Investigation of a novel series of 2-hydroxyisoquinoline-1,3(2 H,4 H)-diones as human immunodeficiency virus type 1 integrase inhibitors

Article

pubs.acs.org/jmc

Investigation of a Novel Series of 2‑Hydroxyisoquinoline-

1,3(2H,4H)‑diones as Human Immunodeﬁciency Virus Type 1

Integrase Inhibitors

Virginie Suchaud,^†,‡Fabrice Bailly,*^,†,‡Cedric Lion,^†,‡Christina Calmels,^∥Marie-Line Andreola,^∥

́

Frauke Christ,^§Zeger Debyser,^§and Philippe Cotelle^†,‡

^†Universite

^‡Equipe d'Accueil de Chimie Molec

́

Lille Nord de France, F-59000 Lille, France

́

ulaire et Formulation, Universite 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 Pathogen

F-33076 Bordeaux, France

́ ́ ́ ́

icite, UMR 5234 CNRS, Universite Bordeaux Segalen, 146 Rue Leo Saignat,

ABSTRACT: We report herein further insight into the biological activities displayed by a series of 2-hydroxyisoquinoline-

1,3(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 anti-

HIV potencies, with advantageous therapeutic indexes going close to those of the clinically used raltegravir and retained

potencies against a panel of IN mutants.

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 drug-

resistant 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

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 syn-

drome (AIDS) and 2.5 million (2.2−2.8 million) new

infections.¹Since 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

Received: January 21, 2014

Published: May 3, 2014

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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.³Raltegravir (Figure 1) resulted from intensive

States.¹³Nevertheless, 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.¹⁵⁻¹⁷For our part, our team has been working for

several years on the development of 2-hydroxyisoquinoline-

1,3(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.¹⁸A pioneer series was

constituted by compounds variously substituted at position 7

(Chart 1) with IC₅₀values against integrase ranging from 0.09

to 18.8 μM. Two hits were discovered with submicromolar IC₅₀

values (R⁷= NHCOCH₂Ph, IC₅₀= 90 nM; R⁷

=

NHCOCH₂(4-F)Ph, IC₅₀= 130 nM). Unfortunately these

compounds exhibited high cellular toxicity, which limited their

application as antiviral agents.¹⁹Second, 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.²⁰Then 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.²¹This 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.²²A 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.²³Compound 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.²³All

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

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.^3,4It 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).^5,6Elvitegravir (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 pharmacoen-

hancer cobicistat and HIV-1 RT by the two NRTIs

emtricitabine and tenofovir disoproxil fumarate.^7,8Although

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.^9,10Dolutegravir (S/GSK1349572; Figure 1), although

apparently superior to raltegravir, exhibits signiﬁcant resistance

overlap¹⁰⁻¹²and has been recently approved in the United

a

Chart 1. Synoptic View of Hit-to-Lead Optimization Process

a

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

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a

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

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%).

a

Scheme 2. Synthesis of Precursor 4

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%).

comparative evaluation of a novel series of 2-hydroxyisoquino-

line-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.

attempts to use the crude organic solution for the following

coupling step with O-benzylhydroxylamine were also un-

successful. 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

a

Scheme 3. Synthesis of Precursors 9−16

CHEMISTRY

■

Scheme 1 shows the synthetic route derived from our

previously reported method,²²which was used for preparation

of target 2-hydroxyisoquinoline-1,3(2H,4H)-diones. N-Benzy-

loxymalonamides II (4 and 29−42) were cyclized in mild

alkaline conditions to aﬀord the corresponding 2-benzyloxy-4-

methoxycarbonylisoquinoline-1,3(2H,4H)-dione ester precur-

sors 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-1,3-

diones²⁴and was used only for the synthesis of 4 (Scheme 2).

4-Hydroxyhomophthalic acid 1,¹⁹obtained 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).

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.^25,26This

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

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

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a

Scheme 4. Synthesis of Precursors 20−28

a

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%).

tetraﬂuoroborate, cuprous halides, and cyanide to aﬀord the 5-

halogeno and 5-cyano derivatives 20−23 (Scheme 4). Reaction

of aniline 19 with several acid chlorides also gave amides 24−

28, where R₅is 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-benzyloxymono-

malonamide derivatives 29−42 by reﬂux with O-benzylhydrox-

ylamine 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 p-

ﬂuorobenzylaminocarbonyl 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-hydroxyisoquinoline-

1,3(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).

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).

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 IC₅₀values similar to our previously

reported hit 101 (IC₅₀= 56 nM).^22,23Regarding 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).

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)²⁷and was later shown to

invade a pocket at the protein−DNA interface that is natively

occupied by the 3′-terminal base of viral DNA.²¹In 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,²²the

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Table 1. Inhibition of HIV-1 IN and RNase H Activities, Antiviral Activity, and Cytotoxicity of Substituted 2-

a

Hydroxyisoquinoline-1,3(2H,4H)-diones 79−101 and Raltegravir

a

b

Concentration required to inhibit by 50% the in vitro overall integrase activity. Eﬀective concentration required to reduce HIV-1-induced

c

d

cytopathic eﬀect by 50% in MT-4 cells. Cytotoxic concentration required to reduce by 50% MT-4 cell viability. Therapeutic index, deﬁned by

CC₅₀/EC₅₀ratio. Percentage inhibition of HIV-1 RNase H activity at 10 μM concentration. Major tautomeric form present in DMSO solution.

e

f

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

(IC₅₀= 0.36 μM),²²with an IC₅₀value 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

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detection of any antiviral activity in cell culture. This

phenomenon was observed before with pioneer 7-substituted

compounds lacking the carboxamido side chain¹⁸at 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 EC₅₀values 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 EC₅₀values 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.²²The

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 EC₅₀values (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]

previous docking protocol,²²developed on the 3S3M crystallo-

graphic structure of dolutegravir in the PFV IN intasome active

site²⁸by use of the CCDC Gold software³³and the CHEMPLP

scoring function,³⁴was 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-

Figure 2. Proposed binding modes A and B of our 2-hydroxyisoquino-

line-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.²²Whereas conformer A

retains it, conformer B requires the rupture of this intra-

molecular 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)²³identiﬁed 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 function³⁴were also

optimized in order to prevent aberrant binding modes and

improve cluster size.

inhibited HIV-1 replication in the micromolar range (EC₅₀

=

0.95 μM). In comparison with our precedent hit compound

101 (EC₅₀= 2.34 μM),^22,23the antiviral activities of 80, 81, and

96−98 are at least 1 log unit lower and 80, with an EC₅₀value

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 (21−86-

fold)²²but 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

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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).

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).

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-

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.

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.

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 EC₅₀values 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 cross-

resistance 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 EC₅₀values of 80 and

101²³tested 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 EC₅₀for 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,²⁹the 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

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Table 2. Cross-Resistance (x-fold Change in EC₅₀) of 101²³

and 80 with Raltegravir

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

Cerivastatin

a

virus strain

E92Q

101

integrase 80

RAL

compd

1.0 μM

30.0 μM

100.0 μM

IC₅₀(μM)

b

1

2

6

5

9

Cytotoxicity (Cell Number)

Q148H

80

−20.1

12.6

nd

−7.4

nd

c

N155H

7

cerivastatin

nd

0.96

d

G140S/Q148H

362

Intracellular Free Calcium

a

80

−1.3

nd

18.8

nd

18.3

nd

Raltegravir.

cerivastatin

0.34

b

Nuclear Size

23.8

with raltegravir, which displayed a 7- and 362-fold change in

EC₅₀for the same mutants (Table 2).

80

2.0

nd

22.1

nd

cerivastatin

nd

0.13

d

Preliminary ADMETox Evaluation of 80. The discrep-

ancy 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 P_app(Caco-2 cells, pH 6.5/7.4)

below 0.1 × 10⁻⁶cm/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 P450-

mediated 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 IC₅₀values for each test.

Membrane Permeability

80

0

23.9

nd

6.7

nd

cerivastatin

nd

0.80

b

Mitochondrial Membrane Potential

80

8.4

nd

−23.8

nd

22.4

nd

cerivastatin

0.60

a

b

Concentration required to inhibit a−e. Percentage reduction relative

to untreated control. Not determined. Percentage increase relative to

untreated control.

c

d

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 IC₅₀value of 24 nM.

CONCLUSIONS

■

The 2-hydroxyisoquinoline-1,3(2H,4H)-dione scaﬀold previ-

ously 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 anti-

HIV 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

c

compd

CYP1A2

CYP2C9

CYP2C19

CYP2D6

CYP3A4

P-gp inhibition

a

b

80

1.4

76.5

11.4

nd

106^o

−5.1, −3.2, 7.6

d

furafyline

1.4

nd

d

sulfaphenazole

tranylcypromine

quinidine

nd

0.18

nd

d

nd

3.1

nd

d

nd

0.017

nd

d

ketoconazole

nd

0.28

a

b

c

Percentage inhibition of control values at a 10.0 μM dose. Not determined. P-gp inhibition at 1.0, 30.0, and 100.0 μM in MDR1−MDCKII

d

system. IC₅₀values (micromolar).

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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 Na₂SO₄and

concentrated in vacuo. After column chromatography of the residue

(eluent petroleum ether/AcOEt 70/30), 4 was obtained as a yellow oil

integrase inhibitor retained antiviral eﬃcacies against ralte-

gravir-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.

1

(40%). H 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,

3

4

CH), 7.03 (dd, 1H, H₄, J_H4−H3= 8.5 Hz, J_H4−H6= 2.6 Hz), 7.23 (s,

5H, H_Ar), 7.39 (d, 1H, H₆, ⁴J_H6−H4= 2.6 Hz), 7.50 (d, 1H, H₃, ³J_H3−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 (C_IV), 128.4 (2CH), 128.6 (CH),

129.3 (2CH), 129.9 (C_IV), 133.0 (CH), 135.2 (C_IV), 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-dimethoxy-

1,3-dioxopropan-2-yl)benzoic Acid (9). A solution of 2-bromo-4-

ﬂuorobenzoic 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 MgSO₄and 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

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 chromato-

graphic analyses were performed on plastic sheets precoated with silica

gel 60F254 (Merck). SiO₂, 40−63 mesh (Merck), or 30 μm HP-

Silprepacked 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) measure-

ments 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.

1

127−130 °C. H NMR (300 MHz, acetone-d₆): δ = 3.61 (s, 6H,

3

2OCH₃), 5.84 (s, 1H, CH), 7.21 (dd, 1H, H₃, J_H3−F= 10.1 Hz,

⁴J_H3−H5= 2.6 Hz), 7.28 (ddd, 1H, H₅, J_H5−F= 8.7 Hz, J_H5−H6= 8.1

Hz, ⁴J_H5−H3= 2.7 Hz), 8.19 (dd, 1H, H₆, ³J_H6−H5= 8.8 Hz, ⁴J_H6−F= 6.6

Hz). ¹³C NMR (75 MHz, acetone-d₆): δ = 53.0 (2OCH₃), 54.9 (CH),

115.3 (d, CH, ²J_C−F= 21.5 Hz), 117.9 (d, CH, ²J_C−F= 23.7 Hz), 126.3

3

4

3

(d, C₁, J_C−F= 3.4 Hz), 134.7 (d, CH, J_C−F= 9.4 Hz), 138.3 (d, C₂,

³J_C−F= 8.9 Hz), 165.3 (C₄, J_C−F= 251.7 Hz), 167.6 (CO), 169.0

1

(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

Synthesis of Precursor 4. Methyl 5-Methoxy-2-(2-methoxy-2-

oxoethyl)benzoate (2). Thionyl chloride (1.5 mL, 20.4 mmol) was

added dropwise to a cooled solution of 2-carboxymethyl-5-

hydroxybenzoic acid¹⁹1 (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, ³J_H4−H3= 8.4 Hz, ⁴J_H4−H6

= 2.7 Hz), 7.15 (d, 1H, H₃, ³J_H3−H4= 8.4 Hz), 7.36 (d, 1H, H₆, ⁴J_H6−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 (C_IV), 130.4 (C_IV), 133.3 (CH), 158.6 (C₅), 167.2 (CO), 172.3

(CO).

Methyl 2-{1-[(Benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2-

yl}-5-methoxybenzoate (4). A solution of freshly distilled diisopropyl-

amine (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

1

(80%), mp 127−131 °C. H NMR (300 MHz, acetone-d₆): δ = 3.73

(s, 6H, 2OCH₃), 5.20 (s, 1H, CH), 7.30 (t, 1H, H₅, ³J_H5−F= ³J_H5−H4

=

9.0 Hz), 7.34 (d, 1H, H₃, ³J_H3−H4= 7.8 Hz), 7.59 (td, 1H, H₄, ³J_H4−H3

= ³J_H4−H5= 8.1 Hz, ⁴J_C4−F= 5.7 Hz). ¹³C NMR (75 MHz, acetone-d₆):

2

δ = 52.6 (2OCH₃), 54.4 (CH), 116.1 (d, C₅, J_C−F= 22.3 Hz), 122.2

2

4

(d, C₁, J_C−F= 16.5 Hz), 125.8 (d, C₃, J_C−F= 3.3 Hz), 132.3 (d, C₄,

³J_C−F= 9.2 Hz), 134.2 (d, C₂, ³J_C−F= 2.7 Hz), 160.2 (C₆, ¹J_C−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

1

(45%). H NMR (300 MHz, acetone-d₆): δ = 3.77 (s, 6H, 2OCH₃),

5.93 (s, 1H, CH), 8.32−8.35 (m, 3H, H_Ar). ¹³C NMR (75 MHz,

acetone-d₆): δ = 52.9 (2OCH₃), 54.4 (CH), 123.4 (CH), 125.4 (CH),

132.7 (CH), 136.1 (C_IV), 136.6 (C_IV), 150.0 (C_IV, 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-4-

triﬂuoromethylbenzoic 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 MgSO₄and

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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, ³J_H5−F= 8.7 Hz, ³J_H5−H6

= 7.7 Hz, ⁴J_H5−H3= 2.6 Hz), 7.13 (dd, 1H, H₃, ³J_H4−F= 9.7 Hz, ⁴J_H3−H5

(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−

3

4

= 2.6 Hz), 8.03 (dd, 1H, H₆, J_H6−H5= 8.8 Hz, J_H6−F= 6.0 Hz); ¹³C

NMR (75 MHz, CDCl₃): δ = 52.4 (OCH₃), 53.0 (2OCH₃), 54.5

2

(CH), 115.3 (d, CH, J_C−F= 21.3 Hz), 117.5 (d, CH, J_C−F= 23.5

4

3

Hz), 125.4 (d, C₁, J_C−F= 3.2 Hz), 137.5 (d, CH, J_C−F= 8.9 Hz),

3

1

138.3 (d, C₂, J_C−F= 8.9 Hz), 164.7 (d, C₄, J_C−F= 250.7 Hz), 166.4

1

(CO), 168.5 (2CO). ESI-MS: m/z = 285 (M + H)⁺.

65 °C. H NMR (300 MHz, CDCl₃): δ = 3.73 (s, 6H, 2OCH₃), 3.90

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 work-

up, 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.

¹H NMR (300 MHz, CDCl₃): δ = 3.61 (s, 6H, 2OCH₃), 3.76 (s, 3H,

(s, 3H, OCH₃), 5.81 (s, 1H, CH), 7.61 (d, 1H, H₃, ³J_H3−H4= 8.6 Hz),

3

4

8.31 (dd, 1H, H₄, J_H4−H3= 8.6 Hz, J_H4−H6= 2.2 Hz), 8.80 (dd, 1H,

H₆, J_H6−H4= 2.2 Hz). ¹³C NMR (75 MHz, CDCl₃): δ = 52.9

4

(2OCH₃), 54.7 (OCH₃), 59.7 (CH), 125.0 (CH), 126.8 (CH), 131.1

(C_IV), 132.5 (CH), 140.3 (C_IV), 146.9 (C_IV, 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-aminoben-

zoate (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,

OCH₃), 4.95 (s, 1H, CH), 6.98 (t, 1H, H₅, ³J_H5−F= ³J_H5−H4= 9.0 Hz),

3

7.14 (d, 1H, H₃, J_H3−H4= 7.8 Hz), 7.31 (td, 1H, H₄, J_H4−H5

=

³J_H4−H3= 8.0 Hz, J_H4−F= 5.8 Hz). ¹³C NMR (75 MHz, CDCl₃): δ =

4

2

52.3 (OCH₃), 52.6 (2OCH₃), 54.4 (CH), 115.9 (d, C₅, J_C−F= 22.3

2

4

Hz), 120.9 (d, C₁, J_C−F= 15.7 Hz), 125.4 (d, C₃, J_C−F= 3.3 Hz),

132.0 (d, C₄, ³J_C−F= 9.2 Hz), 133.7 (d, C₂, ³J_C−F= 2.3 Hz), 160.2 (d,

C₆, ¹J_C−F= 252.0 Hz), 165.0 (CO), 167.8 (2CO). ESI-MS: m/z = 285

(M + H)⁺.

3

1H, CH), 6.84 (d, 1H, H₃, J_H3−H4= 8.3 Hz), 7.21 (dd, 1H, H₄,

4

³J_H4−H3= 8.3 Hz, J_H4−H6= 2.2 Hz), 7.32 (d, 1H, H₆, J_H6−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 (C_IV), 130.1 (C_IV), 131.0

(CH), 146.6 (C₅), 167.5 (CO), 169.6 (2CO). ESI-MS: m/z = 282 (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−

1

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

72 °C. H 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, ³J_H6−H5= 8.6 Hz),

8.12 (dd, 1H, H₅, ³J_H5−H6= 8.6 Hz, ⁴J_H5−H3= 2.1 Hz), 8.21 (d, 1H, H₃,

⁴J_H3−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 (C_IV), 136.0 (C_IV), 149.6 (C_IV, C₄), 165.7 (CO), 167.7 (2CO).

ESI-MS: m/z = 312 (M + H)⁺.

Methyl 4-Triﬂuoromethyl-2-(1,3-dimethoxy-1,3-dioxopropan-2-

yl)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

1

acid), yellow oil. H 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

4

³J_H3−H4= 8.3 Hz), 7.78 (dd, 1H, H₄, J_H4−H3= 8.3 Hz, J_H4−H6= 1.7

3

4

Hz), 8.27 (d, 1H, H₆, ⁴J_H6−H4= 1.9 Hz). ¹³C NMR (75 MHz, CDCl₃):

(td, 1H, H₄, J_H4−H3= J_H4−F= 8.3 Hz, J_H4−H6= 2.5 Hz), 7.36 (dd,

1H, H₃, ³J_H3−H4= 8.3 Hz, ⁴J_H3−F= 5.6 Hz), 7.58 (dd, 1H, H₆, ³J_H6−F

=

δ = 52.37 (OMe), 52.66 (2OMe), 54.47 (CH), 122.56 (q, CF₃, ¹J_C−F

=

9.7 Hz, J_H6−H4= 2.5 Hz). ¹³C NMR (75 MHz, acetone-d₆): δ = 53.0

4

267.0 Hz), 127.80 (q, CH, ³J_C−F= 3.8 Hz), 128.80 (q, CH, ³J_C−F= 3.8

2

(OCH₃), 53.1 (2OCH₃), 55.0 (CH), 118.2 (d, CH_Ar, J_C−F= 24.0

Hz), 130.16 (C_IV), 130.41 (q, C₅, J_C−F= 33.0 Hz), 131.06 (C₃),

2

Hz), 120.2 (CH, J_C−F= 21.3 Hz), 131.5 (C_IV), 132.8 (C_IV), 133.4

138.24 (C_IV), 165.92 (CO), 168.72 (2CO). ESI-MS: m/z = 335 (M +

(C₃, ³J_C−F= 8.2 Hz), 162.6 (C₅, ¹J_C−F= 246.0 Hz), 166.9 (CO), 169.3

H)⁺.

(2CO); ESI-MS: m/z = 285 (M + H)⁺.

Synthesis of Precursors 20−28. Methyl 2-Fluoro-5-nitro-

benzoate (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

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%).

¹H NMR (300 MHz, CDCl₃): δ = 3.80 (s, 6H, 2OCH₃), 3.92 (s, 3H,

1

light yellow needles: yield 90%, mp 47−49 °C. H NMR (300 MHz,

3

CDCl₃): δ = 4.01 (s, 3H, OCH₃), 7.35 (t, 1H, H₃, J_H3−H4= J_H3−F

=

9.2 Hz), 8.44 (dt, 1H, H₄, ³J_H4−H3= 9.2 Hz, ⁴J_H4−F= ⁴J_H4−H6= 3.5 Hz),

8.87 (dd, 1H, H₆, J_H6−F= 5.9 Hz, J_H6−H4= 2.7 Hz). ¹³C NMR (75

4

2

MHz, CDCl₃): δ = 47.7 (OCH₃), 113.2 (d, C₃, J_C−F= 25.1 Hz),

114.5 (d, C₁, ²J_C−F= 12.0 Hz), 122.9 (d, C₆, ³J_C−F= 3.3 Hz), 124.3 (d,

C₄, ³J_C−F= 10.9 Hz), 138.6 (C₅), 157.4 (d, COOCH₃, ³J_C−F= 3.8 Hz),

1

159.8 (d, C₂, J_C−F= 269.0 Hz).

3

OCH₃), 5.76 (s, 1H, CH), 7.40 (d, 1H, H₃, J_H3−H4= 8,3 Hz), 7.54

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

3

4

(dd, 1H, H₄, J_H4−H3= 8.3 Hz, J_H4−H6= 1.7 Hz), 8.03 (d, 1H, H₆,

⁴J_H6−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 (C_IV), 132.8 (2CH),

4649

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

Journal of Medicinal Chemistry

Article

133.2 (C_IV), 133.5 (C_IV), 166.1 (CO), 168.5 (2CO). ESI-MS: m/z =

301 and 303 (M + H)⁺.

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

NaHCO₃and 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, ³J_H3−H4= 8.5

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

1

hydrochloric acid, giving a white solid (61%). H NMR (300 MHz,

CDCl₃): δ = 3.80 (s, 6H, 2OCH₃), 3.92 (s, 3H, OCH₃), 5.74 (s, 1H,

3

CH), 7.34 (d, 1H, H₃, ³J_H3−H4= 8.3 Hz), 7.69 (dd, 1H, H₄, J_H4−H3

=

3

Hz), 7.55 (ddd, 1H, H_Ar, J_H−H= 7.7 Hz, ³J_H−H= 4.8 Hz, ⁴J_H−H= 1.2

8.3 Hz, J_H4−H6= 1.5 Hz), 8.18 (d, 1H, H₆, J_H6−H4= 1.5 Hz). ¹³C

NMR (75 MHz, CDCl₃): δ = 52.7 (OCH₃), 53.0 (2OCH₃), 54.2

(CH), 122.2 (C₅), 131.0 (C_IV), 131.9 (CH), 133.3 (C_IV), 133.9 (CH),

135.5 (CH), 166.1 (CO), 168.5 (2CO); ESI-MS: m/z = 345 and 347

(M + H)⁺.

4

Hz), 7.97 (td, 1H, H_Ar, ³J_H−H= 7.7 Hz, ⁴J_H−H= 1.6 Hz), 8.06 (dd, 1H,

3

4

3

H₄, J_H4−H3= 8.5 Hz, J_H4−H6= 2.4 Hz), 8.34 (d, 1H, H_2′, J_{H2′−H3′}

=

7.7 Hz), 8,45 (d, 1H, H₆, ⁴J_H6−H4= 2.4 Hz), 8.66 (d, 1H, H_5′, ³J_{H5′−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 (C_IV), 130.2 (C_IV), 131.1 (CH), 138.7

(CH), 138.8 (C₅), 148.9 (CH), 150.0 (C_1′), 163.4 (CO), 167.2 (CO),

168.9 (2CO). ESI-MS: m/z = 387 (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/

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−

1

128 °C. H 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, ³J_H3−H4= 8.2 Hz), 7.33 (s, 5H, H_Ar), 7.82 (dd, 1H, H₄, ³J_H4−H3

= 8.5 Hz, ⁴J_H4−H6= 1.9 Hz), 8.24 (d, 1H, H₆, ⁴J_H6−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 (C_IV), 128.8 (2CH), 129.6 (2CH), 130.2 (C_IV), 131.2

(CH), 136.1 (C_IV), 139.5 (C₅), 167.1 (CO), 168.9 (2CO), 170.1

(CO). ESI-MS: m/z = 400 (M + H)⁺.

1

v) as eluent gave 23 as yellow crystals (51%), mp 83 °C. H NMR

Methyl 2-(1,3-Dimethoxy-1,3-dioxopropan-2-yl)-5-[2-(thiophen-

2-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

(300 MHz, CDCl₃): δ = 3.78 (s, 6H, 2OCH₃), 3.93 (s, 3H, OCH₃),

3

5.81 (s, 1H, CH), 7.59 (d, 1H, H₃, J_H3−H4= 8,2 Hz), 7.82 (dd, 1H,

H₄, ³J_H4−H3= 8.2 Hz, ⁴J_H4−H6= 1.8 Hz), 8.32 (d, 1H, H₆, ⁴J_H6−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 (C_IV), 131.5 (CH), 134.6

(CH), 135.3 (CH), 139.0 (C_IV), 165.5 (CO), 167.9 (2CO). ESI-MS:

m/z = 292 (M + H)⁺.

1

°C. H 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,

H_thiophen), 7.32−7.34 (m, 2H, H_thiophenand H₃), 7.70 (dd, 1H, H₄,

4

³J_H4−H3= 8.5 Hz, J_H4−H6= 1.9 Hz), 8.02 (d, 1H, H₆, J_H6−H4= 1.9

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

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 (C_IV), 129.8 (C_IV), 130.7

(CH), 135.5 (C_IV), 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-nitroben-

zoate (29). A solution of intermediate 18 (0.31 g, 1.0 mmol) and O-

benzylhydroxylamine (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, H_Ar), 7.75 (d, 1H, H₃, ³J_H3−H4= 8.6 Hz), 8.32 (dd,

1H, H₄, ³J_H4−H3= 8.6 Hz, ⁴J_H4−H6= 2.2 Hz), 8.75 (dd, 1H, H₆, ³J_H6−H5

3

(s, 1H, CH), 7.26 (d, 1H, H₃, J_H3−H4= 8.5 Hz), 7.80 (dd, 1H, H₄,

4

³J_H4−H3= 8.5 Hz, J_H4−H6= 1.9 Hz), 8.20 (d, 1H, H₆, J_H6−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 (C_IV), 129.6 (C_IV), 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

1

chloride) to give a white powder (67%), mp 135−137 °C. H NMR

4

= 8.6 Hz, J_H6−H4= 2.2 Hz), 9.62 (s, 1H, NH). ¹³C NMR (75 MHz,

(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, H_Ar), 7.58−7.61 (m,

3H, H_Ar), 8.02−8.06 (m, 3H, H_Ar), 8.49 (s, 1H, H_Ar), 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 (C_IV), 129.6 (C_IV), 130.7 (CH), 131.9 (CH), 134.4

(C_IV), 139.1 (C₅), 165.8 (CO), 166.7 (CO), 168.5 (2CO). ESI-MS:

m/z = 386 (M + H)⁺.

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 (C_IV),

133.7 (CH), 135.0 (C_IV), 141.3 (C_IV), 147.2 (C_IV, 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-2-

yl}-5-ﬂuorobenzoate (30). White powder (30%), mp 118−119 °C.

¹H 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, H_Ar),

7.49−7.52 (m, 2H, H_Ar), 7.64−7.67 (m, 1H, H_Ar), 11.51 (s, 1H, NH).

¹³C NMR (75 MHz, DMSO-d₆): δ = 52.2 (CH), 52.9 (OCH₃), 53.1

(OCH₃), 77.3 (OCH₂), 117.3 (d, CH, ²J_C−F= 23.5 Hz), 119.8 (d, CH,

²J_C−F= 20.7 Hz), 128.8 (2CH), 128.9 (CH), 129.5 (2CH), 131.4 (d,

Methyl 2-(1,3-Dimethoxy-1,3-dioxopropan-2-yl)-5-picolinamido-

benzoate (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

C₂, ⁴J_C−F= 3.3 Hz), 131.9 (d, C₁, ³J_C−F= 7.1 Hz), 132.3 (d, C₃, ³J_C−F

8.2 Hz), 136.1 (C_IV), 161.4 (d, C₅, J_C−F= 244.4 Hz), 164.6 (CO),

=

1

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166.3 (d, CO, ⁴J_C−F= 2.7 Hz), 169.1 (CO). ESI-MS: m/z = 376 (M +

H)⁺.

Methyl 2-{1-[(Benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2-

yl}-5-chlorobenzoate (31). White powder (35%), mp 112−113 °C.

¹H 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,

H_Ar), 7.53 (m, 2H, H₄and H₃), 7.78 (s, 1H, H₆), 10.40 (s, 1H, NH).

¹³C 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 (C_IV), 132.0 (CH), 132.3 (CH), 133.2 (C_IV), 134.0

(C_IV), 135.9 (C_IV), 164.5 (CO), 166.0 (CO), 168.5 (CO). ESI-MS:

m/z = 392 and 394 (M + H)⁺.

H_Ar), 7.60 (dd, 1H, H_Ar, ³J_H−H= 6.1 Hz, ³J_H−H= 6.7 Hz), 7.71 (d, 1H,

H_Ar, ³J_H−H= 7.9 Hz), 7.94−8.04 (m, 2H, H_Ar), 8.38 (d, 1H, H_Ar, ³J_H−H

= 7.9 Hz), 8.55 (s, 1H, H_Ar), 8.67 (d, 1H, H₆, ⁴J_H6−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 (CH_A),

124.7 (CH), 127.6 (CH), 128.78 (2CH), 128.84 (CH), 129.5 (2CH),

130.1 (C_IV), 130.3 (CH), 130.5 (C_IV), 136.2 (C_IV), 138.4 (C₅), 138.7

(CH), 149.0 (CH), 150.1 (C_1′), 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-2-

yl}-5-(2-phenylacetamido)benzoate (38). Light yellow powder

1

(25%), mp 65−70 °C. H 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,

Methyl 2-{1-[(Benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2-

yl}-5-bromobenzoate (32). White powder (35%), mp 99−101 °C.

¹H NMR (300 MHz, CDCl₃): δ = 3.64 (s, 3H, OCH₃), 3.77 (s, 3H,

OCH₂), 5.13 (s, 1H, CH), 7.33−7.39 (m, 11H, H₃and 10H_Ar), 7.76

3

4

(dd, 1H, H₄, J_H4−H3= 8.6 Hz, J_H4−H6= 2.3 Hz), 8.23 (d, 1H, H₆,

⁴J_H6−H4= 2.3 Hz), 10.46 (s, 1H, NH), 11.47 (s, 1H, NH). ¹³C NMR

OCH₃), 4.81 (s, 2H, OCH₂), 5.12 (s, 1H, CH), 7.24 (m, 5H, H_Ar),

3

7.45 (d, 1H, H_Ar, ³J_H−H= 8.6 Hz), 7.62 (d, 1H, H_Ar, J_H−H= 8.6 Hz),

(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 (C_IV), 130.1 (C_IV),

130.5 (CH), 136.1 (C_IV), 136.2 (C_IV), 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-2-

yl}-5-[2-(thiophen-2-yl)acetamido]benzoate (39). Yellow powder

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 (C_IV),

128.5 (2CH), 128.7 (CH), 129.3 (3CH), 130.39 (C_IV), 130.40 (C_IV),

133.6 (CH), 135.0 (C_IV), 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-2-

yl}-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, H_Ar), 7.75 (d, 1H,

1

(27%), mp 67−70 °C. H 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, H_Ar), 7.31 (m, 5H,

H_Ar), 7.41−7.43 (m, 2H, H_Ar), 7.93 (s, 1H, H_Ar), 8.13 (s, 1H, H_Ar),

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 (C_IV), 129.3 (3CH), 130.1 (C_IV),

132.5 (CH), 135.0 (C_IV), 135.5 (C_IV), 137.6 (C_IV), 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-2-

yl}-4-ﬂuorobenzoate (40). Four hours of reﬂux; white solid (20%),

3

H_Ar, J_H−H= 8.5 Hz), 7.79 (d, 1H, H_Ar, J_H−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, ¹J_C−F= 270.7

Hz), 127.80 (q, CH, ³J_C−F= 3.8 Hz), 128.5 (2CH), 128.7 (CH), 129.0

(q, CH, ³J_C−F= 3.8 Hz), 129.3 (2CH), 129.5 (C_IV), 130.4 (q, C₅, ²J_C−F

= 33.0 Hz), 132.9 (C₃), 135.0 (C_IV), 138.5 (C_IV), 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-2-

yl}-5-cyanobenzoate (34). White powder (37%), mp 142−146 °C.

¹H NMR (300 MHz, acetone-d₆): δ = 3.56 (s, 3H, OCH₃), 3.72 (s,

1

mp 104−107 °C. H 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),

3H, OCH₃), 4.76 (s, 2H, OCH₂), 5.34 (s, 1H, CH), 7.18−7.27 (m,

5H, H_Ar), 7.69 (d, 1H, H₃, ³J_H3−H4= 8.2 Hz), 7.85 (dd, 1H, H₄, ³J_H4−H3

= 8.2 Hz, ⁴J_H4−H6= 1.8 Hz), 8.12 (d, 1H, H₆, ⁴J_H6−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 (C_IV), 132.6

(CH), 134.7 (CH), 135.9 (CH), 136.5 (C_IV), 140.6 (C_IV), 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-di-

oxopropan-2-yl}benzoate (35). White powder (28%), mp 60−62 °C.

¹H NMR (300 MHz, DMSO-d₆): δ = 2.06 (s, 3H, CH₃), 3.63 (s, 3H,

3

4

7.10 (td, 1H, H₅, J_H5−F= J_H5−H6= 8.7 Hz, J_H5−H3= 2.5 Hz), 7.41

3

4

(dd, 1H, H₃, J_H3−F= 9.6 Hz, J_H3−H5= 1.8 Hz), 8.00 (dd, 1H, H₆,

³J_H6−H5= 8.6 Hz, ⁴J_H6−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,

2

2H, OCH₂), 115.2 (d, CH, J_C−F= 21.4 Hz), 119.1 (d, CH, J_C−F

=

4

23.3 Hz), 125.0 (d, C₁, J_C−F= 3.2 Hz), 128.5 (2CH), 128.6 (CH),

129.2 (2CH), 133.3 (d, C₆, ³J_C−F= 8.9 Hz), 135.0 (C_IV), 137.8 (d, C₂,

³J_C−F= 8.9 Hz), 164.9 (CO), 164.7 (C₄, J_C−F= 253.5 Hz), 167.2

1

(CO), 168.8 (CO). ESI-MS: m/z = 376 (M + H)⁺.

Methyl 2-{1-[(Benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2-

yl}-6-ﬂuorobenzoate (41). Six hours of reﬂux; white solid (38%),

OCH₃), 3.79 (s, 3H, OCH₃), 4.81 (s, 2H, OCH₂), 5.11 (s, 1H, CH),

1

7.36 (s, 6H, H₃and H_Ar), 7.73 (dd, 1H, H₄, ³J_H4−H3= 8.5 Hz, ⁴J_H4−H6

=

mp 156−160 °C. H NMR (300 MHz, DMSO-d₆): δ = 3.68 (s, 3H,

1.9 Hz), 8.20 (d, 1H, H₆, ⁴J_H6−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 (C_IV),

129.6 (C_IV), 129.9 (CH), 135.7 (C_IV), 138.7 (C₅), 164.4 (CO), 166.7

(CO), 168.7 (2CO). ESI-MS: m/z = 415 (M + H)⁺.

OCH₃), 3.84 (s, 3H, OCH₃), 4.73 (s, 1H, CH), 4.83 (s, 2H, OCH₂),

7.31−7.37 (m, 7H, H_Ar), 7.60 (td, 1H, H₄, ³J_H4−H5= ³J_H4−H3= 8.0 Hz,

⁴J_H4−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,

²J_C−F= 21.8 Hz), 120.8 (d, C₁, ²J_C−F= 15.5 Hz), 125.6 (d, C₃, ⁴J_C−F

=

3.0 Hz), 128.4 (2CH), 128.5 (CH), 129.1 (2CH), 132.6 (d, C₄, ³J_C−F

3

= 9.2 Hz), 134.6 (d, C₂, J_C−F= 2.2 Hz), 135.7 (C_IV), 159.5 (d, C₆,

Methyl 5-Benzamido-2-{1-[(benzyloxy)amino]-3-methoxy-1,3-di-

oxopropan-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,

H_Ar), 7.56 (m, 3H, H_Ar), 7.99 (m, 3H, H_Ar), 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 (C_IV),

130.2 (C_IV), 130.4 (CH), 132.3 (CH), 134.9 (C_IV), 136.2 (C_IV), 139.2

(C₅), 164.9 (CO), 166.2 (CO), 167.2 (CO), 169.4 (CO). ESI-MS: m/

z = 491 (M + H)⁺.

¹J_C−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-2-

1

yl}-4-nitrobenzoate (42). White solid (20%), mp 133−137 °C. H

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, H_Ar),

8.13 (d, 1H, H₅, ³J_H5−H6= 8.6 Hz), 8.23 (d, 1H, H₆, ³J_H6−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 (C_IV),

134.9 (C_IV), 136.7 (C_IV), 149.7 (C_IV, C₄), 164.2 (CO), 166.4 (CO),

168.6 (CO). ESI-MS: m/z = 403 (M + H)⁺.

Methyl 2-{1-[(Benzyloxy)amino]-3-methoxy-1,3-dioxopropan-2-

yl}-5-picolinamidobenzoate (37). White powder (20%), mp 207−

1

208 °C. H NMR (300 MHz, CDCl₃): δ = 3.75 (s, 3H, OCH₃), 3.88

Cyclization of N-Benzyloxymalonamides 4 and 29−42.

(s, 3H, OCH₃), 4.90 (s, 2H, OCH₂), 5.27 (s, 1H, CH), 7.33 (s, 5H,

Methyl 2-(Benzyloxy)-7-methoxy-1,3-dioxo-1,2,3,4-tetrahydroiso-

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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 Na₂SO₄and 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, H_Ar), 7.55−7.56 (m, 2H,

H_Ar). 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 (C_IV),

127.1 (2CH), 127.6 (CH), 128.9 (2CH), 130.8 (CH), 133.2 (C_IV),

137.1 (C_IV), 159.5 (CO), 161.0 (CO), 164.0 (CO), 169.9 (CO). ESI-

MS: m/z = 356 (M + H)⁺.

temperature; white powder (90%), 100% enol form, mp 169−172 °C.

¹H NMR (300 MHz, CDCl₃): δ = 4.14 (s, 3H, OCH₃), 5.28 (s, 2H,

OCH₂), 7.42−7.44 (m, 3H, H_Ar), 7.62−7.64 (m, 2H, H_Ar), 7.82 (dd,

1H, H₆, ³J_H6−H5= 8.7 Hz, ⁴J_H6−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 (C_IV), 133.28 (CH), 135.4

(CH), 136.4 (C_IV), 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-tetrahydroi-

soquinoline-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, H_Ar), 7.54−7.57 (m, 2H, H_Ar), 7.89 (d, 1H, H₃,

4

³J_H3−H4= 8.6 Hz), 8.40 (d, 1H, H₆, J_H6−H4= 1.9 Hz), 11.34 (s, 1H,

Methyl 2-(Benzyloxy)-3-hydroxy-7-nitro-1-oxo-1,2-dihydroisoqui-

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 (C_IV), 126.3

(C_IV), 127.9 (CH), 128.4 (2CH), 129.0 (CH), 129.5 (2CH), 134.2

(C_IV), 139.8 (C₅), 160.4 (CO), 163.0 (CO), 167.3 (CO), 168.8 (CO).

ESI-MS: m/z = 383 (M + H)⁺.

noline-4-carboxylate (44). Yellow solid (89%), mp 190−196 °C,

1

100% enol form. H NMR (300 MHz, CDCl₃): δ = 4.17 (s, 3H,

OCH₃), 5.31 (s, 2H, OCH₂), 7.45 (m, 3H, H_Ar), 7.63 (m, 2H, H_Ar),

8.43 (dd, 1H, H₆, ³J_H6−H5= 9.4 Hz, ⁴J_H6−H8= 2.0 Hz), 8.60 (d, 1H, H₅,

4

³J_H5−H6= 9.4 Hz), 9.26 (d, 1H, H₈, J_H8−H4= 1.9 Hz). ¹³C NMR (75

MHz, CDCl₃): δ = 53.7 (OCH₃), 79.4 (OCH₂), 84.5 (C_IV), 121.2

(C_IV), 124.6 (CH), 125.6 (CH), 127.4 (CH), 128.7 (2CH), 129.6

(CH), 130.1 (2CH), 133.0 (C_IV), 138.0 (C_IV), 144.0 (C_IV, 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-tetrahydroiso-

quinoline-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,

Methyl 7-Benzamido-2-(benzyloxy)-1,3-dioxo-1,2,3,4-tetrahy-

droisoquinoline-4-carboxylate (51). Orange powder (83%), 100%

1

keto form, mp 185−187 °C. H 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, H_Ar), 7.57−7.75 (m, 5H, H_Ar), 7.91−8.02 (m, 3H, H_Ar), 8.42

(d, 1H, H_Ar, ³J_H−H= 8.0 Hz), 8.62 (d, 1H, H₈, ⁴J_H8−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 (C_IV), 126.5 (CH), 127.5

(C_IV), 128.2 (2CH), 128.4 (CH), 128.9 (2CH), 129.0 (2CH), 129.4

(CH), 130.0 (2CH), 132.4 (CH), 134.7 (C_IV), 134.8 (C_IV), 140.2

(C₇), 160.9 (CO), 163.5 (CO), 166.3 (CO), 167.8 (CO). ESI-MS: m/

z = 445 (M + H)⁺.

H_Ar), 7.55−7.67 (m, 4H, H_Ar), 7.86 (d, 1H, H₈, ³J_H8−F= 8.2 Hz). ¹³

C

NMR (75 MHz, DMSO-d₆): δ = 54.1 (CH₃), 54.3 (C₄), 78.0 (OCH₂),

2

114.9 (d, CH_Ar, J_C−F= 24.0 Hz), 122.3 (d, CH_Ar, J_C−F= 22.4 Hz),

127.5 (C_IV), 128.9 (2CH), 129.2 (C_IV), 129.5 (CH), 130.0 (2CH),

3

130.7 (d, C₅, J_C−F= 8.2 Hz), 134.6 (C_IV), 160.1 (CO), 162.3 (d, C₇,

Methyl 2-(Benzyloxy)-1,3-dioxo-7-picolinamido-1,2,3,4-tetrahy-

¹J_C−F= 245.7 Hz), 163.2 (CO), 167.5 (CO). ESI-MS: m/z = 344 (M

droisoquinoline-4-carboxylate (52). Beige powder (51%), 100%

1

+ H)⁺.

keto form, mp 177−180 °C. H 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

Methyl 2-(Benzyloxy)-7-chloro-1,3-dioxo-1,2,3,4-tetrahydroiso-

quinoline-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, H_Ar),

7.58 (m, 2H, H_Ar), 8.08 (s, 1H, H₈), 8.43 (d, 1H, H_Ar, ³J_H−H= 8.8 Hz).

¹³C NMR (75 MHz, DMSO-d₆): δ = 53.0 (OCH₃), 54.2 (C₄), 78.0

(OCH₂), 126.6 (CH), 127.2 (C_IV), 128.9 (3 CH), 129.5 (CH), 130.1

(2CH), 131.8 (C_IV), 133.6 (CH), 134.7 (C_IV), 134.7 (C_IV), 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-tetrahydroiso-

quinoline-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,

H_Ar), 7.76 (d, 1H, H_Ar, ³J_H−H= 8.2 Hz), 7.94 (d, 1H, H_Ar, ³J_H−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 (C_IV), 126.6

(CH), 128.9 (3 CH), 129.5 (CH), 129.6 (CH), 130.0 (CH), 133.4

(C_IV), 134.6 (C_IV), 136.3 (CH), 161.4 (CO), 163.0 (CO), 167.2

(CO). ESI-MS: m/z = 404 and 406 (M + H)⁺.

3

(m, 4H, H_Ar), 7.56−7.65 (m, 3H, H_Ar), 7.98 (t, 1H, H_Ar, J_H−H= 7.6

Hz), 8.36 (d, 1H, H_Ar, ³J_H−H= 7.7 Hz), 8.41−8.49 (m, 2H, H_Ar), 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 (C_IV), 126.9 (CH), 127.7 (CH), 127.8 (C_IV), 128.3 (CH), 130.0

(2CH), 129.4 (CH), 130.1 (2CH), 134.7 (C_IV), 138.7 (CH), 139.5

(C_IV), 149.0 (CH), 150.0 (C_1′), 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-tet-

rahydroisoquinoline-4-carboxylate (53). White powder (80%),

1

100% keto form, mp 187−196 °C. H NMR (300 MHz, DMSO-

d₆): 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, H_Ar), 7.54−7.56 (m, 3H, H_Ar),

3

7.92 (d, 1H, H_Ar, J_H−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 (C_IV), 127.1

(CH), 128.5 (CH), 128.8 (2CH), 128.9 (2CH), 129.6 (3CH), 130.0

(2CH), 134.7 (C_IV), 136.1 (C_IV), 136.3 (C_IV), 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)-7-triﬂuoromethyl-1,3-dioxo-1,2,3,4-tetra-

hydroisoquinoline-4-carboxylate (48). Five minutes of stirring at

room temperature; white powder (76%), 100% enol form, mp 160−

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, H_Ar), 7.41 (m, 5H, H_Ar),

1

164 °C. H NMR (300 MHz, CDCl₃): δ = 4.10 (s, 3H, OCH₃), 5.25

3

(s, 2H, OCH₂), 7.40−7.43 (m, 3H, H_Ar), 7.62 (dd, 2H, H_Ar, J_H−H

=

6.6 Hz, ⁴J_H−H= 2.9 Hz), 7.79 (dd, 1H, H₆, ³J_H6−H5= 8.9 Hz, ⁴J_H6−H8

3

1.9 Hz), 8.50 (d, 1H, H₅, ³J_H5−H6= 8.9 Hz), 8.63 (s, 1H, H₈), 15.91 (s,

7.55−7.56 (m, 2H, H_Ar), 7.92 (d, 1H, H_Ar, J_H−H= 7.7 Hz), 8.42 (s,

1H, OH). ¹³C NMR (75 MHz, CDCl₃): δ = 53.5 (OCH₃), 79.3

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 (C_IV), 125.7 (CH), 127.0 (CH), 127.2 (CH), 127.3

(C_IV), 128.6 (CH), 128.9 (2CH), 129.4 (CH), 130.0 (2CH), 134.7

(C_IV), 137.1 (C_IV), 140.0 (C_IV), 160.8 (CO), 163.4 (CO), 167.7

(CO), 169.1 (CO). ESI-MS: m/z = 465 (M + H)⁺.

1

(OCH₂), 84.2 (C₄), 121.0 (C_IV), 123.5 (q, CF₃, J_C−F= 234.7 Hz),

125.1 (CH), 125.8 (CH, J_C−F= 4.1 Hz), 126.5 (q, C₇, J_C−F= 33.6

Hz), 128.7 (2CH), 129.5 (CH), 129.6 (CH, J_C−F= 3.2 Hz), 133.2

3

2

3

(C_IV), 135.6 (C_IV), 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-tetrahydroiso-

quinoline-4-carboxylate (49). Ten minutes of stirring at room

Methyl 2-(Benzyloxy)-6-ﬂuoro-1,3-dioxo-1,2,3,4-tetrahydroiso-

quinoline-4-carboxylate (55). Ten minutes of stirring at room

4652

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

Journal of Medicinal Chemistry

Article

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, ³J_H7−F= ³J_H7−H8= 9.1 Hz, ⁴J_H7−H5= 2.3 Hz),

7.40−7.42 (m, 3H, H_Ar), 7.61−7.64 (m, 2H, H_Ar), 8.06 (dd, 1H, H₅,

³J_H5−F= 12.3 Hz, ⁴J_H5−H7= 2.3 Hz), 8.38 (dd, 1H, H₇, ³J_H7−F= 8.9 Hz,

³J_H7−H8= 6.4 Hz), 15.74 (br s, 1H, OH). ¹³C NMR (75 MHz, CDCl₃):

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, DMSO-

d₆): δ = 4.48 (d, 2H, CH₂, ³J_H−H= 5.6 Hz), 5.06 (s, 2H, OCH₂), 7.15

3

(t, 2H, H_3′and H_5′, J_H−H= J_H−F= 8.8 Hz), 7.40 (m, 5H, H_Ar), 7.62

3

4

2

(m, 2H, H_Ar), 8.01 (dd, 1H, H₆, J_H6−H5= 9.7 Hz, J_H6−H8= 2.3 Hz),

8.78 (d, 1H, H₈, ⁴J_H8−H6= 2.3 Hz), 9.40 (d, 1H, H₅, ³J_H5−H6= 9.7 Hz),

10.35 (t, 1H, NH, ³J_H−H= 5.6 Hz). ¹³C NMR (75 MHz, DMSO-d₆): δ

δ = 53.9 (OCH₃), 79.6 (OCH₂), 111.1 (d, CH, J_C−F= 26.6 Hz),

2

4

113.6 (d, CH, J_C−F= 23.9 Hz), 126.1 (d, C_8a, J_C−F= 3.2 Hz), 129.5

3

(2CH), 130.2 (CH), 130.9 (2CH), 132.2 (d, C₈, J_C−F= 10.9 Hz),

= 41.2 (CH₂), 76.6 (OCH₂), 90.2 (C₄), 114.9 (d, C_3′and C_5′, ²J_C−F

=

3

135.4 (C_IV), 137.9 (d, C_4a, J_C−F= 8.9 Hz), 159.1 (CO), 165.7 (C₆,

¹J_C−F= 247.5 Hz), 167.2 (CO), 174.4 (CO). ESI-MS: m/z = 344 (M

20.7 Hz), 115.8 (C_IV), 123.6 (CH), 124.1 (CH), 124.6 (CH), 128.2

3

+ H)⁺.

(2CH), 128.5 (CH), 129.21 (2CH), 129.22 (d, C_2′and C_6′, J_C−F

=

7.6 Hz), 135.3 (C_IV), 137.0 (d, C_1′, ⁴J_C−F= 3.3 Hz), 137.7 (C_IV), 144.5

(C₇), 159.2 (CO), 161.0 (d, C_4′, ¹J_C−F= 240.0 Hz), 161.5 (CO), 167.8

(CO). ESI-MS: m/z = 464 (M + H)⁺.

Methyl 2-(Benzyloxy)-8-ﬂuoro-1,3-dioxo-1,2,3,4-tetrahydroiso-

quinoline-4-carboxylate (56). One hour of stirring at room

temperature; white solid (70%), 75% enol/25% keto form, mp 182−

1

N-(4-Fluorobenzyl)-2-(benzyloxy)-7-ﬂuoro-1,3-dioxo-1,2,3,4-tet-

185 °C. H NMR (300 MHz, DMSO-d₆): δ = 3.76 (s, 3H, OCH₃

rahydroisoquinoline-4-carboxamide (60). White powder (75%),

keto), 4.05 (s, 3H, OCH₃enol), 4.99 (s, 1H, CH₄keto), 5.14 (s, 2H,

1

OCH₂keto), 5.23 (s, 2H, OCH₂enol), 7.00 (dd, 1H, H₇enol, ³J_H7−F

=

100% keto form, mp 215−217 °C. H NMR (300 MHz, DMSO-

d₆): δ = 4.32 (d, 1H, CH₂, ²J_H−H= 15.6 Hz, ³J_H−H= 5.3 Hz), 4.33 (d,

1H, CH₂, ²J_H−H= 15.6 Hz, ³J_H−H= 5.3 Hz), 5.02 (d, 1H, OCH₂, ²J_H−H

= 9.5 Hz), 5.03 (d, 1H, OCH₂, ²J_H−H= 9.5 Hz), 5.20 (s, 1H, H₄), 7.18

3

10.5 Hz, J_H7−H8= 8.8 Hz), 7.25 (dd, 1H, H₇keto, J_H7−F= 11.0 Hz,

³J_H7−H8= 8.2 Hz), 7.37−7.45 (m, H_Ar, H_3′, H_4′, H_5′enol + keto, H₆

keto), 7.52−7.68 (m, H_Ar, H_2′, H_6′enol + keto, H₅keto, H₆enol), 8.24

(d, 1H, H₅enol, ³J_H5−H6= 8.6 Hz), 15.76 (s, 1H, OH). ¹³C NMR (75

MHz, DMSO-d₆): δ = 53.3 (OCH₃enol), 54.0 (OCH₃keto), 54.6 (d,

3

(t, 2H, H_3′and H_5′, J_H−H= J_H−F= 8.5 Hz), 7.30 (dd, 2H, H_2′and

H_6′, ³J_H−H= 8.2 Hz, ⁴J_H−F= 5.7 Hz), 7.42−7.52 (m, 4 H, H_Ar), 7.56−

3

4

7.58 (m, 2H, H_Ar), 7.64 (t, 1H, H₆, J_H−H= J_H−F= 8.5 Hz), 7.84 (d,

1H, H_Ar, ³J_H−H= 8.5 Hz), 9.33 (t, 1H, NH, ³J_H−H= 5.3 Hz). ¹³C NMR

(75 MHz, DMSO-d₆): δ = 42.0 (CH₂), 55.1 (C₄), 77.4 (OCH₂), 114.1

C₄keto, J_C−F= 2.0 Hz), 79.6 (OCH₂keto), 78.9 (OCH₂enol), 83.7

4

3

(d, C₄enol, J_C−F= 2.4 Hz), 110.4 (d, C_4a, J_C−F= 5.2 Hz), 111.5 (d,

2

C₇enol, J_C−F= 21.1 Hz), 117.7 (d, C₇keto, J_C−F= 21.4 Hz), 119.9

(d, C₅enol, ⁴J_C−F= 4.4 Hz), 123.4 (d, C₅keto, ⁴J_C−F= 4.1 Hz), 128.50

(2CH keto), 128.55 (2CH enol), 129.2 (CH keto, C_4′), 129.4 (CH

enol, C_4′), 130.0 (2CH keto), 130.1 (2CH enol), 133.50 (C_1′enol),

2

(d, CH_Ar, J_C−F= 24.0 Hz), 115.2 (d, C_3′and C_5′, J_C−F= 21.3 Hz),

121.6 (d, CH_Ar, ²J_C−F= 22.9 Hz), 127.3 (d, C_8a, ³J_C−F= 7.6 Hz), 128.4

(2CH), 128.9 (CH), 129,2 (d, C₅, J_C−F= 8.2 Hz), 129.3 (d, C_2′and

C_6′, J_C−F= 8.2 Hz), 129.4 (2CH), 130.9 (d, C_4a, J_C−F= 2.7 Hz),

134.3 (C_IV), 134.6 (d, C_1′, J_C−F= 2.7 Hz), 160.1 (d, C₁, J_C−F= 2.7

3

4

133.51 (d, C_8a, J_C−F= 26.5 Hz), 134.3 (d, C₆enol, ³J_C−F= 10.7 Hz),

2

4

3

135.3 (C_1′keto), 135.7 (d, C₆keto, J_C−F= 10.2 Hz), 155.4 (d, CO₁

enol, J_C−F= 4.9 Hz), 157.4 (d, CO₁keto, J_C−F= 5.1 Hz), 161.9

1

3

Hz), 161.3 (d, C₇, J_C−F= 241.1 Hz), 161.5 (C_4′, J_C−F= 244.9 Hz),

164.2 (CO), 165.9 (CO). ESI-MS: m/z = 437 (M + H)⁺.

1

(CO), 162.6 (CO, J_C−F= 267.0 Hz), 162.8 (CO, J_C−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-dihydroisoqui-

noline-4-carboxylate (57). Yellow solid (68%), mp 118−125 °C,

N-(4-Fluorobenzyl)-2-(benzyloxy)-7-chloro-1,3-dioxo-1,2,3,4-tet-

rahydroisoquinoline-4-carboxamide (61). White powder (81%),

1

100% keto form, mp 216−218 °C. H NMR (300 MHz, DMSO-

2

3

1

d₆): δ = 4.31 (dd, 1H, CH₂, J_H−H= 15.4 Hz, J_H−H= 5.0 Hz), 4.32

(dd, 1H, CH₂, ²J_H−H= 15.4 Hz, ³J_H−H= 5.0 Hz), 5.01 (d, 1H, OCH₂,

²J_H−H= 14.9 Hz), 5.02 (d, 1H, OCH₂, ²J_H−H= 14.9 Hz), 5.20 (s, 1H,

100% enol form. H NMR (300 MHz, CDCl₃): δ = 4.17 (s, 3H,

OCH₃), 5.29 (s, 2H, OCH₂), 7.43 (m, 3H, H_Ar), 7.62 (m, 2H, H_Ar),

8.10 (dd, 1H, H₇, ³J_H7−H8= 8.7 Hz, ⁴J_H7−H5= 1.8 Hz), 8.54 (d, 1H, H₈,

3

4

³J_H8−H7= 8.7 Hz), 9.33 (d, 1H, H₅, J_H5−H7= 1.8 Hz), 15.9 (s, 1H,

H₄), 7.18 (t, 2H, H_3′and H_5′, J_H−H= J_H−F= 8.3 Hz), 7.30 (dd, 2H,

H_2′and H_6′, ³J_H−H= 7.6 Hz, ⁴J_H−F= 4.5 Hz), 7.42−7.47 (m, 4H, H_Ar),

OH). ¹³C NMR (75 MHz, CDCl₃): δ = 53.9 (OCH₃), 79.4 (OCH₂),

84.4 (C_IV, C₄), 118.4 (CH), 120.2 (CH), 124.8 (C_IV), 128.8 (2CH),

129.7 (CH), 130.3 (2CH), 133.2 (C_IV), 133.9 (C_IV), 151.2 (C_IV, 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-4-

carboxamide (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

3

7.56−7.57 (m, 2H, H_Ar), 7.84 (d, 1H, H_Ar, J_H−H= 8.2 Hz), 8.05 (s,

3

1H, H₈), 9.33 (t, 1H, NH, J_H−H= 5.0 Hz). ¹³C NMR (75 MHz,

DMSO-d₆): δ = 42.0 (CH₂), 55.3 (C₄), 77.4 (OCH₂), 115.2 (d, C_3′

2

and C_5′, J_C−F= 21.3 Hz), 127.1 (C_IV), 127.2 (CH), 128.4 (2CH),

128.8 (CH), 128.9 (CH), 129.2 (d, C_2′and C_6′, ³J_C−F= 8.2 Hz), 129.4

(2CH), 133.1 (C_IV), 133.5 (C_IV), 134.0 (CH), 134.3 (C_IV), 134.5 (d,

4

1

C_1′, J_C−F= 2.7 Hz), 160.0 (CO), 161.3 (d, C_4′, J_C−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-tet-

rahydroisoquinoline-4-carboxamide (62). White powder (77%),

1

100% keto form, mp 171−177 °C. H NMR (300 MHz, DMSO-

2

3

d₆): δ = 4.33 (dd, 1H, CH₂, J_H−H= 15.4 Hz, J_H−H= 5.4 Hz), 4.34

(dd, 1H, CH₂, ²J_H−H= 15.4 Hz, ³J_H−H= 5.4 Hz), 5.02 (d, 1H, OCH₂,

1

give a white powder (69%), 100% keto form, mp 194−196 °C. H

²J_H−H= 9.1 Hz), 5.03 (d, 1H, OCH₂, J_H−H= 9.1 Hz), 5.20 (s, 1H,

2

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,

H_Ar), 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,

3

H₄), 7.18 (t, 2H, H_3′and H_5′, J_H−H= J_H−F= 8.6 Hz), 7.31 (dd, 2H,

H_2′and H_6′, ³J_H−H= 8.0 Hz, ⁴J_H−F= 5.7 Hz), 7.39−7.43 (m, 4 H, H_Ar),

3

7.56−7.58 (m, 2H, H_Ar), 7.95 (d, 1H, H_Ar, J_H−H= 8.0 Hz), 8.18 (s,

1H, H₈), 9.34 (t, 1H, NH, J_H−H= 5.4 Hz). ¹³C NMR (75 MHz,

3

2

C_3′and C_5′, J_C−F= 20.7 Hz), 121.4 (CH), 126.3 (C_IV), 126.6 (C_IV),

128.1 (CH), 128.4 (2CH), 128.9 (CH), 129.2 (d, C_2′₄and C_6′, ³J_C−F

=

DMSO-d₆): δ = 42.5 (CH₂), 55.8 (C₄), 77.9 (OCH₂), 115.7 (d, C_3′

2

and C_5′, J_C−F= 20.7 Hz), 121.8 (C₇), 127.8 (C_IV), 128.9 (2CH),

8.2 Hz), 129.4 (2CH), 134.4 (C_IV), 134.7 (d, C_1′, J_C−F= 2.7 Hz),

129.4 (CH), 129.5 (CH), 129.7 (d, C_2′and C_6′, ³J_C−F= 8.2 Hz), 129.9

1

159.0 (CO), 160.8 (CO), 161.3 (d, C_4′, J_C−F= 241.1 Hz), 164.5

4

(CO), 166.3 (CO). ESI-MS: m/z = 449 (M + H)⁺.

(2CH), 130.6 (CH), 134.4 (C_IV), 134.8 (C_IV), 135.0 (d, C_1′, J_C−F

=

1

N-(4-Fluorobenzyl)-2-(benzyloxy)-3-hydroxy-7-nitro-1-oxo-1,2-di-

hydroisoquinoline-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

3.3 Hz), 137.3 (CH), 160.2 (CO), 161.8 (d, C_4′, J_C−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-dioxo-

1,2,3,4-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,

3

H₄), 7.18 (t, 2H, H_3′and H_5′, J_H−H= J_H−F= 8.6 Hz), 7.33 (m, 2H,

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1

H_2′and H_6′), 7.42−7.44 (m, 3H, H_Ar), 7.56−7.58 (m, 2H, H_Ar), 7.69

161.8 (d, C_4′, J_C−F= 241.0 Hz), 163.5 (CO), 164.9 (CO), 166.7

(CO). ESI-MS: m/z = 539 (M + H)⁺.

3

(d, 1H, H_Ar, J_H−H= 7.5 Hz), 8.15 (d, 1H, H_Ar, J_H−H= 7.2 Hz), 8.33

(s, 1H, H₈), 9.41 (m, 1H, NH). ¹³C NMR (75 MHz, DMSO-d₆): δ =

N-(4-Fluorobenzyl)-2-(benzyloxy)-1,3-dioxo-7-phenylacetamido-

1,2,3,4-tetrahydroisoquinoline-4-carboxamide (68). White powder

(80%), 100% keto form, mp 225−229 °C. ¹H NMR (300 MHz,

2

42.2 (CH₂), 55.9 (C₄), 77.6 (OCH₂), 115.3 (d, C_3′and C_5′, J_C−F

=

1

21.8 Hz), 123.6 (CF₃, J_C−F= 270.8 Hz), 124.6 (CH), 126.4 (C_IV),

2

128.3 (CH), 128.5 (2CH), 129.0 (CH), 129.3 (d, C_2′and C_6′, ³J_C−F

=

DMSO-d₆): δ = 3.69 (s, 2H, CH₂), 4.31 (dd, 1H, CH₂, J_H−H= 14.6

Hz, ³J_H−H= 5.7 Hz), 4.32 (dd, 1H, CH₂, ²J_H−H= 14.6 Hz, ³J_H−H= 5.7

Hz), 5.00 (s, 2H, CH₂), 5.13 (s, 1H, H₄), 7.17 (t, 2H, H_3′and H_5′,

³J_H−F= ³J_H−H= 8.6 Hz), 7.26−7.37 (m, 8H, H_Ar), 7.41−7.43 (m, 3H,

H_Ar), 7.55−7.58 (m, 2H, H_Ar), 7.92 (d, 1H, H_Ar, ³J_H−H= 8.3 Hz), 8.36

8.2 Hz), 129.5 (2CH), 130.6 (CH), 134.4 (C_IV), 134.5 (C_IV), 139.0

1

(C_IV), 160.0 (CO), 161.3 (d, C_4′, J_C−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-tet-

(s, 1H, H₈), 9.27 (t, 1H, NH, ³J_H−H= 5.7 Hz), 10.55 (s, 1H, NH). ¹³

C

rahydroisoquinoline-4-carboxamide (64). Two hours of reﬂux; oﬀ-

1

white powder (70%), 100% keto form, mp 189−191 °C. H NMR

NMR (75 MHz, DMSO-d₆): δ = 42.4 (CH₂), 43.7 (CH₂), 55.7 (C₄),

77.8 (OCH₂), 115.6 (d, C_3′and C_5′, J_C−F= 21.3 Hz), 118.2 (CH),

125.1 (CH), 126.1 (C_IV), 127.1 (CH), 127.8 (CH), 128.8 (2CH),

128.9 (2CH), 129.3 (CH), 129.5 (C_IV), 129.6 (2CH), 129.7 (d, C_2′

2

(300 MHz, acetone-d₆): δ = 4.31−4.32 (m, 2H, CH₂), 4.93 (d, 1H,

OCH₂, ²J_H−H= 9.3 Hz), 4.99 (d, 1H, OCH₂, ²J_H−H= 9.3 Hz), 5.23 (s,

3

1H, H₄), 6.95 (t, 2H, H_3′and H_5′, J_H−H= J_H−F= 8.8 Hz), 7.22 (dd,

3

2H, H_2′and H_6′, J_H−H= 8.4 Hz, ⁴J_H−F= 5.5 Hz), 7.25−7.31 (m, 3H,

3

and C_6′, J_C−F= 7.6 Hz), 129.9 (2CH), 134.9 (C_IV), 135.2 (d, C_1′,

H_Ar), 7.48−7.50 (m, 2H, H_Ar), 7.63 (d, 1H, H₅, ³J_H5−H6= 8.1 Hz), 7.96

⁴J_C−F= 2.7 Hz), 136.1 (C_IV), 139.6 (C_IV), 161.3 (CO), 161.8 (d, C_4′,

¹J_C−F= 241.1 Hz), 164.9 (CO), 166.6 (CO), 170.0 (CO). ESI-MS: m/

3

4

(dd, 1H, H₆, J_H6−H5= 8.7 Hz, J_H6−H8= 1.6 Hz), 8.36 (d, 1H, H₈,

⁴J_H8−H6= 1.3 Hz), 9.35 (br s, 1H, NH). ¹³C NMR (75 MHz, acetone-

d₆): δ = 42.0 (CH₂), 55.9 (C₄), 77.5 (OCH₂), 110.4 (C₇), 115.2 (d,

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).

2

C_3′and C_5′, J_C−F= 21.1 Hz), 117.8 (CN), 123.0 (C_IV), 126.5 (C_IV),

1

128.3 (2CH), 129.2 (3CH), 129.3 (4CH), 134.0 (CH), 134.6 (C_IV),

Beige powder (64%), 100% keto form, mp 240−243 °C. H NMR

1

140.4 (C_IV), 161.3 (d, C_4′, J_C−F= 237.7 Hz), 163.7 (CO), 165.1

(300 MHz, DMSO-d₆): δ = 3.92 (s, 2H, CH₂), 4.31 (dd, 1H, CH₂,

²J_H−H= 19.5 Hz, ³J_H−H= 5.8 Hz), 4.32 (dd, 1H, CH₂, ²J_H−H= 19.5 Hz,

(CO), 168.2 (CO). ESI-MS: m/z = 444 (M + H)⁺.

³J_H−H= 5.8 Hz), 5.00 (d, 1H, OCH₂, J_H−H= 15.0 Hz), 5.01 (d, 1H,

2

N-(4-Fluorobenzyl)-7-acetamido-2-(benzyloxy)-1,3-dioxo-1,2,3,4-

tetrahydroisoquinoline-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, ²J_{H−3 H}= 14.6 Hz, ³J_H−H= 5.4

2

OCH₂, J_H−H= 15.0 Hz), 5.13 (s, 1H, H₄), 6.98−7.01 (m, 2H, H_Ar),

3

7.18 (t, 2H, H_3′and H_5′, J_H−H= J_H−F= 8.9 Hz), 7.30 (dd, 2H, H_Ar,

³J_H−H= 8.2 Hz, J_H−F= 5.9 Hz), 7.36−7.43 (m, 5H, H_Ar), 7.56−7.59

3

2

Hz), 4.38 (dd, 1H, CH₂, J_H−H= 14.6 Hz, J_H−H= 5.4 Hz), 5.02 (s,

3

(m, 2H, H_Ar), 7.90 (d, 1H, H_Ar, J_H−H= 8.3 Hz), 8.36 (s, 1H, H_Ar),

2H, OCH₂), 5.18 (s, 1H, H₄), 7.23 (t, 2H, H_3′and H_5′₃, ³J_H−H= ³J_H−F

=

9.28 (t, 1H, NH, J_H−H= 5.8 Hz), 10.59 (s, 1H, NH). ¹³C NMR (75

3

8.9 Hz), 7.34−7.63 (m, 8H, H_Ar), 7.94 (d, 1H, H_Ar, J_H−H= 8.0 Hz),

8.41 (s, 1H, H₈), 9.33 (t, 1H, NH, ³J_H−H= 5.4 Hz), 10.36 (s, 1H, NH).

¹³C NMR (75 MHz, DMSO-d₆): δ = 24.5 (CH₃), 42.4 (CH₂), 55.7

MHz, DMSO-d₆): δ = 38.0 (CH₂), 42.5 (CH₂), 55.7 (C₄), 77.8

(OCH₂), 115.6 (d, C_3′and C_5′, J_C−F= 21.3 Hz), 118.3 (CH), 125.2

2

(CH), 125.7 (CH), 126.2 (C_IV), 127.0 (CH), 127.2 (CH), 127.8

(CH), 128.9 (2CH), 129.3 (CH), 129.6 (C_IV), 129.7 (d, C_2′and C_6′,

2

(C₄), 77.8 (OCH₂), 115.6 (d, C_3′and C_5′, J_C−F= 21.8 Hz), 118.1

(CH), 125.0 (CH), 126.1 (C_IV), 127.8 (CH), 128.9 (2CH), 129.2

³J_C−F= 8.2 Hz), 129.9 (2CH), 134.9 (C_IV), 135.2 (d, C_1′, J_C−F= 3.3

4

3

(C_IV), 129.4 (CH), 129.7 (2CH), 129.8 (d, C_2′and C_6′, J_C−F= 8.2

1

Hz), 137.2 (C_IV), 139.5 (C_IV), 161.3 (CO), 161.8 (d, C_4′, J_C−F

241.1 Hz), 165.0 (CO), 166.7 (CO), 169.0 (CO). ESI-MS: m/z = 558

=

Hz), 131.0 (C_IV), 135.0 (C_IV), 139.8 (C_IV), 161.4 (CO), 161.8 (d, C_4′,

¹J_C−F= 240.5 Hz), 165.0 (CO), 166.7 (CO), 169.0 (CO). ESI-MS: m/

(M + H)⁺.

z = 476 (M + H)⁺.

N-(4-Fluorobenzyl)-2-(benzyloxy)-6-ﬂuoro-1,3-dioxo-1,2,3,4-tet-

rahydroisoquinoline-4-carboxamide (70). White powder (56%),

N-(4-Fluorobenzyl)-7-benzamido-2-(benzyloxy)-1,3-dioxo-

1,2,3,4-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, H_3′and H_5′, ³J_H−H= ³J_H−F= 8.7 Hz), 7.31−7.58 (m,

11H, H_Ar), 8.01−8.04 (m, 2H, H_Ar), 8.14 (s, 1H, H_Ar), 8.57 (s, 1H,

H_Ar), 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, C_3′

1

100% keto form, mp 193−195 °C. H NMR (300 MHz, DMSO-

2

3

d₆): δ = 4.29 (dd, 1H, CH₂, J_H−H= 15.2 Hz, J_H−H= 5.6 Hz), 4.36

(dd, 1H, CH₂, ²J_H−H= 15.2 Hz, ³J_H−H= 5.6 Hz), 4.98 (d, 1H, OCH₂,

²J_H−H= 9.9 Hz), 5.03 (d, 1H, OCH₂, J_H−H= 9.9 Hz), 5.20 (s, 1H,

2

H₄), 7.18 (t, 2H, H_3′and H_5′, ³J_H−H= ³J_H−F= 8.8 Hz), 7.21−7.27 (m,

2H, H_Ar), 7.30 (dd, 2H, H_2′and H_6′, ³J_H−H= 7.8 Hz, ⁴J_H−F= 5.7 Hz),

7.41−7.48 (m, 4H, H_Ar), 7.55−7.57 (m, 2H, H_Ar), 8.17 (dd, 1H, H₈,

³J_H8−H7= 8.4 Hz, ⁴J_H8−F= 5.7 Hz), 9.33 (t, 1H, NH, ³J_H−H= 5.3 Hz).

¹³C NMR (75 MHz, DMSO-d₆): δ = 42.1 (CH₂), 55.6 (C₄), 77.4

(OCH₂), 113.4 (d, CH_Ar, ²J_C−F= 23.8 Hz), 115.1 (d, C_3′and C_5′, ²J_C−F

2

and C_5′, J_C−F= 21.3 Hz), 119.5 (CH), 126.1 (C_IV), 126.3 (CH),

127.6 (CH), 128.2 (2CH), 128.9 (2CH), 129.0 (2CH), 129.3 (CH),

3

129.7 (d, C_2′and C_6′, J_C−F= 8.2 Hz), 129.9 (2CH), 132.4 (CH),

4

134.8 (C_IV), 134.9 (C_IV), 135.1 (C_IV), 135.2 (d, C_1′, J_C−F= 2.7 Hz),

2

139.7 (C₇), 161.4 (CO), 161.8 (d, C_4′, ¹J_C−F= 241.0 Hz), 165.0 (CO),

= 21.5 Hz), 116.1 (d, CH_Ar, J_C−F= 22.3 Hz), 122.1 (C_IV), 128.3

3

166.2 (CO), 166.7 (CO). ESI-MS: m/z = 538 (M + H)⁺.

(2CH), 128.8 (CH), 129.30 (d, C_2′and C_6′, J_C−F= 7.9 Hz), 129.35

(2CH), 131.4 (d, C₈, ³J_C−F= 9.9 Hz), 134.4 (C_IV), 134.6 (C_IV), 137.6

(d, C_4a, ³J_C−F= 10.1 Hz), 161.3 (d, C_IV, ¹J_C−F= 240.8 Hz), 161.8 (C_IV,

¹J_C−F= 251.1 Hz), 164.0 (CO), 165.6 (CO), 166.8 (CO). ESI-MS: m/

N-(4-Fluorobenzyl)-2-(benzyloxy)-1,3-dioxo-7-picolinamido-

1,2,3,4-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, ²J_H₃_−H= 19.7 Hz, ³J_H−H= 5.7 Hz),

z = 437 (M + H)⁺.

2

4.34 (dd, 1H, CH₂, J_H−H= 19.7 Hz, J_H−H= 5.7 Hz), 5.02 (d, 1H,

N-(4-Fluorobenzyl)-2-(benzyloxy)-8-ﬂuoro-1,3-dioxo-1,2,3,4-tet-

rahydroisoquinoline-4-carboxamide (71). Same protocol as for the

synthesis of 58 except 1.2 equiv of 4-ﬂuorobenzylamine and 15 h of

OCH₂, ²J_H−H= 15.0 Hz), 5.03 (d, 1H, OCH₂, ²J_H−H= 15.0 Hz), 5.17

3

(s, 1H, H₄), 7.18 (t, 2H, H_3′and H_5′, J_H−H= J_H−F= 8.9 Hz), 7.31

(dd, 2H, H_2′and H_6′, ³J_H−H= 8.3 Hz, ⁴J_H−F= 5.5 Hz), 7.40−7.44 (m,

4 H, H_Ar), 7.57−7.59 (m, 2H, H_Ar), 7.72 (dd, 1H, H_Ar, ³J_H−H= 7.1 Hz,

³J_H−H= 5.1 Hz), 8.10 (t, 1H, H_Ar, ³J_H−H= 7.7 Hz), 8.16−8.21 (m, 2H,

1

reﬂux; brown solid (78%), 100% keto form, mp 156−158 °C. H

2

NMR (300 MHz, DMSO-d₆): δ = 4.28 (dd, 1H, CH₂, J_H−H= 15.1

Hz, ³J_H−H= 5.6 Hz), 4.32 (dd, 1H, CH₂, ²J_H−H= 15.2 Hz, ³J_H−H= 5.6

Hz), 4.97 (d, 1H, OCH₂, ²J_H−H= 9.6 Hz), 5.02 (d, 1H, OCH₂, ²J_H−H

=

3

H_Ar), 8.77−8.79 (m, 2H, H_Ar), 9.30 (t, 1H, NH, J_H−H= 5.7 Hz),

9.6 Hz), 5.22 (s, 1H, H₄), 7.18 (t, 2H, H_3′and H_5′, ³J_H−H= ³J_H−F= 8.9

Hz), 7.23−7.30 (m, 3H, H_Ar), 7.33−7.46 (m, 4H, H_Ar), 7.51−7.60 (m,

11.06 (s, 1H, NH). ¹³C NMR (75 MHz, DMSO-d₆): δ = 42.5 (CH₂),

55.7 (C₄), 77.8 (OCH₂), 115,6 (d, C_3′and C_5′, ²J_C−F= 21.3 Hz), 119.8

(CH), 123.1 (CH), 126.1 (C_IV), 126.7 (CH), 127.6 (CH), 127.7

3

4

2H, H_Ar), 7.8 (td, 1H, H₆, J_H6−H7= J_H6−H5= 8.1 Hz, J_H6−F= 5.2

3

(CH), 128.9 (2CH), 129.3 (CH), 129.7 (d, C_2′and C_6′, J_C−F= 8.2

Hz), 9.36 (t, 1H, NH, J_H−H= 5.7 Hz). ¹³C NMR (75 MHz, DMSO-

3

Hz), 129.9 (2CH), 130.2 (C_IV), 134.9 (C_IV), 135.2 (d, C_1′, ⁴J_C−F= 2.7

d₆): δ = 42.1 (CH₂), 55.6 (C₄), 77.4 (OCH₂), 114.0 (d, C_4a, J_C−F

=

Hz), 138.7 (CH), 138.9 (C_IV), 149.0 (CH), 150.0 (C_1′), 161.3 (CO),

5.2 Hz), 115.2 (d, C_3′and C_5′, ²J_C−F= 21.5 Hz), 116.6 (d, C₇, ²J_C−F

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21.0 Hz), 123.0 (d, C₅, J_C−F= 2.7 Hz), 128.4 (2CH), 128.9 (CH),

N-(4-Fluorophenethyl)-2-(benzyloxy)-3-hydroxy-7-nitro-1-oxo-

1,2-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, ³J_H−H= 6.8 Hz), 3.48 (t, 2H, ³J_H−H= 6.8

Hz), 5.03 (s, 2H, OCH₂), 7.13 (t, 2H, H_3′and H_5′,³J_H−H= ³J_H−F= 8.8

Hz); 7.30−7.34 (m, 2H, H_Ar), 7.39−7.45 (m, 3H, H_Ar), 7.61−7.63 (m,

2H, H_Ar), 7.99 (d, 1H, H₆, ³J_H6−H5= 9.4 Hz), 8.75 (s, 1H, H₈), 9.32 (d,

3

129.30 (d, C_2′₄and C_6′, J_C−F= 8.0 Hz), 129.5 (2CH), 134.5 (C_1″),

3

134.6 (d, C_1′, J_C−F= 2.9 Hz), 135.8 (d, C₆, J_C−F= 10.0 Hz), 136.9

3

1

(C_8a), 157.8 (d, CO₁, J_C−F= 4.6 Hz), 161.4 (d, C_IV, J_C−F= 242.8

1

Hz), 161.5 (d, C_IV, J_C−F= 262.0 Hz), 163.7 (CO), 165.7 (CO). ESI-

MS: m/z = 437 (M + H)⁺.

N-(4-Fluorobenzyl)-2-(benzyloxy)-3-hydroxy-6-nitro-1-oxo-1,2-di-

hydroisoquinoline-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, H_3′and H_5′, J_H−H= J_H−F= 8.8 Hz), 7.43−7.46 (m, 5H, H_Ar),

7.59 (m, 2H, H_2′and H_6′), 8.15 (dd, 1H, H₇, ³J_H7−H8= 9.7 Hz, ⁴J_H7−H5

= 2.3 Hz), 8.34 (d, 1H, H₈, ³J_H8−H7= 9.7 Hz), 9.40 (d, 1H, H₅, ⁴J_H5−H7

= 2.3 Hz), 10.31 (m, 1H, NH). ¹³C NMR (75 MHz, DMSO-d₆): δ =

3

1H, H₅, J_H5−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),

2

115.4 (d, C_3′and C_5′, J_C−F= 20.7 Hz), 116.3 (C_IV), 124.1 (CH),

124.6 (CH), 125.1 (CH), 128.7 (2CH), 129.0 (CH), 129.8 (2CH),

130.9 (d, C_2′and C_6′, ³J_C−F= 7,6 Hz), 135.7 (C_IV), 136.6 (d, C_1′, ⁴J_C−F

3

= 1.6 Hz), 138.2 (C_IV), 144.8 (C₇), 159.6 (CO), 161.2 (d, C_4′, ¹J_C−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-1,2-

dihydroisoquinoline-4-carboxamide (78). Yellow solid, yield 74%;

2

42.1 (NHCH₂), 76.4 (OCH₂), 86.8 (C₄), 115.0 (d, C_3′and C_5′, J_C−F

1

mp 180 °C, 100% enol form. H NMR (300 MHz, DMSO-d₆): δ =

= 20.2 Hz), 121.0 (C_IV), 121.5 (CH), 123.0 (CH), 128.3 (3CH),

129.3 (5CH), 134.2 (C_IV), 134.4 (C_IV), 136.0 (C_IV), 150.3 (C₆), 159.2

(CO), 161.3 (d, C_4′, ¹J_C−F= 235.5 Hz), 163.8 (CO), 165.2 (CO). ESI-

MS: m/z = 464 (M + H)⁺.

N-Hexyl-2-(benzyloxy)-3-hydroxy-7-nitro-1-oxo-1,2-dihydroiso-

quinoline-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,

3.74 (s, 3H, OCH₃), 4,41 (s, 2H, CH₂), 5.03 (s, 2H, OCH₂), 6,90 (d,

2H, H_Ar,³J_H−H= 8.3 Hz), 7.28 (d, 2H, H_Ar, ³J_H−H= 8.3 Hz), 7.40−7.42

(m, 3H, H_Ar), 7.59−7.62 (m, 2H, H_Ar), 8.00 (dd, 1H, H₆, ³J_H6−H5= 9.8

Hz, ⁴J_H6−H8= 2.0 Hz), 8.76 (d, 1H, H₈, ⁴J_H8−H6= 2.0 Hz), 9.38 (d, 1H,

3

H₅, J_H5−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 (C_3′and C_5′),

116.6 (C_IV), 124.2 (CH), 124.6 (CH), 125.2 (CH), 128.7 (2CH),

129.0 (CH), 129.2 (C_2′and C_6′), 129.7 (2CH), 132.8 (C_IV), 135.7

(C_IV), 138.4 (C_IV), 144.8 (C₇), 158.6 (CO), 159.6 (CO), 161.9 (CO),

168.2 (CO). ESI-MS: m/z = 476 (M + H)⁺.

3

Deprotection of Precursors 58−78. N-(4-Fluorobenzyl)-2-

hydroxy-7-methoxy-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-car-

boxamide (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, ²J_H−H=14.6 Hz, ³J_H−H= 5.0

Hz), 4.30 (dd, 1H, CH₂, ²J_H−H=14.6 Hz, ³J_H−H= 5.0 Hz), 5.06 (s, 1H,

H₄), 7.17 (t, 2H, H_3′and H_5′, ³J_H−H= ³J_H−F= 8.9 Hz), 7.26−7.32 (m,

2H, CH₂, J_H−H= 6.3 Hz), 5.03 (s, 2H, OCH₂), 7.40−7.42 (m, 2H,

3

H_Ar), 7.61−7.63 (m, 3H, H_Ar), 7.97 (d, 1H, H₆, J_H6−H5= 10.3 Hz),

8.74 (s, 1H, H₈), 9.32 (d, 1H, H₅, J_H5−H6= 10.3 Hz). ¹³C NMR (75

3

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 (C_IV),

124.1 (CH), 124.6 (CH), 125.1 (CH), 128.7 (2CH), 128.9 (CH),

129.7 (2CH), 135.7 (C_IV), 138.3 (C_IV), 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-dihydroiso-

quinoline-4-carboxamide (74). Yellow solid, yield 77%, mp 169−171

1

°C, 100% enol form. H NMR (300 MHz, DMSO-d₆): δ = 5.10 (s,

4H, H_Ar), 7.52 (d, 1H, H₈, ⁴J_H8−H6= 2.3 Hz), 9.25 (t, 1H, NH, ³J_H−H

=

2H, OCH₂), 6.98 (t, 1H, H_Ar,³J_H−H= 7.2 Hz), 7.30 (t, 2H, H_Ar, ³J_H−H

= 7.9 Hz), 7.40−7.47 (m, 3H, H_Ar), 7.65−7.69 (m, 4H, H_Ar), 8.08 (dd,

5.1 Hz), 10.61 (s, 1H, NOH). ¹³C NMR (75 MHz, DMSO-d₆): δ =

1H, H₆, ³J_H6−H5= 9.8 Hz, ⁴J_H6−H8= 2.5 Hz), 8.79 (d, 1H, H₈, ⁴J_H8−H6

=

41.9 (CH₂), 54.5 (C₄), 55.5 (OCH₃), 110.9 (CH), 115.1 (d, C_3′and

2

2.5 Hz), 9.42 (d, 1H, H₅, J_H5−H6= 9.8 Hz), 12,41 (s, 1H, OH). ¹³C

NMR (75 MHz, DMSO-d₆): δ = 76.7 (OCH₂), 90.1 (C₄), 116.5

(C_IV), 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 (C_IV), 138.4

(C_IV), 140.2 (C_IV), 144.3 (C₇), 159.0 (CO), 161.8 (CO), 166.0 (CO).

ESI-MS: m/z = 432 (M + H)⁺.

3

C_5′, J_C−F= 21.3 Hz), 121.0 (CH), 126.4 (C_IV), 126.5 (C_IV), 128.0

(CH), 129.1 (d, C_2′and C_6′, ³J_C−F= 8.2 Hz), 134.7 (d, C_1′, ⁴J_C−F= 2.7

Hz), 158.9 (CO), 161.2 (d, C_4′, ¹J_C−F= 240.6 Hz), 161.4 (CO), 164.8

(CO), 166.4 (CO). ESI-MS: m/z = 359 (M + H)⁺. HRMS calcd for

C₁₈H₁₅N₂O₅, 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-Benzyl-2-(benzyloxy)-3-hydroxy-7-nitro-1-oxo-1,2-dihydroiso-

N-(4-Fluorobenzyl)-2,3-dihydroxy-7-nitro-1-oxo-1,2-dihydroiso-

quinoline-4-carboxamide (80). Intermediate 59 (0.46 g, 1.0 mmol)

was dissolved in a minimum of CH₂Cl₂at −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

quinoline-4-carboxamide (75). Orange solid, yield 62%, mp 178−181

1

°C, 100% enol form. H NMR (300 MHz, DMSO-d₆): δ = 4.49 (s,

2H, CH₂), 5.04 (s, 2H, OCH₂), 7.31−7.44 (m, 8H, H_Ar), 7.61 (d, 2H,

H_Ar, ³J_H−H= 7.3 Hz), 8.00 (dd, 1H, H₆, ³J_H6−H5= 9.5 Hz, ⁴J_H6−H8= 1.8

Hz), 8.76 (d, 1H, H₈, ⁴J_H8−H6= 1.8 Hz), 9.38 (d, 1H, H₅, ³J_H5−H6= 9.5

Hz). ¹³C NMR (75 MHz, DMSO-d₆): δ = 42.6 (CH₂), 77.1 (OCH₂),

90.5 (C₄), 116.5 (C_IV), 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

(C_IV), 138.4 (C_IV), 141.0 (C_IV), 144.7 (C₇), 159.6 (CO), 162.0 (CO),

168.3 (CO). ESI-MS: m/z = 446 (M + H)⁺.

3

= 4.46 (d, 2H, NHCH₂, J_H−H= 5.0 Hz), 7.14 (t, 2H, H_3′and H_5′,

N-(4-Fluorophenyl)-2-(benzyloxy)-3-hydroxy-7-nitro-1-oxo-1,2-

dihydroisoquinoline-4-carboxamide (76). Beige solid, yield 89%, mp

3

³J_H−H= J_H−F= 8.6 Hz), 7.36 (dd, 2H, H_2′and H_6′, J_H−H= 8.8 Hz,

⁴J_H−F= 5.7 Hz), 7.98 (dd, 1H, H₆, ³J_H6−H5= 9.6 Hz, ⁴J_H6−H8= 2.6 Hz),

8.75 (d, 1H, H₈, ⁴J_H8−H6= 2.3 Hz), 9.38 (d, 1H, H₅, ³J_H5−H6= 9.7 Hz),

10.07 (s, 1H, OH), 10.28 (t, 1H, NH, ³J_H−H= 5.0 Hz). ¹³C NMR (75

MHz, DMSO-d₆): δ = 41.4 (CH₂), 89.7 (C₄), 115.0 (d, C_3′and C_5′,

²J_C−F= 21.3 Hz), 116.0 (C_IV), 123.7 (CH), 123.8 (CH), 124.2 (CH),

129.2 (d, C_2′and C_6′, ³J_C−F= 8.2 Hz), 136.7 (C_IV), 138.3 (C_IV), 143.0

(C₇), 157.5 (CO), 159.5 (CO), 161.1 (d, C_4′, ¹J_C−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.

1

189−190 °C, 100% enol form. H NMR (300 MHz, DMSO-d₆): δ =

5.11 (s, 2H, OCH₂), 7.11−7.13 (m, 2H, H_Ar), 7.31−7.43 (m, 5H,

H_Ar), 7.65−7.71 (m, 2H, H_Ar), 8.09 (d, 1H, H₆, ³J_H6−H5= 9.8 Hz), 8.80

(s, 1H, H₈), 9.42 (d, 1H, H₅, J_H5−H6= 9.8 Hz). ¹³C NMR (75 MHz,

3

2

DMSO-d₆): δ = 77.2 (OCH₂), 90.3 (C₄), 115.6 (d, C_3′and C_5′, J_C−F

= 21.3 Hz), 117.0 (C_IV), 121.3 (d, C_2′and C_6′, ³J_C−F= 7.6 Hz), 124.2

(CH), 124.4 (CH), 125.5 (CH), 128.8 (2CH), 129.0 (CH), 129.8

(2CH), 135.7 (C_IV), 137.1 (C_IV), 138.9 (C_IV), 144.9 (C₇), 156.2

(CO), 159.6 (CO), 161.0 (d, C_4′, ¹J_C−F= 252.0 Hz), 166.5 (CO). ESI-

MS: m/z = 450 (M + H)⁺.

4655

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

Journal of Medicinal Chemistry

Article

N-(4-Fluorobenzyl)-2,3-dihydroxy-6-nitro-1-oxo-1,2-dihydroiso-

quinoline-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,

(CH₂), 55.1 (C₄), 107.1 (d, CH, ²J_C−F= 24.0 Hz), 113.2 (d, CH, ²J_C−F

2

= 23.1 Hz), 115.1 (d, C_3′and C_5′, J_C−3F= 21.1 Hz), 122.2 (d, C_8a,

⁴J_C−F= 1.6 Hz), 129.3 (d, C_2′and C_6′, J_C−F= 8.1 Hz), 131.2 (d, C₈,

4

3

³J_C−F= 9.9 Hz), 134.6 (d, C_1′, J_C−F= 2.6 Hz), 137.4 (d, C_4a, J_C−F

=

4

1

9.6 Hz), 160.1 (d, C₁, J_C−F= 2.7 Hz), 161.5 (C_4′, J_C−F= 244.9 Hz),

1

160.6 (CO), 160.9 (d, C_IV, J_C−F= 240.0 Hz), 161.3 (d, C_IV, J_C−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.

1

100% enol form. H NMR (300 MHz, DMSO-d₆): δ = 4.46 (d, 2H,

NHCH₂, ³J_H−H= 5.5 Hz), 7.14 (t, 2H, H_3′and H_5′,³J_H−H= ³J_H−F= 8.9

Hz), 7.36 (dd, 2H, H_2′and H_6′, ³J_H−H= 8.5 Hz, ⁴J_H−F= 5.8 Hz), 7.94

3

4

(dd, 1H, H₇, J_H7−H8= 9.6 Hz, J_H7−H5= 2.6 Hz), 8.30 (d, 1H, H₈,

³J_H8−H7= 8.8 Hz), 10.00 (d, 1H, H₅, ⁴J_H5−H7= 2.3 Hz). ¹³C NMR (75

MHz, DMSO-d₆): δ = 41.4 (CH₂), 85.8 (C₄), 115.0 (d, C_3′and C_5′,

²J_C−F= 21.4 Hz), 115.3 (CH), 120.1 (CH), 123.6 (C_IV), 128.0 (CH),

N-(4-Fluorobenzyl)-8-ﬂuoro-2-hydroxy-1,3-dioxo-1,2,3,4-tetrahy-

droisoquinoline-4-carboxamide (85). Hydrogenation for 20 min in

THF at room temperature over Pd/C 5%; brown solid (30%), 100%

1

keto form, mp 155−158 °C. H NMR (300 MHz, DMSO-d₆): δ =

129.7 (d, C_2′and C_6′, ³J_C−F= 8.2 Hz), 135.5 (C_IV), 136.3 (C_IV), 149.0

(C₆), 152.5 (CO), 154.0 (CO), 161.9 (d, C_4′, ¹J_C−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.

2

3

4.25 (dd, 1H, CH₂, J_H−3H= 14.9 Hz, J_H−H= 5.2 Hz), 4.33 (dd, 1H,

2

CH₂, J_H−H= 14.9 Hz, J_H−H= 5.2 Hz), 5.16 (s, 1H, H₄), 7.04−7.43

3

(m, 6H, H_Ar), 7.72 (dd, 1H, H₇, J_H7−F= 12.6 Hz, J_H7−H6= 7.4 Hz),

9.32 (br s, 1H, NH), 10.60 (s, 1H, OH). ¹³C NMR (75 MHz, DMSO-

3

d₆): δ = 41.9 (CH₂), 55.1 (C₄), 114.1 (d, C_4a, J_C−F= 5.3 Hz), 115.2

(d, C_3′and C_5′, ²J_C−F= 21.0 Hz), 116.5 (d, C₇, ²J_C−F= 21.2 Hz), 122.8

N-(4-Fluorobenzyl)-7-amino-2-hydroxy-1,3-dioxo-1,2,3,4-tetra-

hydroisoquinoline-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,

(d, C₅, ⁴J_C−F= 3.4 Hz), 129.30 (d, C_2′and C_6′, ³J_C−F= 8.0 Hz), 134.6

4

3

(d, C_1′, J_C−F= 2.7 Hz), 135.5 (d, C₆, J_C−F= 10.2 Hz), 136.8 (C_8a),

158.5 (d, CO₁, ³J_C−F= 4.8 Hz), 161.3 (d, C_IV, ¹J_C−F= 256.1 Hz), 161.4

1

(d, C_IV, J_C−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.

1

100% keto form. H NMR (300 MHz, DMSO-d₆): δ = 4.25 (dd, 1H,

2

3

2

CH₂, J_H−3H= 15.3 Hz, J_H−H= 5.5 Hz), 4.32 (dd, 1H, CH₂, J_H−H

=

N-(4-Fluorobenzyl)-7-chloro-2-hydroxy-1,3-dioxo-1,2,3,4-tetrahy-

droisoquinoline-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, ²J_H−H= 15.5 Hz, ³J_H−H= 6.2 Hz),

4.29 (dd, 1H, CH₂, ²J_H−H= 15.5 Hz, ³J_H−H= 6.2 Hz), 5.16 (s, 1H, H₄),

15.3 Hz, J_H−H= 5.5 Hz), 4.90 (s, 1H, CH), 5.60 (s, 2H, NH₂), 6.85

3

4

(dd, 1H, H₆, J_H6−H5= 8.2 Hz, J_H6−H8= 2.0 Hz), 7.02 (d, 1H, H₅,

³J_H5−H6= 8.2 Hz), 7.17 (t, 2H, H_3′and H_5′, J_H−H= J_H−F= 8.6 Hz),

3

7.25 (d, 1H, H₈, ⁴J_H8−H6= 2.0 Hz), 7.30 (dd, 2H, H_2′and H_6′, ³J_H−H

=

3

8.4 Hz, J_H−F= 5.7 Hz), 9.15 (t, 1H, NH, J_H−H= 5.1 Hz), 10.46 (s,

3

1H, OH). ¹³C NMR (75 MHz, DMSO-d₆): δ = 42.9 (CH₂), 55.4 (C₄),

7.18 (t, 2H, H_3′and H_5′, J_H−H= J_H−F= 9.0 Hz), 7.30 (dd, 2H, H_2′

and H_6′, ³J_H−H= 9.0 Hz, ⁴J_H−F= 5.6 Hz), 7.41 (d, 1H, H_Ar, ³J_H−H= 8.2

Hz), 7.79 (d, 1H, H_Ar, ³J_H−H= 8.2 Hz), 8.02 (s, 1H, H₈), 9.34 (t, 1H,

2

112.3 (CH), 116.1 (d, C_3′and C_5′, J_C−F= 21.3 Hz), 120.6 (CH),

3

121.7 (C_IV), 126.7 (C_IV), 128.2 (CH), 130.2 (d, C_2′and C_6′, J_C−F

=

8.2 Hz), 135.9 (d, C_1′, ⁴J_C−F= 2.7 Hz), 149.7 (C₇), 162.3 (d, C_4′, ¹J_C−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.

NH, J_H−H= 6.2 Hz). ¹³C NMR (75 MHz, DMSO-d₆): δ = 42.5

3

(CH₂), 55.3 (C₄), 115.6 (d, C_3′and C_5′, ²J_C−F= 21.3 Hz), 127.5 (CH),

127.7 (C_IV), 129.2 (CH), 129.7 (d, C_2′and C_6′, ³J_C−F= 8.2 Hz), 133.5

(C_IV), 133.8 (C_IV), 134.1 (CH), 135.1 (C_IV), 161.0 (CO), 161.8 (d,

C_4′, ¹J_C−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-ﬂuoro-2-hydroxy-1,3-dioxo-1,2,3,4-tetrahy-

droisoquinoline-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, ²J_H−H= 15.6 Hz, ³J_H−H= 5.3 Hz),

4.31 (dd, 1H, CH₂, ²J_H−H= 15.6 Hz, ³J_H−H= 5.3 Hz), 5.15 (s, 1H, H₄),

N-(4-Fluorobenzyl)-7-bromo-2-hydroxy-1,3-dioxo-1,2,3,4-tetra-

hydroisoquinoline-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, ²J_H−H= 15.3 Hz, ³J_H−H= 5.1 Hz),

4.29 (dd, 1H, CH₂, ²J_H−H= 15.3 Hz, ³J_H−H= 5.1 Hz), 5.14 (s, 1H, H₄),

3

7.17 (t, 2H, H_3′and H_5′, J_H−H= J_H−F= 8.6 Hz), 7.30 (dd, 2H, H_2′

and H_6′, ³J_H−H= 8.6 Hz, ⁴J_H−F= 5.7 Hz), 7.44 (dd, 1H, H₅, ³J_H5−H6

=

4

3

8.0 Hz, J_H5−F= 5.1 Hz), 7.60 (td, 1H, H₆, ³J_H6−H5= J_H₄_6−F= 8.0 Hz,

3

⁴J_H6−H8= 1.9 Hz), 7.79 (dd, 1H, H₈, J_H8−F= 9.1 Hz, J_H8−H6= 1.9

3

7.18 (t, 2H, H_3′and H_5′, J_H−H= ³J_H−F= 8.6 Hz), 7.28−7.36 (m, 3H,

H_Ar), 8.06 (d, 1H, H_Ar, ³J_H−H= 8.2 Hz), 8.14 (s, 1H, H₈), 9.33 (t, 1H,

3

Hz), 9.33 (t, 1H, NH, J_H−H= 5.3 Hz). ¹³C NMR (75 MHz, DMSO-

3

NH, J_H−H= 5.1 Hz). ¹³C NMR (75 MHz, DMSO-d₆): δ = 42.5

2

d₆): δ = 42.4 (CH₂), 55.2 (C₄), 114.3 (d, CH_Ar, J_C−F= 23.5 Hz),

(CH₂), 55.4 (C₄), 115.6 (d, C_3′and C_5′, ²J_C−F= 21.3 Hz), 121.6 (C₇),

2

115.6 (d, C_3′and C_5′, J_C−F= 21.3 Hz), 121.7 (d, CH_Ar, J_C−F= 22.4

Hz), 127.9 (d, C_8a, ³J_C−3F= 8.2 Hz), 129.6 (d, 1H, C₅, ³J_C−F= 8.2 Hz),

129.7 (d, C_2′and C_6′, J_C−F= 8.2 Hz), 131.2 (d, C_4a, ⁴J_C−F= 3.3 Hz),

135.1 (d, C_1′, ⁴J_C−F= 3.3 Hz), 161.1 (d, C₁, ⁴J_C−F= 2.2 Hz), 161.7 (d,

127.9 (C_IV), 129.4 (CH), 129.7 (d, C_2′and C_6′, ³J_C−F= 8.2 Hz), 130.5

4

(CH), 134.2 (C_IV), 135.1 (d, C_1′, J_C−F= 2.2 Hz), 136.9 (CH), 160.9

(CO), 161.8 (d, C_4′, ¹J_C−F= 240.6 Hz), 164.9 (CO), 166.3 (CO). ESI-

MS: 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.

1

C₇, J_C−F= 241.1 Hz), 162.0 (C_4′, J_C−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)-2-hydroxy-7-triﬂuoromethyl-1,3-dioxo-

1,2,3,4-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, ²J_H−H= 15.4 Hz, ³J_H−H= 5.8 Hz),

4.35 (dd, 1H, CH₂, ²J_H−H= 15.4 Hz, ³J_H−H= 5.8 Hz), 5.28 (s, 1H, H₄),

7.18 (t, 2H, H_3′and H_5′, ³J_H−H= ³J_H−F= 8.7 Hz), 7.33 (m, 2H, H_2′and

H_6′), 7.68 (d, 1H, H_Ar, ³J_H−H= 8.2 Hz), 8.16 (d, 1H, H_Ar, ³J_H−H= 8.2

N-(4-Fluorobenzyl)-6-ﬂuoro-2-hydroxy-1,3-dioxo-1,2,3,4-tetrahy-

droisoquinoline-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, H_3′and H_5′, H₅, H₇), 7.28−7.30 (m, 2H, H_2′and H_6′), 8.13

3

4

3

(dd, 1H, H₈, J_H8−H7= 8.8 Hz, J_H8−F= 6.0 Hz), 9.31 (m, 1H, NH),

Hz), 8.34 (s, 1H, H₈), 9.44 (t, 1H, NH, J_H−H= 5.2 Hz). ¹³C NMR

10.367 (br s, 1H, OH). ¹³C NMR (75 MHz, DMSO-d₆): δ = 42.0

(75 MHz, DMSO-d₆): δ = 42.2 (CH₂), 55.5 (C₄), 115.3 (d, C_3′and

4656

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Journal of Medicinal Chemistry

Article

2

1

C_5′, J_C−F= 21.8 Hz), 123.6 (CF₃, J_C−F= 270.8 Hz), 124.4 (CH),

³J_H−H= 8.3 Hz), 7.69−7.73 (m, 1H, H_Ar), 8.07−8.20 (m, 3H, H_Ar),

2

3

126.5 (C_IV), 128.2 (CH), 128.9 (C₇, J_C−F= 32.5 Hz), 129.3 (d, C_2′

and C_6′, J_C−F= 8.2 Hz), 130.2 (CH), 134.6 (C_1′, J_C−F= 2.6 Hz),

8.74−8.78 (m, 2H, H_Ar), 9.32 (t, 1H, NH, J_H−H= 5.6 Hz), 11.02 (s,

3

4

1H, NH). ¹³C NMR (75 MHz, DMSO-d₆): δ = 42.4 (CH₂), 55.3 (C₄),

1

2

139.0 (C_IV), 160.0 (CO), 161.4 (d, C_4′, J_C−F= 241.5 Hz), 164.4

115.6 (d, C_3′and C_5′, J_C−F= 21.3 Hz), 119.6 (CH), 123.1 (CH),

(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.

126.2 (C_IV), 126.4 (CH), 127.5 (CH), 127.6 (CH), 129.7 (d, C_2′and

C_6′, ³J_C−F= 8.5 Hz), 130.1 (C_IV), 135.3 (d, C_1′, ⁴J_C−F= 3.3 Hz), 138.7

1

(CH), 138.8 (C₇), 149.0 (CH), 150.0 (C_1″), 161.7 (d, C_4′, J_C−F

=

241.0 Hz), 161.9 (CO), 163.4 (CO), 165.2 (CO), 166.8 (CO). ESI-

MS: 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)-7-cyano-2,3-dihydroxy-1-oxo-1,2-dihydroiso-

quinoline-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),

N-(4-Fluorobenzyl)-2-hydroxy-1,3-dioxo-7-phenylacetamido-

1,2,3,4-tetrahydroisoquinoline-4-carboxamide (93). Intermediate 68

was hydrogenated over Pd/C 5% as in synthesis of 79 to give a gray

3

7.14 (t, 2H, H_3′and H_5′, J_H−H= J_H−F= 8.9 Hz), 7.36 (dd, 2H, H_2′

and H_6′, ³J_H−H= 8.3 Hz, ⁴J_H−F= 5.8 Hz), 7.51 (dd, 1H, H₆, ³J_H6−H5

=

1

9.1 Hz, ⁴J_H6−H8= 2.1 Hz), 8.22 (d, 1H, H₈, ⁴J_H8−H6= 2.0 Hz), 9.44 (d,

solid (83%), mp 203−204 °C, 100% keto form. H NMR (300 MHz,

DMSO-d₆): δ = 3.67 (s, 2H, CH₂), 4.29 (d, 2H, CH₂, ³J_H−H= 5.8 Hz),

3

1H, H₅, J_H5−H6= 9.1 Hz), 10.35 (br s, 1H, NH), 10.10 (br s, 1H,

3

OH). ¹³C NMR (75 MHz, DMSO-d₆): δ = 42.0 (CH₂), 88.1 (C₄),

99.6 (C₇), 115.5 (d, C_3′and C_5′, ²J_C−F= 21.1 Hz), 117.1 (CN), 120.8

(C_IV), 124.6 (CH), 129.6 (d, C_2′and C_6′, ³J_C−F= 8.2 Hz), 132.0 (CH),

132.2 (CH), 137.6 (C_IV), 142.0 (C_IV), 157.3 (CO), 161.2 (CO), 161.5

(d, C_4′, ¹J_C−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.

5.08 (s, 1H, H₄), 7.17 (t, 2H, H_3′and H_5′, J_H−H= J_H−F= 8.8 Hz),

7.25−7.35 (m, 8H, H_Ar, H₅, H_2′, and H_6′), 7.87 (dd, 1H, H₆, ³J_H6−H5

8.4 Hz, ⁴J_H6−H8= 2.2 Hz), 8.33 (d, 1H, H₈, ⁴J_H8−H6= 2.2 Hz), 9.25 (t,

1H, NH, ³J_H−H= 5.8 Hz), 10.51 (s, 1H, NH), 10.61 (s, 1H, OH). ¹³

=

C

NMR (75 MHz, DMSO-d₆): δ = 42.4 (CH₂), 43.7 (CH₂), 55.2 (C₄),

115.6 (d, C_3′and C_5′, J_C−F= 20.7 Hz), 118.1 (CH), 124.8 (CH),

2

126.2 (C_IV), 127.1 (CH), 127.7 (CH), 128.8 (2CH), 129.3 (C_IV),

3

129.6 (2CH), 129.7 (d, C_2′and C_6′, J_C−F= 8.2 Hz), 135.2 (d, C_1′,

⁴J_C−F= 2.7 Hz), 136.1 (C_IV), 139.6 (C_IV), 161.7 (d, C_4′, ¹J_C−F= 241.1

N-(4-Fluorobenzyl)-7-acetamido-2-hydroxy-1,3-dioxo-1,2,3,4-tet-

rahydroisoquinoline-4-carboxamide (90). Intermediate 65 was

treated with boron trichloride as in synthesis of 81 to give a beige

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.

1

solid (43%), 100% keto form, mp 227−230 °C. H NMR (300 MHz,

2

DMSO-d₆): δ = 2.08 (s, 3H, CH₃), 4.29 (dd, 1H, CH₂, J_H−H= 15.5

Hz, ³J_H−H= 5.6 Hz), 4.30 (dd, 1H, CH₂, ²J_H−H= 15.5 Hz, ³J_H−H= 5.6

N-(4-Fluorobenzyl)-2-hydroxy-1,3-dioxo-7-[2-(thiophen-2-yl)-

acetamido]-1,2,3,4-tetrahydroisoquinoline-4-carboxamide (94). In-

termediate 69 was treated with boron trichloride as in synthesis of 81

3

Hz), 5.08 (s, 1H, H₄), 7.17 (t, 2H, H_3′and H_5′, J_H−H= J_H−F= 8.9

Hz), 7.28−7.32 (m, 3H, H₅, H_2′, and H_6′), 7.84 (dd, 1H, H₆, ³J_H6−H5

=

1

8.5 Hz, ⁴J_H6−H8= 2.2 Hz), 8.32 (d, 1H, H₈, ⁴J_H8−H6= 2.2 Hz), 9.27 (t,

to give a green powder (61%), mp 183−186 °C, 100% keto form. H

1H, NH, J_H−H= 5.6 Hz), 10.27 (s, 1H, NH). ¹³C NMR (75 MHz,

3

NMR (300 MHz, DMSO-d₆): δ = 3.91 (s, 2H, CH₂), 4.30 (dd, 1H,

2

3

2

CH₂, J_H−H= 15.6 Hz, J_H−H= 5.7 Hz), 4,31 (dd, 1H, CH₂, J_H−H

=

DMSO-d₆): δ = 24.5 (CH₃), 42.4 (CH₂), 55.2 (C₄), 115.6 (d, C_3′and

3

2

15.6 Hz, J_H−H= 5.7 Hz), 5.09 (s, 1H, C₄), 6.99−7.00 (m, 2H, H_Ar),

7.17 (t, 2H, H_3′and H_5′, J_H−H= J_H−F= 8.8 Hz), 7.31 (dd, 2H, H_2′

C_5′, J_C−F= 21.3 Hz), 117.9 (CH), 124.7 (CH), 126.2 (C_IV), 127.6

3

(CH), 129.1 (C_IV), 129.7 (d, C_2′and C_6′, J_C−F= 8.7 Hz), 135.2 (d,

3

4

1

and H_6′, J_H−H= 8.6 Hz, J_H−F= 5.1 Hz), 7.41−7.42 (m, 2H, H_Ar),

7.86 (d, 1H, H_Ar, ³J_H−H= 8.6 Hz), 8.34 (s, 1H, H₈), 9.27 (t, 1H, NH,

³J_H−H= 5.7 Hz), 10.56 (s, 1H, NH). ¹³C NMR (75 MHz, DMSO-d₆):

C_1′, J_C−F= 2.2 Hz), 139.7 (C₇), 161.7 (C_4′, J_C−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.

δ = 38.0 (CH₂), 42.4 (CH₂), 55.2 (C₄), 115.5 (d, C_3′and C_5′, ²J_C−F

=

21.3 Hz), 118.1 (CH), 124.9 (CH), 125.7 (CH), 126.2 (C_IV), 127.0

(CH), 127.2 (CH), 128.9 (CH), 129.5 (C_IV), 129.7 (d, C_2′and C_6′,

N-(4-Fluorobenzyl)-7-benzamido-2-hydroxy-1,3-dioxo-1,2,3,4-

tetrahydroisoquinoline-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,

4

³J_C−F= 8.2 Hz), 135.2 (d, C_1′, J_C−F= 2.7 Hz), 137.2 (C_IV), 139.4

1

(C_IV), 1613.7 (d, C_4′, J_C−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.

2

3

DMSO-d₆): δ = 4.32 (d, 1H, CH₂, J_H−H=15.2 Hz, J_H−H= 5.7 Hz),

4.33 (d, 1H, CH₂, ²J_H−H= 15.2 Hz, ³J_H−H= 5.7 Hz), 5.12 (s, 1H, H₄),

3

7.18 (t, 2H, H_3′and H_5′, J_H−H= J_H−F= 8.9 Hz), 7.32 (dd, 2H, H_2′

and H_6′, J_H−H= 8.9 Hz, J_H−F= 5.6 Hz), 7.37 (d, 1H, H₅, J_H5−H6

3

4

3

=

N-Hexyl-2,3-dihydroxy-7-nitro-1-oxo-1,2-dihydroisoquinoline-4-

carboxamide (95). Intermediate 73 was treated with boron trichloride

as in synthesis of 80 to give a yellow powder (20%), mp 114−116 °C,

8.5 Hz), 7.56−7.62 (m, 3H, H_Ar), 7.99−8.01 (m, 2H, H_Ar), 8.11 (dd,

1H, H₆, ³J_H6−H5= 8.5 Hz, ⁴J_H6−H8= 2.2 Hz), 8.53 (d, 1H, H₈, ⁴J_H8−H6

=

3

1

2.2 Hz), 9.29 (t, 1H, NH, J_H−H= 5.7 Hz), 10.56 (s, 1H, NH), 10.63

100% enol form. H NMR (300 MHz, DMSO-d₆): δ = 0.87 (m, 3H,

(s, 1H, OH). ¹³C NMR (75 MHz, DMSO-d₆): δ = 42.4 (CH₂), 55.3

CH₃), 1.30 (m, 6H, 3CH₂), 1.48 (m, 2H, CH₂), 3.24 (t, 2H, CH₂,

2

³J_H−H= 5.1 Hz), 7.99 (d, 1H, H₆, ³J_{H6−H 5}= 9.5 Hz), 8.74 (s, 1H, H₈),

(C₄), 115.6 (d, C_3′and C_5′, J_C−F= 21.3 Hz), 119.4 (CH), 126.0

9.23 (d, 1H, H₅, ³J_H5−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 (C_IV), 124.1 (CH), 124.3 (CH), 124.6 (CH),

138.7 (C_IV), 143.2 (C_IV), 157.9 (CO), 161.0 (CO), 167.9 (CO). ESI-

MS: 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.

(CH), 126.2 (C_IV), 127.5 (CH), 128.2 (2CH), 129.0 (2CH), 129.7 (d,

C_2′and C_6′,³J_C−F= 8.2 Hz), 132.4 (CH), 134.1 (C_IV), 134.8 (C_IV),

1

135.2 (C_IV), 139.6 (C₇), 161.8 (C_4′, J_C−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-1,2,3,4-

tetrahydroisoquinoline-4-carboxamide (92). Intermediate 67 was

treated with boron trichloride as in synthesis of 81 to give a yellow

N-Phenyl-2,3-dihydroxy-7-nitro-1-oxo-1,2-dihydroisoquinoline-4-

carboxamide (96). Intermediate 74 was treated with boron trichloride

as in synthesis of 80 to give a yellow powder (32%), mp 162−165 °C,

1

powder (33%), mp 211−212 °C, 100% keto form. H NMR (300

100% enol form. H NMR (300 MHz, DMSO-d₆): δ = 6.98 (t, 1H,

2

3

MHz, DMSO-d₆): δ = 4.31 (dd, 1H, CH₂, J_H−H= 15.6 Hz, J_H−H

=

H_4′, J_H−H= 7.4 Hz), 7.29 (t, 2H, H_3′and H_5′, J_H−H= 7.6 Hz), 7.65

5.6 Hz), 4.32 (dd, 1H, CH₂, ²J_H−H= 15.6 Hz, ³J_H−H= 5.6 Hz), 5.14 (s,

(d, 2H, H_2′and H_6′, J_H−H= 7.8 Hz), 8.07 (d, 1H, H₆, J_H6−H5= 9.8

Hz), 8.79 (s, 1H, H₈), 9.40 (d, 1H, H₅, J_H5−H6= 9.8 Hz), 12.43 (s,

3

1H, C₄), 7.18 (t, 2H, C_3′and C_5′, J_H−H= J_H−F= 8.6 Hz), 7.31 (dd,

3

4

2H, H_2′and H_6′, J_H−H= 8.6 Hz, J_H−F= 5.7 Hz), 7.38 (d, 1H, H_Ar,

1H, OH). ¹³C NMR (75 MHz, DMSO-d₆): δ = 90.3 (C₄), 116.9 (C_1′),

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Article

119.6 (2CH), 119.6 (CH), 124.2 (2CH), 125.0 (CH), 129.2 (2CH),

139.1 (C_IV), 140.7 (C_IV), 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.

Gaussian 09 package.³¹Our 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 3.5.³²Magnesium cations were set to allow

octahedral geometry. Docking calculations were carried out with the

CCDC GOLD 5.2 docking suite.³³The 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 software³⁵after the method was validated by

superimposing the best pose for 101 with its crystal structure

extracted from PDB 4IKF.

N-Benzyl-2,3-dihydroxy-7-nitro-1-oxo-1,2-dihydroisoquinoline-4-

carboxamide (97). Intermediate 75 was treated with boron trichloride

as in synthesis of 80 to give a yellow powder (25%), mp 164−166 °C,

1

100% enol form. H NMR (300 MHz, DMSO-d₆): δ = 4.49 (s, 2H,

3

CH₂), 7.25−7.34 (m, 5H, H_Ar), 8.02 (d, 1H, H₆, J_H6−H5= 9.1 Hz),

3

8.76 (s, 1H, H₈), 9.24 (d, 1H, H₅, J_H5−H6= 9.1 Hz). ¹³C NMR (75

MHz, DMSO-d₆): δ = 42.6 (CH₂), 90.2 (C₄), 116.5 (C_IV), 124.1

(CH), 124.2 (CH), 124.7 (CH), 127.0 (CH), 127.7 (2CH), 128.8

(2CH), 138.7 (C_IV), 140.9 (C_IV), 143.4 (C₇), 158.0 (CO), 161.1

(CO), 167.9 (CO). ESI-MS: m/z = 356 (M + H)⁺. HRMS calcd for

C₁₇H₁₃N₃O₆, 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-dihydroiso-

quinoline-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, H_3′and H_5′₄, ³J_H−H= ³J_H−F= 8.3 Hz), 7.66 (dd, 2H, H_2′

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′-ACTGCTAGAGATTTTCCA-

CACTGACTAAAAGGGTC-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′-GACCCTTTTAGTCAGTGTGGAAAATCTC-

TAGCAGT-3′. For the strand transfer assay, a precleaved oligonucleo-

tide 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 β-

mercaptoethanol, 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-(2-

hydroxyethyl)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.³⁶Escherichia 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.

3

and H_6′, J_H−H= 8.3 Hz, J_H−F= 5.3 Hz), 8.06 (d, 1H, H₆, J_H6−H5

=

9.8 Hz), 8.78 (s, 1H, H₈), 9.40 (d, 1H, H₅, ³J_H5−H6= 9.8 Hz), 12.46 (s,

1H, OH). ¹³C NMR (75 MHz, DMSO-d₆): δ = 90.1 (C₄), 115.7 (d,

C_3′and C_5′, ²J_C−F= 21.8 Hz), 116.9 (C_IV), 121.2 (d, C_2′and C_6′, ³J_C−F

= 6.5 Hz), 124.2 (2CH), 125.0 (CH), 137.1 (C_IV), 139.0 (C_IV), 143.6

(C₇), 156.2 (CO), 159.3 (CO), 160.1 (d, C_4′, ¹J_C−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-dihydroi-

soquinoline-4-carboxamide (99). Intermediate 77 was treated with

boron trichloride as in synthesis of 80 to give a yellow powder (18%),

1

mp 153 °C, 100% enol form. H NMR (300 MHz, DMSO-d₆): δ =

2.79 (t, 2H, CH₂, ³J_H−H= 7.3 Hz), 3.48 (t, 2H, CH₂, ³J_H−H= 7.3 Hz),

3

7.11 (t, 2H, H_3′and H_5′, J_H−H= J_H−F= 7.5 Hz), 7.30 (dd, 2H, H_2′

3

4

3

and H_6′, J_H−H= 7.3 Hz, J_H−F= 6.0 Hz), 7.97 (d, 1H, H₆, J_H6−H5

=

9.8 Hz), 8.73 (s, 1H, H₈), 9.35 (d, 1H, H₅, J_H5−H6= 9.8 Hz). ¹³C

3

NMR (75 MHz, DMSO-d₆): δ = 34.9 (CH₂), 40.7 (CH₂), 89.9 (C₄),

2

115.2 (d, C_3′and C_5′, J_C−F= 20.7 Hz), 116.3 (C_IV), 124.0 (CH),

124.6 (CH), 128.2 (CH), 130.4 (d, C_2′and C_6′, ³J_C−F= 7.6 Hz), 136.2

(C_IV), 136.5 (C_IV), 142.8 (C₇), 157.9 (CO), 160.4 (CO), 160.9 (d,

1

C_4′, J_C−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-dihydroi-

soquinoline-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, H_Ar, ³J_H−H

=

8.1 Hz), 7.26 (d, 2H, H_Ar, ³J_H−H= 8.1 Hz), 8.00 (d, 1H, H₆, ³J_H6−H5

3

9.2 Hz), 8.75 (s, 1H, H₈), 9.28 (d, 1H, H₅, J_H5−H6= 9.2 Hz). ¹³C

NMR (75 MHz, DMSO-d₆): δ = 42.1 (CH₂), 55.5 (OCH₃), 90.2 (C₄),

114.2 (C_3′and C_5′), 116.6 (C_IV), 124.1 (CH), 124.2 (CH), 124.7

(CH), 129.1 (C_2′and C_6′), 132.6 (C_IV), 138.8 (C_IV), 143.2 (C_IV),

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.

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.³⁸Viral p24 antigen production was determined 30 h

postinfection by ELISA (Innogenetics, Belgium). Compounds were

added at 50 and 100 times their EC₅₀as 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

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.¹⁹Predictions of the ionization

state by use of the SPARC online calculator³⁰indicate 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

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Journal of Medicinal Chemistry

Article

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 bro-

mide (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.³⁹For the drug susceptibility assays,

MT-4 cells were infected with 100−300 times CCID₅₀of 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.

NNRTI, nonnucleoside reverse transcriptase inhibitor; 3′-P, 3′-

processing; P_app, 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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AUTHOR INFORMATION

Corresponding Author

*Telephone +33 320 33 72 31; fax +33 320 33 63 09; e-mail

fabrice.bailly@univ-lille1.fr.

■

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

This work was ﬁnancially supported by grants from le Minister

■

̀

e

de l’Enseignement Super

́

ieur et de la Recherche Franca

̧

ise 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.

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 Admin-

istration; FI, fusion inhibitor; HAART, highly active anti-

retroviral 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;

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4660

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

Article Doi

Investigation of a novel series of 2-hydroxyisoquinoline-1,3(2 H,4 H)-diones as human immunodeficiency virus type 1 integrase inhibitors

DOI: 10.1021/jm500109z

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Article abstract of DOI:10.1021/jm500109z

Full text of DOI:10.1021/jm500109z

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