3194
E. A. Litzinger et al. / Bioorg. Med. Chem. 14 (2006) 3185–3198
product was further purified by HPLC. 1H NMR
(500 MHz, D2O) d 3.87 (m, 1H), 3.34 (t, J = 7.0 Hz,
2H), 2.85 (t, J = 6.5 Hz, 2H), 2.76 (t, J = 6.5 Hz, 2H),
2.13 (s, 3H), 1.95 (m, 2H), 1.82–1.73 (m, 2H). HRMS
(ESI) (m/z): M+H+ calcd for C8H18N3O2S 234.1268,
found 234.1276.
1.6 equiv of di-tert-butyl dicarbonate (0.96 g,
7.33 mmol) and 0.3 equiv of 4-(dimethylamino)pyridine
(DMAP, 0.10 g, 0.82 mmol) in tert-butanol (10 mL).
The solvent was removed in vacuo, and the DMAP
was removed by eluting the residue through a short sil-
ica column with 4:1 hexanes/ethyl acetate to obtain the
ester in a 93% yield (1.10 g, 2.53 mmol). Hydrogenation
of the benzyl carbamate as described before gave Na-
Boc-L-Orn-OtBu that was used immediately.
5.4.6. N5-(1-Imino-2-aminoethyl)-L-ornithine (11). Com-
pound 21 (0.11 g, 0.26 mmol) was added to a 30%
HBr/acetic acid solution (3 mL) under an argon atmo-
sphere. The solid did not dissolve immediately and
was left to stir overnight. The desired compound was
precipitated out of solution upon the addition of cold
diethyl ether (50 mL), filtered, and washed again with
ether to give 11 in a quantitative yield (48.0 mg,
0.26 mmol) as a hygroscopic orange solid. A portion
of the product was purified by HPLC. 1H NMR
(400 MHz, D2O) d 4.12 (t, J = 6.0 Hz, 1H), 4.10 (s,
2H), 3.43 (t, J = 6.8 Hz, 2H), 3.06 (t, J = 7.2 Hz, 1H),
2.10–1.78 (m, 4H), 1.95 (m, 2H), 1.82–1.73 (m, 2H).
HRMS (ESI) (m/z): M+H+ calcd for C7H17N4O2
189.2275, found 189.2376.
The di-protected ornithine (1.30 g, 4.53 mmol) was dis-
solved in anhydrous methanol (12.5 mL), and the imidic
ester (0.47 g, 1.57 mmol) was added. The solution was
left to stir at room temperature for 48 h, after which
time cold diethyl ether (100 mL) was added and the
solution was chilled at 4 ꢁC overnight. The next day a
brown oil was retrieved from the sides of the flask,
which was dried in vacuo to give 23 (0.63 g, 1.23 mmol,
80%). 1H NMR (500 MHz, CD3OD) d 7.76 (d,
J = 5.5 Hz, 2H), 7.28 (d, J = 5.5 Hz, 2H), 4.1 (m, 1H),
3.33 (m, 2H), 2.98 (m, 2H), 2.43 (t, J = 6.4 Hz, 2H),
2.35 (s, 3H), 1.78–1.64 (m, 4H), 1.45 (s, 9H), 1.41 (s,
9H). HRMS (ESI) (m/z): M+H+ calcd for C24H41N4O6S
513.2747, found 513.2756.
5.4.7. 3-N-(p-Ts)-Aminopropionitrile (22). In accordance
with the procedure of Rapoport and co-workers,26 3-
aminopropionitrile fumarate (1.31 g, 12.57 mmol) was
dissolved in water (14 mL). The solution was adjusted
to pH 10 with 1 M NaOH and then tosyl chloride
(2.66 g, 13.96 mmol) in ethyl acetate (6 mL) was added.
After being stirred overnight, the layers were separated,
and the aqueous layer was neutralized with 1 M HCl
and extracted with ethyl acetate. The organic layer
was dried over MgSO4 and the solvent was removed
in vacuo. The product was recrystallized from etha-
nol/water to give 2.19 g (78%) of 22. 1H NMR
(500 MHz, CDCl3) d 7.76 (d, J = 8.1 Hz, 2H), 7.34
(d, J = 8.0 Hz, 2H), 5.07 (br s, 1H), 3.26 (q, J = 6.6,
2H), 2.59 (t, J = 6.6, 2H), 2.45 (s, 3H). HRMS (ESI)
(m/z): M+H+ calcd for C10H12N2O6S 225.0698, found
225.0702.
5.4.9. N5-(1-Imino-3-aminopropyl)-L-ornithine (12). Into
a pressure tube that had been flame-dried and cooled
under vacuum were added phenol (1.00 g, 10.64 mmol),
23 (0.19 g, 0.37 mmol), and a minimal amount (<1 mL)
of anhydrous methanol. Once the solids had dissolved,
30% HBr in acetic acid (10 mL) was added, and the tube
was sealed and heated for 5 h at 85–90 ꢁC. After cooling
overnight, water (30 mL) was added, the solution cooled
on ice, and then extracted with ethyl acetate until the
aqueous layer was of a clear brown color; 12 (62 mg,
0.31 mmol, 84%) was obtained as a hygroscopic orange
solid. A portion of the product was further purified by
HPLC. 1H NMR (400 MHz, D2O)
d
4.12 (t,
J = 6.0 Hz, 1H), 3.35 (t, J = 10.4 Hz, 2H), 3.04 (t,
J = 7.8 Hz, 2H), 2.91 (t, J = 8.0, 2H), 2.02 (m, 2H),
1.82 (m, 2H). HRMS (ESI) (m/z): M+H+ calcd for
C8H19N4O2 203.1508, found 203.1512.
5.4.8. N1-Boc-N5-(1-Imino-3-N-(p-Ts)-aminopropyl)-L-
ornithine tert-butyl ester (23). Compound 22 (0.55 g,
6.90 mmol) was added to anhydrous, reagent-grade eth-
anol (7.0 mL, 120.03 mmol) and 1M HCl/ethyl ether
(12 mL) and then chilled at 0 ꢁC. Once all of the 22
had dissolved, HCl gas was bubbled through the solu-
tion for 1 h and then left to stir another 5 h at 0 ꢁC.
The reaction mixture was stored at 4 ꢁC overnight with-
out stirring. After this time, ethyl ether was added to
precipitate the product, which was filtered and washed
again with ether and dried to give the imidic ester
(1.70 g, 5.54 mmol, 80%), which was used in the next
5.4.10. 3,30-Dithiobis(N-[tert-butyl N4-Boc-4-S-butano-
ate]propionamidine) dihydrochloride (25). Diethyl 3,30-
dithiobis(propionimidate) dihydrochloride was syn-
thesized from 3,30-dithiobispropionitrile (1.53 g,
8.91 mmol) and ethanol (1.4 mL, 24.00 mmol) as de-
scribed by Johnston and Gallagher28 to give the disulfide
product in a quantitative yield (3.00 g, 8.87 mmol),
which was used in the next step without additional puri-
fication. 1H NMR (400 MHz, D2O)
d 4.45 (q,
J = 7.2 Hz, 2H), 3.11–3.08 (m, 4H), 1.45 (t, J = 6.8 Hz,
3H).
1
step without further purification. H NMR (400 MHz,
DMSO) d 12.45 (br s, 1H), 11.25 (br s, H1), 8.09 (t,
J = 6.4 Hz, 1H), 7.79 (d, J = 8.0 Hz, 2H), 7.53 (d,
J = 7.6 Hz, 2H), 4.46 (q, J = 7.6 Hz, 2H), 3.16 (q,
J = 6.0 MHz, 2H), 2.86 (t, J = 6.80 MHz, 2H), 2.62 (s,
3H), 1.44 (t, J = 7.2 Hz, 3H).
Na-Boc-L-Orn-OtBu (1.11 g, 3.83 mmol), prepared as
described above for 23, was thoroughly dried and then
dissolved in anhydrous ethanol (5 mL) under argon.
To this was added the imidic ester (0.65 g, 1.93 mmol),
and the resulting suspension was stirred for 72 h.
Absolute ethanol (6 mL) was added, and the solution
was stirred for 24 h more. At the end of this time,
cold ethyl ether was added and the solution was
Na-Boc-L-Orn(Z)-OH was converted to the tert-butyl
ester by the method of Takeda et al.27 Briefly, Na-Boc-
L-Orn(Z)-OH (1 g, 2.73 mmol) was stirred for 2 h with