Structural sweet spot for A1 adenosine receptor activation by truncated (N)-methanocarba nucleosides: Receptor docking and potent anticonvulsant activity

Article

pubs.acs.org/jmc

Structural Sweet Spot for A₁Adenosine Receptor Activation by

Truncated (N)-Methanocarba Nucleosides: Receptor Docking and

Potent Anticonvulsant Activity

Dilip K. Tosh,^†,§Silvia Paoletta,^†,§Francesca Deﬂorian,^†Khai Phan,^†Steven M. Moss,^†Zhan-Guo Gao,^†

Xiaohui Jiang,^‡and Kenneth A. Jacobson*^,†

^†Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney

Diseases, and ^‡Anticonvulsant Screening Program, Oﬃce of Translational Research, National Institute of Neurological Disorders and

Stroke, National Institutes of Health, Bethesda, Maryland 20892, United States

S

* Supporting Information

ABSTRACT: A₁adenosine receptor (AR) agonists display antiischemic and

antiepileptic neuroprotective activity, but peripheral cardiovascular side eﬀects

impeded their development. SAR study of N⁶-cycloalkylmethyl 4′-truncated (N)-

methanocarba-adenosines identiﬁed 10 (MRS5474, N⁶-dicyclopropylmethyl, K_i=

47.9 nM) as a moderately A₁AR-selective full agonist. Two stereochemically

deﬁned N⁶-methynyl group substituents displayed narrow SAR; groups larger than

cyclobutyl greatly reduced AR aﬃnity, and those larger or smaller than cyclopropyl

reduced A₁AR selectivity. Nucleoside docking to A₁AR homology model

characterized distinct hydrophobic cyclopropyl subpockets, the larger “A” forming

contacts with Thr270 (7.35), Tyr271 (7.36), Ile274 (7.39), and carbon chains of

glutamates (EL2) and the smaller subpocket “B” forming contacts between TM6

and TM7. 10 suppressed minimal clonic seizures (6 Hz mouse model) without

typical rotarod impairment of A₁AR agonists. Truncated nucleosides, an appealing

preclinical approach, have more druglike physicochemical properties than other

A₁AR agonists. Thus, we identiﬁed highly restricted regions for substitution around N⁶suitable for an A₁AR agonist with

anticonvulsant activity.

xtracellular adenosine acts through four subtypes of G-

protein-coupled adenosine receptors (ARs), i.e., at A₁-,

conformation, depending on the fusion position of the

cyclopropane ring. This replacement, which is highly preferred

in AR binding over the isomer of the opposite South (S)

conformation, maintains or enhances aﬃnity at A₁- and A₃ARs

with respect to the ribosides, but decreases aﬃnity at A_2AAR.¹⁶

Another widely explored modiﬁcation, truncation at the 4′-

carbon, i.e. removal of the 5′-group, is generally tolerated in

A₃AR binding for various chemical series, including 4′-thio

(e.g., 1a, Chart 1) and (N)-methanocarba analogues (e.g.,

2a).^17,18However, the observed eﬀects of this truncation on the

relative eﬃcacy of nucleoside derivatives to activate ARs are

variable for the diﬀerent AR subtypes and chemotypes. For

example, truncation in the 4′-thio adenosine series tends to

produce nucleosides that antagonize the A₃AR and in some

cases may additionally agonize the A_2AAR, such as 1b.¹⁹

Truncation in the (N)-methanocarba-adenosine series results in

nucleosides that typically lose 2 orders of magnitude of aﬃnity

at the A₁- and A_2AAR, depending on the N⁶-substituent and

display a wide range of eﬃcacies at the A₁- and A₃AR, e.g. A₃AR

antagonist 2a.^18,20Focusing on the neuroprotective A₁AR, we

scanned a wide range of N⁶-substituents known to enhance

E

A_2A-, A_2B-, and A₃AR subtypes.¹Endogenous adenosine begins

to activate the A₁AR at low concentrations (∼10 nM) to induce

cytoprotective and anti-ischemic functions. Full or partial

agonists of the A₁AR are being considered for treatment of

various conditions, including seizures, stroke, diabetes,

cardioprotection, and cardiac arrhythmias.²⁻⁴A₁AR agonists

are highly neuroprotective in ischemic^5,6and epileptic⁷⁻⁹

models. A₁AR agonists are also being explored for anti-

depressant,¹⁰antianxiety,¹¹and other neuropsychiatric eﬀects.

A₁AR agonists are also useful for pain,¹²due to their

presynaptic action to decrease the release of excitatory

neurotransmitters in the brain.¹³However, peripheral cardio-

vascular side eﬀects have prevented the introduction of A₁AR

agonists for treating disorders of the central nervous system

(CNS).¹⁴

Structural modiﬁcation of adenosine to achieve selectivity in

activating one or more AR subtypes has centered on the

adenine C2- and N⁶-positions and on the ribose moiety.^1,15

Modiﬁcations of the ribose moiety, such as 5′-amides and

alternate carbocyclic ring systems, are especially useful for

enhancing AR aﬃnity. For example, replacement of the ribose

tetrahydrofuryl ring with a methanocarba ([3.1.0]-

bicyclohexane) ring system can enforce a North (N)-envelope

Received: July 5, 2012

Published: August 24, 2012

This article not subject to U.S. Copyright.

Published 2012 by the American Chemical

Society

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Chart 1. Structures of Representative 4′-Truncated Adenosine Analogues and Their Receptor Binding Aﬃnities in the Series of

a

4′-Thioribosides (1a,b) and Ring-Constrained (N)-Methanocarba Nucleosides (2a,b)

a

K_ivalues in nM in binding to the hARs are indicated.^17,18,20,44

A₁AR aﬃnity²¹and concluded that only certain N⁶-cyclo-

alkylmethyl and dicycloalkylmethyl groups (e.g., 2b) maintain

selectivity and agonist eﬃcacy at this AR subtype.²⁰

A goal of this study was to characterize the structure−activity

relationship (SAR) of an expanded set of closely related and

stereochemically deﬁned N⁶-cycloalkylmethyl and dicycloalkyl-

methyl 4′-truncated (N)-methanocarba adenosine derivatives

as human (h) A₁AR agonists. Both binding and eﬃcacy studies

were performed on the novel derivatives, and selected

nucleosides (including ribosides for comparison) were

examined in in vivo anticonvulsant models.

15) or cycloalkyl group (16−19) or a phenyl ring (20, 21).

The stereochemistry at the methynyl carbon was clearly

deﬁned. A few previously reported N⁶-unsubstituted (3) and

N⁶-alkyl/cycloalkyl derivatives (4−6, 8, and 9)²⁰were included

for comparison in the biological assays. This ﬁne-tuning at the

N⁶-position was followed by several modiﬁcations at the C2-

position, incorporating 2-H, 2-iodo, and 2-hydrazino sub-

stitutions (37, 40). Additionally, 2-pyrazolyl substitutions (42,

43) were patterned after a set of A₁AR selective riboside

agonists reported by Elzein et al.²³

The synthetic route to the truncated 2-chloro derivatives

involved nucleophilic displacement by the appropriate amine of

a 6-chloroadenine group in an 2′,3′-isopropylidene-protected

precursor 22a (Scheme 1). A 2-chloro substitution of the

adenine ring has been shown to increase aﬃnity at either or

both A₁AR and A₃AR, and in some cases, to alter AR eﬃcacy.²⁴

Finally, while maintaining constant the most eﬀective N⁶-

substituent dicyclopropylmethyl, substitution of the C2-

position was varied by the synthetic routes shown in Scheme

1 (2-iodo, 37b) and Scheme 2 (2-H, 37a; 2-hydrazino, 40; 2-

pyrazolo, 42, 43). The attempted synthesis of the 2-hydrazino

derivative 40 by acid treatment to remove the isopropylidene

group of the protected intermediate 39 resulted in decom-

position. However, its preparation by hydrazine treatment of

the unprotected nucleoside 10 was successful. Condensation of

hydrazine compound 39 with methyl 2-formyl-3-oxopropionate

under reﬂux conditions²³gave the pyrazole derivative 41, which

underwent an acid hydrolysis in the presence of Dowex 50 to

provide compound 42. Hydrolysis of the methyl ester of

compound 42 in the presence of 1 N NaOH aﬀorded acid

derivative 43.

Pharmacological Evaluation. Binding aﬃnity at three

hAR subtypes was measured in assays using standard agonist

radioligands (47, [³H]R-PIA; 48, [³H]CGS21680; 49, [¹²⁵I]I-

AB-MECA) and membrane preparations from Chinese hamster

ovary (CHO) cells (A₁AR and A₃AR) or human embryonic

kidney (HEK) 293 cells (A_2AAR) stably expressing a hAR

subtype (Table 1).¹⁸Because activity within the class of (N)-

methanocarba nucleosides was previously noted to be very

weak or absent at the hA_2BAR,^16,25we did not include this

receptor in the initial pharmacological screen. Known potent

A₁AR agonists, i.e., ribosides (44a, CPA; 44b, CCPA; 45,

NECA; 46, ADAC), were included for comparison.²⁰

Finally, receptor homology modeling and ligand docking

were used to gain insight into the structural basis for AR

recognition and activation in this series of closely related N⁶-

derivatives. The modeling was based on an agonist-bound

A_2AAR X-ray structure recently reported by Xu et al.²²The

interaction of ligands with the G_i-coupled A₁AR was compared

to the G_i-coupled A₃AR, at which the aﬃnity in this nucleoside

series was also dramatically modulated. Pharmacological

properties have been related to speciﬁc binding site

interactions, especially in a small and sterically restricted region

of the hydrophobic pocket, where the N⁶-group has been

proposed to reside. The loss of interactions in the ribose 5′-

region was compensated by structural characteristics of a few

N⁶-substituents in the 4′-truncated analogues. Thus, the present

study had three objectives: to more clearly deﬁne the narrow

structural limits for binding and full activation of the A₁AR

within the N⁶-cycloalkylmethyl series, to correlate these

ﬁndings with molecular modeling based on an X-ray structure

of the A_2AAR, and to characterize anticonvulsant activity known

to be associated with the A₁AR in the brain. Because the

requirements for A₁AR selectivity and full agonist eﬃcacy in

this series were found to be highly restricted, we identiﬁed a

structural sweet spot within the SAR of 4′-truncated nucleo-

sides.

RESULTS

■

Chemical Synthesis. In an eﬀort to increase A₁AR aﬃnity

and selectivity, we explored a set of N⁶-substitutions of 4′-

truncated (N)-methanocarba adenosines (Table 1), initially

containing 2-chloro, that expanded upon our previous

communication on an overlapping series of N⁶-modiﬁed

truncated nucleosides.²⁰The present series included sub-

stitution of a N⁶-methynyl group that was either chiral or

achiral, e.g. 7, 12−21, including the N⁶-dicyclopropylmethyl

derivative 10 (associated previously with moderate A₁AR

selectivity) and the N⁶-dicyclopentylmethyl 11 derivative. In

many cases, a N⁶-cyclopropylmethyl group was further

substituted on the α-methynyl carbon with an acyclic (12−

In the series of 2-chloro derivatives, the A₁AR aﬃnity (K_i≥

50 nM) of 5′-truncated (N)-methanocarba adenosine deriva-

tives (7−21) was often greater than the aﬃnity at the A₃AR (K_i

typically ≥500 nM). The N⁶-dicyclopropylmethyl derivative 10

was the most potent in binding to the A₁AR with a K_ivalue of

47.9 nM and 10-fold selectivity compared to the A₃AR.²⁰

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Table 1. In Vitro Potency of a Series of Truncated (N)-Methanocarba Adenosine Derivatives in Binding to Three Subtypes of

hARs and Relative Eﬃcacy at hA₁AR

a

Using CHO or HEK293 (A_2Aonly) cells stably expressing a hAR (Supporting Information); aﬃnity was expressed as K_ivalue (n = 3−5) or percent

inhibition of radioligand binding at 10 μM. The radioligands used were [³H]-N⁶-R-phenylisopropyladenosine 47, [³H]-2-[p-(2-carboxyethyl)phenyl-

ethylamino]-5′-N-ethylcarboxamidoadenosine 48, or [¹²⁵I]-N⁶-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide 49, respectively, unless

noted. Compounds 3−6 and 8−10 were prepared previously.⁴⁴6 is a diastereomeric mixture. Maximal eﬃcacy (at 10 μM) in an A₁AR functional

b

c

d

assay unless noted was determined by inhibition of forskolin-stimulated cyclic AMP production in AR-transfected CHO cells, expressed as percent

inhibition (mean standard error, n = 3−5) in comparison to eﬀect (100%) of full agonist 44a at 10 μM. ND, not determined Tosh et al.²⁰or Gao

e

et al.²¹Gao et al.⁵⁹Klotz et al.²⁵Muller and Jacobson⁶⁰Klutz et al.⁶¹Functional assay in guanine nucleotide binding. Structures as shown:

f

g

h

i

j

̈

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a

Scheme 1. Synthesis of N⁶-Substituted 4′-Truncated Derivatives in the Ring-Constrained (N)-Methanocarba Adenosine Series

a

Intermediate 22a was prepared as described.⁴⁴Reagents and conditions: (a) RNH₂, Et₃N, MeOH, rt; (b) Dowex 50, MeOH/H₂O, rt. Compound

24 was used for the following step without isolation.

Nevertheless, cycloalkyl derivative 7 was nonselective, and a

cyclopropyl/ethyl derivative 12 displayed considerable aﬃnity

(K_i= 150 nM) in A₃AR binding. In comparing pairs of pure

diastereoisomers diﬀering only in the chirality of the α-

methynyl carbon of the N⁶-group, compounds 12, 17, 19, and

20 were more potent in A₁AR aﬃnity than the opposite

isomers, i.e., 13, 16, 18, and 21, respectively. N⁶-Cyclopropyl/

isopropylmethyl diastereoisomers 14 and 15 diﬀered by only 2-

fold in A₁AR binding aﬃnity. The novel N⁶-(S)-

((cyclopropylcyclobutyl)methyl) derivative 17 was 23-fold

selective for human A₁AR (K_i= 120 nM) in comparison to

A₃AR and 53-fold in comparison to A_2AAR. Other potent

analogues in hA₁AR binding were (R)-cyclopropyl/ethyl 12 (K_i

= 68 nM) and (R)-cyclopropyl/isopropyl 14 (K_i= 76 nM)

derivatives. Compounds 14 and 15, closely related in structure

to 10 except for lacking a bond to close one of the cyclopropyl

rings, were also moderately selective for the A₁AR. Analogues

containing a phenyl (20, 21) group on the N⁶-methynyl

substituent bound weakly at the three ARs, and a dicyclopentyl

analogue (11) inhibited less than 50% binding at 10 μM.

By introducing subtle structural changes, we attempted to

indirectly characterize the environment of the receptor binding

site surrounding each substituent on the chiral or prochiral N⁶-

methynyl carbon atom. A graphical comparison of the most

potent truncated adenosine derivatives arranged by rank order

of hA₁AR binding aﬃnity, and illustrating hA₁AR/hA₃AR

selectivity, is shown in Figure 1. We delineated separate binding

preferences for the N⁶-methynyl substituents, i.e., R²and R³in

Table 1, and the conformational eﬀects of these groups.

Compounds 10, 12, 14, 13, 17, and 15 displayed similar A₁AR

aﬃnities in the range of 50−150 nM, but diﬀered in degree of

hA₁AR/hA₃AR selectivity. Dimethyl analogue 8 was an A₃AR

ligand (K_i= 12 nM) with moderate selectivity in comparison to

A₁AR; diethyl analogue 9 showed comparable aﬃnity at A₁and

A₃ARs. When R³was enlarged from a 3-membered to a 4-

membered ring (17), A₁AR aﬃnity was better preserved than

when the same change occurred at R²(16). Reducing the

rigidity of R³better preserved the A₁AR selectivity than

reducing the rigidity of R²(cf. 12−15). Introduction of a

methylene group that separates a cyclopropyl substituent from

the N⁶-methynyl atom (18 and 19) did not maintain A₁AR

aﬃnity. Thus, the optimal R³substituent, i.e., cyclopropyl,

could be reduced in size or rigidity to ethyl or isopropyl with

only a minor reduction of A₁AR aﬃnity. Also, an enlargement

of R³to cyclobutyl reduced aﬃnity by only 2.5-fold. However,

when the R²substituent was made smaller than the cyclopropyl

ring, a more substantial loss of A₁AR aﬃnity occurred.

Therefore, these two subpockets surrounding the N⁶-

substituent had slightly diﬀerent steric requirements. A greater

than one bond deviation or C-atom alteration from the size of

cyclopropyl either dramatically reduced the AR aﬃnity or the

A₁AR selectivity.

Replacement of 2-Cl on compound 10 with 2-H in 37a was

tolerated at the A₁AR, but aﬃnity at the A₃AR increased 28-

fold. Substitution of 2-chloro of 10 with iodo greatly reduced

aﬃnity and selectivity in 37b. Eﬀorts to combine the N⁶-

dicyclopropylmethyl group with large C2 substituents based on

the C2-pyrazolyl derivatives, known to be compatible with

A₁AR aﬃnity, produced only weak nonselective ligands 42 and

43. The 2-hydrazino derivative 40 was also nonselective, and 10

remained the optimal A₁AR-selective structure.

Functional data determined at a single concentration (10

μM) in an assay of adenylate cyclase (A₁AR-induced inhibition

of cyclic AMP production in CHO cells stably expressing the

receptor²⁶) are reported in Table 1. The potent and selective

agonist 44a was used as the standard full agonist, and the

nonselective AR agonist 5′-N-ethylcarboxamidoadenosine 45

was also a full agonist in this assay. Most of the N⁶-

cycloalkylmethyl analogues were partial agonists of the A₁AR.

However, a concentration−response analysis for 10 and 45 in

A₁AR-mediated inhibition of cyclic AMP indicated that EC₅₀

values were 40.7 19.7 and 10.2 3.3 nM, respectively, which

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Scheme 2. Substitution of the C2-Position of Truncated Nucleosides Containing N⁶-Dicyclopropylmethyl Substitution, i.e., the

a

Most Eﬀective for Preservation of hA₁AR Binding Aﬃnity, Selectivity, and Eﬃcacy

a

Reagents and conditions: (a) RNH₂, DIPEA, i-PrOH, reﬂux; (b) Dowex 50, MeOH/H₂O, rt; (c) hydrazine, reﬂux; (d) methoxycarbonyl

malondialdehyde, EtOH, reﬂux; (e) 1 N NaOH, MeOH, rt.

was in close agreement with their A₁AR binding aﬃnities.

Compound 13 was of intermediate maximal eﬃcacy (57.9% of

full agonist) at the A₁AR based on results at a single saturating

concentration. Other truncated analogues that proved to be of

low eﬃcacy in activation of the A₁AR, but still within the range

of 30−40%, were 12, 14, and 16 (for which aﬃnity at the A₁AR

exceeded other AR subtypes). Curiously, a phenyl/cyclopropyl

analogue 20 did not activate the A₁AR at 10 μM, a

concentration that exceeded its K_ivalue by 13-fold, suggesting

possible antagonism. Compound 10 was tested for functional

activity in stimulation of adenylate cyclase through the hA_2BAR

expressed in CHO cells and was found to be nearly inactive

(13.7 4.4% of activity of full agonist 45 at 10 μM). Thus, the

A₁AR selectivity of 10 was maintained within the entire AR

family.

reported homology models of the hA₁- and hA₃ARs,²⁰built

using a recently reported agonist-bound A_2AAR crystallographic

structure (PDB ID: 3QAK) as a template,²²were used to

perform docking simulations of each of the compounds 8−21.

In the A_2AAR crystal structure, the agonist ligand contained a

bulky N⁶-substituent, which opened EL3 to allow binding of

other N⁶-substituted nucleosides. The thermostabilized agonist-

bound A_2AAR structure of Lebon et al.,²⁷in which EL3 is closer

to EL2 and which contains mutations in the transmembrane

(TM) region, including the ribose binding site, was not used as

a template. In our previous study, the docking pose of

compound 10 at the hA₁AR was compared with that of 5′-N-

ethylcarboxamidoadenosine 45.²⁰Here we give a more detailed

description of the binding mode of this new series of truncated

(N)-methanocarba nucleosides in terms of their aﬃnity and

selectivity proﬁles.

Molecular Modeling. The binding at ARs of the newly

synthesized truncated (N)-methanocarba nucleosides was also

evaluated through molecular modeling studies. Previously

A docking pose of compound 10 inside the hA₁AR binding

site (Figure 2, panel A) featured most of the main receptor−

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Figure 1. Graphical comparison of the most potent truncated adenosine derivatives arranged by rank order of hA₁AR binding aﬃnity (ranging from

48 to 270 nM). The lower plots represent the hA₁AR selectivity in comparison to the hA₃AR and maximal eﬃcacy at the hA₁AR (data in Table 1).

ligand interactions observed in the agonist-bound hA_2AAR

crystal structures.^22,27These interactions, involving both the

adenine core and the ribose ring, were noted in the docking

poses of this new series of truncated (N)-methanocarba

nucleosides at both the hA₁- and hA₃ARs. In particular, the

3′- and 2′-hydroxyl groups formed H-bonds with residues

(using a GPCR numbering convention²⁸) at positions 7.42

(Thr277 in hA₁AR and Ser271 in hA₃AR) and 7.43 (His278 in

hA₁AR and His272 in hA₃AR), respectively. The side chain of

asparagine 6.55 (Asn254 in hA₁AR and Asn250 in hA₃AR)

strongly interacted with these compounds through two H-

bonds involving the 6-amino group and the N⁷atom of the

adenine ring. Moreover, the adenine core was anchored inside

the binding site by a π−π stacking interaction with a

phenylalanine in EL2 (Phe171 in hA₁AR and Phe168 in

hA₃AR) and strong hydrophobic contacts with leucine 6.51

(Leu250 in hA₁AR and Leu246 in hA₃AR) and isoleucine 7.39

(Ile274 in hA₁AR and Ile268 in hA₃AR).

Two key interactions observed for the crystallographic poses

of 45-like agonists (5′-N-ethylcarboxamido derivatives) at the

hA_2AAR were necessarily missing in these truncated derivatives.

In the hA_2AAR X-ray structures, Thr88 (3.36) and His250

(6.52) coordinated, through H-bonding interactions, the 5′-

CO-NH-alkyl groups of the cocrystallized agonists 45 and 6-

(2,2-diphenylethylamino)-9-((2R,3R,4S,5S)-5-(ethylcarbamo-

yl)-3,4-dihydroxytetrahydrofuran-2-yl)-N-(2-(3-(1-(pyridin-2-

yl)piperidin-4-yl)ureido)ethyl)-9H-purine-2-carboxamide (50,

UK-432097).^22,27The threonine at position 3.36 is conserved

among all four AR subtypes, while the histidine at position 6.52

is conserved in hA_2A-, hA_2B-, and hA₁ARs, but is substituted

with a serine in the hA₃AR. Therefore, these conserved residues

were predicted to also be important in agonist binding at the

hA₁AR. The diﬀerence at position 6.52 could additionally be

related to the diﬀerent behavior of the truncated ring-

constrained nucleosides at A₁- and A₃ARs, as previously

hypothesized.²⁰

Moreover, the hydrophilic region of the receptor associated

with ribose binding is key to the activation process, which likely

involves essential residues of TM3, TM6, and TM7 throughout

the AR family, as observed in the A_2AAR.^22,27Thus, the loss of

the 5′-substituent is expected to aﬀect AR eﬃcacy, which it

clearly does at the A₃AR, as observed for other previously

reported truncated (N)-methanocarba nucleosides.³¹However,

the eﬀect at the A₁AR is highly variable with relative maximal

eﬃcacies of this series ranging from low to high (80−100% in

compounds 5 and 10).

The structural basis for the full agonism of 10, in contrast to

closely related compounds that have greatly reduced eﬃcacy at

this subtype, is diﬃcult to identify but might be related to some

interactions formed at the entrance of the binding site, i.e., the

N⁶-binding region, that are crucial in orienting and stabilizing

the compound inside the cavity.

A detailed analysis of ligand interactions with the upper

region of the binding site has helped to clarify the aﬃnity and

selectivity proﬁles of this new series of truncated (N)-

methanocarba derivatives. As shown in Figure 2 (panel B),

the orientation of the N⁶-dicyclopropylmethyl substituent of

compound 10 allowed us to identify two distinct upper

subpockets in the hA₁AR. The larger of the two subpocket,

designated “A”, corresponds to the main entrance of the

binding site and accommodated one cyclopropyl ring to form

hydrophobic contacts with Thr270 (7.35), Tyr271 (7.36), and

Ile274 (7.39) and the carbon chains of Glu170 (EL2) and

Glu172 (EL2). The other cyclopropyl ring of 10 perfectly ﬁts a

smaller side subpocket, designated “B”, which was located

between TM6 and TM7 and delimited by Leu253 (6.54),

Thr257 (6.58), Thr270 (7.35), and at the bottom by Leu250

(6.51). Subpocket A corresponds also to the region that

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Figure 2. (A) Side view and (B) top view of the docking pose of compound 10 (in magenta) inside the binding site of the hA₁AR model. Side chains

of some amino acids important for ligand recognition and H-bonding interactions are highlighted. Hydrogen atoms are not displayed. The Connolly

surface of some amino acids surrounding the binding site is displayed. Surface color indicates hydrophobic regions (green), mildly polar regions

(blue) and H-bonding regions (magenta). The boundaries of the two identiﬁed subpockets are highlighted (larger subpocket A in yellow and smaller

subpocket B in orange). R²(Table 1) is predicted to occupy subpocket A, and R³is predicted to occupy subpocket B.

accomodates the extended N⁶and C2 groups of 50 in the

A_2AAR crystal structure.

(7.35), respectively, giving rise to again a diﬀerent scenario.

Consequently, at the hA₃AR there was no side pocket B

between TM6 and TM7 able to accommodate a cyclopropyl

ring, and this determined a diﬀerent orientation of the N⁶-

substituent inside the cavity (Figure 3). In fact, the upper

region of the hA₃AR binding site was overall more hydrophobic

as compared to that of the hA₁AR, but its shape was more

suitable to accommodate unbranched hydrophobic substitu-

ents. Due to the more diﬃcult ﬁt of the branched N⁶-group in

this region, the position of compound 10 was slightly shifted

within the hA₃AR binding site and so it established weaker

interactions with the key residues deeper in the cavity. This

ﬁnding can explain the low aﬃnity for the hA₃AR of the present

set of (N)-methanocarba nucleosides bearing α-branched N⁶-

With respect to this N⁶-region, docking results showed some

diﬀerences between hA₁- and hA₃ARs in the binding of these

new truncated (N)-methanocarba derivatives at ARs that could

explain their diﬀerent aﬃnity proﬁles at these receptors. In fact,

the orientation and the interactions of the N⁶-substituents were

particular for each receptor subtype, mainly due to diﬀerences

between the residues present in the upper region of the binding

site. In particular, three residues delimiting subpocket B in the

hA₁AR are substituted in the hA₃AR with amino acids bearing

bulkier side chains, namely Ile249 (6.54), Ile253 (6.58), and

Leu264 (7.35). While at the hA_2AAR, these residues are

substituted with Ile252 (6.54), Thr256 (6.58), and Met270

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Figure 3. Top view of the docking pose of compound 10 (in magenta) inside the binding site of the hA₃AR model. Side chains of amino acids at the

entrance of the binding site are highlighted and their Connolly surface is displayed. Surface color indicates hydrophobic regions (green), mildly polar

regions (blue), and hydrogen-bonding regions (magenta). Hydrogen atoms are not displayed.

substituents, with aﬃnity generally decreasing with an increase

in volume of the groups on the branches. This view is also

consistent with the enhanced hA₃AR aﬃnity associated with

reported truncated (N)-methanocarba analogs having un-

branched hydrophobic substituents, such as benzyl, at the N⁶-

position (also compare compounds 5 and 6).²⁰

possesses higher aﬃnity at the receptor (e.g., 19, cyclo-

propylmethyl group in R², as compared to 18, cyclo-

propylmethyl group in R³).

Anticonvulsant Testing. Three A₁AR agonists were

examined in models of electrically and chemically induced

seizures (Table 2).²⁹The anticonvulsant activity of 2-chloro-

Overall, increasing or decreasing the size of the groups on the

α-carbon from cyclopropyl decreased hA₁AR aﬃnity. Thus, the

steric hindrance of these larger groups disfavored the precise

conformational arrangement necessary to occupy the two

subpockets. Pocket A, although larger in its opening to the

extracellular side of the receptor, did not tolerate deviation

from R²= cyclopropyl, while pocket B could accommodate R³

= cyclobutyl or ethyl with some cost in aﬃnity.

Table 2. Anticonvulsant Activity in Mice of A₁AR Agonists

behavioral

toxicity TD₅₀,

mg/kg

b

6 Hz model MES model

scMET model

a

compd

ED₅₀, mg/kg (dose, mg/kg) (dose, mg/kg)

d

c

10

>30

2.74

1/4 (3)

no protection

(3)

c

44b

46

0.84

0.12

0.03

1/4 (1)

4/8 (2)

1/4 (1)

5/8 (1)

e

0.14

Moreover, docking results and conformational analysis of the

dihedral N⁶-Cα bond angles showed that the preferred binding

conformation at the hA₁AR for this series of truncated (N)-

methanocarba nucleosides placed the hydrogen atom on the α-

carbon pointing toward TM7, even though an alternative

conformation, with the hydrogen oriented toward TM5, was

also found (Figure S1, Supporting Information). Therefore, in

its preferred conformation, the R³group on the Cα occupied

the smaller subpocket B, while the R²group was located in the

larger subpocket A. Depending on the groups attached to the

α-carbon and their steric complementarity with the two

subpockets, two opposite diastereoisomers bound with diﬀerent

strength to the receptor binding site and so possessed diﬀerent

binding aﬃnity. In fact, if a small group (e.g., ethyl) with low

complementarity with the larger subpocket A was present on

the Cα, then the most potent diastereoisomer was the one that

can more easily accommodate that group in subpocket B

(compound 12, ethyl group in R³, as compared to compound

13, ethyl group in R²). On the other hand, if an extended group

(e.g., cyclopropylmethyl) was present on the Cα, then the

isomer able to locate this group in the larger subpocket A

a

b

Administered ip. Qualitative results, expressed as number of animals

c

protected from convulsions/total number tested. Measured at 1 h

(time of peak of eﬀect) postinjection, dose range for 10 was 0.75−10

mg/kg. No rotarod toxicity at 30 mg/kg. Measured at 4 h (time of

d

e

peak of eﬀect) post injection.

N⁶-cyclopentyladenosine (44b, CCPA) was studied previ-

ously,^7,9and 44b was included in the present study as a potent

reference A₁AR agonist that does not distinguish between

central and peripheral action. N⁶-[4-[[[4-[[[(2-Aminoethyl)-

amino]carbonyl]methyl]anilino]carbonyl]methyl]phenyl]-

adenosine (46, ADAC) is a potent A₁AR agonist that has been

shown to be neuroprotective in various models.^6,30Full agonist

10 displayed eﬃcacy in a seizure model in mice without the

toxicity observed in the active dose range (tested up to 30 mg/

kg, ip). Other A₁AR agonists began to show toxicity, i.e., the

side eﬀects evident in the rotarod performance test (i.e.,

inability to remain on the rotarod) overlapping the active dose

range.

The 6 Hz minimal clonic seizure model is an acute

electroshock seizure test that produces a seizure with a limbic

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Table 3. Summary of the Characteristics of the Two Pockets in A₁AR Surrounding Substituents of the N⁶-Methynyl Carbon

Atom, Based on Observed SAR and Predictions from Molecular Modeling (see Table 1 for deﬁnition of R²and R³)

characteristic

pocket A

pocket B

size

larger (extends upward, but has narrow dimensions

smaller

near N⁶-methynyl atom)

location

TM7−EL2

between TM6 and TM7

a

contact residues

Thr270 (7.35), Tyr271 (7.36), Ile274 (7.39), Glu170 Leu253 (6.54), Thr257 (6.58), Thr270

(EL2), Glu172 (EL2)

site of PhCH₂binding

yes

(7.35), Leu250 (6.51)

b

correspondence to N⁶-group of 47

site of CH₃binding

c

present in A₃AR?

no

R³

correspondence to R (assuming most favorable docking with R²

N⁶−C-H toward TM7)

preferred group

c-Pr

yes

accommodates group smaller than c-Pr? (Et)

accommodates group less rigid than c-Pr? (i-Pr)

accommodates group larger than c-Pr? (c-Bu)

no (exact steric complementarity is important)

no

d

no

yes (only c-Bu)

a

b

c

The residue numbering convention of Ballesteros and Weinstein is used.²⁸47, (R)-N⁶-phenylisopropyladenosine. Consistent with lower aﬃnity

d

of N⁶-α-branched analogues at the A₃AR. No clear explanation from modeling, except that the dimensions near the N⁶-methynyl atom might be

sterically restrictive. A larger group is tolerated, with intermediate aﬃnity, only if there is an α-CH₂spacer (19).

phenotype and displays a unique pharmacological proﬁle to

established antiepileptic drugs. In particular, the 6 Hz seizure is

uniquely sensitive to the antiepileptic drug levetiracetam and

partially resistant to the Na⁺channel blockers.³¹Three A₁AR

agonists, the two reference compounds 44b and 46 and the

truncated derivative 10, displayed eﬃcacy in the 6 Hz model.

The activities of 10 and 44b were quantiﬁed at 1 h

postadministration of the compound, which was also equal to

the time of peak eﬀect (TPE). Complete dose−response curves

using ﬁve diﬀerent doses each of 10, 44b, and 46 indicated

ED₅₀values of 2.74, 0.12, and 0.03 mg/kg, respectively.

Compound 10 protected in four out of eight mice at 1.5 mg/kg.

Compared to the activity in the 6 Hz model, these compounds

had no or minimal eﬀect in the traditional seizure models, the

maximal electroshock (MES) model, or the subcutaneous

metrazol (pentylenetetrazol) model (scMET) at the same

dosing range.

In the minimal behavioral toxicity test using the rotarod, the

three compounds showed signiﬁcant diﬀerences. The toxicity of

44b was observed roughly in the same dose range (0.84 vs 0.12

mg/kg) as its protection in the 6 Hz model and at the same

time point (1 h postinjection). Animals became lethargic and

were unable to stay on the rotarod following ip drug

administration, as exhibited with other A₁AR agonists.³²For

compound 10, no toxicity (zero out of eight mice) was

observed at all doses tested up to 30 mg/kg, the highest dose

tested, which was nearly completely protective (seven out of

eight animals) in the 6 Hz model. Therefore, the therapeutic

window for 10 appeared to be superior to that of 44b.

Compound 10 was also tested in the corneal kindled mouse

model to examine its eﬀect on focal seizures. In a qualitative

new analogues on the previous observation that 10 had 10-fold

A₁AR selectivity in comparison to the A₃AR and 74-fold of

selectivity vs A_2AAR. Moreover, this analogue was a full agonist

at the hA₁AR. The ﬁne-tuning of the structure of 10 now

indicates that even minor adjustments of the structure cause a

loss of potency, selectivity, or eﬃcacy at the hA₁AR.

Our binding results at the hA₁AR showed that only Cl was

preferred at the adenine C2-position, and N⁶-substituents other

than dicyclopropylmethyl displayed inferior pharmacological

proﬁles. Separate substituents (R²and R³) of the Cα methynyl

group had distinct and very narrow SAR requirements. Groups

that were much larger or smaller than cyclopropyl were not

compatible with A₁AR aﬃnity, selectivity, and eﬃcacy.

Therefore, we describe the compound with optimal pharmaco-

logical properties, compound 10, as a structural sweet spot for

potent and selective activation of the A₁AR.

Molecular docking studies of these truncated (N)-meth-

anocarba nucleosides at the hA₁AR highlighted how a precise

complementarity in the N⁶-region was needed to determine a

good aﬃnity and selectivity proﬁle and to compensate for the

missing anchoring eﬀect of the ribose 5′-region. In fact, a

detailed modeling analysis of the upper part of the hA₁AR

binding site predicted two diﬀerent subpockets, A and B, able

to accommodate the N⁶-substituents of these derivatives, and it

seemed that an optimal occupancy of both subpockets was

required for enhanced aﬃnity at this receptor subtype. In

particular, the smaller subpocket B can readily accommodate up

to a cyclobutyl group, while subpocket A can ﬁt substituents

that are extended in the direction of EL2. The steric restriction

of the subpockets can be the reason for the null aﬃnity at

hA₁AR of compound 11 bearing two cyclopentyl groups, as

they are too bulky to ﬁt in either pocket. On the other hand,

smaller groups on the α-carbon of the N⁶-substituent, such as

methyl or diethyl (compounds 8 and 9, respectively), even

though they can occupy the pockets, possess lower

complementarity as compared to the cyclopropyl group and

consequently have no selectivity and lower aﬃnity at the A₁AR.

The identiﬁcation of these two subpockets and the binding

conformation proposed here are also in agreement with the

binding data of others known A₁AR agonists.²¹For example,

the moderately selective A₁AR agonist (R)-(−)-N⁶-(2-

phenylisopropyl)adenosine (47) is more potent than the

corresponding opposite isomer (S-PIA). A hypothetical binding

test, an EC₅₀( SE) of 1.79

0.71 mg/kg (n = 8) was

determined at 1 h postinjection. This unique response proﬁle of

compound 10 makes it as an attractive candidate to treat drug-

resistant epilepsy.

DISCUSSION

■

Our previous closely related communication²⁰demonstrated

that the aﬃnity of truncated ring-constrained analogues, in

comparison to ribosides, was less well preserved at the A₁AR

than at the A₃AR. However, certain analogues, most notably the

dicyclopropylmethyl analogue 10, were relatively well preserved

in binding and activation of the A₁AR. In this study, we built

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mode for these adenosine derivatives, similar to the one

proposed for our new rigid, truncated nucleosides, would

readily place the phenylmethyl group of the R isomer in the

larger subpocket A and the methyl group in the smaller

subpocket B. Thus, there is a close correspondence of our new

ﬁndings to the previously explored preference for N⁶-Cα-

branched R isomers in comparison to the corresponding S

isomers.³³A summary of the characteristics of the two pockets

surrounding substituents of the N⁶-methynyl carbon atom is

provided in Table 3.

cardiovascular eﬀects. In previous studies of the activity of

A₁AR agonists in the CNS, only a small fraction of a

peripherally administered agent crossed the blood−brain

barrier (BBB).³⁸However, a similar attempt to alter the

biodistribution by removing the 2′-hydroxyl group of 44a did

not enhance brain uptake.³⁵

The peripheral side eﬀects of exogenously administered

A₁AR agonists that do not distinguish between the brain and

periphery have impeded the development of such agents for the

treatment of epileptic seizures. Recently, it was proposed that

the antiseizure eﬀect of endogenous adenosine could be

boosted indirectly by inhibiting formation of the neurabin-

RGS4 complex for “ﬁne-tuning adenosine receptor function in

the nervous system”.³⁹Other approaches to solve this problem

involved the design of ligands that favor the CNS over the

periphery, such as N⁶-[R-(2-benzothiazolyl)thio-2-propyl]-2-

chloroadenosine (NNC-21-0136, K_d= 1.16 nM at rat A₁AR),

which showed eﬃcacy in in vivo stroke models and were

reduced in their accompanying cardiovascular side eﬀects.⁵

Prodrug approaches and localized adenosine delivery have also

been explored.^12,40

The physicochemical properties of nucleosides that act as AR

agonists often lead to limited in vivo bioavailability and reduced

passage across the BBB. The cLog P of compound 10 is 1.41

(more favorable than cLog P of 0.14 for A₁AR-selective riboside

and prototypical agonist 44a), with the optimal for small

molecular pharmaceutical substances being typically 2−3.⁴¹

Also, the total polar surface area (tPSA) for 10 and 44a are

calculated to be 92.8 and 122 Å², respectively. Most druglike

small molecules have a PSA smaller than 120 Å²; thus, 10 is also

preferred by this criterion. Compound 10 has fewer hydroxyl

groups than 44a, which would favor bioavailability in brain, and

the molecular weight of 10 (376 Da) is comfortably within the

most desirable range for pharmaceuticals.⁴¹Therefore, by

several criteria 10 is more druglike than 44a.

We speculate on the basis for the apparent lack of peripheral

side eﬀects of compound 10 up to 30 mg/kg. Both 44b and 10

evidently enter the bloodstream and pass the BBB into the

brain. Although 44b was ∼58-fold more potent than 10 in

binding at the A₁AR, 10 had better physicochemical properties

(cLogP, tPSA, H-bond donors, molecular weight), so it might

penetrate the BBB better than 44b. The bioavailability in the

brain of peripherally administered 10, i.e. whether its altered

physicochemical properties may facilitate its passage across the

BBB, is undetermined. Normally a drug travels from the

bloodstream into brain, such that when the A₁AR in brain is

activated, the A₁AR in heart should be activated within the

same dose range as with 44b. For 10, the fact that no adverse

eﬀects were observed even at 30 mg/kg (ten times the ED₅₀)

suggests that the free concentration of 10 in plasma might be

reduced relative to standard A₁AR agonists, possibly due to

plasma protein binding. If the free drug concentration in blood

would be lower, its ability to cross the BBB may remain

unaﬀected or even increased. This phenomenon has been

observed for a number of CNS drugs.⁴²This hypothesis could

be tested by pharmacokinetic and plasma protein binding

assays.

Complementary anchoring of the N⁶-substituent inside the

two subpockets seems important in orienting and stabilizing the

compound inside the cavity. It could also help in keeping the

adenine-methanocarba moiety in an eﬃcacious active con-

formation, able to form strong H-bonds and hydrophobic

interactions with residues in TM6, TM7, and EL2, while

compensating for the missing interactions due to the lack of a

5′-substituent.

In in vivo testing, compound 10 was the only A₁AR agonist

examined here that displayed a clear separation of anti-

convulsant activity and toxicity. For example, a prototypical

A₁AR agonist 44b displayed toxicity in the rotarod assay (two

out of eight animals) at a dose of 0.1 mg/kg. At a dose of 0.5

mg/kg 44b, three out of eight animals displayed toxicity.

Another known A₁AR agonist, 46, was more potent than 44b in

both in vivo anticonvulsant activity and toxicity, but it also

failed to demonstrate a separation of the two activities.

The unique response proﬁle of compound 10 (inactive in

MES and scMET, active in 6 Hz and corneal kindling models)

and its novel mechanism of action through the A₁AR make it a

potential candidate to treat drug-resistant epilepsy. Traditional

antiepileptic drugs (AEDs) carbamazepine, lamotrigine,

phenytoin and topiramate are Na⁺channel blockers, which

have strong eﬃcacy in the MES model.³⁴They are either

inactive or only partially eﬃcacious in the 6 Hz model. On the

other hand, newer AEDs having diﬀerent mechanisms of action,

such as levetiracetam and retigabine, are potent and eﬃcacious

in the 6 Hz model, which makes the 6 Hz model a model for

identifying compounds that potentially target drug-resistant

epilepsy.³¹The kindling models are useful in searching for

drugs to treat complex partial seizures, because kindled seizures

not only provide an experimental model of focal seizures but

also a means of testing drugs to stop seizure spread and

generalization from a focus.³⁵The corneal kindled mouse

model demonstrates a pharmacological proﬁle consistent with

the traditional hippocampal kindled rat model, while it requires

no implantation surgery and less compound quantity for

testing.³⁶The unique activities of compound 10 in the 6 Hz

and corneal kindled mouse model and its overcoming the

limitations of other A₁AR agonists (i.e., clear separation of

anticonvulsant activity and toxicity) make it an attractive AED

candidate for additional testing.

Many of the eﬀorts to develop A₁AR agonists for peripheral

applications, such as treating cardiac arrhythmias, have tried to

limit central nervous system (CNS) penetration to avoid

centrally mediated side eﬀects. Conversely, other envisioned

applications of A₁agonists, i.e., for neurodegenerative and

neurological disorders, depend on brain entry. A₁AR agonists

have distinct neuroprotective and antinociceptive proper-

ties,^5,6,12and activation of the A₁AR by endogenous adenosine

mediates the protective eﬀects of fractalkine/CX3CL1.³⁷

However, the clinical development of previous generations of

such agents has been limited by side eﬀects, including

A₁AR agonists hold interest therapeutically for their cardio-

and neuroprotective, antiarrhythmic, antiseizure, antilipolytic,

antiglaucoma, and anxiolytic actions. Some of the novel

derivatives were partial A₁AR agonists, which are of interest

for both cerebroprotective and cardiovascular application,

depending on levels of endogenous adenosine and on receptor

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reserve.^3,4,43It is conceivable that the expanded range of

physical properties in the present series of truncated derivatives

would oﬀer pharmacokinetic advantages. Therefore, this

approach is appealing for preclinical development. This

hypothesis will have to be evaluated in further in vivo studies.

raphy. The resulting compound 24 was dissolved with methanol (0.6

mL) and water (0.3 mL). Dowex 50 (4 mg) was added to the solution

and stirring continued at room temperature overnight. After complete

reaction of the starting material, the reaction mixture was ﬁltered and

the ﬁltrate was evaporated under vacuum. The residue was puriﬁed by

ﬂash silica gel column chromatography (CH₂Cl₂:MeOH = 30:1) to

1

give the desired compound 11 (3.4 mg, 64%) as a syrup: H NMR

EXPERIMENTAL PROCEDURES

(CD₃OD, 400 MHz) δ 8.17 (s, 1H), 4.80 (s, 1H), 4.71 (t, J = 6.0 Hz,

1H), 4.40 (t, J = 6.8 Hz, 1H), 3.91 (d, J = 6.4 Hz, 1H), 2.20−1.18 (m,

1H), 1.98−1.96 (m, 1H), 1.81−1.74 (m, 4H), 1.69−1.53 (m, 10H),

1.42−1.29 (m 4H), 0.92−0.90 (m, 1H), 0.81−0.74 (m, 1H); HRMS

calcd for C₁₉H₂₅ClN₅O₂(M + H)⁺390.1697, found 390.1708.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((R)-1-cyclopropylpropylamino)-

9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (12). Dowex 50 resin (5

mg) was added to a solution of compound 25 (10 mg, 0.024 mmol) in

methanol (0.5 mL) and water (0.5 mL) and the mixture stirred at

room temperature overnight. The reaction mixture was ﬁltered on a

Celite bed, the ﬁltrate was evaporated under vacuum, and the residue

was puriﬁed by ﬂash silica gel column chromatography

(CH₂Cl₂:MeOH = 30:1) to give the desired compound 12 (6.1 mg,

■

Chemical Synthesis. General Methods. All reagents and solvents

(regular and anhydrous) were of analytical grade, obtained from

commercial suppliers and used without further puriﬁcation. All amines

were purchased from Asiba Pharmatech (Edison, NJ), except 2,2-

dicyclopropylethylamine, which was obtained from Ryan Scientiﬁc,

Inc. (Mount Pleasant, SC), and dicyclopropylmethylamine, which was

obtained from J&W PharmLab (Levittown, PA). Compounds 22a and

35 were synthesized as reported.⁴⁴Reactions were conducted under an

atmosphere of nitrogen whenever anhydrous solvents were used. All

reactions were monitored by thin-layer chromatography (TLC) using

silica gel coated plates with a ﬂuorescence indicator which were

visualized (a) under UV light, (b) by dipping in a mixture of

anisaldehyde (2.5 mL)/concentrated H₂SO₄(5 mL)/methanol (425

mL), or (c) by dipping the plate in a solution of ninhydrin (0.3 g in

100 mL EtOH, containing AcOH, 1.3 mL) followed by heating. Silica

gel column chromatography was performed with silica gel (SiO₂, 200−

400 mesh, 60 Å) using moderate air pressure. Evaporation of solvents

was carried out under reduced pressure at a temperature below 50 °C.

After column chromatography, appropriate fractions were pooled,

evaporated, and dried at high vacuum for at least 12 h to give the

desired products in high purity. ¹H NMR spectra were recorded with a

Bruker 400 MHz NMR spectrometer. Chemical shifts are reported in

parts per million (ppm) relative to tetramethylsilane or using

deuterated solvent as the internal standard (δH: CDCl₃7.26 ppm).

ESI-high resolution mass spectroscopic (HRMS) measurements were

performed on a proteomics-optimized Q-TOF-2 (Micromass-Waters)

using external calibration with polyalanine. Observed mass accuracies

are those expected on the basis of the known performance of the

instrument as well as the trends in masses of standard compounds

observed at intervals during the series of measurements. Reported

masses are observed masses uncorrected for this time-dependent drift

in mass accuracy. TLC analysis was carried out on glass sheets

precoated with silica gel F₂₅₄(0.2 mm) from Sigma-Aldrich (St. Louis,

MO). The purity of ﬁnal nucleoside derivatives was checked using a

Hewlett−Packard 1100 HPLC equipped with a Zorbax SB-Aq 5 μm

analytical column (50 × 4.6 mm; Agilent Technologies Inc., Palo Alto,

CA). The mobile phase was a linear gradient solvent system of 5 mM

TBAP (tetrabutylammonium dihydrogenphosphate)−CH₃CN from

80:20 to 0:100 in 13 min; the ﬂow rate was 0.5 mL/min. Peaks were

detected by UV absorption with a diode array detector at 230, 254,

and 280 nm. All derivatives tested for biological activity showed >95%

purity by HPLC analysis (detection at 254 nm).

1

69%) as a syrup: H NMR (CD₃OD, 400 MHz) δ 8.15 (s, 1H), 4.79

(s, 1H), 4.71 (t, J = 5.6 Hz, 1H), 3.89 (d, J = 6.8 Hz, 1H), 3.66 (br s,

1H), 2.00−1.97 (m, 1H), 1.84−1.65 (m, 3H), 1.34−1.28 (m, 2H),

1.01 (t, J = 7.6 Hz, 3H), 0.80−0.74 (m, 1H), 0.60−0.58 (m, 1H),

0.45−0.42 (m, 2H), 0.36−0.32 (m, 1H); HRMS calcd for

C₁₇H₂₃ClN₅O₂(M + H)⁺364.1540, found 364.1538.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((S)-1-cyclopropylpropylamino)-

9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (13). Dowex 50 resin (3

mg) was added to a solution of compound 26 (7 mg, 0.017 mmol) in

methanol (0.3 mL) and water (0.3 mL) and the mixture stirred at

room temperature overnight. The reaction mixture was ﬁltered on a

Celite bed, the ﬁltrate was evaporated under vacuum, and the residue

was puriﬁed by ﬂash silica gel column chromatography

(CH₂Cl₂:MeOH = 30:1) to give the desired compound 13 (4.3 mg,

1

68%) as a syrup: H NMR (CD₃OD, 400 MHz) δ 8.15 (s, 1H), 4.79

(s, 1H), 4.71 (t, J = 5.6 Hz, 1H), 3.89 (d, J = 6.8 Hz, 1H), 3.67 (br s,

1H), 2.02−1.98 (m, 1H), 1.86−1.59 (m, 3H), 1.32−1.24 (m, 2H),

1.01 (t, J = 7.6 Hz, 3H), 0.78−0.76 (m, 1H), 0.59−0.57 (m, 1H),

0.45−0.42 (m, 2H), 0.34−0.32 (m, 1H); HRMS calcd for

C₁₇H₂₃ClN₅O₂(M + H)⁺364.1540, found 364.1535.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((R)-1-cyclopropyl-2-methylpropy-

lamino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (14). Dowex 50

resin (7 mg) was added to a solution of compound 27 (10.71 mg,

0.025 mmol) in methanol (0.6 mL) and water (0.5 mL) and the

mixture stirred at room temperature overnight. The reaction mixture

was ﬁltered on a Celite bed, the ﬁltrate was evaporated under vacuum,

and the residue was puriﬁed by ﬂash silica gel column chromatography

(CH₂Cl₂:MeOH = 35:1) to give the desired compound 14 (7.82 mg,

1

82%) as a syrup: H NMR (CD₃OD, 400 MHz) δ 8.17 (s, 1H), 4.79

(s, 1H), 4.71 (t, J = 5.6 Hz, 1H), 3.89 (d, J = 6.4 Hz, 1H), 3.62 (t, J =

7.2 Hz, 1H), 2.07−1.97 (m, 2H), 1.88−1.64 (m, 1H), 1.36−1.31 (m,

1H), 1.08−1.05 (m, 8H), 0.80−0.74 (m, 1H), 0.66−0.61 (m, 1H),

0.45−0.36 (m, 2H); HRMS calcd for C₁₈H₂₅ClN₅O₂(M + H)⁺

378.1697, found 378.1691.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-(2,2-dicyclopropylethylamino)-

9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (7). Dowex 50 resin (8

mg) was added to a solution of compound 23 (12 mg, 0.027 mmol) in

methanol (0.5 mL) and water (0.5 mL) and the mixture stirred at

room temperature overnight. The reaction mixture was ﬁltered on a

Celite bed, the ﬁltrate was evaporated under vacuum, and the residue

was puriﬁed by ﬂash silica gel column chromatography

(CH₂Cl₂:MeOH = 35:1) to give the desired compound 7 (8.2 mg,

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((S)-1-cyclopropyl-2-methyl-pro-

pylamino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (15). Dowex

50 resin (5 mg) was added to a solution of compound 28 (6.8 mg,

0.016 mmol) in methanol (0.5 mL) and water (0.5 mL) and the

mixture stirred at room temperature overnight. The reaction mixture

was ﬁltered on a Celite bed, the ﬁltrate was evaporated under vacuum,

and the residue was puriﬁed by ﬂash silica gel column chromatography

(CH₂Cl₂:MeOH = 35:1) to give the desired compound 15 (4.8 mg,

1

74%) as a syrup: H NMR (CD₃OD, 400 MHz) δ 8.15 (s, 1H), 4.79

(s, 1H), 4.71 (t, J = 5.6 Hz, 1H), 4.90 (d, J = 6.8 Hz, 1H), 3.72 (d, J =

6.4 Hz, 2H), 2.01−1.96 (m, 1H), 1.71−1.64 (m, 1H), 1.34−1.30 (m

2H), 0.78−0.72 (m, 2H), 0.51−0.42 (m, 5H), 0.28−0.17 (m, 4H);

HRMS calcd for C₁₉H₂₅ClN₅O₂(M + H)⁺390.1697, found 390.1708.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-(dicyclopentylmethylamino)-9H-

purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (11). Dicyclopentylmethyl-

amine (1.0 mg, 0.024 mmol) and triethylamine (0.1 mL, 0.16 mmol)

were added to a solution of compound 22a (4.2 mg, 0.012 mmol) in

methanol (0.8 mL), and the mixture was stirred at room temperature

overnight. The reaction mixture was evaporated under vacuum and the

residue was roughly puriﬁed by ﬂash silica gel column chromatog-

1

81%) as a syrup: H NMR (CD₃OD, 400 MHz) δ 8.17 (s, 1H), 4.79

(s, 1H), 4.71 (t, J = 5.6 Hz, 1H), 3.89 (d, J = 6.4 Hz, 1H), 3.63 (t, J =

7.2 Hz, 1H), 2.09−1.96 (m, 2H), 1.86−1.64 (m, 1H), 1.34−1.30 (m,

1H), 1.08−1.05 (m, 8H), 0.80−0.74 (m, 1H), 0.65−0.61 (m, 1H),

0.45−0.36 (m, 2H); HRMS calcd for C₁₈H₂₅ClN₅O₂(M + H)⁺

378.1697, found 378.1694.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((R)-cyclopropylcyclobutylmethy-

lamino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (16). Dowex 50

8085

dx.doi.org/10.1021/jm300965a | J. Med. Chem. 2012, 55, 8075−8090

Journal of Medicinal Chemistry

Article

resin (4 mg) was added to a solution of compound 29 (5.9 mg, 0.013

mmol) in methanol (0.4 mL) and water (0.4 mL) and the mixture

stirred at room temperature for 7 h. The reaction mixture was ﬁltered

on a Celite bed, the ﬁltrate was evaporated under vacuum, and the

residue was puriﬁed by ﬂash silica gel column chromatography

(CH₂Cl₂:MeOH = 30:1) to give the desired compound 16 (3.9 mg,

74%) as an oil: ¹H NMR (CD₃OD, 400 MHz) δ 8.15 (s, 1H), 4.79 (s,

1H), 4.71 (t, J = 5.6 Hz, 1H), 3.89 (d, J = 6.4 Hz, 1H), 3.73 (br s, 1H),

2.69−2.64 (m, 1H), 2.10−2.07 (m, 1H), 2.01−1.91 (m, 4H), 1.89−

1.80 (m, 1H), 1.69−1.65 (m, 1H), 1.34−1.30 (m, 3H), 0.95−0.91 (m,

1H), 0.78−0.74 (m, 1H), 0.56−0.53 (m, 1H), 0.40−0.36 (m, 2H);

HRMS calcd for C₁₉H₂₅ClN₅O₂(M + H)⁺390.1697, found 390.1711.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((S)-cyclopropylcyclobutylmethy-

lamino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (17). Dowex 50

resin (6 mg) was added to a solution of compound 30 (7.95 mg, 0.018

mmol) in methanol (0.6 mL) and water (0.4 mL) and the mixture

stirred at room temperature for 7 h. The reaction mixture was ﬁltered

on a Celite bed, the ﬁltrate was evaporated under vacuum, and the

residue was puriﬁed by ﬂash silica gel column chromatography

(CH₂Cl₂:MeOH = 30:1) to give the desired compound 17 (5.3 mg,

74%) as an oil: ¹H NMR (CD₃OD, 400 MHz) δ 8.15 (s, 1H), 4.79 (s,

1H), 4.71 (t, J = 5.6 Hz, 1H), 3.89 (d, J = 6.4 Hz, 1H), 3.73 (br s, 1H),

2.69−2.62 (m, 1H), 2.12−2.07 (m, 1H), 2.03−1.88 (m, 4H), 1.89−

1.80 (m, 1H), 1.69−1.65 (m, 1H), 1.34−1.31 (m, 3H), 0.95−0.91 (m,

1H), 0.78−0.76 (m, 1H), 0.56−0.53 (m, 1H), 0.40−0.36 (m, 2H);

HRMS calcd for C₁₉H₂₅ClN₅O₂(M + H)⁺390.1697, found 390.1697.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((R)-1,2-dicyclopropylethylami-

no)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (18). Dowex 50

resin (3 mg) was added to a solution of compound 31 (4.94 mg,

0.018 mmol) in methanol (0.3 mL) and water (0.3 mL) and the

mixture stirred at room temperature for 7 h. The reaction mixture was

ﬁltered on a Celite bed, the ﬁltrate was evaporated under vacuum, and

the residue was puriﬁed by ﬂash silica gel column chromatography

(CH₂Cl₂:MeOH = 35:1) to give the desired compound 18 (3.4 mg,

resin (7 mg) was added to a solution of compound 34 (8.6 mg,

0.019 mmol) in methanol (0.8 mL) and water (0.4 mL) and the

mixture stirred at room temperature for 7 h. The reaction mixture was

ﬁltered on a Celite bed, the ﬁltrate was evaporated under vacuum, and

the residue was puriﬁed by ﬂash silica gel column chromatography

(CH₂Cl₂:MeOH = 30:1) to give the desired compound 21 (6.2 mg,

1

79%) as a syrup: H NMR (CD₃OD, 400 MHz) δ 8.17 (s, 1H), 7.48

(d, J = 7.6 Hz, 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.24 (t, J = 7.2 Hz, 1H),

4.78 (s, 2H), 4.71 (t, J = 5.2 Hz, 1H), 3.88 (t, J = 7.6 Hz, 1H), 2.02−

1.95 (m, 1H), 1.68−1.65 (m, 1H), 1.43−1.30 (m, 2H), 0.79−0.73 (m,

1H), 0.65−0.60 (m, 2H), 0.54−0.47 (m, 2H); HRMS calcd for

C₂₁H₂₃ClN₅O₂(M + H)⁺412.1540, found 412.1544.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-(2,2-dicyclopropylethylamino)-

9H-purin-9-yl)-2,3-O-(isopropylidene)bicyclo[3.1.0]hexane (23). 2,2-

Dicyclopropylethylamine (38 mg, 0.30 mmol) and triethylamine (0.12

mL, 0.84 mmol) were added to a solution of compound 22a (20.91

mg, 0.061 mmol) in methanol (1.5 mL), and the mixture was stirred at

room temperature overnight. The reaction mixture was evaporated

under vacuum and the residue was puriﬁed by ﬂash column

chromatography (hexane:ethyl acetate = 1:1) to give the desired

product 23 (22 mg, 84%) as a syrup: ¹H NMR (CD₃OD, 400 MHz) δ

8.14 (s, 1H), 5.36 (t, J = 6.0 Hz, 1H), 4.97 (s, 1H), 4.69 (d, J = 7.2 Hz,

1H), 3.73 (d, J = 6.4 Hz, 2H), 2.09−2.03 (m, 1H), 1.76−1.71 (m,

1H), 1.52 (s, 3H), 1.25 (s, 3H), 0.95−0.90 (m, 2H), 0.75−0.72 (m,

2H), 0.51−0.42 (m, 5H), 0.28−0.17 (m, 4H); HRMS calcd for

C₂₂H₂₉ClN₅O₂(M + H)⁺430.2010, found 430.2013.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((R)-1-cyclopropylpropylamino)-

9H-purin-9-yl)-2,3-O-(isopropylidene)bicyclo[3.1.0]hexane (25).

(R)-1-Cyclopropylpropylamine hydrochloride (16.16 mg, 0.11

mmol) and triethylamine (0.11 mL, 0.82 mmol) were added to a

solution of compound 22a (20.33 mg, 0.05 mmol) in methanol (1.5

mL), and the mixture was stirred at room temperature overnight. The

reaction mixture was evaporated under vacuum and the residue was

puriﬁed by ﬂash column chromatography (hexane:ethyl acetate = 1:1)

1

78%) as a syrup: H NMR (CD₃OD, 400 MHz) δ 8.15 (s, 1H), 4.79

to give the desired product 25 (18.7 mg, 78%) as a foamy syrup: H

(s, 1H), 4.71 (t, J = 5.6 Hz, 1H), 3.89 (d, J = 6.8 Hz, 1H), 3.80 (br s,

1H), 2.02−1.97 (m, 1H), 1.70−1.61 (m, 2H), 1.34−1.30 (m, 3H),

1.10−1.08 (m, 1H), 0.91−0.76 (m, 2H), 0.62−0.58 (m, 1H), 0.47−

0.36 (m, 4H), 0.17−0.07 (m, 2H); HRMS calcd for C₁₉H₂₅ClN₅O₂

(M + H)⁺390.1697, found 390.1691.

NMR (CD₃OD, 400 MHz) δ 8.13 (s, 1H), 5.36 (t, J = 6.4 Hz, 1H),

4.96 (s, 1H), 4.69 (d, J = 7.2 Hz, 1H), 3.66 (br s, 1H), 2.07−2.04 (m,

1H), 1.86−1.79 (m, 1H), 1.77−1.70 (m, 2H), 1.52 (s, 3H), 1.25 (s,

3H), 1.03−0.99 (m, 4H), 0.95−0.90 (m, 2H), 0.62−0.56 (m, 1H),

0.45−0.41 (m, 2H), 0.36−0.33 (m, 1H); HRMS calcd for

C₂₀H₂₇ClN₅O₂(M + H)⁺404.1853, found 404.1855.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((S)-1,2-dicyclopropylethylami-

no)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (19). Dowex 50

resin (5 mg) was added to a solution of compound 32 (7.6 mg,

0.019 mmol) in methanol (0.5 mL) and water (0.5 mL) and the

mixture stirred at room temperature for 7 h. The reaction mixture was

ﬁltered on a Celite bed, the ﬁltrate was evaporated under vacuum, and

the residue was puriﬁed by ﬂash silica gel column chromatography

(CH₂Cl₂:MeOH = 35:1) to give the desired compound 19 (5.2 mg,

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((S)-1-cyclopropylpropylamino)-

9H-purin-9-yl)-2,3-O-(isopropylidene)bicyclo[3.1.0]hexane (26). (S)-

1-Cyclopropylpropylamine hydrochloride (32.8 mg, 0.11 mmol) and

triethylamine (0.11 mL, 0.82 mmol) were added to a solution of

compound 22a (20.43 mg, 0.05 mmol) in methanol (1.5 mL), and the

mixture was stirred at room temperature overnight. The reaction

mixture was evaporated under vacuum and the residue was puriﬁed by

ﬂash column chromatography (hexane:ethyl acetate = 1:1) to give the

1

76%) as a syrup: H NMR (CD₃OD, 400 MHz) δ 8.15 (s, 1H), 4.79

1

(s, 1H), 4.71 (t, J = 5.6 Hz, 1H), 3.89 (d, J = 6.8 Hz, 1H), 3.80 (br s,

1H), 2.00−1.97 (m, 1H), 1.69−1.61 (m, 2H), 1.34−1.30 (m, 3H),

1.10−1.08 (m, 1H), 0.89−0.75 (m, 2H), 0.60−0.59 (m, 1H), 0.47−

0.36 (m, 4H), 0.17−0.07 (m, 2H); HRMS calcd for C₁₉H₂₅ClN₅O₂

(M + H)⁺390.1697, found 390.1697.

desired product 26 (16.6 mg, 80%) as a syrup: H NMR (CD₃OD,

400 MHz) δ 8.13 (s, 1H), 5.36 (t, J = 6.4 Hz, 1H), 4.96 (s, 1H), 4.69

(d, J = 7.2 Hz, 1H), 3.67 (br s, 1H), 2.09−2.03 (m, 1H), 1.88−1.79

(m, 1H), 1.77−1.72 (m, 2H), 1.52 (s, 3H), 1.25 (s, 3H), 1.03−0.99

(m, 4H), 0.96−0.89 (m, 2H), 0.61−0.56 (m, 1H), 0.47−0.41 (m, 2H),

0.36−0.30 (m, 1H); HRMS calcd for C₂₀H₂₇ClN₅O₂(M + H)⁺

404.1853, found 404.1854.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((R)-1-cyclopropyl-2-methylpropy-

lamino)-9H-purin-9-yl)-2,3-O-(isopropylidene)bicyclo[3.1.0]hexane

(27). (R)-1-Cyclopropyl-2-methylpropylamine hydrochloride (18.22

mg, 0.12 mmol) and triethylamine (0.11 mL, 0.85 mmol) were added

to a solution of compound 22a (20.78 mg, 0.06 mmol) in methanol

(1.5 mL), and the mixture was stirred at room temperature overnight.

The reaction mixture was evaporated under vacuum and the residue

was puriﬁed by ﬂash column chromatography (hexane:ethyl acetate =

1:1) to give the desired product 27 (19 mg, 75%) as a syrup: ¹H NMR

(CD₃OD, 400 MHz) δ 8.15 (s, 1H), 5.36 (t, J = 6.0 Hz, 1H), 4.97 (s,

1H), 4.70 (d, J = 6.4 Hz, 1H), 3.62 (t, J = 7.2 Hz, 1H), 2.08−2.03 (m,

1H), 1.76−1.72 (m, 1H), 1.52 (s, 3H), 1.25 (m, 4H), 1.08−1.05 (m,

7H), 0.96−0.88 (m, 2H), 0.65−0.61 (m, 1H), 0.45−0.36 (m, 2H);

HRMS calcd for C₂₁H₂₉ClN₅O₂(M + H)⁺418.2010, found 418.2004.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((R)-cyclopropylphenylmethyla-

mino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (20). Dowex 50

resin (5 mg) was added to a solution of compound 33 (6.6 mg, 0.014

mmol) in methanol (0.6 mL) and water (0.4 mL) and the mixture

stirred at room temperature for 7 h. The reaction mixture was ﬁltered

on a Celite bed, the ﬁltrate was evaporated under vacuum and the

residue was puriﬁed by ﬂash silica gel column chromatography

(CH₂Cl₂:MeOH = 30:1) to give the desired compound 20 (4.8 mg,

1

80%) as a syrup: H NMR (CD₃OD, 400 MHz) δ 8.16 (s, 1H), 7.48

(d, J = 7.6 Hz, 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.26 (t, J = 7.6 Hz, 1H),

4.78 (s, 2H), 4.69 (t, J = 5.6 Hz, 1H), 3.88 (t, J = 7.2 Hz, 1H), 2.01−

1.95 (m, 1H), 1.68−1.63 (m, 1H), 1.42−1.30 (m, 2H), 0.79−0.73 (m,

1H), 0.65−0.62 (m, 2H), 0.54−0.51 (m, 2H); HRMS calcd for

C₂₁H₂₃ClN₅O₂(M + H)⁺412.1540, found 412.1533.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((S)-cyclopropylphenylmethylami-

no)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (21). Dowex 50

8086

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Journal of Medicinal Chemistry

Article

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((S)-1-cyclopropyl-2-methylpropy-

lamino)-9H-purin-9-yl)-2,3-O-(isopropylidene)bicyclo[3.1.0]hexane

(28). (S)-1-Cyclopropyl-2-methylpropylamine hydrochloride (16.5

mg, 0.12 mmol) and triethylamine (0.12 mL, 0.84 mmol) were

added to a solution of compound 22a (20.85 mg, 0.06 mmol) in

methanol (1.5 mL), and the mixture was stirred at room temperature

overnight. The reaction mixture was evaporated under vacuum and the

residue was puriﬁed by ﬂash column chromatography (hexane:ethyl

acetate = 1:1) to give the desired product 28 (19.8 mg, 78%) as a

0.09−0.07 (m, 1H); HRMS calcd for C₂₂H₂₉ClN₅O₂(M + H)⁺

430.2010, found 430.2002.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((R)-N-cyclopropyl-N-phenylme-

thylamino)-9H-purin-9-yl)-2,3-O-(isopropylidene)bicyclo[3.1.0]-

hexane (33). (R)-N-Cyclopropyl-N-phenylmethylamine hydrochlor-

ide (22 mg, 0.12 mmol) and triethylamine (0.11 mL, 0.84 mmol) were

added to a solution of compound 22a (20.68 mg, 0.06 mmol) in

methanol (1.5 mL), and the mixture was stirred at room temperature

overnight. The reaction mixture was evaporated under vacuum and the

residue was puriﬁed by ﬂash column chromatography (hexane:ethyl

acetate = 1:2) to give the desired product 33 (22.15 mg, 81%) as a

1

syrup: H NMR (CD₃OD, 400 MHz) δ 8.15 (s, 1H), 5.36 (t, J = 6.0

Hz, 1H), 4.97 (s, 1H), 4.71 (d, J = 6.4 Hz, 1H), 3.61 (t, J = 7.2 Hz,

1H), 2.08−2.04 (m, 1H), 1.76−1.71 (m, 1H), 1.51 (s, 3H), 1.25 (m,

4H), 1.08−1.05 (m, 7H), 0.96−0.88 (m, 2H), 0.65−0.61 (m, 1H),

0.45−0.36 (m, 2H); HRMS calcd for C₂₁H₂₉ClN₅O₂(M + H)⁺

418.2010, found 418.2017.

1

syrup: H NMR (CD₃OD, 400 MHz) δ 8.15 (s, 1H), 7.48 (d, J = 7.6

Hz, 2H), 7.33 (t, J = 7.6 Hz, 2H), 2.24 (t, J = 7.2 Hz, 1H), 5.35 (t, J =

6.0 Hz, 1H), 4.95 (s, 1H), 4.69 (d, J = 7.2 Hz, 1H), 3.60 (br s, 1H),

2.06−2.03 (m, 1H), 1.74−1.71 (m, 1H), 1.51 (s, 3H), 1.43−1.37 (m,

1H), 1.24 (s, 3H), 0.94−0.89 (m, 2H), 0.65−0.63 (m, 2H), 0.53−0.46

(m, 2H); HRMS calcd for C₂₄H₂₇ClN₅O₂(M + H)⁺452.1853, found

452.1858.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((R)-cyclopropylcyclobutylmethy-

lamino)-9H-purin-9-yl)-2,3-O-(isopropylidene)bicyclo[3.1.0]hexane

(29). (R)-Cyclopropylcyclobutylmethylamine hydrochloride (28.3 mg,

0.17 mmol) and triethylamine (0.11 mL, 0.81 mmol) were added to a

solution of compound 22a (19.92 mg, 0.05 mmol) in methanol (1.5

mL), and the mixture was stirred at room temperature overnight. The

reaction mixture was evaporated under vacuum and the residue was

puriﬁed by ﬂash column chromatography (hexane:ethyl acetate = 1:1)

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((S)-N-cyclopropyl-N-phenylme-

thylamino)-9H-purin-9-yl)-2,3-O-(isopropylidene)bicyclo[3.1.0]-

hexane (34). (S)-N-Cyclopropyl-N-phenylmethylamine hydrochloride

(32.8 mg, 0.17 mmol) and triethylamine (0.11 mL, 0.84 mmol) were

added to a solution of compound 22a (20.43 mg, 0.06 mmol) in

methanol (1.5 mL), and the mixture was stirred at room temperature

overnight. The reaction mixture was evaporated under vacuum and the

residue was puriﬁed by ﬂash column chromatography (hexane:ethyl

acetate = 1:2) to give the desired product 34 (22.1 mg, 82%) as a

1

to give the desired product 29 (20.8 mg, 83%) as a syrup: H NMR

(CD₃OD, 400 MHz) δ 8.15 (s, 1H), 5.36 (t, J = 6.0 Hz, 1H), 4.96 (s,

1H), 4.69 (d, J = 7.2 Hz, 1H), 3.85 (br s, 1H), 2.72−2.67 (m, 1H),

2.10−2.89 (m, 6H), 1.87−1.71 (m, 2H), 1.52 (s, 3H), 1.25 (s, 3H),

0.96−0.89 (m, 3H), 0.58−0.53 (m, 1H), 0.39−0.36 (m, 3H); HRMS

calcd for C₂₂H₂₉ClN₅O₂(M + H)⁺430.2010, found 430.2018.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((S)-cyclopropylcyclobutylmethy-

lamino)-9H-purin-9-yl)-2,3-O-(isopropylidene)bicyclo[3.1.0]hexane

(30). (S)-Cyclopropylcyclobutylmethylamine hydrochloride (35.8 mg,

0.22 mmol) and triethylamine (0.14 mL, 1.03 mmol) were added to a

solution of compound 22a (25.2 mg, 0.73 mmol) in methanol (1.5

mL), and the mixture was stirred at room temperature overnight. The

reaction mixture was evaporated under vacuum and the residue was

puriﬁed by ﬂash column chromatography (hexane:ethyl acetate = 1:1)

1

syrup: H NMR (CD₃OD, 400 MHz) δ 8.15 (s, 1H), 7.48 (d, J = 7.2

Hz, 2H), 7.33 (t, J = 7.6 Hz, 2H), 2.24 (t, J = 7.2 Hz, 1H), 5.35 (t, J =

6.8 Hz, 1H), 4.95 (s, 1H), 4.69 (d, J = 7.2 Hz, 1H), 3.60 (br s, 1H),

2.06−2.03 (m, 1H), 1.75−1.71 (m, 1H), 1.51 (s, 3H), 1.42−1.37 (m,

1H), 1.24 (s, 3H), 0.96−0.88 (m, 2H), 0.66−0.62 (m, 2H), 0.54−0.43

(m, 2H); HRMS calcd for C₂₄H₂₇ClN₅O₂(M + H)⁺452.1853, found

452.1856.

Molecular Modeling. hA₁AR and hA₃AR Homology Models.

Previously reported molecular models of the hA₁AR and hA₃AR, built

using the alignment and the homology modeling tools implemented in

the program Molecular Operating Environment (MOE),⁴⁵were used

in this study. Both models were built using as template the crystal

structure of the human A_2AAR cocrystallized with the agonist 50 (PDB

ID: 3QAK),²²as described by Tosh et al.²⁰In particular, hA₁AR and

hA₃AR sequences were retrieved from the UniProt database⁴⁶and

aligned against the sequence of the A_2AAR template, taking into

account the highly conserved residues in each TM domain. Then,

homology models were built using the automated Homology

Modeling protocol implemented in the MOE suite.

1

to give the desired product 30 (26 mg, 82%) as a syrup: H NMR

(CD₃OD, 400 MHz) δ 8.13 (s, 1H), 5.36 (t, J = 6.0 Hz, 1H), 4.96 (s,

1H), 4.69 (d, J = 7.2 Hz, 1H), 3.84 (br s, 1H), 2.71−2.67 (m, 1H),

2.09−2.89 (m, 6H), 1.87−1.71 (m, 2H), 1.51 (s, 3H), 1.25 (s, 3H),

0.95−0.88 (m, 3H), 0.58−0.54 (m, 1H), 0.37−0.36 (m, 3H); HRMS

calcd for C₂₂H₂₉ClN₅O₂(M + H)⁺430.2010, found 430.2008.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((R)-1,2-dicyclopropylethylami-

no)-9H-purin-9-yl)-2,3-O-(isopropylidene)bicyclo[3.1.0]hexane (31).

(R)-1,2-Dicyclopropylethylamine hydrochloride (28.4 mg, 0.17 mmol)

and triethylamine (0.11 mL, 0.82 mmol) were added to a solution of

compound 22a (20.0 mg, 0.05 mmol) in methanol (1.5 mL), and the

mixture was stirred at room temperature overnight. The reaction

mixture was evaporated under vacuum and the residue was puriﬁed by

ﬂash column chromatography (hexane:ethyl acetate = 1:1) to give the

desired product 31 (18.8 mg, 75%) as a foamy syrup: ¹H NMR

(CD₃OD, 400 MHz) δ 8.13 (s, 1H), 5.36 (t, J = 6.4 Hz, 1H), 4.96 (s,

1H), 4.69 (d, J = 7.2 Hz, 1H), 3.80 (br s, 1H), 2.06−2.03 (m, 1H),

1.73−1.57 (m, 3H), 1.51 (s, 3H), 1.25 (s, 3H), 1.09−1.06 (m, 1H),

0.94−0.85 (m, 3H), 0.60−0.58 (m, 1H), 0.44−0.35 (m, 5H), 0.17−

0.16 (m, 1H), 0.09−0.07 (m, 1H); HRMS calcd for C₂₂H₂₉ClN₅O₂

(M + H)⁺430.2010, found 430.2025.

Molecular Docking of Truncated Methanocarba Derivatives in

the hA₁AR and hA₃AR Models. Compounds structures were built

using the builder tool implemented in the MOE suite⁴⁸and subjected

to energy minimization using the MMFF94x force ﬁeld, until a rms

gradient of 0.05. The minimized conformations of each compound

were used as a starting point for the docking study. The ﬂexible

docking of the ligands in the hA₁AR and hA₃AR models was

performed by means of the Glide⁴⁷package part of the Schrodinger

̈

suite.⁴⁸

The docking site was deﬁned with key residues in the binding

pocket of the hA₁AR and hA₃AR models, namely Phe(EL2),

Asn(6.55), Trp(6.48) and His(7.43), and a 20 Å × 20 Å × 20 Å

box was centered on those residues. Docking of the ligands was

performed in the rigid binding site of the models with Glide using the

XP (extra precision) procedure.

The top scoring docking conformations were retained and subjected

to the receptor sampling by means of the Reﬁnement module in

Prime.⁴⁹The Prime side chain sampling was performed on all the

residues within a 6 Å of the ligand. The reﬁned model for each ligand

was chosen as the ﬁnal binding conformation.

(1R,2R,3S,4R,5S)-4-(2-Chloro-6-((S)-1,2-dicyclopropylethylami-

no)-9H-purin-9-yl)-2,3-O-(isopropylidene)bicyclo[3.1.0]hexane (32).

(S)-1,2-Dicyclopropylethylamine hydrochloride (31.5 mg, 0.19 mmol)

and triethylamine (0.12 mL, 0.91 mmol) were added to a solution of

compound 22a (22.21 mg, 0.05 mmol) in methanol (1.5 mL), and the

mixture was stirred at room temperature overnight. The reaction

mixture was evaporated under vacuum and the residue was puriﬁed by

ﬂash column chromatography (hexane:ethyl acetate = 1:1) to give the

1

desired product 32 (21.5 mg, 77%) as a syrup: H NMR (CD₃OD,

400 MHz) δ 8.13 (s, 1H), 5.36 (t, J = 6.4 Hz, 1H), 4.96 (s, 1H), 4.69

(d, J = 7.2 Hz, 1H), 3.80 (br s, 1H), 2.07−2.03 (m, 1H), 1.74−1.58

(m, 3H), 1.51 (s, 3H), 1.25 (s, 3H), 1.09−1.05 (m, 1H), 0.95−0.84

(m, 3H), 0.60−0.58 (m, 1H), 0.44−0.35 (m, 5H), 0.17−0.15 (m, 1H),

In Vivo Testing. Animals and Test Substances Used for Seizure

Testing. Adult male CF No. 1 albino mice (26−30 g, 6 Hz; 18−25 g

all other tests) were obtained from Charles River, Portage, MI.

Animals were housed in an Association for Assessment and

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Accreditation of Laboratory Animal Care, International (AAALAC)-

accredited temperature and humidity controlled facility and main-

tained on a standard 12 h:12 h light−dark cycle (lights on at 0600)

with free access to standard laboratory chow (Prolab RMH 3000) and

water ad libitum. All animal experiments were performed in

accordance with the guidelines set by National Institutes of Health

and the University of Utah Institutional Animal Care and Use

Committee (IACUC) committee. All animals were allowed free access

to both food and water except when they were removed from their

cages for the experimental procedure. Except for the kindling studies,

animals were used once. All animals were euthanized in accordance

with the Institute of Laboratory Resources policies on the humane care

of laboratory animals.

slope of the regression line, and the SEM of the slope were then

calculated by a computer program based on the method described by

Finney.⁵⁸

Compound Administration. The test compound was administered

at a concentration that permitted optimal accuracy of dosing without

the volume contributing excessively to total body ﬂuid. Thus, test

compounds are administered to mice in a volume of 0.01 mL/g of

body weight. Compound 10 or other AR agonist was initially dissolved

in DMSO (50 mg/mL) as a stock solution. To prepare the

formulation for testing, an appropriate amount of stock solution was

ﬁrst diluted in DMSO to achieve 10% DMSO (v/v) in the ﬁnal

volume. Then, 30% PEG400 (J. T. Baker) was gradually added to the

aqueous DMSO solution to make the ﬁnal formulation.

Anticonvulsant Tests. In vivo anticonvulsant activity was

established by both electrical and chemoconvulsant seizure tests that

have been described previously.⁵⁰⁻⁵²The electrical tests used were the

maximal electroshock (MES) seizure test, the 6 Hz minimal clonic

seizure test, and the corneal kindled mouse test. The chemical test was

the sc metrazol seizure tests. TPE is deduced from data generated in

initial qualitative test procedures. Five groups of four animals each are

administered the test compound, and each group is tested at one of

ﬁve diﬀerent time intervals: 0.25, 0.5, 1, 2, and 4 h. The time point at

which the compound produces the most activity/toxicity was chosen

as TPE.

MES Test and 6 Hz Test. For the MES and 6 Hz tests, a drop of

anesthetic/electrolyte solution (0.5% tetracaine hydrochloride in 0.9%

saline) was applied to the eyes of each animal prior to placement of the

corneal electrodes. The electrical stimulus in the MES test was 50 mA,

60 Hz, for mice. Abolition of the hind leg tonic extensor component of

the seizure was used as the end point.

The ability of the test substance to prevent seizures in mice induced

by 6 Hz corneal stimulation (32 mA, 3 s duration) was evaluated at a

convulsive current that evokes a seizure in 97% of the population

tested (CC₉₇). Six hertz seizures are characterized by a minimal clonic

phase that is followed by stereotyped, automatiztic behaviors described

originally as being similar to the aura of human patients with partial

seizures.^53,54Animals not displaying this behavior were considered

protected.

Corneal-Kindled Mouse Model of Partial Seizures. Mice were

kindled according to the methods described by Matagne and

Klitgaard.⁵⁵Brieﬂy, mice were stimulated twice daily with a corneal

stimulation of 3 mA for 3 s for an average of 12 days. Prior to each

stimulation, a drop of 0.9% saline containing 0.5% tetracaine

hydrochloride (Sigma-Aldrich, St. Louis, MO) was applied to the

cornea to ensure local anesthesia and good electrical conductivity.

Stimulations were at least 4 h apart. Animals were considered kindled

when they displayed ﬁve consecutive stage 5 seizures according to the

Racine scale.⁵⁶At the completion of the kindling acquisition, mice

were permitted at least a 3-day stimulation-free period prior to any

drug testing. Mice were stimulated once the day before drug testing to

ensure they had achieved and maintained a kindled state. On the day

of the drug study, corneal kindled mice (n = 4 or 8) received a single ip

dose of test compound. Mice were challenged with the corneal

kindling stimulus of 3 mA for 3 s at TPE after test compound

administration. Mice were scored as protected (seizure score of 3) or

not protected (seizure score ≥ 4) based on the Racine scoring.⁵⁶

Minimal Behavioral Toxicity Tests. Minimal behavioral toxicity was

identiﬁed in mice by the rotarod performance test.⁵⁷When a mouse is

placed on a 1-in. knurled rod that rotates at a speed of 6 rpm, the

animal can maintain its equilibrium for long periods of time. The

animal was considered toxic if it fell oﬀ this rotating rod three times

during a 1-min period.

ASSOCIATED CONTENT

* Supporting Information

Synthetic procedures for compounds 37 and 39−43,

spectroscopic characterization, in vitro bioassay procedures,

anticonvulsant data, and a modeling ﬁgure. This material is

available free of charge via the Internet at http://pubs.acs.org.

■

S

AUTHOR INFORMATION

Corresponding Author

*E-mail: kajacobs@helix.nih.gov. Tel.: 301-496-9024. Fax: 301-

480-8422.

■

Author Contributions

^§These authors contributed equally to this work.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank Prof. Ray Stevens and Dr. Vsevolod Katrich (Scripps

Res. Inst., La Jolla, CA) for helpful discussions, Dr. Noel

Whittaker (NIDDK) for mass spectral determinations, and

Intramural Research Program of the NIH, NIDDK for support.

The in vivo antiseizure studies conducted at the University of

Utah were supported by NINDS, NIH Contract No.

HHSN271201100029C.

ABBREVIATIONS USED

■

ADAC, N⁶-[4-[[[4-[[[(2-aminoethyl)amino]carbonyl]methyl]-

anilino]carbonyl]methyl]phenyl]adenosine; AR, adenosine re-

ceptor; cyclic AMP, adenosine 3′,5′-cyclic phosphate; BBB,

blood−brain barrier; CCPA, 2-chloro-N⁶-cyclopentyladenosine;

CHO, Chinese hamster ovary; GPCR, G-protein-coupled

receptor; HEK, human embryonic kidney; ip, intraperitoneal;

MES, maximal electroshock; MRS5127, (1′S,2′R,3′S,4′R,5′S)-

4′-[2-chloro-6-(3-iodobenzylamino)purine]-2′,3′-O-

dihydroxybicyclo[3.1.0]hexane; NECA, 5′-N-ethylcarboxami-

doadenosine; PIA, N⁶-phenylisopropyladenosine; scMET,

subcutaneous metrazol model; TM, transmembrane domain;

TPE, time of peak eﬀect; tPSA, total polar surface area.

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Article Doi

Structural sweet spot for A1 adenosine receptor activation by truncated (N)-methanocarba nucleosides: Receptor docking and potent anticonvulsant activity

DOI: 10.1021/jm300965a

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