Triazine-Substituted and Acyl Hydrazones: Experiment and Computation Reveal a Stability Inversion at Low pH

Article abstract of DOI:10.1021/acs.molpharmaceut.5b00205

Condensation of a hydrazine-substituted s-triazine with an aldehyde or ketone yields an equivalent to the widely used, acid-labile acyl hydrazone. Hydrolysis of these hydrazones using a formaldehyde trap as monitored using HPLC reveals that triazine-subst

Full text of DOI:10.1021/acs.molpharmaceut.5b00205

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Article
Triazine-Substituted and Acyl Hydrazones: Experiment
and Computation Reveal a Stability Inversion at Low pH
Kun Ji, Changsuk Lee, Benjamin G. Janesko, and Eric E. Simanek
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00205 • Publication Date (Web): 15 Jun 2015
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Triazine-Substituted and Acyl Hydrazones: Experiment and
Computation Reveal a Stability Inversion at Low pH
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Kun Ji, Changsuk Lee, Benjamin G. Janesko* and Eric E. Simanek*
Department of Chemistry, Texas Christian University, Fort Worth TX 76129
KEYWORDS: Triazine, hydrazine, hydrazone, pH labile, release, computation
ABSTRACT: Condensation of a hydrazineꢀsubstituted [s]ꢀtriazine with an aldehyde or ketone yields an equivalent to the widelyꢀ
used, acidꢀlabile acyl hydrazone. Hydrolysis of these hydrazones using a formaldehyde trap as monitored using HPLC reveal that
triazineꢀsubstituted hydrazones are more labile than acetyl hydrazones at pH >5. The reactivity trends mirror the corresponding
acetylhydrazones, with hydrolysis rates increasing along the series (aromatic aldehyde < aromatic ketone < aliphatic ketone ).
Computational and experimental studies indicate a reversal in stability around the triazine pKa, pH~5. Protonation of the triazine
moiety retards acidꢀcatalyzed hydrolysis of triazinylhydrazones in comparison to acetyl hydrazone analogues. This behavior
supports mechanistic interpretations suggesting that resistance to protonation of the hydrazoneꢀN1 is the critical factor in affecting
reaction rate.
Labile bonds in general, and hydrazones in particular, find diverse roles in chemistry ranging from materials to medical sciences.
Hydrazones have been employed in the creation of dynamic combinatorial libraries (1) and as reagents and auxiliaries in organic
synthesis (2). Fragrant aldehydes and ketones have been incorporated into materials as proꢀperfumes using hydrazone chemistry
(3). Hydrazones can be bioactive in their own right (4) including uses as antiꢀtrypanasomals (5) and tools for molecular biology
(6). The lability of hydrazones in acid inspires the application of these groups as linkers in drug conjugates. Calicheamicin is
conjugated to an oxidized antibody through an acylhydrazone in Mylotarg (7). Conjugates of other drugs have been reported
including doxorubicin (8). Hydrazones have been used as the basis for switches, sensors, and other materials (9). While the
literature is replete with examples of aliphatic, acyl and aromatic hydrazine derivatives of triazines and resulting hydrazones, the
mechanism of hydrazone hydrolysis is unreported (10).
Our longstanding interest in triazine chemistry(11) led us to examine whether hydrazine derivatives of triazines—soꢀcalled
triazinylhydrazines—might mirror acylhydrazines in hydrazone formation and hydrolysis. A priori, it was unclear whether such
hydrazones would be more or less stable than the acyl analogues. To start, a fourꢀstep synthesis of triazinylhydrazine 1 (Scheme 1)
was adopted. The aminoethoxyethanol substituents were included to enhance water solubility. The moderate reactivity of BOCꢀ
NHNH2 (BOC is tꢀbutoxycarbonyl) led to its installation on a dichlorotriazine intermediate that could be subsequently elaborated to
1 as shown. In contrast, reaction of cyanuric chloride with BOCꢀNHNH2 led to a mixture of products including the desired monoꢀ
addition product, the diꢀaddition product and other impurities that were not identified.(12) Similarly, the elevated temperatures
required for installation of BOCꢀNHNH2 (or hydrazine) on a suitably derivatized monochlorotriazine led to the desired compound,
but also additional impurities identified in HPLC chromatograms. Our interest in applying poly(dichlorotriazines) as intermediates
in dendrimer synthesis or on other scaffolds makes this seemingly laborious synthesis generally applicable, if not desirable (13).
o
Scheme 1. Synthesis of triazinylhydrazine 1. a) Aminoethoxyethanol, THF, DIPEA, ꢀ15 to 0 C, 1h. b) BOCꢀNHNH2, THF,
DIPEA, RT 12h. c) Aminoethoxyethanol, dioxane, DIPEA, 80 oC, 12h. d) 4M HCl:MeOH, 12h.
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We examined a suite of five aldehydes and ketones, aꢀe (Chart 1). We denote the hydrazone formed by the condensation of a 1 and
a as 1-a, and the corresponding acetylhydrazone as Ac-a. The alphabetical ordering of these compounds reflects the measured
stabilities to hydrolysis (vide infra).
1
2
3
4
5
Chart 1. Aldehydes and ketones used in this study.
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Condensation of 1 or acetyl hydrazine, AcNHNH2, to form the corresponding hydrazone was accomplished readily by mixing
with a 3ꢀfold excess of a-e and 0.5 equivalents of acetic acid at room temperature in ethanol overnight followed by
1
chromatographic purification. Structures were validated with H and 13C NMR spectroscopy as well as mass spectrometry. All
compounds showed a single peak in HPLC chromatograms with a unique retention time. Hydrolysis rates were measured in a
mixed solvent system to account for the varying hydrophobicity of these molecules. Briefly, 50mM stock solutions of 1-a through
1-e were prepared in methanol (Ac-a through Ac-e were prepared in THF) and diluted to 10mM with disodium phosphate/citric
acid buffers to reach pH of 5.2, 6.8, and 8.0. Hydrolysis was monitored in the presence of 10x excess formaldehyde as a trap (14)
using HPLC (Supporting Information). Hydrolysis was pronounced at pH 5.2. Figure 1a shows the data derived from HPLC traces.
Comparatively, acyl hydrazones are more stable than the triazinyl hydrazone counterpart. The stability increases with hydrazones
a<b<c<d<e.
Figure 1. Hydrolysis profiles at pH 5.2 (left) and 6.8 (right).
Table 1. Firstꢀorder reaction constants and half lives, and DFT computed N1 proton affinities ꢁEprot relative to Ac-b. Changes in
ꢁEprot upon triazine protonation are in parenthesis. The values of k are reported as (x103 minꢀ1) and for t1/2 as (minꢀ1) and for ꢁEprot
as (kcal/mol)
pH 4.0
pH 5.2
pH 6.8
ꢁEprot
Cmpd
1-a
Ac-a
1-b
Ac-b
1-c
Ac-c
1-d
k
39
20
t1/2
18
34
130
370
530
1400
770
k
4.7
2.6
0.3
0.1
0.09
0.04
0.059
0.046
0.014
0.027
t1/2
150
270
2310
ꢀ
ꢀ
8.0
30
1.9
8.6
1.8
2.9
0.7
3.1
ꢀ
ꢀ
87
23
360
81
390
240
990
224
4.4 (ꢀ5.0)
1.7
2.3 (ꢀ4.3)
0.0
1.0 (ꢀ4.1)
ꢀ0.7
ꢀ1.3 (ꢀ4.2)
ꢀ3.0
5.2
1.9
1.3
0.5
0.9
0.4
0.3
0.2
6900
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Ac-d
1-e
Ac-e
1700
2300
3500
ꢀ0.1 (ꢀ4.4)
ꢀ1.9
At pH 6.8, similar trends are observed over 72 h as shown in Figure 1b with most showing less than 20% hydrolysis over this
period. At pH 8.0, neglible hydrolysis was observed over 72h: only 1-a and Ac-a show measurable hydrolysis rates (0.0006 minꢀ1
and 0.0001 minꢀ1, respectively). Firstꢀorder reaction rate constants and half lives are shown in Table 1.
The accepted mechanism for hydrazone hydrolysis starts with protonation of nitrogen N1 to yield I (Scheme 2).(15) Addition of a
molecule of water yields carbinolamine intermediate, II. Proton migration (III) leads to CꢀN bond cleavage to complete the
hydrolysis. Subject to historical and contemporary inquiry (14ꢀ17), this mechanism is consistent with catalysis observed in acid.
Three different interpretations have been invoked to rationalize differences in rates of hydrolyses of different hydrazones (and
oximes and imines). These include a thermodynamic argument on ground state stabilities (16), resonance stabilization arguments
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focusing on the reduction of electrophilicity of C1 (17), and stabilities derived from a resistance to protonation of N1 (Figure 3)
recently invoked by Kalia and Raines to explain relative stabilities of hydrazones and oximes (14).
1
2
3
4
Scheme 2. Mechanism of hydrolysis. R = acetyl or triazinyl.
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Table 1 includes density functional theory (DFT) calculations (18ꢀ20) of proton affinities of N1 of the hydrazone. These values,
ꢁEprot, are assigned relative to Ac-b. Positive values correspond to stable proton binding. Calculations use the B3LYP exchangeꢀ
correlation functional(18), the 6ꢀ31+G(d,p) basis set (19), the SMD continuum model for aqueous solvent (20), and the Gaussian
09 suite of programs (21). Other computational details, and energies & geometries of all species, are reported as Supporting
Information. The measured rates of hydrolysis for Ac-a ꢀ Ac-e correlate with the computed N1 proton affinities, consistent with the
work of Kalia and Raines.(14) For example, ketoneꢀderived Ac-a has a shorter halfꢀlife and larger proton affinity than Ac-b, while
aldehydeꢀderived Ac-c, Ac-d, and Ac-e have longer halfꢀlives and smaller (more negative) proton affinities than Ac-b. Ac-c has a
larger proton affinity and shorter halfꢀlife than Ac-d, consistent with resonance electron donation from the paraꢀmethoxy group to
N1 increasing proton affinity.
Table 1 also shows that the triazinyl hydrazones 1-a - 1-e all have shorter halfꢀlives at pH 5, and larger N1 proton affinities, than
the corresponding acetyl hydrazones. Computation clearly supports an interpretation that resistance of N1 to protonation is the key
predictor of the stability of hydrazones to hydrolysis. Consistent with this analysis, computational models of simple imines
(H2NMe) and oximes (H2NOMe) derived from b (PhMeC=NMe and PhMeC=NOMe) give a large computed N1 proton affinity
14.9 kcal/mol (relative to Ac-b) for the rapidly hydrolyzed imine, and a small proton affinitiy ꢀ1.5 kcal/mol for the stable oxime
(14).
Triazinyl hydrazones, unlike acyl hyrazones, imines and oximes, however, offer additional basic sites on the triazine. Kalia and
Raines note that protonation of N1 is disfavored across a range of pH because the pKa of N1 is <0.7. Accordingly, activated
species will exist at very low concentrations in the pH range of interest. The pKa of protonated triazines is ~5 (22), thus protonated
triazines must be considered at low pH. Triazine protonation should promote hydrolysis by increasing the electrophilicity of C1
through induction, but counterproductively, should increase the resistance of N1 to protonation (Scheme 3), the perceived critical
event in hydrolysis.
Scheme 3. A protonated triazine (pKa~5) could increase electrophility of C1 through induction (increasing rate) or increase
resistance to N1 protonation (decreasing rate).
Computation bears this prediction out. Table 1 shows that paraꢀprotonation of the 1-a - 1-e triazine reduces the predicted N1 proton
affinities by 4ꢀ5 kcal/mol. Orthoꢀprotonation of 1ꢀd reduces the predicted N1 proton affinities by 6ꢀ10 kcal/mol, due in part to steric
clashes between orthoꢀNꢀH and N1ꢀH protons. We note that orthoꢀprotonation could conceivably accelerate hydrolysis proceeding
without N1 protonation, by increasing the electrophilicity of carbon C1. Test calculations suggest that this is a small effect. The
reaction of the C1 carbon of molecule 1-d with OH(ꢀ) is made 45 kcal/mol more favorable by N1 protonation, but only 11 kcal/mol
more favorable by ortho protonation.
Figure 3. Hydrolysis profiles at pH 4 in the presence of 10ꢀfold molar excess of formaldehyde.
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To explore this effect, we measured hydrolysis of 1-b and Ac-b at pH 4. Indeed, an inversion in stability is observed with Ac-b
hydrolyzing more rapidly than 1-b (Figure 3). Specifically, at pH 4, the rate constants (k x 103 minꢀ1) for 1-b, Ac-b, 1-c, and Ac-c
are 8.4, 2.7, 1.9, and 0.9, respectively. The halfꢀlives (min) for 1-b, Ac-b, 1-c, and Ac-c are 83, 25, 364, and 81, respectively.
In summary, the data here support the Kalia/Raines mechanism for hydrolysis. This “resistance to protonation” hypothesis is
further supported by the inversion of stability seen in triazinyl and acetyl hydrazones at low pH. This inversion in stability occurs at
a pH that is biologically relevant—that associated with the cellular uptake and the endosome. Whether it can be exploited
advantageously for the delivery of drugs or other agents remains to be seen.
Supporting Information. Synthetic details, characterization data including spectra and kinetic studies, computational details, and
computed structures and energies are provided. This material is available free of charge via the Internet at http://pubs.acs.org.
ACKNOWLEDGMENT
We thank the Robert A. Welch Foundation (Aꢀ0008) and the DOD (W81XWHꢀ12ꢀ1ꢀ0338) for support.
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