Novel glucagon receptor antagonists with improved selectivity over the glucose-dependent insulinotropic polypeptide receptor

Article abstract of DOI:10.1021/jm7015599

Optimization of a new series of small molecule human glucagon receptor (hGluR) antagonists is described. In the process of optimizing glucagon receptor antagonists, we counter-screened against the closely related human gastric inhibitory polypeptide receptor (hGIPR), and through structure activity analysis, we obtained compounds with low nanomolar affinities toward the hGluR, which were selective against the hGIPR and the human glucagon-like peptide-1 receptor (hGLP-1R). In the best cases, we obtained a >50 fold selectivity for the hGluR over the hGIPR and a >1000 fold selectivity over the hGLP-1R. A potent and selective glucagon receptor antagonist was demonstrated to inhibit glucagon-induced glycogenolysis in primary rat hepatocytes as well as to lower glucagon-induced hyperglycemia in Sprague - Dawley rats. Furthermore, the compound was shown to lower blood glucose in the ob/ob mouse after oral dosing.

Full text of DOI:10.1021/jm7015599

J. Med. Chem. 2008, 51, 5387–5396
5387
Novel Glucagon Receptor Antagonists with Improved Selectivity over the Glucose-Dependent
Insulinotropic Polypeptide Receptor
Ja´nos T. Kodra,*,† Anker Steen Jørgensen,‡ Birgitte Andersen,§ Carsten Behrens,| Christian Lehn Brand,§
Inger Thøger Christensen,† Mette Guldbrandt,§ Claus Bekker Jeppesen,§ Lotte B. Knudsen,§ Peter Madsen,† Erica Nishimura,§
Christian Sams,‡ Ulla G. Sidelmann,| Raymon A. Pedersen, Francis C. Lynn, and Jesper Lau†
Departments of Diabetes Protein Engineering, Diabetes Biology & Pharmacology, Medicinal Chemistry, Protein Engineering, NoVo Nordisk
A/S, NoVo Nordisk Park, DK-2760 MåløV, Denmark
ReceiVed December 13, 2007
Optimization of a new series of small molecule human glucagon receptor (hGluR) antagonists is described.
In the process of optimizing glucagon receptor antagonists, we counter-screened against the closely related
human gastric inhibitory polypeptide receptor (hGIPR), and through structure activity analysis, we obtained
compounds with low nanomolar affinities toward the hGluR, which were selective against the hGIPR
and the human glucagon-like peptide-1 receptor (hGLP-1R). In the best cases, we obtained a >50 fold
selectivity for the hGluR over the hGIPR and a >1000 fold selectivity over the hGLP-1R. A potent and
selective glucagon receptor antagonist was demonstrated to inhibit glucagon-induced glycogenolysis in primary
rat hepatocytes as well as to lower glucagon-induced hyperglycemia in Sprague-Dawley rats. Furthermore,
the compound was shown to lower blood glucose in the ob/ob mouse after oral dosing.
Introduction
secretion.12 In a recent study, a long-acting GIP analogue has
been shown to decrease nonfasting blood glucose, improve
glucose tolerance, and enhance insulin secretion in ob/ob mice.13
The role of GIP in type 2 diabetes is however still an issue of
debate.14 Nevertheless, GIP does stimulate first phase insulin
secretion in type 2 diabetes and thus it would most likely not
be a desirable property of an antidiabetic agent to inhibit the
GIP receptor.
GLP-1 is an important incretin and is the basis for two new
emerging drug classes for the treatment of type-2 diabetes, the
GLP-1 analogues and the dipeptidyl peptidase IV (DPP-IV)
inhibitors. Thus, glucagon receptor antagonists must be selective
against the GLP-1 receptor.
A novel type of orally bioavailable small-molecule glucagon
receptor antagonist, based on ꢀ-alanine urea derivatives, was
recently published.15 We have found that this series of com-
pounds was capable of binding to and antagonize the GIP
receptor function. Other series reported by Merck have dem-
onstrated selectivity of antagonism of the hGluR over the
hGIPR.16,17
We have addressed this selectivity issue of antagonism of
the hGluR versus the hGIPR and have optimized the previously
published series of small-molecule hGluR antagonists containing
a ꢀ-alanine motif to obtain improved binding selectivity over
the hGLP-1R and particularly the hGIPR. This was ac-
complished by changing the ꢀ-alanine motif to an isoserine (R-
hydroxy-ꢀ-alanine) motif.
Blood glucose levels are maintained via a tightly controlled
balance between insulin and a number of counter-regulatory
hormones. Glucagon is a 29 amino acid peptide secreted from
the R-cells in response to low plasma levels of glucose.
Glucagon is a timely and potent activator of hepatic glucose
production, whereas the role of insulin in the liver is to inhibit
hepatic glucose production and induce hepatic glucose utilization
and storage. Inappropriately, high glucose production from the
liver is believed to be an important contributor to the develop-
ment of hyperglycemia in type 2 diabetes. This is presumably
a result of hepatic insulin resistance in combination with lack
of suppression of glucagon secretion from the R-cells in response
to elevated glucose. In type 2 diabetes, the glucagon level is
elevated relative to the blood glucose and insulin levels.
Therefore, new therapeutic agents, capable of blocking the effect
of glucagon on hepatic glucose production, has attracted
attention for treatment of hyperglycemia in type 2 diabetes.1–6
The receptor for the gastric inhibitory polypeptide (GIP) and
glucagon-like peptide-1 (GLP-1) receptors belong to the same
family of G-protein coupled seven-transmembrane domain
receptors as the glucagon receptor and share structural similari-
ties. The primary role of GIP in healthy subjects is to stimulate
glucose-dependent insulin secretion.7 In addition, GIP has been
shown to have an inhibitory effect on ꢀ-cell apoptosis8 and has
been suggested to play a role in lipid metabolism.9 GIP receptor
knockout mice have been demonstrated to be protected from
high-fat diet-induced obesity.10
Chemistry
In humans with type 2 diabetes, the insulinotropic effect of
GIP is reduced,11 but bolus administration of GIP to patients
with type 2 diabetes has been shown to stimulate insulin
In general, the hGluR antagonists were synthesized as outlined
in Scheme 1. The 4-formyl benzoic acid methyl ester was
reacted with various amines in a reductive amination to give
the secondary amine intermediates 1. For the reaction with
4-tert-butyl-cyclohexyl amine the pure trans-4-[(4-tert-butyl-
cyclohexylaminomethyl]benzoic acid methyl ester of which was
obtained as described previously.15 The secondary amines 1
were reacted with various phenyl isocyanates and subsequently
treated with NaOH to give the benzoic acid ureas derivatives
* To whom correspondence should be addressed. Phone: +4544434611.
Fax: +45663450. E-mail: jtk@novonordisk.com
†
Department of Diabetes Protein Engineering, Novo Nordisk A/S.
‡
Department of Medicinal Chemistry, Novo Nordisk A/S.
Department of Diabetes Biology and Pharmacology. Novo Nordisk A/S.
Department of Protein Engineering, Novo Nordisk A/S.
§
|
Department of Physiology, University of British Columbia.
10.1021/jm7015599 CCC: $40.75
2008 American Chemical Society
Published on Web 08/16/2008

5388 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Kodra et al.
Scheme 1. General Reaction Scheme for Synthesis of Glucagon Antagonistsa
a Reagent and conditions: (a) R2-NH2, methanol, 60 min, reflux; (b) NaBH3CN, acetic acid, methanol, 60 min, rt; (c) R3-NCO, dichloromethane, 16 h,
rt; (d) 4 N NaOH in ethanol (1:3), 16 h, rt; (e) EDAC/HOBt, DMF, 30 min, then MeOOCCH(R1)CH2NH2 ·HCl, diisopropylethylamine, 16 h, rt; (f) 2 N
NaOH in ethanol (1:8), 1 h, rt.
Scheme 2. Synthesis of (R)-Isoserinea
a Reagent and conditions: (a) 2,2-dimethoxypropane, toluene, reflux, 2 h (81%); (b) DPPA, triethylamine, toluene, 85 °C; (c) benzyl alcohol, toluene, 85
°C, 17 h, (40%); (d) 1 N HCl in acetonitrile (1:1), 3 h, 40°C (87%); (e) H2/Pd-C, 17 h, rt, (92%); (f) thionylchloride, methanol, -30°C, (99%).
2, which could be transformed into a range of test compounds
using standard chemistries. Initially the racemic mixture of
commercially available (R,S)-isoserine protected as ethyl ester
was coupled to the benzoic acid derivatives 2 to give 3, which
was either hydrolyzed to the test compound 4 and tested as a
racemic mixture or 3 was separated by chiral HPLC into the
pure (R) and (S) forms of the ethyl ester were converted into
the carboxylic acids by saponification to give the (R) and (S)
forms of the hGluR antagonists, respectively (Scheme 1).
For larger scale preparation, a suitable synthesis for the
enantiomers of isoserine was necessary. Enantioselective syn-
thesis of (S)-isoserine as well as enzymatic methods have been
described earlier18,19 and also the conversion of L-malic acid
into (S)-isoserine have previously been described.20 We modified
a procedure from Burger et al.20 for the synthesis the both
enantiomers of isoserine from commercially available malic
acid. Because both enantiomers of malic acid are available with
an eantiomeric purity above 95%, the method could be used
for the synthesis of both (R)- and (S)-isoserine. The conversion
of D-malic acid to (R)-isoserine and the corresponding methyl
ester hydrochloride is outlined in Scheme 2.
D-Malic acid was converted into 2-[(4R)-2,2-dimethyl-5-oxo-
1,3-dioxolana-4-yl]acetic acid 5 by refluxing with an excess of
2,2-dimethoxypropane in toluene and isolated in a reasonably
pure form by simple evaporation. The Curtius rearrangement
of 5 was carried out with diphenylphosphoryl azide (DPPA)
and triethylamine in toluene. The intermediate isocyanate was
not isolated but trapped with benzyl alcohol to give the
benzyloxycarbonylamino intermediate 6. Crude 6 was treated
with 1 N HCl in acetonitrile to give crystalline benzyloxycar-
bonyl protected (R)-isoserine 7, which was purified by recrys-
tallization from toluene. The pure (R)-isoserine 8 was obtained
by removal of the benzyloxycarbonyl with hydrogenolysis (10%
Pd/C, H2, ethanol). The pure (R)-isoserine was protected as the
methyl ester using SOCl2 in methanol.
Biological Methods
The biological methods are described in detail in the
Experimental Section.
Receptor Assays. Receptor assays were carried out using
plasma membranes purified from BHKa cells expressing either
the cloned human glucagon GIP or GLP-1 receptor. The hGluR
binding assay was done as previously published.21 Binding
assays for the cloned human GLP-1 and GIP receptors were
done similarly using BHK cell lines expressing the cloned
human GIP and GLP-1 receptor, and the assay for the isolated
rat glucagon receptor followed same protocol using rat liver
membrane preparations.
Primary Hepatocyte Assays. As a model system for hepatic
glucose production, rat hepatocytes were isolated and cultured
as described earlier.22 The hepatocytes were loaded with
glycogen, and the effect of the glucagon antagonists on
glucagon-induced glycogenolysis was examined.
Pharmacokinetics. The compounds were dosed iv and po
to fasted male Sprague-Dawley rats weighing ∼200 g. The
compounds were dissolved in 5% ethanol, 20% propyleneglycol,
10% hydroxypropyl-ꢀ-cyclodextrin, and phosphate buffer pH
7.5-8.0. Postdose ethylenediaminetetraacetic acid (EDTA)
blood samples were collected by heart puncture under carbon
dioxide anesthesia at time 5, 15, 30, 60, 120, 240, and 360 min
for the iv dosing and at time 15, 30, 60, 90, 120, 240, and 360
min for the po dosing. Plasma samples were prepared by
centrifugation (3000g, 10 min) and stored at 20 °C pending
analysis. Plasma samples were analyzed by high turbulence
liquid chromatography (HTLC) combined with tandem mass
spectrometry (MS/MS). The PK analysis was performed using
noncompartmental methods.
a Abbreviations: BHK, baby-hamster kidney; iv, intravenous; po, oral;
sc, subcutaneous; PK, pharmacokinetics; t1/2, half-life; Fpo, oral bioavail-
ability; SAR, structure activity relationship.

Glucagon Receptor Antagonists with ImproVed SelectiVity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5389
but showed a reduced affinity toward the hGIPR (IC50 ) 701
nM) corresponding to a 54-fold selectivity. It was concluded
that by introducing a hydroxy group next to the carboxylic acid
moiety in the ꢀ-alanine of our hGluR antagonist would be
neutral toward the hGluR receptor binding affinity while it
would impair the binding to the hGIPR. Separation of 14 into
the two enantiomers revealed an affinity difference of the two
enatiomers and subsequent independent synthesis of the R-
isomer and S-isomer showed that the R-isomer had the highest
IC50 (15 3.9 nM and 16 51 nM). The R-isomer also had
improved selectivity, IC50 for the GIP receptor were 359 and
445 nM for 15 and 16, respectively, equivalent to approximate
10-fold increased selectivity.
Methylation of the hydroxyl group 20 seriously diminished
the receptor affinity toward the hGluR (IC50 ) 693 nM). The
isoserine derivatives 15 and 18 also showed low affinity for
the hGLP-1R. In the case of 15, the hGluR/hGLP-1R ratio was
4300-fold compared to the ꢀ-alanine derivative 13 (hGluR/
hGLP-1-R ) 400-fold). These data suggest that the introduction
of the isoserine improves the selectivity toward the GLP-1 and
GIP receptors.
Focused libraries were synthesized, and it was revealed that
the SAR was similar to what was observed in the ꢀ-alanine
series.15 The compounds 21-36 in Table 2 showed that the
combination of R2 being a bulky lipophilic moiety with the
combination of R3 being a phenyl group with electron with-
drawing or lipophilic substituents gave fairly potent glucagon
receptor antagonists. The best substituents for R2 were cyclo-
hexylphenyl and cyclohexenylpheny and the best for R3 were
3,5-di-CF3, 3,5-dichloro, and 3,4-CF2-O-CF2-O. In contrast to
R3 being an unsubstituted phenyl group (27), the potency
dropped 10-fold compared to when a Cl was added in the
metaposition of R3 (28).
Figure 1
Glucagon Challenged Rat. To demonstrate acute in vivo
efficacy, the effect of the compounds on glucagon-stimulated
hyperglycemia was studied in rats. Nonfasted male Sprague-
Dawley rats (200 g) were maintained in the anaesthetized state
during the test by sc administration of a 1:1 mixture of Hypnorm
(fentanyl, 0.05 mg/mL, and fluanizone, 2.5 mg/mL, Janssen
Pharma Ltd., Copenhagen, Denmark) and Dormicum (Mida-
zolam, 1.25 mg/mL, Roche, Basel, Switzerland). Test com-
pounds were administered intravenously. A catheter was inserted
in a jugular vein for administration of compounds. Ap-
proximately 60 min after initiation of anesthesia, test compounds
(0, 3, and 10 mg/kg) and glucagon (3 µg/kg) were administered
with 5 min interval, respectively. Samples for determination of
blood glucose concentrations were taken from the tail tip 25
and 5 min prior to administration of the compound to represent
average basal values and again 10 min after administration of
glucagon (time for peak response of glucagon). The results were
expressed as δ values calculated as the value obtained 10 min
after glucagon administration minus the average of the two basal
values. This method was adapted from the method previously
published21
In all cases, the receptor binding affinities for the hGluR were
always higher than the binding affinities for the hGIPR, although
in the case of 29, the difference was only 2-fold.
The best selectivity was seen for 36, where the hGluR/hGIPR
ratio was 94-fold.
Acute Effect on Blood Glucose in ob/ob Mice. Male ob/ob
mice (11-13 weeks of age) used for the studies were selected
from a larger group of mice having the highest blood glucose
(BG) levels. Selected mice were randomized into treatment
groups having matching BG levels. Compounds (0 and 100 mg/
kg) were administered orally by gavage in the morning, and
blood samples (5 µL) for determination of BG were obtained
Pharmacokinetic Studies in Rats. The most potent isoserine
compounds that showed hGluR selectivity above 10-fold over
the hGIPR were tested for pharmacokinetic properties. The
plasma half-lives and oral availability in rats were tested, and
several compounds showed oral bioavailability. Compound 15
showed an oral bioavailability of >30% and a t1/2 (iv) of 53
min and compound 22 had an acceptable profile with an oral
bioavailability of >20% and t1/2 (iv) ) 72 min. In comparison
the ꢀ-alanine 13 has been reported to have a Fpo ) 69% and
t1/2 ) 82 min in rats,15 and it was our general observation that
the introduction of the hydroxyl group seemed to reduced the
oral bioavailability as well as the plasma half-life of this series
of compounds (Table 3).
Cultured Primary Hepatocyte Studies. To examine a
biological end-point of glucagon receptor inhibition in a cellular
system, the effects of the isoserine compound 15 and the
ꢀ-alanine compound 13 on glucagon-stimulated glucose produc-
tion from primary rat hepatocytes in culture were measured and
compared. As can be seen in Figure 2, inhibition of glucagon-
stimulated glycogenolysis was observed with both compounds
with IC50 values of approximately 820 ( 250 nM for 13 and
200 ( 40 nM for 15. These values are considerably higher than
those obtained in the binding assays for the rat receptor (IC50:
ratGlucR, 13 ) 36 nM, 15 ) 43 nM), but this difference may
be due to the fact that these compounds bind to albumin and
the hepatocyte culture medium contains 0.1% HSA (human
1
approximately /2 h predose and at 2, 4, 6, and 24 h postdose.
The mice had no access to food the first 6 h after dosing and
subsequently had free access to food. Food and water intake
were measured.
Results and Discussion
We have recently described a new type of orally available
glucagon receptor antagonists based on ꢀ-alanine derivatives.15
The ꢀ-alanine hGluR antagonist 10 (Figure 1) was selective
over the hGLP-1R but displayed equally high binding affinity
for the hGIPR as for the hGluR.
In our search for selective hGluR antagonists, we found that
the ꢀ-alanine moiety was very sensitive toward receptor affinity
as reported earlier15 as well as selectivity. The ꢀ-alanine urea
derivative 11 could be made more selective when the ꢀ-alanine
was replaced with (R,S)-isoserine 12 (Table 1). We reported
previously that the cyclehexenyl-3,5 dichlorophenyl urea ꢀ-ala-
nine derivative 13 lowered blood glucose in a murine type 2
diabetes model.15 Estimated from the IC50 values 13 has a 5-fold
greater affinity for the hGluR (IC50 ) 12 nM) than for the
hGIPR (IC50 ) 64 nM). The racemic (R,S)-isoserine 14
displayed a similar affinity toward the hGluR (IC50 ) 14 nM)

5390 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Kodra et al.
Table 1. Glucagon/GIP Receptor Potency Dependence on Stereochemistry
serum albumin), as well as the fact that primary hepatocytes
are metabolic active. There was no observed effect of these
compounds at concentration up to 25 µM on basal or adrenaline-
stimulated glycogenolysis, indicating that the observed effect
is mediated by specific inhibition of the hepatic glucagon
receptor. In addition, both 15 and 13 increased lactate produc-
tion, indicating an increased flux through the glycolysis. These
hepatocyte studies most importantly demonstrate that the
glucagon receptor antagonists have the expected biological
effect.
The compound dose-dependently inhibited the glucagon-
stimulated rise in blood glucose (Figure 3). The minimum
effective iv dose for 15 was 3 mg/kg.
Acute Studies in ob/ob Mice. The leptin deficient, obese
model of type 2 diabetes, the ob/ob mouse, is characterized by
hyperglycemia and hyperinsulinemia. In an oral single-dose
study with no access to food for 6 h, 15 reduced the observed
hyperglycemia (Figure 4).
Receptor Selectivity and GIP Antagonism. To directly test
the effect of these compounds on GIP stimulated insulin
secretion, studies using the isolated perfused rat pancreas were
carried out.23 The results presented in Figure 5 demonstrate that
Pharmacodynamic Studies. To examine the effects in vivo,
compound 15 was tested in a glucagon-challenged rat model.

Glucagon Receptor Antagonists with ImproVed SelectiVity
Table 2. SAR Table of the Isoserine Glucagon Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5391
10, which is a potent GluR antagonist as well as potent GIPR
antagonist (IC50: ratGlucR ) 23 nM, ratGIP ) 42 nM, ratGlucR/
ratGIPR ) 1.7-fold), was able to inhibit GIP stimulated insulin
secretion from the isolated perfused rat pancreas (Figure 5a).
However in comparison 15 (IC50: ratGlucR ) 43 nM, ratGIP
) 501, ratGlucR/ratGIPR ) 12-fold) and 13 (IC50: ratGlucR
) 36 nM, ratGIPR ) 216, ratglucR/ratGIPR ) 6-fold), which
are just as potent glucagon antagonists but with reduced affinity
for the GIP receptor, did not inhibit GIP stimulated insulin
secretion, even at a 10-fold higher concentration than used for
10 (Figure 5).
series15 in many cases lead to decreased GIP receptor affinity.
Glucagon receptor antagonists containing isoserines have been
Conclusion
GIP is an incretin hormone that stimulates first-phase insulin
secretion. GIP receptor inhibition is not a desirable extra effect
for a glucagon receptor antagonist. We have previously reported
a ꢀ-alanine series of orally bioavailable glucagon receptor
antagonist,15 and this series has now been optimized to obtain
a better selectivity especially over the GIP receptor with the
same high affinity for the glucagon receptor. 15 was orally
bioavailable, and furthermore, we found that substituting the
ꢀ-alanine with an isoserine on the compounds from a previous
Figure 2. Inhibition of glucagon-stimulated glycogenolysis in cultured
rat hepatocytes. For glycogenolysis experiments, glycogen-filled hepa-
tocytes were stimulated with 0.5 nM glucagon and increasing concen-
trations of 15 (2) or 13 (9) as indicated, and the glucose production
was measured in the medium after 60 min of stimulation.

5392 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Kodra et al.
Figure 3. Effect of 15 in glucagon challenged rats. Anesthetized
animals were given an intravenous dose of the compounds as shown 5
min prior to a 3 µg/kg glucagon load. The blood glucose values for
each individual rat are given. ) p e 0.001
) p e 0.0005.
**
***
Figure 4. The acute effects of a single oral dose of 15 in ob/ob mice.
These animals had no access to food during the first 6 h following
dosing with 100 mg/kg. Blood glucose is presented as the change from
baseline.
reported previously;24,25 however, no details regarding SAR or
selectivity toward hGIPR have been reported previously. We
found in our series the (R)-form was the more potent of the
two enantiomers. The SAR of the (R)-isoserine ureas was similar
to what was observed with the previous reported ꢀ-alanine urea
series where optimal binding was obtained with electron
withdrawing groups on the distal aromatic group in combination
with cyclohexyl or cyclohexenyl on the proximal aromatic
group. Up to 100-fold selectivity for glucagon receptor over
the GIP receptor was obtained in one case (38), and in all cases,
the introduction of a hydroxyl group on the ꢀ-alanine moiety
gave compounds that showed selectivity for the glucagon
receptor. The most promising compound, 15, which was a potent
glucagon receptor antagonist with a 54-fold selectivity over the
GIP receptor, inhibited glucagon-induced glycogenolysis pri-
mary rat hepatocytes and inhibited glucagon-induced hyperg-
lycemia in Sprague-Dawley rats; in addition, 15 acutely
decreased hyperglycemia in ob/ob mice when dosed orally. The
physiological advantage of selective glucagon receptor antago-
nism was demonstrated by the observation that compound 15
is able to inhibit glucagon stimulated rise in blood glucose
(Figure 3) but does not inhibit GIP stimulated insulin secretion
from the rat pancreas (Figure 5). An approximately 10-fold
reduction in GIP receptor affinity resulted in minimal effect on
insulin secretion.
Figure 5. Isolated rat pancreas were perfused with 80 mg/dL glucose
during the period 1-4 min and switched to 160 mg/dL glucose during
the period 5-50 min. GIP (500pM) was infused during 15-34 min to
stimulate insulin secretion, and compound 10 (a), 13 (b), and 15 (c)
were infused during the period 5-50 min (n ) 4 for each group).
Table 3. PK Properties of Compound 15, 18, 22, 23, 24, 25, 35, and 36
in Ratsa
compound
t1/2 (min, iv)
Fpo (%)
15
18
22
23
24
35
36
53
41
72
46
53
43
55
32
11
21
14
7
8
10
a Rats were dosed 4 mg/kg of compound po and 2mg/kg iv.
) 0 ppm). Elemental analyses were performed by the microana-
lytical laboratories at Novo Nordisk A/S, Denmark, and University
of Southern Denmark. Column chromatography was performed on
silica gel 60 (40-63 µm). Melting points were determined in open
capillary tubes on a Bu¨chi 535 apparatus and are uncorrected.
Chemicals and solvents used were commercially available and were
used without further purification. Yields refer to pure materials and
are not optimized. The preparation methods are illustrated by single
representative experimental procedures.
Experimental Section
General Synthesis. 1H NMR spectra were recorded in deuterated
solvents at 200, 300, or 400 MHz (DRX 200, DRX 300, and AMX2
400 from Bruker Instruments, respectively). Chemical shifts are
reported as δ values (ppm) relative to internal tetramethylsilane (δ
(RS)-Isoserine Ethyl Ester Hydrochloride. Dry ethanol (40 mL)
was cooled on an ice bath, and thionyl chloride (4 mL) was added
dropwise, maintaining the temperature below 5 °C. To this cold

Glucagon Receptor Antagonists with ImproVed SelectiVity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5393
solution was added (RS)-isoserine (2.5 g, 23.79 mmol), and stirring
was continued until a homogeneous solution was obtained. The
ice bath was removed and stirring was continued for 17 h at room
temperature. The solution was concentrated in vacuo to afford 4.0 g
(R)-Isoserine Ethyl Ester Hydrochloride. The compound was
prepared in analogy with the method outlined above for (RS)-
isoserine ethyl ester hydrochloride.
(R)-Isoserine Methyl Ester Hydrochloride. To methanol (2000
mL) thionyl chloride (209 mL) was added dropwise while the
temperature was maintained below -10 °C. After the addition of
the thionyl chloride, the mixture was stirred while the temperature
was kept below -30 °C. To this cold solution was added
(R)-isoserine (120 g, 1.14 mol) while temperature was kept below
-30 °C. When all the (R)-isoserine had dissolved, the cooling was
removed and the mixture was stirred overnight at room temperature.
The solvent was removed in vacuo followed by coevaporation with
toluene. The remaining crystals were washed with toluene (750
mL) for 30 min, filtered, and washed with n-hexane (300 mL). The
crystals dried in vacuo to yield 176 g (99%) of (R)-isoserine methyl
ester hydrochloride. Anal. calcd for C4H9NO3: C, 30.88; H, 6.48;
N, 9.00. Found: C, 30.98; H, 6.60; N, 8.94. mp 113-114 °C. [R]D
) +20.75 (c ) 1, H2O at 20 °C).
1
(100%) of (RS)-isoserine ethyl ester hydrochloride as an oil. H
NMR (DMSO-d6): δ 1.22 (t, 3H), 3.00 (dm, 2H), 4.15 (q, 2H),
4.40 (dd, 1H), 6.30 (br s, 1H), 8.32 (br s, 2H). 13C NMR (DMSO-
d6): δ 14.7 (q), 42.4 (t), 61.7 (t), 67.7 (d), 171 (s).
(R)-Isoserine Hydrochloride. To a suspension of D-(+)-malic
acid (15.0 g, 0.1119 mol) in dry toluene (150 mL) was added 2,2-
dimethoxypropane (50 mL, 0.392 mol). The mixture was refluxed
at 100 °C for 2 h and evaporated in vacuo. The residue was
dissolved in diethyl ether (150 mL) and subjected to flash column
chromatography using diethyl ether as eluent (200 mL). The pure
fractions were pooled and evaporated in vacuo, and the residue
was stirred in n-hexane. The precipitate was collected, washed with
n-hexane, and dried to afford 15.7 g (81%) of (R)-(2,2-dimethyl-
5-oxo-[1,3]dioxolan-4-yl)acetic acid (5) as a solid. 1H NMR
(acetone-d6): δ 1.57 (ds, 6H), 2.85 (m, 2H), 4.80 (dd, 1H), 11.0
(br s, 1H). 13C NMR (acetone-d6): δ 25.9 (q), 26.8 (q), 36.2 (t),
71.5 (d), 111.2 (s), 170.7 (s), 172.7 (s). Anal. calcd for C7H10O5:
C, 48.28; H, 5.79. Found: C, 48.31; H. 6.09. mp 113-114 °C.
(S)-Isoserine Ethyl Ester Hydrochloride. The compound was
prepared in analogy from (S)-isoserine, which was prepared from
L-(-)-malic acid similar to that described above.
Test compounds were in general prepare as illustrated with the
following representative example.
A mixture of (R)-(2,2-dimethyl-5-oxo-[1,3]dioxolan-4-yl)acetic
acid as a solid (5) (10.0 g, 57.41 mmol), triethylamine (10 mL,
68.89 mmol), and diphenylphosphoryl azide (14 mL, 63.15 mmol)
in dry toluene (100 mL) was heated and stirred at 85 °C. When the
gas evolution had ceased, stirring was continued for an additional
hour. Dry benzyl alcohol (6.3 mL, 63.15 mmol) was added, and
heating was continued for 17 h. After evaporation in vacuo, the
residue was partitioned between dichloromethane, water, and brine.
The aqueous phase was further extracted twice with dichlo-
romethane. The combined organic phases were washed twice with
saturated sodium hydrogen carbonate. After drying (magnesium
sulfate), filtration, and concentration in vacuo of the organic phase,
the residue was subjected to flash column chromatography with
dichloromethane as eluent. This afforded 6.4 g (40%) of (R)-(2,2-
dimethyl-5-oxo-[1,3]dioxolan-4-ylmethyl)carbamic acid benzyl ester
(6) as an oil. 1H NMR (Acetone-d6): δ 1.56 (s, 6H), 3.61 (m, 2H),
4.64 (dd, 1H), 5.08 (dd, 2H), 6.51 (br s, 1H), 7.29-7.38 (m, 5H).
(R)-3-{4-[1-(4-Cyclohexylphenyl)-3-(3-methoxy-5-trifluorom-
ethylphenyl)ureidomethyl]benzoylamino}-2-hydroxypropionic
acid (25). 4-Formylbenzoic acid methyl ester (6.65 g, 40.5 mmol)
was dissolved in hot methanol (175 mL). To this mixture,
4-cyclohexylaniline (7.1 g, 40.5 mmol) was added. To the resulting
suspension, more methanol (75 mL) was added and the mixture
was heated at reflux for 1 h. After cooling to 0 °C, the mixture
was filtered and the solid was washed with ice-cold methanol and
dried in vacuo at 40 °C for 16 h to afford 10.95 g of 4-[(4-
cyclohexylphenylimino)methyl]benzoic acid methyl ester. This
compound (10.93 g, 34 mmol) was suspended in methanol (200
mL) and glacial acetic acid (27 mL) was added followed by sodium
cyano borohydride (1.9 g, 30 mmol) in small portions. The mixture
was stirred at room temperature for 1 h and concentrated in vacuo.
The residue was dissolved in DCM (200 mL) and washed with
5% aqueous sodium carbonate (5 × 80 mL), dried (magnesium
sulfate), and concentrated in vacuo. The residue was added ethyl
acetate (100 mL) and n-heptane (200 mL) and concentrated in vacuo
to half the original volume. The solid was filtered, washed with
n-heptane, and dried in vacuo at 40 °C for 16 h to afford 9.52 g
(87%) of 4-((4-cyclohexylphenylamino)methyl)benzoic acid methyl
ester (1). 1H NMR (DMSO-d6): δ 1.2-1.4 (5H, m), 1.65 (5H, m),
2.30 (1H, t), 3.84 (3H, s), 4.30 (2H, d), 6.18 (1H, t), 6.50 (2H, d),
6.87 (2H, d), 7.49 (2H, d), 7.92 (2H, d);
To a solution of (R)-(2,2-dimethyl-5-oxo-[1,3]dioxolan-4-yl-
methyl)carbamic acid benzyl ester (6) (6.0 g, 25.08 mmol) in
acetonitrile (100 mL) was added hydrochloric acid (1 N, 100 mL).
The mixture was stirred for 3 h at 40 °C and concentrated in vacuo
to half the original volume. The solid was collected by filtration
and washed with water. The crude product was stirred for 5 min in
acetone (100 mL) and filtered. Toluene was added to the clear and
colorless filtrate, and the whole was concentrated in vacuo until a
precipitate was obtained. The precipitate was collected by filtration
and dried to afford 4.45 g (87%) of (R)-3-benzyloxycarbonylamino-
4-[1-(Cyclohexylphenyl)-3-(3-methoxy-5-trifluoromethylphe-
nyl)ureidomethyl]benzoic Acid (2). 5-Methoxy-3-(trifluoromethyl)-
aniline (2.0 g, 10.5 mmol) was dissolved in ethyl acetate (10 mL)
and dry HCl in ethyl acetate (15 mL) was added and the solvent
was removed in vacuo. The solid was coevaporated with toluene
(3 × 15 mL). Toluene (75 mL) and diphosgene (13 mL) were
added, and the reaction mixture was refluxed under a nitrogen
atmosphere for 2.5 h. Excess diphosgene was removed in vacuo,
and the clear oil was coevaporated with toluene. The residue was
dissolved in DCM (75 mL), and 4-((4-cyclohexylphenylamino)-
methyl)benzoic acid methyl ester (2.3 g, 7.1 mmol) was added.
The reaction mixture was stirred overnight at room temperature,
the solvent was removed in vacuo, and the residual oil was purified
by column chromatography on silica gel, eluting with a mixture of
heptane and ethyl acetate (7:3) to obtain 3 g (78%) of 4-[1-
(cyclohexylphenyl)-3-(3-methoxy-5-trifluoromethylphenyl)ure-
1
2-hydroxypropionic acid (7). H NMR (acetone-d6): δ 3.45 (ddd,
1H), 3.58 (ddd, 1H), 4.29 (dd, 1H), 5.08 (s, 2H), 6.41 (br s, 1H),
7.29-7.38 (m, 5H). 13C NMR (acetone-d6): δ 45.1 (t), 66.4 (t),
70.4 (d), 128.3 (d), 128.9 (d), 138.0 (d), 157.2 (s), 173.8 (s). Anal.
calcd for C11H13NO5: C, 55.23; H, 5.48; N, 5.85. Found: C, 55.35;
H, 5.72; N, 5.82. mp 131-132 °C.
(R)-3-Benzyloxycarbonylamino-2-hydroxypropionic acid (7) (4.4
g, 18.39 mmol) was dissolved in absolute ethanol (150 mL). Under
a nitrogen atmosphere palladium on activated carbon (10%, 0.5 g)
was added, and the mixture was hydrogenated at 1 atm for 17 h.
The catalyst was filtered off and washed with water. The combined
filtrate and washings were concentrated to about 20 mL by
evaporation in vacuo. A precipitate was obtained by dropwise
addition of methanol (100 mL). The precipitate was filtered off,
washed with methanol, and dried to afford 1.78 g (92%) of (R)-
isoserine (8) as a solid. 1H NMR (D2O): δ 3.07 (dd, 1H), 3.30 (dd,
1H), 4.19 (dd, 1H). 13C NMR (D2O): δ 43.0 (t), 68.9 (d), 177.5
(s). Anal. calcd for C3H7NO3: C, 34.29; H, 6.71; N 13.33. Found:
C, 34.35; H, 6.83; N, 13.19. mp 200-201 °C. [R]D ) +33.05 (c
) 1, H2O at 20 °C)
1
idomethyl]benzoic acid methyl ester as an oil. H NMR (DMSO-
d6): δ 1.22 (broad, 1H), 1.37 (broad, 4H), 1.7 (broad, 1H), 1.79
(broad, 4H), 3.77 (s, 3H), 3.83 (s, 3H), 4.98 (s, 2H), 6.81 (s, 1H),
7.18 (d, 2H), 7.23 (d, 2H), 7.42 (m, 3H), 7.51 (s, 1H), 7.90 (d,
2H), 8.53 (s, 1H), 10.01 (s, 1H).
4-[1-(Cyclohexylphenyl)-3-(3-methoxy-5-trifluoromethylphenyl)-
ureidomethyl]benzoic acid methyl ester (3.0 g) was dissolved in
absolute ethanol (50 mL), sodium hydroxide (4 N, 15 mL) was

5394 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Kodra et al.
added, and the reaction mixture was stirred at room temperature
for 16 h. The organic solvent was removed in vacuo, and additional
water (50 mL) was added, pH was adjusted with hydrochloric acid
(4 N) to acidic reaction, and then ethyl acetate (200 mL) was added.
The organic phase was washed with water (5 × 50 mL), dried
(magnesium sulfate), filtered, and evaporated in vacuo. The residue
was recrystallized from acetonitrile (25 mL) to afford 1.83 g (63%)
of 4-[1-(cyclohexylphenyl)-3-(3-methoxy-5-trifluoromethylphenyl)-
aqueous solution of sodium chloride (4 × 100 mL), dried
(magnesium sulfate), and concentrated in vacuo to give 15.0 g
(90%) of 1-methylsulfanyl-2-trifluoromethoxybenzene.
1-Methylsulfanyl-2-trifluoromethoxybenzene (15.0 g, 72 mmol)
was dissolved in dichloromethane (200 mL) and m-chloroperoxy-
benzoic acid (39.0 g, 216 mmol) was added in small portions during
30 min. The reaction mixture was then allowed to stand overnight.
Dichloromethane (200 mL) was added, followed by slow addition
of sodium hydroxide (2 N, 200 mL). The organic phase was
separated and washed with sodium hydroxide (2 N, 3 × 150 mL),
dried (magnesium sulfate), and concentrated in vacuo to give 15.8 g
(91%) of 1-methylsulfonyl-2-trifluoromethoxybenzene 1H NMR
(CDCl3): δ 8.11 (d, 1H), 7.71 (t, 1H), 7.48 (m, 2H) 3.23(s 1H).
Anal. calcd for C8H7F3O3S: C, 40.0; H, 2.94. Found: C, 40.20; H,
2.90. mp 148-150 °C.
1-Methylsulfonyl-2-trifluoromethoxybenzene (15.7 g, 65 mmol)
was dissolved in concentrated sulfuric acid (27 mL), and the solution
was heated to 40 °C. Nitric acid (100%, 27 mL) was added dropwise
over 45 min. The reaction mixture was allowed to stand overnight
at 60 °C, cooled, and then poured on crushed ice (300 mL). The
precipitated product was isolated by filtration, washed with water
(10 × 50 mL), and dried (magnesium sulfate), affording 17.5 g
(94%) of 3-methylsulfonyl-4-trifluoromethoxynitrobenzene. 1H
NMR (DMSO-d6): δ 8.69 (d, 1H), 8.64 (d, 1H), 7.95 (d, 1H) 3.45
(s 3H). Anal. calcd for C8H6F3NO5S: C, 33.69; H, 2.12; N, 4.91.
Found: C, 33.90; H, 2.07; 4.91. mp 102-104 °C.
1
ureidomethyl]benzoic acid (2) as crystals. H NMR (DMSO-d6):
δ 1.22 (m, 1H), 1.37 (m, 4H), 1.70 (m, 1H), 1.79 (m, 4H), 3.77 (s,
3H), 4.95 (s, 2H), 6.81 (s, 1H), 7.18 (d, 2H), 7.23 (d, 2H), 7.40 (d,
2H), 7.42 (s, 1H), 7.51 (s, 1H), 7.89 (d, 2H), 8.55 (s, 1H), 12.90
(s, 1H). Anal. calcd for C29H29F3N2O4: C, 66.15; H, 5.55; N, 5.32.
Found: C, 66.65; H, 5.70; N, 5.33. mp 148-150 °C.
4-[1-(Cyclohexylphenyl)-3-(3-methoxy-5-trifluoromethylphenyl)-
ureidomethyl]benzoic acid (2) (420 mg, 0.8 mmol) was dissolved
in DMF (10 mL), and then 1-hydroxybenzotriazole (160 mg, 1.2
mmol) and N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hy-
drochloride (230 mg, 1.2 mmol) were added. The reaction mixture
was allowed to stand for 30 min, then (R)-isoserine ethyl ester (260
mg, 1.2 mmol) and diisopropylethylamine (210 µL, 1.2 mmol)
dissolved in DMF (5 mL) were added and the reaction mixture
was stirred at room temperature for 16 h. Water (50 mL) and ethyl
acetate (100 mL) were added and the organic phase was washed
with water (5 × 50 mL), dried (magnesium sulfate), filtered, and
evaporated in vacuo. The residue was purified by column chroma-
tography on silica gel, eluting with a mixture of heptane and ethyl
acetate (1:3) to afford 510 mg (99%) of (R)-3-{4-[1-(4-cyclohexyl-
phenyl)-3-(3-methoxy-5-trifluoromethylphenyl)ureidomethyl]ben-
zoylamino}-2-hydroxypropionic acid ethyl ester (3) as an amor-
3-Methylsulfonyl-4-trifluoromethoxynitrobenzene (17.5 g, 61
mmol) was dissolved in methanol (400 mL), followed by addition
of palladium on carbon (10%, 50% water, 3.2 g). The reaction
mixture was hydrogenated for 17 h at 1 atm of hydrogen, filtered,
and concentrated in vacuo to give 14.3 g (92%) of 3-methylsulfonyl-
1
phous solid. H NMR (DMSO-d6): δ 1.12 (t, 3H), 1.22 (m, 1H),
1
1.37 (m, 4H), 1.70 (m, 1H), 1.79 (broad, 4H), 3.41 (m, 1H), 3.52
(m, 1H), 3.77 (s, 3H), 4.06 (q, 2H), 4.21 (q, 1H), 4.95 (s, 2H),
5.68 (d, 1H), 6.81 (s, 1H), 7.18 (d, 2H), 7.23 (d, 2H), 7.33 (d, 2H),
7.42 (s, 1H), 7.51 (s, 1H), 7.77 (d, 2H), 8.47 (t, 1H), 8.53 (s, 1H).
(R)-3-{4-[1-(4-Cyclohexylphenyl)-3-(3-methoxy-5-trifluorometh-
ylphenyl) ureidomethyl]benzoylamino}-2-hydroxypropionic acid
ethyl ester (3) (510 mg, 0.8 mmol) was dissolved in ethanol (15
mL) and sodium hydroxide (2 N, 2 mL) was added. The reaction
mixture was stirred at room temperature for 60 min. Then ethanol
was removed in vacuo, water (50 mL) was added, and pH was
adjusted with 4 N hydrochloric acid to acidic reaction. Filtration
and washing with water (5 × 5 mL) and drying in vacuo afforded
4-trifluoromethoxyaniline. H NMR (DMSO-d6): δ 7.26 (d, 1H),
7.14 (d, 1H), 6.85 (dd, 1H) 5.89 (s, 2H) 3.21(s, 3H). Anal. calcd
for C8H8F3NO3S: C, 37.65; H, 3.16; N, 5.49. Found: C, 37.65; H,
3.14; N, 5.45. mp 106-109 °C.
To 3-methylsulfonyl-4-trifluoromethoxyaniline (2.0 mmol, 500
mg) dissolved in ethyl acetate (6 mL) was added 3 N hydrochloric
acid in ethyl acetate (5 mL) followed by concentration in vacuo.
The residue was treated with toluene (3 × 5 mL) and each time
concentrated in vacuo. To the residue was added toluene (10 mL)
and diphosgene (6 mmol, 0.73 mL) under a nitrogen atmosphere,
and the suspension was gently refluxed for 2 h. Additional
diphosgene (6 mmol, 0.73 mL) was added, and refluxing was
continued overnight. The reaction mixture was concentrated in
vacuo to afford 3-methylsulfonyl-4-trifluoromethoxyphenyl isocy-
anate, which was used directly in the subsequent step to make 36
according to the method described for compound 25.
3-Amino-2(R)-methoxypropionic Acid. To an ice-cooled solu-
tion of methanol (250 mL) was added acetyl chloride (12.5 mL),
and the solution was stirred for 1 h at 0 °C. (R)-Malic acid (20.0
g) was added, and the solution was stirred for 16 h at room
temperature. Solvent was removed by evaporation in vacuo, leaving
a quantitative yield of (R)-2-hydroxysuccinic acid dimethyl ester
as an oil. 1H NMR (CDCl3): δ 4.52 (dd, 1H), 3.80 (s, 3H), 3.71 (s,
3H), 3.55 (bs, 1H), 2.88 (dd, 1H), 2.80 (dd, 1H).
1
460 mg (94%) of 25 as a crystalline solid. H NMR (DMSO-d6):
δ 1.22 (m, 1H), 1.37 (m, 4H), 1.70 (m, 1H), 1.79 (broad, 4H),
3.37 (m, 1H), 3.51 (m, 1H), 3.77 (s, 3H), 4.09 (t, 1H), 4.95 (s,
2H), 6.80 (s, 1H), 7.18 (d, 2H), 7.23 (d, 2H), 7.33 (d, 2H), 7.42 (s,
1H), 7.51 (s, 1H), 7.77 (d, 2H), 8.47 (t, 1H), 8.53 (s, 1H). Anal.
C30H31N3O51.25 H2O: C, H, N. mp 117-121 °C.
(S)-3-{4-[1-(4-Cyclohex-1-enylphenyl)-3-(3,5-dichlorophenyl)-
ureidomethyl]benzoylamino}-2-hydroxypropionic acid (16). A
750 mg racemic mixture of (RS)-3-{4-[1-(4-cyclohex-1-enylphenyl)-
3-(3,5-dichlorophenyl)ureidomethyl]benzoylamino}-2-hydroxypro-
pionic acid ethyl ester was eluted on a OD PREP-HPLC (50 mm
× 500 mm) from Daicell with absolute ethanol/heptane 4:96 with
a flow at 120 mL/min. The fastest eluting fraction was isolated
and solvent removed in vacuo. The (S)-3-{4-[1-(4-Cyclohex-1-
enylphenyl)-3-(3,5-dichlorophenyl)ureidomethyl]benzoylamino}-2-
hydroxypropionic acid ethyl ester was hydrolyzed with NaOH
similar to what has been described for the procedure above. Anal.
calcd for C30H29Cl2N3O5 ·3/4H2O: C, 60.46; H, 5.16; N, 7.05. Found:
C, 60.75; H, 5.54; N, 6.78.
The above (R)-2-hydroxysuccinic acid dimethyl ester was
redissolved in methyl iodide (100 mL), freshly prepared silver oxide
(30.2 g) was added, and the mixture was stirred for 24 h at room
temperature. The reaction mixture was diluted with acetonitrile (200
mL) and filtered through celite to remove silver salts and excess
silver hydroxide. The filtrate was taken to dryness to leave (R)-2-
1
methoxysuccinic acid dimethyl ester as an oil (23.2 g, 88%). H
NMR (CDCl3): δ 4.20 (dd, 1H), 3.78 (s, 3H), 3.71 (s, 3H), 3.48 (s,
(R)-3-{4-[1-(4-Cyclohexen-1-ylphenyl)-3-(3-methanesulfonyl-
4-trifluoromethoxyphenyl)ureidomethyl]benzoylamino}-2-hy-
droxypropionic acid (36). To a solution of methyl iodide (59.0 g,
0.41 mol) in DMF (150 mL) was added potassium carbonate (23.0
g, 0.16 mol). 2-(Trifluoromethoxy)thiophenol (16.0 g, 0.08 mol)
was added in portions during 30 min. The reaction mixture was
then stirred vigorously overnight. Water (250 mL) was added. The
reaction mixture was extracted with ethyl acetate (2 × 150 mL).
The combined organic phases were washed with a 50% saturated
3H), 2.80 (dd, 2H).
The above (R)-2-methoxysuccinic acid dimethyl ester was
suspended in 2 N aqueous hydrochloric acid and heated to reflux
for 30 min to give a clear solution. Upon evaporation of solvent in
vacuo, a quantitative yield of 2(R)-methoxysuccinic acid was
obtained as oil. The oil was redissolved in acetic anhydride (120
mL) and heated to 110 °C for 2 h. Solvent was removed by rotary
evaporation to leave oil. Ice-cooled methanol (150 mL) was added,

Glucagon Receptor Antagonists with ImproVed SelectiVity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5395
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E.; Karam, J. H.; Forsham, P. H. Prevention of Human Diabetic
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and the mixture was stirred for 3 h at 0 °C followed by 16 h at
room temperature. The solvent was removed to leave of (R)-2-
methoxysuccinic acid 1-methyl ester (5.0 g, 30.8 mmol, 24%). 1H
NMR (CDCl3): δ 10.30 (bs, 1H), 4.19 (dd, 1H), 3.80 (s, 3H), 3.50
(s, 3H), 2.86 (dd, 1H), 2.78 (dd, 1H).
This was dissolved in thionyl chloride (16 mL) and heated to
reflux for 2 h. Thionyl chloride was removed in vacuo, followed
by coevaporation with acetonitrile. The neat acid chloride was
dissolved in toluene (50 mL). Trimethylsilylazide (5.0 mL, 38.2
mmol) was added, and the mixture was heated to 100 °C overnight.
Then tert-butanol (30 mL) was added, and heating was continued
for an additional 16 h. The reaction mixture was cooled, and
insoluble material was removed by filtration. The organic phase
was washed with water (100 mL), saturated sodium hydrogen
carbonate solution (100 mL), 10% citric acid solution (100 mL),
water (100 mL), and saturated sodium chloride solution (100 mL),
then dried over anhydrous sodium sulfate. The solvent was removed
in vacuo. The residual oil was further purified by column chroma-
tography using 20% ethyl acetate/heptane as eluent. Pure fractions
(TLC plates were stained with ammonium molybdate/cerium
sulfate/sulfuric acid) were pooled and solvent removed in vacuo
to afford. 3-tert-butoxycarbonylamino-2(R)-methoxypropionic acid
methyl ester (600 mg, 9%). 1H NMR (CDCl3): δ 6.93 (t, 1H), 3.83
(t, 1H), 3.64 (s, 3H), 3.25 (s, 3H), 3.18 (dd, 2H), 1.36 (s, 9H).
3-tert-Butoxycarbonylamino-2(R)-methoxypropionic acid methyl
ester (500 mg, 2 mmol) was dissolved in 10% TFA in DCM (20
mL), and the reaction mixture was stirred at 30 min at ambient
temperature. The solvent was removed in vacuo, and the residue
coevaporated twice from 30 mL of 1 N hydrochloric acid in ether
to afford 320 mg (88%) of 3-amino-2(R)-methoxypropionic acid
methyl ester hydrochloride. 1H NMR (CDCl3): δ 8.25 (s, 3H), 4.21
(dd, 1H), 3.71 (s, 3H), 3.40 (s, 3H), 3.15 (m, 1H), 2.98 (m, 1H).
3-Amino-2(R)-methoxypropionic acid was used in the subsequent
step to make 20 according to the method described for compound
25.
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modified Krebs-Ringer bicarbonate buffer containing 3% dextran
and 0.2% bovine serum albumin (Fraction V, RIA grade, Sigma),
gassed with 95% O2/5% CO2 to achieve pH 7.4, was perfused at a
rate of 4 mL/min into the abdominal aorta. The portal venous
outflow was collected at 1 min intervals. Following a 10 min
equilibration period, 80 mg/dL glucose was infused from periods
1-4 min, after which the perfusate was switched to 160 mg/dL
glucose from 5 to 50 min ( the compounds to be tested. During
the period from 15 to 34 min, 500 pM GIP was introduced through
a sidearm infusion. Insulin levels in the collected perfusate was
measured by radioimmunoassay as previously described.28
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Acknowledgment. The skillful technical assistance of An-
nette Nielsen, Paw Bloch, Jette Bolding, Dorthe E. Jensen, Claus
B. Jensen, Ingrid Sveistrup, Karin H. Albrechtsen, Mette
Winther, Brian S. Hansen, Jeanette Hansen, Pia Justesen, Helle
Bach, Bent Høegh, Carsten Christensen, Stina Hansen, Dr.
Magali Zundel and AnnBritt Nielsen is gratefully acknowledged.
Supporting Information Available: Characterization data for
for compounds 11-36. This material is available free of charge
via the Internet at http://pubs.acs.org.
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