Non-benzimidazole containing inhibitors of respiratory syncytial virus

Article abstract of DOI:10.1016/j.bmcl.2012.11.062

Several non-benzimidazole containing inhibitors of respiratory syncytial virus are described. Core template modification, analysis of antiviral activity, physicochemistry and optimisation of properties led to the thiazole-imidazole 13, that showed a good potency and pharmacokinetic profile in the rat.

Full text of DOI:10.1016/j.bmcl.2012.11.062

Bioorganic & Medicinal Chemistry Letters xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Non-benzimidazole containing inhibitors of respiratory syncytial virus
David C. Pryde a, , Thien-Duc Tran a, Iain Gardner b, Helen Bright c, Paul Stupple a, Sebastien Galan a,
⇑
Liam Alsop a, Lesa Watson a, Donald S. Middleton a, Satish Dayal b, Michelle Platts a, Edward J. Murray c,
Tanya Parkinson c, Robert Webster b
a Worldwide Medicinal Chemistry, Pfizer Global R and D, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
b Pharmcokinetics, Dynamics and Metabolism, Pfizer Global R and D, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
c Antiinfectives Biology, Pfizer Global R and D, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Several non-benzimidazole containing inhibitors of respiratory syncytial virus are described. Core tem-
plate modification, analysis of antiviral activity, physicochemistry and optimisation of properties led to
the thiazole-imidazole 13, that showed a good potency and pharmacokinetic profile in the rat.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 10 September 2012
Revised 16 November 2012
Accepted 18 November 2012
Available online xxxx
Keywords:
RSV
Virus
Benzimidazole
Antiviral
Heterocycle
Human respiratory syncytial virus (RSV) is an important patho-
gen that affects the respiratory system.1 It is a negative sense RNA
virus of the Paramyxoviridae family responsible for a range of
respiratory tract diseases in people of all ages and geographical
locations.2 RSV infection is usually restricted to being an infection
of the upper respiratory tract that causes generally mild symptoms
but in the immunocompromised and other susceptible populations
such as newborn babies and the elderly, can progress to infect the
lower respiratory tract and lead to serious secondary infections
such as bronchiolitis and pneumonia.3 The first step in the infec-
tion of host cells by RSV involves fusion via the RSV F protein,
which initiates a series of conformational changes that ultimately
allow fusion of the viral and cellular membranes to take place.4
The literature describes a number of small molecule inhibitors
of the RSV F protein.5 Most small molecule F protein inhibitors,
for example compounds 1–3 ( Fig. 1),6 are based on a common
benzimidazole core template, and were generally identified from
viral replication assays and subsequently found to generate resis-
tance mutations mapping to the F protein. The most advanced of
these, 1, demonstrates excellent antiviral potency in a moderately
lipophilic structure, consistent with a high ligand efficiency (LE) of
0.38 and lipophilic efficiency (LipE) of 5.5.
compounds have been shown to be efficacious in rodent RSV infec-
tion models when administered up to 24 h post-infection.8,6a
While no small molecule F-protein inhibitors have progressed to
late stage clinical studies, there is a marketed humanised monoclo-
nal antibody (Palivizumab/Synagis) which targets the F protein and
is effective in reducing viral load and subsequent morbidity and
mortality associated with infection,9 validating this mechanistic
approach for RSV therapy.
In this manuscript, we describe structure–activity relationships
that allowed us to identify several novel, non-benzimidazoles
based on the structures contained in Figure 1 that retained good
levels of RSV antiviral activity and for some examples, an encour-
aging pharmacokinetic profile.
The substituted benzimidazole analogues described in several
papers and reviews by the Bristol-Myers Squibb group5 and exem-
plified by the alcohol 1 showed good antiviral potency. In in vitro
metabolism studies the compound was shown to be metabolized
in liver microsomes from mouse and rat (Clintapp 53 and 66 ll/
min/mg protein, respectively). In incubations with rat liver micro-
somes three mono-hydroxylated oxidative metabolites of 1 were
identified. In human liver microsomes turnover of 1 could not be
observed under standard assay conditions (Clintapp < 8
protein) but in human hepatocytes measurable clearance was ob-
served (Clintapp 11
l/min/106 cells) indicating that the compound
ll/min/mg
In some cases, direct binding to the F protein has also been
demonstrated using radiolabelled compounds.7 Several of these
l
was also potentially metabolized by non-P450 drug metabolizing
enzymes in human. In addition compound 1 had a short half-life
following intravenous dosing to the rat (T1/2 0.4 h). We were
⇑
Corresponding author. Tel.: +44 1304 643687.
E-mail address: David.Pryde@pfizer.com (D.C. Pryde).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.062
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2
D. C. Pryde et al. / Bioorg. Med. Chem. Lett. xxx (2012) xxx–xxx
N
a
b
N
NH
N
N
N
N
N
N
N
EtO
N
O
HO
O
Br
Rat pharmacokinetics
Clint 61mL/min/kg
14
AV IC50 13nM
O
15
16
LE 0.38, LipE 5.5
F 13%
1
LogD 1.7
H
N
Vdss 0.5L/kg
Cl
HLM <8 µL/min/mg
RRCK 14x10-6 cm/s T1/2 0.4h
N
O
c
d,e
N
OH
18
O
O
19
17
N
N
OMe
H
N
N
N
N
MeO2C
H
N
N
N
N
2
O
OH
NH
N
N
N
N
OH
N
N
3
N
N
f
Figure 1. Some literature RSV fusion inhibitors.
21
20
interested in exploring novel structural modifications that would
allow for enhanced antiviral potency and/or pharmacokinetic
improvements over 1. In particular, we aimed to improve lipophilic
efficiency (LipE)10 to maximise our chances of combining high
potency with a low LogD at which we could achieve appropriate
metabolic stability. We aimed to optimise substituents in the lipo-
philic regions of 1 (i.e., the benzo ring of the benzimidazole and the
cyclopropyl region of the imidazolone), by either changing the nat-
ure of the lipophilicity, or by adding a polar group, without com-
promising ligand efficiency (LE)11 or greatly increasing molecular
size.12
OH
OH
Scheme 2. Reagents and conditions: (a) (i) t-BuOK, THF, rt, 1 h; (ii) i-PrI, rt, 18 h;
(iii) LiAlH4, THF, 0 °C, 3 h, 76% (over 3 steps); (b) PBr3, toluene, 120 °C, 1 h, 98%; (c)
(i) Et3N, DCM, rt, 3 h; (ii) NH2NH2.H2O, EtOH, rt, 18 h, 61% (over 2 steps); (d) NaH,
DMF, rt, 18 h, 74% (mixture N1/N2); (e) LiAlH4, THF, 0 °C, 2 h, 82% (mixture N1/N2);
(f) DDQ, dioxane, 110 °C, 18 h, 10% (+30% of the N1 diastereoisomer).
expression of efflux transporters) and human liver microsomal sta-
bility measurement.
All compounds synthesised through the programme were pro-
filed for antiviral activity, lipophilicity, passive permeability
(through MDCK cells grown from clones selected to have low
Representative syntheses of the compounds contained within
this paper are shown in Schemes 1 and 2. For example, biaryl tar-
gets such as the imidazole–thiazole 13 were accessed through
building up the imidazole ring as described in Scheme 1.
4-Methoxy-3-nitro-pyridine 4 was subjected to displacement
with cyclopropylamine followed by nitro group reduction of 5
and cyclisation with 1,10-carbonyldiimidazole to give the imidazo-
lone derivative 6. 2-Acetyl-thiazole 7 was oxidised to the dihydr-
oxyketone 8, and then converted to the substituted imidazole 9
using benzyloxyacetaldehyde and ammonium hydroxide. 9 was
alkylated with the bromide 11 prepared from 1-bromo-butanol
10 to give the intermediate 12, which was then taken through to
the final target 13 by following the steps outlined in the schemes
above. The second scheme, Scheme 2, shows a method of synthes-
ising an indazole template analogue. Commercially available
indazole-3-carboxlic acid ethyl ester 14 was alkylated with isopro-
pyliodide and reduced to give the alcohol 1513 which was then
brominated to give 16. In parallel, N-morpholino-1-cyclohexene
17 was reacted with hydrazine and methyl-4-(chloroformyl)-buty-
rate 18 to give the tetrahydro-indazole 19.14 This was coupled to
the bromide 16 and the ester group reduced to give the alcohol
20. Treatment of 20 with DDQ then afforded the fully aromatic
indazole 21. It should be noted that the alkylation step produced
both N1 and N2 substituted indazoles, in favour of the undesired
N1 isomer, but that both isomers were readily separable by column
chromatography at the final step. Further details of the synthesis of
other examples from this paper are contained in the Supplemen-
tary data.
N
N
N
a
b
N
O
OMe
N
N
H
NO2
NO2
4
6
5
O
N
O
N
N
S
N
OBn
d
c
S
OH
NH
S
HO
8
9
7
O
e
Br
Br
N
f
OH
O
11
10
N
N
OBn
N
S
N
S
N
N
g,h,i
N
N
12
O
13
O
O
OH
Scheme 1. Reagents and conditions: (a) c-PrNH2, EtOH, 80 °C; (b) (i) H2, Pd(OH)2/C,
EtOH; (ii) CDI, THF, 0 °C to rt, 49% over 3 steps; (c) HBr (48% aq), DMSO, 60 °C, 48%;
(d) benzyloxyacetaldehyde, NH4OH, EtOH, rt, 25%; (e) tBuCOCl, TEA, DCM, 0 °C–rt,
75%. (f) Cs2CO3, DMF, rt, quant; (g) BBr3, DCM, ꢀ50 °C, 57%; (h) 6, DIAD, PPh3, THF,
0 °C–rt, 30%; (i) NaOH, H20/THF, rt, 64%.
Having established robust synthetic routes to access a variety of
target structures, our investigations started by expanding the
benzimidazolone substituent to incorporate larger groups, or those
featuring hydrogen bonding functionality. One of the first
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D. C. Pryde et al. / Bioorg. Med. Chem. Lett. xxx (2012) xxx–xxx
3
O
O
group to give the indazolone 29 was well-tolerated, while reduc-
tion of the indazole to the semi-saturated pyrazole bicycle 30 lost
potency.
N
N
N
OMe
OMe
N
N
Compounds 32 and 33 finally confirmed that the connectivity of
the substituents on the core template was quite specific; the
modest structural changes in these analogues were not tolerated.
Nonetheless, we had quickly established some novel chemistry
starting points, with compounds such as 27 in particular showing
good antiviral activity. We remained concerned about the high
lipophilicity of 27 and its very modest LipE and the next set of tar-
gets sought to address this.
N
N
N
N
N
N
O
O
22
LogD 3.4
AV IC50 0.5nM
23
LogD -0.5
AV IC50 55nM
LE 0.36, LipE 5.9
RRCK 36x10-6cm/s
HLM 44µL/min/mg
LE 0.30, LipE 7.7
RRCK 1x10-6cm/s
HLM 15µL/min/mg
OH
OH
We next returned to analogues based on the imidazole 23.Sim-
ple structural changes to the benzimidazole core generally met
with mixed results. Reducing the benzimidazole to the partially
saturated analogue 34 (Fig. 4) gave a threefold increase in activity
with an accompanying small increase in lipophilicity and an
increased metabolic liability. Paring back the benzimidazole ring
Figure 2. Early substituted imidazolone hits.
O
N
O
N
OMe
to
a dichloro-imidazole 35 was some eightfold less potent,
N
N
N
although ligand binding efficiency was maintained and physico-
chemical properties were now much more drug-like than the
starting 23. Smaller imidazole substituents, or the introduction of
polar moieties as seen in the methyl ether 36 were generally
poorly tolerated, consistent with the imidazole group binding in
a lipophilic environment.
N
N
25
N
AV IC50 >20000nM
24
AV IC50 >14100nM
LogD 1.5
RRCK 2x10-6cm/s
HLM 11 µL/min/mg
LogD 2.0
RRCK 6x10-6cm/s
HLM 110 µL/min/mg
OH
OH
Having established that a non-benzimidazole core template was
viable, and single heterocycles were suitable scaffolds for new RSV
inhibitors, imidazole SAR was developed further. Compounds that
were of intermediate lipophilicity between 35 and 36 were pro-
posed, for example the ester 37 (Fig. 5) and the pyridyl-imidazole
38. This latter compound in particular combined good potency and
reasonable permeability in a more polar structure with excellent
stability in human liver microsomes and hepatocytes (Clintapp
N
N
N
N
<8 ll/min/mg and 2.2 l
l/min/106 cells, respectively). Despite the
improved metabolic stability in human in vitro systems this com-
pound was still metabolized relatively rapidly by rat liver micro-
21
AV IC50 219nM
LogD 4.4
LE 0.34, LipE 2.3
somes (Clintapp 44 ll/min/mg) and in an in vivo pharmacokinetic
study in the rat showed high plasma clearance (Cl = 95 ml/min/
kg) and a comparable half-life to 1 (T1/2 = 0.4 h).
OH
Figure 3. Truncated imidazolone analogues.
Nevertheless, encouraged by the potency and physicochemistry
of the pyridyl analogue 38, we embarked on an SAR investigation
around this lead to improve its pharmacokinetic profile. Analogues
of the free hydroxyl group of 38 were prepared in an attempt to
improve the permeability of the series, and are presented in Table
2. Several heterocycles were inserted in this position, with some
subtle SAR observed. While the oxazole 42 lost some 10-fold
potency, the isoxazole 39 was some twofold more active, and the
isomeric isoxazole 41 was some fourfold more potent. The
methyl-pyridyl analogue 40 was of intermediate potency, IC50
96 nM.
Ultimately, no compounds from this effort showed significant
improvements in the combined properties of permeability in the
MDCK model, potency and stability in human liver microsomes
and no further compounds were made in this avenue of
investigation.
We next turned our attention to the pyridyl ring as shown in
Table 3. Substituted 5-membered heterocycles, for example, the
methyl substituted pyrazole 43, imidazole 46 and the thiadiazole
48 were all weak in the antiviral assay. Similar 6-membered het-
erocycles to the starting pyridine showed comparable levels of
activity, with the pyrimidine 44 and the 2-pyridyl 45 having an
IC50 of some 230 nM. Some of the most interesting compounds
came from making simple 5-membered unsubstituted heterocyclic
substituents. The oxazole 47 had an IC50 of 95 nM, while the thia-
zole 13 had an IC50 of 48 nM, some fivefold improvement over the
starting pyridine, which took the ligand efficiency of this com-
pound to 0.35 and the LipE to over 5.
compounds synthesised was the piperidine-carbamate 22 (Fig. 2)
that displayed a significant jump in antiviral activity, albeit in a
more lipophilic template which introduced metabolic liability. By
then trimming back the benzimidazole ring to a simple dimethyl
imidazole 23, a much more polar compound was accessed that
showed excellent lipoidal lipophilic binding efficiency, and left
significant headroom for adjusting polarity to design back in mem-
brane permeability.
By retaining the benzimidazole ring of 22, but trimming back
the benzimidazolone heterocycle, the pyrazole 24 (Fig. 3) was also
obtained, which lost all potency. The truncated indazole piperidine
25 was also inactive in the antiviral assay. Taking the inactive 25 as
a starting point and building back in larger heterocycle substitu-
ents provided access to moderately potent inhibitors exemplified
by the bis-indazole 21.
Encouraged by the moderate levels of potency in this com-
pound, we explored the central indazole ring to assess how much
more broadly tolerated different bicyclic heterocycles might be,
and how physicochemistry and ADME properties were affected.
These data are shown in Table 1. Simple insertion of an oxygen
atom into the side chain of 21 gave the ether 26 of some fivefold
weaker potency than 21. Subsequent lengthening of the side chain
of 26 by a single methylene group to 27 provided a significant
increase in potency, while the truncated side chain in 28 was lar-
gely equipotent with the starting 21. An amide linking group in
the side chain of 31 was significantly weaker. Inserting an oxo
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D. C. Pryde et al. / Bioorg. Med. Chem. Lett. xxx (2012) xxx–xxx
Table 1
Indazole analogues of compound 21
N
N
Compd
Structure
AV IC50 (nM)a
LE (LipE)
0.30 (2.1)
MWt.
364
LogDb
HLMc
246
(ll/min/mg)
MDCK (Papp ꢁ 10ꢀ6 cm/s)d
N
N
N
O
26
992
3.5
16
OH
N
O
27
65
0.35 (3.3)
378
3.9
ND
ND
HO
N
N
28
277
0.34 (2.7)
348
3.8
78
14
OH
O
N
N
N
N
29
169
0.33 (3.9)
378
2.9
462
32
OH
N
30
409
0.30 (2.2)
367
3.6
>320
12
OH
N
O
HN
31
2930
0.26 (3.1)
391
2.4
ND
ND
HO
N
N
32
22700
0.23 (1.0)
362
4.4
242
8
OH
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D. C. Pryde et al. / Bioorg. Med. Chem. Lett. xxx (2012) xxx–xxx
5
Table 1 (continued)
Compd
Structure
AV IC50 (nM)a
LE (LipE)
0.26 (1.0)
MWt.
348
LogDb
HLMc
(l
l/min/mg)
MDCK (Papp ꢁ 10ꢀ6 cm/s)d
HO
33
13300
3.9
58
26
N
N
a
Values are means of at least two experiments. For details of the assay used, see Ref. 15.
LogD measured at pH 7.4 in octanol/neutral buffer.
b
c
Details of the HLM assay used have been published previously.16
d
Methodological details for the MDCK permeability assay have been published previously.17 ND = not determined.
Table 2
Analogues of the hydroxyalkyl side chain of 38
N
N
O
N
Cl
Cl
N
N
N
N
N
N
N
N
O
O
N
N
N
N
N
34
35
AV IC50 4nM
AV IC50 109nM
LogD 1.8
LogD 2.0
LE 0.41, LipE 6.6
RRCK 4x10-6cm/s
HLM 65 µL/min/mg
LE 0.36, LipE 5.7
RRCK 13x10-6cm/s
HLM <8 µL/min/mg
Ar
OH
OH
Compd Ar
AV
LE
MWt. LogDb HLMc
MDCK
IC50
(LipE)
(ll/min/ (Papp ꢁ
(nM)a
mg)
10ꢀ6 cm/
s)d
N
OMe
O
36
N
AV IC50 6090nM
N
N
N
LogD 0.2
0.30
(4.9)
LE 0.26, LipE 5.9
RRCK 1x10-6cm/s
HLM 9 µL/min/mg
39
40
139
96
427
437
1.4
1.5
17
4
2
N
O
OH
0.29
(4.4)
32
Figure 4. Imidazole core analogues.
N
0.31
(5.4)
N
41
42
41
427
413
1.4
0.9
ND
10
3
N
N
O
N
N
MeO2C
N
N
N
N
N
N
N
O
O
0.25
(3.6)
O
2230
ND
38
37
N
AV IC50 265nM
AV IC50 12400nM
LogD 1.2
a
LogD 0.8
Values are means of at least two experiments. For details of the assay used, see
Ref. 15.
LE 0.31, LipE 5.2
RRCK 4x10-6cm/s
HLM <8 µL/min/mg
LE 0.05, LipE 2.8
RRCK 2x10-6cm/s
HLM 9 µL/min/mg
b
LogD measured at pH 7.4 in octanol/neutral buffer.
OH
OH
Details of the HLM assay used have been published previously.16
c
d
Methodological details for the MDCK permeability assay have been published
Figure 5. Substituted imidazole analogues.
previously.17 ND = not determined.
The metabolic stability of compounds 38, 44, 45 and 13 were
further investigated in human hepatocytes. All compounds had
l/min/million cells) in this
in vitro system suggesting that the free hydroxyl group was not
extensively glucuronidated.
and MDCK permeability against cLogP is shown in Figure 6 and
illustrates that for a cLogP of below 2 permeability is consistently
low and metabolic stability is greatest, as expected, but that above
2 being able to retain good microsomal stability and permeability
is much less predictable. It is also clear from the plot that there
were no examples from within the series that showed high micro-
somal stability above cLogP of 2. Our attention therefore focussed
on the more polar examples from the series, and based on
compound potency, moderate permeability and low metabolic rate
low rates of metabolism (Clintapp 62.2
l
Permeability in the MDCK assay17 was uniformly low to moder-
ate in this set of relatively polar structures. The permeability of the
compounds contained in Table 1 were notably higher, but also
notably more lipophilic and less metabolically stable. It became
apparent at this stage of our investigations that balancing perme-
ability and stability in a potent structure would be a significant
challenge in the series. Plotting human liver microsomal stability
in rat liver microsomes in vitro (Clintapp 21.1 ll/min/mg protein)
compound 13 was selected for an in vivo pharmacokinetic study
in the rat (Fig. 7).
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D. C. Pryde et al. / Bioorg. Med. Chem. Lett. xxx (2012) xxx–xxx
Table 3
Imidazole heterocycle analogues
N
N
Ar
N
N
N
O
OH
Compd
Ar
AVa IC50 (nM)
265
LE (LipE)
0.31 (5.2)
MWt.
390
LogDb
HLMc
<8
(l
L/min/mg)
MDCK (Papp ꢁ 10ꢀ6 cm/s)d
Hheps (
l
L/min/106 cells
N
38
1.2
4
<2.2
N
N
43
44
>20000
235
<0.22 (<3.9)
0.30 (5.3)
393
405
0.9
0.6
<8
<8
2
1
ND
N
<2.2
N
N
N
N
45
233
0.30 (5.0)
404
1.4
<8
5
<2.2
46
13
47
10400
58
0.23 (4.7)
0.35 (5.3)
0.34 (5.9)
393
396
380
0.8
1.3
0.6
<8
<8
<8
1
4
1
ND
N
<2.2
ND
S
N
95
O
N
N
48
5110
0.25 (4.1)
411
0.9
<8
1
ND
S
a
b
c
Values are means of at least two experiments. For details of the assay used, see Ref. 15.
LogD measured at pH 7.4 in octanol/neutral buffer.
Details of the HLM assay used have been published previously.16
Methodological details for the MDCK permeability assay have been published previously.17 ND = not determined.
d
N
N
S
N
N
Rat pharmacokinetics
Clint 48mL/min/kg
F 34%
Vdss 3.5L/kg
N
AV IC50 58nM
N
O
LE 0.35, LipE 5.3
LogD 1.3
HLM <8µL/min/mg
RRCK 4x10-6cm/s
T1/2 1.5h
13 AV IC50 58nM
OH
Figure 7. Selected in vitro and in vivo data for compound 13.
oral dosing in the rat the compound had a bioavailability of 34%,
showing good absorption (>60%) despite the relatively modest
permeability observed in in vitro cell-based experiments. Good
exposure was also seen following oral dosing in the mouse. While
no data was generated with any of the compounds described in
this study to confirm their pharmacological target, it is assumed
that they are acting upon the F protein by analogy to the com-
pounds depicted in Figure 1. Further experiments to evaluate the
in vivo efficacy of 13 against RSV will be reported in due course.
Figure 6. HLM stability (left y-axis) and MDCK permeability (right y-axis) plotted
against cLogP (x-axis) for the compounds described in this Letter.
The compound had a comparable clearance (48 ml/min/kg) but
a larger Vdss (3.5 L/kg) and longer half-life (1.5 h) than compound
1. About 16% of the dose of compound 13 was recovered un-
changed in the urine of the rats showing that in vivo it is cleared
by a mixture of metabolic and non-metabolic pathways. Following
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D. C. Pryde et al. / Bioorg. Med. Chem. Lett. xxx (2012) xxx–xxx
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R.; Bonfanti, J.-F.; Bruinzeel, W.; Cummings, M. D.; Van Vlijmen, H.; Andries, K.
PNAS 2010, 107, 308.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.11.
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glutamine, 0.1 mM non-essential amino acids, 1 U/ml penicillin and 0.1 mg/ml
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ll of cell/virus suspension was added to each well of
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Please cite this article in press as: Pryde, D. C.; et al. Bioorg. Med. Chem. Lett. (2012), http://dx.doi.org/10.1016/j.bmcl.2012.11.062