Understanding the mechanism of sweet taste: Synthesis of ultrapotent guanidinoacetic acid photoaffinity labeling reagents

Article abstract of DOI:10.1021/jm960349q

Azido-functionalized analogs of potently sweet guanidinoacetic acids have been synthesized for use as sweetener receptor photoaffinity labeling reagents. These compounds have been synthesized using readily available starting materials. One of the azido-la

Full text of DOI:10.1021/jm960349q

J . Med. Chem. 1996, 39, 4167-4172
4167
Un d er sta n d in g th e Mech a n ism of Sw eet Ta ste: Syn th esis of Ultr a p oten t
Gu a n id in oa cetic Acid P h otoa ffin ity La belin g Rea gen ts1
Srinivasan Nagarajan,*,† Michael S. Kellogg,† and Grant E. DuBois‡
The NutraSweet Company, 601 E. Kensington Road, Mt. Prospect, Illinois 60056
Go¨ran Hellekant
Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, Wisconsin 53706
Received May 13, 1996X
Azido-functionalized analogs of potently sweet guanidinoacetic acids have been synthesized
for use as sweetener receptor photoaffinity labeling reagents. These compounds have been
synthesized using readily available starting materials. One of the azido-labeled guanidinoacetic
acids has been evaluated in an electrophysiological model in the Rhesus monkey. We found
that the photoaffinity-labeling reagent caused irreversible inhibition in electrophysiological
response to sweeteners upon exposure of the monkey tongue to a combination of the reagent
and UV light.
The perception of sweet taste is assumed to be
initiated by the binding of the sweet stimuli to receptor
molecules on the surface of the tongue. Results of many
experiments strongly argue for sweet taste mediation
by multiple molecular recognition units. These multiple
molecular recognition units may be different receptors,
although other rationales are also possible. Attempts
have been made in the past to isolate receptors from
bovine, rodent, and even primate sources.2 These efforts
have been difficult due to the lack of potent agonists or
antagonists. The identification by Nofre and Tinti3 of
the guanidinoacetic acid class of sweeteners, with some
representatives exhibiting potencies in excess of 100 000
times that of sucrose, suggested to us that preparation
of the necessary molecular probes may now be feasible.
We decided to investigate the guanidinoacetic acid
sweetener receptor by employing photoaffinity-labeling
techniques.4 We disclose here the synthesis of four
photoaffinity-labeling reagents and preliminary results
from labeling experiments.
Our photoaffinity-labeling reagents are based on the
templates of guanidinoacetic acids 1 and 2. The selec-
tion of 1 is based on the finding that it is potently active
in both primates and rodents5 and the selection of 2
based on the very high potency in humans.1 These gua-
nidinoacetic acids 1 and 2 have been estimated to be
28 0003 (vs 2% standard solution of sucrose) and 200 000§
(vs 2% sucrose) times as potent as sucrose, respectively.6
* Author to whom correspondence should be addressed.
† Present address: G. D. Searle, 700 Chesterfield Village Parkway,
AA2I, St. Louis, MO 63198.
‡ Present address: The Coca-Cola Co., Corporate Research and
Development, 1 Coca-Cola Plaza, Atlanta, GA 30313.
§ The determination of sweetness potencies is most accurately
A useful photoaffinity probe must exhibit a high level
of activity. Thus the position of attachment of the
photosensitive functionality to the structural core must
not significantly affect sweetness potency. On the basis
of the known structure-activity relationships (SAR) of
the guanidinoacetic acids, we felt that sweetness po-
tency would be maintained by substitution of a photo-
labile azide moiety for the nitrile groups of 1 and 2. This
speculation is based on the correlation between sweet-
ness potencies and the Hammet σ constants of groups
substituted in this position and the σ constant (+0.42)
of the azido group.7 Thus, we elected to synthesize 3
and 4. Synthesis of reagents with photolabel substitu-
accomplished by paired-difference testing of
a narrow range of
sweetener concentrations relative to the sucrose reference. Such testing
must be done with a large panel (e.g., 40-50 subjects), the members
of which have demonstrated proficiency in sweetness intensity scaling.
However, for experimental compounds on which limited safety assess-
ment work has been completed, we employ an estimation procedure
based on small panels (e.g., three or four subjects). Subjects are trained
to recognize the sweetness intensities of sucrose reference in the range
of 2-10%. Subjects then rate the sweetness intensities of several
dilutions of the sweetener where the dilutions are chosen to span the
sweetness intensities of the sucrose reference of interest. For the case
of 2, a concentration of 0.10 ppm was judged to be approximately
equivalent to a 2.0% sucrose reference [Pw(2)] and is calculated to be
200000× sucrose. There are two special considerations which compli-
cated the sweetness potency of 2. (1) It is well-known that guanidi-
noacetic acid compounds often exist in equilibrium with acyl guanidine
cyclic dehydration products (e.g., creatine/creatinine equilibrium; see:
Merck Index, 10th ed.; Windholz, M., Ed.; Merck & Co.: Rahway, NJ ,
1983; p 2551). For this reason, 2 was dissolved under very mild
conditions: Specifically, a 25 mg/25 mL solution was prepared in 1/1
EtOH/H2O with minimal warming. Aqueous dilutions were made as
soon as complete dissolution was achieved and they were then tasted
immediately. (2) The guanidinoacetic acid 2 exhibits a sweetness which
is notably slow in onset relative to sucrose, particularly when evaluated
at low levels of sweetness intensity. For 2 at 0.10 ppm, no sweetness
is noted for the first ∼10-15 s, after which a gradual rise in sweetness
is observed, maximizing at ∼30-45 s. Thus it is difficult for tasters to
decide when the sweetness maxima has been reached, and in fact,
unless the solution is held in the mouth for the requisite time, tasters
report the observation of no sweetness at all.
X Abstract published in Advance ACS Abstracts, September 15, 1996.
S0022-2623(96)00349-4 CCC: $12.00 © 1996 American Chemical Society

4168 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Nagarajan et al.
Sch em e 1
Sch em e 2
tion at the ortho or meta positions of the anilines was
not pursued since the ortho analogs are not sweet3 and
the precursors for the meta analogs are not readily
available. Substitution on the acetic acid moiety re-
duces sweetness potency drastically and was also not
considered.3 From inspection of the SAR of guanidi-
noacetic acid sweeteners, it is also clear that the
substitution at the hydrophobic moiety may be varied
a great deal without significantly affecting sweetness
activity.3 Thus, in addition to azide- substituted guani-
dinoacetic acids 3 and 4 we also elected to synthesize 5
and 6 with the expectation that they also would be
potently sweet.
Photolabeling reagent 3 was synthesized as shown in
Scheme 1 by starting with commercially available p-azi-
dophenyl isothiocyanate (7). Reaction of 7 with (S)-R-
phenethylamine (8) gave thiourea 10. The thiourea 10
was converted to isothiourea 12 by treatment with
methyl iodide. Reaction of isothiourea with sodium salt
of glycine afforded the desired guanidinoacetic acid 3
in 68% overall yield. The synthesis of 4 was ac-
complished in 42% overall yield as illustrated in Scheme
1, employing a similar route starting from benzhydryl-
amine.
The reagents 5 and 6 in which the azido group is
located in the 4- and 3- positions, respectively, of the
benzhydryl portion of guanidine 2 were synthesized
(Scheme 2), employing a procedure similar to that
described above. The corresponding 4- and 3-aminoben-
zhydrylamines (16) and (17) were synthesized from
commercially available 4-aminobenzophenone and 3-ami-
nobenzophenone, respectively, via reduction of their
oximes. Reaction of these amines with p-cyanophenyl
isothiocyanate provided the amino-substituted thioureas
18 and 19. These aromatic amines were transformed
to the azides 20 and 21 by diazotization, followed by
reaction with sodium azide. From the azidothioureas,
the guanidinoacetic acids 5 and 6 were then synthesized
via corresponding isothioureas 22 and 23 as was ac-
complished earlier for 3 and 4 (Scheme 2). The taste
activities of aryl azides 3-6 show that our initial
assumptions were indeed correct. We found that all four
compounds synthesized are sweet. The sweetness po-
tencies of 3-6 were estimated, in humans, to be as
follows: 3, 1000 (1%); 4, 30 000 (3%); 5, 30 000 (3%);
and 6, 30 000 (3%) times that of sucrose. The sweetness
potencies were determined by a trained human taste
panel, and the percentages in parentheses are the

Understanding the Mechanism of Sweet Taste
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4169
sucrose reference concentrations judged to be equisweet
with the X-fold (X ) potency) more dilute guanidinoace-
tic acid sweeteners.
The in vitro photolysis of guanidinoacetic acid 4 was
examined to demonstrate that the reagent is photolabile
and to determine the minimum time required for
photolysis. The in vitro photolysis of 4 was conducted
with a 450 W medium-pressure mercury vapor lamp in
ethanol in the presence of dimethylamine in the hope
of trapping the nitrene intermediate.8 The consumption
of 4 was complete in less than 2 min. NMR and mass
spectral analysis of the photolysis mixture demonstrated
the presence of a nitrene-dimethylamine adduct.
Receptor-labeling studies in animals to date have
been primarily limited to 4. The details of these
experiments will be described elsewhere. Monkeys were
used in these experiments since the sense of taste in
nonprimates differs markedly from that of humans.9a
The sweet compounds synthesized and used in this
study are sweet to old world monkeys and to humans.
The taste nerve impulses have been recorded from the
chorda tympani proper (CT) nerve, which mediates
much of the sweet taste response following application
of the sweeteners on the tongue. The nerve impulses
can be measured by employing electrophysiological
methods. The protocols for surgically exposing the
chorda tympani nerve in anesthetized animals and
measuring and recording nerve responses have been
developed in our laboratories (Wisconsin; see Methods).9
We then attempted to photolable the guanidinoacetic
acid receptor on the tongue of live anesthetized mon-
keys. The experimental protocol we employed to assess
the effects of in vivo photolabeling with 4 on the
electrophysiological responses to sweeteners constituted
the following steps: (1) A range of sweet-tasting stimuli
were applied to the tongue of the animal and the
summated nerve rsponses determined; (2) guanidi-
noacetic acid 4 was placed on the tongue, after which
the tongue was irradiated for 40 s [employing a hand
held pencil UV light (254 nm) with provisions for
limiting exposure to other parts of the animal and the
user]. The UV lamp was held at 1 cm above the tongue
over the area to be irradiated; (3) step 2 was repeated
two times; (4) the tongue was washed, and the sum-
mated CT response to the sweet-tasting stimuli were
again determined.
F igu r e 1. Nerve response in monkey for aspartame before
and after irradiation of the tongue with UV light and 4.
F igu r e 2. Nerve response in monkey for ACE-K before
irradiation of the tongue, after irradiation with a mixture of
4 and 24, and followed by irradiation with 4 alone.
response over a range of concentrations for both sweet-
eners was observed after the photolabeling. An experi-
ment was designed to saturate all the putative sweet
receptors in the tongue by exposing it to high concentra-
tions of 24 [sweetness potency ) 30 000 (compared to
3% sucrose), synthesized as shown in Scheme 1 by
starting from (R,S)-R-(3-pyridyl)benzylamine]. The com-
pound 24 was chosen because of its relatively high water
solubility compared to other sweeteners in this class.
When irradiation of the tongue was attempted with 4
(10 mg/L) in the presence of excess 24 (100 mg/L), no
labeling of the tongue was observed as indicated by the
unchanged nerve response for ACE-K. However, when
the tongue was exposed to the combination of 4 (10 mg/
L) and UV light, in the same experiment, a significant
drop in response was observed for ACE-K (Figure 2).
We have shown from this experiment that 24 which is
structurally similar to 4 competes effectively with 4 and
prevents the photolabeling of the tongue.
Concentration-response functions were determined
for the taste stimulants aspartame (APM) and ac-
esulfame-K (ACE-K) by using solutions these sweeten-
ers of varying concentrations. The concentration-
response functions were determined for aspartame
(Figure 1) and acesulfame-K (Figure 2) before and after
photoaffinity labeling. A decrease in summated nerve
We performed several control experiments. Exposing
the tongue to UV light alone did not cause any decrease
of response to taste stimulants (to show that exposure

4170 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Nagarajan et al.
detail elsewhere.9 We have made use of this technique to
perform our photoaffinty-labeling experiments in Rhesus
monkeys.
of tongue to UV (3 × 40 s) light does not cause damage).
Repeated application of the photoaffinity-labeling re-
agent, or the application of preirradiated reagent, did
not cause any decrease in response to taste stimulants
(to show that either the reagent itself or the product of
irradiation does not cause irreversible inhibition of taste
response). Application of a potently sweet non-photo-
sensitive guanidine (2) followed by exposure to UV light
(to show that the guanidineacetic acid class of sweeten-
ers do not provide active intermediates upon exposure
to UV light) did not reduce the response in monkeys
for the various stimulants. These control experiments
clearly demonstrated that only the combination of the
photolabel and the UV light affected the ability of these
monkeys to respond to various stimulants.
In conclusion, we have found that the photoaffinity-
labeling reagent caused irreversible inhibition in re-
sponse for the sweeteners aspartame and acesulfame-
K. Further work is needed to show that the photo-
labeling is taking place at the putative sweet receptor.
Tritium-labeled 4 has recently been synthesized in our
laboratories,10 and the labeling experiments in tongue
preparations to isolate the photolabeled macromolecules
are in progress.
N-(4-Azid op h en yl)-N′-(d ip h en ylm eth yl)th iou r ea (11).
A mixture of p-azidophenyl isothiocyanate (7, 0.50 g, 2.84
mmol) and benzhydrylamine (9, 0.52 g, 2.84 mmol) in aceto-
nitrile (10 mL) was stirred at room temperature for 2 h, and
the precipitate was filtered. The solid was recrystallized from
hexane-ethyl acetate to afford 0.92 g (90%) of the desired
product as pale orange powder: mp 175 °C dec; 1H NMR
(CDCl3) δ 6.97 (s, 1H), 7.01 (d, 2H, J ) 8.3 Hz), 7.17-7.37 (m,
12H); 13C NMR (CDCl3) δ 61.5, 119.6, 125.9, 127.2, 128.3,
133.6, 137.7, 140.6, 180.1; IR (Nujol) 2126 cm-1
(C20H17N5S) C, H, N.
.
Anal.
N-(4-Azid op h en yl)-N′-[(S)-r-p h en et h yl]t h iou r ea (10).
This was prepared using the above procedure with p-azi-
dophenyl isothiocyanate and (S)-R-phenethylamine: yield 95%;
1
mp 117 °C; H NMR (CDCl3) δ 1.53 (d, 3H, J ) 6.9 Hz), 4.79
(br s, 1H), 7.0 and 7.17 (AB quartet, 4H, J ) 8.7 Hz), 7.28-
7.36 (m, 5H); 13C NMR (CDCl3) δ 21.5, 54.1, 120.2, 126.0, 126.5,
126.6, 127.5, 128.7, 138.6, 141.9, 179.4. Anal. (C15H15N5S) C,
H, N.
N -(4-Azid op h e n yl)-N ′-(d ip h e n ylm e t h yl)-S -m e t h yl-
isoth iou r ea (13). Iodomethane (1.01 g, 7.11 mmol) was added
to a solution of the thiourea (11, 0.85 g, 2.37 mmol) in acetone
(15 mL), and the reaction mixture was stirred at ambient
temperature for 18 h. The reaction mixture was concentrated,
and the residue was partitioned between dichloromethane (50
mL) and sodium hydroxide (1 N, 20 mL). The organic layer
was separated, dried (MgSO4), and concentrated to afford 0.88
Exp er im en ta l Section
1
g (99%) of the desired product as an oil: H NMR (CDCl3) δ
Gen er a l. Melting points were obtained on a Thomas-
Hoover Unimelt capillary apparatus and are not corrected. IR
spectra were obtained on a Perkin Elmer model 881 instru-
ment. NMR spectra were obtained on a General Electric QE
300 instrument using tetramethylsilane as an internal stan-
dard. Mass spectrum (FAB) was obtained using a MAT 90
instrument. Microanalysis was performed by the Midwest
Microlabs, Indianapolis, IN 46250. Chromatography refers to
flash chromatography in silica gel. A hand held pencil UV
light (Spectronics Model 11SC-1, 254 nm) with provisions for
limiting exposure to other parts of the animal and the user
was employed for the animal experiments.
Meth od s. The technique to expose the CT nerve will be
briefly desribed here: Monkeys (M. mulatta) were anesthetized
with an im injection of ketamine (400 mg/animal) and atropine
(0.5 mg/animal). They were then intubated and maintained
on a mixture of oxygen and halothane. Fluid was replenished
with iv 5% dextrose and lactated Ringers solution. The right
CT nerve was exposed through an incision along the man-
dibular angle between the rostal lobes of the partoid gland
and the mandibular bone. First, the tissue attached to the
mandibular angle was sectioned. Then blunt dissection was
used to follow the caudiomedial side of the pterygoid muscle
down to its origin at the pterygoid plate of the skull and to
the CT nerve. After the experiment, the wound was closed
with chrome nylon. The animals recovered, and the nylon
sutures were removed after 7-10 days.
The nerve impulse activity was recorded with a PAR 113
amplifier, monitored over a loud speaker and an oscilloscope,
and fed into a recorder (Gould ES 1000) and an IBM PC-AT
computer via a DAS-Keithly interface. The activity in the
whole nerve preparation was displayed as a summated signal
through an absolute value integrator. The summated signals
and a code related to each tastant on the tongue was fed in to
the computer. The computer program sampled the summated
response before, during, and after stimulation and displayed
it on an oscilloscope. The stimulation time and the sequence
of the tastants can be modified and controlled by the computer.
The stimulus name, its order, the maximum amplitude and
surface area of each response, the level of nerve activity before
each stimulation, and the time for each stimulation was
continually printed out during the experiment. An early
version of the stimulation equipment and recording technique
used has been described. The parameters measured on the
summated nerve trace has also been defined and discussed in
2.15 (s, 3H), 6.22 (s, 1H), 6.80, 6.90 (AB quartet, 4H, J ) 8.3
Hz), 7.18-7.36 (m, 10H); IR (Nujol) 2118 cm-1
(C21H19N5S) C, H, N.
.
Anal.
N-(4-Azidoph en yl)-N′-[(S)-r-ph en eth yl]-S-m eth ylisoth io-
u r ea (12). Using the above procedure thiourea 10 was
converted to isothiourea 12: yield 88%; 1H NMR (CDCl3) δ 1.51
(d, 3H, J ) 6.5 Hz), 2.24 (s, 3H), 4.63 (m, 1H), 6.84 and 6.93
(AB, 4H, J ) 8.7 Hz), 7.23-7.37 (m, 5H); 13C NMR (CDCl3) δ
14.2, 22.7, 52.2, 119.5, 119.6, 123.4, 126.0, 127.3, 128.7, 134.1,
143.7, 146.8. Anal. (C16H17N5S) C, H, N.
N-(4-Azid op h en yl)-N′-(d ip h en ylm eth yl)gu a n id in oa ce-
tic Acid (4). A solution of glycine (0.531 g, 7.078 mmol) and
NaOH (0.283 g, 7.078 mmol) in water (5 mL) was added to a
solution of the isothiourea (13, 0.88 g, 2.359 mmol) in ethanol
(25 mL), and the mixture was heated at reflux for 6 h and
then concentrated. The residue was dissolved in water (50
mL), and the solution was washed with CH2Cl2 (20 mL). The
pH of the aqueous layer was adjusted to 6. The precipitate
formed was collected, dried, and recrystallized from ethanol-
water to afford 0.440 g (47%) of the desired product as a pale
yellow powder: mp 167-168 °C dec; 1H NMR (DMSO-d6) δ
3.53 (s, 2H), 5.96 (s, 1H), 7.03 (s, 4H), 7.31 (s, 10H); 13C NMR
(DMSO-d6) δ 47.6, 59.5, 119.7, 120.2, 125.2, 127.3, 127.5, 127.8,
128.9, 129.0, 141.7, 153.9, 172.1; IR (Nujol) 2120 cm-1. Anal.
(C22H20N6O2‚0.75H2O) C, H, N.
N-(4-Azid op h en yl)-N′-[(S)-r-p h en eth yl]gu a n id in oa ce-
tic a cid (3). Using the above procedure, isothiourea 12 was
converted to 13: yield 81%; mp 184-186 °C dec; 1H NMR
(DMSO-d6) δ 1.43 (d, 3H), 3.52 (s, 2H), 4.82 (m, 1H), 7.03 and
7.12 (AB, 4H, J ) 8.7 Hz), 7.22-7.33 (m, 5H); IR (Nujol) 2120
cm-1. Anal. (C17H18N6O2) C, H, N.
p-Am in oben zh yd r yla m in e (16). Syn th esis of p-Am i-
n oben zh yd r yl Ketoxim e. A mixture of p-aminobenzophe-
none (50.0 g, 253.8 mmol) and hydroxylamine hydrochloride
(35.25 g, 506 mmol) in pyridine (600 mL) was heated at reflux
for 48 h. The pyridine was removed in vacuo, and the residue
was partitioned between water (400 mL) and dichloromethane
(1 L). The organic layer was dried (MgSO4) and concentrated
to afford 52 g (97%) of the desired oxime as a crystalline solid.
Red u ction of th e Ketoxim e. Shiny pieces of sodium (36.0
g, 1.5 mol) were added to a hot solution of the oxime (52.0 g,
0.245 mmol) in dry ethanol (600 mL). After all the sodium
had dissolved, the reaction mixture was cooled, diluted with
water (1 L), and extracted with ether (3 × 600 mL). The
combined organic layers were dried (MgSO4) and concentrated

Understanding the Mechanism of Sweet Taste
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4171
to afford 44.0 g (91%) of the desired amine as an oil. The
diamino compound was carried to the next stage without
further purification.
m -Am in oben zh yd r yla m in e (17) was synthesized using
a similar procedure starting with m-aminobenzophenone:
yield 84%; 1H NMR (CDCl3) δ 5.06 (s, 1H), 6.49-7.39 (m, 9H).
The diamino compound was carried to the next stage without
further purification.
dissolved in water (50 mL), and solution was washed with CH2-
Cl2 (20 mL). The pH of the aqueous layer was adjusted to 6.
The precipitate was collected, dried, and recrystallized from
ethanol-water to afford 3.0 g (90%) of the desired product as
pale yellow powder: mp 150-152 °C dec; 1H NMR (DMSO-
d6) δ 3.65 (s, 2H), 5.99 (s, 1H), 6.96 (d, 2H, J ) 8.5 Hz), 7.13
(d, 2H, J ) 8.5 Hz), 7.21-7.30 (m, 7H), 7.56 (d, 2H, J ) 8.6
Hz); 13C NMR (DMSO-d6) δ 47.5, 59.7, 106.0, 119.4, 119.6,
122.2, 127.5, 128.2, 128.3, 129.1, 129.3, 133.8, 139.2, 140.6,
N-(4′-Azid ob en zh yd r yl)-N′-(4-cya n op h en yl)t h iou r ea
(20): Syn t h esis of N-(4′-Am in ob en zh yd r yl)-N′-(4-cy-
a n op h en yl)th iou r ea (18). A mixture of p-cyanophenyl
isothiocyanate (35.55 g, 222.2 mmol) and p-aminobenzhydry-
lamine (44.0 g, 222.2 mmol) in acetonitrile (900 mL) was
heated at reflux for 2 h, and the reaction mixture was
concentrated. The residue was purified by chromatography
(ethyl acetate-hexane, 1:1) to afford the desired thiourea as
a pale yellow powder.
Con ver sion of N-(4′-Am in ob en zh yd r yl)-N′-(4-cya n o-
p h en yl)th iou r ea (18) to N-(4′-Azid oben zh yd r yl)-N′-(4-
cya n op h en yl)t h iou r ea (20). A solution of sodium nitrite
(2.10 g, 30.60 mmol) in water (50 mL) was added to a cooled
(0-5 °C) suspension of the thiourea (10.0 g, 27.90 mmol) in
glacial acetic acid (100 mL). The color of the reaction mixture
changed to deep yellow. After 15 min at this temperature, a
solution of sodium azide (10.0 g, 153.85 mmol) in water (50
mL) was added over a period of 10 min. The color of the
reaction mixture changed slowly to off-white, and the stirring
was continued for an additional 1 h. Water (200 mL) was
added to the reaction mixture, and the precipitate was filtered,
washed with water, and dried. Chromatography of the solid
(ethyl acetate-hexane, 3:7) afforded 5.2 g (49%) of the desired
azido compound as a gum which slowly solidified: mp 75-76
°C; 1H NMR (CDCl3) δ 6.95 (d, 2H, J ) 8.5 Hz), 7.08 (d, 1H, J
) 7.6 Hz), 7.18-7.33 (m, 7H), 7.40 (d, 2H, J ) 8.7 Hz), 7.50
(d, 2H, J ) 8.7 Hz); 13C NMR (CDCl3) δ 64.6, 79.5, 80.0, 110.9,
121.5, 122.3, 125.8, 130.3, 131.0, 131.8, 131.9, 136.2, 144.8,
182.5; IR (Nujol) 2122, 2227 cm-1. Anal. (C21H16N6S) C, H,
N.
144.1, 154.5, 172.2; IR (Nujol) 2120, 2226 cm-1
.
Anal.
(C23H19N7O2‚0.25H2O) C, H, N.
N-(3′-Azid ob en zh yd r yl)-N′-(4-cya n op h en yl)gu a n id i-
n oa cetic Acid (6). With isothiourea 23 as the starting
material, the guanidineacetic acid 6 was synthesized in 89%
yield: mp 144-145 °C dec; 1H NMR (DMSO-d6) δ 3.58 (s, 2H),
6.00 (s, 1H), 6.93 (d, 1H, J ) 7.9 Hz), 6.99 (s, 1H), 7.04 and
7.52 (AB, 4H, J ) 8.4 Hz), 7.11 (d, 1H, J ) 7.8 Hz), 7.2-7.32
(m, 5H); 13C NMR (DMSO-d6) δ 47.5, 59.7, 104.8, 118.2, 118.5,
119.7, 122.4, 124.4, 127.6, 128.0, 128.1, 128.9, 129.1, 130.7,
133.7, 140.1, 141.0, 143.7, 154.3, 172.4; IR (Nujol) 2120, 2220
cm-1. Anal. (C23H19N7O2‚H2O) C, H, N.
N-(p-Cya n op h en yl)-N′-[(R,S)-r-(3-p yr id yl)ben zyl]th io-
u r ea (25). A mixture of p-cyanophenyl isothiocyanate (18.8
g, 117.5 mmol) and (R,S)-R-(3-pyridyl)benzylamine11 (21.6 g,
117.4 mmol) in acetonitrile (400 mL) was stirred at ambient
temperature for 6 h to give a white precipitate. The precipitate
was filtered, washed with ether, and dried to afford 37.9 g
(94%) of the desired product as a white powder: mp 169 °C;
1H NMR (CDCl3) δ 6.84 (s, 1H), 7.25-7.38 (m, 6H), 7.53 and
7.85 (AB, 4H, J ) 8.5 Hz), 7.63 (m, 1H), 8.47 (dd, 1H, J ) 1.5
and 4.8 Hz), 8.56 (d, 1H, J ) 2.1 Hz); 13C NMR (CDCl3) δ 58.6,
105.8, 118.4, 121.1, 123.2, 127.5, 128.5, 132.2, 135.1, 136.9,
140.0, 143.4, 147.9, 148.4, 179.7. Anal. (C20H16N4S) C, H, N.
N-(p -Cya n op h en yl)-S-m et h yl-N′-[(R,S)-r-(3-p yr id yl)-
ben zyl]isoth iou r ea (26). Iodomethane (5.0 g, 35.21 mmol)
was added to a solution of the thiourea 25 (4.0 g, 11.63 mmol)
in acetone (100 mL), and the reaction mixture was stirred at
ambient temperature for 18 h. The reaction mixture was
concentrated, and the residue was partitioned between dichlo-
romethane (100 mL) and sodium hydroxide (1 N, 50 mL). The
organic layer was separated, dried (MgSO4), and concentrated
to afford a solid. Trituration of the solid with ethyl acetate
followed by concentration afforded a residue. Chromatography
(ethyl acetate) of the residue afforded 0.77 g (19%) of the
desired product as an oil: 1H NMR (CDCl3) δ 2.26 (s, 3H), 6.27
(s, 1H), 6.86 (d, 1H, J ) 7.4 Hz), 7.22-7.38 (m, 6H), 7.46 (d,
2H, J ) 8.4 Hz), 7.57 (s, 1H, J ) 7.9 Hz), 8.47 (d, 1H, J ) 4.7
Hz), 8.56 (d, 1H, J ) 1.6 Hz); 13C NMR (CDCl3) δ 14.3, 58.4,
105.2, 119.5, 122.4, 123.3, 127.4, 127.9, 128.8, 132.8, 134.8,
137.0, 140.3, 148.5, 148.7, 152.0. Anal. (C21H18N4S) C, H, N.
N-(3′-Azid ob en zh yd r yl)-N′-(4-cya n op h en yl)t h iou r ea
(21): Syn t h esis of N-(3′-Am in ob en zh yd r yl)-N′-(4-cy-
a n op h en yl)th iou r ea (19). This was synthesized starting
with m-aminobenzhydrylamine and employing the procedure
1
described above: mp 90 °C; H NMR (CDCl3) δ 6.7 (m, 1H),
6.87 (s, 1H), 6.93-7.02 (m, 2H), 7.20-7.35 (m, 6H), 7.48 (AB,
4H, J ) 1.8 Hz); 13 C NMR (CDCl3) δ 62.0, 108.2, 118.2, 118.5,
118.8, 123.0, 124.1, 127.7, 128.4, 129.2, 130.4, 133.6, 140.8,
142.0, 179.7; IR (Nujol) 2120, 2223 cm-1. Anal. (C21H16N6S)
C, H, N.
N-(4′-Azid oben zh yd r yl)-N′-(4-cya n op h en yl)-S-m eth yl-
isoth iou r ea (22). Iodomethane (4.4 g, 30.1 mmol) was added
to a solution of the thiourea (20, 4.0 g, 10.42 mmol) in acetone
(100 mL), and the reaction mixture was stirred at ambient
temperature for 18 h. The reaction mixture was concentrated,
and the residue was partitioned between dichloromethane (100
mL) and sodium hydroxide (1 N, 20 mL). The organic layer
was separated, dried (MgSO4), and concentrated to afford 3.40
g (82%) of the desired product as an oil which solidified upon
cooling: mp 109-110 °C; 1H NMR (CDCl3) δ 2.26 (s, 3H), 6.18
(s, 1H), 6.86 & 7.35 (AB, 4H, J ) 8.1 Hz), 7.01, 7.50 (AB, 4H,
J ) 8.5 Hz), 7.23-7.47 (m, 5H); 13C NMR (CDCl3) δ 14.4, 59.9,
105.5, 119.3, 119.7, 122.7, 127.4, 127.8, 128.9, 133.1, 138.2,
139.3. Anal. (C22H18N6S) C, H, N.
N-(p-Cyan oph en yl)-N′-[(R,S)-r-(3-pyr idyl)ben zyl]gu an i-
d in oa cetic Acid (24). A solution of glycine (0.566 g, 7.53
mmol) and NaOH (0.30 g, 7.53 mmol) in water (4 mL) was
added to a solution of the isothiourea 26 (0.90 g, 2.51 mmol)
in ethanol (40 mL), and the mixture was heated at reflux for
6 h and then concentrated. The residue was dissolved in water
(50 mL) and washed with CH2Cl2 (50 mL), and the pH of the
aqueous phase was adjusted to 6. The aqueous solution was
lyophilized, and the solid obtained was triturated with ethanol.
Concentration of the ethanolic solution afforded 0.8 g (82%)
1
N-(3′-Azid oben zh yd r yl)-N′-(4-cya n op h en yl)-S-m eth yl-
isoth iou r ea (23). With thiourea 21 as the starting material,
using the procedure above, 23 was synthesized: yield 71%;
of the desired product as a yellow solid: mp 126 °C dec; H
NMR (DMSO-d6) δ 3.82 (s, 2H), 6.24 (s, 1H), 7.19 and 7.59
(AB quartet, 4H, J ) 8.5 Hz), 7.23-7.36 (m, 6H), 7.61 (d, 1H,
J ) 6.5 Hz), 8.4 (dd, 1H, J ) 4.5, 1.1 Hz); 13C NMR (DMSO-
d6) δ 56.2, 58.0, 106.5, 109.1, 122.4, 127.6, 128.3, 129.1, 133.7,
135.6, 136.1, 139.6, 143.0, 149.0, 154.6, 170.9. Anal. Calcd
1
mp 135 °C; H NMR (CDCl3) δ 2.27 (s, 3H), 6.18 (s, 1H), 6.87
(d, 2H, J ) 8.2 Hz), 6.93-7.06 (m, 3H), 7.23-7.36 (m, 6H),
7.59 (d, 2H, J ) 2 Hz); 13C NMR (CDCl3) δ 17.2, 63.1, 108.3,
120.7, 120.9, 122.4, 125.5, 126.6, 130.2, 130.8, 131.7, 132.9,
135.9, 143.3. Anal. (C22H18N6S) C, H, N.
for
C22H19N6O2: MW, 385.1617. Found: 386.1627 (HR-
FABMS, M + 1).
N-(4′-Azid ob en zh yd r yl)-N′-(4-cya n op h en yl)gu a n id i-
n oa cetic Acid (5). A solution of glycine (3.0 g, 23.44 mmol)
and NaOH (0.94 g, 23.44 mmol) in water (10 mL) was added
to a solution of the isothiourea (22, 3.0 g, 7.812 mmol) in
ethanol (100 mL), and the reaction mixture was heated at
reflux for 6 h and then concentrated. The residue was
Ack n ow led gm en t. The authors gratefully acknowl-
edge Dr. J . Christopher Culberson for the computer
programs used in the collection and the analysis of the
data from the electrophysiological experiments.

4172 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Nagarajan et al.
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J M960349Q