Chlorogenic acid and synthetic chlorogenic acid derivatives: Novel inhibitors of hepatic glucose-6-phosphate translocase

Article abstract of DOI:10.1021/jm9607360

The enzyme system glucose-6-phosphatase (EC 3.1.3.9) plays a major role in the homeostatic regulation of blood glucose. It is responsible for the formation of endogenous glucose originating from gluconeogenesis and glycogenolysis. Recently, chlorogenic acid was identified as a specific inhibitor of the glucose-6-phosphate translocase component (Gl-6-P translocase) of this enzyme system in microsomes of rat liver. Glucose 6- phosphate hydrolysis was determined in the presence of chlorogenic acid or of new synthesized derivatives in intact rat liver microsomes in order to assess the inhibitory potency of the compounds on the translocase component. Variation in the 3-position of chlorogenic acid had only poor effects on inhibitory potency. Introduction of lipohilic side chain in the 1-position led to 100-fold more potent inhibitors. Functional assays on isolated perfused rat liver with compound 29i, a representative of the more potent derivatives, showed a dose-dependent inhibition of gluconeogenesis and glycogenolyosis, suggesting glucose-6-phosphatase as the locus of interference of the compound for inhibition of hepatic glucose production also in the isolated organ model. Gl-6-P translocase inhibitors may be useful for the reduction of inappropriately high rates of hepatic glucose output often found in non-insulin-dependent diabetes.

Full text of DOI:10.1021/jm9607360

© Copyright 1997 by the American Chemical Society
Volume 40, Number 2
J anuary 17, 1997
Expedited Articles
Ch lor ogen ic Acid a n d Syn th etic Ch lor ogen ic Acid Der iva tives: Novel In h ibitor s
of Hep a tic Glu cose-6-p h osp h a te Tr a n sloca se
Horst Hemmerle,* Hans-J oerg Burger, Peter Below, Gerrit Schubert, Robert Rippel, Peter W. Schindler,
Erich Paulus, and Andreas W. Herling
Hoechst AG, Hoechst Marion Roussel, 65926 Frankfurt am Main, Germany
Received October 21, 1996X
The enzyme system glucose-6-phosphatase (EC 3.1.3.9) plays a major role in the homeostatic
regulation of blood glucose. It is responsible for the formation of endogenous glucose originating
from gluconeogenesis and glycogenolysis. Recently, chlorogenic acid was identified as a specific
inhibitor of the glucose-6-phosphate translocase component (Gl-6-P translocase) of this enzyme
system in microsomes of rat liver. Glucose 6-phosphate hydrolysis was determined in the
presence of chlorogenic acid or of new synthesized derivatives in intact rat liver microsomes in
order to assess the inhibitory potency of the compounds on the translocase component. Variation
in the 3-position of chlorogenic acid had only poor effects on inhibitory potency. Introduction
of lipohilic side chain in the 1-position led to 100-fold more potent inhibitors. Functional assays
on isolated perfused rat liver with compound 29i, a representative of the more potent
derivatives, showed a dose-dependent inhibition of gluconeogenesis and glycogenolyosis,
suggesting glucose-6-phosphatase as the locus of interference of the compound for inhibition
of hepatic glucose production also in the isolated organ model. Gl-6-P translocase inhibitors
may be useful for the reduction of inappropriately high rates of hepatic glucose output often
found in non-insulin-dependent diabetes.
In tr od u ction
version, termed the flexibility-substrate transport model
by Berteloot et al.,7 postulates that Gl-6-Pase is a
membrane-spanning pore-forming protein with the cata-
lytic site located within a water-filled pore accessible
to the substrate from the cytoplasmic surface of the
membrane. According to this model, Gl-6-Pase activity
in intact microsomes is constrained by the membrane,
and translocation of glucose 6-phosphate into the lumen
of the endoplasmic reticulum is not required. However,
Countaway et al., investigating the catalytic turnover
number for the enzyme, did not find any evidence for a
constrained activity of Gl-6-Pase in intact microsomes.8
The enzyme system glucose-6-phosphatase (Gl-6-
Pase) (EC 3.1.3.9) is known to play a major role in the
homeostatic regulation of blood glucose.1,2 It is respon-
sible for the formation of endogenous glucose originating
from gluconeogenesis and glycogenolysis. Gl-6-Pase is
localized within the membranes of the endoplasmic
reticulum. Upon disruption of membranes of microso-
mal vesicles from rat liver with optimal concentrations
of detergents, higher activity is found compared to
untreated vesicles. In order to explain the molecular
basis of this latent activity, and the role of the mem-
brane for Gl-6-Pase function, two major hypotheses have
been put forward during the last two decades.3,4
Alternative to the conformational model, the substrate
transport model proposed by Arion and co-workers9,10
and supported by the results of numerous kinetic
studies from the same group8,11,12 postulates a catalytic
component with its active site oriented toward the
lumen of the endoplasmic reticulum, a glucose-6-
The conformational model, suggested first by the
groups of Nordlie5 and Schulze,6 and its most recent
X Abstract published in Advance ACS Abstracts, J anuary 1, 1997.
S0022-2623(96)00736-4 CCC: $14.00 © 1997 American Chemical Society

138 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2
Hemmerle et al.
challenged based on methodological grounds,27 even
“normal” rates of hepatic glucose production are evi-
dence for an abnormal response of the liver in the face
of the elevated fasting plasma glucose concentrations
generally found in overt NIDDM.
Thus, hepatic glucose production might be an attrac-
tive target for therapeutic intervention in order to
reduce fasting blood glucose in NIDDM. Recently
published results suggest that even intensive pharma-
cological interventions with insulin or sulfonylureas in
NIDDM do not reach the goal of obtaining near normal
glycemia,28 clearly justifying the search for additional
therapeutical approaches.
Previous attempts to alter hepatic glucose output and
fasting plasma glucose levels in patients with NIDDM
by inhibiting specifically the gluconeogenic pathway
have failed,29 presumably due to the existence of a
functioning autoregulatory mechanism in the liver
involving compensatory glycogenolysis.30
Therefore, we reasoned that simultaneous targeting
of gluconeogenesis and glycogenolysis with an inhibitor
of Gl-6-P translocase should result in a reduction of
hepatic glucose production. Complete absence of func-
tional Gl-6-Pase activity, as evidenced in human glyco-
gen storage disease type I, results in severe hypoglyce-
mia.2,31
Our aim was to find more active derivatives of CHA
which inhibit microsomal Gl-6-P translocase and, fur-
thermore, are active in functional assays of hepatic
glucose production. In this study, we report the first
structure-activity relationships for inhibition of Gl-6-P
translocase by synthetic analogues of this natural
product. In addition, inhibition of glucose production
by a synthesized CHA analogue was evaluated in
functional assays on the isolated perfused rat liver.
F igu r e 1. Structural formulas of chlorogenic acid (CHA, 1),
(-)-quinic acid (2), and 3,4-dihydroxycinnamic acid (3).
phosphate translocase (Gl-6-P translocase) T1, facilitat-
ing the movement of glucose 6-phosphate through the
membrane, Pi released at the luminal surface is believed
to equilibrate via a second translocase T2. While the
catalytic component13,14 and the phosphate transporter
T215 have been recently isolated and/or cloned, evidence
for the Gl-6-P translocase is still circumstantial. How-
ever, recent findings that the Gl-6-Pase gene of a
glycogen storage disease type Ib patient is normal,16 and
light-scattering experiments by Fulceri et al.,17 which
provided further evidence for the translocation of glu-
cose 6-phosphate into the lumen of the ER, are more
consistent with the substrate transport model.
Using the nomenclature of the substrate transport
model, the catalytic unit of disrupted microsomes has
been shown to be a relatively nonspecific hydrolase of
a variety of organic phosphate esters, whereas glucose
6-phosphate is the only physiological sugar phosphate
known to be transported by the Gl-6-P translocase T1,18
which makes this transporter an interesting and logical
pharmacological target.
Ch em istr y
Chlorogenic acid 1 (CHA, Figure 1) is the ester
product of (-)-quinic acid (2) and 3,4-dihydroxycinnamic
acid (3). Our first aim was to find an efficient and
flexible way to synthesize analogues of 1. For variation
of the cinnamic moiety, we used the readily available
3,4-O-cyclohexylidene lactone 4, which could be synthe-
sized in one step by acidic catalysis starting from quinic
acid (2) and cyclohexanone.32 In compound 4 all func-
tional groups are protected except the hydroxy group
in position 1. After transformation of this alcohol to
SEM ether 5, the lactone moiety was hydrolyzed with
an equimolar amount of sodium hydroxide to 6. The
sodium salt 6 was the key intermediate for all variations
in position 3 of (-)-quinic acid. Most of the examined
acylation conditions yielded the lactone 5 as the main
product. This side reaction can be explained by transfer
of the activated acyl group to the carboxylate of 6
resulting in a mixed anhydride, which can readily
cyclize. The exceptions are acyltriazolides or acylimi-
dazolides. Activation of carboxylic acids 7 was achieved
by carbonylditriazole or carbonyldiimidazole, and these
derivatives reacted rapidly at 5 °C with the sodium salt
of 6. The ester 9 was obtained in 40-85% yield after
purification on silica gel. Cleavage of the cyclohexyl-
idene and SEM protecting groups 9 was performed with
2 N hydrochloric acid in dioxane and yielded 10 in 70-
90% (Scheme 1).
Recently, we identified chlorogenic acid 1 (CHA,
Figure 1) as a new inhibitor of microsomal Gl-6-Pase
activity.19 CHA, first described in 1920,20 plays an
important role in plant metabolism.21,22 In the litera-
ture, mainly antioxidant properties have been described
for CHA, which presumably can be ascribed to its
polyhydroxyphenylic nature.23,24
Detailed kinetic studies of the interaction of CHA with
the microsomal Gl-6-Pase system of rat liver suggested
Gl-6-P translocase as the targeted function,12 whereas
the catalytic unit and the phosphate translocase were
not affected even at millimolar concentrations of the
compound. Thus, CHA is the first specific inhibitor of
Gl-6-P translocase. Other described inhibitors of Gl-6-P
translocase (e.g. Hg2+, pyridoxal phosphate, NEM,
certain sulfhydryl poisons, diazobenzene sulfonate, to-
syllysine, chloromethyl ketone, and diethyl pyrocarbon-
ate; for a recent review and literature references of
translocase inhibitors, see ref 12), in addition to inhibit-
ing Gl-6-Pase activity of intact microsomes, show some
degree of inhibition of the phosphohydrolase in deter-
gent disrupted microsomes and hence are not well
suited for specific pharmacological targeting of the Gl-
6-P translocase activity.
In non-insulin-dependent diabetes mellitus (NIDDM),
excessive endogenous hepatic glucose production has
been described to contribute to fasting hyperglyce-
mia.25,26 Although, this contention has recently been
When R1 in acid 7 contained one or more hydroxy
groups, these were protected as SEM ether. Table 1

Chlorogenic Acid Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2 139
Sch em e 1a
a
(a) Cyclohexanone, H2SO4, 75%; (b) [(trimetylsilyl)ethoxy]methyl chloride (SEM-Cl), diisopropylethylamine, 98%; (c) NaOH, dioxane,
95%; (d) NaH, DMF, 40-85%; (e) aqueous hydrochloric acid, dioxane, 70-90%.
precursors 15a -17a .38,39 Diisobutylaluminum hydride
Ta ble 1. Variation of Cinnamic Moiety of Chlorogenic Acid
reduction of lactone 5 followed by Horner-Emmons
reaction with triethyl phosphonoacetate led to 19, which
was hydrogenated under normal pressure in the pres-
ence of rhodium on alumina to yield 20 (Scheme 2).
Subsequent use of the same reaction sequence il-
lustrated in Scheme 1 resulted in homologue 21. We
also synthesized the shorter homologue 27. In this case
we used the known ketone 2240 in which we protected
the hydroxy group with dihydropyran and then added
the lithium enolate of tert-butyl acetate at -78 °C
(Scheme 3). The stereoselectivity of this addition was
3:1 isomer 24 vs isomer 25, respectively. The configu-
ration of 24 was confirmed by X-ray crystallography.46
We selected the hydroxy group in position 1 of CHA
for further variations of the lead structure. From a
synthetic chemical point of view, this position can be
modified easily by alkylation of the unprotected tertiary
hydroxy group in lactone 4. Thus, we deprotonated 4
with sodium hydride in dimethylformamide and added
an activated alkyl halide. Table 3 contains a selection
of the obtained products. Sterically hindered or unac-
tivated alkyl halides gave very poor yields of 29 or no
reaction at all.
With the 4-chlorophenylpropyl side chain in position
lists some examples synthesized according to the de-
scribed procedure.
1, considerable variation of the ester side chain in
position 3 was tolerated, as shown by the examples in
Table 4.
Analysis of the structure-activity relationship for
inhibition of glucose-6-phosphate hydrolysis in intact
microsomes shows that a single hydroxy group in the
para position of the phenyl ring in CHA (1) is sufficient
for activity. Hence, further studies were performed
using the p-hydroxyphenyl side chain of 10b.
Similar to building block 4, we used the known
shikimic acid derivative 11a 33 (Table 2) to synthesize
the dehydrated CHA analogue 11.34 The precursor 12a
of 12 was obtained through radical deoxygenation of 4.35
For hydrolytically stable analogues of 11 we used the
known building block 13a .36 Reaction of this amine 13a
at 60 °C with the imidazolide 8, and removal of the
protecting groups as described in Scheme 1 produced
the desired amide 13.
Biologica l Resu lts a n d Discu ssion
Effect of Ch lor ogen ic Acid a n d Ch lor ogen ic
Acid Der iva tives on Micr osom a l Glu cose-6-p h os-
p h a ta se Activity. The inhibitory activity of CHA and
synthesized CHA analogues was evaluated by their
ability to inhibit glucose 6-phosphate hydrolysis in
highly intact microsomal preparations obtained from
livers of 20-h fasted rats. Gl-6-P translocase imposes
significant rate limitations on the Gl-6-Pase activity of
intact microsomes, whereas under these conditions
other translocase components do not play a significant
role for the steady state hydrolysis of glucose 6-phos-
phate by the system.10 Measuring of the effects of test
compounds on the Gl-6-Pase activity of intact mi-
crosomes can thus be conveniently used to assess their
potency as inhibitors of the Gl-6-P translocase compo-
nent.
To investigate the influence of the carboxylic group
in CHA (1), we esterified this compound with diaz-
omethane to yield the ester 14.37 As further examples,
we synthesized 15-17 from the already published

140 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2
Ta ble 2. Variations of the Cyclohexyl Moiety of CHA
Hemmerle et al.
a
b
d
Reference 33. Reference 35. c Reference 36. Reference 38. e Reference 39.
Sch em e 2a
a
(a) 1.1 equiv of 1.2 M DIBAH solution in toluene, 85%; (b) triethyl phosphonoacetate, NaH, THF, 82%; (c) H2, Rh/Al2O3, ethyl acetate,
94%; further steps as described in Scheme 1.
Cinnamic acid 3 and quinic acid (2) alone did not
inhibit the Gl-6-Pase activity of intact microsomes (data
not shown). Variation of the ester residue in position 3
of CHA (1) had little effect on inhibitory activity.
Compound 11 had nearly the same inhibitory activity
as CHA (Table 2), demonstrating that the OH group in
position 1 is also not essential for inhibition. This result
could also be confirmed by the deoxygenated CHA

Chlorogenic Acid Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2 141
Sch em e 3a
a
(a) DHP, PPTS, CH2Cl2, 95%; (b) LDA, tert-butyl acetate, THF, 55%; (c) PPTS, MeOH; (d) 2,2-dimethoxypropane, PPTS; further steps
as described in Scheme 1.
Ta ble 3. Alkylated Derivatives of CHA
Ta ble 4. Variation of Ester Moiety of CHA Analogue
analogue 12 (IC50 ) 230 µM). The examples 15-17
illustrate the role of hydroxy groups in 1. Compounds
21 and 27 were only weak inhibitors of glucose 6-phos-
phate hydrolysis with apparent IC50 values above 1 mM.
The examples given in Table 2 and Schemes 2 and 3
illustrate the importance of the carboxylic acid and ring
hydroxy groups in CHA (1) for the inhibitory activity.
The methyl ether 29a had nearly the same activity as
the free alcohol 10b, but the corresponding ethyl
analogue 29b showed only little effect on glucose
6-phosphate hydrolysis. Introduction of aromatic sub-
stituents into the side chain increased inhibitory activ-
F igu r e 2. Inhibitory effect of 29i on fructose-induced hepatic
glucose output (gluconeogenesis) in isolated perfused livers
from starved rats. Values are mean ( SEM, n ) 4-8.
ity, with an optimal chain length of four atoms between
the aromatic and the cyclohexyl ring. Variations of
substituents on the aromatic ring resulted in more
potent inhibitors. Introduction of the 4-chlorophenyl-
propyl side chain led to compound 29i, which was 100
times more potent than 1.

142 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2
Hemmerle et al.
In those livers with 0.1, 1, and 3 mM compound 29i
included, glucose production decreased significantly,
again in a concentration-dependent manner (Figure 3),
also indicating that compound 29i is able to reach the
target enzyme system in a complex in vitro system with
an intact intra-organ architecture. Similar series of
experiments with addition of CHA to the perfusate did
not show any effect of the compound on perfusate
glucose formation (data not shown). The more lipophilic
character of compound 29i compared to that of CHA
might be beneficial with respect to the uptake of this
organic anion into the hepatocytes. On the other hand,
concentrations of CHA sufficient for inhibition might not
be reached at the intracellular target site, perhaps due
to an inability of the more hydrophilic CHA to cross the
cytoplasmic membrane.
The observations described above for compound 29i
are consistent with an inhibitory activity of this CHA
derivative at the level of Gl-6-Pase also in the intact
organ model. Concomitant inhibition of glycogenolysis
and fructose-stimulated gluconeogenesis by the CHA
derivative 29i strongly supports inhibition of Gl-6-Pase
via T1 over other possible sites of inhibition of hepatic
glucose output. The availability of inhibitors of Gl-6-P
translocase active in an intact organ model will allow
the study of metabolic directive effects of specific inhibi-
tion of Gl-6-Pase activity under numerous experimental
conditions. Possibilities for further optimization of the
lead structure 29i are currently being investigated.
F igu r e 3. Inhibitory effect of 29i on hepatic glucose ouput
(glycogenolysis) in isolated perfused livers from fed rats.
Values are mean ( SEM, n ) 4-8.
Gl-6-P translocase specific inhibitors have to fulfill the
additional general criteria established previously9 in
that they inhibit glucose 6-phosphate hydrolysis in
intact microsomes, but not in detergent-disrupted mi-
crosomal vesicles. This has been convincingly demon-
strated for CHA (1).12 At a concentration of 1 mM, none
of the CHA derivatives inhibiting glucose 6-phosphate
hydrolysis in intact microsomes to various extents
(Tables 1-4) inhibited glucose 6-phosphate hydrolysis
in fully disrupted microsomes obtained by short treat-
ment of the vesicles with an optimal concentration of
detergent (data not shown), conditions under which the
hydrolysis of glucose 6-phosphate is not restricted by
substrate transport. Thus, the more active derivatives
of CHA, like CHA itself, appear to be specific inhibitors
of the translocase component of the Gl-6-Pase system;
albeit definite evidence of specificity for Gl-6-P trans-
locase will require the exclusion of an effect of the
analogues on the pyrophosphatase activity in intact
microsomes as has been already demonstrated for
CHA,12 experimental conditions where only T2, but not
T1 exerts a rate limiting influcence on the hydrolysis
of pyrophosphate. These new compounds could have a
significant potential for the elucidation of the structure
and function of the Gl-6-Pase system.
Exp er im en ta l Section
Gen er a l Meth od s. Reactions with materials sensitive to
air or moisture were run in dry-glass apparatus under an
argon atmosphere with dry solvents. Melting points were
determined on a Bu¨chi capillary melting point apparatus
(according to Dr. Tottoli) and are uncorrected. 1H NMR
spectra were recorded on a Bruker AM 270 spectrometer.
Significant 1H NMR data are tabulated in the following
order: multiplicity (s, singlet; d, doublet; t, triplet; q, quartet;
m, multiplet), number of protons, coupling constant(s) in hertz.
FAB mass spectra were obtained on a Kratos MS 902 in a
3-nitrobenzylic alcohol matrix (optionally in the presence of
LiCl) using xenon as the target gas. DCI mass spectra were
determined on a Kratos MS 80 RFA using isobutane as reagent
gas. High-resolution FAB MS: mass spectrometer ZAB2-SEQ
(VG Analytical) with data system 11-250J , equipped with the
standard VG FAB ion source and a cesium ion gun operated
at 35 kV. A methanolic sample solution was added to a 10%
solution of polyethylene glycol 1000 or PEG 1000 dimethyl
ether, respectively, in 3-nitrobenzyl alcohol. Resolution M/∆M
) 5000, 30 s linear voltage scan over a mass range of 100 Da,
raw data accumulation (MCA) of 15-17 scans. Mass deter-
mination by software interpolation between two PEG reference
signals; reproducibility better than 0.002 Da (n ) 4). Flash
chromatography was carried out on E. Merck silica gel 60
(0.04-0.063 mm). Thin-layer chromatography was performed
on silica gel F254 plates from E. Merck. Visualization was done
using phosphomolybdic acid/cerium(IV) sulfate/H2SO4 and
heating on a 100 °C plate. Elemental analyses were performed
by the Analytical Laboratories, Hoechst AG.
Effects of a Ch lor ogen ic Acid Der iva tive on
Glu cose P r od u ction in Isola ted P er fu sed Ra t Liv-
er s. An expected and important consequence of block-
ing Gl-6-P translocase by derivatives of CHA would be
the inhibition of hepatic glucose production.
The results of liver perfusion experiments with gly-
cogen-depleted rat livers perfused with glucose-free
medium containing 10 mM fructose, and glycogen-rich
livers with glucose-free medium without any additives
are presented in Figures 2 and 3, respectively. With
glycogen-depleted livers, perfusate glucose formation
was significantly stimulated in the 60 min following
addition of fructose to the system (Figure 2). Addition
of 0.1, 1, and 3 mM compound 29i, as a representative
example for a highly active CHA derivative with a
lipophilic side chain at position 1, caused a concentra-
tion-dependent decrease of the hepatic glucose output
(Figure 2), confirming preliminary findings in isolated
hepatocytes which suggested an inhibitory effect on
substrate-stimulated glucose production (H.-J . Burger,
unpublished data).
Syn th eses of Com p ou n d s. (A) P r ep a r a tion of 4 fr om
2. A 163.3 g (0.85 mol) portion of compound 2 was suspended
in 186 mL (1.8 mol) of cyclohexanone, and 0.5 mL of concen-
trated sulfuric acid was added. The mixture was then heated
slowly to a heating bath temperature of 200 °C, and a water/
cyclohexanone azeotrope was removed by distillation. After
the azeotrope no longer distilled over, the light brown reaction
solution was stirred at a bath temperature of 200 °C for a
further 2 h. The reaction solution was then allowed to cool to
70 °C, and 10 g of sodium hydrogen carbonate was added. The
mixture was then treated with 700 mL of ethyl acetate, and
Perfusion experiments with glycogen-rich livers showed
net glucose production for the first 80 min (Figure 3).

Chlorogenic Acid Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2 143
the organic phase was washed with water and saturated
sodium chloride solution. The organic phase was then con-
centrated in vacuo. The light yellow residue was crystallized
from 2-propanol/water, 1:1. A yield of 142.1 g (75%) of lactone
4 was obtained as colorless crystals.
(H) P r ep a r a tion of 19 fr om 18 (Step b, Sch em e 2). A
7.5 g (33.5 mmol) portion of triethyl phosphonoacetate was
added dropwise at 0 °C under argon atmosphere to a suspen-
sion of 0.9 g (29.9 mmol) of 80% sodium hydride in 200 mL of
anhydrous tetrahydrofuran. The reaction mixture was slowly
allowed to warm to room temperature, and then 7.7 g (19.9
mmol) of 18 dissolved in 20 mL of anhydrous tetrahydrofuran
was added dropwise at -30 °C. This solution was stirred at
-20 to -30 °C for 24 h and then treated with 100 mL of
saturated ammonium chloride solution. It was necessary to
maintain the low temperature, since at higher temperatures,
intramolecular Michael addition of the free hydroxy group
occurred which resulted in the formation of a cyclic ether. The
combined organic phases were washed with saturated sodium
chloride solution and dried using magnesium sulfate. After
concentration in vacuo, the residue was purified on silca gel
(eluent: ethyl acetate/n-heptane, 1:1), and 7.5 g (82%) of 19
was obtained as a colorless oil.
(B) P r ep a r a tion of 5 fr om 4. A 38.14 g (0.15 mol) portion
of hydroxy lactone 4 was dissolved in 180 mL of dichlo-
romethane, 53.0 mL (0.3 mol) of diisopropylamine was added,
and 45.0 mL (0.254 mol) of [(trimethylsilyl)ethoxy]methyl
chloride was added dropwise at room temperature to this
solution, and it was stirred at reflux temperature for 6 h. The
reaction solution was then added to a saturated ammonium
chloride solution and extracted with ethyl acetate. The
combined organic phases were extracted using cold 1 N
potassium hydrogen sulfate solution at about 6 °C and dried
using sodium sulfate. After concentration in vacuo, a light
yellow residue was obtained which was crystallized from
heptane/ethyl acetate, 6:1. A 57.0 g (98%) yield of 5 was
obtained.
(C) P r ep a r a tion of 6 fr om 5. A 1.38 g (3.6 mmol) portion
of 5 was dissolved in 8 mL of dioxane, 3.8 mL of 1 N sodium
hydroxide solution was added at room temperature, and the
resulting reaction mixture was stirred and after 2 h concen-
trated in vacuo. A 1.2 g yield of 6 as an amorphous solid was
obtained.
(I) P r ep a r a tion of 20 fr om 19. To a solution of 1.0 g (2.2
mmol) of 19 in 50 mL of ethyl acetate was added 100 mg of
O
(5% Rh). The mixture was shaken at 25 °C and
Rh/Al2
3
normal pressure under a hydrogen atmosphere for 3 h. The
catalyst was filtered off, and the filtrate was concentrated in
vacuo. A 0.95 g (94%) yield of 20 as a colorless solid was
obtained.
(J ) P r ep a r a tion of 21 fr om 20. 21 was prepared from 20
according the synthesis procedures from 5 to 10 (yield: 37%).
(K) P r ep a r a tion of 23 fr om 22. A 20.0 g (84.4 mmol)
portion of compound 22 was dissolved in 200 mL of anhydrous
dichloromethane and treated at 25 °C with 14.9 g (176.8 mmol)
of dihydropyran and 200 mg of pyridinium p-toluenesulfonate.
This solution was stirred for 12 h, 500 mL of ethyl acetate
was then added, and the organic phase was washed with
sodium chloride solution. The organic phase was dried using
magnesium sulfate and concentrated in vacuo. A 26.0 g (95%)
yield of 23 as a colorless solid was obtained.
(L) P r ep a r a tion of 24 a n d 25 fr om 23. A 3.66 g (36.0
mmol) portion of diisopropylamine was dissolved in 100 mL
of anhydrous tetrahydrofuran, and 25 mL of 1.5 M n-butyl-
lithium solution in hexane was added dropwise at -20 °C
under an argon atmosphere. The reaction solution was
allowed to warm to 0 °C and was then cooled again to -60 °C.
A 4.1 g (35.3 mmol) portion of tert-butyl acetate dissolved in
20 mL of anhydrous tetrahydrofuran was slowly added drop-
wise at this temperature. The solution was stirred at -60 °C
for 30 min, and subsequently 10.0 g (32.2 mmol) of 23 dissolved
in 30 mL of anhydrous tetrahydrofuran was added dropwise
at -60 °C. After being stirred for 1 h at the same temperature,
the reaction mixture was hydrolyzed using saturated sodium
hydrogen carbonate solution. The mixture was extracted using
ethyl acetate. The combined organic phases were washed with
saturated sodium chloride solution and dried using magnesium
sulfate. After concentration, 11.9 g (87%) of 24 and 25 was
obtained in a ratio of 3:1 as a light brown oil. The separation
of the isomers was achieved by chromatography on silica gel
(eluent: ethyl acetate/heptane, 1:2). We obtained 24 in 55%
yield.
For proofing the relative configuration of the new stereo-
center in 24, we cleaved the protecting groups by treatment
with p-toluenesulfonic acid in methanol and acylated the
deprotected intermediate with acetic anhydride in dichlo-
romethane with triethylamine and 4-(dimethylamino)pyridine
as bases to obtain the tetraacetate 24a as colorless crystals.
(M) P r ep a r a tion of 26 fr om 24. A 11.9 g (27.9 mmol)
portion of 24 was dissolved in 200 mL of methanol, and 1.8 g
of pyridinium p-toluenesulfonate was added. The mixture was
heated at reflux temperature for 1 h, and the reaction mixture
was then concentrated in vacuo. The residue was dissolved
in 200 mL of anhydrous dichloromethane, and 8.6 g (93.5
mmol) of dimethoxypropane was added. After 72 h at room
temperature, the solution was concentrated in vacuo again and
the residue was purified by chromatography on silica gel
(eluent: ethyl acetate/n-heptane, 1:1). A 6.6 g (82%) yield of
26 was obtained.
(D) P r ep a r a tion of 8 fr om 7 (Com m on P r oced u r e). A
27 mmol portion of carboxylic acid 7 was dissolved in 35 mL
of anhydrous dimethylformamide. A solution of 27 mmol of
carbonyldiimidazole (or carbonylditriazole) dissolved in 35 mL
of anhydrous dimethylformamide was added dropwise at room
temperature. After 2 h this solution was ready for use in the
following reaction.
(E) P r ep a r a tion of 9 fr om 6 a n d 8 (Com m on P r oce-
d u r e). A 25 mmol portion of sodium hydride was added at
room temperature to a solution of 21 mmol of sodium salt 6 in
50 mL of anhydrous dimethylformamide. This suspension was
stirred for 1 h at room temperature before the solution of
imidazolide 8 was added at 0-5 °C. After 2 h at 0-5 °C the
reaction mixture was added to saturated ammonium chloride
solution. The mixture was acidified to pH 4 by addition of 1
N potassium hydrogen sulfate and extracted using ethyl
acetate. The combined organic phases were washed succes-
sively with saturated ammonium chloride solution, water, and
saturated sodium chloride. The organic phase was dried using
sodium sulfate and concentrated in vacuo. The oily residue
was purified on silica gel.
(F ) P r ep a r a tion of 10 fr om 9 (Com m on P r oced u r e). A
52 mmol portion of cyclohexylidene ketal 9 was dissolved in
130 mL of dioxane and treated with 95 mL (0.19 mol) of 2 N
hydrochloric acid at room temperature with stirring. The
mixture was stirred at room temperature for 20 h. Then the
clear solution was adjusted to pH 3-4 using 2 N sodium
hydroxide and concentrated in vacuo. The solid residue was
suspended in ethyl acetate/methanol, 3:1, and the insoluble
sodium chloride was filtered off. The filtrate was concentrated
again, and the residue was purified on silica gel.
According to these procedures we synthesized the com-
pounds 10a -e, which were characterized by NMR, MS, and
C, H, N analyses. Yields: (6 f 10a ) 54%, (6 f 10b) 61%, (6
f 10c) 43%, (6 f 10d ) 48%, (6 f 10e) 57%.
Compounds 11-17 were prepared according to the steps d
and e (Scheme 1) starting from the known building blocks
11a -17a . Yields: (11a f 11) 77%, (12a f 12) 65%, (13a f
13) 49%, (15a f 15) 81%, (16a f 16) 34%, (17a f 17) 23%.
(G) P r ep a r a tion of 18 fr om 5. A 15.0 g (39 mmol) portion
of 5 was dissolved in 200 mL of anhydrous toluene, and 38
mL (43 mmol) of 1.2 M diisobutylaluminum hydride solution
in hexane was added dropwise at -70 °C. The reaction
mixture was allowed to warm to 0 °C in the course of 2 h and
was hydrolyzed using 10 mL of saturated sodium hydrogen
carbonate solution, and 10 mL of 1 N sodium hydroxide
solution and 10 mL of water were added successively. The
reaction mixture was vigorously stirred at room temperature
for 30 min, and finally the solution was concentrated. A 12.9
g (85%) yield of 18 was as a colorless oil which crystallized at
0 °C was obtained. Mp: 20-25 °C.
(N) P r ep a r a tion of 27 fr om 26. According to the synthe-
sis of 10 from 6 the methylene-prolonged CHA analogue 27

144 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2
Hemmerle et al.
could be obtained with the only modification that aqueous
trifluoracetic acid (95%) was used to cleave the protecting
groups, especially the tert-butyl ester moiety, to yield 27 in
81%.
Krebs-Ringer-Bicarbonate buffer (100 mL total volume). The
buffer consists of 137 mM NaCl, 2.7 mM KCl, 11.9 mM
NaHCO3, 0.72 mM NaH2PO4, 1.8 mM CaCl2, and 0.5 mM
MgCl2 without glucose and was supplemented with 30% (v/v)
washed bovine erythrocytes and 1.6% (w/v) bovine serum
albumin. Routinely, isolated perfused livers from four rats
were prepared for one experiment. Samples of the perfusate
were taken in 10 min intervals for the determination of glucose
and in 30 min intervals for the determination of lactate
dehydrogenase activity using standard enzymatic procedures.45
The measured values for glucose are cumulative values during
a perfusion of up to 2 h.
To study the effect of a test compound on the gluconeoge-
netic process, livers from rats starved for 24 h were used,
assuming that under these conditions there is only a neglect-
able content of glycogen. Hepatic glucose production derived
from gluconeogenesis was induced by adding fructose at 10
mM to the perfusate 30 min after the start of perfusion. Ten
minutes later the test compound (29i) was added to the
perfusate with concentrations ranging from 0.1 to 3 mM.
To study the effect of a test compound on the glycogenolytic
process, livers from rats with access to food ad libitum prior
to the beginning of the experiment were used, assuming that
under these conditions liver glycogen stores are full. The test
compound was added to the perfusate at the start of the
perfusion experiment at concentrations ranging from 0.1 to 3
mmol/L. Perfusate lactate dehydrogenase activities measured
for the estimation of the integrity of the livers during the
perfusion period did not significantly differ between livers
perfused in the absence of or with test compound (data not
shown).
(O) P r ep a r a tion of 28 fr om 4. The introduction of the
side chains 28a -i into hydroxy lactone 4 was achieved by
deprotonation with 1.5 equiv of sodium hydride in anhydrous
dimethylformamide and subsequent treatment with the cor-
responding alkyl halides at 0-10 °C. The alkylated products
28a -i were transformed to 29 according to the procedure
described in Scheme 1. Yields: (4 f 29a ) 23%, (4 f 29b) 27%,
(4 f 29c) 17%, (4 f 29d ) 27%, (4 f 29e) 19%, (4 f 29f) 24%,
(4 f 29g) 17%, (4 f 29h ) 13%, (4 f 29i) 26%, (4 f 29j) 11%.
(P ) P r ep a r a tion of 30. According to the synthesis of 29
we obtained the esters 30a -f. Yields: (4 f 30a ) 18%, (4 f
30b) 21%, (4 f 30c) 15%, (4 f 30d ) 19%, (4 f 30e) 13%.
Micr osom a l Gl-6-P a se Activity Assa y. Microsomes were
prepared by differential centrifugation from 10% (w/v) liver
homogenates obtained from 20-h-fasted male Wistar rats
(160-180 g body weight, Hattersheim, FRG) as has been
reported in detail previously.41 Protein concentration of the
microsomal fraction was determined with the bicinchoninic
acid method.42 Intactness of the preparations, assessed by
measuring the hydrolysis of 1 mM mannose 6-phosphate,43 was
usually above 97%. Optimally detergent disrupted microsomes
were prepared according to published procedures43 by exposing
thawed microsomes to concentrations of detergent Triton
X-100 which resulted in the maximal release of latent activity
assessed by determination of the intactness of the vesicles.43
Glucose 6-phosphate hydrolysis was determined in untreated
and optimally detergent disrupted microsomes at 22 °C using
a colorimetric assay described elsewhere43 with some modifica-
tions for microtiter plates. Briefly, 100 µg of microsomal
protein were incubated for 10 min at 22 °C in a total of 100
µL of assay buffer (250 mM sucrose, 50 mM HEPES, pH 7.0)
containing 1 mM glucose 6-phosphate in the presence or
absence of test compound (0.1-1000 µM). The reaction was
started by the addition of microsomes. Plates for untreated
and disrupted microsomes were run in parallel. The reaction
was stopped by addition of 200 µL of phosphate color reagent,43
and the formation of inorganic phosphate was quantified
colorimetrically after incubation at 37 °C for at least 30 min
by reading the extinction at 630 nm. For each concentration
of test compound, background extinction was determined in
parallel incubations by adding phosphate color reagent before
addition of the microsomes. After correction for background
extinction, the extinction obtained in the presence of a given
concentration of inhibitor was compared to the extinction of
control incubations with only vehicle to determine the percent
inhibition. Stock solutions of test compounds were prepared
in MeOH and diluted with assay buffer. The resulting
maximal methanol concentration of 1.25% (v/v) was without
any effect on the intactness of microsomes or the phosphatase
activity (data not shown).
IC50 values of test compounds were routinely determined,
where appropriate, by nonlinear least-squares analysis of the
inhibition values. In case 50% inhibition was not reached at
a 1 mM concentration of test compound, activities are listed
as percent inhibition at 1 mM. IC50 values and inhibition
values are representative values of at least two independently
performed experiments with a variability of the inhibition
values between experiments of less than 10%.
Glu cose Ou tp u t of Isola ted P er fu sed Ra t Liver s. The
procedure was performed as described previously44 with the
following specifications: Male Sprague-Dawley rats (Moel-
legard, Denmark; 250-300 g body weight) were anaesthetized
with pentobarbital sodium (60 mg/kg ip). The liver was
exposed by longitudinal midline and transverse subcostal
incisions, and the portal vein was cannulated with a venous
cannula. The liver was infused immediately with oxygenated
saline containing heparin (70 units/ml) at 37 °C. The vena
cava caudalis was opened to allow a continuous flow of the
saline/heparin solution for about 2 min. Then the liver was
transferred into a heated (37 °C) perfusion chamber and
perfused via the portal vein in a recirculating manner at a
constant flow of 35 mL/min with continuously oxygenated
Ack n ow led gm en t. We would like to express our
thanks to the collegues of the Hoechst Marion Roussel
research divisions who contributed to the work. We also
would like to thank Prof. William J . Arion for his critical
reading of the manuscript.
Su p p or tin g In for m a tion Ava ila ble: Analytical data for
structure confirmation and purity criteria (20 pages). Order-
ing information is given on any current masthead page.
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