Fluorinated dihydroquinolines as potent n-NOS inhibitors

Article abstract of DOI:10.1016/j.bmcl.2003.11.022

Fluorinated dihydroquinolines showed reduced basicity of the amidine function. Their syntheses and potencies as neuronal nitric oxide synthase (n-NOS) inhibitors are reported.

Full text of DOI:10.1016/j.bmcl.2003.11.022

Bioorganic & Medicinal Chemistry Letters 14 (2004) 743–746
Fluorinated dihydroquinolines as potent n-NOS inhibitors
Stefan Jaroch,a,* Hartmut Rehwinkel,a Peter Holscher,a Detlev Sulzle,b
Gerardine Burton,c Margrit Hillmann,c Fiona M. McDonaldc and Heribert Miklautzd
aMedicinal Chemistry, Corporate Research, Research Center Europe, Schering AG, D-13342-Berlin, Germany
bComputational Chemistry, Corporate Research, Research Center Europe, Schering AG, D-13342-Berlin, Germany
cNeurology Research, Corporate Research, Research Center Europe, Schering AG, D-13342-Berlin, Germany
dPhysical Chemistry, Analytical Development, Schering AG, D-13342-Berlin, Germany
Received 20 August 2003; revised 29 October 2003; accepted 12 November 2003
Dedicated to Professor Wolfgang Steglich on the occasion of his 70th birthday.
Abstract—Fluorinated dihydroquinolines showed reduced basicity of the amidine function. Their syntheses and potencies as neu-
ronal nitric oxide synthase (n-NOS) inhibitors are reported.
# 2003 Elsevier Ltd. All rights reserved.
Nitric oxide synthases (NOS) catalyze the formation of
nitric oxide (NO) through conversion of arginine into
citrulline. NO acts as a messenger in diverse tissues,
however, elevated NO levels in the brain may be asso-
ciated with neurotoxicity, especially in the wake of an
ischemic event.1 Therefore, inhibition of the neuronal
isoform, n-NOS, may be regarded as a possible ther-
apeutic strategy to treat neurodegenerative diseases.2
Figure 1.
Besides n-NOS, two further enzyme isoforms are
known, inducible nitric oxide synthase (i-NOS), which is
predominantly expressed in macrophages and enacts
host defense in the immune system, and endothelial
NOS (e-NOS) expressed in the lining of blood vessels
and playing a pivotal role in vascular homeostasis.
When designing n-NOS inhibitors, selectivity for n-NOS
relative to e-NOS is of paramount importance in order
to avoid adverse cardiovascular effects. In contrast,
additional i-NOS inhibition appears to be advanta-
geous, as increased i-NOS expression has also been
observed during cerebral ischemia.3
crucial. Brain penetration is linked to the basicity of
agents, with too basic compounds prevailing as proto-
nated species at physiological pH (7.4) and thus being
unable to pass the BBB. According to the Henderson–
Hasselbalch equation, for our lead 1a with a pKa value6
of 9.7 only a small fraction of compound (fu=0.5%) is
unprotonated at pH 7.4.7 In an effort to increase the
amount of non-ionized species, fluorine was introduced
at the benzene core and at the 3a-position to lower the
pKa of the amidine function.8
Recently, we described dihydroquinoline 1a and deriva-
tives, for example 1b, as novel n-NOS inhibitors (Fig.
1).4 Introduction of an amine-containing side-chain
gave particularly potent and selective inhibitors, for
example 2.5 Since the compounds are targeted to the
brain, passage through the blood brain barrier (BBB) is
The syntheses of analogues fluorinated at the benzene
core have been described previously4 and are given for
compounds 8a–c in Scheme 1. Boronic acids 4 were
obtained through ortho-lithiation of N-pivaloyl fluoro-
anilines 3. Suzuki coupling with triflate 5 and acidic
hydrolysis gave quinolones 6. Reduction to dihy-
droquinolones 7 was accomplished with magnesium,
predominantly yielding the cis-stereoisomer. The
sequence was completed by a two-step conversion of
lactams 7 into the amidines 8.
* Corresponding author. Tel.: +49-30-468-12146; fax: +49-30-469-
92146; e-mail: stefan.jaroch@schering.de
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.022

744
S. Jaroch et al. / Bioorg. Med. Chem. Lett. 14 (2004) 743–746
Scheme 1. (a) t-BuCOCl, Py; (b) n-BuLi, THF, B(OMe)3; aq HCl; (c) 5, Pd(PPh3)4, Na2CO3, DME–H2O; (d) concd HCl; (e) Mg, MeOH; (f) NCS,
DMF; (g) Lawesson’s reagent, DME; (h) NH3, MeOH.
Scheme 2. (a) Boc2O, DMAP, THF; (b) (PhSO2)2NF, KHMDS or LiNEt2, THF, À78 ꢀC; (c) CF3CO2H, CH2Cl2; (d) Lawesson’s reagent, THF; (e)
NH3, MeOH.
Introduction of a fluoro-substituent at C-3a was rea-
lized through an electrophilic fluorination reaction
(Scheme 2). Lactams 9 were N-protected with Boc2O,
deprotonated at C-3a, and subjected to N-fluoro-
benzenesulfonimide treatment. Removal of the protect-
ing group and thionation gave thiolactams 12, with the
stereochemical assignment being confirmed by X-ray
analysis of 12a. Finally, ammonolysis delivered the
amidines 13.
Based on these results, we set out to further explore the
3a-fluoro series by attaching aminoalkyl residues, that is
chlorobenzylaminopropyl,
methylaminoethyl,
and
methylaminomethyl, at the 7-position (cf. 2). The 3a-
fluoro analogue of 2, 19, was prepared as described in
Scheme 3. Acrylester 145 was transformed into propyl
alcohol 15 and protected as tert-butyldimethylsilyl (TBS)
ether. The lactam was tert-butoxy carbonylated and
fluorinated to 16. Removal of both the Boc and TBS
protecting groups was accomplished with trifluoroacetic
acid leading to trifluoroacetate 17a and alcohol 17b. The
former was converted into the latter by treatment with
First, we examined the basicity of analogues carrying a
fluorine atom at the benzene ring in close proximity to
the amidine. Literature data indicate that the basicity of
fluoro anilines is most markedly influenced by fluoro
substitution in close proximity, that is, at the ortho-
position, to the amino group.9 Indeed, as can be derived
from Table 1, the pKa is reduced by an order of magni-
tude when a 6-fluoro substituent is introduced (8a). A
chloro substituent in para-position (cf. 8b) leads to a fur-
ther decrease, which is more pronounced when a second
fluorine is added (cf. 8c). Fluorination at C-3a was even
more effective in decreasing the basicity: 13a was less
basic than difluoro analogue 8c. Addition of a chlorine
at C-8 (13b) again lowered the pKa by further 0.3 units,
and combination of 3a- and 6-fluorination resulted in
compound 13c, which is three orders of magnitude less
basic than the original lead, 1a, and remains pre-
dominantly unprotonated at physiological pH
(fu(7.4)=86%). In terms of potency, the core fluorinated
analogues 8b and 8c are less potent inhibitors of n-NOS
than 1a, whereas 8a is equipotent.4 Equally, 13a is as
potent and selective as its non-fluorinated counterpart
1a, which shows that reduced amidine basicity does not
affect potency. However, substitution at the benzene
core in the 3a-fluoro series is not as well tolerated as in
the non-fluorinated series: The 8-chloro analogue 13b is
2-fold less potent than 1b and the potency of difluoro
analogue 13c drops 3-fold with respect to 1a and 8a.
Table 1. Inhibition of NOS isoforms by 3a-fluorinated and non-
fluorinated dihydroquinolines
b
Compd
R
X pKa fu(7.4)c(%)
IC50 (mM)a
n-NOS e-NOS i-NOS e/nd i/nd
Selectivity
1a
1b
H
8-Cl
H
H
9.7
9.4
0.5
1
0.16
0.14
3.3
6.2
2.7 21 17
5.7 44 41
8a
8b
8c
6-F
H
8-Cl, 6-F H 8.2
8.5
7
14
20
0.17
0.29 14
0.68 20
5.8
2.3 34 14
8.1 48 28
6,7-F2
H
8.0
5.1 29
8
13a
13b
13c
H
8-Cl
6-F
F
F
F
7.9
7.6
6.6
24
39
86
0.10
0.29 14
0.59 22
2.7
2.1 27 21
8.1 48 28
25
42 37
a NOS activity was determined with recombinant human enzyme based
on the method given in ref 10.
b The pKa of the corresponding acid was determined by photometric
titration.11
c See ref 7.
d e/n represents IC50(e-NOS)/IC50(n-NOS) and i/n represents IC50(i-
NOS)/IC50(n-NOS).

S. Jaroch et al. / Bioorg. Med. Chem. Lett. 14 (2004) 743–746
745
Scheme 3. (a) Mg, MeOH; (b) NaOH, EtOH–THF; H2SO4; (c) ClCO2Et, Et3N, THF; (d) NaBH4, MeOH; (e) TBSCl, imidazole, DMF; (f) Boc2O,
DMAP, THF; (g) (PhSO2)2NF, KHMDS, THF; (h) CF3CO2H, CH2Cl2; (i) K2CO3, MeOH; (j) (COCl)2, DMSO, Et3N, CH2Cl2; (k)
3-ClC6H4CH2NH2, NaBH(OAc)3, AcOH, (CH2Cl)2; (l) Boc2O, cat DMAP, CH2Cl2; (m) Lawesson’s reagent, THF; (n) NH3, MeOH; (o) HCl,
dioxane.
Scheme 4. (a) TBSCl, imidazole, DMF; (b) Boc2O, DMAP, CH2Cl2; (c) (PhSO2)2NF, KHMDS, THF; (d) CF3CO2H, CH2Cl2; (e) K2CO3, MeOH;
(f) MeSO2Cl, Et3N, CH2Cl2; (g) MeNH2, MeOH; (h) Boc2O, cat DMAP, CH2Cl2; (i) Lawesson’s reagent, THF; (j) NH3, MeOH; (k) HCl, dioxane.
Table 2. Inhibition of NOS isoforms by 3a-fluorinated 7-(aminoalkyl)dihydroquinolines
b
Compd
R
X
pKa
IC50 (mM)a
e-NOS
Selectivity
n-NOS
i-NOS
e/nc
i/nc
2
19
25a
25b
3-ClBnNHCH2CH2CH2
3-ClBnNHCH2CH2CH2
MeNHCH2CH2
H
F
F
F
9.9
8.0
7.9
n.d.
0.042
0.31
0.39
0.66
9.4
38
79
66
0.58
2.2
1.9
1.4
224
123
203
100
14
7
5
MeNHCH2
2
a NOS activity was determined with recombinant human enzyme based on the method given in ref 10.
b The pKa of the amidine was determined by photometric titration; n.d. means not determined.11
c e/n represents IC50(e-NOS)/IC50(n-NOS) and i/n represents IC50(i-NOS)/IC50(n-NOS).
potassium carbonate in methanol. Swern oxidation,
reductive amination and Boc protection of the second-
ary amine yielded lactam 18 which was transformed into
the final product 19 through the standard operations.5
roquinolines is critical; a drop in potency for all fluori-
nated derivatives is observed, though an interesting
selectivity profile with >100-fold selectivity against e-
NOS and a decent inhibitory potency against i-NOS is
maintained. The 3a-fluoro analogue 19 loses out 8-fold
compared to 2 and is threefold less potent than the
unsubstituted 13a. Truncation of the side chain as in
25a and 25b does not improve potency, but for 25a an
enhanced selectivity versus e-NOS is observed.
Methylaminomethyl and methylaminoethyl analogues
25a and 25b were synthesized in a similar fashion to 19
(Scheme 4). The methylamino group was introduced
through a nucleophilic displacement reaction of mesyl-
ates 23 with methylamine.
In summary, fluorination at 3a proves to be an efficient
means of reducing the basicity of dihydroquinolines and
maintaining potent n-NOS inhibition. Substitution at
the benzene ring in the 3a-fluoro series is not as broadly
tolerated as in the non-fluorinated series. In particular,
The biological data for the fluorinated 7-(aminoalkyl)-
dihydroquinolines are given in Table 2 compared to the
non-fluorinated analogue 2. The data confirm the trend
that substitution at the benzene core of 3a-fluorodihyd-

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S. Jaroch et al. / Bioorg. Med. Chem. Lett. 14 (2004) 743–746
the introduction of amine-containing side chains, while
not improving potency, gives highly selective n-NOS
inhibitors.
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Acknowledgements
The dedicated and skillful technical assistance of Mrs.
Barbel Bennua-Skalmowski and Mr. Detlev Schmidt is
gratefully acknowledged.
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