Ethnobotanical-directed discovery of the antihyperglycemic properties of cryptolepine: Its isolation from Cryptolepis sanguinolenta, synthesis, and in vitro and in vivo activities

Article abstract of DOI:10.1021/jm9704816

Using an ethnobotanical approach in combination with in vivo-guided fractionation as a means for lead discovery, cryptolepine was isolated as an antihyperglycemic component of Cryptolepis sanguinolenta. Two syntheses of cryptolepine, including an unambiguous synthesis, are reported. The hydroiodide, hydrochloride, and hydrotrifluoromethanesulfonate (hydrotriflate) salts of cryptolepine were synthesized, and a comparison of their spectral properties and their in vitro activities in a 3T3-L1 glucose transport assay is made. Cryptolepine and its salt forms lower blood glucose in rodent models of type II diabetes. While a number of bioactivities have been reported for cryptolepine, this is the first report that cryptolepine posesses antihyperglycemic properties.

Full text of DOI:10.1021/jm9704816

8
94
J . Med. Chem. 1998, 41, 894-901
Eth n obota n ica l-Dir ected Discover y of th e An tih yp er glycem ic P r op er ties of
Cr yp tolep in e: Its Isola tion fr om Cr yptolepis sa n gu in olen ta , Syn th esis, a n d in
Vitr o a n d in Vivo Activities†
Donald E. Bierer,* Diana M. Fort, Christopher D. Mendez, J ian Luo, Patricia A. Imbach, Larisa G. Dubenko,
Shivanand D. J olad, R. Eric Gerber, J oane Litvak, Qing Lu, Pingsheng Zhang, Michael J . Reed, Nancy Waldeck,
§
Reimar C. Bruening, Ben K. Noamesi, Richard F. Hector, Thomas J . Carlson, and Steven R. King
Shaman Pharmaceuticals, Inc., 213 East Grand Avenue, South San Francisco, California 94080
Received J uly 24, 1997
Using an ethnobotanical approach in combination with in vivo-guided fractionation as a means
for lead discovery, cryptolepine was isolated as an antihyperglycemic component of Cryptolepis
sanguinolenta. Two syntheses of cryptolepine, including an unambiguous synthesis, are
reported. The hydroiodide, hydrochloride, and hydrotrifluoromethanesulfonate (hydrotriflate)
salts of cryptolepine were synthesized, and a comparison of their spectral properties and their
in vitro activities in a 3T3-L1 glucose transport assay is made. Cryptolepine and its salt forms
lower blood glucose in rodent models of type II diabetes. While a number of bioactivities have
been reported for cryptolepine, this is the first report that cryptolepine posesses antihyperg-
lycemic properties.
In tr od u ction
model led to the discovery of an indoloquinoline alkaloid,
cryptolepine (1), as the active constituent.
1
8,19
The enormous biodiversity of the tropical rainforest
has provided indigenous cultures around the globe with
a deep and diversified knowledge about the use of plants
for medicinal purposes. As part of our effort to access
and record these oral healing traditions before they
disappear,1 we were interested in the bioactive con-
stituent present in Cryptolepis sanguinolenta respon-
sible for its observed use for treating associated symp-
toms of diabetes.3 Cryptolepis sanguinolenta (Lindl.)
Schlechter, a member of the Asclepiadaceae family and
Periplocoideae subfamily, is a shrub indigenous to
Cryptolepine is a rare example of a natural product
whose synthesis was reported prior to its isolation from
nature. Cryptolepine was first synthesized in 1906 by
2
0-22
Fichter and co-workers.
Its isolation was first
2
3
reported by Clinquart in 1929 from Cryptolepis trian-
gularis N. E. Br. and soon thereafter by Delvaux.2
Cryptolepine was first isolated from C. sanguinolenta
by Gell e´ rt from root samples obtained in Nigeria27 and
again by Dwuma-Badu and co-workers from root samples
,2
4-26
2
8,29
obtained in Ghana.
Cryptolepine has also been
isolated as the major alkaloid from Sida sp. (Malvaceae),
4
tropical West Africa with a history of ethnomedical use.
medicinal plants used by indigenous practitioners of Sri
In Guinea Bissau, the roots are sold locally, its decoction
is used in the treatment of hepatitis, and its leaves have
been used for the treatment of malaria or powdered as
a cicatrizant of wounds.9-12 Root decoctions have been
3
0
Lanka. Cryptolepine and its hydrochloride salt pos-
sess a number of reported bioactivities, including anti-
3
0,31
32-36
37-39
microbial,
antibacterial,
antiinflammatory,
3
9-41
antipyretic,41 antimuscarinic,
noradrenergic receptor antagonis-
42
antihypertensive,
antithrombotic,
used in traditional West African medicine as a remedy
4
3-46
for colic and stomach aches,1
3,14
and in Ghana aqueous
3
9,47
and vasodilative40,48 properties. Cryptolepine
tic,
also possesses significant antimalarial activity
is being pursued in some West African nations for
extracts of the roots and stems have been used clinically
4
9-51
and
for over two decades in the therapies of malaria and of
urinary and upper respiratory tract infections.1
5-17
Our
5
2,53
possible therapeutic use.
own ethnobotanical field research indicated that the
aqueous extract of the root of C. sanguinolenta is used
by traditional healers in Ghana to treat a variety of
symptoms that could occur in a diabetic patient includ-
ing fungal infections, pain, and inflammation. An
alcohol extract of the root is also used in Ghana as a
tonic to strengthen the metabolism. On the basis of this
information, a 40-kg sample of C. sanguinolenta roots
was collected. In vivo-guided fractionation using a non-
insulin-dependent diabetes mellitus (NIDDM) mouse
We wish to report a new bioactivity for cryptolepine,
namely, its ability to lower blood glucose in NIDDM
animal models. We report its isolation from the roots
of C. sanguinolenta and three syntheses of cryptolepine
and its salt forms.
Isola tion
For initial screening purposes a dichloromethane
extract and a hot water extract obtained from the roots
of C. sanguinolenta were prepared and tested at 1 g/kg
in genetically altered obese diabetic mice (designated
C57BL/Ks-db/db, or db/db). Both extracts lowered blood
glucose levels approximately 100 mg/dL 24 h postdose
with no significant effect on food intake relative to the
control. Using in vivo testing as a guide for fraction-
†Dedicated to Professor Henry Rapoport on the occasion of his 79th
birthday.
*
Address correspondence to this author at Shaman Pharmaceuti-
cals. E-mail: dbierer@shaman.com.
§
Professor Ben K. Noamesi is at the University of Science and
Technology, Kumasi, Ghana.
S0022-2623(97)00481-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 02/24/1998

Antihyperglycemic Properties of Cryptolepine
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 6 895
Sch em e 1a
tolepine to its free base 1.27 We preferred an alternative
to this standard method. Small amounts of unreacted
quindolines 8 or 23 were generally present as an
impurity (1-2%) following alkylation with methyl iodide
or methyl triflate, which could not be removed by this
extractive method. Conversion to the free base with
concomitant removal of the quindoline impurity was
conveniently accomplished in the following manner. The
cryptolepine salt was adsorbed onto sodium carbonate
and loaded onto a short column of basic alumina as a
dry powder. Elution with ethanol-free chloroform58
removed the quindoline impurity. Elution with a 1-2%
methanol in chloroform solution eluted the cryptolepine
free base (1) as a purple band, which could be isolated
by simple evaporation of the purple fractions. Alter-
natively, this purple solution was treated with an
ethereal 1 M HCl solution to provide cryptolepine
hydrochloride (4) as a bright-yellow solid.
Recognizing that the above synthetic route contained
a regioisomeric ambiguity, we also synthesized cryptol-
epine (1) by a route which definitively established the
location of the methyl substituent at N-5. Full details
are described in the Supporting Information section. The
N-methyl regioisomer of cryptolepine (9) was also
synthesized (Scheme 1), and the spectral data for this
compound are also shown in the Supporting Information
section along with the spectral data for salts 2-4. The
a
(
a) KOH, H2O, N2; (b) Ph2O, 255 °C, 6 h; (c) CH3I, MeOH,
bomb, 120 °C; (d) KOH, BaO; (e) CH3I; (f) Na2CO3.
ation, the dichloromethane fraction was extracted fur-
ther with aqueous Na2CO3, resulting in a solid residue.
This material was extracted with 90% ethanol and
purified over Dowex 50 × 8-400. Following HPLC, a
pure alkaloid was obtained and identified as cryptol-
epine (1) on the basis of one-bond and long-range proton-
detected heteronuclear correlation experiments (HMQC
and HMBC). These assignments are in agreement with
those reported in the literature (see Supporting Infor-
1
13
H and C NMR spectral data for synthetic 1 matched
the spectral data from the natural product 1. Co-NMR
and co-HPLC experiments on the synthetic and natural
cryptolepine provided further structural confirmation.
2
9,54,55
mation).
For further studies, scale up using an
alternative procedure led to a yield of 1.5 g/kg (see the
Experimental Section).
Syn th esis
In Vitr o Stu d ies
Because our planned biological evaluation using in
vivo models required multigram quantities of cryptol-
epine, we explored a synthetic approach as an alterna-
tive to isolation of the natural product. Our first
Cryptolepine and its salt forms were tested for their
ability to stimulate glucose transport in 3T3-L1 adipo-
cytes, a recognized in vitro model that represents an
important mode of action for glucose utilization and
5
6
approach utilized the procedures of Holt and Petrow
and Deguitis and Ezyarskaite57 with some modification
Scheme 1). Reaction of 3-indolyl acetate with isatin
59
disposal in mammals.
Cryptolepine (1) showed an
increasing ability to stimulate glucose transport begin-
ning at 3 µM with increasing concentration. Hydro-
chloride salt 4 gave a similar result in this assay.
Hydroiodide salt 2 showed increasing activity at 3 and
(
under an inert atmosphere gave quindoline-11-carboxy-
lic acid (7) in 75% yield. Decarboxylation in diphenyl
ether gave quindoline (8) in 90% yield. Alkylation of
the N-5 nitrogen with methyl iodide was accomplished
1
0 µM, but the activity at higher concentrations leveled
off, possibly due to iodide toxicity. Regioisomer 9 was
inactive in this assay, indicating that the location of the
methyl group at the N-5 postion was important for
bioactivity. The results are shown in Figure 1.
The ability of cryptolepine hydrochloride (4) to en-
hance the effect that insulin has on 2-deoxyglucose
uptake in 3T3-L1 adipocytes was also measured, and
the result is shown in Figure 2. Glucose transport
stimulation was shown to increase beginning at 10 µM
with increasing concentration.
2
0
using the method of Fichter and Boehringer.
This
method required the use of a bomb but gave good yields
of the hydroiodide salt of cryptolepine. A more conve-
nient method amenable to larger scale reactions in-
volved the use of methyl triflate as the alkylating agent,
which was accomplished at room temperature and
afforded near quantitative yields of the hydrotrifluo-
romethanesulfonate (hydrotriflate) salt of cryptolepine.
This procedure was the method of choice for preparing
multigram quantities of cryptolepine hydrotriflate and,
ultimately, cryptolepine.
In Vivo Stu d ies (d b/d b Mice)
Standard basic extraction methods could be used to
convert the hydroiodide and hydrotriflate salts of cryp-
Cryptolepine lowered mean plasma glucose levels in
db/db mice 20% from predose levels 3 h postdose and

8
96 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 6
Bierer et al.
F igu r e 1. In vitro effect of chronic treatment of cryptolepine
1), hydroiodide salt 2, hydrochloride salt 4, and regioisomer
on glucose transport in 3T3-L1 adipocytes (absence of
(
9
insulin).
F igu r e 3. In vivo effects of cryptolepine hydrochloride (4) on
plasma glucose concentrations in db/db mice.
Ta ble 2. Effects of Cryptolepine Hydrochloride (4) on Body
Weight and Food Intake in db/db Mice
mean body weight
food intake
(g/mouse)
(g/mouse/day)
treatment
vehicle
0 h
24 h
48 h
0-24 h 24-48 h
38.8 ( 0.2 38.6 ( 0.2 39.3 ( 0.2
5.2
3.3
2.4
7.0
3.8
2.4
2
3
0 mg/kg 38.0 ( 0.4 37.9 ( 0.4 37.4 ( 0.5
0 mg/kg 37. ( 0.8 36.9 ( 0.8 35.5 ( 0.9
Cryptolepine hydrochloride (4) was chosen for further
study and was effective at lowering mean plasma
glucose levels 16% and 47% from predose levels, 3 days
postdose, respectively, at 20 and 30 mg/kg (Figure 3).
At these doses, the reduction in food consumption was
6
0
not as significant but was still apparent (Table 2). To
determine the euglycemic effect that cryptolepine pos-
sessed, we tested hydrochloride salt 4 in C57BL/ks lean
mice at 30 mg/kg/day. After day 3, there was a 10%
drop in mean plasma glucose concentration with a
similar reduction in food consumption that was observed
in the db/db mice at 30 mg/kg/day (Table 3).
F igu r e 2. In vitro effect of chronic treatment of cryptolepine
hydrochloride (4) on glucose transport in 3T3-L1 adipocytes
(presence of insulin).
Ta ble 1. Effects of Cryptolepine and Its Salts on Plasma
Glucose, Body Weight, and Food Intake in db/db Mice
To further separate the anorexigenic effect from the
antihyperglycemic effect of cryptolepine hydrochloride
plasma glucose
food intake
(
% change of
predose)
mean body weight
(g/mouse)
(g/mouse/
day)
(4), we performed two experiments. In the first experi-
ment, db/db mice were divided into a control group and
a treatment group. After the mice were dosed with
vehicle or 30 mg/kg hydrochloride salt 4, respectively,
food was removed from both groups and plasma glucose
levels were measured over time. From the data shown
in Figure 4, a statistically significant difference in
plasma glucose levels was observed at 3, 6, and 12 h
postdose. In the second experiment, db/db mice were
divided into a control group given free access to food
and two pair-fed groups treated with either vehicle or
30 mg/kg hydrochloride salt 4. Plasma glucose concen-
trations were measured initially and 3 days postdose.
While glucose concentrations were lower than control
in both pair-fed groups, mice receiving cryptolepine
hydrochloride treatment had significantly lower plasma
glucose concentrations than did the vehicle-treated
treatment
vehicle
3 h
-3.6
24 h
0 h
24 h
48 h
8.6 41.3 ( 0.6 41.5 ( 0.8
4.0
1.0
0.9
1.0
4.4
1
4
2
9
, 100 mg/kg -19.7 -30.0 39.4 ( 1.5 38.7 ( 1.4
, 100 mg/kg -16.4 -43.1 41.0 ( 0.5 39.8 ( 0.5
, 100 mg/kg -11.5 -22.5 42.7 ( 1.3 41.9 ( 1.6
, 100 mg/kg
15.3
14.1 41.4 ( 1.3 42.0 ( 1.5
reached 30% 24 h postdose at 100 mg/kg. Hydroiodide
salt 2 and hydrochloride salt 4 lowered mean plasma
glucose levels 3 h postdose and reached 22.5% and
4
3.1% reductions, respectively, from predose levels 24
h postdose at 100 mg/kg. These effects were ac-
companied by significant reductions in food consumption
and mean body weight (Table 1). In this model, regioi-
somer 9 was inactive at 100 mg/kg, indicating the
importance of the N-5 methyl substituent.
6
0
group (Table 4).

Antihyperglycemic Properties of Cryptolepine
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 6 897
Ta ble 3. Effects of Cryptolepine Hydrochloride (4) on Plasma Glucose, Body Weight, and Food Intake in Normal (C57BL/ks) Mice
plasma glucose (mg/dL)
mean body weight (g/mouse)
24 h
food intake (g/mouse/day)
treatment
vehicle
predose
day 3
0 h
48 h
0-24 h
24-48 h
184.5 ( 3.7
184.5 ( 4.7
189.1 ( 7.0
170.7 ( 4.4
24.3 ( 0.5
23.7 ( 0.5
24.6 ( 0.5
23.3 ( 0.6
24.6 ( 0.5
22.7 ( 0.5
4.1
2.7
4.2
2.3
a
3
0 mg/kg
a
p < 0.05 vs vehicle (Student’s t-test, n ) 8).
F igu r e 5. In vivo effects of cryptolepine hydrochloride (4) on
serum glucose concentrations in fructose-fed STZ-treated rats.
F igu r e 4. In vivo time course study of cryptolepine hydro-
chloride (4) in db/db mice in the absence of food.
Ta ble 4. Pair-Fed Experiment of Cryptolepine Hydrochloride
(4) in db/db Mice
plasma glucose concentrationsb
3 days postdose (mg/dL)
treatmenta
control
pair-fed control
pair-fed 4, 30 mg/kg
520 ( 20
439 ( 28
358 ( 23
c
a
n ) 8. b Initial plasma glucose concentrations on all groups
c
were 495 ( 36 mg/dL. p < 0.05.
In Vivo Stu d ies (F r u ctose-F ed STZ-Tr ea ted
Ra ts)
F igu r e 6. In vivo effects of cryptolepine hydrochloride (4) on
serum triglyceride concentrations in fructose-fed STZ-treated
rats.
The effect that cryptolepine hydrochloride had on
serum glucose levels in a fructose-fed STZ-treated rat
model of NIDDM was investigated. The fructose-fed
STZ-treated rats used in this study had significantly
elevated glucose and triglyceride concentrations com-
pared to chow-fed controls (Figures 5 and 6, p < 0.05).
Cryptolepine hydrochloride (4) (30 mg/kg) lowered
serum glucose concentrations in fructose-fed STZ-
treated rats by 16%, 34%, and 45% on days 1, 2, and 3
of dosing, respectively (Figure 5, p < 0.05 on days 2 and
Ta ble 5. Effect of Cryptolepine Hydrochloride (4) on Food
Consumption and Body Weight in Fructose-Fed STZ-Treated
Rats
food
consumption
body weight, (g)
treatment group
predose
day 3
∆
(g/day)
chow
207 ( 6 244 ( 2 37 ( 5
193 ( 5 211 ( 6 18 ( 3
201 ( 4 223 ( 4 21 ( 2
22 ( 1
25 ( 1
18 ( 2
fructose/STZ
fructose/STZ +
cryptolepine
HCl, 30 mg/kg
3
). In addition, 4 lowered serum triglyceride concentra-
tions by 30%, 39%, and 69% on days 1, 2, and 3 of
dosing, respectively (Figure 6, p < 0.05 at all time
points). Glucose and triglyceride lowering was ac-
companied by a significant decline in food consumption
Con clu sion
Using an ethnobotanical approach and in vivo-guided
fractionation, the active component from the roots of C.
sanguinolenta was found to be cryptolepine (1). Two
syntheses, including an unambiguous synthesis which
(29%, p < 0.05), but body weight gain over the course
of the experiment was not altered by the cryptolepine
hydrochloride treatment (Table 5).

8
98 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 6
Bierer et al.
provides structural proof, are reported. Three salt forms
of cryptolepine (2-4) were prepared, and their spectral
data were compared to that of the parent free base. The
antihyperglycemic properties of cryptolepine and its salt
forms are reported and measured in NIDDM animal
models and using an in vitro glucose transport assay.
Hydrochloride salt 4 demonstrated greater activity in
vivo and in vitro than hydroiodide salt 2. When
compared to cryptolepine (1), hydrochloride salt 4
showed a similar in vitro profile but displayed greater
efficacy in vivo. The presence of the methyl substituent
at N-5 appears to be critical for activity, as the N-10
methyl regioisomer 9 was inactive in vitro and in vivo.
While cryptolepine hydrochloride (4) lowers blood glu-
cose in NIDDM animals models with reduced food
intake, the results of a fasting time course study and a
pair-fed study in db/db mice seem to indicate that this
antihyperglycemic effect is separate from the anorexi-
nitrogen at room temperature for 3 days. The reaction mixture
was diluted with water (250 mL), and then oxygen was bubbled
through the mixture while it was heated for 20 min at 75-80
°
C. The reaction mixture was filtered hot, the filtrate was
diluted with ethanol (750 mL) to dissolve the formed precipi-
tate, and then concentrated HCl was added to adjust the pH
of the solution to 4. The precipitate was filtered, washed with
hot water and ethanol, and then dried (Na
g (75%) of the title compound, mp >250 °C (lit. mp 330-337
C dec): 1H NMR (DMSO-d
2 4
SO ), yielding 28.2
5
6
°
7
)
6
) δ 11.39 (s, 1H, NH), 9.14 (d, J )
.6, 1H), 8.35 (d, J ) 8.0, 1H), 8.25 (d, J ) 7.2, 1H), 7.77 (d, J
1
3
8.4, 1H), 7.67 (m, 4H), 7.32 (dd, J ) 8.0, 8.0, 1H); C NMR
) δ 167.94, 147.15, 144.43, 143.42, 132.12, 130.20,
29.41, 126.37, 125.77, 125.27, 123.82, 121.22, 120.60, 120.09,
(DMSO-d
6
1
+
112.47; MS (EI, m/z) 262 (M ). Anal. (C16
C, H, N.
H
10
N
2
O
2
2
‚0.5H O)
Qu in d olin e (8). A mixture of 7 (29.2 g, 0.111 mol) and
diphenyl ether (300 mL) was refluxed for 6 h, cooled to 30 °C,
and then diluted with petroleum ether (250 mL). The pre-
cipitate which formed was filtered and washed with petroleum
ether, yielding 22.1 g (91%) of the title compound. A portion
of this material was further purified by LPLC (ethyl acetate-
6
0
genic effect. The results described herein provided the
6
1
57
1
basis for a cryptolepine analogue program. Linkage
of these biological studies on cryptolepine hydrochloride
hexane, 1:3), mp 249-251 °C (lit. mp 251-252 °C): H NMR
(DMSO-d ) δ 11.43 (s, 1H, NH), 8.36 (d, J ) 7.8, 1H), 8.29 (s,
H), 8.19 (d, J ) 8.4, 1H), 8.10 (d, J ) 8.2, 1H), 7.67-7.53 (m,
6
1
4
1
1
2
(4) and results of a human study involving the aqueous
H), 7.27 (dd, J ) 7.4, 7.4, 1H); 13C NMR (DMSO-d
) δ 145.72,
6
extract of C. sanguinolenta will be published else-
44.03, 143.39, 132.44, 129.68, 128.68, 127.48, 126.71, 126.01,
1
9
where.
24.84, 121.34, 120.97, 119.33, 112.99, 111.49; MS (EI, m/z)
+
18 (M ).
Exp er im en ta l Section
1
0-Meth ylqu in d olin iu m Hyd r och lor id e a n d 10-Meth -
ylqu in d olin e (9). A mixture of powdered KOH (1.0 g, 17.8
mmol), BaO (2.8 g, 17.8 mmol), acetone (50 mL), and quindo-
line (8) (1 g, 4.58 mmol) was refluxed for 1 h and cooled to
room temperature, and then methyl iodide (1.42 mL, 3.25 g,
Isola tion of Cr yp tolep in e (1). Dry powdered roots (1.12
kg) of C. sanguinolenta were percolated in 1% acetic acid in
water (10 L) at room temperature for 48 h. The filtered
aqueous extract was extracted with chloroform (3 × 5 L), and
the chloroform layer was separated and then discarded. The
remaining aqueous extract was basified to pH 9 and extracted
three times with chloroform (3 × 5 L). The chloroform layer
was separated and concentrated under reduced pressure to
dryness to yield 2.24 g of a crude alkaloid extract. The crude
alkaloid extract was purified by a chromatotron (2-mm Si gel
plates, solvent system: toluene-ethyl acetate-diethylamine-
methanol, 60/25/10/5) to yield 1.5 g (0.13%) of cryptolepine as
2
2.9 mmol) was added. The mixture was refluxed for 4 h,
cooled, and filtered, and the filtrate was evaporated to dryness.
The residue was extracted with ether (2 × 50 mL), and the
4
ether solution was washed with water, dried (MgSO ), and
then filtered. A stream of HCl gas passed through the
solution, and the solid product was collected to give, after
drying, 0.9 g (73.2%) of 10-methylquindolinium hydrochloride
as a yellow solid.
2
7
The 10-methylquindolinium hydrochloride obtained above
dark-purple needles, mp 178-179 °C (lit. mp 175-178 °C).
2 3
(250 mg, 0.9 mmol) was shaken with an aqueous 5% Na CO
Spectral and chromatographic data of cryptolepine agree with
2
9,54,55
solution (50 mL), extracted with ethyl ether (2 × 50 mL), and
reported values.
found 232.0994.
Gen er a l Exp er im en ta l. Benzene and toluene were dis-
tilled from CaH . Anhydrous DMF, dioxane, and methanol
12 2
HRMS (EI) calcd for C16H N 232.1000,
purified by LPLC (ethyl acetate-hexane, 1:6) to give 100 mg
(
45.5%) of 9, mp 115-116 °C: see Supporting Information for
+
NMR and UV data; MS (EI, m/z) 232 (M ). Anal. (C16
12 2
H N )
2
C, H, N.
were obtained from Aldrich. Methyl triflate was distilled prior
to use and stored in a Schlenk flask under nitrogen. All other
reagents were used as received. Moisture-sensitive reactions
were done under a nitrogen atmosphere, using dry solvents;
air-sensitive reactions were done under a nitrogen atmosphere.
Thin-layer chromatography for synthetic intermediates was
performed on E. Merck 230-400-mesh, 200-µm, silica gel
plates. Thin-layer chromatography for cryptolepine was per-
formed on E. Merck neutral alumina plates. Low-pressure
liquid chromatography (LPLC) for cryptolepine intermediates
was performed on E. Merck 230-400-mesh silica gel using
nitrogen pressure unless otherwise noted. Chromatography
on cryptolepine and its salts was done on Fisher Activity I
5
-Meth ylqu in d olin iu m Hyd r oiod id e (2) (Cr yp tolep in e
Hyd r oiod id e). A mixture of quindoline (8) (2.0 g, 9.2 mmol),
methanol (5 mL), and methyl iodide (880 µL, 14.1 mmol) was
heated in a Parr bomb at 120 °C for 4 h. After cooling, the
brown precipitate (2.5 g, 75.5%) was filtered and washed with
ether. Recrystallization from water gave 1.65 g (50%) of 2 as
2
7
bright-yellow crystals, mp 283.5-284 °C (lit. mp 284-286
°
C): see Supporting Information for NMR and UV data; MS
+
(
EI, m/z) 232 (M ). Anal. (C16
5-Meth ylqu in d olin e (1) (Cr yp tolep in e). Hydroiodide 2
(1.5 g, 4.2 mmol) was shaken with a 5% solution of Na CO
13 2
H N I) C, H, N.
2
3
(100 mL) and then extracted with chloroform (2 × 200 mL).
Sodium carbonate was added to the solution, the mixture was
concentrated, and the adsorbed product was loaded onto a
short column of basic alumina. Elution with chloroform to
elute the quindoline impurity followed by further elution with
1-2% methanol in chloroform gave 0.50 g (51.2%) of 1 as a
5
8
1
basic alumina using ethanol-free chloroform (see note).
H
1
3
and C NMR were recorded at 400 and 100 MHz, respectively,
with NMR shifts being expressed in ppm downfield from
internal TMS. NMR assignments were determined on the
basis of COSY, HMQC, and HMBC experiments, and NMR
coupling constants are reported in hertz. Mass spectrometry
was performed on a Kratos MS 50 spectrometer. Melting
points are uncorrected.
2
7
purple solid, mp 178-180 °C (lit. mp 175-178 °C): see
Supporting Information for NMR and UV data. Anal.
(C16
H
12
N
2
2
‚1.5H O) C, H, N.
Qu in d olin e-11-ca r boxylic Acid (7). In a three-necked
flask containing indolyl acetate (25 g, 0.143 mol) was added,
under nitrogen with shaking, a cooled solution of isatin (21.25
g, 0.144 mol) in aqueous KOH (125.5 g of KOH in 575 mL of
water). The reaction mixture was stirred vigorously under
Cr yp tolep in e Hyd r otr iflu or om eth a n esu lfon a te (3). A
suspension of 8 (11 g, 50.4 mmol) in anhydrous toluene (100
mL) was treated with methyl triflate (11.0 g, 97.2 mmol, 16
mL) at room temperature for 24 h. The mixture was diluted
with ether (200 mL) and filtered, and then the solid product

Antihyperglycemic Properties of Cryptolepine
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 6 899
was washed with ether and dried, yielding 18.3 g (94.8%) of 3
as a yellow solid, mp 241-243 °C: see Supporting Information
incubated for 30 min at 37 °C. To assess the effects of the
compounds on glucose transport, 2-deoxy-D-glucose uptake (a
nonmetabolizable analogue of glucose) was measured in the
presence of insulin stimulation. Glucose transport assays were
+
for NMR and UV data; MS (EI, m/z) 232 (M ). Anal.
(
C
17
H
13
N
2
F
3
O
3
S) C, H, N.
-Meth ylqu in d olin iu m Hyd r och lor id e (4) (Cr yp tol-
ep in e Hyd r och lor id e). Cryptolepine salt 3 (18.3 g) was
shaken with 5% Na CO solution (500 mL) and extracted with
3
5
initiated by the addition of 2-deoxy-D-[ H]glucose (0.5 mCi/mL,
100 mM final concentrations) to each well followed by incuba-
tion for 10 min at 22 °C. Assays were terminated by aspirating
the media and rapidly washing the monolayer two times with
ice-cold phosphate-buffered saline solution. Cell monolayers
were solubilized in 0.1 N NaOH and transferred to scintillation
vials, and radioactivity was determined by liquid scintillation
counting. All data were corrected for nonspecific hexose
uptake determined in parallel samples treated for 5 min with
200 mM cytochalasin B.
2
3
chloroform (3 L). The chloroform extract was dried, concen-
trated to a small volume (∼150 mL), and then acidified with
a 1 M solution of HCl in ether to give, after filtration and
2
7
drying, 11.4 g (84.4%) of 4, mp 268 °C (lit. mp 263-265 °C):
see Supporting Information for NMR and UV data; MS (EI,
+
m/z) 232 (M ). Anal. (C16
5
H
13
N
2
2
Cl‚1.5H O) C, H; N: calcd,
.45; found, 4.89.
In Vitr o Stu d ies. P r ep a r a tion of th e 3T3-L1 Ad ip o-
In Vivo Stu d ies Usin g d b/d b Mice. Genetically altered
obese diabetic mice (designated C57BL/Ks-db/db) were pur-
chased from the J ackson Laboratory (Bar Harbor, ME) and
served as experimental animals. Male animals between the
ages of 8 and 9 weeks were employed in the studies described
here. Animals were housed (4 mice/cage) under standard
laboratory conditions at 22 °C and given Purina rodent chow
and water ad libitum. Prior to treatment, blood was collected
from the tail vein of each animal. Mice that had plasma
glucose levels between 350 and 600 mg/dL were used. Each
treatment group consisted of eight mice that were distributed
so that mean glucose levels were equivalent in each group at
the start of the study. Diabetic designated C57BL/Ks-db/db
mice were dosed orally by gavage once daily for 1 or 2 days
with either vehicle, the experimental compound administered
at 30 or 100 mg/kg qd, or metformin at 250 mg (1510 mmol)/
kg qd (positive control). Metformin (1,1-dimethylbiguanide)
was purchased from Sigma Chemical Co. (St. Louis, MO).
Compounds were delivered in a vehicle formulation appropri-
ate for each compound, including components such as 0.25%
cytes. Murine 3T3-L1 preadipocytes (American Type Culture
Collection CL 173) were maintained in Dulbecco’s modified
Eagles medium (DMEM) containing 10% (v/v) supplemented
calf serum, antibiotics, and 25 mM glucose. Cells were seeded
in 24-well cluster plates (10 000 cells/well), grown to confluence
(
typically 5 days), and induced to differentiate 2 days post-
confluence (day 0) according to the standard protocol of Frost
5
9
and Lane. Following differentiation, adipocytes were main-
tained in DMEM containing 10% fetal bovine serum and
provided with fresh medium every 2-3 days. Adipocytes
employed in this study were used on days 7-10 postdifferen-
tiation. On the day before the experiment, adipocytes were
washed with phosphate-buffered saline and switched to serum-
free DMEM medium. Concentrated stock solutions of the test
compounds were freshly prepared in DMSO and diluted into
culture medium. The final concentration of DMSO was 0.5%
(
v/v) which was also included in basal and insulin controls.
In Vitr o Glu cose Tr a n sp or t Assa y (w ith ou t exog-
3
en ou sly a d d ed in su lin ): [ H]-2-Deoxy-D-glu cose Up ta k e
in Differ en tia ted 3T3-L1 Ad ip ocytes. Adipocytes were
treated (in triplicate) for 18 h with a test compound at 0.1,
(w/v) carboxymethylcellulose, 1% (v/v) Tween 60, and up to
1
0% (v/v) dimethyl sulfoxide (DMSO) in a volume of 10 mL/
0
.3, 1, 3, 10, 30, and 100 µM final concentrations. Following
kg. Blood was sampled from the tail vein 3 h postdosing and
analyzed for plasma glucose levels. Individual body weights
and mean food consumption (each cage) were also measured
after 24-27 h. Plasma glucose levels were determined colo-
rimetrically using a glucose oxidase assay (Sigma Chemical
Co.). Significant differences between groups (comparing com-
pound-treated to vehicle-treated) were evaluated using analy-
sis of variance and Fisher’s post-hoc test.
overnight (18 h) treatment, the cell monolayers were washed,
and the medium was switched to Krebs-Ringer Hepes (KRH)
buffer. Compound-treated adipocytes were given the insulin
vehicle KRH/1% bovine serum albumin (KRH/1% BSA) (con-
taining no insulin). The final concentration of KRH/1% BSA
was 4%, which was also included in the basal control. For
the insulin control adipocytes, a concentrated porcine insulin
stock was freshly diluted into KRH/1% BSA. The final
concentration of insulin in the insulin control was 0.5 nM in
In Vivo Stu d ies Usin g F r u ctose-F ed STZ-Tr ea ted Ra ts.
Male Sprague-Dawley rats at 8 weeks of age were purchased
from Charles River Laboratories (Hollister, CA) and served
as the experimental animals. Animals were housed (4 rats/
cage) under standard laboratory conditions at 22 °C and
provided with Purina rodent chow and water ad libitum.
Animals were injected with streptozotocin (STZ; 55 mg/kg iv;
Sigma Chemical Co.) and placed on a diet containing 60%
fructose (Harlan Teklad, Madison, WI) for 1 week. Nonin-
jected animals maintained on normal chow diet (n ) 19) served
as nondiabetic controls. Prior to treatment, blood was collected
from the tail vein of each animal. Rats that had serum glucose
levels between 250 and 350 mg/dL were considered diabetic
and were sorted into two groups (n ) 14) with equivalent
glucose concentrations. Diabetic rats were dosed orally by
gavage once daily for 3 days with either vehicle or cryptolepine
hydrochloride (4) (30 mg/kg). Control nondiabetic animals
were dosed with vehicle alone. Cryptolepine hydrochloride
was administered in 2% (v/v) DMSO in a volume of 10 mL/kg.
Blood was sampled from the tail vein 3 h postdosing on each
dosing day and analyzed for glucose and triglyceride levels.
Individual body weights and food consumption (each cage)
were also measured each day. Serum glucose (glucose oxidase
method) and triglyceride (GPO-trinder method) levels were
determined colorimetrically using Sigma diagnostic kits (Sigma
Chemical Co.). Significant differences between groups (com-
paring compound-treated to vehicle-treated) were evaluated
using analysis of variance and Fisher’s post-hoc test. A p value
less than 0.05 was considered significant.
4
% KRH/1% BSA. The plates were then incubated for 30 min
at 37 °C. To assess the effects of the compounds on glucose
transport, 2-deoxy-D-glucose uptake (a nonmetabolizable ana-
logue of glucose) was measured in the absence of insulin
stimulation. Glucose transport assays were initiated by the
3
addition of 2-deoxy-D-[ H]glucose (0.5 mCi/mL, 100 mM final
concentrations) to each well followed by incubation for 10 min
at 22 °C. Assays were terminated by aspirating the media
and rapidly washing the monolayer two times with ice-cold
phosphate-buffered saline solution. Cell monolayers were
solubilized in 0.1 N NaOH and transferred to scintillation
vials, and radioactivity was determined by liquid scintillation
counting. All data were corrected for nonspecific hexose
uptake determined in parallel samples treated for 5 min with
2
00 mM cytochalasin B.
In Vitr o Glu cose Tr a n sp or t Assa y (w ith exogen ou sly
3
a d d ed in su lin ): [ H]-2-Deoxy-D-glu cose Up ta k e in Dif-
fer en tia ted 3T3-L1 Ad ip ocytes. Adipocytes were treated
(
1
(
in triplicate) for 18 h with a test compound at 0.1, 0.3, 1, 3,
0, 30, and 100 µM final concentrations. Following overnight
18 h) treatment, the cell monolayers were washed, and the
medium was switched to KRH buffer. Basal control adipocytes
were given the insulin vehicle KRH/1% BSA. The final
concentration of KRH/1% BSA was 4%. A concentrated
porcine insulin stock was freshly diluted into KRH/1% BSA
and given to the compound-treated adipocytes and to the
insulin control adipocytes. The final concentration of insulin
was 0.5 nM in 4% KRH/1% BSA. The plates were then

9
00 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 6
Ack n ow led gm en t. The authors wish to thank Zhi
Bierer et al.
(11) Feijao, R. O. In Elucidario Fitologico; Instituto Botanico de
Lisboa: Lisbon, 1961; Vol. 2, p 258.
J un Ye, J ohn Kuo, J ian Lu Chen, and Nigel Parkinson
for their assistance in obtaining NMR and mass spectral
data; Rowena Richter for botanical assistance; J erry
Reaven for valuable scientific discussions; Michael
Ceniceros and Ray Cooper for editorial assistance;
Dominic Brignetti for assistance with biological figures;
Dr. D. nii-Amon-Kotei from the University of Science
and Technology, Kumasi, Ghana, and Dr. G. K. Noamesi
from the University of Wisconsin for their scientific
collaboration; and Professor Henry Rapoport at the
University of California, Berkeley for valuable consulta-
tion. We also wish to thank the traditional healers,
communities, botanists, physicians, government agen-
cies, and nongovernmental agencies from Nigeria,
Guinea, Cameroon, Zaire, Tanzania, Ghana, and Congo
that have contributed to diabetes research at Shaman
Pharmaceuticals.62
(
12) Silva, O.; Duarte, A.; Cabrita, J .; Pimentel, M.; Diniz, A.; Gomes,
E. T. Antimicrobial Activity of Guinea-Bissau Traditional Rem-
edies. J . Ethnopharmacol. 1996, 50, 55-59.
(
13) Oliver-Bever, B. E. P. Medicinal Plants in Tropical West Africa;
Cambridge University Press: Cambridge, 1986; pp 18, 41, 131.
(14) Watt, J . M.; Breyer-Brandwijk, M. G. The Medicinal and
Poisonous Plants of Southern and Eastern Africa; E.&S. Liv-
ingstone Ltd.: Edinburgh, 1962; pp 128-129.
(
15) Boakye-Yiadom, K. Antimicrobial Properties of Some West
African Plants. Quart. J . Crude Drug Res. 1979, 17, 78-80.
16) Boye, G. L.; Ampofo, O. In Proceeding of the First International
Symposium on Cryptolepine; Boakye-Yiadom, K., Bamgbose, S.
O. A., Eds.; University of Science and Technology: Ghana, 1983;
pp 37-40.
(
(
17) Boye, G. L. Antimalarial Action of Cryptolepis sanguinolenta
Extract. Proceedings of the International Symposium on East-
West Medicine, Oct. 10-11, 1989, Seoul, Korea; 1990, pp 243-
251.
(18) Analytical quantification of the aqueous extract administered
to indigenous humans revealed that the dose corresponded to
0
.11 mg/kg/day cryptolepine.
(19) Results of a human study conducted in Ghana on an aqueous
extract administered to NIDDM patients will be published
elsewhere. See: Luo, J .; Fort, D. M.; Carlson, T. J .; Noamesi,
B.; nii-Amon-Kotei, D.; King, S. R.; Tsai, J .; Quan, J .; Hobensack,
C.; Lapresca, P.; Waldeck, N.; Mendez, C. D.; J olad, S. D.; Bierer,
D. E.; Reaven, G. M. Cryptolepine, A Potentially Useful New
Antihyperglycemic Agent Isolated from Cryptolepis sanguino-
lenta: An Example of the Ethnobotanical Approach to Drug
Discovery. Diabetic Med. 1998, in press.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for N-(2-carboxyphenyl)glycine (12), 1-acetyl-3-ac-
etoxyindole (13), 1-acetyl-3-oxoindole (14), oxoindole 16, 10-
acetylquindoline (17), and quindolinium salt 18; an experi-
mental procedure for quindoline (8) from 17; alternative
experimental procedures for cryptolepine hydroiodide (2),
cryptolepine hydrotrifluoromethanesulfonate (3), and cryptol-
epine hydrochloride (4); a scheme describing the unambiguous
(20) Fichter, F.; Boehringer, R. Ueber Chindolin. (Over Quindoline.)
Chem. Ber. 1906, 39, 3932-3942.
(21) Fichter, F.; Probst, H. Zur Kenntnis des Methyl-chindolanols.
1
13
synthesis of cryptolepine (1); tables of H and C NMR data
for cryptolepine (1), its salts 2-4, and regioisomer 9 (Tables 6
and 7); a table of melting points and UV data (Table 8) for
cryptolepine (1), its salts 2-4, and regioisomer 9; and elemen-
tal analyses for compounds 1-4, 7, and 9 (10 pages). Ordering
information is given on any current masthead page.
(
1
To the Knowledge of the Methyl-Quindolanols.) Chem. Ber.
907, 40, 3478.
(
22) Fichter, F.; Rohner, F. U¨ ber einige Derivate des Chindolins.
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3499.
(
23) Clinquart, E. Sur la composition chimique de Cryptolepis trian-
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Refer en ces
(
1) Bierer, D. E.; Carlson, T. J .; King, S. R. Integrating Indigenous
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(
26) Clinquart isolated a purple compound (mp 160 °C), assumed to
(
(
(
2) Carlson, T. J .; Cooper, R.; King, S. R.; Rozhon, E. J . Modern
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N. E. Br. collected in Kisantu (Belgian Congo). He named this
17 2 4
compound cryptolepine and proposed the formula C14H N O .
2
group, from the roots of C. triangularis
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C
17
H
16
N
2
O. The accepted formula for cryptolepine,
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1
997, 40, 614-617.
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(
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(
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(58) This procedure does not work with chloroform that has been
stablized with ethanol.
(
(
(
59) Frost, S. C.; Lane, M. D. Evidence for the Involvement of Vicinal
Sulfhydryl Groups in Insulin-activated Hexose Transport by
3
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