Facile chemical syntheses of porphobilinogen analogues: A four-step synthesis to iso-porphobilinogen

Article abstract of DOI:10.1055/s-2001-16752

iso-Porphobilinogen was prepared in four good yielding steps, as well as a group of iso-porphobilinogen analogues, using a modification of Zav'yalov's method.

Full text of DOI:10.1055/s-2001-16752

PAPER
1627
Facile Chemical Syntheses of Porphobilinogen Analogues: A Four-Step
Synthesis to iso-Porphobilinogen
S
ynthese
s
of Porph
w
obilinogen Analo
a
gues i-Ming Cheung, Peter M. Shoolingin-Jordan*
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Biomedical Sciences Buil-
ding, Bassett Crescent East, Southampton SO16 7PX, UK
Fax +44(0)2380594459; E-mail: pmsj@soton.ac.uk
Received 1 March 2001; revised 28 May 2001
prepared in only four steps, avoiding several tedious, low
Abstract: iso-Porphobilinogen was prepared in four good yielding
yielding reactions used in earlier methods7 (Scheme 1).
steps, as well as a group of iso-porphobilinogen analogues, using a
modification of Zav’yalov’s method.
Diethyl 4-oxopimelate was transformed into diethyl 3-
formyl-4-oxoheptanedioate (1) which was then con-
densed with potassium aminoacetate. The resulting salt
was heated in acetic anhydride and the pyrrole ring was
constructed after decarboxylation and dehydration. By
varying the amino acid (Scheme 1), various ‘iso-PBG’ an-
alogues were synthesised. Compound 4d was converted
into iso-PBG lactam 5 by reacting with propylamine. The
subsequent hydrolysis to iso-PBG 6 with base has been re-
ported in the literature7a (Scheme 2).
Key words: iso-porphobilinogen, 5-aminolevulinic acid, tetrapyr-
roles, diethyl opsopyrroledicarboxylate, porphobilinogen
Porphobilinogen (PBG), a key building block in the bio-
synthesis of porphyrins, chlorophylls and corrins1 is
formed in a single step from two molecules of 5-aminole-
vulinic acid in a reaction catalysed by 5-aminolevulinic
acid dehydratase.2 Four molecules of PBG are subse-
quently polymerised by PBG deaminase to preuroporphy-
rinogen, a highly labile 1-hydroxymethylbilane. This is
then rapidly cyclised, with rearrangement, by uroporphy-
rinogen (III) synthase3 to give the ubiquitous tetrapyrrole
macrocycle, uroporphyrinogen (III).
The initial enamine intermediate 2 formed was thought to
have tautomerised to a relatively stable , -unsaturated-
1
-amino ketone. This was evident by analysing the H
NMR (CD3OD) of the crude samples 3a, 3b, 3c, 3e, in
which a singlet was observed at = 7.66, 7.72, 7.78 and
7.68 in each case, respectively. The protons of phthalim-
ide obscured the singlet shown by compound 3d.
Because of the essential role that tetrapyrroles play in me-
tabolism, there is a considerable amount of interest in the
design of inhibitors active against enzymes from this bio-
synthesis pathway. The current study focuses on the poly-
merase, PBG deaminase, an enzyme that recognises
pyrroles with paired acetate and propionate functions at
the -positions. Thus, iso-porphobilinogen4 and opsopyr-
roledicarboxylic acid5 have both been shown to exhibit
competitive inhibition against the deaminase. We report
here on the synthesis of a group of PBG analogues that
possess these key properties together with a variety of car-
boxy substitutions at the C2 position of 3-(4-carboxyme-
In the reaction from 3c to 4c, the original synthetic strate-
gy was to place a benzyloxymethyl group at the C2-posi-
tion of the resulting pyrrole ring. However, during the
reaction at 130 °C, the benzyloxy group reacted with the
solvent, acetic anhydride, forming an acetate group. In the
reaction from 3e to 4e, the benzylsulfanyl group did not
react with the solvent, hence, the resulting pyrrole re-
tained the benzylsulfanylmethyl group instead of a thioac-
etate.
The starting amino acid 10 for the preparation of 4d (R =
CH2Pht) is not commercially available and was obtained
from compound 7 (Scheme 3). The amide 7 was convert-
ed first to the amine 8 according to the literature8 and then
to compound 99 by reacting with N-ethoxycarbon-
ylphthalimide. Hydrogenation of 9 subsequently afforded
10.
thyl-1H-pyrrol-3-yl)propionic
acid
(opsopyrroledi-
carboxylic acid). The substituted groups at C2 include ac-
etoxymethyl, phthalimidomethyl, benzylsulfanylmethyl
and aminomethyl moieties. Our approach has been to
adopt existing methodologies6 for pyrrole synthesis to
produce a group of iso-porphobilinogen analogues with
potential for use as pseudo-substrates or inhibitors that
can not only be used to investigate the remarkable en-
zyme-catalysed polymerisation process but that could be
adapted for use as inhibitors with potential in agriculture
or medicine.
From the above results, various ‘iso-PBG’ analogues were
synthesised conveniently and efficiently by the condensa-
tion between compound 1 and the potassium salts of vari-
ous amino acids. Compounds 4a and 4b can be prepared
on gram scales that may provide some alternative facile
synthetic routes to porphobilinogen in a larger scale.
Compounds 4c, 4d, 4d and 4e can be used to synthesise
related dipyrromethanes, tripyrromethanes, and for the
syntheses of uroporphyrinogen and uroporphyrin isomers.
In the method adopted, 3-(2-aminomethyl-4-carboxyme-
thyl-1H-pyrrol-3-yl)propionic acid (6, iso-PBG), can be
Synthesis 2001, No. 11, 28 08 2001. Article Identifier:
1437-210X,E;2001,0,11,1627,1630,ftx,en;E02301ss.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0039-7881

1628
K.-M. Cheung, P. M. Shoolingin-Jordan
PAPER
Scheme 1 Reagents and conditions: (i) Na/HCO2Et/benzene; (ii) MeOH/H2NCH(R)CO2 K+; (iii) Ac2O/130 °C
–
Scheme 2 Reagents and conditions: (i) Propylamine, MeOH, 10 d; (ii) 2 M KOH, then AcOH
peak at = 4.8 as reference. The NMR spectra were recorded on a
Bruker AC300 instrument. Melting points were determined with an
Electrothermal apparatus and are uncorrected. IR spectra were re-
corded on a Philips Pye Unicam SP3-200 spectrophotometer. Elec-
trospray mass spectrometry data, ES (MS), were collected using a
Quattro II triple-quadrapole (VG Biotech, Altringham, Cheshire,
UK). High-resolution accurate mass data were recorded on a VG
Analytical 70-250-SE normal geometry double focussing mass
spectrometer. Compound 1,6 4a,6 88 and 99 were synthesised ac-
cording to methods in the literature. Petroleum ether refers to the
fraction boiling at 40–60 °C.
N- -Benzyloxycarbonyl-L-asparagine, O-benzyl-L-serine and S-
benzyl-L-cysteine were purchased from Novabiochem and diethyl
4-oxopimelate from Aldrich Chemical Co. 1H and 13C NMR spectra
were recorded in CDCl3, DMSO-d6 and DMF-d7 with TMS as inter-
nal reference and 2 M NaOD/D2O with assignment of the DOH
Amino Acid Potassium Salts; General Procedure
Equal mol-equivalents of the corresponding amino acid and KOH
were mixed in H2O and the H2O was then evaporated in a rotary
evaporator. The remaining potassium salt was dried in vacuo for 5
h prior to use.
Pyrroles 4; General Procedure
A mixture of mol-equivalents of potassium amino acid salt and di-
ethyl 3-formyl-4-oxoheptanedioate (1) in MeOH (60 mL) was
Scheme 3 Reagents and conditions: (i) (CF3CO2)2IC6H5/pyridine/
DMF/H2O; (ii) N-ethoxycarbonylphthalimide/1.8% Na2CO3; (iii)
10% Pd-C, EtOH
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PAPER
Syntheses of Porphobilinogen Analogues
1629
stirred at 20 °C (water bath) for 48 h. MeOH was then evaporated
and Ac2O (60 mL) was added. The mixture was stirred at r.t. for 2 h
and 130 °C (oil bath) for 3 h. Evaporation of the Ac2O gave a dark
oil. CHCl3 (250 mL) was added to dissolve the oil and the solution
was filtered. After the filtrate was evaporated, the dark syrup was
passed through an alumina column, eluting with CHCl3 unless oth-
erwise stated.
Ethyl 3-[1-Acetyl-2-(1,3-dioxo-1,3-dihydroisoindol-2-
ylmethyl)-4-ethoxycarbonylmethyl-1H-pyrrol-3-yl]propionate
(4d) and Ethyl 3-[2-(1,3-Dioxo-1,3-dihydroisoindol-2-
ylmethyl)-4-ethoxycarbonylmethyl-1H-pyrrol-3-yl]propionate
(4d )
A mixture of potassium L-2-amino-3-(1,3-dioxo-2,3-dihydro-1H-2-
isoindolyl)propionate (0.55 g, 2.0 mmol) and 1 (0.55 g, 2.1 mmol)
in MeOH (25 mL) was used for the reaction. After evaporation of
CHCl3, the dark syrup was passed through an alumina column, elut-
ing with EtOAc. The fast moving fractions were collected. After
evaporation of the solvent, a yellow oil (0.2 g, 23%) was obtained.
The 1H NMR revealed a mixture of compounds 4d and 4d in a ratio
of 1.0:1.4. They were separated by preparative TLC using EtOAc–
petroleum ether (1:2) as eluent. The Rf values of compounds 4d and
4d were 0.21 and 0.33, respectively.
Ethyl 3-(1-Acetyl-4-ethoxycarbonylmethyl-1H-pyrrol-3-
yl)propionate (4a)
A mixture of potassium 2-aminoacetate (3.18 g, 28 mmol) and 1
(7.26 g, 28 mmol) in MeOH (60 mL) was used for the reaction.
Fractions at Rf 0.70 (alumina) were collected. After evaporation of
the solvent, a brown oil (4.3 g, 51%) was obtained. It was further
purified by chromatography on a silica gel column eluting with
CHCl3–EtOAc (5:1). The fractions at Rf 0.53 (silica gel) were col-
lected and the solvent evaporated.
Compound 4d
IR (film): = 3030, 2980, 1790 (weak), 1740 (br), 1200, 1050, 950
IR (film): = 3000, 2950, 1730 (br), 1190, 1030, 910 cm–1.
cm–1.
1H NMR (CDCl3): = 7.20 (1 H, s), 7.08 (1 H, s), 4.16 (4 H, m),
3.44 (2 H, s), 2.74 (2 H, t, J = 7.5 Hz), 2.60 (2 H, t, J = 7.5 Hz), 2.47
(3 H, s), 1.25 (6 H, m).
13C NMR (CDCl3): = 172.9, 171.3, 167.2, 126.9, 120.4, 118.3,
116.4, 60.7, 60.5, 37.1, 33.9, 31.1, 27.9, 22.1, 20.4, 14.2.
1H NMR (CDCl3): = 7.82 (2 H, m), 7.70 (2 H, m), 7.13 (1 H, s),
5.12 (2 H, s), 4.15 (2 H, m), 4.10 (2 H, m), 3.48 (2 H, s), 2.90 (2 H,
t, J = 7.5 Hz), 2.55 (2 H, t, J = 7.5 Hz), 2.50 (3 H, s), 1.25 (6 H, m).
13C NMR (CDCl3): = 173.1, 171.7, 169.3, 168.4, 134.2, 132.5,
128.5, 125.2, 123.5, 120.7, 119.1, 61.5, 60.9, 34.8, 31.4, 24.3, 19.8,
14.6.
HRMS: m/z Calcd for C15H21NO5 : 295.1420, found: 295.1432.
ES (MS): m/z (%) = 477.2 (100, [M + Na]+), 317.3 (18, [M + Na –
PhtCH2]).
Ethyl 3-(1-Acetyl-4-ethoxycarbonylmethyl-2-methyl-1H-
pyrrol-3-yl)propionate (4b)
A mixture of potassium DL-2-aminopropionate (4.67 g, 37 mmol)
and 1 (9.52 g, 37 mmol) in MeOH (60 mL) was used for the reac-
tion. Fractions at Rf 0.73 (alumina) were collected. After evapora-
tion of the solvent, a brown oil (6.3 g, 65 %) was obtained that was
further purified by chromatography on a silica gel column eluting
with CHCl3–EtOAc (5:1). The fractions at Rf 0.64 (silica gel) were
collected and the solvent evaporated.
HRMS: m/z Calcd for C24H26N2O7: 454.1740, found: 454.1725.
Compound 4d was obtained as a solid and recrystallised from
MeOH at –20 °C; mp 99–100 °C.
IR (nujol): = 3440, 3020, 2985, 1780 (weak), 1740 (br), 1725 (br),
1190, 1090, 1045, 940 cm–1.
1H NMR (CDCl3): = 8.65 (1 H, s), 7.84 (2 H, m), 7.75 (2 H, m),
6.68 (1 H, d, J = 2 Hz), 4.82 (2 H, s), 4.14 (4 H, m), 3.43 (2 H, s),
2.92 (2 H, t, J = 7.5 Hz), 2.53 (2 H, t, J = 7.5 Hz), 1.28 (6 H, m).
13C NMR (CDCl3): = 173.2, 172.4, 168.5, 134.1, 132.0, 123.6,
123.4, 119.3, 117.0, 114.4, 60.7, 60.3, 35.8, 32.5, 31.3, 19.2, 14.2.
ES (MS): m/z (%) = 435.4 (100, [M + Na]+), 412.8 (30, [M]+).
IR (film): = 2990, 2940, 1720 (br), 1180, 1030, 940 cm–1.
1H NMR (CDCl3): = 6.97 (1 H, s), 4.18 (4 H, m), 3.44 (2 H, s),
2.68 (2 H, t, J = 7.5 Hz), 2.49 (3 H, s), 2.42 (2 H, t, J = 7.5 Hz), 2.41
(3 H, s), 1.25 (6 H, m).
13C NMR (CDCl3): = 172.9, 171.6, 169.1, 129.2, 122.8, 118.7,
117.8, 60.9, 60.4, 34.8, 31.1, 24.1, 19.2, 14.2, 13.4.
HRMS: m/z Calcd for C22H24N2O6: 412.1634, found: 412.1644.
HRMS: m/z Calcd for C16H23NO5: 309.1576, found: 309.1566.
Ethyl 3-(1-Acetyl-2-benzylsulfanylmethyl-4-ethoxycarbonyl-
methyl-1H-pyrrol-3-yl)-propionate (4e)
Ethyl 3-(2-Acetoxymethyl-1-acetyl-4-ethoxycarbonylmethyl-
1H-pyrrol-3-yl)-propionate (4c)
A mixture of potassium L-2-amino-3-benzylsulfanylpropionate
(1.18 g, 4.7 mmol) and 1 (1.22 g, 4.7 mmol) in MeOH (10 mL) was
used for the reaction. The fractions at Rf 0.85 were collected (alu-
mina; elution with EtOAc). The dark oil obtained (0.9 g) was puri-
fied by chromatography on a silica gel column using EtOAc–
petroleum ether (1:2) as eluent. The fractions at Rf 0.7 (silica gel)
were collected and the solvent evaporated. The liquid obtained was
further purified by another silica gel column eluting with EtOAc–
petroleum ether–CHCl3 (1:2:0.5). The fractions at Rf 0.59 were col-
lected (silica gel) and the solvent evaporated to give a yellow oil
(0.17 g, 8%).
A mixture of potassium L-2-amino-3-benzyloxypropionate (1.81 g,
7.8 mmol) and 1 (1.87 g, 7.2 mmol) in MeOH (10 mL) was used for
the reaction. The fast moving fractions were collected (alumina).
The dark oil obtained (0.92 g) was further purified by chromatogra-
phy on a silica gel column eluting with CHCl3–EtOAc (5:1). The
fractions at Rf 0.37 (silica gel) were collected and the solvent evap-
orated to afford a yellow oil (0.1 g, ca 4%).
IR (film): = 3020, 2970, 1755, 1745, 1275, 1200, 1040 cm–1.
1H NMR (CDCl3): = 7.15 (1 H, s), 5.34 (2 H, s), 4.18 (4 H, m),
3.45 (2 H, s), 2.80 (2 H, t, J = 7.5 Hz), 2.54 (3 H, s), 2.45 (2 H, t, J
= 7.5 Hz), 2.05 (3 H, s), 1.30 (6 H, m).
13C NMR (CDCl3): = 172.4, 171.2, 170.9, 168.2, 128.6, 126.0,
121.0, 118.8, 61.1, 60.5, 57.0, 35.2, 30.9, 23.9, 21.0, 19.1, 14.2.
IR (film): = 3050, 2960, 1730 (br), 1185, 1100, 1035, 960, 870
cm–1.
1H NMR (CDCl3): = 7.32 (5 H, m), 7.02 (1 H, s), 4.14 (4 H, m),
4.06 (2 H, s), 3.76 (2 H, s), 3.42 (2 H, s), 2.80 (2 H, t, J = 7.5 Hz),
2.58 (2 H, t, J = 7.5 Hz), 2.52 (3 H, s), 1.28 (6 H, m).
HRMS: m/z Calcd for C18H25NO7: 367.1631, found: 367.1631.
Anal. Calcd. For C18H25NO7 (367.4): C 58.84 H 6.86 N 3.81, found
C 58.26 H 6.85 N 3.38.
13C NMR (CDCl3): = 172.8, 171.5, 169.0, 139.0, 129.0, 128.6,
127.0, 120.0, 118.7, 61.2, 60.6, 37.2, 35.0, 31.2, 27.6, 24.4, 19.3,
14.3 (2 CH3).
HRMS: m/z Calcd for C23H29NO5S: 431.1766, found: 431.1763.
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K.-M. Cheung, P. M. Shoolingin-Jordan
PAPER
Ethyl (6-Oxo-1,4,5,6,7,8-hexahydropyrrolo[2,3-c]azepin-3-
yl)acetate (5)
the precipitates. After filtration and evaporation of the solvents, a
white solid (0.5 g, 82%) was obtained, which was washed with
EtOAc and dried; mp >220 °C (dec.).
To a solution of 4d (50 mg, 0.12 mmol) in MeOH (4 mL) was add-
ed propylamine (0.3 mL). After stirring the mixture for 10 d at 25
°C, the solvent was evaporated. The residue was passed through a
small silica gel column and eluted with MeOH–CHCl3 (1:6). The
fractions at Rf 0.6 were collected and the solvent evaporated to yield
a light yellow solid (12 mg, 42%). It was recrystallised from EtOH
at –20 °C; mp 158–159 °C.
IR (nujol): = 3400–2500, 2960, 1790, 1735, 1725, 1640 (br), 1080
cm–1.
1H NMR (2 M NaOD/D2O): = 7.10–7.30 (4 H, m), 3.05–3.35 (3
H, m).
13C NMR (2 M NaOD/D2O): = 182.9, 178.5, 175.6, 140.0, 136.9,
132.9, 131.9, 130.5, 129.7, 58.2, 46.9.
IR (nujol): = 3390, 3200, 1745, 1655 (br), 1200, 1180, 1120, 1045
cm–1.
1H NMR (DMF-d7): = 8.40 (1 H, s), 7.25 (1 H, s), 6.50 (1 H, d, J
= 2.2 Hz), 4.28 (2 H, d, J = 5.5 Hz), 4.10 (2 H, q, J = 7.5 Hz), 3.35
(2 H, s), 2.70 (4 H, br s), 1.15 (3 H, t, J = 7.5 Hz).
13C NMR (DMF-d7): = 175.0, 171.1, 125.3, 115.8, 113.8, 113.4,
59.3, 37.4, 32.9, 30.4, 20.0, 13.1.
Acknowledgement
This work was supported by the EPSRC, the BBSRC and by the
Wellcome Trust. We also wish to thank for the Chemical Database
Service provided by the EPSRC at Daresbury.10 We are grateful to
Joan Street for providing the NMR service and Dr. J. Langley for
the high resolution mass spectroscopy (HRMS) in the Chemistry
Department at the University of Southampton.
HRMS: m/z Calcd for C12H16N2O3: 236.1161, found: 236.1165.
Anal. Calcd for C12H16N2O3 (236.3): C 61.00 H 6.83 N 11.86, found
C 60.44 H 6.64 N 11.30.
References
L-2-Benzyloxycarbonylamino-3-(1,3-dioxo-1,3-dihydro-
isoindol-2-yl)propionic Acid (9)
(1) (a) Cookson, G. H.; Rimington, C. Biochem. J. 1953, 57,
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L-3-Amino-2-benzyloxycarbonylaminopropanoic acid 8 (0.83 g,
3.5 mmol) was stirred with N-ethoxycarbonylphthalimide (0.8 g,
3.7 mmol) in 1.8% aq Na2CO3 solution (20 mL) at r.t. The suspen-
sion became a yellow solution after 4 h. The reaction mixture was
washed with Et2O (3 30 mL). The aqueous phase was then adjust-
ed to pH 2–3 and extracted with EtOAc (3 50 mL). After drying
the extract (Na2SO4) and evaporation of the solvent, the white solid
(0.96 g, 75%) obtained was washed with petroleum ether and dried;
mp 140.5–142 °C.
IR (nujol): = 3340, 3300–2600, 2960, 1790 (weak), 1755 (sh),
1740 (sh), 1725 (sh), 1700 (br), 1545, 1265, 1200, 1070 cm–1.
1H NMR (DMSO-d6): = 13.1 (1 H, br s), 7.90 (4 H, m), 7.30 (5 H,
m), 5.00 (2 H, s), 4.35 (1 H, m), 3.95 (2 H, m).
13C NMR (DMSO-d6): = 171.2, 167.6, 155.9, 136.8, 134.5, 131.5,
128.3, 127.7, 127.4, 123.1, 65.4, 51.7, 38.3.
(8) Waki, M.; Kitajima, Y.; Izumiya, N. Synthesis 1981, 266.
(9) Funabashi, Y.; Tsubotani, S.; Koyama, K.; Katayama, N.;
Harada, S. Tetrahedron 1993, 49, 13.
L-2-Amino-3-(1,3-dioxo-1,3-dihydroisoindol-2-yl)propionic
Acid (10)
(10) The United Kingdom Chemical Database Service: Fletcher,
D. A.; McMeeking, R. F.; Parkin, D. J. Chem. Inf. Comput.
Sci. 1996, 36, 746.
Compound 9 (0.9 g, 2.5 mmol) was dissolved in warm EtOH (20
mL) and 10% Pd-C (0.1 g) was added. The mixture was hydroge-
nated at 60 °C for 2 d, when white precipitates were observed during
the reaction. Concd. ammonia (20 mL) was then added to dissolve
Synthesis 2001, No. 11, 1627–1630 ISSN 0039-7881 © Thieme Stuttgart · New York