Table Of ContentTRIAZENES
Chemical, Biological, and
Clinical Aspects
TRIAZENES
Chemical, Biological, and
Clinical Aspecls
Edited by
Tullio Giraldi
University of Trieste
Trieste, Italy
Thomas A. Connors
Medical Research Council
Toxicology Unit, Carshalton
Surrey, United Kingdom
and
Giuseppe Carlei
Division of Medical Oncology
Ospedale S Maria della Misericordia
Udine, Italy
SPRINGER SCIENCE+BUSINESS MEDIA, LLC
Llbrary of Congress Cataloglng-In-Publlcatlon Data
InternatIonal Conference on Trlazenes: Chemlcal. Blologlcal. and
Cllnlcal Aspects (1989 : Trleste. Italyl
Trlazenes : chemlcal. blologlcal. and cllnlcal aspects I edlted by
Tulllo Glraldl. Thomas A. Connors. and Gluseppe Cartel.
p. cm.
"Proceedlngs of an InternatIonal Conference an Trlazanas:
Che.lcal. Blologlcal, and Cllnlcal Aspacts, held Nova.bar 28-29,
1989, In Trleste, Italy"--T.p. verso.
Includes blbllographlcal references and Index.
ISBN 978-1-4613-6710-9 ISBN 978-1-4615-3832-5 (eBook)
DOI 10.1007/978-1-4615-3832-5
1. Trlazenes--Therapeutlc use--Testlng--Congresses.
2. Antlneoplastlc agents--Congrasses. 1. Glraldl, Tulll0.
II. Connors, Tholas A., 1934- III. Cartal, Glusappa.
IV. Tltle.
[DNLM: 1. Trlazenes--congresses. QO 305.A9 161t 19891
RC271.T68158 1989
616.99'4061--dc20
ONLMI OLC
for Ltbrary of Congress 90-14271
CIP
Proceedings of an International Conference on Triazenes: Chemical,
Biological, and Clinical Aspects, held November 28 - 29, 1989,
in Trieste, Italy
ISBN 978-1-4613-6710-9
© 1990 Springer Science+Business Media New York
Originally published by Plenum Press, New York in 1990
Softcover reprint of the hardcover 1s t edition 1990
Ali rights reserved
No part of this book may be reproduced, stored in a retrieval system, or transmitted
in any form or by any means, electronic, mechanical, photocopying, microfilming,
recording, or otherwise, without written permis sion from the Publisher
INTRODUCTION
More than 25 years have elapsed since the development of
the seminal idea which led to the synthesis of dimethyl triazenes
as antitumor agents. The original suggestion of Shealy et ale
was to use 4-imidazone-carboxarnide as the carrier of a nitro
gen-containing cytotoxic function. 5-diazoimidazole-4-carbox
amide (diazo-IC) was synthesized and tested in mice as a
potential inhibitor of de novo purine biosynthesis. Its lack
of antitumor action was attributed to its polarity and to the
resulting poor uptake of this hydrophilic chemical. Diazo-IC
was then coupled with dimethylamine, yielding 5, (3,3-dimethyl
l-triazeno)imidazole-4-carboxamide) (DTIC) with the intention
of obtaining a less polar and more lipophil~c prod rug which
might release diazo-IC intracellularly. Preliminary tests
showed that DTIC had good antiumor activity in experimental
systems. Further tests demonstrated a broad spectrum of action
against rodent tumors, and clinical trials indicated activity
against human malignancies. Subsequent clinical use of DTIC
has demonstrated its usefulness against malignant melanoma, for
which it is currently the drug of choice, and its effective
ness in combination chemotherapy in the treatment of other
human cancers. Because of its antitumor activity the mechan
ism of action of DTIC has been investigated in some detail.
The original rationale for its development, that is, the
hydrolysis in vivo to diazo-IC, has been shown not to be in
volved in th-e-mechansims of action. DTIC requires metabolic acti
vation before it exerts its biological effects. Biotransfor
mation of DTIC is via hepatic oxidative N-demethylation, form
ing the corresponding monomethy derivative MIC [5-(5-methyl
l-triazeno)imidazole-4-carboxamide], which is a reactive meta
bolite methylating cellular components. Other products of
biotransformation have been identified, particularly for aryl
dimethyltrazenes, which may also be relevant to their in vivo
effects. This pharmacological evidence may explain other---
biological properties of DTIC such as its mutagenic effects in
both prokaryotic and eukaryotic cells. Also in tumor-bearing
animals it appears to stimulate immune responses as a result
of chemical xenogenization of the tumor cells. It may also
modify the malignant properties of tumor cells markedly, reduc
ing their metastatic potentials in a relatively stable fashion.
These biological and pharmacological properties are not
unique for imidazole dimethyltriazene but are also seen with
aryldimethyltriazene, which permits an easier and more detailed
examination of structure-activity relationships. In this con
nection, of a large series of benzenoid derivatives CBIO-277
[1-p-(3,3-dimethyl-I-triazeno)benzoic acid sodium salt] has
v
interesting properties and is currently on clinical trial in
England. Another approach arising for studies of DTIC analo
gues has led to the synthesis of chemicals with cyclic moie
ties, such as mitozolomide and temozolomide, which can yield
methyl- and S-chloroethyltriazene in vivo.
This volume contains contributions by the participants
in the "International Conference on Triazenes: Chemical,
Biological, and Clinical Aspects," which was held in Trieste
on November 28-29, 1989. Synthesis, chemical properties,
biotransformation, and pharmacokinetics in animals and humans
are reported in the following chapters. Similarly, antitumor
effects in animals and man are illustrated, together with
other biological and pharmacological properties. Studies on
imidazotetrazines and antitumor melamines, such as hexa-and
penta-methylmelamine, are included because of the analogies
of these compounds to triazenes. One chapter reports the
work of a workshop session devoted to the alkylation of ~
guanine in the mechanism of action of triazenes, and another
summarizes the main aspects of the poster session.
The Editors wish to express their appreciation to the
contributors to this volume, and to all of the participants
in the Conference. Special acknowledgments are due to the
following agencies which supported the meeting, thus making
this volume possible: Universita di Trieste, Regione Autonoma
Friuli - Venezia Giulia, and Consiglio Nazionale delle Ricer
che. The patronage of Societa Italiana di Farmacologia (SIF),
Associazione Italiana di Oncologia Medica (AI OM), and Societa
Italiana di Cancerologia (SIC) is also gratefully acknowledged.
The Editors wish to express particular recognition of the in
valuable contribution provided by Dr. Sonia Zorzet during the
organization of the Conference and the preparation of this
volume.
Tullio Giraldi
Thomas A. Connors
Giuseppe Cartei
CONTENTS
Triazenes: Synthesis and Chemical Properties........... 1
K. Vaughan
Mechanisms of the Biological Actions of Triazenes...... 15
M. J. Tisdale
Triazenes and Triazene N-Oxides: Antitumour Action
in Animal Tumour Systems......................... 23
D. E. V. Wilman
Antimestastatic Action of Triazene Derivatives......... 45
T. Giraldi, L. Perissin, S. Zorzet,
and V. Rapozzi
Effects of Triazenes on Immune Responses............... 63
S. D'Atri, L. Tentori, M. Tricarico,
and E. Bonmassar
Xenogenization of Experimental Tumors by Triazene
Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . . 79
P. Puccetti, L. Romani, U. Grohmann, R. Bianchi,
P. Fuschiotti, M. Allegrucci and M. C. Fioretti
The Me~abo1ism of Antineoplastic Triazenes............. 91
A. Gescher, L. L. H. Tsang and J. A. Slack
Notes on the Metabolism, Pharmacokinetics and Mode
of Action of N-Methyl and N-Ethyl-Triazenes
in Relation to Their Pharmacological Activity.... 97
M. D'Incalci, T. Colombo, P. Farina, C. Mannironi,
P. Taverna and C.V. Catapano
Clinical Use of Triazenes.............................. 109
S. Eckhardt
Clinical Studies with the p-Carboxyl-Dimethyl-
Phenyl-Triazene CB10-277......................... 119
D. R. Newell, B. J. Foster, J. Carmicheal,
A. L. Harris, K. Jenns, L. Gumbrell,
and A. H. Calvert
Triazenes: Therapeutic Considerations and
Perspectives. . • . • . . . . . • • . . • . . . • . . . . • • • . • • • • . . . • • 133
G. Cartei
vii
Antitumor Imidazotetrazines: Prodrugs Targeted to
the Major Groove of DNA......................... 145
M.F.G. Stevens
06-Alkylguanine-DNA-Alkyltransferase Gene
Expression and the Cytotoxicity of Triazenes..... 161
G. P. Margison, L. C. Harris, L. Cenakova,
V. Veckova, J. Brozmanova, K. Kleibl,
and M. Skorvaga
N-Methylmelamines, a Unique Class of Anti-Tumour
Agents? ............................ .-... . . .... . . . 173
I. R. Judson
Experimental Background and Early Clinical Studies
with Imidazotetrazine Derivatives................ 185
E. S. Newlands, G. R. P. Blackledge, J. Slack,
N. S. A. Stuart and M. F. G. Stevens
'06-Alkylguanine-DNA-Alkyltransferase:
Significance, Methods of Measurement and
Some Human Tumor and Normal Tissue Levels' ....... 195
(Contributions of the Workshop)
G.P. Margison, P.J. O'Connor, D.P. Cooper, J.
Davies, C.N. Hall, S.M.S. Redmond, K. Buser,
T. Cerny, L. Citti and M. D'Incalci
Summary of Poster-Sessions............................. 207
E. Bonmassar, A. Gescher, D. R. Newell,
and M. F. G. Stevens
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Index..... ........ . ... .... . . . . .... .. . . . ... . . ..... . .... . 219
viii
TRIAZENES: SYNTHESIS AND CHEMICAL PROPERTIES
Keith Vaughan
Department of Chemistry, Saint Mary's
University, Halifax, N.S., Canada B3H 3C3
INTRODUCTION
It is a great pleasure to participate in this, the first
international conference brought together to discuss the Chemical,
Biological and Clinical Aspects of Triazenes. This paper deals mainly
with the chemistry of triazenes; the biological properties of these
compounds will be discussed in detail in other chapters of these
proceedings. However, where appropriate, the anti-tumour activity
of relevant compounds will be described briefly here.
Interest in the chemistry of triazenes has been sustained
largely by the significant in vivo anti-tumour activity of the 3,3-
dimethyltriazenes, ArN=N-NMe2. It is important to describe briefly
the known metabolism of these triazenes in order to appreciate the
design of chemical experiments aimed at the development of new
triazenes with improved activity.
It is generally accepted that 3,3-dimethyltriazenes require
metabolic activation to a cytotoxic species and that the course of the
metabolism involves oxidation to give the hydroxymethyltriazene,
ArN=N-NMe-CH20H (I) [Scheme 1]. Loss of formaldehyde from I
affords the "monomethyltriazene", ArN=N-NHMe (II), which is
presumed to be the active metabolite due to its proclivity for
methylation of DNA. The thrust of this paper is to review the
chemistry of the triazenes of type I and II, which have been
synthesised and scrutinised in this laboratory during the last ten
years.
Triazenes, Edited by T. Giraldi et at.
Plenum Press, New York, 1990
Schemel TRIAZENE METABOLISM
mTICi
DACARBAZINE)
MONOMETHYLTRIAZENES
The chemistry of the monoalkyltriazenes, ArN=N-NHR, has been
reviewed (1). Diazonium coupling of the arene diazonium salt with a
primary aliphatic amine (equation i) is a convenient source of the
monoalky I triazene:
(i) ArN2+ + RNH2 - ArN=N-NHR + H+
However, this apparently simple approach can often lead to
higher nitrogen homologues, the pentaazadienes, ArN=N-NR-N=NAr.
The formation of the monoalkyltriazene in reaction (i) is favoured by
the presence of electron withdrawing substituents in Ar (2).
Monoalkyltriazenes are not stable in aqueous media and
undergo degradation to give a mixture of triazene and non-triazene
products (3). The principal products of the hydrolysis of II are the
arylamine, ArNH2, the diaryltriazene, ArN=N-NHAr, and the 1,3-
diaryl-3-alkyltriazene, ArN=N-NRAr. These products are indicative
of the tautomeric nature of the monoalkyltriazene:
-
ArN=N-NHR ..... ArNH-N=N-R
IIa lIb
Nuclear magnetic resonance studies of these triazenes confirm
the tautomeric behaviour in solution and, furthermore, show that the
position of the equilibrium is influenced by the nature of
substitutents in the aryl moiety (4). Thus 3-methyl-l-p-tolyltriazene
in deuteriochloroform exists almost exclusively as the "conjugated"
tautomer (IIa); the chemical shift of the methyl group in this
tautomer is close to the N-methyl chemical shift of a 3,3-dimethyl
triazene and at low temperature the N-methyl proton resonance of
IIa becomes a doublet due to spin-spin coupling to the adjacent NH
proton. In contrast, the NMR spectrum of 3-methyl-l-p-cyano
phenyltriazene shows a very broad room temperature N-methyl
2
signal which resolves into a singlet (for lIb) and a doublet (for IIa);
apparently the presence of an electron-withdrawing substituent in
Ar shifts the equilibrium towards the "unconjugated" tautomer (lIb).
Quite a novel efect is seen when a nitro-group is positioned ortho- to
the triazene moiety; in this case, only the presence of lIb is indicated
by a singlet methyl resonance in the NMR. Presumably hydrogen
bonding between the nitro and the NH groups locks the molecule into
the "unfavourable" tautomeric form.
The tautomeric behaviour of .monoalkyltriazenes in solution
would suggest that the biological activity might be influenced by the
facility of nucleophilic displacement at tautomer lIb. However, there
is no evidence to suggest that the anti-tumour activity of the 3,3-
dimethyltriazene is affected by the electronic character of the
substituent in the I-aryl moiety.
The requirement for metabolic activation of the
dimethyltriazene may be a factor in limiting the clinical efficacy of
these drugs. It has been suggested (5) that metabolism of N-alkyl
compounds is species dependent. The plasma half-life of the
dimethyltriazene, OTIC (III), is significantly greater in the rat than in
the mouse, and greater still in man. The poor clinical activity of OTIC
may be due at least in part to the low level of metabolism in man. A
possible solution to this problem is to devise a pro-drug for OTIC, one
that avoids the requirement for oxidative metabolism. A possible
pro-drug candidate is a monomethyltriazene; however, the poor
stability of monomethyltriazenes in aqueous solution has precluded
such application.
An alternative candidate for a pro-drug is the
hydroxymethyltriazene, or one of its derivatives, and the remainder
of this paper will explore the chemistry of the
hydroxymethyltriazenes and their known derivatives.
HYDRO~ETHYLTRLAZENES
Until relatively recently, these triazenes were thought to exist
only as transient metabolic intermediates and that their chemical
isolation would not be feasible. It turns out that, under appropriate
conditions, the synthesis of a hydroxymethyltriazene is straight
forward. Oiazonium coupling with an aqueous mixture of methyl
amine and formaldehyde (equation ii) affords the crystalline
hydroxymethyltriazene, which once isolated is a stable crystalline
solid (6).
(ii)
3
