J. J. Swidorski et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
to discover compounds with improved virological profiles.
V370A-containing virus was used since this is the most common
polymorphic variation in the Gag protein in the SP1 region
observed in HIV-1 subtype B-infected patients that leads to
through this kind of structural modification.22,23 Entries 8–10
exemplify the initial alkyl-linked heterocyclic amide modifications
examined and the results indicate that improved potency against
BVM-resistant viruses can be achieved. Each heterocycle showed
similar potency toward the WT virus as 1, but with only a 1 to 6-
fold decline in potency against the V370A virus. The potency shift
in the presence of HSA was only evaluated for 9 because the series
reduced susceptibility (50-fold) to 2.15 In addition, a
DV370-con-
taning virus was used as it is a common polymorphic variation in
non-subtype B viruses, although it is only present in a small per-
centage of the subtype B patient population (0.9%). Additionally,
lacked significant inhibition of the
DV370 virus; however, the
D
V370 is one of the most BVM-resistant single polymorphic
serum shift for 9 was less than two-fold, preserving the improve-
ment observed with 1 when compared to 2. Unfortunately, 9
showed an increase in in vitro cytotoxicity, precluding it from fur-
ther studies. Additional methyl-linked heterocycles were synthe-
sized, but these failed to further improve the antiviral profile
(data not shown). Extending the carboxylic acid moiety further
from the core, as in 11 and 12, was detrimental to potency against
both WT and V370A viruses, although to a lesser extent with the
ethyl-linked 12. However, compounds 11 and 12 were associated
with reduced in vitro cytotoxicity compared to heterocycles 8–
10. Replacing the carboxylic acid terminus with an alcohol, as
exemplified by ethanolamide 13, resulted in improved activity
toward both WT and V370A virus, although this compound still
variations (>190-fold resistance), and improved potency against
this virus concomitantly improved potency toward other
clinically-relevant polymorphisms.16 In vitro potency was
measured utilizing a multiple cycle assay in MT-2 cells with an
NL4-3-derived virus expressing the Renilla luciferase gene incorpo-
rated as a marker for virus growth (NLRepRlucP373S). V370A and
D
V370 viruses were identical to the WT virus except for a single
amino acid substitution (V370A) or deletion (
position of the Gag protein.
DV370) at the 370
Carboxylic acid 36 was modified through one of two routes to
give the C-28 amides and acyl sulfonamides (Scheme 1). The first
route employed oxalyl chloride to form the acid chloride 4, which
was reacted with a series of amines to give the C-28 amides. The
second route utilized HATU as the amide coupling agent with a
reactant amine. Amide formation using both of these procedures
was followed by hydrolysis of the C-3 benzoate ester using either
NaOH or LiOH with heating at 50–85 °C in 1,4-dioxane and H2O.
Acidification and purification delivered the compounds compiled
in Tables 1 and 4.17
failed to address the more challenging
DV370 virus. Capping the
alcohol as a methyl ether (14) resulted in a further decline in
potency against both WT and V370A viruses. Small alkyl amides,
including methyl amide 15, were explored next. WT virus inhibi-
tory activity was acceptable; however, the V370A-containing virus
was more than 25-fold less susceptible to 15 than WT virus. The
piperidinamide 16 exhibited greatly reduced activity toward both
WT and V370A viruses while also showing increased cytotoxicity.
However, an enhancement in potency toward both BVM-resistant
test viruses was achieved when a basic dimethylamine functional-
ity was incorporated into the amide moiety, as exemplified by the
ethyl-linked compound 17. In addition to improved WT potency,
both polymorphic viruses were inhibited with EC50 values below
35 nM, suggesting that polarity and/or basicity in the side chain
was essential in this portion of the molecule in order to enhance
antiviral coverage, especially in regard to the more challenging
The compounds compiled in Table 1 represent a selection of the
C-28 amide and acyl sulfonamide side chains that were examined
to screen for inhibition of WT and the two polymorphism-contain-
ing viruses. Initially, we screened C-28 acyl sulfonamides since this
motif mimics the acidic nature of the carboxylic acid of 1. Unfortu-
nately, acyl sulfonamides 5–7 demonstrated reduced WT potency
compared to 1 and insufficient potency against
DV370 virus to
be of further interest. The next avenue of exploration focused on
heterocycles extending from the C-28 amide of the triterpenoid
core as a means of improving potency and influencing ADME
(absorption, distribution, metabolism and excretion) properties.
While other labs have reported on the effects of modifying the
C-28 amide in combination with the dimethyl succinate side chain
of 2,18–21 at the time this work was conducted, there were no Let-
ters suggesting that C28 amide derivatives would confer improved
potency toward BVM-resistant viruses; however, several recent
reports have suggested that BVM-resistant viruses can be inhibited
D
V370 polymorph. In addition, potency in the presence of human
serum albumin was also largely preserved, with a less than 4-fold
serum shift for 17 compared to 9-fold for 1. However, incorporat-
ing a basic amine into the side chain also led to an increase in the
in vitro cytotoxicity (3 lM for 17 compared to 27 lM for 1), per-
haps not entirely surprising since basic amines have been associ-
ated with promiscuity.24
Since 17 exhibited the preferred virological profile, SARs of the
amine-containing side chain were probed more deeply in order to
identify compounds with ADME properties suitable for progressing
into in vivo studies. Capping the amine as a terminal amide, exem-
plified by 18–20, retained antiviral activity toward WT virus and
showed only modest changes toward V370A virus compared to
H
H
OH
Cl
17; however, potency toward the
DV370 virus declined by 7- to
H
H
a
O
O
65-fold compared to 17. Less basic amines were also examined,
including the morpholine-containing side chain in 21; however,
potency and in vitro cytotoxicity were not improved when com-
pared to 17. When both methyl groups of 17 were eliminated, as
in the primary amine 22, inhibitory potency against both WT and
H
H
3
4
H
H
MeO2
C
MeO2C
d
b
H
R1
N
V370A viruses were retained, but inhibition of the
was reduced by 23-fold compared to 17. The tertiary amide 23
showed similar potency toward WT and V370 virus but almost
DV370 virus
H
R1
N
c
H
R2
H
R2
O
D
O
H
H
a 5-fold decline in potency toward the V370A virus when com-
pared to progenitor 17. Nevertheless, 23 offered an alternative to
the free NH of the secondary amide of 17, which may influence
the permeability of the molecule. The three carbon side chain
homolog 24 maintained potency but in vitro CC50 values were
6-fold lower than for 17. Installing a gem dimethyl group in the
H
H
HOOC
MeO2
C
Scheme 1. Synthesis of C-28 amides and acyl sulfonamides derived from the C-3
benzoic acid-modified triterpenoid 3. Reagents and conditions: (a) oxalyl chloride,
DCM, rt, 2–5 h, 100%; (b) DIEA, DMAP, amine reactant, DCM, rt; (c) NaOH or LiOH
monohydrate, 1,4-dioxane, H2O, 50–85 °C; (d) HATU, THF, DIEA, amine reactant, rt.
linker element
a- to the amine gave the tertiary amine 25, a