Wake promoting agents: Search for next generation modafinil, lessons learned: Part III

Article abstract of DOI:10.1016/j.bmcl.2012.04.031

In searching for a next generation molecule to the novel wake promoting agent modafinil (compound 1), a series of fluorene-derived wakefulness enhancing agents were developed and evaluated in rat. Extensive pharmacokinetic studies of a potent member of the series (compound 15) revealed that the wake promotion activity of the analog was likely due to an active metabolite (compound 3).

Full text of DOI:10.1016/j.bmcl.2012.04.031

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3751–3753
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Wake promoting agents: Search for next generation modafinil, lessons
learned: Part III
Derek Dunn, Greg Hostetler, Mohamed Iqbal, Val R. Marcy, Yin Guo Lin, Bruce Jones, Lisa D. Aimone,
⇑
John Gruner, Mark A. Ator, Edward R. Bacon, Sankar Chatterjee
Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380-4245, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In searching for a next generation molecule to the novel wake promoting agent modafinil (compound 1),
a series of fluorene-derived wakefulness enhancing agents were developed and evaluated in rat. Exten-
sive pharmacokinetic studies of a potent member of the series (compound 15) revealed that the wake
promotion activity of the analog was likely due to an active metabolite (compound 3).
Ó 2012 Elsevier Ltd. All rights reserved.
Received 13 February 2012
Revised 22 March 2012
Accepted 3 April 2012
Available online 13 April 2012
Keywords:
Wake-promoting agent
Modafinil
Transporters
O
S
O
Disorders of ‘wakefulness’ including states of impaired alertness,
vigilance and attention affect millions of individuals. Relatively few
pharmacotherapies are available to treat such symptoms. Modafinil
(compound 1, Fig. 1), a novel agent, pharmacologically distinct from
classical stimulants (caffeine, amphetamine, and methylphenidate),
improves wakefulness in a variety of species and is efficacious in
humans with few peripheral or central side effects.1 While the
precise mode of action of modafinil has yet to be well-defined, due
to its modest binding activity, mechanistic studies have suggested
that dopamine (DAT) and norepinephrine transporters (NET) con-
tribute to modafinil’s wake-promoting pharmacology with a causal
or indirect relationship.2–4
O
S
O
NH2
Previous work
NH2
This work
1
2
Cl
O
S
O
NR1R2
A
Figure 1. Chemical structures of compound 1, compound 2, and current series (A).
While modafinil continues to be evaluated for expanded clinical
applications, a few aspects of its overall profile have served as the
cornerstone for efforts to identify a follow-on molecule. Modafinil
demonstrates modest inhibition of CYP2C19 (IC50 = 11 lM) but
shows virtually no interaction with CYP3A4 and CYP2D6. Since
clinical studies demonstrated that human plasma concentrations
can reach levels greater than 30 lM at efficacious dose, the poten-
tial for drug–drug interactions is possible which will become
important as modafinil is used as an adjunctive therapy in patients
with psychiatric and/or neurological disorders. Furthermore, it was
anticipated that a follow-on molecule from a different structural
scaffold but having a modafinil-like (or better) profile, distinct
from classical psychostimulants, might shed more light in elucidat-
ing the mechanism of action of modafinil. Also, a molecule of this
class might help to elucidate wake promoting mechanisms in gen-
eral, especially contributory roles played by various transporters.
Previously, our laboratories disclosed biphenyl-derived wake pro-
moting agents, exemplified by structure 2 (Fig. 1).5 In this Letter,
we report another aspect of our discovery effort disclosing a series
of fluorene-based wake promoting agents (in rat) exemplified by
generic structure A (Fig. 1).
Scheme 1 depicts the general synthetic scheme that was uti-
lized to generate the target compounds. Coupling of compounds
3a–b and 4 in acidic media produced generic compound 5 that
on basic hydrolysis followed by treatment with chloroacetic acid
generated carboxylic acid 6. Compound 6, in turn, was converted
to amide of generic structure 7. Compound 7, on controlled oxida-
tion, generated target compounds 8–19.
⇑
Corresponding author.
E-mail address: Sankar.Chatterjee24@gmail.com (S. Chatterjee).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.031

3752
D. Dunn et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3751–3753
O
S
O
S
X
X
O
OH
a
+
S
NH2
NH
NR1R2
H2N
NH2
OH
+
15
Figure 2. Metabolism of compound 15 in rat.
21
3
5
3 a) X = H
b) X = Me
4
b
O
X
e
O
O
S
X
Y
S
in literature. Thus the exploration began with locking two phenyl
rings of the parent molecule via a central five-member ring main-
taining the distant sufinylacetamide moiety, thought to be unique
for a CNS drug. As shown in Table 1, the initial analog 8 displayed
similar wake promoting activity of parent compound 1. However,
compound 8 did not display any significant DAT inhibitory activity,
bringing the contributory role of DAT in the wake promoting activ-
ity of this compound into question (vide infra). The corresponding
methyl analog, compound 9 bearing a quaternary carbon center at
the fluorene scaffold was comparatively less active in the wake
promotion assay. This suggested a detrimental effect of steric
congestion on activity from this site. While modification of the pri-
mary carboxamide moiety of compound 8 to a secondary methyl-
amide, as in compound 10, was slightly detrimental, extension of
the methyl chain to an ethanol moiety (compound 11) produced
wake promoting activity raising the possibility of a beneficial inter-
action from a distant site. While conversion of the primary carbox-
amide of compound 8 to a linear tertiary carboxamide (compound
12) was not beneficial, introduction of a cyclic amine moiety in the
region was significantly productive (compounds 13, 14 and 15;
note these compounds were screened at 30 mg/kg ip since residual
waking activity beyond 3 h was observed at the higher dose).
Hypothesizing additional steric bulk in the carboxamide area
might be beneficial, the series was further extended by including
amino acid residues for example compounds 16, 17, 18, and 19.
Note the last three compounds were tested as a mixture of diaste-
reomers. All four compounds displayed superior activity in the
NR1R2
c
or
d
Y = OH
Y = NR1R2
6
8 - 19
7
Scheme 1. Reagents and conditions: (a) 48% HBr, H2O, mixing at 60 °C followed by
reflux, 0.5 h, 90%; (b) (i) 10 N NaOH, 80 °C, 1 h; (ii) ClCH2COOH, reflux, 2 h, 80% over
two steps; (c) for Y = NH2: (i) SOCl2, C6H6, reflux, 3 h; (ii) 28% NH4OH, CH2Cl2, 0 °C –
rt, 0.5 h, 70% over two steps; (d) for Y = NR1R2: HATU, HOAt, NMM, DMF, 0 °C to rt,
overnight, 50–70%; (e) 30% H2O2, gl. acetic acid, rt, 2 h, 80–90%.
At the outset of our synthetic program, no well-defined molec-
ular target(s)4b,c and no historical database of modafinil’s wake
promotion structure–activity relationships existed in the litera-
ture.6 Thus the members of the new series were initially evaluated
in a DAT binding assay (rat).7 Subsequently cumulative wake-pro-
moting activity in rats [i.e., total time (min) awake over a period of
3 h after dosing (3 h AUC) at 100 mg/kg ip] was utilized as the
biological activity of the new analogs following our previously
published procedure.5 Compounds were also evaluated in
a
CYP2C19 enzyme inhibitory assay. Table 1 displays the biological
data for modafinil, analogs 8–19 and compound 3 (vide infra).
As mentioned previously, at the outset of our synthetic pro-
gram, no known molecular target describing modafinil-SAR existed
Table 1
Biological data of new analogs
O
S
X
O
NR1R2
8 - 19
Compound
X
NR1R2
DAT IC50
(l
M)
Wake 3 h AUC (min)a
2C19 IC50 (lM) or %Inhib. at 10 lM
1b
Modafinil
NH2
NH2
NHMe
NH(CH2)2OH
NEt2
N-Morpholinyl
N-Piperazinyl
N-(4-Acetyl)-piperazinyl
N-Glycinamide
(S)-N-Alaninylamide
(S)-N-Serinylamide
(S)-N-Prolinylamide
—
3.70
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9
117 13⁄
11
8b
H
Me
H
H
H
H
H
H
H
H
H
H
—
116 8⁄
>100
26%
48%
>100
>100
30%
>100
>100
>100
>100
>100
>100
—
9b
71
5
10b
11b
12b
13b,c
14b,c
15b,c
16b
17d
18d
19d
3
83 6⁄
167 5⁄
99 5⁄
158 10⁄
119 17
146 12
140 10⁄
151 14⁄
136 14⁄
169 13⁄
163 3⁄
Vehiclee
—
—
65
9
—
a
b
c
Mean SEM; ⁄P <0.05 versus within-experiment vehicle control.
Tested as a mixture of enantiomers.
Tested at a dose of 30 mg/kg.
Tested as a mixture of diastereomers.
Average of vehicle group: mean and SEM values (N = 11) for compounds shown.
d
e

D. Dunn et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3751–3753
3753
Table 2
Pharmacokinetic parameters
Pharmacokinetic parameters
Plasma
Brain
Compound 15
Compound 3
Compound 21
Compound 15
Compound 3
Compound 21
Cmax, ng/g
tmax, h
AUC0-t, ng h/g
Mean SEM, n = 3
6286 2973
0.8 0.2
7501 2657
263 44
255 70
1.2 0.4
939 214
416 272
0.8 0.2
295 252
4384 844
0.8 0.2
11639 2773
971 309
0.8 0.2
2349 665
2
0
1081 149
Dosed as compound 15 at 116 mg/kg ip
should be noted that compound 3 maintains some DAT binding
activity, though less active than modafinil, raising the possibility
of involvement of this transporter in its wake promoting activity
in rats. However, no additional study was carried out with com-
pound 3, as a more efficacious molecule (compound 2, Fig. 1), ac-
tive both in rat and dog wake promotion assays, had already
been identified.
In this Letter, we disclosed a series of fluorene-derived wakeful-
ness promoting agents (in rat) in search of a next generation mol-
ecule to modafinil. From this work, representative compound 15
was advanced for additional profiling. However, compound 15
lacked wake promoting activity in dogs. Investigative work re-
vealed that the wake promoting activity of compound 15 in rats
might have been due to the reactive metabolite 3.
10000
1000
100
10
Compound 15
Compound 3
Compound 21
1
0
1
2
3
4
5
6
7
8
Time (h)
Acknowledgments
Figure 3. Brain levels in rats dosed with compound 15 116 mg/kg ip, 0.5%
methylcellulose, 0.2% tween 80.
Authors wish to acknowledge the scientific support and encour-
agement of Drs. Donna Bozyczko-Coyne, John P. Mallamo and Jeffry
L. Vaught during the course of this research. Authors sincerely
thank the reviewers for their constructive criticism and valuable
suggestions to improve the quality of the manuscript.
wake promotion assay. As shown in Table 1, compounds 8–19 did
not display any significant DAT inhibitory activity, compared to
modafinil’s modest activity in the same assay. Similarly, most of
them (except 9, 10 and 13) did not offer any unwarranted 2C19-
liability.
References and Notes
1. Modafinil had been introduced in the market by Cephalon, Inc., Frazer, PA under
the trade name ProvigilÒ. Detailed information on the drug could be found in the
website www.provigil.com (accessed on February 12, 2012).
2. Andersen, M. L.; Kessler, E.; Murnane, K. S.; McClung, J. C.; Tufik, S.; Howell, L. L.
Psychopharmacology 2010, 210, 439; (b) Wisor, J. P.; Nishino, S.; Sora, I.; Uhl, G.
L.; Mignot, E.; Edgar, D. M. J. Neurosci. 2001, 21, 1787.
3. Volkow, N. D.; Fowler, J. S.; Logan, J.; Alexoff, D.; Zhu, W.; Telang, F.; Wang, G. J.;
Jayne, M.; Hooker, J. M.; Wong, C.; Hubbard, B.; Carter, P.; Warner, D.; King, P.;
Shea, C.; Xu, Y.; Muench, L.; Apelskog-Torres, K. JAMA 2009, 301, 1148.
4. (a) Madras, B. K.; Xie, Z.; Lin, Z.; Jassen, A.; Panas, H.; Lynch, L.; Johnson, R.; Livni,
E.; Spencer, T. J.; Bonab, A. A.; Miller, G. M.; Fischman, A. J. J. Pharmacol. Exp. Ther.
2006, 319, 561; (b) Nishino, S.; Mao, S.; Sampathkumaran, R.; Shelton, J. Sleep
Res. Online 1998, 1, 49; (c) Mignot, E.; Nishino, S.; Guilleminault, C.; Demont, W.
C. Sleep 1994, 17, 436.
5. Dunn, D.; Hostetler, G.; Iqbal, M.; Messina-McLaughlin, P.; Reiboldt, A.; Lin, Y. G.;
Gruner, J.; Bacon, E. R.; Ator, M. A.; Chatterjee, S. Bioorg. Med. Chem. Lett., in
press.
6. After our work was completed, following publication describing several
modafinil analogs appeared in the literature: Cao, J.; Prisinzano, T. E.;
Okunola, O. M.; Kopajtic, T.; Shook, M.; Katz, J. L.; Newman, A. M. ACS Med.
Chem. Lett. 2011, 2, 48.
7. Protocol for DAT binding assay: Frozen striata (Pel-Freez) were homogenized in
10 volumes of buffer (10 mM NaPO4 pH 7.4; 140 mM NaCl) and centrifuged at
10,000 g for 10 min (4 °C). The pellet was suspended and centrifuged again with
the remaining pellet suspended to a final concentration of 1 mg tissue/ml. 3H-
WIN-35428 (Perkin–Elmer) was added to a final concentration of 4 nM and
incubated for 120 min at room temperature. Reactions were terminated by
From this series, representative compound 15 was advanced for
additional profiling as well as measuring its wake promotion activ-
ity in a higher species. However, the compound displayed a lack of
wake promoting activity in dogs. Subsequent detailed pharmacoki-
netic work in rats revealed that compound 15 underwent metabo-
lism generating compound 3 (major metabolite) and compound
21 (minor metabolite) in rats (Fig. 2, NR1R2 = N-(4-Acetyl)-
piperazinyl).
After ip dosing of compound 15 in rats, plasma levels of the two
metabolites were ꢀ4% of the level of the parent compound as mea-
sured by Cmax and 13% as represented by the AUC. However, in
brain the parent was extensively converted to compound 3 and
to a lesser extent to compound 21 (Table 2 and Fig. 3). Cmax concen-
trations of compound 3 in brain were over 10-fold that of the par-
ent compound with an AUC ca 40 times that of compound 15. To a
lesser extent, compound 21 was also formed in the brain following
administration of compound 15.
As shown in Table 1, subsequent screening in wake promoting
assay in rats, compound 3 (100 mg/kg ip) was found to be active.
Thus, it is likely that the wake promoting activity of compound
15 in rats might have arisen from the activity of compound 3. On
the other hand, such metabolism might be minimal in dogs. It
filtration. Nomifensine (20 lM) was used to determine nonspecific binding.