Discovery of Small-Molecule Cyclic GMP-AMP Synthase Inhibitors

Article abstract of DOI:10.1021/acs.joc.9b02666

Cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) (cGAS), a cytosolic DNA sensor, plays an important role in the type I interferon response. DNA from either invading microbes or self-origin triggers the enzymatic activity of cGAS. Aberrant activation of cGAS is associated with various autoimmune disorders. Only one selective probe exists for inhibiting cGAS in cells, while others are limited by their poor cellular activity or specificity, which underscores the urgency for discovering new cGAS inhibitors. Here, we describe the development of new small-molecule human cGAS (hcGAS) inhibitors (80 compounds synthesized) with high binding affinity in vitro and cellular activity. Our studies show CU-32 and CU-76 selectively inhibit the DNA pathway in human cells but have no effect on the RIG-I-MAVS or Toll-like receptor pathways. CU-32 and CU-76 represent a new class of hcGAS inhibitors with activity in cells and provide a new chemical scaffold for designing probes to study cGAS function and development of autoimmune therapeutics.

Full text of DOI:10.1021/acs.joc.9b02666

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Article
Discovery of Small Molecule Cyclic GMP-AMP Synthase Inhibitors
Rosaura Padilla-Salinas, Lijun Sun, Rachel Anderson, Xikang
Yang, Shuting Zhang, Zhijian J. Chen, and Hang Hubert Yin
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b02666 • Publication Date (Web): 12 Dec 2019
Downloaded from pubs.acs.org on December 12, 2019
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Discovery of Small Molecule Cyclic GMP-AMP Synthase Inhib-
itors
Rosaura Padilla-Salinas1, Lijun Sun2, Rachel Anderson1, Xikang Yang3, Shuting Zhang3, Zhijian J. Chen2, *, and
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Hang H. Yin1,3, 4, *
1Department of Chemistry and Biochemistry and Biofrontiers Institute, University of Colorado Boulder, Boulder, CO 80309-USA
2Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, 75390-9148, US; b Howard Hughes
Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148; c Department of Immunology, Univer-
sity of Texas Southwestern Medical Center, Dallas, TX, 75390-9148; d Animal Resource Center, University of Texas Southwestern
Medical Center, Dallas, TX, 75390-9 148
3School of Pharmaceutical Sciences, Tsinghua-Peking University Center of Life Science, Tsinghua University, Beijing 100082, China
4Lead contact
*Correspondence: yin_hang@tsinghua.edu.cn (H. H. Y.) and zhijian.chen@utsouthwestern.edu (Z. J. C.)
Abstract
Cyclic GMP-AMP (cGAS), a cytosolic DNA sensor, plays an important role in the type-I-interferon response. DNA from either invading mi-
crobes or self-origin triggers the enzymatic activity of cGAS. Aberrant activation of cGAS is associated with various autoimmune disorders.
Only one selective probe exists for inhibiting cGAS in cells while others are limited by their poor cellular activity or specificity, which under-
scores the urgency for discovering new cGAS inhibitors. Here, we describe the development of new small molecule human cGAS (hcGAS)
inhibitors (80 compounds synthesized) with high binding affinity in vitro and cellular activity. Our studies show CU-32 and CU-76, selectively
inhibit the DNA pathway in human cells but had no effect on RIG-I-MAVS or Toll-like Receptor pathways. CU-32 and CU-76 represent a new
class of hcGAS inhibitors with activity in cells and provide a new chemical scaffold for designing probes to study cGAS function and develop-
ment of autoimmune therapeutics.
cGAS-STING signaling, inflammation, type-I interferon, autoimmune, cyclic GMP-AMP synthase, inhibitor
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INTRODUCTION
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In the cytosol, microbial RNA with specific features can be recognized by Retinoic acid-Inducible Gene I (RIG-I) or Melanoma Differentiation-
Associated protein 5 (Mda5), which activate Mitochondrial Anti-Viral Signaling protein (MAVS). As an adaptor protein, MAVS recruits and acti-
vates Tank-Binding Kinase 1 (TBK1) complex and Inhibitor of kB Kinase (IKK) complex. On the other hand, cytosolic DNA is detected by the
cyclic GMP-AMP synthase (cGAS), a primary DNA sensor that belongs to the nucleotidyltransferase enzyme family.1, 2, 3 Upon dsDNA recognition,
cGAS catalyzes the formation of cyclic dinucleotide, cyclic GMP-AMP (cGAMP or c[G(2’,5’)pA(3’,5’)p]). As a second messenger, cGAMP binds
to the adaptor protein stimulator of interferon genes (STING) and triggers its cellular trafficking and activation of TBK1 and IKK complexes. In both
RIG-I/MAVS and cGAS/STING pathways, TBK1 and IKK activate the transcription factors Interferon regulatory factor 3 (IRF3) and Nuclear Factor
kappa-light-chain-enhancer of activated B cells (NF-kB), which are essential for induction of type I interferons and other inflammatory cytokines.
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Unlike many other nucleic acid sensors, cGAS does not distinguish self- from non-self-DNA, therefore aberrant accumulation of self-DNA in the
cytoplasm can induce unwanted immune response.2 Normal cells deploy multiple DNases including Trex1 to keep cytoplasm clear of DNA. In the
absence of functional Trex1, endogenous DNA can activate cGAS-STING pathway.4 Recent studies in mice and humans have shown that dysregu-
lation of the cGAS-STING pathway leads to uncontrolled chronic inflammation and autoimmunity.3, 4, 5 For example, multiple mutations in Trex1
gene are associated with Aicardi-Goutiés syndrome (AGS). Mice deficient in the TREX1-gene develop lupus-like syndrome and die prematurely
from multi-organ inflammation.6, 7 Gain-of-function mutations within STING in human patients are linked to early onset STING-associated vascu-
lopathy, an autoinflammatory disease.8 Lastly, cGAS-STING signaling has also been shown to promote cancer growth and metastasis through mod-
ulation of the tumor microenvironment.9
Crystal structures of the cGAS dimer bound to dsDNA have provided valuable insight into the activation mechanism of cGAS. However, in order
to interrogate the physiological function of cGAS in a cellular context, chemical tools that selectively modulate cGAS enzymatic activity would be
of great value. The central role of cGAS-STING pathway in inflammation, autoimmunity, cancer, and tumor progression has spurred intensive
investigations toward the identification and characterization of small molecule inhibitors for cGAS, including RU.521, PF-06928215, suramin, and
X6. Although these modulators all have demonstrated antagonistic effects on cGAS, the inhibitors are also associated with drawbacks that may limit
their utility as cellular chemical tools. The compound RU.521 is a potent small molecule inhibitor of murine cGAS (mcGAS), and PF-06928215
only inhibits human cGAS (hcGAS) but lacks cellular activity. Suramin, an approved drug, and amino acridine, X6 were also identified as viable
cGAS inhibitors.10-11 Unlike RU.521 and PF-06928215, suramin and X6 inhibit hcGAS and mcGAS, respectively, through the displacement of DNA
from cGAS.12,13 Although suramin is active in human cells, the inhibitor suffers from off-target effects through inhibition of the Toll-like receptor
(TLR) 3 dsRNA sensing pathway. This year, a new post translational modification for regulation of cGAS was discovered and led to reprofiling of
aspirin as a potential cGAS inhibitor in humans (vide infra).14 Indeed, only few small molecule cGAS inhibitors exist, highlighting the urgent need
to discover new chemical scaffolds that can selectively inhibit hcGAS in cells.
Inspired by the crystal structure studies describing the activation loop and the mutagenesis experiments demonstrating the essential role of the
two DNA binding surfaces and the protein-protein interface (PPI) of cGAS for IRF3 activation and IFN-b induction, we decided to take an innovative
rational approach to target the PPI of hcGAS as a means to inhibit its enzymatic activity.15 In this manuscript, we describe the successful identification
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of a new chemical scaffold and the development of a new small molecule inhibitor targeting PPI of hcGAS with in vitro and cellular activity in
human monocyte THP-1 cells.
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RESULTS and DISCUSSION
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Strategy for discovering a small molecule hcGAS inhibitor. Crystallographic studies of the 2:2 mcGAS complex with DNA revealed several
key residues involved in the PPI and cGAS-DNA interaction for mcGAS and are shown in Figure 1A.15 Notably, point mutations studies demonstrated
that Lys335 (Lys347 in human cGAS) is essential and involved in mediating the formation of the cGAS dimer. cGAS activity was also abolished in
cGAS mutants with point mutations of both Lys335 and Lys382 (Lys 394 in humans), demonstrating their critical role for cGAS function. Further-
more, a group demonstrated hcGAS can be inhibited by aspirin mediated acetylation of either Lys384, Lys394, or Lys494 in patient cells.14 These
results highlighted a potential druggable pocket and motivated us to develop a new approach directly targeting the PPI of cGAS using a virtual screen
with a grid-ligand binding box that incorporated Lys335 and Lys382, See Figure 1B. Importantly, these recent findings also provide additional
evidence for targeting residues, in particular Lys384, Lys394, or Lys494, involved in DNA binding to cGAS.
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Figure 1. Residues mediating important cGAS-cGAS and cGAS-DNA interactions and the design of an unprecedented strategy for inhibiting hcGAS.
(A) Schematic of mcGAS and dsDNA interaction surfaces. The residues highlighted in red are involved in the dimer interface of cGAS and mediate
cGAS-DNA interactions. (B) Close-up view of the grid generated on the PPI of mcGAS. For the virtual high throughput screen of the Maybridge
and Enamine drug databases, the grid box was generated to target the PPI of mcGAS with incorporation of important residues mediating the PPI
interactions.
Virtual screen results and discovery of a small molecule hcGAS inhibitor. Using the strategy described above, we performed a high throughput
virtual screen (HTVS) of drug-like libraries against the cGAS/dsDNA complex to identify novel small molecule hcGAS inhibitor. An in-silico screen
of the Maybridge (53,000 compounds) and Enamine (1.7 million compounds) Hit finder libraries using the Glide 5.6 program was conducted using
reported co-crystal structures of recombinant mcGAS, since hcGAS-DNA complex was unknown until recently.16 Based on the findings described
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above, we generated the grid on the PPI with incorporation of the residues involved in the dsDNA binding site (crystal structure PDB ID: 4O6A),
see Figure 1B.17,18
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We identified ten small molecules hits from the in-silico screen, see Supplemental Figure S1 and Table S1. The selection of the candidate mole-
cules was based on four criteria: (1) predicted binding energy and spatial complementarity; (2) reasonable chemical structures found in the dsDNA-
binding site of cGAS; (3) existence of at least one hydrogen bond between the ligand and one of the dsDNA-recognizing residues on the cGAS
surface; (4) drug-like properties analysis. Drug-like properties considered in this study follow Lipinski’s rule of five and include properties such as
molecular weight, hydrogen bond or, hydrogen bond acceptor, Lipophilicity (log P), and human oral absorption. From the initial hits validated, only
one hit inhibited hcGAS, Z918, at 100 µM in the in vitro activity assay, see Supplemental Figure S1.19 Notably, Z918 was the only hit with a
heterocyclic core similar to PF-06928215. Preliminary in vitro data for Z918 displayed sufficient inhibitory activity against hcGAS to advance
potency optimization. Additionally, all efforts have only focused on targeting the catalytic pocket of cGAS or the dsDNA/cGAS protein-nucleic acid
interface through the displacement of dsDNA. In contrast, our innovative approach targets the PPI of hcGAS and provides an opportunity to possibly
overcome the selectivity shortcomings of current cGAS inhibitors via the discovery of a new scaffold and a druggable binding pocket.
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Synthesis overview of the designed hcGAS inhibitor 1 and related molecules. To rapidly discover a novel potent hcGAS inhibitor, we used
synthetic routes amenable to high throughput synthesis and designed versatile intermediates that could be diversified to different classes of target
compounds. Methyl 4-amino-6-[(4-fluorophenyl)amino]-1,3,5-triazine-2-carboxylate (1) was synthesized using a reported one-pot cyclisation con-
densation reaction of the N'-(azaniumylmethanimidoyl)-N-(4-fluorophenyl)guanidine chloride with dimethyl oxalate See Figure 1A.18 This strategy
was robust for the synthesis of several 4-amino-6-(arylamino)-1,3,5-triazine-2-carboxylate derivatives with various substitutions of different size and
electron withdrawing (EWG)/donating (EDG) capabilities on the N2-phenyl ring. Reduction of compound 1 with lithium aluminum hydride (LiAlH4)
afforded 2a in 30–78% yield, See Figure 1A. See Supplemental Experimental Procedures and Scheme S1–S8 for the synthesis of 7–15 compounds.
Rapid diversification of the 6-position of the 1,3,5-triazine core was achieved using temperature controlled nucleophilic aromatic substitution (SNAr)
of the -Cl atoms on commercially available cyanuric chloride, see Scheme 1B. This synthetic route was appealing because it was a scalable route
that provided access to multiple symmetrical and unsymmetrical substituted analogs in 3 steps. Based on the SAR results described below we only
pursued unsymmetrical 2,4,6-substitued triazine analogs with a focus on analogs bearing a 2º aniline and -NH2 group at the 2- and 4-positions
respectively. Intermediates 4 and 7 were synthesized from commercially available cyanuric chloride (3) and 2-azido-4,6-dichloro-1,3-5-triazine (6)
via SNAr with varying nucleophiles in moderate yields, see Scheme 1B. These compounds were then further functionalized with different commer-
cially available building blocks for the synthesis 27–33 targets, see Supplemental Experimental Procedures and Supplemental Scheme S11–S12.
Overall, the modular synthetic approaches allowed us to diversify the core scaffold, culminating in the synthesis of 80 distinct novel analogues and
the discovery of two new leads and an alternative scaffold displaying similar activity compared to top inhibitors, see SAR discussion of bioactivity
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results below and Supplemental Experimental Procedures, Supplemental Scheme S9–S13 for additional syntheses, and Supplemental Table S3–S5
for additional SAR.
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9
A
O
H
N
H
N
1. NaOMe, MeOH
2. Dimethyl
oxalate
N
N
MeO
HO
F
LiAlH4
NH
NH
N
N
N
N
• HCl
NH2
F
F
THF
3, h rt
N
H
N
H
MeOH, reflux
NH2
NH2
51%
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30–78%
1
2a
3 M HCl
90 ºC
49%
NH
F
N
+
NH2
N
H
NH2
B
NH-Heterocycle
Z
H
N
X
R2
Base
N
N
N
R2
=
N
DMSO
120ºC
29–46%
N
N
I
28
31, X = C = Z = N
32, X = Z = H
33, X = N, Z = C
NH2
O
a) aniline
Na2CO3
H
N
H
H
N
N
Cl
Cl
N
NC
N
N
N
Cl
BF3OEt2
MeO
NaCN
N
N
N
N
N
N
N
N
CH2Cl2 0ºC–rt
87%
MeOH
reflux
DMSO
120ºC
47–70%
F
F
F
R1
R1
R1
b) HNR1R2
47–66%
Cl
23–45%
8, X = F, R1 = -NMe2
3
4
5
64, X = F, R1 = -NMe2
66, X = F, R1 = -NHMe
62, X = F, R1 = Cl
9, X = F, R1 = -NHMe
63, X = F, R1 = -NMe2
65, X = F, R1 = -NHMe
N
N
(a) K2CO3
Acetone
40ºC, 83%
H
H
N
N3
N
N
N
N
N3
N
Cl
R1
CuI
N
N
N
N
N
N
I
I
(b) NH4OH,
THF, reflux
59%
DMSO
5–31%
R3
NH2
NH2
Cl
6
7
F
43
29, R3 = -NH2
42, R3 = -SO2NH2
Scheme 1. Synthesis of 1, 2a and related compounds. (A) Schematic representation of the one-step condensation cyclisation reaction for the synthesis
of 4-amino-6-(arylamino)-1,3,5-triazine-2-carboxylate derivatives. (B) Schematic representation of select 6-subsituted 4-amino-6-(arylamino)-1,3,5-
triazine derivatives. (C) Synthetic routes for targets are summarized in the supplemental information (see Supplemental Experimental Procedures
and Supplemental Scheme S1–S14.)
Discovery of a small molecule inhibitor targeting hcGAS. Initially thirteen analogs were designed to identify key structural motifs contributing
to the active pharmacophore and structure activity relationship (SAR) trends to improve the bioactivity of the putative lead, Z918. The ester func-
tionality was identified as a metabolically unstable group as it can be hydrolyzed by an esterase. To test this concept, we first synthesized the primary
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alcohol, 2a, the hydrolysis product of Z918 and –OMe (2b) as the H-bonding control for 2a. A 20-fold increase in potency compared to Z918 was
observed with 2a in vitro, Figure 2. We hypothesized the lower in vitro activity of Z918 could be due to poor binding caused by the steric bulk of
the large hydrophilic anthranilic acid motif. The amine (2c) and ester (1) functional groups at the 6-position were also examined. Compound 1 with
the ester functional group was identified as the most active inhibitor, and Figure 2.
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2
3
4
5
6
7
8
9
A
B
key disconnection
O
H
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O
1
N
N
H
N
O
6
2
X
Z918
IC50 ~ 100 µM
MeO
N
N
5
3
F
NH2
F
4
F
NH2
hydrophilic
hydrophobic
NH2
> 20 fold
improvement
11 X = C IC50 = >1000 µM
12 X = N IC50 = >1000 µM
O
H
N
H
N
N
N
R
MeO
Me
N
N
N
N
F
F
R
N
N
NH2
NH2
N
N
2a R = -OH, IC50 = 4.3±12 µM
2b R = -OMe, weak
2c R = -NH2, IC50 = 2.3 ±1.1 µM
1
F
IC50 = 1.9±0.21 µM
NH2
13 R = -CO2Me, IC50 = 278±91 µM
14 R = -CH2OH, IC50 = 502±637 µM
O
O
H
H
N
N
X
R
N
R
MeO
N
N
N
N
Substitutions
resulting in
0% inhibition
F
F
NH2
8 (CU-45) X = N, R = -NMe2
9 X = N, R = -NHMe
10 X = C, R = -H, IC50 > 100 µM
15 R= -NH2CH2Ph, IC50 = 3.4±0.35 µM
16 R= -NH2, IC50 = 2.3±1.4 µM
Figure 2. Preliminary SAR studies for hcGAS inhibitors. (A) Targets synthesized to identify key protein binding structural motifs. (B) Structures of
additional derivatives designed to probe inhibitor interactions.
We then focused on systematically altering the 4-amino group and the heterocyclic core, see Figure 2A and 2B. Interestingly pyrimidine 10,
lacking the 4-NH2 group was active at 100 µM, implying a critical role for the 4-NH2 group. Replacement of the 1,3,5-triazine core with a benzene
ring (11) abrogated the bioactivity, indicating an electron deficient heterocyclic core is necessary. We ruled out 4-aminopyridine 12 (>1000 µM) and
pyrimidine 10 (>100 µM) scaffolds because inhibition was only observed at very high concentrations. Thus, the methylated derivatives of 8 (CU-45)
and 9 were prepared to further investigate the impact of the amine substitution. Methylation of the 4-NH2 group resulted in complete loss of activity.
These results implied a functional group with a combination of H-bond donating/accepting capabilities at the 4-position is critical to the bioactivity.
We rationalized the loss in activity for 8 and 9 could be due to steric effects. Because the 4-NH2 group was key to the activity, we prepared an
additional derivative with a tertiary aniline at two position, compound (13), to systematically establish the importance of the presence of the -NH
bond. We also observed a significant decrease in inhibitory activity for compound 13 (IC50 = 278±91 µM, see Figure 2B.). These results helped us
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identify an additional position where a combination of H-bond donating/accepting capabilities is critical to the bioactivity. Compound 1 was advanced
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to chemical optimization due to its high activity and ease of synthesis.
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Improving CU-1 potency through SAR. We first prepared several 4-amino-6-(arylamino)-1,3,5-triazine-2-carboxylate derivatives with various
substitutions of different size and electron withdrawing (EWG)/donating (EDG) capabilities on the N2-phenyl ring, See Scheme 1 for synthesis.
Keeping the 2-phenylamino ring, we replaced the -F with -H (17) and -CF3 (18) and this decreased inhibitory activity indicating electronics is the
dominating factor rather than size. Therefore, we examined other halogens and introduced EWG at the para-position. Substitution of -F with -I led
to 3 orders of magnitude increase in potency with compound (19) (IC50 = 0.45 µM), Table 1. By contrast, substitution at the meta- (20) and ortho-
positions (21) with -I decreased potency demonstrating the para- position is optimal. EWG (-NO2, -CN, and -CF3) and electron donating groups (-
OH and -OMe) with H-bond donating/accepting capabilities were less active, Supplemental Table S3. Replacement of the 2-phenylamino ring with
other heteroaromatic groups such quinoline, did not improve the activity. Unlike fluorine, iodine can engage in halogen bonding, an electrostatically
driven interaction.18 To further explore this, we examined the ethynyl group, as previous investigators have shown it can be used as an iodine bi-
oisostere.21 The ethynyl group is a nonclassical bioisostere that has a polarized –CH moiety, and it is a weak hydrogen bond donor.24 For our inhibitors,
replacement of -I with the ethynyl moiety (24) did not improve the potency, which explained that halogen bonding may not be the dominating factor.18
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Table 1. SAR Analysis for in vitro inhibition of cGAS, Related to Supplemental Table S3.
O
H
N
N
MeO
Ar
N
N
NH2
Compound
Ar
Compound
Ar
IC50 (µM)
5.9 ± 1.20
14.9 ± 1.40
0.45 ± 0.04
IC50 (µM)b
8.7 ± 0.69
4.5 ± 0.33
17
18
C6H5
22
23
4-OH-C6H4
4-OMe-C6H4
4-CF3-C6H4
4-I-C6H4
19 (CU-32)
24
25
1.4 ± 0.03
1.5 ± 0.07
F
20
21
3-I-C6H4
2-I-C6H4
3.9 ± 0.16
F
F
F
13.1 ± 1.00
26 (CU-76)
I
0.24 ± 0.01
F
Ar defined as aryl group. IC50 defined as half maximal inhibitory concentration.
a In vitro IC50 derived from dose-response curve for the measurement of ATP consumption
from cGAS-mediated 2’,3’-cGAMP synthesis. b See Supplemental Figure S7 for curves used to generate the reported IC50 values.
To improve the cellular activity of CU-32, we modified the 6-position of the 1,3,5-triazine core and the 3- and 5- positions of the NH-phenylamino
motif. Replacement of the ester group with heterocycles, such as 1,3,4-oxaziazole (27), N- pyrazole (28), and aryl-1,2,3-triazoles (29 and 30) only
showed modest inhibitory activity while benzimidazole (31) and indole (32) were inactive, see Figure 3 and Supplemental Table S4 for chemical
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structures. Finally, 3,4,5-trisubstituted and 3,5-disubstituted phenylamino rings were examined to thoroughly explore additional substitutions on the
NH-phenylamino motif. We introduced two F- atoms at the 3- and 5-positions of the NH-phenylamino motif and a ~3-fold increase of inhibitory
potency with compound CU-76 (25) was achieved showing a low micromolar IC50 (0.24±0.01 µM) value. Derivatives with 3,5-disubstition only
showed modest activity suggesting tri-substitution with the I- atom is favorable, see Supplemental Table S5. 2,4,6-trisubstituted analogs were not
examined because ortho- substituents decreased potency (20). Improving the inhibitory activity to the nanomolar range is desirable and would be
beneficial for low dosing in the long-term treatment of chronic inflammation pathologies. We believe the micromolar affinities of the triazole inhib-
itors (compound 29 and 43) can be further improved to nanomolar range by focusing SAR efforts on 2-aminopenyl-1H-1,2,3-trizaole motif and
installing groups that increase solubility.
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H
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IC50 = 0.23±0.05 µM
• Benzimidazole (31), indole (32), and indazole (33) were inactive
Figure 3. SAR in vitro results for analogs bearing an NH-heterocycles at the six position, Related to Supplemental Table S4. (A) Key targets
synthesized to identify protein binding structural motifs. (B) Structure of additional derivatives designed to probe inhibitor interactions. See Supple-
mental Table S4 for chemical structure of compounds 31–33 and Supplemental Experimental Procedures.
DNA intercalation studies. Selected target compounds from the SAR studies were tested in a high throughput fluorescence polarization (FP)
assay for their capacity to intercalate DNA following the protocol developed for RU.521.9 The five compounds tested showed 0% DNA intercalation
compared to mitoxantrone, a known DNA intercalator, see Supplemental Figure S2. The biological investigation for CU-32 and CU-76 was priori-
tized based on stability, potency, and lack of DNA interaction for further testing in cellular assays.
CU-32 analogs selectively inhibit cGAS pathway in human cells. To evaluate cellular activity of selected cGAS inhibitors, we first examined
their effects on DNA-induced IRF3 dimerization, which is a hallmark of its activation. Transfection of interferon-stimulatory DNA (ISD), a 45-
basepair dsDNA that specifically triggers cGAS-STING pathway, led to strong dimerization of IRF3, which was reduced by both CU-32 and CU-
76 in a dose-dependent manner (Figure 4A, upper panel). In sharp contrast, CU-32 and CU-76 had no effect on Sendai virus-induced IRF3 activation,
which is mediated through RIG-I-MAVS pathway, indicating the specificity of these compounds (Figure 4A lower panel). To evaluate their effect
on the biological outcome of DNA pathway, we used ELISA to measure IFN-b production from these cells following ISD transfection or Sendai
virus infection. CU-32 and CU-76 suppressed levels of IFN-b in the media dose-dependently; however, IFN-b levels I response to Sendai virus were
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not affected (Figure 4B), confirming the effectiveness and specificity of these compounds. Furthermore, we also confirmed the inhibitory activity of
CU-32 and analogs was not the result of toxicity, as the top inhibitors had no effect on cell viability up to 30 µM, with only partial toxicity at 300
µM (Supplemental Figure S3).
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As part of the SAR study, the carboxylic acid derivatives of CU-1 and CU-32 were also prepared and determined to have an in vitro IC50 value
of 3.8±1.9 µM (1b) and 0.59±0.3 µM (19b), see Figure 4A. for chemical structures. The in vitro inhibitory activity of two amides were determined
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for compounds 15 at 3.4±0.35 µM and 16 at IC50 = 2.3±1.4 µM, see Figure 2B. for chemical structures. Utilizing a prodrug strategy to optimize
effectiveness and “drug like” properties, such as permeability and target selectivity, is a possibility based on the in vitro activities for the carboxylic
acid derivatives of 1b and 19b.24 However, 1b did not display antagonistic activity toward the cGAS-STING pathway IRF dimerization assay (See
Supplemental Figure S4). Thus, we cannot effectively conclude the active drug is the carboxylic acid of the corresponding methyl ester inhibitor (1)
since two amide derivatives also inhibited hcGAS in vitro and lacked cellular activity. We speculate the carboxylic acid (1b) and amides (15 and 16)
lack cellular activity due to poor permeability caused by the 4-NH2 and 2-COOH functional groups.
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Figure 4. CU-76 and CU-32 selectively inhibit the cytosolic DNA sensing, but not the RNA sensing pathway. (A) The interferon regulatory factor
3 (IRF3) dimerization assay with THP-1 cells was used for evaluating cellular inhibitory activity of CU-76 and CU-32. Inhibition of IRF3 activation
induced by ISD (upper panel) and Sendai virus (SeV, lower panel) by CU-32 and CU-76 in human monocyte THP-1 cells. Readout of cellular
dimerization is measured by Western Blotting to detect the interferon regulatory factor 3 dimer (IRF)2 dimer (0% inhibition = 100% IRF3 dimeriza-
tion). The (IRF)2 dimer was not detected at 30 and 100 µM for CU-76 and at 100 µM for CU-32 confirming the compounds effectiveness for
inhibiting the cGAS-STING pathway. At 10, 30, and 100 µM CU-76 and CU-32 did not reduce dimerization of IRF3 induced by the RIG-I-MAVs
pathways confirming the specificity of these compounds for the DNA pathway. (B) Effects of CU-76 and CU-32 treatment on the production of
interferon beta-1alpha (IFN-b) (ELISA measurements) in THP-1 cells stimulated with interferon-stimulatory DNA (ISD) (upper panel and Sendai
virus (Sev). CU-32 and CU-76 (10, 30, and 100 µM) suppressed levels of IFN-b in media for ISD stimulated THP-1 cells in a dose dependent manner
further supporting the compounds specificity for the DNA pathway. The levels of IFN-b in media were not reduced in THP-1 cells stimulated with
Sev showing CU-32 and CU-76 (10, 30, and 100 µM) do not display off-target effects for the RNA pathway.
Recent structural and molecular docking studies of the human cGAS-DNA complex defined the species-specificity observed with RU.521 and
PF-06928215 small molecule inhibitors.16 We assessed the in vitro and cellular inhibitory activity of CU-32 and CU-76 towards mcGAS. Both CU-
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32 (IC50 = 0.66±0.10 µM) and CU-76 (IC50 = 0.27±0.06 µM) inhibited the enzymatic activity of mcGAS in vitro (Figure 5A). In contrast to the effect
in THP-1 cells, CU-32 and CU-76 were much less effective to suppress DNA-dependent activation of interferon reporter in RAW cells (see Figure
5B and Supplemental Figure S5).
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Figure 5. Effect of CU-32 and CU-76 on mouse cGAS. (A) In vitro concentration-dependent inhibition of mcGAS enzymatic activity in an ATP
consumption assay by CU-32 and CU-76. The IC50 value reported represents the mean value. (B) RAW-ISG-luc cells were transfected with inter-
feron-stimulatory DNA (ISD) or infected with Sendai virus (SEV) for 16 h in the presence of serial concentrations of compounds or DMSO, followed
by measurement of ISRE reporter expression using luminescence. CU-32 and CU-76 were ineffective for suppressing the enzymatic activity of
mcGAS in RAW 264.7 cells.
CU-32 does not inhibit Toll-like receptor pathways. To further investigate the selectivity, we tested the effect of CU-32 on activation of TLR
pathways, which are membrane localized pathogen recognition receptors of the innate immune system.26 Various TLRs recognize different viral or
bacterial membrane component or nucleic acids. We used human embryonic kidney (HEK) cell lines each ectopically expressing a TLR together
with NF-κB-inducible SEAP (secreted embryonic alkaline phosphatase). Each cell line was stimulated with respective TLR ligands including
poly(I:C) for TLR3, LPS for TLR4, R848 and ssRNA27 for TLR7/8, and CpG-ODN for TLR9, in the presence of CU-32 or DMSO, and activation
of TLR signaling was evaluated by measurement of SEAP activity in the media. As shown in Supplemental Figure S6A and Figure S6B., none of
the TLR pathways was inhibited by CU-32 at concentrations up to 50 µM. This compound also had no effect on R848-induced Nitric Oxide (NO)
production in Raw cells, see Supplemental Figure S6C. These results further underline the selectivity of CU-32.
Molecular docking studies. Because X-ray crystallographic studies did not successfully generate a complex structure, we conducted molecular
docking studies to gain insight into the binding mode of CU-76. We used mcGAS-DNA (PDB ID: 4O6A) for molecular docking analysis with active
(CU-76) and inactive cGAS inhibitors (CU-45). Based on previously reported crystallographic studies, we utilized relevant key amino acids located
on the mcGAS-mcGAS binding interface and DNA binding sites (see Figure 1).30 The molecular docking analysis of CU-76 against mcGAS-DNA
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complex show the inhibitors may bind in the groove aside the Zn loop (see Figure 6A and Supplemental Figure S8. We speculate the insertion of our
molecules aside the Zn loop disturbs the interface of the dimer, thus inhibiting the dimerization through an allosteric effect (conformational change).
We also hypothesize the 4-NH2 (donor) may have an H-bond interaction with GLU386, Figure 6A. In our model, CU-45 cannot interact with GLU-
386 mainly because there is not a H (donor) on the N-atom and due to steric clash with the methyl groups, see Figure 6B. The in vitro results for CU-
45 and 9 (0% cGAS inhibition) are consistent with our hypothesis. Determining the pKa values of LYS394 and LYS347 would be useful for probing
the electrostatic environment of cGAS and provide insights on how distinct protonation states of ionizable residues impact the enzyme active site.
Ongoing efforts include determining how binding affinities of our inhibitors change the protonation states of both native LYS394 and LYS347 as
well as the corresponding acylated residues. We provide an estimation of the pKa values of LYS394 and LYS347 to be £ 10.8 since pKa measure-
ments and calculations are experimentally demanding and difficult.[31] The molecular docking studies also indicate CU-76 and analogs may bind to
different pocket compared to other cGAS inhibitors.16 Of course, further clarification of the inhibitory mechanism is necessary. Co-crystallizations
studies are needed to provide insights on the specific binding mode.
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Figure 6. Molecular docking studies for CU-45 and CU-76 cGAS inhibitors. (A) Close-up view of the binding site of CU-76 on the cGAS dimer
interface. CU-76 may potentially disrupt the interface of the mcGAS dimer by inserting aside the Zn loop. (B) Schematic representation of residues
around the molecules (left, using MOE v.2014.0901) and close-up view of the binding site on the protein interface (right, using PYMOL v2.0.7) are
shown. For CU-45, methylation of –NH2 abrogates H-bond interaction with GLU386. In the right pictures, contacts within 3 Å are shown, See
Supplemental Figure S8A and S8B and Supplemental Experimental Procedures.
To rule out disruption of the cGAS-DNA complex as an alternative inhibition mode, we performed electrophoretic mobility shift assay (EMSA)
in the presence and absence of CU-32 and CU-76.12,13 cGAS protein caused mobility shift of ISD (lane2) and reduction of the unbound DNA. This
effect was reversed by adding Quinacrine (lane 2-4), a compound known to disrupt cGAS:DNA binding32, see Supplemental Figure S9. In contrast,
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either CU-32 (lane 6-8) or CU-76 (lane9-11) had no effect on cGAS-caused shift. This result indicates CU-32 and CU-76 do not disrupt cGAS:DNA
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intercalation.
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CONCLUSION
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In conclusion, we have successfully discovered and characterized a novel small molecule inhibitor, with a distinct chemical scaffold, targeting
hcGAS with high binding affinity in vitro. Our cellular studies indicate CU-32, CU-76, and analogs selectively inhibit the DNA pathway representing
a new class of hcGAS inhibitors with cellular activity in human THP-1 cells. Using mouse and human cell lines, we determined the inhibitory activity
of CU-32 is only specific for hcGAS versus mcGAS in cells. Further studies are needed to gain insights on the cellular activity differences. Lastly,
CU-32 and CU-76 are selective for the cytosolic DNA pathway over other NA-sensing pathways such as RIG-I-MAVS and endosomal TLRs. In
general, our discovered small molecule inhibitors provide a new chemical scaffold for developing hcGAS inhibitors with potential therapeutic appli-
cations and a much-needed small molecule chemical probe for studying cGAS biology and cGAS related disorders in human cells.
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EXERIMENTAL SECTION
Virtual screening procedure. High throughput virtual screening (HTVS) was performed against the cGAS/dsDNA complex structure. The Enamine
drug database (1.3 million small molecules) and Maybridge library (50,000 small molecules) was docked into the dsDNA-binding domain of cGAS (PDB:
4O6A). Glide maestro protocol was used for the virtual screening using Schrodinger software. The grid was generated on the protein-protein interface with
incorporation of important residues involved in dsDNA binding, See Figure 1.
The protocol includes addition of hydrogens, restrained energy-minimizations of the protein structure with the Optimized Potentials for Liquid Simula-
tions-All Atom (OPLS-AA) force field, and finally setting up the Glide grids using the Protein and Ligand Preparation Module. All compounds were first docked
and ranked using High Throughput Virtual Screening Glide, continued with standard precision docking (SP) Glide for the top 10,000 compounds. To reduce
the number of compounds in the library, after performing HTVS screening, the remaining 10% was docked using the more accurate and computationally inten-
sive SP docking, after which the remaining 10% was docked using Extra-precision. The top ranked compounds were re-ranked by predicted binding energy. The
compounds were filtered by Lipkinski’s rule of five and reactive functionality. It performed docking of the drug compounds in the different phases like HTVS,
SP, XP (Extra-precision).
Selection of the candidate molecules was based on four criteria: (1) predicted binding energy and spatial complementarity; (2) reasonable chemical struc-
tures found in the dsDNA-binding site of cGAS; (3) existence of at least one hydrogen bond between the ligand and one of the dsDNA-recognizing residues on
the cGAS surface; (4) drug-like properties analysis. Drug-like properties follow Lipinski’s rule of five and include properties such as molecular weight, hydrogen
bond or, hydrogen bond acceptor, Lipophilicity (log P), and human oral absorption. Ten of these molecules were selected by chemical and geometrical proper-
ties for experimental evaluation (Table 1).
cGAS in vitro assay. The initial hits and all cGAS inhibitors described herein were evaluated using an in vitro cGAS assay. Briefly, serial dilutions of
compounds in DMSO were added to a reaction mixture containing 20mM Tris-Cl, 5mM MgCl2, 0.2mg/ml bovine serum albumin (BSA), 0.01mg/ml Herring
testis DNA (HT-DNA), 0.1mM GTP, 0.006mM ATP, and 30nM human cGAS protein, incubated at 37°C for 20min. Remaining ATP levels was measured by
adding 40µl of KinaseGlo (Promega) and reading luminescence. Reactions omitting cGAS and reactions without compounds but DMSO were considered
100% and 0% inhibition, respectively. IC50 values were deduced from non-linear fitting of [inhibitor] vs response in Prism 8.
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Fluorescence polarization assay (FP) for DNA intercalations studies. Select target compounds from structure activity relationship analysis
(SAR) studies were tested in a fluorescence polarization (FP) assay for their ability to intercalate DNA following the protocol developed for known cGAS
inhibitors.12a The assay was performed in a final volume of 30 μl in 384-solid bottom opaque plates. Ten microliters of HEN buffer (10mM HEPES pH 7.5, 1mM
EDTA pH 7.5, 100mM NaCl) were dispensed per well using an Eppendorf multichannel pipette. Compounds were dissolved in DMSO and dispensed per well
(10 μL dosing volume) using Eppendorf multichannel pipette. Ten microliters of a solution of 150 nM acridine orange in HEN buffer was dispensed per well
using Eppendorf multichannel pipette. Subsequently, 10 μL of a solution of 45-bp dsDNA at μgml−1, see Supplemental Table S5, was dispensed per well using
Eppendorf multichannel pipette. The liquid was collected at the well bottom using centrifugation for 30 s at 180 x g. Mitoxantrone, a known DNA intercalator,
was used at 50 μM as a positive control, while DMSO alone was used as negative control. FP was measured using a Synergy H2 plate reader. The data was
analyzed and plotted using Microsoft Excel.
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Secreted embryonic alkaline phosphatase (SEAP) assay. Cell Culture and SEAP Assay: Commercially available human embryonic kidney
(HEK) 293 cells expressing human Toll-like receptor (hTLR) gene and an inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene were used
to evaluate compound potency on TLR pathways. The SEAP reporter gene is fused to five NF-κB and AP-1 sites. Stimulation of the hTLR is induced with
natural ligand or small molecule chemical ligand (R848, Invivogen). This activates NF-kb and AP-1, which induces the production of SEAP protein. Growth
media for cell maintenance was prepared using DMEM media with 10% FBS, 1% L-glutamine, 1% Penicillin/Streptomycin and supplemental antibiotics (10
μg/mL blasticidin and 100 μg/mL zeocin) per manufacture’s recommendations.
Un-supplemented test media was prepared using DMEM media with 10% FBS (deactivated), 1% L-glutamine, and 1% Penicillin/Streptomycin (Note: sup-
plemental antibiotics were not added). 100,000 cells/well or 70,000 cells/well were plated in a tissue culture treated 96-well (Costar 3596) in un-supplemented
DMEM test media. Cells were then treated with appropriate concentration of compound, natural TLR ligand (5 μg/mL Poly(I:C), 20 ng LPS, 1 μg/mL R848,
1 μg/mL CpG-ODN, or ssRNA/LyoVec, (Invivogen). The cells were incubated for 18–20 hours and assayed for NF-kb signaling using a SEAP assay. Quanti-
Blue (Invivogen) medium for quantification of alkaline phosphatase was used to monitor expression of SEAP via detection of SEAP reporter protein secreted
by cells. The compounds were considered active if they decreased SEAP levels as indicated by a decrease in absorbance at 620 nm. The data was normalized as
[(raw data – untreated cells)/(ligand + solvent control – untreated cells)]. Ligand + solvent is 100% activation, and untreated cells are 0% activation. The result
of one representative biological replicate for three independent days is plotted with the error bars representing the standard deviation of three technical replicates
for one independent biological replicate. The result of one representative biological replicate for three independent days is plotted with the error bars represent-
ing the standard deviation of three technical replicates for one independent biological replicate.
Nitric oxide (NO) assay. Raw 264.7 cells were plated on day one at 375,000 cells/mL in a tissue culture treated 96-well plate. The cells were plated in
supplemented RPMI medium (10% fetal bovine serum, 1% L-glutamine, 1% Penicillin/Streptomycin) and incubated at 37 ºC. On day two, supplemented media
was removed from the cells, and the unsupplemented RPMI was added (100 μL). The cells were treated with 1 μgmL-1 R848 (90 μL) (Invivogen) and varying
concentrations of the appropriate organic compound (10 μL). The final volume in each well was 200 μL. The 96-well plate was incubated with the organic
compound for 18–24 hours at 37 ºC.
On day three, a solution of 0.05 mgmL-1 2,3-diaminonapthalene (DAN, Sigma Aldrich) in 0.62 M HCl was prepared. The 96-well plate was removed from
the incubator and 90 μL of media from each well was transferred to a black 96-well plate (ThermoScientific), respectively. Followed by the addition of
DAN/HCl solution (10 μL/well) each well with. The plate was covered with aluminum foil and shaken at room temperature for 15–20 minutes. The plate was
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quenched with 3 M aqueous NaOH (5 μL/ well). A BioTek Synergy HTX Multi-mode reader or a Beckman Coulter DTX 880 Multimode Detector were used
to quantify the results. Samples were excited at 360 nm and emission was measured at 430 nm. The data was normalized as (well raw data – untreated cells)/(lig-
and + solvent control – untreated cells) such that ligand + solvent is 100% activation, and untreated cells are 0% activation. The experiment was conducted with
a minimum of three biological replicates, in triplicate.
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The NO assay uses an aryldiazonium intermediate to convert 2,3-diaminonapthalene to fluorescent 1(H)-naphthotriazole in the presence of NO. As NO is
produced in the TLR inflammatory response, this readout provides information on the extent of TLR signaling.
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Cell culture and interferon regulatory factor (IRF)-Lucia assay. Commercially available Raw-Dual cells (Invivogen) transfected with IRF-
Luc/KI-[macrophage inflammatory protein-2 (MIP-2)]-secreted embryonic alkaline phosphatase (SEAP) reporter genes and an inducible Lucia luciferase gene
(Luc) were used to evaluate compound potency for murine macrophages. The Lucia luciferase gene is under the control of an ISG54 minimal promoter with
IFN-stimulated response elements. Stimulation of cGAS was induced with G3YSD, a cGAS agonist (Invivogen). This activates the IRF pathway, which induces
the production of the Luciferase protein. Growth media for cell maintenance was prepared using DMEM media with 10 % FBS 1% L-glutamine, 1% Penicil-
lin/Streptomycin and supplemental antibiotics (100 μgmL-1 normocin and 200 μgmL-1 zeocin) per manufacture’s recommendations to select for cGAS and IRF-
Lucia/KI-[MIP-2]SEAP reporter expression.
Un-supplemented test media was prepared using DMEM media with 10% FBS (heat deactivated), 1% L-glutamine, and 1% Penicillin/Streptomycin (Note:
supplemental antibiotics were not added). 100,000 cells/well were plated in a tissue culture treated 96-well (Costar 3596) in un-supplemented DMEM test
media. Cells were then treated with appropriate concentration of compound, and 1 μgmL-1 G3YSD ligand. The cells were incubated for 18–20 hours and assayed
for IRF signaling using a Lucia luciferase assay. Quanit-Luc (Invivogen) medium for quantification of luciferase was used to monitor the expression of luciferase
via detection of Lucia luciferase reporter protein secreted by cells. The compounds were considered active if they decreased luciferase levels as indicated by a
decrease in luminescence relative light units (RLU). The data was normalized with 100% untreated cells as the negative control and 100% cells treated with
cGAS ligand (1 μgmL-1 G3YSD) as the positive control. All data for cell-based assays is represented as the average and standard deviation of three biological
replicates, unless otherwise noted.
Molecular docking calculations for cGAS inhibitors. The crystal structure of murine cGAS (mcGAS) –DNA(2:2) complex (PDB: 4O6A) was
used. Similar to the approach described above for the HTVS, we processed 4O6A prior to docking and only the monomer mcGAS was retained. The DNA and
water molecules were removed. The compounds were first optimized using the GaussView v.5.0.9 and Gaussion v.9.5(Method: b3lyp, Basic set: 6-31+g(d,p),
pseudo potential for I: sdd). We prepared the ligands and protein receptor with AutoDockTools-1.5.6 (added hydrogens and gasteiger charges, set rotatable
bonds for ligands etc.). The GridBox was generated at the cGAS-cGAS interface, Zn loop (K382 E386) and α7 helix(K335) involved (Supplemental Figure
S8A.). The docking parameters were all set to default (Number of Genetic Algorithm Runs: 50). In the first-round of docking, the protein structure was set to
be rigid. We then selected one reasonable candidate pose from the results based on the predicted binding energy and spatial complementarity. To further im-
prove docking accuracy, redocking was conducted with select residues (Lys335, Lys382, Glu386) in the GridBox being flexible. Using the docking results, CU-
76 was overlapped with mcGAS–DNA(2:2) complex together (Supplemental Figure S8B.).
Quantification and statistical analysis. Statistical differences were performed using one-way ANOVA with the Turkey method for comparisons of
experimental group against the control group. All statistical analysis was performed using OriginPro, and a P value of < 0.05 was considered statistically signifi-
cant.
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General Chemical Synthesis Considerations
Unless otherwise noted, all non-aqueous reactions were run under an atmosphere of dried nitrogen in dried glassware. All reagents were reagent grade and
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used without further purification. Moisture sensitive reagents were added via syringe. All chemicals were obtained from Sigma-Aldrich, Acros, or Strem unless
otherwise noted. Flash column chromatography was performed using EM Reagents Silica Gel 60 (230-400). Analytical thin-layer chromatography (TLC) was
performed using EM Reagents 0.25 mm silica gel 254-F plates. Visualization was accopmlished with UV light, p-anisaldehyde stain, and/or iodine.
1H NMR and 13C NMR spectra were recorded on Brüker 400 MHz Fourier transform NMR spectrometer. Chemical shifts are reported relative to the
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solvent resonance peak δ 7.27 (CDCl3), 3.31 (CD3OD), and 2.51 (DMSO-d6) for H and δ 77.23 (CDCl3), 49.15 (CD3OD), and 39.51 (DMSO-d6) for 13C
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NMR. The 13C NMR spectra were recorded at 101 MHz in CDCl3, CD3OD, or DMSO-d6 as the internal standards, respectively. Data are reported as follows:
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, bs = broad singlet, m = multiplet), coupling constants, and number of protons. Mass
spectrometry was performed at the mass-spectrometry facility of the Biofrontiers Institute at the University of Colorado Boulder. High resolution mass spectra
were obtained using a Waters Synapt G2 QToF HR-MS using an ESI ionization mode. Infrared spectra are reported in cm-1 and recorded using a Agilent Cary
630 FT/IR instrument and opitcal rotations were measured on JASCO P-1030 and are reported as an average of data points. The compounds were purified
using flash column chromatography (ACI systems, Biotage). Unless otherwise noted, all yields refer to isolated yields, and product purity was determined by 1H
NMR spectroscopy and Agilent HPLC 1200 series instrument with an Eclipse XDB-C18 column. All samples tested were > 95% pure based on HPLC and/or
1H NMR analysis.
Note: Characterization and spectral data for the intermediates used for the synthesis of inhibitors are described after characterization data for the inhibitors
described in the main text.
Numbering System
The numbering system described below has been used in the Supplementary Information and article for NMR assignments and discussion of 1,3,5-triazine
derivatives.
General Procedure B: Preparation of arylbiguanide hydrochloride salt:19, 27, 28
Dicyandiamide (0.1 mmol) and the correspoding aniline (0.1 mmol) were weighed into a microwave vial followed by the addition of 3 M HCl. The vial
was sealed with a microwave cap and stirred at rt for 24-36 hours. The arylbiguanide hydrochloride salt was filtered and washed with cold H2O (3 x 5 mL). The
arylbuiguanide hydrochloride salts were used without further purification or were purified by trituration with Et2O or MeOH. All salts were characterized by
1
HRMS and/or H NMR. Note: For previously synthesized arylbiguanide hydrochloride salts, only the characterization data for the corresponding 4-amino-
1,3,5-carboxylates is provided. Characterizaiton data for all new arylbiguanide hydrochloride salts and the corresponding 4-amino-1,3,5-carboxylates is included
herein.
General Procedure C: Preparation of arylbiguanide free base:20
The arylbiguanide salt (1.0 mmol) in anhydrous ethanol or methanol (4.4 M) was added to a mixture of sodium ethoxide (1.2 mmol) in anhydrous ethanol
(0.34 M). After stirring the solution for 3 h at rt, the mixture was filtered through a pad of celite. The filtrate was concentrated by rotary evaporation. The residue
was dissolved in hot ethanol and filtered through a pad of celite. The filtrate was concentrated by rotary evaporation to afford the desired arylbiguanide base and
was used without further purification.
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General Procedure D: Preparation of 4-amino-1,3,5-carboxylates:20
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Dimethyl oxalate (3.0 mmol) was added to the arylbiguanide base in anhydrous MeOH (0.27 M). After stirring the solution at 35 ºC for 1 h, the solution
was heated to reflux and stirred overnight. The reaction mixture was cooled to rt and allowed to stand for 3 h. The crystals were collected by filtration, washed
with cold methanol, and dried under vacuum. The solid was recrystallized with hot methanol (or isopropanol) or chromatographed to afford the product as
crystalline solids.
Methyl 4-amino-6-((4-fluorophenyl)amino)-1,3,5-triazine-2-carboxylate (1)20: Following the general free base procedure C, a mixture of 1-carbamim-
idamido-N-(4-fluorophenyl)methanimidamide hydrochloride (400 mg, 1.73 mmol) and NaOEt (118 mg, 1.73 mmol) was stirred in EtOH (0.34 M) at rt for 3
h. Following the general procedure D, a mixture of dimethyloxalate (538 mg, 5.19 mmol) and the arylbiguanide base in anhydrous MeOH (0.27 M) was stirred
at 25 ºC for 3 h and then refluxed overnight. The mixture was cooled to rt and the precipitate was collected by filtration. The solid was recrystallized with
isopropanol and stored at -20 ºC for 5 d to afford a white solid (231 mg, 97% pure) in 51% yield: m.p. = 226–227 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 10.5
(br, 1H), 7.78 (m, 2H), 7.62 (m, 1H), 7.49 (m, 1H), 7.13 (t, J = 8.9 Hz, 2H), 3.82 (s, 3H,); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.2, 164.2, 164.0, 163.9,
158.7 (d, 1JCF = 239.2 Hz), 135.8 (d, 4JCF = 2.5 Hz), 121.7 (d, 3JCF = 3.2 Hz), 115.1 (d, 2JCF = 22.0 Hz), 52.5; 19F NMR (DMSO-d6, 365 MHz) δ -121.0; IR (film)
v 3238 3096, 1748, 1644, 1212, 787; HRMS (ESI-TOF) M/Z: m/z: [M+H]+ Calcd for C11H11FN5O2 264.0897; Found 264.0894.
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General procedure A for lithium aluminum hydride (LiAlH4) reduction:
(4-Amino-6-((4-fluorophenyl)amino)-1,3,5-triazin-2-yl)methanol (2a): To a solution of methyl 4-amino-6-((4-fluorophenyl)amino)-1,3,5-triazine-2-
carboxylate (250 mg, 0.950 mmol) in anhydrous THF (2.0 mL) was added dropwise to a slurry of LiAlH4 in anhydrous THF (9.5 mL) under a N2 atmosphere.
After stirring the mixture at rt for 3 h, the reaction was quenched utilizing the Fieser protocol. The mixture was diluted with diethyl ether and cooled to 0 ºC.
Water (0.250 mL) was slowly added to the mixture and allowed to stir for 5 minutes. Next, 15% aqueous NaOH (0.250 mL) was added followed by water ( 3 x
0.75 mL) and then stirred for 15 min. Sodium sulfate was added and the mixture was stirred for another 15 min, filtered, and concentrated by rotary evaporation
to afford a white solid (4%, 97% pure; Note: LiBH4 and NaBH4 can also be used, 15 and 63-77% yield respectively): m.p. = 223-224 ºC; 1H NMR (DMSO-d6,
400 MHz) δ 9.59 (br, 1H), 7.79 (dd, J = 10.5, 5.0 Hz, 2H), 7.10 (J = 8.9 Hz, 2H) 7.10 (br s, 2H), 4.86 (t, J = 6.1 Hz, 1H), 4.21 (d, J = 6.1 Hz, 2H); 13C{1H }
NMR (DMSO-d6, 101 MHz) δ 177.0, 166.6, 164.0, 157.4 (d, 4JCF = 238.6 Hz), 136.3 (3JCF = 2.6 Hz), 121.4 (2JCF = 7.6 Hz), 114.9 (1JCF = 238 Hz), 63.8; IR (film)
v 3499, 3324, 3134, 1670, 1555, 1506, 1212, 1093, 825; 19F NMR (DMSO-d6, 365 MHz) δ -121.8; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C10H11FN5O,
236.0944; Found 236.0948.
N2-(4-Fluorophenyl)-6-(methoxymethyl)-1,3,5-triazine-2,4-diamine (2b): The general procedure was followed using 1-carbamimidamido-N-(4-
fluorophenyl)methanimidamide hydrochloride (1.83 g, 7.9 mmol), NaOMe (513 mg, 9.5 mmol), and in anhydrous MeOH (23 mL). The corresponding ar-
ylbiguanide base, ethylmethoxyacetate (0.95 mL, 8.06 mmol), and 15 mL MeOH were added and then heated at reflux for 24 h. The reaction mixture was cooled
to rt and concentrated to afford a white solid. The solid was purified by column chromatography (1–5% MeOH:CH2Cl2) to afford a white solid (75 mg, 97%
pure) in 4% yield: m.p. = 185–188 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 9.60 (s, 1H), 7.80 (dd, J = 9.1, 5.0 Hz, 2H), 7.10 (t, J = 8.9 Hz, 2 H), 7.10 (overlapping
br s, 2H), 4.18 (s, 2H), 3.36 (s, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 174.4, 166.7, 164.1, 157.4 (d, 1JCF = 238. 7 Hz), 136.2 (d, 4JCF = 2.4 Hz), 121.4 (d,
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3JCF = 7.5 Hz), 114.9 (d, JCF = 22.0 Hz), 73.8, 58.2; 19F NMR (DMSO-d6, 365 MHz) δ -121.3; IR (film) v 3353, 3160, 1201, 1119; HRMS (ESI-TOF) m/z:
[M+H]+ Calcd for C11H13FN5O 250.1099; Found 250.1103.
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6-(Aminomethyl)-N2-(4-fluorophenyl)-1,3,5-triazine-2,4-diamine (2c): Following the general procedure
A using 4-amino-6-((4-fluoro-
phenyl)amino)-1,3,5-triazine-2-carboxamide (240 mg, 0.967 mmol) and LiAlH4 (110 mg, 2.90 mmol) in anhydrous THF (0.1 M). the reaction was stirred at
rt and monitored by TLC. After 6.5 h, the reaction was quenched using the Feiser protocol to afford a yellow residue. The residue was purified using column
chromatography (1–5% MeOH:CH2Cl2) to afford a white residue (81 mg, 95% pure) in 23–36% yield (Note: Heating the reaction > rt leads to reduction of
amine to afford N2-(4-fluorophenyl)-6-methyl-1,3,5-triazine-2,4-diamine and/or decomposition byproducts. At rt, baseline decomposition is observed by
TLC. For this reason, the reaction should not be stirred > 7 h at rt. The yields vary significantly between reactions. Additionally, reduction of the corresponding
azide derivative using H2 Pd/C at rt for 1 h affords N2-(4-fluorophenyl)-6-methyl-1,3,5-triazine-2,4-diamine instead of 1c in quantitative yields.): 1NMR
(DMSO-d6, 400 MHz) δ 9.59 (s, 1H), 7.80 (dd, J = 8.9, 5.1 Hz, 2H), 7.10 (t, J = 8.7 Hz, 4H), 4.87 (t, J = 6.0 Hz, 2H), 4.21 (d, J = 5.9 Hz, 2H); 13C{1H } NMR
(DMSO-d6, 101 MHz) δ 176.9, 166.5, 163.9, 156.1 (1JCF = 239.1 Hz) 136.2 (4JCF = 2.3 Hz), 121.2 (d, 3JCF = 7.4 Hz), 114.7 (d, 2JCF = 21.9 Hz); 19F NMR (DMSO-
d6, 365 MHz) δ -121.9; IR (film) v 3547, 3428, 1718, 1502, 1204; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C10H12FN6 235.1102; Found 235.1107.
Methyl 4-(dimethylamino)-6-((4-fluorophenyl)amino)-1,3,5-triazine-2-carboxylate (8): 4-(Dimethylamino)-6-((4-fluorophenyl)amino)-1,3,5-tria-
zine-2-carbonitrile (37 mg, 0.143 mmol) was added to a flame dried round bottom flask fitted with a reflux condenser. Anhydrous methanol (0.317 mL) was
added to the reaction vessel followed by the addition of BF3•OEt2 (0.572 mmol). The reaction mixture was refluxed overnight and a white precipitate. The
reaction mixture was cooled to ambient temperature and concentrated by rotary evaporation. The residue was re-dissolved in CH2Cl2 and washed with H2O.
The organic mixture was dried with Na2SO4, filtered, and concentrated by rotary evaporation to afford a white solid. The solid was purified by flash column
chromatography (100% CH2Cl2 –5% MeOH:CH2Cl2) to afford a white solid (19 mg, 95% pure) in 45% yield: 1H NMR (DMSO-d6, 400 MHz) δ 9.55 (s, 1H),
7.77 – 7.71 (m, 2H), 7.52 (br, 2H), 7.13 (td, J = 9.1, 2.5 Hz, 2H), 3.84 (s, 3H), 3.16 – 3.08 (m, 6H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 159.9, 155.7,
154.7, 154.3, 145.7 (1JCF = 211.5 Hz) 125.8, (4JCF = 2.4 Hz), 110.9, (3JCF = 7.0 Hz), 104.5 (2JCF = 22.1 Hz), 43.1, 25.5 (d); 19F NMR (DMSO-d6, 365 MHz) δ -
121.3; IR (film) v 3408, 1659, 1003; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C13H15FN5O2 292.1205; Found 292.1210.
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Methyl 4-[(4-fluorophenyl)amino]-6-(methylamino)-1,3,5-triazine-2-carboxylate (9): 4-[(4-Fluorophenyl)amino]-6-(methylamino)-1,3,5-triazine-
2-carbonitrile (0.614 mmol) was weighed into a flame dried around bottom flask and dissolved with anhydrous MeOH (0.25 M). Then freshly distilled BF3OEt2
(4.91 mmol) was added and refluxed. After 12 h, the mixture was cooled to rt and diluted with H2O (2 mL). The mixture was extracted with CH2Cl2 (3 x 5 mL).
The organic mixture was dried with Na2SO4, filtered, and concentrated by rotary evaporation to afford a solid. The solid was chromatographed (1-20%
MeOH:CH2Cl2) to afford a white solid (39 mg, 96% pure) in 23% yield: m.p. = 151–152 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 9.55 (s, 1H), 7.89 – 7.68 (m, 2
H), 7.43 (s, 1H), 7.20 – 6.98 (m, 2 H), 3.83 (d, J = 16.7 Hz, 3 H), 2.81 (dd, J = 12.7, 4.7 Hz, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 170.5 (d, 3JCF = 39.2
Hz), 167.2, 164.9, (d, 2JCF = 48.2 Hz), 157.4 (d, 1JCF = 238.6 Hz), 121.4 (d, 4JCF = 7.3 Hz), 144.9 (d, 3JCF = 22.1 Hz), 114.8 (d, 3JCF = 22.0 Hz), 53.5, 27.3 (d); 19
F
NMR (DMSO-d6, 365 MHz) δ -122.0; HRMS (ESI-TOF) m/z: [M-H]- Calcd for C12H11FN5O2 276.0897; Found 276.0900.
6-((4-Fluorophenyl)amino)pyrimidine-4-carboxylic acid (10): General pyrimidine synthesis procedure was followed using pyrimidine-4-carboxylate
(209 mg, 1.21 mmol) and 4-fluoroaniline (0.116 mL, 1.21 mmol) in 2-propanol (2.0 mL, 0.58 M) and 37% HCl (2.18 mmol, 0.214 mL). The reaction was
stirred at 100 ºC for 19 h. The product hydrolyzed quantitatively to the corresponding carboxylic acid. Purification by column chromatography (eluent 10%
MeOH:CH2Cl2) provided 5 (157 mg, 92% pure) in 52% yield as a yellow solid: m.p. = 226–227 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 11.33 (s, 1H), 8.82 (d,
J = 0.9 Hz, 1H), 7.76 (dd, J = 9.0, 4.9 Hz), 7.51 (s, 1H), 7.36 – 7.19 (m, 2H), 3.88 (3H); 19F NMR (DMSO-d6, 365 MHz) δ 119.5; HRMS (ESI-TOF) m/z:
[M–H]-1 Calcd for C11H7FN3O2 232.0522; Found 232.0523.
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To a solution of 6-((4-fluorophenyl)amino)pyrimidine-4-carboxylic acid in anhydrous methanol was added concentrated H2SO4 (20 μL). The resulting
mixture was heated at reflux overnight. The mixture was concentrated by rotary evaporation, and the residue was purified by flash column chromatography (5%
MeOH:CH2Cl2) to afford methyl 6-((4-fluorophenyl)amino)pyrimidine-4-carboxylate (5) (46 mg, 95% pure) in 43% yield as a white solid: 1H NMR (DMSO-
d6, 400 MHz) δ 10.02 (s, 1H), 8.77 – 8.66 (m, 1H), 7.72 (dd, J = 9.0, 4.9 Hz, 2H), 7.37 (d, J = 1.2 Hz, 1H), 7.30 – 7.12 (m, 2H), 3.88 (s, 3H); 13C{1H } NMR
(DMSO-d6, 101 MHz) δ 164.7, 161.0, 158.0 (d, 1JCF = 239.3 Hz), 158.5, 152.6, 135.5 (d, 4JCF = 2.4 Hz),122.0 (d, 3JCF = 7.9 Hz), 115.5 (d, 2JCF = 22.4 Hz), 107.7,
52.7; 19F NMR (DMSO-d6, 365 MHz) δ -119.5; IR (film) v 3324, 1566, 843; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C12H11FN3O2 248.083; Found
248.0837.
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Methyl 3-amino-5-((4-fluorophenyl)amino)benzoate (11): A mixture of methyl 3-((4-fluorophenyl)amino)-5-nitrobenzoate (44) ( 150.1 mg, 0.517
mmol) and 10 mol% Pd/C ( 1.7 mg, 0.0517 mmol) in anhydrous methanol (3.7 mL, 0.14 M) was stirred at rt under 1.1 atm of H2 for 12 h. The reaction progress
was monitored by TLC using 5% MeOH:CH2Cl2. After 12 h, the catalyst was removed by filtration using Celite® and methanol. The filtrate was concentrated
by rotary evaporation to afford a brown solid. The solid was purified using column chromatography (1–5% MeOH:CH2Cl2) to afford a light brown solid (92
mg, 99%) 54% yield: m.p. = 135–136 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 8.01 (br, 1H), 7.07 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 5.0 Hz, 2H), 7.76 (dd, J = 3.6,
1.6 Hz, 1H), 6.61 (dd, J = 3.5, 1.6 Hz, 1H), 6.47 (t, J = 2.1 Hz, 1H), 5.28 (br, 2H) 3.75 (s, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 166.9, 156.5 (d, 1JCF
=
236.3 Hz), 149.8, 145.0, 139.5 (d, 4JCF = 2.0 Hz), 130.8, 119.6 (d, 3JCF = 7.7 Hz), 115.7 (d, 2JCF = 22.2 Hz), 106.4, 104.9, 104.8, 51.8; 19F NMR (DMSO-d6, 365
MHz) δ -123.5; IR (film) v = 3413, 3435, 1711, 1506, 1219, 769, 521; HRMS (ESI-TOF) m/z : [M+H]+ Calcd for C14H14FN2O2 261.1034; Found 261.1039.
Methyl 4-amino-6-((4-fluorophenyl)amino)picolinate (12): A mixture of methyl 6-((4-fluorophenyl)amino)-4-nitropicolinate (46) (16.0 mg, 0.0549
mmol) and 20 mol% Pd/C (1.2 mg, 0.0110 mmol) in anhydrous methanol (0.8 mL, 0.07 M) was stirred at rt under 1.1 atm H2 pressure. TLC was used to
monitor the reaction progress. (5% MeOH:CH2Cl2). After 2 h, the catalyst was removed by filtration using Celite®. The solid residue was washed with methanol
and the filtrate was concentrate by rotary evaporation to afford a purple residue. The residue was purified by flash column chromatography (gradients 100%
CH2Cl2 to 0.5, 1, and 5 % MeOH:CH2Cl2) to afford a solid in 71% yield (10.1 mg). 1H NMR (Methanol-d4, 400 MHz) δ 7.40 (dd, J = 9.2, 4.8 Hz, 2H), 7.04-
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6.96 (m, 2H), 6.88 (d, J = 1.9 Hz, 1H), 6.14 (d, J = 1.9 Hz, 1H), 3.87 (s, 3H); 13C{1H } NMR (Methanol-d4, 101 MHz) δ 167.8, 159.7 (d, JCF = 240.1 Hz),
158.9, 158.2, 147.3, 138.9 (d, 4JCF = 2.6 Hz), 123.0 (d, 3JCF = 7.7 Hz), 116.4 (d, 2JCF = 22.6 Hz), 106.5, 95.0, 52.8; 19F NMR (Methanol-d4, 365 MHz) δ -123.76;
HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C13H13FN3O2 262.0987; Found 262.0993.
Methyl 4-amino-6-((4-fluorophenyl)(methyl)amino)-1,3,5-triazine-2-carboxylate (13): Following the general free base procedure C, a mixture of aryl
biguanide salt (61) (2.5 g, 10.0 mmol) and NaOEt (885 mg, 13.0 mmol) was stirred in EtOH (0.3 M) at rt for 3 h. Following the general procedure D, a mixture
of dimethyloxalate (3.5 g, 30.0 mmol) and the arylbiguanide base in anhydrous MeOH (0.27 M) was stirred at 25 ºC for 1h and then refluxed overnight. The
mixture was cooled to rt and the crude product was collected by filtration. The crude product was purified with column chromatography (5% MeOH:CH2Cl2)
to afford a white solid (784 mg, 92% pure) in 50% yield (over two steps): m.p. = 176–178 ºC; 1H NMR (DMSO-d6, 400 MHz) 7.40 (s, 1H), 7.40 – 7.33 (m,
2H), 7.30 – 7.18 (m, 3H), 3.78 (s, 3H), 3.39 (s, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.2, 165.9, 164.63, 164.57, 160.5 (d, 1JCF = 243.6 Hz), 140.7
(d, 4JCF = 2.8 Hz), 129.4 (d, 3JCF = 8.6 Hz), 116.1 (d, 2JCF = 22.6 Hz) 52.9, 38.5; 19F NMR (DMSO-d6, 365 MHz) δ -116.2; IR (film) v 3517, 3137, 1610, 1510,
1219, 799; HRMS (ESI-TOF) m/z: [M+H] +Calcd for C12H13FN5O2 278.1048; Found 278.1051.
(4-Amino-6-((4-fluorophenyl)(methyl)amino)-1,3,5-triazin-2-yl)methanol (14):30 To a solution of methyl 4-amino-6-((4-fluorophenyl)(me-
thyl)amino)-1,3,5-triazine-2-carboxylate, 13, (500 mg, 1.90 mmol) in anhydrous THF (0.2 M) was added LiBH4 (1.0 mL, 2.08 mmol) at 0 ºC. The
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reaction was heated at 50 ºC and monitored by TLC. After 12 h, the reaction was quenched with MeOH (1.5 mL) at 0 ºC and concentrated to afford a white
solid. The crude solid was purified using column chromatography (1–5% MeOH:CH2Cl2) to afford the title compound as a white solid (217 mg, 99% pure) in
48% yield: m.p. = 193–195 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 7.43–7.27 (m, 2H), 7.28–7.09 (m, 2H), 6.92 (s, 2H), 4.65 (t, J = 5.9 Hz, 1H), 4.12 (d, J = 5.9
Hz, 2H), 3.38 (s, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 176.4, 166.4, 165.2, 159.8 (d, 1JCF = 242.5 Hz), 140.7 (d, 4JCF = 3.1 Hz), 128.9 (d, 3JCF = 8.5 Hz),
115.5 (d, 2JCF = 22.4 Hz), 63.7, 37.7; 19F NMR (DMSO-d6, 365 MHz) δ -116.8; IR (film) v 3391, 3152, 1670, 1543, 1368, 832; HRMS (ESI-TOF) m/z: [M+H]+
Calcd for C11H13FN5O 250.1099; Found 250.1104.
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4-Amino-N-benzyl-6-((4-fluorophenyl)amino)-1,3,5-triazine-2-carboxamide (15)32: A mixture of methyl 4-amino-6-((4-fluorophenyl)amino)-1,3,5-
triazine-2-carboxylate (1) (0.570 mmol), benzyl amine (0.855 mmol), and 20 mol% (0.114 mmol) glacial acetic acid in dioxane (8 mL) was heated at reflux.
The reaction was monitored with TLC (5% MeOH:CH2Cl2). After 19 h, the mixture was cooled to rt and concentrated by rotary evaporation. The residue was
poured onto ice, and the resulting precipitate was collected by vacuum filtration. The solid was recrystallized from MeOH to afford a white solid in 64% yield
(123 mg, 96% pure): m.p. = 243–244 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 9.94 (s, 1H), 8.79 (t, J = 6.3 Hz, 1H), 7.94 – 7.64 (m, 2H), 7.48 – 7.37 (m, 2H),
7.37 – 7.29 (m, 4H), 7.18 – 7.05 (m, 2H), 4.44 (d, J = 6.2 Hz, 2H); 13C NMR (DMSO-d6, 101 MHz) δ 167.0, 166.3, 164.3, 163.1, 157.7 (d, 1JCF= 240.1 Hz),
139.0, 135.9 (d,3JCF = 2.6 Hz), 128.4, 127.5, 126.7, 121.7 (m), 115.0 (d, 2JCF = 22.2 Hz), 42.4; 19F NMR (DMSO-d6, 365 MHz) δ -120.7; IR (film) v 3491, 3294,
1677, 1629, 1510, 1231, 832; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C17H16FN6O 339.1365; Found 339.1370.
4-Amino-6-((4-fluorophenyl)amino)-N-methyl-1,3,5-triazine-2-carboxamide (16): Methyl-4-amino-6-((4-fluorophenyl)amino-1,3,5-triazine-2-car-
boxylate (1) (300 mg, 1.14 mmol) was weighed into a glass tube followed by the addition of anhydrous MeOH (0.11 M). Then 0.5 M NH3 (39. 9 mL, 39.9
mmol) in THF was added to the mixture. The glass tube was sealed with a screw cap and heated at 120 ºC for 24 h. The reaction mixture was cooled to rt and
concentrated by rotary evaporation to afford a white solid. The solid was recrystallized (1:1 petroleum ether:diethyl ether) to afford a white solid in 96% yield
(272 mg, 97% pure): m.p. = > 260 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 9.85 (s, 1H), 7.79 (dd, J = 9.0, 5.0 Hz, 2H), 7.67 (br s, 2H), 7.34 (br s, 2H), 7.21 –
7.04 (m, 2H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.1, 166.5, 164.9, 164.4, 157.6 (d, 1JCF = 239.1 Hz), 135.9 (d, 4JCF = 2.6 Hz), 121.7 (d, 3JCF = 8.7 Hz),
115.0 (d, 2JCF = 22.2 Hz); 19F NMR (DMSO-d6, 365 MHz) δ -121.3; IR (film) v 3551, 3435, 1637, 1558; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for
C10H10FN6O 249.0895; Found 249.0900.
Preparation of cGAS Inhibitors – see General Procedures B, C, and D
Methyl 4-amino-6-(phenylamino)-1,3,5-triazine-2-carboxylate (17): Following the general free base procedure C, a mixture of 1-carbamimidamido-N-
phenylmethanimidamide hydrochloridei (1.0 g, 4.681 mmol) and NaOEt (318 mg, 4.68 mmol) was stirred in EtOH (0.34 M) at rt for 3 h. Following the general
procedure D, a mixture of dimethyloxalate (1.6 g, 14.0 mmol) and the arylbiguanide base in anhydrous MeOH (0.27 M) was stirred at 25 ºC for 3 h and then
refluxed overnight. The mixture was cooled to rt and the precipitate was collected by filtration. The solid was triturated with MeOH to afford a white solid (534
mg, 99% pure) in 46% yield: m.p. = 206–207 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 9.98 (br, 1H), 7.79 (d, J = 8.0 Hz, 2H) ,7.60 (br, 1H), 7.47 (br, 1H, NH2),
7. 30 (d, J = 7.3 Hz, 1H), 7.27 (d, J =7.4 Hz, 1H), 7.03–6.98 (m, 1H), 3.83 (s, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.1, 164.3, 163.9, 139.4, 128.5,
122.5, 120.0, 52.5; IR (film) v 3484, 3350, 1737, 1644, 1242; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C11H12N5O2 246.0986; Found 246.0991.
Methyl 4-amino-6-((4-(trifluoromethyl)phenyl)amino)-1,3,5-triazine-2-carboxylate (18): Following the general free base procedure C, a mixture of
1-carbamimidamido-N-[4-(trifluoromethyl)phenyl]methanimidamidei hydrochloride (1.3g, 4.75 mmol) and NaOEt ( 323 mg, 4.75 mmol) was stirred in
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MeOH (30 mL) at rt for 22 h. Following the general procedure D, a mixture of dimethyloxalate ( 1.7 g, 14.3 mmol) and the arylbiguanide base in anhydrous
MeOH (0.20 M) was stirred at 35 ºC for 1.5 h and then refluxed overnight. The mixture was cooled to rt and the crude product was collected by filtration and
washed with cold MeOH. The crude product was recrystallized with MeOH to afford the title compound as a bright yellow solid in 30% yield (450 mg, 97%
pure): m.p. = 243–245 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 10.39 (s, 1H), 8.03 (d, J = 8.5 Hz, 2H), 7.82–7.77 (m, 1H), 7.64 (d, J = 8.7 Hz, 2H), 3.85 (s, 3H);
13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.6, 165.0, 164.5, 164.2, 143.7, 126.2 (q, 3JCF3 = 7.7 Hz), 123.6, 122.7 (d, 2JCF3 = 32.0 Hz), 120.0, 53.0 two unresolved
quartet; 19F NMR (DMSO-d6, 365 MHz) δ -60.05; IR (film) v 3506, 3119, 1748, 1543, 1227, 1111, 791; HRMS (ESI-TOF) m/z: [M+H]+Calcd for
1
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60
C
12H11F3N5O2 314.0860; Found 314.0858.
Methyl 4-amino-6-((4-iodophenyl)amino)-1,3,5-triazine-2-carboxylate (19): The general procedure C was followed using 1-carbamimidamido-N-(4-
iodophenyl)methanimidamide hydrochloride (48) (4.0 g, 11.8 mmol), sodium methoxide (829 mg, 15.34 mmol), and dimethyloxalate (4.2 g, 35.4 mmol) in
anhydrous MeOH (35 mL and 20 mL, respectively) at rt to reflux for 24 h. The precipitate was filtered and triturated with MeOH to afford a white solid (1.32
g, 99% pure) in 30% yield: m.p. = 224–225 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 10.10 (br, 1H), 7.64–7.60 (m, 4H), 7.52 (br s, 1H), 3.83 (s, 3H); 13C{1H }
NMR (DMSO-d6, 101 MHz) δ 167.6, 164.7, 164.4, 164.3, 139.8, 137.5, 122.6, 86.3, 53.0; IR (film) v 3327, 3182 3327, 1752, 1666, 1536, 1219, 795, 504; HRMS
(ESI-TOF) m/z: [M+H]+ Calcd for C11H11IN5O2 371.9952; Found 371.9959.
4-Amino-6-((4-iodophenyl)amino)-1,3,5-triazine-2-carboxylic acid (19b): Aqueous 1 M NaOH (2.16 mmol) was added to a solution of methyl 4-
amino-6-((4-iodophenyl)amino)-1,3,5-triazine-2-carboxylate (19) (800 mg, 2.16 mmol) in ethanol (10.8 mL). After heating at reflux for 3 h, the reaction mix-
ture was cooled to rt. Aqueous 1 M HCl (0.540 mL, 4 M HCl in dioxane) was added slowly. The mixture was stirred for 2 h at ambient temperature. After
allowing the solution to stand for approximately 30 min, the yellow precipitate was filtered and washed with water and acetone several times. The yellow solid
was dried under vacuum to afford 4-amino-6-((4-iodophenyl)amino)-1,3,5-triazine-2-carboxylic acid in (885 mg, 97% pure) in quantitative yield. The product
was used without purification: m.p. = 257–258 ºC; H NMR (DMSO-d6, 400 MHz) δ 10.05 (s, 1H), 7.63 (q, J = 8.9 Hz, 4H), 7.55–7.19 (m, 1H); 13C{1H }
1
NMR (DMSO-d6, 101 MHz) δ 166.9, 165.4, 165.0, 164.2, 139.4, 137.0, 122.1, 85.8; IR (film) v 3320, 3137, 1681, 1491, 1622, 1566, 1331, 776, 631; HRMS
(ESI-TOF) m/z: [M–H]- Calcd for C10H7IN5O2 355.9644; Found 355.9645.
Methyl 4-amino-6-((3-iodophenyl)amino)-1,3,5-triazine-2-carboxylate (20): The general procedure C was followed using 1-carbamimidamido-N-(3-
iodophenyl)methanimidamide hydrochloride (49) (300 mg, 0.883 mmol), NaOMe (62.2 mg, 1.15 mmol), and 2.6 mL in anhydrous MeOH. Following the
general procedure D, the corresponding arylbiguanide base, dimethyl oxalate (313 mg, 2.65 mmol) 5.3 mL MeOH at reflux for 12 h. The reaction mixture was
cooled to rt and concentrated to afford a grey-purple solid. The solid was purified by column chromatography (1–20% MeOH:CH2Cl2) to afford a white solid
(154 mg, 92% pure) in 47% yield: m.p. = 212–213 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 10.10 (s, 1H), 8.14 (s, 1H), 7.89 (s, 1H), 7.71 (s, 1H), 7.58 (s, 1H),
7.36 (dt, J = 8.0, 1.1 Hz, 1H), 7.09 (t, J = 8.1 Hz, 1H), 3.84 (s, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.5, 164.7, 163.9, 163.7, 138.9, 133.2, 130.5,
129.3, 119.9, 94.3, 53.8; IR (film) v 3473, 3149, 1744, 1528, 1219, 1011; HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C11H10 IN5O2Na 393.9777; Found
393.9773.
Methyl 4-amino-6-((2-iodophenyl)amino)-1,3,5-triazine-2-carboxylate (21): The general procedure C was followed using 1-carbamimidamido-N-(2-
iodophenyl)methanimidamide hydrochloride (50) (400 mg, 1.08 mmol), NaOMe (75.7 mg, 1.40 mmol), and 2.7 mL in anhydrous MeOH. The corresponding
arylbiguanide base, dimethyl oxalate (383 mg, 3.24 mmol) 1.8 mL MeOH at reflux 18 h. The reaction mixture was cooled to rt. The solid was precipitated and
purified by washing several times with H2O and Et2O to afford a white solid (122 mg) in 38% yield: m.p. = 215–216 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 9.35
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(s, 1H, NH), 7.96 – 7.83 (m, 1H), 7.48 – 7.37 (m, 2H), 7.32 (s, 1H), 7.02 (ddd, J = 7.9, 6.8, 2.2 Hz, 1H), 3.82 (s, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz)
δ 167.6, 165.8, 164.8, 164.5, 140.4, 139.4, 129.3, 129.0, 128.4, 99.7, 52.9; IR (film) v 3499, 3361, 1741, 1536, 1264, 1007; HRMS (ESI-TOF) m/z: [M+H]+
Calcd for C11H11IN5O2 371.9957; Found 371.9953.
1
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7
8
Methyl 4-amino-6-((4-hydroxyphenyl)amino)-1,3,5-triazine-2-carboxylate (22): The general procedure C was followed using 1-carbamimidamido-
N-(4-hydroxyphenyl)methanimidamide hydrochloride (51) (700 mg, 3.05 mmol), NaOMe (200 mg, 3.7 mmol), and 7.6 mL in anhydrous MeOH. The corre-
sponding arylbiguanide base, dimethyl oxalate (1.1 g, 9.2 mmol) 10 mL MeOH at reflux 12 h. The solid was precipitated and purified by flash column chroma-
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1
tography (1–20% MeOH:CH2Cl2) to afford a white solid (283 mg, 95% pure) in 36% yield: m.p. = 228–229 ºC; H NMR (DMSO-d6, 400 MHz) δ 9.76 (s,
2H), 9.18 (s, 1H), 7.51 (s, 2H), 7.36 (s, 2H), 6.69 (d, J = 8.9 Hz, 2H), 3.82 (s, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.6, 164.5, 164.2, 153.6, 131.2,
122.5, 115.4, 100.0, 52.9; IR (film) v 3461, 3394, 3163, 1737, 1219, 1022, 638, 612; HRMS (ESI-TOF) m/z: [M+Li]+ Calcd for C11H11N5O3Li 268.1022;
Found 268.1029.
Methyl 4-amino-6-((4-methoxyphenyl)amino)-1,3,5-triazine-2-carboxylate (23): Following the general free base procedure C a 1-carbamimidamido-
N-(4-methoxyphenyl)methanimidamide hydrochloridei (200 mg, 0.821 mmol) and NaOEt (67 mg, 0.985 mmol) was stirred in EtOH (0.34 M) at rt for 3 h.
Following the general procedure D, a mixture of dimethyloxalate (291 mg, 2.46 mmol) and the arylbiguanide base in anhydrous MeOH (0.27 M) was stirred
at 25 ºC for 1 h and then refluxed overnight. The mixture was cooled to rt and the precipitate was collected by filtration. The solid was triturated with MeOH to
afford a white solid (136 mg) in 60% yield: m.p. = 245–246 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 9.86 (br, 1H), 7.64 (br, 2H), 7.52 (1H), 7.40 (br, 1H), 6.86
(d, J = 9.1 Hz, 2H), 3.81 (s, 1H), 3.72 (s, 1 H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.2, 164.5, 164.0, 163.8, 155.0, 132.3, 121.7, 113.7, 55.2, 52.4; IR
(film) v 3413, 3160, 1744, 1510, 1223, 1029; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C12H14N5O3 276.1091; Found 276.1096.
Methyl 4-amino-6-((4-ethynylphenyl)amino)-1,3,5-triazine-2-carboxylate (24)34: To a dry THF solution of Methyl 4-amino-6-((4-((trimethylsi-
lyl)ethynyl)phenyl)amino)-1,3,5-triazine-2-carboxylate (45 mg, 0.132 mmol) was added a solution of tetrabutylammonium fluoride (45 mg, 0.172 mmol)
dropwise at 25 ºC under N2. The mixture was stirred at rt and monitored by TLC. After 3.5 h, the mixture was concentrated by rotary evaporation and dissolved
with CH2Cl2 and washed with H2O. The organic mixture was dried with Na2SO4, filtered, and concentrated by rotary evaporation to afford a white solid. The
1
solid was purified by flash column chromatography (1–5% MeOH:CH2Cl2) to afford a white solid (24 mg, 97% pure) in 61% yield: m.p. = 204–205 ºC; H
NMR (DMSO-d6, 400 MHz) δ 10.20 (s, 1H), 7.85 (d, J = 8.6 Hz, 2H), 7.70 (br s, 1H), 7.57 (br s, 1H), 7.47 – 7.29 (d, J = 9.7 Hz, 2H), 4.09 (s, 1H), 3.84 (s,
3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.6, 164.7, 164.4, 164.3, 140.6, 132.6, 120.0, 115.6, 84.3, 80.2, 53.0; IR (film) v 3327, 3201, 2109, 1744, 1532,
1231, 1011; HRMS (ESI-TOF) m/z: [M+H]+ Calcd C13H12N5O2 270.0991; Found 270.0987.
Methyl 4-amino-6-((3,4,5-trifluorophenyl)amino)-1,3,5-triazine-2-carboxylate (25): The general procedure B was followed using 1-carbamimidam-
ido-N-(3,4,5-trifluorophenyl)methanimidamide hydrochloride (58) (400 mg, 1.49 mmol), NaOMe (97 mg, 1.79 mmol), and 3.7 mL in anhydrous MeOH.
The corresponding arylbiguanide base, dimethyl oxalate (531 mg, 4.5 mmol) 5.0 mL MeOH at reflux 12 h. The solid was precipitated and purified by flash
1
column chromatography (1–20% MeOH:CH2Cl2) to afford a white solid (283 mg, 93% pure) in 23% yield: m.p. = 230–232 ºC; H NMR (DMSO-d6, 400
MHz) δ 10.40 (s, 1H), 7.86 – 7.74 (m, 4H), 3.84 (s, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.1, 164.2, 163.8, 163.6, 151.2 (m, JCF,) 148.7 (dd, 3JCF
9.7, 5.7 Hz), 136.0 (td, 3a, 3b, 4 CF 12.4, 12.2, 3.6 Hz) 132.7 (d, 1JCF = 242.9 Hz), 103.6 (d, 2b
=
J
J
CF = 24.8 Hz), 52.5; 19F NMR (DMSO-d6, 365 MHz) δ -135.0, -135.1;
IR (film) v 3506, 3346, 3301, 1744, 1528, 1029, 791 cm-1; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C11H9F3N5O2 300.0708; Found 300.0716.
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Methyl 4-amino-6-[(3,5-difluoro-4-iodophenyl)amino]-1,3,5-triazine-2-carboxylate (26): The general procedure C was followed using 1-carbamim-
idamido-N-(3,5-difluoro-4-iodophenyl)methanimidamide hydrochloride (59) (65 mg, 0.173 mmol), NaOMe (28 mg, 0.519 mmol), and 0.340 mL in anhy-
drous MeOH. The corresponding arylbiguanide base, dimethyl oxalate (20.4 mg, 0.173 mmol) 0.340 mL MeOH at reflux for 12 h. The reaction mixture was
cooled to rt and concentrated to afford a grey purple solid. The solid was purified by column chromatography (1–20% MeOH:CH2Cl2) to afford a white yellow
solid (14.3 mg, 96% pure) in 20% yield: 1H NMR (DMSO-d6, 400 MHz) δ 7.85 (s, 1H), 7.69 (d, J = 9.3 Hz, 2H), 7.24 (br, 2H), 3.84 (s, 3H); 13C{1H } NMR
1
2
3
4
5
6
7
8
9
1
3
2
2
(DMSO-d6, 101 MHz) δ 170.8, 168.0, 165.2, 163.0 (dd, JCF = 239.6 Hz), 160.7, 142.9 (t, JCF = 14.0 Hz), 102.3 (dd, JCF = 30.5 Hz), 61.4 (t, JCF = 31.0 Hz),
53.7; 19F NMR (DMSO-d6, 365 MHz) δ -94.0; HRMS (ESI-TOF) m/z: [M+H]+Calcd for C11H9F2IN5O2 407.9769; Found 407.9778 (+2.2 ppm off).
N-2-(4-Iodophenyl)-6-(1,3,4-oxadiazol-2-yl)-1,3,5-triazine-2,4-diamine (27)34: Triethyl orthofomate (0.216 mL) was added to a mixture of FeCl3 (1.8
mg, 0.011 mmol), L-proline (1.2 mg, 0.011 mmol) and Et3N (3 µL, 0.022 mmol), and the resulting solution was stirred for 1 h at rt. The hydrazide substrate
was added, and the mixture was stirred at 80 ºC for 12 h. After cooling, the reaction mixture was washed with Et2O (3 x 0.5 mL). The organic layer was dried
over MgSO4 and evaporated. The residue was subjected to column chromatography on silica gel (10% MeOH:CH2Cl2) to afford a white solid (6 mg) in 29%
yield: 1H NMR (DMSO-d6, 400 MHz) δ 10.53 (s, 1H), 10.04 (s, 1H), 7.62 (m, 4H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167. 3, 166.2, 164.6, 158.7, 139.8
158.2, 137.5, 122.8, 86.4.
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N2-(4-Iodophenyl)-6-(1H-pyrazol-1-yl)-1,3,5-triazine-2,4-diamine (28): Pyrazole (22 mg, 0.317 mmol), 6-chloro-N2-(4-iodophenyl)-1,3,5-triazine-
2,4-diamine (100 mg, 0.288 mmol), and K2CO3 (44 mg, 0.318 mmol) were weighed into a microwave vial with a stir bar. Anhydrous DMSO (1.0 mL) was
added to the sealed vial. The reaction mixture was stirred at 120 ºC overnight. The reaction mixture was diluted with EtOAc (5 mL) and 5% aq LiCl (5 x 5 mL).
The organic mixture was separated, and the aqueous mixture was extracted with EtOAc (3 x 5 mL). The combined organic mixtures were washed with 5% aq
LiCl (5 x 5 mL). The organic mixture was dried with Na2SO4, filtered, and concentrated by rotary evaporation to afford a light brown residue. The residue was
purified by flash column chromatography (5% MeOH:CH2Cl2) to afford a white solid (40 mg, 99% pure) in 46% yield: m.p. = 204–205 ºC; 1H NMR (DMSO-
d6, 400 MHz) δ 9.97 (br s, 1H), 8.50 (dd, J = 2.7, 0.7 Hz, 1H), 7.86–7.80 (m, 1H), 7.69 (d, J = 8.8 Hz, 2H), 7.63 (d, J = 8.9 Hz, 2H), 7.56 (s, 1H), 6.58 (dd, J =
2.7, 1.6 Hz, 1H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.9, 165.2, 162.0, 143.6, 140.0, 137.5, 129.6, 122.7, 109.0, 86.1; IR (film) v 3324, 3189, 1607, 1394;
HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C12H11IN7 380.0121; Found 380.0114.
6-(4-(2-Aminophenyl)-1H-1,2,3-triazol-1-yl)-N2-(4-iodophenyl)-1,3,5-triazine-2,4-diamine (29): 6-Azido-N2-(4-iodophenyl)-1,3,5-triazine-2,4-di-
amine (72) (50 mg, 0.141 mmol) and copper iodide (8.1 mg, 0.042 mmol) were in anhydrous DMSO (0.214 mL) under N2. After 15 min, 2-ethynyl aniline
(48 μL, 0.423 mmol) was added to the mixture and stirred at rt for 2 h. The reaction progress was monitored using TLC until completion. The reaction mixture
was diluted with 10% NH3 aq (1 mL), and the mixture was extracted with EtOAc (3 x 2 mL). The combined organic mixture was washed several times with H2O
and then dried with Na2SO4, filtered, and concentrated by rotary evaporation to afford a brown residue. The residue was purified by flash column chromatog-
raphy (50–75% EtOAc:Hexanes) to afford a white solid (20 mg, 97% pure) in 29% yield: 1H NMR (DMSO-d6, 400 MHz) δ 10.25 (broad s, 1H), 8.93 (s, 1H),
7.82 (broad s, 2H), 7.67 (p, J = 9.1 Hz, 5H), 7.10 (ddd, J = 8.4, 7.2, 1.6 Hz, 1H), 6.82 (dd, J = 8.3, 1.2 Hz, 1H), 6.66 (td, J = 7.4, 1.2 Hz, 1H), 6.00 (broad s, 2H);
13C{1H } NMR (DMSO-d6, 101 MHz) δ 165.0, 163.5, 161.0, 146.4, 140.3, 139.7, 137.6, 129.6, 128.0, 123.0, 119.7, 116.8, 113.0, 103.8, 86.7; IR (film) v 3335,
1607,1439, 1234; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C17H15IN9 472.0495; Found 472.0497.
6-(Azidomethyl)-N2-(4-iodophenyl)-1,3,5-triazine-2,4-diamine (30): To a solution of 6-(azidomethyl)-N2-(4-iodophenyl)-1,3,5-triazine-2,4-
diamine (73) (40 mg, 0.109 mmol) and CuI (6.2 mg, 0.0327 mmol) in DMSO (0.33 mL) was added 2-erthynyl aniline (37.2 µL, 0.327
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mmol). The mixture was stirred at rt. After 5 h, the mixture was diluted with brine (1.0 mL) and extracted with EtOAc (2.0 mL X 3).
The organic mixture was washed with 5% aq LiCl (2.0 mL). The organic mixture was dried with Na2SO4, filtered, and concentrated by
rotary evaporation to afford a brown solid. The solid was purified by column chromatography (1–5% MeOH: CH2Cl2) to afford a light
brown solid (22 mg) in 42% yield: 234–235 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 9.66 (s, 1H), 8.60 (s, 1H), 7.50 (dd, J = 7.8, 1.6 Hz, 1H), 7.46 (br s, 5
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H), 7.31 (s, 1H), 7.05 (ddd, J = 8.4, 7.1, 1.5 Hz, 1H), 6.80 (d, J = 8.1 Hz, 1 H), 6.61 (ddd, J = 8.2, 7.2, 1.2 Hz, 1H), 6.26 (s, 2 H), 5.50 (s, 2H); 13C{1H } NMR
(DMSO-d6, 101 MHz) δ 172.4, 167.0, 164.4, 147.9, 146.1, 140.0, 137.3, 128.9, 128.0, 123.4, 122.5, 116.4, 116.2, 113.1, 85.9, 54.5; IR (film) v 3286, 3186, 1618,
1525, 1402; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C18H17IN9 486.0573; Found 486.0658.
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6-(1H-Benzo[d]imidazol-1-yl)-N2-(4-iodophenyl)-1,3,5-triazine-2,4-diamine (31): Benzimidazole (34 mg, 0.288 mmol), 6-chloro-N2-(4-iodo-
phenyl)-1,3,5-triazine-2,4-diamine (100 mg, 0.288 mmol), and K2CO3 (51.7 mg, 0.374 mmol) were weighed into a microwave vial with a stir bar. Anhydrous
DMSO (0.96 mL) was added to the sealed vial. The reaction mixture was stirred at 120 ºC overnight. The reaction mixture was diluted with EtOAc (5 mL) and
5% aq LiCl (5 x 5 mL). The organic mixture was separated, and the aqueous mixture was extracted with EtOAc (3 x 5 mL). The combined organic mixtures
were washed with 5% aq LiCl (5 x 5 mL). The organic mixture was dried with Na2SO4, filtered, and concentrated by rotary evaporation to afford a light brown
residue. The residue was purified by flash column chromatography (5% MeOH:CH2Cl2) to afford a white solid (57 mg, 95% pure) in 46% yield: m.p. = > 260
ºC; 1H NMR (DMSO-d6, 400 MHz) δ 9.90 (broad s, 1H), 8.94 (s, 1H), 8.70 (broad s, 1H), 7.81–7.73 (m, 1H), 7.67 (m, 4H), 7.61 (broad s, 1H), 7.46–7.32
(m, 2H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.6, 164.8, 162.0, 145.0, 142.0, 139.8, 137.6, 132.0, 124.7, 124.1, 123.0, 120.4, 116.9, 85.6; IR (film) v
3491, 3305, 1547, 1465, 1204; HRMS (ESI-TOF) m/z: [M-H]– Calcd for C16H11IN7 428.0121; Found 428.0127.
6-(1H-Indol-1-yl)-N2-(4-iodophenyl)-1,3,5-triazine-2,4-diamine (32): Indole (33.7 mg, 0.288 mmol), 6-chloro-N2-(4-iodophenyl)-1,3,5-triazine-2,4-
diamine (100 mg, 0.288 mmol), and K2CO3 (51.7 mg, 0.374 mmol) were weighed into a microwave vial with a stir bar. Anhydrous DMSO (1.0 mL) was added
to the sealed vial. The reaction mixture was stirred at 120 ºC overnight. The reaction mixture was diluted with EtOAc (5 mL) and 5% aq LiCl (5 x 5 mL). The
organic mixture was separated, and the aqueous mixture was extracted with EtOAc (3 x 5 mL). The combined organic mixtures were washed with 5% aq LiCl
(5 x 5 mL). The organic mixture was dried with Na2SO4, filtered, and concentrated by rotary evaporation to afford a light brown residue. The residue was purified
by flash column chromatography (1% MeOH:CH2Cl2) to afford a white solid (35.3 mg, 99% pure) in 29% yield: m.p. = 163–164 ºC; 1H NMR (Methanol-d4,
400 MHz) δ 8.84 (d, J = 8.4 Hz, 1H), 8.19 (d, J = 3.7 Hz, 1H), 7.68 – 7.60 (m, 2H), 7.60 – 7.51 (m, 3H), 7.25 (ddd, J = 8.4, 7.2, 1.4 Hz, 1H), 7.17 (td, J = 7.5,
1.1 Hz, 1H), 6.64 (dd, J = 3.8, 0.7 Hz, 1H); 13C{1H } NMR (Methanol-d4, 101 MHz) δ 169.4, 166.5, 164.8, 141.0, 138.7, 137.0, 132.9, 126.6, 124.5, 124.0, 123.2,
121.7, 118.4, 107.7, 86.3; IR (film) v 3487, 3260, 1607, 1555, 1450, 1216; HRMS (ESI-TOF) m/z: [M-H]– Calcd for C17H12IN6 427.0168; Found 427.0204.
6-(1H-Indazol-1-yl)-N2-(4-iodophenyl)-1,3,5-triazine-2,4-diamine (33): Indazole (34 mg, 0.288 mmol), 6-chloro-N2-(4-iodophenyl)-1,3,5-triazine-
2,4-diamine (100 mg, 0.288 mmol), and K2CO3 (44 mg, 0.318 mmol) were weighed into a microwave vial with a stir bar. Anhydrous DMSO (1.0 mL) was
added to the sealed vial. The reaction mixture was stirred at 120 ºC overnight. The reaction mixture was diluted with EtOAc (5 mL) and 5% aq LiCl (5 x 5 mL).
The organic mixture was separated, and the aqueous mixture was extracted with EtOAc (3 x 5 mL). The combined organic mixtures were washed with 5% aq
LiCl (5 x 5 mL). The organic mixture was dried with Na2SO4, filtered, and concentrated by rotary evaporation to afford a light brown residue. The residue was
purified by flash column chromatography (gradient 100% CH2Cl2 to 5% MeOH:CH2Cl2) to afford a white solid (52 mg, 99%) in 42% yield: m.p. = 246–248
ºC; 1H NMR (DMSO-d6, 400 MHz) δ 9.90 (s, 1H), 8.90 (d, J = 8.4 Hz, 1H), 8.43 (d, J = 0.9 Hz, 1H), 7.89 (dt, J = 7.9, 1.0 Hz, 1H), 7.80 – 7.70 (m, 2H), 7.70
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– 7.63 (m, 2H), 7.63 – 7.52 (m, 2H), 7.36 (ddd, J = 7.9, 7.0, 1.0 Hz, 2H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.8, 165.1, 163.7, 140.2, 139.6, 139.1,
137.5, 128.5, 126.5, 123.6, 122.8, 121.6, 116.9, 86.0; IR (film) v 3491, 3305, 1547, 1465, 1424, 746; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C16H13IN7
430.0277; Found 430.0271.
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Methyl 4-amino-6-((4-chlorophenyl)amino)-1,3,5-triazine-2-carboxylate (34): Following the general free base procedure C, a mixture of 1-carbamim-
idamido-N-(4-chlorophenyl)methanimidamide hydrochlorideii (8.9 g, 35.8 mmol) and NaOEt ( 2.4 mg, 35.8 mmol) was stirred in MeOH (70 mL) at rt for 22
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h. Following the general procedure D, a mixture of dimethyloxalate ( 8.5 g, 71.6 mmol) and the arylbiguanide base (7.6 g, 35.8 mmol) in anhydrous MeOH
(0.25 M) was stirred at 35 ºC for 1.5 h and then refluxed overnight. The mixture was cooled to rt and the crude product was collected by filtration and washed
with cold MeOH. The crude solid was triturated with MeOH to afford a white solid (7.3 g, 94% pure) in 73% yield: m.p. = 256–249 ºC; 1H NMR (DMSO-d6,
400 MHz) δ 10.17 (br, 1H), 7.84 (d, J = 8.5 Hz, 2H), 7.70 (br, 1H), 7.56 (br, 1H), 7.34 (d, J = 8.9 Hz, 2H), 3.84 (s, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz)
δ 167.1, 164.3, 163.9, 163.8, 138.5, 128.4, 126.1, 121.2, 52.5; IR (film) v 3476, 3145, 1748, 1528, 1245; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for
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C
11H11ClN5O2 280.0596; Found 280.0608.
Methyl 4-amino-6-((4-bromophenyl)amino)-1,3,5-triazine-2-carboxylate (35): Following the general free base procedure C, a mixture of N-(4-bro-
mophenyl)-1-carbamimidamidomethanimidamide hydrochloridei (12.4 g, 42.5 mmol) and NaOEt ( 2.9 g, 42.5 mmol) was stirred in EtOH (70 mL) at rt for
22 h. Following the general procedure D, a mixture of dimethyloxalate ( 9.1 g, 76.9 mmol) and the arylbiguanide base (9.9 g, 38.5 mmol) in anhydrous MeOH
(0.21 M) was stirred at 35 ºC for 1 h and then refluxed overnight for ~2.5 d. After 28 h at -20 ºC, the precipitate was collected by filtration and washed with cold
MeOH to afford a white solid (8.6 g) in 69% (crude). The solid was recrystallized using MeOH to afford title compound as a white solid (218 mg, 99% pure) in
18% yield: m.p. = 224–226 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 10.17 (br, 1H, NH), 7.79 (d, J = 8.5 Hz, 2H), 7.70 (s, 1H), 7.60 (br s, 1H), 7.46 (d, J = 8.9
Hz, 2H), 3.83 (s, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.1, 164.3, 163.9, 163.8, 138.9, 131.3, 121.8, 114.1, 52.5; IR (film) v 3473, 3145, 1748, 1618,
1249, 825, 519; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C11H11BrN5O2 324.0091; Found 324.0095.
Methyl 4-amino-6-((4-nitrophenyl)amino)-1,3,5-triazine-2-carboxylate (36): Following the general free base procedure C a mixture of 1-carbamimid-
amido-N-(4-nitrophenyl)methanimidamide hydrochloride (52) (15.8 g, 61.1 mmol) and NaOEt ( 4.2 g, 61.1 mmol) was stirred in EtOH (100 mL) at rt for 22
h. Following the general procedure D, a mixture of dimethyloxalate ( 574 mg, 2.43 mmol) and the arylbiguanide base (540 mg, 2.43 mmol) in anhydrous MeOH
(0.12 M) was stirred at 35 ºC for 0.5 h and then refluxed overnight for ~2.5 d. After 24 h at -20 ºC, a precipitate formed. The crude product was collected by
filtration and washed with cold MeOH to afford a tan powder (130 mg, 94% pure) in 18% yield. The solid was used without further purification: m.p. = > 260
ºC; 1H NMR (DMSO-d6, 400 MHz) δ 10.71 (br s, 1H), 8.19 (d, J = 9.4 Hz, 2H), 8.09 (d, J = 9.4 Hz, 2H), 7.91 (br s, 1H), 7.74 (br s, 1H), 3.86 (s, 3H); 13C{1H}
NMR (DMSO-d6, 101 MHz) δ 167.6, 164.9, 164.5, 164.0, 146.6, 141.7, 125.2, 119.5, 53.1; IR (film) v 3413, 3368, 1744, 1644, 1502, 1335; HRMS (ES-TOF)
M/Z: [M+H]+ Calcd for C11H11N6O4 291.0837; Found 291.0842.
Methyl 4-amino-6-((4-cyanophenyl)amino)-1,3,5-triazine-2-carboxylate (37):The general procedure C was followed using 1-carbamimidamido-N-(4-
cyanophenyl)methanimidamide hydrochloride (53) (496 mg, 2.08 mmol), NaOMe (145.9 mg, 2.70 mmol), and 6.9 mL in anhydrous MeOH. Following the
general procedure D, a mixture of the corresponding arylbiguanide base, dimethyl oxalate (737 mg, 6.24 mmol) 4.0 mL MeOH was stirred at 35 ºC for 0.5 h
and then refluxed for 22 h. The reaction mixture was cooled to rt. The solid was precipitated and purified by flash column chromatography (5% MeOH:CH2Cl2)
to afford a white solid (99.8 mg, 97% pure) in 18% yield: m.p. = > 260 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 10.47 (s, 1H), 8.03 (d, J = 8.9 Hz, 2H), 7.82 (s,
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1H), 7.75 (d, J = 8.8 Hz, 2H), 7.70 (s, 1H), 3.85 (s, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.6, 164.9, 164.5, 164.1, 144.4, 133.4, 120.0, 119.8, 104.2,
53.1; IR (film) v 3402, 3357, 2229, 1733, 1655, 1238, 795, 609; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C12H11N6O2 271.0944; Found 271.0944.
Methyl-4-amino-6-((4-((trimethylsilyl)ethynyl)phenyl)amino)-1,3,5-triazine-2-carboxylate (38): Methyl 4-amino-6-((4-iodophenyl)amino)-1,3,5-
triazine-2-carboxylate (19) (75 mg, 0.202 mmol), PdCl2(PPh3)2 (76 mg, 0.108 mmol), and CuI (10 mg, 0.054 mmol) were weighed into a microwave vial with
a stir bar. The flask was sealed and placed under vacuum then backfilled with N2. Then, trimethylsilyl acetylene (0.157 mL, 0.592 mmol), Et3N (0.524 mL, 3.77
mmol), and THF (1.8 mL) were added, and the mixture was stirred at rt overnight. After 18 h, the mixture was diluted with EtOAc (1.0 mL) and washed with
brine (3 mL x 2). The organic mixture was dried with Na2SO4, filtered, and concentrated by rotary evaporation to afford a brown solid. The solid was purified
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by column chromatography (1–5% MeOH:CH2Cl2) to afford a white solid (118 mg, 99% pure) in 64% yield: m.p. = 204–206 ºC; H NMR (DMSO-d6, 400
MHz) δ 10.22 (s, 1H), 7.85 (d, J = 8.6 Hz, 2H), 7.71 (s, 1H, NH2), 7.40 – 7.34 (m, 2H), 3.84 (s, 3H), 0.23 (s, 6H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ
167.7, 164.7, 164.4, 164.2, 140.7, 132.5, 119.9, 116.0, 106.2, 93.4, 53.0, 0.50; IR (film) v 3510, 3126, 1748, 1532, 1413, 1227; HRMS (ESI-TOF) m/z: [M+H]+
Calcd for C16H20N5O2Si 342.1386; Found 342.1389.
Methyl 4-amino-6-((4-(cyclopropylethynyl)phenyl)amino)-1,3,5-triazine-2-carboxylate (39): Methyl 4-amino-6-((4-iodophenyl)amino)-1,3,5-tria-
zine-2-carboxylate (19) (50 mg, 0.135 mmol), PdCl2(PPh3)2 (19 mg, 0.027 mmol), and CuI (2.6 mg, 0.014 mmol) were weighed into a microwave vial with a
stir bar. The flask was sealed and placed under vacuum then backfilled with N2. Then, ethynylcyclopropane (12.6 µL, 0.149 mmol), Et3N (0.131 mL, 0.945
mmol), and THF (0.43 mL) were added, and the mixture was stirred at rt overnight. After 18 h, the mixture was diluted with EtOAc (1.0 mL) and washed with
brine (3 mL x 2). The organic mixture was dried with Na2SO4, filtered, and concentrated by rotary evaporation to afford a brown solid. The solid was purified
by column chromatography (1–5% MeOH:CH2Cl2) to afford a white solid (15 mg, 96% pure) in 36% yield: 1H NMR (DMSO-d6, 400 MHz) δ 10.11 (s, 1H),
7.79 (d, J = 8.3 Hz, 2H), 7.70 – 7.40 (m, 2H), 7.28 (d, J = 8.4 Hz, 2H), 3.84 (s, 3H), 1.52 (tt, J = 8.3, 5.0 Hz, 1H), 0.99 – 0.79 (m, 2H), 0.72 (dt, J = 5.1, 3.0 Hz,
2H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.1, 164.2, 163.9, 163.8, 139.1, 131.6, 119.5, 116.8, 92.6, 75.8, 52.4, 8.3; IR (film) v 3298, 3130, 1748, 1510;
HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C16H16N5O2 310.1304; Found 310.1310.
Methyl 4-amino-6-[(6-iodopyridin-3-yl)amino]-1,3,5-triazine-2-carboxylate (40): The general procedure C was followed using 1-Carbamimidamido-
N-(6-iodopyridin-3-yl)methanimidamide hydrochloride (54) (787 mg, 2.31 mmol), NaOMe (374 mg, 6.93 mmol), and 4.6 mL in anhydrous MeOH. Follow-
ing the general procedure D, the corresponding arylbiguanide base, dimethyl oxalate (818 mg, 6.93 mmol) and 9.0 mL MeOH were stirred at 35 ºC for 3 h and
then heated at reflux for 12 h. The reaction mixture was cooled to rt and concentrated to afford a grey purple solid. The solid was purified by column chroma-
tography (1–20% MeOH:CH2Cl2) to afford a light purple solid (95 mg, 95% pure) in 11% yield: m.p. = 244–246 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 10.3
(br s, 1H), 8.87 (s, 1H), 7.91 (dd, J = 8.6, 2.9 Hz, 1H), 7.74 (d, J = 8.7 Hz, 2H), 7.62 (br s, 1H), 3.84 (s, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.3,
164.6, 164.2, 163.9, 143.0, 136.8, 134.1, 129.2, 109.2, 52.8; IR (film) v 3484, 3353, 3171, 1748, 1227, 944; HRMS (ESI-TOF) m/z: [M+H]+ Calcd
forC10H10IN6O2 372.9910; Found 372.9903.
Methyl 4-amino-6-(quinolin-6-ylamino)-1,3,5-triazine-2-carboxylate (41):The general procedure C was followed using 1-carbamimidamido-N-(quin-
olin-6-yl)methanimidamide hydrochloride (55) (250 mg, 0.944 mmol), NaOMe (153 mg, 2.83 mmol), and 2.7 mL in anhydrous MeOH. The corresponding
arylbiguanide base and dimethyl oxalate (3.34 mg, 2.83 mmol) in anhydrous 3.5 mL MeOH at reflux for 22 h. The reaction mixture was cooled to rt. The precip-
itate was filtered, washed with MeOH, and dried under vacuum to afford a white solid (164 mg, 99% pure) in 59% yield: m.p. = 258–260 ºC; 1H NMR (DMSO-
d6, 400 MHz) δ 10.28 (s, 1H), 8.77 (dd, J = 4.2, 1.7 Hz, 1H), 8.63 (s, 1H), 8.26–8.17 (m, 1H), 8.02 (dd, J = 9.1, 2.4 Hz, 1H), 7.94 (d, J = 9.1 Hz, 1H), 7.75 (br
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s, 1H), 7.60 (br s, 1H), 7.50 (dd, J = 8.3, 4.2 Hz, 1H), 3.87 (s, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.7, 164.9, 164.5, 164.3, 149.2, 144.9, 137.9.
135.7, 129.6, 128.8, 124.6, 122.2, 115.9, 53.0; IR (film) v 2784, 1756, 1644, 1208; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C14H13N6O2 297.1095; Found
297.1103.
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N-[2-(1-{4-amino-6-[(4-iodophenyl)amino]-1,3,5-triazin-2-yl}-1H-1,2,3-triazol-4-yl)phenyl]methanesulfonamide (42): 6-Azido-N2-(4-iodo-
phenyl)-1,3,5-triazine-2,4-diamine (72) (37 mg, 0.104 mmol), Et3NH (14.5 µL, 0.104 mmol) and copper iodide (5.9 mg, 0.031 mmol) were in anhydrous
DMSO (0.416 mL) under N2. After 15 min, N-(2- N-(2-ethynylphenyl)methanesulfonamide (20.3 mg, 0.104 mmol) was added to the mixture and stirred at rt
for 2 h. The reaction progress was monitored using TLC until completion. The reaction mixture was diluted with 10% NH3 aq (1 mL), and the mixture was
extracted with EtOAc (3 x 2 mL). The combined organic mixture was washed several times with H2O and then dried with Na2SO4, filtered, and concentrated
by rotary evaporation to afford a brown residue. The residue was purified by flash column chromatography (5-100% EtOAc:Hexanes) to afford a white solid (5
mg, 90% pure) in 5% yield: 1H NMR (DMSO-d6, 400 MHz) δ 13.01 (s, 1H), 9.58 (s, 1H), 8.04 (dq, J = 8.4, 0.9 Hz, 1H), 7.79 – 7.66 (m, 3H), 7.63 – 7.57 (m,
2H), 7.37 – 7.25 (m, 2H), 7.11 (s, 2H), 6.75 (d, J = 0.8 Hz, 1H), 3.88 (s, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.5, 164.9, 164.3, 146.4, 140.4, 137.4,
135.9, 128.2, 125.1, 123.8, 122.7, 122.3, 114.5, 95.6, 85.6, 55.8; HRMS (ESI-TOF) m/z: [M-] Calcd for C10H8Cl2N4 253.0038; Found 253.0048.
6-(4-(4-Fluorophenyl)-1H-1,2,3-triazol-1-yl)-N2-(4-iodophenyl)-1,3,5-triazine-2,4-diamine (43): 6-Azido-N2-(4-iodophenyl)-1,3,5-triazine-2,4-di-
amine (72) (50 mg, 0.141 mmol) and copper iodide (8.1 mg, 0.0423 mmol) were in anhydrous DMSO (0.214 mL) under N2. After 15 min, 1-ethylnyl-4-
fluorobenzene (49 μL, 0.423 mmol) was added to the mixture and stirred at rt for 2 h. The reaction progress was monitored using TLC until completion. The
reaction mixture was diluted with 10% NH3 aq (1 mL), and the mixture was extracted with EtOAc (3 x 2 mL). The combined organic mixture was washed
several times with H2O and then dried with Na2SO4, filtered, and concentrated by rotary evaporation to afford a yellow residue. The residue was purified by flash
column chromatography (50–75% EtOAc:Hexanes) to afford a white solid (21 mg, 97% pure) in 31% yield: 1H NMR (DMSO-d6, 400 MHz) δ 10.22 (s, 1H),
9.06 (s, 1 H), 8.07 (dd, J = 8.5, 5.5 Hz, 2H), 7.78 (s, 2H), 7.68 (q, J = 8.7 Hz, 4H), 7.41 – 7.27 (m, 2H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.9, 165.0,
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163.8 (d, JCF= 246.6 Hz), 161.0, 146.1, 139.6, 137.6, 128.3 (d, JCF= 7.9 Hz), 126.8 (d, JCF = 3.0 Hz), 122.9, 119.6, 116.4 (d, JCF = 21.7 Hz), 86.7; 19F NMR
(DMSO-d6, 365 MHz) δ -113.1; IR (film) v 3461, 3286, 1644, 1540, 1417, 1238, 1011; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C17H13FIN8 475.0292;
Found 475.0259.
Methyl 4-amino-6-((3,5-difluorophenyl)amino)-1,3,5-triazine-2-carboxylate (44): Following the general free base procedure C, a mixture of 1-car-
bamimidamido-N-(3,5-difluorophenyl)methanimidamide hydrochloride1 (152 mg, 0.607 mmol) and NaOEt (41 mg, 0.607 mmol) was stirred in EtOH (5
mL) at rt for 24 h. Following the general procedure D, a mixture of dimethyloxalate ( 143 mg, 1.21 mmol) and the arylbiguanide base (129 mg, 0.607 mmol) in
anhydrous MeOH (0.11 M) was stirred at 25 ºC for 0.5 h and then refluxed for 44 h. After 20 h at -20 ºC, the precipitate was collected by filtration. The solid
was recrystallized using MeOH to afford a white solid (15 mg, 98% pure) in 8% yield: 1H NMR (DMSO-d6, 400 MHz) δ 10.39 (s, 1H), 7.81 (s, 1H), 7.73 (s,
1H), 7.67 – 7.44 (m, 2H), 6.83 (tt, J = 9.3, 2.4 Hz, 1H), 3.84 (s, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.6, 164.8, 164.3, 164.1, 162.9 (dd, 1JCF = 242.8
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Hz), 142.6 (t, 3JCF = 14.3 Hz), 102.8 (d, 2JCF = 29.8 Hz), 97.7 (t, 2JCF = 26.6 Hz), 53.1; 19F NMR (DMSO-d6, 365 MHz) δ -109.6; IR (film) v 3491 3294, 1756,
1555, 1242, 1037; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C11H10F2N5O2 282.0798; Found 282.0801.
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Methyl 4-amino-6-((3,5-bis(trifluoromethyl)phenyl)amino)-1,3,5-triazine-2-carboxylate (45): Following the general free base procedure C, a mix-
ture of N-[3,5-bis(trifluoromethyl)phenyl]-1-carbamimidamidomethanimidamide hydrochloride (56) (5.6 g, 16.1 mmol) and NaOEt ( 1.1 g, 16.1 mmol) was
stirred in EtOH (30 mL) at rt for 22 h. Following the general procedure D, a mixture of dimethyloxalate ( 3.8 g, 32.2 mmol) and the arylbiguanide base (5.1 g,
16.1 mmol) in anhydrous MeOH (0.21 M) was stirred at 25 ºC for 2 h and then refluxed overnight for 21 h. After 48 h at -20 ºC, the precipitate was collected
by filtration. The solid was recrystallized from MeOH to afford a white solid (2.9 g, 99% pure) in 47% yield: 1H NMR (DMSO-d6, 400 MHz) δ 10.62 (s, 1H),
8.51 (s, 2H), 7.88 (s, 1H), 7.74 – 7.63 (m, 2H), 3.85 (s, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.1, 164.6, 163.9, 163.5, 141.6, 130.7 (q, 3JCF = 32.7
Hz), 123.4 (q, 1JCF = 272.7 Hz), 114.8 (m), 52.7; IR (film) v 3286, 3156, 1733, 1543, 1278; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C13H10F6N5O2 382.0734;
Found 382.0738.
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Methyl 4-amino-6-((3,5-dimethylphenyl)amino)-1,3,5-triazine-2-carboxylate (46): Following the general free base procedure C, a mixture of 1-car-
bamimidamido-N-(3,5-dimethylphenyl)methanimidamide hydrochloride (57) (226 mg, 0.933 mmol) and NaOEt ( 64 mg, 0.933 mmol) was stirred in EtOH
(5 mL) at rt for 22 h. Following the general procedure D, a mixture of dimethyloxalate ( 3.8 g, 32.2 mmol) and the arylbiguanide base (5.1 g, 16.1 mmol) in
anhydrous MeOH (0.21 M) was stirred at 25 ºC for 1 h and then refluxed for 24 h. The precipitate was collected by filtration and recrystallized using MeOH to
afford a white solid (53 mg, 99% pure) in 21% yield: m.p. = 214–215 ºC; 1H NMR (DMSO-d6, 400 MHz) δ 9.87 (br, 1 H), 7.60 (br, 1 H), 7.41 (s, 2 H), 6.66
(s, 1 H), 3.83 (s, 3 H), 2.25 (s, 6 H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 167.1, 164.7, 164.3, 164.0, 139.2, 137.4, 124.2, 117.9, 52.5, 21.2; IR (film) v 3346,
3193, 1748, 1547, 1242, 795, 605; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C13H16N5O2 274.1299; Found 274.1308.
Methyl 4-amino-6-((3-fluoro-4-iodophenyl)amino)-1,3,5-triazine-2-carboxylate (47): The general procedure C was followed using 1-carbamimidam-
ido-N-(3-fluoro-4-iodophenyl)methanimidamide hydrochloride (60) (200 mg, 0.559 mmol), NaOMe (118 mg, 2.18 mmol), and 1.6 mL in anhydrous MeOH.
The corresponding arylbiguanide base, dimethyl oxalate (198 mg, 1.68 mmol) 1.8 mL MeOH at reflux 18 h. The reaction mixture was cooled to rt. The solid
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was precipitated and purified by washing several times with H2O to afford a white solid (72 mg, 97% pure) in 33% yield: H NMR (DMSO-d6, 400 MHz) δ
10.29 (s, 1H), 8.07 – 7.99 (m, 1H), 7.74 (s, 1H), 7.70 (dd, J = 8.7, 7.6 Hz, 2H), 7.64 (s, 1H), 7.33 (dd, J = 8.6, 2.4 Hz, 1H), 3.84 (s, 6H); 13C{1H } NMR (DMSO-
d6, 101 MHz) δ 167.0, 164.2, 163.8, 163.6, 160.1 (4JCF = 239.6 Hz), 142.1 (d, 3JCF = 10.7 Hz), 138.9 (d, 4JCF = 3.3 Hz), 117.9, 107.3 (d, 2JCF = 29.6 Hz), 72.8 (d,
2JCF = 26.2 Hz), 53.0 19F NMR (DMSO-d6, 365 MHz) δ -94.60; IR (film) v 3335, 3197, 1748, 1540, 1223, 791; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for
C11H10FIN5O2 389.9863; Found 389.9872.
Characterization data for arylbiguanide salts and key intermediates
1-Carbamimidamido-N-(4-iodophenyl)methanimidamide hydrochloride (48): The general procedure B was followed using 4-iodoaniline (18.8 g, 86
mmol) and dicyandiamide (7.2 g, 86 mmol) in 28.6 mL 3 M HCl at 90 ºC for 48 h. The precipitate was then filtered to afford a grey purple solid. The solid was
triturated with Et2O and then rinsed several times with H2O ( 5 x 5 mL) to afford a grey purple solid (23.2 g) in 79%: 1H NMR (DMSO-d6, 400 MHz) δ 9.93
(d, J = 3.1 Hz, 1H), 7.62 (d, J = 8.7 Hz, 2H), 7.40 (s, 3H), 7.23 (d, J = 8.8 Hz, 2H), 7.10 (s, 2H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 161.3, 154.7, 138.7,
137.2, 122.7, 86.7; HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C8H11ClIN5Na 361.9645; Found 361.9635.
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1-Carbamimidamido-N-(3-iodophenyl)methanimidamide hydrochloride (49): The general procedure B was followed using 3-iodoaniline (500 mg,
2.28 mmol) and dicyandiamide (192 mg, 2.28 mmol) in 0.76 mL 3 M HCl at 100 ºC for 24 h. The reaction mixture was concentrated by rotary evaporation and
triturated with Et2O to afford a grey purple solid (606 mg) in 78% yield: 1H NMR (DMSO-d6, 400 MHz) δ 10.00 (s, 1H), 7.83 (s, 1H), 7.44 (s, 3H), 7.38 (d, J
= 7.9 Hz, 2H), 7.10 (dd, J = 14.7, 6.7 Hz, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 161.4, 154.7, 140.4, 131.5, 130.6, 128.5, 119.7, 94.4; HRMS (ESI-TOF)
m/z: [M]+ Calcd for C8H11IN5 304.0059; Found 304.0066.
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1-Carbamimidamido-N-(2-iodophenyl)methanimidamide hydrochloride (50): The general procedure B was followed using 2-iodoaniline (1.0 g, 4.57
mmol) and dicyandiamide (384 mg, 4.57 mmol) in 1.5 mL 3 M HCl at 100 ºC for 48 h. The reaction mixture was concentrated by rotary evaporation and
triturated with Et2O to afford a grey purple solid (953 mg) in 56% yield: 1H NMR (DMSO-d6, 400 MHz) δ 7.75 (s, 1H), 7.47 (d, J = 1.6 Hz, 1H), 6.66 (d, J =
8.4 Hz, 1H), 6.52 – 6.37 (m, 1H), 4.63 (s, 3H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 161.8, 156.6, 139.2, 128.9, 128.1, 127.7, 121.2, 96.5; HRMS (ESI-
TOF) m/z: [M]+ Calcd for C8H11IN5 304.0059; Found 304.0058.
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1-Carbamimidamido-N-(4-hydroxyphenyl)methanimidamide hydrochloride (51):36 Into a microwave vessel was added 4-aminophenol (649 mg, 5.95
mmol), dicyandiamide (500 mg, 5.95 mmol), and (0.83 mL, 6.55 mmol). Then acetonitrile (7.9 mL) was added and the mixture was stirred at 150 ºC. After 4.5
hours, a purple precipitate formed. The precipitate was dissolved in MeOH and stirred for 15 min, then concetrated by rotary evaporation to afford a purple
solid. The solid was purified by trituration with Et2O and provided the title compound (1.38 mg) in quantitative yield as a purple solid: 1H NMR (DMSO-d6,
400 MHz) δ 9.39 (s, 1H), 9.23 (d, J = 2.6 Hz, 1H), 7.09 (d, J = 8.8 Hz, 2H), 7.08-7.07 (m, 3H), 6.95 (s, 2 H), 6.72 (d, J = 8.8 Hz, 2H); 13C{1H } NMR (DMSO-
d6, 101 MHz) δ 161.0, 156.8, 154.8, 129.7, 124.6, 115.7; HRMS (ESI-TOF) m/z: [M]+ Calcd for C8H12N5O 194.1042; Found 194.1048.
1-Carbamimidamido-N-(4-nitrophenyl)methanimidamide hydrochloride (52): The general procedure B was followed using 4-nitroanline (15.4 g,
111.8 mmol) and dicyandiamide (9.40 g, 111.8 mmol) in 37.2 mL 3 M HCl at 90 ºC for 4 d and 16 h. The reacttion mixture was cooled for 6 h. The precipitate
was then filtered and triturated with Et2O (3 x) to afford an orange brown solid (15.7 mg) in 55% yield: 1H NMR (DMSO-d6, 400 MHz) δ 10.51 (s, 1H), 8.19
(d, J = 9.3 Hz, 2H), 7.66 (d, 9.3 Hz, 2H), 7.67-7.66 (br overlapping, 3H), 7.25 (br, 2H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 161.8, 153.6, 141.5, 124.8,
119.1, 117.5; HRMS (ESI-TOF) m/z: [M]+ Calcd for C8H11N6O2 223.1304; Found 223.0943.
1-Carbamimidamido-N-(4-cyanophenyl)methanimidamide hydrochloride (53):37 The general procedure B was followed using 4-aminobenzonitrile
(1.0 g, 8.47mmol) and dicyandiamide (712 mg, 8.47 mmol) in 2.8 mL 3 M HCl and 10 mL MeCN at 100 ºC for 2 d. The precipitate was filtered and used
without further purification to afford a grey solid (1.2 g) in 60% yield: 1H NMR (DMSO-d6, 400 MHz) δ 10.22 (s, 1H), 7.80 – 7.69 (d, J = 8.9 Hz, 2H), 7.60 (d,
J = 8.5 Hz, 2H), 7.18 (broad s, 2H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 162.2, 154.3, 144.1, 133.5, 120.2, 119.7, 104.6; HRMS (ESI-TOF) m/z: [M]-
Calcd for C8H11N6O2 223.0943; Found 223.0948.
N'-(Azaniumylmethanimidoyl)-N-(6-iodopyridin-3-yl)guanidine chloride (54): The general procedure B was followed using 5-amino-2-iodopyridine
(500 mg, 2.27 mmol) and dicyandiamide (191 mg, 2.27 mmol) in 0.91 mL 3 M HCl and 0.91 mL H2O at 100 ºC for 24 h. The reaction mixture was concentrated
by rotary evaporation and triturated with Et2O to afford a brown solid (787 mg) in 98%: 1H NMR (DMSO-d6, 400 MHz) δ 8.42 (s br, 1H), 7.76–7.73 (m, 1H),
7.59–7.49 (m, 1H), 7.23–7.17 (m, 5H 13C{1H } NMR (DMSO-d6, 101 MHz) δ 161.6, 154.4, 143.1, 136.1, 134.1, 130.0; HRMS (ESI-TOF) m/z: [MH]+ Calcd
for C7H10IN6 305.0012; Found 305.0007.
1-Carbamimidamido-N-(quinolin-6-yl)methanimidamide hydrochloride (55): The general procedure B was followed using quinolin-6-amine (1.0g,
6.94 mmol) and dicyandiamide (584 mg, 6.95 mmol) in 2.31 mL 3 M HCl at 100 ºC for 36 h. The precipicate was filtered, washed with H2O, and purified by
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trituration (MeOH) provided 50 (802 mg) in 44% yield as a brown solid: 1H NMR (DMSO-d6, 400 MHz) δ 10.05 (s, 1H), 8.81 (dd, J = 4.6, 2.2 Hz, 1H), 8.30
(d, J = 8.4 Hz, 1H), 8.03 – 7.94 (m, 2H), 7.78 (dd, J = 9.0, 2.4 Hz, 1H), 7.55 – 7.48 (m, 1H), 7.44 (s, 2H), 7.17 (s, 2H); 13C{1H } NMR (DMSO-d6, 101 MHz)
δ 161.5, 154.9, 148.6, 144.0, 137.0, 135.7, 128.9, 128.3, 124.8, 121.8, 116.0; HRMS (ESI-TOF) m/z: [M+] Calcd for C11H13N6 229.1202; Found 229.1207.
N-[3,5-Bis(trifluoromethyl)phenyl]-1-carbamimidamidomethanimidamide hydrochloride (56): The general procedure B was followed using 3,5-
bis(trifluoromethyl)aniline (5 mL, 32.0 mmol) and dicyandiamide (2.69 g, 36.1 mmol) in 10.7 mL 3 M HCl at 90 ºC for 21 h. The reaction mixture was cooled
for 2.5 h. The precipitate was then filtered and triturated with Et2O to afford a white powder (5.6 g) in 74%: 1H NMR (DMSO-d6, 400 MHz) δ 10.40 (s, 1H),
8.07 (s, 1H), 7.72 (d, J = 1.9 Hz, 1H), 7.61 (s, 2H), 7.23 (s, 1H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 161.9, 154.0, 141.5, 130.6 (q, 2JCF = 32.6 Hz), 123.2
(q, 1JCF = 272.6 Hz), 119.8 (d, 3JCF = 4.4 Hz), 115.2 (t, 4JCF = 3.8 Hz); HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C10H10F6N5 314.0840; Found 314.0840.
1-Carbamimidamido-N-(3,5-dimethylphenyl)methanimidamide hydrochloride (57): The general procedure B was followed using 3,5-dimethylaniline
(0.187 mL, 1.5 mmol) and dicyandiamide (126 mg, 1.5 mmol) in 0.5 mL 3 M HCl at 90 ºC for 48 h. The precipitate was then filtered to afford a tan solid (284
mg) in 78%: 1H NMR (DMSO-d6, 400 MHz) δ 9.75 (s, 1H), 7.31 (s, 3H), 7.10 (s, 1H), 6.99 (s, 2H), 6.69 (s, 1H), 2.22 (s, 6H); 13C{1H } NMR (DMSO-d6,
101 MHz) δ 161.0, 155.4, 138.5, 137.6, 124.9, 118.5, 21.0; HRMS (ESI-TOF) m/z: [M]+ Calcd for C10H16N5 206.1406; Found 206.1402.
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1-Carbamimidamido-N-(3,4,5-trifluorophenyl)methanimidamide hydrochloride (58): The general procedure
B was followed using 3,4,5-
trifluoroaniline (875 mg, 5.95 mmol) and dicyandiamide (500 mg, 5.95 mmol) in 1.98 mL 3 M HCl and 6.0 mL acetonitrile at 150 ºC for 14 h. The precipitate
was filtered, washed with H2O and purified by trituration with Et2O provided the title compound (1.06 mg, 93% pure) in 67% yield as a light brown solid: 1H
NMR (DMSO-d6, 400 MHz) δ 7.36–7.32 (m, 2H), 7.11–7.07 (m, 5H) 6.34–6.30 (s, 1H); 13C{1H } NMR (DMSO-d6, 101 MHz) 158.1, 156.1, 154.1, 110.6 (d,
2JCF = 23.1 Hz), 104.5 (d, 2JCF = 24.0 Hz); HRMS (ESI-TOF) m/z: [M]+ Calcd for C8H9F3N5 232.0810; Found 232.0818.
1-Carbamimidamido-N-(3,5-difluoro-4-iodophenyl)methanimidamide hydrochloride (59): The general procedure B was followed using 3,5-
difluoro-4-iodoaniline (148 mg, 0.581 mmol) and dicyandiamide (132 mg, 0.581 mmol) in 0.193 mL 3 M HCl and 0.193 mL H2O at 100 ºC for 48 h. The
reaction mixture was concentrated by rotary evaporation and triturated with Et2O to afford a brown solid (91.8 mg) in 42%: 1H NMR (DMSO-d6, 400 MHz) δ
10.29 (s, 1 H), 7.66 (d, J = 10.1 Hz, 1 H), 7.05 (s, 1 H), 6.97 (s, 1 H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 166.2, 161.8 (d, 4JCF = 239.8 Hz), 162.8 (d, 4JCF
= 238.9 Hz), 158.5, 143.0 (d, 2JCF = 13.9 Hz), 102.6 (d, 2JCF = 30.3 Hz); HRMS (ESI-TOF) m/z: [M]+ Calcd for C8H9F2IN5 339.9871; Found 339.9876.
1-Carbamimidamido-N-(3-fluoro-4-iodophenyl)methanimidamide hydrochloride (60): The general procedure B was followed using 3-fluoro-4-
iodoaniline (500 mg, 2.11 mmol) and dicyandiamide (177 mg, 2.11 mmol) in 0.7 mL 3 M HCl and 1.0 mL acetonitrile at 100 ºC for 18 h. The precipitate was
filtered, washed with H2O and purified by trituration with Et2O provided the title compound (406 mg) in 54% yield as a grey purple solid: 1H NMR (DMSO-
d6, 400 MHz) δ 10.29 (s, 1H), 7.71 (dd, J = 8.6, 7.5 Hz, 1H), 7.50 (d, J = 2.4 Hz, 4H), 7.15 (s, 2H), 6.98 (dd, J = 8.7, 2.3 Hz, 1H); 13C{1H } NMR (DMSO-d6,
101 MHz) δ 160.9 (d, 1JCF = 240 Hz), 161.5, 154.3, 141.2 (d, 3JCF = 10.3 Hz), 138.6 (d, 4JCF = 3.4 Hz), 117.8 (d, 4JCF = 2.9 Hz), 107.2 (d, 2JCF = 29 Hz), 73.0 (d,
2JCF = 26.2 Hz); HRMS (ESI-TOF) m/z: [M+Li]+ Calcd for C8H10ClIFN5Li 363.9814; Found 363.3083.
N'-(zaniumylmethanimidoyl)-N-(4-fluorophenyl)-N-methylguanidine chloride (61):The general procedure
B was followed using 4-
fluoromethylaniline (0.5 mL, 4.4 mmol) and dicyandiamide (370 mg, 4.4 mmol) in 1.5 mL 3 M HCl at 90 ºC for 24 h. The reacttion mixture was cooled for 1 h
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at rt. The precipitate was filtered and triturated with Et2O to afford a dark brown solid in 98% yield (992 mg): H NMR (DMSO-d6, 400 MHz) δ 7.43 – 7.34
(m, 2H), 7.31 (d, J = 13.4 Hz, 2H), 7.23 (dd, J = 9.9, 7.7 Hz, 2H), 7.06 (s, 4H, s), 3.26 (s, 3 H); 13C{1H } NMR (DMSO-d6, 101 MHz) δ 163.5, 161.0 (d, 1JCF
=
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