SAR of psilocybin analogs: Discovery of a selective 5-HT2C agonist

Article abstract of DOI:10.1016/j.bmcl.2005.06.104

An SAR study of psilocybin and psilocin derivatives reveals that 1-methylpsilocin is a selective agonist at the h5-HT_2C receptor. The corresponding phosphate derivative, 1-methylpsilocybin, shows efficacy in an animal model for obsessive-compul

Full text of DOI:10.1016/j.bmcl.2005.06.104

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4555–4559
SAR of psilocybin analogs: Discovery of a selective 5-HT2C agonist
Howard Sard,a,* Govindaraj Kumaran,a Cynthia Morency,a Bryan L. Roth,b
Beth Ann Toth,b Ping Hec and Louis Shusterc
aOrganix, Inc., 240 Salem Street, Woburn, MA 01801, USA
bCase Western Reserve University Medical School, 10900 Euclid Avenue, Cleveland, OH 44106, USA
cTufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA
Received 2 June 2005; revised 28 June 2005; accepted 29 June 2005
Available online 2 August 2005
Abstract—An SAR study of psilocybin and psilocin derivatives reveals that 1-methylpsilocin is a selective agonist at the h5-HT2C
receptor. The corresponding phosphate derivative, 1-methylpsilocybin, shows efficacy in an animal model for obsessive–compulsive
disorder, as does 4-fluoro-N,N-dimethyltryptamine. These results suggest a new area for development of novel 5-HT2C agonists with
applications for drug discovery.
Ó 2005 Elsevier Ltd. All rights reserved.
Psilocybin (1), a hallucinogenic component of the sacred
Mexican mushroom Psilocybe mexicana, and its metabo-
lite, psilocin (2), are both potent agonists at the 5-HT2a
and 5-HT2c receptors (Fig. 1). In recent years, several case
reports of the efficacy of psilocybin in the treatment of
obsessive–compulsive disorder (OCD) have been pub-
lished.1 As a result, an FDA-approved clinical trial for pa-
tients suffering from OCD is now underway.2 The
hallucinogenic activity of psilocybin and psilocin is be-
lieved to be largely due to activation of 5-HT2A receptors,
while the anti-OCD activity is associated with agonist
activity at 5-HT2C. Thus, it is believed that a selective
5-HT2C agonist would have considerable potential for
treatment ofOCDandotherindications, suchasobesity.3,4
N(CH3)2
OR
N
H
1: R = P(O)(OH)2
2: R = H
Figure 1.
We examined the influence of structural modification at
a number of sites in the psilocybin structure. The com-
pounds prepared (3–17)8 are shown in Figure 2. Known
N-methylated derivatives 3 and 4 were synthesized
following published procedures.5,9,10 Treatment of 4-
benzyloxyindole with oxalyl chloride and then with dim-
ethylamine gave the 3-substituted oxamide. Amide
reduction (LAH), N-methylation (NaH, MeI), and
hydrogenation (H2, Pd(OH)2) afforded N-methylpsilo-
cin, 3. Phosphorylation followed by debenzylation gave
N-methylpsilocybin, 4. Targets 5, 6, 7,9 8, and 911 were
prepared following the same general route (Scheme 1).
Analogs 10,12 11, and 1213 were obtained similarly upon
treatment of the intermediates formed from 4-ben-
zyloxyindole and oxalyl chloride with the appropriate
secondary amines, followed by LAH reduction and de-
benzylation. The synthesis of 13 followed the reported
We recently began an initial structure–activity relation-
ship (SAR) study of some psilocin and psilocybin deriv-
atives with the goal of obtaining selectivity for the
5-HT2C receptor. Only very limited analog work has
been reported in the psilocybin area,5 and to our knowl-
edge, no pharmacological testing of psilocybin or psilo-
cin derivatives has been published since the discovery of
5-HT2 receptor subtypes.6,7 Although the amino acid
sequence of the 5HT2C receptor has been determined,
little data are available regarding its three-dimensional
structure3 and therefore, our program has initially
relied on an empirical approach.
*
Corresponding author. Tel.: +1 781 932 4142, fax: +1 781 933
6695; e-mail: sard@organixinc.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.104

4556
H. Sard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4555–4559
R2
N(CH3)2
N(CH3)2
10: R2 =
11: R2 =
N
N
N CH3
R
OH
R
R1
N
R1
N
H
N
H
3: R = 4-OH, R1 = CH3
8: R = OH, R1 = CH3
9: R = F, R1 = H
12: R2 =
4: R = 4-OP(O)(OH)2, R1 = CH 3
5: R = 4-OH, R1 = (CH2)3CH3
6: R = 4-OP(O)(OH)2, R1 = (C H2)3CH3
7: R = 6-OH, R1 = H
N
N(CH3)2
CH3
N(CH3)2
OH
OH
OH
(CH2)n
N
N
N
H
CH2CH2N(CH3)2
R
17
16
13: n = 1, R = H
14: n = 1, R = CH3
15: n = 3, R = H
Figure 2.
N(CH3)2
N(CH3)2
HO
N(CH3)2
OH
O
PhCH2
O
P
OCH2Ph
O
OH
a, b
c, d
e,f
N
H
N
H
N
R
N
R
4, 6
3, 5
Scheme 1. Reagents: (a) (COCl)2, (CH3)2NH (71%); (b) LAH (73%); (c) NaH, CH3I (73%), CH3CH2CH2CH2I (48%); (d) H2, Pd/C (3: 78%, 5: 67%);
(e) LDA (PhCH2O)2P(O)OH; (f) H2, Pd/C (4: 12%, 6: 26%).
route14 using a Mannich reaction of formaldehyde and 4-
benzyloxyindole to give the intermediate, 4-benzyloxy-3-
dimethylaminomethylindole, followed by debenzylation.
N-Methylation of 4-benzyloxy-3-dimethylaminomethy-
lindole and debenzylation afforded target 14 (Scheme 2).
Compound 159 was obtained from 4-benzyloxyindole
upon treatment of the magnesium salt with 3-chloropro-
pionyl chloride, to give an intermediate chloroketone.
After reaction with dimethylamine, an intermediate de-
benzylated ketone was the major product isolated. LAH
reduction provided 15 (Scheme 3). A similar sequence
using 2-chloropropionyl chloride afforded 16;9 however,
in this case debenzylation required a separate step
(Scheme 4). Alkylation of 4-benzyloxyindole with N,N-
dimethyl-2-chloroethylamine and debenzylation gave 17.9
N(CH3)2
The receptor binding of these 15 analogs to the seroto-
nin receptor subtypes 5-HT2A, 5-HT2B, and 5-HT2C
was then determined (Table 1).15 Functional assays of
selected compounds were also carried out (Table 2).16
OCH2Ph
OH
a, b (13)
N
H
N
R
or a, c, b (14)
1-Methylpsilocin, 3, displays selective binding at both
the INI and VGI isoforms of the h5-HT2C receptor
(7.0 and 33 nM, respectively) as compared to the h5-
HT2A receptor (900 nM). Functional assays reveal
that 3 is an agonist at both receptor subtypes with
13, 14
Scheme 2. Reagents: (a) (CH3)2NH, H2CO (82%); (b) H2, Pd/C (13:
74%, 14: 73%); (c) NaH, CH3I (34%).

H. Sard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4555–4559
4557
OCH2Ph
OH
OH
COCH2CH2N(CH3)2
b
CH2CH2CH2N(CH3)2
a
N
H
N
H
N
15
H
Scheme 3. Reagents: (a) MeMgI, ClCH2CH2COCl, (CH3)2NH (46%); (b) LAH (12%).
N(CH3)2
N(CH3)2
PhCH2
PhCH2
OH
OCH2Ph
O
O
COCH(CH3)N(CH3)2
c
a
b
N
H
N
H
N
H
N
H
16
Scheme 4. Reagents: (a) MeMgI, CH3CHClCOCl, (CH3)2NH (16%); (b) LAH (27%); (c) H2, Pd/C (55%).
Table 1. Receptor binding (Ki) in nM of psilocybin analogs
Compound
r5-HT2A
h5-HT2A
h5-HT2B
r5-HT2C
h5-HT2C INI
h5-HT2C VGI
33
540 320
14
3
4
590 80
>10,000
200 16
>10,000
>10,000
1683 486
1008 114
6903 1692
8367 1676
ND
900 17
>10,000
38 1.7
5500 3000
5.8 0.5
170 40
6300 3500
ND
48 5.7
>10,000
15.7 0.5
5700 880
19,000 5600
1276 177
359 125
>10,000
1446 312
ND
7.0 1.6
>10,000
4.4 1.1
73 21
6
5
310 30
3000 800
7600 1500
122 37
335 66
9753 4089
ND
4
6
210 70
6800 3000
539 277
84 12
7
>10,000
ND
82 34
8
9
8.39 0.85
116 95
5566 3440
423
10
11
12
13
14
15
16
17
103 63
468 450
275 72
24 0.8
87 22
ND
ND
429 137
923 224
498 185
588 219
982 169
ND
1772 1079
12.6 1.8
125 74
84 64
834 257
ND
ND
1242 295
1242
98 66
49 11
ND
ND
ND
1114 41
126 19
2182 848
ND
>10,000
745 316
ND
ND
4200 788
9351 5551
Data represent means SD of computer-derived affinities from three or more separate experiments; ND, not determined.
Table 2. Functional assay (EC50) in nM of psilocybin and analogs
Compound
5-HT2A
5-HT2B
5-HT2C
1
2
3475 2904 (31 8%)
24 2 (43 17%)
633 1.14 (31 2.9%)
32 29.7 (0.12 0.065%)
Antagonist
74 (24%)
58 (45%)
506 164 (51 3%)
30 18 (51 37%)
12 1.5 (45 5.5%)
3
Inverse agonist
Inverse agonist
Antagonist
5
595 42.5 (83 12.9%)
Antagonist
6
9
13
949 1.04 (49 2%)
Antagonist
1180 316 (38 1.82%)
Antagonist
99 168 (93 49%)
Antagonist
Data represent mean EC50 values for activation of phosphoinositide hydrolysis in cells expressing human 5-HT2A, 5-HT2B or 5-HT2C-INI receptors,
relative to serotonin at 100%. When SD is given N P 3.
considerable selectivity (EC50 at 5-HT2C = 12 nM, EC50
at 5-HT2A = 633 nM). Evaluation of the affinity of 3 for
the 5-HT2B receptor was also carried out, as agonist
activity at 5-HT2B is strongly associated with heart valve
toxicity.17,20 It was gratifying to find that although high
affinity for the 5-HT2B receptor was found (38 nM), a
functional assay revealed that 3 is an inverse agonist
at this receptor subtype. The observed selectivity of
compound 3 for the 5-HT2C receptor proved to be
remarkably sensitive to structural variation. An increase
in the size of the 1-substituent of 3 to n-butyl (5) affor-
ded a compound that was a much weaker agonist at
the 5-HT2C receptor in the functional assay used.21
Modification of the 4-hydroxy group (7), the 2-position
(8), and the diethylamino substituent (10, 11, and 12) all
resulted in much less potent binding at the 5-HT2C
receptor and less selectivity over the 5-HT2A receptor.
However, the 4-fluoro analog
9 showed modest

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H. Sard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4555–4559
functional activity as an agonist at the 5-HT2C receptor
with some selectivity over the 5-HT2A and 5-HT2B
receptors (about 10-fold each). A shortening in the
length of the linker at the 3-position of the indole ring
to a methylene group (13) gave an antagonist at the
5-HT2A and 5-HT2C receptors, while 14 (N-methyl,
methylene linker), 15 (propylene linker), and 16 (a-
methyl) all showed reduced affinity and selectivity for
the 5-HT2C receptor. Finally, 17 (side chain moved to
the 1-position) was also quite inactive in binding at the
5-HT2C receptor. The psilocybin analogs (4, 6) gave very
low binding as compared to their psilocin analogs (3, 5).
However, metabolic dephosphorylation of psilocybin to
psilocin is known to readily occur in vivo22 and thus,
these psilocybin derivatives could act as prodrugs for
their psilocin analogs. In fact, 4 shows improved in vivo
activity as compared to 3 (vide infra, Fig. 3).
sociated scratching in the mice was thought to indicate
their action on 5-HT receptors, and a study was devised
as an animal model for OCD.25 Selected results are
shown in Figure 3. Psilocybin strongly inhibits scratch-
ing in this model, and more so than psilocin; however,
psilocin shows much greater functional activity at the
5-HT2C receptor. This difference may be due to active
transport of psilocybin across the blood–brain barrier
prior to dephosphorylation. N-Methyl derivatives 3
and 4 show comparable activity, but only at higher con-
centrations. Compound 4, like psilocybin, dramatically
inhibits scratching. N-Butylpsilocin, 5, however, is near-
ly inactive, in contrast to 3. Thus, positive effects seen in
this mouse model are consistent with 5-HT2C agonist
activity. Finally, 4-fluoro-N,N-dimethyltryptamine, 9,
also exhibits strong activity, which is comparable to that
of psilocybin and compound 4, despite displaying only
modest agonist activity at the 5-HT2C receptor
(99 nM).26 One possible explanation for these results is
that relatively lipophilic 9 may more readily penetrate
the blood–brain barrier, and thus affording the observed
in vivo activity.
We next examined selected analogs in a mouse model for
OCD. Serotonin produces an itching sensation when
applied to the human skin and has been suggested to
be involved in pruritic diseases. Further research dem-
onstrated that an ip injection of 5-HT into the rostral
back of the mouse elicits scratching with the hind paws,
which is itch-associated rather than a pain response.23
The 5-HT action is at least partly mediated by 5-HT2
receptors in the skin, as shown by blocking with specific
antagonists.24 Psilocin and its analogsÕ effect on itch-as-
Potent and selective 5-HT2C agonists may have applica-
tion in other therapeutic areas besides OCD, including
appetite suppression,7 AlzheimerÕs disease,27 and epilep-
sy.28 Further studies are underway to more fully develop
this class of compounds.29
1600
1400
1200
1000
800
600
400
200
0
a
b
#
1000
#
#
800
#
control
psilocybin
0.5mg/kg
600
400
200
0
control
#
psilocin 0.5mg
*
5
10
15 20
25
30
5
5
5
10 15 20 25 30
Time after 5-HT(min)
Time after 5-HT(min)
c
800
600
400
200
0
2000
1500
1000
500
0
d
#
#
#
control
control
*
#
compound 4
5mg/kg
compound 3
5mg/kg
*
#
*
5
10 15 20 25 30
10 15 20 25 30
Time after 5-HT(min)
Time after 5-HT(min)
e
1000
800
600
400
200
0
f
2000
1500
1000
500
0
#
#
#
control
control
compound 5
5mg/kg
#
compound 9
5mg/kg
#
*
10 15 20 25 30
5
10 15 20 25 30
Time after 5-HT(min)
Time after 5-HT(min)
Figure 3. Inhibition of serotonin-induced scratching by psilocybin and analogs (a) N = 7, (b) N = 5, (c) N = 12, (d) N = 8, (e) N = 5, (f) N = 5;
#P < 0.01, *P < 0.05.

H. Sard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4555–4559
4559
overnight in inositol-free DMEM without serum. The next
day, [3H]inositol phosphate accumulation assays were
performed in a modified Krebs-bicarbonate buffer. Kact
(nmol/L) and percent Vmax (relative to 5-HT) values were
calculated.18,19
Acknowledgments
This work was partially supported by grants from the
National Institute of Mental Health (1R43MH63529
to H.S. and K02MH01366 to B.L.R.).
17. Rothman, R.; Baumann, M.; Savage, J.; Rauser, L.;
McBride, A.; Hufeisen, S.; Roth, B. L. Circulation 2000,
102, 2836.
References and notes
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Pharmacol. 1997, 52, 259.
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30, 359.
19. Roth, B. L.; Choudhary, M.; Khan, N.; Uluer, A.
J. Pharmacol. Exp. Ther. 1997, 280, 576.
2. Private communication from Dr. Francisco Moreno,
University of Arizona, Tucson.
3. Roth, B. L.; Shapiro, D. Expert Opin. Ther. Targets 2001,
5, 685.
4. Vickers, S.; Clifton, P.; Dourish, C.; Tecott, L. Psycho-
pharmacology (Berlin) 1999, 143, 309.
5. Hofmann, A.; Heim, R.; Brack, A.; Kobel, H.; Frey, A.;
Ott, H.; Petrzilka, Th.; Troxler, F. Helv. Chim. Acta 1959,
42, 1557.
20. Fitzgerald, L.; Burn, T.; Brown, B.; Patterson, J.; Corjay,
M.; Valentine, P.; Sun, J.-H.; Link, J.; Abbaszade, I.;
Hollis, J.; Largent, B.; Hartig, P.; Hollis, G.; Meunier,
P.; Robichaud, A.; Robertson, D. Mol. Pharmacol. 2000,
57, 75.
21. In recent preliminary results, analogs of compounds 3 and
5 with N-substitution intermediate in size between methyl
and n-butyl also show selective agonism at the 5-HT2C
receptor.
6. Cerletti, A.; Taeschler, M.; Weidmann, H. Adv. Pharma-
col. B 1968, 6, 233.
7. Fitzgerald, L.; Ennis, M. In Annual Reports in Medicinal
Chemistry; Doherty, A., Ed.; Academic Press: New York,
2002; Vol. 37, pp 21–30.
22. Jacob, P., III; Shulgin, A. T. In Lin, G. C., Glennon, R.
A., Eds.; NIDA Research Monograph 146 (Hallucino-
gens, an Update); 2000, p 74.
23. Kuraishi, Y.; Nagasawa, T.; Hayashi, K.; Satoh, M. Eur.
J. Pharmacol. 1995, 275, 229.
8. Compounds 3–17 were homogeneous on TLC, displayed
the expected H NMR and mass spectral data, and gave
24. Yamaguchi, T.; Nagasawa, T.; Satoh, M.; Kuraishi, Y.
Neurosci. Res. 1999, 35, 77.
1
satisfactory combustion analyses.
9. Troxler, F.; Seeman, F.; Hofmann, A. Helv. Chim. Acta
1959, 42, 2073.
10. Nichols, D. E.; Frescas, S. Synthesis 1999, 935.
11. Bentov, M.; Pelchowicz, Z.; Levy, A. Isr. J. Chem. 1964, 2,
25.
12. Repke, D.; Ferguson, W.; Bates, D. J. Heterocycl. Chem.
1981, 18, 175.
13. Repke, D.; Ferguson, W.; Bates, D. J. Heterocycl. Chem.
1977, 14, 71.
25. The subjects were male Swiss–Webster mice, 4–6 weeks
old, weighing 25–45 g. Mice were housed five per cage,
given free access to standard mouse food and water
except during experiments, and maintained in a temper-
ature-controlled room (70 °F). Serotonin and all test
drugs were made up with ascorbic acid (1 mg/mL) to
protect against oxidation. Two mice, one a control and
the other experimental, were tested each time. Each
mouse was separately placed into a plexiglas box. The
control mouse was injected with 10 mg/kg ascorbic acid
in saline solution (0.9% NaCl) intraperitoneally (ip). For
the experimental mouse, test compounds in saline plus
ascorbic acid were injected ip. After 5 min, 0.1 mL of
0.4 mg/mL of serotonin in ascorbic acid in saline solution
was injected subcutaneously (sc) to the rostral back of
each mouse. After the injection of serotonin, the cumu-
lative number of scratches was recorded every 5 min for
30 min.
26. In other experiments, psilocin produced significant inhi-
bition of scratching at a dose of 0.85 mg/kg. Compound 9
at 2 mg/kg was not active.
27. Wurtman, R.; Arjona, A.; Anibal, A.; Lee, R.; Pooler, A.
Brain Res. 2002, 951, 135.
28. Isaac, M. Curr. Top. Med. Chem. 2005, 5, 59.
29. Compounds 3 and 9 bind only at micromolar levels to the
dopamine D2, D3, and D4 receptors (data not shown).
30. Choudhary, M.; Craigo, S.; Roth, B. Mol. Pharmacol.
1992, 42, 627.
14. McCormick, K.; Kobayashi, K.; Goldin, S.; Reddy, N.;
Meinwald, J. Tetrahedron 1993, 49, 11155.
15. 5-HT2A and 5-HT2C radioligand binding assays were
performed using [3H]ketanserin and [3H]mesulergine,
respectively.30 5-HT2B radioligand binding assays were
3
performed using H-LSD.31 For initial screens, 10 lM of
each compound (dissolved in 10% DMSO) was incubated
with the appropriate receptor preparation and percent
inhibition was determined for duplicate determinations
each performed in duplicate. Where >50% inhibition of
specific binding was measured, Ki determinations were
then measured by competition binding assays in which
concentrations from 1 to 100,000 nM were incubated in
duplicate. For each Ki value, the data represent
means SD of computer-derived estimates for N = 4
separate determinations, as previously detailed.17–19
16. Phosphoinositide hydrolysis assays were performed with
stably (5-HT2A, 5-HT2C) or transiently (5-HT2B) expressed
receptors plated in 24-well culture plates. Transfected cells
were loaded with [3H]inositol (15 Ci/mmol, 1 mCi/mL)
31. Setola, V.; Dukat, M.; Glennon, R.; Roth, B. Mol.
Pharmacol. 2005, 68, 20.