Comparison of the Non-Nucleoside Reverse Transcriptase Inhibitor Lersivirine with its Pyrazole and Imidazole Isomers

Article abstract of DOI:10.1111/j.1747-0285.2011.01113.x

Lersivirine is a potent non-nucleoside reverse transcriptase inhibitor with exceptional mutant resilience. Here, we compare the pharmacological and pharmacokinetic profile of lersivirine with its pyrazole and imidazole isomers and briefly explore the prof

Full text of DOI:10.1111/j.1747-0285.2011.01113.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01113.x
Chem Biol Drug Des 2011; 77: 393–397
Research Letter
Comparison of the Non-Nucleoside Reverse
Transcriptase Inhibitor Lersivirine with its
Pyrazole and Imidazole Isomers
Lyn H. Jones1,*, Gill Allan2, Romuald
Corbau3, Donald S. Middleton1, Charles E.
Mowbray1, Sandra D. Newman1, Chris
Phillips1, Rob Webster2 and Mike Westby3
to the peptide back bone of RT (7,8). Additionally, the Y181 residue
adopts the more unusual 'down' position, similar to that present in
the apo-structure, that may explain the resilience of lersivirine to
the clinically relevant Y181C mutation (3,6).
1Sandwich Chemistry, World Wide Medicinal Chemistry, Pfizer Ltd.,
Ramsgate Road, Sandwich CT13 9NJ, UK
These key interactions suggest other templates that are able to
present these essential fragments in an appropriate geometry may
retain the impressive potency and mutant profile of lersivirine. We
have previously described the creation of the imidazole isomer 1 of
lersivirine (Figure 2), which retains these important structural ele-
ments and preserves the desirable potency for the RT enzyme and
activity against the clinically relevant mutations K103N and Y181C
(9,10).
2Pharmacokinetic, Dynamics and Metabolism, Pfizer Ltd., Ramsgate
Road, Sandwich CT13 9NJ, UK
3Antiviral Biology, Pfizer Ltd., Ramsgate Road, Sandwich CT13 9NJ,
UK
*Corresponding author: Lyn H. Jones, lyn.jones@pfizer.com
Lersivirine is
a potent non-nucleoside reverse
transcriptase inhibitor with exceptional mutant
resilience. Here, we compare the pharmacological
and pharmacokinetic profile of lersivirine with its
pyrazole and imidazole isomers and briefly explore
the profile of these series. This work establishes
lersivirine as the outstanding molecule in this set.
The pyrazole isomer of lersivirine (2) was also prepared as there
would appear to be little difference in the appropriate presentation
of the optimized interacting fragments (Figure 2). Moreover, the N1
pyrazole atom does not make any specific interactions with the
enzyme, as expected, and we therefore presumed that moving this
atom to other locations in the five-membered ring would be toler-
ated. The preparation of 2 progressed smoothly via the unoptimized
route shown in Scheme 1 in just 4 steps. Commercially available
Key words: drug design, drug discovery, structure-based drug design
Received 13 October 2010, revised 21 February 2011 and accepted for
publication 21 February 2011
Reverse transcriptase (RT) is a DNA polymerase that is essential to
the infectious life cycle of HIV, and inhibition of this enzyme has
shown utility in the treatment of HIV. Non-nucleoside RT inhibitors
(NNRTIs) are non-competitive in that they bind to an allosteric site
close to, but distinct from the catalytic site of the enzyme. NNRTIs
hinder the protein domain movements required by RT to efficiently
synthesize new DNA (1). Because this site is not present in the RT
enzyme of normal cells, NNRTIs are more specific for the viral
enzyme. NNRTIs are particularly vulnerable to the development of
viral resistance caused by mutations in RT that can retain viable
enzymatic function.
The NNRTI lersivirine has recently emerged as an exciting clinical
candidate for the treatment of HIV that possesses excellent efficacy
against NNRTI-resistant viruses (2,3). The crystal structure of lersivi-
rine with the RT enzyme can help rationalize the relationship
between protein–ligand molecular interactions and mutant resil-
ience (Figure 1). Key features include an edge-to-face p-interaction
between the dicyanophenyl ring in lersivirine and the immutable
Trp229 residue (4–6) and hydrogen bonding from the hydroxyl group
Figure 1: Crystal structure of lersivirine with wild-type reverse
transcriptase (PDB 2WON). Key interactions highlighted (distances
in ꢀ). Residues Y181 (red), Y188 (dark blue) and W229 (purple) are
shown (3).
393

Jones et al.
CN
CN
CN
N
NC
NC
NC
N
O
O
O
N
OH
OH
OH
N
N
N
lersivirine
1
2
Figure 2: NNRTI isomers lersivirine, imidazole 1 and pyrazole 2.
NaH,THF, 0oC
then nBuLi
then PhCH2OCH2Cl
EtNHNH2.oxalate
EtOH, reflux
EtO
EtO
O
Ph
48%
28%
O
O
O
O
4
3
CN
NC
ArF, K2CO3
DMF, 85oC
4
3
HO
O
O
Ph
O
Ph
N
N
69%
N
N
5
6
CN
N
CN
N
NC
Cl
BCl3.SMe2
DCM
O
O
OH
OH
75%
N
N
7
2
Scheme 1: Preparation of pyrazole 2 and structure of congener 7.
ketoester 3 was doubly deprotonated and reacted with benzylchlo-
romethyl ether to provide 4 that was cyclized with ethyl hydrazine
to furnish 5. The key step was arylation of hydroxy-pyrazole 5 using
3,5-dicyano fluorobenzene, a disconnection similar to that used to
prepare 1 and other NNRTIs in our laboratory.6,9,10
appear to be an attractive alternative to the preparation of these
derivatives.
The RT and antiviral (AV) potency for the three isomers is shown in
Table 1. Although all isomers are potent inhibitors of RT, lersivirine
possesses the highest AV potency and when normalized for lipophi-
The structure of 2 was unambiguously determined using 1H-13C
HMBC NMR correlation experiments (see Experimental). Interest-
ingly, similar pyrazole NNRTIs were reported by Roche, although
compound 2 was not exemplified.a The synthetic route to these
molecules also used the cyclization of hydrazines with b-ketoest-
ers, and then Vilsmeier conditions installed a C4 formyl group with
concomitant chlorination of the hydroxy-pyrazole. Phenol displace-
ment of the chloride, conversion of the aldehyde to the C4 alkyl
and C3 modification provided a variety of pyrazole NNRTIs. For
example, compound 7 (Scheme 1) was prepared in 10 steps from
sodium diethyloxaloacetate. Therefore, our short route would
licity, it also has the highest lipophilic efficiency (LipE = )LogIC50 –
LogD) (11,12). All three compounds exhibited activity against the
K103N and Y181C mutations, which were within tenfold of their
wild-type potencies (2,3,9,10).
Figure 3 shows an overlay of the heterocyclic rings of the three iso-
mers: lersivirine (from the cocrystal structure with wild-type RT) and
the energy minimized structures of imidazole 1 and pyrazole 2.b
The similarity of the conformations, and no doubt similar interac-
tions with the RT enzyme, is reflected in the comparable potency
and mutant resilience of these isomers.
394
Chem Biol Drug Des 2011; 77: 393–397

NNRTI Lersivirine with its Pyrazole and Imidazole Isomers
Table 1: Pharmacology and pharmacokinetics of lersivirine, 1
and 2
CN
CN
Cl
NC
Lersivirine (2,3)
1
2
O
O
OH
OH
LogD
1.8
89
>120
119
4
1.6
1.8
N
N
N
N
HLM T1 ⁄ 2 (mins)
HHeps T1 ⁄ 2 (mins)
wt RT IC50 (nM)a
wt AV IC50 (nM)b
AV LipE
>120
>120
78
10
6.4
99
4.3
0.50
0.25
Positive
>120
>120
250
29
5.7
216
6.4
0.34
0.22
Negative
8
7
wt AV IC50 3.9 nM
wt AV IC50 2.9 nM
6.6
LogD 2.2
LipE 6.2
LogD 2.8
LipE 5.7
Rat Clu (mL ⁄ min per Kg)c
Rat Vdu (L ⁄ Kg)d
Rat T1 ⁄ 2 (h)e
101
13.8
1.6
0.15
Negative
HLM T1/2 52 min
HHeps T1/2 105 min
HLM T1/2 51 min
HHeps T1/2 25 min
Solubility (mg ⁄ mL)
Reactive metabolite
Figure 4: Aryloxy-pyrazole Non-nucleoside RT inhibitors 7 and
8.
AV, antiviral; HLM, human liver microsomes; RT, Reverse transcriptase.
aGeomean for n = 3.
bLersivirine, 1 and 2 were not cytotoxic, CC50 > 100 lM.
c
clearance, but a low volume of distribution translates to only a
moderate improvement in half-life over 2. However, lersivirine com-
bines low clearance with a high volume, resulting in a significantly
longer half-life in rat over 1 and 2 (14). These data clearly estab-
lish lersivirine as the superior NNRTI isomer.
rat iv pk, 2 mg ⁄ kg dose, mean for n = 2, Clu = unbound clearance.
dVdu = unbound volume of distribution. T1 ⁄ 2 = half-life.
e
For completeness, we used the hydroxy-pyrazole arylation chemistry
shown in Scheme 1 to prepare the chloro and isopropyl derivatives
7 and 8, respectively (Figure 4) because structure–activity relation-
ships from the imidazole series suggest these congeners could be
more potent than 2 (9). Structural information also guides these
changes because both the ethyl- and cyano-substituents reside in
hydrophobic pockets that can accommodate a moderate increase in
size and fragmental lipophilicity. As expected, these derivatives pos-
sess superior potency to 2, and although 8 possesses a superior
lipophilic efficiency to 2 and 7, the higher lipophilicity also drives
poorer metabolic stability, thus ruling out further progression of
these specific compounds. We have recently reviewed the aromatic
chloride to nitrile medicinal chemistry transformation, and the result
here (no improvement in LipE from 2 to 7) is not surprising (15).
However, we believe further elaboration of this series, and the gen-
eral learnings outlined above could provide exciting starting points
in the creation of mutant resilient inhibitors.
Figure 3: Overlay of cocrystal structure of lersivirine (green)
from Figure 1 (protein removed for clarity) with minimized structures
of imidazole 1 (cyan) and pyrazole 2 (pink).
Conclusions
In conclusion, we have prepared potent NNRTI isomers that possess
a balanced profile against clinically relevant mutant enzyme. Pyraz-
ole 2 was prepared in four steps and satisfied a key feature of our
design in the NNRTI area to keep synthetic complexity to a mini-
mum so as not to negatively impact cost of goods and to facilitate
analogue generation. A comparison of the pharmacological and
pharmacokinetic profiles of the three isomers establishes lersivirine
to be the exceptional molecule in this set. Interestingly, subtle
structural differences to the NNRTI template for these derivatives
translate to significantly different medicinal chemistry profiles. Ini-
tial elaboration of the pyrazole series exemplified by 2, 7 and 8
suggests that this series warrants further investigation. Ongoing
clinical trials are investigating the potential of lersivirine for the
treatment of HIV-infected patients (16).
The polarity of these compounds translates to impressive aqueous
solubility and metabolic stability, as demonstrated by half-life in
human liver microsomes (HLM) and human hepatocytes (HHeps,
Table 1). We have previously reported that imidazole 1 formed a
reactive metabolite conjugate with glutathione following its incuba-
tion with HLM (9), a feature that has been linked with hepato- and
idiosyncratic toxicity (13). It is noticeable that the pyrazoles lersivi-
rine and 2 do not form reactive metabolite conjugates in this
manner.
Intravenous rat pharmacokinetic experiments demonstrate clear dif-
ferences between the isomers (Table 1). The high clearance of pyr-
azole 2 confers a short half-life in rat. Imidazole 1 has lower
Chem Biol Drug Des 2011; 77: 393–397
395

Jones et al.
Figure 5: HMBC experiment of 2.
(MgSO4) filtered and concentrated under reduced pressure to yield 5
as a white solid (690 mg, 48%), which was used without further puri-
fication: APCI LCMS m ⁄ z 275 [M+H]+ (>95% pure). 5 (340 mg,
1.2 mmol) was dissolved in dimethylformamide (DMF) (3 mL) potas-
sium carbonate (170 mg, 1.2 mmol); then, 3,5-dicyanofluorobenzene
(210 mg, 1.44 mmol) was added, and the mixture was stirred at
85 ꢀC for 30 min. Water was added and extracted with EtOAc, dried
(MgSO4), filtered, and concentrated under reduced pressure. The resi-
due was purified by FCC (20% EtOAc ⁄ pentane) to yield 6 as a colour-
less oil that crystallized on standing (155 mg, 69%): mp 83–85 ꢀC; 1H
NMR (400 MHz, CDCl3) 7.68 (s, 1H), 7.40 (s, 2H), 7.35 (m, 4H), 7.31
(m, 1H), 4.58 (s, 2H), 3.91 (q, 2H), 3.80 (t, 2H), 2.92 (t, 2H), 2.20 (q,
2H), 1.31 (t, 3H), 0.98 (t, 3H); APCI MS m ⁄ z 401 [M+H]+.
Experimental
General
Unless otherwise stated, all reactions were carried out under a
nitrogen atmosphere, using commercially available anhydrous sol-
vents. Thin-layer chromatography was performed on glass-backed
precoated Merck silica gel (60 F254) plates, and FCC (flash column
chromatography) was carried out using 40- to 63-lm silica gel.
NMR spectra were carried out on a Varian Mercury 400 and a Var-
ian Inova 500 spectrometer in the solvents specified. LC ⁄ MS were
recorded on a Waters Micromass ZQ using atmospheric pressure
chemical ionization (APCI). Other abbreviations are used in conjunc-
tion with standard chemical practice.
6 (155 mg, 0.39 mmol) was dissolved in dichloromethane (DCM)
(5 mL) cooled to 0 ꢀC, BCl3.SMe2 (1 M in DCM, 0.39 mL, 0.39 mmol)
was added dropwise and allowed to warm slowly to room tempera-
ture. The mixture was then cooled to 0 ꢀC; 2N NaOH aq (10 mL) was
added and stirred for 30 min. The mixture was extracted with DCM,
dried (MgSO4), filtered and concentrated under reduced pressure. The
residue was triturated with diisopropylether to yield 2 as a white
crystalline solid (90 mg, 75%); mp 120–122 ꢀC; Found C, 65.69; H,
5-[2,4-diethyl-5-(2-hydroxy-ethyl)-2H-pyrazol-3-
yloxy]-isophthalonitrile (2)
NaH (60% in oil, 660 mg, 16.5 mmol) was suspended in THF (6 mL)
and cooled to -5 ꢀC before the dropwise addition of commercially
available ketoester 3 (2.4 g, 15 mmol) dissolved in THF (10 mL). After
n
10 min, BuLi (1.6 M, 10.3 mL, 16.5 mmol) was added dropwise and
after a further 10 min, benzylchloromethyl ether (2.1 mL, 15 mmol) in
THF (10 mL) was added. After 2 h, the reaction mixture was
quenched with brine, acidified with 2N HCl aq. and extracted with
diethyl ether, dried (MgSO4) filtered, concentrated under reduced
pressure and the residue purified using FCC (10% EtOAc ⁄ pentane) to
1
5.82; N, 17.95. calc. for C17H18N4O2: C, 65.79; H, 5.85; N, 18.05; H
NMR (400 MHz, CDCl3) 7.68 (s, 1H), 7.42 (s, 2H), 4.00 (t, 2H), 3.89
(q, 2H), 2.83 (t, 2H), 2.20 (q, 2H), 1.35 (t, 3H), 1.00 (t, 3H); APCI MS
m ⁄ z 311 [M+H]+; HMBC (Figure 5); chemical stability of 2: pH ⁄ %
remaining 1.2 ⁄ 95, 4.8 ⁄ 99, 7.2 ⁄ 94, 9.8 ⁄ 36, 12.7 ⁄ 0.
1
yield 4 as a colourless oil (1.4 g, 28%); H NMR (400 MHz, CDCl3)
7.30 (m, 5H), 4.49 (s, 2H), 4.15 (q, 2H), 3.75 (t, 2H), 3.39 (t, 1H), 2.81 (t,
2H), 1.92 (m, 2H), 1.31 (t, 3H), 0.92 (t, 3H); APCI MS m ⁄ z 279 [M+H]+.
Acknowledgments
4 (1.4 g, 5.3 mmol) was dissolved in ethanol (40 mL), ethylhydrazine
oxalate (960 mg, 6.3 mmol) was added, and the mixture was heated
under reflux for 9 h. The solvent was removed under reduced pres-
sure, and the residue dissolved in EtOAc, washed with water, dried
We thank Torren Peakman and Michael Kinns for running NMR
experiments. We also thank Ian Burr, Alex Martin and Amy Thomas
for determining the potencies of these compounds.
396
Chem Biol Drug Des 2011; 77: 393–397

NNRTI Lersivirine with its Pyrazole and Imidazole Isomers
11. Ryckmans T., Edwards M.P., Horne V., Correia A., Owen D.R.,
Thompson L.R., Tran I., Tutt M.F., Young T. (2009) Rapid assess-
ment of a novel series of selective CB2 agonists using parallel
synthesis protocols: a lipophilic efficiency (LipE) analysis. Bioorg
Med Chem Lett;19:4406–4409.
12. Leeson P.D., Springthorpe B. (2007) The influence of drug-like
concepts on decision-making in medicinal chemistry. Nat Rev
Drug Disc;6:881–890.
13. Evans D.C., Watt A.P., Nicoll-Griffith D.A., Baille T.A. (2004)
Drug-protein adducts: an industry perspective on minimising the
potential for drug bioactivation in drug discovery and develop-
ment. Chem Res Toxicol;17:3–16.
14. Allan G., Davis J., Dickins M., Gardner I., Jenkins T., Jones H.,
Webster R., Westgate H. (2008) Pre-clinical pharmacokinetics of
UK-453,061, a novel non-nucleoside reverse transcriptase inhibi-
tor (NNRTI), and use of in silico physiologically based prediction
tools to predict the oral pharmacokinetics of UK-453,061 in man.
Xenobiotica;38:620–640.
15. Jones L.H., Summerhill N., Swain N., Mills J.E. (2010) Aromatic
chloride to nitrile transformation: medicinal and synthetic chem-
istry. Med Chem Comm;1:309–318.
16. Fꢁtkenheuer G., Straszewski S., Plettenburg A., Hackman F., Layton
G., McFadyen L., Davis J., Jenkins T.M. (2009) Activity, pharmaco-
kinetics and safety of lersivirine (UK-453,061), a next generation
non-nucleoside reverse transcriptase inhibitor, during 7-day mono-
therapy in HIV-1-infected patients. AIDS;818:2115–2122.
References
1. Ivetac A., McCammon J.A. (2009) Elucidating the inhibition
mechanism of HIV-1 non-nucleoside reverse transcriptase inhibi-
tors through multicopy molecular dynamics simulations. J Mol
Biol;388:644–658.
2. Mowbray C.E., Burt C., Corbau R., Gayton S., Hawes M., Perros
M., Tran I., Price D.A., Quinton F.J., Selby M.D., Stupple P.A.,
Webster R., Wood A. (2009) Pyrazole NNRTIs 4: selection of UK-
453,061 (lersivirine) as a development candidate. Bioorg Med
Chem Lett;19:5857–5860.
3. Corbau R., Mori J., Phillips C., Fishburn L., Martin A., Mowbray
C.E., Panton W., Smith-Burchnell C., Thornberry A., Ringrose H.,
Knçchel T., Irving S., Westby M., Wood A., Perros M. (2010) Ler-
sivirine: a non-nucleoside reverse transcriptase inhibitor with
activity against drug-resistant human immunodeficiency virus-1.
Antimicrob Agents Chemother;54:4451–4463.
4. Pelemans H., Esnouf R., De Clercq E., Balzarini J. (2000) Muta-
tional analysis of Trp-229 of human immunodeficiency virus type
1 reverse transcriptase (RT) identifies this amino acid residue as
a prime target for the rational design of new non-nucleoside RT
inhibitors. Mol Pharm;57:954–960.
5. Fattorusso C., Gemma S., Butini S., Huleatt P., Catalanotti B.,
Persico M., De Angelis M. et al. (2005) Specific targeting highly
conserved residues in the HIV-1 reverse transcriptase primer grip
region. design, synthesis and biological evaluation of novel,
potent and broad spectrum NNRTIs with antiviral activity. J Med
Chem;48:7153–7165.
Notes
6. Jones L.H., Allan G., Barba O., Burt C., Corbau R., Dupont T.,
Knçchel T. et al. (2009) Novel indazole non-nucleoside reverse
transcriptase inhibitors using molecular hybridisation based on
crystallographic overlays. J Med Chem;52:1219–1223.
7. Ren J., Nichols C., Bird L.E., Fujiwara T., Sugimoto H., Stuart
D.I., Stammers D.K. (2000) Binding of the second generation
non-nucleoside inhibitor S-1153 to HIV-1 reverse transcriptase
aDunn, J.P., Hogg, J.H., Mirzadegan, T., Swallow, S. (2004) Prepara-
tion of pyrazole derivatives as non-nucleoside reverse transcriptase
inhibitors for the treatment of HIV disorders and compositions
thereof. WO ⁄ 2004 ⁄ 074257.
bThe unconstrained minimisations were performed using a limited-
memory Truncated-Newton minimiser with a termination energy
threshold of 0.5 kcal ⁄ mol.
involves extensive main chain hydrogen bonding.
Chem;275:14316–14320.
J Biol
8. Lansdon E.B., Brendza K.M., Hung M., Wang R., Mukund S., Jin
D., Birkus G., Kutty N., Liu X. (2010) Crystal structures of HIV-1
reverse transcriptase with etravirine (TMC125) and rilpivirine
(TMC278): implications for drug design. J Med Chem;53:4295–
4299.
Supporting Information
Additional Supporting Information may be found in the online ver-
sion of this article:
9. Jones L.H., Allan G., Corbau R., Hay D., Middleton D.S., Mow-
bray C.E., Newman S., Perros M., Randall A., Vuong H., Webster
R., Westby M., Williams D. (2008) Optimization of 5-aryloxyimi-
dazole non-nucleoside reverse transcriptase inhibitors. Chem
Med Chem;3:1756–1762.
10. Jones L.H., Dupont T., Mowbray C.E., Newman S. (2006) A con-
cise and selective synthesis of novel 5-aryloxyimidazole NNRTIs.
Org Lett;8:1725–1727.
Appendix S1. The syntheses of compounds 7 and 8.
Please note: Wiley-Blackwell is not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
corresponding author for the article.
Chem Biol Drug Des 2011; 77: 393–397
397