Discovery and optimization of pyridazinone non-nucleoside inhibitors of HIV-1 reverse transcriptase

Article abstract of DOI:10.1016/j.bmcl.2008.06.072

A series of benzyl pyridazinones were evaluated as HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). Several members of this series showed good activity against the wild-type virus and NNRTI-resistant viruses. The binding of inhibitor 5a to HIV-RT was analyzed by surface plasmon resonance spectroscopy. Pharmacokinetic studies of 5a in rat and dog demonstrated that this compound has good oral bioavailability in animal species. The crystal structure of a complex between HIV-RT and inhibitor 4c is also described.

Full text of DOI:10.1016/j.bmcl.2008.06.072

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4352–4354
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of pyridazinone non-nucleoside inhibitors
of HIV-1 reverse transcriptase
a
a
a
a
a
Zachary K. Sweeney a, , James P. Dunn , Yu Li , Gabrielle Heilek , Pete Dunten , Todd R. Elworthy ,
Xiaochun Han a, Seth F. Harris a, Donald R. Hirschfeld a, J. Heather Hogg a, Walter Huber b,
Ann C. Kaiser a, Denis J. Kertesz a, Woongki Kim a, Taraneh Mirzadegan a, Michael G. Roepel a,
Y. David Saito a, Tania M. P. C. Silva a, Steven Swallow a, Jahari L. Tracy a, Armando Villasenor a,
Harit Vora a, Amy S. Zhou a, Klaus Klumpp a
*
a Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
b F. Hoffmann-La Roche Ltd., Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of benzyl pyridazinones were evaluated as HIV-1 non-nucleoside reverse transcriptase inhibitors
(NNRTIs). Several members of this series showed good activity against the wild-type virus and NNRTI-
resistant viruses. The binding of inhibitor 5a to HIV-RT was analyzed by surface plasmon resonance spec-
troscopy. Pharmacokinetic studies of 5a in rat and dog demonstrated that this compound has good oral
bioavailability in animal species. The crystal structure of a complex between HIV-RT and inhibitor 4c is
also described.
Received 1 April 2008
Revised 20 June 2008
Accepted 24 June 2008
Available online 28 June 2008
Keywords:
Human immunodeficiency virus
Ó 2008 Elsevier Ltd. All rights reserved.
HIV
NNRTI
Non-nucleoside
Reverse transcriptase
Chemotherapy for the treatment of HIV infection often includes
administration of an allosteric inhibitor of the viral reverse trans-
criptase (HIV-RT). These medicines, known collectively as non-
nucleoside reverse transcriptase inhibitors (NNRTIs), bind to the
transcriptase enzyme in a hydrophobic cavity adjacent to the poly-
merase catalytic site.1,2 Association of an NNRTI with the reverse
transcriptase is thought to inhibit chain elongation by altering
the motions of the protein residues that interact with the nucleic
acid chain.3 Some NNRTIs have also been found to influence dimer-
ization of the HIV-RT subunits,4 HIV-RT RNase activity,5–7 and the
effectiveness of chain termination by deoxynucleoside drugs.8
Currently marketed NNRTIs include efavirenz, nevirapine,
delavirdine, and etravirine. Efavirenz and nevirapine have good
pharmacokinetic profiles and effectively inhibit replication of the
wild-type virus,9 but they are less effective against several com-
monly observed mutant viruses.10 Etravirine shows improved
potency against many NNRTI-resistant viruses,11,12 but must be
administered twice daily and is approved for use only in patients
infected with HIV-1 strains resistant to an NNRTI and other antiret-
roviral agents. There is therefore a need for new NNRTIs that are
active against virus strains resistant to current NNRTIs and have
pharmacokinetic properties suitable for once-daily dosing.13
Our search for novel NNRTIs commenced with a high-through-
put screen of the Roche compound library. From this screen, pyrid-
azinone 1 was discovered to inhibit the activity of wild-type HIV-1
reverse transcriptase in an isolated enzyme assay (Table 1).14 This
compound was also found to be active against HIV replication in
MT4 cells infected with wild-type HIV-1.15 Subsequent studies
Table 1
Potency of lead compound 1a
O
N
NH
Cl
O
O
1
Assay
WT
K103N
Y181C
IC50
EC50
0.40
0.08
0.26
0.07
0.36
0.21
* Corresponding author. Tel.: +1 650 855 6376; fax: +1 650 852 1311.
E-mail address: zachary.sweeney@roche.com (Z.K. Sweeney).
a
IC50 and EC50 in lM.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.072

Z. K. Sweeney et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4352–4354
4353
Table 2
Table 4
SAR of phenyl-benzofuran linkera
Improvements in Potencya
Cl
X
N
R3
NH
O
N
NH
O
O
Cl
O
R4
2
Compound
X
IC50
CYP3A4 IC50
Compound
R3
R4
WT
K103N
Y181C
2a
2b
2c
CO
CH2
O
8.2
3.9
1.0
0.6
0.18
0.41
4a
4b
4c
4d
H
H
F
H
Me
H
0.52
0.36
0.10
0.02
3.35
0.92
0.43
0.05
5.49
2.13
0.5
F
Me
0.21
a
IC50 (lM) versus the wild-type enzyme and for inhibition of recombinant
a
CYP3A4 using BFC as a fluorogenic substrate.
WT, K103N, and Y181C refer to IC50 values in lM versus the indicated enzyme.
Table 3
SAR of central phenyl ring substitutiona
Cl
O
N
NH
O
R1
R2
3
Compound
R1
R2
IC50
3a
3b
3c
3d
H
H
Me
Cl
H
Me
H
21
14
0.64
0.10
H
a
IC50 (lM) versus the wild type enzyme.
indicated that 1 inhibited the replication of the clinically relevant
K103N and Y181C mutant viruses,16 and this compound was cho-
sen as a lead molecule for our discovery program.
Initial modifications focused on the influence of the linker be-
tween the terminal phenyl ring and the benzofuran portion of
the inhibitors. As shown in Table 2, diaryl ether compounds proved
to be somewhat more potent than their benzophenone analogs.
However, the benzofuran lead compounds were found to be uni-
formly strong inhibitors of CYP3A4. A series of diaryl ethers that
did not contain the benzofuran moiety were therefore prepared
in an effort to discover NNRTIs that would not inhibit cytochrome
p450 enzymes (Table 3). Excision of the furan hetereocycle (3a)
resulted in a severe loss in activity, but analogs with R1 = Cl or
Me (3c–d) maintained good potency. These compounds were also
relatively weak inhibitors of CYP3A4 (e.g., 3d, CYP3A4
Figure 1. Inhibitor 4c bound to wild-type HIV-RT. Only key protein residues are
shown.
pocket defined by the side chains of V106 and V179. The terminal
phenyl ring engages in hydrophobic interactions with Y188, Y181,
and W229. The methyl group of 4d appears to interact with P225,
which is located on a flexible loop region.
In the course of our optimization program, it became apparent
that appropriate substitution of the terminal phenyl ring improved
the activity of the pyridazinone NNRTIs in the cellular assay. These
modifications also strongly affected the potency of the inhibitors
when they were tested against NNRTI-resistant mutant viruses.
Analogs containing a 3,5-disubstituted aromatic ring had low
nanomolar activity against the wild-type virus and a number of
resistant mutant viruses (Table 5). These compounds maintained
their potency in tests conducted in the presence of additional hu-
man serum.20
Inhibitor 5a was selected for further characterization. Surface
plasmon resonance experiments21 showed that binding of this
inhibitor to the wild-type reverse transcriptase and to the K103N
and Y181C mutant viruses was rapid and reversible. For the
wild-type enzyme, the dissociation constant (KD) was calculated
to be 8.4 Â 10À9 MÀ1 (kon = 1.0 Â 105 MÀ1 sÀ1, koff = 8.4 Â 10À4 sÀ1).
None of the major cytochrome P450 enzyme isoforms were
strongly inhibited by this compound (Table 6).22 NNRTI 5a was
well absorbed when dosed orally in rats (5 mg/kg), with a Cmax
IC50 = 4.0 lM).
As we had identified a series of compounds that did not strongly
interfere with CYP function, our attention turned to improving the
potency of the pyridazinone inhibitors. Substitution of the pyridaz-
inone ring revealed that addition of a methyl group in the 4-posi-
tion could improve activity against the mutant viruses (Table 4 4a
vs 4b). Fluorination of the central ring also improved potency (4c).
Compound 4d, which contains both fluorine and methyl substitu-
tions, was 25-fold more potent than precursor 4a in testing against
both the wild-type and mutant viruses.
Crystallographic studies of a complex of 4c with the wild-type
reverse transcriptase enzyme provided insight into the likely bind-
ing mode of this series of inhibitors (Fig. 1).17–19 A bidentate hydro-
gen-bonding interaction between the pyridazinone ring and the
K103 amide backbone anchors the compound to the NNRTI-bind-
ing pocket. The central ring of the inhibitor is positioned by the
bridging methylene group to be nearly perpendicular to the plane
of the pyridazinone ring, and the chlorine atom occupies a small
of 2.3 lM and an AUC of 22.4 lM h. Following intravenous

4354
Z. K. Sweeney et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4352–4354
Table 5
Antiviral activity of pyridazinones 5a–c
F
NC
O
N
NH
Cl
O
R
CH3
5
Compound
R5
WT
WT + serum
K103N
Y181C
G190A
K103N/L100I
K103N/Y181C
Efavirenz
—
F
OMe
CN
0.001
0.001
0.002
0.001
0.019
0.011
0.015
0.007
0.034
0.002
nt
0.002
0.005
0.009
0.005
0.006
0.001
0.001
0.001
>0.50
0.003
0.002
0.007
0.083
0.041
0.031
0.019
5a
5b
5c
0.002
WT, K103N, Y181C, G190A, K103N/L100I, and K103N/Y81C refer to EC50 values in lM versus the wild-type virus and the 103, 181, 190, 103 + 100, and 103 + 181 mutant
viruses, respectively. WT + serum refers to experiments performed in the presence of 40% human serum.
Table 6
availability and low clearance in animals, and do not inhibit major
CYP enzymes. Further optimization of these inhibitors is described
in the following letter.
CYP inhibition and pharmacokinetics for 5a P450 inhibition (lM)
3A4
24
1A2
>50
2D6
>50
2C9
8
2C19
21
Pharmacokinetics
Rat
Dog
Acknowledgments
Cmax
(
l/mL)
2.3
22.4
3.8
1.73
61
1.47
8.8
6.4
2.06
34
AUC (
l
M h/mL)
We thank Grace Lam, Witold Woroniecki, Jessica Brilliant, and
Andre Desrosier for their analytical expertise.
t1/2 (h)
Vdss
F%
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(3.8 h), and the apparent bioavailability was determined to be 61%.
Similar experiments in dogs showed that the compound had good
stability and bioavailability in this species as well (Table 6).
The synthesis of inhibitor 5a is outlined in Scheme 1.23 Treat-
ment of difluoronitrophenyl acetate with the potassium salt of 3-
cyano, 5-fluorophenol provided the diaryl ether in excellent yield.
Reduction of the nitro group with iron was followed by a Sandmey-
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sodium enolate of the aryl acetate to 3,6-dichloro-4-methylpyrid-
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2:1 mixture of regioisomers. Decarboxylation of the ester followed
by hydrolysis of the chloropyridazine in refluxing acetic acid gave
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14. Enzymatic assay: IC50 values were obtained from a scintillation counting assay
using poly-rA primer and tritiated dTTP.
15. Replication assay: EC50 values were obtained following incubation of
compound with infected MT4 cells in the presence of 10% fetal bovine
serum. The assay assesses the reduction in the cytopathic effect of HIV-1 on
MT4 cells in the presence of HIV-RT inhibitors. Each value represents the
average of at least two independent assays.
In summary, we have discovered a series of pyridazinones that
display excellent potency against a wild-type HIV-RT and several
NNRTI-resistant mutant viruses. These compounds have good bio-
16. Demeter, L. M.; Shafer, R. W.; Meehan, P. M.; Holden-Wiltse, J.; Fischl, M. A.;
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17. In addition to the marketed NNRTI delavirdine, there have been several recent
examples of NNRTIs that bind to the K103 residue. For a very recent closely
related series see: Tucker, T. J. et al Bioorg. Med. Chem. Lett. 2008, 18, 2959.
18. This type of linked triaryl motif can also be found in the aminopyrimidine
series of inhibitors that interact with the backbone of K101. See: Das, K.;
Bauman, J. D.; Clark, A. D., Jr.; Frenkel, Y. V.; Lewi, P. J.; Shatkin, A. J.; Hughes, S.
H.; Arnold, E. Proc Natl. Acad. Sci. U.S.A. 2008, 105, 1466.
F
OH
NC
O
a
CO2Et
O2N
NC
F
F
19. Atomic coordinates the structure of 4c bound to HIV-RT were deposited with
the RCSB Protein Data Bank (PDB) under the Access Code 3DI6.
20. For these tests, the replication assay was conducted with additional 40%
human serum.
21. Surface plasmon resonance experiments were performed using HIV-RT protein
immobilized to the CM5 sensor chip via an amide-coupling reaction. The
binding experiments were performed using Hepes buffer (10 mM Hepes (pH
8.0), 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20, 1% DMSO) as the running
buffer.
b, c
F
NC
O
d
CO2Et
5a
Cl
F
22. CYP inhibition experiments were determined using 7-benzyloxy-4-
(trifluoromethyl)-coumarin as a fluorogenic substrate.
Scheme 1. Reagents and conditions: (a) KOtBu, Ethyl (2, 3-difluoro-4-nitro-
phenyl)acetate, THF, 92%; (b) Fe, NH4Cl, EtOH, H2O, 93%; (c) tBuONO, CuCl2, CH3CN,
63%; (d) i—NaH, 3,6-dichloro-4-methylpyridazine, DMF; ii—LiOH, THF, H2O,
iii—NaOAc, HOAc, 30%.
23. For a more detailed description of the synthesis of this compound and other
pyridazinone inhibitors see: Dunn, J. P.; Dymock, B. W.; Mirzadegan, T.;
Sjogren, E. B.; Swallow, S.; Sweeney, Z. K. WO 2004/085406; Kertesz, D. J.;
Martin, M.; Palmer, W.S. US 2005/234236.