An Improved, Practical, and Scalable Five-Step Synthesis of Psilocybin

Article abstract of DOI:10.1055/s-0039-1691565

Described herein is an improved synthesis of 3-[2-(dimethylamino)ethyl]-1 H -indol-4-yl dihydrogen phosphate (psilocybin). The protocol outlines: synthesis of multigram quantities of psilocybin, identification of critical in-process parameters, and isolation of psilocybin without the use of chromatography, TLC, or aqueous workup. The synthesis furnishes psilocybin in five steps in 23percent overall yield from an inexpensive acetoxyindole starting material. With specific focus on process control and impurity fate and removal, the improved procedure is amenable to providing high-quality psilocybin.

Full text of DOI:10.1055/s-0039-1691565

SYNTHESIS
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X
© Georg Thieme Verlag Stuttgart · New York
2020, 52, A–G
psp
A
en
A. M. Sherwood et al.
PSP
Synthesis
An Improved, Practical, and Scalable Five-Step Synthesis of Psilo-
cybin
Alexander M. Sherwood
Poncho Meisenheimer
Gary Tarpley
O
N
P
HO
O
OH
Robert B. Kargbo*
0
0
0
0-
0
0
0
2-5
5
3
9-6
3
4
3
N
Usona Institute, 2780 Woods Hollow Road, Madison,
WI 53711, USA
H
No chromatography, TLC
or aqueous work-up
Gramsscale reactions
Psilocybin
(Magic mushroom)
robert.kargbo@usonainstitute.org
Received: 02.11.2019
Accepted after revision: 12.12.2019
Published online: 08.01.2020
duce a large antidepressant effect that may last up to six
months.7 Rates of adherence to psilocybin-assisted therapy
when administered in supervised and monitored in clinical
settings are relatively high compared to standard therapy.8
DOI: 10.1055/s-0039-1691565; Art ID: ss-2019-n0488-psp
Abstract Described herein is an improved synthesis of 3-[2-(dimethyl-
amino)ethyl]-1H-indol-4-yl dihydrogen phosphate (psilocybin). The
protocol outlines: synthesis of multigram quantities of psilocybin, iden-
tification of critical in-process parameters, and isolation of psilocybin
without the use of chromatography, TLC, or aqueous workup. The syn-
thesis furnishes psilocybin in five steps in 23% overall yield from an inex-
pensive acetoxyindole starting material. With specific focus on process
control and impurity fate and removal, the improved procedure is
amenable to providing high-quality psilocybin.
N
O
N
H
+ N
O
N
N
P
O–
H
HO
O
O
N
N
N
H
H
H
HN
1
2
3
4
Psilocybin
Lysergic acid
diethylamide
(LSD)
N,N-Dimethyltrptamine 5-Methoxy-N,N-dimethyltryptamine
(DMT)
(5-CH3O-DMT)
Key words psilocybin, psilocin, phosphorylation, hydrogenolysis, re-
duction, quenching method, major depressive disorder
Figure 1 Representative structures of indole-based psychedelics
Psilocybin (1) is a psychoactive natural product found in
polyphyletic group of fungi that has been used as an en-
theogen and hallucinogenic drug.1 When ingested, psilocy-
bin (1) functions as a prodrug that is rapidly dephosphory-
lated to psilocin (7).2 Psilocin, which is the pharmacologi-
cally active ingredient is structurally related to a number of
similar compounds depicted in Figure 1 such as lysergic
acid diethylamide [LSD (2)], N,N-dimethyltryptamine [DMT
(3)], and 5-methoxy-N,N-dimethyltryptamine [5-CH3O-
DMT (4)], which are all known to induce similar psychoac-
tive effects in humans. A common feature to all of these
compounds is their capacity to occasion mystical experi-
ences, which has been correlated with positive therapeutic
outcomes.3 Since the 1960s when Sandoz laboratories sup-
plied psilocybin, the psychiatric community has reported
numerous studies into the therapeutic use of psilocybin.4 In
particular, studies linking psilocybin and activity in seroto-
nergic neurons5 and the sensory cortex in general suggest-
ed potential implications for treating depression.6 In con-
trast to standard antidepressants, which are often taken
daily, a single dose of psilocybin has been reported to pro-
The prevalence of depression has risen in every coun-
try9 and major depressive disorders (MDD) is currently the
leading cause of disability worldwide.10 An estimated 60–
70% of suicides in the United States are from people consid-
ered clinically depressed.11 Suicide is one of the top ten
causes of death in the United States.12 This troubling statis-
tic is exacerbated, in part, by opioid addiction, drug over-
doses, and MDD. The current treatment for addiction, de-
pression, and anxiety often fail to affect a meaningful quali-
ty of life. Consequently, the increase in anecdotal and
scientific reports suggesting that psychedelics have thera-
peutic potential in treatment of these indications have gar-
nered interest in pursuing regulatory changes to enable fur-
ther research of their clinical use.13
To further scientific and clinical research on psychedel-
ics, we embarked on clinical development of psilocybin for
the treatment of MDD in participants considered medically
healthy and in patients with potentially life-threatening
cancer diagnosis. As a result, a practical large-scale
synthesis of 1 was needed. Though there were literature
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–G
Georg Thieme Verlag KG, Rüdigerstraße 14, 70469 Stuttgart, Germany

B
A. M. Sherwood et al.
PSP
Synthesis
precedents that described the synthesis of 1, it became crit-
ical to modify the procedures in order to provide sufficient
quantities of compound with characterization data and
quality to meet strict regulatory requirements and support
clinical operations.
obtain 7 with subsequent phosphorylation by TBPP. When
benzylated compound 9 was subjected to hydrogenolysis,
compound 1 was obtained cleanly.
Starting from acetoxyindole (5), we found that con-
trolling residual oxalyl chloride in the initial acylation reac-
tion was crucial for the stability of intermediate 10 (Scheme
2). In the presence of traces of oxalyl chloride, intermediate
10 decomposes rapidly, which was undesirable for a large-
scale campaign where storage of intermediates may last
from few hours to days. Additionally, removal of excess ox-
alyl chloride mitigates a side reaction during the dimethyl-
amine addition, which gave a tetramethyloxamide side-
product along with compound 6, which was difficult to re-
move during purification. Furthermore, the presence of te-
tramethyloxamide during the lithium aluminum hydride
(LiAlH4) reduction of compound 6 to compound 7, caused
excess consumption of LiAlH4, which led to incomplete re-
action and by-products. Using optimized conditions, we
found thorough washing of the glyoxal chloride with hep-
tane/MTBE gave clean yellow solid 10 that was stable and
stored for several days without decomposition.
To date, several syntheses of 1 have been reported.14
Most commonly, the classical Speeter–Antony tryptamine
synthesis (STS) has been used to produce psilocin (7) reli-
ably.15 The subsequent phosphorylation of psilocin (7) has
been historically problematic, owing in part to its labile na-
ture. The use of a benzyl-protected phosphorylating re-
agent and deprotection has been demonstrated in all previ-
ous literature reports. Hofmann first reported the use of
O,O-dibenzylphosphoryl chloride in the penultimate phos-
phorylation step. More recently, Nichols alternatively used
tetrabenzyl pyrophosphate (TBPP), citing various undesir-
able properties of O,O-dibenzylphosphoryl chloride.16 Fur-
thermore, refinement of the Nichols’ procedure by Shirota
was published several years later.17 In this report, Shirota
identified and characterized an unexpected rearrangement
product formed by the migration of the O-benzyl group to
the dimethylamine functionality (Scheme 1) resulting in a
zwitterionic isomer. The limited solubility of zwitterionic
intermediate 9 allowed isolation by trituration and simple
filtration, which circumvented a difficult chromatographic
purification at this step.
Cl
N
O
O
OAc
OAc
OAc
oxalyl
chloride
O
O
2 M Me2NH in THF
pyridine
Et2O, 0 °C–rt, 3 h
N
N
N
H
H
H
5
10
6
N
N
O
Scheme 2 Synthesis of 3-[2-(dimethylamino)-2-oxoacetyl]-1H-indol-
4-yl acetate (6)
OAc
OAc
OH
O
1. Acylation
Reduction
N
N
N
H
2. Dimethyl
addition
H
H
In the reaction of dimethylamine with glyoxal chloride
10, ketoamide 6 was conveniently isolated directly from the
reaction mixture as a crude solid, which was reslurried
with water to remove pyridine salts, followed by heptane
and ethyl acetate to remove any additional organic impuri-
ties. This process ultimately provided 6 in over 80% yield
and 96% purity from 4-acetoxy indole (5). In this form, 6
has been stored for up to several months under ambient
conditions with no apparent degradation. Though this ma-
terial was of sufficient purity to be used in subsequent
steps, 6 could be further purified by recrystallization from
isopropyl alcohol to afford 70% of a crystalline solid in >99%
purity. Compound 6 displays the characteristics of possibly
appropriate starting material in current Good Manufactur-
ing Practices (cGMP) because it is readily purifiable, highly
stable, and easily characterizable.
Initial attempts at reduction of compound 6 using
LiAlH4 in refluxing THF for 2 hours showed a single chro-
matographic peak by LCMS with the expected protonated
product mass (205), but also an intense signal with 203
(Scheme 3). Attempted isolation of product from this reac-
tion gave an impure material with only low levels of expect-
ed signals for compound 7 detectable by NMR. Upon further
investigation using analytical HPLC with an optimized
5
6
7
Phosphorylation
O
O
P
O–
O
P
+ N
N
N
Bn
P
Bn
HO
BnO
O
O
O
O
Benzyl
Debenzylation
HO
BnO
migration
Recrystallization
N
N
N
H
H
H
1
9
8
Scheme 1 General synthetic approaches to psilocybin (1)
In our hands, exact reproduction of the procedure by
Shirota fell short of the expected yield and purity. Conse-
quently, we have developed a suitable procedure, which
synthesized sufficient quantities of 1 in high purity (99.9%)
to support our clinical operations. This report describes po-
tential problems in each step and defines critical process
parameters that inform large-scale synthesis of the final ac-
tive pharmaceutical ingredient (API, 1) in high purity with-
out chromatographic purification.
The general synthetic approach to 1 remained consis-
tent with the previously reported route (Scheme 1), utiliz-
ing the classical STS approach from 4-acetoxyindole (5) to
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–G

C
A. M. Sherwood et al.
PSP
Synthesis
gradient, an intermediate with similar retention was de-
tected alongside 7. Under prolonged refluxing in THF (>24
h), this intermediate eventually disappeared and a signifi-
cant amount of product 7 was seen. Due to stability con-
cerns, prolonged refluxing in THF was ultimately undesir-
able. Attempts to add excess LiAlH4 during the reaction to
affect completion was unfruitful. Fortunately, 2-methyltet-
rahydrofuran (2-CH3-THF), with a higher boiling point com-
pared to THF, was found to be an acceptable substitute sol-
vent. With good in process control (IPC) by HPLC for reac-
tion monitoring in place, it was determined that after
LiAlH4 addition at 0 °C and subsequent warming to ambient
temperature, only the major intermediate and approxi-
mately 5% of 7 was present in the reaction mixture. The re-
action was intentionally stalled at this stage to isolate the
intermediate. We found the intermediate impurity to pref-
erentially retain on silica gel and aluminum cake compared
to 7. We isolated and analyzed the intermediate by 1H and
13C NMR and found it to be the -hydroxy compound 11
(Scheme 3).
than a few hours led to decomposition of the product. It
was found that a careful wash with 7 N NH3 in methanol
and DCM was sufficient to free the product from the alumi-
num salts in the filter cake. With this protocol (Procedure
A), compound 7 was obtained in high yield and purity, but
was found to darken quickly despite retaining purity by
HPLC.
An alternative quench protocol employing wet THF and
silica gel (Procedure B) was later identified to be superior to
the Fieser process. Using this protocol, 7 was obtained by
simple filtration as a white solid of higher purity compared
to material isolated by the Fieser workup, which typically
required reslurry to obtain higher yield.
The next step of the phosphorylation-rearrangement
protocol in the conversion of 7 into 9 via intermediate 8,
originally described by Shirota was not consistently repro-
ducible on scale. Careful monitoring of the reaction prog-
ress by LCMS rather than TLC16 was advantageous in estab-
lishing conditions for this reaction that were reproducible
and scalable. With the proper IPC in place, it became possi-
ble to accurately monitor reaction progress and side reac-
tions. Careful control of the temperature profile of the reac-
tion was found to be most critical in the smooth conversion
of 7 into 9 while minimizing unwanted side reactions given
the highly reactive nature of both the tetrabenzyl pyro-
phosphate (TBPP) and the lithium salt of 7 (Scheme 4).
N
N
O
HO
OAc
HO
O
LiAlH4, 2-CH3-THF
0 °C–rt
N
N
H
H
6
11
O
O
P
+ N
N
N
N
Bn
P
O–
Bn
N
Bn
O
O
HO
OH
O
O
nBuLi, TBPP
O
DCM
HO
reflux
Bn
THF
reflux
–78 to –20 °C
N
N
H
N
N
H
H
H
N
8
7
12
9
7
Scheme 4 Synthesis of zwitterionic intermediate 9
Scheme 3 Synthesis of key psilocin intermediate 7
The detected mass signal of 203 (12) from the LCMS
analysis of 7 in the crude reaction mixture could be ex-
plained by possibly the in-source elimination of the rela-
tively labile hydroxyl group (Scheme 3) during the LCMS
run. Furthermore, this elimination process may represent
the most likely rate limiting step in the conversion of 11
into 7, given the difficulty of LiAlH4 directly reducing a hy-
droxyl group. Refluxing for 3 hours in the higher-boiling 2-
CH3-THF solvent was sufficient to give a conversion of >95%
by HPLC. This was a significant breakthrough that led to
compound 7 in purity of >99% after the requisite workup
procedure. In our hands, compound 7 was found to be un-
stable if not isolated cleanly in a timely manner as the re-
sidual byproduct from the LiAlH4 reduction may contribute
to its decomposition.
We found that holding the reaction at –78 °C during bu-
tyllithium addition followed by TBPP addition and allowing
the mixture to stir for 1.5 hours prior to temperature rise to
–20 °C led to clean conversion. The reaction was quenched
with ethyl acetate at –20 °C rather than the reported 0 °C,
and filtration of the precipitated inorganic solids drowned
out compound 8 in the filtrate. The intermediate 8 was sus-
pended in DCM and heated with heat gun for 5 minutes and
then slowly cooled to room temperature prior to being held
at 0 °C. This process led to complete conversion of 8 into the
zwitterionic compound 9. On the other hand, in earlier trial
reactions where impure intermediate 8 was utilized, the re-
arrangement reaction gave incomplete conversions into 9
and took several days to accomplish. Furthermore, if the hot
solution was quickly cooled to room temperature, a suspen-
sion with very small particle size was obtained, which pro-
longed filtration time. We obtained complete conversion to
crude solid 9, and further trituration with DCM afforded
pure compound 9. The color of compound 9 was affected by
traces of compound 7, which is prone to oxidation and can
Several standard quenching protocols for the LiAlH4 re-
ductions were attempted. The Fieser workup18 gave higher
purity, however, recovery of the product was very low and
much of the material became trapped in the filter cake. Fur-
thermore, holding the product on the filter cake for more
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–G

D
A. M. Sherwood et al.
PSP
Synthesis
give variable color. In addition, excess TBPP was detrimental
to the reaction as it produced phosphate by-products, in-
cluding dibenzyl phosphate (DBP) that not only impeded
conversion into 9 but also affected other downstream pro-
cesses.
emerged as the most attractive. Zwitterionic 1 was virtually
insoluble in acetone, which had the additional benefit of
also solubilizing minor nonpolar impurities.
Precipitation of 1 from the aqueous reaction mixture
using acetone typically provided a semi-crystalline solid in
about 90% recovery and 97–98% purity. Encouraged by the
anti-solvent properties of acetone and given the moderate
solubility of 1 in pure water, we investigated the solvent
pair as a recrystallization solvent mixture. It was deter-
mined that 80 volumes of a mixture of 30% water in acetone
could completely solubilize 1 at reflux. Upon slowly cooling
back 0 °C over 6 hours, large colorless crystals formed to
provide 1 in 70–80% recovery; HPLC purity analysis of this
crystalline solid was typically >99%. Finally, to provide ma-
terial suitable for clinical use that is free from any inorganic
by-products or trace solvent residues, a final recrystalliza-
tion using pure water was employed, which typically recov-
ered 50–60% product. Full certificate of analysis of this ma-
terial indicated no detectible impurity.
In summary, we have shown the investigative large-
scale synthesis of psilocybin with the goal of identifying
and addressing in-process control and critical process pa-
rameters like temperature, appropriate solvent, base and so
forth, for each step in the synthesis. Furthermore, identifi-
cation of critical parameters enabled complete conversions
of the reactions with minimal side or by-products, which
allowed simple purification of product via trituration, fil-
tration, and recrystallization techniques. Finally, the reli-
able synthesis of compound 7 provides an attractive combi-
nation of synthetic/biocatalysis, in which the direct enzy-
matic phosphorylation of 7 to psilocybin (1) is envisioned.19
Such synthetic/biocatalytic investigation is ongoing and the
report will be presented in due course.
Furthermore, we examined the timing of the reaction,
which required several hours from –78 °C to –20 °C, based
on our initial findings. We questioned why the very reactive
reactants would require such a lengthy timing for comple-
tion. To investigate that question, we removed the dry-ice
bath after the addition of the TBPP and then rinsed out the
dry-ice coating around the round-bottomed flask. We ob-
served suspension of the TBPP at –78 °C, which was indica-
tive of partial solubility of the TBPP at that temperature. We
monitored the reaction as it warms up relatively quickly
and also removed any ice buildup around the round-bot-
tomed flask. The disappearance of the suspended TBPP cor-
related to the conversion into 8. At –50 °C, when we ob-
served no traces of suspended TBPP, the reaction was com-
plete in less than 30 minutes. This implies that a solution of
TBPP can be added at –50 °C rather than addition of solid
TBPP according literature precedents. Controlled addition
of substrate is always a preferred protocol on scale, if possi-
ble, since it can minimize isothermic reactions. More so,
addition of a solution of TBPP makes the protocol amenable
to flow chemistry, where both reactants could come in at –
50 °C and then a quench line with EtOAc could safely deliver
the desired product.
With pure 9 in hand, catalytic hydrogenolysis of 9 to 1
was relatively straightforward. Catalytic hydrogenolysis of 9
was done using 10 wt% Pd/C catalyst under a balloon of hy-
drogen (1 atm). The reaction was typically complete in 30
minutes (Scheme 5). Compound 1 had low solubility in
methanol and during the reaction precipitated from the re-
action mixture. Prior to filtration of the catalyst, dilution of
the reaction mixture with water helped to solubilize the
product.
Reactions were performed using commercially obtained solvents. Un-
less otherwise stated, all commercially obtained reagents were used
as received. 1H and 13C NMR spectra were recorded on a Varian Mer-
cury 400 (at 400 MHz and 100 MHz, respectively) and are reported
relative to internal CHCl3 (1H,  = 7.26), DMSO-d6 (1H,  = 2.50),
O
O
P
+
N
N
Bn
P
O–
Bn
HO
O
O
O
HO
CD2HOD (1H,  = 3.31), and CDCl3 13C,  = 77.0), DMSO-d6 13C,  =
( (
1. Pd/C, CH3OH, H2
39.5), CD3OD (13C,  = 49.0). Data for 1H NMR spectra are reported as
follows: chemical shift ( ppm) [multiplicity, coupling constant (Hz),
integration]. Standard multiplicity and qualifier abbreviations are
used. High-resolution mass spectra were obtained on AB Sciex Triple-
TOF 5600+. Preparative HPLC was performed with Waters 2535 Qua-
ternary Gradient Module utilizing XBridge PREP C18 Column 5 m
(30 mm × 250 mm). Analytical HPLC was performed with an Agilent
1100 Series HPLC utilizing Phenomenex Synergi™ 2.5 m MAX-RP
100Å columns (4.6 mm × 50 mm).
2. Recrystallization
(acetone/water)
N
N
H
H
9
1
Scheme 5 Synthesis of 3-[2-(dimethylamino)ethyl]-1H-indol-4-yl dihy-
drogen phosphate [psilocybin (1)]
As part of the development of the large-scale manufac-
ture of 1, isolation processes that avoid concentration of
solutions to dryness was preferable. This is due to ease of
transfer and secondly, stability data indicated degradation
of psilocybin (1) to psilocin (7) under prolonged thermal
condition in solution. A series of water-miscible anti-sol-
vents were screened that could be used to precipitate 1
from the aqueous solution. Of the solvents tested, acetone
3-[2-(Dimethylamino)-2-oxoacetyl]-1H-indol-4-yl Acetate (6)
A 2000 mL, four-necked, round-bottomed flask was equipped with an
overhead stirrer, J-Kem temperature controller, a 250 mL dropping
funnel, and rubber septum through which a positive pressure of dry
N2 was inserted. The septum was removed and the flask was charged
sequentially with 1H-indol-4-yl acetate (5; 50.1 g, 285 mmol, 1 equiv)
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–G

E
A. M. Sherwood et al.
PSP
Synthesis
and anhyd Et2O (700 mL). The flask was resealed with the septum and
is flushed with N2. The suspension was stirred for 10 min and then
cooled to 0 °C in an ice-water bath for 30 min. The dropping funnel
was charged with a solution of oxalyl chloride (37.1 mL, 428 mmol,
1.5 equiv) in Et2O (60 mL). The oxalyl chloride solution was added
dropwise at a rate sufficient to keep the temperature at or below 5 °C,
to minimize formation of dimer and other possible by-product. As the
addition progressed, a yellow slurry of 10 was formed and when the
addition was completed, the mixture was stirred for 4 h. The comple-
tion of the reaction was assayed by heating an aliquot of the reaction
mixture in MeOH and checking by LCMS the conversion of compound
10 into the corresponding methyl ester [3-(2-methoxy-2-oxoacetyl]-
1H-indol-4-yl acetate]. After this time, heptane (400 mL) was added
and the mixture stirred for 30 min at 0 °C. The yellow solid obtained
was quickly filtered and rinsed successively with heptane (2 × 300
mL), which was quickly used in the next step. Rinsing the solid with
heptane removes excess oxalyl chloride, which prevents the forma-
tion of tetramethyloxalylamide in the formation of 6. Tetramethyl-
oxalylamide becomes a detrimental impurity, which is difficult to re-
move in the synthesis of 6. Compound 10 was quickly dissolved in
THF (500 mL) and cooled to 0 °C. A 2.0 M solution of dimethylamine
in THF (175 mL) was added dropwise at a rate sufficient to maintain
temperature below 5 °C in order to minimize side reactions. After the
addition was complete, pyridine (46 mL) in of THF (100 mL) was add-
ed dropwise and the mixture was stirred well for 60 min. Heptane
(600 mL) was added and the flask contents were suction filtered via a
Büchner funnel. The filtered residue was transferred into a round-
bottomed flask and deionized H2O (1000 mL) was added, stirred for
30 min, and filtered via Büchner funnel. The off-white solid was tritu-
rated sequentially for 40 min in EtOAc (600 mL) and heptane (400
mL). The slurry was filtered via Büchner funnel and the solid was
dried in an oven at 40 °C overnight to afford 6 as a light-yellow solid;
yield: 66.1 g (81%); mp 205–207 °C.
The reaction mixture was assayed by analytical HPLC, which showed
completion of >90% to product peak 7. The heating mantle was re-
moved, and the flask was allowed to cool to 50 °C. The flask was again
chilled to 20 °C. The reaction was quenched by sequential addition of
3 drops of aq 1 M NaOH and 3 drops of deionized H2O. The mixture
was diluted with THF (500 mL) and stirred for 20 min. The mixture
was filtered via Büchner funnel and the filtrate was kept under N2.
The filter cake was quickly reslurried with 200 mL of [10% solution of
(7% ammonia in MeOH) in CH2Cl2] and THF (500 mL). The filtrates
were then combined and concentrated to give a green solid. The solid
was triturated with 1:1 EtOAc/heptane (50 mL), then filtered via
Büchner funnel. The dark green solid was dried in an oven at 40 °C
overnight to provide dry psilocin (7) as a dark green solid; yield: 20.7
g (91%); mp 167–169 °C.
IR (KBr): 3398, 3264, 2952, 1574, 1467, 1353, 1261, 1041, 736 cm–1
.
1H NMR (400 MHz, CDCl3):  = 13.60 (s, 1 H), 8.05 (s, 1 H), 7.08 (t, J =
7.8 Hz, 1 H), 6.91–6.82 (m, 2 H), 6.60 (d, J = 7.7 Hz, 1 H), 3.01–2.94 (m,
2 H), 2.76–2.69 (m, 2 H), 2.40 (s, 6 H).
13C NMR (100 MHz, CDCl3):  = 152.12, 139.07, 123.54, 120.88,
117.52, 114.56, 106.46, 102.43, 61.69, 45.35, 25.19.
HRMS (ESI): m/z calcd for C12H16N2O (M + H)+: 205.1335; found:
205.1338.
Compound 11 (-Hydroxypsilocin)
Intermediate 11 was isolated by performing essentially the same re-
action sequence described for compound 7 on a smaller scale except
it was stirred at rt instead of reflux for 4 h. Following quench, the fil-
trate was concentrated, and the residue was purified by flash column
chromatography (SiO2, 100:10:1 CH2Cl2/CH3OH/NH4OH) to provide 11
as a gray solid; mp 172–174 °C.
IR (KBr): 3374, 3257, 1468, 1359, 1242, 1052, 1001, 739 cm–1
.
1H NMR (400 MHz, DMSO-d6):  = 10.79 (s, 1 H), 7.10 (d, J = 2.4 Hz, 1
H), 6.88 (t, J = 7.7 Hz, 1 H), 6.80 (dd, J = 8.0, 1.1 Hz, 1 H), 6.32 (dd, J =
7.5, 1.0 Hz, 1 H), 5.04 (dd, J = 7.6, 5.0 Hz, 1 H), 3.34 (s, 1 H), 2.58 (dd,
J = 12.5, 7.6 Hz, 1 H), 2.45 (dd, J = 12.5, 5.0 Hz, 1 H), 2.24 (s, 6 H).
13C NMR (100 MHz, DMSO-d6):  = 150.83, 139.01, 122.74, 120.50,
117.25, 116.10, 103.98, 103.26, 67.93, 65.88, 46.18.
IR (KBr): 3236, 1738, 1625, 1428, 1412, 1362, 1217, 854, 772 cm–1
.
1H NMR (400 MHz, CDCl3):  = 10.63 (s, 1 H), 7.53–7.47 (m, 1 H), 7.21–
7.11 (m, 1 H), 7.07 (d, J = 8.1 Hz, 1 H), 6.93 (d, J = 7.7 Hz, 1 H), 3.02 (d,
J = 1.4 Hz, 3 H), 2.92 (d, J = 1.4 Hz, 3 H), 2.51 (s, 3 H).
13C NMR (100 MHz, CDCl3):  = 185.38, 170.91, 168.47, 144.26,
139.23, 138.22, 124.72, 118.25, 115.99, 113.44, 110.83, 37.45, 34.20,
21.55.
HRMS (ESI): m/z calcd for C12H16N2O2 (M + H – H2O)+: 203.1179;
found: 203.1186.
HRMS (ESI): m/z calcd for C14H14N2O4 (M + H)+: 275.1026; found:
Procedure B: The reduction step was carried out using essentially the
same protocol described in Procedure A with 6 (40.21 g, 135.2 mmol)
and 2.3 M LiAlH4 in 2-CH3-THF (188.1 mL, 432.5 mmol). The reaction
was quenched by dropwise addition of THF/H2O (27:100, 50 mL) at a
rate that kept the temperature below 30 °C. Anhyd Na2SO4 (100 g) was
added followed by silica gel (50 g) and DCM (400 mL). The mixture
was stirred for 10 min and was filtered via Büchner funnel. The filter
cake was washed with DCM/CH3OH mixture (9:1, 1500 mL). The fil-
trates were then combined and concentrated to give a light green sol-
id. The solid was triturated with 1:1 EtOAc/heptane (50 mL), then fil-
tered via Büchner funnel. The off-white solid was dried in an oven at
40 °C overnight to provide dry psilocin (7) as an off-white solid; yield:
21.6 g (77%); 99.2% HPLC purity.
275.1028.
3-[2-(Dimethylamino)ethyl]-1H-indol-4-ol (Psilocin, 7)
Procedure A: A 2000 mL, four-necked, round-bottomed flask was
equipped with an overhead stirrer, J-Kem temperature controller, a
250 mL dropping funnel, and rubber septum through which a positive
pressure of dry N2 was inserted. The septum was removed and the
flask charged sequentially with 3-[2-(dimethylamino)-2-oxoacetyl]-
1H-indol-4-yl acetate (6; 31.5 g, 115 mmol) and 2-CH3-THF (1000
mL). The flask was immersed in an ice-bath at 0 °C and a solution of
2.3 M LiAlH4 in 2-CH3-THF (140 mL, 322 mmol) was added through
the 250 mL dropping funnel. The dropping funnel was rinsed with ad-
ditional 2-CH3-THF (20 mL). The LiAlH4 solution was added dropwise
at a rate to maintain a temperature below 20 °C. After the addition,
the ice-water bath was removed and the mixture stirred for 30 min.
Analytical HPLC indicated a single major peak, which has been found
to be the intermediate compound 11 (-hydroxypsilocin intermedi-
ate). The light-yellow solution was heated to reflux (80 °C) with a
heating mantle and became ivory-colored after 3 h. Accumulation of
yellow solids was observed on the sides of the round-bottomed flask.
Characterization consistent with report in Procedure A.
Benzyl {3-[2-(Benzyldimethylammonio)ethyl]-1H-indol-4-yl}
Phosphate (9)
A 2000 mL, four-necked, round-bottomed flask was equipped with an
overhead stirrer, J-Kem temperature controller, a 100 mL dropping
funnel, and rubber septum through which a positive pressure of dry
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–G

F
A. M. Sherwood et al.
PSP
Synthesis
N2 inserted. The septum was removed and the flask was charged se-
quentially with psilocin (7; 10.3 g, 60.2 mmol) and anhyd THF (500
mL). The mixture was stirred for 15 min and the flask was immersed
in a solid CO2/acetone cooling bath at –78 °C. When the internal tem-
perature of the reaction reached –67 °C, a solution of 2.5 M BuLi in
hexanes (28.9 mL, 72.3 mmol) was added dropwise over a period of a
few min and maintained the internal temperature reading below
–60 °C. After stirring the olive-green colored reaction mixture for 10
min, tetrabenzyl pyrophosphate (35.7 g, 66.2 mmol) was added in
one portion and the mixture was stirred well. After 1.5 h, the solid
CO2/acetone cooing bath was removed and the temperature was al-
lowed to slowly rise to –25 °C over 2 h, at which time LCMS showed
completion of the reaction to compound 15 with no trace of com-
pound 10 in the reaction mixture. Amino bound silica gel (30 g) was
added in one portion and the reaction was diluted with EtOAc (600
mL). The dark mixture was filtered through a pad of Celite and
washed with EtOAc (400 mL). The filter cake was reslurried for 10 min
with EtOAc (400 mL) and again filtered. The combined filtrates were
concentrated and transferred into a 500 mL single-necked round-
bottomed flask. The gray oil was redissolved in DCM (100 mL) and
heated with a heat gun to boiling for 5 min. The flask was allowed to
reach rt and then held at 4 °C overnight. The crude grayish-colored
zwitterion precipitate 9 was filtered via Büchner funnel, then tritu-
rated with DCM (4 × 100 mL). The zwitterion precipitate 9 was trans-
ferred into a 250 mL single-necked round-bottomed flask and thor-
oughly dried in the vacuum oven at 40 °C overnight to provide a light-
purple solid; yield: 19.2 g (63%); mp 226–228 °C.
deionized H2O (~50 mL) gave a white solid, which was dried in an
oven at 60 °C for two days to provide 1 as a white solid; yield: 4.9 g
(49%); 99.9% HPLC purity; mp 221–223 °C.
IR (KBr): 3365, 3172, 1098, 1040, 919, 856, 811 cm–1
.
1H NMR (400 MHz, D2O):  = 7.20 (d, J = 7.9 Hz, 1 H), 7.11 (s, H-2, 1 H),
7.09 (t, J = 8.0 Hz, 1 H, H-6), 6.95 (d, J = 8.0 Hz, 1 H), 3.39 (t, J = 8.0, Hz,
2 H, H2-2′), 3.23 (t, J = 8.0 Hz, 2 H), 2.82 (s, 6 H).
13C NMR (100 MHz, D2O + 1 drop of CH3OH):  = 146.4 (d, J = 6 Hz, C,
split, C-4), 139.4 (C, C-7a), 124.8 (CH, C-6), 123.3 (CH, C-2), 119.1 (d,
J = 6 Hz, C, split, C-3a), 109.1 (d, J = 3 Hz, CH, split, C-5a), 108.6 (C, C-3),
108.4 (CH, C-7), 59.7 (CH2, C-2′), 43.4 [2 × CH3, N(CH3)2], 22.4 (CH2, C-
1′).
For a comparison of 1H and 13C NMR spectra data of 1 with the litera-
ture values, see the Supporting Information.
HRMS (ESI): m/z calcd for C12H17N2O4P (M + H)+: 285.0999; found:
285.0999.
Acknowledgment
The authors wish to thank Dr. Nicholas V. Cozzi, Dr. David E. Nichols,
and Dr. Paul Daley for their invaluable insights towards the develop-
ment of this work.
Supporting Information
IR (KBr): 3031, 2936, 2872, 1233, 1067, 1006, 856, 738, 697 cm–1
.
Supporting information for this article is available online at
1H NMR (400 MHz, DMSO-d6):  = 11.06 (d, J = 2.5 Hz, 1 H), 7.60–7.54
(m, 2 H), 7.53–7.40 (m, 3 H), 7.36–7.25 (m, 4 H), 7.28–7.19 (m, 1 H),
7.09 (t, J = 1.6 Hz, 1 H), 7.01 (dd, J = 14.4, 7.8 Hz, 2 H), 6.91 (t, J = 7.8
Hz, 1 H), 4.85 (d, J = 6.4 Hz, 2 H), 4.68 (s, 2 H), 3.72–3.64 (m, 2 H),
3.41–3.26 (m, 3 H), 3.13 (s, 6 H).
https://doi.org/10.1055/s-0039-1691565.
S
u
p
p
orit
n
gInformati
o
n
S
u
p
p
orit
n
gInformati
o
n
References
13C NMR (100 MHz, DMSO-d6):  = 148.32, 148.25, 139.99, 139.91,
139.00, 133.47, 130.56, 129.25, 128.88, 128.50, 127.47, 123.60,
122.15, 119.67, 119.60, 109.09, 109.07, 108.14, 106.29, 67.00, 66.90,
66.85, 65.45, 49.59, 20.74.
(1) (a) Rucker, J. J. H.; Iliff, J.; Nutt, D. J. Neuropharmacology 2018,
142, 200. (b) Kyzar, E. J.; Nichols, C. D.; Gainetdinov, R. R.;
Nichols, D. E.; Kalueff, A. V. Trends Pharmacol. Sci. 2017, 38, 992.
(c) Matsushima, Y.; Eguchi, F.; Kikukawa, T.; Matsuda, T.
Inflamm. and Regen. 2009, 29, 47.
HRMS (ESI): m/z calcd for C26H29N2O4P (M + H)+: 465.1938; found:
(2) (a) Dinis-Oliveira, R. J. Drug Metab. Rev. 2017, 49, 84. (b) The
LD50/ED50 ratio for aspirin is 199 and 641 for psilocin, which
compares favorably to presently approved drugs, see: Palenicek,
T.; Horacek, J. Eur. Neuropsychopharm. 2014, 24, 342.
(3) Halberstadt, A. L.; Vollenweider, F. X.; Nichols, D. E. Behavioral
Neurobiology of Psychedelic Drugs, 1st ed; Springer: Berlin, 2018.
(4) (a) Thomas, K.; Malcolm, B.; Lastra, D. J. Psychoactive Drugs
2017, 49, 446. (b) Hasler, F.; Bourquin, D.; Brenneisen, R.; Bär,
T.; Vollenweider, F. X. Pharmaceut. Acta Helv. 1997, 72, 175.
(5) Trulson, M. E.; Heym, J.; Jacobs, B. L. Brain Res. 1981, 215, 275.
(6) Roiser, J. P.; Rees, G. Curr. Biol. 2012, 22, 231.
465.1935.
Psilocybin (1)
Into a 2000 mL round-bottomed flask was added 9 (16.9 g, 35.6
mmol) followed by CH3OH (1200 mL). The mixture was degassed and
refilled with N2. 10% Pd/C (1.1 g) was added and the mixture was de-
gassed and refilled with a H2 balloon at 1 atm. The reaction mixture
was stirred overnight at rt. LCMS showed completion of the reaction
with no starting material remaining (Subsequent reactions revealed
that the hydrogenolysis was complete after 30 min). The flask was de-
gassed, refilled with N2 and the suspension was filtered through a pad
of Celite via Büchner funnel. The filter pad was washed with CH3OH
(500 mL) and the purple colored filtrate was concentrated and dried
overnight under vacuum to give 10.7 g of crude 1 (106%). The crude
solid was suspended in i-PrOH (200 mL) and boiled for 30 min, then
filtered hot (50 to 60 °C). The collected solid was washed with ace-
tone to give a pale purple colored solid. The purple solid was then
suspended in 25% CH3OH/i-PrOH and boiled for 30 min and filtered
hot, washing with 25% CH3OH/i-PrOH to give a light purple solid.
Finally, the solid was recrystallized from 30% H2O in acetone and
filtered to give light blue needles. Further recrystallization from 30%
acetone/water gave colorless needles. A final recrystallization from
(7) Brown, R. T.; Nicholas, C. R.; Cozzi, N. V.; Gassman, M. C.;
Cooper, K. M.; Muller, D.; Thomas, C. D.; Hetzel, S. J.; Henriquez,
K. M.; Ribaudo, A. S. Clin. Pharmacokinet. 2017, 56, 1543.
(8) (a) Grob, C. S.; Danforth, A. L.; Chopra, G. S.; Hagerty, M.; McKay,
C. R.; Halberstadt, A. L.; Greer, G. R. Arch. Gen. Psychiatry 2011,
68, 71. (b) Griffiths, R. R.; Johnson, M. W.; Carducci, M. A.;
Umbricht, A.; Richards, W. A.; Richards, B. D.; Cosimano, M. P.;
Klinedinst, M. A. J. Psychopharmacol. 2016, 30, 1181. (c) Ross, S.;
Bossis, A.; Guss, J.; Agin-Liebes, G.; Malone, T.; Cohen, B.;
Mennenga, S. E.; Belser, A.; Kalliontzi, K.; Babb, J.; Su, Z.; Corby,
P.; Schmidt, B. L. J. Psychopharmacol. 2016, 30, 1165.
(d) Carhart-Harris, R. L.; Bolstridge, M.; Rucker, J.; Day, C. M. J.;
Erritzoe, D.; Kaelen, M.; Bloomfield, M.; Rickard, J. A.; Forbes, B.;
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–G

G
A. M. Sherwood et al.
PSP
Synthesis
Feilding, A.; Taylor, D.; Pilling, S.; Curran, V. H.; Nutt, D. J. Lancet
Psychiatry 2016, 3, 619. (e) Carhart-Harris, R. L.; Goodwin, G. M.
Neuropsychopharmacology 2017, 42, 2105. (f) Idell, R. D.;
Florova, G.; Komissarov, A. A.; Shetty, S.; Girard, R. B. S.; Idell, S.
Med. Hypotheses 2017, 100, 46.
6208. (c) Hofmann, A.; Frey, A.; Ott, H.; Petrzilka, T.; Troxler, F.
Experientia 1958, 15, 397. (d) Ono, M.; Shimamine, M.;
Takahashi, K. Bull. Nat. Inst. Hygienic Sci. 1973, 91, 39.
(15) (a) Bartolucci, S.; Mari, M.; Di Gregorio, G.; Piersanti, G. Tetrahe-
dron 2016, 72, 2233. (b) Gathergood, N.; Scammells, P. J. Org.
Lett. 2003, 5, 921. (c) Sakagami, H.; Ogasawara, K. Heterocycles
1999, 51, 1131. (d) Furman, S.; Nissim-Bardugo, E.; Zeeli, S.;
Weitman, M.; Nudelman, A.; Finkin-Groner, E.; Moradov, D.;
Shifrin, H.; Schorer-Apelbaum, D.; Weinstock, M. Bioorg. Med.
Chem. Lett. 2014, 24, 2283. (e) Hu, C.; Qin, H.; Cui, Y.; Jia, Y. Tet-
rahedron 2009, 65, 9075. (f) Troxler, F.; Seemann, F.; Hofmann,
A. Helv. Chim. Acta 1959, 42, 2073. (g) Hofmann, A.; Frey, A.; Ott,
H.; Petrzilka, T.; Troxler, F. Experientia 1958, 14, 397.
(9) Weissman, M. M.; Wickramaratne, P.; Greenwald, S.; Hsu, H.;
Ouellette, R.; Robins, L. N.; Hallmayer, J. JAMA 1992, 268, 3098.
(10) (a) World Health Organization; Depression and Other Common
Mental Disorders (2017, February); WHO: Genf, 2017;
http://www.who.int/mediacentre/factsheets/fs369/en/.
(b) Kessler, R. C.; Berglund, P.; Demler, O.; Jin, R.; Koretz, D.;
Merikangas, K. R.; Water, E. E.; Wang, P. S. JAMA 2003, 289,
3095.
(11) Sudak, H. S. Suicide, In Kaplan & Sadock’s Comprehensive Text-
book of Psychiatry, 8th ed., Vol II; Sadock, B. J.; Sadock, V. A., Ed.;
Lippincott, Williams & Wilkins: New York, 2005, 2442–2453.
(12) Akiskal, H. S. Mood Disorders: Historical Introduction and Con-
ceptual Overview, In Kaplan & Sadock’s Comprehensive Textbook
of Psychiatry, 8th ed., Vol. I; Sadock, B. J.; Sadock, V. A., Ed.; Lip-
pincott, Williams & Wilkins: New York, 2005, 1559–1575.
(13) Nichols, D. E.; Johnson, M. W.; Nichols, C. D. Clin. Pharmacol.
Ther. 2017, 101, 209.
(16) Reaction monitoring by TLC for this reaction was ambiguous.
Even though it showed apparent completion, as it was difficult
to distinguish mixtures of O,O-dibenzylpsilocybin and O-mono-
benzylpsilocybin, see: Nichols, D. E.; Frescas, S. Synthesis 1999,
935.
(17) Shirota, O.; Hakamata, W.; Goda, Y. J. Nat. Prod. 2003, 66, 885.
(18) Fieser, L. F.; Fieser, M. Reagents for Organic Synthesis, Vol. 1;
Wiley: New York, 1967, 581–595.
(19) (a) Blei, F.; Baldeweg, F.; Fricke, J.; Hoffmeister, D. Chem. Eur. J.
2018, 24, 10028. (b) Fricke, J.; Blei, F.; Hoffmeister, D. Angew.
Chem. Intl. Ed. 2017, 56, 12352. (c) Lenz, C.; Wick, J.;
Hoffmeister, D. J. Nat. Prod. 2017, 80, 2835.
(14) (a) Hofmann, A.; Heim, R.; Brack, A.; Kobel, H.; Frey, A.; Ott, H.;
Petrzilka, T.; Troxler, F. Helv. Chim. Acta 1959, 42, 1557.
(b) Speeter, M. E.; Anthony, W. C. J. Am. Chem. Soc. 1954, 76,
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–G