Antimycobacterial and H1-antihistaminic activity of 2-substituted piperidine derivatives

Article abstract of DOI:10.1016/j.bmc.2008.10.042

2-Substituted derivatives of the antihistaminic agents Bamipine, Diphenylpyraline and of their 1-phenyl analogues were tested for their antimycobacterial and H₁-antagonistic activities. They are strong H₁-receptor antagonists and also inhibit the growth of mycobacterials with a maximum MIC of 6.25 μg/mL against Mycobacterium tuberculosis H₃₇Rv. H₁-receptor antagonistic potency was slightly decreased by substitution in ring position 2 and distinctly diminished by N-aryl substitution. The antimycobacterial potency of Diphenylpyraline was in general increased by substitution in ring position 2, whereas only a few Bamipine derivatives showed markedly improved activity. A correlation between the two activities was not detected for those compounds.

Full text of DOI:10.1016/j.bmc.2008.10.042

Bioorganic & Medicinal Chemistry 16 (2008) 10326–10331
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antimycobacterial and H1-antihistaminic activity of 2-substituted
piperidine derivatives
a
a
a
a
b
Robert Weis a, , Klaus Schweiger , Johanna Faist , Erich Rajkovic , Andreas J. Kungl , Walter M. F. Fabian ,
*
Walter Schunack c, Werner Seebacher a
a Institute of Pharmaceutical Sciences, Pharmaceutical Chemistry, University of Graz, Universitätsplatz 1, A-8010 Graz, Austria
b Institute of Chemistry, University of Graz, Heinrichstraße 28, A-8010 Graz, Austria
c Institute of Pharmacy, Free University of Berlin, Königin-Luise-Straße 2 + 4, D-14195 Berlin, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
2-Substituted derivatives of the antihistaminic agents Bamipine, Diphenylpyraline and of their 1-phenyl
analogues were tested for their antimycobacterial and H1-antagonistic activities. They are strong H1-
receptor antagonists and also inhibit the growth of mycobacterials with a maximum MIC of 6.25
Received 11 July 2008
Accepted 17 October 2008
Available online 22 October 2008
l
g/mL against Mycobacterium tuberculosis H37Rv. H1-receptor antagonistic potency was slightly decreased
by substitution in ring position 2 and distinctly diminished by N-aryl substitution. The antimycobacterial
potency of Diphenylpyraline was in general increased by substitution in ring position 2, whereas only a
few Bamipine derivatives showed markedly improved activity. A correlation between the two activities
was not detected for those compounds.
Keywords:
Antihistamines
Antimycobacterials
Piperidines
Ó 2008 Elsevier Ltd. All rights reserved.
Tuberculosis
1. Introduction
Compounds with alkyl substituents in ring position 2 showed high-
er activity.7
With more than 1.6 million deaths and 8.8 million new cases
being reported per year tuberculosis is the leading infectious cause
of death today.1 Due to the spreading of multi-drug-resistant and
extensively drug-resistant strains, new antimycobacterial drugs
with alternative mechanism of action are urgently needed for the
therapy of tuberculosis.2 A number of compounds with H1-recep-
tor antagonistic activity were far more active against Mycobacte-
rium tuberculosis than against other bacterials.3–5 Since histamine
is a growth-promotor for mycobacterials and the growth inhibition
was blocked by the addition of histamine, the antimycobacterial
activity of antihistaminics has been argued to be linked to their
histamine-receptor antagonistic potency.4,5 We modified the struc-
tures of two of those antihistaminic compounds, Diphenylpyraline
(1) and Bamipine (2), in ring positions 1 and 2 (Fig. 1).
In the present paper, the synthesis of some new analogues of
Bamipine will be described, which were prepared to complete the
series for the purpose of comparison. The antimycobacterial activ-
ities and the cytotoxic properties of all 2-substituted derivatives of
Bamipine (2) were examined and compared to those of compounds
1 and 2 in order to elucidate structure–activity relationships. In
addition, the H1-antihistaminic activity of selected compounds
was tested and investigated for a possible correlation between
those activities.
2. Results
2.1. Chemistry
A great number of derivatives of Diphenylpyraline and Bamipine
derivatives with different substitution on the ring nitrogen or at
the aromatic ring system have been reported, whereas substitution
of the piperidine ring was restricted to a few methyl analogues.
Recently, we reported the synthesis of 2-alkyl and 2-aryl substi-
tuted derivatives of 1 and 2 and their 1-phenyl analogues which
have been prepared via new pathways.6–8 The Diphenylpyraline
derivatives were tested against Mycobacterium tuberculosis H37Rv.
Considering that enhanced lipophilicity was argued to increase
the antimycobacterial activity of antihistamines,4 we prepared
Me
N
Me
N
H
OCH(Ph)2
H
NBzlPh
1
2
* Corresponding author. Tel.: +43 316 380 5379; fax: +43 316 380 9846.
E-mail address: robert.weis@uni-graz.at (R. Weis).
Figure 1. Structures of Diphenylpyraline (1) and Bamipine (2).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.042

R. Weis et al. / Bioorg. Med. Chem. 16 (2008) 10326–10331
10327
2-alkyl and 2-aryl derivatives of 1 and 2 as well as the correspond-
ing 1-phenyl analogues. In contrast to the formerly reported syn-
theses of Diphenylpyraline or Bamipine derivatives our method is
based on the conversion of easily available 4-aminodihydropyri-
dine-2(1H)-thiones to 2-substituted piperidines. The syntheses of
the Diphenylpyraline derivatives 3–7 and of the Bamipine deriva-
tives 8–10 have already been reported.6–8
The new 2,2-dimethyl analogues 11–13 of Bamipine were ob-
tained by means of slightly modified procedures. The corresponding
N-arylbenzylamines9 were fused with the 4-hydroxypyridine-
2(1H)-thione 14,10 giving the 4-aminodihydropyridine-2(1H)-thi-
ones 15, 16 in good yields. Their S-methyl derivatives 17, 18 were
further methylated yielding the 1-methylpyridinium salts 19, 20.
Compounds 19, 20 were converted to the 4-anilino piperidines 21,
22 via hydrogenation with Raney nickel W-211 at 45 psi. Final N-
alkylation with benzyl chloride gave the Bamipine derivatives 11,
12 (Scheme 1).
The corresponding 1-phenyl analogue 13 was synthesized from
the 1-substituted 4-anilinodihydropyridine-2(1H)-thione 23,12
which was converted to its isomeric methoiodides cis-24 and
trans-24. Those were hydrogenated giving the 4-anilino-1-phen-
ylpiperidine 25, which was alkylated with benzyl chloride afford-
ing the 1-phenyl substituted analogue 13 of compound 11.
The discrimination between the isomeric compounds cis-24 and
trans-24 was accomplished via NMR experiments. In the 1H NMR
spectrum the resonance of the 5-H of cis-24 was shifted to lower
frequencies due to shielding of the phenyl ring. The assignment
was confirmed by an NOE from the 5-H of cis-24 to aromatic ortho
protons.
The structures of all new piperidine derivatives were established
by NMR spectroscopy as outlined in Ref. 8 All of the new piperidine
derivatives gave NOEs between the axial protons in ring positions 3
and 5 as well as between 4 and 6. The preference for the chair
conformation of the piperidine rings was confirmed by the charac-
teristic coupling constants of the corresponding ring protons in the
1H NMR spectra. The distinction between the 1H- and 13C-reso-
nances of axial and equatorial methyl groups in ring position 2 of
the piperidine derivatives succeeded by means of NOE experiments.
Through-space interactions from the axial protons in ring positions
4 or 6 to protons of the axial 2-methyl groups were observed.
analogues trans-5 and 7 were among the least active compounds.
For the most active compound 13 a minimum inhibitory concentra-
tion (MIC) of 6.25
mined (Table 1).
lg/mL against M. tuberculosis H37Rv was deter-
With the exception of cis-8 all of the tested compounds were
less cytotoxic than Bamipine (2). Fortunately the cytotoxicity of
the most active compound 13 is comparable to that of Diphenylpyr-
aline (1) which is still used in the therapy of rhinitis. However,
compared to the antimycobacterial drugs in use (INH, Rifampicin)
the cytotoxicity of the prepared compounds needs further
improvement.
The H1-receptor antagonist potencies of compounds 1, 2, 4, 11
and 13 were examined. Diphenylpyraline (1) was slightly more
potent than Bamipine (2). Their 2,2-dimethyl derivatives 4 and 11
were still potent H1-receptor antagonists, but the dimethylation
in position 2 led to loss of activity of about 0.6 log units retaining
the Diphenylpyraline analogue 4 as the more effective compound.
Both substances showed competitive antagonism at concentra-
tions between 0.3 Â 10À7 mol/L and 1 Â 10À6 mol/L shifting the
dosage–response curve parallel to the right. At concentrations
between 0.3 and 1 Â 10À5 mol/L the dosage–response curves were
depressed indicating non-competitive antagonism. Only slight
activity (Àlog KB = 5) was detectable for the 1-phenyl compound
13 at 0.3 Â 10À4 mol/L (Table 1). For compounds 1, 2, 4, 11 and
13 molecular modeling studies were undertaken in order to inves-
tigate the reasons for the decreased H1-receptor antagonist
potency of the prepared 2-substituted and especially the 1-phenyl
analogues of compounds 1 and 2. Neutral molecules as well as pro-
tonated and diprotonated dications were considered. The con-
former populations were not affected by dimethylation at C-2.
The loss of activity of about 0.6 log units (1 vs 4, 2 vs 11) can be
attributed to steric hindrance and to the changes in the molecular
electrostatic and lipophilic potential. Thus, the drop in H1-receptor
antagonist potency in 4 and 11 can be attributed to a ‘‘shielding”
effect of the 2,2-dimethyl moiety of the positive charge at the pro-
tonated N-1 center. The very low H1-antihistaminic activity (loss of
>3 log units) of the 1-phenyl compound 13 can be explained by a
combination of increased ‘‘shielding” of N-1 and diminished basi-
city (For computational details see Supplementary material).
In both series a 1-phenyl derivative was the most active anti-
mycobacterial agent. Since those compounds possess negligible
H1-antihistaminic activity the presumed linkage between H1-anti-
histaminic and antimycobacterial activity has not been confirmed.
2.2. Antimycobacterial and antihistaminic activity, cytotoxicity
The antimycobacterial activity and the cytotoxicity of Bamipine
(2) and its derivatives 8–13 have been determined and are com-
pared to those of the corresponding Diphenylpyraline analogues 1,
3–7 in Table 1, which in addition contains the H1-antihistaminic
activity of selected compounds.
4. Conclusion
The H1-receptor antagonistic activities of Diphenylpyraline and
Bamipine were slightly decreased by substitution in ring position
2 and nearly lost by replacement of the 1-methyl by a 1-phenyl
group. In contrast the antimycobacterial activity was nearly always
increased by this derivatization. The supposed correlation of anti-
mycobacterial and H1-receptor antagonistic activity of piperidine
derivatives was not observed for the tested analogues of Diphenyl-
pyraline and Bamipine. Moreover, the compound with the highest
3. Discussion
The antimycobacterial activities of Diphenylpyraline (1) and
Bamipine (2) were only slightly changed by dimethylation in posi-
tion 2 (compounds 4, 11 and 12). However, an additional equatorial
isopropyl substituent increased the activity remarkably, as was ob-
served for compounds cis-3 and cis-8. In the 1-phenyl series the
influence of the substitution was not the same in both compared
series. The Diphenylpyraline derivatives cis-5 and cis-6 (36–39%
activity against Mycobacterium tuberculosis H37Rv (MIC: 6.25 lg/
mL) exhibits neglectable H1-antihistaminic activity indicating an
alternative mechanism of action.
5. Experimental
inhibition at 6.25
lg/mL) showed improved potency and trans-6
(75% inhibition at 6.25
l
g/mL) had significantly increased activity.
5.1. Instrumentation and chemicals
Their corresponding analogues in the Bamipine series cis-9, cis-10
and trans-10 were less or slightly more active than their parent
compound 2. However, compounds trans-9 (52% inhibition at
Melting points were obtained on a melting point apparatus Dr.
Tottoli (Büchi 510) or on a digital melting point apparatus Electro-
thermal IA 9200 and are uncorrected. IR spectra: infrared spec-
trometer system 2000 FT (Perkin-Elmer). NMR spectra: Varian
6.25
lg/mL) and 13 (91% inhibition at 6.25 lg/mL) were the most
active 1-phenyl derivatives of 2, whereas their Diphenylpyraline

10328
R. Weis et al. / Bioorg. Med. Chem. 16 (2008) 10326–10331
H
N
H
N
Ph
Me
Me
Me
Me
Me
S
S
N
S
i
Me
NBzlR4
15,16
OH
14
NHPh
23
vi
ii
Ph
Ph
Me
Me
Me
Me
Me
MeS
N
MeS
N
MeS
N
Me
+
I
I
NBzlR4
17,18
N
N
H
Ph
Ph
H
trans-24
cis-24
vii
iii
R1
N
Me
N
I
Me
Me
Me
MeS
iv
Me
H
21,22,25
NHR4
NBzlR4
19,20
v
R1
N
R1
R1
N
R2
R3
R2
R3
Me
Me
N
H
OCH(Ph)2
H
NBzlPh
8-10
H
NBzlR4
11-13
3-7
R2 R3 R4
R1
Compound
cis-3, cis-8
Me
Me
Ph
H
iPr ---
Me ---
iPr ---
Me
4
cis-5, cis-9
H
trans-5, trans-9
cis-6, cis-10
Ph iPr H ---
Ph Ph ---
Ph Ph ---
Me
H
trans-6, trans-10
H
7
Ph
Me ---
11, 21
Me --- --- Ph
Me --- --- Ar
Ph --- --- Ph
12, 22
13, 25
15, 17, 19
16, 18, 20
---
---
--- --- Ph
--- --- Ar
Ar=4-methoxyphenyl; Bzl=benzyl
aReagents and reaction conditions: (i) NHBzlPh or NHBzlAr, 120 °C, 16h; (ii) (1) CH3I, CHCl3,
room temperature (rt), 16 h, (2) NaOH, rt, 1 h; (iii) CH3I, CHCl3, rt, 16 h; (iv) Raney nickel W-
2, EtOH, rt; (v) BzlCl, NaNH2, PhMe, 110 °C, 6 h; (vi) CH3I, CHCl3, rt, 16 h; (vii) Raney nickel
W-2, ethanol, 30-45 psi (H2), rt, 16 h.
Scheme 1.

R. Weis et al. / Bioorg. Med. Chem. 16 (2008) 10326–10331
10329
Table 1
56.00 (NCH2), 99.61 (C-3), 114.79, 126.76, 127.23, 128.71, 128.80,
136.46, 137.09, 157.97 (aromatic C), 151.77 (C-4), 189.16 (C-2).
Anal. Calcd for C20H22N2S: C, 71.56; H, 6.86; N, 7.95; S, 9.10. Found:
C, 71.51; H, 6.99; N, 7.90; S, 8.95.
Antimycobacterial activity (Alamar Blue Assay), cytotoxicity and H1-receptor antag-
onist activity
Compound
% Inhibition MIC
Cytotoxicity
(LC50)
M)a
H1-receptor
6.25
l
g/mLa
l
g/mLa
lg/mL guinea-pig
(
l
ileum (ÀlogKB)b
5.2.1.2. General procedure for the synthesis of 1-unsubstituted
2,2-dimethyl-6-methylthio-2,3-dihydropyridines (17, 18). To an
ice-cooled solution of the dihydropyridinethiones 15, 16 in CHCl3
(80 mL), a solution of iodomethane in CHCl3 (20 mL) was added
through a dropping funnel within 1 h. The reaction mixture was
stirred for 16 h at room temperature, and the solvent was removed
in vacuo. The residue was triturated with ethyl acetate, filtered
with suction, dried with Na2SO4 and recrystallized from CHCl3/
ethyl acetate. The salts were stirred in 1 M NaOH (90 mL, 90 mmol)
for 1 h. Then the suspension was extracted with ether repeatedly.
The combined organic layers were washed three times with H2O
and dried with Na2SO4. The solvent was removed in vacuo and
the residue was recrystallized.
Diphenylpyraline (1)
cis-3
4
cis-5
trans-5
cis-6
trans-6
7
Bamipine (2)
cis-8
cis-9
trans-9
cis-10
trans-10
11
12
13
5
68
18
36
7
39
75
3
29
66
7
52
37
22
31
26
91
—
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
6.25
107.59
98.06
104.67
69.38
97.06
77.82
111.67
65.08
64.08
35.93
126.91
>28
>28
>28
69.46
81.96
107.84
8.79
n.t.
8.19
n.t.
n.t.
n.t.
n.t.
n.t.
8.55
n.t.
n.t.
n.t.
n.t.
n.t.
7.93
n.t.
5.0
5.2.1.2.1. 4-(N-Benzylanilino)-2,2-dimethyl-6-methylthio-2,3-dihy-
dropyridine (17). Compound 15 (9.67 g, 30 mmol) gave with
iodomethane (5.1 g, 36 mmol) pale yellow crystals of 17. Mp
89 °C (EtOH); yield: 7.80 g (77%). IR (KBr) 2970, 1604, 1544,
Isoniazid (INH)
Rifampicin
0.025–0.2 >200
0.06–0.5 >200
n.t.
n.t.
—
a
1496, 1397, 1098, 764, 705 cmÀ1 1H NMR (DMSO-d6, 200 MHz)
.
Data for compounds 1, 3–7 were taken from Ref. 7.
n.t., not tested.
b
d(ppm) 1.06 (s, 6H, (CH3)2), 2.10 (s, 2H, 3-H), 2.17 (s, 3H, SCH3),
4.85 (s, 1H, 5-H), 4.86 (s, 2H, NCH2), 7.18–7.41 (m, 10H, aromatic
H). 13C NMR (DMSO-d6, 50 MHz) d(ppm) 11.23 (SCH3), 28.42
((CH3)2), 38.15 (C-3), 55.53 (C-2), 55.72 (NCH2), 91.86 (C-5),
126.10, 126.64, 126.89, 127.05, 128.60, 129.45, 137.74, 144.37
(aromatic C), 150.74 (C-4), 160.10 (C-6). Anal. Calcd for
C21H24N2S: C, 74.96; H, 7.19; N, 8.32; S, 9.53. Found: C, 75.15; H,
7.49; N, 8.35; S, 9.46.
Gemini 200, Varian Inova 400 (298 K) 5 mm tubes, TMS as internal
standard. 1H- and 13C-resonances were assigned using 1H,1H- and
1H,13C-correlation spectra. Microanalyses: EA 1108 CHNS-O appa-
ratus (Carlo Erba) at the Microanalytical Laboratory at the Institute
of Physical Chemistry, University of Vienna; Hydrogenations were
performed in a Parr hydrogenation apparatus shaker type 3911.
Chromatography: TLC: plates (Merck) silica gel 60 F254
.
5.2.1.2.2.
4-(N-Benzyl-p-anisidino)-2,2-dimethyl-6-methylthio-
2,3-dihydropyridine (18). Compound 16 (10.58 g, 30 mmol) gave
with iodomethane (5.1 g, 36 mmol) pale yellow crystals of 18.
Mp 120 °C (EtOH/H2O); yield: 8.40 g (76%). IR (KBr) 2966, 1603,
5.2. Syntheses
5.2.1. Synthesis of dihydropyridine derivatives
1541, 1509, 1249, 1093, 764, 738 cmÀ1 1H NMR (DMSO-d6,
;
5.2.1.1. General procedure for the synthesis of 4-(N-Benzyla-
rylamino)-6,6-dimethyl-5,6-dihydropyridine-2(1H)-thiones (15,
16). Compound 1410 was fused with the corresponding N-arylb-
enzylamine for 16 h at 120 °C in an argon atmosphere. Then the
mixture was cooled to room temperature and triturated with EtOH
(15 mL). The precipitate was filtered with suction and dried. Then
it was stirred for 1 h at 60 °C in CHCl3 (100 mL). The suspension
was cooled to room temperature and filtered. The filtrate was con-
centrated and recrystallized from EtOH.
400 MHz) d(ppm) 1.15 (s, 6H, (CH3)2), 2.06 (s, 2H, 3-H), 2.31 (s,
3H, SCH3), 3.78 (s, 3H, OCH3), 4.73 (s, 2H, NCH2), 4.94 (s, 1H,
5-H), 6.82–7.32 (m, 10H, aromatic H). 13C NMR (DMSO-d6,
100 MHz) d(ppm) 11.98 (SCH3), 28.25 ((CH3)2), 38.54 (C-3),
55.38 (OCH3), 55.76 (C-2), 56.25 (NCH2), 91.40 (C-5), 114.37,
126.77, 127.08, 128.53, 137.40, 137.54 (aromatic C), 151.47 (C-
4), 157.86 (aromatic C), 161.71 (C-6). Anal. Calcd for C22H26N2OS:
C, 72.09; H, 7.15; N, 7.64; S, 8.75. Found: C, 72.08; H, 7.32; N,
7.60; S, 8.66.
5.2.1.1.1. 4-(N-Benzylanilino)-6,6-dimethyl-5,6-dihydropyridine-
2(1H)-thione (15). Compound 14 (7.86 g, 50 mmol) gave with
N-phenylbenzylamine (10.1 g, 55 mmol) pale yellow crystals of
15. Mp 214 °C; yield: 10.7 g (66%). IR (KBr) 2960, 1554, 1495,
5.2.1.3. 4-Anilino-6,6-dimethyl-1-phenyl-5,6-dihydropyridine-
2(1H)-thione (23). Compound 23 was prepared from 2,2-dime-
thyl-N-phenyl-6-phenylimino-3,6-dihydro-2H-thiopyran-4-amine
by a reported procedure.12 Mp 214 °C (mp 214 °C12). IR (KBr) 2982,
1448, 1390, 1112, 1103, 703 cmÀ1 1H NMR (DMSO-d6, 400 MHz)
.
d(ppm) 1.16 (s, 6H, (CH3)2), 2.30 (s, 2H, 5-H), 4.93 (s, 2H, NCH2),
5.36 (s, 1H, 3-H), 7.25–7.43 (m, 10H, aromatic H), 8.64 (s, 1H,
NH). 13C NMR (DMSO-d6, 100 MHz) d(ppm) 26.84 ((CH3)2), 38.75
(C-5), 52.91 (C-6), 55.86 (NCH2), 100.60 (C-3), 126.64, 127.00,
127.24, 127.42, 128.73, 129.70, 137.13, 143.86 (aromatic C),
151.01 (C-4), 189.26 (C-2). Anal. Calcd for C20H22N2S: C, 74.49; H,
6.88; N, 8.69; S, 9.94. Found: C, 74.44; H, 6.76; N, 8.73; S, 9.68.
1574, 1529, 1492, 1394, 1147, 693 cmÀ1 1H NMR (DMSO-d6,
;
200 MHz) d(ppm) 1.22 (s, 6H, (CH3)2), 2.73 (s, 2H, 5-H), 6.08 (s,
1H, 3-H), 7.08–7.42 (m, 10H, aromatic H), 8.82 (s, 1H, NH) ppm.
13C NMR (DMSO-d6, 50 MHz) d(ppm) 26.42 ((CH3)2), 41.76 (C-5),
58.43 (C-6), 100.26 (C-3), 122.25, 123.99, 126.93, 128.43, 129.27,
129.86, 139.32, 142.98 (aromatic C), 146.32 (C-4), 191.53 (C-2)
ppm.
5.2.1.1.2.
4-((N-Benzyl)-p-anisidino)-6,6-dimethyl-5,6-dihydro-
pyridine-2(1H)-thione (16). Compound 14 (7.89 g, 50 mmol) gave
with N-(4-methoxyphenyl)benzylamine (10.1 g, 55 mmol) pale
yellow crystals of 16. Mp 208 °C; yield: 8.89 g (50%). IR (KBr)
5.2.1.4. General procedure for the synthesis of 1-substituted
2,2-dimethyl-6-methylthio-2,3-dihydropyridines (19, 20, cis-24,
trans-24). To a solution of compounds 17, 18, 23 in CHCl3
(60 mL), a solution of iodomethane in CHCl3 (10 mL) was added
through a dropping funnel within 1 h. The reaction mixture was
stirred for 16 h at room temperature. The solvent was evaporated
in vacuo and the residue was triturated with EtOH/ethyl acetate.
The crystallizate was sucked off and recrystallized.
2967, 1565, 1509, 1446, 1397, 1110, 720 cmÀ1 1H NMR (DMSO-
.
d6, 200 MHz) d(ppm) 1.16 (s, 6H, (CH3)2), 2.27 (s, 2H, 5-H), 3.73
(s, 3H, OCH3), 4.85 (s, 2H, NCH2), 5.31 (s, 1H, 3-H), 6.91–7.37 (m,
9H, aromatic H), 8.54 (s, 1H, NH). 13C NMR (DMSO-d6, 50 MHz)
d(ppm) 26.88 ((CH3)2), 38.70 (C-5), 52.82 (C-6), 55.45 (OCH3),

10330
R. Weis et al. / Bioorg. Med. Chem. 16 (2008) 10326–10331
5.2.1.4.1. 4-(N-Benzylanilino)-1,2,2-trimethyl-6-methylthio-2,3-
5.2.2.2.1. (RS)-( )-4-Anilino-1,2,2-trimethylpiperidine (21). Com-
pound 19 (2.39 g, 5 mmol) was hydrogenated with Raney nickel
W-2.11 The crude base of 21 contained N-phenylbenzylamine as
by-product which was removed by distillation prior to the prepara-
tion of the hygroscopic dihydrochloride. Mp 220 °C (propan-2-ol/
acetone); yield: 1.1 g (73%). IR (KBr) 2939, 2618, 1647, 1603,
dihydropyridiniumiodide (19). Compound 17 (6.73 g, 20 mmol)
gave with iodomethane (14.2 g, 100 mmol) yellow crystals of 19.
Mp 162 °C (EtOH/ethyl acetate); yield: 9.2 g (96%). IR (KBr) 2972,
1561, 1496, 1338, 1276, 1242, 1080, 697 cmÀ1 1H NMR (DMSO-
;
d6, 200 MHz) d(ppm) 1.28 (s, 6H, (CH3)2), 2.51 (s, 3H, SCH3), 2.63
(s, 2H, 3-H), 3.21 (s, 3H, NCH3), 5.27 (s, 2H, NCH2), 5.64 (s, 1H,
5-H), 7.25–7.57 (m, 10H, aromatic H). 13C NMR (DMSO-d6,
50 MHz) d(ppm) 15.65 (SCH3), 23.16 ((CH3)2), 33.37 (NCH3),
39.96 (C-3), 57.40 (NCH2), 60.32 (C-2), 88.52 (C-5), 127.30,
127.55, 127.94, 128.88, 130.06, 135.16, 141.73 (aromatic C),
159.45 (C-4), 173.20 (C-6). Anal. Calcd for C22H27IN2S: C, 55.23;
H, 5.69; I, 26.52; N, 5.86; S, 6.70. Found: C, 55.27; H, 5.78; I,
26.72; N, 5.69; S, 6.65.
1476, 1385, 1320, 1222, 1173, 1124, 1042, 758, 692 cmÀ1 1H
.
NMR (CDCl3, 400 MHz) d(ppm) 1.03 (s, 3H, CH3ax), 1.17 (s, 3H,
CH3eq), 1.28 (dd, J = 12.5, 12.0 Hz, 1H, 3-Hax), 1.43 (dddd, J = 12.2,
12.2, 12.2, 4.8 Hz, 1H, 5-Hax), 1.89 (ddd, J = 12.5, 3.5, 2.7 Hz, 1H, 3-
Heq), 2.05–2.11 (m, 1H, 5-Heq), 2.27 (s, 3H, NCH3), 2.55 (ddd,
J = 12.2, 12.2, 3.1 Hz, 1H, 6-Hax), 2.71 (ddd, J = 12.2, 4.8, 2.7 Hz,
1H, 6-Heq), 3.44–3.52 (m, 1H, 4-Hax), 6.58 (d, J = 8.0 Hz, 2H, o-aro-
matic H), 6.67 (t, J = 8.0 Hz, 1H, p-aromatic H), 7.16 (t, J = 8.0 Hz,
2H, m-aromatic H). 13C NMR (CDCl3, 100 MHz) d(ppm) 14.47
(CH3ax), 30.19 (CH3eq), 33.61 (C-5), 37.32 (NCH3), 46.74 (C-3),
47.25 (C-4), 49.99 (C-6), 54.13 (C-2), 113.15 (o-aromatic C),
117.21 (p-aromatic C), 129.29 (m-aromatic C), 147.08 (i-aromatic
C). Anal. Calcd for C14H24Cl2N2 Â0.5 H2O: C, 56.00; H, 8.39; Cl,
23.61; N, 9.33. Found: C, 55.80; H, 8.49; Cl, 23.25; N, 9.16.
5.2.1.4.2. 4-(N-Benzyl-p-anisidino)-1,2,2-trimethyl-6-methylthio-
2,3-dihydropyridiniumiodide (20). Compound 18 (10.58 g, 20
mmol) gave with iodomethane (5.1 g, 100 mmol) yellow crystals
of 20. Mp 177 °C (EtOH/ethyl acetate); yield: 9.0 g (89%). IR (KBr)
2973, 1566, 1496, 1346, 1294, 1251, 1171, 1112, 1083, 698 cmÀ1
.
1H NMR (DMSO-d6, 400 MHz) d(ppm) 1.28 (s, 6H, (CH3)2), 2.51 (s,
3H, SCH3), 2.60 (s, 2H, 3-H), 3.20 (s, 3H, NCH3), 3.78 (s, 3H,
OCH3), 5.20 (s, 2H, NCH2), 5.62 (s, 1H, 5-H), 7.00–7.39 (m, 9H, aro-
matic H). 13C NMR (DMSO-d6, 100 MHz) d(ppm) 15.66 (SCH3),
23.06 ((CH3)2), 33.25 (NCH3), 39.39 (C-3), 55.54 (OCH3), 57.52
(NCH2), 60.10 (C-2), 88.16 (C-5), 114.93, 127.48, 127.75, 128.37,
128.69, 134.25, 134.96, 158.95 (aromatic C), 159.76 (C-4), 172.92
(C-6). Anal. Calcd for C23H29IN2OS: C, 54.33; H, 5.75; I, 24.96; N,
5.51; S, 6.31. Found: C, 53.96; H, 5.80; I, 24.94; N, 5.33; S, 6.27.
5.2.1.4.3. 2,2-Dimethyl-6-methylthio-N,1-diphenyl-2,3-dihydro-
pyridin-4(1H)-iminiumiodide (cis-24, trans-24). Compound 23
(6.2 g, 20 mmol) gave with iodomethane (3.4 g, 24 mmol) a mix-
ture of cis-24 and trans-24. Mp 191 °C (CHCl3/ethyl acetate); yield:
8.8 g (98%). IR (KBr) 2975, 1606, 1573, 1489, 1474, 1459, 1436,
1212, 1182, 1174, 759, 702 cmÀ1. cis-24 (main component): 1H
NMR (DMSO-d6, 400 MHz) d(ppm) 1.32 (s, 6H, (CH3)2), 2.36 (s,
3H, SCH3), 3.16 (s, 2H, 3-H), 5.73 (s, 1H, 5-H), 7.30–7.60 (m, 10H,
aromatic H), 11.10 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz)
d(ppm) 15.77 (SCH3), 24.64 ((CH3)2), 41.12 (C-3), 61.03 (C-2),
85.93 (C-5), 124.10, 127.53, 129.38, 129.93, 130.00, 130.32,
136.61, 137.54 (aromatic C), 159.42 (C-4), 175.56 (C-6). trans-24
(minor constituent): 1H NMR (DMSO-d6, 400 MHz) d(ppm) 1.22
(s, 6H, (CH3)2), 2.51 (s, 3H, SCH3), 3.16 (s, 2H, 3-H), 5.96 (s, 1H, 5-
H), 7.30–7.60 (m, 10H, aromatic H), 11.10 (s, 1H, NH). 13C NMR
(DMSO-d6, 100 MHz) d(ppm) 15.92 (SCH3), 24.64 ((CH3)2), 37.58
(C-3), 60.84 (C-2), 89.78 (C-5), 124.69, 127.36, 129.49, 129.75,
130.25, 135.97, 137.66 (aromatic C), 162.39 (C-4), 173.27 (C-6).
Anal. Calcd for C20H23IN2S: C, 53.34; H, 5.15; I, 28.17; N, 6.22; S,
7.12. Found: C, 53.26; H, 5.11; I, 28.05; N, 6.01; S, 7.02.
5.2.2.2.2. (RS)-( )-4-(p-Anisidino)-1,2,2-trimethylpiperidine
(22). Compound 20 (2.54 g, 5 mmol) was hydrogenated with
Raney nickel W-2.11 The crude base of 22 contained N-phenylben-
zylamine as by-product which was removed by distillation prior to
the preparation of the hygroscopic dihydrochloride. Mp 217 °C
(ethyl acetate); yield: 1.1 g (68%). IR (KBr) 2928, 2624, 1647, 1598,
1476, 1384, 1321, 1220, 1171, 1114, 1041, 756, 690 cmÀ1. 1H NMR
(CDCl3, 400 MHz) d(ppm) 0.99 (s, 3H, CH3ax), 1.14 (s, 3H, CH3eq),
1.22 (dd, J = 12.6, 12.0 Hz, 1H, 3-Hax), 1.37 (dddd, J = 12.2, 12.2,
12.2, 4.4 Hz, 1H, 5-Hax), 1.86 (ddd, J = 12.6, 3.2, 2.1 Hz, 1H, 3-Heq),
2.02–2.08 (m, 1H, 5-Heq), 2.24 (s, 3H, NCH3), 2.51 (ddd, J = 12.3,
12.3, 2.7 Hz, 1H, 6-Hax), 2.67 (ddd, J = 12.3, 4.4, 2.7 Hz, 1H, 6-Heq),
3.22 (br, 1H, NH), 3.32–3.41 (m, 1H, 4-Hax), 3.72 (s, 1H, OCH3), 6.55
(d, J = 8.9 Hz, 2H, o-aromatic H), 6.75 (d, J = 8.9 Hz, 2H, m-aromatic
H). 13C NMR (CDCl3, 100 MHz) d14.24 (CH3ax), 30.07 (CH3eq), 33.61
(C-5), 37.13 (NCH3), 46.75 (C-3), 48.14 (C-4), 49.84 (C-6), 53.79 (C-
2), 55.54 (OCH3), 114.68 (o-aromatic C), 114.74 (m-aromatic C),
141.08 (i-aromatic C), 151.85 (p-aromatic C). Anal. Calcd for
C15H26Cl2N2O Â0.25 H2O: C, 55.30; H, 8.20; Cl, 21.76; N, 8.60. Found:
C, 55.29; H, 8.20; Cl, 21.81; N, 8.50.
5.2.2.2.3. (RS)-( )-4-Anilino-2,2-dimethyl-1-phenylpiperidine
(25). A mixture of cis-24 and trans-24 (2.25 g, 5 mmol) was
hydrogenated with Raney nickel W-211 giving an oily residue,
which was converted to the dihydrochloride of 25. Mp 237 °C
(EtOH/ethyl acetate); yield: 1.3 g (74%). IR (KBr) 3058, 2662,
2502, 1598, 1488, 1429, 764, 703 cmÀ1
.
1H NMR (CDCl3,
400 MHz) d(ppm) 1.08 (s, 3H, CH3eq), 1.10 (s, 3H, CH3ax), 1.45
(dd, J = 12.6, 11.4 Hz, 1H, 3-Hax), 1.49 (dddd, J = 12.0, 12.0, 11.7,
4.4 Hz, 1H, 5-Hax), 1.89 (ddd, J = 12.6, 3.6, 2.4 Hz, 1H, 3-Heq),
2.16–2.22 (m, 1H, 5-Heq), 2.94 (ddd, J = 12.0, 4.4, 3.0 Hz, 1H, 6-
Heq), 3.44 (br, 1H, NH), 3.48 (ddd, J = 12.0, 12.0, 2.7 Hz, 1H, 6-
Hax), 3.60–3.69 (m, 1H, 4-Hax), 6.62–7.29 (m, 10H, aromatic H).
13C NMR (CDCl3, 100 MHz) d(ppm) 17.26 (CH3ax), 32.21 (CH3eq),
34.33 (C-5), 47.50 (C-3, C-6), 47.67 (C-4), 55.26 (C-2), 113.23,
117.24, 124.48, 127.87, 127.94, 129.33, 147.16, 149.72 (aromatic
C). Anal. Calcd for C19H26Cl2N2: C, 64.59; H, 7.42; Cl, 20.07; N,
7.93. Found: C, 64.48; H, 7.67; Cl, 19.96; N, 7.79.
5.2.2. Piperidines
5.2.2.1. Diphenylpyraline analogues (3–7). Their syntheses
have already been reported.7
5.2.2.2. General procedure for the synthesis of 1-substituted 4-
anilinopiperidines 21, 22, 25. Ten grams of freshly prepared
Raney nickel W-211 were added to a solution of compounds 19, 20
or a mixture of cis-24 and trans-24 in 80 mL of EtOH. The mixture
was shaken for 16 h at room temperature in hydrogen atmosphere
at 45 psi in a shaker apparatus. The catalyst was sucked off through
a sintered disc filter funnel and the residue rewashed with 100 mL of
EtOH. The solvent was removedin vacuoand the residuedissolved in
CHCl3 and H2O. The organic layers were separated and the aqueous
phase was extracted once with CHCl3. The combined organic layers
were dried with Na2SO4 and the solvent evaporated. The oily base
was converted to the dihydrochloride with an ethanolic solution of
HCl. The solvent was removed and the residue recrystallized.
5.2.2.3. Bamipine analogues (8–10). Their syntheses have al-
ready been reported.8
5.2.2.4. General procedure for the synthesis of 2,2-dimethyl-
substituted analogues of Bamipine (11–13). In an atmosphere
of nitrogen NaNH2 was added to a solution of the corresponding
4-anilinopiperidine in 30 mL of benzene. The mixture was stirred
on an oil-bath at 110 °C until the evolution of NH3 ceased. Then

R. Weis et al. / Bioorg. Med. Chem. 16 (2008) 10326–10331
10331
benzyl chloride was added dropwise. After 6 h the mixture was
5.3. Biological tests
cooled, diluted with 100 mL of benzene, poured into a separatory
funnel and shaken twice with H2O. The organic layer was dried
over K2CO3 and the solvent removed in vacuo. Excess benzyl chlo-
ride was removed beyond 70 °C in high vacuum. The oily base was
converted to the dihydrochlorides with an ethanolic solution of
HCl. The solvent was removed and the residue recrystallized.
5.2.2.4.1. (RS)-( )-4-(N-Benzylanilino)-1,2,2-trimethylpiperidine
(11). The reaction of compound 21 (0.55 g, 2.5 mmol), NaNH2
(0.12 g, 3.1 mmol) and benzyl chloride (0.40 g, 3.1 mmol) gave an
oily residue, which was converted to the dihydrochloride of 11.
Mp 211 °C (2-propanol/acetone); yield: 0.70 g (73%). IR (KBr)
2927, 2375, 1630, 1605, 1493, 1477, 1435, 1392, 1379, 1221,
5.3.1. Antimycobacterial activity
The primary screening of the antimycobacterial activity of all
synthesized analogues of Diphenylpyraline and Bamipine was con-
ducted in vitro at a concentration of 6.25 lg/mL against Mycobacte-
rium tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium
using a broth microdilution assay, the Microplate Alamar Blue As-
say (MABA).13 Isoniazid (INH) was used as standard. Compounds
effecting >90% inhibition were retested at lower concentrations
to determine their minimum inhibitory concentration. The MIC is
defined as the lowest concentration effecting a reduction in fluo-
rescence of 90% relative to controls.
1168, 1119, 1042, 748, 699 cmÀ1 1H NMR (CDCl3, 400 MHz)
.
d(ppm) 1.08 (s, 3H, CH3ax), 1.14 (s, 3H, CH3eq), 1.57 (dd, J = 12.5,
12.5 Hz, 1H, 3-Hax), 1.68 (ddd, J = 12.5, 3.6, 2.3 Hz, 1H, 3-Heq),
1.73 (dddd, J = 12.2, 12.2, 12.2, 4.7 Hz, 1H, 5-Hax), 1.79–1.85 (m,
1H, 5-Heq), 2.24 (s, 3H, CH3), 2.57 (ddd, J = 12.2, 12.2, 3.1 Hz, 1H,
6-Hax), 2.67 (ddd, J = 12.2, 4.7, 2.7 Hz, 1H, 6-Heq), 4.02–4.10 (m,
1H, 4-Hax), 4.42–4.51 (2d, J = 18.0 Hz, 2H, NCH2), 6.67–7.29 (m,
10H, aromatic H). 13C NMR (CDCl3, 100 MHz) d(ppm) 13.91 (CH3ax),
30.60 (C-5), 30.75 (CH3eq), 37.33 (NCH3), 42.58 (C-3), 49.02 (NCH2),
50.33 (C-6), 51.83 (C-4), 54.18 (C-2), 112.77, 116.51, 126.16,
126.37, 128.35, 129.19, 140.72, 149.27 (aromatic C). Anal. Calcd
for C21H30Cl2N2: C, 66.13; H, 7.93; Cl, 18.59; N, 7.35. Found: C,
65.88; H, 7.64; Cl, 18.34; N, 7.10.
5.3.2. Cytotoxicity
The cytotoxic properties of all Bamipine analogues were de-
tected indirectly via cell proliferation of a HEK-293 cell line (hu-
man embryonic kidney 293 cells) using a colorimetric assay
based on the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) to a colored formazan product by
proliferating cells.14 INH was used as standard. The data for the
Diphenylpyraline derivatives were taken from Ref. 7 which contains
a detailed description of the test method.
5.3.3. Antihistaminic activity
The H1-receptor antagonist potency of selected compounds was
determined in whole segments of the guinea-pig ileum as previ-
ously described.15
5.2.2.4.2. (RS)-( )-4-(N-Benzyl-p-anisidino)-1,2,2-trimethylpiperi-
dine (12). The reaction of compound 22 (0.64 g, 2.5 mmol),
NaNH2 (0.12 g, 3.1 mmol) and benzyl chloride (0.40 g, 3.1 mmol)
gave an oily residue, which was converted to the dihydrochloride
of 12. Mp 223 °C (EtOH/ethyl acetate); yield: 0.67 g (65%). IR
(KBr) 2919, 2365, 1601, 1495, 1482, 1458, 1430, 1400, 1375,
Acknowledgments
.
1221, 1174, 1120, 1041, 753, 696 cmÀ1 1H NMR (CDCl3,
Antimycobacterial data were provided by the Tuberculosis
Antimicrobial Aquisition and Coordinating Facility (TAACF)
through a research and development contract with the US National
Institute of Allergy and Infectious Diseases.
400 MHz) d(ppm) 1.08 (s, 3H, CH3ax), 1.18 (s, 3H, CH3eq), 1.63
(dd, J = 12.3, 12.3 Hz, 1H, 3-Hax), 1.66–1.88 (m, 3H, 3-Heq, 5-H),
2.28 (s, 3H, NCH3), 2.59 (ddd, J = 12.6, 11.8, 3.7 Hz, 1H, 6-Hax),
2.72–2.79 (m, 1H, 6-Heq), 3.72 (s, 1H, OCH3), 3.83–3.92 (m, 1H, 4-
Hax), 4.33, 4.42 (2d, J = 17.2 Hz, 2H, NCH2), 6.67–7.27 (m, 10H, aro-
matic H). 13C NMR (CDCl3, 100 MHz): d(ppm) 14.44 (CH3ax), 30.07
(C-5), 30.28 (CH3eq), 37.15 (NCH3), 42.31 (C-3), 49.91 (NCH2), 50.40
(C-6), 52.93 (C-4), 55.01 (C-2), 55.64 (OCH3), 114.60, 115.81,
126.34, 126.47, 128.27, 140.84, 143.46, 151.87 (aromatic C). Anal.
Calcd for C22H32Cl2N2O: C, 64.23; H, 7.84; Cl, 17.23; N, 6.81. Found:
C, 64.44; H, 7.62; Cl, 17.27; N, 6.68.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.10.042.
References and notes
1. Am Ende, C. W.; Knudson, S. E.; Liu, N.; Childs, J.; Sullivan, T. J.; Boyne, M.; Xua,
H.; Gegina, Y.; Knudson, D. L.; Johnson, F.; Peloquin, C. A.; Slayden, R. A.; Tonge,
P. J. Bioorg. Med. Chem. Lett. 2008, 18, 3029.
2. World Health Report 2008, www.who.int/tb/publications/global_report/2008/
pdf/fullreport.pdf.
3. Meindl, W. Arch. Pharm. 1988, 321, 473.
4. Meindl, W. Arch. Pharm. 1989, 322, 493.
5. Meindl, W. Arch. Pharm. 1987, 320, 475.
6. Weis, R.; Kungl, A. J.; Seebacher, W. Tetrahedron 2003, 59, 1403.
7. Weis, R.; Faist, J.; di Vora, U.; Schweiger, K.; Brandner, B.; Kungl, A. J.;
Seebacher, W. Eur. J. Med. Chem. 2008, 43, 872.
8. Weis, R.; Seebacher, W. Tetrahedron 2003, 59, 1395.
9. Willson, F. G.; Wheeler, T. S.. In Organic Syntheses Collect; Gilman, H., Ed.; Wiley:
New York, 1932; Vol. I, pp 102–104.
10. Zigeuner, G.; Schweiger, K.; Fuchsgruber, A. Monatsh. Chem. 1981, 112,
187.
11. Mozingo, R.. In Organic Syntheses Collect; Horning, E. C., Ed.; Wiley: New York,
1955; Vol. III, pp 181–183.
5.2.2.4.3. (RS)-( )-4-(N-Benzylanilino)-2,2-dimethyl-1-phenylpi-
peridine (13). The reaction of compound 25 (0.7 g, 2.5 mmol),
NaNH2 (0.12 g, 3.1 mmol) and benzyl chloride (0.40 g, 3.1 mmol)
gave an oily residue, which was converted to the dihydrochloride
of 13. Mp 207 °C (propan-2-ol); yield: 0.62 g (56%). IR (KBr)
3049, 2992, 2930, 2355, 1599, 1497, 1468, 1440, 1407, 1379,
1200, 1155, 1128, 1037, 769, 700 cmÀ1
.
1H NMR (CDCl3,
400 MHz) d(ppm) 1.06 (s, 3H, CH3), 1.17 (s, 3H, CH3), 1.75 (dd,
J = 12.5, 12.5 Hz, 1H, 3-Hax), 1.77–1.86 (m, 1H, 3-Heq, 5-Hax),
1.92–1.97 (m, 1H, 5-Heq), 2.92 (ddd, J = 12.0, 4.2, 3.0 Hz, 1H,
6-Heq), 3.51 (ddd, J = 12.0, 12.0, 2.9 Hz, 1H, 6-Hax), 4.19–4.27 (m,
1H, 4-Hax), 4.48–4.59 (2d, J = 17.9 Hz, 2H, NCH2), 7.09–7.30 (m,
15H, aromatic H). 13C NMR (CDCl3, 100 MHz) d(ppm) 16.88 (CH3),
31.39 (C-5), 32.61 (CH3), 43.22 (C-3), 47.99 (C-6), 49.12 (NCH2),
52.20 (C-4), 55.58 (C-2), 112.83, 116.55, 124.52, 126.17, 126.41,
127.84, 127.95, 128.39, 129.23, 140.69, 149.25, 149.69 (aromatic
C). Anal. Calcd for C26H32Cl2N2: C, 70.42; H, 7.27; Cl, 15.99; N,
6.32. Found: C, 70.15; H, 7.29; Cl, 15.16; N, 6.14.
12. Schweiger, K. Monatsh. Chem. 1983, 114, 581.
13. Collins, L.; Franzblau, S. G. Antimicrob. Agents Chemother. 1997, 41, 1004.
14. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
15. Pertz, H.; Elz, S. J. Pharm. Pharmacol. 1995, 47, 310.