Design, synthesis, biochemical, and antiviral evaluations of C6 benzyl and C6 biarylmethyl substituted 2-hydroxylisoquinoline-1,3-diones: Dual inhibition against HIV reverse transcriptase-associated RNase H and polymerase with antiviral activities

Article abstract of DOI:10.1021/jm501132s

Reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not targeted by current chemotherapy against human immunodeficiency virus (HIV). Although numerous chemotypes have been reported to inhibit

Full text of DOI:10.1021/jm501132s

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Article
Design, Synthesis, Biochemical and Antiviral Evaluations of C6
Benzyl and C6 Biarylmethyl Substituted 2-Hydroxylisoquinoline-1,3-
diones: Dual Inhibition against HIV Reverse Transcriptase-
Associated RNase H and Polymerase with Antiviral Activities
Sanjeev Kumar V. Vernekar, Zheng Liu, Eva Nagy, Lena Garvis Miller, Karen A. Kirby, Daniel J.
Wilson, jayakanth kankanala, Stefan G. Sarafianos, Michael A Parniak, and Zhengqiang Wang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501132s • Publication Date (Web): 18 Dec 2014
Downloaded from http://pubs.acs.org on December 22, 2014
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Design, Synthesis, Biochemical and Antiviral Evaluations of C6
Benzyl and C6 Biarylmethyl Substituted 2-
Hydroxylisoquinoline-1,3-diones: Dual Inhibition against HIV
Reverse Transcriptase-Associated RNase H and Polymerase
with Antiviral Activities
a
a
b
b
c
Sanjeev Kumar V. Vernekar , Zheng Liu , Eva Nagy , Lena Miller , Karen A. Kirby ,
a
a
c
b
Daniel J. Wilson , Jayakanth Kankanala , Stefan G. Sarafianos , Michael A. Parniak
a
and Zhengqiang Wang*
a
Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, MN 55455, USA
b
Department of Microbiology & Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219,
USA
c
Department of Molecular Microbiology and Immunology and Department of Biochemistry, University of Missouri School
of Medicine, Christopher S. Bond Life Sciences Center, Columbia, MO 65211, USA
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Abstract
Reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally
encoded enzymatic function not targeted by current chemotherapy against human
immunodeficiency virus (HIV). Although numerous chemotypes have been reported to
inhibit HIV RNase H biochemically, few show significant antiviral activity against HIV.
We report herein the design, synthesis and biological evaluations of a novel variant of 2-
hydroxyisoquinoline-1,3-dione (HID) scaffold featuring a crucial C-6 benzyl or
biarylmethyl moiety. The synthesis involved a recently reported metal-free direct
benzylation between tosylhydrazone and boronic acid, which allowed the generation of
structural diversity for the hydrophobic aromatic region. Biochemical studies showed that
the C-6 benzyl and biarylmethyl HID analogues, previously unknown chemotypes,
consistently inhibited HIV RT-associated RNase H and polymerase with IC s in low to
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sub- micromolar range. The observed dual inhibitory activity remained uncompromised
against RT mutants resistant to non-nucleoside RT inhibitors (NNRTIs), suggesting the
involvement of binding site(s) other than the NNRTI binding pocket. Intriguingly, these
same compounds inhibited the polymerase, but not the RNase H function of Moloney
Murine Leukemia Virus (MoMLV) RT and also inhibited E. coli RNase H. Additional
biochemical testing revealed a substantially reduced level of inhibition against HIV
integrase. Molecular docking corroborates favorable binding of these analogues to the
active site of HIV RNase H. Finally, a number of these analogues also demonstrated
antiviral activity at low micromolar concentrations.
Introduction
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1
HIV infects an estimated 35 million people worldwide. With the lack of effective
2
,3
4-6
vaccines and challenges in achieving viral eradication, managing HIV infection
continues to rely heavily on antivirals for prophylaxis and therapy. Anti-HIV drugs
targeting all three virally encoded enzymes: RT, integrase (IN) and protease, as well as
viral entry proteins and cellular co-receptors, provide a large repertoire for the highly
active antiretroviral therapy (HAART). Although largely efficacious, these regimens can
be plagued by the emergency of resistant HIV mutants. Therefore, less explored and
unvalidated viral targets key to HIV replication have become increasingly attractive for
developing antivirals with novel mechanism of action to inhibit resistant viral strains.
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,8
One such target is the RT associated RNase H activity. RT has two domains with
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distinct enzymatic functions essential for HIV replication: a polymerase domain that
carries out both RNA dependent DNA polymerization and DNA dependent DNA
polymerization; and an RNase H domain that selectively degrades RNA from the
RNA/DNA heteroduplex intermediate during reverse transcription. Current FDA-
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approved nucleoside RT inhibitors (NRTIs) and non-nucleoside RT inhibitors
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(NNRTIs) all target the DNA polymerase function of RT; inhibitors of RT-associated
RNase H have yet to make it to the development pipeline.
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Fig. 1. Major chemotypes reported as HIV RNase H active site inhibitors. Chemotypes
4–7 reflect a pharmacophore model consisting of a chelating triad (magenta) and an aryl
or biaryl moiety (cyan) connected through a methylene or amino linker.
The critical role of RNase H in HIV replication has long been recognized and efforts in
targeting RNase H for antiviral development have identified a few active site inhibitor
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chemotypes (Fig. 1),
including HID (1), -thujaplicinol (2), furan-2-carboxylic
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acid carbamoylmethyl ester (3), diketoacid (4), the Gilead pyrimidinol carboxylic acid
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(
5), the Merck naphthyridinone (6) and the GSK pyridopyrimidinone (7).
These
chemotypes all have a chelating triad (magenta) for competitive binding to the active site
divalent metals. Structurally more elaborate chemotypes (4–7) also feature a hydrophobic
aromatic moiety, typically an aryl (4–5) or biaryl (6–7), connected to the chelating core
through a methylene or amino linker, conferring potent and selective RNase H inhibition.
The biaryl substituent proved to be particularly effective as compounds 6–7 are among
1
8,19
the very few RNase H inhibitors that demonstrate potent antiviral activity.
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Fig. 2. Design of a novel HID scaffold 9 based on the pharmacophore model of 4–7.
We are particularly interested in the HID chelating core because we have previously
constructed C6 / C7 aryl-substituted HID scaffolds for inhibiting hepatitis C virus
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NS5B. Other variants of HID have also been explored as HIV IN inhibitors.
Klumpp et al first reported the ability of HID (1) to inhibit HIV, but not the E. coli RNase
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H, albeit without antiviral activity in cell-based assays (Fig. 2). Improved inhibitory
profile, including anti-HIV activity, was achieved by Billamboz et al through C4
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carboxylate substitution (Fig. 2, compound 8). As aforementioned, the best RNase H
inhibitors known reflect a pharmacophore model that features a biaryl moiety. This
pharmacophore model prompted us to design a previously unknown variant of HID (Fig.
2
, chemotype 9). We report herein the chemical synthesis, biochemical and aniviral
evaluations, and molecular modeling of 9.
Results and Discussion
Chemistry. The synthetic chemistry for constructing HID ring has been well
established. The synthesis typically involves a Hurtley reaction for parent HID (1) or C4
2
6,27
carboxylated HID (8).
A synthetic handle on C6 / C7 position, particularly a halogen
21,27
or amino group, also allowed variation of HID through similar synthetic routes.
This
general strategy, however, proved unsuccessful toward the synthesis of our newly
designed HID chemotype 9. The C6 benzylation in this case turned out to be a major
synthetic hurdle. After several unsuccessful attempts, we were able to work out a
synthetic route that allowed the synthesis of a library of twenty 6-benzyl or biarylmethyl
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substituted 2-hydroxyisoquinoline-1,3(2H,4H)-dione analogues in 9–10 steps (Scheme
2
8
1
). Key to this approach was the adaptation of a one-pot benzylation procedure for the
formation of C-C bond reported by Valdés and co-workers, which involves a reductive
coupling of aldehyde 12 with a boronic acid in the presence of a base (steps c–d, Scheme
1). The employment of various boronic acids in this step generated the structural diversity
for the C6 benzyl series of analogues. Another prominent feature of this synthesis is the
masking of the requisite dicarboxylic acid (17) with a alcohol handle (12) to facilitate the
key benzylation. The diversity for the second series, the C6 biarylmethyl HID, was
introduced much later in the synthesis (step h, Scheme 1) via Suzuki coupling.
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a
Scheme 1 . Synthesis of C6 arylmethyl HID analogues 20a–t.
a
Reagents and conditions: a) NaBH , THF, rt, 12 h, 87%; b) n-BuLi, DMF, THF, -78
4
ºC–rt, overnight, 76%; c) TsNHNH , toluene, 80 ºC, 2 h; d) boronic acid, K CO , 1,4-
2
2
3
dioxane, 110 ºC, 3–5 h, 40–55%; e) PPTS, toluene, reflux, 6–12 h, 75–90%; f) OsO4,
NMO, t-BuOH/ acetone/ H O, rt, 2–6 h, 60–81%; g) NaIO , RuCl , CH CN / CCl / H O,
2
4
3
3
4
2
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rt, 2–4 h, 67–85%; h) ArB(OH) , Pd(PPh ) , K CO , EtOH/ H O, microwave, 120–150
2
3 4
2
3
2
ºC, 20–30 min, 60–75%; i) NH OTHP, CDI, toluene, reflux, 12 h; j) p-TSA hydrate,
2
MeOH, 2–3 h, rt, 35–55% over two steps.
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The detailed synthesis is depicted in Scheme 1. To begin the synthesis, commercially
available 5-bromoindan-1-one (10) was reduced to alcohol 11 with NaBH , followed by a
4
direct formylation with N,N-dimethylformamide (DMF) and n-BuLi to afford aldehyde
1
2 in good yield. The subsequent benzylation was carried out via a two-step sequence:
the formation of tosylhydrazone 13 by treating aldehyde 12 with tosylhydrazine, and the
reductive coupling of 13 with various boronic acids to produce 5-arylmethylindanones
2
8
(
14a–g). Although this two-step benzylation process worked with only moderate yields,
it did tolerate a range of functional groups. The elaboration from alcohols 14a–g to
diacids 17a–g was achieved in three steps: first, a dehydration by treating with
pyridinium p-toluenesulfonate (PPTS) at reflux toluene furnished alkenes 15a–g in good
2
9,30
to excellent yields; second, an Upjohn dihydroxylation
of alkenes 15a–g yielded diols
16a–g under ambient temperature; and third, an oxidative cleavage of 1,2 diols 16a–g
31
with NaIO and RuCl resulted in intermediate homophthalic acid derivatives 17a–g in
4
3
good yields. At this point, a series of C6 biarylmethyl diacids (18h–t) were obtained via
Suzuki coupling from 4-Br intermediate diacid 17e. Finally, the target scaffold 9 as
represented by analogues 20a–t was constructed by the condensation of diacids 17a–g
and 18h–t with O-tetrahydropyran (THP) protected hydroxylamine in the presence of
carbonyldiimidazole (CDI) in refluxing toluene. The isolation of cyclized products 19a–t
from the reaction mixture proved to be rather straightforward by flash column
chromatography. This cyclization followed by the deprotection of THP under p-
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toluenesulfonic acid (p-TSA) afforded target compounds 20a–t in 35-55% yield over two
steps.
Biology. All newly synthesized analogues were evaluated biochemically for inhibition in
RNase H and/or polymerase assays of HIV RT, MoMLV RT, and E. coli RNase H, as
well as in an HIV IN strand transfer assay. Antiviral activity was assessed in cell-based
assays and antiviral EC50 and cytoxicity CC50 values were generated for selected
analogues.
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All new C6 benzyl and C6 biarylmethyl HID analogues potently inhibit HIV RT
associated RNase H and polymerase. RNA cleavage by RNase H is required at multiple
stages of reverse transcription and could involve at least three distinct modes of RNase H
1
2
cleavages : the random internal cleavages which likely represent the majority of RNase
H event during reverse transcription; the DNA 3’ end directed and polymerase dependent
cleavages which allow specific RNA cuts 17—18 nucleotides downstream from the
polymerase active site; and the RNA 5’ end directed cleavages which degrade the
recessed RNA template. The RNase H inhibitory activity of our compounds was assessed
using three different oligonucleotide duplexes as described in Materials and Methods.
Substrate HTS-1 is a short (18 bp) duplex that measures internal cleavages and is highly
32-34
sensitive to inhibitors;
substrate HTS-2 assesses DNA 3’ end directed cleavages; and
substrate HTS-3 evaluates RNA 5’ end directed cleavages. Detailed assay results are
summarized in Table 1. Overall, our newly synthesized HID analogues, both the C6
benzyl series (20a–h) and C6 biarylmethyl series (20i–t), potently inhibited all three
forms of RNase H cleavage with virtually equal potencies. Interestingly, while almost all
compounds within the C6 benzyl series (20a–g) were active in low micromolar range,
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quite a few analogues of the C6 biarylmethyl series (20i, 20l, 20o, 20r and 20t)
demonstrated submicromolar activities, suggesting that the additional aromatic ring can
be advantegous for the biochemical inhibitory activity. Another key observation was the
relatively flat structure-activity-relationship (SAR) around the C6 benzyl series where all
analogues exhibited a similar IC50 value (1.2—1.6 M) except for the 4-cyclopropyl
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derivative (20h, IC = 0.6 M) and the 4-Cl analogue (20d, IC = 3.9 M). By contrast,
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the C6 biarylmethyl series showed greater variation in IC values with two notable SAR
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trends: 1) a fluoro group on the terminal aromatic ring did not enhance biochemical
inhibition (20k and 20q vs 20i), which represents a significant departure from the
canonical IN inhibitor pharmacophore model where such a fluoro group typically benefit
target binding substantially; 2) a strong electron-withdrawing group with H-bonding
ability, such as cyano (20l) sulfonamide (20o) and pyridine (20t) appeared to confer the
most potent biochemical inhibition. These observations corroborate an inhibitor binding
mode where the terminal aromatic group makes critical contacts with the RNA / DNA
substrate. Further biochemical evaluation with a reconstituted and catalytically active
RNase H domain confirmed the observed potency and SAR trends (Table 1, RNase H
Fragment).
Table 1. Biochemical inhibitory activity of compounds 20a–t against HIV RT RNase H
and polymerase.
a
Full-Length RT RNase H IC50 (M)
RNase H
Fragment
IC (M)
RT pol
e
Cpd
R
b
c
d
IC (M)
5
0
HTS-1
HTS-2
HTS-3
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20a
0b
0c
20d
0e
0f
0g
0h
H
4-CH
4-F
1.3 ± 0.1
1.2 ± 0.1
1.3 ± 0.1
3.9 ± 0.2
1.4 ± 0.2
1.5 ± 0.1
1.6 ± 0.3
1.4 ± 0.1
1.3 ± 0.1
0.80 ± 0.1
3.7 ± 0.3
1.3 ± 0.2
1.1 ± 0.3
1.5 ± 0.05
1.1 ± 0.05
1.1 ± 0.2
1.2 ± 0.1
4.0 ± 0.15
1.1 ± 0.1
2.0 ± 0.2
1.0 ± 0.1
1.1 ± 0.05
0.9 ± 0.1
0.5 ± 0.1
2.9 ± 0.1
1.0 ± 0.1
1.2 ± 0.1
1.2 ± 0.2
1.7 ± 0.3
2.0 ± 0.3
2.3 ± 0.7
3.8 ± 0.3
1.5 ± 0.1
3.3 ± 1.3
2.3 ± 0.1
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4-Cl
4-Br
2
2
4-CF
3
2
2,4-F
2
2
0.60 ± 0.1 0.80 ± 0.05 0.90 ± 0.05 0.90 ± 0.05
1.3 ± 0.2
1.6 ± 0.7
1.5 ± 0.3
2
0i
0.80 ± 0.1
2.7 ± 0.6
0.60 ± 0.1 0.90 ± 0.05 0.50 ± 0.15
0j
2.0 ± 0.3
4.9 ± 1.1
2.7 ± 0.9
5.0 ± 1.9
0.70 ± 0.2
2.8 ± 0.2
20k
5.4 ± 0.8
0.90 ± 0.2
3.0 ± 0.4
3.7 ± 0.1
0.75 ± 0.1
2.5 ± 0.6
2
0l
0.90 ± 0.2
3.1 ± 0.3
0.90 ± 0.4
2.0 ± 0.2
0.50 ± 0.2
1.0 ± 0.1
2
0m
2
0n
1.2 ± 0.2
1.3 ± 0.5
1.4 ± 0.1
1.2 ± 0.05
1.8 ± 0.7
0.5 ± 0.1
2
0o
0.40 ± 0.1
0.50 ± 0.2
0.50 ± 0.1
0.40 ± 0.15
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2
0p
0q
2.4 ± 0.2
1.5 ± 0.5
5.1 ± 0.6
2.2 ± 0.6
2.1 ± 0.5
0.90 ± 0.15
8.7 ± 0.9
2.9 ± 0.4
1.4 ± 0.2
5.6 ± 0.3
1.2 ± 0.15
1.5 ± 0.15
0.70 ± 0.05
2.5 ± 0.3
2.6 ± 0.2
2.0 ± 0.1
6.5 ± 0.6
1.9 ± 0.2
10
11
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
0r
20s
1.8 ± 0.3
0.70 ± 0.05
2
0t
0.50 ± 0.05 1.3 ± 0.15
1.0 ± 0.1
0.80 ± 0.1
0.50 ± 0.2
0.9 ± 0.1
>25
1
--
1.2 ± 0.1
1.0 ± 0.2
0.60 ± 0.05
a
IC : concentration of a compound producing 50% inhibition, expressed as mean ±
5
0
standard deviation from at least three independent experiments.
b
Substrate that measures internal cleavage.
c
Substrate that measures DNA 3’ end directed cleavage.
d
Substrate that measures RNA 5’ end directed cleavage.
e
Reconstituted HIV RNase H domain.
In parallel to the RNase H assays, all compounds were also tested in a classic RT
3
polymerase assay with poly(A) as template, poly(dT) as primer and [ H]dTTP. The
results are listed in Table 1. Interestingly, our compounds inhibited polymerase with
potencies nearly equal to RNase H inhibition while a control compound (1) showed a
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selective profile towards RNase H inhibition consistent with previous report. Selected
analogues were further tested against NNRTI-resistant HIV RT single (Y181C) and
double (L100I/K103N) mutants. These assays yielded IC values against both RNase H
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and polymerase largely in range with those of WT RT (Table 2), strongly suggesting that
our compounds do not occupy the NNRTI pocket. Although the exact mechanism of
polymerase inhibition is not clear, it is conceivable that, with the chelating triad, these
compounds could compete for polymerase active site binding. It is also possible that they
may bind at an unknown site of RT and affect both polymerase and RNase H activities.
Nevertheless, previous work on similar HID scaffolds has reported selective inhibition
1
3,26
23
22,27
against HIV RNase H,
IN or dual inhibition against both.
The unique and potent
dual inhibition of our compounds against RT RNase H and polymerase will add to the
activity profile of these important compounds and may contribute to achieving the elusive
antiviral activity for RNase H-targeting HID compounds.
Table 2. Biochemical inhibitory activity of selected compounds against RNase H and
polymerase functions of NNRTI-resistant HIV RT mutants.
a
a
Y181C mutant IC50 (M)
L100I/K103N mutant IC50 (M)
Cpd
R
RNase H
HTS-1
RNase H
HTS-2
RNase H
RNase H
HTS-2
pol
pol
b
b
b
b
HTS-1
2
0d
4-Cl
7.0 ± 0.9
0.9 ± 0.5
9.0 ± 0.3
1.1 ± 0.6
5.3 ± 1.0
8.2 ± 0.3
0.7 ± 0.1
8.7 ± 2.5
1.0 ± 0.4
5.1 ± 0.4
0.5 ± 0.1
2
0i
0.7 ± 0.2
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0k
4.7 ± 3.4
1.3 ± 0.8
3.8 ± 0.5
7.2 ± 4.8
1.8 ± 1.3
3.9 ± 1.0
1.7 ± 0.1
0.8 ± 0.3
1.7 ± 0.3
2.5 ± 0.2
0.9 ± 0.2
1.3 ± 0.3
2.5 ± 0.6
0.8 ± 0.2
1.9 ± 0.1
1.5 ± 0.1
0.3 ± 0.1
1.0 ±0.1
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59
60
20l
2
0m
2
0n
0.8 ± 0.1
1.4 ± 0.4
0.7 ± 0.2
0.3 ±0.1
1.2 ± 0.1
1.4 ± 0.1
0.3 ± 0.1
0.5 ± 0.1
0.2 ±0.05
2
0o
0.2 ± 0.05 0.3 ± 0.1
0.4 ± 0.05
a
IC : concentration of a compound producing 50% inhibition, expressed as mean ±
5
0
standard deviation from at least three independent experiments.
b
RNase H substrates as described in Materials and Methods.
To further establish the biochemical inhibitory profile of our compounds, we also tested
selected compounds against E. coli RNase H and MoMLV RT RNase H and polymerase
activities. As shown in Table 3, all analogues demonstrated single digit M activity
against E. coli RNase H. For MoMLV RT, no inhibitory activity was detected against
RNase H and low M activity was observed against polymerase (Table 3). These results
suggest that dual inhibition of both RT functions can be achieved with HIV RT only.
Table 3. Biochemical inhibitory activity of selected compounds against E. coli RNase H
and MoMLV RT RNase H and polymerase activities.
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MoMLV RT
pol
E. coli
RNase H
MoMLV RT
RNase H
Compd
R
a
IC50 (M)
IC (M)
IC (M)
50
50
20a
H
6.5 ± 1.3
4.5 ± 1.2
7.2 ± 1.2
>30
>30
>30
3.2 ± 1.1
7.1 ± 1.1
6.9 ± 1.2
2
0d
0f
0h
4-Cl
2
4-CF
3
2
3.2 ± 1.3
3.4 ± 1.3
>30
>30
3.1 ± 1.1
13 ± 1.2
20i
2
0k
2.9 ± 1.3
3.2 ± 1.2
3.1 ± 1.3
>30
>30
>30
11 ± 1.2
8.5 ± 1.2
11 ± 1.2
20l
2
0m
2
0n
7.2 ± 1.2
2.5 ± 1.3
>30
>30
3.9 ± 1.1
2.4 ± 1.1
20o
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0t
4.0 ± 1.3
>30
2.8 ± 1.4
a
IC : concentration of a compound producing 50% inhibition, expressed as mean ±
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standard deviation from two independent experiments of duplicates.
Some C6 benzyl and C6 biarylmethyl HID analogues moderately inhibit HIV IN. RNase
H enzymes form a signature fold at the active site that defines a whole family of highly
35
homologous enzymes termed as retroviral integrase superfamily (RISF). Members of
this family of enzymes, including HIV IN, adopt a very similar active site fold as RNase
H. As a result, achieving selective RNase H inhibition over IN inhibition represents a
major challenge in RNase H inhibitor discovery. To gauge the selectivity profile of our
new HID chemotype, we tested all compounds in an HIV IN strand transfer (ST) assay.
The results are summarized in Table 4. Notably, with the exception of 20f which did not
show any activity at concentrations up to 100 M, all compounds within the C6 benzyl
HID series (20a–h) demonstrated significant activity against HIV IN at low micromolar
concentrations with IC values ranging between 3.8 M and 36 M. This observation
5
0
conforms to the pharmacophore model of IN ST inhibitors where a chelating triad and a
36
terminal benzyl group are the two major structural determiants. Nevertheless, most
compounds within this series inhibited IN with a considerably higher IC than that for
5
0
RNase H inhibition. Furthermore, the IN inhibitory activity was drastically reduced when
a second aromatic ring was itroduced to the benzyl terminus resulting in the biarylmethyl
HID series (20i–t). Seven out of the twelve analogues within this series showed no
activity against IN at concentrations up to 100 M, leading to excellent selectivity toward
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5
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RNase H inhibition (Table 4, compounds 20i–m, 20q and 20s). Others (20n–p, 20r and
2
0t) inhibited IN in low micromolar range, yet still with a sizable nominal selectivity
(3.8–16 fold, Table 4) toward RNase H inhibition. Although intractable variables
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
involved in biochemical assays render it unreliable to quantitatively correlate distinct
biochemical assays (IN vs RNase H and pol), the general lack of IN inhibition and the
potent anti-RNase H and anti-polymerase activities of the biarylmethyl series could
indicate that it is possible to develop RT dual inhibitors without inhibiting IN based on
this particular chemotype.
Table 4. Biochemical inhibitory activity of compounds 20a–t against HIV IN.
b
RNase H
IN IC50
c
Compd
R
SI
a
IC50 (M)
(M)
20a
H
4-CH
4-F
1.3 ± 0.1
1.2 ± 0.1
1.3 ± 0.1
3.9 ± 0.2
1.4 ± 0.2
1.5 ± 0.1
1.6 ± 0.3
3.8 ± 0.7
36 ± 15
36 ± 25
4.4 ± 0.5
28 ± 6.4
>100
2.9
30
2
0b
0c
20d
0e
0f
0g
0h
3
2
28
4-Cl
4-Br
1.1
20
2
2
4-CF
3
>67
13
2
2,4-F
20 ± 5.5
2
0.60 ± 0.1
0.80 ± 0.1
3.8 ± 1.6
>100
6.3
2
0i
>130
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0j
2.7 ± 0.6
5.4 ± 0.8
>100
>100
>37
>19
2
0k
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20l
0.90 ± 0.2
3.0 ± 0.4
>100
>100
>110
>33
2
0m
2
0n
1.2 ± 0.2
11 ± 7.4
9.2
20o
0.40 ± 0.1
2.4 ± 0.2
1.5 ± 0.5
0.57 ± 0.22
2.2 ± 0.6
3.6 ± 1.5
9.1 ± 2.7
>100
9.0
3.8
>67
16
2
0p
20q
2
0r
9.1 ± 2.6
>100
2
0s
>45
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2
0t
0.50 ± 0.05
5.7 ± 2.4
11
a
b
c
Data with HTS-1 as substrate.
Expressed as mean ± standard deviation from at least three independent experiments.
Selectivity index defined by IC IN / IC RNase H.
0
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0
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50
50
Many C6 benzyl and C6 biarylmethyl HID analogues inhibit HIV-1 in low micromolar
range. To assess the ability of our new C6 benzyl and biarylmethyl HID chemotypes in
inhibiting HIV replication in cell culture, we have carried out a single replication cycle
3
7,38
antiviral assay.
This assay quantitatively measures HIV infection in indicator cells
(
P4R5) through the expression of a Tat-dependent reporter -galactosidase) and quickly
determines the infectivity / inhibition with only one replication cycle by colorimetric
analysis after incubation with a -galactosidase substrate. We tested all twenty newly
synthesized HID analogues in this antiviral assay, and nine of them inhibited HIV
replication in low micromolar range (Table 5), with compound 20i being the most potent
with an antiviral EC of 3.0 M. Although compounds of a few chemotypes have been
5
0
reported to biochemically inhibit HIV RNase H, a limited number demonstrated
significant antiviral activities, which signifies the challenge for small molecules to
compete against much larger RNA / DNA substrates for active site binding. Achieving
antiviral activity with RNase H active site inhibitors entails exceptionally tight binding
20
only achieved with a few compounds. The consistent and low micromolar antiviral
activity observed with our compounds may reflect the benefits of additional polymerase
inhibition in overcoming the biochemical barrier of competing against endogenous
substrates.
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a
Table 5. Antiviral potency of selected analogues .
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Anti-HIV
CC50
Compd
R
TI
b
EC (M) (M)
50
2
0c
4-F
13
> 50
50
> 4
8
2
0f
0i
4-CF
3
6.2
2
3.0
13
50
17
2
0j
> 50
> 4
c
2
0k
10
13
10
13
13
n.d.
--
20l
> 50
n.d.
> 4
--
2
0m
2
0q
> 50
> 50
> 4
> 4
20s
a
All values are averages of two separate determinations.
b
50 μM was the highest concentration tested
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c
n.d., not determined
RNase H Active Site Binding. To confirm the RNase H active site binding mode of our
new chemotypes, we have conducted molecular modeling where a selected analogue 20i
was docked along with the potent GSK-5724 (7) into the crystal structure of full length
0
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RT co-crystallized with napthyridinone based scaffold MK2 (PDB code: 3LP1). The
full length RT consists of two domains, the p66 subunit with fingers (blue), palm (red),
thumb (green), connection (yellow) and RNase H (magenta) subdomains, and the p51
subunit (orange) as shown in Fig. 3, left. The above RT structure was subjected to
analysis and found that the napthyridinone was bound to the active site of RNase H
which is ~50 Å from the NNRTI site. The docking analysis was performed using Glide
40
(
Schrodinger Inc). The predicted binding modes of both the compounds within the
active site of RNase H were shown in Fig. 3, right.
Fig. 3 Binding mode of compound 20i. Left: structure of full length RT with two subunits
p66 and p51 (orange). p66 subunit comprises fingers (blue), palm (red), thumb (green),
connection (yellow) and RNase H (magenta) domains. Compounds 7 (green) and 20i
(
cyan) are docked into the RNase H active site. Right: a close-up view of RNase H active
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site with predicted binding mode of compound 7 (green) and 20i (cyan). Metal cofactors
2
+
(
Mn ) are colored in grey with the active site residues (D443, E478, D498 and D549) in
4
1
yellow. Pictures were generated using PyMol.
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The predicted binding mode of compound 7 within the active site of RNase H suggests a
potential interaction between the pyridopyrimidinone core (chelating triad) and the two
2
+
metal cofactors (Mn ) which are coordinated to the active site acidic residues D443,
E478, D498 and D549. The binding pocket is lined up with the residues G444, S449,
A538, H539, W535, V552, S553 of the p61 subunit and N265, W266, F346 and W535 of
the p51 subunit. The biaryl moiety is extended into the small hydrophobic region of the
RNase H which could potentially interact with the protein or nucleic acid substrate. A
similar binding mode was postulated for compound 20i within the active site of RNase H
2+
where the chelating triad in HID interacts with both the metal cofactors (Mn ), while the
CH and C-H groups at the C4 and C5 position of compound 20i respectively interacts
2
with the small hydrophobic residue A538 at the beginning of the pocket close to H539.
The biaryl linker extends from the C6 position into the pocket lined up with hydrophobic
residues W535 of the p66 subunit and W266, F346, W426 of the p51 subunit. The
predicted binding mode of both the compounds (7 and 20i) appears to satisfy the typically
required chelating triad interaction with both the metal cofactors and the biaryl linker
extends into the hydrophobic pocket (Fig. 3, right), hence corroborating the active site
inhibition mechanism against RNase H. As for polymerase binding, the uncompromised
biochemical inhibition against NNRTI-resistant RT mutants strongly indicates that the
NNRTI binding pocket is not occupied, consistent with our inability to stably dock our
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HID analogues into the NNRTI-binding pocket (data not shown). The exact binding
mode for inhibition of RT polymerase activity is still unclear.
Conclusions
0
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0
New HID chemotypes featuring a C-6 benzyl or biarylmethyl moiety were designed and
synthesized as inhibitors of HIV RT associated RNase H domain. Key to the synthesis
was the adaptation of a metal-free reductive benzylation and the masking of the requisite
dicarboxylic acid. Primary biochemical assays with WT and NNRTI-resistant HIV RT
showed that all analogues of our new chemotypes were dually active against RNase H
and polymerase in sub- to low micromolar range. Additional testing demonstrated that
our compounds also inhibit E. Coli RNase H, as well as the polymerase, but not the
RNase H, of MoMLV RT. Some analogues were also found active against HIV IN in a
strand transfer assay, though the vast majority of the newly synthesized compounds,
particularly the 6-biarylmethyl series, demonstrated a discernible preference toward
RNase H and polymerase inhibition. Importantly, many of these new analogues inhibited
HIV-1 in cell culture at low micromolar concentrations. Although the polymerase
inhibition mechanism is not clear, molecular docking corroborates a mechanism of
active site binding for RNase H inhibition. Collectively these studies established our new
C6 benzyl and biarylmethyl scaffolds as potent dual inhibitors of HIV RT RNase H and
polymerase, and that the additional polymerase inhibition may benefit active site RNase
H inhibitors in achieving antiviral activity.
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General Procedures. All commercial chemicals were used as supplied unless otherwise
indicated. Dry solvents were either purchased (toluene and dioxane) or dispensed under
argon from an anhydrous solvent system with two packed columns of neutral alumina or
molecular sieves (THF and DMF). Flash chromatography was performed on a Teledyne
Combiflash RF-200 with RediSep columns (silica) and indicated mobile phase. All
moisture sensitive reactions were performed under an inert atmosphere of ultra-pure
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argon with oven-dried glassware. H and C NMR spectra were recorded on a Varian
600 MHz spectrometer. Mass data were acquired on an Agilent TOF II TOS/MS
spectrometer capable of ESI and APCI ion sources. Analysis of sample purity was
performed on a Varian Prepstar SD-1 HPLC system with a Phenomenex Gemini, 5
micron C18 column (250mm x 4.6 mm). HPLC conditions: solvent A = H O, solvent B =
2
MeCN; flow rate = 1.0 mL/min; compounds were eluted with a gradient of 5%
MeCN/H O to 100% MeCN for 25 min. Purity was determined by total absorbance at
2
2
54 nm. All tested compounds have a purity ≥ 96.
General procedure 1 for the one-pot reductive coupling of aldehydes with boronic
acids (14a-g). To a solution of aldehyde 12 (17.4 mmol, 1.0 equiv.) in toluene (15 mL)
was added TsNHNH (1.34 g, 7.4 mmol, 1.0 equiv.) and stirred at 80 °C for 2-3 h. The
2
reaction was monitored by TLC. After complete consumption of aldehyde, solvent was
removed under vacuo. Tosylhydrazone was further dried under high vacuum for several
hours and used in the next step without further purification. To a solution of
tosylhydrazone in dioxane (20 ml) was added potassium carbonate (1.02 g, 11.1 mmol,
1
.5 equiv.), and boronic acid (11.1 mmol, 1.5 equiv.). The reaction mixture was refluxed
for 2-4 h, and monitored by TLC. The solvent was removed under reduced pressure and
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saturated solution of NaHCO was added and extracted with DCM (3 x 50 mL). The
3
combined organic layers were dried over Na SO , filtered and concentrated. The crude
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4
product was purified by flash column chromatography (EtOAc/hexane; 1:6) to afford
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desired compound (14a-g) as a solid.
1
5-Benzyl-2,3-dihydro-1H-inden-1-ol (14a). Yield 54%. H NMR (CDCl , 600 MHz) δ
3
7
.32-7.26 (m, 4H), 7.22-7.20 (m, 2H), 7.09-7.08 (m, 2H), 5.22 (t, J = 6.6 Hz, 1H), 3.98
(s, 2H), 3.04-2.99 (m, 1H), 2.80-2.75 (m, 1H), 2.50-2.45 (m, 1H), 1.97-1.94 (m, 1H),
1.61 (s, 1H, OH).
General procedure 2 for the synthesis of alkene (15a-g). To a solution of alcohol (3.45
mmol, 1.0 equiv.) in toluene (20 mL) was added PPTS (10.37 mmol, 3.0 equiv.). The
reaction mixture was refluxed for 6-10 h and monitored by TLC, cooled to room
temperature. The saturated NaHCO solution was added, and the aqueous layer was
3
extracted with EtOAc (3 x 25 mL). The combined organic layers were dried over
Na SO , filtered and concentrated. The crude product was purified by flash column
2
4
chromatography (EtOAc/hexane; 1:99) to afford desired alkene (15a-g) as a solid.
1
6-Benzyl-1H-indene (15a). Yield 90%. H NMR (CDCl , 600 MHz) δ 7.23-7.17 (m,
3
4
3
H), 7.14-7.12 (m, 3H), 7.03 (d, J = 7.8 Hz, 1H), 6.76-6.75 (m, 1H), 6.40-6.39 (m, 1H),
.93 (s, 2H), 3.25 (s, 2H).
General procedure 3 for the synthesis of diol (16a-g). To a solution of alkene (2.81
mmol, 1.0 equiv.) in (acetone/t-butanol/water; 3:3:4; 20 mL) was added osmium tetroxide
(2.5% solution in t-BuOH, 0.28 mmol, 0.1 equiv.) and N-methylmorpholineoxide (4.22
mmol, 1.5 equiv.) and stirred at room temperature for 4-10 h, and the reaction was
monitored by TLC. After complete consumption of the starting material sodium sulfite (4
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equiv.) was added and stirred at room temperature for 1 h more. The solvent was
evaporated and the aqueous layer was extracted with EtOAc (3 x 25 ml). The combined
organic layers were dried over Na SO , filtered and concentrated. The crude product was
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purified by flash column chromatography (EtOAc/hexane; 1.5:3.5) to afford desired diol
(16a-g) as a solid.
1
5
-Benzyl-2,3-dihydro-1H-indene-1,2-diol (16a). Yield 81%. H NMR (CDCl , 600
3
MHz) δ 7.34 (d, J = 7.8 Hz, 1H), 7.29-7.26 (m, 3H), 7.21-7.17 (m, 2H), 7.11 (d, J = 7.8
Hz, 1H), 7.06 (s, 1H), 4.97 (d, J = 4.8 Hz, 1H), 4.50-4.48 (m, 1H), 3.96 (s, 2H), 3.08 (dd,
J = 6.0 Hz, J = 16.2 Hz, 1H), 2.90 (dd, J = 4.2 Hz, J = 16.2 Hz, 1H), 2.27 (s, 2H, 2-OH).
General procedure 4 for the synthesis of dicarboxylic acid (17a-g). The suspension of
diol (1.86 mmol, 1.0 equiv.) in CH CN/CCl /H O (2:2:3; 20 mL) was sonicated to make
3
4
2
a clear solution. After the addition of NaIO (11.2 mmol, 6.0 equiv.) the reaction mixture
4
was stirred for 10 min, then treated with RuCl ·xH O (0.19 mmol, 0.1 equiv.). The
3
2
reaction mixture was stirred for 30-40 min, and monitored by TLC. The mixture was
diluted with water and extracted with CH Cl (3 x 25 mL), and the combined organic
2
2
layers were dried over Na SO , filtered and concentrated to give a black residue, then
2
4
Et O was added to the residue and filtered through a short pad of celite. The filtrate was
2
concentrated to provide a brown oil and was triturated with ether to get dicarboxylic acid
(17a-g) as a solid.
1
4
-Benzyl-2-(carboxymethyl)benzoic acid (17a). Yield 81%. H NMR (CD OD, 600
3
MHz) δ 7.93 (d, J = 8.4 Hz, 1H), 7.27-7.25 (m, 2H), 7.21-7.18 (s, 4H), 7.15 (s, 1H), 4.00
s, 2H), 3.96 (s, 2H).
(
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General procedure 5 for Suzuki coupling (18h-t). The mixture of di-acid 17e (150 mg,
0
.43 mmol), boronic acid (0.86 mmol, 2.0 equiv.), K CO (1.7 mmol, 4.0 equiv.),
2 3
EtOH/H O (1:1, 4.0 mL) and Pd[P(Ph) ] (30 mg) were microwaved at 120 ºC for 20-30
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min. The reaction was monitored by TLC and MS. The black residue formed was filtered,
the filtrate was concentrated under reduced pressure to remove EtOH. The aqueous
mixture was washed with Et O, and then acidified to pH 3. The white precipitate was
2
obtained via filtration, dried under high vacuum overnight, and used in the next step
without further purification.
1
4
-([1,1'-Biphenyl]-4-ylmethyl)-2-(carboxymethyl)benzoic acid (18i). Yield 75%. H
NMR (DMSO, 600 MHz) δ 7.83 (d, J = 7.8 Hz, 1H), 7.61 (d, J = 7.2 Hz, 2H), 7.57 (d, J
= 8.4 Hz, 2H), 7.42 (t, J = 7.8 Hz, 2H), 7.33-7.31 (m, 3H), 7.25-7.23 (m, 2H), 3.99 (s,
2
H), 3.88 (s, 2H).
General procedure 6 for cyclization and deprotection (20a-t). A solution of
dicarboxylic acid (0.28 mmol, 1.0 equiv.) and NH OTHP (0.34 mmol, 1.2 equiv.) in
2
toluene (15 mL) was refluxed for 5 minutes. To the mixture, a solution of CDI in DCM
(0.28 mmol, 1.0 equiv.) was added dropwise. The suspension turned clear and stirred at
reflux for 12 h, a black solid separated from solution, and the reaction monitored by TLC
and MS. The reaction mixture was passed through a short pad of silica gel which then
rinsed with (EtOAc/hexane; 1:3), the combined filtrate was evaporated to dryness. The
product was used for the next reaction without further purification. The cyclized product
was dissolved in MeOH (5.0 mL) and treated with pTSA hydrate (1.0 equiv.) and stirred
at room temperature for 2-3 h. Upon the disappearance of starting material by TLC, the
mixture was evaporated to dryness to get pale yellow solid. The solid obtained was
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triturated with water and then with ether, and dried at room temperature to afford a
desired pure compound (20a-t) as a solid.
6
-Benzyl-2-hydroxyisoquinoline-1,3(2H,4H)-dione (20a). Yield 55% (for two steps
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from 17a). H NMR (CD OD, 600 MHz) δ 8.02 (d, J = 8.4 Hz, 1H), 7.32 (d, J = 7.8 Hz,
3
1
3
1H), 7.28-7.25 (m, 2H), 7.22-7.18 (m, 4H), 4.84 (s, 2H), 4.03 (s, 2H); C NMR (CD OD,
3
1
50 MHz) δ 167.1, 162.2, 148.2, 139.9, 134.4, 128.6, 128.2, 128.1, 128.0, 127.5, 126.0,
-
1
22.8, 47.2, 41.2; HRMS-ESI(-) m/z calcd for C H NO 266.0817 [M-H] , found
16
12
3
266.0808.
2
-Hydroxy-6-(4-methylbenzyl)isoquinoline-1,3(2H,4H)-dione (20b). Yield 54% (for
1
two steps from 17b). H NMR (CD OD, 600 MHz) δ 8.02 (d, J = 8.4 Hz, 1H), 7.30-7.29
3
1
3
(m, 1H), 7.20 (s, 1H), 7.09-7.08 (m, 4H), 4.15 (s, 2H), 3.98 (s, 2H), 2.27 (s, 3H); C
NMR (CD OD, 150 MHz) δ 167.1, 162.2, 148.5, 136.8, 135.7, 134.3, 128.8, 128.5,
3
-
1
28.1, 127.4, 122.8, 40.8, 19.6; HRMS-ESI(-) m/z calcd for C H NO 280.0974 [M-H] ,
17 14
3
found 280.0961.
6
-(4-Fluorobenzyl)-2-hydroxyisoquinoline-1,3(2H,4H)-dione (20c). Yield 51% (for
1
two steps from 17c). H NMR (DMSO, 600 MHz) δ 10.31 (s, 1H, OH), 7.92 (d, J = 7.8
Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.28-7.26 (m, 2H), 7.23 (s, 1H), 7.10 (t, J = 9.0 Hz,
1
3
2
H), 4.19 (s, 2H), 3.99 (s, 2H); C NMR (DMSO, 150 MHz) δ 166.7, 162.0, 147.6,
135.4, 131.1, 128.5, 128.3, 128.0, 123.6, 115.8, 115.6, 47.5, 37.3; HRMS-ESI(-) m/z
-
calcd for C H NFO 284.0723 [M-H] , found 284.0722.
1
6
11
3
6
-(4-Chlorobenzyl)-2-hydroxyisoquinoline-1,3(2H,4H)-dione (20d). Yield 51% (for
1
two steps from 17d). H NMR (CD OD, 600 MHz) δ 8.03 (d, J = 8.4 Hz, 1H), 7.31 (d, J
3
13
=
3.9 Hz, 1H), 7.27-7.26 (m, 2H), 7.21-7.18 (m, 3H), 4.84 (s, 2H), 4.02 (s, 2H); C NMR
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(
CD OD, 150 MHz) δ 167.1, 162.1, 147.6, 138.7, 134.5, 131.9, 130.2, 128.3, 128.2,
3
1
27.9, 127.5, 123.0, 48.1, 40.4; HRMS-ESI(-) m/z calcd for C H ClNO 300.0427 [M-
16 11 3
-
H] , found 300.0420.
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-(4-Bromobenzyl)-2-hydroxyisoquinoline-1,3(2H,4H)-dione (20e). Yield 48% (for
1
two steps from 17e). H NMR (CD OD, 600 MHz) δ 7.95 (d, J = 7.8 Hz, 1H), 7.32-7.33
3
(m, 2H), 7.34-7.22 (m, 1H), 7.13 (s, 1H), 7.05 (d, J = 7.8 Hz, 2H), 4.75 (s, 2H), 3.92 (s,
1
2
H); H NMR (DMSO, 600 MHz) δ 10.39 (s, 1H, OH), 8.01 (d, J = 7.8 Hz, 1H), 7.55 (d,
J = 7.8 Hz, 2H), 7.40 (d, J = 8.4 Hz, 1H), 7.30-7.27 (m, 3H), 4.27 (s, 2H), 4.06 (s, 2H);
13
C NMR (CD OD, 150 MHz) δ 167.4, 162.2, 147.5, 139.3, 134.5, 131.3, 130.5, 128.3,
3
1
28.3, 127.9, 127.5, 125.5, 123.0, 119.8 48.1, 40.4; HRMS-ESI(-) m/z calcd for
-
C H BrNO 343.9922 [M-H] , 343.9911.
16
11
3
2
4
7
4
-Hydroxy-6-(4-(trifluoromethyl)benzyl)isoquinoline-1,3(2H,4H)-dione (20f). Yield
1
9% (for two steps from 17f). H NMR (CD OD, 600 MHz) δ 8.06 (d, J = 8.4 Hz, 1H),
3
.58 (d, J = 7.8 Hz, 2H), 7.42 (d, J = 7.8 Hz, 2H), 7.35 (d, J = 7.8 Hz, 1H), 7.25 (s, 1H),
13
.84 (s, 2H), 4.14 (s, 2H); C NMR (CD OD, 150 MHz) δ 162.1, 147.0, 144.6, 134.6,
3
129.2, 128.3, 128.0, 127.6, 125.1, 123.2, 47.1, 40.4; HRMS-ESI(-) m/z calcd for
-
C H F NO 334.0691 [M-H] , found 334.0679.
17
11
3
3
6
-(2,4-Difluorobenzyl)-2-hydroxyisoquinoline-1,3(2H,4H)-dione (20g). Yield 50%
1
(for two steps from 17g). H NMR (CD OD, 600 MHz) δ 8.04 (d, J = 7.2 Hz, 1H), 7.32-
3
13
7
.28 (m, 2H), 7.22 (s, 1H), 6.97-6.89 (m, 2H), 4.17 (s, 2H), 4.04 (s, 2H); C NMR
(CD OD, 150 MHz) δ 162.1, 146.5, 134.5, 131.8, 131.7, 128.2, 127.7, 127.3, 123.1,
3
1
3
11.0 110.9, 103.4, 103.2, 103.0, 33.6; HRMS-ESI(-) m/z calcd for C H F NO
16 10 2 3
-
02.0629 [M-H] , 302.0616.
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-(4-Cyclopropylbenzyl)-2-hydroxyisoquinoline-1,3(2H,4H)-dione (20h). Yield 54%
1
(for two steps from 18h). H NMR (CDCl , 600 MHz) δ 8.11 (d, J = 8.4 Hz, 1H), 7.24 (d,
3
J = 8.4 Hz, 1H), 7.09 (s, 1H), 7.05 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 8.4 Hz, 2H), 4.12 (s,
10
11
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14
15
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2
H), 3.99 (s, 2H), 1.88-1.85 (m, 1H), 0.96-0.93 (m, 2H), 0.68-0.65 (m, 2H); HRMS-ESI(-
-
) m/z calcd for C H NO 306.1130 [M-H] , found 306.1115.
1
9
16
3
6
-([1,1'-Biphenyl]-4-ylmethyl)-2-hydroxyisoquinoline-1,3(2H,4H)-dione (20i). Yield
1
4
7% (for two steps from 18i). H NMR (CDCl , 600 MHz) δ 8.15 (d, J = 7.8 Hz, 1H),
3
7.57-7.54 (m, 4H), 7.43 (t, J = 7.8 Hz, 2H), 7.38-7.34 (m, 2H), 7.24 (d, J = 7.8 Hz, 2H),
13
7
1
.15 (s, 1H), 4.14 (s, 2H), 4.09 (s, 2H); C NMR (CDCl , 150 MHz) δ 163.5, 160.1,
3
48.4, 140.6, 139.7, 138.2, 133.3, 129.3, 129.2, 129.0, 128.8, 127.8, 127.5, 127.3, 126.9,
-
122.0, 41.5, 36.4; HRMS-ESI(-) m/z calcd for C H NO 342.1130 [M-H] , found
22
16
3
3
2
4
7
42.1111.
-Hydroxy-6-(4-(thiophen-2-yl)benzyl)isoquinoline-1,3(2H,4H)-dione (20j). Yield
1
7% (for two steps from 18j). H NMR (CDCl , 600 MHz) δ 8.14 (d, J = 7.2 Hz, 1H),
3
.56 (d, J = 7.8 Hz, 2H), 7.35 (d, J = 8.4 Hz, 1H), 7.29-7.28 (m, 2H), 7.18 (d, J = 7.2 Hz,
13
2H), 7.12 (s, 1H), 7.07-7.06 (m, 1H), 4.14 (s, 2H), 4.05 (s, 2H); C NMR (CDCl , 150
3
MHz) δ 163.5, 160.2, 148.2, 143.8, 138.4, 133.3, 129.4, 129.2, 128.9, 128.0, 127.8,
1
26.3, 124.8, 123.1, 122.0, 44.6, 41.5, 36.4; HRMS-ESI(-) m/z calcd for C H NSO
20 14 3
-
348.0694 [M-H] , found 348.0677.
6
-((4'-Fluoro-[1,1'-biphenyl]-4-yl)methyl)-2-hydroxyisoquinoline-1,3(2H,4H)-dione
1
(20k). Yield 45% (for two steps from 18k). H NMR (CDCl /DMSO, 600 MHz) δ 8.20
3
(d, J = 7.2 Hz, 1H), 7.62-7.58 (m, 5H), 7.45 (d, J = 6.6 Hz, 1H), 7.35 (d, J = 6.6 Hz, 2H),
13
7
.21-7.19 (m, 2H), 4.23 (s, 2H), 4.17 (s, 2H); C NMR (CDCl /DMSO, 150 MHz) δ
3
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