Self-immolative base-mediated conjugate release from triazolylmethylcarbamates

Article abstract of DOI:10.1039/c5ob00984g

A range of carbamate functionalized 1,4-disubstituted triazoles featuring a base sensitive trigger residue, plus a model aromatic amine reporter group, were prepared via copper(i) catalysed azide-alkyne cycloaddition and evaluated for their self-immolativ

Full text of DOI:10.1039/c5ob00984g

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Self-immolative base-mediated conjugate release
from triazolylmethylcarbamates†
Cite this: DOI: 10.1039/c5ob00984g
Christopher A. Blencowe,a David W. Thornthwaite,b Wayne Hayes*a and
Andrew T. Russell*a
A range of carbamate functionalized 1,4-disubstituted triazoles featuring a base sensitive trigger residue,
plus a model aromatic amine reporter group, were prepared via copper(I) catalysed azide–alkyne cyclo-
addition and evaluated for their self-immolative characteristics. This study revealed a clear structure–reac-
tivity relationship, via Hammett analysis, between the structure of the 1,4-disubstituted triazole and the
rate of self-immolative release of the amine reporter group, thus demonstrating that under basic con-
ditions this type of triazole derivative has the potential to be employed in a range of chemical release
systems.
Received 14th May 2015,
Accepted 7th July 2015
DOI: 10.1039/c5ob00984g
www.rsc.org/obc
It is well-established that the activity of a compound (e.g. drug et al. to the directed, iron-catalysed, arylation of arenes and
or fragrance, referred to here as a ‘reporter’ group) can be atte- alkenes, conferring both high regio- and diastereoselectivity,
nuated by attachment to a protecting moiety (referred to here and facilitating traceless cleavage under mild conditions.20 In
as a ‘trigger’ group).1–4 The spatial proximity of trigger and contrast, self-immolative triazole linkers designed to degrade
reporter groups can be significant, especially for scissile bonds under alkaline conditions have not been reported to date. In
requiring enzyme mediated cleavage.5,6 This steric effect can the light of the development of the efficient copper(I) catalyzed
be negated by inserting a linker unit between trigger and [3 + 2] cycloaddition between azides and alkynes, 1,2,3 tri-
reporter moieties, of which ‘self-immolative’ linkers have azoles have been studied extensively for a wide range of appli-
become an important development in recent years, especially cations in recent years.21,22 This paper reports the synthesis of
in the area of polymeric drug delivery systems.7–11 In this a range of novel degradable triazoles via a copper-catalysed
approach, the linker forms a scissile bond with the trigger azide–alkyne cycloaddition approach and details the study of
moiety and a stable bond to the reporter group – the latter unit their self-immolative ability under basic conditions. This
is released via solvolysis upon removal of the trigger group linker technology was designed with a broad application spec-
which effects rapid irreversible disassembly of the three com- trum in mind, focussing initially on the controlled release of
ponents through a cascade of elimination processes. This amines. In this study, we demonstrate that the generic 1H-tri-
phenomenon has been observed for polysubstituted, electron- azole 1 (Fig. 1) is capable of displaying self-immolative charac-
rich aromatic species that feature an electron-releasing substi- teristics upon formation of the corresponding triazolyl anion.
tuent in conjugation with a suitable leaving group at a benzylic Furthermore, we show that the rate of self-immolative elimin-
position and has been extended recently to heterocyclic ation was tunable by the degree and nature of substitution at
systems.12–15 In particular, 1,4-disubstituted triazoles exhibit the triazole α-methine position (1, R1 and R2).
self-immolative characteristics under acidic conditions16 and
To facilitate this study, triazoles featuring both the base
have been exploited for the controlled release of alcohols,17 labile pivaloyloxymethyl moiety and the alkaline stable benzyl
amines18 and anilines.19 Recently, an acid-sensitive triazole- protecting group were prepared and evaluated – the latter to
based self-immolative linker has been applied by Ackermann demonstrate the importance of triazolyl anion formation as a
aDepartment of Chemistry, University of Reading, Reading, Berkshire RG6 6AD, UK.
E-mail: a.t.russell@reading.ac.uk; Fax: +44 (0)118 378 6331;
Tel: +44 (0)118 378 6234
bUnilever Research and Development, Quarry Road East, Bebington, Wirral CH63
3JW, UK. E-mail: david.thornthwaite@unilever.com; Fax: +44 (0)151 641 1852;
Tel: +44 (0)151 641 3257
†Electronic supplementary information (ESI) available: Experimental and
characterization data of triazoles 6a–6j and 7a–7d and additional mechanistic
details. See DOI: 10.1039/c5ob00984g
Fig. 1 Self-immolative 1H-triazole.
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Scheme 1 Synthesis of POM protected triazoles 6a–6j and benzyl protected triazoles 7a–7d. Reagents and conditions: (i) p-nitrophenylchlorofor-
mate, pyridine, CH2Cl2, rt, 16–24 h, (ii) NHMeBn, pyridine, CH2Cl2, rt, 12–16 h, (iii) 2, CuI (5–10 mol%), pyridine, rt, 2–12 h, (iv) 3, CuI (5–10 mol%),
pyridine, rt, 2–12 h.
prerequisite for self-immolative elimination. Pivaloyloxymethyl
(POM) azide23 2 and benzyl azide 3 were prepared from their
corresponding halides. N-Methylbenzylamine was chosen as
the model reporter group as the N-methyl substituent could be
used as a marker in the degradation studies and removed any
ambiguity regarding the site of deprotonation. To elucidate
structure–reactivity relationships in the degradation of the tria-
zole conjugates, a structurally diverse range of alkyl- and aryl-
substituted propargyl N-methylbenzylcarbamate precursors
were targeted. Propargyl alcohols 4d–4j were synthesized by
Grignard reaction of ethynyl magnesium bromide and the
corresponding aldehydes via a known method (Scheme 1).24
The corresponding triazoles were prepared in acceptable
yields (54–90%) following the method devised by Bertrand and
Gesson.16 In each case, the formation of the triazole adduct
was followed by 1H NMR spectroscopy which revealed the
characteristic downfield shift of the acetylene proton upon aro-
matization. In addition, AB system resonances correlating to
the benzyl- and POM- methylene protons, respectively, were
also observed as a result of the asymmetric centre in triazole 6
or 7 (other than 6/7c).
Fig. 2 1H NMR spectra showing self-immolative release from triazolyl
anion intermediate from 6d; (a) t = 5 min. (b) t = 15 min. (c) t = 46 min.
(d) t = 102 min.
1H NMR spectroscopy was also used to monitor the kinetics
of the self-immolative processes and provided an insight to the
degradation pathways. A non-aqueous polar protic system
(sodium methoxide-d3 in methanol-d4) was chosen in conjunc-
tion with TMS as the calibrant for the degradation studies. De
Groot et al. confirmed the self-immolative elimination of 2,6-
bis(hydroxymethyl)phenol based linkers in an analogous
manner.25 The excess amount of NaOMe-d3 used was based
upon the observations of Sharpless et al., who determined that
2.2 equivalents of base were required to effect efficient pivaloyl-
oxymethyl deprotection.23 In the case of triazoles 6a–6j a
common degradation profile was observed spectroscopically
(see Fig. 2).
1H NMR spectra obtained were interpreted by reference to
chemical shift data of standards (such as (1H-triazol-4-yl)phe-
nylmethyl methyl ether-d3, (1H-triazol-4-yl)phenylmethyl
alcohol, methyl N-methylbenzyl-carbamate and N-hydroxy-
methyl-N-methylbenzylcarbamate) in order to determine the
presence of the triazole linker fragment and amine reporter.
The carbamate anion 8 was also identified by an HMBC spec-
trum. For triazoles 6a–6j the deprotection of the POM group
was found to be rapid and complete within the timescale
required to make the first measurement (ca. 4 minutes). Using
triazole 6d as a representative example (Fig. 2), formation of
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the triazolyl anion in situ was characterized by an upfield shift
of the triazole proton from rotameric resonances at δH 8.02
and 7.85 ppm, respectively, into the aromatic region (ca. δH
7.30 ppm) and the singlet α-methine proton resonance at δH
6.99 ppm (see ESI, Fig. S27†). Rotameric resonances for the
N-methylene (AB pattern) and N-methyl protons were observed at
δH 4.65–4.47, and 2.94–2.86 ppm, respectively, their positions
not affected by deprotection. Degradation of the triazolyl
anion fragment was observed by the decrease in intensity
and disappearance of the singlet and rotameric resonances
corresponding to the α-methine and N-methyl protons over the
course of approximately two hours.
Table 1 Rate constants and half-lives for triazoles 6a–6j
Triazole
α-Substituent
t1/2 (minutes)
k0 (min−1
)
6a
6b
6g
6e
6d
6f
6i
6j
6h
6c
CH2
Me
86 413a 2493
534.9 19.9
87.2 1.0
27.1 0.6
15.3 1.3
13.2 0.8
9.5 0.2
8.1 0.7
5.4 0.3
<2
8.02 × 10−6 2.4 × 10−7
0.0016 0.0006
0.0084 0.0002
0.0253 0.0004
0.0439 0.002
0.0582 0.002
0.0727 0.003
0.0856 0.008
0.1426 0.021
NDb
[m-F]Ph
[p-Br]Ph
Ph
[p-F]Ph
2-Naph
p-biPh
[p-Me]Ph
Gem-diMe
a Calculated based on rate constant. Errors expressed as first standard
deviation of values measured in triplicate. b Not determined.
We observed transient species following self-immolative
elimination of the triazolyl anion, for example, solvolysis at the
α-methine position led to the release of the N-methyl-
benzylcarbamate anion 8, which had characteristic resolution
of the rotameric methylene resonances to a sharp singlet in methanol-d4 resulted in slow deuteration of the triazole ring
resonance at δH 4.50 ppm. This intermediate anion then col- at the C-5 position (see ESI, Fig. S30†).
lapsed to afford the reporter unit, N-methylbenzylamine and
The nature of the substituent at the α-methine position of
carbon dioxide. The former reacted further with formaldehyde the triazole had a profound effect upon the fragmentation rate
generated as a by-product of POM deprotection, to afford N- of the α-methine-carbamate C–O bond (see Table 1).18
hydroxymethyl-N-methylbenzylamine (Scheme 2). For compari-
In order for degradation of the protected triazoles to
son, benzyl protected triazoles 7a–7d were evaluated under the occur at rates amenable to applications in controlled delivery
same conditions. Cleavage of the carbamate linker or release systems, secondary or tertiary triazole α-carbon linkages were
of N-methylbenzylamine was not observed, even after several required. The use of variously substituted phenyl groups
months. In this case treatment of triazoles 7a–7d with NaOMe-d3 offered potential to fine-tune such release and so were exam-
Scheme 2 Proposed mechanism for the self-immolative elimination of N-methylbenzylamine from triazoles 6a–6j.
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confirmed, the latter of which existed predominantly in the
methoxymethanol-d3 hemiacetal form. As the reaction pro-
gressed, N-methylbenzylamine was liberated, however, as a
result of the ‘closed system’, reaction of this amine with for-
maldehyde led to the formation of N-hydroxymethyl-N-methyl-
benzylamine. The very low reactivity of 7a–d implies that self-
immolative elimination was facilitated by the formation of
triazole anion 9 (1,2,3-triazole pKa 9.4).30 The proposed inter-
mediate formed from 9 was not detected, presumably being
quenched rapidly, with restoration of aromaticity, by metha-
nol/methoxide addition at the electron deficient α-methine
position to afford triazolyl anion intermediate 10. Using
triazole 6d as a representative example, the identity of the
product fragment was confirmed by NMR doping experiments
(see ESI, Fig. S28 and S29†). The relative stability of the
N-methylbenzylcarbamate anion 8 under basic conditions
meant that it could be observed and characterized by NMR
spectroscopy (ESI, Fig. S31†). Subsequent decarboxylation
led to the expulsion of carbon dioxide, which was quenched
with methoxide to afford methylcarbonate anion, and
liberation of the reporter group N-methylbenzylamine.
The presence of formaldehyde-methoxymethanol-d3 formed
via POM deprotection, resulted in further reaction of N-methyl-
benzylamine to give N-hydroxymethyl-N-methylbenzylamine.
The absence of the hydroxy analogue of 10 strongly
suggested that direct attack of methoxide at the carbamate
carbonyl carbon, by a ‘BAC2’ type mechanism, could be dis-
counted. Hence, from the Hammett analysis and degradation
profiles observed spectroscopically, it was concluded that a
single mechanism is operative.
Fig. 3 Hammett plot of the logarithm of relative rate vs. σ+ values.
ined as part of this study. The inclusion of a phenyl substitu-
ent provided an immediately measurable degradation rate,
whilst the presence of electron releasing or electron-withdraw-
ing substituents resulted in further incremental changes.18,19
Under these conditions the gem-dimethyl derivative 6c
underwent such rapid self-immolative elimination that accu-
rate kinetic data was not obtained by the 1H NMR spectro-
scopic assay. In the case of aryl substituted triazoles 6d–6j,
their degradation rates were correlated with their Hammett
substituent constants σ+ (Fig. 3), σ and σ-values.26–28 Using
these values and the rate constants obtained, Hammett analy-
sis afforded a graph whose slope gave the reaction constant
ρ, which is a measure of the susceptibility of the reaction to
electronic effects.
Excellent correlation was obtained when the modified
Hammett equation (Brown σ+ values) was used, to account for
the through conjugation of the aromatic substituents to
the reaction centre. The negative value obtained for ρ (−1.67)
indicated that the self-immolative elimination is enhanced by
electron-releasing substituents, whilst electron-withdrawing
substituents have the opposite effect.29 The negative sign
being consistent with a transition state where the degree of
negative charge is decreasing. The linearity of the Hammett
plot implied that a common rate determining step is observed
for all triazoles across the series. The relative rate obtained for
triazole 6f with respect to triazole 6d also provided important
information regarding the electronic character of the tran-
sition state. The overall effect of the fluorine (χ = 3.98) substitu-
ent can change significantly depending on the electronics of
the transition state, and provide useful insight regarding
the reaction mechanism. The rate enhancement observed for
triazole 6f suggested that there is a decrease in negative
charge in the transition state of the rate limiting step, which
correlated with the interpretation of the Hammett plot. In the
case of triazole 6i, in lieu of a known literature value for σ+,
the Hammett plot plus the experimentally determined rate
constant was used to define this parameter (σ+ = 1.20).
Conclusions
We have demonstrated that triazolylmethylcarbamates are sus-
ceptible to base mediated self-immolative elimination. The
stability of triazoles 7a–7d confirmed that formation of the
triazolyl anion is a prerequisite step in this degradation
process. A structure–reactivity relationship study highlighted
that the reaction proceeds with reduction of negative charge
in the transition state of the rate limiting step and degrades
via an ‘E1cb’ type 1,4-elimination. By altering the nature of
the triazole α-methine substituent, predictable changes in
degradation rate were realized and quantified. Work investi-
gating the scope of this triazole linker for the controlled
release of alcohols under acidic/basic conditions, and the
synthesis and evaluation of self-immolative triazole–polymer
conjugates, will be the subject of further publications.
Acknowledgements
From the identity of intermediate and product fragments, a
detailed mechanism could be postulated (Scheme 2, also see
ESI, Fig. S32†). From rapid methanolysis of the POM group,
the formation of methyl pivalate-d3 and formaldehyde were
We gratefully acknowledge EPSRC, Unilever and the University
of Reading for funding (PhD studentship for CAB). The
authors also acknowledge the University of Reading for access
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