Chemoenzymatic synthesis and cannabinoid activity of a new diazabicyclic amide of phenylacetylricinoleic acid

Article abstract of DOI:10.1016/j.bmcl.2010.04.074

Endocannabinoids (eCBs) are endogenous neuromodulators of synaptic transmission. Their dysfunction may cause debilitating disorders of diverse clinical manifestation. For example, drug addiction, lack of sex desire, eating disorders, such as anorexia or bulimia and dyssomnias. eCBs also participate in the regulation of core temperature and pain perception. In this context, it is important to recognize the utility of cannabinoid receptor 1 (CB1R) agonists, natural as Δ⁹-tetrahydrocannabinol (THC) or synthetic as Nabilone as useful drugs to alleviate this kind of patients' suffering. Therefore, we have developed a new drug, (R,Z)-18-((1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)-18-oxooct adec-9-en-7-yl phenylacetate (PhAR-DBH-Me), that appears to bind and activate the CB1R. This diazabicyclic amide was synthesized from phenylacetylricinoleic acid and (1S,4S)-2,5-diazabicyclo[2.2.1]heptane. To test its cannabinergic properties we evaluated its effects on core temperature, pain perception, and the sleep-waking cycle of rats. Results indicate that 20 and 40 mg/kg of PhAR-DBH-Me readily reduced core temperature and increased pain perception threshold. In addition, 20 mg/kg increased REM sleep in otherwise normal rats. All these effects were prevented or attenuated by AM251, a CB1R antagonist. Place preference conditioning studies indicated that this molecule does not produce rewarding effects. These results strongly support that PhAR-DBH-Me possesses cannabinoid activity without the reinforcement effects.

Full text of DOI:10.1016/j.bmcl.2010.04.074

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3231–3234
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chemoenzymatic synthesis and cannabinoid activity of a new diazabicyclic
amide of phenylacetylricinoleic acid
Manuel López-Ortíz a, Andrea Herrera-Solís b, Axel Luviano-Jardón a, Nidia Reyes-Prieto b, Ivan Castillo c,
b,
Ivan Monsalvo a, Patricia Demare a, Mónica Méndez-Díaz b, Ignacio Regla a,c, , Oscar Prospéro-García
*
*
a Facultad de Estudios Superiores Zaragoza, Universidad Nacional Autónoma de México (UNAM), Batalla del 5 de Mayo y Fuerte de Loreto, Iztapalapa 09230, Mexico, D.F., Mexico
b Laboratorio de Canabinoides, Grupo de Neurociencias, Facultad de Medicina, UNAM, Circuito Exterior, Ciudad Universitaria, 04510 Mexico, D.F., Mexico
c Instituo de Química, UNAM, Circuito Exterior, Ciudad Universitaria, 04510 Mexico, D.F., Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Endocannabinoids (eCBs) are endogenous neuromodulators of synaptic transmission. Their dysfunction
may cause debilitating disorders of diverse clinical manifestation. For example, drug addiction, lack of
sex desire, eating disorders, such as anorexia or bulimia and dyssomnias. eCBs also participate in the
regulation of core temperature and pain perception. In this context, it is important to recognize the utility
of cannabinoid receptor 1 (CB1R) agonists, natural as
Nabilone as useful drugs to alleviate this kind of patients’ suffering. Therefore, we have developed a
new drug, (R,Z)-18-((1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)-18-oxooctadec-9-en-7-yl
Received 5 March 2010
Revised 14 April 2010
Accepted 16 April 2010
Available online 21 April 2010
D
9-tetrahydrocannabinol (THC) or synthetic as
Keywords:
Cannabinoids
phenylacetate (PhAR-DBH-Me), that appears to bind and activate the CB1R. This diazabicyclic amide
was synthesized from phenylacetylricinoleic acid and (1S,4S)-2,5-diazabicyclo[2.2.1]heptane. To test its
cannabinergic properties we evaluated its effects on core temperature, pain perception, and the sleep-
waking cycle of rats. Results indicate that 20 and 40 mg/kg of PhAR-DBH-Me readily reduced core
temperature and increased pain perception threshold. In addition, 20 mg/kg increased REM sleep in
otherwise normal rats. All these effects were prevented or attenuated by AM251, a CB1R antagonist. Place
preference conditioning studies indicated that this molecule does not produce rewarding effects. These
results strongly support that PhAR-DBH-Me possesses cannabinoid activity without the reinforcement
effects.
Core temperature
Pain regulation
Sleep-waking cycle
Ó 2010 Elsevier Ltd. All rights reserved.
Endocannabinoids (eCBs) are molecules synthesized by neurons
of all mammalians and insects known.1 eCBS modulate the synap-
tic transmission participating in the generation of several types of
behavior.2 Therefore, their dysfunction may cause debilitating dis-
orders of diverse clinical manifestation. For example, drug addic-
tion,3 lack of sex desire and eating disorders,4 such as anorexia or
bulimia5 and dyssomnias.6 Additionally, the activation of the can-
nabinoid receptor 1 (CB1R) modulates pain perception and core
temperature.7 Mounting evidence indicates that in schizophrenia
eCBs may be dysfunctional.8
Figure 1. Chemical structures of THC and Nabilone.
Marijuana derivatives, such as
D
9-tetrahydrocannabinol (THC,
Fig. 1) commercially available as DronabinolÒ or THC plus cannabi-
diol (SativexÒ) and several synthetic compounds as Nabilone
(CesametÒ), among others, produce several therapeutic effects.9,10
For example, they are appetite stimulants in patients suffering
from wearing-off caused by acquired immunodeficiency syndrome
Scheme 1. Chemical synthesis of (1S,4S)-2-methyl-2,5-diazabicyclo[2.2.1]heptane
(1) from trans-4-hydroxy-(S)-proline. Reagents and conditions: (a) TsCl, Na2CO3,
H2O, 94%; (b) NaBH4, BF3ꢀEt2O, THF, 85%; (c) TsCl, C5H5N, toluene, 20 h, 83%; (d)
PhCH2NH2, toluene, reflux, 96%; (e) HBr 40%, 96%; (f) HCO2H, HCOH, MeOH, reflux,
73%; (g) H2, Pd/C, MeOH 80%.
* Corresponding authors. Tel.: +52 55 5623 0795 (I.R.); +52 55 5623 2509
(O.P.-G.).
E-mail addresses: regla@unam.mx (I. Regla), opg@unam.mx (O. Prospéro-
García).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.074

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M. López-Ortíz et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3231–3234
or chemotherapy. Cannabinoids can also be used as antiemetic
drugs in patients under chemotherapy.11 Pain is another indication
for this class of compounds.9,12 There is information indicating its
successful use to control glaucoma.13 Likewise, there is mounting
experimental evidence indicating that cannabinoids may be useful
to control epilepsy,14 anxiety,15 and insomnia.6 Despite these
advances, the toxic or lethal effects caused by cannabinoids have
not been discussed thus far.16 In addition, blocking of the CB1R
receptor by drugs such as SR141716a, may induce side effects that
compromise patients’ health or life, as has been suggested by the
experience gained with the use of SR141716a for food intake con-
trol in obese patients.17 Therefore, it is important to recognize the
Scheme 2. Chemoenzymatic synthesis of compound 4. Reagents and conditions: (a) Novozym 435, water/acetone 6:4, phosphate buffer 100 mM pH 7.5, 37 °C, 1 h,
quantitative yield; (b) (1) PvCl, CH2Cl2; (2) diamine 1, 80%.
Figure 2. This figure depicts the effect of compound 4 at three doses on pain perception as evaluated with the hot-plate paradigm. Such an effect has been blocked by AM251.
p <0.05, compared to control.
Figure 3. This figure illustrates the effect of three doses of compound 4 on the core temperature. There is a reduction of about 1 °C in 15 min after the ip injection. This effect
remains for 30 more minutes, and is hampered by AM251. p <0.05 compared to control.

M. López-Ortíz et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3231–3234
3233
Figure 4. These graphs illustrate the effect of compound 4 on the sleep-waking cycle of rats during 4 h. In A the effect on REM sleep is depicted. Likewise for NonREM sleep in
B and for waking in C. Observe the important REM sleep inducing effect caused by compound 6 compared to control. This effect is blockaded with AM251, a selective CB1
antagonist. p <0.05.
therapeutic potential of cannabinoids and develop new drugs with
reduced side effects, and with potential clinical applications in the
treatment of several diseases.
Regarding the sleep-waking cycle, results indicate that 20 mg/
kg of 4 increased REM (rapid eye movement) sleep total time dur-
ing 4 h, with a simultaneous tendency to increase NREMS total
time. A tendency to reduce waking-up was also documented. These
changes in REM sleep were blocked by AM251 (see Fig. 4). These
PhAR-DBH-Me-induced changes have been observed with agonists
and antagonists of the CB1R receptor.
Finally, PhAR-DBH-Me (4) was tested at the dose of 20 mg/kg to
evaluate its capacity to induce addiction. A conditioned place pref-
erence paradigm was utilized to determine if 4 is capable of induc-
ing signs of dependence. Data indicated that 4 failed to induce
signs of dependence (see Fig. 5).
We have recently synthesized a new diazabicyclic amide
derivative of phenylacetylrincinoleic acid (R,Z)-18-((1S,4S)-5-
methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)-18-oxooctadec-9-en-
7-yl phenylacetate (PhAR-DBH-Me, 4), and tested its effects on some
physiological variables, revealing that it possesses cannabinoid
activity and that such an effect is mediated by the CB1R. This new
compound 4 has been prepared starting from trans-4-hydroxy-(S)-
proline and castor oil, both compounds acting as natural sources of
chirality. In the first step, (1S,4S)-2-methyl-2,5-diazabicy-
clo[2.2.1]heptane (1) was synthesized according to the procedure
recently described by Regla, Juaristi and co-workers (Scheme 1).18
The synthetic strategy for the preparation of 4 is outlined in
Scheme 2. The preparation of methyl 12-phenylacetylricinoleate
(2) took place with the recently reported conditions.19 A key step
in the synthetic methodology was the development of a new strat-
egy for the regioselective chemoenzymatic hydrolysis of 4 to obtain
12-phenylacetylricinoleic acid (3), a described precursor for the syn-
thesis of 12-acyl substituted ricinoleylamides.20,21 Compound 4 was
obtained in 70% yield by the mixed anhydride method. Thus, treat-
ment of 3 with pivaloyl chloride in a mixture of Et3N and dichloro-
methane at 0 °C for 15 minutes to produce the corresponding
mixed anhydride was followed by reaction with diamine 1.22
Our physiological and behavioral22 results indicate that three
doses of PhAR-DBH-Me (4) reduced core temperature by about
1.5–2.0 °C; such doses also increased rats’ latency to exhibit signs
of discomfort in the hot-plate test. Moreover, AM251 successfully
blocked these effects; however, AM251 did not cause any effect
by itself at the dose levels tested. These results suggest that
PhAR-DBH-Me (4) is a CB1R agonist (see Figs. 2 and 3).
In conclusion, PhAR-DBH-Me (4) is able to induce effects similar
to those observed with well-known cannabinoids. Moreover, such
effects are prevented by the administration of the CB1R antagonist
Figure 5. This figure illustrates the absence of rewarding effect of compound 4. As
it can be seen, compound 4 does not modify the time spend in the compartment A
(associated to the administration of this drug).

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M. López-Ortíz et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3231–3234
2 g, (4.8 mmol) of 3 and 667 lL (4.8 mmol) of Et3N were dissolved in CH2Cl2
(25 mL). The reaction mixture was cooled to 0 °C in an ice bath and pivaloyl
AM251. Further experiments will clarify if this drug induces ad-
verse side effects.
chloride (591 L, 4.8 mmol) was added and stirred at the same temperature for
l
15 min. Afterwards, (1S,4S)-2-methyl-2,5-diazabicyclo[2.2.1]heptane (537 mg,
4.8 mmol) was added and stirring continued for 15 min at 0 °C. The organic layer
was washed with water (3 ꢂ 10 mL), dried with anhydrous Na2SO4 and
Acknowledgements
concentrated under reduced pressure to obtain
a liquid, which after
This work was partially supported by Grant 24768 from CONA-
CyT and DGAPA UNAM PAPIIT IN211509. I.R. thanks the Instituto
de Química for the fellowship ‘Cátedra Especial Jesús Romo Arm-
ería’, granted by the Instituto de Química-UNAM. Authors are also
grateful to Q. Ma de los Angeles Peña, Q. Elizabeth Huerta, and QFB.
Rocío Patiño-Maya from Instituto de Química, UNAM for spectro-
scopic analysis, and to Novozymes-México for the generous gift
of CaLB.
chromatographic purification (CH2Cl2/MeOH 95:5) afforded 1.76 g (74%) of
PhAR-DBH-Me (4) as a colorless oil. 1H NMR (300 MHz, C2D2Cl4) ꢃ130 °C:
d = 0.90 (t, J = 7.2 Hz, 3H), 1.28–1.35 (m, 16 H), 1.58–1.66 (m, 4 H), 1.77 (d,
J = 10.5, 1H), 2.02 (c J = 6.9, 2H), 2.11–2.24 (m, 2H), 2.19 (t, J = 7.8 Hz, 2H), 2.57 (s,
3H), 2.91–3.03 (m, 2H), 3.31 (d, J = 10.8 Hz, 2H), 3.58 (s, 2H), 3.71 (br s, 2H), 3.82
(d, J = 12, 2H), 4.88 (q, J = 6.6 Hz, 1H), 5.31–5.34 (m, 1H), 5.44–5.48 (m, 1H), 7.26–
7.29 (m, 5H). ½a 2D0
ꢁ
= ꢃ4.2 (c 1, MeOH). IR mmax (film) cmꢃ1: 3441, 3062, 2928,
2855, 1731, 1644, 1430, 1255, 1161, 1028, 763, 723. MS (EI) m/z (%) = 511 (100)
[M+], 375 (64), 113 (18), 82 (55), 68 (9). HRMS-FAB+: m/z calcd for C32H50N2O3
[M+H]+: 511.3900; found: 511.3888.
Experimental methods for the physiological and behavioral evaluations: Subjects:
Adult Wistar rats, weighting 250–350 g were used in this study. All animals were
housed individually in Plexiglas cages. They were maintained at an ambient
temperature of 22 1 °C and a controlled 12:12 h light–dark cycle (08:00 am–
08.00 pm; lights off at 08:00 am) throughout the study. Food and water were
available ad libitum. Animals were treated according to the Norma Oficial
Mexicana (NOM-062-ZOO-199), the Guide for Care and Use of Laboratory
Animals established by the National Institutes of Health, and the European
Community Council Directive 86/609/ EEC. Additionally, our protocol was
approved by the Research and Ethics Committee of the Facultad de Medicina,
Universidad Nacional Autónoma de México (UNAM). Every effort was made to
minimize the number of animals used and their potential suffering. Effects on
core temperature and pain perception: Seventy rats were used in this part of the
study. They were randomly assigned to seven groups to determine a PhAR-DBH-
Me (4) dose–response curve (0, 10, 20 and 40 mg/kg ip). Two additional groups
(n = 10, each group) received 20 or 40 mg/kg of PhAR-DBH-Me (4) 15 min after
the administration of AM251 (3 mg/kg, ip), a selective CB1 receptor (CB1R)
antagonist to block the effect. One last group was included to test the effect of the
AM251 alone. Core temperature was measured by using a rectal probe and pain
perception by using the hot plate method. Sleep-waking cycle recordings: (a)
Surgery: Rats (n = 6 per group) were stereotaxically implanted under anesthesia
(cocktail: 66 mg/kg ketamine, 0.26 mg/kg xylazine, and 1.3 mg/kg
acepromazine) with two electrodes inserted into the hippocampus (P = 4.0,
L = 2.5, V = 2.5) according to the Paxinos and Watson atlas23 for
electroencephalographic (EEG) recordings. Two additional screw electrodes
were implanted into the frontal bones for grounding the animal. Two twisted
wire electrodes were placed into the neck musculature for electromyographic
(EMG) recordings. (b) Sleep recordings: After surgery, animals were monitored
and provided with the proper veterinary aid to speed their recovery up. They
were allowed to recover for 10 days. Upon the completion of this period, rats
were habituated to the recording conditions for 24 h. Once the habituation
period was completed, rats were divided into different experimental groups.
Vehicle group (control) was administered with two ip injections. First injection
was DMSO (1 ml/kg), the vehicle for AM251 and 15 min later vegetal oil (1 ml/
kg), the vehicle for PhAR-DBH-Me (4). PhAR-DBH-Me group was injected with
DMSO and 15 min later with PhAR-DBH-Me (4). Group AM251, was injected
with this drug and 15 min later injected with vegetal oil. Finally, the
AM251 + PhAR-DBH-Me group, was injected with these two drugs 15 min
apart and in this sequence. All injections were performed at the beginning of the
dark phase of the light-dark cycle. Immediately after the second administration,
the sleep-waking cycle was recorded for 12 h. The EEG and EMG signals were
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18. Melgar, R.; González, R.; Olivares, J. L.; González, V.; Romero, L.; Ramírez, M.;
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19. Castillo, E.; Regla, I.; Demare, P.; Luviano-Jardón, A.; López-Munguía, A. Synlett
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20. Appendino, G.; Cascio, M. G.; Bacchiega, S.; Moriello, A. S.; Minassi, A.; Thomas,
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22. General: 1H and 13C NMR spectroscopy was carried out on a JEOL Eclipse
instrument at 200 and 300 MHz, with TMS as an internal standard and CDCl3 as
solvent. The IR spectra were carried out on a Bruker spectrophotometer Tensor
amplified with
a Grass Model 7 polygraph, Amplifier Model 7P511, in a
frequency range of 1–30 Hz, and 30–100 Hz, respectively. Signals were
acquired and analyzed with the ICELUSÒ software. Data analysis: Polygraphic
recordings were analyzed every 12 s and classified according to the following
vigilance stages: wakefulness (W), slow wave sleep (SWS), and rapid eye
movement sleep (REMS). Electrophysiological criteria were used to define these
stages of vigilance as follows: W was characterized by the EEG expressing mixed
low fast voltage and theta activity, as well as high muscle activity. In SWS, the
EEG showed delta waves and decreased EMG amplitude. Finally, in REMS the EEG
expressed theta activity without EMG activity (postural atonia). The time spent
in W, SWS, and REMS per hour was calculated during 12 h period. SWS and REM
sleep latency was also calculated by measuring the time elapsed from the start of
the sleep recording to the first SWS bout. REMS latency was considered from the
first SWS bout to the first REMS bout. Frequency and average duration of REMS
bouts were also calculated. Statistics: Results of REMS, SWS, and W were
compared by a mixed analysis of variance (ANOVA) with a Greenhouse-Geisser
correction, and subsidiary ANOVAs to detect changes per hour with an LSD post
hoc test used only for specific comparison when indicated by mixed ANOVA.
Sleep latencies and average duration of REMS episodes were analyzed with one
way ANOVA and post hoc LSD. Finally, frequency of REMS was analyzed with
Kruskal–Wallis test with post hoc U-Mann-Whitney and Bonferroni correction.
23. Paxinos, G.; Watson, C. The Rat Brain in Stereotaxic Coordinates, Second ed.;
Academic Press: San Diego, 1986.
27. The ½a 2D0
ꢁ
values were determined on a 341 Perkin–Elmer polarimeter, at 1 dm
cell length. HRMS was determined on a JEOL JMS-SX102A instrument. Silica gel
chromatography: 70–230 mesh. Multiplicity keys: s = singlet, d = doublet,
t = triplet, c = quartet, q = quintet, m = multiplet, br = broad, dd = doublet of
doublets. Typical procedure for the enzymatic hydrolysis for the preparation of
phenylacetylricinoleic acid (3). To a solution of methyl 12-phenylacetylricinoleate
(7 g, 16.25 mmol) in acetone (360 mL), were added water (120 mL), phosphate
buffer 100 mM pH 7.5 (240 mL) and Novozym 435 (3.6 g (10 mg/mL). The
reaction mixture was incubated for 1 h at 37 °C and 250 rpm, monitoring the
reaction by TLC (hexane/EtOAc 9:1). The mixture was filtered through Celite and
concentrated under reduced pressure in order to remove acetone. The aqueous
layer was extracted with dichloromethane (3 ꢂ 50 mL), the organic layer was
dried with anhydrous Na2SO4 and concentrated under reduce pressure to yield
6.7 g of pure 3 as a colorless oil in 99% yield. 1H NMR (200 MHz, CDCl3): d = 0.86
(t, J = 6.2, 3H), 1.2–1.35 (m, 16H), 1.49 (m, 2H), 1.59–1.66 (m, 2H), 1.96–1.99 (m,
2H), 2.24–2.27 (m, 2H), 2.34 (t, J = 7.4 Hz, 2H), 3.59 (s, 2H), 4.87 (q, J = 6.2 Hz, 1H),
5.21–5.33 (m, 1H), 5.37–5.50 (m, 1H), 7.23–7.32 (m, 6H), 9.44 (br s, 1H). 13C NMR
(50 MHz, CDCl3): d = 14.0, 22.4, 24.6, 25.1, 27.2, 28.9, 29.0, 29.1, 29.4, 31.6, 31.8,
33.5, 33.9, 41.7, 74.4, 124.1, 126.9, 128.4, 129.1, 132.5, 134.2, 171.3, 179.9.
(R,Z)-18-((1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)-18-oxooctadec-9-
en-7-yl phenylacetate (4, PhAR-DBH-Me): In a three-necked round-bottom flask,