Sulfate esters of morphine derivatives: Synthesis and characterization

Article abstract of DOI:10.1016/j.ejps.2010.10.007

Sixteen 3-O- and 6-O-sulfate esters of morphine, codeine and some of their N-methyl quaternary derivatives were synthesized by means of sulfation with pyridine-SO₃ complex and sulfuric acid/N,N′- dicyclohexylcarbodiimide. Complete ¹H- and ¹³C-NMR assignments are given for each of the synthesized compounds based on one- and two-dimensional homo- and heteronuclear measurements. Comparative analysis of chiral properties by circular dichroism and optical rotatory dispersion revealed characteristic differences in the spectra due to changes in charge, polarity and intramolecular association by strong hydrogen bonds in aqueous solution. The synthesized sulfate esters are prospective peripheral analgesics lacking central side effects and are also useful as reference substances for various analytical studies involving sulfate ester metabolites.

Full text of DOI:10.1016/j.ejps.2010.10.007

European Journal of Pharmaceutical Sciences 42 (2011) 65–72
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Sulfate esters of morphine derivatives: Synthesis and characterization
András Váradia,∗, András Gergelya, Szabolcs Bénia, Péter Jankovicsb, Béla Noszála, Sándor Hosztafia
a Department of Pharmaceutical Chemistry, Semmelweis University, Ho˝gyes Endre u., 9. H-1092 Budapest, Hungary
b National Institute of Pharmacy, Zrínyi u., 3. H-1051 Budapest, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Sixteen 3-O- and 6-O-sulfate esters of morphine, codeine and some of their N-methyl quaternary
derivatives were synthesized by means of sulfation with pyridine–SO3 complex and sulfuric acid/N,Nꢀ-
dicyclohexylcarbodiimide. Complete 1H- and 13C-NMR assignments are given for each of the synthesized
compounds based on one- and two-dimensional homo- and heteronuclear measurements. Comparative
analysis of chiral properties by circular dichroism and optical rotatory dispersion revealed characteristic
differences in the spectra due to changes in charge, polarity and intramolecular association by strong
hydrogen bonds in aqueous solution. The synthesized sulfate esters are prospective peripheral anal-
gesics lacking central side effects and are also useful as reference substances for various analytical studies
involving sulfate ester metabolites.
Received 20 September 2010
Received in revised form 13 October 2010
Accepted 20 October 2010
Available online 27 October 2010
Keywords:
Morphine
Sulfate ester
NMR
© 2010 Elsevier B.V. All rights reserved.
CD
ORD
1. Introduction
metabolites and to synthesize new analogous sulfate esters to
better understand their physicochemical properties. Despite the
Central and peripheral opioid receptors are primary targets
for pain management. Therapeutic potential and demand of opi-
oid analgesics have initiated tremendous scientific efforts, which
have resulted in the development of a number of new opioid anal-
gesics and an expansion of knowledge on the opioid pharmacology
(Eguchi, 2004). The structural features of morphine skeleton have
long been the basis of successful drug developments. There is,
however, a great need for morphine-like molecules with improved
efficacy, in vivo half lives, bioavailability and reduced side-effects
such as tolerance liability, depressed respiration, and reduced gas-
trointestinal motility. The prospect for highly polar or charged
opioid analogues with limited brain penetration to produce a
peripherally mediated analgesic effect in inflammatory conditions
has long been a pharmacologic desire and has recently been studied
in several laboratories (Guan et al., 2008; Smith, 2008; Hernández
et al., 2009; Obara et al., 2009; Zheng, 2010). Such highly polar,
zwitterionic molecules are found among the naturally occurring
metabolites (i.e. glucuronides and sulfates) of morphine and its
derivatives (Milne et al., 1996; Wittwer and Kern, 2006; Coller
et al., 2009). Morphine-3-O-sulfate and naloxone-3-O-sulfate have
been isolated from biological samples (Ober and Fujimoto, 1972;
Yeh et al., 1977). It is therefore important to characterize known
apparent inability of sulfate metabolites to cross the blood–brain
barrier due to the presence of the charged sulfate ester group,
several studies have indicated that some sulfate esters of mor-
phine derivatives are of pharmacological interest (Mori et al., 1972;
Brown et al., 1985; Preechagoon et al., 1998a,b; Zuckerman et al.,
1999). Introduction of a sulfate ester group at the C-6 hydroxyl
has been shown to significantly increase the analgesic potency of
morphine (Brown et al., 1985). The present paper focuses on sulfate
esters that may be effective as peripherally acting opiate analgesics.
For the synthesis of sulfate esters a number of approaches have
been reviewed (Al-Horani and Desai, 2010). The most common
methods are the reaction of the phenol or the alcohol with chloro-
sulfonic acid in pyridine and with trimethylamine–SO3 complex or
pyridine–SO3 complex in DMF, 1,4-dioxane or pyridine (Kaspersen
and Van Boeckel, 1987). Successful syntheses of sulfate esters using
pyridine–SO3 complex are reported for morphine derivatives, car-
bohydrates and steroids (McKenna and Norymberski, 1957; Popek
and Lis, 2002; Crooks et al., 2006). While some of the compounds
are known, this paper provides detailed NMR and CD spectroscopic
analysis of a large set of sulfate esters which could serve as basis
for further physicochemical studies (Visky et al., 2000; Mazák et al.,
2009) and as a tool for the identification of sulfate metabolites in
various biological samples.
2. Results and discussion
∗
Corresponding author. Tel.: +36 1 2170891; fax: +36 1 2170891.
E-mail addresses: varadian@gytk.sote.hu (A. Váradi), gerand@gytk.sote.hu
(A. Gergely), jankovics.peter@ogyi.hu (P. Jankovics), nosbel@gytk.sote.hu
(B. Noszál), shosztafi@gytk.sote.hu (S. Hosztafi).
The synthesis of sixteen sulfate esters of morphine and codeine
derivatives was accomplished. The reaction involves sulfation
0928-0987/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2010.10.007

66
A. Váradi et al. / European Journal of Pharmaceutical Sciences 42 (2011) 65–72
Scheme 1. Synthesis of morphine monosulfate esters (4,8,11,12). (a) Acetic anhydride, NaHCO3, H2O, r.t., 1 h. (b) Pyridine-SO3, pyridine, 60 ◦C, 3.5 h. (c) 10% aq. NaOH, MeOH,
r.t., 1 h. (d) Hydroxylamine hydrochloride, EtOH, 60 ◦C, 45 min. (e) MeI, acetone, 40 ◦C, 4 h. (f) 20% aq. K2CO3 solution, r.t., 1 h.
of the respective C-6 or C-3 hydroxyl functions, upon stir-
ring the appropriate morphine derivative with 3 equivalents of
pyridine–SO3 complex in dry pyridine at 60 ◦C for 3.5 h.
heating the morphine derivatives with small excess methyl iodide
in acetone. Due to the limited solubility of the quaternary com-
pounds in pyridine, sulfation was carried out in pyridine/DMF
2:1 mixture at 100 ◦C. Deacetylation of the quaternary morphine
and dihydromorphine sulfate esters was achieved by 20% aqueous
potassium carbonate solution.
A set of sulfate esters of morphine 3-O-ethers has been synthe-
sized. A new and potentially pharmacologically active morphine
derivative, 3-O-isopropylmorphine (20) was prepared by heat-
ing morphine (1) with 2-iodopropane in the presence of sodium
ethoxide in dry ethanol (Scheme 2). Isocodeine, the 6␤ epimer of
codeine was prepared by Mitsunobu reaction (Simon et al., 1991).
Direct sulfation using the general method described above yielded
the desired sulfate esters of codeine (15), dihydrocodeine (16),
their quaternary derivatives (17, 18), ethylmorphine (19), 3-O-
isopropylmorphine (21) and isocodeine (22).
The reaction of morphine (1) with 6 equivalents of pyridine–SO3
complex, along with the esterification of both morphine-3-O-
sulfate (8) and morphine-6-O-sulfate (4) by pyridine–SO3 have
afforded mixtures of the diester morphine-3,6-O-disulfate (23)
and the monoester, respectively. Since most physicochemical
properties of these highly polar, zwitterionic substances are
very similar and are therefore inseparable by common tech-
Morphine and dihydromorphine have two hydroxyl groups (C-3
phenolic and C-6) that can be readily sulfated. The reactivity differ-
ence of the two hydroxyl groups is not enough, however, for direct
regioselective sulfation to produce the monoesters. In order to
obtain the sulfate esters (4, 8–10), we used acetyl protecting groups
at the C-3 phenolic or C-6 hydroxyl groups. Selective acetylation of
the phenolic hydroxyl moiety was achieved upon stirring morphine
(1) with acetic anhydride in aqueous medium in the presence of
excess sodium hydrogen carbonate (Welsh, 1954). For the synthesis
of morphine-3-O-sulfate (8) protection of the C-6 hydroxyl group
was achieved by selective hydrolysis of 3,6-O-diacetylmorphine (5)
with hydroxylamine hydrochloride in ethanol (Wright, 1941). The
sulfation of the appropriately acetylated substances was achieved
by the general method using pyridine–SO3 with subsequent alka-
line hydrolysis of the protecting group (Scheme 1).
The same pathway was followed for the synthesis of the respec-
tive dihydromorphine analogues (9, 10). During the synthesis of the
quaternary 6-O- and 3-O-sulfate esters of morphine (11, 12) and
dihydromorphine (13, 14) introduction of the appropriate acetyl
protecting group was followed by methylation of the N-atom upon

A. Váradi et al. / European Journal of Pharmaceutical Sciences 42 (2011) 65–72
67
Scheme 2. Synthesis of 3-O-isopropylmorphine-6-O-sulfate (21). (a) 2-iodopropane, NaOEt, EtOH, reflux, 4 h. (b) Pyridine-SO3, pyridine, 60 ◦C, 3.5 h.
niques (i.e. recrystallization, chromatography) we synthesized
the new diester morphine-3,6-O-disulfate by direct sulfation
of morphine (1) by concentrated sulfuric acid in DMF using
N,Nꢀ-dicyclohexylcarbodiimide as the coupling agent (Linder and
Fishman, 1973) (Scheme 3).
tives. The N-methyl shifts for the zwitterionic sulfate esters are
significantly higher than those of the respective salts in which the
N-atom is also protonated. In quaternary sulfate esters the shifts for
both the axial and the equatorial N-methyl (Iorio et al., 1984) groups
were higher than those of the respective unesterified substances,
nevertheless, the difference was less significant.
2.1. Spectral analyses
In 13C-NMR spectra, the sulfation of the phenolic hydroxyl
group causes downfield shifts for C-2 and C-4 (from +3.34 ppm to
+7.20 ppm). The signals for C-3 were shifted upfield by approx-
imately 3.20 ppm. In contrast, esterification of the C-6 hydroxyl
group results in downfield shifts for C-6 and slight upfield shifts for
C-7 and C-8. Slight upfield shifts could also be observed for C-14 and
the carbon of the N+-methyl group in all of the studied substances.
A second set of signals for H-6–H-9 and N-Me with an inte-
gral ratio of approximately 1:4 was observed in the 1H spectra
of 7-8 unsaturated 6-O-sulfates containing tertiary N-atom (4, 15,
19, 21, 22). The respective peaks merged upon heating the sam-
ple suggesting a slow and hindered conformational equilibrium of
the rings C and D between the boat/chair and skew-boat/skew-
chair conformations as previously observed in solid state 13C-NMR
investigations (Kottayil, 1993). Intensive cross-peaks in the NOESY
spectrum of (4) between the hydrogen atoms of the phenolic
hydroxyl, the protonated nitrogen atom and water may suggest
the presence of a water molecule bound to both groups and also to
the sulfate ester by strong hydrogen bonds.
The structural properties of the synthesized compounds were
studied by MS, NMR, UV, CD and ORD spectroscopy. All NMR sig-
nals in the 1H and 13C spectra of the synthesized derivatives were
completely assigned based on one- and two-dimensional homo-
and heteronuclear NMR experiments (COSY, TOCSY, HSQC, HMBC
and NOESY) (Tables 1 and 2). The effect of the sulfate ester groups
on the chemical shifts of several atoms due to changes in charge
distribution, steric interactions and ring conformation was studied.
For comparison, assignment of some of the unesterified substances
was also done based on spectra recorded in DMSO-d6.
2.2. NMR spectroscopy
In the 1H-NMR spectra of sulfate esters, the aromatic H-1 and H-
2 appear as doublets due to spin-spin 3-bond coupling. Significant
downfield shifts were observed in 3-O-esters (8, 10, 12, 14) for H-1
(about +0.20 ppm) and for H-2 (from +0.53 ppm to +0.70 ppm). This
is attributed to steric crowding and decreased electron density of
the aromatic ring, due to the electron-withdrawing effect of the
sulfate group (Brown et al., 1985).
2.3. Chiroptical spectroscopy
Significant downfield shifts in 6-O-esters were observed for H-
7, H-8, H-14, H-5 and H-6, the latter being most significant for most
of the studied 6-O-esters. In isocodeine-6-O-sulfate (22) the sulfate
ester moiety is axial, causing increased steric crowding and larger
chemical shifts for H-5, H-7 and H-8 relative to those of codeine-6-
sulfate (15). The signal for H-6 was detected as a broad, irresolvable
multiplet in the spectra of the described sulfate esters; this is due
to the electromagnetic effect of the charged sulfate ester moiety.
For H-9 and H-16 remarkably high downfield shifts were observed
in all of the studied substances (from +0.50 ppm to +0.90 ppm).
The signals for N-methyl hydrogens were shifted downfield
by approximately +0.60 ppm compared to the unesterified deriva-
Concerning structural elements of chiral properties, the circu-
lar dichroism (CD) and optical rotatory dispersion (ORD) methods
have proven to be sensitive tools in the analysis of morphine
derivatives (Bowen, 1980; Crone and Purdie, 1981; Han and Purdie,
1986; Purdie et al., 1989; Gergely et al., 2004). Morphine sulfate
esters contain one substituted benzene ring as UV chromophore,
two hydroxyls (phenolic and cycloaliphatic) capable of forming
hydrogen bond interactions and an ether group. The highly polar,
permanently charged zwitterionic character could affect chiral
properties by changing solvatation, forming hydrogen bonds or salt
bridges between the charged functional groups.
Fig. 1 shows the CD, ORD and UV spectra of selected 3-O-sulfate
and 6-O-sulfate derivatives. CD and ORD spectra show remarkable
differences that can be considered as consequences of the geomet-
ric factors responsible for intensifying Cotton effects of ␲–␲* and
n–␲* transitions originating from the different substituents and
conformational states of the morphine C and D rings. The UV spec-
tra of the 6-O-sulfates (4, 11, 15, 22) are identical to morphine
(1) (ꢀmax = 285.2 nm), but hypsochromic shifts are detected in the
spectrum of morphine-3-O-sulfate (8) (␭max = 281.8 nm).
Upon comparing the CD spectral properties of morphine with
morphine O-sulfates, similarities are found in the spectra of 6-O-
sulfate derivatives (1, 4, 11, 15, 22, 23) but characteristic differences
are observed in the spectra of 3-O-sulfate derivatives (8, 12). The
studied compounds show low negative Cotton effect at the 1Lb
Scheme 3. Synthesis of morphine-3,6-O-disulfate ester (23). (a) N,Nꢀ-
dicyclohexylcarbodiimide, cc. sulfuric acid, DMF, 0 ◦C, 15 min.

68
A. Váradi et al. / European Journal of Pharmaceutical Sciences 42 (2011) 65–72
Table 1
1H-NMR assignments of the synthesized compounds.
Compound
1H-NMR
H1a
H2a
H5b
H6c
H7d
H8e
H9f
H10g
H14h
H15i
H16j
NMe
OR
4
8
9
6.46
6.56
6.55
6.56
6.97
6.66
4.97
4.87
4.78
4.59
4.11
4.23
5.79
5.62
5.31
5.22
4.16
4.11
3.84
2.75
2.76
2.82
3.21
3.25
3.17
2.83
2.82
2.42
1.96
1.95
1.81
2.21
2.19
2.02
2.86
2.84
2.74
3.32
3.30
3.20
2.91
2.91
2.87
1.00eq
1.55ax
1.36eq
1.53ax
5.76
0.82ax
1.64eq
1.09ax
1.25eq
5.29
10
11
12
13
14
6.64
6.43
6.55
6.52
6.65
6.96
6.56
6.97
6.66
6.98
4.62
4.95
4.84
4.77
4.60
3.94
4.83
4.15
4.45
4.20
3.79
4.10
4.10
3.78
3.81
2.81
2.81
2.82
2.92
2.89
3.16
3.46
3.51
3.40
3.43
1.91
3.47
3.41
2.28
2.85
1.66
1.84
1.86
1.68
1.60
2.00
2.58
2.55
2.44
2.32
2.59
3.14
3.13
3.01
2.89
3.15
3.36
3.40
3.24
3.28
2.82
3.27eq
3.41ax
3.28eq
3.38ax
3.22eq
3.32ax
3.20eq
3.32ax
2.93
5.60
5.22
0.96eq
1.59ax
1.45eq
1.54ax
5.76
1.12eq
1.51ax
5.74
0.89ax
1.59eq
1.09ax
1.24eq
5.29
0.83ax
1.59eq
5.29
15
16
6.59
6.67
6.75
6.84
5.00
4.80
4.62
4.28
4.17
3.86
2.78
2.85
3.26
3.18
2.87
2.42
1.96
1.80
2.24
2.03
2.86
2.71
3.31
3.20
3.73k
3.79k
2.86
17
18
6.56
6.65
6.74
6.85
5.00
4.79
4.84
4.47
4.11
3.81
2.84
2.95
3.49
3.44
3.49
3.04
1.86
1.69
2.60
2.42
3.13
2.98
3.37
3.27
3.28eq
3.40ax
3.22eq
3.33ax
2.91
2.44
2.91
2.91
2.88
3.73k
3.80k
1.07eq
1.56ax
5.79
5.69
5.79
5.94
5.75
5.54
1.17eq
1.30ax
1.16eq
1.30ax
5.53
0.84ax
1.55eq
5.32
5.29
5.32
5.65
5.30
5.25
0.90ax
1.38eq
0.89ax
1.39eq
5.24
19
20
21
22
6.56
6.54
6.56
6.64
6.53
6.46
6.53
6.73
6.66
6.70
6.79
7.14
6.61
6.68
4.98
4.87
4.98
5.10
4.97
4.68
4.46
4.60
4.17
4.59
4.49
4.61
4.11
3.81
4.17
3.35
4.17
4.16
4.15
4.11
2.93
2.79
2.30
2.79
2.78
2.84
2.23
2.27
3.26
3.04
3.25
3.26
3.26
2.92
2.83
2.85
2.66
2.85
3.11
2.95
2.55
2.10
1.98
1.87
1.97
1.94
1.91
1.62
1.44
2.23
2.06
2.22
2.15
2.25
1.98
1.79
2.85
2.40
2.86
2.78
2.89
2.22
2.06
3.32
2.59
3.31
3.31
3.29
2.45
2.35
4.01l 1.29m
4.52n 1.29o 1.34o
4.57n 1.21o
3.75k
23
Codeine
Dihydrocodeine
2.30
2.25
3.72k
3.76k
Dihydromorphine
6.41
6.34
6.52
6.44
4.44
4.66
3.80
4.09
2.92
3.24
2.24
2.20
2.82
2.88
2.10
2.53
1.44
1.62
1.78
1.97
2.08
2.23
2.35
2.44
2.25
2.29
Morphine
a
d, J1,2 ∼ 8.0 Hz, 1 H.
b
d, J56 ∼ 6.0 Hz, 1 H.
c
br d, J6,7 ∼ 2.0 Hz, 1 H.
d
e
f
7–8 unsaturated: d, J7,8 ∼ 9.5 Hz, 1 H; 7–8 saturated: dd, J7eq
,
,
7ax ∼ 14.5 Hz, J7eq
,
8eq ∼ 3.0 Hz, 2 H.
7–8 unsaturated: d, J7,8 ∼ 9.5 Hz, 1 H; 7–8 saturated: dd, J8eq
d, J8,9 ∼ 4.5 Hz, 1 H.
8ax ∼ 14.5 Hz, J7eq
,8eq ∼ 3.0 Hz, 2 H.
g
h
i
dd, J10a
s, 1 H.
,10b ∼ 20.0 Hz, J9,10 ∼ 5.5 Hz, 2 H.
dd, J15a
dd, J16a
s, 3 H, OCH3.
,
,
∼ 13.0 Hz, J1516 ∼ 3.5 Hz, 2 H.
15b ∼ 13.0 Hz, J15,16 ∼ 3.5 Hz, 2 H.
j
16b
k
l
m, 1 H, OCH2CH3.
m
n
o
t, J = 7.0 Hz, 3 H, OCH2CH3.
m, 1 H, OCH(CH3)2.
d, J = 5.0 Hz, 6 H, OCH(CH3)2.
absorption band around 286 nm (−75 to −13 deg cm2 dmol−1),
medium intensive positive molar ellipticity at the 1La absorption
band between 242 and 246 nm (+250 to +450 deg cm2 dmol−1) for
6-O-sulfates, between 235 and 236 nm for the 3-O-sulfates (+120
to +160 deg cm2 dmol−1) and very intensive 1B absorption band
between 210 and 218 nm (−540 to −1300 deg cm2 dmol−1) in the
CD spectra. The most remarkable differences are found in the high
intensity 1B absorption bands. The absolute values of these neg-
ative ellipticities increase in the order 1 < 8 < 12 < 23 < 4 < 22 < 15.
3-O-sulfation caused moderate hypsochromic shift as compared
to morphine in the same absorption band. The peak wavelengths
detected for the 1B band are in the following order: 8 ≈ 12
(211.0 nm) < 22 (213.4 nm) < 23 (215.5 nm) < 1 ≈ 4 (217.0 nm) < 15
(217.4 nm). While ꢀ0 (wavelength with zero ellipticity) occurred
at the same wavelengths for (1, 4, 11, 15, 22, 23) (231 nm and
263 nm), ꢀ0 values for (8, 12) are shifted towards shorter wave-
lengths (220 nm) between the 1B and 1La absorption bands, and
longer wavelengths (276 nm) between 1La and 1Lb bands. These
characteristic spectral features make 3-O-sulfates distinguishable
from 6-O-sulfates.
The ORD spectra of morphine and selected morphine O-
sulfate derivatives were compared. Morphine sulfates exhibit
broad maxima around 245–260 nm and intensive minima around
280–300 nm. The only remarkable difference is found in the spec-
trum of (22), the 6␤ epimer of (15). On the basis of the ORD spectral
similarities the absolute stereochemistry of asymmetric centers in
morphine sulfates are identical to those of their parent molecules
and have not changed under the conditions of the different sulfa-
tion reactions.
Remarkable changes in intensities and the shape of CD and ORD
curves suggest that interactions between the C-3 and C-6 groups
may be attributed to modification or stabilization of the C-ring con-
formations and hindered rotation of the sulfate ester group around
the O-S bond. The more polar sulfate group in morphine deriva-
tives causes expansion in solvate shell, which could fold back to
the 3-hydroxyl group of morphine derivatives by insertion of water
molecule or ammonium complex formation. This type of associa-
tion was supported by NOE experiments, where correlation was
found between the C-3 hydroxyl, C-6 sulfate and water. Order of
hypsochromic shifts in the CD spectra could also provide expla-

A. Váradi et al. / European Journal of Pharmaceutical Sciences 42 (2011) 65–72
69
Table 2
13C-NMR assignments of the synthesized compounds.
Compound 13C-NMR
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
NMe
OR
4
8
119.10 117.22 139.18 146.39 89.13 70.56 132.59 125.42 59.63 20.88 121.80 128.71 41.70 38.28 32.71 46.20 40.55
118.93 123.29 134.94 150.24 89.97 65.13 135.83 124.29 59.64 20.90 127.36 129.47 40.80 37.73 32.25 46.20 40.33
9
10
11
118.95 117.82 138.83 146.04 87.62 71.11
118.47 124.54 133.72 150.26 88.99 65.01
118.95 117.26 139.39 146.18 89.54 70.26 132.62 125.46 68.75 23.31 121.12 128.84 41.53 33.04 29.18 54.98 49.54ax
20.30
26.84
19.65 60.31 20.30 122.16 127.63 41.36 33.62 34.64 46.02 40.49
16.82 60.24 20.35 123.45 129.18 41.59 30.91 33.50 46.70 40.04
53.60eq
118.91 123.47 135.01 150.04 90.36 65.09 135.52 124.60 68.78 23.55 126.72 129.47 40.73 32.75 28.95 55.11 49.65ax
53.56eq
12
13
14
118.82 117.93 139.04 146.03 87.63 70.39
20.17
19.58 69.27 22.21 124.60 127.99 41.22 27.60 30.51 54.12 49.26ax
53.15eq
17.83 69.95 23.30 124.33 128.18 41.08 32.83 30.33 56.34 49.95ax
53.60eq
118.48 124.49 134.19 150.25 89.07 64.86
27.13
15
16
17
119.26 113.99 141.92 147.31 89.51 70.43 132.59 125.52 59.54 20.88 123.85 128.94 41.58 38.18 32.62 46.39 40.61
119.12 115.53 141.53 147.14 87.75 70.98 21.21 19.27 60.33 20.30 124.18 127.91 41.20 34.20 34.15 46.20 40.50
119.09 114.05 142.00 147.13 89.92 70.10 132.67 125.46 68.69 23.31 129.06 132.67 41.39 32.96 29.06 54.92 49.58ax
53.61eq
55.97a
56.57a
55.99a
18
19
20
118.95 115.60 141.56 146.98 88.16 70.58
21.28
19.71 69.67 22.64 123.67 128.00 40.89 28.61 30.81 54.55 49.52ax
53.71eq
56.56a
119.17 115.18 140.96 147.45 89.43 70.43 132.69 125.41 59.53 20.86 123.83 128.93 41.57 38.18 32.59 46.35 40.56
64.15b
14.84c
72.45d
22.38e
22.55e
71.11d
22.09e
22.13e
56.20a
119.80 117.79 140.17 147.86 91.20 66.49 133.47 128.46 59.02 20.66 127.87 131.43 42.96 40.90 35.93 46.60 43.20
21
118.19 119.31 139.51 148.39 89.40 70.48 132.66 125.45 59.54 20.97 124.27 129.24 41.52 38.19 30.60 46.38 40.56
22
23
119.53 114.46 141.98 145.68 90.57 70.32 130.04 130.63 59.57 20.56 123.94 128.63 41.85 37.35 33.03 46.94 40.53
118.42 121.94 135.13 149.53 89.38 70.59 132.48 125.64 59.38 24.31 126.50 128.91 42.87 35.00 32.85 45.90 40.06
118.35 113.21 141.23 147.19 91.96 66.44 133.33 128.37 57.86 20.07 127.37 131.14 42.91 40.52 35.47 45.86 42.74
Codeine
Dihydro-
codeine
Dihydro-
morphine
Morphine
56.01a
56.23a
117.97 113.96 140.81 146.99 90.34 65.93
25.82
19.36 58.85 19.65 127.13 130.34 41.85 38.37 37.18 46.00 42.73
117.97 116.67 137.95 146.01 89.93 66.09
25.59
19.47 58.90 19.59 125.18 130.06 42.00 38.18 37.23 46.02 42.75
118.47 116.25 138.40 146.19 91.42 66.34 133.34 128.40 57.97 20.06 125.46 130.94 42.91 40.56 35.48 45.95 42.77
a
OCH3.
OCH2CH3.
OCH2CH3.
OCH(CH3)2.
b
c
d
e
OCH(CH3)2.
nation to these strong hydrogen–bond interactions. Significant
differences were not found in the spectra of the quaternary com-
pounds (11, 12) suggesting that methylation of the nitrogen does
not influence ring conformation or intramolecular interactions.
bonate (17.00 g) was added. To the resultant suspension 4 × 1.5 ml
acetic anhydride was added in 10 min intervals, and then stirred for
another 15 min. The aqueous phase was then extracted with chlo-
roform and dried. After evaporation of chloroform the pale yellow
oil product 2 was dissolved in 10 ml dry pyridine. To the resul-
tant solution pyridine-SO3 complex (1.43 g, 9.00 mmol) was added
and stirred for 3.5 h at 60 ◦C. The crude 3 was crystallized from
hot water. 3 was dissolved in a mixture of 30 ml methanol and
5 ml 10% aqueous NaOH and stirred for 1 h at room temperature.
The solution was neutralized with concentrated acetic acid and the
methanol was then evaporated under reduced pressure. The crude
product was filtered and washed with methanol and crystallized
from hot water to yield 4 (colorless crystals).
3. Experimental
3.1. Materials and methods
The reagents were purchased from Aldrich and used without
further purification. Solvents were freshly distilled prior to use.
Organic solutions were dried over anh. Na2SO4. 1H- (600 MHz)
and 13C-NMR (150 MHz) spectra: 600 MHz Varian VNMRS spec-
trometer in DMSO-d6 solutions; ı in ppm rel. to Me4Si as internal
standard. Melting points were taken on a Stuart SMP-3 apparatus.
Mass spectra: Agilent 6410 Triple Quad instrument using elec-
trospray ionization (ESI) and negative polarity. UV, CD and ORD
spectra: Jasco J-810 instrument, pH 7 aqueous solution, concen-
trations 2.5–2.8 × 10−4 mol dm−3. Structures of the synthesized
compounds, results for TLC analyses, melting points, yields and MS
data are shown separately (Table 3).
3.3. Synthesis of morphine-3-O-sulfate (8)
3,6-O-diacetyl-morphine (5) (2.00 g, 5.41 mmol) was dis-
solved in ethanol. To the resultant solution 0.38 g (5.46 mmol)
hydroxylamine-hydrochloride was added and stirred for 45 min
at 60 ◦C. After the addition of water, the ethanol was evaporated
under reduced pressure. The residue was basified (pH 9) with con-
centrated NH4OH solution. The aqueous phase was extracted with
chloroform and the organic phase was dried. The chloroform was
then evaporated under reduced pressure to give 1.55 g 6. 6 was then
sulfated using the method described for 4. The product was crys-
tallized from hot water followed by hydrolysis of the 6-O-acetyl
3.2. Synthesis of morphine-6-O-sulfate (4)
Morphine hydrochloride (1) (1.70 g, 5.28 mmol) was dissolved
in water by stirring at room temperature and sodium hydrogen car-

70
A. Váradi et al. / European Journal of Pharmaceutical Sciences 42 (2011) 65–72
Fig. 1. UV (top), CD (middle) and ORD (bottom) spectra of selected sulfate esters. Solid line: 1, long dash: 4, short dash: 8, dash-dot-dot-dash: 15, dash-dot-dash: 22, dotted
line: 23.

A. Váradi et al. / European Journal of Pharmaceutical Sciences 42 (2011) 65–72
71
Table 3
Structure and physicochemical data of the synthesized compounds.
c
Compound
R1
R2
R3
C7–C8 bond
Total yield [%]
M.p. [◦C]
Rf
ESI-MS
4
8
9
H
SO2O−
H
H
H
H
H
CH3
CH3
CH3
CH3
H
Double
Double
Single
32.4a
58.4a
51.7a
56.9a
69.7
49.5
66.4
63.0
>256b
0.34
0.31
0.32
0.33
0.23
0.15
0.18
0.18
0.24
0.23
0.19
0.14
0.38
0.39
0.25
0.27
365
365
367
367
n/ad
n/ad
n/ad
n/ad
379
381
n/ad
n/ad
393
407
379
445
SO2O−
H
>280b
SO2O−
H
>300b
10
11
12
13
14
15
16
17
18
19
21
22
23
SO2O−
H
Single
288–292b
>284b
SO2O−
H
Double
Double
Single
Single
Double
Single
Double
Single
Double
Double
Double
Double
SO2O−
H
>285b
SO2O−
H
>300b
SO2O−
CH3
CH3
CH3
CH3
Et
>300b
SO2O−
SO2O−
SO2O−
SO2O−
SO2O−
SO2O−
SO2O− (6␤)
SO2O−
36.6a
66.7a
73.5a
90.5
239–241b
270–272b
>285b
H
CH3
CH3
H
H
H
>273b
43.9a
48.3a
68.7a
40.3
>218b
iPr
>225b
CH3
242–247b
175–178
SO2O−
H
a
Recrystallized yields.
Decomposition.
b
c
TLC. Mobile phase: 45:15:10:20 mixture of butan-1-ol, acetone, cc. acetic acid, and water, respectively. Stationary phase: Merck Silica Gel 60 F254.
Quaternary sulfate esters cannot be detected by ESI-MS.
d
protecting group by the method described for 4. The crude product
was filtered and washed with methanol and crystallized from hot
water to give 8 (colorless crystals).
iodopropane (1.3 ml) were added and stirred for 6 h at 60 ◦C. The
ethanol was evaporated under reduced pressure and to the residue
was added 50 ml water and 5 ml 10% aqueous NaOH solution fol-
lowed by extraction with chloroform. The organic phase was dried
and the chloroform evaporated under reduced pressure to yield
2.00 g of 3-O-isopropylmorphine (20) (oil). 20 was then sulfated as
described for 4. The crude product was crystallized from hot water
to yield 21 (pale yellow crystals).
3.4. Synthesis of morphine-3,6-O-disulfate ammonium salt (23)
Morphine (1) (0.175 g, 0.612 mmol) was dissolved in 3 ml of dry
DMF and cooled to 0 ◦C. To this solution 0.20 ml (6 eq.) of con-
centrated sulfuric acid and DCC (1.15 g) were added and stirred
for 15 min at 0 ◦C. The reaction was then basified (pH 9) with 10%
NH4OH and filtered. The filtrate was evaporated in vacuo and the
residue dissolved (3 ml of DMF) and filtered to remove inorganic
residue. To the filtrate 50 ml Et2O was added and the resulting sus-
pension was filtered to give the ammonium salt of 23 (pale yellow
solid).
4. Conclusion
In conclusion, we synthesized a systematic series of sulfate
monoesters of morphine and its derivatives and also, the diester
morphine-3,6-O-disulfate. For monosubstitution, pyridine–SO3
complex was used with moderate yields. In the case of the
diesters the sulfations were performed with DCC/cc. H2SO4 reagent.
Detailed 1H- and 13C-NMR and CD/ORD analyses are presented that
can serve as a basis for further physicochemical investigations or
as a tool for the identification of various sulfate esters in biological
samples. Most of the synthesized sulfate esters have the potential
of selective peripheral analgesic activity.
3.5. Synthesis of codeine-6-O-sulfate (15)
Codeine (0.90 g, 3.00 mmol) was sulfated using the method
described for 4. The product slowly precipitated during the reaction
time as a white powder. Cold water (10 ml) and chloroform (10 ml)
were added to the suspension and was left to stand overnight in
the freezer. The precipitate was collected by filtration and washed
twice with cold water and crystallized from hot water to give 15
(colorless crystals).
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