Pyrazole NNRTIs 3: Optimisation of physicochemical properties

Article abstract of DOI:10.1016/j.bmcl.2009.08.043

Our efforts to reduce overall lipophilicity and increase ligand-lipophilicity efficiency (LLE) by modification of the 3- and 5-substituents of pyrazole 1, a novel non-nucleoside HIV reverse transcriptase inhibitor (NNRTI) prototype were unsuccessful. In c

Full text of DOI:10.1016/j.bmcl.2009.08.043

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5603–5606
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrazole NNRTIs 3: Optimisation of physicochemical properties
b
a
a
a
b
Charles E. Mowbray a, , Romuald Corbau , Michael Hawes , Lyn H. Jones , James E. Mills , Manos Perros ,
*
Matthew D. Selby a, Paul A. Stupple a, Rob Webster c, Anthony Wood a
a Department of Discovery Chemistry, Pfizer Global Research and Development, Sandwich, Kent CT 13 9NJ, UK
b Department of Discovery Biology, Pfizer Global Research and Development, Sandwich, Kent CT 13 9NJ, UK
c Department of Pharmacokinetics, Dynamics and Metabolism Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent CT 13 9NJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Our efforts to reduce overall lipophilicity and increase ligand-lipophilicity efficiency (LLE) by modifica-
tion of the 3- and 5-substituents of pyrazole 1, a novel non-nucleoside HIV reverse transcriptase inhibitor
(NNRTI) prototype were unsuccessful. In contrast replacement of the substituted benzyl group with cor-
responding phenylthio or phenoxy groups resulted in marked improvements in potency, ligand efficiency
(LE) and LLE.
Received 25 June 2009
Revised 7 August 2009
Accepted 8 August 2009
Available online 14 August 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
HIV
Reverse transcriptase
NNRTI
In our previous papers we described the initial design and sub-
sequent optimisation of a new series of pyrazole NNRTIs.1,2 The
early lead compound in this series alcohol 1 was an inhibitor of
wild type (WT) and drug resistant mutant HIV reverse transcrip-
tase (RT) but was relatively lipophilic (clog P 4.3) and consequently
suffered rapid metabolism in human liver microsomes. Our efforts
to date had not managed to improve potency and increase meta-
bolic stability and we recognized the need to improve ligand-lipo-
philicity efficiency (LLE).3 Following our initial survey of the SAR in
the pyrazole series we made a more in depth study of the 3- and 5-
substituents on the pyrazole core (Table 1). The majority of these
compounds were designed to be less lipophilic than the leads 1
and 2 and we anticipated that they would have improved meta-
bolic stability. However all these compounds were significantly
weaker inhibitors of HIV RT and in many cases were essentially
inactive.
We were frustrated by our lack of success in improving potency and
physical properties through modification of the substituents around
the periphery of the benzylpyrazole and so resolved to make more
synthetically challenging5 modifications to the core template itself.
Whilst developing this more challenging chemistry we were able to
probe whether we could disconnect the relationship between lipo-
philicity and potency by preparing some simple 3,5-dimethyl pyra-
zoles shown in Table 2.
We were very excited to discover that phenylthiopyrazole 25
was about five times more potent as an HIV RT inhibitor than the
corresponding benzylpyrazole 24. This improved activity against
the isolated enzyme also resulted in improved antiviral activity.
We recognized that this improvement in potency and ligand effi-
ciency came at the cost of an increase in lipophilicity and so did
not offer an improvement in LLE. Oxidation of the new linking sul-
fur atom to give the corresponding sulfoxide 26 or sulfone 27 pro-
duced significant losses in potency consistent with introducing
polar substituents in a region of the enzyme which had so far fa-
voured lipophilic groups. The phenoxypyrazole 28 proved to be
the most interesting member of this set of structurally similar
compounds being slightly more potent as an HIV RT inhibitor than
the parent benzylpyrazole 24 and also lacking the metabolically
vulnerable, doubly benzylic carbon atom. Removal of this site for
possible oxidative metabolism resulted in increased stability in hu-
man liver microsomes (compare 24 to 28) as shown in Table 5.
Although the ligand efficiency (LE) and LLE of this ether 28 are
not significantly different from those of the parent compound 24
we felt that the observed improved potency and metabolic stability
coupled with the opportunity to explore a wider range of phenyl
group substitution patterns by employing parallel chemistry
Cl
Cl
N
OH
N
1
* Corresponding author. Tel.: +44 1304 648427; fax: +44 1304 651821.
E-mail address: Charles.Mowbray@Pfizer.com (C.E. Mowbray).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.043

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C. E. Mowbray et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5603–5606
Table 1
Table 3
Variation of pyrazole 3- and 5-substituents
Further variation of biaryl linker
Cl
Cl
Cl
Cl
R2
X
N
N
N
N
OH
OH
R1
R2
a
c
Compound
X
clog P
IC50
(l
M)
LE
LLE
AV50 (nM)
Compound
R1
clog P
IC50 (lM)
a
1
CH2
CO
CHOMe
CHOH
S
4.3
3.9
3.6
2.8
5.0
4.7
0.66
2.0
3.0
>100
0.065
0.12
0.41
0.36
0.34
—
0.48
0.46
1.86
1.77
1.89
—
2.17
2.23
14
94
341
—
0.63
3.3
Efavirenz
1
2
3
4
5
6
7
—
—
Et
3.7
4.3
4.2
3.5
3.2
3.8
3.7
3.7
2.8
2.2
4.9
4.9
2.2
1.6
2.7
3.6
2.2
2.1
3.4
2.7
4.0
2.1
2.6
1.9
0.0084
0.66
1.9
36
6.3
29
30b
31b
32
33
Et
Pri
Me
Me
OMe
OEt
Me
CO2Et
Me
Me
Me
Ph
Me
Me
NH2
NHCH2CH2OMe
NH2CONH2
NHCOMe
NMe2
Me
Me
CO2Et
Me
CH2OCH3
CO2H
Ph
Me
NH2
CONH2
Et
Et
Et
Et
Et
Et
Et
Et
Et
O
54
27
a
Inhibition of wild type HIV RT with a poly(rA) ꢀ300 template, (dT) 16 primer
and dTTP as substrate.3,6
>10
>10
>100
>10
>10
>10
>10
>50
>20
>100
>100
77
>100
9.8
>100
>100
55
b
8
9
Racemic.
c
Antiviral activity in cell culture in SupT 1 cells infected with the RF strain of HIV.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
The metabolic stability of ether 33 was evaluated and compared
to the parent benzylpyrazole 1 (Table 5). The ether 33 demon-
strated improved resistance to oxidative metabolism when as-
sessed in human liver microsomes. However in human
hepatocytes ether 33 was rapidly metabolized by glucuronidation
of the primary alcohol as previously demonstrated for members
of the benzyl pyrazole series.1 As our earlier efforts to modify or re-
place the alcohol side chain in lead 1 had generally resulted in a
loss of activity2 we believed that the best design strategy to reduce
the rate of glucuronidation of lead ether 33 was to reduce the over-
all lipophilicity of the compound.7 We recognized the dominant
contribution of the 3,5-dichlorophenyl group to the overall lipo-
philicity of ether 33 and resolved to seek alternative, less lipophilic
replacements. To enable this exploration of SAR we developed a
synthetic route amenable to parallel chemistry6 shown in Scheme
1 and using readily available phenols we subsequently prepared
compounds 34–66 bearing a wide variety of substitution patterns
with reduced lipophilicities shown in Table 4.
NHCO2Et
NHCOEt
NME2
NHSO2Me
NHCO-3-pyridyl
NHCOCH2OMe
Et
a
Inhibition of wild type HIV RT with a poly(rA) ꢀ300 template, (dT) 16 primer
and dTTP as substrate.4
Table 2
Initial variation of biaryl linker
Cl
Cl
A few of the substitution patterns tested in this study such as 3-
Cl, 2,5-diF, 2,6-diF, 3,5-diF and 3,5-diMe retained submicromolar
activity against HIV RT but were weaker inhibitors of HIV RT than
the parent 33 and still had clog P values in the range 3.2–4.3. In
contrast the 3-CN compound 52 was only a threefold weaker inhib-
itor of HIV RT and crucially was two log units less lipophilic than
the parent 33. This marked improvement in lipophilic efficiency
can also be seen in the plot of Àlog(RT IC50) against clog P for the
pyrazole series (Fig. 1) and the jump in LLE from 2.23 for parent
compound 33 to 3.77 for the new 3-cyanophenoxypyrazole 52.
X
N
N
OH
a
c
Compound
X
clog P
IC50
(
lM)
LE
LLE
AV50 (nM)
24
25
26b
27
28
CH2
S
SO
SO2
O
3.3
4.0
2.1
2.3
3.6
2.1
0.36
>10
15
0.42
0.47
—
0.32
0.44
2.41
2.47
—
2.55
2.33
1100
80
—
—
157
1.1
a
Inhibition of wild type HIV RT with a poly(rA) ꢀ300 template, (dT) 16 primer
and dTTP as substrate.4,6
b
Racemic.
Cl
c
a
Antiviral activity in cell culture in SupT 1 cells infected with the RF strain of HIV.
O
O
O
b
O
warranted further exploration. This decision was further supported
by synthesis and profiling of the homologous set of 3,5-diethylpy-
razoles 29–33 shown in Table 3.
X
The improvements seen for thioether 25 and ether 28 were mag-
nified in this series of diethylpyrazoles. The phenylthiopyrazole 32
and phenoxypyrazole 33 were approximately 10 times and five
times more potent HIV RT inhibitors than the benzylpyrazole 1,
respectively. These improvement in HIV RT inhibition again deliv-
ered compounds with correspondingly improved antiviral activity
in cell culture. Other linking groups such as ketone, methoxymeth-
ylene and hydroxymethylene were explored in compounds 29–31
and although modest potency was retained for some of these, none
of them seemed to offer the promise of ether 33.
X
c
O
O
N
N
OH
O
O
34-66
Scheme 1. General synthesis of ethers 34–36. Reagents and conditions: (a) Bu4NBr,
TMS-Cl, DMSO, MeCN; (b) Cs2CO3, appropriate phenol, acetone; (c) 2-hydroxyeth-
ylhydrazine, AcOH.

C. E. Mowbray et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5603–5606
5605
Table 4
Modification of pyrazole 4-substituent
a
Compound
X
clog P IC50
LE
LLE
l
M
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
3,5-diCl
2-Cl
3-Cl
4.7
3.7
4.0
4.0
4.3
4.5
4.5
4.2
4.6
3.2
3.4
3.4
3.3
3.4
3.4
3.2
3.5
3.6
4.3
2.7
2.7
1.8
2.8
3.9
2.8
2.7
3.2
3.3
3.7
3.8
0.12
3.8
0.56
>50
4.3
>90
1.2
6.1
31
3.3
1.2
>100
2.6
>40
0.81
0.96
>50
0.60
0.51
0.35
>50
>100
>100
24
35
>100
31
>100
3.2
>100
0.46 2.23
0.38 1.68
0.44 2.28
4-Cl
—
—
2,3-diCl
2,4-diCl
2,5-diCl
2,6-diCl
3,4-diCl
2-F
3-F
4-F
2,3-diF
2,4-diF
2,5-diF
2,6-diF
3,4-diF
3,5-diF
3,5-diMe
3-CN
4-CN
3-CONH2
3-F-4-CN
4-F-3-Me
4-F-3-CN
2,3-DiF-4-CN
2-Cl-4-CN
3-Cl-4-CN
2,6-diMe-4-CN
3,5-diMe-4-CN
0.36 1.03
—
—
0.39 1.46
0.35 0.98
0.3
0.38 2.28
0.41 2.52
À0.06
—
—
0.37 2.31
—
—
0.41 2.74
0.4
—
2.87
—
Figure 1. Plot of Àlog(RT IC50) against clog P for pyrazole series. The 45° lines
indicate equal values of LLE.
0.41 2.67
0.42 2.03
0.43 3.77
ity. We were very pleased to observe a dramatic reduction in the
rate of glucuronidation of 3-cyanophenoxyprazole 52 compared
to 3,5-dichlorophenoxypyrazole 33 resulting in a 20-fold lower
clearance rate in human hepatocytes. These results vindicated
our design goal of lowering overall lipophilicity to reduce the rate
of glucuronidation in this series.
In our initial design of the early pyrazole NNRTIs we had man-
aged to retain excellent activity against HIV RT bearing important
drug resistance mutations from clinically significant drug resistant
viruses.1 Throughout the development of this series of inhibitors
we regularly checked to make sure that new leads retained this
broad spectrum of activity as is shown for selected compounds
in Table 6. It can be seen that the excellent spectrum of activity
of early lead 1 is retained as the atom linking the phenyl group
to the pyrazole core is changed from carbon to sulfur (compounds
25 and 32) or oxygen (compounds 33 and 52).
—
—
—
—
—
—
0.31 0.72
0.28 1.62
—
—
0.29 1.33
—
—
0.33 1.8
—
—
N
63
64
65
66
1.8
2.3
2.3
1.7
>100
>100
4.2
—
—
O
N
OH
N
Table 5
N
Microsomal stability and binding of key compounds
—
—
O
N
OH
OH
a
Compound
HLM T1/2 (min)
Predicted Clu from
human hepatocytesa,b
(mL/min/kg)
Microsomal
free fractiona (%)
N
1
18
39
92
89
27
—
—
—
1000
50
24
—
—
13
80
N
24
28
33
52
0.38 3.12
O
N
N
a
The methods for determination of in vitro metabolism and microsomal binding
have recently been separately published.8
b
Clu is unbound clearance.
N+
O
>100
—
—
O
Table 6
N
N
OH
IC50 fold-resistance of selected pyrazoles versus HIV RT enzymesa bearing NNRTI
resistance mutations cf. wild type
a
Inhibition of wild type HIV RT with a poly(rA) ꢀ300 template, (dT) 16 primer
Efavirenz
1
25
32
33
52
and dTTP as substrate.6
K103N
Y181C
F227L
V106A
Y188C
K101E
P236L
V108I
L100I
44
2.2
0.4
1.8
0.8
3.8
2.8
1.0
—
0.8
1.1
9.0
1.8
0.6
5.1
0.2
5.5
—
2.5
1.7
—
—
—
—
—
—
—
1.3
1.5
13
2.9
0.7
4.6
0.9
5.0
10
1.3
3.8
7.1
3.8
0.2
4.6
0.3
5.9
—
2.3
1.5
4.7
15
0.3
9.4
0.4
11
We were keen to evaluate any improvement in stability to-
wards oxidative metabolism and glucuronidation of this less lipo-
philic lead. The apparent stability of 3-cyanophenoxyprazole 52 in
human liver microsomes was actually worse than the parent 3,5-
dichlorophenoxypyrazole 33 (T1/2 = 27 versus 89 min) however
we believe that this is actually due to a reduction in microsomal
binding as seen in Table 5. This reduction in microsomal binding
is likely to be a further consequence of the reduction of lipophilic-
2.9
11
L234I
—
—
—
11
—
a
Inhibition of wild type and mutated HIV RT with a poly(rA) ꢀ300 template, (dT)
16 primer and dTTP as substrate.4,6

5606
C. E. Mowbray et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5603–5606
Stupple, P. A.; Webster, R.; Wood A. Bioorg. Med. Chem. Lett. 2009, in press.
At this stage in our work we felt that the profile of the nitrile 52
doi:10.1016/j.bmcl.2009.08.039.
represented a significant step towards our goal of identifying a no-
vel NNRTI combining excellent antiviral activity with a high quality
pharmacokinetic, pharmaceutical and safety profile. We aimed to
further improve both antiviral activity and the pharmacokinetic
profile of this series and our efforts towards this goal are described
in the next paper in this series.
2. The second paper in this series ‘Pyrazole NNRTIs 2: Exploring the Dependency of
Potency on Lipophilicity’: Burt, C.; Corbau, R.; Mills, J.; Mowbray, C. E.; Perros,
M.; Tran, I.; Price, D.A.; Selby, M. D.; Stupple, P. A.; Webster, R.; Wood, A.
Manuscript in preparation.
3. (a) Leeson, P. D.; Springthorpe, B. Nat. Rev. Drug Disc. 2007, 6, 881; (b) The same
concept was independently proposed by researchers at Pfizer and termed lipE.
Ryckmans, T.; Edwards, M. P.; Horne, V. A.; Monica Correia, A.; Owen, D. R.;
Thompson, L. R.; Tran, I.; Tutt, M. F.; Young, T. Bioorg. Med. Chem. Lett. 2009, 15,
4406.
4. (a) Corbau, R. G.; Mowbray, C. E.; Perros, M.; Stupple, P. A.; Wood, A. World
Patent Application WO 200204424.; (b) Corbau, R. et al. Poster Presentation, 47th
Interscience Conference on Antimicrobial Agents and Chemotherapy, September
17–20, 2007, Chicago.; (c) Corbau, R. et al. Antimicrob. Agents Chemother. 2009, in
press.
5. Synthetic routes to compounds described in this paper have already been
outlined4,6 and will be discussed in more detail elsewhere.
6. Jones, L. H.; Mowbray, C. E.; Price, D. A.; Selby, M. D.; Stupple, P. A. World Patent
Application WO 2002085860.
Acknowledgements
We would like to thank Dave Beal, Chris Carr, Faye Quinton and
Isabelle Tran for compound synthesis, Lesley Fishburn and Alex
Martin for HIV RT testing, Julie Mori and Caroline Smith-Burchnell
for antiviral testing and Gill Allan for metabolism studies.
References and notes
7. van de Waterbeemd, H.; Smith, D. A.; Beaumont, K.; Walker, D. J. Med. Chem.
2001, 44, 1313.
8. Allan, G.; Davis, J.; Dickins, M.; Gardner, I.; Jenkins, T.; Jones, H.; Webster, R.;
Westgate, H. Xenobiotica 2008, 38, 620.
1. First paper in this series ‘Pyrazole NNRTIs 1: Design and Initial Optimization of a
Novel Template’: Mowbray, C. E.; Burt, C.; Corbau, R.; Perros, M.; Tran, I.;