Synthesis of indolizine derivatives with selective antibacterial activity against Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejps.2006.09.006

1-Substituted indolizines with activity against Mycobacterium tuberculosis have been synthesized. The most active compounds carry an hydroxyphenylmethyl- or hydroxyalkyl substituent in the indolizine 1-position. The alkyl chain should be moderately long (C-5 or C-6). Aryl groups in the 2- and 3-position of the indolizine are also required. Removal of the 3-substituent resulted in significant loss of activity. A nitrile substituent in the 7-position is beneficial for both chemical stability and bioactivity. The compounds studied display a narrow antibacterial spectrum and appear to be quite selective antimycobacterial compounds. Moderate activity against certain pathogenic protozoa was also observed.

Full text of DOI:10.1016/j.ejps.2006.09.006

european journal of pharmaceutical sciences 3 0 ( 2 0 0 7 ) 26–35
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ejps
Synthesis of indolizine derivatives with selective
antibacterial activity against Mycobacterium tuberculosis
Lise-Lotte Gundersena,∗, Colin Charnockb, Ayele Hailu Negussiea, Frode Risea,
Solomon Teklua
a
Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo, Norway
Faculty of Health Sciences, Oslo University College, PO Box 4 St. Olavs plass, N-0130 Oslo, Norway
b
a r t i c l e i n f o
a b s t r a c t
Article history:
1-Substituted indolizines with activity against Mycobacterium tuberculosis have been syn-
thesized. The most active compounds carry an hydroxyphenylmethyl- or hydroxyalkyl
substituent in the indolizine 1-position. The alkyl chain should be moderately long (C-5
or C-6). Aryl groups in the 2- and 3-position of the indolizine are also required. Removal
of the 3-substituent resulted in significant loss of activity. A nitrile substituent in the
7-position is beneficial for both chemical stability and bioactivity. The compounds stud-
ied display a narrow antibacterial spectrum and appear to be quite selective antimy-
cobacterial compounds. Moderate activity against certain pathogenic protozoa was also
observed.
Received 1 March 2006
Received in revised form
1 August 2006
Accepted 26 September 2006
Published on line 1 October 2006
Keywords:
Indolizine
© 2006 Elsevier B.V. All rights reserved.
Antimicrobial
Tuberculosis
1.
Introduction
The lead compound was ( )-1-(hydroxyphenylmethyl)-2,3-
diphenylindolizine-7-carbonitrile (compound 2a, Scheme 1
There are approximately 1.6 million deaths worldwide annu-
ally from tuberculosis (TB). Two out of three HIV infected
individuals in sub-Saharan Africa are co-infected with TB,
which is the actual cause of death for 30% of the AIDS
patients worldwide. The standard drug combinations used
to treat TB today, must be taken for 6–9 months. There has
been no major breakthrough in TB drug development for
the past 30 years and at the same time multi-drug resis-
tant TB (MDR-TB) is a growing problem (Global Alliance,
2005).
below). We are now reporting the synthesis of a series of 1-(1-
hydroxyalkyl)indolizine-7-carbonitriles as well as analogues
of the lead compound with other substituents in the indolizine
3- and 7-positions. The target compounds and synthetic inter-
mediates have been screened as antimycobacterials and activ-
ities against other microorganisms have been investigated
as well. No structures related to those discussed herein are
currently used to treat bacterial infections. The seemingly
unavoidable development of resistance against any antibac-
terial compound used clinically, calls for the development
of new agents with novel mechanisms and chemical struc-
tures. For treatment of mycobacterial infections, organism-
specific agents are recommended (Brown and Wright,
2005).
From screening of indolizine derivatives, first studied as
antioxidants (Gundersen et al., 2003a; Nasir et al., 1998; Teklu
et al., 2005a,b; Østby et al., 2000, 2001), we identified certain
compounds as antimycobacterials (Gundersen et al., 2003b).
∗
Corresponding author. Tel.: +47 22857019; fax: +47 22855507.
E-mail address: l.l.gundersen@kjemi.uio.no (L.-L. Gundersen).
0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2006.09.006

european journal of pharmaceutical sciences 3 0 ( 2 0 0 7 ) 26–35
27
Scheme 1 – Reagents and conditions: (i) n-BuLi, RCHO, THF, −78 ◦C; (ii) CH3I, NaH, THF; (iii) TMS(CH2)2OCH2Cl (SEM-Cl),
(i-Pr)2NEt, CH2Cl2; (iv) RꢀꢀLi, THF, −78 ◦C; (v) MgBr2, CH3NO2, Et2O; (vi) ZnBr2, CH3OH, CH2Cl2; (vii) n-BuLi (10 equiv.), PhCHO,
THF, −78 ◦C; (viii) CDCl3.
eluting with EtOAc–hexane (1:29) followed by EtOAc–hexane
(1:9).
2.
Materials and methods
The 1H NMR spectra were acquired on a Bruker Avance DRX
500 spectrometer, a Bruker Avance DPX 300 spectrometer or
a Bruker Avance DPX 200 at 500, 300 or 200 MHz, respectively.
The 1H decoupled 13C NMR spectra were recorded at 125, 75 or
50 MHz using the above-mentioned spectrometers. MS spec-
tra under electron impact conditions were recorded with a
VG Prospec instrument at 70 eV ionizing voltage, and are pre-
sented as m/z (% rel. int.). Elemental analyses were performed
by Ilse Beetz Mikroanalytisches Laboratorium, Kronach, Ger-
many. As is typical of the class, some of the indolizines pre-
pared in this work analyze poorly by this microanalytical
method; however, chromatographic and spectrometric anal-
ysis demonstrated that these compounds were greater than
97% pure. Melting points are uncorrected. MnO2 was prepared
according to literature procedure (Pratt and McGovern, 1964).
THF, diethyl ether, benzene and toluene were distilled from Na
or Na/benzophenone, and dichloromethane from CaH2. The
following compounds were prepared by literature methods: 1a
and 2a (Østby et al., 2000), 1b–d and 2b–d (Teklu et al., 2005a)
2e, 2l and 3a (Gundersen et al., 2003b). Clog P values were
calculated using MaclogP 4.0 from Biobyte Corp., Claremont,
USA. Activity against Mycobacterium tuberculosis and VERO cells
(Gundersen et al., 2003b; Collins and Franzblau, 1997; Orme et
al., 2001) other antibacterial activities (Vorland et al., 1998),
and antiprotozoal activities (WHO, 2004) were determined as
reported before.
2.1.1. 1-(1-Hydroxypropyl)-2,3-diphenylindolizine-7-
carbonitrile (2f)
Yield 85%, yellow crystals, mp 145–146 ◦C. 1H NMR (300 MHz,
CDCl3) ı 0.91 (t, J 7.4 Hz, 3H, CH3), 1.8–2.0 (m, 2H, CH2), 2.21 (br
s, 1H, OH), 4.90 (t, J 6.8 Hz, 1H, CHOH), 6.50 (dd, J 7.3 and 1.6 Hz,
1H, H-6), 7.1–7.3 (m, 10H, 2 × Ph), 8.01 (d, J 7.3 Hz, 1H, H-5), 8.32
(s, 1H, H-8); 13C NMR (75 MHz, CDCl3) ı 10.7 (CH3), 31.7 (CH2),
69.8 (CHOH), 99–134 (aromatic C and CN); MS EI m/z (rel.%): 352
(18, M+), 334 (100); HRMS: found 352.1591, calcd. for C24H20N2O
352.1591.
2.1.2. 1-(1-Hydroxybutyl)-2,3-diphenylindolizine-7-
carbonitrile (2g)
Yield 70%, yellow crystals, mp 155–157 ◦C. 1H NMR (300 MHz,
CDCl3) ı 0.83 (t, J 7.4 Hz, 3H, CH3), 1.32 (m, 2H, CH2), 1.99 (m, 2H,
CH2), 5.02 (t, J 6.8 Hz, 1H, CHOH), 6.53 (dd, J 7.4 and 1.8 Hz, 1H,
H-6), 7.1–7.3 (m, 10H, 2 × Ph), 7.99 (dd, J 7.4 and 0.8 Hz, 1H, H-5),
8.30 (dd, J 1.8 and 0.8 Hz, 1H, H-8); 13C NMR (75 MHz, CDCl3) ı
13.7 (CH3), 19.4 (CH2), 41.0 (CH2), 68.0 (CHOH), 99–134 (aromatic
C and CN); MS EI m/z (rel.%): 366 (13, M+), 348 (100); HRMS:
found 366.1755, calcd. for C25H22N2O 366.1732.
2.1.3. 1-(1-Hydroxypentyl)-2,3-diphenylindolizine-7-
carbonitrile (2h)
Yield 65%, yellow crystals, mp 147–148 ◦C. 1H NMR (300 MHz,
CDCl3) ı 0.82 (t, J 6.8 Hz, 3H, CH3), 1.2–1.3 (m, 4H, 2 × CH2),
1.8–2.0 (m, 2H, CH2), 1.85 (br s, 1H, OH), 5.00 (t, J 6.8 Hz, 1H,
CHOH), 6.53 (dd, J 7.4 and 1.7 Hz, 1H, H-6), 7.1–7.3 (m, 10H,
2 × Ph), 7.67 (dd, J 7.4 and 0.6 Hz, 1H, H-5), 8.28 (br s, 1H, H-
8); 13C NMR (75 MHz, CDCl3) ı 14.4 (CH3), 22.8 (CH2), 28.8 (CH2),
39.0 (CH2), 68.7 (CHOH), 99–134 (aromatic C and CN); MS EI m/z
(rel.%): 380 (28, M+), 323 (100); HRMS: found 380.1898, calcd. for
C26H24N2O 380.1897.
2.1.
General procedure for the synthesis of
compounds (2)
All novel compounds 2 were prepared by lithiation of the
appropriate bromoindolizine 1 followed by reaction with alde-
hyde as described for 2a before (Østby et al., 2000). The
products were isolated by flash chromatography on silica gel

28
european journal of pharmaceutical sciences 3 0 ( 2 0 0 7 ) 26–35
450.2645, calcd. for C31H34N2O 450.2656; anal. found: C, 82.38;
H, 7.60. C31H34N2O requires C, 82.63; H, 7.61%.
2.1.4. 1-(1-Hydroxy-3-methylbutyl)-2,3-
diphenylindolizine-7-carbonitrile (2i)
Yield 66%, yellow crystals, mp 170–171 ◦C. 1H NMR (300 MHz,
acetone-d6) ı 0.74 (d, J 6.6 Hz, 6H, 2 × CH3), 1.5–1.7 (m, 2H, CH2),
1.91 (m, 1H, CH), 4.31 (br s, 1H, OH), 5.11 (m, 1H, CHOH), 6.67
(dd, J 7.4 and 1.8 Hz, 1H, H-6), 7.2–7.4 (m, 10H, 2 × Ph), 8.11 (dd,
J 7.4 and 0.8 Hz, 1H, H-5), 8.42 (dd, J 1.8 and 0.8 Hz, 1H, H-
8); 13C NMR (75 MHz, acetone-d6) ı 22.1 (CH3), 25.6 (CH), 48.9
(CH2), 66.2 (CHOH), 99–135 (aromatic C and CN); MS EI m/z
(rel.%): 380 (16, M+), 323 (100); HRMS: found 380.1882, calcd.
for C26H24N2O 380.1897; anal. found: C, 82.81; H, 6.46; N, 6.96.
C26H24N2O requires C, 82.07; H, 6.36; N, 7.36%.
2.2.
1-[2-(Trimethylsilyl)ethoxymethoxy]-
phenylmethyl-2,3-diphenylindolizine-7-carbonitrile (3b)
2-(Trimethylsilyl)ethoxymethyl chloride (0.8 mL, 4.7 mmol)
was added drop-wise to
a
stirring mixture of 1-
(hydroxyphenylmethyl)-2,3-diphenylindolizine-7-carbonitrile
2a (378 mg, 0.945 mmol), and ethyl(diisopropyl)amine (0.5 mL,
2.8 mmol) in dry CH2Cl2 (5 mL) under N2 at ambient tem-
perature. The resulting mixture was stirred for 3 days, and
evaporated in vacuo. The product was isolated by flash chro-
matography on silica gel eluting with EtOAc–hexane (1:79);
yield 493 mg (98%), yellow wax. 1H NMR (300 MHz, CDCl3) ı
−0.06 (s, 9H, TMS), 0.82 (m, 2H, CH2Si), 3.59 (m, 2H CH2O),
4.76 (m, 2H CH2O), 5.09 (s, 1H, PhCHO), 6.05 (dd, J 7.4 and
1.7 Hz, 1H, H-6), 7.1–7.3 (m, 15H, 3 × Ph), 8.00 (d, J 7.4 Hz, 1H,
H-5), 8.10 (br s, 1H, H-8); 13C NMR (75 MHz, CDCl3) ı −1.84
(TMS), 17.9 (CH2Si), 65.3 (CH2O), 72.7 (CH2O), 92.5 (PhCHO),
99–142 (aromatic C and CN); MS EI m/z (rel.%): 530 (32, M+),
383 (100).
2.1.5. 1-(1-Hydroxy-2,2-dimethylpropyl)-2,3-
diphenylindolizine-7-carbonitrile (2j)
Yield 40%, yellow crystals, mp 95–97 ◦C. 1H NMR (300 MHz,
acetone-d6) ı 0.77 (s, 9H, t-Bu), 4.50 (d, J 3.7 Hz, 1H, OH), 5.00 (d, J
3.7 Hz, 1H, CHOH), 6.63 (dd, J 7.4 and 1.8 Hz, 1H, H-6), 7.2–7.4 (m,
10H, 2 × Ph), 8.12 (dd, J 7.4 and 0.8 Hz, 1H, H-5), 8.41 (dd, J 1.8 and
0.8 Hz, 1H, H-8); 13C NMR (75 MHz, acetone-d6) ı 26.9 (3 × CH3),
38.5 (C in t-Bu), 76.3 (CHOH), 99–136 (aromatic C and CN); MS
EI m/z (rel.%): 380 (1, M+), 323 (100); HRMS: found 380.1872,
calcd. for C26H24N2O 380.1897; anal. found: C, 82.38; H, 6.68.
C26H24N2O requires C, 82.07; H, 6.36%.
2.3.
General procedure for the synthesis of
compounds (4)
2.1.6. 1-(1-Hydroxyhexyl)-2,3-diphenylindolizine-7-
carbonitrile (2k)
A hexane solution of the required alkyllithium (2 equiv.) was
added drop-wise to a solution of compound 3 (0.6 mmol) in
dry THF (5 mL), under N2 at −78 ◦C. The resulting mixture
was stirred for 2 h at −78 ◦C and quenched by the addition of
sat. aq. NH4Cl (1 mL). Diethyl ether (20 mL) and EtOAc (20 mL)
were added and the phases separated. The organic layer was
washed with water (3 × 20 mL), dried (MgSO4) and evaporated
in vacuo. The product was isolated by flash chromatography on
silica gel.
Yield 88%, yellow crystals, mp 57–59 ◦C. 1H NMR (200 MHz,
CDCl3) ı 0.81 (t, J 6.2 Hz, 3H, CH3), 1.20 (m, 6H, 3 × CH2), 1.7–2.0
(m, 3H, OH and CH2), 5.02 (t, J 6.8 Hz, 1H, CHOH), 6.54 (dd,
J 7.4 and 1.6 Hz, 1H, H-6), 7.1–7.3 (m, 10H, 2 × Ph), 8.02 (d, J
7.4 Hz, 1H, H-5), 8.30 (br s, 1H, H-8); 13C NMR (50 MHz, CDCl3)
ı 13.9 (CH3), 22.5 (CH2), 26.0 (CH2), 31.5 (CH2), 38.8 (CH2), 68.2
(CHOH), 98–134 (aromatic C and CN); MS EI m/z (rel.%): 394
(4, M+), 333 (100); HRMS: found 394.2056, calcd. for C27H26N2O
394.2054.
2.3.1. [1-(Methoxyphenylmethyl)-2,3-diphenylindolizin-
1-yl]ethanone (4a)
2.1.7. 1-(1-Hydroxyoctyl)-2,3-diphenylindolizine-7-
carbonitrile (2m)
EtOAc–hexane (1:79) was used for flash chromatography; yield
147 mg (57%), yellow wax. 1H NMR (200 MHz, acetone-d6) ı 2.41
(s, 3H, COCH3), 3.36 (s, 3H, OCH3), 5.66 (s, 1H, PhCHO), 7.08 (dd,
J 7.5 and 1.9 Hz, 1H, H-6), 7.2–7.4 (m, 15H, 3 × Ph), 8.07 (dd, J
7.5 and 0.9 Hz, 1H, H-5), 8.24 (dd, J 1.9 and 0.9 Hz, 1H, H-8);
13C NMR (50 MHz, acetone-d6) ı 25.7 (CH3), 56.9 (OCH3), 79.1
(PhCHO), 109–144 (aromatic C), 195.1 (CO); MS EI m/z (rel.%): 431
(51, M+), 400 (100); HRMS: found 431.1870, calcd. for C30H25NO2
431.1885.
Yield 74%, yellow crystals, mp 84–85 ◦C. 1H NMR (300 MHz,
acetone-d6) ı 0.83 (t, J 6.9 Hz, 3H, CH3), 1.1–1.4 (m, 10H, 5 × CH2),
1.9–2.0 (m, 2H, CH2), 4.00 (br s, 1H, OH), 4.99 (d, J 6.7 Hz, 1H,
CHOH), 6.58 (dd, J 7.4 and 1.8 Hz, 1H, H-6), 7.2–7.4 (m, 10H,
2 × Ph), 8.12 (dd, J 7.4 and 0.9 Hz, 1H, H-5), 8.43 (dd, J 1.8 and
0.9 Hz, 1H, H-8); 13C NMR (75 MHz, acetone-d6) ı 14.2 (CH3), 23.1
(CH2), 26.8 (CH2), 29.78 (CH2), 29.81 (CH2), 32.4 (CH2), 39.6 (CH2),
68.0 (CHOH), 99–135 (aromatic C and CN); MS EI m/z (rel.%): 422
(11, M+), 333 (100); HRMS: found 422.2358, calcd. for C29H30N2O
422.1250.
2.3.2. [1-(Methoxyphenylmethyl)-2,3-diphenylindolizin-
1-yl]pentanone (4b)
2.1.8. 1-(1-Hydroxydecyl)-2,3-diphenylindolizine-7-
carbonitrile (2n)
EtOAc–hexane (1:79) was used for flash chromatography; yield
193 mg (68%), yellow wax. 1H NMR (300 MHz, CDCl3) ı 0.86 (t, J
7.3 Hz, 3H, CH3), 1.3–1.4 (m, 2H, CH2), 1.5–1.6 (m, 2H, CH2), 2.70
(t, J 7.5 Hz, 2H, CH2), 3.35 (s, 3H, OCH3), 5.57 (s, 1H, PhCHO), 7.17
(dd, J 7.5 and 1.8 Hz, 1H, H-6), 7.2–7.3 (m, 15H, 3 × Ph), 7.98 (d,
J 7.5 Hz, 1H, H-5), 8.02 (s, 1H, H-8); 13C NMR (75 MHz, CDCl3) ı
13.9 (CH3), 22.5 (CH2), 27.3 (CH2), 37.3 (CH2), 56.9 (OCH3), 78.5
(PhCHO), 109–142 (aromatic C) 198.4 (CO); MS EI m/z (rel.%): 473
(53, M+), 442 (100); HRMS: found 473.2361, calcd. for C33H31NO2
473.2356.
Yield 66%, yellow wax. 1H NMR (300 MHz, acetone-d6) ı 0.86
(t, J 6.8 Hz, 3H, CH3), 1.2–1.3 (m, 14H, 7 × CH2), 1.9–2.0 (m, 2H,
CH2), 4.41 (br s, 1H, OH), 5.01 (d, J 6.8 Hz, 1H, CHOH), 6.62 (dd,
J 7.4 and 1.8 Hz, 1H, H-6), 7.2–7.4 (m, 10H, 2 × Ph), 8.11 (dd, J
7.4 and 0.8 Hz, 1H, H-5), 8.42 (dd, J 1.8 and 0.8 Hz, 1H, H-8); 13
C
NMR (75 MHz, acetone-d6) ı 14.2 (CH3), 23.1 (CH2), 26.7 (CH2),
29.82 (2 × CH2), 30.06 (2 × CH2), 68.0 (CHOH), 99–135 (aromatic
C and CN); MS EI m/z (rel.%): 450 (1, M+), 432 (100); HRMS: found

european journal of pharmaceutical sciences 3 0 ( 2 0 0 7 ) 26–35
29
pound 6 (93 mg, 37%) was also isolated]. 1H NMR (300 MHz,
CDCl3) ı 0.94 (t, J 7.3 Hz, 3H, CH3), 1.3–1.4 (m, 2H, CH2), 1.61
(m, 2H, CH2), 2.68 (t, J 7.5 Hz, 2H, CH2), 6.25 (br s, 1H, PhCHO),
7.07 (dd, J 7.5 and 1.8 Hz, 1H, H-6), 7.2–7.5 (m, 15H, 3 × Ph), 7.91
(d, J 0.9 Hz, 1H, H-8), 8.00 (dd, J 7.5 and 0.9 Hz, 1H, H-5); 13C
NMR (75 MHz, CDCl3) ı 13.9 (CH3), 22.5 (CH2), 27.2 (CH2), 37.3
(CH2), 69.0 (CHOH), 109–144 (aromatic C), 198.4 (CO); MS EI m/z
(rel.%): 459 (100, M+), 443 (63); HRMS: found 459.2178, calcd.
for C32H29NO2 459.2185; anal. found: C, 83.95; H, 6.41; N, 3.10.
C32H29NO2 requires C, 83.63; H, 6.41; N, 3.05%.
2.3.3. (1-[2-(Trimethylsilyl)ethoxymethoxy]phenylmethyl-
2,3-diphenylindolizin-1-yl)ethanone (4c)
EtOAc–hexane (1:4) was used for flash chromatography; yield
220 mg (67%), yellow wax. 1H NMR (300 MHz, CDCl3) ı −0.05
(s, 9H, TMS), 0.83 (t, J 8.3 Hz, 2H, CH2Si), 2.47 (s, 3H, COCH3),
3.61 (m, 2H CH2O), 4.79 (m, 2H CH2O), 6.16 (s, 1H, PhCHO), 7.08
(dd, J 7.4 and 1.4 Hz, 1H, H-6), 7.2–7.4 (m, 15H, 3 × Ph), 8.01 (d, J
7.5 Hz, 1H, H-5), 8.24 (br s, 1H, H-8); 13C NMR (75 MHz, CDCl3) ı
−1.47 (TMS), 18.0 (CH2Si), 25.5 (CH3), 65.3 (CH2O), 72.6 (CH2O),
92.5 (PhCHO), 109–134 (aromatic C) 195.6 (CO); MS EI m/z (rel.%):
547 (27, M+), 400 (100).
2.5.2. Method B
2.3.4. (1-[2-(Trimethylsilyl)ethoxymethoxy]phenylmethyl-
2,3-diphenylindolizin-1-yl)pentanone (4d)
n-BuLi (1.7 mL, 1.6 M in hexane, 2.7 mmol) was added
drop-wise to a stirred solution of the bromoindolizine 1a
(0.27 mmol) under N2 at −78 ◦C. The mixture was stirred at
−78 ◦C for an additional 25 h before benzaldehyde (0.11 mL,
1.08 mmol) was added. The resulting mixture was stirred at
−78 ◦C for 1 h, EtOAc (20 mL), diethyl ether (20 mL), and sat.
aq. NH4Cl (20 mL) were added and the phases were separated.
The organic layer was washed with water (2 × 30 mL), dried
(MgSO4) and evaporated in vacuo. The product was isolated by
flash chromatography eluting with EtOAc–hexane (1:29) fol-
lowed by EtOAc–hexane (1:9); yield 93 mg (75%).
EtOAc–hexane (1:4) was used for flash chromatography; yield
307 mg (87%), yellow wax. 1H NMR (300 MHz, CDCl3) ı −0.04 (s,
9H, TMS), 0.8–0.9 (m, 5H, CH2Si and CH3), 1.3–1.5 (m, 2H, CH2),
1.6–1.8 (m, 2H, CH2), 2.83 (t, J 6.2 Hz, 2H, CH2CO), 3.59 (m, 2H,
CH2O), 4.82 (m, 2H, CH2O), 6.19 (s, 1H, PhCHO), 7.10 (dd, J 7.5 and
1.8 Hz, 1H, H-6), 7.1–7.4 (m, 15H, 3 × Ph), 8.02 (d, J 7.5 Hz, 1H, H-
5), 8.28 (br s, 1H, H-8); 13C NMR (75 MHz, CDCl3) ␦ −1.52 (TMS),
13.9 (CH3), 17.9 (CH2Si), 22.4 (CH2), 27.2 (CH2), 37.2 (CH2), 65.2
(CH2O), 72.5 (CH2O), 92.4 (PhCHO), 109–142 (aromatic C) 198.2
(CO); MS EI m/z (rel.%): 589 (31, M+), 442 (100).
2.6.
[1-(2-Nitro-1-phenylethyl)-2,3-diphenylindolizin-
2.4.
[1-(Hydroxyphenylmethyl)-2,3-diphenylindolizin-
1-yl]pentanone (6)
1-yl]ethanone (5a)
The product was isolated in 37% when compound 5b was pre-
pared according to Method A. 1H NMR (300 MHz, acetone-d6) ı
0.93 (t, J 7.3 Hz, 3H, CH3), 1.36 (m, 2H, CH2), 1.62 (m, 2H, CH2),
2.92 (t, J 7.4 Hz, 2H, CH2), 5.21 (dd, J 11.6 and 6.4 Hz, PhCH), 5.43
(m, 2H, CH2NO2), 7.10 (dd, J 7.5 and 1.7 Hz, 1H, H-6), 7.2–7.4 (m,
15H, 3 × Ph), 8.07 (d, J 7.5 Hz, 1H, H-5), 8.22 (br s, 1H, H-8); 13C
NMR (75 MHz, acetone-d6) ı 14.3 (CH3), 23.1 (CH2), 27.7 (CH2),
37.9 (CH2), 40.9 (CH2NO2), 78.5 (PhCH), 109–141 (aromatic C)
197.9 (CO); MS EI m/z (rel.%): 502 (97, M+), 442 (100).
A solution of ZnBr2 (4.49 g, 22.0 mmol) and CH3OH (0.40 mL,
10 mmol) in CH2Cl2 (4 mL) was added to a solution of the SEM-
ether 4c (273 mg, 0.50 mmol) in CH2Cl2 (1 mL). The resulting
mixture was stirred under N2 for 30 min at ambient temper-
ature, diluted with CH2Cl2 and washed with water (40 mL).
The aq. layer was extracted with EtOAc (3 × 10 mL), the com-
bined organic layers were washed with brine (20 mL), dried
(MgSO2) and evaporated in vacuo. The products were sep-
arated by flash chromatography on silica gel eluting with
EtOAc–hexane (1:79), EtOAc–hexane (1:29), and EtOAc–hexane
(1:9); yield 50 mg (24%) of 5a, yellow wax [compound 4a (75 mg,
35%) was also isolated]. 1H NMR (300 MHz, acetone-d6) ı 2.36 (s,
3H, COCH3), 6.16 (s, 1H, CHOH), 7.04 (dd, J 7.5 and 1.8 Hz, 1H, H-
6), 7.2–7.4 (m, 15H, 3 × Ph), 8.05 (d, J 7.5 Hz, 1H, H-5), 8.19 (s, 1H,
H-8); 13C NMR (75 MHz, acetone-d6) ı 25.5 (CH3), 69.1 (CHOH),
109–146 (aromatic C), 195.0 (CO).
2.7.
1-(1-Hexen-1-yl)-2,3-diphenylindolizine-7-
carbonitrile (7)
Compound 2k (50 mg, 0.14 mmol) was kept in CDCl3 for 13 days
and the product was isolated by flash chromatography on sil-
ica gel eluting with EtOAc–hexane (1:9); 34 mg (72%), yellow
crystals, mp 132–133 ◦C. 1H NMR (300 MHz, CDCl3) ı 0.92 (t, J
7.1 Hz, 3H, CH3), 1.40 (m, 4H, 2 × CH2), 2.20 (m, 2H, CH2), 5.96
(dt, J 16.0 and 7.0 Hz, 1H, CH ), 6.38 (dt, J 16.0 and 1.4 Hz, 1H,
CH ), 6.50 (dd, J 7.4 and 1.8 Hz, 1H, H-6), 7.2–7.4 (m, 10H, 2 × Ph),
7.91 (dd, J 7.5 and 0.8 Hz, 1H, H-5), 8.12 (br s, 1H, H-8); 13C NMR
(75 MHz, CDCl3) ı 13.9 (CH3), 22.1 (CH2), 31.6 (CH2), 33.3 (CH2),
99–134 (aromatic C, CN and CH CH); MS EI m/z (rel.%): 376
(67, M+), 333 (100); HRMS: found 376.1942, calcd. for C27H24N2
376.1953.
2.5.
[1-(Hydroxyphenylmethyl)-2,3-diphenylindolizin-
1-yl]pentanone (5b)
2.5.1. Method A
A solution of MgBr2 (1.54 g, 8.50 mmol) and CH3NO2 (0.92 mL,
17 mmol) in diethyl ether (4 mL) was added to a solution
of the SEM-ether 4d (295 mg, 0.50 mmol) in diethyl ether
(1 mL). The resulting mixture was stirred under N2 for 16 h at
ambient temperature, diluted with diethyl ether and washed
with water (40 mL). The aq. layer was extracted with EtOAc
(3 × 10 mL), the combined organic layers were washed with
brine (20 mL), dried (MgSO2) and evaporated in vacuo. The prod-
ucts were separated by flash chromatography on silica gel
eluting with EtOAc–hexane (1:79), EtOAc–hexane (1:29), and
EtOAc–hexane (1:9); yield 94 mg (41%) of 5b, yellow wax [com-
2.8.
N-(Carboxymethyl)pyridinium chloride (9)
A mixture of pyridine (8.10 mL, 100 mmol) and chloroacetic
acid (9.45 g, 100 mmol) in EtOAc (20 mL) was stirred at reflux
for 2 h. After cooling to ambient temperature the product was
filtered off, washed with EtOAc, and dried; yield 16.95 g (98%),
colorless solid. 1H NMR (300 MHz, DMSO-d6) ı 5.78 (s, 2H, CH2),

30
european journal of pharmaceutical sciences 3 0 ( 2 0 0 7 ) 26–35
8.25 (m, 2H, H-3 and H-5), 8.65 (t, J 7.7 Hz, 1H, H-4), 9.22 (d, J
6.1 Hz, 2H, H-2 and H-6); 13C NMR (75 MHz, DMSO-d6) ı 60.6
(CH2), 127.7, 146.3, 146.4, 167.5 (CO2H).
MS EI m/z (rel.%): 221 (73, M+), 144 (100); anal. found: C,
81.77; H, 4.89; N, 6.58. C15H11NO requires C, 81.43; H, 5.01;
N, 6.33%.
2.9.
4-Cyano-N-methylpyridinium chloride (10)
2.12. (2-Methylindolizin-1-yl)(phenyl)methanone (12b)
A
mixture of 4-cyanopyridine (10.41 g, 100 mmol) and
The title compound was synthesized from 2-methylindo-
lizine-1-carbonitrile 11b (312 mg, 2.00 mmol) and phenylmag-
nesium bromide as described for the synthesis of compound
12a above. Yield 432 mg (92%), yellow wax. Spectral data were
in good agreement with those reported before (Sashida et al.,
1988).
chloroacetic acid (9.45 g, 100 mmol) in acetone (20 mL) was
stirred at reflux for 24 h. After cooling to ambient temperature
the product was filtered off, washed with EtOAc, and dried;
yield 8.93 g (58%), colorless solid.
2.10. General procedure for the synthesis of
compounds (11)
2.13. Phenyl(2-phenylindolizin-1-yl)methanone (12c)
and 3-(1-benzoyl-2-phenylindolizin-3-yl)-1,3-
diphenylpropan-1-one (13)
A mixture of N-(carboxymethyl)pyridinium chloride 9 (692 mg,
4.00 mmol), the required ␣,␤-unsaturated nitrile (20 mmol), tri-
ethylamine (0.66 mL, 4.8 mmol) and MnO2 (2.78 g, 23 mmol)
in toluene (40 mL) was stirred under N2 at 90 ◦C for 18 h.
After cooling to ambient temperature, the mixture was fil-
tered through Celite, and the Celite was washed with ace-
tone. The combined filtrates were evaporated in vacuo and
the product isolated by flash chromatography on silica gel
eluting with EtOAc–hexane (1:29) followed by EtOAc–hexane
(1:9).
The title compounds were synthesized from N-(carboxy-
methyl)pyridinium chloride 9 (346 mg, 2.00 mmol) and 1,3-
diphenyl-2-propen-1-one, essentially as described for the syn-
thesis of compounds 11 above. Yield 380 mg (64%) of 12c and
30 mg (3%) of 13.
2.13.1. Phenyl(2-phenylindolizin-1-yl)methanone (12c)
Yellow crystals, mp 132–133 ◦C [mp literature (Sashida et al.,
1988) 133–135 ◦C].
2.10.1. Indolizine-1-carbonitrile (11a)
Yield 408 mg (72%), colorless crystals, mp 52–53 ◦C [mp litera-
ture (Zhang et al., 2000) 52–53 ◦C].
2.13.2. 3-(1-Benzoyl-2-phenylindolizin-3-yl)-1,3-
diphenylpropan-1-one (13)
Pink crystals, mp 81–82 ◦C. 1H NMR (500 MHz, acetone-d6) ı 3.80
(m, 1H, HA in CH2), 4.21 (m, 1H, HB in CH2), 5.52 (m, 1H, CH),
6.70 (m, 1H, H-6), 7.01 (m, 3H, Ph), 7.07–7.11 (m, 4H, Ph and
H-7), 7.15–7.21 (m, 4H, Ph), 7.27 (m, 4H, Ph), 7.44–7.55 (m, 3H,
Ph and H-8), 7.85 (m, 2H, Ph), 8.00 (m, 2H, Ph), 8.09 (d, J 7.3 Hz,
1H, H-5); 13C NMR (125 MHz, acetone-d6) ı 36.9 (CH) 41.2 (CH2),
113–142 (aromatic C), 197.6 (CO), 197.7 (CO); MS EI m/z (rel.%):
505 (42, M+), 386 (100).
2.10.2. 2-Methylindolizine-1-carbonitrile (11b)
Yield 344 mg (55%), colorless crystals, mp 102–103 ◦C [mp liter-
ature (Hurst et al., 1965) 100–101 ◦C].
2.10.3. 2-Phenylindolizine-1-carbonitrile (11c)
Yield 410 mg (47%), pale yellow crystals, mp 106–107 ◦C. 1H
NMR (300 MHz, CDCl3) ı 6.64 (m, 1H, H-6), 6.96 (m, 1H, H-7),
7.2–7.4 (m, 6H, Ph and H-3), 7.49 (d, J 9.0 Hz, 1H, H-8), 7.71
(d, J 7.4 Hz, 1H, H-5); 13C NMR (75 MHz, DMSO-d6) ı 79.5 (CN),
111–139 (aromatic C); MS EI m/z (rel.%): 218 (100, M+); anal.
found: C, 82.87; H, 4.60; N, 12.44. C15H10N2 requires C, 82.55;
H, 4.62; N, 12.84%.
2.14. 1-Benzoyl-2-phenylindolizine-7-carbonitrile (12d)
A mixture of methyl 1-benzoyl-7-cyano-2-phenyl-indolizine-
3-carboxylate 15 (109 mg, 0.28 mmol) and NaCl (33 mg,
0.57 mmol) in DMSO (10 mL) and water (0.02 mL) was stirred at
150 ◦C for 20 h, cooled to ambient temperature and poured into
sat. aq. NH4Cl (30 mL). The resulting mixture was extracted
with EtOAc (30 mL) and the organic extract was washed with
water (2 × 30 mL), dried (MgSO4) and evaporated in vacuo. The
product was purified by flash chromatography on silica gel
eluting with EtOAc–hexane (1:9) followed by EtOAc–hexane
(1:4); yield 82 mg (90%) yellow crystals, mp 232–234 ◦C [mp lit-
erature (Tamura et al., 1972) 233.5–235 ◦C].
2.11. Indolizin-1-yl(phenyl)methanone (12a)
A stirred mixture of indolizine-1-carbonitrile 11a (284 mg,
2.00 mmol) and phenylmagnesium bromide (10 mL, 10 mmol,
1 M sol. in THF) in dry benzene (4 mL) was heated at reflux
under N2-atm. for 24 h. After cooling to ambient temperature,
sat. aq. NH4Cl (2 mL) was added and the mixture was extracted
with EtOAc (30 mL) and Et2O (30 mL). The combined organic
extracts were washed with water (2 × 30 mL), dried (MgSO4)
and evaporated in vacuo. The product was purified by flash
chromatography on silica gel eluting with EtOAc–hexane–Et3N
(10:10:1); yield 407 mg (92%), yellow wax. 1H NMR (500 MHz,
CDCl3) ı 6.57 (t, J 6.6 Hz, 1H, H-6), 6.83–6.86 (m, 2H, H-2 and
H-7), 7.20 (d, J 2.4 Hz, 1H, H-3), 7.38–7.42 (m, 3H, Ph), 7.54 (m,
2H, Ph), 7.88 (d, J 9.1 Hz, 1H, H-8), 7.91 (d, J 6.6 Hz, 1H, H-
5); 13C NMR (125 MHz, CDCl3) ı 111.76, 111.82, 112.9, 116.4,
120.0, 120.7, 125.6, 127.5, 128.1, 129.2, 133.1, 142.2, 173.8 (CO);
2.15. 4-Cyano-1-(methoxycarbonylmethyl)pyridinium
bromide (14)
4-Cyanopyridine 8b (3.06 g, 29.4 mmol) and methyl bromoac-
etate (7.25 mL, 29.4 mmol) in acetone (50 mL) was heated at
reflux for 24 h and cooled to ambient temperature. The solid
was filtered off, washed with acetone and dried; yield 7.53 g
(93%), colorless crystals, mp 181–183 ◦C.

european journal of pharmaceutical sciences 3 0 ( 2 0 0 7 ) 26–35
31
2.16. Methyl 1-benzoyl-7-cyano-2-phenyl-indolizine-
3-carboxylate (15)
2.19. Determination of antifungal activity against
Candida krusei (ATCC 6258)
The title compound was synthesized from 4-cyano-1-
(methoxycarbonyl)pyridinium bromide 14 (1.02 g, 4.0 mmol)
and 1,3-diphenyl-2-propen-1-one (4.17 g, 20.0 mmol), essen-
tially as described for the synthesis of compounds 11 and 12c
above. The product was purified by flash chromatography on
silica gel eluting with EtOAc–hexane (1:9); yield 836 mg (55%)
colorless crystals, mp 180–181 ◦C. 1H NMR (200 MHz, CDCl3) ı
3.79 (s, 3H, CH3), 7.19–7.41 (m, 9H, Ph and H-6), 7.55–7.59 (m,
2H, Ph), 8.52 (dd, J 1.8 and 0.94, 1H, H-8), 9.80 (dd, J 7.4 and
0.94 Hz, 1H, H-5); 13C NMR (50 MHz, CDCl3) ı 51.5 (CH3), 108–139
(aromatic C and CN), 161.8 (CO2), 192.1 (CO); MS EI m/z (rel.%):
380 (100, M+), 303 (53); anal. found: C, 75.87; H, 3.95; N, 7.39.
C24H16N2O3 requires C, 75.78; H, 4.24; N, 7.36%.
C. krusei (ATCC 6258) was subcultured twice on Sabouraud
dextrose agar plates (Oxoid, Basingstoke, UK) and incubated
at 35 ◦C for 24 h prior to testing. Testing was performed
according to the guidelines of the NCCLS document M27-A
(NCCLS, 1997). Analytical grade powders of amphotericin B
(Sigma–Aldrich, St. Louis, MO) and the indolizine agents to
be tested were prepared as described above. Stock solutions
of the powders were prepared in DMSO (Sigma–Aldrich) at
1.6 mg/mL. These were then diluted with RPMI 1640 medium
(with l-glutamine but without bicarbonate; Sigma–Aldrich)
buffered to pH 7.0 with 0.176 M morpholinopropanesulfonic
acid (MOPS, Sigma–Aldrich). The final concentration range
tested was 0.0625–32 ␮g/mL. Testing was performed in 96-well
microtitration plates (Nalge Nunc International, Denmark).
Yeast inocula were prepared in sterile water and were diluted
in RPMI 1640 medium to give a final inoculum concentration
in wells of 5 × 102 to 2.5 × 103 colony forming units (CFU)/mL.
The plates were lidded, and incubated at 35 ◦C. Endpoints were
read visually at 48 h. The minimum inhibitory concentration
(MIC) for amphotericin (control) and indolizines was taken
to be the lowest drug concentration that prevented any dis-
cernible growth.
2.17. 1-[Hydroxy(phenyl)methyl]-2-phenylindolizine-7-
carbonitrile (16)
Sodium borohydride (17 mg, 0.45 mmol) was added care-
fully to a stirred solution of 1-benzoyl-2-phenylindolizine-7-
carbonitrile 12d (72 mg, 0.22 mmol) in abs. EtOH (8 mL), and the
resulting mixture was stirred at ambient temperature under
N2-atm. for 48 h before sat. aq. NH4Cl (2 mL) was added. The
mixture was extracted with EtOAc (20 mL) and Et2O (20 mL),
and the combined organic extracts were washed with water
(3 × 20 mL), dried (MgSO4) and evaporated in vacuo. The prod-
uct was purified by flash chromatography on silica gel elut-
ing with EtOAc–hexane (1:29) followed by EtOAc–hexane (1:9);
yield 65 mg (85%), yellow wax. 1H NMR (300 MHz, acetone-d6) ı
5.08 (d, J 4.0 Hz, OH), 6.30 (d, J 4.0 Hz, CHOH), 6.68 (m, 1H, H-6),
7.18–7.56 (m, 10H, Ph), 7.84 (s, 1H, H-3), 7.88 (s, 1H, H-8), 8.32 (m,
1H, H-5); 13C NMR (75 MHz, acetone-d6) ı 68.6 (CHOH), 108–139
(aromatic C and CN); MS EI m/z (rel.%): 324 (100, M+), 247 (89);
anal. found: C, 81.40; H, 5.30; N, 8.38. C22H16N2O requires C,
81.46; H, 4.97; N, 8.64%.
3.
Results and discussion
3.1.
Chemistry
We have previously synthesized 2,3-diphenylindolizin-1-ols
by cyclization between diphenylcyclopropenone and a pyri-
dine (Gundersen et al., 2003a,b; Nasir et al., 1998; Teklu et al.,
2005a,b; Østby et al., 2000, 2001). We have generally chosen to
start from 4-cyanopyridine because of high regioselectivity in
the cyclization reaction as well as reasonable stability of the
intermediate 1-indolizinol formed. Other pyridines may give
mixtures of isomeric 1- and 3-indolizinols when reacted with
cyclopropenones (Wadsworth et al., 1986), and indolizinols
without an electron withdrawing substituent in the 7-position
often decompose readily by oxidative processes (Wadsworth
et al., 1989). We have also shown that 1-indolizinols can be
further functionalized in the 1-position, for instance to give
the bromides 1 (Scheme 1). These bromides were treated with
n-BuLi and reacted with aldehydes to give a series of com-
pounds 2, structurally related to the antimycobacterial lead
compound 2a (Scheme 1).
The cyanoindolizines could be transformed to the corre-
sponding ketones by addition of organolithium reagents to
the nitrile. The nitrile 2a was converted to the O-methylated
derivative 3a and reacted with MeLi or n-BuLi to give the
ketones 4a and 4b. Protection of the relatively hindered OH-
group in 2a by silylation with TBDMS-Cl failed, but the com-
pound could easily be protected as the SEM-ether 3b. How-
ever, deprotection of SEM-ethers is often troublesome, and the
original cleavage procedure employing tetrabutylammonium
fluoride (TBAF) in THF (Lipshutz and Pegram, 1980) failed to
give compounds 5a or 5b. The methyl ketone 5a was isolated
in only 13% yield when a modified procedure with TBAF in
2.18. 1-(Methoxyphenylmethyl)-2-phenylindolizine-7-
carbonitrile (17)
Sodium hydride (11.4 mg, 0.48 mmol) was added to
a
solution of 1-[hydroxy(phenyl)methyl]-2-phenylindolizine-7-
carbonitrile 16 (70 mg, 0.22 mmol) in dry THF (5 mL) and the
resulting mixture was stirred at ambient temperature under
N2-atm. for 1.5 h, then iodomethane (30 ␮L, 0.48 mmol) was
added. After stirring for an additional 36 h, the reaction mix-
ture was quenched by the addition of sat. aq. NH4Cl (2 mL)
and extracted with EtOAc (20 mL) and Et2O (20 mL). The com-
bined organic extracts were washed with water (3 × 20 mL),
dried (MgSO4) and evaporated in vacuo. The product was
purified by flash chromatography on silica gel eluting with
EtOAc–hexane (1:29); yield 61 mg (80%), yellow wax. 1H NMR
(200 MHz, acetone-d6) ı 3.34 (s, 3H, CH3), 5.81 (s, 1H, CHO), 6.71
(m, 1H, H-6), 7.18–7.48 (m, 10H, Ph), 7.86 (s, 1H, H-3), 8.01 (br s,
1H, H-8), 8.33 (dd, J 7.2 and 0.8 Hz, 1H, H-5); 13C NMR (75 MHz,
acetone-d6) ı 56.8 (CH3), 78.9 (CHO), 100–143 (aromatic C and
CN); MS EI m/z (rel.%): 338 (40, M+), 307 (100); anal. found: C,
81.82; H, 5.40; N, 8.15. C23H18N2O requires C, 81.63; H, 5.36; N,
8.28%.

32
european journal of pharmaceutical sciences 3 0 ( 2 0 0 7 ) 26–35
Table 1 – Antimycobacterial activity against Mycobacterium tuberculosis and cytotoxic activity against VERO cells for
indolizines 1–7a
No.
R1
R2 = R3
R7
% Inhibition of M.
tuberculosis at
6.25 ␮g/mL
MIC M. tuberculosis
H37Rv (␮g/mL)b
IC50 VERO
cells (␮g/mL)
concentration
1a
1b
1c
1d
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
3a
4a
4b
5a
5b
6
Br
Br
Br
Br
Ph
p-F-C6H4
p-Cl-C6H4
p-CH3-C6H4
Ph
p-F-C6H4
p-Cl-C6H4
p-CH3-C6H4
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
COCH3
COBu-n
COCH3
COBu-n
COBu-n
CN
69c
58
100
52
100c
35
86
100
78c
96
84
99
0
94
96
90c
57
0
74c
80
81
18
n.d.
n.d.
3.13
n.d.
6.25c
n.d.
n.d.
3.13
n.d.
>6.25
n.d.
3.13
n.d.
6.25
6.25
>6.25c
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
8.62
n.d.
>10d
n.d.
n.d.
5.61
n.d.
n.d.
n.d.
7.56
n.d.
5.99
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
CH(OH)Ph
CH(OH)Ph
CH(OH)Ph
CH(OH)Ph
CH(OH)CH3
CH(OH)Et
CH(OH)Pr-n
CH(OH)Bu-n
CH(OH)Bu-i
CH(OH)Bu-t
CH(OH)Pent-n
CH(OH)Hex-c
CH(OH)Hept-n
CH(OH)Non-n
CH(OCH3)Ph
CH(OCH3)Ph
CH(OCH3)Ph
CH(OH)Ph
CH(OH)Ph
C(CH2NO2)Ph
(E) CH CHBu-n
40
16
46
7
a
For a general structure and definition of R1, R2, R3 and R7, see Fig. 1.
MIC rifampin 0.25 ␮g/mL.
Data from literature Gundersen et al. (2003b).
b
c
d
Low solubility precluded determination of exact IC50
.
according to the “Lipinski rule of five,” a compound with
good oral bioavailability generally has log P < 5 (Lipinski, 2000;
Lipinski et al., 2001). Clog P were found to be 5.95 and 6.69,
respectively, for the active compounds 2a and 2h. As dis-
cussed below, a fairly lipophilic substituent in the indolizine
1-position is required for antimycobacterial activity. Analo-
gous indolizines without aryl groups in the 2- or 3-position
would have substantially reduced lipophilicity, and we found
such compounds highly interesting synthetic targets.
DMPU in the presence of molecular sieves was tried (Lipshutz
and Miller, 1989). SEM ethers have been cleaved with MgBr2
in diethyl ether/CH3NO2, or with ZnBr2 in CH2Cl2/CH3OH
(Vakalopoulos and Hoffmann, 2000). Both methods gave the
ketones 5a and 5b, but only in moderate yields. Unexpected
reactions between the co-solvents, CH3NO2 or CH3OH, took
place, and compound 6 or compounds 4a or 4b were formed
in various amounts depending on variations in reaction con-
ditions.
Whereas indolizines carrying two identical aryl groups
in the 2- and 3-positions are accessible from diarylcyclo-
propenones as discussed above, alternative synthetic strate-
gies were required, when we decided to attempt synthe-
sis of compound 2a analogues without two identical aryls
in the indolizine 2- and 3-positions. In order to construct
indolizines with an unsubstituted C-3 (Shipman, 2001), we
chose to follow the route depicted by Zhang et al., where N-
(carboxymethyl)pyridinium salts were reacted with electron
deficient alkenes in the presence of MnO2 (Zhang et al., 2000).
Pyridine 8a was N-alkylated with chloroacetic acid to give the
pyridinium salt 9 (Scheme 2). When the same reaction was
attempted on the pyridine 8b, carrying an electron withdraw-
ing substituent, decarboxylation of the pyridinium salt formed
initially appeared to be unavoidable, and we were only able
to isolate compound 10. The salt 9 was cyclized with ␣,␤-
When the bromide 1a was reacted with a large excess
of n-BuLi, both reaction with the cyanide and metal-halogen
exchange took place, and the butyl ketone 5b was isolated in
high yield after trapping with benzaldehyde. Unfortunately,
the methyl ketone 5a was not available by this short route.
Reacting the bromide 1a with excess MeLi followed by ben-
zaldehyde resulted only in the formation of 2a, which was
isolated in 88% yield.
The aldehyde adducts 2, with R = n-alkyls, slowly elimi-
nated water when left in CDCl3 solution. For instance the
E-alkene 7 was formed quantitatively after a few days in this
weakly acidic solvent.
Even though several compounds
2 exhibit profound
inhibitory activity against M. tuberculosis in vitro (see Table 1
below), a draw back with these compounds is their lipophilic-
ity. An antituberculosis drug should be taken orally, and,

european journal of pharmaceutical sciences 3 0 ( 2 0 0 7 ) 26–35
33
Scheme 2 – Reagents and conditions: (i) ClCH2CO2H, EtOAc, ꢀ; (ii) R2CH CHCN, MnO2, Et3N, PhCH3, 90 ◦C; (iii) PhMgBr,
PhCH3, 80 ◦C; (iv) PhCH CHCOPh, MnO2, Et3N, PhCH3, 90 ◦C; (v) BrCH2CO2CH3, acetone, ꢀ; (vi) NaCl, H2O, DMSO, 150 ◦C; (vii)
NaBH4, EtOH; (viii) CH3I, NaH, THF.
unsaturated nitriles to give compounds 11. Reaction between
PhMgBr and the nitriles 11a or 11b gave the corresponding
ketones 12a or 12b in excellent yields. For the synthesis of
the ketone 12c we chose a shorter route. The pyridinium
salt 9 was reacted directly with the required ␣,␤-unsaturated
phenyl ketone (chalcone). In addition to the desired 12c, a
minor amount of compound 13 was also formed. Compounds
11 (Ceder and Sharif, 1987; Hurst et al., 1965; Mahon et al.,
1992; Pauls and Kroehnke, 1977; Zhang et al., 2000) and 12b–d
(Dainis, 1972; Sashida et al., 1988; Tamura et al., 1972) have
been reported before. The syntheses described herein are gen-
erally shorter and give higher total yields. Unfortunately, none
of the phenyl ketones 12a–c, could be reduced to benzyl alco-
hols and we were not able to isolate analogues of compound
2a without substituents in both 3- and 7-position. Although
attempts to isolate any pure products from the reductions of
12a–c failed, NMR and MS data indicated that dimerization
between two indolizines had taken place.
Indolizines without substituents in the 7-position are
prone to dimerization (Wadsworth et al., 1989), and we chose
to continue our attempts to synthesize indolizines without
C-3 substituents, with the cyano group in the 7-position hop-
ing that this structural element would stabilize the target
compounds. As mentioned above, alkylation of the cyanopy-
ridine 8b with chloroacetic acid gave only the decarboxylated
product 10. Instead compound 8b was N-alkylated with the
methyl ester of bromoacetic acid to give the pyridinium salt
14 which cyclized with chalcone in the presence of MnO2 to
the indolizinyl ketone 15. Attempts to remove the ester func-
tion in the 3-position by basic ester hydrolysis followed by
decarboxylation were not successful; the nitrile group was
also affected under the hydrolytic conditions. On the other
hand, the ester was readily removed in only one step by the
Krapcho demethoxycarbonylation reaction (Krapcho, 1982).
7-Cyanoindolizinyl ketone 12d could be reduced by sodium
borohydride. The alcohol 16 was isolated in 85%, but a grad-
ual color change of the compound indicated limited stability,
and since we had previously found that the ether 3a was only
slightly less active as an antimycobacterial agent than the free
alcohol 2a (Gundersen et al., 2003b) the alcohol 16 was also
converted to the stable methyl ether 17. Clog P values for both
16 (Clog P = 3.85) and 17 (Clog P = 4.66) were lower than for the
active antimycobacterial 2a (Clog P = 5.95).
3.2.
Antimicrobial activity
The indolizines synthesized were screened for inhibitory
activity against M. tuberculosis H37Rv. The general structure of
the compounds examined, is shown in Fig. 1 and the antimy-
cobacterial activities are given in Tables 1 and 2. The com-
pounds were first screened at concentration of 6.25 ␮g/mL.
MIC values as well as toxicity against VERO cells were deter-
mined whenever the inhibition of M. tuberculosis growth in the
initial screening was 90% or higher. Some previously published
results (Gundersen et al., 2003b) are also included in Table 1
for comparison.
Because we previously found moderate inhibitory activity
against M. tuberculosis for the bromoindolizine 1a (Gundersen
et al., 2003b), we also included the bromides 1b–d in this
Fig. 1 – General structure for all indolizines in
Tables 1 and 2.

34
european journal of pharmaceutical sciences 3 0 ( 2 0 0 7 ) 26–35
Table 2 – Activity against M. tuberculosis for indolizines 11–13 and 15–17a
No. R1 R2 R3
R7
% Inhibition of M. tuberculosis
at 6.25 ␮g/mL concentration
11a
11b
11c
12a
12b
12c
12d
13
CN
CN
CN
COPh
COPh
COPh
COPh
COPh
COPh
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CN
H
CN
CN
CN
6
0
39
0
8
30
0
45
9
8
CH3
Ph
H
CH3
Ph
Ph
Ph
Ph
Ph
Ph
CHPhCH2COPh
CO2CH3
H
H
15
16
17
CH(OH)Ph
CH(OCH3)Ph
13
a
For a general structure and definition of R1, R2, R3 and R7, see Fig. 1.
study. Compound 1c with p-chlorophenyl substituents in the
indolizine 2- and 3-position is actually among the most active
indolizines studied to date. The activities found for 1b and
1d were quite similar to those reported for 1a. Unfortunately,
the lack of readily available diarylcyclopropenones as starting
materials has so far precluded a more extensive screening of
2,3-diaryl-1-bromoindolizines.
Table 3 – Antimicrobial activity against and Candida
krusei, Plasmodium falciparum, Leishmania donovani,
Trypanosoma cruzi, and Trypanosoma brucei rhodesiense
Compound
2a
MIC C. krusei (␮g/mL)a
MIC P. falciparum ␮g/mL)b
MIC L. donovani (␮g/mL)c
MIC T cruzi (␮g/mL)d
MIC T. b. rhodesiense (␮g/mL)e
>32
2.3
2.3
3.2
12
Compounds 2b–d are close analogues of the lead structure
2a, but with p-substituted aryls in the 2- and 3-positions. The
antimycobacterial activity was somewhat enhanced with a
methyl substituent (2d) and reduced with fluorine (2b). Screen-
ing of the alkyl aldehyde adducts 2e–2n revealed that the
optimal side chain substituent (R in compounds 2, Scheme 1)
may very well be an alkyl group. The optimum chain length
was found to be approximately 4 carbons for the n-alkyls (2h).
Bulky substituents except for the isobutyl group (2i) were also
tolerated. The (E)-alkene 7 formed by elimination of water
from 2k, was only moderately active against M. tuberculosis at
6.25 ␮g/mL concentration, confirming that the oxygen in the
1-substituent is important for inhibitory activity (Gundersen
et al., 2003b). Conversion of the 7-cyano group in compound
2a into an acyl group (5a or 5b) resulted in somewhat reduced
antimycobacterial activity. The O-methylated analogues of the
ketones (4a or 4b) were, however appeared to be equally active
as the corresponding nitrile 3a in the bioassay used. Unfor-
tunately, the most active antimycobacterial indolizines were
also relatively toxic towards VERO cells (Table 1), and further
studies on antimycobacterial indolizines should be directed
towards the development of safer drug candidates.
Compound 16, the C-3 unsubstituted analogue of the
lead compound 2a, was completely without antimycobacte-
rial activity at 6.25 ␮g/mL concentration (Table 2). However, as
discussed above, there are indications of limited stability for
16 and the lack of activity may also be the result of decompo-
sition. Hence we also prepared the more stable methyl ether
17. Comparison of inhibitory activity against M. tuberculosis
for compound 17 (13%) and compound 3a (74%), indicate that
a substituent in the indolizine 3-position is required. None
of the synthetic intermediates 11, 12, 13 or 15 exhibited pro-
found antimycobacterial activity at 6.25 ␮g/mL concentration
(Table 2).
a
MIC amphotericin B 1.0 ␮g/mL.
MIC chloroquine 0.04 ␮g/mL.
MIC miltefosine 0.24 ␮g/mL.
MIC benznidazole 0.25 ␮g/mL.
b
c
d
e
MIC melarsoprol 0.005 ␮g/mL.
Eschericha coli, None of the compounds examined, showed
activity against these organisms at the highest concentra-
tion, 32 ␮g/mL, tested (data not shown). For comparison Gen-
tamycin exhibited MIC = 0.03 ␮g/mL against S. aureus and
MIC = 0.125 ␮g/mL against E. coli. These findings points towards
a rather narrow spectrum for the indolizines as antimicrobial
compounds.
Finally the indolizine 2a was tested for inhibitory activity
against certain pathogenic protozoa as well as the yeast C.
krusei (Table 3). Protozoal infections affect a huge number of
individuals, especially in developing countries, and there is an
urgent need for effective drugs against several parasites caus-
ing tropical diseases (Lemke, 2002). Compound 2a was mod-
erately active against Plasmodium falciparum (malaria), Leish-
mania donovani (viceral leishmaniasis/Kala azar), Trypanosoma
cruzi (Chagas disease) and Trypanosoma brucei rhodesiense
(African trypanosomiasis/sleeping sickness). Compound 2a as
well as compounds 2d, 2h, 2k and 2l (data not shown), were
inactive towards C. krusei at 32 ␮g/mL concentration.
Acknowledgements
Quota Program Scholarships to AHN and ST are grate-
fully acknowledged. Antimycobacterial data were provided
by the Tuberculosis Antimicrobial Acquisition and Coordi-
nating Facility (TAACF) through a research and development
contract with the US National Institute of Allergy and Infec-
Selected indolizines (2a, 2d, 2h, 2j, 3a and 16) were also
screened against other bacteria, Staphylococcus aureus and

european journal of pharmaceutical sciences 3 0 ( 2 0 0 7 ) 26–35
35
Nasir, A.I., Gundersen, L.-L., Rise, F., Antonsen, Ø., Kristensen, T.,
tious Diseases. We are grateful for all help provided by Dr.
Cecil Kwong and co-workers. Other antibacterial data were
provided by University Hospital of North Norway, Depart-
ment of Microbiology, Tromsø, Norway and antiprotozoal
activities were determined by The Special Programme for
Research and Training in Tropical Diseases (TDR) and the
Swiss Tropical Institute. C. krusei ATCC 6528 was a kind gift
from J.E. Cutler at the Department of Microbiology, Montana
State University, Bozeman, Montana 59717–3520. The Nor-
wegian Research Council is acknowledged for partial financ-
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