Opiate receptor binding properties of morphine-, dihydromorphine-, and codeine 6-O-sulfate ester congeners

Article abstract of DOI:10.1016/j.bmcl.2006.05.060

A series of 3-O-acyl-6-O-sulfate esters of morphine, dihydromorphine, N-methylmorphinium iodide, codeine, and dihydrocodeine were prepared and evaluated for their ability to bind to μ-, δ-, κ₁-, κ₂-, and κ₃-opiate receptors. Several compounds exhibited good affinity for the μ-opiate receptor. Morphine-3-O-propionyl-6-O-sulfate had four times greater affinity than morphine at the μ-opiate receptor and was the most selective compound at this receptor subtype.

Full text of DOI:10.1016/j.bmcl.2006.05.060

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4291–4295
Opiate receptor binding properties of morphine-, dihydromorphine-,
and codeine 6-O-sulfate ester congeners
Peter A. Crooks,a,* Santosh G. Kottayil,b Abeer M. Al-Ghananeem,a
Stephen R. Byrnc and D. Allan Butterfieldb
aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA
bDepartment of Chemistry, University of Kentucky, Lexington, KY 40536-0082, USA
cDepartment of Industrial and Physical Pharmacy, School of Pharmacy and Pharmaceutical Sciences, Purdue University,
575 Stadium Mall Drive, West Lafayette, IN 47907-2091, USA
Received 14 March 2006; revised 13 May 2006; accepted 18 May 2006
Available online 13 June 2006
Abstract—A series of 3-O-acyl-6-O-sulfate esters of morphine, dihydromorphine, N-methylmorphinium iodide, codeine, and dihy-
drocodeine were prepared and evaluated for their ability to bind to l-, d-, j1-, j2-, and j3-opiate receptors. Several compounds
exhibited good affinity for the l-opiate receptor. Morphine-3-O-propionyl-6-O-sulfate had four times greater affinity than morphine
at the l-opiate receptor and was the most selective compound at this receptor subtype.
Ó 2006 Elsevier Ltd. All rights reserved.
Opium contains a number of alkaloids, of which only a
few, that is, morphine, codeine, noscapine, and papaver-
ine, have found clinical use.1 Metabolites of codeine,
other than CYP2D6-dependent formation of morphine,
have also been reported to have central nervous system
effects.2
as an analgesic when both compounds are administered
by the icv route in mice.
Structural analogs of morphine that contain a 3-hy-
droxy group in their structure are generally 30- to 100-
fold more potent than their corresponding 3-methoxy
analogs at the l-receptor, and these compounds general-
ly exhibit greater efficacy (e.g., morphine produced 2-
fold greater maximal stimulation than its 3-methoxy
analog, codeine).11 Furthermore, analgesic potency has
been shown to increase by sulfation of the 6-hydroxy
group of morphine.6,10 Morphine-6-O-sulfate (3a,
M6S) has been shown to displace [3H]-morphine and
[3H]-leucine enkephalin binding in rat brain membranes
with Ki values of 1.8 and 4.8 nM, respectively;12 this
study indicated that 6-O-sulfation of morphine results
in reduced affinity for the l-receptor but enhanced affin-
ity for the j-receptor.
Metabolic conjugation (phase II metabolism) usually
terminates the pharmacological action of a drug; howev-
er, in the case of morphine it has been observed that the
metabolite morphine-6b-glucuronide (M6G) is almost
100-fold more potent than morphine when administered
via the intracerebroventricular (icv) route in animals.3
The 3-O- and 6-O-sulfate conjugates of morphine (1)
have been known for many years.4–6 Morphine-3-O-sul-
fate (2, M3S) is a known phase II metabolite of mor-
phine,7 and has been detected in rat tissues after
administration of morphine.8 However, morphine-6-O-
sulfate (3a, M6S) has not been detected as a biotransfor-
mation product of morphine.7,9 While M3S has been
reported by several groups4–6 to have little or no in vivo
analgesic potency, Brown et al.10 have reported this
compound to be three times more potent than morphine
M6S is an effective analgesic, with a 30-fold greater
potency than morphine in the mouse radiant heat tail-
flick assay, and is similar in potency to the active mor-
phine metabolite, morphine-6b-glucuronide (M6G).13
3-O-acylation of the M6S molecule affords more lipo-
philic derivatives that are considered to be prodrugs of
M6S.14,15 These O-acylated derivatives were found to
be relatively stable in phosphate-buffered saline over
the pH range 6–8, and were slowly hydrolyzed in blood
and brain homogenate to M6S.16 It has been demon-
Keywords: Morphine analogs; Sulfate conjugates; Opiate receptors.
*
Corresponding author. Tel.: +1 859 257 1718; fax: +1 859 257
7585; e-mail: pcrooks@email.uky.edu
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.060

4292
P. A. Crooks et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4291–4295
strated that the 3-O-acetyl derivative of M6S (M3A6S,
3b) exhibits enhanced in vivo duration of antinocicep-
tive activity compared to both M6S and morphine,
when given subcutaneously (sc) to rats, and has greater
potency and duration of action than morphine when
given to rats via the icv route.16,17
M3A6S demonstrated the presence of two independent
zwitter-ionic molecules per asymmetric unit.17 The ste-
reotopic crystal structures of the two molecules indi-
cated that they differ from one another in the
relative conformation of the C- and D-rings. Similar
to the parent morphine molecule,19 one of the struc-
tures incorporates the C- and D-rings in their boat
and chair conformations, respectively. However, in
the second structure, rings C and D exist in the
skew-boat and skew-chair forms. The presence of
two independent molecules in the crystal structure of
M3A6S provides an explanation for the observation
of the presence of several doublets in the solid state
13C NMR spectrum of this molecule.17
The relative stability and interesting antinociceptive pro-
file of M3A6S and other structurally related O-acyl ana-
logs of M6S prompted us to undertake a comprehensive
structure–affinity study of a number of 3-O-acyl deriva-
tives of M6S and other congeners (Fig. 1)18 to determine
the affinity of these compounds for opiate receptors.
Compounds 3a–i were prepared from 6-O-sulfation of
codeine or the appropriate 3-O-acyl analog of mor-
phine, with pyridine-SO3 reagent (Scheme 1).
Hartley guinea pigs were decapitated and their brains
were quickly removed and weighed. The brains were
then homogenized in 50 mM Tris–HCl buffer, pH
7.7 (about 25 ml/brain), using a Polytron. The homog-
enate was centrifuged at 40,000g for 15 min, re-ho-
mogenized, and centrifuged. The final pellet was
suspended in Tris–HCl, pH 7.7, at a final concentra-
tion of 6.67 mg original wet weight of tissue per mil-
liliter, except for tissue prepared for NalBzOH
(naloxone benzoylhydrazone) binding, which was sus-
pended in buffer containing 5 mM EDTA. The 6-O-
sulfate congeners of morphine, dihydromorphine, co-
deine, and dihydrocodeine in Table 1 were evaluated
for their binding affinities for l-, d-, j-1, j-2, and
The dihydro analogs 4a–d were synthesized in a simi-
lar manner from dihydrocodeine or the appropriate 3-
O-acyl analog of dihydromorphine. The betaines 3g
and 3h were obtained from pyridine-SO3 sulfation of
3-O-acetyl-N-methylmorphinium iodide and 3-O-ben-
zoyl-N-methylmorphinium iodide, respectively. M3S
(2) was prepared from direct sulfation of morphine,
and M6S (3a) and DM6S (4a) were prepared from
deacetylation of 3-O-acetylmorphine-6-O-sulfate and
3-O-acetyl-dihydromorphine-6-O-sulfate, respectively,
in MeOH–NaOH. X-ray crystallographic studies on
CH
N
3
H
CH
3
N
O
O
HO
OH
O SO
3
OH
CH
1
2
M3S
Morphine
2
2
R
CH
R
3
3
N
N
O
O
1
1
R
OSO
3
R
OSO
3
4
3
1
2
a) R = OH, R = H (DM6S)
1
2
1
2
a) R = OH, R = H (M6S)
b)R = OCOCH , R = H (DM3A6S)
3
1
2
1
2
b) R = OCOCH , R = H (M3A6S)
c) R = OCOC H , R = H (DM3B6S)
3
6
5
2
1
2
1
c) R = OCOCH CH , R = H (M3Pr6S)
d) R = OCH , R = H (DC6S)
2
3
3
1
2
d) R = OCOCH(CH ) , R = H (M3IBu6S)
3 2
1
2
e) R = OCOC(CH ) , R = H (M3P6S)
3 3
2
1
f) R = OCOC H , R = H (M3B6S)
6
5
1
2
g) R = OCOCH , R = CH (MM3A6S)
3
3
1
2
h) R = OCOC H , R = CH (MM3B6S)
6
5
3
1
2
i) R = OCH , R = H (C S)
3
6
Figure 1. Structure of morphine, dihydromorphine, and codeine 6-O-sulfate ester congeners.

P. A. Crooks et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4291–4295
4293
CH3
N
H
CH3
N
pyridine-SO3
o
pyridine, 55 C
O
RO
OH
O
RO
OSO3
R = R'CO
R = CH
3
CH3I/CH2Cl2
RT, 12-14 h
[R = CH3CO]
5% MeOH
aq. NaOH, RT 1 h
H3C
CH3
N
I
Pd-C, 5%
H , 50psig
2
H
CH3
N
O
CH3
RO
OH
N
pyridine-SO3
O
o
pyridine, 55 C
HO
OSO3
O
RO
OH
pyridine-SO3
H3C
CH3
N
o
pyridine, 55 C
H
CH3
N
O
RO
OSO3
O
RO
OSO3
5% MeOH
aq. NaOH, RT 1 h
[R = CH CO]
3
H
CH3
N
O
HO
OSO3
Scheme 1. Synthesis of 3-O-acyl derivatives of morphine, dihydromorphine, and codeine 6-O-sulfate ester congeners.
j-3 opiate receptors utilizing guinea pig brain homog-
enate preparations. Around 1 nM of morphine, Met-
enkephalin, DAMGO (a l-selective agonist),
DPDPE-Cl (a d-selective agonist), U69,593 (a j-1-se-
lective agonist), and NalBzOH (a j-3-selective agonist)
were utilized as standard radioligands at these opiate
receptors. Affinities at j-2 receptors were determined
utilizing [3H]-bremazocine in the presence of 100 nM
DAMGO, U69,593, and DSLET.
mined by incubating in the presence of 1 lM of the
‘cold’ unlabeled counterpart of each labeled ligand, ex-
cept that 10 lM NalBzOH was used for the j-3 receptor
assay. The samples were then filtered through glass fiber
filters on a 48-well Brandel cell harvester. The filters
were washed three times with 3 ml of buffer. Filters were
incubated overnight with 5 ml of scintillation cocktail
before counting.
Results are reported in terms of IC50 (concentration of
test compound that produces 50% inhibition of labeled
ligand binding). Ki values (inhibitory dissociation
The guinea pig brain suspension (1.8 ml) was incubated
in 50 mM Tris–HCl. Nonspecific binding was deter-

4294
P. A. Crooks et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4291–4295
Table 1. Inhibitory effects (Ki values) of opiate receptor ligands and morphine-6-O-sulfate congeners on the binding of titrated ligands to l-, d-, and
j-receptors in guinea pig brain homogenates
Compound
Ki; nM SEM (Hill slope)
j-1
l
d
j-3
Morphine (1)
DAMGO
DAMGO
2.5 0.3 (1)
58 3.8 (0.9)
33.9 4.1 (0.8)
1841 230 (0.8)
1841 230 (0.8)
13.9 4.1 (0.7)
26.9 6.3 (0.8)
26.9 6.3 (0.8)
1.1 0.2 (1)
1.1 0.2 (1)
180 1.2 (1)
127 13.9 (0.9)
127 13.9 (0.9)
0.3 0.07 (1)
DPDPE-C
U69
NalBzOH
>10,000
1028 219 (0.8)
1099 0.2 (0.9)
0.3 0.07 (1)
692 97 (0.9)
0.2 0.01 (1)
6.9 1.5 (0.9)
101 0.7 (1)
1358 118 (0.8)
1.4 0.13 (0.8)
1.1 0.2 (0.9)
0.7 0.05 (0.9)
0.4 0.1 (0.9)
4467 865 (0.8)
Met-Enkephalin
M3S (2)
M6S (3a)
277 29 (0.9)
1694 287 (0.7)
6.3 0.3 (0.9)
5.5 1.2 (0.8)
19.9 16 (0.8)
266 68.5 (0.7)
11.2 3.16 (0.7)
513 14.7 (0.8)
>10,000
18 0.4 (1)
>10,000
0.9 0.01 (1)
0.8 0.01 (1)
0.6 0.15 (1)
19.8 0.6 (1)
0.7 0.15 (1)
36.9 1.3 (1)
1.5 0.25 (0.9)
13.7 0.55 (0.8)
61.8 13.15 (0.9)
34.2 12.1 (0.9)
444 169 (1)
38.7 7.95 (0.9)
196 57.7 (0.8)
1192 697 (0.8)
1165 185 (0.6)
252 38.6 (0.9)
4558 229 (0.8)
1940 198 (0.8)
M3A6S (3b)
M3Pr6S (3c)
M3IBu6S (3d)
M3P6S (3e)
M3B6S (3f)
DM6S (4a)
DM3A6S (4b)
DM3B6S (4c)
MM3A6S (3g)
MM3B6S (3h)
C6S (3i)
154 16.4 (0.4)
40 1.9 (0.9)
584 28.8 (0.6)
15.2 4.25 (0.9)
600 150 (1.2)
18.8 3.35 (0.9)
325 71.2 (0.9)
1598 422 (0.8)
842 189 (1)
>10,000
3129 368 (1.3)
>10,000
7
0.2 (0.7)
838 744 (0.8)
822 83.1 (0.9)
400 58.4 (0.8)
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
414 140 (0.8)
1152 184 (0.9)
455 224 (0.8)
760 83.4 (1.2)
DC6S (4d)
constant) are derived from the following equation:
Ki = IC50/1 + [L]/Kd. The Kd values were obtained by
computer analysis of detailed self-inhibition curves for
each of the labeled ligands (L), using the curve-fitting
program LIGAND.
MM3B6S, had low affinity in all the opiate receptor as-
says employed.
The above data clearly demonstrate that 3-O-acylation
of M6S and related compounds gives rise to molecules
with high affinity for the l-receptor. The 3-O-propionyl
analog, M3Pr6S, was the most potent l-receptor ligand
in the series, exhibiting a Ki of 600 nM. M3Pr6S exhib-
ited a profile similar to morphine, but was slightly more
potent at l-receptors. It is unlikely that these com-
pounds are hydrolyzing to M6S in the binding assay
buffer, since previous studies have shown that M3A6S,
the compound likely to be the most labile 3-O-acyl ana-
log of M6S, hydrolyzes relatively slowly to M6S in phos-
phate-buffered saline over the pH range 6.0–8.0 and is
not converted to morphine under these conditions.16
Interestingly, the rates of enzymatic hydrolysis of
M3A6S in blood and brain were relatively faster than
in buffer, and hydrolysis in rat brain was considerably
faster than in blood.16 Thus, these 3-O-acyl derivatives
of M6S, although originally considered to be prodrugs,
also exhibit high affinity for l-receptors. This may ex-
plain the relatively prolonged duration of analgesia pro-
duced by these compounds in in vivo experiments in the
rat when compared to morphine. This is likely due to
these active 3-O-acyl compounds having plasma half-
lives greater than 1 h, and to the fact that subsequent
metabolism by plasma esterases will afford the 3-O-
deacylated active parent compound.
The compounds listed in Table 1 exhibited no affinity
for j-2 receptors (data not shown) and only weak affin-
ity at j-1 receptors was observed. Sulfation of the 6-hy-
droxy group of morphine afforded the compound M6S,
which exhibited slightly improved affinity over mor-
phine for l-, d-, and j-3-receptors, and reduced affinity
at j-1-receptors. The 7,8-dihydro analog, DM6S, had
similar activity to M6S.
Sulfation of the 3-hydroxy group of morphine afforded
M3S, which had little or no affinity at any of the opiate
receptors examined. Both codeine 6-O-sulfate (C6S) and
its dihydro derivative, DC6S, exhibited a significant loss
in affinity compared to M6S at all opiate receptors.
However, M3A6S, the 3-O-acetylated derivative of
M6S, exhibited comparable Ki values to M6S at l-
and j-3-receptors, but showed decreased affinity for d-
receptors, whereas the dihydro derivative, DM3A6S,
and the quaternary ammonium analog, MM3A6S both
had reduced affinity compared to M3A6S in all the opi-
ate receptor assays employed. The 3-O-propionyl
(M3Pr6S), 3-O-isobutyryl (M3IBu6S), and 3-O-pivaloyl
(M3P6S) analogs of M6S all showed high affinity for l-
receptors and low affinity for j-1 receptors. The sterical-
ly hindered ester, M3P6S, exhibited a very similar recep-
tor affinity profile to the parent compound, M6S. Both
the benzyl ester, M3B6S, and its dihydro derivative,
DM3B6S, were less potent than their respective parent
compounds, M6S and DM6S, at all the opiate receptors
examined. Interestingly, the betaines, MM3A6S and
Acknowledgment
This research was supported by a Grant from National
Institute on Drug Abuse (Medications Development
Division Contract No. 271-89-8159).

P. A. Crooks et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4291–4295
4295
12. Oguri, K.; Yamada, M. I.; Shigezane, J.; Hirano, T.;
Yoshimura, H. Life Sci. 1987, 41, 1457.
References and notes
13. Zuckerman, A.; Bolan, E.; de Paulis, T.; Schmidt, D.;
Spector, S.; Pasternak, G. W. Brain Res. 1999, 842, 1.
14. Houdi, A. A.; Kottayil, S.; Crooks, P. A.; Butterfield, D.
A. Pharmacol., Biochem. Behav. 1996, 53, 665.
15. Kottayil, S.; Butterfield, D. A.; Crooks, P. A. 202nd
National Meeting of the American Chemical Society,
1991; MEDI 149.
16. Preechagoon, D.; Brereton, I.; Staatz, C.; Prankerd, R.
Int. J. Pharm. 1998, 163, 177.
17. Kottayil, S. G. Ph.D. Thesis, University of Kentucky,
1993.
1. Sjoegren, P.; Zylicz, Z. Pol. Med. Paliatywna 2004, 3, 101.
2. Loetsch, J.; Skark, C.; Schmidt, H.; Rohrbacher, M.;
Hofmann, U.; Schwab, M.; Geisslinger, G. Clin. Pharma-
col. Ther. 2006, 79, 35.
3. Paul, D.; Standifer, K. M.; Inturrisi, C. E.; Pasternak, G.
W. J. Pharmacol. Exp. Ther. 1989, 251, 477.
4. Shimomura, K.; Kamata, O.; Ueka, S.; Ida, S.; Oguri, K.;
Yoshimura, H. Tohoku J. Exp. Med. 1971, 105, 45.
5. Mori, M.; Oguri, K.; Yoshimura, H.; Shimomura, K.;
Kamata, O.; Ueka, S. Life Sci. 1972, 11, 525.
6. Watrous, W. M.; Fujimoto, J. M.; Haarstad, V. B.
Pharmacologist 1968, 10, 173.
7. Yeh, S. Y.; Chernov, H. I.; Woods, L. A. J. Pharm. Sci.
1971, 60, 469.
8. Donnerer, J.; Cardinale, J. C.; Lisek, C. A.; Jardine, I.;
Spector, S. Pharmacol. Exp. Ther. 1987, 242, 583.
9. Nagano, E.; Yamada, H.; Oguri, K. Life Sciences 2000,
67, 2453.
18. Of the 13 compounds listed in Figure 1, M3S,5 M6S,16
M3A6S,16 M3Pr6S,16 DM6S,20 C6S,13 and DC6S13 have
been previously characterized in the literature. The char-
acterization data for the following compounds: M3IBu6S,
M3P6S, M3B6S, DM3A6S, DM3B6S, MM3A6S, and
MM3B6S are reported in Ref. 21.
19. Fridrichsons, J.; Mackay, M. F.; Mathieson, A. M.
Tetrahedron Lett. 1968, 24, 2887.
20. Brock, C. P.; Kottayil, S.; Butterfield, D. A.; Crooks, P. A.
Acta Crystallogr., Sect. C 1996, C52, 122.
10. Brown, C. E.; Roerig, S. C.; Burger, V. T.; Cody, R. B.;
Fujimoto, J. M. J. Pharm. Sci. 1985, 74, 821.
11. Thompson, C. M.; Wojno, H.; Greiner, E.; May, E. L.;
Rice, K. C.; Selley, D. E. J. Pharmacol. Exp. Ther. 2004,
308, 547.
21. Crooks, P. A.; Houdi, A. A.; Kottayil, S. G.; Butterfield,
D. A. U.S. Patent 6,403,602, 2002.