N-Benzyl-N-(pyrrolidin-3-yl)carboxamides as a new class of selective dual serotonin/noradrenaline reuptake inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.06.078

The structure-activity relationship and the synthesis of novel N-benzyl-N-(pyrrolidin-3-yl)carboxamides as dual serotonin (5-HT) and noradrenaline (NA) monoamine reuptake inhibitors are described. Compounds such as 18 exhibited dual 5-HT and NA reuptake i

Full text of DOI:10.1016/j.bmcl.2008.06.078

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4308–4311
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Bioorganic & Medicinal Chemistry Letters
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N-Benzyl-N-(pyrrolidin-3-yl)carboxamides as a new class of selective dual
serotonin/noradrenaline reuptake inhibitors
a
a
b
a
a
Florian Wakenhut a, , Paul V. Fish , M. Jonathan Fray , Ian Gurrell , James E. Mills , Alan Stobie ,
*
Gavin A. Whitlock a
a Department of Genitourinary Chemistry, Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
b Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Sandwich Laboratories, 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:
The structure–activity relationship and the synthesis of novel N-benzyl-N-(pyrrolidin-3-yl)carboxamides
as dual serotonin (5-HT) and noradrenaline (NA) monoamine reuptake inhibitors are described. Com-
pounds such as 18 exhibited dual 5-HT and NA reuptake inhibition, good selectivity over dopamine
(DA) reuptake inhibition and drug-like physicochemical properties consistent with CNS target space.
Compound 18 was selected for further preclinical evaluation.
Received 23 May 2008
Revised 24 June 2008
Accepted 25 June 2008
Available online 28 June 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
N-benzyl-N-(pyrrolidin-3-yl)carboxamides
Serotonin reuptake
Noradrenaline reuptake
Metabolic profile
Metabolic stability
CYP2D6 inhibition
Lipophilicity
SNRI
Selective inhibition of serotonin (5-HT) and noradrenaline (NA)
reuptake constitutes an attractive dual pharmacology approach to
the treatment of a number of diseases. For example, venlafaxine 1
and duloxetine 2 have become established drugs for the treatment
of depression.1,2 Duloxetine has also shown clinical efficacy for the
treatment of diabetic neuropathic pain3 and stress urinary inconti-
nence (SUI).4
As part of our research efforts to identify new dual serotonin
and noradrenaline reuptake inhibitors (SNRI) as potential drug
candidates, we recently reported several new templates5–8 that
delivered potent selective dual serotonin/noradrenaline reuptake
inhibitors (SNRI), and notably a 3-amino-pyrrolidine template that
furnished compound 3 (Fig. 1). Unfortunately, when the metabolic
profile of compound 3 was investigated, we found that it was
mainly metabolized by the cytochrome P450 enzyme CYP2D6
through oxidation of the benzylic ring (Fig. 2).
leading to compound 4. This strategy was successful and N-dealky-
lation was identified to be the major route of metabolism for com-
pound 4. However, the metabolizing enzyme had also swapped in
the process and this metabolic pathway was now mainly mediated
by CYP2D6 and not CYP3A4, as for compound 3, resulting in no
mitigation of the drug–drug interaction risk.
We therefore decided to try blocking the N-dealkylation path-
way and to do so we elected to replace the 4-tetrahydropyranyl
group with carboxamides (Fig. 4). The structure–activity relation-
ship of these amides 8–29 is reported in this letter.
Test compounds were conveniently prepared from commer-
cially available homochiral N-BOC 3-amino pyrrolidine 5 by the
general method described in Scheme 1. Standard reductive amina-
tion with the appropriate benzaldehydes followed by acylation of
the resulting amines 6 afforded amides 7. Finally HCl or TFA depro-
tection of the N-BOC amines provided required targets 8–29. It is
worth noting that this synthetic route was highly amenable to par-
allel synthesis and this greatly facilitated SAR exploration. Initial
investigations focused on the variation of the carboxamide group
(R1) with the benzylic substitution (R) maintained as 2,3-di-Cl
and 2,4-di-Cl (Table 1). A series of analogues 8–21 were prepared
with R1 alkyls of increasing size. Interestingly, both benzylic sub-
stitution patterns afforded good dual serotonin/noradrenaline
reuptake inhibition although 2,4-di-Cl analogues generally exhib-
As a consequence of the potential for drug–drug interactions
and the risk of overexposure in poor metabolizers,9 the progression
of compound 3 was halted. In order to block the CYP2D6 mediated
oxidation pathway, our initial strategy was to replace the 2,3-di-
Me benzylic substituents of compound 3 with 2,3-di-Cl (Fig. 3)
* Corresponding author. Tel.: +44 1304 640950; fax +44 1304 651987.
E-mail address: florian.wakenhut@pfizer.com (F. Wakenhut).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.078

F. Wakenhut et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4308–4311
4309
H
N
O
NHMe
S
N
HO
O
NMe2
Me
MeO
1 venlafaxine
Figure 1. Structures of duloxetine, venlafaxine and recently disclosed SNRI 3.
Me
3
2 duloxetine
Having identified the isopropyl group as our preferred R1 sub-
stituent a broader set of R substituents with different substitution
patterns was investigated (compounds 22–28). This analysis affor-
ded additional compounds with excellent SNRI activity and good
selectivity against dopamine reuptake activity (compound 24).
Interestingly, it also demonstrated that, depending on the choice
of R substituents, the series could deliver selective inhibitors of
the noradrenaline transporter (NRI) as illustrated by compound
22 with a single 2-Ph substituent, or selective inhibitors of the
serotonin transporter (SRI), for example, compound 26 with a 2-
F, 4-CF3 substitution pattern. Finally, it is worth noting that both
aminopyrrolidine enantiomers possessed SNRI activity but that
the (S) enantiomer provided more balanced SNRI activities (com-
pounds 18 and 29).
H
N
O
N
CYP3A4
Minor Pathway
CYP2D6
Major Pathway (>70%)
Me
Me
3
H
N
H
N
O
HN
N
Based on that observation, assuming that the remaining sub-
structures overlap, there are two distinct overlaps possible for
the pyrrolidine portion of the two enantiomers. In an overlap dri-
ven by H-bonding complementarity (Fig. 5), in this case, the pro-
jection of the N–H moieties to a common acceptor atom position,
the rings do not sterically overlap perfectly, but certainly should
be able to occupy the same pocket.
Me
Me
Me
Me
Oxidation
Figure 2. Metabolic profile of compound 3.
In an overlap driven by steric complementarity (Fig. 6), the two
nitrogen atoms do not superimpose, and it is assumed that the pre-
sumably acidic moiety of the transporter can flex to accommodate
these alternative positions.
From these experiments, compounds 11, 18 and 24 emerged as
preferred compounds, exhibiting a superior combination of dual
SNRI activity and good selectivity against dopamine reuptake
activity. The three compounds also exhibited attractive drug-like
physicochemical properties consistent with CNS target space11 as
illustrated in Table 2.
H
N
H
N
O
CYP2D6
Major Pathway (>80%)
HN
Cl
N
Cl
4
Cl
Cl
Additional screening in in vitro ADME assays (Table 3) demon-
strated that compounds 11, 18 and 24 all exhibited good metabolic
stability in human liver microsomes, therefore predicting low
in vivo clearance. To further rank them, the three analogues were
tested in a human hepatocyte assay and compound 18 was found
to be the more metabolically robust, therefore, predicting lowest
clearance. All three compounds were weak CYP2D6 inhibitors with
compound 24 being the most potent. This may be attributed to its
higher lipophilicity as it is well established that lipophilic com-
pounds tend to have an increased risk of off-target promiscuity.12
Compounds 11, 18 and 24 all exhibited good membrane perme-
ability in the CaCO-2 cell line,13 therefore, predicting good oral
absorption.
Figure 3. Metabolic profile of compound 4.
H
H
N
O
N
O
R1
N
N
R
R
Compounds 8-29
Figure 4. Structure of new amino-pyrrolidine carboxamides 8–29.
Compounds 11, 18 and 24 were tested for their ability to block
the hERG channel, as it is well established that compounds that
block this ion channel have the potential to cause QT prolongation
and cardiac arrhythmia in man.14 The three analogues were found
to be selective with compound 24 being the least selective proba-
bly also as a result of its higher lipophilicity.
Based on its promising profile, including notably exquisite met-
abolic stability and weak CYP2D6 inhibition, compound 18 was se-
lected to further evaluate the series.
ited slightly weaker activity for the 5-HT transporter, as can be
seen when comparing compounds 19 and 12. From this analysis,
the isopropyl group emerged as our preferred R1 substituent and
compounds 11 and 18 gave potent SNRI activity, and excellent
selectivity against dopamine reuptake activity (DRI). Replacing
the alkyl groups with either a phenyl group (compound 17) or a
more polar branched 4-tetrahydropyranyl group (compound 21)
resulted in a drop in both SRI and NRI activities.

4310
F. Wakenhut et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4308–4311
O
O
H
O
O
N
O
O
O
N
N
O
R1
N
N
a
b
c
R1
N
HN
H2N
5 (
R
R
R
8-29
S
) enantiomer
6
7
Scheme 1. Reagents and conditions: (a) Benzaldehyde, toluene, Dean–Stark apparatus, reflux, 18 h then NaBH4, MeOH, RT, 2 h; (b) R1COCl, dioxane, Et3N, 70 °C, 2 h; (c) TFA,
CH2Cl2, RT, 2–18 h or 4 N HCl in dioxane, rt, 2–18 h.
Table 1
In vitro inhibition of human monoamine reuptake,a,b and clogP for compounds 8–29
H
N
H
N
O
O
R1
N
N
R
Cl
8-28
29
Cl
Compound
R
R1
Stereochemistry
5-HT IC50 (nM)
NA IC50 (nM)
DA IC50 (nM)
clogP
3
8
9
9
7
60
31
70
28
31
92
39
25
64
94
23
727
NTc
NT
NT
>40,000
NT
NT
10,200
NT
NT
NT
1270
NT
NT
NT
NT
2,4-di-Cl
2,4-di-Cl
2,4-di-Cl
2,4-di-Cl
2,4-di-Cl
2,4-di-Cl
2,4-di-Cl
2,4-di-Cl
2,4-di-Cl
2,4-di-Cl
2,3-di-Cl
2,3-di-Cl
2,3-di-Cl
2,3-di-Cl
2-Phenyl
2-Cl
Ethyl
Propyl
Butyl
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
R
25
72
33
13
93
41
27
38
41
195
12
17
26
123
>400
91
5
3.1
3.6
4.1
3.4
4
3.7
4.4
3.1
3.4
4.2
3.3
3.9
3.8
2
3.5
2.7
3.8
4
2.9
2.8
2.2
3.3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Isopropyl
Isobutyl
Tertbutyl
Neopentyl
Cyclopropyl
Cyclobutyl
Phenyl
Isopropyl
Isobutyl
Tertbutyl
4-Tetrahydropyranyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
62
>322
300
14
114
16
29
>400
98
NT
2,3,4-tri-Cl
2,3,5-tri-Cl
2-F, 4-CF3
2-Cl, 4-F
2-F,4-F
5870
21,600
NT
NT
NT
44
24
161
>400
5
Isopropyl
Isopropyl
Isopropyl
Isopropyl
>400
37
2,3-di-Cl
1120
a
See Ref. 10 for complete details of assay conditions.
Monoamine reuptake IC50 are geometric means of a minimum of two experiments. Differences of <2-fold should not be considered significant.
NT denotes not tested.
b
c
Figure 5. Overlap of compounds 18 and 29 driven by H-bonding complementarity.
Figure 6. Overlap of compounds 18 and 29 driven by steric complementarity.

F. Wakenhut et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4308–4311
4311
Table 2
To summarise, we have described the discovery of novel amides
derived from the 3-amino-pyrrolidine template we previously re-
ported6 as potent SNRIs. More specifically, compound 18 exhibited
good SNRI activity in combination with good selectivity against the
dopamine transporter and no significant off-target pharmacology.
Compound 18 also exhibited a very encouraging ADME profile
and pharmacokinetics potentially compatible for bid dosing.
As a result compound 18 was selected for further evaluation in
preclinical disease models. The results of these studies will be re-
ported in future publications.
Physicochemical properties of compounds 11, 18 and 24a
11
18
24
MW
clogP
LogD7.4
HBD/HBA count
pKa
315
3.4
0.8
1/3
ND
32
315
3.3
0.8
1/3
9.4
32
348
3.8
1.3
1/3
ND
32
TPSA
a
ND denotes not determined.
Acknowledgements
Table 3
ADME profiles and K+ channel affinities of compounds 11, 18 and 24a
We Carol Bains, Gerwyn Bish, Helena Barker, Timothy Buxton,
Edelweiss Evrard, Arnaud Lemaitre, Debbie Lovering, Edward Peg-
den, Bhairavi Patel, and Melanie Skerten for compound synthesis.
We are also grateful to Stephen Phillips and Donald Newgreen
and their teams (Discovery Biology) for screening data, and to Neil
Attkins, Anthony Harrison, Christopher Kohl and Nicola Lindsay
(PDM) for ADME studies.
11
18
24
HLM, t1/2 (min)
h.heps, Clint (
CYP2D6 inhibition, IC50
>120
4.8
21.8
16/24
40,200
>120
2.7
27.1
19/28
24,700
>120
8.8
17.6
l
l/min/million cells)
(lM)
CaCO-2, AB/BA at 10
lM
15/18
K+, hERG binding IC50 (nM)
>6870b
a
Maximum measurable half life (t1/2) was 120 min in human liver microsomes.
Geometric mean of nine experiments, two of which >10,000 nM.
b
References and notes
1. (a) Jann, M. W.; Slade, J. H. Pharmacotherapy 2007, 11, 1571; (b) Montgomery, S.
Int. J. Psychol. Clin. Pract. 2006, 10(Suppl. 2), 5; (c) Jackson, S. Curr. Med. Res.
Opin. 2005, 21, 1669.
Table 4
Pharmacokinetics of compound 18 in doga
2. (a) Wernicke, J. F.; Iyengar, S.; Ferrer-Garcia, M. D. Curr. Drug Ther. 2007, 2, 161;
(b) Bauer, M.; Moeller, H-J.; Schneider, E. Expert Opin. Pharmacother. 2006, 7,
421.
3. (a) Kajdasz, D. K.; Iyengar, S.; Desaiah, D. Clin. Ther. 2007, 29, 2536; (b) Thor, K.
B.; Kirby, M.; Viktrup, L. Int. J. Clin. Pract. 2007, 61, 1349.
4. (a) Mariappan, P.; Alhasso, A.; Ballantyne, Z.; Grant, A.; N’Dow, J. Eur. Urol.
2007, 51, 67; (b) Steers, W. D.; Herschorn, S.; Kreder, K. J.; Moore, K.; Strohbehn,
K.; Yalcin, I.; Bump, R. C. BJU Int. 2007, 100, 337.
Blood parameter
Dog (n = 2)
IV
Dose (mg/kg)
t1/2 (h)
Cls (ml/min/kg)
Vd (L/kg)
0.01
5.7
1.1
0.5
5. (a) Fray, M. J.; Bish, G.; Brown, A. D.; Fish, P. V.; Stobie, A.; Wakenhut, F.;
Whitlock, G. A. Bioorg. Med. Chem. Lett. 2006, 16, 4345; (b) Fray, M. J.; Bish, G.;
Fish, P. V.; Stobie, A.; Wakenhut, F.; Whitlock, G. A. Bioorg. Med. Chem. Lett.
2006, 16, 4349.
6. Fish, P. V.; Fray, M. J.; Stobie, A.; Wakenhut, F.; Whitlock, G. A. Bioorg. Med.
Chem. Lett. 2007, 17, 2022.
7. Whitlock, G. A.; Blagg, J.; Fish, P. V. Bioorg. Med. Chem. Lett. 2008, 18, 596.
8. Fish, P. V.; Deur, C.; Gan, X.; Greene, K.; Hoople, D.; MacKenny, M.; Para, K. S.;
Reeves, K.; Ryckmans, T.; Stiff, C.; Stobie, A.; Wakenhut, F.; Whitlock, G. A.
Bioorg. Med. Chem. Lett. 2008, 18, 2562.
9. Lee, J. T.; Kroemer, H. K.; Silberstein, D. J.; Funck-Brentano, C.; Lineberry, M. D.;
Wood, A. J.; Roden, D. M.; Woosley, R. L. New Engl. J. Med. 1990, 322, 100.
10. The assays were a modification of those described by Blakely, R. D.; Clark, J. A.;
Rudnick, G.; Amara, S. G. Anal. Biochem. 1991, 194, 302. HEK293 cells
Oral
Dose (mg/kg)
t1/2 (h)
F (%)
0.013
5.7
91
a
Dog liver microsomes t1/2 = 35 min.
Compound 18 exhibited no significant inhibition of other
CYP450 enzymes (1A2, 2C9, 2C19, IC50s >30 M; 3A4 IC50
17.9 M). We were also pleased to find that it demonstrated
>200-fold selectivity for serotonin and noradrenaline-reuptake
inhibition when assessed against the CEREP/bioprintTM panel of
receptors, enzymes and ion channels.
When we investigated the metabolic profile of compound 18,
we were pleased to find that our strategy of blocking the route of
metabolism had been successful. Indeed, the compound was so
metabolically stable in in vitro microsomal preparations that no
measurable turnover was observed. Although this result was very
encouraging, it does not guarantee that compound 18 will not be
a CYP2D6 substrate in human and differences in exposures be-
tween poor and extensive metabolizers need to be monitored in
clinical studies.
l
l
expressing
a single human amine transporter protein (7500 cells/well in
Millipore 96-well filter bottom plates) were pre-incubated at 25 °C for 5 min
with assay buffer containing vehicle (DMSO in water) or test compound.
Uptake of neurotransmitter into the cells was initiated by the addition of
tritiated 5-HT (50 nM), NA (200 nM) or DA (200 nM) substrates, the samples
were shaken in an incubator at 25 °C for 5 min (5-HT, DA) or 15 min (NA). The
assays were stopped by an ice-cold buffer wash followed by filtration. The
filters were then dried before measuring the amount of radioactivity taken up
into the cells by scintillation counting. Potency of test compounds was
quantified as IC50 values, that is, concentration required to inhibit the specific
uptake of radiolabelled substrate into the cells by 50% relative to maximum
(vehicle only) over a 10-point dose–response range.
11. Mahar Doan, K. M.; Humphreys, J. E.; Webster, L. O.; Wring, S. A.; Shampine, L.
J.; Serabjit-Singh, C. J.; Adkison, K. K.; Polli, J. W. J. Pharm. Exp. Ther. 2002, 303,
1029.
Finally, pharmacokinetic data for compound 18 were generated
in the dog following intravenous and oral administration (Table 4).
Compound 18 exhibited an encouraging pharmacokinetic profile
with a combination of very low clearance, low volume of distribu-
tion, long half-life and very high bioavailability. Based on these
data, compound 18 was predicted to exhibit good pharmacokinet-
ics in humans.
12. Leeson, P. D.; Springthorpe, B. Nat. Rev. Drug Discov. 2007, 6, 881.
13. CaCO-2: Human colon adenocarcinoma cell line. Flux across cells was
measured at 10
lM substrate concentrations. Figures quoted correspond to
the flux rates (Papp  10À6 cm sÀ1) for apical to basolateral (AB) and basolateral
to apical (BA) directions. See: Van de Waterbeemd, H.; Smith, D. A.; Beaumont,
K.; Walker, D. K. J. Med. Chem. 2001, 44, 1313 and references therein.
14. (a) Fermini, B.; Fossa, A. A. Nat. Rev. Drug Discov. 2003, 2, 439; (b) Keating, M. T.;
Sanguinetti, M. C. Cell 2001, 104, 569.