11-Ketotigogenin cellobioside (pamaqueside): A potent cholesterol absorption inhibitor in the hamster

Full text of DOI:10.1021/jm960123n

© Copyright 1996 by the American Chemical Society
Volume 39, Number 10
May 10, 1996
Communications to the Editor
Sch em e 1a
11-Ketotigogen in Cellobiosid e
(P a m a qu esid e): A P oten t Ch olester ol
Absor p tion In h ibitor in th e Ha m ster 1
Peter A. McCarthy,* Michael P. DeNinno,
Lee A. Morehouse, Charles E. Chandler,
Faan Wen Bangerter, Theresa C. Wilson,
Frank J . Urban, Stanley W. Walinsky,
Patricia G. Cosgrove, Kimberly Duplantier,
J ohn B. Etienne, Michael A. Fowler,
J ohn F. Lambert, J ohn P. O’Donnell,
Susan L. Pezzullo, Harry A. Watson, J r.,
Robert W. Wilkins, Lawrence M. Zaccaro, and
Michael P. Zawistoski
Central Research, Pfizer, Inc., Eastern Point Road,
Groton, Connecticut 06340
Received February 7, 1996
Heart attacks are a major cause of death in Western
industrialized countries. In the United States alone,
over 500 000 people die of heart attacks each year. The
underlying disease, coronary heart disease, is estimated
to cost the U.S. economy more than $60 billion each
year. There is a straightforward relationship between
coronary heart disease death and serum cholesterol: the
higher the serum cholesterol level, the higher the death
rate.2 Further studies have shown that dietary or
pharmacological reduction in serum cholesterol levels
reduces coronary heart disease mortality3 and total
mortality.4
In recent years, HMG-CoA reductase inhibitors such
as lovastatin, pravastatin, simvastatin, and fluvastatin
have been introduced to combat elevated serum choles-
terol. These agents have proven to be efficacious,
convenient, and, for the most part, safe; however, safety
concerns still exist.5 For these reasons, there is need
for new cholesterol-lowering agents. Ideally, a new
agent should have a safety profile superior to that of
the HMG-CoA reductase inhibitors. This may alter the
benefit-risk analysis in mild hypercholesterolemics in
favor of treatment. A new agent should also have a
complementary mechanism-of-action and safety profile
to allow for combination therapy with LDL-lowering
agents (HMG-CoA reductase inhibitors) and HDL-
a
(a) Br2, HCl, MeOH, CH2Cl2, 59%; (b) NaOH, t-BuOH, H2O;
(c) Ac2O, pyridine; H2O, 93% from 4; (d) Ca, NH3, THF; bromoben-
zene, H2O; THF, MeOH, H2O, 87%; (e) heptaacetylcellobiosyl
bromide, ZnF2, CH3CN; CH2Cl2, 93%; (f) NaOMe, MeOH, THF,
57%.
elevating agents (fibrates and niacin). Herein we com-
municate our preclinical data on pamaqueside (1, CP-
148,623), an agent which may demonstrate this profile.
Alfalfa saponins provided the genesis for our ap-
proach. Many lines of research have shown that certain
alfalfa saponin subfractions inhibit cholesterol absorp-
tion in animals,6 probably through a nonsystemic mech-
S0022-2623(96)00123-9 CCC: $12.00 © 1996 American Chemical Society

1936 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10
Communications to the Editor
anism of action.7 Tiqueside (2, CP-88,818) is a synthetic
saponin with a structure based upon naturally-occurring
saponins. As summarized elsewhere, 2 is an efficacious
inhibitor of cholesterol absorption in several species.8
In humans dosed 4 g/day for 12 weeks, 2 was well-
tolerated and lowered LDL cholesterol levels by more
than 20%.9 These effects were associated with a de-
crease in fractional cholesterol absorption and increased
excretion of fecal neutral sterols. The bioavailability of
2 in dogs was low (1.7%), but not insignificant.10
Although these results were encouraging, 2 appeared
limited by its weak potency and its higher than neces-
sary bioavailability.
F igu r e 1. Hamsters (n ) 4/dose group) were pre-fed a high-
cholesterol diet for 3 d and then fasted overnight. They were
dosed with drug followed immediately by a liquid diet bolus
containing [3H]cholesterol. Cholesterol absorption was deter-
mined by hepatic 3H content 24 h later. Data on 1 are a
compilation of three different dose-response experiments and
are given as the mean ( SD for each dose group. Data on 2
are given as the mean ( SD of eight experiments.
Through exploration of the structure-activity rela-
tionship of 2, we came to focus on C-ring-oxygenated
spirostane glycosides such as 1. The synthesis of 1
(Scheme 1) begins with a precedented11 four-step ketone
transposition to convert hecogenin (3) into 11-ketotigo-
genin (7).12 Bromination of 3 under carefully controlled
conditions provides 4 without contamination of the
11,23-dibromo side product.11a The 11-bromide of 4 is
hydrolyzed, and the resulting ketol rearranges under
the reaction conditions to the more stable ketol repre-
sented by structure 5. Acetylation of the C-3 and C-12
hydroxy groups provides 6. This material must be
scrupulously dried prior to calcium and ammonia treat-
ment, or else the desired reduction product 7 is con-
taminated with a hydrolysis product 5. We have
investigated a variety of methods for glycosidic cou-
pling.13 One of the better procedures involves a zinc
fluoride14 coupling of 7 with cellobiosyl bromide15 which
provides high â-selectivity. Deprotection of 8 gives
compound 1.
We tested the pharmacological effects of these agents
in the hamster. Hamsters have a more human-like
lipoprotein profile than other rodents and respond to
dietary cholesterol and fat in a manner similar to
humans. To assess the ability of these agents to acutely
inhibit cholesterol absorption, we used the procedure
of Harwood.8 Briefly, cholesterol-fed hamsters are
adminstered a bolus of radiolabeled cholesterol in liquid
diet with or without test compound. Twenty-four hours
later, the animals are euthanized and cholesterol ab-
sorption is estimated by comparing liver counts with
those of control animals. In this model, 1 was found to
be approximately 25 times more potent than 2 (Figure
1).
F igu r e 2. Hamsters (n ) 6/group) were fed a 0.2% cholesterol,
0.1% cholic acid diet or a cholesterol-free diet for 4 days with
1 or 2 mixed in the chow. Liver was homogenized prior to
extraction, and data was expressed per milligram of protein.
Values are the mean ( SD. *p < 0.05 versus cholesterol-fed
controls (Student-Newman Keuls Test).
advantages for 1 were indeed observed for both hepatic
cholesterol accumulation (Figure 2) and plasma non-
HDL cholesterol levels (Figure 3). Given the close
structural relationship of these two compounds, the
increased potency is remarkable.
Since these agents are believed to act in the intestinal
lumen, absorption of any drug poses an unnecessary
burden on the patient. For this reason, the bioavail-
ability of 2, although quite low, could stand improve-
ment. From our experience with 2, we found that the
dog was a good pharmacokinetic model for humans, so
we chose to compare 1 and 2 in the dog. As shown in
Table 1, even at a higher dose, 1 demonstrated dramati-
cally lower systemic exposure than 2. 1 demonstrated
a 4-fold lower volume of distribution and 2-fold higher
clearance, resulting in a 10-fold shorter half-life than
2. The bioavailability of 1 was more than 40-fold lower
than that of 2. The lower bioavailability of 1 coupled
with its improved potency should lead to a much lower
systemic drug burden to the patient.
Next, we wanted to demonstrate that the cholesterol
absorption inhibition potency advantage would translate
into a similar potency advantage for lowering hepatic
and plasma cholesterol levels. These parameters were
evaluated in a 4-day dosing protocol in cholesterol-fed
hamsters. In this subchronic model, similar potency
In summary, compared with 2, 1 is 10-30-fold more
potent and substantially less bioavailable. On the basis
of these data, 1 offers a new mechanistic class of
hypocholesterolemic agents with the potential for an

Communications to the Editor
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10 1937
(5) For example, see: Mevacor in Physician’s Desk Reference, 49th
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(6) (a) Price, K. R.; J ohnson, I. T.; Fenwick, G. R. The Chemistry
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Tigogenin Cellobioside on Intestinal Uptake of Cholesterol,
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Malinow, M. R.; Elliott, W. H.; McLaughlin, P.; Upson, B. Effects
of Synthetic Glycosides on Steroid Balance in Macaca fascicu-
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(7) Stedronsky, E. R. Interaction of Bile Acids and Cholesterol with
Non-systemic Agents Having Hypocholesterolemic Properties.
Biochim. Biophys. Acta 1994, 1210, 255-287.
F igu r e 3. Hamsters (n ) 6/group) were fed a 0.2% cholesterol,
0.1% cholic acid diet for 4 days with 1 or 2 mixed in the chow.
Non-HDL values are calculated by difference (TPC minus HDL
cholesterol). Values are the mean ( SD. *p < 0.05 versus
cholesterol-fed controls (Student-Newman Keuls Test).
(8) Harwood, H. J ., J r.; Chandler, C. E.; Pellarin, L. D.; Bangerter,
F. W.; Wilkins, R.W.; Long, C. A.; Cosgrove, P. C.; Malinow, M.
R.; Marzetta, C. A.; Pettini, J . L.; Savoy, Y. E.; Mayne, J . T.
Pharmacological Consequences of Cholesterol Absorption Inhibi-
tion: Alteration in Cholesterol Metabolism and Reduction in
Plasma Cholesterol Concentration Induced by the Synthetic
Saponin â-Tigogenin Cellobioside (CP-88,818; Tiqueside). J .
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Ta ble 1. Comparison of the Pharmacokinetics of 1 and 2 in
Fed Beagle Dogs
(9) Dujovne, C. A.; Harris, W. S.; Gelfand, R. A.; Morehouse, L. A.;
McCarthy, P. A.; Chandler, C. E.; DeNinno, M. P.; Harwood, H.
J ., J r.; Newton, F. A.; Shear, C. L. Investigations of the Effects
of Synthetic Saponins on Cholesterol Absorption and Serum
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J ., Sniderman, A., Eds; Elsevier Science: New York, 1995; pp
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(10) Inskeep, P. B.; Connolly, A. G.; Cole, M. J .; Luther, E. W.; Biehl,
M. L.; Marzetta, C. A.; Savoy, Y. E.; Silber, B. M. Pharmacoki-
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(11) (a) Phillipps, G. H.; Nice, F. A.; Graham, W.; Wetherill, L. A.
U.S. Patent 3,178,418, 1965. (b) Elks, J .; Phillipps, G. H.;
Walker, T.; Wyman, L. J . Studies in the Synthesis of Cortisone.
Part XV. Improvements in the Conversion of Hecogenin into
3â: 12â-Diacetoxy-5R:25D-spirostan-11-one and a Study of the
Isomeric 11:12 Ketols. J . Chem. Soc. 1956, 4330-4344. (c)
Chapman, J . H.; Elks, J .; Phillipps, G. H.; Wyman, L. J . Studies
in the Synthesis of Cortisone. Part XVI. A New Method of
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(12) 11-Ketotigogenin is also commercially available from Steraloids
Inc., Wilton, NH.
parameter
dose, mg/kg
1
2
500
375
Cmax, g/mL
0.11 ( 0.04
2.15 ( 1.03
0.04 ( 0.02
4.5
0.5 ( 0.1
1.4 ( 0.5
2.93 ( 1.39
186 ( 100
1.7 ( 0.8
37.1
2.1( 0.6
0.6 ( 0.1
AUC, ghr/mL
bioavailability, %
half-life,a,b
h
volumess,a L/kg
clearance,a mL/min/kg
a
b
Calculated following iv administration of 1.5 mg/kg. Har-
monic mean; 0.693/mean Kel.
improved safety profile by virtue of a non-systemic
mechanism of action. 1 has been advanced to clinical
evaluation, and further research on this new mecha-
nistic class has been pursued. Further results will be
reported in due course.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and spectral data for the preparation of compound 1
and details of the pharmacology and pharmacokinetic studies
(10 pages). Ordering information is given on any current
masthead page.
(13) Urban, F. J .; Moore, B. S.; Breitenbach, R. Synthesis of Tigogenyl
â-O-Cellobioside Heptaacetate and Glycoside Tetraacetate Via
Schmidt’s Trichloroacetimidate Method; Some New Observa-
tions. Tetrahedron Lett. 1990, 31, 4421-4424.
(14) Walinsky, S. W.; Lambert, J . F.; Goggin, K. D. Zinc Fluoride-
Activated Glycoside Syntheses. Tigogenyl â-O-Cellobioside as a
Novel Cholesterol Lowering Agent. Abstracts from the 208th
ACS National Meeting, Washington, DC, August 1994; CARB
11.
(15) Heptaacetylcellobiosyl bromide may be purchased from the
Sigma Chemical Co., St. Louis, MO. See also: Goggin, K. D.;
Hammen, P. D.; Knuktson, K. L.; Lambert, J . F.; Walinsky, S.
W.; Watson, H. A. Commercial Synthesis of R-D-Cellobiosyl
Bromide Heptaacetate. J . Chem. Tech. Biotechnol. 1994, 60,
253-256.
Refer en ces
(1) Portions of this research were presented at Xth International
Symposium on Atherosclerosis, Montreal, Canada, October
9-14, 1994; abstract pages 88, 309, 310, and 315.
(2) Martin, M. J .; Hulley, S. B.; Browner, W. S.; Kuller, L. H.;
Wentworth, D. Serum Cholesterol, Blood Pressure, and Mortal-
ity: Implications from a Cohort of 361,663 Men Lancet 1986,
No. 2, 933-936.
(3) Lipid Research Clinics Program. The Lipid Research Clinics
Coronary Primary Prevention Trial Results. J . Am. Med. Assoc.
1984, 251, 351-364 and 365-374.
(4) Scandinavian Simvastatin Survival Study Group. Randomised
Trial of Cholesterol Lowering in 4444 Patients with Coronary
Heart Disease: The Scandinavian Simvastatin Survival Study
(4S). Lancet 1994, 344, 1383-1389.
J M960123N