Discovery and crystallography of bicyclic arylaminoazines as potent inhibitors of HIV-1 reverse transcriptase

Article abstract of DOI:10.1016/j.bmcl.2015.06.074

Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that incorporate a 7-indolizinylamino or 2-naphthylamino substituent on a pyrimidine or 1,3,5-triazine core. The most potent compounds show below 10 nanomolar activity towards wild-type HIV-1 and variants bearing Tyr181Cys and Lys103Asn/Tyr181Cys resistance mutations. The compounds also feature good aqueous solubility. Crystal structures for two complexes enhance the analysis of the structure-activity data.

Full text of DOI:10.1016/j.bmcl.2015.06.074

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and crystallography of bicyclic arylaminoazines as potent
inhibitors of HIV-1 reverse transcriptase
a
b
a
b
b
Won-Gil Lee , Kathleen M. Frey , Ricardo Gallardo-Macias , Krasimir A. Spasov , Albert H. Chan ,
b,
⇑
a,
⇑
Karen S. Anderson , William L. Jorgensen
a
Department of Chemistry, Yale University, New Haven, CT 06520-8107, USA
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520-8066, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that incorporate a 7-in-
dolizinylamino or 2-naphthylamino substituent on a pyrimidine or 1,3,5-triazine core. The most potent
compounds show below 10 nanomolar activity towards wild-type HIV-1 and variants bearing Tyr181Cys
and Lys103Asn/Tyr181Cys resistance mutations. The compounds also feature good aqueous solubility.
Crystal structures for two complexes enhance the analysis of the structure–activity data.
Ó 2015 Published by Elsevier Ltd.
Received 17 June 2015
Accepted 22 June 2015
Available online xxxx
Keywords:
HIV-1 reverse transcriptase
NNRTI
Structure-based drug design
Protein crystallography
Drug solubility
CN
Non-nucleoside inhibitors of HIV-1 reverse transcriptase
NNRTIs) are an essential element of anti-HIV therapy. Efavirenz
1
H
CN
(
(
HN
1) and rilpivirine (2) are components of the one-a-day triple-com-
2
CN
bination therapies Atripla and Complera. In spite of their success,
there is a need for continued development of NNRTIs in view of the
rapid mutation of the virus, varying resistance and side effect pro-
files, and evolving risks of long-term treatment.3
N
N
O
O
O
O
N
6
N
H
N
NH
–5
H
O
5
CN
N
O
HN
CF3
CN
Cl
N
2
O
To this end, we have explored multiple series of NNRTIs. The work is
challenging owing to the needs for potency towards wild-type HIV-
N
N
H
O
N
H
1
and multiple commonly observed viral variants, and for good
1
pharmacological properties. Dosage (600 mg/day), resistance, and
CNS side effects are problematic for efavirenz, while poor solubility
and virologic failure are issues with rilpivirine. Significant head-
Cl
CN
F
2,4
N
CN
way in potency was made for our series with the discovery of cate-
6
chol diethers. Assays tested activity against wild-type HIV-1 and
O
O
O
O
the most common clinically observed viral variants, which contain
Tyr181Cys (Y181C) and Lys103Asn (K103N) point mutations in
the reverse transcriptase enzyme. EC50 values as low as 0.055 nM
were obtained for inhibition of viral replication in human T-cells
N
NH
N
NH
H
O
F
O
O
O
3
4
6
infected with the wild-type virus. Overall, 3 is among the best
examples with EC50 values of 0.31, 46, and 24 nM towards wild-
type HIV-1, and virus containing the Y181C mutation and the par-
ticularly challenging K103N/Y181C double variant. Though the
⇑
Corresponding authors.
E-mail addresses: karen.anderson@yale.edu (K.S. Anderson), william.jorgense-
n@yale.edu (W.L. Jorgensen).
http://dx.doi.org/10.1016/j.bmcl.2015.06.074
0
960-894X/Ó 2015 Published by Elsevier Ltd.
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2
W.-G. Lee et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
activity of 3 towards the variants is less than for 1 or 2, the com-
pound has remarkable aqueous solubility and relatively low toxicity
Table 2
Anti-HIV-1 activities (EC50) and cytotoxicity (CC50)a
Compd Rb
X
Y
Z
EC50
WT
EC50
EC50 K103N/ CC50
Y181C
towards human T-cells (CC50 = 18
lM), as summarized in Table 1.
However, 3 like rilpivirine incorporates a cyanovinyl group,
Y181C
which is rarely found in drugs owing to concerns for Michael
7a
7b
7c
H
H
H
H
H
H
H
Cl
H
Mo
H
H
H
H
CH
H
H
H
200
30
NA
510
26
0.57
1600
98
NA
>100,000
additions that might lead to undesirable covalent modifications
CH Me
CH
CH Me Me 1.0
CH
CH
N
N
N
N
CH
CH
6200
170
39
4600
NA
32
NA
2200
NA
520
130
6.9
790
50
7.0
67
65
150
98
72,000
>100,000
10,100
76,000
1000
16,000
38,000
20,000
10,000
>100,000
>100,000
7800
45,000
4800
9500
7500
>100,000
1200
of proteins or nucleic acids.7 Thus, less reactive alternatives were
H
Me 1.4
7
7
7
d
e
f
pursued by incorporating the cyanovinyl fragment into a 6:5 or
F
F
H
F
200
1.4
6
:6 bicyclic ring system.7,8 Resultant notable examples include 4
and 5. The indolizine 4 shows excellent potency towards the
wild-type virus and the double variant, low cytotoxicity, and good
aqueous solubility; however, it and the other 6:5 bicyclics are
7g
7h
Me Me 0.52
Me Me 14000 NA
F
H
H
H
7.1
7
7
8
i
j
a
F
2.7
Me 1900
24
Me 7.0
230
NA
170
8.5
2.2
70
6.0
1.3
41
7.0
NA
58
160
36
7
oddly much less active towards the Y181C single variant. This
H
8
problem was largely overcome with 2-naphthyl catechol diethers.
8b
8c
For example, 5 has all three EC50 values below 20 nM and shows no
CH Me Me 2.9
8d
CH
CH
N
N
N
N
N
N
N
H
F
F
F
6.0
1.8
cytotoxicity, but its solubility is only 4.3 lg/mL, which is at the
9
8e
H
H
bottom of the range normally observed for oral drugs.
8
8
f
g
Me Me 1.1
Me Me 2.3
F
H
H
H
Simultaneously, analysis of crystal structures and molecular
modeling led us to realize that the solubility of anilinylazines such
as 2 could be enhanced without unacceptable loss of potency by
judicious placement of a polar substituent that would project into
the entrance channel of the NNRTI binding site.10 This led to dis-
covery of 6, a morpholinylpropoxytriazinyl analogue of 2, which
has excellent potency and greatly improved solubility
Cl
H
Mo
Mo
Mo
Mo
8h
8i
F
H
1.2
120
8
8
8
j
k
l
Me 21
36
5900
3800
17,000
F
180
16
Me Me 17
a
EC50 and CC50 in nM from infected T-cell asssays. NA = not active (EC50 >CC50).
Mo = N-morpholinylpropoxy as in 6.
b
1
1
(
14.2 lg/mL). Nevertheless, further efforts were deemed desir-
able to replace the cyanovinyl group in 6 and also to seek addi-
tional gains for potency and solubility. As described here, success
was obtained by combining the 6:5 and 6:6 bicyclic notion with
anilinyl pyrimidine or triazine cores in 7 and 8.
Compounds listed in Table 2 were prepared as summarized in
Schemes 1–3. The final products come from Pd-catalyzed amina-
tion of chloroazines (Scheme 1).11 The morpholinylpropoxy ana-
logues were derived from the 7 and 8 triazinyl chlorides (X = N,
R = Cl) by reaction with 3-morpholinopropan-1-ol and NaH in
Y
N
CN
Y
N
HN
N
CN
a
Z
CN
H N
X
N
2
Cl
N
Z
+
CN
R
N
H
X
N
7
R
N
H
1
,4-dioxane for 16 h at 90 °C. The requisite 7-aminoindolizines
+
a
Y
CN
CN
were derived from substituted pyridines, which after oxidation,
and nitration, underwent a Baylis–Hillman reaction, cyclization,
and reduction to yield the desired intermediates.7 For the
Y
CN
HN
N
H N
2
Z
2
-aminonaphthalenes, acetals 9 were prepared by alkylation of
X
N
Z
dimethyl 2-(2,2-diethoxyethyl)malonate with benzyl bromides.
Cyclization to the methyl naphthoate was followed by conversion
of the ester progressively to the aldehyde and nitrile. Finally, the
R
N
H
8
2
-amino group was introduced by Pd-catalyzed reaction of the bro-
mides with t-butyl carbamate, followed by acid-catalyzed removal
of the Boc group.
Scheme 1. Synthesis of 7 and 8 where X = CH, N; R = H, Cl; Y, Z = H, F, Me. Reagents
2 3
and conditions: (a) BINAP, Pd (dba) , 1,4-dioxane, 90 °C, 12–24 h.
In the same manner as in previous reports,6–8,10,11 activities
against the IIIB strain of HIV-1 were measured using MT-2 human
T-cells; EC50 values are obtained as the dose required to achieve
O-
Y
Y
Y
N+
c, d
N
N
a, b
O
5
0% protection of the infected cells by the MTT colorimetric
O2N
2
O N
Z
Z
Z
Y
Y
Table 1
e
N
f, g
N
Anti-HIV-1 activities (EC50), cytotoxicity (CC50), and aqueous solubilitya
CN
O2N
CN
H2N
Compd
EC50 WT
EC50 Y181C
EC50
K103N/Y181C
CC50
S (lg/mL)
Z
OH
Z
1
2
3
4
5
6
2.0
10
0.65
46
250
19
12
0.57
7.1
2.2
1.3
30
2.0
24
10
15
1.3
39
32
6.9
7.0
15,000
8000
18,000
50,000
>100,000
4500
10,100
16,000
7800
68.0
Scheme 2. Synthesis of substituted indolizines. Reagents and conditions: (a) m-
CPBA, CHCl , rt, 18 h; (b) HNO , H SO , 95 °C, 4–16 h; (c) (i) Ac O, 110 °C, 4 h; (ii)
0.67
0.31
0.40
0.53
1.2
1.0
0.52
2.9
0.02b
510
43.8
4.3
3
3
2
4
2
HCl, 70 °C, 3 h; (d) SeO , 1,4-dioxane, 90 °C, 16 h; (e) acrylonitrile, DABCO, neat, rt,
2
1.5 h; (f) Ac O, 100–135 °C, o/n; (g) Pd/C, H , MeOH, rt, 12 h.
2
2
14.2
8.2
33.1
12.2
28.7
7
7
8
8
d
g
c
f
method. Simultaneously, CC50 values for inhibition of MT-2 cell
growth by 50% are obtained. The analyses used triplicate samples
at each concentration. The identity of all assayed compounds was
1.1
9500
1
13
a
confirmed by H and C NMR and high-resolution mass spectrom-
etry; purity was >95% as judged by high-performance liquid
EC50 and CC50 in nM from infected T-cell assays. Data for 1–6 from Refs. 6–8.
Ref. 15.
b
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W.-G. Lee et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
O
The same pattern is found for fluorination with 7e and 7f, but to
a lesser extent. This observation is readily attributed to the but-
tressing by the added groups that forces the indolizine ring to be
perpendicular to the azine ring, yielding better preorganization
for binding (Fig. 1). The effects are larger for the bulkier methyl
groups. 7g, the triazine analogue of 7d, is also impressive with
EC50 values of 0.52, 7.1, and 32 nM towards the wild-type virus
and the two variants. Less potency is again found with the difluoro
analogue 7i, and a chlorine (7h) or a morpholinylpropoxy group
O
Y
Y
O
a
O
Br
CH(OEt)2
Br
Z
9
Z
O
Y
CN
Y
b, c
d, e
H N
Br
2
Z
Z
(7j) at the 6-position of the triazine ring are not well tolerated
owing to steric interference with the Lys101-Glu138 salt bridge.
The parent naphthyl pyrimidine 8a is significantly more potent
than the corresponding indolizine 7a. This appears to be due to
improved aryl-aryl contacts with Tyr181 and Trp229 (Figs. 1 and
Scheme 3. Synthesis of substituted naphthalenes. Reagents and conditions: (a) aq
0% H SO
, MeOH, DDQ, 0 °C to rt, 1 h; (b) DIBAL, THF, À78 °C to rt, o/n; (c) PCC,
DCM, rt, 2 h; (d) NaN , TfOH, ACN, rt, 1 h; (e) (i) t-butyl carbamate, Pd (dba)
ÁCHCl
xantphos, Cs CO , toluene, 100 °C, 16 h; (ii) 2 N HCl, 1,4-dioxane, 60 °C, 2 h.
8
2
4
3
2
3
3
,
2
3
2). Methylation or fluorination at the 1- and 3-positions again pro-
vides significant gains in inhibitory activity (8b–8e). The dimethyl
pyrimidine and triazine analogues 8c and 8f are the best new com-
pounds for potency with sub-10 nM results for all three viral forms.
Addition of a chlorine at the 6-position of the triazine diminishes
activity, though not as much as in the indolizine series. The diflu-
oro analogue 8h shows no cytotoxicity, but it performs less well
than 8f for the HIV-1 variants. A methyl group is expected to better
fill the space vacated upon the Tyr181Cys exchange than a fluorine.
Finally, the morpholinylpropoxy analogue 8l is interesting; the
added substituent is much better tolerated than for 7j. An explana-
tion is not obvious from the crystal structures as C6 of the triazine
is 3.61 and 4.06 Å away from the closest oxygen of Glu138 and
ammonium nitrogen of Lys101 for 7g and 3.29 and 4.14 Å for 8f.
Aqueous solubilities for the most potent new compounds were
determined and are compared with the previously reported values
for 1–6 in Table 1. The results for the triazines 7g and 8f, 33 and
chromatography. Aqueous solubilities were also measured as pre-
viously described using shake-flask procedure in Britton-
Robinson buffer at pH 6.5 and 25 °C.
collected using a 0.2 m Pall Life Sciences Acrodisc syringe filter,
a
10–12
The supernatant was
l
and analyzed by UV–vis spectrophotometry (Agilent 8453).
Interpretation of the structure-activity data was facilitated by
obtaining crystal structures of several compounds in complex with
HIV-1 RT. Following previously reported procedures,7
,8,10,11,13
crys-
tals were obtained with recombinant RT enzyme that diffracted to
1
4
2
.6–2.8 Å on beamlines at the Brookhaven NSLS or Argonne APS.
For 7g (Fig. 1), the indolizine ring is positioned in the NNRTI bind-
ing site as expected from the earlier studies to make aryl-aryl
interactions with Tyr181, Tyr188, and Trp229. The plane of the
indolizine ring is perpendicular to the anilinyltriazine fragment
and there is a characteristic hydrogen bond between the anilinyl
1
0,11,13,15
NH and the backbone carbonyl group of Lys 101 (2.59 Å).
29 lg/mL, are well within the normal range observed for oral
In this structure, Tyr181 and Tyr188 are in the ‘up–up’ conforma-
tion, that is, typical for anilinylazines, and Glu138 and Lys101 are
forming a salt-bridge. A rendering of the crystal structure of 8f
with HIV-1 RT, which illustrates the orientation of the substituted
naphthalene ring system and the cluster of aromatic residues, is
provided in Figure 2.
9
drugs. The corresponding pyrimidines, 7d and 8c, are 2-4 fold less
soluble than the triazines. The pyrimidines are also somewhat
more cytotoxic than the triazines (lower CC50 values). The pro-
15
foundly low solubility of rilpivirine (0.02 lg/mL) is striking. The
7
much higher solubility of the indolizines was expected, but the
similarly higher solubility of the naphthalene analogues is surpris-
Turning to the data in Table 2, the parent indolizinyl pyrimidine
8
ing; it must reflect subtleties in crystal packing.
7
a is a modest inhibitor of the wild-type virus (200 nM) and is not
active towards the two variants. Progressive methylation at the
- and 8-positions (7b–7d) dramatically enhances the potency.
In summary, exploration of alternatives for the cyanovinyl
group in catechol diethers like 3, led to indolizine and naphthalene
6
7,8
containing alternatives including 4 and 5. Attempts at improving
1
0
the solubility of our own anilinylazines as well as rilpivirine led
1
1
to 6. Merger of these series has now brought us to 7 and 8. The
Figure 1. Rendering from the crystal structure of 7g bound to WT HIV-RT. Some
residues are omitted for clarity. Coordinates have been deposited in the PDB as
structure 5C24.
Figure 2. Rendering from the 5C25 crystal structure of 8f bound to WT HIV-RT.
Some residues are omitted for clarity.
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W.-G. Lee et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
naphthyl azines 8c and 8f are particularly notable; they have
sub-10 nM potency towards wild-type HIV-1 and viral variants
containing the clinically important Y181C and K103N/Y181C
mutations, greater activity than efavirenz particularly towards
the K103N-bearing variant, normal cytotoxicity, and solubility,
that is, ca. 1000-fold greater than for rilpivirine.
6. Bollini, M.; Domaoal, R. A.; Thakur, V. V.; Gallardo-Macias, R.; Spasov, K. A.;
Anderson, K. S.; Jorgensen, W. L. J. Med. Chem. 2011, 54, 8582.
7.
Lee, W.-G.; Gallardo-Macias, R.; Frey, K. M.; Spasov, K. A.; Bollini, M.; Anderson,
K. S.; Jorgensen, W. L. J. Am. Chem. Soc. 2013, 135, 16705.
8. Lee, W.-G.; Frey, K. M.; Gallardo-Macias, R.; Spasov, K. A.; Bollini, M.; Anderson,
K. S.; Jorgensen, W. L. ACS Med. Chem. Lett. 2014, 5, 1259.
9. Jorgensen, W. L.; Duffy, E. M. Adv. Drug Deliv. Rev. 2002, 54, 355.
10. 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,
5
209.
Acknowledgements
1
1. Bollini, M.; Cisneros, J. A.; Spasov, K. A.; Anderson, K. S.; Jorgensen, W. L. Bioorg.
Med. Chem. Lett. 2013, 23, 5213.
Gratitude is expressed to the National Institutes of Health
AI27690, AI44616, GM32136, GM49551) for research support
12. Baka, E.; Comer, J. E. A.; Takács-Novák, K. J. Pharm. Biomed. Anal. 2008, 46, 335.
13. Frey, K. M.; Puleo, D. E.; Spasov, K. A.; Bollini, M.; Jorgensen, W. L.; Anderson, K.
S. J. Med. Chem. 2015, 58, 2737.
(
and fellowship support for K. M. F. (AI104334). Receipt of reagents
through the NIH AIDS Research and Reference Reagent Program,
Division of AIDS, NIAID, NIH is also greatly appreciated. For beam-
line access, we thank the National Synchrotron Light Source and
the Northeastern Collaborative Access Team, which is funded by
the NIH (P41 GM103403). This research used resources of the
Advanced Photon Source, a U.S. Department of Energy facility at
Argonne National Laboratory under Contract DE-AC02-
1
4. Recombinant RT52A enzyme was expressed, purified, and co-crystallized with
0.5
HEPES (pH 7.0), 100 mM ammonium sulfate, 15 mM magnesium sulfate, and
mM spermine-HCl using hanging drop vaporization. Crystals were then
lM rilpivirine in an optimized condition of 16–20% v/v PEG 8000, 50 mM
5
transferred to a stabilizing solution containing 22% PEG 8000 and compounds
7g and 8f were soaked overnight (final concentration of 0.5 mM). Soaked
crystals were then flash cooled in liquid nitrogen in a stabilizing solution
containing 27% ethylene glycol. Diffraction data for the RT:7g complex was
collected at NSLS on beam line X29A; data for RT:8f was collected at APS on
beam line 24-ID-E through NE-CAT. Diffraction data for the RT:7g complex was
processed using HKL2000; diffraction data for the RT:8f complex was
processed using XDS. Phases were obtained using difference Fourier methods
for the RT:7g complex or molecular replacement with Phaser for RT:8f.
Structures were refined using Phenix until acceptable geometry and
refinement statistics were achieved. Atomic coordinates and structure factors
are deposited in the PDB with codes 5C24 (RT:7g) and 5C25 (RT:8f).
0
6CH11357.
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Please cite this article in press as: Lee, W.-G.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.06.074