Elucidation of fluorine's impact on p: K a and in vitro Pgp-mediated efflux for a series of PDE9 inhibitors

Article abstract of DOI:10.1039/c8md00114f

P-Glycoprotein (Pgp)-mediated cellular efflux is recognized as a common challenge in CNS drug discovery. In this study, the influence of replacing a hydrogen atom with fluorine on the pK_a and Pgp-mediated efflux is elucidated for a series of PDE9 inhibitors. The PDE9 inhibitors with and without fluorine were synthesized using a novel condensation-oxidation approach, providing access to several analogues, all from the same stereoenriched aldehyde building block. The incorporation of fluorine was found to influence two acid-base functionalities concomitantly, both of which were involved in Pgp-recognition. By methylating the acidic functionality, it was possible to isolate the effect responsible for lowering the Pgp-mediated efflux.

Full text of DOI:10.1039/c8md00114f

View Article Online
View Journal
MedChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: K. Fjelbye, M.
Marigo, R. Clausen, E. B. Jørgensen, C. T. Christoffersen and K. Juhl, Med. Chem. Commun., 2018, DOI:
10.1039/C8MD00114F.
This is an Accepted Manuscript, which has been through the
Volume 7 Number 1 January 2016 Pages 1–204
Royal Society of Chemistry peer review process and has been
accepted for publication.
MedChemComm
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Broadening the field of opportunity for medicinal chemists
www.rsc.org/medchemcomm
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
Themed issue: Antibiotic Resistance
ISSN 2040-2503
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
rsc.li/medchemcomm

Please do not adjust margins
MedChemComm
Page 1 of 4
View Article Online
DOI: 10.1039/C8MD00114F
MedChemComm
RESEARCH ARTICLE
Elucidation of fluorine’s impact on pKa and in vitro Pgp-mediated
efflux for a series of PDE9 inhibitors
Received 00th January 20xx,
Accepted 00th January 20xx
Kasper Fjelbye, a, b Mauro Marigo, a Rasmus P. Clausen, b Erling B. Jørgensen, a Claus T.
Christoffersen, a Karsten Juhl a,
*
DOI: 10.1039/x0xx00000x
P-glycoprotein (Pgp)-mediated cellular efflux is recognized as a common challenge in CNS drug discovery. In this study, the
influence of replacing a hydrogen atom with fluorine on the pKa and Pgp-mediated efflux is elucidated for a series of PDE9
inhibitors. The PDE9 inhibitors with and without fluorine were synthesized using a novel condensation-oxidation approach,
providing access to several analogues, all from the same stereoenriched aldehyde building block. The incorporation of
fluorine was found to influence two acid-base functionalities concomitantly, both of which were involved in Pgp-
recognition. By methylating the acidic functionality, it was possible to isolate the effect responsible for lowering the Pgp-
mediated efflux.
www.rsc.org/
carbaldehydes,13 we set out to study the influence of H/F
replacement on the activity, pKa and Pgp-mediated efflux of a
series of phosphodiesterase 9 (PDE9) inhibitors (Figure 1).
1. Introduction
Medicinal chemists involved in drug discovery increasingly
appreciate the changes of key properties afforded by the
introduction of fluorine in their lead compounds. This
remarkable element has been shown to affect the electron
density of neighboring functional groups, altering the
compound’s pKa, conformation, solubility, permeability and
binding affinity.1-4 Additionally, fluorine has also been used in
strategic replacements for metabolically liable hydrogen
O
acidic
site
R:
N
N
HN
N
N
Me
N
H
PF-04447943
O
N
basic
site
H / F exchange:
∆ in acidity and basicity
∆ in Pgp-mediated efflux
R
atoms, improving half-life significantly.5 Introducing fluorine
,
6
to modulate the basicity of simple aliphatic amines generally
results in a significant shift in pKa, if installed at either the β or Fig. 1 Series of PDE9 inhibitors. R = aromatic or heteroaromatic group.
γ-position.78 In the area of CNS drug design, among the most
These compounds containing the 3,4-substituted pyrrolidine
crucial parameters to optimize are passive permeability and
scaffold constitute good candidates for such examination, as
Pgp-mediated efflux, and to de-risk complications associated
the cellular flux ratios within the series vary significantly
with poor brain exposure, the efflux ratio should generally be <
,
10
2.5.9 To control a compound’s Pgp-recognition, of particular
relevance to medicinal chemists are the hydrogen bonding
capabilities, LogD, topological Polar Surface Area (tPSA) and
depending on the nature of the R group and is thus likely to be
effected by fluorine.14 The PDE enzymes are involved in the
hydrolysis and break-down of the cyclic nucleotides cAMP and
cGMP that serve as key intracellular initiators of numerous
signaling processes in the nervous system. Of all the PDE
enzyme families, PDE9 has the highest affinity for cGMP, and
inhibition of the PDE9 enzyme to increase cGMP levels has
basicity.11 For a chemical series with a consistent number of
hydrogen bond donors/acceptors, the individual H-bond
strengths become particularly important. Thus, being able to
introduce a fluorine atom at a specific position can play a
pivotal role in modulating these core parameters without
drastically altering molecular size. Encouraged by our recent
diastereodivergent fluorination of branched pyrrolidinyl
,
12
,16
been reported to enhance cognitive function in rodents.15
The 3,4-substituted pyrrolidine-based PDE9 inhibitors in Figure
1 without fluorine were originally discovered by Pfizer,14 and
the pyrimidinylated analogue PF-04447943 was advanced into
phase 2 trials to assess its application in mild to moderate
probable Alzheimer’s Disease treatment.
As will become evident from this study, introduction of
fluorine had a moderate effect on PDE9 inhibition of these
compounds – PDE9 IC50 values varied from unchanged to a
10-fold increase (i.e. reduced potency). Contrary to our
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 2 of 4
View Article Online
DOI: 10.1039/C8MD00114F
ARTICLE
Journal Name
expectations, the introduction of fluorine led to a significant The preparation of
1
and
2
allowed subsequent synthesis of a
increase in Pgp mediated efflux in some cases – probably due range of final products
8
-11 using a two-step sequence in
to increased acidity of the amide moeity. In other cases where which the Boc-protecting group was removed using HCl in
the acidic site was blocked by methylation, the Pgp mediated methanol followed by reductive amination with commercially
efflux was reduced by fluorination in the
regard to the amine moiety.
β
-position with available aldehydes (Table 1). To our satisfaction, the
conversion of common intermediates and to the
1,
2
,
6
7
respective final products were successful and moderate yields
were obtained for the two-step processes.
2. Chemistry
3. Results & Discussion
Synthetic access to the fluorinated PDE9 inhibitor analogues in
a convenient manner relied on the formation of a common The condensation-oxidation methodology (scheme 1)
fluorinated intermediate and the non-fluorinated that combined with the reductive amination chemistry provided us
could be converted directly into the final products (Scheme 1). with several candidates for in vitro examination, featuring only
Inspired by Verhoest et al., a pyrazolyl amide building block minor structural variance to aid isolating the impact of H/F
1
2
3
could be condensed with a carbonyl derivative to furnish the replacement. The pKa’s for both the acidic and basic sites for
pyrazolopyrimidinone core.14 We hypothesized that a β- compounds 8a-f were experimentally determined. The acidic
fluoropyrrolidinyl carbaldehyde
organocatalysis could be used as starting point for the pKa’s below 10, being even lower than phenol, indicating a
condensation-oxidation approach with
to directly provide strong hydrogen-bond capability for an N-H donor (Table 1)18
4, prepared by stereoselective functionality found in the pyrazolopyrimidinone cores had
3
the bicyclic core. This strategy was based on findings by Togo The difference in pKa between 9a and its fluorinated analogue
et al. in which ethylenediamine was condensed with a series of 8a falls well within the interval expected for small cyclic
aldehydes and oxidized to the corresponding imidazolines amines. Similar comparisons through the complete list of
using molecular iodine and potassium carbonate.17 This direct compounds and their fluorinated analogues 8a-f reveals a
approach furnished the desired common intermediate
14% yield along with range of byproducts. Further (acidic)), and shifts for the basic pyrrolidines between 1.1-3.3
optimization led to the use of Ti(OEt)4 to promote units (pKa (basic)). Throughout the series , the fluorinated
condensation followed by filtration and oxidation with I2 at 80 analogues generally had lower potencies. In half the cases, the
°C for 30 min. Thus, the yield increased to 36% for and the Pgp-efflux ratios were significantly affected by the presence of
protocol also successfully transformed the non-fluorinated fluorine; for 8a 8b and 8e, the efflux ratios significantly
increased, whereas they remained unchanged for 8c 8d and
8f The introduction of fluorine simultaneously affects
1 in systematic decrease in acidic pKa between 1.2-1.7 (pKa
a
8-9
1
,
aldehyde
5
into
2
in 56% yield (Scheme 1).
,
.
pyrrolidine-basicity and the lactam acidity - parameters most
likely involved in either increasing or decreasing Pgp-
recognition, respectively. For a structurally advanced scaffold
as studied herein featuring several functional groups, the
outcome of such opposing effects might be difficult to predict
a priori or quantitatively rationalize posteriori. Thus, we were
interested in isolating and evaluating the effect responsible for
decreasing the efflux ratio. For this reason, we chose to study
Scheme 1
Reagents and conditions: a: Ti(OEt)4, THF (0.5 M), 75 °C, 24 h then
the pairs of N-methylated lactams 10e
hydrogen bond donor. These compounds were chosen as both
fluorinated non-methylated analogues 8e had high flux ratios
-f and 11e-f lacking the
filtration and b: I2 (1.5 eq), DMF (0.15 M), 80 °C, 30 min. X = H (56%), F (36%).
Having established a route to
methylated lactams and could be prepared using NaH and
MeI in DMF (Scheme 2).
1 and 2, the corresponding N-
-f
6
7
and thus, a potential decrease in Pgp-efflux would likely be
noticeable.
Scheme 2
Reagents and conditions: (a) NaH, MeI, DMF, 0 °C, 1.5 h. X = H, (28%), F
(13%).
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 3 of 4
View Article Online
DOI: 10.1039/C8MD00114F
Journal Name
ARTICLE
Table 1. Data for the fluorinated (8, 10) and non-fluorinated PDE9 inhibitors (9, 11) a, b, c, d, e
Comp.
R
X
Y
MDCK-
Δ
pKa (acidic)
pKa (basic)
IC50 a(PDE9)
Yield
group
MDR1 ratio
9a
8a
9b
8b
H
F
H
H
H
H
1.3
6.9
1.9
4.2
9.6
8.4
9.6
8.4
6.8
5.6
7.0
5.9
49 nM
200 nM
14 nM
n/a
62%
36%
47%
↑
H
F
↑
≈
220 nM
9c
8c
9d
H
F
H
H
H
1.3
0.9
1.4
10.1
8.4
8.4
5.9
7.9
750 nM
590 nM
17 nM
33%
33%
63%
H
9.7
≈
8d
F
H
1.4
8.5
6.5
65 nM
40%
9e
8e
H
F
H
H
2.7
7.1
9.7
8.4
n/a
n/a
9.5
8.4
n/a
n/a
8.8
5.5
6.8
5.2
7.7
4.9
6.4
4.9
59 nM
180 nM
50%
35%
55%
48%
33%
75%
59%
48%
↑
↓
≈
11e
10e
9f
H
F
Me
Me
H
3.1
66% (10µM)
22% (10µM)
36 nM
1.8
H
F
15.5
16.0
4.7
8f
H
210 nM
11f
10f
H
F
Me
Me
850 nM
↓
2.6
14% (10µM)
a IC50: potency in the PDE9 assay, values in % correspond to the inhibition at 10 µM.19 b MDCK-MDR1: the Madin Darby canine kidney permeability assay with a multi-
drug resistant gene coding for Pgp over-expression. The efflux ratio is calculated as basolateral-apical/apical-basolateral. See supporting information for further
c
d
e
details.20 n/a = not applicable. Δ reflects the direction of the change in MDCK-MDR1 efflux ratio after H/F exchange. Compounds 9a, b, d-f have previously been
prepared through a different route.14
Not surprisingly, a major loss of activity throughout the N- and 11e-f to be 1.5 and 1.6 pKa units. It was particularly
methyl series was observed, which is rationalized by the loss of interesting that within this N-methyl lactam series, 10e-f and
a key hydrogen-bond interaction between the lactam and 11e-f, the Pgp-mediated efflux was significantly lowered from
glutamine-453 in the binding pocket of the PDE9 enzyme.14 3.1 to 1.8 and 4.7 to 2.6 for the fluorinated compounds,
Despite the expected loss in activity, further elucidation of placing them into the acceptable region for CNS candidates.
how pKa changes, within such structurally similar compounds, Thus, in support of our hypothesis that two opposing effects
influence the Pgp-mediated efflux continued to pique our were affecting the MDCK-MDR1 ratio, removing the acidic
interest. The differences in pyrrolidine basicities were functionality by methylation indeed led fluorine to decrease
determined between each of the respective compounds 10e-f the Pgp-mediated efflux for both 10e and 10f. The prediction
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 4 of 4
View Article Online
DOI: 10.1039/C8MD00114F
ARTICLE
Journal Name
of how H/F replacement exactly impacts a compound’s 4. Müller, K.; Faeh, C.; Diederich, F. Science 2007, 317, 1881-
pharmacokinetic and pharmacodynamic properties is
a
1886.
complex matter. However, during the final stages of lead
optimization in drug discovery, where the general molecular
framework is established, possessing the synthetic means for
5.
Gleeson, P.; Bravi, G.; Modi, S.; Lowe, D. Bioorganic Med.
Chem. 2009, 17, 5906-5919.
introducing a fluorine atom can be a resourceful means to 6. Dossetter, A. G. Bioorganic Med. Chem. 2010, 18, 4405-
balance the properties. In addition, e.g. for biological targets in
the periphery, it is desired to prevent the active compound
4414.
7.
Morgenthaler, M.; Schweizer, E.; Hoffmann-Röder, A.;
Benini, F.; Martin, R. E.; Jaeschke, G.; Wagner, B.; Fischer,
H.; Bendels, S.; Zimmerli, D.; Schneider, J.; Diederich, F.;
Kansy, M.; Müller, K. ChemMedChem 2007, 2, 1100-1115.
from entering the brain. For compounds, similar to
8 and 9,
fluorine incorporation might even be a viable strategy to
decrease brain exposure by increased Pgp-mediated efflux.
However, prediction of fluorine’s impact a priori might be
simpler in cases where fluorine only affects either a hydrogen 8. Hicken, E. J.; Marmsater, F. P.; Munson, M. C.; Schlachter,
bond donor or acceptor for compounds facing the hurdle of
Pgp-efflux.
S. T.; Robinson, J. E.; Allen, S.; Burgess, L. E.; DeLisle, R. K.;
Rizzi, J. P.; Topalov, G. T.; Zhao, Q.; Hicks, J. M.; Kallan, N.
C.; Tarlton, E.; Allen, A.; Callejo, M.; Cox, A.; Rana, S.;
Klopfenstein, N.; Woessner, R.; Lyssikatos, J. P. ACS Med.
Chem. Lett. 2014, 5, 78-83.
Conclusion
In conclusion, several fluorinated and non-fluorinated drug-like
9.
Raub, T. J. Mol. Pharmaceutics 2006, 3, 3-25.
compounds were synthesized from a single aldehyde
diastereoselective fluorination protocol to give 4 13
followingly a novel condensation-oxidation approach to yield
the common precursors and . These precursors were
then converted into the final compounds 11 that were used
5
using a
10. Hitchcock, S. A. Curr. Opin. Chem. Biol. 2008, 12, 318-323.
,
and
11. Wager, T. T.; Villalobos, A.; Verhoest, P. R.; Hou, X.; Shaffer,
1,
2
6,7
C. L. Expert Opin. Drug Discov. 2011, 6, 371-381.
8-
12. Wager, T. T.; Chandrasekaran, R. Y.; Hou, X.; Troutman, M.
D.; Verhoest, P. R.; Villalobos, A.; Will, Y. ACS Chem.
Neurosci. 2010, 1, 420-434.
to elucidate the impact of fluorine incorporation on pKa and
Pgp-mediated efflux. The consistent trend observed in the pKa
shifts for
9 and 11 after H/F replacement to give 8 and 10, and
the relationship between pKa, the presence of a hydrogen
bond donor and the efflux ratio aid the general understanding
of the possibilities and limitations afforded by fluorine in drug
discovery. Future strategies in CNS drug design may benefit
from such findings towards a more effective optimization
process in the search for compounds that can successfully
progress towards clinical candidate status.
13. Fjelbye, K.; Marigo, M.; Clausen, R. P.; Juhl, K. Org. Lett.
2016, 18, 1170-1173.
14. Verhoest, P. R.; Fonseca, K. R.; Hou, X.; Proulx-LaFrance, C.;
Corman, M.; Helal, C. J.; Claffey, M. M.; Tuttle, J. B.;
Coffman, K. J.; Liu, S.; Nelson, F.; Kleiman, R. J.; Menniti, F.
S.; Schmidt, C. J.; Vanase-Frawley, M.; Liras, S. J. Med.
Chem. 2012, 55, 9045-9054.
15. Fisher, D. A.; Smith, J. F.; Pillar, J. S.; Denis, S. H. S.; Cheng,
Conflicts of interest
J. B. J. Biol. Chem. 1998, 273, 15559-15564.
The authors declare no competing interests.
16. Hutson, P. H.; Finger, E. N.; Magliaro, B. C.; Smith, S. M.;
Converso, A.; Sanderson, P. E.; Mullins, D.; Hyde, L. A.;
Eschle, B. K.; Turnbull, Z.; Sloan, H.; Guzzi, M.; Zhang, X.;
Wang, A.; Rindgen, D.; Mazzola, R.; Vivian, J. A.; Eddins, D.;
Uslaner, J. M.; Bednar, R.; Gambone, C.; Le-Mair, W.;
Marino, M. J.; Sachs, N.; Xu, G.; Parmentier-Batteur, S.
Neuropharmacology 2011, 61, 665-676.
Acknowledgements
The work was financially supported by the Ministry of Higher
Education and Science and H. Lundbeck A/S. We would like to
also thank Dr. Henrik Pedersen from H. Lundbeck A/S with
analytical assistance in HR MS acquisition.
17. Ishihara, M.; Togo, H. Synlett 2006, 2006, 227-230.
18. Gilli, P.; Pretto, L.; Bertolasi, V.; Gilli, G. Acc. Chem. Res.
2009, 42, 33-44.
References
1.
Meanwell, N. A. Chem. Res. Toxicol. 2011, 24, 1420-1456.
19. Human PDE9A2 enzyme (GenBank Accession No.
NM_001001567), full length with N-terminal GST-tag, MW
= 87.7 kDa, expressed in a Baculovirus infected Sf9 cell
expression system (BPS Bioscience, San Diego, CA, USA).
2.
Hopkins, A. L.; Keseru, G. M.; Leeson, P. D.; Rees, D. C.;
Reynolds, C. H. Nat. Rev. Drug Discov. 2014, 13, 105-121.
3.
Wager, T. T.; Kormos, B. L.; Brady, J. T.; Will, Y.; Aleo, M. D.;
Stedman, D. B.; Kuhn, M.; Chandrasekaran, R. Y. J. Med.
Chem. 2013, 56, 9771-9779.
20. Provided by WuXi AppTec, Shanghai, China.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins