Synthesis and characterization of lithocholic acid derived dipyrromethanes: Precursors for pyrrole-steroidal macrocycles

Article abstract of DOI:10.1016/j.molstruc.2004.02.014

Three steroidal dipyrromethanes, 3,3,24,24-tetrakis(pyrrol-2-yl)-5β- cholane 1, 3,3-bis(pyrrol-2-yl)-5β-cholan-24-oic acid 2, and methyl 3,3-bis(pyrrol-2-yl)-5β-cholan-24-oate 3, have been prepared from 3α-hydroxy-5β-cholan-24-oic acid (lithocholic acid) 4 in good overall yields. The structures of 1-3 have been fully characterized by ¹H, ¹³C, PFG DQF ¹H-¹H COSY, ¹H- ¹H ROESY, ¹³C DEPT-135, PFG ¹H-¹³C HMQC, PFG ¹H-¹³C HMBC, and PFG ¹H- ¹⁵N HMBC NMR spectra. Their molecular weights and compositions have been determined by ESI-TOF and EI mass spectra, and elemental analyses. The energetically optimised geometry and isotropic ¹³C NMR chemical shifts of 3,3,24,24-tetrakis(pyrrol-2-yl)-5β-cholane 1 have been calculated by ab initio HF/6-31G* and DFT B3PW91/6-311G* methods.

Full text of DOI:10.1016/j.molstruc.2004.02.014

Journal of Molecular Structure 693 (2004) 81–86
www.elsevier.com/locate/molstruc
Synthesis and characterization of lithocholic acid derived
dipyrromethanes: precursors for pyrrole-steroidal macrocycles
*
Juha Koivukorpi , Arto Valkonen, Erkki Kolehmainen
University of Jyv a¨ skyl a¨ , Department of Chemistry, P.O. Box 35, FIN-40014, University of Jyv a¨ skyl a¨ , Finland
Received 20 January 2004; revised 9 February 2004; accepted 11 February 2004
Abstract
Three steroidal dipyrromethanes, 3,3,24,24-tetrakis(pyrrol-2-yl)-5b-cholane 1, 3,3-bis(pyrrol-2-yl)-5b-cholan-24-oic acid 2, and methyl
,3-bis(pyrrol-2-yl)-5b-cholan-24-oate 3, have been prepared from 3a-hydroxy-5b-cholan-24-oic acid (lithocholic acid) 4 in good overall
3
yields. The structures of 1–3 have been fully characterized by H, C, PFG DQF H– H COSY, H– H ROESY, C DEPT-135, PFG
1
13
1
1
1
1
13
1
13
H– C HMQC, PFG H– C HMBC, and PFG H– N HMBC NMR spectra. Their molecular weights and compositions have been
1
13
1
15
1
3
determined by ESI-TOF and EI mass spectra, and elemental analyses. The energetically optimised geometry and isotropic C NMR
chemical shifts of 3,3,24,24-tetrakis(pyrrol-2-yl)-5b-cholane 1 have been calculated by ab initio HF/6-31G* and DFT B3PW91/6-311G*
methods.
q 2004 Elsevier B.V. All rights reserved.
Keywords: Pyrrole; Dipyrromethane; Lithocholic acid; Multinuclear magnetic resonance; Electrospray ionization and EI mass spectra
1
. Introduction
important precursors in syntheses of calix [4] pyrroles,
porphyrins and other macrocyclic pyrrole derivatives [9].
Dipyrromethanes containing one or two hydrogen atoms at
meso position can be oxidized to dipyrromethenes, which
can chelate transition metal cations [9]. The aim of this
study is to prepare steroidal oligopyrromethanes, which can
be used further as precursors in the syntheses of pyrrole
macrocycles. Molecules containing both steroid and pyrrole
moieties are interesting and potential carriers for drug
molecules. They also have potential to act as prodrugs or
drugs by themselves [8].
Bile acids and their derivatives have been used in the
treatment of bile acid deficiency and liver diseases, as well
as in dissolution of cholesterol gallstones [1]. They can be
used as cholesterol level lowering agents and as carriers for
liver-specific drugs [2]. Previously, we have reported the
synthesis and characterization of a thiophene and piperazine
þ
bearing cholaphane, which has cation (e.g. Ag ) binding
properties [3]. In this work we are extending our studies to
pyrrole-bile acid conjugates, because pyrrole is a biochemi-
cally important structural fragment found in heme, chlor-
ophyll and many alkaloid structures [4]. Additionally
pyrrole derivatives are widely used in the pharmaceutical
industry, for example as anti-inflammatory and anticancer
drugs [5,6]. Porphines are used in photodynamic therapy
2. Experimental
2.1. Spectroscopy
(
PDT) as photosensitizers, which tend to accumulate in
All NMR experiments were run with Bruker Avance DRX
00 FT NMR spectrometer equipped with a z-gradient
diseased tissue. In visible light they react with molecular
oxygen to generate radicals, which inactivate tumour cells
5
accessory and a 5 mm diameter inverse detection probehead
working at 500.13 MHz for proton, 125.77 MHz for carbon-
[7]. A steroid-porhyrin conjugate has also been used for
saccharide binding in protic media [8]. Dipyrromethanes are
1
13
3 and 50.70 MHz for nitrogen-15, respectively. H and C
1
NMR chemical shifts were referenced to the trace signal of
*
Corresponding author. Tel.: þ358-14-260-2684; fax: þ358-14-260-
501.
E-mail address: jkkorpi@cc.jyu.fi (J. Koivukorpi).
1
undeuteriated solvent, d( H) CHCl ¼ 7.26 ppm, and to the
2
3
1
3
signal of solvent, d( C) CDCl ¼ 77.00 ppm, from internal
3
0022-2860/$ - see front matter q 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2004.02.014

82
J. Koivukorpi et al. / Journal of Molecular Structure 693 (2004) 81–86
Fig. 1. Ab initio HF/6-31G* optimized structure of 3,3,24,24-tetrakis(pyrrol-2-yl)-5b-cholane.
1
5
TMS. N NMR chemical shifts were referenced to the
signal of an external nitromethane in 1 mm diameter
capillary inserted coaxially inside the 5 mm NMR tube,
2.2. Calculations
The geometry of 1 was optimized at the ab initio
HF/6-31G*, and NMR shieldings were calculated at the
B3PW91/6-311G* levels of theory using GAUSSIAN 98
[14]. The crystal structure of methyl 3-oxo-5b-cholan-
24-oate was used as a starting structure for the
steroidal part of molecule [15]. The optimized
structure of 1 is shown in Fig. 1. The predicted NMR
1
5
1
13
15
d(CH NO ) ¼ 0.0 ppm. H, C, and N NMR chemical
3
2
1
1
shift assignments were based on PFG DQF H– H COSY
1
10], H– H ROESY [11], C DEPT-135, PFG H– C
1
13
1
13
[
1
13
1
15
HMQC [12], PFG H– C HMBC [13], and PFG H– N
HMBC NMR measurements.
Mass spectrometric measurements were performed
using VG AutoSpec EI mass spectrometer and
Micromass LCT time of flight (TOF) mass spectrometer
with electrospray ionization (ESI). Elemental analysis was
performed using VarioEL III elemental analyzer.
chemical shifts were derived from equation d ¼ s 2 s;
n
where d is the chemical shift, s is the absolute
shielding, and sn is the absolute shielding of the
standard (TMS; 185.76 ppm).
Scheme 1. Synthesis of 1.

J. Koivukorpi et al. / Journal of Molecular Structure 693 (2004) 81–86
83
Scheme 2. Syntheses of 2 and 3.
2
.3. Synthesis
,3,24,24-tetrakis(pyrrol-2-yl)-5b-cholane 1: 3a-
hydroxy-5b-cholan-24-oic acid (lithocholic acid) 4 was
products like tripyrranes. Reaction time was also crucial
because the more reactive aldehyde reacted first with
pyrrole forming dipyrromethane. Thus, when the reaction
mixture was stirred for 3 h, the product was mainly 3-oxo-
24,24-bis(pyrrol-2-yl)-5b-cholane. Increasing the reaction
time to 20 h gave the product where both the aldehyde
and the ketone were reacted. The crude product was
purified by flash chromatography (silica gel, 0.040–
3
used as a starting material in the synthesis of 3,3,24,
2
4-tetrakis(pyrrol-2-yl)-5b-cholane 1. In the first step
LCA (3.28 mmol) was allowed to react with triethylamine
11.8 mmol) and ethyl chloroformate (10.8 mmol) result-
(
ing in a very reactive anhydride intermediate (NaBH is
4
0.063 mm; CH Cl ). The overall yield of 1 was 22%
2 2
not powerful enough to reduce acid directly), which was
further reduced to 5b-cholane-3,24-diol 5 with NaBH4
(0.71 mmol) calculated from lithocholic acid. The reaction
route to 1 is described in Scheme 1.
(
36.1 mmol) [16]. The diol (1.97 mmol) was then oxidized
to 3-oxo-5b-cholan-24-al 6 by using CrO -pyridine (CrO ,
Elemental analysis of 1: C: 81.70, H: 9.59, N: 9.06%.
Calculated: C: 81.31, H: 9.21, N: 9.48%.
3,3-Bis(pyrrol-2-yl)-5b-cholan-24-oic acid 2 was pre-
3
3
2
3.6 mmol; pyridine, 47.3 mmol) [17]. The desired
product was obtained by allowing 3-oxo-5b-cholan-24-al
1.87 mmol) to react with a five-fold excess (20 equiva-
pared by oxidizing 4 (5.43 mmol) with Na Cr O7. 2 H O
2 2 2
(
(5.43 mmol) [19], and formed 3-oxo-5b-cholan-24-oic
acid (5.38 mmol) was stirred for 3 h at r.t. with 10
.
3
lents) of pyrrole (37.4 mmol) using BF OEt as a catalyst
2
.
[18]. Excess pyrrole was the needed in reaction to prevent
the formation of pyrrole macrocycles and other side
equivalents of pyrrole (53.8 mmol) using BF OEt as a
2
3
catalyst. The crude product was purified by flash
Fig. 2. Structures and numbering of 1–3.

8
4
J. Koivukorpi et al. / Journal of Molecular Structure 693 (2004) 81–86
Table 2
chromatography (silica gel, 0.040–0.063 mm; CH Cl :
2
2
13
C NMR chemical shifts
MeOH, 99:1). The overall yield of 2 was 45%
(
2.42 mmol) calculated from 4.
Elemental analysis of 2: C: 76.01, H: 9.44, N: 4.97, O:
Carbon 1 Measured d 1 Calculated d 2 Measured d 3 Measured d
(
ppm)
33.0
(ppm)
(ppm)
(ppm)
9
.58%. Calculated: C: 75.55, H: 9.51, N: 5.50, O: 9.43%,
contains 2 þ H O.
1
2
3
4
5
6
7
8
9
1
1
31.2
28.9
43.5
39.1
40.7
26.8
26.1
36.5
41.2
38.2
21.3
40.2
46.3
56.8
24.2
27.4
58.4
9.7
33.0
31.3
40.6
37.9
39.3
27.1
26.3
35.8
40.4
35.0
20.9
40.2
42.8
56.5
24.2
28.2
56.0
12.1
23.8
35.3
18.3
33.0
31.3
40.5
37.9
39.3
27.1
26.3
35.8
40.4
34.9
20.9
40.2
42.8
56.5
24.2
28.2
56.0
12.0
23.8
35.4
18.3
2
(a)
31.3
Methyl 3,3-Bis(pyrrol-2-yl)-5b-cholan-24-oate 3 was
prepared by the same procedure as 2, except that methyl
lithocholate 7 (1.46 mmol) was used as starting compound
40.6
37.9
39.3
27.2
26.4
35.8
40.4
35.0
20.9
40.2
42.7
56.5
24.2
28.2
56.0
12.0
23.8
35.7
18.7
33.8
[
20]. The overall yield of 3 was 40% (0.59 mmol) calculated
from 7. The synthetic route to 2 and 3 is described in
Scheme 2.
Elemental analysis of 3: C: 77.74, H: 9.66, N: 5.06, O:
0
1
7
.55%. Calculated: C: 77.14, H: 9.61, N: 5.45, O: 7.78%,
contains 2* 3 þ H O
2
12
1
1
1
1
1
3
4
5
6
7
3
. Results and discussion
The ab initio HF/6-31G* optimised minimum energy
18
19
structure of 1 and numbering of 1–3 are shown in Figs. 1
and 2. The optimised structure of 1 was necessary for
determination of proton–proton distances, which were
crucial in estimating intramolecular nuclear Overhauser
effects (NOE) and assigning four pyrrole moieties. Unfortu-
nately, we were not able to grow single crystals suitable for
X-ray structural analyses.
21.3
33.4
13.9
30.9
27.5
35.6
–
20
21
22
23
(
a)
(a)
31.0
31.1
31.0
174.7
51.4
141.3
101.6
107.8
116.3
134.9
106.4
107.9
117.1
–
(a)
(a)
(a)
31.2
38.1
30.8
179.9
–
24
25
a2
a3
a4
a5
b2
b3
b4
b5
c2
c3
c4
c5
d2
d3
d4
d5
–
141.3
101.6
107.8
116.3
134.9
106.4
108.0
117.2
134.0
105.1
108.1
116.8
133.5
105.5
108.1
116.9
139.9
101.5
107.7
114.7
134.3
106.8
107.9
115.6
133.0
103.6
107.9
115.2
131.7
108.9
108.1
116.1
141.3
101.6
107.8
116.3
134.9
106.4
108.0
117.2
–
1
H NMR chemical shifts of the pyrrole moieties of 1–3
1
are presented in Table 1, C NMR chemical shifts of 1–3 in
3
Table 1
1
H NMR chemical shifts of pyrrole rings
(b)
(b)
(b)
(b)
(b)
(b)
Proton 1 d (ppm)
2 d (ppm)
3 d (ppm)
–
–
a-3
a-4
6.04 (m)
6.11
6.04 (m)
6.12
6.04 (m)
6.11
(c)
(d)
–
–
–
–
(
ddd, J < 2:8 Hz each) (ddd, J < 2:7 Hz (ddd, J < 2:8 Hz
–
–
each)
each)
–
–
a-5
6.53
6.53
6.53
(c)
(d)
–
–
(
d, J ¼ 1:35 Hz)
(d, J ¼ 1:51 Hz)
7.59 (s)
6.19 (m)
6.17
(d, J ¼ 1:49 Hz)
7.59 (s)
6.19 (m)
6.17
–
–
a-N
b-3
b-4
7.58 (s)
6.19 (m)
6.17
Superscripts denote pairs of exchangeable assignments.
(ddd, J < 2:8 Hz each) (ddd, J < 2:7 Hz (ddd, J < 2:7 Hz
1
Table 2, and N NMR chemical shifts of 1 in Table 3.
5
each)
each)
b-5
6.67
6.67
6.66
1
In Table 1 the assignments of the H chemical shifts of
(d, J ¼ 1:18 Hz)
(d, J ¼ 1:48 Hz)
(d, J ¼ 1:06 Hz)
1
pyrrole rings were based on H– H Rotating Frame NOESY
1
b-N
c-3
c-4
7.80 (s)
6.10 (m)
6.15
7.82 (s)
7.83 (s)
–
–
–
–
(ROESY with spin lock time 50 ms) measurement of 1.
(ddd, J < 2:8 Hz each)
c-5
6.63
–
–
Table 3
15
(d, J ¼ 1:1:33 Hz)
N NMR chemical shifts of 1
c-N
d-3
d-4
7.78 (s)
6.10 (m)
6.15
–
–
–
–
–
–
Pyrrole
d (ppm)
(
ddd, J < 2:8 Hz each)
6.64
d, J ¼ 1:37 Hz)
7.78 (s)
a
b
c
d
2234,8
2236,8
2234,8
2234,8
d-5
–
–
–
–
(
d-N

J. Koivukorpi et al. / Journal of Molecular Structure 693 (2004) 81–86
85
13
Fig. 3. Differences in calculated C NMR chemical shifts of 1 compared to measured shifts.
NOE connectivities were observed between H-b3 in the
˚
and attached to an acyclic carbon, are more shielded. The
1
3
axial pyrrole b and H-5ax (distance H-b3–H-5 ¼ 2.35 A)
ax
calculated C NMR chemical shifts of the carbons in
pyrroles are generally slightly deshielded, but an exception
is C-3 in pyrrole d, which is shielded in comparison with the
experimental value. The differences between calculated and
˚
and the H-4eq (distance H-b3–H-4 ¼ 2.42 A) of the
eq
cyclohexane ring A of the cholane moiety, while H-a3 in
the equatorial pyrrole a showed NOEs with H-2ax (distance
1
3
˚
H-a3–H-2ax ¼ 2.28 A) and H-4 (distance H-a3–H-
ax
measured C NMR chemical shifts are presented in a
graphical form in Fig. 3.
˚
4ax ¼ 3.58 A) in cyclohexane ring A, respectively
1
5
(the distances are measured from minimum energy structure
of 1, so they are not the shortest possible distances). The
N NMR chemical shifts of 1 have been determined by
1 15
PFG H– N HMBC measurement (evolution delay 100 ms)
1
complete H NMR shift assignment of the steroidal part of 1
They are close to that of pyrrole itself measured in CDCl ,
3
1
5
was not carried out owing to seriously overlapping
resonance lines.
145.2 ppm from d( NH ) ¼ 0.0 ppm [21], which
3
1
3
5
corresponds to 2235.0 ppm from d(CH NO ) ¼ 0.0 ppm.
2
1
The C NMR chemical shift assignments of 1–3 were
3
The ESI-MS measurement of 1 showed the ion
1
3
1
13
based on C DEPT-135, PFG H, C HMQC and PFG
2
m=z ¼ 625.48 [M þ Cl] (M[C H N ] ¼ 590.90). EI
4
0
54
4
1
13
H, C HMBC (with 3.45 ms low pass filter to remove the
þ
mass measurement showed ions m/z ¼ 590 [M] , 523
þ þ þ
effect of direct couplings and 50 ms evolution time for
geminal and vicinal couplings) measurements, and the data
were compared with our previously published data of
[M–pyrrole] , 456 [M–2 pyrrole] , 320 [M–4 pyrrole]
and lower fragments from the steroidal moiety. As EI mass
measurement shows, the first two pyrroles are cleaved one at
the time, and the last two pyrroles are cleaved at the same
time. Probably the pyrroles attached to carbon 3 in steroidal
skeleton are the ones, which are first cleaved one at the time
due to the more sterically hindered structure. The pyrrole
rings attached to the carbon 24 are equal, and so it can be
assumed that they are cleaved at the same time.
1
3
similar compounds [3,15]. The C NMR chemical shift
ranges of two pyrroles at C-3 in 1 are clearly larger than
those attached to C-24. This is mainly due to the effect two
b-carbons (C-2 and C-4) to C-a2 and C-b2 while C-c2 and
C-d2 in the pyrroles at C-24 have only one b-carbon (C-23).
Another reason can be that pyrroles at C-24 have a greater
motional freedom than those at C-3.
The ESI-MS measurement showed following ions
2
1
3
2
for 2: m=z ¼ 489:40 [M-H] , 625.48 [M þ Cl] ,
2
The B3PW91/6-311G*/HF/6-31G* calculated C NMR
chemical shifts of 1 are also presented in Table 2.
The greatest differences between the calculated and
experimental data are 23.63 (C-13) and 4.83 ppm (C-21).
(M[C32
2
H
N
O
] ¼ 490.74) and for 3: m=z ¼ 503.38 [M–
46 2 2
H] , 539.37 [M þ Cl] , (M[C33 O
EI mass measurement showed following ions of interest
H
N
48
] ¼ 504.76).
2
2
1
3
þ
þ
In general, it can be mentioned that the calculated C NMR
chemical shifts of the acyclic carbons (such as the methyls
for 2: m=z ¼ 490 [M] , 423 [M–pyrrole] , and for 3 m=z ¼
þ þ
504 [M] , 437 [M–pyrrole] , and also the lower fragments
from the steroidal moiety. In the cases of 2 and 3 only the
cleavage of one pyrrole ring can be observed in EI mass
spectra.
1
8, 19 and 21 and the side chain) in cholane moiety are
more deshielded than the measured ones, whereas carbons
0, 13 and 17, which are members of the cyclic system,
1

86
J. Koivukorpi et al. / Journal of Molecular Structure 693 (2004) 81–86
4
. Conclusions
[9] A. Montalban, A.J. Herrera, J. Johannsen, J. Beck, T. Godet,
M. Vrettou, A.J.P. White, D.J. Williams, Tetrahedron Lett. 43
2002) 1751.
(
We describe the syntheses and characterization of three
[
10] (a) M. Rance, O.W. Sørensen, G. Bodenhausen, G. Wagner, R.R.
Ernst, K. W u¨ thrich, Biochem. Biophys. Res. Commun. 117
lithocholic acid based dipyrromethanes. Because the
syntheses take place with good overall yields, these
compounds can be used further in syntheses of other
oligopyrroles and complexation studies with transition
metal cations.
(
1984) 479. (b) A. Derome, M. Williamson, J. Magn. Reson. 88
(1990) 177.
[11] A. Bax, D.G. Davis, J. Magn. Reson. 63 (1985) 207.
[
12] (a) A. Bax, R.H. Griffey, B.L. Hawkins, J. Magn. Reson. 55 (1983)
01. (b) A. Bax, S. Subramanian, J. Magn. Reson. 67 (1986) 565.
3
[
[
13] A. Bax, M.F. Summers, J. Am. Chem. Soc. 108 (1986) 2093.
14] Revision A.11, M.J. Frisch, G.W. Trucks, H.B. Schlegel,
G.E. Scuseria, M. A. Robb, J. R. Cheeseman, V. G. Zakrzewski,
J. A. Montgomery Jr., R. E. Stratmann, J. C. Burant, S. Dapprich,
J. M. Millam, A.D. Daniels, K.N. Kudin, M.C. Strain, O. Farkas,
J. Tomasi, V. Barone, M. Cossi, R. Cammi, B. Mennucci, C. Pomelli,
C. Adamo, S. Clifford, J. Ochterski, G.A. Petersson, P.Y. Ayala,
Q. Cui, K. Morokuma, P. Salvador, J.J. Dannenberg, D.K. Malick,
A.D. Rabuck, K. Raghavachari, J.B. Foresman, J. Cioslowski,
J.V. Ortiz, A.G. Baboul, B.B. Stefanov, G. Liu, A. Liashenko,
P. Piskorz, I. Komaromi, R. Gomperts, R.L. Martin, D.J. Fox,
T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara,
M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen, M.W. Wong,
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Acknowledgements
The authors acknowledge Spec. Lab. Tech. Reijo
Kauppinen for his help in running the NMR spectra, Spec.
Lab. Tech. Mirja Lahtiper a¨ for mass spectra, Lab. Tech.
Elina Hautakangas for elemental analysis, and M.Sc. Heikki
Tuononen for his assistance in molecular modeling.
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