Optimization of diarylazines as anti-HIV agents with dramatically enhanced solubility

Article abstract of DOI:10.1016/j.bmcl.2013.06.091

Non-nucleoside inhibitors of HIV-1 reverse transcriptase are reported that have ca. 100-fold greater solubility than the structurally related drugs etravirine and rilpivirine, while retaining high anti-viral activity. The solubility enhancements come from strategic placement of a morpholinylalkoxy substituent in the entrance channel of the NNRTI binding site. Compound 4d shows low-nanomolar activity similar to etravirine towards wild-type HIV-1 and key viral variants.

Full text of DOI:10.1016/j.bmcl.2013.06.091

Accepted Manuscript
Optimization of diarylazines as anti-HIV agents with dramatically enhanced
solubility
Mariela Bollini, José A. Cisneros, Krasimir A. Spasov, Karen S. Anderson,
William L. Jorgensen
PII:
S0960-894X(13)00824-X
http://dx.doi.org/10.1016/j.bmcl.2013.06.091
BMCL 20655
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Please cite this article as: Bollini, M., Cisneros, J.A., Spasov, K.A., Anderson, K.S., Jorgensen, W.L., Optimization
of diarylazines as anti-HIV agents with dramatically enhanced solubility, Bioorganic & Medicinal Chemistry
Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.06.091
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Optimization of diarylazines as anti-HIV agents with
dramatically enhanced solubility
Mariela Bollinia, José A. Cisnerosa, Krasimir A. Spasovb, Karen S. Andersonb*, and
William L. Jorgensena*
aDepartment of Chemistry, Yale University, New Haven, CT 06520-8107, USA
bDepartment of Pharmacology, Yale University School of Medicine, New Haven, CT 06520-8066, USA
Received May 15, 2013; Accepted Month XX, 2013
Abstract— Non-nucleoside inhibitors of HIV-1 reverse transcriptase are reported that have ca. 100-fold greater solubility than the
structurally related drugs etravirine and rilpivirine, while retaining high anti-viral activity. The solubility enhancements come from
strategic placement of a morpholinylalkoxy substituent in the entrance channel of the NNRTI binding site. Compound 4d shows low-
nanomolar activity similar to etravirine towards wild-type HIV-1 and key viral variants. ©2013 Elsevier Science Ltd. All rights reserved.
The use of non-nucleoside inhibitors of HIV-1
reverse transcriptase (NNRTIs) is commonplace for
treatment of HIV infection.1,2 Among the five FDA-
approved drugs in the class, the most recent
introductions have been etravirine and rilpivirine. These
diarylpyrimidines provide much improved performance
in cell assays against variant forms of HIV-1 that
incorporate mutations in the vicinity of the NNRTI
binding site.3,4 The earliest approved NNRTIs,
nevirapine and delavirdine, are debilitated by most
common mutations. Though the second-generation
compound, efavirenz, performs well against variants
bearing the clinically prevalent Tyr181Cys mutation,
resistance arises from other common variants such as
those including Lys103Asn.2-4 The clinical significance
of efavirenz and rilpivirine is particularly great since
they are incorporated into the once-a-day combination
therapies Atripla and Complera, respectively.5 The other
two active components of the pills are the same, the
nucleosides emtricitabine and tenofovir. Though the
performance in cell-based assays is far better for
rilpivirine than for efavirenz, surprisingly more
virologic failure is observed for patients under treatment
with Complera than Atripla.5-7 Thus, from this
observation and the desire to further diminish dosages
and side effects, improvements are still possible for the
NNRTI class.
ramifications including low bioavailability, difficulties
in formulation, and accumulation in fatty tissues.8,9
Most oral drugs have an aqueous solubility (S) in the
range 10-5 to 10-2 M, which for a drug with a molecular
weight of 400, corresponds to 4 to 4,000 µg/mL. It is
very rare for an FDA-approved oral drug to have a
solubility near neutral pH below 10-6 M.9 However,
rilpivirine ―is practically insoluble in water (20 ng/mL
at pH 7.0)‖,4 which translates to an S of 5 x 10-8 M. It
appears to have an unusual absorption mechanism
involving aggregates.10 For etravirine, the solubility is
also ―<<1 µg/mL‖, and extensive formulation work was
needed to bring the daily dosage to 0.4 g per day.11
Furthermore, in view of its low solubility, dapivirine is
being evaluated as a vaginal microbicide.12 This was
A particular issue with aminoazine NNRTIs has
been poor solubility, which often has undesirable

also the fate of UC-781, an earlier, NNRTI with poor
solubility (<30 ng/mL).13 Interestingly, the daily dosage
for nevirapine, like etravirine, is 0.4 g despite the fact
that its potency towards WT HIV-1 is ca. 100-fold less
than for etravirine. An important factor is undoubtedly
that the observed aqueous solubility of nevirapine is 167
µg/mL (10-3.2 M).14 Nevirapine demonstrates that it is
possible to have a viable NNRTI that has an EC50 of ca.
100 nM in cell assays, if the compound has good
solubility and bioavailability.
the dose required to achieve 50% protection of the
infected cells by the MTT colorimetric method. CC50
values for inhibition of MT-2 cell growth by 50% are
obtained simultaneously. Solubility measurements used
a shake-flask protocol with triplicate samples.19 The
compounds were dissolved in Britton-Robinson buffer
and stirred in vials for 48 hours at 25 ºC. The pH of the
buffer solutions was measured by a Corning General
Purpose pH Combination probe (4136L21). The
solution containing excess solid was filtered using a
Whatman Mini-UniPrep syringeless filter device with a
0.45 µm pore size, and the supernatant was analyzed by
UV-vis spectrophotometry (Agilent 8453). Piroxicam
was used as a reference compound; our solubility result
of 7.2 µg/mL compares well with the prior report of 6.4
µg/mL.19
The results of the anti-viral assays are presented in
Table 1. Our synthesized TMC120 (8a) yielded 0.7 nM
potency in the WT assay and 39 nM results for both the
Y181C and K103N/Y181C variants. This can be
compared with previous results of 1.2, 7, and 54 nM
using MT-4 cells.4 The other compound that was
previously reported, the triazine 9a, was found here to
have 2.3, 47, and 90 nM EC50 values, while the MT-4
assays yielded 0.3, 8, and 50 nM.20 Thus, the accord is
reasonable except that it appears that the Y181C
containing strain used here is more challenging.
Appendage of the morpholinoethoxy substituent to
1 (R = H, R’ = Me) to yield 2 causes a 9-fold reduction
in WT potency,15 while the modification of pyrimidine
8a to yield 3a and 3b results in 10- to 20-fold declines.
Though the compounds are still potent NNRTIs towards
WT virus, the EC50 results of 700 and 480 nM for the
Y181C-bearing variant are more problematic. However,
Poor solubility has also been an issue with
aminoazine-containing NNRTIs (1) from our
laboratory. In view of the structural overlap of 1, the
TMC compounds, and UC-781, it is not surprising that
the measured solubility of 1 (R = H, R’ = Me) is 0.1
µg/mL.15 In order to improve the solubility of such
aminoazines, extensive modeling, synthesis, assaying
and crystallography were undertaken that demonstrated
that it was possible to attach a solubilizing substituent as
OR’ that would extend into the entrance channel of the
NNRTI binding site.15,16 Specifically, 2 has S = 42
µg/mL, while retaining an EC50 of 92 nM in a standard
assay using MT-2 cells infected with WT HIV-1.15
In the present communication, results are presented
for applying this strategy to improve the solubility of
diarylpyrimidines and triazines by investigating 3 and 4
(Scheme 1). Synthesis of the compounds proceeded as
indicated via three SNAr reactions.17 The final
intermediate 7 was also reduced to obtain the
Table 1
Anti-HIV-1 Activity (EC50) and cytotoxicity (CC50), μMa
corresponding analogs
8
and
9
lacking the
EC50
morpholinoalkoxy group. The identities of all assayed
K103N/
Y181C CC50
1
compounds were confirmed by H and 13C NMR and
Compnd
n
-
2
3
-
2
3
3
3
3
3
3
3
3
2
3
R’
R
WT
0.0007
0.019
0.0086
0.0023
0.012
0.0081
0.0068
0.0012
Y181C
0.039
0.700
0.480
0.047
0.700
0.310
1.0
0.012
0.350
0.500
0.270
1.100
0.230
1.000
1.200
NA
8ab
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
CN
0.039
0.060
0.038
0.090
0.110
0.031
2.0
9.0
3.1
7.0
95
high-resolution mass spectrometry; purity was >95% as
judged by high-performance liquid chromatography.
Activities against the IIIB and variant strains of
HIV-1 were measured using MT-2 human T-cells, as
previously described.16,18 EC50 values are obtained as
3a
3b
9a
4a
4b
4c
4d
4e
4f
4g
4h
4i
10a
8.0
0.042 >100
Me CVc
F
F
F
F
F
Me
Me
0.0013
0.070
0.050
0.030
0.150
0.020
0.100
0.140
NA >100
0.030
0.005
0.002
4.5
10.0
13.0
1.5
2.2
27.0
4.2
Me 0.00019
Et
i-Pr
c-Pr
CEd
Me
0.0016
0.0022
0.0028
0.005
0.024
0.024
0.11
0.002
0.001
0.00067 0.00065
10b
Me
4.0
nevirapine
efavirenz
etravirine
rilpivirine
0.010
0.008
15
11
8
a
Results using human MT-2 cells. Antiviral and toxicity curves used
triplicate samples at each concentration. NA = not active. 8a =
b
Scheme 1. Synthesis of 3 (X = CH) and 4 (X = N). Reagents: (a) DIPEA,
THF, rt, overnight; (b) DIPEA, THF, 65 oC, 2 days; (c) NaH, THF, rt,
overnight; (d) H2/Pd-C.
TMC120. c CV = E-cyanovinyl. d CE = 2-cyanoethyl.

the impressive performance of 3b towards the
challenging double mutant is notable. The
corresponding triazine 4b fares even better with 8, 310,
and 31 nM EC50 values. Thus, these results encouraged
further study of triazine analogs. Replacement of the 4-
Me substituent of the mesityl group by cyano and
cyanovinyl was explored and provided the remarkably
potent 4d with EC50 results of 1, 12, and 1 nM for the
WT and mutant HIV-1 strains. 4d may be viewed as a
triazine relative of rilpivirine with a strategically added
morpholinopropoxy group. Its activity results are very
similar to those obtained here for etravirine. 2,6-
difluorophenyl alternatives 4e – 4i with various 4-R
groups were also considered, but did not surpass the
overall performance of 4d. However, the 4-methyl
analog 4e was strikingly potent, 190 pM, in the WT
assay. Only one NNRTI with greater anti-HIV activity
has been previously reported.18
The solubility results are summarized in Table 2.
Consistent with the results for 1 and 2,15 the addition of
the morpholinoalkoxy groups has profound effects with
83- and 182-fold increases in S in going from TMC120
(8a) to 3a and 3b. The aqueous solubilty of the
corresponding triazine 9a is also very low (0.2 µg/mL);
large enhancements are again delivered by the
morpholinoalkoxy analogs. Notably, 4d has a solubility
of 14.2 µg/mL, which is 100-fold greater than for
dapivirine (TMC120, 8a) and 59- and 710-fold greater
than the prior reports for rilpivirine.
Figure 1. Computed structure for the complex of 4b with WT HIV-1
reverse transcriptase. Carbon atoms of 4b in yellow. Some residues
removed for clarity.
structure, which has an anilinyltriazine as the ligand.25
Consistent with the modeling and crystallography for
2,15 the contacts in the NNRTI binding site are normal
and the morpholinopropoxy side chain extends past
Glu138 towards Glu28. A salt bridge, which is
sometimes observed between Glu138 and Lys101,
cannot be present to allow the passage. With the (CH2)3
spacer a salt-bridge between Gu138 and the protonated
morpholine is unlikely. However, if Glu28 reoriented, it
could be in close contact with the morpholine terminus.
This suggested possible benefit of replacing the ether
oxygen with a positively charged group. Thus, the
piperidine analogs 10a and 10b were synthesized
(Scheme 2), but they turned out to be 2-3-fold less
potent than the corresponding morpholine analogs 4a
and 4b (Table 1). The intended salt-bridge is largely
solvent-exposed and in competition with the Glu28-
Lys32 interaction.
As demonstrated crystallographically for 2, it is
expected that the morpholinoalkoxy side chains for the
present compounds in complex with HIV-1 reverse
transcriptase extend past Glu138 into the entrance
channel of the NNRTI binding site.15,16 An illustration
for 4b is provided in Figure 1, as created by modeling
with the BOMB and MCPRO programs22,23 using the
OPLS/CM1A force field24 starting with the 1S9E crystal
Scheme 2. Synthesis of 10a and 10b. Reagents: (a) NaH, ACN, 70 ºC,
overnight; (b) TFA, DCM, 0 ºC to rt, 15 min.
In summary, structural analyses suggested the
possibility of appending solubilizing groups to the
diarylazine class of NNRTIs at the 6-position in the
azine ring. The strategy was successful and delivered
promising, new NNRTIs, whose formulation should be
facilitated. Notably, compound 4d has similar potency
as etravirine in infected T-cell assays using WT HIV-1
(IIIB) as well as viral variants that incorporate the two
most commonly found resistance mutations in the RT
enzyme, Tyr181Cys and Lys103Asn.2 However, the
solubility of 4d is ca. 100-fold greater than for the
diarylpyrimidines dapivirine (8a), etravirine, and
Table 2
Aqueous Solubility at pH 6.5 (S)
Compound
S, μg/mL
0.15
12.5
27.3
0.20
4.42
15.3
13.3
Compound
4d
S, μg/mL
14.2
8a
3a
3b
9a
4a
4b
4e
4i
22.9
25.4
nevirapine
efavirenz
etravirine
rilpivirine
167a
68.0
<< 1b
4c
0.02,c 0.24d
a
Ref. 14. b Ref. 11. c Ref. 4, pH 7. d Ref. 21, pH 7.4.

15. Bollini, M.; Frey, K. M.; Cisneros, J. A.; Spasov, K.
A.; Das, K.; Bauman, J. D.; Arnold, E.; Anderson, K.
S.; Jorgensen, W. L. Bioorg. Med. Chem. Lett. 2013,
23, accompanying article.
rilpivirine. Further fine-tuning of properties and
activities can be envisioned now that the benefits of
exploration of the NNRTI entrance channel are clear.
16. Ekkati, A. R.; Bollini, M.; Domaoal, R. A.; Spasov, K.
A.; Anderson, K. S.; Jorgensen, W. L. Bioorg. Med.
Chem. Lett. 2012, 22, 1565.
17. General procedure: 5 (0.34 g, 1.2 mmol) was
dissolved in anhydrous THF (10 mL), following by
addition of the corresponding aniline 6 (1.2 mmol) and
DIPEA (0.18 g, 1.44 mmol). The reaction mixture was
stirred at room temperature or refluxed overnight.
THF was removed under pressure; the crude was
purified on silica gel to afford the corresponding
Acknowledgements
Gratitude is expressed to the National Institutes of
Health (AI44616, GM32136, GM49551) for research
support. Receipt of reagents through the NIH AIDS
Research and Reference Reagent Program, Division of
AIDS, NIAID, NIH is also greatly appreciated.
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a
solution of the
hydroxyalkylmorpholine (12.8 mmol) in anhydrous
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o
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*Graphical Abstract (for review)
HIV-RT
Solubilized
NNRTI