Synthesis and biochemical testing of 3-(carboxyphenylethyl)imidazo[2,1-f] [1,2,4]triazines as inhibitors of AMP deaminase

Article abstract of DOI:10.1021/ml100092a

C-Ribosyl imidazo[2,1-f][1,2,4]triazines and 3-[2-(3-carboxyphenyl)ethyl]- 3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ols represent two classes of known AMP deaminase inhibitors. A combination of the aglycone from the former class with the ribose phosphate mimic from the latter led to the 3-[2-(3-carboxyphenyl)ethyl]imidazo[2,1-f][1,2,4]triazines, which represent a new class of AMP deaminase inhibitors. The best compound, 3-[2-(3-carboxy-5,6,7, 8-tetrahydronaphthyl)ethyl]imidazo[2,1-f][1,2,4]triazine (8), was a good inhibitor of all three human AMPD recombinant isozymes (AMPD1, AMPD2, and AMPD3; IC₅₀ = 0.9-5.7 μM) but a poor inhibitor of the plant recombinant enzyme (Arabidopsis FAC1; IC₅₀ = 200 μM).

Full text of DOI:10.1021/ml100092a

pubs.acs.org/acsmedchemlett
Synthesis and Biochemical Testing of 3-(Carboxyphenylethyl)-
imidazo[2,1-f][1,2,4]triazines as Inhibitors of AMP Deaminase
Stephen D. Lindell,*,† Simon Maechling,† and Richard L. Sabina‡
†
‡
€
Bayer CropScience AG, Werk Hochst, G836, D-65926 Frankfurt am Main, Germany, and Department of Biomedical Sciences,
Oakland University William Beaumont School of Medicine, Rochester, Michigan 48309
ABSTRACT C-Ribosyl imidazo[2,1-f][1,2,4]triazines and 3-[2-(3-carboxyphenyl)-
ethyl]-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ols represent two classes of
known AMP deaminase inhibitors. A combination of the aglycone from the former
class with the ribose phosphate mimic from the latter led to the 3-[2-(3-carboxy-
phenyl)ethyl]imidazo[2,1-f][1,2,4]triazines, which represent a new class of AMP dea-
minase inhibitors. The best compound, 3-[2-(3-carboxy-5,6,7,8-tetrahydronaphthyl)-
ethyl]imidazo[2,1-f ][1,2,4]triazine (8), was a good inhibitor of all three human AMPD
recombinant isozymes (AMPD1, AMPD2, and AMPD3; IC50 = 0.9-5.7 μM) but a poor
inhibitor of the plant recombinant enzyme (Arabidopsis FAC1; IC50 = 200 μM).
KEYWORDS AMP-deaminase, adenylate-deaminase, imidazotriazine, AMPD inhi-
bitor, AMPD inhibition
he synthesis and biological testing of inhibitors of the
enzyme adenosine 50-monophosphate deaminase
T
(AMPD, EC 3.5.4.6) is of interest within both the
agrochemical and the pharmaceutical industries. Inhibition
of AMPD in plants causes an increase in intracellular adenyl-
ate nucleotide concentrations, setting off a chain of physio-
logical events that lead to a strong herbicidal effect.1,2 In
mammals, AMPD inhibition under anoxic conditions leads
to an increased intracellular concentration of adenosine,
which it has been proposed could be beneficial to ischemic
tissue.3,4 Coformycin 50-phosphate (1) is an extremely po-
tent AMPD inhibitor (Ki = 5.5 pM5); however, the physico-
chemical properties of such polar-charged molecules are not
conducive to efficient cellular uptake.3 In an effort to im-
prove bioavailability, Kasibhatla and co-workers succeeded
in replacing the polar ribose phosphate moiety of compound
1 with a much less polar 3-carboxyphenylethyl group, as
exemplified by inhibitors 2 {Ki [porcine heart enzyme
(AMPD3)] = 510 nM6}, 3 [Ki (human AMPD3 recombinant
enzyme) = 15 nM7], and 4 {Ki (human AMPD3 recombinant
enzyme) = 2 nM;7 IC50 [human skeletal muscle enzyme
(AMPD1)] = 500 nM4}. In our own work, we have found that
certain modified C-nucleosides containing pyrazolopyrimi-
dine,8 imidazotriazine,9 and triazolotriazine10,11 aglycones
possess interesting herbicidal activity. It is believed that this
activity is due to inhibition of AMPD, following 50-phosphor-
ylation and covalent hydration of the aglycone ring, which
converts nucleosides of type 5 into the nucleotide inhibi-
tors 6.12 In this paper, we report the synthesis of the novel
AMPD inhibitors 7 and 8, which result from combining the
ribose phosphate mimics from inhibitors 2 and 3 with the
aglycone from our own imidazotriazine inhibitors 5 (R = H;
X=CH).
Received Date: April 30, 2010
Accepted Date: June 9, 2010
Published on Web Date: June 18, 2010
r
2010 American Chemical Society
286
DOI: 10.1021/ml100092a ACS Med. Chem. Lett. 2010, 1, 286–289
|

pubs.acs.org/acsmedchemlett
Scheme 1a
Scheme 2a
a Reagents, conditions, and yields: (i) 1 M NaOH, MeOH, room tem-
perature, 16 h. (ii) Me3SiCH2CH2OH (1.5 equiv), BOP [benzotriazol-l-yl-
oxy-tris-(dimethylamino)-phosphonium hexa-fluorophosphate] (1.2
equiv), Et3N (3.4 equiv), CH2Cl2, room temperature, 16 h (69% for
16; 59% over two steps for 18). TMSE = trimethylsilylethyl.
a Reagents, conditions, and yields: (i) 30% H2O2/H2O, AcOH, 70°C, 5 h,
40%. (ii) NBS (2 equiv), DMF, room temperature, 4 h (85%). (iii) 0.15 M
solution in POCl3, N,N-dimethylaniline (3.5 equiv), reflux, 4 h. (iv) 2 M
NH3/i-PrOH (15 equiv), i-PrOH, 0 °C to room temperature, 16 h (76%
over two steps).
Scheme 3a
Our previous attempts to synthesize inhibitors related to
compounds 7 and 8 had been hampered by the presence of
6- and 8-methylsulfanyl groups in the imidazotriazine ring
system, which interfered with an attempted transition metal-
catalyzed hydrogenation reaction.13 In addition, the car-
boxyl group of the ribose mimic had been protected as a
methyl ester, which we were unfortunately unable to hydro-
lyze at the end of the synthesis without concomitant degra-
dation of the imidazotriazine aglycone. Consequently, in the
current work, we determined to remove the methylsulfanyl
groups early on in the synthesis and to protect the acid as a
more labile trimethylsilylethyl ester. We envisaged accessing
the target molecules 7 and 8 via a convergent route involving
a Heck coupling reaction between a 3-vinylphenycarboxy
ester and a 3-bromoimidazotriazine.
The imidazotriazinone 11 was selected as a promising
sulfur-free starting point for further aglycone elaboration.
This key intermediate can be obtained from the 8-methyl-
sulfanyl imidazotriazine 10, itself accessible in three steps
from the previously described aminotriazine 9,9,14 by
treatment with 30% hydrogen peroxide in acetic acid
(Scheme 1).15 Alternatively, compound 11 can be synthe-
sized in three steps from imidazole 2-carboxylic acid using
the recently published procedure of Babu et al.16 Bromina-
tion of the imidazotriazinone 11 with N-bromosuccinamide
in DMF proceeded in good yield to give the bromide 12.
Chlorination of compound 12 with phosphorus trichloride
oxide and N,N-dimethylaniline yielded the chloride 13,
which was treated with alcoholic ammonia to give the bromo
amine 14 in 76% yield over two steps.
The Heck coupling partners, styrenes 16 and 18, were
simply prepared from the commercially available acid 15
and the previously described methyl ester 1713 as detailed in
Scheme 2. Several different conditions were tried to achieve
the Heck coupling between the styrene 16 and the bromide
14.17,18 Thus, the effect of varying the palladium source
[Pd(OAc)2 and Pd(dba)2], ligand [none, PPh3, and P(o-Tol)3],
base (Et3N, NaHCO3, and K2CO3), and additive (n-Bu4NBr
and n-Bu4NCl) was investigated. The best conditions used
palladium acetate, trio-tolylphosphine, triethylamine, and
tetra-n-butylammonium chloride to give the desired coupling
a Reagents, conditions, and yields: (i) 16 or 18 (3 equiv), Pd(OAc)2 (0.1
equiv), (o-Tol)3P (0.2 equiv), Et3N (3 equiv), n-Bu4NCl (1 equiv), Me2N-
CO Me, 100 °C, 16 h (39% for 19; 41% for 20). (ii) H2, 10% Pd/C, MeOH/
3
DMF 1:1, room temperature, 16 h (66% for 21; 24% for 22). (iii)
n-BuONO (3 equiv), 1,4-dioxane, 80 °C, 5 h (49% for 23); n-BuONO
(10 equiv), 1,4-dioxane, 100 °C, 2 h (38% for 24). (iv) (a) CsF (1.1 equiv),
DMF, 60 °C, 4 h; (b) 4 M HCl/1,4-dioxane (1.1 equiv), 1,4-dioxane (99%
for 7 and 8). TMSE = trimethylsilylethyl.
product 19 in 39% yield (Scheme 3). Repeating the reaction
but with the tetrahydronaphthyl styrene 18 yielded the
coupling product 20 in 41% yield. Subsequent hydrogena-
tion over palladium on charcoal gave the amines 21 and 22,
which were reductively deaminated using n-butylnitrite in
dioxane19 to give the esters 23 and 24. Finally, the trimethyl-
silylethyl esters were cleaved using cesium fluoride followed
byacidification to give the targetacids 7 and 8 in quantitative
yield. Compounds 7 and 8 are actually pro-inhibitors of
AMPD, which need to undergo covalent hydration to give
the proposed active inhibitors 7a and 8a. Enthalpy of hydra-
tion calculations indicated that the imidazotriazine ring sys-
tem should readily undergo covalent addition of water,11 and
NMR experiments conducted on the previously reported9
parent heterocycle 25 provided experimental support for
the required hydration. The proton NMR spectrum of the
r
2010 American Chemical Society
287
DOI: 10.1021/ml100092a ACS Med. Chem. Lett. 2010, 1, 286–289
|

pubs.acs.org/acsmedchemlett
Table 1. Inhibition of Different AMPD Isozymes by Compounds
7 and 8
SUPPORTING INFORMATION AVAILABLE Experimental
details for the synthesis and characterization of compounds 7, 8,
11-14, 18, 20, 22, and 24; protocol describing the preparation of
the Arabidopsis ΔV211M recombinant enzyme; and procedure
used for the AMP deaminase assay and IC50 value determinations.
This material is available free of charge via the Internet at http://
pubs.acs.org.
IC50 (μM)
human AMPD1
human
human
plant AMPD
inhibitor
(ΔM54)20
AMPD2 (1A/2)21 AMPD3 (1b)22 (ΔV211M)
7
8
310
5.7
100
2.3
370
0.9
1400
200
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. Tel: þ49 69 305 22574. Fax: þ49 69 30517768. E-mail:
stephen.lindell@bayercropscience.com.
heterocycle 25 in CDCl3 showed that it existed entirely in the
10π aromatic form 25, whereas in D2O it existed as a 95:5
mixture of 25 and the covalent hydrate 25a. The addition of
1 equiv of DCl to the D2O solution shifted the equilibrium
entirely over in favor of the hydrate 25a.
ACKNOWLEDGMENT The authors would like to acknowledge
Erika Buell for her technical assistance in the IC50 determinations.
REFERENCES
(1)
Dancer, J. E.; Hughes, R. G.; Lindell, S. D. Adenosine-50-
phosphate Deaminase: A Novel Herbicide Target. Plant Phy-
siol. 1997, 114, 119–129.
(2)
Sabina, R. L.; Paul, A.-L.; Ferl, R. J.; Laber, B.; Lindell, S. D.
Adenine Nucleotide Pool Perturbation is a Metabolic Trigger
for AMP Deaminase Inhibitor-Based Herbicide Toxicity. Plant
Physiol. 2007, 143, 1752–1760.
(3)
(4)
Erion, M. D.; Kasibhatla, S. R.; Bookser, B. C.; van Poelje, P. D.;
Reddy, M. R.; Gruber, H. E.; Appleman, J. R. Discovery of AMP
Mimetics that Exhibit High Inhibitory Potency and Specificity
for AMP Deaminase. J. Am. Chem. Soc. 1999, 121, 308–319.
Borkowski, T.; Orlewska, C.; Slominska, E. M.; Yuen, A.;
Lipinski, M.; Rybakowska, I.; Foks, H.; Kaletha, K. K.; Yacoub,
M. H.; Smolenski, R. T. Pharmacological Inhibition of AMP-
Deaminase in Rat Cardiac Myocytes. Nucleosides, Nucleotides
Nucleic Acids 2008, 27, 867–871.
Frieden, C.; Kurz, L. C.; Gilbert, H. R. Adenosine Deaminase
and Adenylate Deaminase: Comparative Kinetic Studies with
Transition State and Ground State Analogue Inhibitors. Bio-
chemistry 1980, 19, 5303–5309.
Kasibhatla, S. R.; Bookser, B. C.; Probst, G.; Appleman, J. R.;
Erion, M. D. AMP Deaminase Inhibitors. 3. SAR of 3-(Car-
boxyarylalkyl)coformycin Aglycon Analogues. J. Med. Chem.
2000, 43, 1508–1518.
Kasibhatla, S. R.; Bookser, B. C.; Xiao, W.; Erion, M. D. AMP
Deaminase Inhibitors. 5. Design, Synthesis and SAR of a
Highly Potent Inhibitor Series. J. Med. Chem. 2001, 44, 613–
618.
Biochemical testing of compounds 7 and 8 for inhibition of
plant Arabidopsis recombinant AMPD and the human AMPD1,
AMPD2, and AMPD3 recombinant isozymes showed inter-
esting differences in inhibition levels (Table 1). In particular,
both compounds were found to be significantly better
inhibitors of all three human isozymes than of plant AMPD.
This selectivity seems reasonable, bearing in mind that the
ribose mimics in inhibitors 7 and 8 come from a research
program directed toward discovering inhibitors of human
AMPD.3,6,7 As expected, by analogy with the literature
inhibitors 2, 3, and 4,7 compound 8 was a more powerful
inhibitor than compound 7. The inhibition levels shown by
compound 8 againstour human AMPD1 (IC50 = 5.7 μM) and
AMPD3 (IC50 = 900 nM) recombinant isozymes are around
an order of magnitude higher than the values reported for
the related inhibitor 4 against the human skeletal muscle
enzyme (AMPD1; IC50 = 500 nM)4 or inhibitor 3 against the
human AMPD3 recombinant enzyme (Ki = 15 nM).7 This
suggests that the hydrated imidazotriazine ring system in
compound 8a binds less strongly to AMPD than the diaze-
pinol ring system in compounds 3 and 4. Nonetheless, the
levels of inhibition achieved in Table 1, taken together with
the improved hydrolytic stability of the imidazotriazine ring
system (as compared to the diazepinol in compounds
1-4),23 mean that inhibitor 8 is an interesting lead structure
worthy of further investigation.
(5)
(6)
(7)
(8)
(9)
Lindell, S. D.; Moloney, B. A.; Hewitt, B. D.; Earnshaw, C. G.;
Dudfield, P. J.; Dancer, J. E. The Design and Synthesis of
Inhibitors of Adenosine 50-Monophosphate Deaminase.
Bioorg. Med. Chem. Lett. 1999, 9, 1985–1990.
Dudfield, P. J.; Le, V.-D.; Lindell, S. D.; Rees, C. W. Synthesis of
C-Ribosyl Imidazo[2,1-f][1,2,4]triazines as Inhibitors of Ade-
nosine and AMP Deaminases. J. Chem. Soc., Perkin Trans. 1
1999, 2929–2936.
(10) Dudfield, P. J.; Le, V.-D.; Lindell, S. D.; Rees, C. W. Synthesis of
C-Ribosyl 1,2,4-Triazolo[3,4-f][1,2,4]triazines as Inhibitors of
Adenosine and AMP Deaminases. J. Chem. Soc., Perkin Trans.
1 1999, 2937–2942.
(11) Bojack, G.; Earnshaw, C. G.; Klein, R.; Lindell, S. D.; Lowinski,
C.; Preuss, R. Design and Synthesis of Inhibitors of Adenosine
and AMP Deaminases. Org. Lett. 2001, 3, 839–842.
(12) Maechling, S; Lindell, S. D. Covalent CdN Bond Hydration in
Heteroaromatic Compounds: Chemical and Biological As-
pects. Targets Heterocycl. Syst. 2006, 10, 66–90.
In summary, the first examples of AMPD inhibitors com-
bining an imidazotriazine ring with a carboxyphenylethyl
ribose mimic, compounds 7 and 8, have been successfully
synthesized in a convergent manner that would be suitable
for analogue synthesis. Biochemical studies indicate that
compound 8 is a good inhibitor of human AMPD3 (IC50
=
900 nM) but only a very modest inhibitor of the plant
enzyme (IC50 = 200 μM).
r
2010 American Chemical Society
288
DOI: 10.1021/ml100092a ACS Med. Chem. Lett. 2010, 1, 286–289
|

pubs.acs.org/acsmedchemlett
(13) Kirkman, J. K.; Lindell, S. D.; Maechling, S.; Slawin, A. M. Z.;
Moody, C. J. Synthesis of 3-(Carboxyarylalkyl)imidazo[2,1-f]-
[1,2,4]triazines as Potential Inhibitors of AMP Deaminase.
Org. Biomol. Chem. 2008, 6, 4452–4459.
(14) Tzeng, C.-C.; Motola, N. C.; Panzica, R. P. Synthesis of certain
5,6-diamino-as-triazines: Precursors for fused heterocyclic
systems. J. Org. Chem. 1983, 48, 1271–1275.
(15) Tominaga, Y.; Matsuoka, Y.; Oniyama, Y.; Uchimura, Y.;
Komiya, H.; Hirayama, M.; Kohra, S.; Hosomi, A. Polarized
Ethylenes. IV. Synthesis of Polarized Ethylenes Using Thioa-
mides and Methyl Dithiocarboxylates and Their Application
to Syntheses of Pyrazoles, Pyrimidines, Pyrazolo[3,4-d]pyri-
midines and 5-Aza[2.2.3]cyclazines. J. Heterocycl. Chem.
1990, 27, 647–660.
(16) Babu, Y. S.; Chand, P.; Kotian, P. L.; Kumar, S. Therapeutic
Agents. PCT Int. Appl. WO 2010/014930 A2.
(17) Heck, R. F. Palladium-catalyzed Vinylation of Organic Ha-
lides. Org. React. 1982, 27, 345–390.
(18) Jeffrey, T. On the Efficiency of Tetraalkylammonium Salts in
Heck Type Reactions. Tetrahedron 1996, 52, 10113–10130.
(19) Nair, V.; Chamberlain, S. D. Reductive Deamination of Ami-
nopurine Nucleosides. Synthesis 1984, 401–402.
(20) Mahnke-Zizelman, D. K.; Sabina, R. L. Localization of
N-Terminal Sequences in Human AMP Deaminase Isoforms
That Influence Contractile Protein Binding. Biochem. Biophys.
Res. Commun. 2001, 285, 489–495.
(21) Haas, A. L.; Sabina, R. L. Expression, Purification and Inhibi-
tion of In-vitro Proteolysis of Human AMPD2 (Isoform L)
Recombinant Enzymes. Protein Expression Purif 2003, 27,
293–303.
(22) Mahnke-Zizelman, D. K.; D'Cunha, J.; Wojnar, J. M.; Brogley,
M. A.; Sabina, R. L. Regulation of Rat AMP Deaminase 3
(Isoform C) by Development and Skeletal Muscle Fibre Type.
Biochem. J. 1997, 326, 521–529.
(23) Bzowska, A.; Lassota, P.; Shugar, D. Phosphorylation of Cofor-
mycin and 20-Deoxycoformycin, and Substrate and Inhibitor
Properties of the Nucleosides and Nucleotides in Several
Enzyme Systems. Z. Naturforsch. 1985, 40c, 710–714.
r
2010 American Chemical Society
289
DOI: 10.1021/ml100092a ACS Med. Chem. Lett. 2010, 1, 286–289
|