Journal of Medicinal Chemistry
ARTICLE
heroin’s psychoactive effects in case of relapse. Toward this
heroin vaccine goal, we emphasize that heroin-like immunocon-
jugate 11b produces high, sustainable titers that can be rapidly
reached via a minimum number of injections. We predict that if
our vaccine provides protection within a clinical setting, then the
recovery process could be more readily managed while requiring
a lessened degree of noninvasive maintenance. In addition, if
sufficiently selective antibody titers as seen in rats can be
translated in humans, this vaccine could readily be used in
tandem with opiate replacement therapy (where available) as a
synergistic treatment option for addicts.
(m, 1H), 5.11 (d, J = 6.6 Hz, 1H), 3.45 (s, 1H), 3.15 (m, 2H), 2.96 (d, J =
18.7 Hz, 1H), 2.77 (m, 1H), 2.67 (m, 1H), 2.54 (m, 2H), 2.36 (m, 1H),
2.32 (m, 1H), 2.27 (s, 3H), 2.12 (s, 3H), 2.08 (m, 1H), 1.87 (m, 1H),
1.55 (m, 4H), 1.44 (s, 9H). 13C NMR 125 MHz (CDCl3) δ 170.6, 168.6,
156.2, 149.5, 132.0, 131.9, 131.5, 129.5, 128.7, 122.2, 119.5, 88.7, 79.2,
68.0, 56.9, 54.3, 44.9, 43.3, 40.4, 40.2, 34.7, 29.3, 28.0, 24.6, 21.7, 20.8,
20.7. High resolution mass spectrometry (ESI) found 527.2761
[calculated for C29H39N2O7 (M + H+) 527.2752]. Purity (HPLC): 94%
(4aR,7S,7aR,12bS)-3-(3-(3-Tritylthio)propanamido)propyl)-
2,3,4,4a,7,7a-hexahydro-1H-4,12-methanobenzofuro[3,2-e]-
isoquinoline-7,9-diyl Diacetate (9). To a solution of Boc-pro-
tected amine 6 (18 mg/0.034 mmol) in CH2Cl2 (1 mL) at room
temperature was added a solution of CH2Cl2:TFA (1 mL:1 mL) in one
portion. The resulting solution was stirred at room temperature for 2 h,
and then the solvents removed under reduced pressure to give 7 as a
yellow solid. This solid was placed under high vacuum overnight,
followed by dissolving in CH2Cl2 (4 mL) and cooling the resulting
solution to 0 °C. Triethylamine (17 μL/0.12 mmol) and activated ester
8 (18 mg/0.04 mmol) were added at the same temperature, and the
resulting solution was stirred at 0 °C for 2 h before allowing to warm to
room temperature overnight. The solution was transferred to a separa-
tory funnel, and washed once with brine (1 ꢁ 4 mL). The aqueous layer
was extracted with CH2Cl2 (2 ꢁ 5 mL), the combined organic layers
were dried with MgSO4, and the solvent removed under reduced
pressure to give the crude product as a viscous oil, which was purified
by preparative TLC (9:1 CHCl3:MeOH) to give the product as an
amorphous solid (18 mg/71% yield from 6). 1H NMR 500 MHz
(CDCl3) δ 7.55ꢀ7.25 (m, 15H), 6.85 (d, J = 8.2 Hz, 1H), 6.65 (d,
J = 8.2 Hz, 1H), 5.70 (m, 2H), 5.45 (dt, J = 2.4, 10.0 Hz, 1H), 5.23 (m,
1H), 5.16 (d, J = 6.6 Hz, 1H), 3.52 (m, 1H), 3.30 (m, 2H), 3.04 (d, J =
18.8 Hz, 1H), 2.81 (m, 1H), 2.72 (m, 1H), 2.63 (m, 2H), 2.57 (t, J = 7.2
Hz, 2H), 2.43 (m, 1H), 2.37 (m, 1H), 2.36 (2, 3H), 2.22 (s, 3H), 2.12 (t,
J = 7.1 Hz, 2H), 2.05 (m, 1H), 1.90 (m, 1H), 1.65 (m, 4H). 13C NMR
125 MHz (CDCl3) δ 171.1, 170.6, 168.6, 149.4, 144.7, 132.0, 131.9,
131.6, 129.7, 129.5, 128.6,128.0 126.8, 122.0, 119.5, 88.7, 68.0, 66.8,
57.0, 54.3, 44.8, 43.4, 40.5, 39.3, 35.9, 35.1, 27.9, 27.5, 24.8, 21.8, 20.7,
20.6. High resolution mass spectrometry (ESI) found 757.3326
[calculated for C46H49N2O6S (M + H+) 757.3306].
N-(3-((4aR,7S,7aR,12bS)-7,9-Dihydroxy-4,4a,7,7a-tetrahy-
dro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)-
propyl-3-(tritylthio)propanamide (10). To a solution of trityl
protected heroin hapten 9 (12 mg/0.15 mmol) in MeOH (2 mL) was
added 0.1 M NaOH (1 mL) at room temperature. The resulting solution
was stirred at room temperature for 45 min before the removal of MeOH
under reduced pressure. The remaining aqueous phase was extracted
with EtOAc (6 ꢁ 5 mL), the combined organics were dried with MgSO4
and the solvent removed under reduced pressure to give the crude
product as a viscous oil which was purified by preparative TLC (9:1
CHCl3:MeOH) to give the pure product as an amorphous solid (9.5
mg/92% yield). 1H NMR 600 MHz (CDCl3) δ 7.40ꢀ7.20 (m, 15H),
6.70 (d, J = 8.1 Hz, 1H), 6.58 (d, J = 8.1 Hz, 1H), 5.78 (br s, 1H), 5.75 (d,
J = 11.0 Hz, 1H), 5.29 (d, J = 9.7 Hz, 1H), 4.96 (d, J = 5.9 Hz, 1H), 4.25
(m, 1H), 3.57 (m, 1H), 3.28 (m, 2H), 3.0 (d, J = 18.5 Hz, 1H), 2.69 (m,
1H), 2.54 (m, 2H), 2.50 (t, J = 7.3 Hz, 2H), 2.40 (m, 1H), 2.30ꢀ2.45 (m,
5H), 2.15 (t, J = 7.1 Hz, 2H), 1.90 (m, 1H), 1.70 (m, 4H). 13C NMR 150
MHz (CDCl3) δ 170.2, 145.3, 144.8, 138.1, 133.1, 130.9, 129.7, 128.1,
128.0, 126.9, 121.9, 120.1, 116.8, 91.8, 66.9, 66.6, 56.7, 54.3, 44.9, 43.8,
40.7, 39.4, 35.9, 30.9, 27.9, 27.4, 22.8, 21.4. High resolution mass
spectrometry (ESI) found 673.3100 [calculated for C42H45N2O4S
(M + H+) 673.3094].
’ EXPERIMENTAL PROCEDURES
NMR spectra were recorded on Bruker spectrometers. Chemical
1
shifts are reported in parts per million (ppm). For H NMR spectra
(CDCl3), the residual solvent peak was used as the reference
(7.26 ppm), while the central solvent peak was used as the reference
(77.0 ppm in CDCl3) for 13C NMR. Preparative HPLC was performed
using a Shimadzu LC-8A system equipped with a Grace-Vydac C18
column (2.2 cm ꢁ 15 cm). All HPLC experiments were monitored at
254 or 214 nm. Analytical thin layer chromatography (TLC) was per-
formed using EMD precoated TLC plates, silica gel 60F-254, 0.25 mm
layer thickness. TLC plates were visualized by exposure to UV light or
submersion in aqueous potassium permanganate followed by heating
on a hot plate. Preparative TLC was performed using EMD Silicagel
60F-254 plates (20 cm ꢁ 20 cm), 0.5 mm thickness. When necessary,
reaction vessels were oven-dried and cooled in a desiccator and
performed under an inert argon atmosphere. Reagents were commercial
grade and used without further purification. Heroin hydrochloride was
obtained from NIDA, 6-acetyl morphine was prepared according to the
literature procedure22 and Leu-enkephalin/endomorphin-1 were pre-
pared by standard solid phase peptide synthesis.23 Analytical HPLC was
used for analysis of compound purity; all purified compounds were of
>95% purity, with the exception of Boc-protected compound 6.
(4aR,7S,7aR,12bS)-3-(3-((tert-Butoxycarbonyl)amino)propyl)-
2,3,4,4a,7,7a-hexahydro-1H-4,12-methanobenzofuro[3,2-e]-
isoquinoline-7,9-diyl Diacetate (6). To a solution of heroin
hydrochloride 3 (21 mg/0.052 mmol) in 4 mL of dry 1,2-dichloroethane
was added NaHCO3 (35 mg/0.42 mmol) and R-chloroethylchlorofor-
mate (93 μL/0.85 mmol) at room temperature. The resulting solution
was heated to reflux for 4 h with monitoring by TLC (9:1 CHCl3:
MeOH). After this time, the solution was cooled and filtered before
removal of the solvent under reduced pressure. The remaining residue
was placed under high vacuum for 2 h, followed by the addition of
CH3CN:H2O (4:1, 0.1% TFA, 2 mL) and stirring for 2 h at room
temperature. Acetonitrile was removed under reduced pressure, and the
aqueous solution lyophilized to give crude norheroin 4 as an amorphous
solid (22 mg/90% crude yield). Crude norheroin 4 (22 mg/0.047
mmol) was dissolved in dry CH2Cl2 (5 mL) and cooled to 0 °C,
followed by the addition of aldehyde 5 (25 μL/0.24 mmol), acetic acid
(14 μL/0.18 mmol), and NaBH(OAc)3 (120 mg/0.6 mmol) at the same
temperature. The solution was allowed to stir at 0 °C for 2 h before
allowing to slowly warm to room temperature overnight. After this time,
additional aldehyde 5 (25 μL/0.24 mmol) and NaBH(OAc)3 (60 mg/
0.3 mmol) were added and the solution stirred at room temperature for
6 h before the addition of water (3 mL). The layers were separated, and
the aqueous layer extracted with CH2Cl2 (3 ꢁ 5 mL). The combined
organic layers were washed with brine (1 ꢁ 5 mL), dried with MgSO4,
and the solvent removed under reduced pressure. The resulting residue
was purified by preparative TLC (9:1 CHCl3:MeOH) to give the pure pro-
duct as an amorphous solid (18 mg/65% yield from 3). 1H NMR 500 MHz
(CDCl3) δ 6.75 (d, J = 8.2 Hz, 1H), 6.56 (d, J = 8.2 Hz, 1H), 5.63 (d, J =
9.9 Hz, 1H), 5.43 (dt, J = 2.5, 10.8 Hz, 1H), 5.24 (br s, 1H), 5.15
(4aR,7S,7aR,12bS)-3-(3-(3-Mercaptopropanamido)propyl-
2,3,4,4a,7a-hexahydro-1H-4,12-methanobenzofuro[3,2-e]-
isoquinoline-7,9-diyl diacetate (1). Five mg of trityl protected
hapten 9 was placed in a round-bottom flask and put under high vacuum
5201
dx.doi.org/10.1021/jm200461m |J. Med. Chem. 2011, 54, 5195–5204