Integrase mutations can reduce the effectiveness of the first-generation FDA-approved integrase strand transfer inhibitors (INSTIs), raltegravir (RAL) and elvitegravir (EVG). The second-generation agent, dolutegravir (DTG), has enjoyed considerable clinical success; however, resistance-causing mutations that diminish the efficacy of DTG have appeared. Our current findings support and extend the substrate envelope concept that broadly effective INSTIs can be designed by filling the envelope defined by the DNA substrates. Previously, we explored 1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides as an INSTI scaffold, making a limited set of derivatives, and concluded that broadly effective INSTIs can be developed using this scaffold. Herein, we report an extended investigation of 6-substituents as well the first examples of 7-substituted analogues of this scaffold. While 7-substituents are not well-tolerated, we have identified novel substituents at the 6-position that are highly effective, with the best compound (6p) retaining better efficacy against a broad panel of known INSTI resistant mutants than any analogues we have previously described.
Integrase mutations can reduce the effectiveness of the first-generation FDA-approved integrase strand transfer inhibitors (INSTIs), raltegravir (RAL) and elvitegravir (EVG). The second-generation agent, dolutegravir (DTG), has enjoyed considerable clinical success; however, resistance-causing mutations that diminish the efficacy of DTG have appeared. Our current findings support and extend the substrate envelope concept that broadly effective INSTIs can be designed by filling the envelope defined by the DNA substrates. Previously, we explored 1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides as an INSTI scaffold, making a limited set of derivatives, and concluded that broadly effective INSTIs can be developed using this scaffold. Herein, we report an extended investigation of 6-substituents as well the first examples of 7-substituted analogues of this scaffold. While 7-substituents are not well-tolerated, we have identified novel substituents at the 6-position that are highly effective, with the best compound (6p) retaining better efficacy against a broad panel of known INSTI resistant mutants than any analogues we have previously described.
HIV-1 integrase (IN)
plays a key role in the viral life cycle,
inserting the double-stranded DNA that is generated by reverse transcription
of the viral RNA genome into the genome of the host cell.[1] Integration is essential for viral replication,
and for this reason, IN is a therapeutic target for the treatment
of HIV infections. To date, three HIV IN antagonists have been approved
for clinical use: raltegravir (RAL, 1), elvitegravir
(EVG, 2), and dolutegravir (DTG, 3) (Figure ).[2−4] These drugs
belong to a class of compounds called integrase strand transfer inhibitors
(INSTIs) because they inhibit DNA strand transfer (ST), the second
step of integration catalyzed by IN, rather than the first step, the
3′-processing reaction (3′-P).[5−8] Development of drug resistance mutations is a common problem in
antiviral therapy and, not surprisingly, mutations affecting the susceptibility
of the virus to RAL and EVG have rapidly emerged.[9−11] However, the
second-generation inhibitor, DTG, retains potency against some but
not all RAL/EVG resistant HIV variants.[12−16] Therefore, the development of new small molecules
that have minimal toxicity and improved efficacy against the existing
resistant mutants remains an important research objective.[17]
Figure 1
HIV-1 integrase inhibitors. Colored areas indicate regions
of intended
correspondence.
HIV-1 integrase inhibitors. Colored areas indicate regions
of intended
correspondence.Retroviral integration
is mediated by IN multimers that are assembled
on the viral DNA ends, forming a stable synaptic complex, also referred
to as the intasome.[18−21] The INSTIs only bind to the active site of IN when the processed
viral DNA ends are appropriately bound to the intasome.[8,22] The way in which INSTIs bind to the intasome was elucidated by solving
crystal structures of the orthologous retroviral IN from the prototype
foamy virus (PFV).[19,23,24] The INSTIs are “interfacial” inhibitors; they bind
to the active site of IN and interact with the bound viral DNA following
the 3′-processing step.[8,19,25] Essential structural features that contribute to the binding of
INSTIs include an array of three heteroatoms (highlighted in red, Figure ) that chelate the
two catalyticMg2+ ions in the IN active site and a halobenzyl
side chain (halophenyl portion highlighted in blue, Figure ) that stacks with the penultimate
nucleotide (a deoxycytidine) at the 3′ end of the viral DNA.[8,19] We have recently shown that the 1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide
motif (4) can serve a useful platform for developing
HIV-1 IN inhibitors that retain high efficacy against the RAL/EVG-resistant
mutants.[26,27] We initially examined the properties of
a series of analogues related to structure 4 by varying
the substituents at the 4-position. Our objective was to identify
compounds that retain efficacy against the mutations Y143R, N155H,
and Q148H/G140S, which have been associated with clinical resistance
to RAL,[27] and some of these mutations also
play a role in the development of resistance against DTG.[28] This approach yielded compounds including 4a–d, which are approximately equivalent
to RAL in their potency against recombinant wild-type (WT) HIV-1 IN
in biochemical assays. However, the small molecules also showed improved
antiviral efficacies against the Y143R and N155H mutants in cell-based
assays.[26,27] Although antiviral efficacies against the
Q148H/G140S double mutant were also improved relative to RAL, the
new compounds were inferior to DTG, prompting us to continue our developmental
efforts.Structural studies using the PFV intasome have revealed
that the
tricyclic system of DTG is sufficiently extended to make contacts
with G187 in the β4−α2 loop of PFV IN (G118 in
IN).[23] It has been argued that the interactions
with this region may contribute to the improved properties of DTG
and other second-generation INSTIs.[4,23,29,30] Therefore, we considered
that adding functionality to either the 6- or 7-positions of 4 could interact with the same region of the catalytic site
(highlighted in green and cyan, respectively, in the structures of
DTG and 4, Figure ). In a preliminary work, we modified the 6-position of 4 and showed that adding linear side chains bearing terminal
hydroxyl groups can improve antiviral efficacies against the Q148H/G140S
double mutant to levels approaching that of DTG.[31] Furthermore, depending on the 6-substituent, compounds
could retain essentially all of their antiviral potency against a
more extensive panel of HIV-1-based vectors that carry the major DTG-resistant
IN mutants, including the G118R, T66I, E92Q, R263K, and H51Y single
mutants and the H51Y/R263K double mutant.[17,32−34] These data have two important implications: First,
6-substituents can have an important role in maintaining antiviral
efficacy against resistant mutant forms of IN. Second, compounds that
are broadly effective against mutant forms of IN bind in ways that
involve substrate mimicry and, when bound, fit within the “substrate
envelope”.[31,35] However, the data were confined
to a small number of 6-modificaitons that had limited chemical diversity.
Given the potential promise of 6-substituents, we felt that it was
important to more thoroughly examine this position and, accordingly,
we describe here an extensive set of 6-modifications (designated as
the 6 series analogues, Figure ). We were also interested in examining substituents
at the 7-position of 4 because these could potentially
afford an alternate way to engage G118 in the β4−α2
loop of IN. We present the first examination of compounds that have
modifications at the 7-position (designated as the 5 series
analogues). To provide a more complete SAR study, we have also introduced
various functionalities at the 4-position in combination with various
6- and 7-substitutents. These efforts have resulted in one of the
most potent and effective compounds that we have developed to date.
This work sheds additional light on enzyme–inhibitor interactions
that can contribute to retention of efficacy against strains of virus
that contain mutant forms of IN resistant to the approved INSTIs.
Results
and Discussion
Chemistry
To prepare our 7-substituted
analogues 5a–i, we employed 2,6-dichloronicotinic
acid (7) as the starting material. The 6-chloro group
of 7 was converted into the final 7-substituent in two
ways. In one route, the 6-chloro moiety was displaced with methoxyl
(KOBu in MeOH),[36] and this was followed by esterification of the 3-carboxy group to
yield 8 (Scheme ). In the second route, the 3-carboxyl group of 7 was first converted directly to the 3-methyl ester. In both cases,
the 2-chloro group was then displaced with BnONH2 to give
the corresponding 2-benzyloxyamine-containing derivatives (10a and 9, respectively). The 6-chloro-substituents of
the second series 9 were transformed into the corresponding
6-piperidinyl and 6-morpholino derivatives (10b and 10c, respectively) by heating with DMF solutions of piperidine
or morpholine. Using a procedure similar to those we previously reported
(Scheme ),[26,27,31] intermediates 10a–c were transformed to the 4-hydroxy (5a–c), 4-amino (5d–f), and 4-[2-(methyl glycinate)] (5g–i) final products.
Reagents and conditions: (i)
KOBu, MeOH, 65 °C; (ii) H2SO4, MeOH; (iii) BnONH2, DIEA, dioxane, 110
°C; (iv) (b) piperidine or (c) morpholine, DMF, 80 °C; (v)
ClCOCH2CO2CH3, TEA, DCM; (vi) NaOMe,
MeOH; (vii) 2,4-diF-BnNH2, DMF, 140 °C; (viii) TsCl,
DIEA, MeCN; (ix) RNH2, DIEA, DMF, 50 °C, R = 2,4-diMeOBn
(a) or CH2CO2Me (b); (x) TFA, DCM; (xi) H2, 10% Pd/C, MeOH.We transformed the
6-bromo analogues (17a–c, Scheme ) into a variety
of 6-substituted INSTIs by coupling alkynes and
alkenes using Sonogashira and Heck coupling chemistries.[31] In addition to the 6-substituted products with
a 4-hydroxyl (6a), we prepared analogues having a free
amine at the 4-position (6b–p) as
well as analogues with 4-[2-(methyl glycinate)]amine (6q) and 4-(2-hydroxyethyl)amine (6r–t) (Scheme ).
Scheme 2
Synthesis of 4,6-Bis-Substituted Analogues 6a–6t
Reagents and conditions: (i)
TsCl, Et3N, CH3CN; (ii) RNH2, DIEA,
DMF, 50 °C, R = 2,4-diMeOBn (DMP) (a), CH2CH2OH (b), or CH2CO2Me (c); (iii) alkyne, Pd(PPh3)2Cl2, DIEA, CuI, DMF, 70 °C or
alkene, Pd(OAc)2, TEA, PPh3, DMF, 100 °C;
(iv) TFA, DCM; (v) H2, 10% Pd/C, MeOH.
Synthesis of 4,6-Bis-Substituted Analogues 6a–6t
Reagents and conditions: (i)
TsCl, Et3N, CH3CN; (ii) RNH2, DIEA,
DMF, 50 °C, R = 2,4-diMeOBn (DMP) (a), CH2CH2OH (b), or CH2CO2Me (c); (iii) alkyne, Pd(PPh3)2Cl2, DIEA, CuI, DMF, 70 °C or
alkene, Pd(OAc)2, TEA, PPh3, DMF, 100 °C;
(iv) TFA, DCM; (v) H2, 10% Pd/C, MeOH.
Evaluation of New Derivatives Using in Vitro IN Catalytic Assays
and Single-Round Virus Replication Assays
We designed compounds 5a–i to examine the effects of substituents
at the 4-position (hydroxyl, amine, or methyl glycinate) in combination
with modifications of the 7-position (methoxyl, piperidinyl, or morpholino).
We evaluated the compounds by in vitro IN activity assays that utilize 32P-radiolabeled oligonucleotides (Table ).[37] We also tested
the potency of the compounds in single-round replication assays that
employ HIV-1 vectors carrying WT IN. We evaluated the antiviral potencies
of a subset of the compounds against HIV-1 vectors carrying the canonical
Y143R, N155H, and Q148H/G140S mutations that are associated with high-level
HIV-1 resistance to RAL and virological failure in patients (Table ).[17,32−34,38,39] As we recently reported, compounds that lack substituents at the
6- and 7-positions and have a hydroxyl group at the 4-position (5′a) showed lower potencies in the ST assay and poorer
antiviral potencies than the corresponding 4-amino-containing compound
(5′d) (Tables and 2).[26,27] In our current work, we found that a compound with a 4-[2-(methyl
glycinate)] modification of the 4-amino group (5′g) retained its enzymatic potency in these assays relative to 5′d. Bulky substituents at the 7-position (5b, 5c, 5e, 5f, 5h. and 5i, Table ) diminished potency by three orders of magnitude relative
to the corresponding unsubsituted analogues (5′a, 5′d, and 5′g, respectively).
Although a 7-methoxyl group was better tolerated (compare with 5a, 5d, and 5g), in all cases there
was a reduction in the antiviral potency (Table ).
Table 1
Inhibitory Potencies
of Compounds
Using an in Vitro IN Assaya
in vitro
(IC50 μM)
no.
R4
R6
R7
3′-P
ST
5′ab
OH
H
H
1.2 ± 0.2
0.055 ± 0.008
5a
OH
H
OCH3
32 ± 4
0.033 ± 0.005
5b
OH
H
piperidinyl
>333
70 ± 16
5c
OH
H
morpholino
>333
1.9 ± 0.2
5′dc
NH2
H
H
2.5 ± 0.3
0.019 ± 0.002
5d
NH2
H
OCH3
18 ± 2
0.011 ± 0.002
5e
NH2
H
piperidinyl
>333
54 ± 18
5f
NH2
H
morpholino
>333
54 ± 10
5′gc
NHCH2CO2CH3
H
H
0.71 ± 0.10
0.021 ± 0.011
5g
NHCH2CO2CH3
H
OCH3
20 ± 2
0.042 ± 0.005
5h
NHCH2CO2CH3
H
piperidinyl
>333
96 ± 40
5i
NHCH2CO2CH3
H
morpholino
>333
6.2 ± 0.8
6a
OH
(CH2)5OH
H
14.3 ± 1.1
0.012 ± 0.003
6′bd
NH2
(CH2)5OH
H
8.2 ± 1.2
0.0027 ± 0.0004
6b
NH2
(CH2)3OAc
H
14.3 ± 2.1
0.010 ± 0.002
6c
NH2
(CH2)6OBz
H
143 ± 14
3.3 ± 0.6
6d
NH2
(CH2)3cHex
H
289 ± 49
3.3 ± 0.5
6e
NH2
(CH2)4Ph
H
42 ± 4
2.0 ± 0.4
6f
NH2
(CH2)2Ph
H
48 ± 4
0.014 ± 0.003
6g
NH2
(CH2)3N(CH3)2
H
3.0 ± 0.4
0.014 ± 0.002
6h
NH2
(CH2)3O(CH2)2OH
H
1.5 ± 0.4
0.0020 ± 0.0004
6i
NH2
(CH2)2CO2H
H
NDe
0.004 ± 0.001
6j
NH2
(CH2)2CON(CH3)2
H
ND
0.008 ± 0.001
6k
NH2
(CH)2CON(CH3)2
H
ND
0.005 ± 0.001
6l
NH2
(CH2)2CONHiPr
H
ND
0.002 ± 0.007
6m
NH2
(CH)2CONHiPr
H
ND
0.006 ± 0.005
6n
NH2
(CH2)2CONH(CH2)2OH
H
ND
0.002 ± 0.001
6o
NH2
(CH)2CONH(CH2)2OH
H
ND
0.007 ± 0.001
6p
NH2
(CH2)2CO2CH3
H
2.9 ± 0.6
0.0031 ± 0.0005
6q
NHCH2CO2CH3
(CH2)5OH
H
2.1 ± 0.5
0.004 ± 0.001
6r
NH(CH2)2OH
(CH2)5OH
H
4.0 ± 0.5
0.0025 ± 0.0002
6s
NH(CH2)2OH
(CH2)5OAc
H
ND
ND
6t
NH(CH2)2OH
(CH2)2CO2CH3
H
3.2 ± 0.3
0.0031 ± 0.0003
Assays were performed using a gel-based
protocol with a Mg2+ cofactor as described.[51]
Data
have been reported previously.[26]
Data have been reported previously.[27]
Data
have been reported previously.[31]
Not determined.
Table 2
Antiviral Potencies in Cells Infected
with HIV-1 Vectors That Carry WT or Resistant IN Mutantsa
EC50 (nM/FCe)
no.
WT
Y143R
N155H
G140S/Q148H
CC50 (μM)
RAL (1)b
4 ± 2
162 ± 16 (41×)
154 ± 33 (39×)
1900 ± 300 (475×)
>250
EVG (2)b
6.4 ± 0.8
7.9 ± 2.3 (1×)
90 ± 18 (14×)
5700 ± 1100 (891×)
>250
DTG (3)b
1.6 ± 0.9
4.3 ± 1.2 (3×)
3.6 ± 1.3 (2×)
5.8 ± 0.5 (4×)
>250
5′ac
6.2 ± 2.9
11 ± 2 (2×)
31 ± 8 (5×)
308 ± 125 (50×)
137 ± 20
5a
215 ± 31
NDf
ND
ND
12 ± 5
5′dc
1.1 ± 0.7
2.5 ± 0.6 (2×)
5.3 ± 2.3 (5×)
35 ± 9 (32×)
>250
5d
30 ± 9
62 ± 26 (2×)
400 ± 101 (13×)
3600 ± 1600 (120×)
2.2 ± 0.4
5′g
3.8 ± 1.2
4.6 ± 2.2 (1×)
19 ± 7 (5×)
36 ± 16 (9×)
>250
5g
32 ± 13
52 ± 13 (2×)
1260 ± 320 (39×)
ND
12 ± 3
6a
24 ± 4
8.3 ± 1.7 (0.3×)
32 ± 3 (1×)
29 ± 10 (1×)
>250
6′bd
1.3 ± 0.2
3.0 ± 0.5 (2×)
2.4 ± 0.8 (2×)
9.4 ± 3.6 (7×)
>250
6b
1.4 ± 0.4
2.9 ± 0.9 (2×)
5.4 ± 2.5 (4×)
13 ± 8 (9×)
>250
6c
4.8 ± 1.6
3.5 ± 1.2 (0.7×)
5.4 ± 1.9 (1×)
21 ± 7.8 (4×)
14.4 ± 4.8
6d
53 ± 14
112 ± 14 (2×)
146 ± 37 (3×)
ND
27.4 ± 3.1
6e
11 ± 2
4.7 ± 0 (0.4×)
39 ± 10 (4×)
128 ± 44 (12×)
13 ± 2
6f
5.6 ± 1.9
5.5 ± 2.4 (1×)
21 ± 5 (4×)
140 ± 34 (25×)
18 ± 2
6g
6.1 ± 2.1
7.0 ± 2.3 (1×)
29 ± 9 (5×)
32 ± 5 (5×)
5.0 ± 1.8
6h
1.6 ± 0.2
1.2 ± 0.6 (0.8×)
4.2 ± 1.5 (3×)
35 ± 3.7 (22×)
>250
6i
1.9 ± 0.5
4.9 ± 1.5 (3×)
10 ± 3 (5×)
195 ± 25 (103×)
>250
6j
1.3 ± 0.5
6 ± 1 (5×)
6.8 ± 1.6 (5×)
16 ± 5 (12×)
>250
6k
1.6 ± 0.9
7.2 ± 0.3 (5×)
15 ± 2 (9×)
58 ± 19 (36×)
>250
6l
5 ± 1.3
8.7 ± 2.2 (2×)
11 ± 4 (2×)
13 ± 1 (3×)
>250
6m
2.1 ± 0.7
11 ± 1 (5×)
12 ± 3 (6×)
286 ± 8 (136×)
>250
6n
263 ± 52
ND
ND
ND
>250
6o
196 ± 32
ND
ND
ND
>250
6p
0.67 ± 0.15
0.67 ± 0.23 (1×)
2.3 ± 0.2 (3×)
5.3 ± 1.8 (8×)
>250
6q
3.3 ± 1.7
3.5 ± 1.6 (1×)
11 ± 2 (3×)
42 ± 2 (13×)
>250
6r
13 ± 4.2
12 ± 2 (0.9×)
15 ± 4 (1×)
16 ± 7 (1×)
>250
6s
5.3 ± 1.2
4.1 ± 1 (0.8×)
10 ± 2 (2×)
5.3 ± 2.1 (1×)
>100
6t
4.1 ± 1
3.8 ± 1.9 (0.9×)
3.9 ± 2.1 (1×)
16 ± 4 (4×)
>250
Cytotoxic concentration resulting
in 50% reduction in the level of ATP in human osteosarcoma (HOS) cells;
EC50 values obtained from cells infected with lentiviral
vector harboring WT or indicated IN mutants as previously reported.
Data has been reported previously.[27]
Data
have been reported previously.[26]
Data have been reported previously.[31]
FC,
fold change relative to WT =
EC50 of mutants/EC50 of WT.
Not determined.
Assays were performed using a gel-based
protocol with a Mg2+ cofactor as described.[51]Data
have been reported previously.[26]Data have been reported previously.[27]Data
have been reported previously.[31]Not determined.Cytotoxic concentration resulting
in 50% reduction in the level of ATP in humanosteosarcoma (HOS) cells;
EC50 values obtained from cells infected with lentiviral
vector harboring WT or indicated IN mutants as previously reported.Data has been reported previously.[27]Data
have been reported previously.[26]Data have been reported previously.[31]FC,
fold change relative to WT =
EC50 of mutants/EC50 of WT.Not determined.We recently described a series of structurally related
4-amino-containing
INSTIs, which possess 6-substituents consisting of primary hydroxyl
or sulfone groups tethered by alkyl chains of various lengths.[31] The chemical diversity of these substituents
was limited. To extend the diversity of the modifications at the 6-position,
our current study examines analogues containing tethered acetoxy (6b), benzyloxy (6c), cyclohexyl (6d), phenyl (6e and 6f), dimethylamino (6g), carboxyl (6i), methyl ester (6p), and carboxamido having a variety of alkylamide groups (6j–6o) groups (Tables and 2). Several of
the resulting compounds retained low-nanomolar IC50 values
in ST reactions. N,N-Dimethylpropylamine
(6g, ST IC50 = 14 nM), propanoic acid (6i), propanoic amides (6j–o), and propanoic
acid methyl ester (6p) as well as the reverse ester 6b displayed IC50 values of 10 nM or less in the
ST assay. Tethering a phenyl ring with an ethylene chain maintained
good inhibitory potency (6f, ST IC50 = 14
nM). However, appending phenyl or cyclohexyl groups by increasing
spacer lengths reduced potency by as much as three orders of magnitude:
three methylenes (cyclohexyl-containing 6d, ST IC50 = 3.3 μM), four methylenes (phenyl-containing 6e, ST IC50 = 2.0 μM), or longer (6c, ST IC50 = 3.3 μM) (Table ). This result contrasts with our previous
finding that appending a phenyl ring using a three-unit chain that
contains a sulfone moiety can result in retention of high potency.
In the latter case, a cocrystal structure with the PFV intasome revealed
that the sulfone group causes the phenyl ring to adopt an unusual
π–π stacking orientation, in which it is folded
up under the naphthyridine ring system and fills the catalytic space
more completely.[31]Most 6-substituted
analogues in our current study showed low nanomolar
potencies against the WT enzyme in the in vitro ST assay and against
a WT HIV-1 vector in the single-round infectivity assay (Tables and 2, respectively). However, for the 6-tethered 2-hydroxyethylamides
(6n and 6o), in vitro ST inhibitory potencies
and antiviral potencies against a vector that replicates using WT
IN differed considerably (IC50 < 10 nM as compared to
EC50 ≥ 200 nM). Additionally, while analogues 6c, 6d, and 6e showed micromolar
ST-inhibitory potencies in the in vitro assay, they were significantly
more potent in the antiviral assay. The reasons for these discrepancies
are not clear. Among the remaining compounds, 6p, with
a 6-(CH2)2CO2CH3 substituent,
exhibited the best antiviral profile against the panel of resistant
mutants (Table ).
This derivative displayed a modest improvement in antiviral potency
against a vector carrying a WT IN (EC50 = 0.67 nM) as compared
to 6′b, which had been the most promising compound
in work we recently reported.[31] Inhibitor 6′b contains a 6-(CH2)5OH group
and a primary amine at the 4-position. Adding 4-[2-(methyl glycinate)]
or 2-hydroxyethyl substituents to the 4-amino group of 6′b did not improve the inhibitory profiles (compounds 6q and 6r, respectively), nor were the antiviral potencies
improved by the addition of a 2-hydroxyethyl substituent to the 4-amino
group of 6p to yield 6t (Table ). Most 6-subsituted analogues
were not cytotoxic within the range tested (Table ). Exceptions were found with 6c, 6d, and 6e, which also showed anomalous
discrepancies between their in vitro and antiviral potencies, and
with 6f and 6g. The reasons for the greater
cytotoxicities of these analogues are not clear.An important
component of our current study is to test the effects
of substituents at the 7-position. Given proximity to the metal-chelating
8-aryl nitrogen, we were uncertain of what the effects such modifications
would have. Bulky groups at the 7-position (5b, 5c, 5e, 5f, 5h, and 5i) resulted in from two to three orders of magnitude loss
of inhibitory potency against the WT enzyme in vitro. These latter
data were consistent with the bulky substituents causing a disruption
of essential interactions with the catalyticmetal ions. Of greater
interest was our finding that 7-OCH3 substituents were
well tolerated against WT enzyme in in vitro assays (see 5a, 5d, and 5g, Table ).
Potencies of the New Compounds Against an
Extended Set of Drug
Resistant IN Mutants
We selected a subset of the analogues
(6b, 6p, 6r, 6s, and 6t) for additional evaluation in single-round
viral replication assays using HIV-1 vectors harboring single G118R,
T66I, E92Q, R263K, and H51Y, or the double H51Y/R263K substitutions
associated with DTG resistance (Table ).[17,32−34,38,39] For comparison, we
also included 6′b, which was one of the most broadly
effective of our previously reported inhibitors.[31] The new compounds showed good antiviral potencies against
the entire panel of mutants. Although several compounds appear to
be promising, the most potent analogue (by a small margin) was the
6-methyl 3-propanoate-containing 6p, which showed a slightly
better inhibitory profile against the first panel of mutants (Table ) while also retaining
greater inhibitory potency than DTG against the second, more extended
panel of mutants (Table ).
Table 3
Antiviral Potencies in Cells Infected
with HIV-1 Vectors That Carry DTG-Resistant IN Mutantsa
EC50 (nM/FCc)
no.
WT
G118R
T66I
E92Q
R263K
H51Y
H51Y/R263K
RAL (1)b
4 ± 2
36 ± 5 (9×)
2.8 ± 0.4 (0.7×)
30 ± 10 (8×)
5.7 ± 2.3 (1×)
3.4 ± 0.2 (0.9×)
6 ± 2.3 (2×)
EVG (2)b
6.4 ± 0.8
21 ± 10 (3×)
66 ± 1 (10×)
154 ± 34 (24×)
10 ± 6 (2×)
4.5 ± 2.1 (0.7×)
53 ± 18 (8×)
DTG (3)b
1.6 ± 0.9
13 ± 5 (8×)
0.9 ± 0.8 (0.6×)
2.3 ± 0.4 (1×)
11 ± 3 (7×)
3.2 ± 0.2 (2×)
16 ± 2 (10×)
6′bb
1.3 ± 0.2
5.3 ± 1.6 (4×)
0.93 ± 0.24 (0.7×)
3.8 ± 2.3 (3×)
2.6 ± 0.1 (2×)
3.8 ± 0.6 (3×)
2.6 ± 1.4 (2×)
6b
1.4 ± 0.4
5.9 ± 1.4 (4×)
0.75 ± 0.07 (0.5×)
1.2 ± 0.1 (1×)
1.8 ± 0.3 (1×)
0.8 ± 0.2 (0.6×)
3.9 ± 1.9 (3×)
6p
0.67 ± 0.15
4.8 ± 1.5 (7×)
0.53 ± 0.06 (0.8×)
2.0 ± 1.1 (3×)
0.5 ± 0.0 (0.7×)
0.63 ± 0.30 (0.9×)
2.4 ± 0.8 (3×)
6r
13 ± 4.2
24 ± 8 (2×)
1.9 ± 0.07 (0.1×)
6.5 ± 0.8 (0.5×)
18 ± 2 (1×)
11 ± 1.4 (0.8×)
31 ± 10 (2×)
6s
5.3 ± 1.2
15 ± 2 (3×)
1.2 ± 0.5 (0.2×)
3.8 ± 1.3 (0.7×)
3.6 ± 0.9 (0.7×)
7.4 ± 0.9 (1×)
15 ± 1.8 (3×)
6t
4.1 ± 1
4.8 ± 0.6 (1×)
0.62 ± 0.15 (0.2×)
2.7 ± 1.2 (0.7×)
6.5 ± 0.8 (2×)
6.9 ± 2.9 (2×)
2.5 ± 0.1 (0.9×)
EC50 values obtained
from cells infected with a lentiviral vector harboring WT or the indicated
IN mutants as previously reported.
Data for these compounds have been
reported previously.[27,31]
FC, fold change relative to WT =
EC50 of mutants/EC50 of WT.
Table 4
Comparison of Antiviral
Potencies
and Fold Improvement of Compounds 6′b and 6p Relative to DTG
EC50 (nM)a (fold improvementb)
integrase
mutants
DTG (3)c
6′bc
6p
WT
1.6 ± 0.9
1.3 ± 0.2 (0.81×)
0.67 ± 0.15 (0.42×)
Y143R
4.3 ± 1.2
3.0 ± 0.5 (0.71×)
0.67 ± 0.23 (0.16×)
N155H
3.6 ± 1.3
2.4 ± 0.8 (0.67×)
2.3 ± 0.2 (0.64×)
G140S/Q148H
5.8 ± 0.5
9.4 ± 3.6 (1.62×)
5.3 ± 1.8 (0.91×)
G118R
13 ± 5
5.3 ± 1.6 (0.41×)
4.8 ± 1.5 (0.37×)
T66I
0.9 ± 0.8
0.93 ± 0.24 (1.03×)
0.53 ± 0.06 (0.59×)
E92Q
2.3 ± 0.4
3.8 ± 2.3 (1.65×)
2.0 ± 1.1 (0.87×)
R263K
11 ± 3
2.6 ± 0.1 (0.24×)
0.5 ± 0.0 (0.05×)
H51Y
3.2 ± 0.2
3.8 ± 0.6 (1.19×)
0.63 ± 0.30 (0.20×)
H51Y/R263K
16 ± 2
2.6 ± 1.4 (0.16×)
2.4 ± 0.8 (0.15×)
EC50 values obtained
from cells infected with a lentiviral vector harboring WT or the indicated
IN mutants as previously reported.
Fold improvement relative to DTG
= EC50 of compounds/EC50 of DTG.
Data for these compounds have been
reported previously.[27,31]
EC50 values obtained
from cells infected with a lentiviral vector harboring WT or the indicated
IN mutants as previously reported.Data for these compounds have been
reported previously.[27,31]FC, fold change relative to WT =
EC50 of mutants/EC50 of WT.EC50 values obtained
from cells infected with a lentiviral vector harboring WT or the indicated
IN mutants as previously reported.Fold improvement relative to DTG
= EC50 of compounds/EC50 of DTG.Data for these compounds have been
reported previously.[27,31]
Crystal Structures of the PFV Intasome in Complex with 5′g, 5g, and 6p
To understand how some of the current analogues interact with the
active site of IN, we soaked PFV intasome crystals in the presence
of 5′g, 5g, or 6p and
refined the resulting structures (Table ). We recently described a crystal structure
of 5′d bound to the active site of PFV intasome.[31] The inhibitor 5′g bound
to PFV IN differs from the previously reported 5′d structure only in having a N-(methyl 2-glycinate)amine
moiety at its 4-position and, predictably, the two compounds bind
to the active site in a very similar fashion. Superposition with the
DTG-bound structure confirmed that 8-naphthyridinenitrogen and 1-N-hydroxyl of 5′g take the place of
the 6-oxo and 7-hydroxyl heteroatoms of DTG. The naphthyridine 2-oxo
carbonyl of 5′g corresponds to the ring 8-oxo
carbonyl of DTG (Figure A).[23] However, the 4-N-(methyl 2-glycinate)amine group of 5′g extends
into the region which is occupied by the first base of the scissile
dinucleotide in viral DNA prior to 3′-P (designated A18 in
the crystal structure, Figure A), which may be related to the ability of the compound to
inhibit the 3′-P reaction.[8] Therefore,
an extended form of substrate mimicry may arise from the modification
at the 4-position of 5′g, which was not seen in
the cocrystal structure of 5′d.[31] The idea that inhibitors that remain within the “substrate
envelope” are particularly effective against resistant forms
of the target enzyme was proposed by Schiffer and colleagues and is
based on the idea that to be able to support viral replication, any
mutant form of a viral enzyme must be capable of binding its normal
substrate(s).[35] Thus, if the bound inhibitor
remains within an envelope defined by the substrate(s), the inhibitor
is expected to retain some efficacy against mutant forms of the enzyme.[40] Even though the original hypothesis was based
on work with protease inhibitors, it seems likely that this principle
can be applied broadly to antiviral compounds that bind at the active
sites of essential viral enzymes. The 4-N-(methyl
2-glycinate) amine group of 5′g is situated near
(within 5 Å) to the side chains of Y212 and P214 (residues corresponding
to Y143 and P145 in HIV-1 IN) and to the adenosine base of the 3′
terminal viral DNA residue (A17), which adopts two alternative conformations
in the crystal structure (Figure B). The interactions with the side chains are much
less extensive in the cases of DTG (3) and 5′d. Extension into this region of the catalytic site is also seen with
several highly potent INSTIs having diverse structures (see Figure S1 in Supporting Information).
Table 5
X-Ray Data Collection and Refinement
Statisticsa
5′g
5g
6p
Data Collection
space group
P41212
P41212
P41212
cell
dimensions a, b, c (Å)
159.6, 159.6, 124.1
158.6, 158.6, 123.1
160.5, 160.5, 123.8
resolution range (Å)
71.36–2.55 (2.62–2.55)
79.29–2.60(2.67–2.60)
71.77–2.77 (2.84–2.77)
Rmerge
0.062 (1.49)
0.025 (0.55)
0.072 (1.075)
I/σ(I)
26.6 (2.0)
19.5 (1.4)
18.9 (2.0)
completeness (%)
99.8 (98.8)
99.9 (99.7)
98.8 (97.4)
redundancy
11.5 (11.0)
14.4 (14.2)
7.2 (7.3)
Refinement
reflections (total/free)
52565/2601
48670/2438
40988/2057
R/Rfree
0.179/0.207
0.176/0.204
0.177/0.205
no. atoms
protein,
DNA
5150
5129
5127
ligand
159
132
146
water
229
109
87
average B-factors (Å2)
71.7
84.3
75.7
protein, DNA
71.3
83.9
74.9
ligands
91.8
117.23
107.3
water
67.2
78.9
68.3
rmsd
bond lengths (Å)
0.038
0.039
0.012
bond angles (deg)
0.56
0.76
1.38
Ramachandran plot (%)
favored
99
97
98
outliers
0
0
0.2
Each structure was determined from
a single crystal. Data for the highest resolution shells are given
in parentheses.
Figure 2
Crystal structures of
PFV intasome-bound inhibitors. (A) Bound 5′g (cream)
with overlays showing the relative positions
of pre-3′-P viral DNA (violet; PDB 4E7I) and DTG (3) (green; PDB 3S3M). The 4-N-(methyl 2-glycinate) moiety of 5′g is circled in red, highlighting its correspondence with the T+2 adenosine (A18) base of pre-3′-P viral DNA. (B) Bound 5′g with its 4-N-(methyl 2-glycinate)
moiety shown as transparent spheres. Atoms within 5 Å (Y212 and
P214 and A17 of 3′-P DNA) are shown in green as a stick diagram.
For both A and B, metal ions are shown as solid blue spheres.
Each structure was determined from
a single crystal. Data for the highest resolution shells are given
in parentheses.Crystal structures of
PFV intasome-bound inhibitors. (A) Bound 5′g (cream)
with overlays showing the relative positions
of pre-3′-P viral DNA (violet; PDB 4E7I) and DTG (3) (green; PDB 3S3M). The 4-N-(methyl 2-glycinate) moiety of 5′g is circled in red, highlighting its correspondence with the T+2 adenosine (A18) base of pre-3′-P viral DNA. (B) Bound 5′g with its 4-N-(methyl 2-glycinate)
moiety shown as transparent spheres. Atoms within 5 Å (Y212 and
P214 and A17 of 3′-P DNA) are shown in green as a stick diagram.
For both A and B, metal ions are shown as solid blue spheres.Prior structural studies using
the PFV intasome showed that the
tricyclic system of DTG makes contacts with G187 in the β4−α2
loop of PFV IN (G118 in IN), and it has been argued that the interactions
with this region may contribute to the improved properties of DTG
and other second-generation INSTIs.[4,23,29,30] As has already been
discussed, we thought that it might be possible to make useful contacts
with the same region of the catalytic site by adding functionality
to the 6- and 7-positions of 4. To this end, we introduced
substituents at the 6- and 7-positions of 4 that could
potentially recapitulate some of the desirable features of DTG (highlighted
in green and cyan, respectively, in the structures of DTG and 4, Figure ). We obtained the crystal structure of 6p bound to
the PFV intasome because it represents the most potent 6-substituted
analogue in our current study. The structure revealed that the 6-methyl
3-propanoate side chain of the compound is situated within the region
defined by an envelope defined by both the pre-3′-P viral DNA
and the host target DNA substrate (Figure ). This is consistent with our recent report
that 6-substituents of the compounds exhibiting the best antiviral
profiles against the panel of resistant mutants bind within an envelope
defined as the confluence of viral DNA substrates.[31] The structure also shows that the methyl carboxylate group
of 6p makes van der Waals interactions with the β4−α2
loop of IN, becoming sandwiched between main chain atoms of Q186 and
G187 (residues corresponding to HIV-1 N117 and G118, respectively)
and the side chain of Y212 (Figure ).
Figure 3
Crystal structures of 6p bound to the PFV
intasome.
Structures of the pre-3′-P viral DNA (violet; PDB 4E7I) and target complex
host DNA substrate (gray; PDB 4E7K) are overlaid. The 6-(methyl 3-propanoate)
group of 6p is circled in red, showing its correspondence
with aspects of both pre-3′-P viral DNA and the host target
DNA. Cofactor Mg2+ ions are shown as blue spheres; the
structures of bound viral DNAs have been omitted for clarity.
Figure 4
Crystal structures of PFV intasome-bound inhibitors.
(A) Bound 5g (orange) with superimposed 6p (cyan) and DTG
(3) (green; PDB 3S3M) showing closest distance to the Gly187 α-methylene
(side chains of Asp186 and Gln187 shown in semitransparent), bound
3′-P DNAs have been omitted for clarity. (B) Superimposed bound
3′-P DNAs associated with A, circled in cyan are the oxygens
in the C16–A17 phosphoryl linkages, whose engagement in electrostatic
interactions with the side chain imidazole nitrogen of the N224H mutant
(corresponding to IN mutant N155H) would be broken on binding of the
INSTIs. (C) Bound 5g with superimposed pre-3′-P
viral DNA (violet; PDB 4E7I) and target complex host DNA substrate (gray; PDB 4E7K), circled in red
is the 7-OCH3, which protrudes outside the substrate envelope.
Metal ions are shown as solid blue spheres.
Crystal structures of 6p bound to the PFV
intasome.
Structures of the pre-3′-P viral DNA (violet; PDB 4E7I) and target complex
host DNA substrate (gray; PDB 4E7K) are overlaid. The 6-(methyl 3-propanoate)
group of 6p is circled in red, showing its correspondence
with aspects of both pre-3′-P viral DNA and the host target
DNA. Cofactor Mg2+ ions are shown as blue spheres; the
structures of bound viral DNAs have been omitted for clarity.Crystal structures of PFV intasome-bound inhibitors.
(A) Bound 5g (orange) with superimposed 6p (cyan) and DTG
(3) (green; PDB 3S3M) showing closest distance to the Gly187 α-methylene
(side chains of Asp186 and Gln187 shown in semitransparent), bound
3′-P DNAs have been omitted for clarity. (B) Superimposed bound
3′-P DNAs associated with A, circled in cyan are the oxygens
in the C16–A17 phosphoryl linkages, whose engagement in electrostatic
interactions with the side chain imidazolenitrogen of the N224H mutant
(corresponding to IN mutant N155H) would be broken on binding of the
INSTIs. (C) Bound 5g with superimposed pre-3′-P
viral DNA (violet; PDB 4E7I) and target complex host DNA substrate (gray; PDB 4E7K), circled in red
is the 7-OCH3, which protrudes outside the substrate envelope.
Metal ions are shown as solid blue spheres.To understand the effects of introducing a 7-OCH3 substituent,
we solved the structure of 5g bound to the PFV intasome
and overlaid the corresponding structure of DTG bound to the PFV intasome
(Figure ). We observed
that the 7-OCH3 group of 5g is approximately
3.6 Å from the G187 residue in the β4−α2 loop,
which compares favorably with DTG and 6p (approximately
3.9 and 3.7 Å, respectively, Figure A). The 7-OCH3 group of 5g did not appear to significantly alter the position of the
metal chelating heteroatoms of 5g relative to 6p. However, it is not immediately obvious why 5g is more
susceptible to the N155H mutation compared to 6p or to
the more closely related 5′g. Because the N155
residue of HIV-1 IN corresponds to N224 in PFV IN, the X-ray crystal
structure with the mutation N224H in PFV IN did show that the side
chain of His224 interacts with a phosphoryl oxygen atom in viral DNA
and that INSTI binding is accompanied by a loss of this contact.[24] The associated energetic penalty may explain
the loss potency of some compounds against N155HHIV-1 IN. In the
cocrystal structure of PFV intasome with 5g, the location
of this phosphoryl oxygen is nearly identical to that seen for 6p, 5′g, and DTG (Figure C). The abilities of some INSTIs to readjust
in the active site of IN may help explain variations among in their
susceptibilities of the different INSTIs to the N155H mutation.[23,24,41]
Conclusions
Earlier,
we reported that the 1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide
scaffold represents a potentially attractive platform for the development
of INSTIs that retain good antiviral efficacy against a panel of viral
vectors carrying resistant mutant forms of IN.[26,27] More recently, we began an exploration of the effects of modifying
the 6-position of our earlier compounds.[31] We found that 6-substituents may play an important role in enhancing
the maintenance of antiviral efficacy against drug-resistant mutant
forms of IN. Furthermore, the most broadly effective compounds are
substrate mimics that fit within the “substrate envelope.″
Our original purpose in synthesizing 6-substituents was to create
additional interactions within areas of the catalytic region accessed
by the third ring of DTG. However, the range of functionality reported
in the prior paper was quite limited, consisting of primary hydroxyl
groups tethered by linear polymethylene chains of varying lengths
and a single tethered phenyl sulfone. We now report a much more extensive
exploration of functionalities at the 6-position as well as the first
examination of derivatives with modifications at the 7-position. We
also tested whether the compounds could be improved by introduced
by varying functionalities at the 4-position.We observed that
−OCH3 groups are somewhat better
tolerated than bulky subsituents at the 7-position when examined in
vitro against WT enzyme. However, compounds with 7-OMe groups showed
significantly reduced antiviral potency when tested against viral
vectors that carry the N155H mutation. We found that a variety of
substituents can be added at the 6-position, which enhance the antiviral
potencies of the compounds against the major RAL-, EVG-, and DTG-resistant
IN mutants. Many of these compounds are not cytotoxic at the tested
concentrations, and the best analogue, 6p, is the most
effective in terms of its ability to inhibit the entire spectrum of
IN mutants of any of the compounds we have described. The crystal
structure of the PFV intasome with 6p bound supports
the idea that interactions with the β4−α2 loop
are important and that compounds that bind within the envelope defined
by both the viral and host DNA substrates helps the compounds retain
broad efficacy. We found that the 4-amine substituent in 5′g accesses a region of the active site of IN that would normally be
occupied by the base of the T+2 adenosine of the viral
DNA prior to the 3′-P cleavage, providing further support that
binding within an envelope defined by the DNA substrates helps the
compounds retain broad efficacy. Thus, our results will inform the
design and development of next-generation INSTIs with broader efficacy
against the known drug resistant mutants.
Experimental
Section
General Procedures
Proton (1H) and carbon
(13C) NMR spectra were recorded on a Varian 400 MHz spectrometer
or a Varian 500 MHz spectrometer and are reported in ppm relative
to TMS and referenced to the solvent in which the spectra were collected.
Solvent was removed by rotary evaporation under reduced pressure,
and anhydrous solvents were obtained commercially and used without
further drying. Purification by silica gel chromatography was performed
using Combiflash with EtOAc–hexanes solvent systems. Preparative
high pressure liquid chromatography (HPLC) was conducted using a Waters
Prep LC4000 system having photodiode array detection and Phenomenex
C18 columns (catalogue no. 00G-4436-P0-AX, 250 mm ×
21.2 mm 10 μm particle size, 110 Å pore) at a flow rate
of 10 mL/min. Binary solvent systems consisting of A = 0.1% aqueous
TFA and B = 0.1% TFA in acetonitrile were employed with gradients
as indicated. Products were obtained as amorphous solids following
lyophilization. Electrospray ionization-mass spectrometric (ESI-MS)
were acquired with an Agilent LC/MSD system equipped with a multimode
ion source. Purities of samples subjected to biological testing were
assessed using this system and shown to be ≥95%. High resolution
mass spectrometric (HRMS) were acquired by LC/MS-ESI using LTQ-Orbitrap-XL
at 30K resolution.
General Procedure I for the Synthesis of
Carboxamides (5a–i and 6a–t)
Benzyl protected compounds (13, 15, 16, and 19)
(0.1 mmol) were
suspended in methanol (10 mL) and ethyl acetate (3 mL). One equivalent
of Pd/C (10%) was added. The mixture was stirred at room temperature
under hydrogen. When the starting material was disappeared (TLC),
the crude mixture was filtered and washed by methanol. The filtrate
was concentrated and purified by HPLC to provide final carboxamides
(5a–i and 6a–t).
Treatment of 19g with
aqueous NaOH and then as outlined in general procedure I and purification
by preparative HPLC (with a linear gradient of 30% B to 50% B over
30 min; retention time = 19.5 min) provided 6i as a white
fluffy solid (80% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.63 (t, J = 5.4
Hz, 1H), 8.68–8.66 (m, 1H), 8.63–8.60 (m, 1H), 8.57
(d, J = 1.9 Hz, 1H), 8.53 (d, J =
1.9 Hz, 1H), 7.40–7.34 (m, 1H), 7.21–7.16 (m, 1H), 7.04–6.99
(m, 1H), 4.47 (d, J = 5.5 Hz, 2H), 2.87 (t, J = 7.5 Hz, 2H), 2.62 (t, J = 7.6 Hz, 2H).
ESI-MS m/z: 419.1 (MH+). HRMS calcd C19H17F2N4O5 [MH+], 419.1162; found, 419.1147.
4-Amino-N-(2,4-difluorobenzyl)-6-(3-(dimethylamino)-3-oxopropyl)-1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide
(6j) and (E)-4-Amino-N-(2,4-difluorobenzyl)-6-(3-(dimethylamino)-3-oxoprop-1-en-1-yl)-1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide
(6k)
Treatment of 18e as outlined
in general procedures I and H and purification by preparative HPLC
(with a linear gradient of 30% B to 45% B over 30 min) provided 6j and 6k as white fluffy solids. For 6j: retention time = 20.7 min. 1H NMR (400 MHz, DMSO-d6) δ 10.62 (t, J = 5.8
Hz, 1H), 10.46 (brs, 1H), 8.56 (d, J = 1.8 Hz, 1H),
8.50 (d, J = 1.8 Hz, 1H), 7.36 (dd, J = 15.4, 8.6 Hz, 1H), 7.20–7.14 (m, 1H), 7.00 (td, J = 8.6, 1.9 Hz, 1H), 4.45 (d, J = 5.7
Hz, 2H), 2.90 (s, 3H), 2.84 (t, J = 7.4 Hz, 2H),
2.74 (s, 3H), 2.65 (t, J = 7.5 Hz, 2H). ESI-MS m/z: 446.2 (MH+). HRMS calcd
C21H22F2N5O4 [MH+], 446.1634; found, 446.1617. For 6k: retention time = 24.1 min. 1H NMR (400 MHz, DMSO-d6) δ 10.63 (s, 1H), 10.53 (t, J = 5.8 Hz, 1H), 8.94 (s, 2H), 7.49 (d, J = 15.4 Hz, 1H), 7.37 (dd, J = 12.2, 5.3 Hz, 1H),
7.31 (d, J = 15.5 Hz, 1H), 7.21–7.15 (m, 1H),
7.01 (td, J = 8.9, 2.3 Hz, 1H), 4.46 (d, J = 5.7 Hz, 2H), 3.14 (s, 3H), 2.89 (s, 3H). ESI-MS m/z: 444.1 (MH+). HRMS calcd
C21H20F2N5O4 [MH+), 444.1478; found, 444.1475.
4-Amino-N-(2,4-difluorobenzyl)-1-hydroxy-6-(3-(isopropylamino)-3-oxopropyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide
(6l) and (E)-4-Amino-N-(2,4-difluorobenzyl)-1-hydroxy-6-(3-(isopropylamino)-3-oxoprop-1-en-1-yl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide
(6m)
Treatment of 18f as outlined
in general procedures I and H and purification by preparative HPLC
(with a linear gradient of 30% B to 45% B over 30 min) provided 6l and 6m as white fluffy solids. For 6l: retention time = 23.0 min. 1H NMR (400 MHz, DMSO-d6) δ 10.62 (t, J = 5.7
Hz, 1H), 10.46 (brs, 1H), 8.50 (d, J = 1.8 Hz, 1H),
8.47 (s, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.36 (dd, J = 15.4, 8.7 Hz, 1H), 7.20–7.14 (m, 1H), 7.02–6.98
(m, 1H), 4.45 (d, J = 5.7 Hz, 2H), 3.76–3.68
(m, 1H), 2.84 (t, J = 7.5 Hz, 2H), 2.36 (t, J = 7.6 Hz, 2H), 0.90 (d, J = 6.6 Hz, 6H).
ESI-MS m/z: 460.2 (MH+). HRMS calcd C22H24F2N5O4 [MH+], 460.1791; found, 460.1772. For 6m: retention time = 27.9 min. 1H NMR (400 MHz,
DMSO-d6) δ 10.63 (s, 1H), 10.53
(t, J = 5.8 Hz, 1H), 8.84 (s, 1H), 8.82 (s, 1H),
8.01 (d, J = 7.7 Hz, 1H), 7.41 (d, J = 15.8 Hz, 1H), 7.37–7.33 (m, 1H), 7.20–7.15 (m, 1H),
7.00 (td, J = 8.6, 2.3 Hz, 1H), 6.66 (d, J = 15.9 Hz, 1H), 4.46 (d, J = 5.7 Hz,
2H), 3.91 (td, J = 13.3, 6.6 Hz, 1H), 1.05 (d, J = 6.6 Hz, 6H). ESI-MS m/z: 458.2 (MH+). HRMS calcd C22H22F2N5O4 [MH+], 458.1634;
found, 458.1628.
4-Amino-N-(2,4-difluorobenzyl)-1-hydroxy-6-(3-((2-hydroxyethyl)amino)-3-oxopropyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide
(6n) and (E)-4-Amino-N-(2,4-difluorobenzyl)-1-hydroxy-6-(3-((2-hydroxyethyl)amino)-3-oxoprop-1-en-1-yl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide
(6o)
Treatment of 18g as outlined
in general procedures I and H and purification by preparative HPLC
(with a linear gradient of 20% B to 50% B over 30 min) provided 6n and 6o as white fluffy solids. For 6n: retention time = 22.1 min. 1H NMR (400 MHz, DMSO-d6) δ 8.56 (d, J = 1.9
Hz, 1H), 8.49 (d, J = 1.8 Hz, 1H), 7.42 (dd, J = 15.4, 8.6 Hz, 1H), 7.25–7.19 (m, 1H), 7.06 (dd, J = 9.4, 7.7 Hz, 1H), 4.50 (s, 2H), 3.32 (t, J = 6.1 Hz, 2H), 3.08 (t, J = 6.1 Hz, 2H), 2.92 (t, J = 7.5 Hz, 2H), 2.48 (t, J = 7.5 Hz, 2H).
ESI-MS m/z: 462.1 (MH+), 484.1 (MNa+). HRMS calcd C21H22F2N5O5 [MH+], 462.1584;
found, 462.1562. For 6o: retention time = 23.9 min. 1H NMR (400 MHz, DMSO-d6) δ
10.63 (s, 1H), 10.53 (t, J = 5.8 Hz, 1H), 8.85 (s,
1H), 8.83 (s, 1H), 8.09 (t, J = 5.8 Hz, 1H), 7.43
(d, J = 15.8 Hz, 1H), 7.36 (dd, J = 15.4, 8.6 Hz, 1H), 7.20–7.15 (m, 1H), 7.03–6.98
(m, 1H), 6.73 (d, J = 15.9 Hz, 1H), 4.65 (brs, 1H),
4.46 (d, J = 5.6 Hz, 2H), 3.40 (brs, 2H), 3.20 (dd, J = 11.9, 6.0 Hz, 2H). ESI-MS m/z: 460.1 (MH+), 482.1 (MNa+). HRMS
calcd C21H20F2N5O5 [MH+], 460.1427; found, 460.1421.
Treatment of 18k as outlined
in general procedure I and purification by preparative HPLC (linear
gradient of 30% B to 50% B over 30 min; retention time = 21.0 min)
provided 6t as a white fluffy solid (20% yield). 1H NMR (500 MHz, DMSO-d6) δ
11.09 (bs, 1H), 10.54 (t, J = 5.7 Hz, 1H), 8.57 (d, J = 1.8 Hz, 1H), 8.43 (d, J = 1.7 Hz, 1H),
7.46 (dd, J = 15.4, 8.6 Hz, 1H), 7.26–7.21
(m, 1H), 7.09–7.05 (m, 1H), 4.50 (d, J = 5.7
Hz, 2H), 3.71 (s, 2H), 3.61 (t, J = 5.1 Hz, 2H),
3.58 (s, 3H), 2.96 (t, J = 7.4 Hz, 2H), 2.73 (t, J = 7.4 Hz, 2H). ESI-MS m/z: 477.1 (MH+). HRMS calcd C23H26F2N4O5 [MH+], 477.1580;
found, 477.1581.
Methyl 2-Chloro-6-methoxynicotinate (8)
Commercially available 2,6-dichloronicotinic acid 7 (3.1
g, 16 mmol) was added to the mixture of potassium t-butoxide (5.7 g, 48 mmol) in methanol (75 mL). The reaction mixture
was stirred (65 °C, 24 h). The reaction mixture was concentrated
and acidified using concentrated aqueous HCl. The crude mixture was
filtered. The formed solid was collected to provide 2-chloro-6-methoxynicotinic
acid as a white solid (2.8 g, 94% yield).[36] [1H NMR (400 MHz, DMSO-d6) δ 13.33 (bs, 1H), 8.19 (d, J = 8.5 Hz, 1H),
6.92 (d, J = 8.5 Hz, 1H), 3.92 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 165.52,
164.54, 147.69, 143.80, 120.02, 109.82, 54.86. ESI-MS m/z: 188.0 (MH+).] 2-Chloro-6-methoxynicotinic
acid (2.8 g, 15 mmol) was suspended in thionyl chloride (20 mL). The
suspension was stirred and refluxed (3 h). The reaction mixture was
concentrated. The residue was mixed with methanol (20 mL). The mixture
was refluxed (3 h) and concentrated. The crude residue was purified
by silica gel chromatography to provide 8 as a colorless
oil (2.5 g, 81% yield). 1H NMR (400 MHz, CDCl3) δ 7.89 (d, J = 8.5 Hz, 1H), 6.48 (d, J = 8.5 Hz, 1H), 3.77 (s, 3H), 3.70 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 164.56, 164.25, 148.96,
142.60, 118.11, 108.96, 54.30, 52.09.
General Procedure A for
the Synthesis of Methyl 2-((Benzyloxy)amino)-nicotinates
(9 and 10a)
2-Chloronicotinates
(7 and 8) (12 mmol) were mixed with O-benzylhydroxylamine (48 mmol) and DIEA (36 mmol). The
reaction mixture was stirred (110 °C, 18 h). The crude mixture
was purified by silica gel chromatography to provide methyl 2-((benzyloxy)amino)-nicotinates
(9 and 10a).
General
Procedure B for the Synthesis of 6-Substituted Methyl
2-((Benzyloxy)amino)-nicotinates (10b and 10c)
Methyl 2-((benzyloxy)amino)-6-chloronicotinate (9) (1 mmol) was dissolved in DMF (1 mL). Piperidine or morphiline
(4 mmol) was added. The mixture was stirred and heated (80 °C,
1 h). The crude mixture was purified by silica gel chromatography
to provide 6-substituted methyl 2-((benzyloxy)amino)-nicotinates (10b and 10c).
General Procedure C for the Synthesis of
Methyl Nicotinates
(11a–c)
Methyl 3-chloro-3-oxopropanoate
(12 mmol) was added dropwise to a solution of methyl 2-((benzyloxy)amino)-nicotinates
(10a–c) (6 mmol) and triethylamine
(12 mmol) in CH2Cl2 (40 mL). The mixture was
stirred at room temperature (1 h). The crude mixture was filtered
and the filtrate was concentrated. The crude residue was purified
by silica gel chromatography to provide methyl nicotinates (11a–c).
Treatment of 10c as outlined
in general procedure C provided 11c as a yellow oil (79%
yield). 1H NMR (400 MHz, CDCl3) δ 8.04
(d, J = 8.8 Hz, 1H), 7.32–7.28 (m, 5H), 6.52
(dd, J = 8.9, 0.9 Hz, 1H), 5.01 (s, 2H), 3.85–3.83
(m, 1H), 3.78 (s, 3H), 3.77–3.75 (m, 4H), 3.72–3.71
(m, 1H), 3.68 (s, 3H), 3.60–3.57 (m, 4H). ESI-MS m/z: 444.2 (MH+).
General Procedure
D for the Synthesis of Methyl Carboxylates
(12a–c)
A solution of sodium
methanolate (16 mmol, 25% in methanol) was added to a solution of
methyl nicotinates (11a–c) (6 mmol)
in methanol (4 mL). The mixture was stirred at room temperature (18
h). The reaction mixture was brought to pH 4 by the addition of aqueous
HCl (2N) and stirred (0 °C, 15 min). The crude suspension was
filtered, and the solid was collected to provide methyl carboxylates
(12a–c).
Treatment of 11c as outlined
in general procedure D provided 12c as a white solid
(84% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.97 (d, J = 9.3 Hz, 1H), 7.53–7.51
(m, 2H), 7.39–7.33 (m, 4H), 6.60 (d, J = 9.3
Hz, 1H), 5.00 (s, 2H), 3.72–3.54 (m, 11H). ESI-MS m/z: 412.2 (MH+).
General Procedure
E for the Synthesis of Carboxyamides (13a–c)
A solution of methyl carboxylates
(12a–c) (0.3 mmol) and (2,4-difluorophenyl)methanamine
(1.5 mmol) in DMF (1 mL) was heated (140 °C, 2 h) in microwave
reactor. The crude mixture was purified by silica gel chromatography
and recrystallized from methanol to provide carboxamides (13a–c).
Treatment of 12c as outlined
in general procedure E provided 13c as a white solid
(89% yield). 1H NMR (400 MHz, CDCl3) δ
10.22 (bs, 1H), 8.08 (d, J = 9.1 Hz, 1H), 7.50–7.48
(m, 2H), 7.33–7.24 (m, 4H), 6.79–6.69 (m, 2H), 6.48
(d, J = 9.1 Hz, 1H), 5.15 (s, 2H), 4.56 (d, J = 6.2 Hz, 2H), 3.73–3.66 (m, 8H). ESI-MS m/z: 523.2 (MH+).
General
Procedure F for the Synthesis of p-Methylbenzenesulfonates
(14a–c)
Compound 4-hydroxyl
analogues (13a–c) (0.5 mmol) were
dissolved in CH3CN (3 mL). DIEA (2.8 mmol), CH2Cl2 (2 mL), and p-methylbenzene-1-sulfonyl
chloride (1.4 mmol) were added. The reaction mixture was stirred at
room temperature (18 h). The mixture crude was purified by silica
gel chromatography to provide p-methylbenzenesulfonates
(14a–c).
General Procedure G for
the Synthesis of 4-Substituted Amines
(15a–f, 17d, and 17e)
A solution of p-methylbenzenesulfonates
(14a–c and 17b) (0.2
mmol), DIPEA (2 mmol), and methyl 2-aminoacetate hydrochloride or
2,4-dimethoxyphenyl)methanamine (1 mmol) in DMF (2 mL) was stirred
and heated (50 °C, 1 h). The crude mixture was purified by silica
gel chromatography to provide 4-substituted amines (15a–f, 17d, and 17e).
Treatment of 14c with (2,4-dimethoxyphenyl)methanamine
as outlined in general procedure G provided 15f as a
white solid which was directly used in next step. 1H NMR
(400 MHz, CDCl3) δ 11.73 (t, J =
6.1 Hz, 1H), 10.75 (t, J = 5.8 Hz, 1H), 7.95 (d, J = 9.2 Hz, 1H), 7.52–7.50 (m, 2H), 7.33–7.21
(m, 5H), 6.80–6.71 (m, 4H), 6.30 (d, J = 9.2
Hz, 1H), 5.16 (s, 2H), 4.63 (d, J = 6.1 Hz, 2H),
4.51 (d, J = 5.8 Hz, 2H), 3.76 (s, 3H), 3.74 (s,
3H), 3.73–3.71 (m, 4H), 3.65–3.63 (m, 4H). ESI-MS m/z: 672.3 (MH+).
General
Procedure H for the Synthesis of Amine Analogues (16a–c, 19b–g)
The 2,4-dimethoxybenzyl protected compounds (15d–f and 18a–h) (0.25 mmol)
were dissolved in CH2Cl2 (2 mL).
TFA (2 mL) was added at room temperature. The reaction mixture was
concentrated. The crude residue was purified by silica gel chromatography
to provide amine analogues (16a–c and 19b–g).
Treatment of 1-(benzyloxy)-6-bromo-3-((2,4-difluorobenzyl)carbamoyl)-2-oxo-1,2-dihydro-1,8-naphthyridin-4-yl
4-methylbenzenesulfonate 17b(31) with 2-aminoethan-1-ol as outlined in general procedure G provided 17e as a white solid (93% yield). 1H NMR (500 MHz,
CDCl3) δ 11.65 (bs, 1H), 10.65 (bs, 1H), 8.64 (d, J = 1.9 Hz, 1H), 8.47 (d, J = 1.9 Hz, 1H),
7.58 (d, J = 6.1 Hz, 2H), 7.31 (t, J = 7.2 Hz, 4H), 6.79–6.73 (m, 2H), 5.15 (s, 2H), 4.55 (d, J = 5.6 Hz, 2H), 3.88–3.86 (m, 2H), 3.78–3.75
(m, 2H). ESI-MS m/z: 559.1, 561.1
(MH+).
General Procedure J for the Synthesis of
6-Alkylated Analogues
(18a, 18b, 18d, 18i, and 18j) Using Sonogashira Reaction
Bromides
(17a and 17c–e) (0.3
mmol) were mixed with bis(triphenylphosphine)palladium(II) dichloride
(9 μmol), DIEA (0.3 mmol), and copper(I) iodide (0.03 mmol)
in DMF (1.5 mL) in a reaction tube. The related terminal alkynes (0.45
mmol) were added. The reaction mixture was flushed with argon and
sealed with a cap and heated (70 °C, 4 h). The crude mixture
was purified by silica gel chromatography to provide 6-alkylated analogue
(18a, 18b, 18d, 18i, and 18j).
General Procedure K for the Synthesis of
6-Alkylated Analogues
(18c, 18e–h, and 18k) Using Heck Reaction
The mixture of bromides
(17c and 17e) (0.2 mmol), alkenes (0.4 mmol),
triethylamine (0.6 mmol), diacetoxypalladium (0.04 mmol), and triphenylphosphine
(0.08 mmol) in DMF (0.5 mL) was flushed with argon and sealed in a
reaction tube. The reaction mixture was microwave-heated (100 °C,
5 h). The crude mixture was purified by silica gel chromatography
to provide 6-alkylated analogues (18c, 18e–h, and 18k).
As previously
described,[43] inhibitors or an equivalent
volume of DMSO were
added to a reaction mixture containing 20 nM DNA substrate 5′ 32P-labeled on the transferred strand and 400 nM IN in 50 mM
MOPS pH 7.2, 7.5 mM MgCl2, and 14 mM 2-mercaptoethanol.
Reactions were allowed to proceed for 2 h at 37 °C and were stopped
by the addition of an equal volume of loading buffer [1% sodium dodecyl
sulfate, 0.025% bromophenol blue, and 0.025% xylene cyanol made in
formamide]. Reaction products, separated in 16% polyacrylamide denaturing
sequencing gels, were visualized using by phosphorimaging using a
Typhoon 8600 instrument (GE Healthcare) and quantified using the ImageQuant
5.1 software (GE Healthcare). Data analyses (linear regression, IC50 determination, and standard deviation) were performed using
Prism 5.0 software from GraphPad.
HIV-1 Vector Constructs
pNLNgoMIVR–ΔEnv.LUC has
been described previously.[44] The IN reading
frame was removed from pNLNgoMIVR–ΔEnv.LUC by digestion with KpnI and SalI and inserted between the KpnI and SalI sites of pBluescript IIKS+.
With this construct as the WT template, the following HIV-1 IN mutants
were prepared using the QuikChange II XL (Stratagene, La Jolla, CA)
site-directed mutagenesis protocol: H51Y, T66I, E92Q, G118R, Y143R,
Q148H, N155H, R263K, G140S + Q148H, and H51Y + R263K. The following
sense and cognate antisense (not shown) oligonucleotides (Integrated
DNA Technologies, Coralville, IA) were used in the mutagenesis: H51Y,
5′-CTAAAAGGGGAAGCCATGTATGGACAAGTAGACTGTA-3′;
T66I, 5′-CCAGGAATATGGCAGCTAGATTGTATACATTTAGAAGGAAAAGTT-3′;
E92Q, 5′-GCAGAAGTAATTCCAGCACAGACAGGGCAAGAAA-3′;
G118R, 5′-GTACATACAGACAATCGCAGCAATTTCACCAGTAC-3′;
G140S, 5′-GGGGATCAAGCAGGAATTTAGCATTCCCTACAATC-3′;
Y143C, 5′-GCAGGAATTTGGCATTCCCCGCAATCCCCAAAGTCAAGGA-3′;
Q148H, 5′-CATTCCCTACAATCCCCAAAGTCATGGAGTAATAGAATCTA-3′;
N155H, 5′-CCAAAGTCAAGGAGTAATAGAATCTATGCATAAAGAATTAAAGAAAATTATAGGACA-3′;
R263K 5′-AAAGTAGTGCCAAGAAAAAAAGCAAAGATCATC-3′.
The double mutation G140S + Q148H was constructed using the previously
generated Q148H mutant and the appropriate oligonucleotides to introduce
the second mutation, G140S. The double mutation H51Y + R263K was constructed
by using the previously generated H51Y mutant and the appropriate
oligonucleotides for the second mutation, R263K. The DNA sequence
of each construct was verified by DNA sequencing. The mutant IN coding
sequences from pBluescript IIKS+ were subcloned into pNLNgoMIVR–ΔEnv.LUC (between the KpnI and SalI sites) to produce the full-length
mutant HIV-1 IN constructs. The mutant forms of the vector were validated
by DNA sequence determination.
Single-Round HIV-1 Infectivity
Assay
Assays were performed
using the humanembryonic kidney cell culture cell line 293 acquired
from the American Type Culture Collection (ATCC) and the humanosteosarcoma
cell line, HOS, obtained from Dr. Richard Schwartz (Michigan State
University, East Lansing, MI) using methods that have been previously
reported.[44,45] The 293 and HOS cells were grown in Dulbecco’s
Modified Eagle’s Medium (Invitrogen, Carlsbad, CA) supplemented
with 5% (v/v) fetal bovine serum, 5% newborn calf serum, and penicillin
(50 units/mL) plus streptomycin (50 μg/mL; Quality Biological,
Gaithersburg, MD). The transfection vector, pNLNgoMIVR–ΔLUC was made from pNLNgoMIVR–ΔEnv.HSA by removing the HSA reporter gene and replacing
it with a luciferase reporter gene, which was inserted between the
NotI and XhoI restriction sites.[44] VSV-g-pseudotyped HIV was produced by transfection of 293 cells.[45] On the day prior to transfection, 293 cells
were plated on 100 mm diameter dishes at a density of 1.5 × 106 cells per plate and transfected with 16 μg of pNLNgoMIVR–ΔLUC and 4 μg of pHCMV-g (obtained from
Dr. Jane Burns, University of California, San Diego) using the calcium
phosphate method. At approximately 6 h after the calcium phosphate
precipitate was added, 293 cells were washed twice with phosphate-buffered
saline (PBS) and incubated with fresh media for 48 h. The virus-containing
supernatants were harvested, clarified by low-speed centrifugation,
filtered, and diluted for use in antiviral infection assays. On the
day prior to the screen, HOS cells were seeded in 96-well luminescence
cell culture plates at a density of 4000 cells in 100 μL per
well. On the day of the assay for cellular cytotoxicity, cells were
treated with compounds using a concentration range from 250 to 0.05
μM and then incubated at 37 °C (48 h). On the day of the
assay for antiviral activity, cells were treated with compounds using
a concentration range from 5 to 0.0001 μM using 11 serial dilutions
and then incubated at 37 °C (3 h). After 3 h, 100 μL of
virus-stock [diluted to achieve a luciferase signal between 0.2 and
1.5 relative luciferase units (RLUs)] was added to each well and incubation
was continued (37 °C, 48 h). Cellular cytotoxicity was measured
by using the ATP Lite luminescence detection system and monitored
by adding 50 μL of cell lysis buffer from the luminescence ATP
detection assay to each well followed by mixing at 700 rpm at room
temperature using a compact thermomixer (5 min). After addition of
50 μL of reconstituted luminescence ATP detection assay reagent
to all wells except for the negative control/background wells, the
plates were mixed at 700 rpm at room temperature using a compact thermomixer
(5 min) and incubated at room temperature to allow time for signal
development (20 min), and cytotoxicity was determined using the microplate
reader. Infectivity was measured using the Steady-lite Plus luminescence
reporter gene assay system (PerkinElmer, Waltham, MA). Luciferase
activity was measured by adding 100 μL of Steady-lite Plus buffer
(PerkinElmer) to the cells, incubating at room temperature (20 min),
and measuring luminescence using a microplate reader. Both cytotoxicity
and antiviral activity were normalized to the cellular cytotoxicity
and infectivity using cells incubated in absence of the respective
target compounds. KaleidaGraph (Synergy Software, Reading, PA) was
used to perform nonlinear regression analysis on the data. Final IC50 values were determined from the fit model.
X-ray Crystallography
PFV intasome crystals were soaked
in the presence of 0.5–1 mM INSTIs in cryoprotection solution
prior to snap freezing in liquid nitrogen as described.[23,24] X-ray diffraction data collected on beamlines I04 and I03 of the
Diamond Light Source (Oxfordshire, UK) were processed using XDS[46] and Aimless.[47] Structures were determined
via rigid-body refinement of model generated from PDB ID 4BDZ by removing small
molecule atoms, and the compounds were fitted into resulting Fo–Fc difference
maps. The models were built in Coot,[48] refined
in Phenix,[49] and validated using MolProbity.[50] Relevant data collection and refinement statistics
are given in Table .
Authors: Allison Ballandras-Colas; Daniel P Maskell; Erik Serrao; Julia Locke; Paolo Swuec; Stefán R Jónsson; Abhay Kotecha; Nicola J Cook; Valerie E Pye; Ian A Taylor; Valgerdur Andrésdóttir; Alan N Engelman; Alessandro Costa; Peter Cherepanov Journal: Science Date: 2017-01-06 Impact factor: 47.728
Authors: Yang Shen; Michael D Altman; Akbar Ali; Madhavi N L Nalam; Hong Cao; Tariq M Rana; Celia A Schiffer; Bruce Tidor Journal: ACS Chem Biol Date: 2013-09-26 Impact factor: 5.100
Authors: Xue Zhi Zhao; Steven J Smith; Daniel P Maskell; Mathieu Metifiot; Valerie E Pye; Katherine Fesen; Christophe Marchand; Yves Pommier; Peter Cherepanov; Stephen H Hughes; Terrence R Burke Journal: ACS Chem Biol Date: 2016-02-05 Impact factor: 5.100
Authors: Jing Tang; Ha T Do; Andrew D Huber; Mary C Casey; Karen A Kirby; Daniel J Wilson; Jayakanth Kankanala; Michael A Parniak; Stefan G Sarafianos; Zhengqiang Wang Journal: Eur J Med Chem Date: 2019-02-02 Impact factor: 6.514
Authors: Ashley N Matthew; Florian Leidner; Gordon J Lockbaum; Mina Henes; Jacqueto Zephyr; Shurong Hou; Desaboini Nageswara Rao; Jennifer Timm; Linah N Rusere; Debra A Ragland; Janet L Paulsen; Kristina Prachanronarong; Djade I Soumana; Ellen A Nalivaika; Nese Kurt Yilmaz; Akbar Ali; Celia A Schiffer Journal: Chem Rev Date: 2021-01-07 Impact factor: 60.622
Figure 1
Scheme 1
Scheme 2
Figure 2
Figure 3
Figure 4