Synthesis of hydantoin analogues of (2S,3R,4S)-4-hydroxyisoleucine with insulinotropic properties

Article abstract of DOI:10.1016/j.bmcl.2008.06.081

The first synthesis of an optically pure (2R,3R,4S)-hydantoin 2, analogue of (2S,3R,4S)-4-hydroxyisoleucine, was achieved in two steps in un-optimized 35% overall yield from previously reported aldehyde synthon 1. (2R,3R,4S)-Hydantoin is stable at acidic pH. This solves the major drawback of (2S,3R,4S)-4-hydroxyisoleucine that easily cyclizes into inactive lactone. Furthermore, (2R,3R,4S)-hydantoin stimulates the insulin secretion by 150% at 25 μM compared with 4-hydroxyisoleucine and insulin secretagogue drug repaglinide. In view of its stability and biological activity, (2R,3R,4S)-hydantoin represents a good candidate for type-2 diabetes management and control.

Full text of DOI:10.1016/j.bmcl.2008.06.081

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4332–4335
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of hydantoin analogues of (2S,3R,4S)-4-hydroxyisoleucine
with insulinotropic properties
Didier Sergent, Qian Wang, N. André Sasaki, Jamal Ouazzani *
Institut de Chimie des Substances Naturelles, CNRS, UPR 2301, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The first synthesis of an optically pure (2R,3R,4S)-hydantoin 2, analogue of (2S,3R,4S)-4-hydroxyisoleu-
cine, was achieved in two steps in un-optimized 35% overall yield from previously reported aldehyde
synthon 1. (2R,3R,4S)-Hydantoin is stable at acidic pH. This solves the major drawback of (2S,3R,4S)-4-
hydroxyisoleucine that easily cyclizes into inactive lactone. Furthermore, (2R,3R,4S)-hydantoin
Received 28 February 2008
Revised 21 June 2008
Accepted 25 June 2008
Available online 28 June 2008
stimulates the insulin secretion by 150% at 25 lM compared with 4-hydroxyisoleucine and insulin secre-
tagogue drug repaglinide. In view of its stability and biological activity, (2R,3R,4S)-hydantoin represents a
good candidate for type-2 diabetes management and control.
This article is dedicated to Professor Pierre
Potier, in memory of his second anniversary.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Type-2 diabetes
Fenugreek
4
-Hydroxyisoleucine
Hydantoin
Insulin secretion
Type-2 diabetes, formerly known as non-insulin dependent dia-
betes mellitus, has reached epidemic proportions with currently
ity to insulin, causes severe lactic acidosis. Thiazolidinediones that
enhance tissues sensitivity and repaglinide, a rapid acting insulin
secretaguogue, both undergo hepatic injury.
5
1
3
80 million persons worldwide. This number is projected to reach
00 million by 2030. Besides increasing morbidity and mortality,
1
Fenugreek (Trigonella foenum-graecum) seeds have been tradi-
type-2 diabetes remains the most costly disease undergoing social,
familial, and professional impacts. All therapeutic protocols and
tionally used in Asia and Africa to decrease hyperglycemia in dia-
betic patients. The non-proteinogenic amino acid, (2S,3R,4S)-4-
2
6,7
trials focus on the prevention of short- and long-term complica-
tions, mainly due to micro- and macro-vascular disorders. Micro-
vascular injuries lead to retinopathy, nephropathy, and neuropathy
in long-term diabetes patients. However, macro-vascular compli-
cations provoke coronary heart disease, stroke, and myocardial
infraction, and represent the major cause of handicap and death.
Type-2 diabetes is a metabolic disorder characterized by at least
three major defects, decreased insulin secretion, altered insulin
efficiency (tissue resistance), and increased release of hepatic glu-
cose. Each of these disorders involves specific physiological targets
justifying the need for large spectrum therapeutic agents.
hydroxyisoleucine, extracted from the seeds of fenugreek, was pro-
ven to possess interesting insulinotropic properties. Indeed,
(2S,3R,4S)-4-hydroxyisoleucine enhance insulin secretion propor-
tionally to glucose concentration without any hypoglycemic risk.8
We have published the first total synthesis of (2S,3R,4S)-4-
9
hydroxyisoleucine. The key steps were the bio-conversion of
ethyl-2-methylacetoacetate to ethyl-(2S,3S)-2-methyl-3-hydroxy-
butanoate and an asymmetric Strecker synthesis that provides the
desired amino acid in a stereoselective manner. The major draw-
back of (2S,3R,4S)-4-hydroxyisoleucine lies in its high propensity
to form a five-membered lactone between the OH group and the
carboxylic group under acidic conditions. It has been demonstrated
that the lactone of 4-hydroxyisoleucine possesses no insulinotropic
Besides insulin, several drugs have proven their efficacy to re-
duce hyperglycemia-induced disorders. Each possesses its own
pharmacological mechanism of action and exhibits diverse toxico-
1
0
activity. In a stomach, it is likely that the pH is low enough to con-
vert most, if not all, of the hydroxyisoleucine into its inactive lac-
tone analogue. In order to solve this problem, we have focused
our effort to develop derivatives of (2S,3R,4S)-4-hydroxyisoleucine
that may have similar pharmacological profiles of those of the par-
ent compound yet are more stable under acidic conditions. We have
synthesized for the first time hydantoin analogues that are stable in
acidic conditions and stimulate insulin secretion.
3
,4
logical risks. For instance, sulfonylureas that stimulate insulin
secretion through interaction with a specific islet cell receptor,
may cause light to severe hypoglycemia. Bisguanide metformin
that suppresses glucose biosynthesis and enhances tissue sensitiv-
*
Corresponding author. Tel.: +33 1 69 82 30 01; fax: +33 1 69 07 72 47.
E-mail address: Jamal.ouazzani@icsn.cnrs-gif.fr (J. Ouazzani).
0
960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.081

D. Sergent et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4332–4335
4333
Figure 1. Synthesis of 5-(2-hydroxy-1-methyl-propyl)-imidazolidine-2,4-dione 3 (hydantoin) from aldehyde 1.
In order to improve our previously reported synthesis of
2S,3R,4S)-4-hydroxyisoleucine, and to avoid the Strecker step,
carboxyl and the hydroxy groups that favors the formation of the
corresponding lactone. In the fenugreek seeds, a small proportion
of 4-hydroxyisoleucine exists as a lactone (5–10%) that accounts
for the aromatic properties of the curry spice (15–30% of fenugreek
seeds powder). Pure 4-hydroxyisoleucine has no particular odor.
Prior to biological evaluations, we studied the benefit of the hydan-
toin form in terms of acidic stability. For the pH stability of hydan-
toin versus 4-hydroxyisoleucine, we measured the integration of
NMR signals of the five-membered lactones. The NMR tubes were
(
we opted for the hydantoin approach. Despite the synthetic chal-
lenge, hydantoin offers various advantages compared to the parent
amino acid. The first advantage is their expected stability in acidic
conditions that overcome the major drawback in hydroxyisoleu-
cine development. The second advantage is the prodrug character
of hydantoins that may generate the parent amino acid through li-
ver hydantoinase hydrolysis.
Hydantoin 2 was prepared from aldehyde 1 through the classi-
2
filled with 0.5 ml of D O containing 10 mg of compound. HCl was
cal Bucherer–Bergs reaction.1
1–13
Aldehyde 1 represents the key
added to the final concentration of 0.5 M and the tubes were kept
at 37 °C during the experiment. NMR spectra were recorded for
every 1 h and compared.
intermediate and could be prepared according to our previously re-
9
14
ported approach or by the method of Cordova (Fig. 1). The
hydantoin 2 is obtained in 70% un-optimized yield and with 90%
diastereomeric excess. The latter was measured by NMR signal
integration of the proton at position 2. The hydantoins reported
in this Letter are members of a new family of compounds that have
never been reported before.
1
8
As shown in Figure 3, Hydantoin is largely more stable than 4-
hydroxyisoleucine. Thus, after 4 h of incubation (histograms and
NMR details), 29% of 4-hydroxyisoleucine is converted to lactone
(Fig. 3a) compared to 5.7% for the hydantoin (Fig. 3b). This is easily
understandable since the hydantoin must undergo hydrolysis be-
fore cyclization to lactone.
Compound 2 was deprotected to give the hydantoin 3 in un-
optimized 50% yield and with 90% isomeric excess (NMR-peak
Biological activity. Stimulation of insulin secretion was evalu-
1
5
19
integration). The two isomers of 3 were converted separately to
their corresponding five-membered lactones and were compared
with the lactones obtained from (2S,3R,4S) and (2R,3R,4S)-4-
hydroxyisoleucine, respectively. Lactonization was conducted in
acidic conditions to exclude any epimerization risk. Analysis of
thus obtained lactones by NMR shows unambiguously that the
minor hydantoin bears the natural (2S,3R,4S) stereochemistry
ated on rat pancreatic Langerhans islets.
(2R,3R,4S) and
(2S,3R,4S)-hydantoins were compared to 4-hydroxyisoleucine and
repaglinide, as positive controls. In each experiment, the insulin
stimulation must be compared to the basal insulin level (untreated
islets). Basal secretion is 45 pg/islet/h for 4-hydroxyisoleucine
experiment and 100 pg/islet/h for repaglinide and hydantoin. In
our conditions, repaglinide, as well as other compounds, failed to
(
2
Fig. 2). The major one has the inverse stereochemistry at position
induce insulin secretion at concentrations below 25 lM. This is
not in agreement with literature. Figure 4 confirms that 4-
hydroxyisoleucine enhances the insulin secretion by the islets,
compared to untreated rats (100 pg/islet/h at 75 lM instead of
2
0
being (2R,3R,4S)-hydantoin.
Major and minor isomers of hydantoin 3 were hydrolyzed,16 to
1
7
give the expected (2R,3R,4S) and (2S,3R,4S)-4-hydroxyisoleucine.
Hydantoin approach is so far the shortest and efficient way to
45 pg/islet/h). This result obtained in vitro by using buffered neu-
tral solution needs to be balanced in case of oral 4-hydroxyisoleu-
cine administration. Stimulation of insulin secretion by 4-
hydroxyisoleucine is not dose dependent and remains constant
(2R,3R,4S)-4-hydroxyisoleucine.
Stability to pH. The stability of 4-hydroxyisoleucine and its insu-
linotropic activity is compromised by the proximity between the
Figure 2. Determination of the absolute configuration of hydantoins.

4
334
D. Sergent et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4332–4335
Figure 3. pH stability of hydantoin (b) versus 4-hydroxyisoleucine (a).
Figure 4. Effects of target compounds on insulin secretion by isolated rat pancreatic islets ( P < 0.01). Islets were prepared according to literature as mentionned.19 Before
experiments, islets were pre-incubated during 60 min at 37 °C in KRB buffer containing 8.3 mM glucose. Twenty-seven islets per well were plated in 6-well plates and then
incubated 60 min with test substances at the desired end concentration. Mean values are from six single-point measurements.
**
from 25 to 200
l
M concentration. This observation is consistent
ural 4-hydroxyisoleucine. The hypoglycemic potential of hydanto-
ins was previously reported for sulfonyl conjugates.
1
0
21
with literature. Repaglinide is more effective at high concentra-
tions leading to a 100% increase in insulin secretion at 200 M. This
2R,3R,4S)-hydantoin appears to be more potent than repaglinide.
A 120% stimulation of insulin secretion is obtained even at
M. (2S,3R,4S)-Hydantoin is less relevant than (2R,3R,4S)-
hydantoin even though it has the same stereochemistry with nat-
l
In conclusion, our preliminary results concerning (2R,3R,4S)-
hydantoin, a stable and potent analogue of (2S,3R,4S)-4-hydroxy-
isoleucine, are convincing and promising. In order to further
confirm the potential of this compound, in vivo tests and mecha-
nistic studies are currently underway.
(
25 l

D. Sergent et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4332–4335
4335
+
1
C
7
60.6, 69.0, 62.1, 44.5, 22.0, 12.9. MS (ES): m/z = 172 [MH ]. HRMS Calcd for
: 171.0770. Found: 171.0777. C (172,18): Calcd C, 48.83; H,
.02; N, 16.27; O, 27.88. Found: C, 48.70; H, 6.91; N, 15.6; O, 26.8. (2R,3R,4S)-
Acknowledgment
7
H
12
2
N O
3
7 12 2 3
H N O
25
We thank Cellis-Pharma (Saint Malo, France) for biological
investigations.
Hydantoin. Mp 207–208 °C, [a]D +116.1 (c 0.5, MeOH), IR: m = 3458, 3177,
2967, 2926, 1709, 1434, 1375, 1341, 1318, 1289, 1244, 1192, 1149, 1116, 1080,
À1
1
1
044, 1003, 919, 870, 760, 638 cm
3
. H NMR (300 MHz, CD OD): d 4.54 (d,
J = 2.4 Hz, 1H), 3.57 (qd, J = 6.2, 8.4 Hz, 1H), 1.94 (dqd, J = 2.4, 6.9, 8.5 Hz, 1H),
References
13
1
.23 (d, J = 5, 6.2 Hz, 3H), 0.83 (d, J = 5, 6.9 Hz, 3H). C NMR (62.5 MHz, CD
3
OD):
d 179.8, 160.6, 69.9, 60.8, 43.6, 22.0, 10.5. MS (ES ): m/z = 172 [MH ]. HRMS
Calcd for C : 171.0770. Found: 171.0767. C (172,18): Calcd C,
+
+
1.
2.
3.
4.
Harris, M. I. Diabetes Care 2000, 23, 754.
7
H
12
N
2
O
3
7 12 2 3
H N O
Stephens, J. M.; Botteman, M. F.; Hay, J. W. J. Manag. Care Pharm. 2006, 12, 130.
Luna, B.; Feinglos, M. K. Am. Fam. Physician 2001, 63, 1747.
Padwal, R.; Majumdar, S. R.; Johnson, J. A.; Varney, J.; Mcalister, F. A. Diabetes
Care 2005, 28, 736.
48.83; H, 7.02; N, 16.27; O, 27.88. Found: C, 48.77; H, 6.88; N, 16.06; O, 27.45.
17. Conversion of 3 to the corresponding 4-hydroxyisoleucine. (2R,3R,4S)-Hydantoin 3
(2.5 g, 14 mmol) was dissolved in 40 ml of 5 N NaOH and refluxed for 17 h. The
mixture is then neutralized by 10 N HCl and evaporated to dryness. The residue
is recovered in hot methanol and dried under reduced pressure.
Recrystallization in ethanol gives 2 g (68% yield) of (2R,3R,4S)-
5.
6.
7.
8.
Waring, S. Medicines 2007, 35, 590.
Eidi, A.; Eidi, M.; Sokhteh, M. Nutr. Res. 2007, 27, 728.
Sharma, R. D. Nutr. Res. 1986, 6, 1353.
Sauvaire, Y.; Petit, P.; Broca, C.; Manteghetti, M.; Baissac, Y.; Fernandez-
Alavarez, J.; Gross, R.; Roye, M.; Leconte, A.; Gomis, R.; Ribes, G. Diabetes 1998,
9
hydroxyisoleucine identical, to those previously reported. The same
procedure gives (2S,3R,4S)-hydroxyisoleucine from (2S,3R,4S)-hydantoin.
18. Stability of (2S,3R,4S)-4-hydroxyisoleucine and (2R,3R,4S)-hydantoin under acidic
conditions (Fig. 3). The two doublet peaks at d1.15 and 0.87 are attributed to the
methyl groups of C-3 and C-4 position of (2S,3R,4S)-4-hydroxyisoleucine,
respectively. When incubated at 37 °C and at pH 1 for 1 h, two new doublet
peaks appear at d 1.35 and 1.06 due to the two methyl groups of newly formed
lactone that is about 10% with respect to 4-hydroxyisoleucine. After 4 h, the
lactone represents 32%. After 20 h at the same conditions, the two doublets of
the methyl groups of lactone become predominant. On the contrary, the
hydantoins of (2R,3R,4S)-4-hydroxyisoleucine 1 are relatively stable under
acidic conditions. The two doublet peaks at d 1.25 and 0.82 attributed to the
two methyl groups of compound 1 are still the major peaks (73%) after 20 h of
incubation at 37 °C and at pH 1.
47, 206.
9
.
Wang, Q.; Ouazzani, J.; Sasaki, N. A.; Potier, P. Eur. J. Org. Chem. 2002, 834.
1
0. Broca, C.; Manteghetti, M.; Gross, R.; Baissac, Y.; Jacob, M.; Petit, P.; Sauvaire,
Y.; Ribes, G. Eur. J. Pharm. 2000, 390, 339.
1
1
1
1. Bucherer, H. T.; Fischbeck, H. T. J. Prakt. Chem. 1934, 140, 69.
2. Mahmoodi, N. O.; Khodaee, Z. ARKIVOC 2007, 29.
3. Synthesis of 2. (2S,3S)-3-(Tetrahydropyranyloxy)-2-methylbutanal
1 (25 g,
1
34 mmol), KCN (44 g, 677 mmol) and NH CO (116 g, 1.2 mol) were
4
3
dissolved in 280 ml of 50% aqueous ethanol. The mixture was stirred for 2
days at 45 °C and evaporated under reduced pressure. The residue was purified
by flash chromatography (silica gel 35–79
2 2 3
l, CH Cl /CH OH 97:3) to provide 3
(
20.5 g, 70% yield) as a mixture of isomers resulting from the presence of an
19. Insulin assay (according to Lacy, P. E.; Kostianovsky, M. Diabetes 1967, 16, 35).
Ò
asymmetric carbon on the THP moiety. This mixture of isomers was used
without further purification for the synthesis of 3.
The insulin secretion was quantified by the ELISA kit ‘‘Insuline de Rat ELIT
ultrasensible” (Eurobio, France) with 5 mL of incubation medium diluted to fit
with the kit instructions. The results of insulin secretion are expressed in pg/
islet/h (n = 6–12 points). Effects of target compounds on insulin secretion by
isolated rat pancreatic islets. Islets were prepared from 28 healthy male
Sprague–Dawley rats. The body weight of rats was between 360 and 390 g.
Tissue was digested with collagenase XI (Sigma, France) at 2.5 mg/ml in Hanks’
balanced salt solution (HBSS) under gentle shaking (120 rpm) for 35 min at
37 °C. The digestion was monitored by microscopic observations and stopped
when the islets were well separated. The suspension was then transferred to a
Histopaque discontinuous gradient (Ref. 1083-1, Sigma, France) and
centrifuged at 4 °C from 25 to 800 g in 16 min. The upper fraction containing
the islets and fat cells was carefully recovered. The islets were then handpicked
and counted in HBBS under light microscope to ensure 100% purity of the
preparation. Four hundred and eighty islets were collected and re-suspended
1
1
4. Cordova, A. Tetrahedron Lett. 2004, 45, 3949.
5. Synthesis of 3. Compound 2 (15 g, 110 mmol) was dissolved in 250 ml of EtOAc
in the presence of p-toluenesulfonic acid (2.5 g). The reaction mixture was
stirred overnight at rt. The precipitate was filtered and the residue was washed
with EtOAc to give 3 (6.5 g, 65% yield). The ratio between the two isomers of 3
1
was determined to be 95 to 5 by H NMR analysis. The major diastereoisomer
(
6 g) was obtained pure by flash chromatography (silica gel 35–70
CH OH 95:5). Its absolute configuration as well as that of the minor isomer
300 mg) was assigned by comparing their corresponding lactones with those
obtained from authentic (2R,3R,4S)- and (2S,3R,4S)-4-hydroxyisoleucine (pure
2 2
l, CH Cl /
3
(
9
1
samples obtained from our previous work), respectively. The H NMR of the
lactones obtained from the major hydantoin and authentic (2R,3R,4S)-4-
hydroxyisoleucine are identical. Likewise, the H NMR spectra of the lactones
1
obtained from the minor hydantoin and authentic (2S,3R,4S)-4-
hydroxyisoleucine match perfectly. In order to avoid epimerization,
transformations of both isomers of 3 to lactones were carried out under
acidic conditions (6 N HCl, 2 h, 70 °C).
in 9.3 ml of KRB, in order to have 10 islets per 190
islets were pre-incubated during 60 min at 37 °C in humidified atmosphere
with 5% CO in KRB buffer containing 8.3 mM glucose. Twenty-seven islets per
lL. Before experiments,
2
well were plated in 6-well plates (the number was checked after plating under
light microscope) and then incubated 60 min with test substances at the
desired end concentration.
2
5
1
6. (2S,3R,4S)-Hydantoin. Mp 148–149 °C, [
a
]
D
À9.2 (c 0.5, MeOH), IR:
m = 3208,
2
8
3
6
966, 2932, 1693, 1407, 1333, 1298, 1242, 1190, 1130, 1079, 993, 973, 944, 908,
64, 748, 717, 683 cm
.86 (qd, J = 6.3, 9.2 Hz, 1H), 1.93 (dqd, J = 3, 7.1, 9.3 Hz, 1H), 1.19 (d, J = 5,
3
.3 Hz, 3H), 0.94 (d, J = 5, 7.1 Hz, 3H). C NMR (62.5 MHz, CD OD): d 178.0,
À1
1
.
H NMR (300 MHz, CD
3
OD): d 4.27 (d, J = 2.9 Hz, 1H),
20. Malaisse, W. J. Eur. J. Clin. Invest. 1999, 29, 21.
21. Ahmad, R.; Jabeen, R.; Zia-ul-Haq, M.; Nadeem, H.; Duddeck, H.; Verspohl, E. J.
Z. Naturforsch. 2000, 55, 203.
1
3