Synthesis and quantitative structure-activity relationship of imidazotetrazine prodrugs with activity independent of O6-methylguanine-DNA- methyltransferase, DNA mismatch repair, and p53

Article abstract of DOI:10.1021/jm401121k

The antitumor prodrug temozolomide is compromised by its dependence for activity on DNA mismatch repair (MMR) and the repair of the chemosensitive DNA lesion, O6-methylguanine (O6-MeG), by O6-methylguanine-DNA-methyltransferase (E.C. 2.1.1.63, MGMT). Tumor response is also dependent on wild-type p53. Novel 3-(2-anilinoethyl)-substituted imidazotetrazines are reported that have activity independent of MGMT, MMR, and p53. This is achieved through a switch of mechanism so that bioactivity derives from imidazotetrazine-generated arylaziridinium ions that principally modify guanine-N7 sites on DNA. Mono- and bifunctional analogues are reported, and a quantitative structure-activity relationship (QSAR) study identified the p-tolyl-substituted bifunctional congener as optimized for potency, MGMT-independence, and MMR-independence. NCI60 data show the tumor cell response is distinct from other imidazotetrazines and DNA-guanine-N7 active agents such as nitrogen mustards and cisplatin. The new imidazotetrazine compounds are promising agents for further development, and their improved in vitro activity validates the principles on which they were designed.

Full text of DOI:10.1021/jm401121k

Subscriber access provided by UNIVERSITY OF CALGARY
Article
Synthesis and Quantitative Structure–Activity Relationship of
Imidazotetrazine Prodrugs with Activity Independent of O6-
Methylguanine-DNA-methyltransferase, DNA Mismatch Repair and p53.
Dimitrios Pletsas, Elrashied A. E. Garelnabi, Li Li, Roger M. Phillips, and Richard Thomas Wheelhouse
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm401121k • Publication Date (Web): 29 Jul 2013
Downloaded from http://pubs.acs.org on August 4, 2013
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Synthesis and Quantitative Structure–Activity
Relationship of Imidazotetrazine Prodrugs with Activity
Independent of O6-Methylguanine-DNA-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
methyltransferase, DNA Mismatch Repair and p53.
†
†§
‡
‡
†
Dimitrios Pletsas, Elrashied A.E. Garelnabi, Li Li, Roger M. Phillips, Richard T. Wheelhouse *
†
‡
School of Pharmacy and Institute of Cancer Therapeutics,
University of Bradford, BRADFORD, BD7 1DP, UK.
ꢀ 1 ꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Abstract
The antitumor prodrug Temozolomide is compromised by its dependence for activity on DNA mismatch
repair (MMR) and the repair of the chemosensitive DNA lesion, O6-methylguanine (O6-MeG), by O6-
methylguanine-DNA-methyltransferase (EC 2.1.1.63, MGMT). Tumor response is also dependent on wild-
type p53. Novel 3-(2-anilinoethyl)-substituted imidazotetrazines are reported that have activity
independent of MGMT, MMR and p53. This is achieved through a switch of mechanism so that bioactivity
derives from imidazotetrazine-generated arylaziridinium ions that principally modify guanine-N7 sites on
DNA. Mono- and bi-functional analogs are reported and a quantitative structure-activity relationship
(QSAR) study identified the p-tolyl-substituted bi-functional congener as optimized for potency, MGMT-
independence and MMR-independence. NCI60 data show the tumor cell response is distinct from other
imidazotetrazines and DNA-guanine-N7 active agents such as nitrogen mustards and cisplatin. The new
imidazotetrazine compounds are promising agents for further development and their improved in vitro
activity validates the principles on which they were designed.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Introduction
The imidazotetrazine prodrug temozolomide 1 (TMZ), a DNA methylating agent, in combination with
radiotherapy, is now first line treatment for glioblastoma multiforme (GBM) in North America and Europe.
However, intrinsic and acquired resistance significantly limits the ultimate efficacy of therapy. In
particular, tumor re-growth up to two years following TMZ treatment is aggressive and resistant to
further TMZ therapy.1-3 This paper reports new compounds of the TMZ class that have activity
independent of the two principal constraints on the ability of a tumor to respond to TMZ therapy: MGMT
activity which directly repairs TMZ GO6-methylation of DNA, and MMR which enables the tumor response
to TMZ therapy.2
ꢀ 2 ꢀ
ACS Paragon Plus Environment

Page 3 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. The mechanism of action of temozolomide 1.
4
At neutral pH, TMZ is relatively unstable (t1/2 = 1.24 h, pH 7.4) and undergoes hydrolytic ring-cleavage
to the open chain triazene 5-(3-methyltriazen-1-yl)- imidazole-4-carboxamide (MTIC, t1/2 = 8 min, pH 7.4),5
which then fragments to the highly reactive electrophile, methyldiazonium (t1/2 = 0.39 sec, pH 7.4).6-8
Methyldiazonium reacts with nucleophilic groups on DNA, resulting in DNA methylation, Scheme 1.
Approximately 70% of the methyl groups transferred to DNA appear at N7-guanine, 10 % at N3-adenine,
and 5 % at O6-guanine sites. The N-methylation products are readily repaired by the base-excision repair
9
pathways so are not major contributors to chemosensitivity. In contrast, O6-MeG lesions are reversed by
direct removal by the MGMT protein. The MGMT gene is silenced by promoter methylation in
1
approximately 35% of GBM. In these tumors, persistent O6-MeG lesions form wobble base-pairs with
thymidine during replication; O6-MeG=T pairings result in futile cycles of mismatch repair, leading to
1
stalled replication forks, DNA double-strand breaks and ultimately cell death. Low levels of MMR
expression (or mutations leading to non-functional MMR) lead to a tolerant phenotype that cannot
respond to TMZ. Consistent with these mechanisms of O6-MeG processing and repair, high-level
expression of the MGMT protein is a major mechanism of inherent TMZ resistance in primary tumors.
Down regulation or mutations of the MMR pathway or up-regulation of MGMT expression are important
2
causes of acquired TMZ resistance in GBM. Herein we report the synthesis, pre-clinical activity in vitro
and QSAR of new imidazotetrazines that have activity independent of MMR and MGMT.
ꢀ 3 ꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Compound Design
The imidazotetrazine bicycle is a prodrug of alkyldiazonium ions which are liberated by pH-dependent
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
hydrolysis. Careful control of these reactive intermediates, in particular the suppression of competing
side reactions, such as hydrolysis, elimination or re-arrangement,10-12 is essential in the design of effective
new agents.13, 14 Furthermore, of the methyl groups transferred from TMZ to DNA, only a small fraction
becomes the therapeutically-beneficial O6-MeG lesion: we sought to achieve therapeutic benefit though
generation of N7-G-adducts, the major products of reaction of TMZ with DNA, thereby making more
efficient use of the imidazotetrazine prodrug. We have designed novel series of mono- (2a–i) and bi-
functional (3a–f) imidazotetrazines in which a 3-(2-anilinoethyl) group was substituted for the 3-methyl
group of TMZ. The new compounds are efficient precursors of aziridinium ions, 4, Scheme 2. This strategy
provides for effective control of the reactivity of incipient diazonium ions using a neighboring group
participation mechanism not available to TMZ. Moreover, aziridinium ions are reactive intermediates of
proven clinical utility, being closely related to those generated by nitrogen mustard drugs, and are
1
5
established as working through N7-guanine adducts. An additional feature of the drug design is the
aniline para-substituent “X” of derivatives 2 and 3 that can be optimized to fine-tune pharmacological
activity. This group affects the electron density at the aniline nitrogen by resonance or inductive effects
and thereby controls the basicity and nucleophilicity of this site: i.e. the propensity either to protonate or
to form an aziridinium ion. In addition, the bi-functional molecules would be expected to generate DNA
cross-links, that would not be processed by MMR or MGMT and so avert those constraints on activity;
1
6
17
previous studies have demonstrated that polar and bulky GO6 adducts cannot be processed by MGMT.
ꢀ 4 ꢀ
ACS Paragon Plus Environment

Page 5 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2. Aziridinium ion formation by imidazotetrazine 2b.15
Synthesis
Scheme 3.
The new imidazotetrazines were prepared by variants of the established TMZ synthesis where a
1
8
diazoimidazole 5 is reacted with an isocyanate 6 to yield the imidazotetrazine, Scheme 3. The target
compounds all included an aminoethyl group in the 3-position. The requisite β-aminoisocyanate
precursors 7 are inherently unstable compounds as they bear incompatible functional groups. In the two
aniline series, the anilines were sufficiently deactivated to allow isolation of the isocyanates without
ꢀ 5 ꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
recourse to protecting group strategies. The formation of the relatively fragile tetrazine ring was usually
planned to be the last step in the synthesis.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 4. Route to bis-imidazotetrazines 3. Reagents and conditions: (i) CuCl, AcOH, Δ, 76–98%; (ii)
2 2
NH NH ·xH
2 2 2
O, IPA, Δ, 64–99%; (iii) NaNO , AcOH, H O, CH
2
Cl , 0 °C; (iv) PhMe, Δ, [>95%]; (v) DMSO, RT, 2
2
d, 6–60% (over two steps)
To prepare the dimers 3a–f, Cu(I)-catalyzed Aza-Michael conjugate addition of methyl acrylate to the
requisite aniline furnished the diesters 8 in good (60–98%) yields, Scheme 4. Conversion to isocyanates 9
was achieved through the intermediate hydrazides 10 and azides 11 followed by Curtius rearrangement.
For the electron rich anilines (X=CH , OMe), hydrazide formation had to be performed under very mildly
3
acidic (0.17 M AcOH) conditions to avoid nitration of the aniline ring (10e → 11f → 3f). This is an
attractive route as the bis-hydrazides are usually solids and easily purified while the Curtius
rearrangement produces pure isocyanates directly, free from contaminating by-products.
ꢀ 6 ꢀ
ACS Paragon Plus Environment

Page 7 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 5 Route to mono-imidazotetrazines 2. Reagents and conditions: (i) CuCl, AcOH, Δ, 58–97%; (ii)
2 2
NH NH ·xH
2
O / IPA, Δ, 44–98%; (iii) NaNO
2
, AcOH, H
2
O, CH
2
Cl , 0 °C; (iv) PhMe, Δ, [>95%]; (v) DMSO, RT,
2
6
–51% over two steps.
The mono-imidazotetrazine analogs 2a–i were similarly prepared from N-methyl anilines, Scheme 5. In
one example, the secondary aniline version 2g (R=H, X=Cl) was achieved using a BOC protection strategy
that exploited the relative acid stability of the imidazotetrazine ring. Carefully controlled diazotization
(12f → 13f → 14f) yielded BOC-protected isocyanatoethylaniline 15f; tetrazine ring closure followed by
final treatment with TFA gave secondary aniline 2g. As a similar yield was obtained omitting the BOC
group, the protection was shown to be redundant. The p-nitro derivative 2e was accessed from hydrazide
13a by simultaneous diazotization and nitration under more vigorous conditions (10 M HCl); the mixture
of regioisomers was carried through to the final step where the p-NO isomer, tetrazine 2e, was isolated
2
by flash column chromatography.
In vitro QSAR: influence of MGMT and MMR
ꢀ 7 ꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1 . In vitro chemosensitivity (IC50 / ꢀM) of new imidazotetrazine compounds against A2780
(
MMR+) and A2780-cp70 (MMR–) cell lines in the absence (MGMT+) and presence (MGMT–) of the MGMT
inactivator PaTrin2 (10 ꢀM). All data are the mean of ≥3 determinations, error bars are the SD; t-test
analysis is presented in Table S3. * IC50 >250 ꢀM. MEL, melphalan; CP, cisplatin.
ꢀ 8 ꢀ
ACS Paragon Plus Environment

Page 9 of 55
Journal of Medicinal Chemistry
1
2
3
Screening of all new compounds for chemosensitizing efficacy was undertaken following a protocol by
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Margisson et al that used the MGMT and MMR-proficient ovarian carcinoma cell line A2780, and its MMR-
1
9
deficient derivative A2780-cp70, Figure 1. To assess dependence of cytotoxicity on MGMT function, each
2
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
cell line was further treated with the MGMT inactivator PaTrin2. The data are presented graphically in
Figure 1 and fully tabulated in Table S1. As expected, sensitivity to TMZ and mitozolomide (MTZ) was
highly dependent on both lack of MGMT activity and presence of wild-type MMR capacity. For TMZ, the
IC50 was >250 ꢀM in both A2780 and A2780-Cp70 cell lines, compared with IC50 = 8.5 and 231 ꢀM
respectively for the same cell lines co-treated with PaTrin2. In contrast, the novel imidazotetrazines were
significantly more potent, with IC50 values in the A2780 wild-type line ranging from 22–73 ꢀM for the
mono-functional (2a–i) and 1–15 ꢀM for the bi-functional (3a–f) compounds. Dependence on MMR can be
examined by comparing the shaded bars with the grey bars (i.e. MMR+/MMR– with MGMT inactivated in
both cases). For temozolomide the IC50 was >27–fold lower in the MMR-proficient cell line. For the new bi-
functional agents 3, this ratio was reduced to 5–5.8-fold and for the mono-functional agents 2, 2.8–10-fold.
The extent of MGMT-mediated resistance can be assessed by comparing the shaded and the black bars (i.e.
MGMT–/MGMT+ with MMR competent in both cases). Here, temozolomide was >30-fold more potent
when MGMT was inactivated whilst for the new agents, this ratio was 0.5–5-fold for the bi-functional and
1.1–2.9-fold for the mono-functional agents. Importantly, in the absence of MMR, all compounds showed
activity greater than temozolomide irrespective of the MGMT status of the cells (white bars and grey bars)
showing that MMR-dependent toxicity and MGMT-mediated resistance are now only minor determinants
of the chemosensitizing effect.
The similarity of response of examples 2d and 2g indicate that the N-methyl aniline is not essential to
activity as the secondary and tertiary aniline compounds are equipotent (see Figure 1 and Table S1).
Retention of the BOC group in analog 2f significantly reduced potency, presumably by impeding the
aziridine/aziridinium formation step. Nitration in the aniline ring, whether as the sole or as an additional
ꢀ 9 ꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
substituent, resulted in reduced potency. This is consistent with electron withdrawal reducing
nucleophilicity of the aniline and hence the propensity for aziridinium ion formation, (for examples,
compare the compound pairs 3e/3f, 2a/2e, and 2b/2h).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To probe the structure-activity relationship and examine MGMT and MMR dependence in more detail,
the results for dimeric compounds 3a-e were presented in Hammett plots, Figure 2. The Hammett
constant σ-p is a structural parameter that measures the electron donating or withdrawing effect of the
substituent “X” on the aniline ring. The plot of IC50 against σ-p is informative in several respects, Figure
2
A. For each cell type, the graph shows a pair of lines, the dotted line being MGMT-inactivated data (i.e.
PaTrin 2 present); the difference between the solid and dotted lines shows the effect of MGMT on the
cellular response. For most analogs this separation is of the same order as the error bars on the data, so in
effect is zero. This is a clear demonstration that these new compounds have activity independent of MGMT
function and is consistent with a mechanism of action independent of guanine-O6 alkylation (or involving
a guanine-O6 adduct that cannot be repaired by MGMT). The separation between the two pairs of lines is
the MMR effect which ranges from about 2–5-fold on the IC50, which is greatly reduced from the >27-fold
effect measured for TMZ.
p-Methyl-substituted analog 3b lies on a minimum in the graph so is optimally potent, with a modestly
electron-donating substituent on the aniline ring, Figure 2A. The data for all analogues except 3e (X =
OMe) lie on exponential curves (see the semi-log plot, Figure 2B). The anomalous data for 3e are likely
due to enhancement of the basicity of the aniline nitrogen lone pair leading to appreciable protonation at
the pH of the experiment, and thereby a reduced propensity to act as a nucleophile in the aziridinium ion-
forming reaction. These data clearly demonstrate the direct effect of electron density at the aniline
nitrogen in determining the in vitro activity of the compounds, so provide further evidence that the
aziridinium ion mechanism occurs in cells.
ꢀ 10 ꢀ
ACS Paragon Plus Environment

Page 11 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢀ 11 ꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
A
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
B
Figure 2. (A, upper panel) Hammett plot of the IC50 data for the dimers 3a–e, in each case the MGMT– data
are shown in dotted lines; the substituents X are shown in circles. (B, lower panel) QSAR Semi-log plot of
ꢀ 12 ꢀ
ACS Paragon Plus Environment

Page 13 of 55
Journal of Medicinal Chemistry
1
2
3
log IC50 vs. Hammett constant (σ-p) for the dimeric imidazotetrazines 3a–e. Regression lines were fitted
omitting the data for OMe derivative 3e.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NCI60 Panel Data
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compounds 2d, and 3c-e were selected for full screening in the NCI 60 cell line panel. Mean graph data
are presented in Figures S1–6 and are summarized in Table 1. These data show that the NCEs have
-4
pharmacological activity distinct from TMZ. TMZ is essentially inactive against the panel (GI50 > 10 M for
5
3
7/60 cell lines, Figure S1). In contrast, the new agents exhibit strong patterns of discrimination over a 2–
-log range of GI50 (Table 1 and Figures S2–5); this is important as it shows that the new compounds
possess a degree of selectivity and are not uniformly cytotoxic against cells grown in culture. These data
compare favorably with the logGI50 mean and range data for established agents CHB, MEL, CP, BCNU and
CCNU; this shows there is good reason to suppose that the new compounds may have an acceptable
therapeutic index and be no more systemically toxic in vivo than these other clinically useful agents.
Log GI50
Log GI50 range
Mean (SD)
2
3
3
3
MEL
CHB
CP
d
c
d
e
–4.69 (0.58)
–5.11 (0.69)
–5.07 (0.52)
–4.83 (0.46)
–4.54 (0.55)
–4.21 (0.56)
–5.40 (1.10)
–4.12 (0.82)
–4.53 (0.48)
2.05
2.81
1.72
1.80
1.88
1.64
2.38
1.86
2.22
BCNU
CCNU
Table 1 Summary of NCI60 statistics for compounds 2d, 3c–e, and established prodrugs for
comparison.
Particular sensitivity is evident in the leukemia, CNS and ovarian sub-panels. In the CNS sub-panel, the
pattern of activity is similar to MTZ and CP, with particular sensitivity in the glioblastoma lines SF295 and
SNB75. In the ovarian sub panel, particular sensitivity is seen in the OVACR-3 and SK-OV-3 lines. Here, in
ꢀ 13 ꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
contrast to the CNS activity, the new compounds show improvement over CP (see Figure S6) this is a
particularly interesting result given the wide clinical use of CP against ovarian adenocarcinoma and the
fact that TMZ lacks clinical activity against ovarian cancer.21
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
COMPARE analysis of mean graph data can be valuable in discerning molecular mechanisms of action.
The similarity in patterns of response is assessed by the Pearson rank correlation coefficient, P: values >
0
.7 are considered highly significant, values 0.6–0.7 less so.22, 23 Matrix COMPARE was valuable in
confirming the novelty of mechanism of action of the new agents. In this method of data handling, the new
compounds were compared with standard agents selected for similarities of chemical structure or
predicted mechanism of action. Agents selected were other imidazotetrazines (TMZ, MTZ), nitrogen
mustard cross linkers (chlorambucil CHB, melphalan MEL), nitrosoureas (1,3-Bis(2-chloroethyl)-1-
nitrosourea BCNU, N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea CCNU) and CP, Table 2. Correlations
amongst the four new imidazotetrazine compounds examined were all strong (0.92 ≤ P ≥ 0.77) and,
interestingly, there was no clear discrimination between the mono- and bi-functional agents: for
monofunctional 2d, 0.83 ≤ P ≥ 0.77 and in particular P = 0.79 with the equivalent p-Cl dimer 3d.
Gratifyingly, the new compounds showed only moderate correlations (0.52 ≤ P ≥ 0.38) with the
established imidazotetrazines TMZ and MTZ, so are distinct as new members of this compound class. The
absence of correlation with MTZ is significant as this compound, exhibits a characteristic pattern of
differential activity across the panel, unlike TMZ.
The putative DNA lesion of the bifunctional agents 3 is a five-atom crosslink, related in structure to
those formed by the nitrogen mustard prodrugs; however, no drugs of this class showed strong
correlations 0.59 ≤ P ≥ 0.29. Notably, there was no similarity to the nitrosoureas which are also diazonium
ion precursors, 0.45 ≤ P ≥ 0.05. The new compounds all showed strong correlations with dacarbazine
(
DTIC, highest dose = 1 ꢀM). This observation is not easy to interpret, as this prodrug requires hepatic
ꢀ 14 ꢀ
ACS Paragon Plus Environment

Page 15 of 55
Journal of Medicinal Chemistry
1
2
3
oxidative demethylation in order to release its active electrophile, so it is somewhat surprising that DTIC
scored so highly against the new imidazotetrazines. Part of the reason for this may lie in the two relatively
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
featureless datasets of DTIC [mean log GI50(SD) = –4.30(0.28), range = 0.99, high conc. = 10 M; mean log
–
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
GI50(SD) = –3.66(0.548), range = 2.55, high conc. = 10 M] and a shared sensitivity in the leukemia sub-
-3
panel. Simple COMPARE using DTIC GI50 (high conc. = 10 M) data as seed in the Standard Agents
database yields no correlations P>0.537.
2
d
3c
0.83 0.79 0.77 0.50
0.92 0.92 0.52
0.81 0.46
0.51
3d
3e
TMZ
MTZ
0.42
0.35
0.38
0.46
DTIC MEL
CHB
0.33
0.33
0.29
0.53
BCNU CCNU
CP
2d
3c
1
0.62
0.72
0.64
0.71
0.35
0.4
0.25
0.32
0.27
0.45
0.08
0.06
0.05
0.10
0.27
0.32
0.35
0.42
0.83
0.79 0.92
0.77 0.92 0.81
1
3
d
e
1
0.36
0.59
3
1
Table 2. Results of Matrix COMPARE (P values) for NCEs 2d, 3c-e with selected standard agents.
Simple COMPARE analysis was performed with the Standard Agents and Molecular Targets databases
using the GI50 data as seed, Table 3. The Standard Agents data again identified similarity with DTIC, as
noted above. In addition, modest correlations with 6MP and 6TG appeared for all four compounds, which
is presumably linked to residual MMR-dependence,24, 25 a property that is shared with MTZ and DTIC. With
the exception of compound 3e, correlations in the Molecular Targets database were not strong. Notable
amongst the correlations of analog 3e are the lymphocyte and leukocyte related proteins LCP1, LRMP,
RASSF5 and LAIR1 which may account for the high sensitivity of the leukemia sub-panel to these new
compounds. Also intriguing are the correlations with the RAS-related proteins ARHGAP4 and RASSF5 and
the moderate inverse correlation with RAB1A, a RAS superfamily GTPase.26
2
7
All of the NCI cell lines are characterized for expression of MGMT and components of MMR. Plots of
protein expression vs. log GI50 were prepared to probe correlation or inverse correlation between GI50 and
ꢀ 15 ꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
expression of MGMT, hMLH1 (the core MMR protein) or MSH6 (the MMR domain that recognizes O6-
alkylG-DNA damage), see Figures S6-8. For each of the compounds 2d, 3c–e, the graphs showed scatters
with no connection between the protein expression levels and GI50: a result consistent with the
independence of activity from MGMT and MMR found in the A2780 cell line screen.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ 16 ꢀ
ACS Paragon Plus Environment

Page 17 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Standard Agents Database
Molecular Targets Database
Pa
DTICb
0.669
0.751
P
P
Strong positive correlationsc
(P)
Strongest negative correlation
(P)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
6TG
6MP
2
d
c
0.563 0.571
0.543 0.524
U2AF1 (0.61)
RAB1A (–0.611)
RAB1A (–0.636)
3
RCSD1 (0.686)
HCLS1 (0.683)
WAS (0.526)
3
d
e
0.717
0.727
0.515 0.527
0.573 0.515
RAB1A (–0.526)
K1AA1191 (–0.64)
RAB1A (–0.5136)
3
HCLS1, WAS, NCKAP1L, LCP1,
LRMP, ARHGAP4, RASSF5, CD53,
LAIR1 (>0.7 for all)
TMZ
MTZ
0.326
0.517 0.418
a
b
c
P, Pearson rank correlation coefficient; in all cases the highest ranking correlation was with DTIC; examples from the 10 top correlations, P
values in parentheses. 6TG, 6-thioguanine; 6MP, 6-mercaptopurine; DTIC, dacarbazine.
Table 3. Selected results from NCI60 screen and COMPARE analysis using the log GI50 data as seed in the Standard Agents and Molecular Targets
databases.
ꢀ
17 ꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Further in vitro evaluation
The new compounds were further assessed in selected drug-resistant cell lines (Tables 4 and 5).
HCT116 is a human colon carcinoma cell line that is deficient in both MGMT and MMR, in consequence
it is insensitive to both TMZ and CP.28-30 Both mono- and bi-functional agent families showed
considerably more potent activity than TMZ and MTZ in this cell line and, in further contrast, activity
was retained in the p53-deficient mutant, Table 5. TMZ activity is known to be p53 dependent.31
Compounds were also evaluated against an isogenic pair of SNB19 GBM cell lines competent in MMR
that either expressed MGMT, SNB19(MGMT), or were negative for MGMT, SNB19(vector). This pair of
cell lines highlights the improvements the new bi-functional agents offer over TMZ. The MGMT-
proficient line being remarkably insensitive to TMZ (IC50 is unattainable) and the new bi-functional
agents showing equivalent activity against the two lines (IC50 6-12 ꢀM) with increased potency against
both SNB19(vector) and SNB19 (MGMT). A2058 is an aggressive melanoma line that is TMZ-sensitive
and even so, the new bi-functional agents show a 10-fold increase in potency.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A2058
>100
5.5
HCT116
20.0
SNB19(vector)
SNB19(MGMT)
2
3
3
g
c
e
77.0
11.0
6.6
>100
12.0
7.0
12.5
2.5
5.5
TMZ
35.5
>250
37.0
>250
Table 4. IC50 (ꢀM) for new compounds in selected TMZ-resistant cell lines using the Cyquant Assay
n=2).
(
ꢀ
18 ꢀ
ACS Paragon Plus Environment

Page 19 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
HCT116+/+a
HCT116–/– a
35.8 (7.1)
9.5 (4.8)
Ratio +/+: –/–
2d
2g
55 (8.2)
13.2 (14.1)
5.5 (1.2)
1.5
1.4
1.1
1.4
1.6
0.8
3.4
6.2
3a
4.8 (2.1)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3b
3c
6.2 (n=2)
28 (8.1)
4.4 (2.7)
17 (5.3)
3e
7.6 (2.1)
9.9 (n=2)
17.9 (8.9)
1.9 (2.5)
MTZ
CP
60 (11.2)
11.8 (n=2)
Table 5. p53 Independence of chemosensitivity of HCT116 cell lines proficient and deficient in p53.
a
IC50, ꢀM (SD). IC50 (ꢀM)
Discussion and Conclusions
The aims of generating new imidazotetrazine prodrugs with activity independent of MGMT and
MMR have been achieved through a rationally-designed switch in molecular mechanism. This
complements other work, where alternative GO6-lesion dependent strategies have been pursued.29
Two new families of mono- and bi-functional 3-anilinoethyl imidazotetrazines have been prepared.
These were designed to shift the mechanism of action from dependence on GO6-DNA modification to
GN7-modification. This was achieved through trapping the diazonium electrophile released on
hydrolysis of the imidazotetrazine prodrug as an aziridinium ion. The mono-functional agent 2b is
efficiently converted to an aziridinium ion (Scheme 2) and 2b and 3b are both DNA GN7-alkylators.15
It is proposed, on the basis of analogy with reactive intermediates generated by nitrogen mustard and
similar prodrugs, that biological activity is likely associated with this lesion. However it is not yet
possible to distinguish definitively between a mechanism of action deriving from GN7-adduct
formation and one proceeding from GO6-adducts that are resistant to MGMT-mediated repair.
ꢀ
19 ꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Screening in A2780 and A2780-cp70 cell lines showed that the new compounds are more potent
than TMZ. The mono-functional agents are approximately equipotent with MTZ and the bi-functional
agents more active. This activity is independent of MGMT activity as determined by use of the MGMT
inactivator PaTrin2. Dependence of the IC50 on MMR is greatly reduced from approximately 30-fold for
TMZ to 2–5-fold for the new bi-functional agents. The Hammett plot, Figure 2, for the bi-functional
agents demonstrates the critical rôle of the para-substituent in directing the reactivity and bioactivity
of the compounds, consistent with the aziridinium ion mechanism occurring in vitro and corroborating
the design principles. The data identified the p-toluidine derivative 3b as the optimal analog for MGMT
and MMR independence and potency.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In the NCI60 screen, the new agents were not uniformly cytotoxic and showed distinct and
individual patterns of response over a near 3-log range of GI50. The patterns of biological response to
the new agents were distinct from standard agents drawn from the imidazotetrazine, nitrogen
mustard and nitrosourea families which, by chemical consideration, may be predicted to share similar
mechanisms of action. For all compounds evaluated in the full 60 cell line screen, there was neither
correlation with MLH1 or MSH6, nor inverse correlation with MGMT protein expression levels.
Correlations in the Molecular Targets database suggest there may be a specific molecular mechanism
that accounts for the sensitivity of the leukemia sub-panel to the new agents and, moreover, that
activity could be linked to RAS family oncogenes.
Collectively, the in vitro data show that the initial aims of improved potency and MGMT and MMR
independence have been achieved. The switch of chemical mechanism has resulted in new agents that
are distinct from existing imidazotetrazines, nitrogen mustards and nitrosoureas. This implies that an
increased diversity of tumor types may now be able to respond to drugs of the imidazotetrazine class.
NCI60 panel data identified CP-resistant ovarian carcinoma cell lines as responsive to the new agents.
Chemosensitivity in selected drug-resistant colon carcinoma and glioma cell lines (HCT116 and
SNB19) showed that there is activity against TMZ- and CP-resistant tumor lines of these types, and
ꢀ
20 ꢀ
ACS Paragon Plus Environment

Page 21 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
moreover, that the HCT116 response is independent of p53 status. The ability of cells to repair
damage by the new agents is linked to the ATR and FANC pathways for the bi-functional and ATM and
32
FANC pathways for mono-functional agents. Mutation in one of these genes would hypersensitize a
tumour to these new agents.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Overall these findings validate our understanding of the underlying chemistry of imidazotetrazine
prodrugs. The members of our compound library are distinctive new agents, worthy of further
development and application against a more diverse range of tumor types than TMZ.
Acknowledgement
This work was supported by grant C14492/A4884 from Cancer Research UK. Undergraduate project
students M. Haroon Ahmad, Nicholas H. Gunn, Nosheen Hanif, Mobeen Mumtaz, Nazia Nazir, Zaynab
Nejadhamzeeigilani and Matthew J. Nelson contributed to the synthetic work. We thank Paul Zavodny
and Paul Kirschmeier, Schering-Plough Corp. Kenilworth, NJ for the data in Table 3; Dr Geoff Wells,
University College London, for advice on handling NCI data; Xin Ivy Meng and Mohammed U. Saleem,
University of Bradford, for the data in Table 5 and some of the data in Figure 1 respectively; Sachin
Korde and Amit Sonawane, University of Bradford, for assistance with HPLC.
ꢀ
21 ꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Experimental
Synthesis
Reagents were purchased from SigmaꢀAldrich, Alfa Aesar and Fluka, solvents from Fisher
Scientific. TLC was performed on highlyꢀpurified silica gel plates with UV indicator (silica gel F245),
manufactured by Merck and visualized under UV light (366 or 254 nm) or stained with iodine.
Melting points were determined with an Electrothermal IA9200 digital melting point apparatus.
Infrared data were obtained using a Perkin Elmer (Paragon 1000) FTꢀIR Spectrophotometer. NMR
spectra were acquired on a JEOL GX270 Delta, or where indicated ECA600, spectrometers
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
13
13
observing H at 270.05 and 600.17 MHz and C at 67.80 and 150.91 MHz respectively.
C
assignments were made with the aid of the DEPT135 experiment. Mass spectra were obtained
from the EPSRC National Mass Spectrometry Service Centre at the University of Swansea, UK,
and the Analytical Centre at the University of Bradford using a Micromass Quattro Ultima mass
spectrometer. Elemental analyses were obtained from the Advanced Chemical and Material
Analysis Unit at the University of Newcastle upon Tyne, UK. All compounds entering biological
evaluation were dried in vacuo for 3–4 days ≤35 °C; ≥95% purity was adjudged by combustion
analysis and hplc, solvation is indicated where appropriate; corresponding analytical hplc
1
chromatograms and highfield H NMR spectra are shown in ESI.
Compounds 2b and 3b were prepared as described in reference 15.
Methyl 3-(4-chlorophenylamino)propanoate 12g
General Method A: Single Conjugate Addition to Anilines.
4ꢀChloroaniline (20.0 g, 157 mmol) was mixed with methyl acrylate (54.0 g, 627 mmol, 4 eq),
cuprous chloride (1.56 g, 15.68 mmol, 0.1 eq) and AcOH (30 mL) and heated under reflux at 160
°C for 2 h. The solvents were removed by evaporation and the residue partitioned between
chloroform (500 mL) and water (500 mL). The organic layer was dried (MgSO
4
) in the presence of
charcoal and filtered through celite. The solvents were removed leaving an oily residue. Petroleum
ꢀ
22 ꢀ
ACS Paragon Plus Environment

Page 23 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
ether (200 mL) was added and heated to reflux. The solvent was decanted and the product
precipitated upon standing. The solid was collected by filtration, washed with petroleum ether and
1
dried to give the ester (23.7 g, 71 %): H NMR (CDCl
3
) 7.11 (d, J = 8.8 Hz, 2H, 2ꢀH & 6ꢀH), 6.55 (d,
J = 8.8 Hz, 2H, 3ꢀH & 5ꢀH), 3.68 (OCH
3
), 3.41 (t, J = 7.2 Hz, 2H, CH N), 2.61 (t, J = 7.2 Hz 2H,
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3
CH CO); C NMR (CDCl ) 172.8 (C=O), 146.1 (Cꢀ1), 129.2 (Cꢀ2 & Cꢀ6), 122.5 (Cꢀ4), 114.3 (Cꢀ3 &
2
3
•+
Cꢀ5), 51.9 (OCH
3
), 39.7 (CH
2
N), 33.6 (CH
2
CO); MS (ES): m/z 214.1 (M+H) ; IR (film) 3400, 1725,
ꢀ1
1
600, 1500, 1435 cm .
15, 33
Methyl 3-(methyl(phenyl)amino)propanoate 12a
1
Prepared by General Method A. Ester 12a was obtained as a colorless oil (13.3 g, 74 %). H
NMR (CDCl
3
) 7.16 (t, 2H, J = 8.6 Hz, 3ꢀH & 5ꢀH), 6.64 (m, 3H, 2ꢀH, 4ꢀH & 6ꢀH), 3.58 (m, 5H, CH
2
N
13
&
CH N), 2.85 (s, 3H, OCH ), 2.49 (t, J = 7.3 Hz, 2H, CH CO) C NMR (CDCl ) 172.8 (C=O),
3 3 2 3
1
48.6 (Cꢀ1), 129.4 (Cꢀ3 & Cꢀ5), 116.9 (Cꢀ4), 112.6 (Cꢀ2 & Cꢀ6), 51.8 (OCH
3
2
), 48.7 (NCH ), 38.3
•+
ꢀ1
(
3 2
NCH ), 31.6 (CH CO); MS (ES): m/z 194.1(M+H) ; νmax (film) 1725s (C=O), 1175m (CꢀO) cm
Methyl 3-((4-fluorophenyl)(methyl)amino)propanoate 12c
Prepared according to General Method A. Ester 12c was obtained as an orange oil (2.22 g,
1
7
3
1
5%). H NMR (CDCl
3
) 6.92 (m, 2H, 3ꢀH & 5ꢀH), 6.66 (m, 2H, m, 2ꢀH & 6ꢀH), 3.65 (s, 3H, OCH
3
),
1
3
.60 (t, J = 7.3 Hz, 2H, NCH ), 2.86 (NCH ), 2.52 (t, J = 7.3 Hz, 2H, CH CO); C NMR (CDCl3)
2
3
2
1
2
72.8 (C=O), 155.7 (d, JCF = 235 Hz, Cꢀ4), 145.5 (Cꢀ1), 115.7 (d, JCF = 22 Hz, Cꢀ3 & Cꢀ5), 114.1
3
(
d, JCF = 7 Hz, Cꢀ2 & Cꢀ6), 51.8 (CH
3
O), 49.5 (CH
2
N), 38.7 (CH
3
N), 31.5 (CH
2
CO); MS (ES): m/z
•+
212.1 (M+H) ; IR (KBr) 3025m (Ar CꢀH), 2950m (CꢀH), 1725s (C=O), 1525m (Ar C=C), 1175m (Cꢀ
ꢀ1
O) cm .
Methyl 3-((4-chlorophenyl)(methyl)amino)propanoate 12d
Prepared according to General Method A. Ester 12d was obtained as a yellow oil (9.1 g, 54%).
1
3
H NMR (CDCl ) 7.15 (d, J = 8.9 Hz, 2H, 3ꢀH & 5ꢀH), 6.63 (d, J = 8.9 Hz, 2H, 2ꢀH & 6ꢀH), 3.63 (s,
ꢀ
23 ꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
3H, OCH
NMR (CDCl
(OCH ), 48.8 (NCH
(Ar CꢀH), 2950m (CꢀH), 1725s (C=O), 1525m (Ar C=C), 1175m (CꢀO) cm .
3
), 3.62 (t, J = 7.2 Hz, 2H, NCH
2
), 2.89 (s, 3H, NCH
3
), 2.54 (t, J = 7.2 Hz, 2H, CH
2
CO); C
3
) 172.6 (C=O), 147.2 (Cꢀ1), 129.1 (Cꢀ3 & Cꢀ5), 121.7 (Cꢀ4), 113.9 (Cꢀ2 & Cꢀ6), 51.9
•+
3
2 3 2
), 38.4 (NCH ), 31.5 (CH CO); MS (ES): m/z 228.1 (M+H) ; IR (KBr) 3025m
ꢀ1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
Methyl 3-((4-methoxyphenyl)(methyl)amino)propanoate 12e
1
According to General Method A. Ester 12e was obtained as a colorless oil (4.72 g, 58%): H
3
NMR (CDCl ) 6.76 (d, 2H, J = 9.0 Hz, 3ꢀH & 5ꢀH), 6.67 (d, 2H, J = 9.0 Hz, 2ꢀH & 6ꢀH), 3.68 (s, 3H,
CH
3
OꢀAr), 3.59 (s, 3H, CH
3
OCO), 3.51 (t, J = 7.3 Hz, 2H, CH
2 3
N), 2.78 (s, 3H, CH N), 2.46 (t, J =
13
7
.3 Hz, 2H, CH CO); C NMR (CDCl ) 172.4 (C=O), 151.6 (Cꢀ4), 143.1 (Cꢀ1), 114.6 & 114.3 (Cꢀ3,
2
3
Cꢀ5 & C2, C6), 55.3 (CH
3
OꢀAr), 51.2 (CH
3
OCO), 49.4 (NCH
2
3 2
), 38.4 (CH N), 30.9 (CH CO); MS
•+
ꢀ1
(
ES): m/z 224.1 (M+H) ; IR (film) 1725s (C=O), 1175m (CꢀO) cm .
Methyl 3-(tert-butoxycarbonyl-(4-chlorophenyl)amino)propanoate 12f
Ester 12g (2.27 g, 10.6 mmol) was mixed with diꢀtertꢀbutyl carbonate (9.3 g, 42.6 mmol, 4 eq) in
the absence of solvent. The mixture was heated at 100 °C for 18 h. The mixture was partitioned
between H O and petroleum ether. The organic phase was washed with H O (5 x 50 mL), dried
2
2
1
and evaporated to give ester 4h as an oil (3.2 g, 97%). H NMR (CDCl
3
) 7.29 (d, J = 8.8 Hz, 2H, 2ꢀ
H & 6ꢀH), 7.11 (d, J = 8.8 Hz, 2H, 3ꢀH & 5ꢀH), 3.91 (t, J = 7.2 Hz, 2H, NCH
2
), 3.53 (OCH
3
), 2.57 (t,
13
J = 7.2 Hz, 2H, CH CO), 1.41 (s, 9H, C(CH ) ); C NMR (CDCl ) 172.3 (OC=O), 154.4 (NC=O),
2
3 3
3
141.0 (Cꢀ1), 130.9 (Cꢀ4), 129.1 (Cꢀ2 & Cꢀ6), 113.7 (Cꢀ3 & Cꢀ5), 79.8 (C(CH
3
)
3
), 51.8 (OCH
3
), 48.2
•+
(
NCH ), 32.8 (CH CO), 28.4 (CH ); MS (ES): m/z 314.1 (M+H) ; IR (film) 1725s (C=O), 1175m (Cꢀ
2 2 3
ꢀ1
O) cm .
3
4, 35
3-(4-Chlorophenylamino)propanehydrazide 13g
General Method B: Preparation of monohydrazides.
ꢀ
24 ꢀ
ACS Paragon Plus Environment

Page 25 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Ester 12g (3.7 g, 11.8 mmol, 1 eq) was mixed with hydrazine hydrate (5.9 g, 118 mmol, 10 eq) in
propanꢀ2ꢀol (10 mL) for 48h at RT. The volatiles were removed with evaporation to leave hydrazide
1
13g as colorless oil (3.5 g, 95%). H NMR (DMSOꢀd
6
) 9.03 (br s, 1H, NHNH
2
), 7.05 (d, 2H, J = 8.9
Hz, 2ꢀH & 6ꢀH), 6.53 (d, 2H, J = 8.8 Hz, 3ꢀH & 5ꢀH), 5.76 (br s, 1H, NHꢀaniline), 4.28 (s, br, 2H,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3
NH ), 3.19 (t, J = 7.2 Hz, 2H, NCH ), 2.41 (t, J = 7.2 Hz, 2H, CH CO); C NMR (DMSOꢀd ) 170.5
2
2
2
6
(
(
C=O), 148.0 (Cꢀ1), 129.1 (Cꢀ3 & Cꢀ5), 119.4 (Cꢀ4), 113.9 (Cꢀ2 & Cꢀ6), 39.9 (CH
2
N), 33.7
•+
ꢀ1
CH
2
CO); MS (ES): m/z 214.1 (M+H) ; IR (KBr) 3300s (NH), 3050m (Ar CꢀH), 1650s (CONH) cm .
3-(Methyl(phenyl)amino)propanehydrazide 13a
Prepared according to General Method B. Hydrazide 13a was obtained as a colorless oil (12.7 g,
1
9
8%). H NMR (CDCl
3
) 7.94 (br s, 1H, NH), 7.20 (t, 2H, J = 7.8 Hz, 3ꢀH & 5ꢀH), 6.69 (m, 3H, 2ꢀH,
13
4
2 2 3 2
ꢀH, 6ꢀH), 3.62 (br, 4H, CH N & NH ), 2.86 (s, 3H, CH N), 2.33 (t, J = 6.8 Hz, 2H, CH CO); C
NMR (CDCl
3
) 172.5 (C=O), 148.7 (Cꢀ1), 129.4 (Cꢀ3 & Cꢀ5), 117.1 (Cꢀ4), 112.8 (Cꢀ2 & Cꢀ6), 49.2
•+
(
NCH ), 38.6 (NCH ), 31.9 (CH CO); MS (ES): m/z 194.1 (M+H) ; IR (KBr) 3300s (NH), 3050m (Ar
2
3
2
ꢀ1
CꢀH), 1650s (CONH) cm .
3-((4-Fluorophenyl)(methyl)amino)propanehydrazide 13c
Prepared according to General Method B. Hydrazide 13c was obtained as a yellow oil (2.16 g,
1
9
7%): H NMR (CDCl
3
) 7.47 (br s, 1H, NH), 6.91 (m, 2H, 3ꢀH & 5ꢀH), 6.66 (m, 2H, 2ꢀH & 6ꢀH), 3.56
(
t, J = 6.8 Hz, 2H, CH
2
N), 3.5 (br s, 2H, NH
2
), 2.83 (s, 3H, NCH
3
2
), 2.34 (t, J = 6.8 Hz, 2H, CH CO);
1
3
1
2
C NMR (CDCl
3
) 172.4 (C=O), 156.0 (d, JCF = 237 Hz, Cꢀ4), 145.6 (Cꢀ1), 115.8 (d, JCF = 22 Hz,
3
Cꢀ3 & Cꢀ5), 114.8 (d, J = 8 Hz, Cꢀ2 & Cꢀ6), 50.1 (CH N), 39.3 (CH N), 31.8 (CH CO); MS (EI):
CF
2
3
2
•+
ꢀ1
m/z 212.1 (M+H) ; IR (KBr) 3300m (NH), 3050m (Ar CꢀH), 1650s (CONH), 1525m (Ar C=C) cm .
3-((4-Chlorophenyl)(methyl)amino)propanehydrazide 13d
Prepared according to General Method B. Hydrazide 13d was obtained as white solid (4.0 g,
1
44%): H NMR (CDCl
3
) 7.15 (d, 2H, J = 8.9 Hz, 3ꢀH & 5ꢀH), 7.03 (s, 1H, NH), 6.62 (d, 2H, J = 8.9
ꢀ
25 ꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Hz, 2ꢀH & 6ꢀH), 3.86 (br s, 2H, NH
2
), 3.63 (t, J = 6.8 Hz, 2H, CH
CO); C NMR (CDCl ) 172.2 (C=O), 147.3 (Cꢀ1), 129.2 (Cꢀ3 & Cꢀ5), 122.1 (Cꢀ4),
114.0 (Cꢀ2 & Cꢀ6), 49.3 (NCH ), 38.8 (NCH
3300m (NH), 3050m (Ar CꢀH), 1650s (CONH), 1525m (Ar C=C) cm .
2 3
N), 2.88 (s, 3H, NCH ), 2.35 (t, J =
1
3
6.8 Hz, 2H, CH
2
3
•+
2
3 2
), 31.8 (CH CO); MS (ES): m/z 226.9 (M+H) ; IR (KBr)
ꢀ1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3-((4-Methoxyphenyl)(methyl)amino)propanehydrazide 13e
Prepared according to General Method B. Hydrazide 13e was obtained as an oil which
1
crystallized upon standing (4.5 g, 95%): H NMR (CDCl
3
) 7.63 (br s, 1H, NH), 6.80 (d, 2H, J = 9.2
Hz, 3ꢀH & 5ꢀH), 6.75 (d, 2H, J = 9.2 Hz, 2ꢀH & 6ꢀH), 3.72 (s, 3H, CH
3
OꢀAr), 3.47 (t, J = 6.6 Hz, 2H,
13
CH N), 3.3 (br s, 2H, NH ), 2.79 (s, 3H, NCH ), 2.33 (t, J = 6.6 Hz, 2H, CH CO); C NMR (CDCl )
2
2
3
2
3
3
172.7 (C=O), 152.9 (Cꢀ4), 143.7 (Cꢀ1), 116.5 (Cꢀ2 & Cꢀ6), 114.9 (Cꢀ3 & Cꢀ5), 55.8 (CH OꢀAr), 50.7
•+
(
CH
2
N), 40.0 (NCH
3
), 31.9 (CH
2
CO); MS (ES): m/z 224.1 (M+H) ; IR (KBr) 3300s (NH), 3050m (Ar
ꢀ1
CꢀH), 1650s (CONH) cm .
tert-Butyl-4-chlorophenyl(3-hydrazinyl-3-oxopropyl)carbamate 13f
Prepared according to General Method B. Hydrazide 13f was obtained as a colorless oil (3.5 g,
1
9
5%). H NMR (CDCl
3
) 7.76 (br s, 1H, NH), 7.22 (d, 2H, J = 7.8 Hz, 2ꢀH & 6ꢀH), 7.04 (d, 2H, J =
), 3.65 (s, br, 2H, NH ), 2.41 (t, J = 6.8 Hz, 2H,
7
.8 Hz, 3ꢀH & 5ꢀH), 3.86 (t, J = 6.8 Hz, 2H, NCH
2
2
13
CH CO), 1.41 (s, 9H, C(CH ) ); C NMR (CDCl ) 173.6 (C=O), 154.3 (NC=O), 141.8 (Cꢀ1), 131.1
2
3 3
3
(
(
Cꢀ4), 129.2 (Cꢀ2 & Cꢀ6), 114.1 (Cꢀ3 & Cꢀ5), 79.8 (C(CH
3
)
3
2 2
), 48.7 (NCH ), 34.2 (CH CO), 20.3
•+
ꢀ1
ArCH
3
); MS (ES): m/z 314.1 (M+H) ; IR (KBr) 3300s (NH), 3050m (Ar CꢀH), 1650s (CONH) cm .
3-(2-(4-Chlorophenylamino)ethyl)-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-
carboxamide 2g
General Method C: Preparation of monoꢀimidazotetrazines
ꢀ
26 ꢀ
ACS Paragon Plus Environment

Page 27 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Hydrazide 13g (1.0 g, 4.68 mmol) was dissolved in dichloromethane (20 mL). Water (20 mL) was
2
added followed by HCl (2.5 mL, 37%). The mixture was cooled in an ice/CaCl bath and a solution
of NaNO (0.39 g, 56.2 mmol, 1.2 eq) in water (10 mL) was added with strong agitation at below –5
2
°C. The ice bath was removed and dichloromethane (20 mL) was added. The reaction mixture was
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
stirred for 40 min and the organic layer was separated, dried over MgSO and evaporated to give
4
the crude azide 14g as an oil, identified by IR. The oil was diluted with toluene (100 mL) and
2
heated under reflux for 1 h under N . The volatile components were removed to give the crude
isocyanate 15g as an oil. Diethylether (150 mL) was added and the mixture heated with strong
agitation. The hot ether solution was decanted leaving an oily residue of impurities behind. The
ether layer was evaporated to leave pure isocyanate as pale yellow oil (IR νmax 2260s). The
18
isocyanate (0.1 g, 0.48 mmol) was mixed with diazoꢀIC 5 (0.07 g, 0.48 mmol, 1 eq) in dry DMSO
(
2
0.3 mL) under N at RT in the absence of light for 24 h. The reaction was quenched with water (10
mL) and the resultant solid collected by filtration and washed with copious amounts of water to
1
leave imidazotetrazine 2g as a brown solid (0.16 g, 29%): m.p. 160–161 °C; H NMR (DMSOꢀd
6
,
6
3
2
00 MHz) 8.84 (s, 1H, imidazole CH), 7.82 & 7.70 (2 × br s, 2H, CONH
2
), 7.62 (d, J = 8.8 Hz, 2H,
), 4.42 (t, J = 6.0 Hz,
2 2 6
H, CH CO), 3.3 (br s, NHꢀaniline & H O); C NMR (DMSOꢀd , 151 MHz) 161.8 (C=O amide),
ꢀH & 5ꢀH), 7.54 (d, J = 8.8 Hz, 2H, 2ꢀH & 6ꢀH), 4.52 (t, J = 6.0 Hz, 2H, NCH
2
13
140.0 (Cꢀ1), 139.4 (C=O tetrazine), 134.4 (Cq tetrazine), 132.7 (Cꢀ4), 131.8 (Cq imidazole), 130.0
(Cꢀ3 & Cꢀ5), 129.5 (CꢀH imidazole), 122.3 (Cꢀ2 & Cꢀ6), 45.9 (CH Nꢀaniline), 42.0 (CH Nꢀtetrazine);
), 1750s (C=O), 1650s (CONH ),
1600m & 1500s (ArꢀH) cm . Anal. C H N O ꢁ0.25H O, CHN.
2
2
•+
MS (ES): m/z 334.1 (M+H) ; IR (KBr) 3450m & 3300 (CONH
2
2
ꢀ1
2
0
19 13
4
2
3-(2-(Methyl(phenyl)amino)ethyl)-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-
carboxamide 2a
Prepared according to General Method C. The product was purified by flash column
chromatography (2% MeOH/CHCl
3
) and imidazotetrazine 2a was obtained as a white solid (0.34 g,
, 600 MHz) 8.77 (s, 1H, imidazole CH), 7.79 & 7,68 (2
1
2
1%): m.p. 186–187 °C. H NMR (DMSOꢀd
6
ꢀ
27 ꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
× br s, 2H, CONH
2
), 7.06 (t, J = 8.6 Hz, 2H, 3ꢀH & 5ꢀH), 6.68 (d, J = 8.6 Hz, 2H, 2ꢀH & 6ꢀH), 6.50 (t,
J = 8.6 Hz, 1H, 4ꢀH), 4.46 (t, J = 6.4 Hz, 2H, NCH ), 3.80 (t, J = 6.4 Hz, 2H, CH CO), 2.91 (s, 3H,
2
2
13
NCH
3
); C NMR (DMSOꢀd
6
, 151 MHz) 161.9 (CONH
2
), 148.9 (Cꢀ1), 139.6 (C=O tetrazine), 134.7
(
(
Cq tetrazine), 131.3 (Cq imidazole), 129.4 (Cꢀ3 & Cꢀ5), 129.2 (CꢀH imidazole), 116.6 (Cꢀ4), 112.4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
•+
Cꢀ2 & Cꢀ6), 50.3 (CH
2
Nꢀaniline), 46.4 (CH
2
Nꢀtetrazine), 38.3 (CH
3
); MS (ES): m/z 314.2 (M+H) ;
•+
ꢀ1
6
27.4 (2M+H) ; IR

2
), 1750s (C=O), 1675s (CONH
2
), 1600m (ArꢀH) cm .
15 7 2
Anal. C14H N O
3-(2-((4-Chlorophenyl)(methyl)amino)ethyl)-4-oxo-3,4-dihydroimidazo[5,1-
d][1,2,3,5]tetrazine-8-carboxamide 2d
Prepared by General Method C. The product was precipitated with water, collected by filtration
and the crude solid dried and suspended in chloroform (20 mL). The impurities were removed by
filtration and the filtrate evaporated to yield imidazotetrazine 2d as a yellow solid (0.084 g, 25%):
1
m.p. 154–155 °C H NMR (DMSOꢀd
6
, 600 MHz) 8.76 (s, 1H, imidazole CH), 7.75 & 7.65 (2 × br s,
2
H, CONH
.6 Hz, 2H, CH
) 161.9 (CONH
imidazole), 129.2 (CꢀH imidazole), 129.1 (Cꢀ3 & Cꢀ5), 120.3 (Cꢀ4), 113.9 (Cꢀ2 & Cꢀ6), 50.4 (CH
2
), 7.07 (d, J = 8.8 Hz, 2H, 3ꢀH & 5ꢀH), 6.66 (d, J = 8.8 Hz, 2H, 2ꢀH & 6ꢀH), 4.43 (t, J =
13
6
2
N), 3.77 (t, J = 6.6 Hz, 2H, CH
2 3
CO), 2.87 (s, 3H, NCH ); C NMR (151 MHz,
DMSOꢀd
6
2
), 147.8 (Cꢀ1), 139.6 (C=O tetrazine), 134.7 (Cq tetrazine), 131.4 (Cq
2
Nꢀ
•+
•+
aniline), 46.3 (CH
2
Nꢀtetrazine), 38.5 (CH
3
); MS (ES): m/z 348.2 (M+H) ; 695.3 (2M+H) ; 717.3
•+
ꢀ1
(2M+Na) ; IR (KBr) 3450m (CONH
2
), 1750s (C=O), 1675s (CONH
2
), 1600m & 1500s (ArꢀH) cm .
Anal. C H ClN O ꢁ0.5 H O, CHN.
1
4
14
7
2
2
tert-Butyl 2-(8-carbamoyl-4-oxoimidazo[5,1-d][1,2,3,5]tetrazin-3(4H)-yl)ethyl(4-
chlorophenyl) carbamate 2f
Prepared according to General Method C. The product was purified by flash column
3
chromatography (10% AcOH/CHCl ) and imidazotetrazine 2f was obtained as a white solid (0.15 g,
1
51%): m.p. 177–178 °C; H NMR (DMSOꢀd , 600 MHz) 8.91 (s, 1H, imidazole CH), 7.83 & 7.70 (2
6
ꢀ
28 ꢀ
ACS Paragon Plus Environment

Page 29 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
× br s, 2H, CONH
2
), 7.40 (d, J = 8.8 Hz, 2H, 3ꢀH & 5ꢀH), 7.32 (d, J = 8.8 Hz, 2H, 2ꢀH & 6ꢀH), 4.40
13
(
t, J = 6.7 Hz, 2H, NCH ), 4.04 (t, J = 6.5 Hz, 2H, CH CO), 1.11 (s, 9H, C(CH ) ); C NMR (DMSOꢀ
2 2 3 3
6 2
d , 151 MHz) 161.8 (CONH ), 154.3 (N(CO)O), 140.8 (Cꢀ1), 139.6 (CO tetrazine), 134.6 (Cq
tetrazine), 131.4 (Cq imidazole), 131.0 (Cꢀ4), 129.4 (CꢀH imidazole), 129.2 & 129.1 (Cꢀ3 & Cꢀ5,Cꢀ2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
&
Cꢀ6), 80.74 (C(CH
3
)
3
), 48.3 & 48.0 (CH
2
Nꢀtetrazine & CH
2
Nꢀaniline), 28.0 (CH
3
); MS (ES): m/z
•+
•+
434.3 (M+H) ; 456.2 (M + Na) ; IR (KBr) 3450m (CONH
2
), 3150m (CONH ), 1750s (C=O), 1700s
2
ꢀ1
1
(
CONH
2
), 1600m & 1500s (ArꢀH) cm . Anal. C18
H20ClN
7
4
O ꢁ /
3
H
2
O, CHN.
3-(2-(Methyl(4-methyl-3-nitrophenyl)amino)ethyl)-4-oxo-3,4-dihydroimidazo[5,1-
d][1,2,3,5]tetrazine-8-carboxamide 2h
Prepared according to General Method C from hydrazide 13b. On completion of the reaction, the
mixture was quenched with propanꢀ2ꢀol (10 mL) and the solid collected by filtration and washed
with propanꢀ2ꢀol then methanol to leave imidazotetrazine 2h as an orange solid (0.43 g, 24%): m.p.
1
133–134 °C. H NMR (DMSOꢀd
6
, 600 MHz) 8.76 (s, 1H, imidazole CH), 7.77 & 7,66 (2 × br s, 2H,
2
CONH ), 7.38 (d, J = 2.0 Hz, 1H, 2ꢀH), 7.20 (d, J = 8.4 Hz, 1H, 5ꢀH), 7.14 (br d, J = 8.4 Hz, 1H, 6ꢀ
H), 4.44 (t, J = 6.2 Hz, 2H, CH Nꢀaniline), 3.45 (t, J = 6.2 Hz, 2H, CH Nꢀtetrazine), 2.75 (s, 3H,
2
2
13
NCH
3
), 2.12 (s, 3H, ArCH
3
); C NMR (DMSOꢀd
6
2
, 151 MHz) 161.9 (CONH ), 143.0 (Cꢀ3), 142.5 (Cꢀ
1), 139.4 (C=O tetrazine), 134.6 (Cq tetrazine), 134.3 (Cꢀ2), 131.4 (Cq imidazole), 131.3 (Cꢀ4),
129.2 (CꢀH imidazole), 125.3 (Cꢀ6), 121.8 (Cꢀ5), 53.4 (CH Nꢀaniline), 46.4 (CH Nꢀtetrazine), 40.7
2
2
•+
•+
(CH
3
N), 20.0 (ArCH
3
); MS (ES): m/z 373.2 (M+H) ; 395.2 (M + Na) . Anal. C15
H
16
N
8
O
4
ꢁ0.15 H
2
O,
CHN.
3
-(2-((4-Methoxy-2-nitrophenyl)(methyl)amino)ethyl)-4-oxo-3,4-dihydroimidazo[5,1-
d][1,2,3,5]tetrazine-8-carboxamide 2i
Prepared according to General Method C from 13e. The product was purified by flash column
chromatography (10% AcOH/CHCl ). The solid was washed with 50% MeOH/H O and
imidazotetrazine 2i was obtained as a purplish red solid (0.21 g, 26%): m.p. 166–168 °C. H NMR
3
2
1
ꢀ
29 ꢀ
ACS Paragon Plus Environment