J o u m l o f Medicinal Chemistrv

d /
0 Copyright 1986 by the American Chemical Society

Volume 29,Number 8 August 1986

Communications to the Editor

The Mechanism of Action of the Gastric Acid We also studied some structural analogues to avoid un-
Secretion Inhibitor Omeprazole necessary isomer problems.
Of crucial importance to this work was the great sim-
Sir: plification of decomposition by adding P-mercaptoethanol
Omeprazole (I), a potent antiulcer agent,'s2 is presently to the acid medium before addition of omeprazole (I).
under extensive clinical evaluation. This compound and This caused only two compounds to form: the sulfide 8
several close analogues are effective inhibitors of gastric and, according to 'H NMFl [PF, salt: 6 (500MHz, CDC1,)
acid secretion in the rat, dog, and by being in- 2.5 (8, 6 H), 2.65 (t, 2 H), 3.6 (t, 2 H),3.85 (s, 3 H),4.2 (s,
hibitors of the gastric H+,K+-ATPase6(unlike acid se- 2 H), 4.3 (s,3 H), 7.05 (dd, 1 H), 7.1 (d, 1 H), 7.6 (d, 1 H),
cretion inhibitors such as cimetidine). The enzyme 8.35 (e, 1 H)], an adduct with P-mercaptoethanol (5).13
H+,K+-ATPaseis responsible for gastric acid production At high dilution M) in dilute hydrochloric acid
and is located in the secretory membranes of the parietal (0.001-0.5M) the conversion of omeprazole (1) into an
~ell.'9~Omeprazole itself is not an active inhibitor of this intermediate 4 (taking 5-15 min to completion at 37 "C)
enzyme, but it is transformed within the acid compart- could be followed kinetically by UV. When a slight excess
ments of the parietal cell into the active inhibitor, close of P-mercaptoethanolwas added to the resulting solution,
to the e n ~ y m e . ~ a very rapid conversion of 4 into the above-mentioned
We now report the isolation and structure of a new adduct 5 could be followed kinetically by UV. The three
sulfenamide (4) from acid decomposition of omeprazole UV spectra recorded before the addition of acid, 15 min
(I) and propose, in contradiction to two recent investiga- after the addition of acid, and after the addition of 8-
tions,l0J1that sulfenamide 4, or the corresponding unstable mercaptoethanol are identical with those presented in ref
sulfenic acid 3, is the active inhibitor formed in vivo from 10. We were also able to obtain crystals for X-ray analysis
omeprazole (1).12 of the P-mercaptoethanol adduct (Clod-salt from metha-
Chemistry. To obtain information about the mecha- nol) generated from the omeprazole analogue 2-[[ (4-
nism of action at a molecular level we undertook an ex- methoxy-3,5-dimethyl-2-pyridinyl)methyl]sulfinyl]-1H-
tensive study of the acid degradation of omeprazole (1). benzimidazole, resulting in the disulfide structure 9,14
containing a pyridinium cation.
PF
Gustavsson, S.; LGijf, L.; Adami, H. 0.;Nyberg, A,; NyrBn, 0.
Lancet 1983,2,124.
Lauritaen, K.;et al. N . Engl. J. Med. 1985,312,958.
Larsson, H.; et al. Gastroenterology 1983,85,900.
Brbdstrom, A.; Lindberg, P.; Junggren, U. Scand. J. Gas-
troenterol. 1985,108 (Suppl.), 15.
Lind, T.; Cederberg, C.; Ekenved, G.; Haglund, U.; Olbe, L.
Cut 1983,24,270.
Fellenius, E.; Berglindh, T.; Sachs, G.; Olbe, L.; Elander, B.;
Sjbtrand, %E.; Wallmark, B. Nature (London) 1981,290,159.
Smolka, A.; Helander, H. F.; Sachs, G. Am. J. Physiol. 1983, / \
245,G589. 9 CH3 CH3
Sachs, G.; Chang, H. H.; Rabon, E.; Schakmann, R.; Lewin, M.; 10
Saccomani, G. J. Biol. Chem. 1976,251,7690.
Wallmark, B.; Brandstrom, A.; Larsson, H. Biochim. Biophys. On decomposition of omeprazole (1) in methanol instead
Acta 1984,778, 549. of water, the intermediate 4 was significantly more stable,
Im, W. B.; Sih, J. C.; Blakeman, D. P.; McGrath, J. P. J. Biol. even at moderate concentrations. When a 0.1 M solution
Chem. 1985,260,4591.
Rackur, G.; Bickel, M.; Fehlhaber, H.-W.; Herling, A.; Hitzel, of 1 in 0.2 M methanolic HC1 was allowed to stand at room
V.; Lang, H.-J.; Rijsner, M.; Weger, R. Biochem. Biophys. Res. temperature for 7 min and then a 50% excess of 50%
Commun. 1985,128, 477. HBF, or 70% HPF, was added, crystals of the corre-
Reported for the first time at the ASPET-ACS meeting in sponding salts of 4 were obtained in a yield of about 45%.
Boston, MA, August 18-22, 1985. See: Brbdstrdm, A. 'H NMR spectra (in acetonitrile) of these salts were
Pharmacologist 1985, 27, 104. Also reported (during the characterized by a very low shift (6 9.3) for the proton in
preparation of this manuscript) at the 3rd SCI-RSC Medicinal
Chemistry Symposium in Cambridge, UK, September 15-18,
1985: Wallmark, B.;BrHndstrom, A.; Lindberg, P. Abstract S (13) Anal. (C19H24N303S2PF6) H,N, C: calcd, 46,26;found, 44.88.
16. At the same symposium the sulfenamide and the &mer- (14) Anal. (C18H22N306S2C1) C, H, N, S. The X-ray investigation
captoethanol adduct structures were presented, independently was performed by Anders Svensson, Leif Andersson, and
of us, also by another group: Figala, V.; et al. Abstract P 20. Lennart Sjolin (unpublished results).

0022-262318611829-1327$01.50/0 0 1986 American Chemical Society

1328 Journal of Medicinal Chemistry, 1986, Vol. 29, No. 8 Communications to t h e Editor

Scheme I

H3C&CH3 H3C&CH3 H3C&CH3

/
OCH3
/
OCH,
2
Q \
OCH, OCH3
1 (orneprazole)
3 4
I
oridolion;
(liver);

H3ca
OCH, OCH,

H3ca
I I

N H3 H3
RS-

OCH, \
OCH3 OCH,
8
7 5: R=HOCH$H,
6: R=enryme

the 6-position of the pyridine ring. An X-ray investigation a C-S bond cleavage. The subsequent formation of the
of a PF6- salt of an analogue to compound 4 revealed the sulfenamide 4 is in accordance with known reactions be-
cyclic sulfenamide structure 10.15 The sulfenamide in- tween sulfenic acids and amines.I7 Likewise, the reaction
termediate 416 from omeprazole (1) is a mixture of two of 4 with 6-mercaptoethanol to form adduct 5 is now easily
isomers, which can easily be seen from the 'H NMR understood, since sulfenamides or sulfenic acid derivatives
spectrum [AuCl,- salt: 6 (500 MHz, CD&N) 2.5 (s, 3 H), in general are known to react with mercaptans to form
2.6 (s, 3 H), 3.9 (s, 3 H), 4.3 ( s , 3 HI, 4.85 (s, 2 H), 7.05 and di~ulfides.'~The adduct 5 may then react with a second
7.15 (2 dd, total 2 H), 7.1 and 7.3 (2 d, total 1H), 7.7 and molecule of 0-mercaptoethanol in a base-catalyzed (un-
7.5 (2 d, total 1 H),9.25 and 9.3 (2 s, total 1 H)]. published results) reaction to form the sulfide 8, probably
The reaction mechanism we propose for the acid via the unstable mercaptan 7, resulting from an S-S bond
transformation of omeprazole (1) to the sulfenamide iso- cleavage during simultaneous formation of the disulfide
mers 4 is outlined in Scheme I. The reaction is reversible of 6-mercaptoethanol.
and goes via the spiro intermediate 2 and the sulfenic acid The introduction of a methyl group in the 6-position of
3. The reversibility was firmly proved by kinetic mea- the pyridine ring of omeprazole analogues results in com-
surements in both directions, i.e., starting from l or 4. An pounds stable in acid solution. This supports the suggested
equilibrium solution contains 1 and 4 in the ratio 1 : l O mechanism, since space-filling models show that the 6-
(HPLC measurements; unpublished results). The for- methyl group will experience a strong steric interference
mation of the spiro intermediate 2 in the ratelimiting step with the imidazole ring, which prevents the formation of
is supported by kinetic measurements. The rate constants the spiro intermediate 2.
obtained for omeprazole analogues are strongly dependent Biochemistry and Pharmacology. When the isomeric
on substituents in the pyridine ring, indicating that a sulfenamide mixture 4 was added to an isolated H+,K+-
positive charge is created on the pyridine nitrogen atom ATPase preparation,@we observed an instantaneous block
in the ratelimiting step. In the decomposition of a 1mM of the enzyme with an IC50value of 0.6 X lo* M. With
solution of 1 in 0.5 mM HCl, it was observed that the pH the concentrations of omeprazole and enzyme used above,
increased a t a rate corresponding to the consumption of the extent of block is increased with increasing time of
one proton per molecule of 1 in the rate-limiting step, in contact with the enzyme and with decreasing pH and will
good agreement with the mechanism proposed. The spiro under no conditions exceed 50%.
intermediate 2 is a dihydrobenzimidazole, with a pro- A mercapto group is known to be involved in the in-
nounced tendency to undergo aromatization, thus forming hibiting reaction,lgand therefore the transformations de-
the sulfenic acid 3 (which we were unable to isolate) by scribed in Scheme I can be considered as a model of the
reactions in the acid compartments of the parietal cell
~ _ -~ _ ~ ~~

(15) Anal. (C17H18N3SPFB)

C, H, N. The X-ray investigation was
performed by Staffan Sundell and Max Lundmark (unpub- (17) Hogg, D. R. In Comprehensive Organic Chemistry;Barton, D.,
lished results). Ullis, W. D., Eds.; Pergamon: London, 1979; Vol. 3, p 261.
(16) The IUPAC names of the two isomers are 2,4-dimethyl-3,9- (18) Saccomani, G.;Stewart, H. B.; Shaw, D.; Lewin, M.; Sachs, G.
and 2,4-dimethyl-3,lO-dimethoxy-5H-pyrido[1',2':4,5]
[ 1,2,3]- Biochem. Biophys. Acta 1977,465, 311.
thiadiazino[2,3-a]benzimidazol-13-iumtetrachloroaurate. (19) Lorentzon, P.; Eklundh, B.; Briindstrom, A.; Wallmark, B.
Anal. (CI7Hl8N3O2S.AuCl4) C, H, N, 0, S. Biochim. Biophys. Acta 1985, 81 7, 25.
 J.Med. Chem. 1986,29,1329-1340 1329

when omeprazole inhibits the H+,K+-ATPase enzyme. dose may complete this cyclic process.
This formation of a disulfide is a fundamentally different Factors contributing to the specificity of the inhibition
mechanism of action of omeprazole than those previously of gastric acid secretion by omeprazole are as follows: (1)
proposed by Im et al.1° and by Rackur et al." It is a well-known fact that weak bases concentrate in
In vitro the enzyme-adduct complex 6 reacts with 0- acidic compartments. Omeprazole is a weak base and
mercaptoethanol to form the sulfide 8 and the enzyme- therefore concentrates in acidic compartment^.^ (2) The
B-mercaptoethanol complex enzyme-SSCH2CH20H, in parietal cell containing the enzyme H+,K+-ATPaseis the
which form the enzyme is still blocked. This complex then only cell in the body with a low pH value. The low pH
slowly reacts with 8-mercaptoethanol to the free enzyme value causes the conversion of omeprazole into the active
enzyme-SH.l9 It is not clear whether, under in vivo con- inhibitor close to the enzyme that produces the acid. (3)
ditions, glutathione or any other endogenous mercaptan The active inhibitors, the sulfenamide 4, and the sulfenic
can form a corresponding enzymemercaptan complex and acid 3 are permanent cations with limited possibilities to
furthermore react in a second step to generate the free penetrate membranes.
enzyme and the disulfide of glutathione. Recovery of the Registry No. 1, 73590-58-6;2, 102283-09-0;4.AuC1,- (3,9-
enzyme activity might require synthesis of the enzyme de (OMe)J, 102283-06-7;4.AuC1,- (3,lO-(OMe)J, 102283-08-9;5,
novo. 102283-11-4;8,73590-85-9; 9,102260-42-4; 10,102260-44-6; HO-
Since the sulfenamide 4 and the sulfenic acid 3 are (CHz)ZSH, 60-24-2.
permanent cations, neither possible active inhibitors can Supplementary Material Available: X-ray data on com-
probably penetrate the secretory membrane of the parietal pounds 9 and 10 (7pages). Ordering information is given on any
cell. This fact and maybe also the permanent-cation current masthead page.
character of the enzyme-inhibitor complex 6 might be
important factors for the in vivo inhibiting effect of
omeprazole (1). (20) Department of Organic Chemistry.
(21) Department of Biology.
The sulfide 8 is formed from omeprazole in isolated
gastric glands? This is probably also the case in the gastric Per Lindberg,*" Peter Nordberg?O Tomas Alminger20
mucosa in vivo. We have also demonstrated that the Arne Brlindstriim,2° Bjorn Wal1mark2'
sulfide 8 in vivo is oxidized to the sulfoxide omeprazole Hassle Gastrointestinal Research Laboratories
(l),thus closing the cyclic reaction process. However, in Departments of Organic Chemistry and Biology
the in vivo situation, due to simultaneous metabolic pro- S-431 83 Malndal, Sweden
cesses, probably only a fraction of the total omeprazole Received December 3, 1985

Art i d e s

Streptonigrin. 1. Structure-Activity Relationships among Simple Bicyclic

Analogues. Rate Dependence of DNA Degradation on Quinone Reduction Potential
Iftikhar A. Shaikh, Francis Johnson,* and Arthur P. Grollman
Departments of Pharmacological Sciences and Chemistry, State University of New York, Stony Brook, New York 11 794.
Received August 23, 1985

A series of simple aza and diaza bicyclic quinones related to the AB ring system of streptonigrin (1)have been
synthesized and tested in vitro for their ability to degrade DNA under conditions similar to those used with the
parent drug. The results obtained from a study of 22 quinones indicate that there is a quantitative linear relationship
between their reduction potentials and the rate at which they degrade DNA under identical conditions in vitro.
Almost all of the synthetic substances were superior to 1 in their DNA-degrading ability.

I. Introduction 0
Streptonigrin (1) is an antitumor agent that has seen
only limited use as an anticancer agent because of its
toxicity1 but continues to receive attention because of
interest in its ability, common to a number of quinone
antibiotics, to degrade2DNA. This ability has been dem-

(1) Teller, M. N.; Wagshul, S. F.; Wolley, G. W. Antibiot. Che-

mother. 1961,11,165. McBride, J. J.; Oleson, T. J.; Wolff, D.
Cancer Res. 1966,26,727.Ebert, P. S.;Chirigos, M. A.; Elle-
worth, P. A. Zbid. 1968,28,363. Kuang, D.T.;Whittington, 1
R. M.; Spencer, H. H.; Patno, M. E. Cancer 1969,23, 597.
(2) For a recent review, see: Lown, J. W. In Molecular Aspects onstrated both in vivo and in vitro. The drug is lethal t o
of Anti-Cancer Drug Action; Neidle, S., Waring, M. J., Eds.; Escherichia coli and to human leucocytes and in both cases
Macmillan: London, 1983;Chapter 9,pp 283-314. DNA degradation3i4is observed. These effects seem to be
0022-2623/86/1829-1329$01.50/0 0 1986 American Chemical Society