Tunable Stability of Imidazotetrazines Leads to a Potent Compound for Glioblastoma

Article abstract of DOI:10.1021/acschembio.8b00864

Even in the era of personalized medicine and immunotherapy, temozolomide (TMZ), a small molecule DNA alkylating agent, remains the standard-of-care for glioblastoma (GBM). TMZ has an unusual mode-of-action, spontaneously converting to its active component via hydrolysis in vivo. While TMZ has been FDA approved for two decades, it provides little benefit to patients whose tumors express the resistance enzyme MGMT and gives rise to systemic toxicity through myelosuppression. TMZ was first synthesized in 1984, but certain key derivatives have been inaccessible due to the chemical sensitivity of TMZ, precluding broad exploration of the link between imidazotetrazine structure and biological activity. Here, we sought to discern the relationship between the hydrolytic stability and anticancer activity of imidazotetrazines, with the objectives of identifying optimal timing for prodrug activation and developing suitable compounds with enhanced efficacy via increased blood-brain barrier penetrance. This work necessitated the development of new synthetic methods to provide access to previously unexplored functionality (such as aliphatic, ketone, halogen, and aryl groups) at the C8 position of imidazotetrazines. Through synthesis and evaluation of a suite of compounds with a range of aqueous stabilities (from 0.5 to 40 h), we derive a predictive model for imidazotetrazine hydrolytic stability based on the Hammett constant of the C8 substituent. Promising compounds were identified that possess activity against a panel of GBM cell lines, appropriate hydrolytic and metabolic stability, and brain-to-serum ratios dramatically elevated relative to TMZ, leading to lower hematological toxicity profiles and superior activity to TMZ in a mouse model of GBM. This work points a clear path forward for the development of novel and effective anticancer imidazotetrazines.

Full text of DOI:10.1021/acschembio.8b00864

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Article
Tunable Stability of Imidazotetrazines Leads
to a Potent Compound for Glioblastoma
Riley L. Svec, Lucia Furiassi, Christine G. Skibinski, Timothy
M. Fan, Gregory J. Riggins, and Paul J Hergenrother
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00864 • Publication Date (Web): 08 Oct 2018
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ACS Chemical Biology
Tunable Stability of Imidazotetrazines Leads to a Potent
Compound for Glioblastoma
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Riley L. Svec†, Lucia Furiassi†, Christine G. Skibinski‡, Timothy M. Fan§#, Gregory J. Riggins‡,
and Paul J. Hergenrother*†#
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†Department of Chemistry, §Department of Veterinary Clinical Medicine, and #Carl R. Woese Institute for Genomic
Biology, University of Illinois at Urbana–Champaign, Urbana, IL 61801, USA.
‡Department of Neurosurgery, Johns Hopkins University School of Medicine, Department of Neurosurgery,
Baltimore, MD, 21287, USA.
* to whom correspondence should be addressed, hergenro@illinois.edu
ABSTRACT
Even in the era of personalized medicine and immunotherapy, temozolomide (TMZ), a small molecule
DNA alkylating agent, remains the standard-of-care for glioblastoma (GBM). TMZ has an unusual
mode-of-action, spontaneously converting to its active component via hydrolysis in vivo. While TMZ
has been FDA approved for two decades, it provides little benefit to patients whose tumors express the
resistance enzyme MGMT and gives rise to systemic toxicity through myelosuppression. TMZ was first
synthesized in 1984, but certain key derivatives have been inaccessible due to the chemical sensitivity
of TMZ, precluding broad exploration of the link between imidazotetrazine structure and biological
activity. Here we sought to discern the relationship between the hydrolytic stability and anticancer
activity of imidazotetrazines, with the objectives of identifying optimal timing for prodrug activation and
developing suitable compounds with enhanced efficacy via increased blood-brain barrier penetrance.
This work necessitated the development of new synthetic methods to provide access to previously
unexplored functionality (such as aliphatic, ketone, halogen, and aryl groups) at the C8 position of
imidazotetrazines. Through synthesis and evaluation of a suite of compounds with a range of aqueous
stabilities (from 0.5 to 40 hours), we derive a predictive model for imidazotetrazine hydrolytic stability
based on the Hammett constant of the C8 substituent. Promising compounds were identified that
possess activity against a panel of GBM cell lines, appropriate hydrolytic and metabolic stability, and
brain-to-serum ratios dramatically elevated relative to TMZ leading to lower hematological toxicity
profiles and superior activity to TMZ in a mouse model of GBM. This work points a clear path forward
for the development of novel and effective anticancer imidazotetrazines.
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INTRODUCTION
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Glioblastoma multiforme (GBM) is the most prevalent, infiltrative, and lethal primary malignant
brain tumor, with only 10% of patients surviving five years.1 The current standard-of-care for GBM is
gross surgical resection followed by radiotherapy combined with temozolomide (TMZ, Scheme 1a), a
small molecule DNA alkylating agent. The antitumor effect of TMZ is ultimately mediated through
methylation of the O6-position of guanine residues and subsequent mismatch repair-dependent cell
death.2–6 Among the beneficial properties of TMZ are favorable pharmacokinetics (including 100% oral
bioavailability7), non-enzymatic prodrug activation, and some accumulation in the brain (cerebral spinal
fluid:blood ratio of 17:83 in human cancer patients8,9). TMZ provides a significant therapeutic benefit to
a subset of GBM patients; for example, in patients whose tumors do not express O6-methylguanine
DNA methyltransferase (MGMT), an enzyme that removes O6-methylguanine lesions, TMZ extends
median survival to approximately two years.10 Even in the era of personalized anticancer therapy, TMZ
remains frontline therapy for oligodendrogliomas, diffuse astrocytic gliomas, and pleomorphic
xanthoastrocytomas in addition to GBM.11 However, given the ineffectiveness of TMZ against tumors
expressing MGMT and the inevitable recurrence of GBM after multimodal combination therapy, there
remains a significant clinical need for better treatment strategies.
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TMZ is a prodrug activated in aqueous solutions that ultimately releases methyldiazonium, the
active alkylating component (Scheme 1a). The half-life of TMZ is ~2 hours in vivo and in aqueous
solutions in vitro, and it has been suggested that the drug has an increased rate of hydrolysis in the
more alkaline environment of gliomas, providing some selectivity for cancerous vs. non-cancerous
cells.12–15 While this 2 hour half-life enables TMZ to reach the central nervous system (CNS) and
release methyldiazonium, there is scarce information on the relationship between half-life and
anticancer activity; specifically, it is unclear if 2 hours is optimal to maximize therapeutic efficacy or if
shorter (or longer) half-lives may bolster its effect. Given the advantageous features of TMZ, we sought
to understand the relationship between its structure, hydrolytic stability, and anticancer activity.
While the amide at C8 had been suggested in the past to be essential for activity,2,16 conflicting
reports have since indicated that alternate functionality may be tolerated at this position.17–19 Indeed,
our own analysis led us to believe that strategic substitutions at C8 could be used to tune the hydrolytic
stability of imidazotetrazines, and that in doing so a suite of compounds with a range of half-lives could
be constructed. In addition to varying the stability of the prodrug, alterations at the C8 position could
lead to compounds that retain the favorable pharmacokinetic properties of TMZ but have increased
CNS penetrance. Conceivably, an imidazotetrazine with enhanced blood-brain barrier (BBB)
penetrance may exhibit lower systemic toxicity and allow for higher and more efficacious dosing
regimens since the dose-limiting toxicity of TMZ (myelosuppression) is not CNS-related.7,20,21 Herein
we describe the development of a model that accurately predicts the hydrolytic stability and half-life of
imidazotetrazines, and we use this model to discover novel imidazotetrazines with exceptional BBB
penetration and superior anticancer activity compared to TMZ, including in a murine model of GBM.
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a)
1
O
2
H2N
O
O
H2N
H2N
N
O
3
4
5
6
8
N
H
N
N
4
H2O
N
N
+
NH2
3
CH3
N
CH3
Apoptosis
N
N2 CH3
+
N
N
CH3
– CO2
NH
NH
TMZ
t1/2 ~ 2 h
MTIC
t1/2 = 8 min
AIC
Methyl diazonium
7
t1/2 = 0.39 s
8
9
O
CH3
N
H2N
b)
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11
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N
+ (CH3)2NH
N
CH3
N
Challenges of replacing
C8 amide
NH
O
1
Imidazotriazene
H2N
Dacarbazine (1962)
Aqueous sensitivity
Base sensitivity
N2
N2
N
N
– VS–
N
O
N
2
H2N
Diazoimidazole
degradation
N
O
+ CH3NCO
N
N
N
Poor substitutes
for CH3NCO
Decomposes
immediately at
room temp
(1961)
Stable > 2.5 yrs
N
CH3
Imidazotetrazine
TMZ (1984)
Scheme 1. (a) Mechanism of temozolomide (TMZ) activation in aqueous solution. (b) The favorable stability of
compound 1 relative to related versions (such as 2) accounts for the incorporation of the amide at C8 of TMZ.
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RESULTS AND DISCUSSION
Construction of C8-substituted imidazotetrazines. The inclusion of an amide at the C8
position of TMZ is largely an artifact of the original synthesis of imidazotriazenes and imidazotetrazines.
Both dacarbazine and TMZ are derived from precursor 4-diazoimidazole-5-carboxamide (1, Scheme
1b). The remarkable stability of this diazo species, reportedly >2.5 years at room temperature,22
permitted its use for exploratory chemistry where other diazoimidazole species (such as 4-
diazoimidazole (2)) simply decomposed.23 Thus, the initial synthesis of dacarbazine in 1962 and TMZ
in 1984 involved the quenching of 1 with dimethylamine24 or the cyclization of 1 with methyl
isocyanate,25 respectively, and the primary amide moiety remained. Over time, there have been
suggestions that this amide is critical for anticancer activity. Such claims were supported by theoretical
studies suggesting that a hydrogen bond donor at C8 is required for activity,2,16 but clouding the picture
is a conflicting structure-activity relationship (SAR) adopted from derivatives of a related compound
(mitozolomide) in non-CNS cancer models.26 There are considerable challenges to establishing a
general synthetic route that can be used to construct novel derivatives at the C8 position; these
synthetic challenges have hindered the development of new imidazotetrazines, and in the absence of
new compounds and biological data, the outdated SAR has persisted.
Key challenges to making novel imidazotetrazines include sensitivity to protic solvents or basic
reagents, instability of intermediate diazo species, and the lack of efficient reagents to install the N3
methyl. The sensitivity of the prodrug to conditions involving base or water (at pH > 6) renders the
tetrazinone unstable to many practical cross-coupling or reducing conditions. Another challenge, as
alluded to above, is the instability of intermediate diazo species. The privileged 4-diazoimidazole-5-
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carboxamide (1, Scheme 1b) readily precipitates out of solution as a pure, stable compound; however,
other 4-diazoimidazoles (such as 2) remain in aqueous solution, are exceptionally prone to
decomposition, and are sensitive to heat, shock, and often light.23 Finally, installation of the N3-methyl
group in the initial route to TMZ was achieved via cyclization with methyl isocyanate.25 Methyl
isocyanate, however, is a poisonous gas and no longer commercially available. As such, alternate
routes27 or less effective alternatives to methyl isocyanate such as N-succinimidyl N-methylcarbamate
or N-methylcarbamic chloride must be used that reduce the yield of the cyclizations.
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To provide access to certain derivatives of the C8 amide, we began by modifying an established
route, largely developed for mitozolomide.28 This sequence begins with a hydrolysis of the amide of
TMZ to carboxylic acid 3 (Scheme 2a), which can then be converted to the acid chloride. From this
intermediate, various nucleophiles may be substituted in high yields. This route was used to synthesize
amide, ester, and thioester derivatives 4-10 (Scheme 2a). Additionally, an established reaction was
employed to install a cyano group (11) directly from TMZ (Scheme 2a).29 The creation of a structurally
diverse panel of C8 analogs, however, would require novel synthetic routes, especially for those with
aliphatic, ketone, halogen, and aryl groups; such substituents have not been described at this position
in the ~35 year history of TMZ. Thus, an aliphatic group at C8 was introduced via diazotization of 5-
amino-4-methylimidazole 12 to diazo species 13 and subsequent cyclization with methyl isocyanate
surrogate N-methylcarbamoyl chloride to afford C8-methyl derivative 14 (Scheme 2b). Although various
amides, esters, and thioamides had been installed at C8, ketones were entirely absent, perhaps
unsurprisingly since initial attempts to use Grignard or alkyllithium reagents led to complete
degradation of the tetrazinone ring. Thus, a stepwise cyclization was utilized to synthesize methyl
ketone derivative 17 from its disubstituted precursor 16, obtained upon hydrolytic degradation of 6-
methylpurine N-oxide (15)30 (Scheme 2c). Bromine and chlorine substituents were directly incorporated
at C8 in moderate yields upon a decarboxylative halogenation of intermediate 3 employing Dess-Martin
periodinane and the respective tetraethylammonium salt (compounds 18 and 19, Scheme 2a). This
strategy had not previously been applied to imidazoles and endows potential points of diversity in
addition to representing novel derivatives themselves. Using 18 as a cross coupling partner, however,
was unsuccessful due to the basic, aqueous conditions required. Instead, a Suzuki coupling fashioned
5-nitro-4-phenylimidazole (21) from the 5-nitro-4-bromoimidazole (20) precursor, which could be
subsequently reduced to corresponding amine 22 and cyclized to the phenyl-substituted
imidazotetrazine as above (Scheme 2d). This method supplied 23 as well as a small series of p-
substituted aryl derivatives 24-26. Finally, heterocyclic compounds 27 and 28 (Scheme 2e) were
synthesized upon cyclization of the C8 amide or thioamide, respectively (Supporting Information); an
analogous route had been utilized to introduce bulkier 4-substituted oxazoles and thiazoles at the C8
position,19 but not smaller methyl groups.
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Scheme 2. Synthesis of novel C8-substituted imidazotetrazines. DMP = Dess-Martin periodinane, SNMC = N-
succinimidyl N-methylcarbamate.
Anticancer activity of C8 substituted imidazotetrazines. With a suite of imidazotetrazines in
hand, each compound was evaluated against a panel of human GBM cell lines (Table 1, Table S1).
Cell lines were selected to include those expressing and lacking MGMT (Figure S1) and, consistent
with literature reports, those with negligible MGMT expression were sensitive to TMZ (IC50 ~50 µM or
less) whereas those with significant MGMT expression were resistant (IC50 >300 µM). Amide-
substituted derivatives 4-8 as well as ester (9) and thioester (10) derivatives had activity comparable to
TMZ in the MGMT-deficient U87 and D54 cell lines. Notably, the retention of activity for disubstituted
amide (5-8) and ester (9) imidazotetrazines confirms that a hydrogen bond donor is not required at C8.
In U118MG and T98G MGMT-expressing GBM cells, more potent activity was observed for these
derivatives compared to TMZ. Ketone analog 17 was also effective against MGMT-deficient cell lines,
demonstrating that an amide is not required at the C8 position. Compounds completely lacking a
carbonyl, such as 14, 19, 23, and 27 proved to be as (or more) potent than TMZ in the absence of
MGMT and significantly more potent in cell lines expressing MGMT. Methyl (14) and phenyl (23)
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substitutions were the most active across all cell lines. Cyano derivative 11 and carboxylic acid
derivative 3 were inactive in all tested cell lines (>7-fold less potent than TMZ), even in the absence of
MGMT. In addition to these canonical adherent GBM cell lines, most analogs were more active than
TMZ in the patient-derived U3054MG GBM cell line cultured under serum-free stem cell conditions.31
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MGMT
9
–
–
+
+
+
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Compound
R
U87
D54
U118MG
322 ± 7
T98G
U3054MG
370 ± 40
TMZ
CONH2
51 ± 8
12 ± 1
660 ± 10
3
COO–
320 ± 7
49 ± 7
130 ± 8
11 ± 1
370 ± 20
280 ± 20
321 ± 8
ND
4 (Me-TMZ)
CONHMe
580 ± 20
290 ± 20
5 (DiMe-TMZ)
CONMe2
CONEt2
CON(n-Bu)2
CO(Prl)
COOEt
COSEt
CN
40 ± 20
80 ± 30
27 ± 6
17 ± 3
66 ± 3
64 ± 21
500 ± 60
3 ± 1
12 ± 5
13 ± 2
11 ± 2
12 ± 4
7 ± 1
130 ± 30
80 ± 10
62 ± 2
250 ± 60
160 ± 40
140 ± 20
186 ± 5
236 ± 10
327 ± 4
>1000
132 ± 6
ND
6
7
ND
8
136 ± 8
180 ± 20
165 ± 4
670 ± 20
7 ± 1
ND
9
ND
10
8 ± 1
ND
11
91 ± 6
6 ± 3
870 ± 60
ND
14
Me
6 ± 1
17 (K-TMZ)
COMe
Cl
44 ± 6
15 ± 4
9 ± 1
18 ± 1
21 ± 4
7 ± 1
115 ± 9
60 ± 20
3 ± 1
240 ± 20
60 ± 10
14 ± 1
125 ± 4
87 ± 4
18 ± 1
123 ± 6
ND
19
23
Ph
27 (Ox-TMZ)
28
5-Me-Oxaz
4-Me-Thiaz
27 ± 4
9 ± 1
9 ± 1
70 ± 20
12 ± 2
100 ± 20
31 ± 4
8 ± 1
Table 1. Panel of C8-substituted imidazotetrazines and associated IC50 values (µM) in multiple GBM cell lines. Cell
lines were incubated with compound for 7 days then viability was assessed using the Alamar Blue assay. Error is
SEM, n≥3. Prl = pyrrolidine. A table with additional compounds (Table S1) and a Western blot for MGMT status of all
cell lines used (Figure S1) can be found in the Supplementary Information.
Hydrolytic stability of C8 substituted imidazotetrazines. The principal aspect governing the
anticancer activity of imidazotetrazines is the hydrolytic activation of the prodrug. As depicted in
Scheme 1a, TMZ has a half-life of ~2 hours in humans.7 This timeline allows the intact prodrug to reach
the brain and release the active methylating component prior to elimination. Beyond TMZ, the
relationship between imidazotetrazine stability and anticancer activity is unknown; that is, while
hydrolytic activation is required for cancer cell death, the optimal timing of this event is unclear both in
vitro and in vivo. Towards this end, the hydrolytic stability of each new compound was assessed in
buffered saline, which mimics in vivo conditions (in pH 7.4 PBS TMZ has a half-life of 119 minutes,
Figure 1). A HPLC assay was developed to quantify the fraction of intact prodrug remaining in solution
after 2 hours at pH 7.4, 37°C. The results of this experiment suggest that electronic substituent effects
at C8 directly translate through the bicycle to C4, the site of hydrolysis. The magnitude of this effect
was dramatic, with stabilities ranging from 0% to 97% remaining after 2 hours depending on the
substituent at the C8 position (Figure 1a). Since the group at C8 appeared to have such a clear
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influence on the aqueous stability of the prodrug, its Hammett constant (σp) was plotted against the
percent remaining after 2 hours. As shown in Figure 1a, an obvious relationship exists between these
two parameters, suggesting that σp can be used to accurately predict the stability of C8-substituted
imidazotetrazines. Among those compounds possessing substituents with similar electronics (0.23 < σp
< 0.50) to a primary amide (σp = 0.36) were amide derivatives 4 and 5, ketone derivative 17, and chloro
derivative 19. Each had measured half-lives within an hour of TMZ in PBS at pH 7.4 (Figure 1b). On
either extreme were cyano analog 11 (σp = 0.66), with a half-life of 0.5 h, and methyl derivative 14 (σp =
–0.17), which remained in its prodrug form the longest with a half-life of 40 hours. The same assay was
used to confirm that hydrolysis remained pH-dependent for C8-substituted derivatives (e.g. K-TMZ 17,
Figure S2).
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23
Compound
t1/2 (h)
(a)
b)
3
18
28
11
0.57 ± 0.03
1.20 ± 0.10
1.98 ± 0.01
4
17 (K-TMZ)
TMZ
24
27
4 (Me-TMZ)
5 (DiMe-TMZ)
25
2.70 ± 0.10
2.80 ± 0.20
2.90 ± 0.30
3.00 ± 0.10
3.10 ± 0.10
9
19
5
17
27 (Ox-TMZ)
TMZ
19
23
14
27 ± 3
40 ± 1
11
σp
Figure 1. Hydrolytic stability of C8-substituted imidazotetrazines. (a) The percentage of compound remaining after 2
hours plotted against the Hammett constant of its C8 substituent. Compounds with hydrolytic stability similar to TMZ
are enclosed in the oval. (b) Half-lives of select C8 derivatives in PBS (pH 7.4, 37°C).
Relationship between hydrolytic stability and anticancer activity. Methyl and phenyl
derivatives 14 and 23 were consistently the most potent compounds in each of the tested cell lines
(Table 1). Interestingly, they also possessed electron-donating substituents and, accordingly, the
greatest aqueous stability (Figure 1), suggesting that a longer-lived prodrug is favorable for efficacy in
cell culture. The opposite effect was observed for compound 11, which was the least stable in solution.
Even in U87 cells lacking MGMT, it exhibited a ten-fold loss of activity compared to TMZ, suggesting
that there is a critical threshold of aqueous stability below which hydrolysis occurs too quickly to
methylate target DNA. Compounds with hydrolytic stabilities similar to TMZ such as 4, 5, 17, 19, and 27
retained activity in culture. Notably, ketone derivative 17 was equipotent to TMZ even with a shorter
aqueous half-life, indicating that compounds with σp ~ 0.50 can still retain marked anticancer activity.
Liver microsome stability. TMZ fortuitously possesses several ideal pharmacokinetic
properties including avoidance of primary metabolism.7 To assess whether modification or replacement
of the amide at C8 would lead to significant metabolic liabilities, the stabilities of select compounds
were assessed after 2 hours in the presence of mouse liver microsomes. Prodrug hydrolysis was
accounted for by including control runs that did not contain liver microsomes. The slightly acidic pH of
the working solution resulted in enhanced stability of TMZ compared to incubation in PBS alone.
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Predictably, TMZ was insensitive to metabolic perturbation as its instability was entirely accounted for
by hydrolysis (Table 2). The addition of methyl(s) to the amide (compounds 4 and 5) resulted in some
susceptibility to the effects of the microsomes, and this effect was amplified for larger amide
substitutions (compound 7), which demonstrated improved aqueous stability but markedly less stability
in liver microsomes. Ketone 17 and chloro 19 were generally stable to oxidative metabolism,
suggesting that for these compounds hydrolysis could drive the pharmacokinetics in vivo, similar to
TMZ.
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Stability
Compound
(2h, Microsomes) (2h, No Microsomes)
cLogBB
CNS MPO
Propranolol
TMZ
4 (Me-TMZ)
5 (DiMe-TMZ)
6
7
17 (K-TMZ)
19
23
68 ± 2%
87 ± 6%
86 ± 1%
81 ± 2%
81 ± 1%
1 ± 1%
70 ± 1%
91 ± 3%
44 ± 2%
71 ± 1%
102 ± 3%
86 ± 4%
93 ± 1%
92 ± 3%
95 ± 2%
98 ± 3%
77 ± 3%
91 ± 1%
103 ± 5%
95 ± 4%
ND
ND
4.9
5.7
6.0
6.0
5.6
6.0
6.0
5.7
5.9
–1.58
–1.34
–1.18
–1.07
–0.78
–1.08
–0.72
–0.56
–1.19
27 (Ox-TMZ)
Table 2. Metabolic stability, cLogBB, and CNS MPO values for relevant C8 analogs. The metabolic stability was
assessed in mouse liver microsomes. Compounds were incubated in microsomes for 2 h, then the percentage
remaining was quantified relative to t0. Experiments assessing stability in the absence of microsomes were identical
but replaced liver microsomes with PBS. Error is SEM, n≥2. Internal standard = N3-propyl TMZ. CNS MPO = Central
Nervous System Multiparameter Optimization Score.
Blood-brain barrier penetrance. It has been reported that >98% of small molecule drugs do
not penetrate the BBB,32 making TMZ unusual, especially amongst anticancer agents. In humans, TMZ
is rapidly absorbed and reaches the brain in minutes with cerebral spinal fluid concentrations averaging
20% of those in the plasma;8,9 the accumulation of even more drug in the brain by increasing the BBB
penetrance may be a viable strategy to increase efficacy against CNS-based tumors. To predict the
BBB penetrance of the novel imidazotetrazines, logBB values were calculated (cLogBB) based on a
formula utilizing cLogP and total polar surface area.33 When applied across a consistent drug scaffold,
these types of in silico metrics have been used reliably to predict relative changes in BBB penetrance
as well as other biological phenomenon,34–38 though not always reflective of absolute concentrations.
The cLogBB value for TMZ is –1.58 (Table 2). Replacing the primary amide led to marked increases in
the cLogBB and larger predicted brain:blood ratios relative to TMZ. Importantly, cLogBB does not
account for molecular weight, making us wary of analogs with large, hydrophobic functionality (e.g. 7)
even if they possess attractive predicted values. A more comprehensive metric, the CNS
multiparameter optimization (MPO) tool39,40 was also employed to gauge prospective BBB
permeabilities. CNS MPO scores span from 0 to 6.0 based on the optimal ranges of 6 physicochemical
properties. Though TMZ has an agreeable MPO of 4.9, higher scores were achieved for the C8
analogs, which in several cases reached the top desirability value (Table 2). The more favorable
cLogBB and CNS MPO values predicted for the panel suggests that certain derivatives may achieve
higher drug concentrations in the brain than TMZ.
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The BBB penetrance of top compounds (those with favorable anticancer activity, appropriate
hydrolytic and liver microsome stability, and predicted BBB penetrance, Figure 2a) was thus assessed
in vivo. In an initial experiment, Me-TMZ (4) and DiMe-TMZ (5) were tested head-to-head with TMZ to
explore whether alkylation of the C8 amide could confer increased brain:blood ratios. Mice were
administered 25 mg/kg drug intravenously and sacrificed 5 minutes after injection. The serum and
perfused brain samples were immediately acidified to prevent prodrug degradation before the drug
concentration within each compartment was quantitated by LC-MS/MS. After 5 minutes, drug
concentrations in the brain were significantly elevated for analogs Me-TMZ and DiMe-TMZ versus
TMZ, a >3-fold increase in brain:serum ratio for each compound (Figure 2b). The equivalent
brain:serum ratios for Me-TMZ and DiMe-TMZ is likely due to the fast metabolism of the dimethylated
amide to its monomethylated counterpart. This preliminary experiment suggested that other derivatives
with higher predicted BBB penetrance may lead to greater brain permeability in vivo. Accordingly,
compounds Ox-TMZ (27) and K-TMZ (17) were evaluated head-to-head with DiMe-TMZ and TMZ.
After 5 minutes, each derivative had accumulated numerically higher concentrations in the brain than
TMZ (Figure 2c). When paired with the corresponding serum concentrations (Figure 2d), TMZ had a
relative brain:serum ratio of 0.23 ± 0.03 ng/g:ng/mL, comparable to the few other TMZ biodistribution
experiments in murine systems.41,42 Assigning average mouse blood volumes to equate units (see
Supplementary Information), TMZ had an absolute brain:serum ratio of 8:92, while Ox-TMZ and K-TMZ
boasted brain:serum ratios of 55:45 and 69:31, respectively. (Figure 2e). The dramatic differences in
drug partitioning suggest that replacing the amide at C8 is a viable strategy to significantly increase
local drug concentration in the brain relative to the blood, which may increase effectiveness against
brain tumors and also reduce hematological toxicity.
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a)
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K-
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Me-TMZ DiMe-TMZ
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Ox-
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K-
TMZ
TMZ
DiMe-
TMZ
K-
TMZ
Ox-
TMZ
Figure 2. Blood-brain barrier permeability of imidazotetrazines. (a) Structures of lead C8-substituted compounds. (b)
Relevant brain:serum ratios of TMZ, Me-TMZ, and DiMe-TMZ (25 mg/kg) were measured 5 minutes after IV injection
into mice. Values are the fold change of brain:serum ratio relative to TMZ. In a second experiment, brain (c) and
serum (d) concentrations of TMZ and C8 analogs (25 mg/kg) were quantitated 5 minutes after IV injection into mice.
(e) Brain:serum ratios were calculated based on (c) and (d) assuming a mouse blood volume of 58.5 mL/kg. Error is
SEM, number of mice per cohort = 3. Statistical significance was determined by using a two-sample Student’s t-test
(two-tailed test, assuming equal variance).
Assessment of hematological toxicity. The elevated brain concentrations and dramatically
decreased serum concentrations (Figure 2c, 2d) observed upon treatment with Ox-TMZ and K-TMZ
compared to TMZ suggested that these C8-modified imidazotetrazines may attenuate the dose-limiting
hematological toxicity observed for TMZ in the clinic. To test this hypothesis, mice were treated with a
single dose of 125 mg/kg TMZ, Ox-TMZ, or K-TMZ intravenously; this dose of TMZ has been reported
to induce non-lethal toxicity in mice.43,44 Seven days post-treatment, whole blood was collected and
complete blood counts were obtained for each individual mouse. Expectedly, a dose of 125 mg/kg TMZ
led to white blood cell (WBC) depletion relative to control mice (Figure 3a), suggestive of drug-induced
myelosuppression. Both lymphocyte (Figure 3b) and neutrophil (Figure S3a) concentrations were
decreased in TMZ-treated mice. Conversely, treatment with 125 mg/kg of Ox-TMZ or K-TMZ did not
produce myelosuppression. Total WBC, lymphocyte, and neutrophil counts for mice treated with these
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compounds were equivalent to those of control mice. Notably, the novel imidazotetrazines did not give
rise to other hematological symptoms such as red blood cell (RBC) toxicity (Figure S3b) or
thrombocytopenia (Figure S3c) and did not lead to weight loss 7 days post-treatment (Figure S3d).
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TMZ
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K-TMZ
Control
TMZ
Ox-TMZ
K-TMZ
Figure 3. Assessment of the hematological toxicity of imidazotetrazines in vivo. Mice were administered a single IV
dose of 125 mg/kg imidazotetrazine. After 7 days, whole blood was collected and a complete blood count was
obtained for each individual mouse. (a) Total WBC count. Control vs. Ox-TMZ: P = 0.7, Control vs. K-TMZ: P = 0.9. (b)
Lymphocyte concentrations. Control vs. Ox-TMZ: P = 0.5, Control vs. K-TMZ: P = 0.9. Error is SEM, number of mice
per cohort = 4. Statistical significance was determined by using a two-sample Student’s t-test (two-tailed test,
assuming equal variance). The concentrations of other relevant blood constituents can be found in Supplementary
Information Figure S3.
Novel imidazotetrazines induce alkylation-mediated cancer cell death. The cytotoxicity of
TMZ is mediated by methylation of O6 guanine; subsequent single- and double-strand breaks and
apoptosis are facilitated by the mismatch repair system.2–6 To assess if the novel imidazotetrazines kill
through the same mechanism, O6-methylguanine adducts were quantitated in U87 cells treated with
100 or 1000 µM of each imidazotetrazine. After 8 hours of incubation with compound, the genomic
DNA was isolated, quantified, and hydrolyzed to its constituent deoxyribonucleosides, which were
quantitated via LC-MS/MS analysis. Dose dependent increases in the concentration of O6-methylated
deoxyguanosine were observed for TMZ as well as each of the lead compounds (Figure 4a), indicating
that DNA methylation is occurring. Further confirmation that the novel compounds remain DNA
alkylators was obtained upon pre-treatment with MGMT inhibitor O6-benzylguanine (O6BG). O6BG is a
pseudosubstrate for MGMT that quenches cellular stores of the enzyme, leading to the persistence of
O6-methylguanine DNA adducts. Pre-incubation of MGMT-expressing T98G cells with O6BG (100 µM)
led to an eight-fold enhancement in cytotoxicity for TMZ (Figure 4b), consistent with literature
reports.45,46 Similarly, DiMe-TMZ, Ox-TMZ, and K-TMZ demonstrated a significant increase in activity
when administered after O6BG, suggesting that O6-methylguanine lesions are the cause of cell death.
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1
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IC Values (µM)
50
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Compound
(–) O6BG
(+) O6BG
Fold Change
TMZ
660 ± 10
250 ± 60
100 ± 20
240 ± 20
81 ± 8
25 ± 9
5 ± 1
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DiMe-TMZ
Ox-TMZ
K-TMZ
32 ± 4
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µM
µM
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K-TMZ
DiMe-TMZ
TMZ
Figure 4. (a) The concentration of O6-methylguanine was measured in U87 cells (10 µg DNA) after treatment with 100
or 1000 µM of imidazotetrazine for 8 hours. (b) Imidazotetrazines were added to T98G cells with or without 3 h pre-
treatment of O6BG (100 µM). IC50 values after 7 day incubation and fold changes between (+/–) O6BG treatments are
reported. P-values between IC50 values (+/–) O6BG < 0.02 for all compounds. Error is SEM, n≥3. Statistical
significance was determined by using a two-sample Student’s t-test (two-tailed test, assuming equal variance).
Novel imidazotetrazines have superior activity in mouse models of GBM. The increased
BBB penetrance observed for amide derivatives (Me-TMZ and DiMe-TMZ) relative to TMZ suggested
that greater drug concentrations in the brain might lead to greater efficacy in an intracranial tumor
model. GBM oncosphere lines were chosen for these studies as they more accurately recapitulate the
genetic and histopathological features of human GBM than traditional adherent cell lines, which are
passaged in serum and typically grow as compact masses in vivo.47 The Br23c GBM oncosphere cell
line does not express MGMT, was sensitive to TMZ and the novel C8-substituted imidazotetrazines
(Table S2), and was thus chosen as the model system. Mice implanted intracranially with these cells
were administered 15 mg/kg TMZ or the equimolar equivalent of Me-TMZ or DiMe-TMZ once-per-day,
5x/week via oral gavage. As expected, TMZ significantly increased median survival compared with
vehicle (Figure 5a). Mice treated with both Me-TMZ and DiMe-TMZ, however, outperformed TMZ and
increased median survival by 24% and 46%, respectively, suggesting that increasing the BBB-
permeability of imidazotetrazine prodrugs is a viable strategy to improve efficacy. In a second
experiment, K-TMZ was selected for evaluation due to its most favorable brain:blood ratio (Figure 2e).
Mice intracranially implanted with Br23c cells were treated with K-TMZ (via oral gavage), which led to
an extended median survival of more than 50 days past TMZ-treated mice, and showed greater
efficacy even compared to DiMe-TMZ (Figure 5b). Importantly, methyl derivative 14, which has
excellent efficacy in cell culture but an extended (40 hour) half-life in aqueous solution, had no effect in
this in vivo model (Figure S4) suggesting that dramatically elongated half-lives are detrimental in vivo,
likely due to compound clearance prior to hydrolytic activation.
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Median
Survival
Control
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a)
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338 d
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Figure 5. Evaluation of imidazotetrazines in intracranial mouse models of GBM. GBM Br23c oncospheres were
intracranially implanted into female athymic nude mice. Treatment was started 5 days post implantation. (a) Mice
were administered 15 mg/kg TMZ or an equimolar dose of Me-TMZ (16.1 mg/kg) or DiMe-TMZ (17.2 mg/kg) orally once-
per-day, 5x/week for 7 weeks. Control vs. TMZ: P = 0.0014, TMZ vs. DiMe-TMZ: P = 0.061, TMZ vs. Me-TMZ: P = 0.016.
(b) Mice were administered 15 mg/kg TMZ or an equimolar dose of DiMe-TMZ (17.2 mg/kg) or K-TMZ (14.9 mg/kg)
orally once-per-day for 5 total doses. Control vs. TMZ: P = 0.0007, DiMe-TMZ vs. TMZ: P = 0.7, K-TMZ vs. TMZ: P =
0.055. Compounds were formulated in 10% PEG in PBS immediately prior to each treatment. Number of mice per
treatment cohort ≥5. Survival curves were compared using log-rank test.
CONCLUSION
Despite being known since 1984, FDA approved since 1999, and reaching $1 billion in sales in
2009, TMZ remains the only approved imidazotetrazine anticancer drug; this likely stems from the lack
of generalized syntheses for this class of compounds prohibiting conventional medicinal chemistry
campaigns. Here we report new synthetic methods enabling the construction of novel C8-substituted
imidazotetrazines that were previously inaccessible. Evaluation of these compounds in systematic,
head-to-head assays allows us to definitively conclude that the C8 amide is not required for anticancer
activity, and indeed compounds lacking an H-bond donor or acceptor (or both) at C8 can still retain
activity comparable to TMZ against cancer cells in culture. Unmoored from the necessity of an amide at
C8, a panel of imidazotetrazines was synthesized, varying this position. Strikingly, the electronic
properties of the substituent at C8 has a dramatic effect on the activation of the corresponding prodrug,
a previously undefined phenomenon. The relationship derived herein between the hydrolytic stability of
imidazotetrazines and the electronics at C8 allows the stability of the prodrug to be tuned by employing
easily accessible σp values, enabling the rational design of TMZ derivatives that have similar stabilities
in vivo and facilitating investigation into the optimal timing of imidazotetrazine prodrug activation.
From this work it appears that compounds with very short half-lives (such as 11, t1/2=0.57 h)
simply hydrolyze too rapidly, releasing methyl diazonium prior to accumulation in the DNA
microenvironment and diminishing anticancer activity. Thus for activity against cancer cells in culture, a
half-life of 1 h or greater is optimal. Conversely, compounds that have very long half-lives (such as 14
or 23, t1/2 >20 h) can be distinctly more potent than TMZ in cell culture as the prodrug has ample time
to distribute to the nucleus before conversion to the active methylating agent. However, these
compounds with markedly increased hydrolytic stabilities are less likely to be useful in vivo as
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elimination through alternate pathways (excretion of the intact prodrug, oxidative metabolism, etc.) will
occur before activation to the alkylating species. This hypothesis accounts for the lack of in vivo
efficacy of compound 14.
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A hallmark of GBM is its invasion into surrounding brain tissue at an early stage, making cure
via surgical resection unachievable. As such, there is an obvious clinical need for improved compounds
that can reach the entirety of the diffuse tumor in sufficient concentrations to be effective. Importantly,
our data shows that the BBB-penetrance of imidazotetrazines can be improved through modifications
at the C8 position. The dramatically enhanced brain:serum distribution of Ox-TMZ and K-TMZ, in
particular, could provide substantial improvement over TMZ for treatment of CNS cancers. Both of
these compounds retain the favorable features of TMZ (timely prodrug activation, stability to liver
microsomes) while also accumulating higher drug concentration in the brain and reduced concentration
in the blood. We hypothesized that partitioning the imidazotetrazine more to the site of the tumor and
less to the compartment responsible for adverse effects would expand the therapeutic window by
enhancing anticancer activity while simultaneously reducing systemic toxicity. Myelotoxicity occurs in
~20% of TMZ-treated patients, is the major dose-limiting toxicity,48 and is exacerbated in elderly and
female GBM patients.49,50 Ox-TMZ and K-TMZ demonstrated significantly less in vivo toxicity to WBCs
compared to TMZ, likely a direct result of the increased partitioning to the CNS. Imidazotetrazines such
as these with lower toxicity profiles could permit elevated dosing schedules and additional anticancer
efficacy, and/or make this drug class accessible to more patients.
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Other imidazotetrazines of various composition in the literature have failed to improve median
survival head-to-head compared to TMZ in preclinical models, despite promising results in cell
culture.42,51,52 To our knowledge, only one derivative has outperformed TMZ in an intracranial murine
model of GBM, bestowing a modest 10% increase in median survival.53 Clearly, the interplay between
retaining the favorable properties that have kept TMZ as frontline treatment for GBM and modulating its
structure is not trivial. The data reported herein now suggest that imidazotetrazines may be
substantially modified without losing these advantages, and indeed such new compounds can have
dramatically enhanced in vivo efficacy. TMZ remains the gold-standard for treating the most aggressive
brain tumors, shows promise against brain metastases from other cancers,54 and its predictable activity
(based on clinical biomarkers) has recently led to advocation for an expanded use of TMZ in the
management of diverse cancer types.55 As such, the novel imidazotetrazines reported here could hold
considerable promise for treatment of GBM and other cancers.
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ASSOCIATED CONTENT
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The Supplementary Information is available free of charge at the ACS Publications website.
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Supplementary Tables S1–S2, Western blot for MGMT for all cell lines used, Supplementary Figures
S1–S3, experimental details for mouse liver microsome assay, in vivo BBB permeability and
hematological toxicity studies, O6-MedG quantitation, and in vivo mouse models. Synthetic procedures
and characterization for C8 analogs.
AUTHOR INFORMATION
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Corresponding Author
*hergenro@illinois.edu
Author Contributions
Funding Sources
No competing financial interests have been declared.
ACKNOWLEDGMENTS
We thank L. Li (Metabolomics Center, Roy J. Carver Biotechnology Center, UIUC) for LC-MS/MS
analysis and Charlie Pierce for preliminary stability data. We are grateful to the National Institutes of
Health R21-CA195149 (to P.J.H. and G.J.R.) and R01-CA190223 (to G.J.R.) for support of this work.
R.L.S. is a member of the NIH Chemistry-Biology Interface Training Grant (T32-GM070421). L.F. was
a visiting student from the Department of Biomolecular Sciences, University of Urbino “Carlo Bo”,
Piazza Rinascimento 6, I-61029 Urbino, Italy.
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versus Radiotherapy Alone on Survival in Glioblastoma in a Randomised Phase III Study: 5-Year Analysis
of the EORTC-NCIC Trial. Lancet Oncol. 10, 459–466.
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