SAR and lead optimization of an HIV-1 Vif-APOBEC3G axis inhibitor

Article abstract of DOI:10.1021/ml300037k

We describe structure-activity relationship and optimization studies of RN-18, an HIV-1 Vif-APOBEC3G axis inhibitor. Targeted modifications of RN-18 ring C, ring B, ring A, bridge A-B, and bridge B-C were performed to identify the crucial structural features, which generated new inhibitors with similar (4g and 4i) and improved (5, 8b, and 11) activities. Two potent water-soluble RN-18 analogues, 17 and 19, are also disclosed, and we describe the results of pharmacological studies with compound 19. The findings described here will be useful in the development of more potent Vif inhibitors and in the design of probes to identify the target protein of RN-18 and its analogues.

Full text of DOI:10.1021/ml300037k

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Letter
SAR and Lead Optimization of an HIV-1 Vif-APOBEC3G Axis Inhibitor
Idrees Mohammed, Maloy K Parai, Xinpeng Jiang, Natalia
Sharova, Gati Krushna Singh, Mario Stevenson, and Tariq M. Rana
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/ml300037k • Publication Date (Web): 14 May 2012
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SAR and Lead Optimization of an HIV-1 Vif-APOBEC3G Axis
Inhibitor
Idrees Mohammed,[a]† Maloy K. Parai,[a] Xinpeng Jiang,[a] Natalia Sharova,[b] Gatikrushna Singh,[a] Mario
Stevenson,[b] Tariq M. Rana*[a]
[a] Program for RNA Biology, Sanford-Burnham Medical Research Institute, La Jolla, California 92037 (USA)
[b] Division of Infectious Diseases, Miller School of Medicine, University of Miami, Miami, FL 33136 (USA)
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ABSTRACT: We describe structure-activity relationship and optimization studies of RN-18, an HIV-1 Vif-APOBEC3G axis inhibitor.
Targeted modifications of RN-18 ring-C, ring-B, ring-A, bridge A-B, and bridge B-C were performed to identify the crucial structural features,
which generated new inhibitors with similar (4g and 4i) and improved (5, 8b, and 11) activities. Two potent water-soluble RN-18 analogues,
17 and 19, are also disclosed, and we describe the results of pharmacological studies with compound 19. The findings described here will be
useful in the development of more potent Vif inhibitors and in the design of probes to identify the target protein of RN-18 and its analogues.
KEYWORDS: RN-18, HIV-1 Vif-APOBEC3G axis inhibition, structure-activity relationship and optimization, pharmacological studies
Chemotherapies that target inhibition of HIV-1 proteins, in
particular HIV-1 protease1 and reverse transcriptase2, have been of
immense help in prolonging the lives of patients with acquired
immunodeficiency syndrome (AIDS). However, the high rate of
HIV-1 replication has led to the emergence of drug resistant strains
the antiviral activity was Vif specific. RN-18 showed the greater
potency (IC50 4.5 µM in CEM cells) and specificity (IC50 >100 µM
in MT4 cells) among the two compounds and was therefore
selected for further study.5
Based on this discovery, we have initiated two projects: (1) lead
optimization studies of RN-18 to improve its activity and
pharmacological profile, and (2) the identification and validation of
RN-18 target protein. In this communication, we describe our
initial efforts in the area of structure-activity relationships (SAR)
and optimization of RN-18.
For convenience, we refer to the structural components of RN-18
as ring-A, ring-B, ring-C, bridge A-B (thioether linker), and bridge
B-C (amide linker). RN-18 (1) was synthesized by a conventional
acid chloride route (Scheme 1A). S-arylation of 4-
nitrofluorobenzene with thiosalicylic acid under basic and reflux
conditions yielded intermediate acid 2, which was converted to
corresponding acyl chloride. This was reacted with o-anisidine in
that remain
a major challenge in the field of anti-HIV
chemotherapy. Considerable effort is being focused on
understanding the structural basis of HIV-1 multidrug resistance
(MDR)3 and to develop new inhibitors that are effective in HIV-1
MDR mutants.4 In this regard, we recently reported the discovery
of small molecule inhibitors of HIV-1 viral infectivity factor (Vif).5
HIV-1 mainly infects CD4+ T lymphocyte in vivo; thus, if the
infection is not controlled the immune system becomes seriously
weakened and AIDS develops. Efficient HIV-1 replication requires
expression of Vif protein to counter the activity of APOBEC3G
(A3G), a DNA-editing cytidine deaminase expressed in certain
host cells. A3G catalyzes critical hypermutations in the viral DNA
and acts as an innate weapon against retroviruses.6 Cells that
express A3G are ‘non-permissive’ for viral replication in which
HIV-1 must express Vif in order to replicate. In contrast, HIV-1
replication is Vif-independent in host cells that do not express A3G
(permissive cells). Vif protein hinders A3G packaging into virions
and promotes ubiquitin-proteosomal degradation of A3G.7
Therefore, the development of small molecules that inhibit Vif-
mediated degradation of A3G in non-permissive T cells is an
exciting component of the anti-HIV drug discovery program.
We previously disclosed the structures of 25 Vif antagonists (RN-
1 to RN-25) and reported their median inhibitory concentrations
(IC50) for inhibition of A3G degradation in non-permissive cells.
The compounds were then examined for their capacity to inhibit
Vif activity and the replication of wild-type HIV-1 in A3G-
expressing non-permissive H9 and CEM cells or in permissive
MT4 and CEM-SS cells. Among the 25 compounds, RN-18 and
RN-19 exhibited potent antiviral activity in the non-permissive H9
and CEM cells, but not in MT4 or CEM-SS cells, confirming that
the presence of Et3N base to yield the lead molecule
1
quantitatively. In the same way, a series of substituted anilines (3a-
k, Table 1S in the supporting information) were reacted with the
acyl chloride of intermediate 2 in a parallel synthetic format to
generate a variety of ring-C substituted analogues, 4a-k (see Table
1 for structures and activities of 4f, 4g, 4i and Table 2S for 4a-e, 4h,
and 4j-k). In the ring-C studies, we noticed that methoxy at R1 is
preferred for the antiviral activity. However, from the list of 4a-k
RN-18 ring-C analogues (Table 1 and Table 2S), 2-iodo analogue
4g and methanesulfonate analogue 4i showed IC50 values of 4.3 µM
and 5.6 µM, respectively, which are similar to the lead molecule.
Similarly, from three R2 substitutions tested (methyl, 4a; bromo,
4e; and methyl carboxylate, 4f) in the presence of methoxy as the
R1 substituent on ring-C, only the methyl carboxylate functionality
(4f) retained appreciable activity (IC50 of 10 µM).
We next performed SAR studies associated with bridge A-B. The
thioether (sulfide) linker present in the lead molecule was oxidized
directly using a published method8 to afford sulfone 5 (Scheme
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1B). Compound 1 was treated with potassium permanganate
separately coupled with o-anisidine in basic conditions to obtain
adsorbed on manganese dioxide, which yielded sulfone 5 as the sole
product. Interestingly, we found the activity of the sulfone (5) was
approximately five-fold greater than the sulfide (1), with the
sulfone 5 exhibiting an IC50 of 1.1 µM. To continue SAR on bridge
A-B, the thioether linker was extended by a CH2 attached to ring-B
(Scheme 4S). However, this extension of bridge A-B reduced the
potency of the compound to an IC50 of 51 µM. Similarly, changes
involving amide or urea linkers at bridge A-B and reverse amides at
bridge B-C yielded relatively inactive compounds (Scheme 5S and
Table 3S).
intermediates 7a and 7b. Compounds 7a and 7b were then S-
arylated with 4-nitrothiophenol in the absence of base, yielding
RN-18 pyridine analogues 8a and 8b. Substitution of CH with N
adjacent to the thioether-linked carbon in ring-B was well tolerated
(Table 1, 8a and 8b); in the case of compound 8b, which has a
methyl group at position 6 of the pyridine ring, the activity was
improved to an IC50 of 1.5 µM. Of note, we discovered an
interesting reaction while attempting S-arylation of compounds 7a
and 7b in the presence of potassium carbonate base in refluxing
DMF. We observed formation of an unexpected compound instead
of the desired products 8a and 8b, which could be explained by a
cascade reaction involving simultaneous steps of amide hydrolysis,
C-S bond fission, and N-arylation (see Scheme 10S in the
supporting information file). Further exploration of this new
reaction is in progress.
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Scheme 1a
Scheme 2a
aReagents and conditions: (a) Methyl 4-mercaptobenzoate, cat. CuI,
K2CO3, DMF, 110 oC, 8 h. (b) SOCl2, cat. DMF, benzene, 80 oC. (c) o-
o
anisidine, Et3N, benzene, 80 C, 6 h. (d) KMnO4/ manganese dioxide,
CH2Cl2, room temperature, 12 h. (e) (CH3)3SnOH, 1,2-
dichloroethane, 80 oC, 6 h, room temperature, 5 h. (f) choline base
45% in methanol, EtOAc or acetone, room temperature.
A potent compound (11) was obtained by changing the point of
attachment of the thioether link (bridge A-B) from the ortho
position (as in RN-18) to the para position of ring-B and by placing
a chloro group at the ortho position of ring-B (Scheme 1D). The
synthetic scheme for 11 was started by selective mono S-arylation
of commercially available methyl 2-chloro-4-iodobenzoate with 4-
nitrothiophenol in the presence of copper (I) iodide as a catalyst
and potassium carbonate9, which yielded the intermediate ester 9.
The methyl ester was hydrolyzed using lithium hydroxide
monohydrate to afford S-arylated chloro acid 10, which was then
coupled with o-anisidine through the acid chloride route to obtain
the RN-18 analogue 11. The antiviral studies of analogue 11
showed an improved IC50 of 2.5 µM. The activity profile of 11
prompted us to examine whether ring-A and bridge A-B were
required at all for antiviral activity. To address this, we synthesized
12 (Scheme 1E) by coupling commercially available 2-
chlorobenzoic acid with o-anisidine using the acid chloride route.
However, 12 did not exhibit antiviral activity selectively in non-
aReagents and conditions: (a) 4-Fluoronitrobenzene, K2CO3, DMF,
o
o
120 C, 8 h. (b) SOCl2, cat. DMF, benzene, 80 C, 2 h. (c) o-anisidine,
o
Et3N, benzene, 80 C, 5 h. (d) 3a-k (see Table-1S in the supporting
information), Et3N, Benzene, 80 oC, 5 h. (e) KMnO4/manganese
dioxide, CH2Cl2, room temperature, 12 h. (f) 4-nitrothiophenol, DMF,
100-120 oC. (g) 4-nitrothiophenol, K2CO3, 5 mol% CuI, DMF, 100 oC,
6 h. (h) lithium hydroxide monohydrate, THF/MeOH/H2O (4:2:1),
R.T., 5 h.
To perform SAR studies associated with ring-B, we initially
synthesized two pyridine derivatives as outlined in Scheme 1C.
Commercially available 2-chloronicotinic acid 6a and 2-chloro-6-
methylnicotinic acid 6b were converted to acyl chlorides and
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permissive H9 cells. Similarly, SAR involving methyl or benzyl
H9
RN-18, 1
MT-4
RN-18, 1
moieties on ring-B and sulfonamide, amide, urea, or amine linkages
for bridge A-B resulted in compounds with either very low or no
activity (Scheme 6S and Table 3S).
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Table 1: Antiviral activities of selected compounds
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Figure 1: Vif antagonists inhibit HIV-1 replication in non-permissive H9 cells
but not in permissive MT4 cells. H9 and MT4 cells (2 x 105 cells per well in 48-
well plates) were treated overnight with various concentrations of Vif
antagonists (activity graphs are shown here for RN-18 (A), 5 (B), 8a (C), 8b
(D), and 19 (E)) before infection with an X4-tropic HIV-1 variant (HIV-1LAI).
Cells were maintained in the presence or absence of Vif antagonists for 10-15
days. Viral replication was monitored every other day by measuring reverse
transcriptase (RT) activity in culture supernatants.
Studies on ring-A were directed mainly at finding a suitable
replacement for the nitro functionality present in the lead molecule
1. To address this, we explored methyl carboxylate, carboxylic acid,
and choline carboxylate groups on ring-A, due to their structural
similarity with nitro functionality. 2-iodobenzoic acid was S-
arylated with methyl 4-mercaptobenzoate using copper (I) iodide
as a catalyst, yielding an intermediate carboxylic acid 13. This was
coupled with o-anisidine using the acid chloride route to afford
methyl benzoate derivative 14 (Scheme 2A). Compound 14 was
hydrolyzed using an excess of trimethyltin hydroxide in refluxing
1,2-dichloroethane solvent10 leading to the formation of carboxylic
acid 16 (Scheme 2B). Compound 16 was converted to a water-
soluble choline salt by treating with choline hydroxide (available
commercially in methanol) in ethyl acetate to afford compound 17.
N.A. = No Activity
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Compounds 14 and 16 did not exhibit antiviral activity, but
maintained for further 10-15 days (Figure 1). Viral replication was
interestingly, the choline salt 17 showed potent antiviral activity
with an IC50 of 1.0 µM. Prompted by the activity profile of sulfone
5, compound 14 was similarly oxidized using potassium
permanganate adsorbed on manganese dioxide to obtain sulfone
ester 15. Compound 15 was then hydrolyzed using trimethyltin
hydroxide to afford carboxylic acid 18, which was converted to the
water-soluble choline salt 19 (Scheme 2A and 2B). Compound 19
also showed considerable antiviral activity, with an IC50 of 10 µM.
Further SAR was performed involving: (i) nitro functionality of
ring-A and sulfone as bridge A-B, which generated two compounds
(Scheme 7S); (ii) combinatorial changes involving methoxy or
trifluoromethoxy groups on ring-C; sulfonamide, amide, urea, or
amine linkers as bridge A-B; and sulfonamide, amide or urea linkers
as bridge B-C, which generated eight compounds, (Scheme 8S),
and finally (iii) 7-methoxy-1H-indole and benzo[d]imidazole-2-
amine were used as ring-C moieties (Scheme 9S). However, none
of the compounds generated by these three schemes were active in
the antiviral assays.
monitored every second day by measuring reverse transcriptase
(RT) activity in the culture supernatants. For this, 1/3 of the
culture supernatant was replaced with an equivalent volume of
fresh medium containing the appropriate compounds every second
day. In all experiments, RN-18 (1) was used as a positive control
and cells cultured without compound served as negative controls.
Measurements of antiviral activity in cultured cells were repeated at
least 3 times (5-6 times for active compounds) and the IC50 values
were calculated using GraFit software. The IC50 values for selected
compounds of interest are presented in Table 1. Figure 1 shows the
concentration-response curves for inhibition of viral replication by
RN-18, and compounds 5, 8a, 8b, and 19. Figure 1A demonstrates
that RN-18 inhibited HIV-1 replication in non-permissive H9 cells
but not in permissive MT4 cells. Similarly, selective antiviral
activity was exhibited by the RN-18 analogues 5, 8a, 8b, and 19
(Figure 1B, C, D, and E, respectively). Antiviral activities of other
RN-18 analogues of interest are presented in Table 1. Several
compounds (5, 8b, 11, and 17) showed superior antiviral activities
in H9 cells compared with the lead molecule. Compound 12
showed no activity in non-permissive H9 cells.
Having demonstrated the antiviral activity of several novel Vif
antagonists, we wished to determine if the analogues could
upregulate A3G and downregulate Vif, as was shown for RN-18. To
do this, 293FT cells co-expressing hemagglutinin (HA)-tagged
A3G and green fluorescent protein (GFP)-tagged Vif or ΔVif were
grown alone or in the presence of the small molecule Vif
antagonists (50 µM) for 16 h (see supporting information for
methods). The cell extracts were then analyzed by immunoblotting
with anti-HA-A3G, anti-GFP-Vif, and anti-glyceraldehyde 3-
phosphate dehydrogenase (anti-GAPDH) antibodies (Figure 2).
In control cells grown in the absence of Vif antagonists (lane b),
HA-A3G was downregulated by GFP-Vif protein. In cells treated
with compounds 8b, 5, 17, and 11 (lanes e, f, g, and i, respectively),
HA-A3G expression was upregulated and GFP-Vif was
downregulated, as was observed with cells incubated with RN-18
(lane c). Compound 12 (lane h) had no effect on A3G or Vif
expression. Similarly, A3G and Vif levels were unaffected by
choline chloride (lane d), suggesting that the antiviral activity of
the choline carboxylate salts 17 and 19 was not due to choline
itself.
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PBS(1X)
- -
+ +
-
+
-
+
- -
+
+
-
+
+
-
+
-
DMSO(1%)
GFP-Vif
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+
+
-
+ + + + + + +
-
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-
- - - -
∆Vif
HA-A3G
+ + + + + + + + +
IB
Anti-HA-A3G
Anti-GFP-Vif
Anti-GAPDH
Lane
a
b
c
d
e
f
g
h
i
The SAR trends presented in this manuscript are derived from
cell-based antiviral studies. We plan to perform SAR based on the
RN-18 target protein once the identification and validation of the
target is complete. We expect to report those results in the near
future. The presence of three aromatic rings was found to be a
common structural feature of all the active analogues. A methoxy
group at the ortho position of ring-C is preferred for activity;
however, iodo (4g) and methane sulfonate (4i) groups at the ortho
position were also tolerated without lowering the activity or
specificity. Other substitutions on ring-C significantly lowered or
completely eliminated the antiviral activity, with the exception of
methyl carboxylate (4f) at the para position. Antiviral activity was
retained when the phenyl ring-B was replaced with a pyridine ring
(8a) and the presence of a methyl group at 6 position of the
pyridine ring (8b) improved its activity. Replacement of the sulfide
Figure 2: Vif antagonist small molecules enhance A3G levels and reduce Vif
expression. 293FT cells co-expressing HA-tagged A3G and GFP-tagged Vif or
ΔVif were grown for 16 h in the presence of RN-18, 1 (lane c), choline chloride
(lane d), 8b (lane e), 5 (lane f), 17 (lane g), 12 (lane h), and 11 (lane i), or in
the absence of compounds (lane a and b). Cell extracts were analyzed by
immunoblotting with anti-HA-A3G, anti-GFP-Vif, and anti-GAPDH antibodies
(see S.I. for details)
The antiviral activities of the RN-18 analogues were measured
with wild-type HIV-1 in H9 cells (non-permissive) and MT-4 cells
(permissive). H9 and MT4 cells (2 x 105 cells per well in 48-well
plates) were treated overnight with differing concentrations of Vif
antagonists (1 to 50 µM) in Roswell Park Memorial Institute
(RPMI) medium containing 10% fetal bovine serum. The cells
were then infected with an X4-tropic HIV-1 variant (HIV-1LAI) and
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Structure of Substituted Bis-tetrahydrofuran (Bis-THF)-Derived Potent
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bridge A-B (1) with sulfone (5) increased the potency about five-
fold. Bridge B-C SAR showed that amide functionality is essential
and its replacement with reverse amide or sulfonamides, led to an
overall decrease in activity. Although our SAR results with ring-A
are not yet conclusive, the carboxylic salts of choline (17 and 19)
showed promising results.
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protein. Nature 2002, 418, 646-650. (b) Mehle, A.; Strack, B.; Ancuta, P.;
Zhang, C.; McPike, M.; Gabuzda, D. J. Biol. Chem. 2004, 279, 7792-7798.
8. Shabani, A.; Mirzaei, P.; Naderi, S.; Lee, D. G. Green oxidations. The use
of potassium permanganate supported on manganese dioxide. Tetrahedron
2004, 60, 11415-11420.
9. (a) Kwong, F. Y.; Buchwald, S. L. A General, Efficient, and Inexpensive
Catalyst System for the Coupling of Aryl Iodides and Thiols. Org. Lett.
2002, 4, 3517-3520. (b) Sperotto, E.; van Klink, G. P. M.; de Vries, J. G.;
van Koten, G. Ligand-Free Copper-Catalyzed C-S Coupling of Aryl Iodides
and Thiols. J. Org. Chem. 2008, 73, 5625-5628.
We have performed some initial pharmacological studies with 19
and experiments with 17 are currently in progress. These studies
revealed both merits and demerits of 19 (see supporting
information for details). Merits include aqueous solubility
(moderately soluble at the pH tested: 5.0, 6.2, and 7.2), metabolic
stability (hepatic microsome stability), plasma stability, and lack of
cytotoxicity (>50 µM). Demerits include poor membrane
permeability (moderately permeable at pH 5.0 and poorly
permeable at pH 6.2 and 7.4), and its significant plasma protein
binding. The poor membrane permeability and high plasma protein
binding of 19 suggest that bioavailability of the compound may be
compromised. Nonetheless, this water-soluble, non-toxic, and
metabolically stable compound represents as a starting point for
further development.
In conclusion, this SAR study generated several interesting RN-18
analogues (4f, 4g, 4i, 5, 8a, 8b, 11, 17, and 19). We briefly
described some pharmacological characteristics of 19 and similar
studies are underway for several compounds, including 17. Our
current studies are directed towards finding a better replacement of
the amide functionality present in RN-18 and a detailed study for
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SAR of ring-A.
A report of the ongoing studies will be
communicated in the near future.
ASSOCIATED CONTENT
Experimental procedures, characterization data, and a brief summary of
the pharmacological studies are given in the supporting information
file. This information is available free of charge via the Internet at
http://pubs.acs.org.
10. Nicolaou, K. C.; Estrada, A. A.; Zak, M.; Lee, S. H.; Safina, B. S. A Mild
and Selective Method for the Hydrolysis of Esters with Trimethyltin
Hydroxide. Angew. Chem. Int. Ed. 2005, 44, 1378-1382.
AUTHOR INFORMATION
Corresponding Author
*Prof. Dr. Tariq M. Rana
Program for RNA Biology,
Sanford-Burnham Medical Research Institute
La Jolla, California 92037 (USA)
Tel: +1 858 795 5325; Fax: +1 858 795 5387
†Notes
This author’s name appeared as Mohd. Idrees prior to this publication.
REFERENCES
1. Ghosh, A. K.; Martyr, C. D.; Steffey, M.; Wang, Y. –F.; Agniswamy, J.;
Amano, M.; Weber, I. T.; Mitsuya, H. Design, Synthesis, and X-ray
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SAR and Lead Optimization of an HIV-1 Vif-APOBEC3G Axis Inhibitor
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