz), 137.3 (CH), 160.2 (CO), 161.8 (d, C4′, 1JC−F = 241.1 Hz), 164.5 (CO), 166.1 (CO). ESI-MS: m/z = 497 and 499 (M + H)+. N-(4-Fluorobenzyl)-2-(benzyloxy)-7-trifluoromethyl-1,3-dioxo1,2,3,4-tetrahydroisoquinoline-4-carboxamide (63). White powder (64%), 100% keto form, mp 198−200 °C. 1H NMR (300 MHz, DMSO-d6): δ = 4.48 (m, 2H, CH2), 5.05 (m, 2H, OCH2), 5.34 (s, 1H, H4), 7.18 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.6 Hz), 7.33 (m, 2H,

temperature; white solid (67%), 100% enol form, mp 154−159 °C. 1H NMR (300 MHz, CDCl3): δ = 4.08 (s, 3H, OCH3), 5.24 (s, 2H, OCH2), 7.04 (td, 1H, H7, 3JH7−F = 3JH7−H8 = 9.1 Hz, 4JH7−H5 = 2.3 Hz), 7.40−7.42 (m, 3H, HAr), 7.61−7.64 (m, 2H, HAr), 8.06 (dd, 1H, H5, 3 JH5−F = 12.3 Hz, 4JH5−H7 = 2.3 Hz), 8.38 (dd, 1H, H7, 3JH7−F = 8.9 Hz, 3 JH7−H8 = 6.4 Hz), 15.74 (br s, 1H, OH). 13C NMR (75 MHz, CDCl3): δ = 53.9 (OCH3), 79.6 (OCH2), 111.1 (d, CH, 2JC−F = 26.6 Hz), 113.6 (d, CH, 2JC−F = 23.9 Hz), 126.1 (d, C8a, 4JC−F = 3.2 Hz), 129.5 (2CH), 130.2 (CH), 130.9 (2CH), 132.2 (d, C8, 3JC−F = 10.9 Hz), 135.4 (CIV), 137.9 (d, C4a, 3JC−F = 8.9 Hz), 159.1 (CO), 165.7 (C6, 1 JC−F = 247.5 Hz), 167.2 (CO), 174.4 (CO). ESI-MS: m/z = 344 (M + H)+. Methyl 2-(Benzyloxy)-8-fluoro-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylate (56). One hour of stirring at room temperature; white solid (70%), 75% enol/25% keto form, mp 182− 185 °C. 1H NMR (300 MHz, DMSO-d6): δ = 3.76 (s, 3H, OCH3 keto), 4.05 (s, 3H, OCH3 enol), 4.99 (s, 1H, CH4 keto), 5.14 (s, 2H, OCH2 keto), 5.23 (s, 2H, OCH2 enol), 7.00 (dd, 1H, H7 enol, 3JH7−F = 10.5 Hz, 3JH7−H8 = 8.8 Hz), 7.25 (dd, 1H, H7 keto, 3JH7−F = 11.0 Hz, 3 JH7−H8 = 8.2 Hz), 7.37−7.45 (m, HAr, H3′, H4′, H5′ enol + keto, H6 keto), 7.52−7.68 (m, HAr, H2′, H6′ enol + keto, H5 keto, H6 enol), 8.24 (d, 1H, H5 enol, 3JH5−H6 = 8.6 Hz), 15.76 (s, 1H, OH). 13C NMR (75 MHz, DMSO-d6): δ = 53.3 (OCH3 enol), 54.0 (OCH3 keto), 54.6 (d, C4 keto, 4JC−F = 2.0 Hz), 79.6 (OCH2 keto), 78.9 (OCH2 enol), 83.7 (d, C4 enol, 4JC−F = 2.4 Hz), 110.4 (d, C4a, 3JC−F = 5.2 Hz), 111.5 (d, C7 enol, 2JC−F = 21.1 Hz), 117.7 (d, C7 keto, 2JC−F = 21.4 Hz), 119.9 (d, C5 enol, 4JC−F = 4.4 Hz), 123.4 (d, C5 keto, 4JC−F = 4.1 Hz), 128.50 (2CH keto), 128.55 (2CH enol), 129.2 (CH keto, C4′), 129.4 (CH enol, C4′), 130.0 (2CH keto), 130.1 (2CH enol), 133.50 (C1′ enol), 133.51 (d, C8a, 2JC−F = 26.5 Hz), 134.3 (d, C6 enol, 3JC−F = 10.7 Hz), 135.3 (C1′ keto), 135.7 (d, C6 keto, 3JC−F = 10.2 Hz), 155.4 (d, CO1 enol, 3JC−F = 4.9 Hz), 157.4 (d, CO1 keto, 3JC−F = 5.1 Hz), 161.9 (CO), 162.6 (CO, 1JC−F = 267.0 Hz), 162.8 (CO, 1JC−F = 262.5 Hz), 163.4 (CO), 166.4 (CO), 173.3 (CO). ESI-MS: m/z = 344 (M + H)+. Methyl 2-(Benzyloxy)-3-hydroxy-6-nitro-1-oxo-1,2-dihydroisoquinoline-4-carboxylate (57). Yellow solid (68%), mp 118−125 °C, 100% enol form. 1H NMR (300 MHz, CDCl3): δ = 4.17 (s, 3H, OCH3), 5.29 (s, 2H, OCH2), 7.43 (m, 3H, HAr), 7.62 (m, 2H, HAr), 8.10 (dd, 1H, H7, 3JH7−H8 = 8.7 Hz, 4JH7−H5 = 1.8 Hz), 8.54 (d, 1H, H8, 3 JH8−H7 = 8.7 Hz), 9.33 (d, 1H, H5, 4JH5−H7 = 1.8 Hz), 15.9 (s, 1H, OH). 13C NMR (75 MHz, CDCl3): δ = 53.9 (OCH3), 79.4 (OCH2), 84.4 (CIV, C4), 118.4 (CH), 120.2 (CH), 124.8 (CIV), 128.8 (2CH), 129.7 (CH), 130.3 (2CH), 133.2 (CIV), 133.9 (CIV), 151.2 (CIV, C7), 157.6 (CO), 164.1 (CO), 173.1 (CO). ESI-MS: m/z = 371 (M + H)+. Synthesis of Carboxamides 58−78. N-(4-Fluorobenzyl)-2(benzyloxy)-7-methoxy-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4carboxamide (58). A solution of intermediate 43 (0.35 g, 1.0 mmol) and 4-methoxybenzylamine (0.68 g, 5.0 mmol) in toluene (15.0 mL) was refluxed for 15 h in a Dean−Stark apparatus. After cooling, the solution was concentrated in vacuo. The residue was dissolved in EtOAc. The organic layer was washed with 2.0 M HCl and dried over Na2SO4. After concentration in vacuo, the residue was triturated with ether and the precipitate was filtered and dried at room temperature to give a white powder (69%), 100% keto form, mp 194−196 °C. 1H NMR (300 MHz, DMSO-d6): δ = 3.86 (s, 3H, OCH3), 4.31 (s, 2H, CH2), 5.01 (s, 2H, OCH2), 5.12 (s, 1H, H4), 7.19−7.56 (m, 12H, HAr), 9.28 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 41.9 (CH2), 54.9 (C4), 55.6 (OCH3), 77.4 (OCH2), 111.1 (CH), 115.1 (d, C3′ and C5′, 2JC−F = 20.7 Hz), 121.4 (CH), 126.3 (CIV), 126.6 (CIV), 128.1 (CH), 128.4 (2CH), 128.9 (CH), 129.2 (d, C2′ and C6′, 3JC−F = 8.2 Hz), 129.4 (2CH), 134.4 (CIV), 134.7 (d, C1′, 4JC−F = 2.7 Hz), 159.0 (CO), 160.8 (CO), 161.3 (d, C4′, 1JC−F = 241.1 Hz), 164.5 (CO), 166.3 (CO). ESI-MS: m/z = 449 (M + H)+. N-(4-Fluorobenzyl)-2-(benzyloxy)-3-hydroxy-7-nitro-1-oxo-1,2-dihydroisoquinoline-4-carboxamide (59). A solution of intermediate 44 (0.37 g, 1.0 mmol) and 4-fluorobenzylamine (0.62 g, 5.0 mmol) in toluene (15.0 mL) was refluxed for 15 h in a Dean−Stark apparatus. After cooling, the solution was concentrated in vacuo. The residue was dissolved in EtOAc. The organic layer was washed with 2.0 M HCl and dried over Na2SO4. During the washings, an amount of intermediate 4653



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161.8 (d, C4′, 1JC−F = 241.0 Hz), 163.5 (CO), 164.9 (CO), 166.7 (CO). ESI-MS: m/z = 539 (M + H)+. N-(4-Fluorobenzyl)-2-(benzyloxy)-1,3-dioxo-7-phenylacetamido1,2,3,4-tetrahydroisoquinoline-4-carboxamide (68). White powder (80%), 100% keto form, mp 225−229 °C. 1H NMR (300 MHz, DMSO-d6): δ = 3.69 (s, 2H, CH2), 4.31 (dd, 1H, CH2, 2JH−H = 14.6 Hz, 3JH−H = 5.7 Hz), 4.32 (dd, 1H, CH2, 2JH−H = 14.6 Hz, 3JH−H = 5.7 Hz), 5.00 (s, 2H, CH2), 5.13 (s, 1H, H4), 7.17 (t, 2H, H3′ and H5′, 3 JH−F = 3JH−H = 8.6 Hz), 7.26−7.37 (m, 8H, HAr), 7.41−7.43 (m, 3H, HAr), 7.55−7.58 (m, 2H, HAr), 7.92 (d, 1H, HAr, 3JH−H = 8.3 Hz), 8.36 (s, 1H, H8), 9.27 (t, 1H, NH, 3JH−H = 5.7 Hz), 10.55 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 42.4 (CH2), 43.7 (CH2), 55.7 (C4), 77.8 (OCH2), 115.6 (d, C3′ and C5′, 2JC−F = 21.3 Hz), 118.2 (CH), 125.1 (CH), 126.1 (CIV), 127.1 (CH), 127.8 (CH), 128.8 (2CH), 128.9 (2CH), 129.3 (CH), 129.5 (CIV), 129.6 (2CH), 129.7 (d, C2′ and C6′, 3JC−F = 7.6 Hz), 129.9 (2CH), 134.9 (CIV), 135.2 (d, C1′, 4 JC−F = 2.7 Hz), 136.1 (CIV), 139.6 (CIV), 161.3 (CO), 161.8 (d, C4′, 1 JC−F = 241.1 Hz), 164.9 (CO), 166.6 (CO), 170.0 (CO). ESI-MS: m/ z = 552 (M + H)+. N-(4-Fluorobenzyl)-2-(benzyloxy)-1,3-dioxo-7-[2-(thiophen-2-yl)acetamido]-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (69). Beige powder (64%), 100% keto form, mp 240−243 °C. 1H NMR (300 MHz, DMSO-d6): δ = 3.92 (s, 2H, CH2), 4.31 (dd, 1H, CH2, 2 JH−H = 19.5 Hz, 3JH−H = 5.8 Hz), 4.32 (dd, 1H, CH2, 2JH−H = 19.5 Hz, 3 JH−H = 5.8 Hz), 5.00 (d, 1H, OCH2, 2JH−H = 15.0 Hz), 5.01 (d, 1H, OCH2, 2JH−H = 15.0 Hz), 5.13 (s, 1H, H4), 6.98−7.01 (m, 2H, HAr), 7.18 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.9 Hz), 7.30 (dd, 2H, HAr, 3 JH−H = 8.2 Hz, 3JH−F = 5.9 Hz), 7.36−7.43 (m, 5H, HAr), 7.56−7.59 (m, 2H, HAr), 7.90 (d, 1H, HAr, 3JH−H = 8.3 Hz), 8.36 (s, 1H, HAr), 9.28 (t, 1H, NH, 3JH−H = 5.8 Hz), 10.59 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 38.0 (CH2), 42.5 (CH2), 55.7 (C4), 77.8 (OCH2), 115.6 (d, C3′ and C5′, 2JC−F = 21.3 Hz), 118.3 (CH), 125.2 (CH), 125.7 (CH), 126.2 (CIV), 127.0 (CH), 127.2 (CH), 127.8 (CH), 128.9 (2CH), 129.3 (CH), 129.6 (CIV), 129.7 (d, C2′ and C6′, 3 JC−F = 8.2 Hz), 129.9 (2CH), 134.9 (CIV), 135.2 (d, C1′, 4JC−F = 3.3 Hz), 137.2 (CIV), 139.5 (CIV), 161.3 (CO), 161.8 (d, C4′, 1JC−F = 241.1 Hz), 165.0 (CO), 166.7 (CO), 169.0 (CO). ESI-MS: m/z = 558 (M + H)+. N-(4-Fluorobenzyl)-2-(benzyloxy)-6-fluoro-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (70). White powder (56%), 100% keto form, mp 193−195 °C. 1H NMR (300 MHz, DMSOd6): δ = 4.29 (dd, 1H, CH2, 2JH−H = 15.2 Hz, 3JH−H = 5.6 Hz), 4.36 (dd, 1H, CH2, 2JH−H = 15.2 Hz, 3JH−H = 5.6 Hz), 4.98 (d, 1H, OCH2, 2 JH−H = 9.9 Hz), 5.03 (d, 1H, OCH2, 2JH−H = 9.9 Hz), 5.20 (s, 1H, H4), 7.18 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.8 Hz), 7.21−7.27 (m, 2H, HAr), 7.30 (dd, 2H, H2′ and H6′, 3JH−H = 7.8 Hz, 4JH−F = 5.7 Hz), 7.41−7.48 (m, 4H, HAr), 7.55−7.57 (m, 2H, HAr), 8.17 (dd, 1H, H8, 3 JH8−H7 = 8.4 Hz, 4JH8−F = 5.7 Hz), 9.33 (t, 1H, NH, 3JH−H = 5.3 Hz). 13 C NMR (75 MHz, DMSO-d6): δ = 42.1 (CH2), 55.6 (C4), 77.4 (OCH2), 113.4 (d, CHAr, 2JC−F = 23.8 Hz), 115.1 (d, C3′ and C5′, 2JC−F = 21.5 Hz), 116.1 (d, CHAr, 2JC−F = 22.3 Hz), 122.1 (CIV), 128.3 (2CH), 128.8 (CH), 129.30 (d, C2′ and C6′, 3JC−F = 7.9 Hz), 129.35 (2CH), 131.4 (d, C8, 3JC−F = 9.9 Hz), 134.4 (CIV), 134.6 (CIV), 137.6 (d, C4a, 3JC−F = 10.1 Hz), 161.3 (d, CIV, 1JC−F = 240.8 Hz), 161.8 (CIV, 1 JC−F = 251.1 Hz), 164.0 (CO), 165.6 (CO), 166.8 (CO). ESI-MS: m/ z = 437 (M + H)+. N-(4-Fluorobenzyl)-2-(benzyloxy)-8-fluoro-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (71). Same protocol as for the synthesis of 58 except 1.2 equiv of 4-fluorobenzylamine and 15 h of reflux; brown solid (78%), 100% keto form, mp 156−158 °C. 1H NMR (300 MHz, DMSO-d6): δ = 4.28 (dd, 1H, CH2, 2JH−H = 15.1 Hz, 3JH−H = 5.6 Hz), 4.32 (dd, 1H, CH2, 2JH−H = 15.2 Hz, 3JH−H = 5.6 Hz), 4.97 (d, 1H, OCH2, 2JH−H = 9.6 Hz), 5.02 (d, 1H, OCH2, 2JH−H = 9.6 Hz), 5.22 (s, 1H, H4), 7.18 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.9 Hz), 7.23−7.30 (m, 3H, HAr), 7.33−7.46 (m, 4H, HAr), 7.51−7.60 (m, 2H, HAr), 7.8 (td, 1H, H6, 3JH6−H7 = 3JH6−H5 = 8.1 Hz, 4JH6−F = 5.2 Hz), 9.36 (t, 1H, NH, 3JH−H = 5.7 Hz). 13C NMR (75 MHz, DMSOd6): δ = 42.1 (CH2), 55.6 (C4), 77.4 (OCH2), 114.0 (d, C4a, 3JC−F = 5.2 Hz), 115.2 (d, C3′ and C5′, 2JC−F = 21.5 Hz), 116.6 (d, C7, 2JC−F =

H2′ and H6′), 7.42−7.44 (m, 3H, HAr), 7.56−7.58 (m, 2H, HAr), 7.69 (d, 1H, HAr, 3JH−H = 7.5 Hz), 8.15 (d, 1H, HAr, 3JH−H = 7.2 Hz), 8.33 (s, 1H, H8), 9.41 (m, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 42.2 (CH2), 55.9 (C4), 77.6 (OCH2), 115.3 (d, C3′ and C5′, 2JC−F = 21.8 Hz), 123.6 (CF3, 1JC−F = 270.8 Hz), 124.6 (CH), 126.4 (CIV), 128.3 (CH), 128.5 (2CH), 129.0 (CH), 129.3 (d, C2′ and C6′, 3JC−F = 8.2 Hz), 129.5 (2CH), 130.6 (CH), 134.4 (CIV), 134.5 (CIV), 139.0 (CIV), 160.0 (CO), 161.3 (d, C4′, 1JC−F = 240.0 Hz), 163.9 (CO), 165.3 (CO). ESI-MS: m/z = 487 (M + H)+. N-(4-Fluorobenzyl)-2-(benzyloxy)-7-cyano-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (64). Two hours of reflux; offwhite powder (70%), 100% keto form, mp 189−191 °C. 1H NMR (300 MHz, acetone-d6): δ = 4.31−4.32 (m, 2H, CH2), 4.93 (d, 1H, OCH2, 2JH−H = 9.3 Hz), 4.99 (d, 1H, OCH2, 2JH−H = 9.3 Hz), 5.23 (s, 1H, H4), 6.95 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.8 Hz), 7.22 (dd, 2H, H2′ and H6′, 3JH−H = 8.4 Hz, 4JH−F = 5.5 Hz), 7.25−7.31 (m, 3H, HAr), 7.48−7.50 (m, 2H, HAr), 7.63 (d, 1H, H5, 3JH5−H6 = 8.1 Hz), 7.96 (dd, 1H, H6, 3JH6−H5 = 8.7 Hz, 4JH6−H8 = 1.6 Hz), 8.36 (d, 1H, H8, 4 JH8−H6 = 1.3 Hz), 9.35 (br s, 1H, NH). 13C NMR (75 MHz, acetoned6): δ = 42.0 (CH2), 55.9 (C4), 77.5 (OCH2), 110.4 (C7), 115.2 (d, C3′ and C5′, 2JC−F = 21.1 Hz), 117.8 (CN), 123.0 (CIV), 126.5 (CIV), 128.3 (2CH), 129.2 (3CH), 129.3 (4CH), 134.0 (CH), 134.6 (CIV), 140.4 (CIV), 161.3 (d, C4′, 1JC−F = 237.7 Hz), 163.7 (CO), 165.1 (CO), 168.2 (CO). ESI-MS: m/z = 444 (M + H)+. N-(4-Fluorobenzyl)-7-acetamido-2-(benzyloxy)-1,3-dioxo-1,2,3,4tetrahydroisoquinoline-4-carboxamide (65). Beige powder (78%), 100% keto form, mp 218−220 °C. 1H NMR (300 MHz, DMSO-d6): δ = 2.10 (s, 3H, CH3), 4.37 (dd, 1H, CH2, 2JH−H = 14.6 Hz, 3JH−H = 5.4 Hz), 4.38 (dd, 1H, CH2, 2JH−H = 14.6 Hz, 3JH−H = 5.4 Hz), 5.02 (s, 2H, OCH2), 5.18 (s, 1H, H4), 7.23 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.9 Hz), 7.34−7.63 (m, 8H, HAr), 7.94 (d, 1H, HAr, 3JH−H = 8.0 Hz), 8.41 (s, 1H, H8), 9.33 (t, 1H, NH, 3JH−H = 5.4 Hz), 10.36 (s, 1H, NH). 13 C NMR (75 MHz, DMSO-d6): δ = 24.5 (CH3), 42.4 (CH2), 55.7 (C4), 77.8 (OCH2), 115.6 (d, C3′ and C5′, 2JC−F = 21.8 Hz), 118.1 (CH), 125.0 (CH), 126.1 (CIV), 127.8 (CH), 128.9 (2CH), 129.2 (CIV), 129.4 (CH), 129.7 (2CH), 129.8 (d, C2′ and C6′, 3JC−F = 8.2 Hz), 131.0 (CIV), 135.0 (CIV), 139.8 (CIV), 161.4 (CO), 161.8 (d, C4′, 1 JC−F = 240.5 Hz), 165.0 (CO), 166.7 (CO), 169.0 (CO). ESI-MS: m/ z = 476 (M + H)+. N-(4-Fluorobenzyl)-7-benzamido-2-(benzyloxy)-1,3-dioxo1,2,3,4-tetrahydroisoquinoline-4-carboxamide (66). Beige powder (54%), 100% keto form, mp 242−244 °C. 1H NMR (300 MHz, DMSO-d6): δ = 4.33 (s, 2H, CH2), 5.03 (s, 2H, OCH2), 5.18 (s, 1H, H4), 7.20 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.7 Hz), 7.31−7.58 (m, 11H, HAr), 8.01−8.04 (m, 2H, HAr), 8.14 (s, 1H, HAr), 8.57 (s, 1H, HAr), 9.30 (s, 1H, NH), 10.62 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 42.5 (CH2), 55.7 (C4), 77.8 (OCH2), 115.6 (d, C3′ and C5′, 2JC−F = 21.3 Hz), 119.5 (CH), 126.1 (CIV), 126.3 (CH), 127.6 (CH), 128.2 (2CH), 128.9 (2CH), 129.0 (2CH), 129.3 (CH), 129.7 (d, C2′ and C6′, 3JC−F = 8.2 Hz), 129.9 (2CH), 132.4 (CH), 134.8 (CIV), 134.9 (CIV), 135.1 (CIV), 135.2 (d, C1′, 4JC−F = 2.7 Hz), 139.7 (C7), 161.4 (CO), 161.8 (d, C4′, 1JC−F = 241.0 Hz), 165.0 (CO), 166.2 (CO), 166.7 (CO). ESI-MS: m/z = 538 (M + H)+. N-(4-Fluorobenzyl)-2-(benzyloxy)-1,3-dioxo-7-picolinamido1,2,3,4-tetrahydroisoquinoline-4-carboxamide (67). Beige powder (64%), 100% keto form, mp 240−243 °C. 1H NMR (300 MHz, DMSO-d6): δ = 4.33 (dd, 1H, CH2, 2JH−H = 19.7 Hz, 3JH−H = 5.7 Hz), 4.34 (dd, 1H, CH2, 2JH−H = 19.7 Hz, 3JH−H = 5.7 Hz), 5.02 (d, 1H, OCH2, 2JH−H = 15.0 Hz), 5.03 (d, 1H, OCH2, 2JH−H = 15.0 Hz), 5.17 (s, 1H, H4), 7.18 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.9 Hz), 7.31 (dd, 2H, H2′ and H6′, 3JH−H = 8.3 Hz, 4JH−F = 5.5 Hz), 7.40−7.44 (m, 4 H, HAr), 7.57−7.59 (m, 2H, HAr), 7.72 (dd, 1H, HAr, 3JH−H = 7.1 Hz, 3 JH−H = 5.1 Hz), 8.10 (t, 1H, HAr, 3JH−H = 7.7 Hz), 8.16−8.21 (m, 2H, HAr), 8.77−8.79 (m, 2H, HAr), 9.30 (t, 1H, NH, 3JH−H = 5.7 Hz), 11.06 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 42.5 (CH2), 55.7 (C4), 77.8 (OCH2), 115,6 (d, C3′ and C5′, 2JC−F = 21.3 Hz), 119.8 (CH), 123.1 (CH), 126.1 (CIV), 126.7 (CH), 127.6 (CH), 127.7 (CH), 128.9 (2CH), 129.3 (CH), 129.7 (d, C2′ and C6′, 3JC−F = 8.2 Hz), 129.9 (2CH), 130.2 (CIV), 134.9 (CIV), 135.2 (d, C1′, 4JC−F = 2.7 Hz), 138.7 (CH), 138.9 (CIV), 149.0 (CH), 150.0 (C1′), 161.3 (CO), 4654



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21.0 Hz), 123.0 (d, C5, 4JC−F = 2.7 Hz), 128.4 (2CH), 128.9 (CH), 129.30 (d, C2′ and C6′, 3JC−F = 8.0 Hz), 129.5 (2CH), 134.5 (C1″), 134.6 (d, C1′, 4JC−F = 2.9 Hz), 135.8 (d, C6, 3JC−F = 10.0 Hz), 136.9 (C8a), 157.8 (d, CO1, 3JC−F = 4.6 Hz), 161.4 (d, CIV, 1JC−F = 242.8 Hz), 161.5 (d, CIV, 1JC−F = 262.0 Hz), 163.7 (CO), 165.7 (CO). ESIMS: m/z = 437 (M + H)+. N-(4-Fluorobenzyl)-2-(benzyloxy)-3-hydroxy-6-nitro-1-oxo-1,2-dihydroisoquinoline-4-carboxamide (72). Same protocol as for the synthesis of 59 except 2.0 equiv of amine and 1 h of reflux; white solid (61%), mp 224−228 °C, 100% enol form. 1H NMR (300 MHz, DMSO-d6): δ = 4.24 (m, 2H, NHCH2), 4.94 (s, 2H, OCH2), 7.15 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.8 Hz), 7.43−7.46 (m, 5H, HAr), 7.59 (m, 2H, H2′ and H6′), 8.15 (dd, 1H, H7, 3JH7−H8 = 9.7 Hz, 4JH7−H5 = 2.3 Hz), 8.34 (d, 1H, H8, 3JH8−H7 = 9.7 Hz), 9.40 (d, 1H, H5, 4JH5−H7 = 2.3 Hz), 10.31 (m, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 42.1 (NHCH2), 76.4 (OCH2), 86.8 (C4), 115.0 (d, C3′ and C5′, 2JC−F = 20.2 Hz), 121.0 (CIV), 121.5 (CH), 123.0 (CH), 128.3 (3CH), 129.3 (5CH), 134.2 (CIV), 134.4 (CIV), 136.0 (CIV), 150.3 (C6), 159.2 (CO), 161.3 (d, C4′, 1JC−F = 235.5 Hz), 163.8 (CO), 165.2 (CO). ESIMS: m/z = 464 (M + H)+. N-Hexyl-2-(benzyloxy)-3-hydroxy-7-nitro-1-oxo-1,2-dihydroisoquinoline-4-carboxamide (73). A solution of intermediate 44 (0.37 g, 1.0 mmol) and hexylamine (5.0 mmol) in toluene (15.0 mL) was refluxed for 12 h in a Dean−Stark apparatus. The workup is identical to that reported for synthesis of 59 to give an orange solid, yield 97%, mp 155−156 °C, 100% enol form. 1H NMR (300 MHz, DMSO-d6): δ = 0.88 (m, 3H, CH3), 1.31 (m, 6H, CH2), 1.49 (m, 2H, CH2), 3.24 (t, 2H, CH2, 3JH−H = 6.3 Hz), 5.03 (s, 2H, OCH2), 7.40−7.42 (m, 2H, HAr), 7.61−7.63 (m, 3H, HAr), 7.97 (d, 1H, H6, 3JH6−H5 = 10.3 Hz), 8.74 (s, 1H, H8), 9.32 (d, 1H, H5, 3JH5−H6 = 10.3 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 14.4 (CH3), 22.6 (CH2), 26.9 (CH2), 29.9 (CH2), 31.5 (CH2), 38.9 (CH2), 77.1 (OCH2), 90.6 (C4), 116.4 (CIV), 124.1 (CH), 124.6 (CH), 125.1 (CH), 128.7 (2CH), 128.9 (CH), 129.7 (2CH), 135.7 (CIV), 138.3 (CIV), 144.6 (C7), 159.6 (CO), 161.8 (CO), 168.2 (CO). ESI-MS: m/z = 440 (M + H)+. N-Phenyl-2-(benzyloxy)-3-hydroxy-7-nitro-1-oxo-1,2-dihydroisoquinoline-4-carboxamide (74). Yellow solid, yield 77%, mp 169−171 °C, 100% enol form. 1H NMR (300 MHz, DMSO-d6): δ = 5.10 (s, 2H, OCH2), 6.98 (t, 1H, HAr,3JH−H = 7.2 Hz), 7.30 (t, 2H, HAr, 3JH−H = 7.9 Hz), 7.40−7.47 (m, 3H, HAr), 7.65−7.69 (m, 4H, HAr), 8.08 (dd, 1H, H6, 3JH6−H5 = 9.8 Hz, 4JH6−H8 = 2.5 Hz), 8.79 (d, 1H, H8, 4JH8−H6 = 2.5 Hz), 9.42 (d, 1H, H5, 3JH5−H6 = 9.8 Hz), 12,41 (s, 1H, OH). 13C NMR (75 MHz, DMSO-d6): δ = 76.7 (OCH2), 90.1 (C4), 116.5 (CIV), 119.3 (2CH), 121.9 (CH), 123.8 (CH), 123.9 (CH), 124.9 (CH), 128.3 (3 CH), 128.7 (2CH), 129.3 (2CH), 135.2 (CIV), 138.4 (CIV), 140.2 (CIV), 144.3 (C7), 159.0 (CO), 161.8 (CO), 166.0 (CO). ESI-MS: m/z = 432 (M + H)+. N-Benzyl-2-(benzyloxy)-3-hydroxy-7-nitro-1-oxo-1,2-dihydroisoquinoline-4-carboxamide (75). Orange solid, yield 62%, mp 178−181 °C, 100% enol form. 1H NMR (300 MHz, DMSO-d6): δ = 4.49 (s, 2H, CH2), 5.04 (s, 2H, OCH2), 7.31−7.44 (m, 8H, HAr), 7.61 (d, 2H, HAr, 3JH−H = 7.3 Hz), 8.00 (dd, 1H, H6, 3JH6−H5 = 9.5 Hz, 4JH6−H8 = 1.8 Hz), 8.76 (d, 1H, H8, 4JH8−H6 = 1.8 Hz), 9.38 (d, 1H, H5, 3JH5−H6 = 9.5 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 42.6 (CH2), 77.1 (OCH2), 90.5 (C4), 116.5 (CIV), 124.2 (CH), 124.5 (CH), 125.2 (CH), 127.1 (CH), 127.8 (3 CH), 128.7 (2CH), 128.8 (2CH), 129.8 (2CH), 135.7 (CIV), 138.4 (CIV), 141.0 (CIV), 144.7 (C7), 159.6 (CO), 162.0 (CO), 168.3 (CO). ESI-MS: m/z = 446 (M + H)+. N-(4-Fluorophenyl)-2-(benzyloxy)-3-hydroxy-7-nitro-1-oxo-1,2dihydroisoquinoline-4-carboxamide (76). Beige solid, yield 89%, mp 189−190 °C, 100% enol form. 1H NMR (300 MHz, DMSO-d6): δ = 5.11 (s, 2H, OCH2), 7.11−7.13 (m, 2H, HAr), 7.31−7.43 (m, 5H, HAr), 7.65−7.71 (m, 2H, HAr), 8.09 (d, 1H, H6, 3JH6−H5 = 9.8 Hz), 8.80 (s, 1H, H8), 9.42 (d, 1H, H5, 3JH5−H6 = 9.8 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 77.2 (OCH2), 90.3 (C4), 115.6 (d, C3′ and C5′, 2JC−F = 21.3 Hz), 117.0 (CIV), 121.3 (d, C2′ and C6′, 3JC−F = 7.6 Hz), 124.2 (CH), 124.4 (CH), 125.5 (CH), 128.8 (2CH), 129.0 (CH), 129.8 (2CH), 135.7 (CIV), 137.1 (CIV), 138.9 (CIV), 144.9 (C7), 156.2 (CO), 159.6 (CO), 161.0 (d, C4′, 1JC−F = 252.0 Hz), 166.5 (CO). ESIMS: m/z = 450 (M + H)+.

N-(4-Fluorophenethyl)-2-(benzyloxy)-3-hydroxy-7-nitro-1-oxo1,2-dihydroisoquinoline-4-carboxamide (77). Yellow solid, yield 81%, mp 171−173 °C, 100% enol form. 1H NMR (300 MHz, DMSO-d6): δ = 2.81 (t, 2H, 3JH−H = 6.8 Hz), 3.48 (t, 2H, 3JH−H = 6.8 Hz), 5.03 (s, 2H, OCH2), 7.13 (t, 2H, H3′ and H5′,3JH−H = 3JH−F = 8.8 Hz); 7.30−7.34 (m, 2H, HAr), 7.39−7.45 (m, 3H, HAr), 7.61−7.63 (m, 2H, HAr), 7.99 (d, 1H, H6, 3JH6−H5 = 9.4 Hz), 8.75 (s, 1H, H8), 9.32 (d, 1H, H5, 3JH5−H6 = 9.4 Hz), 9.77 (s, 1H, OH). 13C NMR (75 MHz, DMSO-d6): δ = 35.4 (CH2), 40.7 (CH2), 77.1 (OCH2), 90.7 (C4), 115.4 (d, C3′ and C5′, 2JC−F = 20.7 Hz), 116.3 (CIV), 124.1 (CH), 124.6 (CH), 125.1 (CH), 128.7 (2CH), 129.0 (CH), 129.8 (2CH), 130.9 (d, C2′ and C6′, 3JC−F = 7,6 Hz), 135.7 (CIV), 136.6 (d, C1′, 4JC−F = 1.6 Hz), 138.2 (CIV), 144.8 (C7), 159.6 (CO), 161.2 (d, C4′, 1JC−F = 242.7 Hz), 161.8 (CO), 168.2 (CO). ESI-MS: m/z = 478 (M + H)+. N-(4-Methoxybenzyl)-2-(benzyloxy)-3-hydroxy-7-nitro-1-oxo-1,2dihydroisoquinoline-4-carboxamide (78). Yellow solid, yield 74%; mp 180 °C, 100% enol form. 1H NMR (300 MHz, DMSO-d6): δ = 3.74 (s, 3H, OCH3), 4,41 (s, 2H, CH2), 5.03 (s, 2H, OCH2), 6,90 (d, 2H, HAr,3JH−H = 8.3 Hz), 7.28 (d, 2H, HAr, 3JH−H = 8.3 Hz), 7.40−7.42 (m, 3H, HAr), 7.59−7.62 (m, 2H, HAr), 8.00 (dd, 1H, H6, 3JH6−H5 = 9.8 Hz, 4JH6−H8 = 2.0 Hz), 8.76 (d, 1H, H8, 4JH8−H6 = 2.0 Hz), 9.38 (d, 1H, H5, 3JH5−H6 = 9.8 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 42.1 (CH2), 55.5 (OCH3), 77.1 (OCH2), 90.5 (C4), 114.2 (C3′ and C5′), 116.6 (CIV), 124.2 (CH), 124.6 (CH), 125.2 (CH), 128.7 (2CH), 129.0 (CH), 129.2 (C2′ and C6′), 129.7 (2CH), 132.8 (CIV), 135.7 (CIV), 138.4 (CIV), 144.8 (C7), 158.6 (CO), 159.6 (CO), 161.9 (CO), 168.2 (CO). ESI-MS: m/z = 476 (M + H)+. Deprotection of Precursors 58−78. N-(4-Fluorobenzyl)-2hydroxy-7-methoxy-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (79). Intermediate 58 (0.10 g) was dissolved in a mixture of DMF (10 mL) and methanol (10 mL) and hydrogenated for 3 h at room temperature over Pd/C 5% (10 mg). The catalyst was filtered and the solvent was removed in vacuo. Trituration of the residue in ether afforded the deprotected compound as a white powder (86%), mp 174−177 °C, 100% keto form. 1H NMR (300 MHz, DMSO-d6): δ = 3.85 (s, 3H, OCH3), 4.29 (dd, 1H, CH2, 2JH−H =14.6 Hz, 3JH−H = 5.0 Hz), 4.30 (dd, 1H, CH2, 2JH−H =14.6 Hz, 3JH−H = 5.0 Hz), 5.06 (s, 1H, H4), 7.17 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.9 Hz), 7.26−7.32 (m, 4H, HAr), 7.52 (d, 1H, H8, 4JH8−H6 = 2.3 Hz), 9.25 (t, 1H, NH, 3JH−H = 5.1 Hz), 10.61 (s, 1H, NOH). 13C NMR (75 MHz, DMSO-d6): δ = 41.9 (CH2), 54.5 (C4), 55.5 (OCH3), 110.9 (CH), 115.1 (d, C3′ and C5′, 2JC−F = 21.3 Hz), 121.0 (CH), 126.4 (CIV), 126.5 (CIV), 128.0 (CH), 129.1 (d, C2′ and C6′, 3JC−F = 8.2 Hz), 134.7 (d, C1′, 4JC−F = 2.7 Hz), 158.9 (CO), 161.2 (d, C4′, 1JC−F = 240.6 Hz), 161.4 (CO), 164.8 (CO), 166.4 (CO). ESI-MS: m/z = 359 (M + H)+. HRMS calcd for C 18 H 15 N2 O 5 , 358.09650; found, 358.09755. Anal. Calcd for C18H15N2O5: C, 60.34; H, 4.22; N, 7.82. Found: C, 60.19; H, 4.21; N, 7.84. N-(4-Fluorobenzyl)-2,3-dihydroxy-7-nitro-1-oxo-1,2-dihydroisoquinoline-4-carboxamide (80). Intermediate 59 (0.46 g, 1.0 mmol) was dissolved in a minimum of CH2Cl2 at −78 °C, and boron trichloride (1.0 M solution in CH2Cl2, 5.0 mL, 5.0 mmol) was added dropwise. The solution was stirred for 1 h at −78 °C and the reaction mixture was allowed to reach 0 °C. Then water (5.0 mL) was added. The first precipitate (impure fraction) was filtered and the filtrate was kept for several hours at room temperature. The second precipitate was filtrated and triturated with ether to give an orange solid (15%), mp 159−162 °C, 100% enol form. 1H NMR (300 MHz, DMSO-d6): δ = 4.46 (d, 2H, NHCH2, 3JH−H = 5.0 Hz), 7.14 (t, 2H, H3′ and H5′, 3 JH−H = 3JH−F = 8.6 Hz), 7.36 (dd, 2H, H2′ and H6′, 3JH−H = 8.8 Hz, 4 JH−F = 5.7 Hz), 7.98 (dd, 1H, H6, 3JH6−H5 = 9.6 Hz, 4JH6−H8 = 2.6 Hz), 8.75 (d, 1H, H8, 4JH8−H6 = 2.3 Hz), 9.38 (d, 1H, H5, 3JH5−H6 = 9.7 Hz), 10.07 (s, 1H, OH), 10.28 (t, 1H, NH, 3JH−H = 5.0 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 41.4 (CH2), 89.7 (C4), 115.0 (d, C3′ and C5′, 2 JC−F = 21.3 Hz), 116.0 (CIV), 123.7 (CH), 123.8 (CH), 124.2 (CH), 129.2 (d, C2′ and C6′, 3JC−F = 8.2 Hz), 136.7 (CIV), 138.3 (CIV), 143.0 (C7), 157.5 (CO), 159.5 (CO), 161.1 (d, C4′, 1JC−F = 241.0 Hz), 167.6 (CO). ESI-MS: m/z = 374 (M + H)+. HRMS calcd for C17H12FN3O6, 373.07101; found, 373.07345. Anal. Calcd for C17H12FN3O6: C, 54.70; H, 3.24; N, 11.26. Found: C, 54.58; H, 3.24; N, 11.28. 4655



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(CH2), 55.1 (C4), 107.1 (d, CH, 2JC−F = 24.0 Hz), 113.2 (d, CH, 2JC−F = 23.1 Hz), 115.1 (d, C3′ and C5′, 2JC−F = 21.1 Hz), 122.2 (d, C8a, 4 JC−F = 1.6 Hz), 129.3 (d, C2′ and C6′, 3JC−F = 8.1 Hz), 131.2 (d, C8, 3 JC−F = 9.9 Hz), 134.6 (d, C1′, 4JC−F = 2.6 Hz), 137.4 (d, C4a, 3JC−F = 9.6 Hz), 160.1 (d, C1, 4JC−F = 2.7 Hz), 161.5 (C4′, 1JC−F = 244.9 Hz), 160.6 (CO), 160.9 (d, CIV, 1JC−F = 240.0 Hz), 161.3 (d, CIV, 1JC−F = 240.7 Hz), 164.4 (CO), 165.8 (CO). ESI-MS: m/z = 347 (M + H)+. HRMS calcd for C17H12F2N2O4, 346.07651; found, 346.07465. Anal. Calcd for C17H12F2N2O4: C, 58.96; H, 3.49; N, 8.09. Found: C, 58.82; H, 3.48; N, 8.08. N-(4-Fluorobenzyl)-8-fluoro-2-hydroxy-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (85). Hydrogenation for 20 min in THF at room temperature over Pd/C 5%; brown solid (30%), 100% keto form, mp 155−158 °C. 1H NMR (300 MHz, DMSO-d6): δ = 4.25 (dd, 1H, CH2, 2JH−H = 14.9 Hz, 3JH−H = 5.2 Hz), 4.33 (dd, 1H, CH2, 2JH−H = 14.9 Hz, 3JH−H = 5.2 Hz), 5.16 (s, 1H, H4), 7.04−7.43 (m, 6H, HAr), 7.72 (dd, 1H, H7, 3JH7−F = 12.6 Hz, 3JH7−H6 = 7.4 Hz), 9.32 (br s, 1H, NH), 10.60 (s, 1H, OH). 13C NMR (75 MHz, DMSOd6): δ = 41.9 (CH2), 55.1 (C4), 114.1 (d, C4a, 3JC−F = 5.3 Hz), 115.2 (d, C3′ and C5′, 2JC−F = 21.0 Hz), 116.5 (d, C7, 2JC−F = 21.2 Hz), 122.8 (d, C5, 4JC−F = 3.4 Hz), 129.30 (d, C2′ and C6′, 3JC−F = 8.0 Hz), 134.6 (d, C1′, 4JC−F = 2.7 Hz), 135.5 (d, C6, 3JC−F = 10.2 Hz), 136.8 (C8a), 158.5 (d, CO1, 3JC−F = 4.8 Hz), 161.3 (d, CIV, 1JC−F = 256.1 Hz), 161.4 (d, CIV, 1JC−F = 246.8 Hz), 163.9 (CO), 165.9 (CO). ESI-MS: m/z = 347 (M + H)+. HRMS calcd for C17H12F2N2O4, 346.07651; found, 346.07720. Anal. Calcd for C17H12F2N2O4: C, 58.96; H, 3.49; N, 8.09. Found: C, 59.11; H, 3.50; N, 8.07. N-(4-Fluorobenzyl)-7-chloro-2-hydroxy-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (86). Intermediate 61 was treated with boron trichloride as in synthesis of 81 to give a beige powder (69%), mp 168−169 °C, 100% keto form. 1H NMR (300 MHz, DMSO-d6): δ = 4.28 (dd, 1H, CH2, 2JH−H = 15.5 Hz, 3JH−H = 6.2 Hz), 4.29 (dd, 1H, CH2, 2JH−H = 15.5 Hz, 3JH−H = 6.2 Hz), 5.16 (s, 1H, H4), 7.18 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 9.0 Hz), 7.30 (dd, 2H, H2′ and H6′, 3JH−H = 9.0 Hz, 4JH−F = 5.6 Hz), 7.41 (d, 1H, HAr, 3JH−H = 8.2 Hz), 7.79 (d, 1H, HAr, 3JH−H = 8.2 Hz), 8.02 (s, 1H, H8), 9.34 (t, 1H, NH, 3JH−H = 6.2 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 42.5 (CH2), 55.3 (C4), 115.6 (d, C3′ and C5′, 2JC−F = 21.3 Hz), 127.5 (CH), 127.7 (CIV), 129.2 (CH), 129.7 (d, C2′ and C6′, 3JC−F = 8.2 Hz), 133.5 (CIV), 133.8 (CIV), 134.1 (CH), 135.1 (CIV), 161.0 (CO), 161.8 (d, C4′, 1JC−F = 240.6 Hz), 164.9 (CO), 166.4 (CO). ESI-MS: m/z = 363 and 365 (M + H)+. HRMS calcd for C17H12ClFN2O4, 362.04696; found, 362.04506. Anal. Calcd for C17H12ClFN2O4: C, 56.29; H, 3.33; N, 7.72. Found: C, 56.46; H, 3.32; N, 7.74. N-(4-Fluorobenzyl)-7-bromo-2-hydroxy-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (87). Intermediate 62 was treated with boron trichloride as in synthesis of 81 to give a pink powder (45%), mp 170−171 °C, 100% keto form. 1H NMR (300 MHz, DMSO-d6): δ = 4.28 (dd, 1H, CH2, 2JH−H = 15.3 Hz, 3JH−H = 5.1 Hz), 4.29 (dd, 1H, CH2, 2JH−H = 15.3 Hz, 3JH−H = 5.1 Hz), 5.14 (s, 1H, H4), 7.18 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.6 Hz), 7.28−7.36 (m, 3H, HAr), 8.06 (d, 1H, HAr, 3JH−H = 8.2 Hz), 8.14 (s, 1H, H8), 9.33 (t, 1H, NH, 3JH−H = 5.1 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 42.5 (CH2), 55.4 (C4), 115.6 (d, C3′ and C5′, 2JC−F = 21.3 Hz), 121.6 (C7), 127.9 (CIV), 129.4 (CH), 129.7 (d, C2′ and C6′, 3JC−F = 8.2 Hz), 130.5 (CH), 134.2 (CIV), 135.1 (d, C1′, 4JC−F = 2.2 Hz), 136.9 (CH), 160.9 (CO), 161.8 (d, C4′, 1JC−F = 240.6 Hz), 164.9 (CO), 166.3 (CO). ESIMS: m/z = 407 and 409 (M + H)+. HRMS calcd for C17H12BrFN2O4, 405.99645; found, 405.99519. Anal. Calcd for C17H12BrFN2O4: C, 50.14; H, 2.97; N, 6.88. Found: C, 50.00; H, 2.96; N, 6.90. N-(4-Fluorobenzyl)-2-hydroxy-7-trifluoromethyl-1,3-dioxo1,2,3,4-tetrahydroisoquinoline-4-carboxamide (88). Hydrogenation in THF for 3 h at room temperature over Pd/C 5%; white powder (80%), 100% keto form, mp 155−157 °C. 1H NMR (300 MHz, DMSO-d6): δ = 4.27 (dd, 1H, CH2, 2JH−H = 15.4 Hz, 3JH−H = 5.8 Hz), 4.35 (dd, 1H, CH2, 2JH−H = 15.4 Hz, 3JH−H = 5.8 Hz), 5.28 (s, 1H, H4), 7.18 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.7 Hz), 7.33 (m, 2H, H2′ and H6′), 7.68 (d, 1H, HAr, 3JH−H = 8.2 Hz), 8.16 (d, 1H, HAr, 3JH−H = 8.2 Hz), 8.34 (s, 1H, H8), 9.44 (t, 1H, NH, 3JH−H = 5.2 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 42.2 (CH2), 55.5 (C4), 115.3 (d, C3′ and

N-(4-Fluorobenzyl)-2,3-dihydroxy-6-nitro-1-oxo-1,2-dihydroisoquinoline-4-carboxamide (81). Intermediate 72 (1.0 mmol) was dissolved in a minimum of CH2Cl2, and boron trichloride (1.0 M solution in CH2Cl2, 6.0 mL, 6.0 mmol) was added dropwise at −78 °C. The solution was stirred for 1 h at −78 °C, and water (20 mL) was slowly added. After 15 min of stirring, the precipitate was filtered and triturated with ether to give an orange solid (56%), mp 210−214 °C, 100% enol form. 1H NMR (300 MHz, DMSO-d6): δ = 4.46 (d, 2H, NHCH2, 3JH−H = 5.5 Hz), 7.14 (t, 2H, H3′ and H5′,3JH−H = 3JH−F = 8.9 Hz), 7.36 (dd, 2H, H2′ and H6′, 3JH−H = 8.5 Hz, 4JH−F = 5.8 Hz), 7.94 (dd, 1H, H7, 3JH7−H8 = 9.6 Hz, 4JH7−H5 = 2.6 Hz), 8.30 (d, 1H, H8, 3 JH8−H7 = 8.8 Hz), 10.00 (d, 1H, H5, 4JH5−H7 = 2.3 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 41.4 (CH2), 85.8 (C4), 115.0 (d, C3′ and C5′, 2 JC−F = 21.4 Hz), 115.3 (CH), 120.1 (CH), 123.6 (CIV), 128.0 (CH), 129.7 (d, C2′ and C6′, 3JC−F = 8.2 Hz), 135.5 (CIV), 136.3 (CIV), 149.0 (C6), 152.5 (CO), 154.0 (CO), 161.9 (d, C4′, 1JC−F = 242.0 Hz), 164.4 (CO). ESI-MS: m/z = 374 (M + H)+. HRMS calcd for C17H12FN3O6, 373.07101; found, 373.07260. Anal. Calcd for C17H12FN3O6: C, 54.70; H, 3.24; N, 11.26. Found: C, 54.72; H, 3.22; N, 11.25. N-(4-Fluorobenzyl)-7-amino-2-hydroxy-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (82). Intermediate 59 (0.46 g, 1.0 mmol) was dissolved in a mixture of ethyl acetate (10 mL) and methanol (5 mL) and hydrogenated for 4 h at room temperature over Pd/C 5% (50.0 mg). The catalyst was filtered and the solvent was removed in vacuo. Trituration of the residue in ether afforded the deprotected compound as a green solid, yield 70%, mp 212−216 °C, 100% keto form. 1H NMR (300 MHz, DMSO-d6): δ = 4.25 (dd, 1H, CH2, 2JH−H = 15.3 Hz, 3JH−H = 5.5 Hz), 4.32 (dd, 1H, CH2, 2JH−H = 15.3 Hz, 3JH−H = 5.5 Hz), 4.90 (s, 1H, CH), 5.60 (s, 2H, NH2), 6.85 (dd, 1H, H6, 3JH6−H5 = 8.2 Hz, 4JH6−H8 = 2.0 Hz), 7.02 (d, 1H, H5, 3 JH5−H6 = 8.2 Hz), 7.17 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.6 Hz), 7.25 (d, 1H, H8, 4JH8−H6 = 2.0 Hz), 7.30 (dd, 2H, H2′ and H6′, 3JH−H = 8.4 Hz, 3JH−F = 5.7 Hz), 9.15 (t, 1H, NH, 3JH−H = 5.1 Hz), 10.46 (s, 1H, OH). 13C NMR (75 MHz, DMSO-d6): δ = 42.9 (CH2), 55.4 (C4), 112.3 (CH), 116.1 (d, C3′ and C5′, 2JC−F = 21.3 Hz), 120.6 (CH), 121.7 (CIV), 126.7 (CIV), 128.2 (CH), 130.2 (d, C2′ and C6′, 3JC−F = 8.2 Hz), 135.9 (d, C1′, 4JC−F = 2.7 Hz), 149.7 (C7), 162.3 (d, C4′, 1JC−F = 241.0 Hz), 163.0 (CO), 166.2 (CO), 168.0 (CO). ESI-MS: m/z = 344 (M + H)+. HRMS calcd for C17H14FN3O4, 343.09683; found, 343.09752. Anal. Calcd for C17H14FN3O4: C, 59.48; H, 4.11; N, 12.24. Found: C, 59.59; H, 4.11; N, 12.27. N-(4-Fluorobenzyl)-7-fluoro-2-hydroxy-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (83). Intermediate 60 was treated with boron trichloride as in synthesis of 81 to give a pink powder (55%), mp 179−180 °C, 100% keto form. 1H NMR (300 MHz, DMSO-d6): δ = 4.30 (dd, 1H, CH2, 2JH−H = 15.6 Hz, 3JH−H = 5.3 Hz), 4.31 (dd, 1H, CH2, 2JH−H = 15.6 Hz, 3JH−H = 5.3 Hz), 5.15 (s, 1H, H4), 7.17 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.6 Hz), 7.30 (dd, 2H, H2′ and H6′, 3JH−H = 8.6 Hz, 4JH−F = 5.7 Hz), 7.44 (dd, 1H, H5, 3JH5−H6 = 8.0 Hz, 4JH5−F = 5.1 Hz), 7.60 (td, 1H, H6, 3JH6−H5 = 3JH6−F = 8.0 Hz, 4 JH6−H8 = 1.9 Hz), 7.79 (dd, 1H, H8, 3JH8−F = 9.1 Hz, 4JH8−H6 = 1.9 Hz), 9.33 (t, 1H, NH, 3JH−H = 5.3 Hz). 13C NMR (75 MHz, DMSOd6): δ = 42.4 (CH2), 55.2 (C4), 114.3 (d, CHAr, 2JC−F = 23.5 Hz), 115.6 (d, C3′ and C5′, 2JC−F = 21.3 Hz), 121.7 (d, CHAr, 2JC−F = 22.4 Hz), 127.9 (d, C8a, 3JC−F = 8.2 Hz), 129.6 (d, 1H, C5, 3JC−F = 8.2 Hz), 129.7 (d, C2′ and C6′, 3JC−F = 8.2 Hz), 131.2 (d, C4a, 4JC−F = 3.3 Hz), 135.1 (d, C1′, 4JC−F = 3.3 Hz), 161.1 (d, C1, 4JC−F = 2.2 Hz), 161.7 (d, C7, 1JC−F = 241.1 Hz), 162.0 (C4′, 1JC−F = 244.4 Hz), 165.0 (CO), 166.5 (CO). ESI-MS: m/z = 347 (M + H)+. HRMS calcd for C17H12F2N2O4, 346.07651; found, 346.07789. Anal. Calcd for C17H12F2N2O4: C, 58.96; H, 3.49; N, 8.09. Found: C, 59.07; H, 3.48; N, 8.11. N-(4-Fluorobenzyl)-6-fluoro-2-hydroxy-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (84). Hydrogenation for 20 min in THF/acetone at room temperature over Pd/C 5%; dark brown solid (67%), 100% keto form, mp 147−150 °C. 1H NMR (300 MHz, DMSO-d6): δ = 4.31−4.34 (m, 2H, CH2), 5.16 (s, 1H, H4), 7.14−7.20 (m, 4H, H3′ and H5′, H5, H7), 7.28−7.30 (m, 2H, H2′ and H6′), 8.13 (dd, 1H, H8, 3JH8−H7 = 8.8 Hz, 4JH8−F = 6.0 Hz), 9.31 (m, 1H, NH), 10.367 (br s, 1H, OH). 13C NMR (75 MHz, DMSO-d6): δ = 42.0 4656



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C5′, 2JC−F = 21.8 Hz), 123.6 (CF3, 1JC−F = 270.8 Hz), 124.4 (CH), 126.5 (CIV), 128.2 (CH), 128.9 (C7, 2JC−F = 32.5 Hz), 129.3 (d, C2′ and C6′, 3JC−F = 8.2 Hz), 130.2 (CH), 134.6 (C1′, 4JC−F = 2.6 Hz), 139.0 (CIV), 160.0 (CO), 161.4 (d, C4′, 1JC−F = 241.5 Hz), 164.4 (CO), 165.7 (CO). ESI-MS: m/z = 487 (M + H)+. HRMS calcd for C18H12F4N2O4, 396.07332; found, 396.07209. Anal. Calcd for C18H12F4N2O4: C, 54.55; H, 3.05; N, 7.07. Found: C, 54.63; H, 3.04, N, 7.05. N-(4-Fluorobenzyl)-7-cyano-2,3-dihydroxy-1-oxo-1,2-dihydroisoquinoline-4-carboxamide (89). Hydrogenation in methanol/EtOAc over 5% Pd/C (1 h); brown powder (35%), 100% enol form, mp 190−195 °C. 1H NMR (300 MHz, DMSO-d6): δ = 4.45 (s, 2H, CH2), 7.14 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.9 Hz), 7.36 (dd, 2H, H2′ and H6′, 3JH−H = 8.3 Hz, 4JH−F = 5.8 Hz), 7.51 (dd, 1H, H6, 3JH6−H5 = 9.1 Hz, 4JH6−H8 = 2.1 Hz), 8.22 (d, 1H, H8, 4JH8−H6 = 2.0 Hz), 9.44 (d, 1H, H5, 3JH5−H6 = 9.1 Hz), 10.35 (br s, 1H, NH), 10.10 (br s, 1H, OH). 13C NMR (75 MHz, DMSO-d6): δ = 42.0 (CH2), 88.1 (C4), 99.6 (C7), 115.5 (d, C3′ and C5′, 2JC−F = 21.1 Hz), 117.1 (CN), 120.8 (CIV), 124.6 (CH), 129.6 (d, C2′ and C6′, 3JC−F = 8.2 Hz), 132.0 (CH), 132.2 (CH), 137.6 (CIV), 142.0 (CIV), 157.3 (CO), 161.2 (CO), 161.5 (d, C4′, 1JC−F = 240.0 Hz), 168.6 (CO). ESI-MS: m/z = 354 (M + H)+. HRMS calcd for C18H12FN3O4; found: 353.08187. Anal. Calcd for C18H12FN3O4: C, 61.19; H, 3.42; N, 11.89. Found: C, 60.99; H, 3.40; N, 11.91. N-(4-Fluorobenzyl)-7-acetamido-2-hydroxy-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (90). Intermediate 65 was treated with boron trichloride as in synthesis of 81 to give a beige solid (43%), 100% keto form, mp 227−230 °C. 1H NMR (300 MHz, DMSO-d6): δ = 2.08 (s, 3H, CH3), 4.29 (dd, 1H, CH2, 2JH−H = 15.5 Hz, 3JH−H = 5.6 Hz), 4.30 (dd, 1H, CH2, 2JH−H = 15.5 Hz, 3JH−H = 5.6 Hz), 5.08 (s, 1H, H4), 7.17 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.9 Hz), 7.28−7.32 (m, 3H, H5, H2′, and H6′), 7.84 (dd, 1H, H6, 3JH6−H5 = 8.5 Hz, 4JH6−H8 = 2.2 Hz), 8.32 (d, 1H, H8, 4JH8−H6 = 2.2 Hz), 9.27 (t, 1H, NH, 3JH−H = 5.6 Hz), 10.27 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 24.5 (CH3), 42.4 (CH2), 55.2 (C4), 115.6 (d, C3′ and C5′, 2JC−F = 21.3 Hz), 117.9 (CH), 124.7 (CH), 126.2 (CIV), 127.6 (CH), 129.1 (CIV), 129.7 (d, C2′ and C6′, 3JC−F = 8.7 Hz), 135.2 (d, C1′, 4JC−F = 2.2 Hz), 139.7 (C7), 161.7 (C4′, 1JC−F = 241.6 Hz), 161.9 (CO), 165.2 (CO), 166.8 (CO), 169.3 (CO). ESI-MS: m/z = 386 (M + H)+. HRMS calcd for C19H16FN3O5, 385.10740; found, 385.10572. Anal. Calcd for C19H16FN3O5: C, 59.22; H, 4.19; N, 10.90. Found: C, 59.30; H, 4.20; N, 10.88. N-(4-Fluorobenzyl)-7-benzamido-2-hydroxy-1,3-dioxo-1,2,3,4tetrahydroisoquinoline-4-carboxamide (91). Intermediate 66 was hydrogenated over Pd/C 5% as insynthesis of 79 to give a gray powder (89%), mp 196−198 °C, 100% keto form. 1H NMR (300 MHz, DMSO-d6): δ = 4.32 (d, 1H, CH2, 2JH−H =15.2 Hz, 3JH−H = 5.7 Hz), 4.33 (d, 1H, CH2, 2JH−H = 15.2 Hz, 3JH−H = 5.7 Hz), 5.12 (s, 1H, H4), 7.18 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.9 Hz), 7.32 (dd, 2H, H2′ and H6′, 3JH−H = 8.9 Hz, 4JH−F = 5.6 Hz), 7.37 (d, 1H, H5, 3JH5−H6 = 8.5 Hz), 7.56−7.62 (m, 3H, HAr), 7.99−8.01 (m, 2H, HAr), 8.11 (dd, 1H, H6, 3JH6−H5 = 8.5 Hz, 4JH6−H8 = 2.2 Hz), 8.53 (d, 1H, H8, 4JH8−H6 = 2.2 Hz), 9.29 (t, 1H, NH, 3JH−H = 5.7 Hz), 10.56 (s, 1H, NH), 10.63 (s, 1H, OH). 13C NMR (75 MHz, DMSO-d6): δ = 42.4 (CH2), 55.3 (C4), 115.6 (d, C3′ and C5′, 2JC−F = 21.3 Hz), 119.4 (CH), 126.0 (CH), 126.2 (CIV), 127.5 (CH), 128.2 (2CH), 129.0 (2CH), 129.7 (d, C2′ and C6′,3JC−F = 8.2 Hz), 132.4 (CH), 134.1 (CIV), 134.8 (CIV), 135.2 (CIV), 139.6 (C7), 161.8 (C4′, 1JC−F = 241.0 Hz), 161.9 (CO), 165.3 (CO), 166.2 (CO), 166.8 (CO). ESI-MS: m/z = 448 (M + H)+. HRMS calcd for C24H18FN3O5, 447.12305; found, 447.12379. Anal. Calcd for C24H18FN3O5: C, 64.43; H, 4.06; N, 9.39. Found: C, 64.32; H, 4.07; N, 9.37. N-(4-Fluorobenzyl)-2-hydroxy-1,3-dioxo-7-picolinamido-1,2,3,4tetrahydroisoquinoline-4-carboxamide (92). Intermediate 67 was treated with boron trichloride as in synthesis of 81 to give a yellow powder (33%), mp 211−212 °C, 100% keto form. 1H NMR (300 MHz, DMSO-d6): δ = 4.31 (dd, 1H, CH2, 2JH−H = 15.6 Hz, 3JH−H = 5.6 Hz), 4.32 (dd, 1H, CH2, 2JH−H = 15.6 Hz, 3JH−H = 5.6 Hz), 5.14 (s, 1H, C4), 7.18 (t, 2H, C3′ and C5′, 3JH−H = 3JH−F = 8.6 Hz), 7.31 (dd, 2H, H2′ and H6′, 3JH−H = 8.6 Hz, 4JH−F = 5.7 Hz), 7.38 (d, 1H, HAr,

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JH−H = 8.3 Hz), 7.69−7.73 (m, 1H, HAr), 8.07−8.20 (m, 3H, HAr), 8.74−8.78 (m, 2H, HAr), 9.32 (t, 1H, NH, 3JH−H = 5.6 Hz), 11.02 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 42.4 (CH2), 55.3 (C4), 115.6 (d, C3′ and C5′, 2JC−F = 21.3 Hz), 119.6 (CH), 123.1 (CH), 126.2 (CIV), 126.4 (CH), 127.5 (CH), 127.6 (CH), 129.7 (d, C2′ and C6′, 3JC−F = 8.5 Hz), 130.1 (CIV), 135.3 (d, C1′, 4JC−F = 3.3 Hz), 138.7 (CH), 138.8 (C7), 149.0 (CH), 150.0 (C1″), 161.7 (d, C4′, 1JC−F = 241.0 Hz), 161.9 (CO), 163.4 (CO), 165.2 (CO), 166.8 (CO). ESIMS: m/z = 449 (M + H)+. HRMS calcd for C23H17FN4O5, 448.11830; found, 448.12016. Anal. Calcd for C23H17FN4O5: C, 61.61; H, 3.82; N, 12.49. Found: C, 61.70; H, 3.80; N, 12.53. N-(4-Fluorobenzyl)-2-hydroxy-1,3-dioxo-7-phenylacetamido1,2,3,4-tetrahydroisoquinoline-4-carboxamide (93). Intermediate 68 was hydrogenated over Pd/C 5% as in synthesis of 79 to give a gray solid (83%), mp 203−204 °C, 100% keto form. 1H NMR (300 MHz, DMSO-d6): δ = 3.67 (s, 2H, CH2), 4.29 (d, 2H, CH2, 3JH−H = 5.8 Hz), 5.08 (s, 1H, H4), 7.17 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.8 Hz), 7.25−7.35 (m, 8H, HAr, H5, H2′, and H6′), 7.87 (dd, 1H, H6, 3JH6−H5 = 8.4 Hz, 4JH6−H8 = 2.2 Hz), 8.33 (d, 1H, H8, 4JH8−H6 = 2.2 Hz), 9.25 (t, 1H, NH, 3JH−H = 5.8 Hz), 10.51 (s, 1H, NH), 10.61 (s, 1H, OH). 13C NMR (75 MHz, DMSO-d6): δ = 42.4 (CH2), 43.7 (CH2), 55.2 (C4), 115.6 (d, C3′ and C5′, 2JC−F = 20.7 Hz), 118.1 (CH), 124.8 (CH), 126.2 (CIV), 127.1 (CH), 127.7 (CH), 128.8 (2CH), 129.3 (CIV), 129.6 (2CH), 129.7 (d, C2′ and C6′, 3JC−F = 8.2 Hz), 135.2 (d, C1′, 4 JC−F = 2.7 Hz), 136.1 (CIV), 139.6 (CIV), 161.7 (d, C4′, 1JC−F = 241.1 Hz), 161.8 (CO), 165.2 (CO), 166.8 (CO), 170.0 (CO). ESI-MS: m/ z = 462 (M + H)+. HRMS calcd for C25H20FN3O5, 467.13870; found, 467.13733. Anal. Calcd for C25H20FN3O5: C, 65.07; H, 4.37; N, 9.11. Found: C, 65.19; H, 4.39; N, 9.09. N-(4-Fluorobenzyl)-2-hydroxy-1,3-dioxo-7-[2-(thiophen-2-yl)acetamido]-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (94). Intermediate 69 was treated with boron trichloride as in synthesis of 81 to give a green powder (61%), mp 183−186 °C, 100% keto form. 1H NMR (300 MHz, DMSO-d6): δ = 3.91 (s, 2H, CH2), 4.30 (dd, 1H, CH2, 2JH−H = 15.6 Hz, 3JH−H = 5.7 Hz), 4,31 (dd, 1H, CH2, 2JH−H = 15.6 Hz, 3JH−H = 5.7 Hz), 5.09 (s, 1H, C4), 6.99−7.00 (m, 2H, HAr), 7.17 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.8 Hz), 7.31 (dd, 2H, H2′ and H6′, 3JH−H = 8.6 Hz, 4JH−F = 5.1 Hz), 7.41−7.42 (m, 2H, HAr), 7.86 (d, 1H, HAr, 3JH−H = 8.6 Hz), 8.34 (s, 1H, H8), 9.27 (t, 1H, NH, 3 JH−H = 5.7 Hz), 10.56 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6): δ = 38.0 (CH2), 42.4 (CH2), 55.2 (C4), 115.5 (d, C3′ and C5′, 2JC−F = 21.3 Hz), 118.1 (CH), 124.9 (CH), 125.7 (CH), 126.2 (CIV), 127.0 (CH), 127.2 (CH), 128.9 (CH), 129.5 (CIV), 129.7 (d, C2′ and C6′, 3 JC−F = 8.2 Hz), 135.2 (d, C1′, 4JC−F = 2.7 Hz), 137.2 (CIV), 139.4 (CIV), 1613.7 (d, C4′, 1JC−F = 240.5 Hz), 161.8 (CO), 165.2 (CO), 166.8 (CO), 168.9 (CO). ESI-MS: m/z = 468 (M + H)+. HRMS calcd for C23H18FN3O5S, 467.09512; found, 467.09696. Anal. Calcd for C23H18FN3O5S: C, 59.10; H, 3.88; N, 8.99. Found: C, 58.97; H, 3.86; N, 8.98. N-Hexyl-2,3-dihydroxy-7-nitro-1-oxo-1,2-dihydroisoquinoline-4carboxamide (95). Intermediate 73 was treated with boron trichloride as in synthesis of 80 to give a yellow powder (20%), mp 114−116 °C, 100% enol form. 1H NMR (300 MHz, DMSO-d6): δ = 0.87 (m, 3H, CH3), 1.30 (m, 6H, 3CH2), 1.48 (m, 2H, CH2), 3.24 (t, 2H, CH2, 3 JH−H = 5.1 Hz), 7.99 (d, 1H, H6, 3JH6−H 5 = 9.5 Hz), 8.74 (s, 1H, H8), 9.23 (d, 1H, H5, 3JH5−H6 = 9.5 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 14.4 (CH3), 22.6 (CH2), 26.8 (CH2), 29.8 (CH2), 31.5 (CH2), 38.8 (CH2), 90.3 (C4), 116.4 (CIV), 124.1 (CH), 124.3 (CH), 124.6 (CH), 138.7 (CIV), 143.2 (CIV), 157.9 (CO), 161.0 (CO), 167.9 (CO). ESIMS: m/z = 350 (M + H)+. HRMS calcd for C16H19N3O6, 349.12739; found, 349.12564. Anal. Calcd for C16H19N3O6: C, 55.01; H, 5.48; N, 12.03. Found: C, 55.09; H, 5.50; N, 12.06. N-Phenyl-2,3-dihydroxy-7-nitro-1-oxo-1,2-dihydroisoquinoline-4carboxamide (96). Intermediate 74 was treated with boron trichloride as in synthesis of 80 to give a yellow powder (32%), mp 162−165 °C, 100% enol form. 1H NMR (300 MHz, DMSO-d6): δ = 6.98 (t, 1H, H4′, 3JH−H = 7.4 Hz), 7.29 (t, 2H, H3′ and H5′, 3JH−H = 7.6 Hz), 7.65 (d, 2H, H2′ and H6′, 3JH−H = 7.8 Hz), 8.07 (d, 1H, H6, 3JH6−H5 = 9.8 Hz), 8.79 (s, 1H, H8), 9.40 (d, 1H, H5, 3JH5−H6 = 9.8 Hz), 12.43 (s, 1H, OH). 13C NMR (75 MHz, DMSO-d6): δ = 90.3 (C4), 116.9 (C1′), 4657



Article

Gaussian 09 package.31 Our previous docking protocol that used the PDB 3S3M crystallographic structure was modified. The cocrystallized ligand was extracted and the protein was prepared by adding hydrogens and removing water molecules and irrelevant heteroatoms in Accelrys DS Visualizer 3.5.32 Magnesium cations were set to allow octahedral geometry. Docking calculations were carried out with the CCDC GOLD 5.2 docking suite.33 The active site was defined as a sphere containing all atoms within 15 Å of the X-ray ligand centroid. A dynamic distance constraint d was introduced between O11 and H27 of the mol2 ligand structure (1.0 Å < d < 2.4 Å), and a library of 34 rotamers was enabled for Arg329. The CHEMPLP fitness function34 was used with modified parameters. After manual editing of atom and bond types in mol2 files, both ligands were submitted to 600 docking runs with 0.75 Å RMSD clustering. The resulting docking poses were analyzed in DS Visualizer and selected poses were rendered with UCSF Chimera software35 after the method was validated by superimposing the best pose for 101 with its crystal structure extracted from PDB 4IKF. Integrase Inhibition. To determine the susceptibility of HIV-1 integrase enzyme to different compounds, an enzyme-linked immunosorbent assay (ELISA) was used. This assay uses a substrate in which one oligonucleotide (5′-ACTGCTAGAGATTTTCCACACTGACTAAAAGGGTC-3′) is labeled with biotin on the 3′ end and the other oligonucleotide is labeled with digoxigenin at the 5′ end. For the overall integration assay, the second 5′-digoxigenin-labeled oligonucleotide is 5′-GACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGT-3′. For the strand transfer assay, a precleaved oligonucleotide substrate [the second oligonucleotide lacks GT (shown in boldface type) at the 3′ end] was used. The integrase was diluted in 750 mM NaCl, 10 mM Tris (pH 7.6), 10% glycerol, 1 mM βmercaptoethanol, and 0.1 mg/mL bovine serum albumin. To perform the reaction, 4 μL of diluted integrase (corresponds to a concentration of wild-type integrase of 1.6 μM) and 4 μL of annealed oligonucleotides (7 nM) were added in a final reaction volume of 40 μL containing 10 mM MgCl2, 5 mM dithiothreitol, 20 mM N-(2hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES, pH 7.5), 0.5% poly(ethylene glycol), and 15% DMSO. As such, the final concentration of integrase in this assay was 160 nM. The reaction was carried out for 1 h at 37 °C. The reaction products were denatured with 30 mM NaOH and detected by ELISA on avidin-coated plates. Reverse Transcriptase RNase H Assay. The substrate for RNase H activity was prepared as previously described.36 Escherichia coli RNA polymerase used single-stranded calf thymus DNA as a template to synthesize complementary 3H-labeled RNA. For RNase H activity, recombinant HIV-1 RT37 (4.5 pmol) was incubated with the appropriate compound for 10 min at 37 °C in 20 μL. The components of the incubation mixture were added to reach a final concentration of 50 mM Tris-HCl (pH 8.0), 10 mM dithiothreitol, 6 mM MgCl2, 80 mM KCl, and the labeled nucleic acid duplex (20 000 cpm) in a final volume of 50 μL. After incubation for 10 min at 37 °C, the reaction was stopped by addition of 1 mL of cold 10% trichloroacetic acid (TCA) containing 0.1 M sodium pyrophosphate, the acid-precipitable material was collected on nitrocellulose filters and washed, the radioactivity was determined, and the radioactivity released from the hybrid was determined by subtraction from the undigested hybrid control. Time of Addition. MT-4 cells (100 000 per well) in a 96-well microtiter plate were infected with HIV-IIIB at a multiplicity of infection of 0.7. Compounds were added at different time points after infection (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, and 25 h) as described previously.38 Viral p24 antigen production was determined 30 h postinfection by ELISA (Innogenetics, Belgium). Compounds were added at 50 and 100 times their EC50 as determined by the drug susceptibility assay (MTT/MT-4). In Vitro Anti-HIV and Drug Susceptibility Assays. The inhibitory effect of antiviral drugs on the HIV-1-induced cytopathic effect (CPE) in human lymphocyte MT-4 cell culture was determined by the MT-4/MTT assay. MT-4 cells were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institutes of Health. The cells were grown in RPMI 1640 medium

119.6 (2CH), 119.6 (CH), 124.2 (2CH), 125.0 (CH), 129.2 (2CH), 139.1 (CIV), 140.7 (CIV), 143.6 (C7), 158.3 (CO), 161.6 (CO), 166.3 (CO). ESI-MS: m/z = 342 (M + H)+. HRMS calcd for C16H11N3O6, 341.06479; found, 341.06342. Anal. Calcd for C16H11N3O6: C, 56.31; H, 3.25; N, 12.31. Found: C, 56.17; H, 3.24; N, 12.34. N-Benzyl-2,3-dihydroxy-7-nitro-1-oxo-1,2-dihydroisoquinoline-4carboxamide (97). Intermediate 75 was treated with boron trichloride as in synthesis of 80 to give a yellow powder (25%), mp 164−166 °C, 100% enol form. 1H NMR (300 MHz, DMSO-d6): δ = 4.49 (s, 2H, CH2), 7.25−7.34 (m, 5H, HAr), 8.02 (d, 1H, H6, 3JH6−H5 = 9.1 Hz), 8.76 (s, 1H, H8), 9.24 (d, 1H, H5, 3JH5−H6 = 9.1 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 42.6 (CH2), 90.2 (C4), 116.5 (CIV), 124.1 (CH), 124.2 (CH), 124.7 (CH), 127.0 (CH), 127.7 (2CH), 128.8 (2CH), 138.7 (CIV), 140.9 (CIV), 143.4 (C7), 158.0 (CO), 161.1 (CO), 167.9 (CO). ESI-MS: m/z = 356 (M + H)+. HRMS calcd for C 17H 13 N 3 O6 , 355.08044; found, 355.08123. Anal. Calcd for C17H13N3O6: C, 57.47; H, 3.69; N, 11.83. Found: C, 57.64; H, 3.67; N, 11.85. N-(4-Fluorophenyl)-2,3-dihydroxy-7-nitro-1-oxo-1,2-dihydroisoquinoline-4-carboxamide (98). Intermediate 76 was treated with boron trichloride as in synthesis of 80 to give a brown powder (40%), mp 178−180 °C; 100% enol form. 1H NMR (300 MHz, DMSO-d6): δ = 7.13 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 8.3 Hz), 7.66 (dd, 2H, H2′ and H6′, 3JH−H = 8.3 Hz, 4JH−F = 5.3 Hz), 8.06 (d, 1H, H6, 3JH6−H5 = 9.8 Hz), 8.78 (s, 1H, H8), 9.40 (d, 1H, H5, 3JH5−H6 = 9.8 Hz), 12.46 (s, 1H, OH). 13C NMR (75 MHz, DMSO-d6): δ = 90.1 (C4), 115.7 (d, C3′ and C5′, 2JC−F = 21.8 Hz), 116.9 (CIV), 121.2 (d, C2′ and C6′, 3JC−F = 6.5 Hz), 124.2 (2CH), 125.0 (CH), 137.1 (CIV), 139.0 (CIV), 143.6 (C7), 156.2 (CO), 159.3 (CO), 160.1 (d, C4′, 1JC−F = 259.0 Hz), 166.3 (CO). ESI-MS: m/z = 360 (M + H)+. HRMS calcd for C16H10FN3O6, 359.05536; found, 359.05353. Anal. Calcd for C16H10FN3O6: C, 53.49; H, 2.81; N, 11.70. Found: C, 53.63; H, 2.81; N, 11.73. N-(4-Fluorophenethyl)-2,3-dihydroxy-7-nitro-1-oxo-1,2-dihydroisoquinoline-4-carboxamide (99). Intermediate 77 was treated with boron trichloride as in synthesis of 80 to give a yellow powder (18%), mp 153 °C, 100% enol form. 1H NMR (300 MHz, DMSO-d6): δ = 2.79 (t, 2H, CH2, 3JH−H = 7.3 Hz), 3.48 (t, 2H, CH2, 3JH−H = 7.3 Hz), 7.11 (t, 2H, H3′ and H5′, 3JH−H = 3JH−F = 7.5 Hz), 7.30 (dd, 2H, H2′ and H6′, 3JH−H = 7.3 Hz, 4JH−F = 6.0 Hz), 7.97 (d, 1H, H6, 3JH6−H5 = 9.8 Hz), 8.73 (s, 1H, H8), 9.35 (d, 1H, H5, 3JH5−H6 = 9.8 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 34.9 (CH2), 40.7 (CH2), 89.9 (C4), 115.2 (d, C3′ and C5′, 2JC−F = 20.7 Hz), 116.3 (CIV), 124.0 (CH), 124.6 (CH), 128.2 (CH), 130.4 (d, C2′ and C6′, 3JC−F = 7.6 Hz), 136.2 (CIV), 136.5 (CIV), 142.8 (C7), 157.9 (CO), 160.4 (CO), 160.9 (d, C4′, 1JC−F = 242.7 Hz), 167.7 (CO). ESI-MS: m/z = 388 (M + H)+. HRMS calcd for C18H14FN3O6, 387.08666; found, 386.08881. Anal. Calcd for C18H14FN3O6: C, 55.82; H, 3.64; N, 10.85. Found: C, 55.66; H, 3.63; N, 10.87. N-(4-Methoxybenzyl)-2,3-dihydroxy-7-nitro-1-oxo-1,2-dihydroisoquinoline-4-carboxamide (100). Intermediate 78 was treated with boron trichloride as in synthesis of 80 to give an orange powder (40%), mp 137 °C, 100% enol form. 1H NMR (300 MHz, DMSO-d6): δ = 3.73 (s, 3H, OCH3), 4.40 (s, 2H, CH2), 6.89 (d, 2H, HAr, 3JH−H = 8.1 Hz), 7.26 (d, 2H, HAr, 3JH−H = 8.1 Hz), 8.00 (d, 1H, H6, 3JH6−H5 = 9.2 Hz), 8.75 (s, 1H, H8), 9.28 (d, 1H, H5, 3JH5−H6 = 9.2 Hz). 13C NMR (75 MHz, DMSO-d6): δ = 42.1 (CH2), 55.5 (OCH3), 90.2 (C4), 114.2 (C3′ and C5′), 116.6 (CIV), 124.1 (CH), 124.2 (CH), 124.7 (CH), 129.1 (C2′ and C6′), 132.6 (CIV), 138.8 (CIV), 143.2 (CIV), 158.0 (CO), 158.5 (CO), 160.9 (CO), 167.8 (CO). ESI-MS: m/z = 386 (M + H)+. HRMS calcd for C18H15N3O7, 385.09100; found, 385.08994. Anal. Calcd for C18H15N3O7: C, 56.11; H, 3.92; N, 10.91. Found: C, 56.20; H, 3.92; N, 10.94. Docking Procedure. In a previous report, we showed by NMR that the 2-hydroxyisoquinoline-1,3-(2H,4H)-dione scaffold complexes magnesium as the enol or enolate form.19 Predictions of the ionization state by use of the SPARC online calculator30 indicate that such enols are deprotonated in aqueous media at physiological pH in the presence of magnesium cations; hence we modeled our ligands as the dianionic enolate form. Compound 80 and 101 were thus created and their geometry minimized at the HF/3-21G level by use of the 4658



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supplemented with 10% fetal calf serum (FCS) and 20 μg/mL gentamicin (RPMI-complete). This assay is based on the reduction of the yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by mitochondrial dehydrogenase of metabolically active cells to a blue formazan derivative, which can be measured spectrophotometrically. The 50% cell culture infective dose (CCID50) of the HIV-1 (IIIB) strain was determined by titration of the virus stock by use of MT-4 cells. The origin of HIV-1 strain IIIB has been described previously.39 For the drug susceptibility assays, MT-4 cells were infected with 100−300 times CCID50 of the virus stock in the presence of 5-fold serial dilutions of the antiviral drugs. The concentration of various compounds achieving 50% protection against the CPE of the different HIV strains, which is defined as EC50, was determined. In parallel, 50% cytotoxic concentration (CC50) was determined. ADMETox Studies. Aqueous solubility (in phosphate-buffered saline, PBS, pH 7.4; Cerep catalogue reference 0435), partition coefficient (log D, n-octanol/PBS, pH 7.4; Cerep catalogue reference 0417), human plasma protein binding (Cerep catalogue reference 2194), A-B permeability coefficient (Papp, Caco-2 cells, pH 6.5/7.4; Cerep catalogue reference 3318), inhibition of P-gp efflux (MDR1− MDCKII, calcein AM substrate; Cerep catalogue reference 1324), metabolic stability in human liver microsomes (Cerep catalogue reference 0416), inhibition of CYP1A2 (recombinant, CEC substrate, Cerep catalogue reference 0389), inhibition of CYP2C9 (recombinant, MFC substrate, Cerep catalogue reference 0412), inhibition of CYP2C19 (recombinant, CEC substrate, Cerep catalogue reference 0390), inhibition of CYP2D6 (recombinant, MFC substrate, Cerep catalogue reference 1338), inhibition of CYP3A4 (recombinant, BFC substrate, Cerep catalogue reference 0391), cardiac toxicity (hErg, automated patch clamp, Cerep catalogue reference 2245), in vitro cytotoxicity panel (cell number, intracellular free calcium, nuclear size, membrane permeability, and mitochondrial membrane potential; http://www.cerep.fr/cerep/users/pages/downloads/Documents/ Marketing/Pharmacology%20&%20ADME/OTP/CytotoxicityPanel. pdf) were determined in standard assays by Cerep, France (www. cerep.fr).

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NNRTI, nonnucleoside reverse transcriptase inhibitor; 3′-P, 3′processing; Papp, apparent permeability coefficient; PI, protease inhibitor; PFV, prototype foamy virus; RAL, raltegravir; RT, reverse transcriptase; SAR, structure−activity relationship; ST, strand transfer; THF, tetrahydrofuran; TOA, time of addition

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REFERENCES

(1) World Health Organization. GLOBAL HIV/AIDS RESPONSE: Epidemic Update and Health Sector Progress toward Universal Access. Progress Report 2011; World Health Organization, Geneva, Switzerland, 2011. (2) Mehellou, Y.; De Clercq, E. Twenty-six years of anti-HIV drug discovery: where do we stand and where do we go? J. Med. Chem. 2010, 53, 521−538. (3) McColl, D. J.; Chen, X. Strand transfer inhibitors of HIV-1 integrase: bringing IN a new era of antiretroviral therapy. Antiviral Res. 2010, 85, 101−118. (4) Summa, V.; Petrocchi, A.; Bonelli, F.; Crescenzi, B.; Donghi, M.; Ferrara, M.; Fiore, F.; Gardelli, C.; Paz, O. G.; Hazuda, D. J.; Jones, P.; Kinzel, O.; Laufer, R.; Monteagudo, E.; Muraglia, E.; Nizi, E.; Orvieto, F.; Pace, P.; Pescatore, G.; Scarpelli, R.; Stillmock, K.; Witmer, M. V.; Rowley, M. Discovery of raltegravir, a potent, selective orally bioavailable HIV-integrase inhibitor for the treatment of HIV-AIDS infection. J. Med. Chem. 2008, 51, 5843−5855. (5) Wainberg, M. A.; Zaharatos, G. J.; Brenner, B. G. Development of antiretroviral drug resistance. N. Engl. J. Med. 2011, 365, 637−646. (6) Marcelin, A. G.; Ceccherini Silberstein, F.; Perno, C. F.; Calvez, V. Resistance to novel drug classes. Curr. Opin. HIV AIDS 2009, 4, 531−537. (7) Marchand, C. The elvitegravir quad pill: the first once-daily dualtarget anti-HIV tablet. Expert Opin. Invest. Drugs 2012, 21, 901−904. (8) Schrijvers, R.; Debyser, Z. Combinational therapies for HIV: a focus on EVG/COBI/FTC/TDF. Expert Opin. Pharmacother. 2012, 13, 1969−1983. (9) Shimura, K.; Kodama, E.; Sakagami, Y.; Matsuzaki, Y.; Watanabe, W.; Yamataka, K.; Watanabe, Y.; Ohata, Y.; Doi, S.; Sato, M.; Kano, M.; Ikeda, S.; Matsuoka, M. Broad antiretroviral activity and resistance profile of the novel human immunodeficiency virus integrase inhibitor Elvitegravir (JTK-303/GS-9137). J. Virol. 2008, 82, 764−774. (10) Menendez-Arias, L. Molecular basis of human immunodeficiency virus type 1 drug resistance: overview and recent developments. Antiviral Res. 2013, 98, 93−120. (11) Garrido, C.; Soriano, V.; Geretti, A. M.; Zahonero, N.; Garcia, S.; Booth, C.; Guttierez, F.; Viciana, I.; de Mendoza, C. Resistance associated mutations to dolutegravir (S/GSK1349572) in HIVinfected patients: impact of HIV subtypes and prior raltegravir experience. Antiviral Res. 2011, 90, 164−167. (12) Canducci, F.; Ceresola, E. R.; Boeri, E.; Spagnuolo, V.; Cossarini, F.; Castagna, A.; Lazzarin, A.; Clementi, M. Cross-resistance profile of the novel integrase inhibitor dolutegravir (S/GSK1349572) using clonal viral variants selected in patients failing raltegravir. J. Infect. Dis. 2011, 204, 1811−1815. (13) Ballantyne, A. D.; Perry, C. M. Dolutegravir: first global approval. Drugs 2013, 73, 1627−1637. (14) Tintori, C.; Demeulemesster, J.; Franchi, L.; Massa, S.; Debyser, Z.; Christ, F.; Botta, M. Discovery of small molecule HIV-1 integrase dimerization inhibitors. Bioorg. Med. Chem. Lett. 2012, 22, 3109−3114. (15) Christ, F.; Shaw, S.; Demeulemeester, J.; Desimmie, B. A.; Marchand, A.; Butler, S.; Smets, W.; Chaltin, P.; Westby, M.; Debyser, Z.; Pickford, C. Small-molecule inhibitors of the LEDGF/p75 binding site of integrase block HIV replication and modulate integrase multimerization. Antimicrob. Agents Chemother. 2012, 56, 4365−4374. (16) Christ, F.; Voet, A.; Marchand, A.; Nicolet, S.; Desimmie, B. A.; Marchand, D.; Bardiot, D.; Van der Veken, N. J.; Van Remoortel, B.; Strelkov, S. V.; De Mayer, M.; Chaltin, P.; Debyser, Z. Rational design of small-molecule inhibitors of the LEDGF/p75-integrase interaction and HIV replication. Nat. Chem. Biol. 2010, 6, 442−448.

AUTHOR INFORMATION

Corresponding Author

*Telephone +33 320 33 72 31; fax +33 320 33 63 09; e-mail [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by grants from le Ministère de l’Enseignement Supérieur et de la Recherche Française and l’Agence Nationale de la Recherche contre le Sida (ANRS). Experiments at KU Leuven were funded by the FP7 project CHAARM and the IWT.

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ABBREVIATIONS USED ADMETox, adsorption, distribution, metabolism, excretion, and toxicity; AZT, zidovudine; BOP, (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; CRI, coreceptor inhibitor; EDG, electron-donating group; EWG, electron-withdrawing group; FDA, Food and Drug Administration; FI, fusion inhibitor; HAART, highly active antiretroviral therapy; hERG, human ether-a-go-go-related gene potassium channel 1; HID, 2-hydroxyisoquinoline-1,3(2H,4H)dione; HIV-1, human immunodeficiency virus type 1; IN, integrase; INI, integrase inhibitor; INSTI, integrase strand transfer inhibitor; LDA, lithium diisopropyl amide; LEDGIN, inhibitor of lens epithelium-derived growth factor binding site in integrase; NRTI, nucleoside reverse transcriptase inhibitor; 4659



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(17) Engelman, A.; Kessl, J. J.; Kvaratskhelia, M. Allosteric inhibition of HIV-1 integrase activity. Curr. Opin. Chem. Biol. 2013, 17, 339−345. (18) Parkes, K. E. B.; Ermert, P.; Fassler, J.; Ives, J.; Martin, J. A.; Merrett, J. H.; Obrecht, D.; Williams, G.; Klumpp, K. Use of a pharmacophore model to discover a new class of influenza endonuclease inhibitors. J. Med. Chem. 2003, 46, 1153−1164. (19) Billamboz, M.; Bailly, F.; Barreca, M. L.; De Luca, L.; Mouscadet, J. F.; Calmels, C.; Andréola, M. L.; Christ, F.; Debyser, Z.; Witvrouw, M.; Cotelle, P. Design, synthesis and biological evaluation of a series of 2-hydroxyisoquinoline-1,3(2H,4H)-diones as dual inhibitors of human immunodeficiency virus type 1 integrase and reverse transcriptase RNase H domain. J. Med. Chem. 2008, 51, 7717− 7730. (20) Billamboz, M.; Bailly, F.; Lion, C.; Calmels, C.; Andréola, M. L.; Witvrouw, M.; Christ, F.; Debyser, Z.; De Luca, L.; Chimirri, A.; Cotelle, P. 2-Hydroxyisoquinoline-1,3(2H,4H)-diones as inhibitors of HIV-1 integrase and reverse transcriptase RNase H domain: influence of the alkylation of position 4. Eur. J. Med. Chem. 2011, 46, 535−546. (21) Hare, S.; Gupta, S. S.; Valkov, E.; Engelman, A.; Cherepanov, P. Retroviral intasome assembly and inhibition of DNA strand transfer. Nature 2010, 464, 232−236. (22) Billamboz, M.; Suchaud, V.; Bailly, F.; Lion, C.; Christ, F.; Debyser, Z.; Cotelle, P. 4-Substituted 2-hydroxyisoquinoline-1,3(2H,4H)-diones as a novel class of HIV-1 integrase inhibitors. ACS Med. Chem. Lett. 2013, 4, 606−611. (23) Desimmie, B. A.; Demeulemeester, J.; Suchaud, V.; Taltynov, O.; Billamboz, M.; Lion, C.; Bailly, F.; Strelkov, S. V.; Debyser, Z.; Cotelle, P.; Christ, F. 2-Hydroxyisoquinoline-1,3(2H,4H)-diones (HIDs), novel inhibitors of HIV integrase with a high barrier to resistance. ACS Chem. Biol. 2013, 8, 1187−1194. (24) Billamboz, M.; Bailly, F.; Cotelle, P. Facile synthesis of 4alkoxycarbonylisoquinoline-1,3-diones and 5-alkoxycarbonyl-2-benzazepine-1,3-diones via a mild alkaline cyclization. J. Heterocycl. Chem. 2009, 46, 392−398. (25) Malamas, M. S.; Hohman, T. C.; Millen, J. Novel spirosuccinimide aldose reductase inhibitors derived from isoquinoline-1,3-diones: 2-[(4-bromo-2-fluorophenyl)methyl]-6-fluorospiro[isoquinoline-4(1H),3′-pyrrolidine]-1,2′,3,5′(2H)-tetrone and congeners. J. Med. Chem. 1994, 37, 2043−2058. (26) Bruggink, A.; Mc Killop, A. A study of the copper-catalysed direct arylation of β-dicarbonyl compounds with 2-bromobenzoic acids. Tetrahedron 1975, 31, 2607−2619. (27) Hazuda, D. J.; Felock, P.; Witmer, M.; Wolfe, A.; Stillmock, K.; Grobler, J. A.; Espeseth, A.; Gabryelski, L.; Schleif, W.; Blau, C.; Miller, M. D. Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells. Science 2000, 287, 646−650. (28) Hare, S.; Smith, S. J.; Metifiot, M.; Jaxa-Chamiec, A.; Pommier, Y.; Hughes, S. H.; Cherepanov, P. Structural and functional analyses of the second-generation integrase strand transfer inhibitor dolutegravir (S/GSK1349572). Mol. Pharmacol. 2011, 80, 565−572. (29) Allen, F. H.; Baalham, C. A.; Lommerse, J. P. M.; Raithby, P. R.; Sparr, E. Hydrogen-bond acceptor properties of nitro-O atoms: a combined crystallographic database and ab initio molecular orbital study. Acta Crystallogr. 1997, B53, 1017−1024. (30) Hilal, S. H.; Karickhoff, S. W.; Carreira, L. A. A rigorous test for SPARC’s chemical reactivity models: estimation of more than 4300 ionization pKas. Quant. Struct.-Act. Relat. 1995, 14, 348−355. (31) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.;

Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision D.01; Gaussian Inc., Wallingford, CT, 2009. (32) Visualizer DS, Release 3.5; Accelrys Software Inc., San Diego, CA, 2012. (33) (a) Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. J. Mol. Biol. 1997, 267, 727−748. (b) Cole, J. C.; Nissink, J. W. M.; Taylor, R. Virtual Screening in Drug Discovery; Shoichet, B., Alvarez, J., Eds.; CRC Press, Taylor & Francis Group: Boca Raton, FL, 2005. (34) Korb, O.; Stützle, T.; Exner, T. Empirical scoring functions for advanced protein-ligand docking with PLANTS. J. Chem. Inf. Model. 2009, 49, 84−96. (35) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605−1612. (36) Moelling, K.; Schulze, T.; Diringer, H. Inhibition of human immunodeficiency virus type 1 RNase H by sulfated polyanions. J. Virol. 1989, 63, 5489−5491. (37) Dufour, E.; Reinbolt, J.; Castroviejo, M.; Ehresmann, B.; Litvak, S.; Tarrago-Litvak, L.; Andreola, M. L. Cross-linking localization of a HIV-1 reverse transcriptase peptide involved in the binding of primer tRNALys3. J. Mol. Biol. 1999, 285, 1339−1346. (38) Pauwels, R.; Andries, K.; Desmyter, J.; Schols, D.; Kulda, M. J.; Breslin, H. J.; Raeymaeckers, A.; Van Gelder, J.; Woestenborghs, R.; Heykants, J. Potent and selective inhibition of HIV-1 replication in vitro by a novel series of TIBO derivatives. Nature 1990, 343, 470− 474. (39) Adachi, A.; Gendelman, H. E.; Koenig, S.; Folks, T.; Willey, R.; Rabson, A.; Martin, M. A. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J. Virol. 1986, 59, 284−291.

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