Synthesis of tritium labelled mecillinam

Article abstract of DOI:10.1002/jlcr.717

The potent β-lactam antibiotic mecillinam has successfully been labelled with tritium. 2,3,4,7-Tetrahydro-1H-azepin-1-carbaldehyde was formed in a ring-closing metathesis reaction and then condensed with trimethylsilyl protected penicillanic acid to give dehydro-mecillinam. Pd/C catalysed tritiation then gave [³H₂]-mecillinam (6-β (([3,4-³H₂]hexahydro-1H-azepin-1-yl)methyleneamino) penicillanic acid) with a radiochemical purity of 96% and a specific activity of 24Ci/mmol. Copyright

Full text of DOI:10.1002/jlcr.717

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 773–779.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.717
Research Article
Synthesis of tritium labelled mecillinam
Sꢀren Melsing Frederiksen and Gunnar Grue-Sꢀrensen*
LEO Pharma, Medicinal Chemistry Research, Industriparken 55,
DK-2750 Ballerup, Denmark
Summary
The potent b-lactam antibiotic mecillinam has successfully been labelled
with tritium. 2,3,4,7-Tetrahydro-1H-azepin-1-carbaldehyde was formed in
a ring-closing metathesis reaction and then condensed with trimethylsilyl
protected penicillanic acid to give dehydro-mecillinam. Pd/C catalysed
tritiation then gave [3H2]-mecillinam (6-b (([3,4-3H2]hexahydro-1H-azepin-
1-yl)methyleneamino)penicillanic acid) with
a radiochemical purity of
96% and a specific activity of 24 Ci/mmol. Copyright # 2003 John Wiley &
Sons, Ltd.
Key Words: mecillinam; tritium; ring-closing metathesis
Introduction
Mecillinam1,2 (10a) is a narrow-spectrum penicillin, selectively active
against Gram-negative organisms and currently primarily used for
treatment of cystitis. For biological studies we needed labelled
mecillinam with a specific activity >10 Ci/mmol. The penicillanic acid
moiety of other penicillins has been labelled with tritium via an
exchange reaction with tritiated water.3,4 This method requires a large
excess of tritium and the specific activities obtained were ꢀ 0.4 Ci/
mmol. Pivmecillinam (pivaloyloxymethyl ester of mecillinam) has been
labelled with carbon-14 in the amidine group5 but the maximal specific
*Correspondence to: Gunnar Grue-Sꢀrensen, LEO Pharma, Medicinal Chemistry Research, 55
Industriparken, DK-2750 Ballerup, Denmark. E-mail: grs@leo-pharma.com
Copyright # 2003 John Wiley & Sons, Ltd.
Received 6 March 2003
Accepted 20 March 2003

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S. M. FREDERIKSEN AND G. GRUE-SØRENSEN
activity of carbon-14 is 0.06 Ci/mmol. To obtain the desired level of
specific activity we decided to introduce tritium via a catalytic tritiation
of a double bond in a tetrahydro-1H-azepin precursor to be synthesized
via a ring-closing metathesis (RCM) reaction.6 Here we report our
synthesis.
Results and discussion
The secondary amine 3 was prepared in 61% yield by alkylation
of allylamine (1) with 5-bromopent-1-ene (2) using sodium iodide
as a catalyst (see Scheme 1). The RCM reaction was unsuccessful
when performed on the amine 3. Formylation of the amine 3 with
formyl acetic anhydride gave the amide 4 in 65% yield (cf. Jahngen
and Rossomando7) and this compound smoothly underwent the RCM
reaction using a Grubbs’ ruthenium-based catalyst in a very dilute
dichloromethane solution (cf. Visser et al.8). Similar observations,
that increased bulkiness around the nitrogen atom will lead to
more favourable transition states in the RCM reaction, have been
reported.6,9 Extensive purification to remove the catalyst finally gave
the N-formyl tetrahydro-1H-azepin 5 in a yield of 24%. The amide
5 was activated by reaction with oxalyl chloride in diethylether.2
The Vilsmeier-type activated amide 6 was not isolated, but, after
removal of the solvent and dissolution in dichloromethane, was
used directly in the following coupling reaction. 6-Aminopenicillanic
acid (7) was protected using 2 equivalents chlorotrimethylsilane
and triethylamine (cf. Grossmann and Hardcastle10) and immediately
coupled with the chloroiminium chloride 6. The coupling was initiated
in dichloromethane at À788C and the temperature was then slowly
raised to ambient level (cf. Lund2). Compounds containing b-lactam
rings are labile when chromatographed on silica gel and dehydro-
mecillinam 9 was therefore purified by preparative reversed phase
HPLC by using an unbuffered eluent of water and acetonitrile to avoid
a salt residue after evaporation of the solvents. The tritiation was
performed by treating dehydro-mecillinam 9 and 10% Pd/C in water
with tritium-gas at 218C/1 atm for 2 h. Both the imino-functionality and
the b-lactam ring were stable under those conditions.11 Purification by
reversed phase HPLC (cf. Strojny and Silva12) afforded the desired
product 10b with 96% radiochemical purity and a specific activity of
24 Ci/mmol.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 773–779

SYNTHESIS OF TRITIUM LABELLED MECILLINAM
775
Scheme 1. Synthesis of [3H]-Mecillinam. Reagents and conditions: (a) NaI,
508C, 90 min; (b) HCOOH, (CH3CO)2O, 558C, 1 h; (c) Bis(tricyclohexylphos-
phine)benzylidene ruthenium(IV) dichloride, CH2Cl2, reflux, 4 h; (d) (COCl)2,
Et2O, 08C, 40 min; (e) TMS-Cl, Et3N, CH2Cl2, room temperature, 75 min;
(f) mixing contents of (d) and (e) in CH2Cl2, À788C to room temperature, 3.5 h;
(g) [3H2], 10% Pd/C, H2O, room temperature, 2 h.
Experimental
General
Preparative HPLC of dehydro-mecillinam was performed using a
Gilson apparatus with a Gilson UV/VIS-155 UV-detector (simulta-
neous detection at 220 and 254 nm). Preparative HPLC of [3H2]-
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S. M. FREDERIKSEN AND G. GRUE-SØRENSEN
mecillinam was performed using a Merck Hitachi apparatus with a
L4250 UV-VIS detector (254 nm). Chemical and radiochemical purity
was determined by HPLC on a Merck Hitachi apparatus (L-6200
pump) on a Spherisorb ODS1 column (150 mm  4.6 mm) with 0.01 M
phosphate buffer (pH 5)/acetonitrile 9:1 as eluent (1 ml/min) and UV-
detection (254 nm) (L-4250 UV-VIS detector) or radioactivity detection
(Packard Flow System Analyzer Model D525F1 with Ultima-Flo M
(Packard) as scintillation liquid). Concentrations and specific activities
were determined by HPLC by comparison of peak areas of radio-
inactive reference compounds. A Packard Tri-Carb 2900TR Liquid
Scintillation Analyzer was used to determine activity in liquid samples
using Pico-Fluor 40 (Packard) as scintillation cocktail. 1H and
13C NMR spectra were obtained on a Bruker ARX300 spectrometer.
Chemical shifts are reported in ppm with tetramethylsilane (TMS) in
CDCl3 or sodium 3-(trimethylsilyl) propionate-2,2,3,3-d4 (TMSP-d4) in
D2O as internal reference. ES Mass spectra were obtained on a VG
Quattro II mass spectrometer in positive ion electrospray mode. IR
spectrum was obtained on a Bruker EQVINOX 55 FT IR spectrometer.
Allyl-pent-4-enyl-amine (3)
5-Bromopent-1-ene (2) (7.55 g, 50.6 mmol) was slowly added to a
vigorously stirred suspension of sodium iodide (534 mg, 3.6 mmol) in
allylamine (1) (14.45 g, 253.1 mmol). The mixture was stirred at 508C for
1.5 h. After cooling potassium carbonate (14 g) was added to the
mixture. After standing for 48 h at 58C water (30 ml) was added and the
potassium carbonate dissolved. After ensuring a pH-level >10 the
solution was extracted with diethyl ether (2 Â 15 ml). The combined
organic layers were washed with brine, dried over sodium sulphate,
filtered and concentrated in vacuo (308C, 800 mbar). The residue was
purified by distillation (41–448C, 13 mbar) which afforded the product
1
(3) in 61% yield (3.84 g). H NMR (CDCl3) d 5.98–5.74 (m, 2H), 5.17
(dq, 1H), 5.08 (dq, 1H), 5.02 (dq, 1H), 4.95 (m, 1H), 3.25 (dt, 2H), 2.62
(bt, 2H), 2.10 (m, 2H), 1.60 (m, 2H), 1.07 (bs, NH). 13C NMR (CDCl3) d
138.5, 137.1, 115.7, 114.6, 52.5, 48.9, 31.6, 29.4. MS: m/z 126.06 [M+].
N-Allyl-N-pent-4-enyl-formamide (4)
Acetic anhydride (5 ml, 53 mmol) was slowly added to a stirred mixture
of formic acid (20 ml, 530 mmol) and allyl-pent-4-enyl-amine (3) (1.0 g,
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS OF TRITIUM LABELLED MECILLINAM
777
7.99 mmol) under argon at room temperature. The reaction was stirred at
558C for 1 hour. After cooling to room temperature the mixture was
concentrated in vacuo. Water (30 ml) was added to the residue and the pH
level was adjusted to 11–12 by addition of 2N sodium hydroxide. The
mixture was extracted with ethyl acetate (2 Â 30 ml). The combined organic
phases were acidified with 2 N hydrochloric acid until pH 5. The solution
was then washed with water (2Â 20 ml) and with brine (20 ml). The solution
was dried over sodium sulphate, filtered and concentrated in vacuo to give
1
the desired product (4) in 65% yield (2.39 g). HNMR (CDCl3) d (two
conformers, 1:1) 8.09 (s, 1H), 5.88–5.66 (m, 2H), 5.24–5.19 (m, 2H), 5.10–
4.94 (m, 2H), 3.95 (m) and 3.83 (m) (2H), 3.31 (m) and 3.23 (bt) (2H), 2.12–
2.00 (m, 2H), 1.70–1.56 (m, 2H). 13CNMR (CDCl3) d (two conformers, 1:1)
162.9, 162.7, 137.6, 137.0, 133.5, 132.5, 118.4, 117.9, 115.8, 115.16, 50.2,
46.3, 44.6, 41.9, 31.0, 30.4, 27.3, 26.3. MS: m/z 154.04 [M+].
2,3,4,7-Tetrahydro-azepin-1-carbaldehyde (5)
Under an argon atmosphere N-allyl-N-pent-4-enyl-formamide (4)
(1.0 g, 6.52 mmol) was diluted with dichloromethane (650 ml) and while
stirring bis(tricyclohexylphosphine)benzylidene ruthenium(IV) dichloride
(107 mg, 0.13 mmol) was added. The solution was refluxed 4 h and then
concentrated in vacuo. The residue was filtered through silica gel (43–60 m)
to remove the catalyst. Ethyl acetate/petroleum ether (b.p. 35–508C) 4:1
was used as eluent. This filtration was repeated. Presence of catalyst was
visible due to strong colourization of the silica gel. The resulting filtrate
was concentrated, redissolved in ethyl acetate (20 ml) and filtered through
a layer of activated carbon. After concentration in vacuo the residue was
purified by flash chromatography using ethyl acetate/petroleum ether
(b.p. 35–508C) 4:1. This gave the desired product (5) in 24% yield
1
(199 mg). H NMR (CDCl3) d (two conformers, 1:1) 8.08 (s) and 8.04 (s)
(1H), 5.87–5.61 (m, 2H), 4.00 (m) and 3.91 (m) (2H), 3.63 (bt) and 3.52
(bt) (2H), 2.30–2.20 (m, 2H), 1.94–1.82 (m, 2H). 13C NMR (CDCl3) d
(two conformers, 1:1) 162.6, 161.9, 132.7, 132.2, 127.4, 126.8, 49.7, 47.1,
44.0, 41.2, 27.7, 27.2, 26.6, 26.0. MS: m/z 126.03 [M+].
1-Chloromethylene-2,3,4,7-tetrahydro-1H-azepin chloride (6)
2,3,4,7-Tetrahydro-azepin-1-carbaldehyde (5) (494 mg, 3.94 mmol) was
diluted in diethylether (10 ml) under argon in a dried flask. The solution
was cooled to 08C and oxalyl chloride (341 ml, 3.94 mmol) diluted in
Copyright # 2003 John Wiley & Sons, Ltd.
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778
S. M. FREDERIKSEN AND G. GRUE-SØRENSEN
diethylether (2 ml) was slowly added. Precipitation was immediately
observed. After 40 min most of the diethylether was removed via a
canula. The residue was then dissolved in dichloromethane (6 ml) and
used immediately as described below.
6-Aminopenicillanic acid trimethylsilyl ester (8)
6-Aminopenicillanic acid (7) (852 mg, 3.94 mmol) was dissolved in
dichloromethane (17 ml) and triethylamine (797 mg, 7.88 mmol) under
argon in a dried flask. After 25 min of stirring 6-aminopenicillanic acid
had dissolved and trimethylsilyl chloride (856 mg, 7.88 mmol) was
added. Some precipitation was observed. The mixture was stirred for
50 min and used immediately as described below.
6-b((2,3,4,7-Tetrahydro-1H-azepin-1-yl)methyleneamino)penicillanic
acid (9)
The 6-aminopenicillanic acid trimethylsilyl ester (8) solution (see above)
was cooled to À788C and while vigorously stirred under argon the
solution of the chloroiminium chloride 6 (see above) (3.94 mmol in 6 ml
dichloromethane) was added via syringe. The reaction slowly became
orange and was stirred for 1.5 h at À788C and then for 2 h at room
temperature. The solution was concentrated to dryness in vacuo. The
residue was purified in portions each containing roughly 50 mg using a
Merck Lichrosper RP-18 250-10 column with water/acetonitrile 85:15 as
eluent (2 ml/min, UV-detection at 220 and 254 nm). Compound 9 eluted
after 17.01 min. Of the total crude product (2.54 g) only a fraction
(357 mg) was purified to give 75 mg (0.23 mmol) of the desired product
1
(9). H NMR (D2O) d (two conformers, 1:1) 8.04 (bs) and 7.99 (bs)
(1H), 6.04–5.88 (m, 1H), 5.81–5.67 (m, 1H), 5.58 (d) and 5.57 (d) (1H),
5.43 (d) and 5.41 (d) (1H), 4.31 (s, 1H), 4.29 (m, 1H), 4.15 (m, 1H), 3.84
(bt, 1H), 3.71 (m, 1H), 2.44–2.26 (m, 2H), 2.11–1.98 (m, 2H), 1.70 (s,
3H), 1.55 (s, 3H). 13C NMR (D2O) d (two conformers, 1:1) 177.1, 175.0,
174.9, 158.7, 157.6, 138.5, 136.1, 126.2, 125.1, 76.4, 69.5, 69.4, 67.8, 67.8,
65.6, 65.2, 59.8, 55.5, 52.3, 48.1, 31.9, 30.0, 29.8, 28.4, 27.5, 25.8. MS: m/
z 324.09 [M+]. IR (0.3% in KBr): 1773 cmÀ1 (C=O of b-lactam-ring).
3
6-b (([3,4- H2]-Hexahydro-1H-azepin-1-yl)methyleneamino)penicilla-
nic acid (10b)
6-b ((2,3,4,7-Tetrahydro-1H-azepin-1-yl)methyleneamino)penicillanic
acid (9) (3.6 mg) was dissolved in water (0.5 ml) and 10% Pd/C-catalyst
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS OF TRITIUM LABELLED MECILLINAM
779
(4 mg) was added. The solution was stirred under a tritium atmosphere
for 2.5 h and the catalyst was removed by filtration. The residue was
initially purified by HPLC using a Merck Lichrosper RP-18 250-10
column using water/acetonitrile 75:25 as eluent (Retention time was
9.9 min) (4 ml/min, UV-detection at 220 nm). The solvent was evapo-
rated in vacuo. The product obtained was 74% pure and was further
purified by HPLC using 0.01 M phosphate buffer (pH 5)/acetonitrile
90:10 as eluent (Retention time was 27.6 min). The solvent was
evaporated in vacuo and the residue redissolved in water (5 ml) to give
the desired product (10b) (13.4 mCi) with 96% radiochemical purity and
a specific activity of 24 Ci/mmol.
Acknowledgements
We are indebted to Grethe Aagaard (NMR), Malene Mohr (MS), Dr.
Karen Margrethe Engell and Peter Anker Jꢀrgensen (HPLC) (Leo
Pharma A/S) and to Dr. Ewald Bannwart (catalytic tritiation) (RC
TRITEC AG, Teufen, Switzerland) for invaluable assistance.
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J Label Compd Radiopharm 2003; 46: 773–779