Scholarly article on Propanoic acid, 2,2-dimethyl-3-[[(4-nitrophenoxy)carbonyl]oxy]-, methyl ester 685544-62-1 from Journal of Medicinal Chemistry p. 2037

J . Med. Chem. 2004, 47, 2037-2061

2037

Discover y of Novel 2,8-Dia za sp ir o[4.5]d eca n es a s Or a lly Active Glycop r otein

IIb-IIIa An ta gon ists^†

Mukund M. Mehrotra,* J ulie A. Heath,^§Mark S. Smyth, Anjali Pandey,^§J ack W. Rose,^§J oseph M. Seroogy,

Deborah L. Volkots,^§Lisa Nannizzi-Alaimo,^§Gary L. Park, J oseph L. Lambing,^§Stanley J . Hollenbach,^§and

Robert M. Scarborough*^,§

Millennium Pharmaceuticals, Inc., 256 E. Grand Avenue, South San Francisco, California 94080

Received J uly 25, 2003

In our efforts to develop orally active GPIIb-IIIa antagonists with improved pharmaceutical

properties, we have utilized a novel 2,8-diazaspiro[4.5]decane scaffold as a template. We describe

here our investigation of a variety of templates including spiropiperidinyl-γ-lactams, spiropi-

peridinylimide, spiropiperidinylureas, and spiropiperidinylhydantoins. With the appropriate

acidic and basic pharmacophores in place, each template yielded analogues with potent GPIIb-

IIIa inhibitory activity. One of the compounds, 59 (CT50787), was also used to demonstrate

for the first time the use of a pharmacological agent which is RIIbâ3 specific to display biological

activity in a lower species such as mouse and to extend bleeding times. Evaluation of the

pharmacokinetic properties of selected compounds from each series in rat, dog, and cynomolgus

monkey has led to the identification of 22 (CT51464), a double prodrug, with excellent

pharmacokinetic properties. It exhibited good pharmacokinetic profile across species (F% )

33 (Cyno), 73 (dog), 22 (rat); t_1/2â) 14.2 h (Cyno), 8.97 h (dog), 1.81 h (rat)). The biologically

active form, 23 (CT50728), displayed inhibition of platelet aggregation in platelet rich plasma

(PRP) with an IC₅₀value of 53 nM in citrate buffer, 110 nM in PPACK anticoagulated PRP,

and 4 nM in solid-phase GPIIb-IIIa competition binding assay (ELISA). Both 23 and 22 were

stable in human liver microsomes, did not inhibit the P450 3A4 isozyme, and had low protein

binding (18.22% for 23) and a desirable log P (0.45 ( 0.06 for 22, and -0.91 ( 0.32 for 23). It

is predicted that the high oral bioavailability for these compounds in multiple species should

translate into lower intra- and intersubject variability in man. The long plasma half-life of the

lead is consistent with once or twice daily administration for chronic therapy. Analogue 22

(CT51464) thus appears to be a promising oral GPIIb-IIIa inhibitor with significantly improved

pharmacokinetic properties over the previously described clinical candidates and may be found

useful in the treatment of arterial occlusive disorders.

In tr od u ction

lished,⁵and GPIIb-IIIa receptor antagonists are con-

sidered to be the most potent antiplatelet drugs available

to the clinician.

Platelet rich clot formation is important in many

vasooclusive disorders such as unstable angina, acute

myocardial infarction, reocclusion after percutaneous

interventions, and stroke.¹Platelet activation is pro-

duced by a wide variety of stimuli, but the final common

event leading to platelet rich thrombus formation is the

binding of the activated platelet integrin GPIIb-IIIa²to

the soluble plasma adhesive proteins fibrinogen (Fg) and

von Willebrand factor (vWf).³Fibrinogen and von Will-

ebrand factor are multivalent and participate in the

aggregation of platelets to form thrombi at the site of

atherosclerotic plaque rupture. If GPIIb-IIIa is blocked

from binding to adhesive proteins, then it should

prevent formation and/or propagation of a platelet

thrombus, no matter what the physiological stimuli was

that initiated GPIIb-IIIa activation.⁴The use of intra-

venous GPIIb-IIIa inhibitors (eptifibatide, tirofiban,

abciximab) in preventing vessel closure after percuta-

neous coronary angioplasty (PTCA) is now well estab-

Since chronic oral administration of GPIIb-IIIa an-

tagonists could potentially address the additional indi-

cations of myocardial infarction and stroke, two of the

most deadly cardiovascular diseases in the Western

World, intense efforts have been devoted to the discov-

ery of orally active GPIIb-IIIa antagonists.^6-9Unfortu-

nately, despite the success of intravenous anti-platelet

GPIIb-IIIa antagonists (ReoPro, Integrilin, and Aggr-

astat)^5,10in preventing cardiac events in the hospital,

several clinical trials involving oral formulations of

“super-aspirins” have failed to show prophylactic benefit

in patients with recurrent vascular events in a long term

dosing environment. Efficacy analysis of the oral agents

orbofiban, xemilofiban, sibrafiban, and lotrafiban (Fig-

ure 1)^8,9,11in the presence or absence of background

aspirin showed no benefit to the composite endpoint of

death, recurrent myocardial infarction, and other recur-

rent ischemic events. Overall, the results have been

quite disappointing. Long-term oral GPIIb-IIIa inhibi-

tion has uniformly failed to provide protection from

ischemic events and was associated with a paradoxical

increase in adverse events. Mortality increased in each

of the five trials (EXCITE, OPUS-TIMI 16, BRAVO,

* To whom correspondence should be addressed. M.M.: Tel: 650-

866-4847. E-mail: mukund_mehrotra@hotmail.com. R.S.: Tel: 650-

246-7444. Fax: 650-615-9023. E-mail: rscarborough@portola.com.

^†Dedicated to Professor E. J . Corey on the occasion of his 75th

birthday.

^§Current address: Portola Pharmaceuticals, Inc., 270 E. Grand Ave,

Suite 22, South San Francisco, CA, 94080.

Published on Web 03/11/2004

2038 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Mehrotra et al.

F igu r e 1. Oral GPIIb/IIIa antagonists that have been studied in advanced stage clinical trials.

SYMPHONY, 2nd SYMPHONY). A combined analysis

reveals a highly significant 35% relative (or 0.7%

absolute) increase in the risk of death in the 45 523

patients studied, with a 2-fold increase in the risk of

bleeding.^9b

leads to cardiac events. It has also been suggested that

GPIIb-IIIa inhibitors may lead to platelet activation

and/or to the release of inflammatory mediators, at least

under certain conditions or at specific drug concentra-

tions.^13bThe proinflammatory trigger of subthreshold

receptor blockade, particularly in the setting of high

arterial shear stress, could be quite eventful. It is

possible that improved pharmacokinetic properties would

therefore lead to a successful oral GPIIb-IIIa inhibitor

drug. Other potential mechanisms by which these drugs

may cause the undesired side effects are also under

investigation. While an in-depth biological examination

of this class of antiplatelet drugs is needed, studies until

recently have continued on two oral GPIIb-IIIa inhibi-

tors, roxifiban and cromafiban, which have different

properties from the earlier oral agents.^9a,12,14

Many reasons for the failure of orally administered

GPIIb-IIIa inhibitors to prevent recurrent cardiac events

have been proposed.^4,12,13One leading explanation is

that the pharmacokinetic properties of the agents tested

so far were suboptimal. Short half-lives of these agents

can result in large peak-to-trough ratios and poor oral

bioavailability can result in significant intra- and in-

terindividual variability of drug in the blood stream of

individual patients. Either excess or insufficient drug

in the bloodstream at any given time point may lead to

undesirable consequences. Excessive drug levels may

prevent the blood from clotting when necessary, such

that bleeding events result. Too little drug may be

ineffective in preventing the platelet aggregation that

The key aim of our investigation has been to identify

active and selective GPIIb-IIIa inhibitors with improved

pharmacokinetic properties. Most synthetic efforts in

Novel 2,8-Diazaspiro[4.5]decanes

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2039

Sch em e 1^aSynthesis of the γ-Spirolactam Scaffolds:

4-(1-Oxo-2,8-Diazaspiro [4.5]dec-2-yl)benzonitrile 11,

4-(3-Oxo-2,8-diazaspiro[4.5]dec-2-yl)benzonitrile 12, and

Spiroimide Scaffold 76

the GPIIb-IIIa inhibitor class have focused on devel-

oping novel templates upon which an acidic and a

basic pharmacophore are appended. Many classes of

GPIIb-IIIa antagonists have been reported which con-

tain constrained templates consisting of monocyclic and/

or fused bicyclic scaffolds.¹¹We have previously reported

the use of spirocyclic scaffolds for designing potent

GPIIb-IIIa antagonists.¹⁵Herein, we describe the opti-

mization of the novel 2,8-diazaspiro[4.5]decane scaffold

for obtaining orally active and selective inhibitors.

Within this spirocyclic framework, highly potent com-

pounds were obtained with favorable pharmacokinetics

in the spirolactam, spirohydantoin, and spirourea tem-

plates. The spirolactam series which has a carbonyl

group adjacent to the spiro ring junction (1-oxo) exhibits

the most favorable pharmacokinetic profiles. Optimized

compounds were evaluated for efficacy and pharmaco-

kinetics in rat, dog, and cynomologous monkey. Syn-

thesis, SAR, and pharmacokinetics of these compounds

are described leading to the identification of the opti-

mized prodrug analogue 22 (CT51464).

Ch em istr y

For the synthesis of the proposed compounds, we first

prepared the diazaspiro benzonitrile scaffolds (11, 12,

18a , 18b, 19a , 19b, and 76), followed by elaboration into

the final targets. The spiropiperidines were first coupled

with various carboxy-containing appendages, followed

by transformation of the benzonitrile functionality into

the corresponding benzamidines.¹⁶

a

Reagents and conditions: (a) ethyl cyanoacetate, Et₃N, CH₂Cl₂,

The synthesis of the γ-spirolactams, 4-(1-oxo-2,8-

diazaspiro[4.5]dec-2-yl)benzonitrile 11, 4-(3-oxo-2,8-

diazaspiro[4.5]dec-2-yl)benzonitrile 12, and spiroimide,

4-(1,3-dioxo-2,8-diazaspiro[4.5]dec-2-yl)benzonitrile 76,

are described in Scheme 1. Starting from the com-

mercially available 1-benzyl-4-piperidone, the two γ-lac-

tam scaffolds were synthesized in 10 steps in an overall

yield of 22%. 1-Benzyl-4-piperidone was first subjected

to Knoevenagel condensation with ethyl cyanoacetate,

followed by reaction of the R,â-unsaturated ester with

KCN to give the dinitrile 1. This was hydrolyzed with

concentrated HCl to yield the diacid 2, which was

subsequently dehydrated with DCC to furnish the

anhydride 3. The anhydride was reacted with 4-cyanoa-

niline to yield the mixture of amide-carboxylic acids,

which were cyclized with NaOAc/Ac₂O to give the imide

4. The imide was reduced nonselectively with NaBH₄

in methanol to give a mixture of hydroxy-spirolactams

5 and 6, which were reduced with NaBH₄/TFA to give

a mixture of the spirolactams 7 and 8 (ca. 1:2 by NMR,

and analytical HPLC, vide infra). This mixture of

isomeric lactams proved difficult to separate by flash

chromatography on large scale, and so it was used in

the next step without purification. The N-benzyl group

was then conveniently deprotected in two steps as

follows. Reaction with 2,2,2-trichloroethyl chloroformate

(TrOCOCl) brought about the protective group exchange

to yield trichloroethyl-carbamates 9 and 10.¹⁷This

mixture of Troc-lactams was more easily separable by

silica gel chromatography, and at this step both the

lactams were fully characterized (¹H NMR). The car-

bamates 9 and 10 were then individually treated with

10% Cd-Pb couple to effect reductive cleavage¹⁸of the

Troc group to give the key γ-spirolactam intermediates

4 Å sieves; (b) KCN, EtOH/H₂O, ∆; (c) concd HCl, ∆; (d) DCC,

DMF; (e) 4-cyanoaniline, TEA, DMF, rt; (f) Ac₂O, NaOAc, ∆; (g)

NaBH₄, MeOH; (h) NaBH₄, TFA; (i) 2,2,2-trichloroethyl chloro-

formate, CH₃CN/CH₂Cl₂; separate isomers by flash chromatogra-

phy; (j) 10% Cd-Pb, THF/NH₄OAc, pH ) 5.

11 and 12, respectively. This high yielding N-debenzy-

lation route was also used for the conversion of 4 to

afford the spiroimide 76.

The synthesis of spirohydantoins, 4-(1-methyl-2,4-

dioxo-1,3,8-triazaspiro[4.5]dec-3-yl)benzonitrile (and its

1-H analogue) 19b,a , and of the spiroureas, 4-(1-methyl-

2-oxo-1,3,8-triaza-spiro[4.5]dec-3-yl)benzonitrile (and its

1-H analogue) 18b,a , are described in Scheme 2.¹⁹

1-Benzyl-4-piperidone was subjected to Strecker condi-

tions by reaction with KCN and either ammonium

chloride or methylamine hydrochloride to give R-amino-

nitriles 13a and 13b, respectively. These were then

coupled with 4-cyanophenyl isocyanate to yield R-urea-

nitriles 14. Cyclization to hydantoins 15 was effected

by treatment with 1 N HCl at reflux. The N-benzyl

group was then removed as described before to afford

the spirohydantoin scaffolds 19a ,b. For the synthesis

of the spirourea template, the hydantoins 15 were first

reduced with NaBH₄/MeOH to give the hydroxy-urea

intermediates 16, followed by treatment with NaBH₄/

TFA as described previously to yield 17. N-Debenzyla-

tion provided the key intermediates 18a ,b.

The spiropiperidines (11, 12, 18a ,b, 19a ,b, and 76)

were coupled with the carboxy-containing appendages

by standard methods (Schemes 3-15). The required

carboxylic acids, acid chlorides, and isocyanates were

either commercially available or were synthesized as

shown (Schemes 5, 7-13, and 15).

2040 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Mehrotra et al.

Sch em e 2^aSynthesis of the Hydantoin and the Urea Scaffolds: 4-(1-Methyl-2,4-dioxo-1,3,8-triazaspiro[4.5]dec-

3-yl)benzonitrile (and its 1-H analog) 19b,a and 4-(1-Methyl-2-oxo-1,3,8-triazaspiro[4.5]dec-3-yl)benzonitrile (and its

1-H analog) 18b,a

a

Reagents and conditions: (a) KCN, RNH₂.HCl, EtOH/H₂O; (b) 4-cyanophenyl isocyanate, Et₃N, CH₂Cl₂; (c) 1 N HCl, ∆; (d) NaBH₄,

MeOH; (e) NaBH₄, TFA; (f) 2,2,2-trichloroethyl chloroformate, CH₃CN/CHCl₃; (g) 10% Cd-Pb, THF/NH₄OAc, pH ) 5.

Sch em e 3^aSynthesis of 23 (CT50728) and Its Double Prodrug 22 (CT51464)

a

Reagents and conditions: (a) CH₂Cl₂, Et₃N, rt.; (b) (i) H₂S, pyridine/Et₃N; (ii) CH₃I, acetone; (iii) NH₄OAc, MeOH; (c) 1 M LiOH,

THF, rt; or 3 N HCl, rt; (d) NH₂OH.HCl, Et₃N, EtOH, rt.

The general synthesis of the compounds with 3-

urea-propionic acid tails (e.g., 23) and their double pro-

drugs (e.g., 22) is described in Scheme 3. The spiro-

piperidines were reacted with ethyl 3-isocyanatopro-

pionate to give rise to the coupled urea compounds. The

benzonitrile functionality was then converted into

amidine either via thio-Pinner reaction^16aor via amid-

oximes.^16bReaction of the benzonitriles with hydroxyl-

amine hydrochloride/Et₃N yielded the amidoxime ethyl

ester double prodrugs. An analogous scheme for the

synthesis of the corresponding amide-linked compounds,

5-oxo-5-azaspiro-1-yl-pentanoic acids (e.g., 28), is de-

scribed in Scheme 4. The spiropiperidines were first

reacted with ethyl succinyl chloride, followed by trans-

formation of the benzonitriles into benzamidines.

Scheme 5 depicts the synthesis of the oxy-acetic acid

analogue 33 and its amidoxime ethyl ester double

prodrug 36. Benzyl glycolate was subjected to a carbene

insertion reaction with tert-butyl diazoacetate under

rhodium(II) acetate dimer catalysis to yield 30a . The

benzyl group was then hydrogenolyzed to yield acid 31,

which was subsequently coupled with spirolactam 11

to afford 32. The benzonitrile function was then trans-

formed into 33 and 36 as described before.

Scheme 6 depicts the synthesis of the substituted

amidine analogues 39b and 40b. The benzonitrile 25

was converted to thiomethylimidate 38 by the thio-

Pinner procedure. Thiomethylimidate 38 was then

treated with a mixture of morpholine and acetic acid

(ca. 1:2) in methanol to afford 39a . When acetic acid

was omitted a poor yield of 39a was obtained and the

major byproduct was the starting nitrile 25, formed via

the base-catalyzed elimination of CH₃SH from 38. The

same protocol was used for the synthesis of the N-

ethylamidine analogue 40a . Treatment of 39a and 40a

with 3 N HCl yielded 39b and 40b.

The synthesis of 44 and 45, the 3-(S)-methyl and

3-(R)-methyl analogues of 28, were achieved by using

the route described in Scheme 7. (R)-1-Ethyl hydrogen

3-methylglutarate was converted to its tert-butyl ester

Novel 2,8-Diazaspiro[4.5]decanes

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2041

Sch em e 4^aSynthesis of 28 (CT50614) and Its Double Prodrug 27 (CT51269)

a

Reagents and conditions: (a) DMF, DIEA, rt.;(b) (i) H₂S, pyridine/Et₃N; (ii) CH₃I, acetone; (iii) NH₄OAc, MeOH; (c) 3 N HCl, rt.; (d)

NH₂OH.HCl, Et₃N, EtOH, rt.

Sch em e 5^aSynthesis of 33 (CT51907) and Its Double

Prodrug 36 (CT52108)

acid was converted to 3-phenylglutaric anhydride by

refluxing in acetic anhydride. This was then reacted

with EtOH/Et₃N to yield 46, which was carried forward

to 48 by procedures described before.

The synthesis of 53, the 3-(S)-methyl analogue of 23,

is shown in Scheme 9. Cbz-D-Alanine was subjected to

Arndt-Eistert homologation conditions to yield 49.^23b

This was debenzylated to give ethyl (S)-3-aminobu-

tanoate 50, which was then converted to its 4-nitrophe-

nyl carbamate derivative 51. The carbamate was re-

acted with the spirolactam 11 to yield 52, which was

then transformed to 53 as described previously.

Scheme 10 describes the synthesis of spirohydantoin

analogue 56. Ethyl 3-amino-3-phenylpropanoate was

reacted with 4-nitrophenyl chloroformate to yield the

4-nitrophenyl carbamate derivative 54. This was then

reacted with 19b to afford 55, which was carried

through to 56 as described previously.

a

Reagents and conditions: (a) CH₂Cl₂, rt.; (b) Pearlman’s

Scheme 11 depicts the synthesis of 59, the 2-R-

(3,5-dimethylisoxazole-4-sulfonamide) analogue of 28.

H-Glu-O-t-Bu was reacted with 3,5-dimethylisoxazole-

4-sulfonyl chloride under Schotten-Baumann condi-

tions to afford the sulfonamide 57. This was coupled

with spirolactam 11 to yield 58, which was converted

to 59 as described before.

catalyst, EtOAc, 40 psi H₂; (c) 11, HBTU, DIEA, DMF; (d) (i) H₂S,

Pyr/Et₃N; (ii) CH₃I, acetone; (iii) NH₄OAc, MeOH;(e) TFA;(f)

NH₂OH‚HCl, Et₃N, EtOH; (g) 3 N HCl.

derivative 41. The ethyl ester was then saponified to

give free acid 42. This was coupled with spirolactam 11

to yield the benzonitrile intermediate 43, which was

then transformed to 44 as described previously. A

similar sequence of reactions provided the (R)-isomer

45.

Scheme 12 describes the synthesis of 63, the R-tosyl-

amide analogue of 23. Ethyl (S)-2-amino-3-(Boc-amino)-

propanoate was reacted with TsCl to yield tosylamide

60. Treatment with TFA yielded amine 61. This was

Scheme 8 describes the synthesis of 48, which is the

3-phenyl-substituted analogue of 28. 3-Phenylglutaric

Sch em e 6^aSynthesis of the Substituted Amidine Analogues 39b and 40b

a

Reagents and conditions: (a) (i) H₂S, pyridine/Et₃N; (ii) CH₃I, acetone; (b) morpholine, AcOH, MeOH, rt.; (c) EtNH₂, AcOH, MeOH,

rt.; (d) 3 N HCl, rt.

2042 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Mehrotra et al.

Sch em e 7^aSynthesis of 44 and 45, the 3-(S)- and 3-(R)-Methyl Analogues of 28

a

Reagents and conditions: (a) DCC, t-BuOH, CH₂Cl₂; (b) 1 M LiOH, THF; then AcOH; (c) 11, HBTU, DIEA, DMF f 43; (d) (i) H₂S,

Pyr/Et₃N; (ii) CH₃I, acetone; (iii) NH₄OAc, MeOH; (e) TFA; (f) 11, EDC‚HCl, HOBt, DMF.

Sch em e 8^aSynthesis of the 3-Phenylpentanedioic Acid

Monoethyl Ester 46 and Its Use in the Synthesis of 48

Sch em e 10^aSynthesis of the 3-(4-Nitrophenoxycarbon-

ylamino)-3-phenylpropionic Acid Ethyl Ester 54 and Its

Use in the Synthesis of 56

a

Reagents and conditions: (a) 4-nitrophenyl chloroformate,

DIEA, CH₂Cl₂; (b) 19b, DIEA, CH₂Cl₂; (c) (i) H₂S, Pyr/Et₃N; (ii)

CH₃I, acetone; (iii) NH₄OAc, MeOH; (d) 1 N LiOH, THF/H₂O.

a

Reagents and conditions: (a) Ac₂O, ∆; (b) EtOH, Et₃N, ∆ ; (c)

11, EDC‚HCl, HOBt, DMF; (d) (i) H₂S, Pyr/Et₃N; (ii) CH₃I, acetone;

(iii) NH₄OAc, MeOH; (e) 1 M LiOH, THF/H₂O.

Sch em e 11^aSynthesis of the 2-(2-Carboxyethyl)-2-(3,5-

dimethylisoxazole-4-sulfonylamino)-3,3-dimethylbutyric

Acid 57 and its Use in the Synthesis of 59 (CT50787)

Sch em e 9^aSynthesis of the 3-(4-Nitrophenoxycarbonyl-

amino)butyric Acid Ethyl Ester 51 and Its Use in the

Synthesis of 53

a

Reagents and conditions: (a) (i) 1 N NaOH, Na₂CO₃, 0 °C; (ii)

2 N HCl to pH ) 3; (b) 11, EDC‚HCl, DMF; (c) TFA; (d) (i) H₂S,

Pyr/Et₃N; (ii) CH₃I, acetone; (iii) NH₄OAc, MeOH.

Sch em e 12^aSynthesis of the 3-Amino-2-(toluene-4-sul--

fonylamino)propionic Acid Ethyl Ester 61 and its Use in

the Synthesis of 63

a

Reagents and conditions: (a)20% Pd(OH)₂/C, EtOAc, 50 psi

H₂; (b) 4-nitrophenyl chloroformate, DIEA, CH₂Cl₂; (c) 11, DIEA,

CH₂Cl₂; (d) (i) H₂S, Pyr/Et₃N; (ii) CH₃I, acetone (iii) NH₄OAc,

MeOH; (e) 3 N HCl.

then reacted with triphosgene, followed by spirolactam

11 to give 62. The benzonitrile functionality was con-

verted to the amidine via the amidoxime route. 62 was

reacted with hydroxylamine hydrochloride/Et₃N to yield

the amidoxime, which was O-acetylated with Ac₂O/

AcOH, and then hydrogenolyzed to yield benzamidine.

Hydrolysis of the ethyl ester with 1 M LiOH then

provided 63.

The synthesis of 69, the R-n-butyl carbamate ana-

logue of 28, was achieved by the route outlined in

Scheme 13. Z-Glu(O-t-Bu)-OH was converted to its ethyl

ester 64, followed by deprotection of the Cbz-group to

a

Reagents and conditions: (a) DIEA, DCM; (b) TFA; (c) 11,

triphosgene, CH₂Cl₂; (d) (i) NH₂OH‚HCl, Et₃N, EtOH; (ii) Ac₂O,

AcOH; (iii) 10% Pd/C, H₂, 40 psi; (e) 1 M LiOH, THF/H₂O.

yield 65. This was reacted with n-butyl chloroformate

to yield the carbamate 66. De-tert-butylation gave 67.

This was then coupled with the spirolactam 11 to yield

Novel 2,8-Diazaspiro[4.5]decanes

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2043

Sch em e 13^aSynthesis of the 2-Butoxycarbonylaminopentanedioic Acid 1-Ethyl Ester 67 and Its Use in the Synthesis

of 69

a

Reagents and conditions: (a) EDC‚HCl, HOBt, EtOH, DMF; (b) 20% Pd(OH)₂/C, 1 atm H₂, EtOH; (c) n-butyl chloroformate, DIEA,

CH₂Cl₂; (d) TFA; (e) 11, HBTU, DIEA, DMF; (f) (i) NH₂OH‚HCl, Et₃N, EtOH; (ii) Ac₂O, AcOH; (iii) 10% Pd/C, 1 atm H₂; (g) 1 M LiOH,

THF/H₂O.

Sch em e 14^aSynthesis of the 5-Azaspiro-1-ylpentanoic

Acid Analogue 72

to mimic the spacing between the basic guanidine and

the acidic carboxylate of the RGD sequence of fibrino-

gen, which has been approximated to be 10-20 Å.²⁰To

explore this, we synthesized amides with C₂to C₅

linkers (Table 1; 77, 78, 28, 79). The C₄-linker was found

to be optimum (28). By comparison, the compound with

C₃-linker (78) was found to be 9 times less potent, and

the longer version with a C₅-linker (79) was about 42

times less potent. This four-atom spacing between

piperidine and carboxylate was found to be optimal in

all the spiro-templates reported herein.

a

Reagents and conditions: (a) DIEA, DMF, rt.; (b) NH₂OH‚HCl,

Et₃N, EtOH; (c) (i) Ac₂O, AcOH; (ii) 10% Pd/C; H₂, 40 psi; (d) 1 N

LiOH, THF/H₂O, rt.

The potent activity of compound 28 confirmed to us

that this novel series merited further investigation. The

compounds of Table 1 were prepared to explore the SAR

of this 1-oxo-γ-spirolactam series. In essence, having

worked out the crucial spacing between the essential

acidic and basic pharmacophores, we undertook the

synthesis of numerous analogues of compound 28 to

optimize its potency and improve in vivo properties.

Sch em e 15^aSynthesis of the 2,2-Dimethylpropionic

Acid Analogue 75

On the basis of our previous observations^20,21as well

as those reported in the literature for other series of

integrin inhibitors, we targeted two regions of the lead

molecule 28 for modification: the central rigid [4.5]-

spirocyclic nucleus and the carboxyl linker unit. In the

spirocyclic nucleus, we investigated the isomeric 3-oxo-

γ-spirolactam, spiropiperidinylimide, spiropiperidiny-

lureas, and spiropiperidinylhydantoins. On the linker

unit, we compared urea with amide linkages to the

piperidine of the scaffold and we made substitutions R

or â to the carboxylate terminus.

Most of the compounds in which the carboxylic acid

appendage is attached to the piperidine via a urea

linkage are generally slightly more potent than the

corresponding amides (28 versus 23 (Table 1); 87 versus

84 (Table 2); 94 versus 89 (Table 3); 101 versus 99

(Table 4); 105 versus 104 (Table 5)). In the literature,

analogues containing substituted â-amino acid units

appended to various scaffolds have been widely studied

as GPIIb-IIIa antagonists, and the â-amino acid unit

has been postulated to mimic the â-carboxyl group of

the aspartic acid residue of the RGD sequence.^6e,7,11

Further lipophilic substitutions at both R- and â-posi-

tions of these units have generally enhanced potency

and PK properties. However, in the 1-oxo-γ-spirolactam

scaffold, a â-position methyl group (53, 81 versus 23

(Table 1)) did not improve potency. This may imply

special conformational requirements for this rigid tem-

plate.

a

Reagents and conditions: (a) 4-nitrophenyl chloroformate,

DIEA, CH₂Cl₂; (b) 12, DIEA, DMF; (c) (i) H₂S, Pyr/Et₃N; (ii) CH₃I,

acetone; (iii) NH₄OAc, MeOH; (d) 1 N LiOH, THF/H₂O.

68 and converted to 69 using previously described

procedures.

Scheme 14 describes the synthesis of 72, which is the

amine analogue of amide 28. The spirolactam 11 was

alkylated with ethyl 5-bromovalerate to yield 70. The

benzonitrile functionality was converted to benzamidine

via amidoxime 71, and then the ester group was sapon-

ified to yield 72.

In Scheme 15 is outlined the synthesis of 75. Methyl

2,2-dimethyl-3-hydroxypropionate was reacted with 4-ni-

trophenyl chloroformate to yield 73. This was coupled

with spirolactam 12 to give 74, which was converted to

75 by previously described procedures.

All the final compounds were purified by RP-HPLC

and isolated either as their trifluoroacetate or HCl salts.

Resu lts a n d Discu ssion

In Vitr o SAR. On the basis of our previous studies

with other spirocyclic scaffolds,¹⁵initially the SAR

investigation focused on optimizing the distance be-

tween the basic amidine functionality and the carboxylic

acid group. This bond distance has been hypothesized

2044 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Mehrotra et al.

Ta ble 1. In Vitro Activity and Pharmacokinetic Parameters for the 1-Oxospirolactam Scaffold-Based Analogues:

4-(1-Oxo-2,8-diazaspiro[4.5]dec-2-yl)benzamidines

a

Inhibition of platelet aggregation induced by 20 µM adenosine 5′-diphosphate (ADP) in citrated human platelet rich plasma (h-PRP).

Inhibition of the binding of fibrinogen to purified human GPIIb-IIIa in a 96-well format (ELISA assay). ^cInhibition of platelet aggregation

b

induced by 20 µM ADP in h-PRP with PPACK (see Experimental Section for details).

Hartman et al., Egbertson et al., and Xue et al.²²have

reported that introduction of a sulfonamide or a car-

bamate substituent R to the carboxylate resulted in very

potent and specific inhibitors of GPIIb-IIIa. These

authors have suggested that this favorable effect is the

result of an interaction with an exosite in the integrin.

These R-substituents might also be functioning as a

surrogate for the R-carboxylic acid of the Asp-residue.

This modification has been applied in our various series

and was not found to be uniformly successful in enhanc-

ing the GPIIb-IIIa inhibitory activity of compounds. In

the 1-oxo-γ-spirolactam series, R-sulfonamides (Table 1;

59, 63) or R-carbamate (69) substituents were close to

equipotent with the parent compounds lacking substit-

uents (23, 28). The result is different from our observa-

tions in the hydantoin series (Table 3; 92, 97, 91) and

Novel 2,8-Diazaspiro[4.5]decanes

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2045

Ta ble 2. In Vitro Activity and Pharmacokinetic Parameters for

the 3-Oxospirolactam Scaffold-Based Analogues:

4-(3-Oxo-2,8-diazaspiro[4.5]dec-2-yl)benzamidines

imide series (Table 5; 106) in which such R-substitution

leads to a significant improvement over the unsubsti-

tuted analogue in hPRP.

Numerous studies of GPIIb-IIIa inhibitors have

suggested that residues at or very near the aspartic acid

of the RGD sequence can also modulate the selectivity

of ligand binding to integrins, and introduction of

aromatic or other hydrophobic residues proximal to

the RGD site can significantly enhance the inhibitory

activity and specificity of the RGD analogues.²³On

the basis of an extensive SAR study at a position â-to

the carboxylic acid terminus in a different series of

GPIIb-IIIa inhibitors, Zablocki et al.²⁴have reported

the discovery of potent platelet aggregation inhibitor,

xemilofiban (Figure 1). We have studied the effect of

â-substitutions to the carboxylic acid in our spiro-

cyclic series by incorporating methyl and phenyl sub-

stituents. In the 1-oxo-γ-spirolactam series, â-substit-

uents were less active than the corresponding com-

pounds lacking substituents (Table 1; 44, 45, 48). Again,

this result is opposite to what is observed in the

hydantoin (93), urea (103), and 3-oxo-γ-spirolactam

series (86). For these, â-substitution resulted in a

a^,b

See Table 1.

Ta ble 3. In Vitro Activity and Pharmacokinetic Parameters for the Spirohydantoin Scaffold-Based Analogues:

4-(2,4-Dioxo-1,3,8-triazaspiro[4.5]dec-3-yl)benzamidines

a,b

See Table 1.

2046 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Mehrotra et al.

Ta ble 4. In Vitro Activity and Pharmacokinetic Parameters for the Spirourea Scaffold-Based Analogues:

4-(2-Oxo-1,3,8-triazaspiro[4.5]decan-2-one-3-yl)benzamidines

a,b

See Table 1.

Ta ble 5. In Vitro Activity and Pharmacokinetic Parameters for

the Spiroimide Scaffold-Based Analogues: 4-(1,3-Dioxo-2,8-

diazaspiro[4.5]decane-1,3-dione-2-yl)-benzamidines

scribed here has led to the expected enhancements in

activity when modifications were made to the com-

pounds.

Changing only the spirocyclic scaffold, a comparison

of the inhibitory activities of compounds 23 (Table 1),

87 (Table 2), 94, 95 (Table 3), 101, 102 (Table 4), and

105 (Table 5) implies that the 1-oxo-γ-spirolactam

scaffold is in the appropriate rigid conformation to

display the acidic and basic pharmacophores for opti-

mum binding.

In Vivo Resu lts. In general, GPIIb-IIIa antagonists

containing both an amidine and a carboxylic acid group

have very low oral bioavailability. This has been at-

tributed both to the strongly basic nature of the amidine

functionality and to the highly polar, acidic nature of

the carboxylic acid. Analogues which display both

groups are expected to behave as zwitterionic species.

In particular, the strongly basic amidine group would

be expected to exist in its ionic form both in the acidic

conditions of the stomach and also in the weakly basic

conditions of the intestine. This lasting ionic form could

prevent the absorption through a passive transport from

the intestine, which is greatly dependent on the lipo-

philicity of the molecule. In addition, the terminal

carboxylic acid may also contribute to poor absorption.

To circumvent these problems, various approaches have

been aimed at both masking the basicity of the amidine

group and enhancing the lipophilicity of the molecule.

When selected compounds from each spirocyclic scaf-

fold were evaluated as their ethyl ester prodrugs in rat,

they exhibited bioavailability of e5%, although some

displayed encouraging elimination half-lives (Tables

1-5). Again, the strongly basic nature of the amidine

group was considered a likely cause of the poor bio-

availability.

a,b

See Table 1.

significant improvement over the unsubstituted ana-

logue in hPRP (Tables 2-4).

Interestingly, all the templates investigated within

the[4.5]-spirocyclic framework have provided potent

compounds. This is consistent with the hypothesis that

GPIIb-IIIa does not require significant interaction with

the central template of inhibitors. Its sole purpose

appears to be providing the scaffold to deliver the acidic

and basic pharmacophores in the correct geometry to

mimic the RGD sequence of fibrinogen receptor, which

is believed to represent the minimal sequence necessary

for binding to GPIIb-IIIa.

Of all the spirocyclic templates that we have inves-

tigated, the 1-oxo-γ-spirolactam template has provided

the most potent GPIIb-IIIa antagonists. Optimum bind-

ing to the RGD binding motif of the fibrinogen binding

site in the basic framework was depicted in the com-

pounds 28 and 23 (Table 1). Attempts to improve their

activity by utilizing the SAR previously documented in

the other GPIIb-IIIa antagonist templates proved fruit-

less. Interestingly, SAR in other spiro-scaffolds de-

In one approach aimed at finding a less basic substi-

tute for the amidine group, GPIIb-IIIa antagonists

where a pyridine or piperidine ring substitutes for the

benzamidine have been explored (Figure 1, ZD-2486,²⁵

lotrafiban,²⁶elarofiban,²⁷UR-12947). However, when

used in the spirocyclic scaffold series, the pyridine-

Novel 2,8-Diazaspiro[4.5]decanes

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2047

Ta ble 6. PK Properties of Active Drugs after Oral

Administration of Double Prodrugs in Rats (1.0 mg/kg)

containing compounds did not show a favorable phar-

macokinetic profile. This was despite retaining equiva-

lent potency compared to the corresponding amidine

analogues.¹⁵

In another approach aimed at enhancing the lipophi-

licity of the overall molecule and also partially masking

the charge on the amidine group, mono- and dialkylated

amidino analogues have been investigated (Figure 1,

NSL-96184, PSA0613). The dialkylated analogues have

been found to retain their activity and exhibit improved

PK profiles.²⁸However, in the 1-oxo-spirolactam series,

both mono- and disubstituted amidino analogues showed

greatly diminished activity (Table 1; 40b and 39b versus

28). This anomaly may also be rationalized by invoking

the spatial-constraints of the centrally rigid spirocyclic

template.

A double prodrug approach relies on the strategy of

masking both polarizable pharmacophore groups in

order to enhance oral bioavailability. Such an approach

has been employed previously to obtain orally bio-

available GPIIb-IIIa antagonists, e.g. lefradafiban

(BIBU-104) (methyl carbamate),²⁹sibrafiban (Ro 48-

3657) (amidoxime), ³⁰gantofiban³¹(EMD-122347) (meth-

yl carbamate) (Figure 1). In the novel amidoxime pro-

drug approach, aimed at lowering the pK_aof the basic

benzamidine, the liver- and/or gut-mediated reduction

of the amidoxime generates the active metabolite. Novel

GPIIb-IIIa double prodrug containing antagonists with

amidoxime and ethyl ester functionalities have been

described which exhibit PK profiles in rats, dogs, and

rhesus monkeys which are independent of the species.

On the basis of these observations, we investigated this

double prodrug strategy for our most potent spirocyclic

compounds. The amidoxime ethyl ester double prodrugs

described here were synthesized using the reaction

sequence shown in Schemes 3 and 4. The pharmacoki-

netic data on the amidoxime ethyl ester double pro-

drugs, representing each spirocyclic scaffold, is depicted

in Table 6. The double prodrugs of the spirourea and

spirohydantoin-containing scaffolds (111, 112, and 113)

did not show a noticeable improvement in bioavailability

over their nonprotected version. However, for the 1-oxo-

spirolactam series, a dramatic improvement was ob-

served by the double prodrug approach. Three of the

best of these (22, 27, and 36; Table 6) were chosen for

further study.

a

Calculated using ACD/LogP version 4.56 (Advanced Chemistry

Development, Inc., 90 Adelaide St. West, Toronto, Canada M5H

3V9). Calculated using MOE (Chemical Computing Group, Inc.,

b

1010 Sherbrooke St. West, #910, Montreal, Canada H3A 2R7).

^cCalculated using the method of Ertl.³⁴

Ta ble 7. Pharmacokinetic Parameters of 23, 28 and 33 after

Oral Administration of 22, 27, and 36 in Rat (1.0 mg/kg)

iv

po

compound

AUC, ng‚h/mL t_{1/2 â}AUC, ng‚h/mL t_1/2F, %

28, CT50614

23, CT50728

33, CT51907

500.6

565.1

651

1.8

1.81

1.94

181.9

126.2

366.6

1.14 36.3

1.42 22.3

1.57 35.9

solvation than the corresponding 1-oxo-carbonyl group

in 22, as is reflected in their polar surface areas, and

this may help explain the distinct differences in their

PK profiles. This is noteworthy because for these two

compounds all other physicochemical parameters are

the same, including the number of rotatable bonds and

the hydrogen-bond donor/acceptor groups.

The pharmacokinetic data, in rats, for the double

prodrugs 22 (CT51464), 27 (CT51269), and 36 (CT52108)

are shown in Table 7. This PK data indicates that all

the double prodrugs are rapidly (T_max) 1 h) and well

absorbed. All the drugs exhibited similar PK profiles.

For example, after oral administration of 22, at a dose

of 1 mg/kg to male rats (n ) 6) and the active drug (23)

as well as amidoxime acid (24) were detected in plasma

(Figure 2). While 23 showed a relatively long elimina-

The higher oral bioavailability observed for the 1-ox-

ospirolactam series over other spirocyclic scaffolds may

be attributed to its higher rigidity, optimal lipophilicity,

and its lower polar surface area (Table 6). Reduced polar

surface area has been suggested to correlate better with

increased permeation rate than does lipophilicity (cLogP),

and this should also help explain the difference in F%

for the compounds within this lactam series which have

similar cLogP values (Table 1).³²

The differences in PK properties of compounds 22,

110, 111, and 112 (Table 6) which differ only in the

nature of the five-membered ring is noteworthy in this

regard. Hydantoin- and urea-containing spiro templates

have relatively more polar surface area and thus they

would be expected to be less able to penetrate mem-

branes. They indeed are less bioavailable. For the 1-oxo-

and 3-oxospirolactams (22 and 110), one would expect

the 3-oxo-carbonyl group in 110 to be more exposed to

tion half-life (t_1/2) 1.81 h, mean), amidoxime 24

disappeared more rapidly (t_1/2â) 0.6 h ( 0.03, mean (

SD).

The double prodrug 22 was absorbed and con-

verted to an active compound in vivo, most probably

â

2048 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Mehrotra et al.

F igu r e 4. Plasma levels of 23 after iv administration of 23

(0.2 mg/kg) and po administration of 22 (2.0 mg/kg) versus

time, as determined by LC/MS/MS in cynomolgus monkeys

(n ) 4). 24 is the corresponding amidoxime acid.

F igu r e 2. Plasma levels of 23 after iv administration of 23

(0.1 mg/kg) and po administration of 22 (1.0 mg/kg) versus

time, as determined by LC/MS/MS in rats (n ) 6). 24 is the

corresponding amidoxime acid.

F igu r e 5. Plasma levels of 28 after iv administration of 28

(0.2 mg/kg) and po administration of 27 (2.0 mg/kg) versus

time, as determined by LC/MS/MS in cynomolgus monkeys

(n ) 4). 29 is the corresponding amidoxime acid.

F igu r e 3. Plasma levels of 23 after iv administration of 23

(0.1 mg/kg) and po administration of 22 (1.0 mg/kg) versus

time, as determined by LC/MS/MS in dogs (n ) 4). 24 is the

corresponding amidoxime acid.

to the amidino acid 24. The metabolic reduction of

benzamidoxime derivatives to benzamidines, in vivo

as well as in vitro, is well documented.³³The clearance

mechanism for 24 has not been determined. Despite the

two metabolic steps involved, the interindividual vari-

ability of the plasma concentration of active compound

23 was low. In either case, the rapid appearance of 24

suggests rapid absorption of prodrug. T_maxfor 24 after

oral administration of 22 was 0.25 h, and that for 29

and 37 after oral administration of 27 and 36, repec-

tively, was e0.5 h. In a separate experiment, the

amidoxime acid 29 exhibited low bioavailability in rats

(F ) 3.7%).

The oral absorption of 22 (CT51464) may be a

complicated process involving multiple absorption sites

in the digestive tract. Metabolism of the double prodrug

in the intestinal tract may also play a role in the

absorption process. The rapid appearance of 24 and the

delayed absorption maxima of 23 (Figure 2) may indi-

cate a two-step absorption process. A portion of the drug

may be absorbed from the stomach and/or proximal

sections of the intestines while the remaining drug is

absorbed in distal portions of the intestine. A second

possibility for the delayed appearance of 23 may be that

rapid absorption of the prodrug occurs but the inter-

mediate product 24 or the active compound 23 forms a

conjugate (glucuronic acid, sulfate, glycine, etc.) which

is subsequently hydrolyzed to give 23.

F igu r e 6. Plasma levels of 33 after iv administration of 33

(0.2 mg/kg) and po administration of 36 (2.0 mg/kg) versus

time, as determined by LC/MS/MS in cynomolgus monkeys

(n ) 4). 37 is the corresponding amidoxime acid.

to the acid seems to be faster than the conversion of

the N-hydroxyamidine to the amidine. The data may

also suggest that the relative rates of absorption of the

different structures may vary by species.

For 28/27 (Figure 5), pharmacokinetic patterns simi-

lar to those described above for rats were observed. The

peak plasma level of amidoxime acid 29 was higher and

was reached earlier (T_maxe 0.5 h) than that of 28 (T_max

) 2 h). For 33/36 (Figure 6), a similar PK profile was

observed. However, for 23/22 (Figure 4), the peak

plasma levels of amidoxime acid 24 were lower than

that of 23, even at 0.5 h when the first samples were

drawn. T_maxfor 24 and 23 were the same (1.5-3.5 h).

The half-life of 28 in cynomolgus monkeys (8.9 h) is

longer than that observed in rats (1.8 h). The affinity

of 28 for the cynomolgus monkey GPIIb-IIIa receptor

is stronger than that for rat (IC₅₀) 0.128 ( 0.031 µM

versus 0.60 ( 0.013 µM), and we and others hypothesize

Figures 4, 5, and 6 show the PK profiles of 23, 28,

and 33 in cynomolgus monkeys after iv administration

of the active drugs 23, 28, and 33 and after oral admin-

istration of their respective double prodrugs (22, 27, and

36). From the in vivo data, the conversion of the ester

Novel 2,8-Diazaspiro[4.5]decanes

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2049

Ta ble 8. Summary of Pharmacokinetic Parameters of 23, 28, and 33 after iv (1 mg/kg) and Oral Administration of 22, 27, and 36 in

Rat, Dog, and Cyno

F, %^a,c

average

t

_1/2â, h^a,b

V_z, L/kg^d

average

Cl, mL/min/kg

oral dose,

mg/kg

prodrug/drug

species

SD

average

SD

average

SD

27/28

rat (6)

1

2

1

2

1

2

36.3

39

-

1.8

10.3

8.9

1.81

8.97

14.2

1.94

10.3

7.51

-

dog (4)

cyno (4)

rat (6)

dog (4)

cyno (4)

rat (6)

16.3

1.2

3.5

2.4

5.75

5.02

1.04

5.26

6.44

6.51

-

1.16

6.83

18.77

22.3

72.9

32.4

35.9

23.3

30.6

22/23

36/33

-

1.89

3.18

-

1.35

2.16

-

4.12

6.30

-

2.93

1.75

-

1.46

2.12

-

0.32

0.22

-

2.98

1.73

-

0.36

0.34

26.1

8.43

5.12

-

3.28

2.70

8.85

10

3.9

3.41

dog (4)

cyno (4)

a

Data are mean ( SD. ^bThe apparent elimination half-life (t_1/2â) was determined from the linear portion of the log plasma concentration-

time profile. ^cThe bioavailability (F) of 23, 28 and 33 after po administration of 22, 27, and 36 was calculated accounting for the differences

d

in molecular weights and doses. The volume of distribution (V_Z) was calculated as dose/(AUCINF* λ_z) where λ_zis the terminal elimination

rate constant. Lambda Z (λ_z) is calculated as the slope of the terminal, linear portion of the log linear plot of plasma concentration versus

time.

Ta ble 9. Activity Summary for Leading Candidates: Comparison to Sibrafiban and Orbofiban

28 CT050614 (µM)

23 CT050728 (µM)

33 CT051907 (µM)

orbofiban (µM)

sibrafiban (µM)

Aggregation Assays (in PRP)

20 µM ADP-citrate

0.054

(( 0.005)

0.168

(( 0.010)

0.123

(( 0.021)

0.368

(( 0.009)

0.175

(( 0.009)

0.464

0.053

(( 0.010)

0.110

(( 0.019)

0.120

(( 0.047)

0.365

(( 0.092)

0.140

(( 0.045)

0.450

0.108

(( 0.011)

0.260

(( 0.029)

0.273

(( 0.080)

0.691

(( 0.034)

0.393

(( 0.049)

0.812

0.068

(( 0.015)

0.137

(( 0.038)

0.166*

(( 0.008)

0.362*

(( 0.013)

n.d.

0.051

(( 0.008)

0.072

(( 0.017)

0.147*

(( 0.017)

0.182*

(( 0.011)

n.d.

20 µM ADP-PPACK

5 µM TRAP6-citrate

5 µM TRAP6-PPACK

4 µg/mL collagen-citrate

4 µg/mL collagen-PPACK

n.d.

(( 0.073)

(( 0.072)

(( 0.187)

Solid-Phase Assays

0.00390

fibrinogen binding to GPIIb-IIIa

0.00367

(( 0.00104)

>50

0.00976

(( 0.0021)

>50

0.00238

(( 0.0032)

n.d.

0.00114

(( 0.0002)

>50

(( 0.00128)

>50

vitronectin binding to R_vâ₃

Cell Adhesion Assays

HUVEC adhesion to vitronectin

HUVEC adhesion to fibronectin

>50

n.d.

>50

*211-47 TRAP was used for these determinations.

that the terminal half-life of the drug may be controlled

to a large extent by its binding affinity to platelets.¹¹

Although this platelet binding hypothesis is in agree-

ment with the half-life data obtained for 28, no direct

evidence exists to prove the hypothesis in this series.

When compound 22 was given orally to fasted male

beagle dogs at 1 mg/kg (n ) 4), a pharmacokinetic

pattern similar to that described above for cynomolgus

monkey was observed (Figure 3). The peak plasma

levels of amidoxime acid 24 were lower than that of 23

even at 0.5 h. For both 23 and 24, the T_maxwas about

5 h. The t_1/2âfor 23 was 8.97 h. Compared to the

experiment in monkeys, the half-life was longer and

bioavailability was higher. The affinity of 23 for the dog

GPIIb-IIIa receptor may be stronger than that for cyno

(IC₅₀) 0.239 ( 0.39 µM versus 0.410 ( 0.152 µM).

The pharmacokinetic data for 22, 27, and 36 is

summarized in Table 8, and the comprehensive in vitro

data in Table 9 along with data for orbofiban and

sibrafiban. The three selected double prodrug analogues

all exhibit good activity in human plasma, have low

protein binding, and are stable in gastric juice, phos-

phate-buffered saline (PBS), and water. They also

exhibit superior stability in human hepatic microsomes

and do not inhibit the P450 3A4 isozyme (data not

shown). All the final candidates were also found to

be negative in preliminary AMES tests. Virtually all

the active compounds in this series demonstrated a

selectivity of >5000-fold for GPIIb-IIIa versus the most

closely related integrin, the vitronectin receptor R_vâ₃.

22 (CT51464) was eventually chosen for further devel-

opment due to its superior bioavailability and half-life.

The relatively high oral bioavailability of 22 may

obviate the problem of large plasma level variability

from patient to patient, which has been postulated to

be a drawback with previous oral GPIIb-IIIa antagonists

that demonstrated no clear therapeutic benefits in

Phase III clinical evaluation. The high bioavailability

of 22 coupled with its long elimination half-life may also

address the problem of a narrow therapeutic index

leading to a narrow window of safety.

In Vivo Dem on str a tion of GP IIb-IIIa Dep en -

d en ce of Mou se Bleed in g Tim e. A platelet-dependent

mouse tail bleeding time model was used to demonstrate

the in vivo antithrombotic capacity of these potent

GPIIb-IIIa antagonists. In this model, 59 (CT50787), a

specific RIIbâ3 (GPIIb-IIIa) antagonist, was adminis-

tered to prevent platelets from aggregating and forming

the platelet plug necessary to promote hemostasis.

GPIIb-IIIa antagonists are generally very species selec-

tive, and of the many spirocyclic analogues that were

tested, 59 was determined to have the highest affinity

2050 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Mehrotra et al.

Ta ble 10. Dose-Dependent Effect of 59 (CT50787) vs DMSO

(Vehicle) on Bleeding Time in Mice

elimination half-life, and metabolic stability. A favorable

biological profile of these compounds as platelet ag-

gregation inhibitors clearly demonstrates the utility of

the spirocyclic structures as central templates for non-

peptide RGD mimics. The easy synthesis of these

templates and the useful oral pharmacokinetic profile

of novel compounds containing these templates may

facilitate their utilization in other drug targets.

treatment regimen,

mg/kg

mean BT

(s)

% of average

vehicle control

n

SD

vehicle control

10

15

3

116.0

96.7

30.4

22.1

-17

69

385

668

40

50

100

4

8

196.5

562.5

890.6

81.4

161.4

26.5

Exp er im en ta l Section

for the mouse GPIIb-IIIa receptor with an in vitro IC₅₀

value of 48 nM.

Unless otherwise noted, all starting materials were obtained

from commercial suppliers and used without further purifica-

tion. All reactions involving oxygen or moisture-sensitive

compounds were performed under a dry argon atmosphere.

All reaction mixtures and chromatography fractions were

analyzed by thin-layer chromatography on 250 mm silica gel

plates and visualized with UV light and I₂stain. Flash column

chromatography was carried out using Merck silica gel 60

To test the effects of 59 on mouse bleeding times, it

was administered iv to female C57/bl mice in 10%

DMSO in water at 10-100 mg/kg via a tail vein.

Bleeding was initiated by transection of the tip of the

tail, which was then immersed in a 37 °C normal saline

bath. The bleeding time was recorded until cessation of

blood flow by visual inspection or up to 15 min (900 s).

Because of the rapid clearance and a short half-life

of 59 in rodents, maintaining an in vivo exposure level

necessary to inhibit platelet aggregation and extend

bleeding times was achieved by administering high

doses via intravenous bolus injection. Table 10 shows

the bleeding times in the presence and absence of 59.

A dose-dependent extension of bleeding time was dem-

onstrated with iv administration of 59 with no effect at

the lowest dose of 10 mg/kg and a near maximal effect,

up to 668% of baseline, at the highest dose examined.

Ex vivo aggregation measurements were not accom-

plished at the end of the bleeding time procedure due

to the low level of drug in the plasma at the end of the

15 min time period.

1

(230-400 mesh). H NMR spectra were recorded on a Varian

Unity+400 instrument. Chemical shifts are expressed in ppm

downfield from internal tetramethylsilane. Apparent multi-

plicities are designated as s, singlet; d, doublet; t, triplet; q,

quartet; m, multiplet; b, broad. Low-resolution mass spectra

were recorded with a HP 1100-MSD LC-MS spectrometer.

High-resolution mass spectra (HRMS) were recorded with a

Sciex Qstar time-of-flight high-resolution mass spectrometer

coupled with an Agilent HPLC. All mass spectra were taken

in the positive ion mode under electrospray ionization (ESI).

Final compounds were purified by reverse-phase high-perfor-

mance liquid chromatography (RP-HPLC) using a Waters

4000Prep, Waters 490E multiwavelength detector, and Vydac

218TP1022 column (10 µm, C₁₈, 22 mm × 250 mm). The new

compounds synthesized were characterized by mass spectrom-

etry (MS) and NMR, and purity was determined by HRMS

and two RP-HPLC systems. We did not obtain melting points

and elemental analyses of these compounds, as most of the

derivatives were amorphous, hygroscopic, and prepared in

small quantities. Purity of the compounds was confirmed by

two diverse RP-HPLC systems using Waters 600 controller,

Waters 996 photodiode array detector, and Keystone Beta

Basic column (C18, 4.6 mm × 50 mm), analytical RP-HPLC

run using 214 nm for detection. RP-HPLC (# 1): Gradient

method utilized 5-85% CH₃CN/9 min in the H₂O mixture with

0.1% trifluoroacetic acid (TFA). RP-HPLC (# 2): Isocratic

method using various % of CH₃CN for 20 min in the H₂O

mixture with 0.1% trifluoroacetic acid (TFA) in order to have

the compound elute at a reasonable time during the run.

The typical experimental procedures, used in this study, for

the conversion of benzonitriles to benzamidines are decribed

below.

Despite the limitation of not directly correlating the

in vivo extension of bleeding time to ex vivo platelet

aggregation inhibition, this experiment shows for the

first time the use of a pharmacological agent which is

RIIbâ3 specific to show extension of bleeding times in

mice.³⁵In addition, the dose dependent extensions of

bleeding time shown in this study validates the platelet

dependence of the mouse bleeding time model.

Con clu sion s

We have described the novel 2,8-diazaspiro[4.5]decane

spirocyclic template for the disposition of pharmacoph-

ores to achieve potent and selective GPIIb-IIIa inhibi-

tion. Structural optimization within this scaffold has led

to the characterization of a 1-oxo-spirolactam 23

(CT50728) as a novel, low molecular weight, selective,

and reversible GPIIb-IIIa antagonist, with an IC₅₀of

0.110 µM for the inhibition of ADP-induced human

platelet aggregation in vitro. While the parent active

compound 23 has negligible oral bioavailability, its

double prodrug 22 (CT51464) exhibits good oral bio-

availability across species (rat, dog, monkey) and is

rapidly metabolized to the active compound in vivo. We

ascribe the excellent bioavailability of 22 to its molec-

ular rigidity, low polar surface area, remarkably good

water solubility as a crystalline hydrochloride salt (>10

mg/mL), and its lipophilicity (log P ) 0.45 ( 0.06). Its

pK_avalues (pK_a1) 11.28, and pK_a2) 4.74) indicate that

the drug remains uncharged over a wide range, and this

could enhance oral absorption.

Typ ica l P r oced u r e A. Ben za m id in es fr om Ben zon i-

tr iles via Th io-P in n er Syn th esis.^16a(a) H₂S gas was bubbled

into a solution of the benzonitrile (1.0 mmol) in 5.0 mL of the

solvent mixture pyridine/Et₃N (9:1) until a deep green color

persisted. The reaction mixture was then stirred overnight at

room temperature, concentrated, and partitioned between

5%MeOH/CH₂Cl₂and H₂O (3 ×). The combined organic extract

was washed with brine, dried over Na₂SO₄, and concentrated

to yield the thioamide as a yellow/green solid. (b) The thio-

amide was then dissolved in acetone (2.50 mL) and treated

with CH₃I (5.0 mmol). The clear solution was stirred overnight

at room temperature, and the resulting yellow suspension was

concentrated to dryness to yield thiomethylimidate as a bright

yellow solid. (c) The thiomethylimidate was mixed with

NH₄OAc (25.0 mmol), MeOH (5.0 mL) was added, and the

reaction mixture was either stirred overnight at room tem-

perature or refluxed for 2.0 h. After TLC/RP-HPLC showed

the complete consumption of the thiomethylimidate, methanol

was removed by rotary evaporation, and the benzamidine was

purified by prep RP-HPLC as either TFA or HCl salt.

Typ ica l P r oced u r e B. Syn th esis of th e N-Su bstitu ted

Ben za m id in es (39a , 40a ) fr om Ben zon itr iles via Th io-

P in n er Rou te. For the synthesis of the N-substituted ben-

Other potent inhibitors in this series also exhibited

the required PK profile, including bioavailability, long

Novel 2,8-Diazaspiro[4.5]decanes

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2051

zamidines (39a and 40a ), in step c of ‘Typical Procedure A,’

appropriate amine (EtNH₂, morpholine) was first treated with

AcOH (2.0 equiv) in MeOH, and then this mixture was added

to the thiomethylimidate. The resulting reaction mixture was

stirred at room temperature, concentrated, and purified by

prep RP-HPLC to afford the N-substituted-benzamidines.

4-(8-Ben zyl-1-oxo-2,8-d ia za sp ir o[4.5]d ec-2-yl)ben zon i-

tr ile (7) a n d 4-(8-Ben zyl-3-oxo-2,8-d ia za sp ir o[4.5]d ec-2-

yl)ben zon itr ile (8). To a suspension of imide 4 (500 mg, 1.39

mmol) in MeOH (30 mL) at 0 °C was added NaBH₄(275 mg,

7.27 mmol) in lots, and the mixture was stirred at room

temperature until TLC showed complete consumption of the

starting material. Then the reaction was quenched with AcOH

and concentrated to dryness to yield a mixture of 5 and 6 (1:2

by NMR, and analytical RP-HPLC).

The whole was then dissolved in TFA (25 mL) at 0 °C and

treated with NaBH₄(250 mg, 6.61 mmol) in lots (caution:

reaction is extremely vigorous!). The reaction mixture was

stirred at room temperature until TLC showed no starting

material, concentrated to dryness, and partitioned between

CH₂Cl₂/1 N NaOH (3 ×). The CH₂Cl₂extract was washed with

brine, dried over Na₂SO₄, and concentrated to yield a colorless

solid. Purification by RP-HPLC yielded 7 (97 mg) and 8 (210

mg), in 64% overall yield.

Typ ica l P r oced u r e C. Ben za m id in es fr om Ben zon i-

tr iles via Ben za m id oxim es.^16b(a) To benzonitrile (10.0

mmol) in EtOH (50.0 mL) was added Et₃N (50.0 mmol),

followed by hydroxylamine hydrochloride (30.0 mmol), and the

reaction mixture was either stirred overnight at room tem-

perature or heated to 60 °C for 5.0 h until RP-HPLC indicated

complete consumption of the benzonitrile. Then the reaction

mixture was concentrated under reduced pressure and purified

by prep RP-HPLC to afford pure benzamidoximes. (b) The

benzamidoxime (5.0 mmol) was dissolved in glacial acetic acid

(25.0 mL), and acetic anhydride (7.50 mmol) was added. After

0.5 h, RP-HPLC indicated complete consumption of the start-

ing material. Then to it was added 10% Pd/C (15 wt %), and

the mixture was shaken on a Parr hydrogenator at 40 psi H₂

for 5.0 h until RP-HPLC indicated no unreacted starting

material. The reaction mixture was filtered through Celite and

concentrated under reduced pressure to yield crude benzami-

dine, which was purified by prep RP-HPLC.

4-(8-Ben zyl-3-oxo-2,8-d ia za sp ir o[4.5]d ec-2-yl)ben zon i-

tr ile (8). ¹H NMR (CDCl₃): δ 7.78 (d, 2H), 7.62 (d, 2H), 7.3

(m, 5H), 3.62 (s, 2H), 3.56 (s, 2H), 2.56 (m, 4H), 2.40 (m, 4H),

1.71 (m, 4H). MS (ES): 346 (M + H)⁺.

2-(4-Cya n op h en yl)-1-oxo-2,8-d ia za sp ir o[4.5]d eca n e-8-

ca r boxylic Acid 2,2,2-Tr ich lor oeth yl Ester (9), a n d 2-(4-

Cya n op h en yl)-3-oxo-2,8-d ia za sp ir o[4.5]d eca n e-8-ca r box-

ylic Acid 2,2,2-tr ich lor oeth yl Ester (10). The mixture of

lactams 7 and 8 (2.30 g, 6.66 mmol) in CH₃CN/CH₂Cl₂(8:2)

(40 mL) was treated with 2,2,2-trichloroethyl chloroformate

(1.20 mL, 8.71 mmol), and the reaction mixture was stirred

overnight at room temperature. Then all the solvents were

evaporated, and the crude product was purified by flash

chromatography (5% CH₃CN/CH₂Cl₂) to afford 9 (792 mg) and

10 (1.633 g); combined yield 85%.

2-(4-Cya n op h en yl)-1-oxo-2,8-d ia za sp ir o[4.5]d eca n e-8-

ca r boxylic Acid 2,2,2-tr ich lor oeth yl Ester (9). ¹H NMR

(CDCl₃): δ 7.79 (d, 2H), 7.61 (d, 2H), 4.73 (q, 2H), 4.05 (m,

2H), 3.81 (t, 2H), 3.30 (m, 2H), 2.12 (m, 2H), 1.96 (m, 2H),

1.56 (m, 2H). MS (ES): 430 (M + H)⁺.

1-Ben zyl-4-cya n om eth ylp ip er id in e-4-ca r bon itr ile (1).

To a stirred solution of 1-benzyl-4-piperidone (50.0 g, 264.20

mmol) and ethyl cyanoacetate (39.0 g, 344.80 mmol) in

CH₂Cl₂(300 mL) was added Et₃N (75.0 mL, 538.10 mmol)

dropwise. Then crushed 4 Å molecular sieves (25 g) were

added, and the mixture was stirred at room temperature until

TLC check showed complete consumption of 1-benzyl-4-pip-

eridone. The reaction mixture was filtered through Celite, and

the filtrate was concentrated to yield (1-benzylpiperidin-4-

ylidine)cyanoacetic acid ethyl ester as a light yellow syrup.

This was then dissolved in EtOH (500 mL) and treated with

a solution of KCN (85.0 g, 1.30 mol) in H₂O (200 mL). The

brown solution was heated to reflux for 2.0 h, then concen-

trated to half its volume, and extracted with EtOAc (3 ×). The

combined EtOAc extract was washed with brine, dried over

Na₂SO₄, and concentrated to yield a brown solid. Flash

chromatography using EtOAc/hexane furnished pure 1, 55.10

g (87%). ¹H NMR (DMSO-d₆): δ 7.26 (m, 5H), 3.43 (s, 2H),

3.04 (s, 2H), 2.80 (d, 2H), 2.04 (m, 2H), 1.86 (d, 2H), 1.61 (m,

2H). MS (ES): 240 (M + H)⁺.

2-(4-Cya n op h en yl)-3-oxo-2,8-d ia za sp ir o[4.5]d eca n e-8-

1

ca r boxylic Acid 2,2,2-tr ich lor oeth yl Ester (10). H NMR

(CDCl₃): δ 7.72 (d, 2H), 7.62 (d, 2H), 4.73 (q, 2H), 3.78 (m,

2H), 3.67 (s, 2H), 3.4 (m, 2H), 2.6 (s, 2H), 1.75 (m, 4H). MS

(ES): 430 (M + H)⁺.

4-(1-Oxo-2,8-d ia za sp ir o[4.5]d ec-2-yl)ben zon itr ile (11).

To a solution of TCE-carbamate 9 (740 mg, 1.718 mmol) in

THF (10 mL), under argon, was added 10 mL of a 1 M solution

of NH₄OAc in H₂O (10 mmol), followed by 10% Cd-Pb couple¹⁸

(1.20 g, 9.0 mmol of Cd). The cloudy suspension was stirred

at room temperature until TLC showed disappearance of

starting material. Then the reaction mixture was filtered

through Celite, the aqueous layer was made basic with 1 N

NaOH, and extracted with CH₂Cl₂(3 ×). The combined CH₂-

Cl₂extract was washed with brine, dried over Na₂SO₄,

concentrated, and chromatographed to yield 11, 325 mg (74%).

¹H NMR (CDCl₃): δ 7.8 (d, 2H), 7.63 (d, 2H), 3.78 (m, 2H), 3.1

(m, 2H), 2.72 (t, 2H), 2.13 (t, 2H), 1.88 (m, 2H), 1.48 (m, 2H).

MS (ES): 256 (M + H)⁺.

1-Ben zyl-4-car boxym eth ylpiper idin e-4-car boxylic Acid

Hyd r och lor id e (2). A suspension of dinitrile 1 (50.0 g, 208.92

mmol) in concentrated HCl (750 mL) was refluxed for 3 days.

Then the pale yellow solution was concentrated to dryness to

afford the diacid 2 as an off-white solid, as its hydrochloride

1

salt, 64.0 g (98%). H NMR (DMSO-d₆): δ 11.0 (m, 2H), 7.60

(s, 2H), 7.45 (s, 3H), 4.24 (dd, 2H), 3.11 (d, 1H), 3.05 (s, 2H),

2.90 (q, 1H), 2.40 (s, 2H), 2.08 (d, 2H), 1.88 (t, 2H). MS (ES):

278 (M + H)⁺.

4-(8-Ben zyl-1,3-d ioxo-2,8-d ia za sp ir o[4.5]d ec-2-yl)ben -

zon itr ile (4). To a stirred solution of the diacid hydrochloride

(2) (4.70 g, 15.0 mmol) in DMF (50 mL) was added DCC (4.10

g, 19.87 mmol), and the slurry was stirred at room temperature

for 2.0 h to furnish the anhydride (3). To it was added

4-aminobenzonitrile (2.20 g, 18.62 mmol) and Et₃N (6.0 mL,

43.0 mmol). The suspension was stirred overnight at room

temperature, filtered through Celite, and concentrated to yield

1-benzyl-4-[(4-cyano-phenylcarbamoyl)-methyl]-piperidine-4-

carboxylic acid as a pale yellow syrup. This was then sus-

pended in acetic anhydride (75 mL), mixed with anhyd NaOAc

(4.50 g, 54.85 mmol), and refluxed for 2.0 h. The reaction

mixture was filtered through Celite, concentrated to dryness,

and partitioned between 5% MeOH-CH₂Cl₂/H₂O (3 ×). The

organic extract was washed with brine, dried over Na₂SO₄, and

concentrated. Flash chromatography using CH₂Cl₂/MeOH

afforded pure 4, as a colorless solid, 3.40 g (63%). ¹H NMR

(DMSO-d₆): δ 7.93 (d, 2H), 7.5 (d, 2H), 7.3 (m, 4H), 7.2 (m,

1H), 3.43 (s, 2H), 2.73 (m, 4H), 2.01 (t, 2H), 1.85 (t, 2H), 1.72

(d, 2H). MS (ES): 360 (M + H)⁺.

4-(3-Oxo-2,8-d ia za sp ir o[4.5]d ec-2-yl)ben zon itr ile (12).

This compound was prepared from TCE-carbamate 10 using

the method described above for the synthesis of 11. Colorless

1

solid, yield 89%. H NMR (DMSO-d₆): δ 7.8 (q, 4H), 3.62 (s,

2H), 2.65 (m, 4H), 2.42 (s, 2H), 1.43 (m, 4H). MS (ES): 256

(M + H)⁺.

1-Ben zyl-4-m eth ylam in opiper idin e-4-car bon itr ile (13b).

To a mixture of 1-benzyl-4-piperidone (47.32 g, 0.25 mol) and

methylamine hydrochloride (16.90 g, 0.25 mol) in 1:1 MeOH/

H₂O (100 mL), under stirring and at 0 °C, was added a solution

of KCN (16.30 g, 0.25 mol) in H₂O (40 mL) dropwise, and the

mixture was stirred overnight at room temperature. It was

then diluted with Et₂O/H₂O (150 mL/75 mL) and transferred

to a separatory funnel. The aqueous layer was extracted with

ether (2 ×), and the combined ether extract was washed with

brine, dried over Na₂SO₄, and concentrated to yield 13b, 53.90

1

g (94%). H NMR (CDCl₃): δ 7.29 (m, 5H), 3.53 (s, 2H), 2.80

2052 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Mehrotra et al.

(d, 2H), 2.50 (s, 3H), 2.34 (d, 2H), 2.0 (d, 2H), 1.71 (m, 2H).

stirred overnight at room temperature and then concentrated

to dryness and partitioned between CH₂Cl₂/1 N NaOH (3 ×).

The CH₂Cl₂extract was washed with brine, dried over Na₂-

SO₄, and concentrated to yield a colorless solid, which was

purified by RP-HPLC to yield 17b as a colorless solid, 562 mg

MS (ES): 230 (M + H)⁺.

1-(1-Ben zyl-4-cya n op ip er id in -4-yl)-3-(4-cya n op h en yl)-

1-m eth ylu r ea (14b). To a solution of 4-cyanophenyl isocyan-

ate (10.60 g, 73.54 mmol) in CH₂Cl₂(75 mL) at 0 °C was added

a solution of 13b (17.0 g, 74.13 mmol) in CH₂Cl₂(35 mL),

followed by Et₃N (14.0 mL, 100.44 mmol). After stirring

overnight at room temperature, the reaction mixture was

concentrated to dryness and recrystallized from CH₂Cl₂/MeOH/

hexane (150/10/100 mL) to afford 14b as a colorless solid, 25.20

g (92%). ¹H NMR (CDCl₃): δ 7.5 (t, 2H), 7.28 (m, 4H), 7.11

(m, 1H), 3.62 (s, 2H), 2.96 (m, 5H), 2.78 (m, 2H), 2.09 (m, 2H),

1.90 (d, 2H). MS (ES): 374 (M + H)⁺.

1

(73%). H NMR (CDCl₃): δ 7.65 (d, 2H), 7.55 (d, 2H), 7.28 (m,

5H), 3.60 (s, 2H), 3.55 (s, 2H), 2.96 (m, 2H), 2.79 (s, 3H), 2.08

(m, 4H), 1.48 (m, 2H). MS (ES): 361 (M + H)⁺.

3-{[2-(4-Cyan oph en yl)-1-oxo-2,8-diazaspir o[4.5]decan e-

8-ca r bon yl]a m in o}p r op ion ic Acid Eth yl Ester (20). To a

stirred solution of 11 (500 mg, 1.958 mmol) in CH₂Cl₂(25 mL)

at 0 °C was added ethyl-3-isocyanatopropionate (300 mg, 2.10

mmol), followed by Et₃N (320 µL, 2.30 mmol), and the mixture

was stirred at room temperature for 9 h. The reaction was then

quenched with H₂O (200 µL), concentrated to dryness, and

purified by flash chromatography (5% MeOH/CH₂Cl₂) to afford

1-(1-Ben zyl-4-cya n op ip er id in -4-yl)-3-(4-cya n op h en yl)-

u r ea (14a ). This compound was prepared starting from

1-benzyl-4-piperidone using the procedures described above for

14b and substituting methylamine hydrochloride with NH₄-

Cl in the first step (Scheme 2), in an overall yield of 74%, as

1

20, 715 mg (92%). H NMR (CDCl₃): δ 7.75 (d, 2H), 7.60 (d,

2H), 5.30 (m, 1H), 4.02 (q, 2H), 3.81 (t, 2H), 3.46 (m, 2H), 3.38

(m, 2H), 3.06 (m, 2H), 2.48 (m, 2H), 2.10 (t, 2H), 1.90 (m, 2H),

1.51 (m, 2H), 1.22 (t, 3H). MS (ES): 399 (M + H)⁺.

1

a colorless solid. H NMR (DMSO-d₆): δ 9.25 (s, 1H), 8.81 (s,

1H), 7.86 (d, 2H), 7.70 (d, 2H), 7.2-7.35 (m, 5H), 3.49 (s, 2H),

2.72 (m, 2H), 2.30 (m, 2H), 2.01 (m, 2H), 1.52 (d, 2H). MS

(ES): 360 (M + H)⁺.

3-{[2-(4-Ca r b a m im id oylp h en yl)-1-oxo-2,8-d ia za sp ir o-

[4.5]d eca n e-8-ca r b on yl]a m in o}p r op ion ic Acid E t h yl

Ester (21). Compound 21 was synthesized from 20 using

Typical Procedure A. (a). H₂S gas was bubbled into a solution

of 20 (125 mg, 0.313 mmol) in 5.0 mL of pyridine/Et₃N (9:1)

until a deep green color persisted. After stirring overnight at

room temperature, the mixture was concentrated and parti-

tioned between 5% MeOH/CH₂Cl₂and H₂O (3 ×). The organic

extract was washed with brine, dried over Na₂SO₄, and

concentrated to yield 3-{[1-oxo-2-(4-thiocarbamoylphenyl)-2,8-

diazaspiro[4.5]decane-8-carbonyl]amino}propionic acid ethyl

ester as a yellow/green solid. (b). This was then dissolved in

acetone (5.0 mL), CH₃I (195 µL, 3.13 mmol) was added, and

the reaction mixture was stirred overnight at room tempera-

ture. The yellow suspension was concentrated to dryness to

afford 3-{[2-(4-methylsulfanylcarbonimidoyl-phenyl)-1-oxo-2,8-

diazaspiro[4.5]decane-8-carbonyl]amino}propionic acid ethyl

ester as a yellow solid. (c). This was then dissolved in MeOH

(5.0 mL) and NH₄OAc (600 mg, 7.78 mmol) was added. The

reaction mixture was stirred overnight at room temperature,

concentrated to dryness, and purified by RP-HPLC to afford

21 as its HCl salt, as a colorless solid, 98 mg (75% overall from

4-(8-Ben zyl-1-m et h yl-2,4-d ioxo-1,3,8-t r ia za sp ir o[4.5]-

d ec-3-yl)ben zon itr ile (15b). 14b (3.0 g, 8.033 mmol) was

suspended in 1 N HCl (100 mL) and refluxed for 16 h. The

clear reaction mixture was then concentrated to dryness,

suspended in CH₂Cl₂, washed with 1 N NaOH and brine, dried

over Na₂SO₄, and concentrated. The crude product was tritu-

rated with ether to afford 15b as a colorless solid, 2.68 g (89%).

¹H NMR (CDCl₃): δ 7.74 (d, 2H), 7.64 (d, 2H), 7.28 (m, 5H),

3.62 (s, 2H), 2.96 (s, 3H), 2.82 (m, 4H), 2.14 (m, 2H), 1.76 (m,

2H). MS (ES): 375 (M + H)⁺.

4-(8-Ben zyl-2,4-d ioxo-1,3,8-t r ia za sp ir o[4.5]d ec-3-yl)-

ben zon itr ile (15a ). This compound was prepared from 14a

using the method described above for 15b, as a colorless solid

1

(80%). H NMR (DMSO-d₆): δ 9.14 (s, 1H), 7.91 (d, 2H), 7.62

(d, 2H), 7.28 (m, 4H), 7.22 (m, 1H), 3.48 (s, 2H), 2.71 (m, 2H),

2.13 (m, 2H), 1.92 (m, 2H), 1.71 (m, 2H). MS (ES): 361 (M +

H)⁺.

4-(1-Met h yl-2,4-d ioxo-1,3,8-t r ia za sp ir o[4.5]d ec-3-yl)-

ben zon itr ile (19b). This compound was synthesized from

15b, using the procedure described above for the preparation

of 11 from 7. (a) To 15b (1.70 g, 4.54 mmol) in CH₃CN/CHCl₃

(20/10 mL) at room temperature was added 2,2,2-trichloroethyl

chloroformate (1.0 mL, 7.264 mmol), and the mixture was

stirred at room temperature for 6 h. Then it was concentrated

to dryness and purified by flash chromatography to afford the

TCE-carbamate intermediate, 3-(4-cyanophenyl)-1-methyl-2,4-

dioxo-1,3,8-triazaspiro[4.5]decane-8-carboxylic acid 2,2,2-trichlo-

roethyl ester, 1.80 g (86%). (b) To a stirred solution of the TCE-

carbamate (1.80 g, 3.91 mmol) in THF (20 mL), under argon,

was added 20 mL of a 1 M solution of NH₄OAc in H₂O (20

mmol), followed by 10% Cd-Pb couple (2.70 g, 20.2 mmol of

Cd). The suspension was stirred overnight at room tempera-

ture and then filtered through Celite, and the aqueous layer

was made basic with 1 N NaOH and extracted with CH₂Cl₂

(3 ×). The CH₂Cl₂extract was washed with brine, dried over

Na₂SO₄, concentrated, and chromatographed to yield 19b, as

a colorless solid, 880 mg (79%).¹H NMR (CD₃OD): δ 7.80 (d,

2H), 7.63 (d, 2H), 3.27 (m, 2H), 2.99 (m, 2H), 2.91 (s, 3H), 1.97

(m, 2H), 1.80 (m, 2H). MS (ES): 285 (M + H)⁺.

4-(8-Ben zyl-1-m eth yl-2-oxo-1,3,8-tr ia za sp ir o[4.5]d ec-3-

yl)ben zon itr ile (17b). The urea 17b was synthesized from

the hydantoin 15b using the previously described procedure

for the synthesis of 7 and 8 from 4. (a) To a suspension of the

hydantoin 15b (800 mg, 2.136 mmol) in MeOH (30 mL) at 0

°C was added NaBH₄(400 mg, 10.57 mmol) in lots, and the

mixture was stirred overnight at room temperature. Then the

reaction was quenched with AcOH (1.0 mL) at 0 °C, and

concentrated to dryness to afford crude 4-(8-benzyl-4-hydroxy-

1-methyl-2-oxo-1,3,8-triazaspiro[4.5]dec-3-yl)benzonitrile 16b.

(b) The crude 16b was then dissolved in TFA (35 mL) at 0 °C

and treated with NaBH₄(400 mg, 10.57 mmol) in lots (cau-

tion: reaction is extremely vigorous!). The reaction mixture was

1

20). H NMR (CD₃OD): δ. 9.11 (s, 1H), 8.68 (s, 2H), 7.98 (d,

2H), 7.83 (d, 2H), 4.13 (q, 2H), 3.94 (m, 2H), 3.45 (t, 2H), 3.11

(m, 2H), 2.54 (m, 2H), 2.22 (t, 2H), 1.83 (m, 2H), 1.61 (m, 2H),

1.24 (t, 3H). MS (ES): 416 (M + H)⁺. Exact mass (FAB, M +

1)⁺calcd, 416.2220; found, 416.2239.

3-{[2-(4-Ca r b a m im id oylp h en yl)-1-oxo-2,8-d ia za sp ir o-

[4.5]d eca n e-8-ca r b on yl]a m in o}p r op ion ic

Acid

(23)

(CT50728). 21 (40 mg, 0.096 mmol) in THF (5.0 mL) was

treated with 1 M LiOH (1.0 mL), and the reaction mixture

was stirred at room temperature for 6 h. Then it was cooled

in ice, acidified with 6 N HCl, concentrated to dryness, and

purified by RP-HPLC to afford 23 as a colorless solid as its

TFA salt, 43 mg (89%). ¹H NMR (CD₃OD): δ 7.92 (d, 2H), 7.79

(d, 2H), 3.87 (m, 3H), 3.38 (t, 2H), 3.30 (m, 1H), 3.05 (t, 2H),

2.47 (m, 2H), 2.18 (t, 2H), 1.80 (m, 2H), 1.55 (d, 2H). MS (ES):

388 (M + H)⁺. Exact mass (FAB, M + 1)⁺calcd, 388.1979;

found, 388.1944.

3-({2-[4-(N-Hyd r oxyca r ba m im id oyl)p h en yl]-1-oxo-2,8-

d ia za sp ir o[4.5]d eca n e-8-ca r bon yl}a m in o)p r op ion ic Acid

Eth yl Ester (22) (CT51464). To 20 (1.13 g, 2.836 mmol) in

EtOH (25 mL) was added Et₃N (1.60 mL, 11.48 mmol),

followed by hydroxylamine hydrochloride (590 mg, 8.49 mmol).

The reaction mixture was then stirred overnight at room

temperature, concentrated, and purified by RP-HPLC to afford

22 as its HCl salt, 1.052 g (79%). ¹H NMR (CD₃OD): δ 7.93

(d, 2H), 7.70 (d, 2H), 4.12 (q, 2H), 3.91 (m, 4H), 3.40 (m, 2H),

3.06 (m, 2H), 2.51 (m, 2H), 2.20 (m, 2H), 1.81 (m, 2H), 1.56

(m, 2H), 1.23 (t, 3H). MS (ES): 432 (M + H)⁺. Exact mass

(FAB, M + 1)⁺calcd, 432.2241; found, 432.2254.

3-({2-[4-(N-Hyd r oxyca r ba m im id oyl)p h en yl]-1-oxo-2,8-

d ia za sp ir o[4.5]d eca n e-8-ca r bon yl}a m in o)p r op ion ic Acid

(24). The ethyl ester 22 (215 mg, 0.498 mmol) was treated with

Novel 2,8-Diazaspiro[4.5]decanes

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2053

3 N HCl (5.0 mL) and the mixture was stirred at room

temperature for 8 h. It was then concentrated, and purified

by RP-HPLC to afford 24 as its HCl salt, 203 mg (92%). ¹H

NMR (CD₃OD): δ 7.88 (d, 2H), 7.67 (d, 2H), 3.88 (t, 4H), 3.38

(t, 2H), 3.05 (t, 2H), 2.48 (t, 2H), 2.18 (t, 2H), 1.80 (t, 2H), 1.53

(d, 2H). MS (ES): 404 (M + H)⁺. Exact mass (FAB, M + 1)⁺

calcd, 404.1928; found, 404.1889.

ter t-Bu toxyca r bon ylm eth oxya cetic Acid (31). The reac-

tion mixture composed of 30a (1.0 g, 3.56 mmol) and 150 mg

of 20% Pd(OH)₂/C (Pearlman’s catalyst) in EtOAc (40 mL) was

shaken on a Parr hydrogenator at 40 psi of H₂for 8 h. It was

then filtered through Celite and concentrated to yield 31, 651

mg (96%). ¹H NMR (CD₃OD): δ 4.11 (s, 2H), 4.04 (s, 2H), 1.43

(s, 9H). MS (ES): 191 (M + H)⁺.

5-[2-(4-Cya n op h en yl)-1-oxo-2,8-d ia za sp ir o[4.5]d ec-8-

yl]-5-oxop en ta n oic Acid Eth yl Ester (25). To 11 (150 mg,

0.587 mmol) in DMF (3.0 mL) at 0 °C was added DIEA (180

µL, 1.03 mmol), followed by ethyl glutaryl chloride (136 mg,

0.761 mmol), and the reaction mixture was stirred at room

temperature for 4 h. Then it was partitioned between CH₂-

Cl₂/satd NaHCO₃. The combined CH₂Cl₂extract was washed

with brine, dried over Na₂SO₄, concentrated, and purified by

flash chromatography to afford 25, 214 mg (92%). ¹H NMR

(CDCl₃): δ 7.78 (d, 2H), 7.62 (d, 2H), 4.25 (m, 1H), 4.07 (q,

2H), 3.87 (m, 1H), 3.80 (m, 2H), 3.25 (m, 1H), 3.12 (m, 1H),

2.36 (m, 4H), 2.10 (m, 2H), 1.92 (m, 4H), 1.54 (m, 2H), 1.21 (t,

3H). MS (ES): 398 (M + H)⁺.

{2-[2-(4-Cya n op h en yl)-1-oxo-2,8-d ia za sp ir o[4.5]d ec-8-

yl]-2-oxo-eth oxy}a cetic Acid ter t-Bu tyl Ester (32). The

reaction mixture composed of 11 (70 mg, 0.274 mmol), 31 (70

mg, 0.368 mmol), HBTU (230 mg, 0.606 mmol), and DIEA (150

µL, 0.86 mmol) in anhyd DMF (5.0 mL) was stirred at room

temperature for 20 h. Then the reaction mixture was parti-

tioned between CH₂Cl₂/satd NaHCO₃. The combined CH₂Cl₂

extract was washed with brine, dried over Na₂SO₄, concen-

trated, and purified by flash chromatography to yield 32, 96

mg (82%). ¹H NMR (CDCl₃): δ 7.78 (d, 2H), 7.63 (d, 2H), 4.15-

4.35 (m, 5H), 4.0 (m, 1H), 3.82 (m, 2H), 3.2-3.4 (m, 2H), 2.13

(q, 2H), 1.95 (m, 2H), 1.59 (m, 2H), 1.46 (s, 9H). MS (ES): 428

(M + H)⁺.

5-[2-(4-Ca r b a m im id oylp h en yl)-1-oxo-2,8-d ia za sp ir o-

[4.5]d ec-8-yl]-5-oxop en ta n oic Acid Eth yl Ester (26). Com-

pound 25 was converted to 26, using Typical Procedure A,

elaborated above for the synthesis of 21 from 20. Overall yield,

{2-[2-(4-Ca r b a m im id oylp h en yl)-1-oxo-2,8-d ia za sp ir o-

[4.5]d ec-8-yl]-2-oxoeth oxy}a cetic Acid (33) (CT51907).

The compound 33 was synthesized from 32 by using Typical

Procedure A, described previously for the synthesis of 21 from

20, followed by deprotection of the tert-butyl ester functionality

by treating with neat TFA, concentration, and RP-HPLC

purification to yield as its HCl salt. Overall yield, 68%. ¹H

NMR (CD₃OD): δ 7.93 (d, 2H), 7.70 (d, 2H), 4.34 (m, 2H), 4.24

(m, 1H), 4.14 (m, 2H), 3.92 (m, 3H), 3.34 (m, 1H), 3.10 (m,

1H), 2.14 (m, 2H), 1.8 (m, 2H), 1.60 (m, 4H). MS (ES): 389 (M

+ H)⁺. Exact mass (FAB, M + 1)⁺calcd, 389.1819; found,

389.1731.

1

68%. H NMR (CD₃OD): δ 7.92 (d, 2H), 7.80 (d, 2H), 4.28 (m,

1H), 4.07 (q, 2H), 3.93 (m, 3H), 3.32 (m, 1H), 3.05 (m, 1H),

2.44 (t, 2H), 2.35 (t, 2H), 2.21 (m, 2H), 1.85 (m, 2H), 1.76 (m,

2H), 1.61 (m, 2H), 1.20 (t, 3H). MS (ES): 415 (M + H)⁺. Exact

mass (FAB, M + 1)⁺calcd, 415.2339; found, 415.2313.

5-[2-(4-Ca r b a m im id oylp h en yl)-1-oxo-2,8-d ia za sp ir o-

[4.5]d ec-8-yl]-5-oxop en ta n oic Acid (28) (CT50614). The

ethyl ester 26 (57 mg, 0.137 mmol) was suspended in 3 N HCl

(2.0 mL) and the reaction mixture was stirred overnight at

room temperature. Then it was concentrated, and purified by

RP-HPLC to afford 28, 49 mg (92%). ¹H NMR (CD₃OD): δ 7.80

(d, 2H), 7.68 (d, 2H), 4.17 (m, 1H), 3.8 (m, 3H), 3.21 (m, 1H),

2.94 (m, 1H), 2.33 (t, 2H), 2.23 (t, 2H), 2.10 (m, 2H), 1.6-1.8

(m, 4H), 1.52 (m, 2H). MS (ES): 387 (M + H)⁺. Exact mass

(FAB, M + 1)⁺calcd, 387.2026; found, 387.2017.

5-{2-[4-(N-Hyd r oxyca r b a m im id oyl)p h en yl]-1-oxo-2,8-

d ia za sp ir o[4.5]d ec-8-yl}-5-oxop en ta n oic Acid Eth yl Es-

ter (27) (CT51269). To 25 (400 mg, 1.006 mmol) in EtOH (10

mL) was added Et₃N (420 µL, 3.01 mmol), followed by

hydroxylamine hydrochloride (174 mg, 2.50 mmol). The mix-

ture was stirred overnight at room temperature, concentrated,

and purified by RP-HPLC to afford 27 as its HCl salt, as a

colorless solid, 390 mg (83%). ¹H NMR (CD₃OD): δ 7.85 (d,

2H), 7.66 (d, 2H), 4.35 (m, 1H), 4.05 (q, 2H), 3.80 (m, 3H), 3.31

(t, 1H), 3.04 (t, 1H), 2.43 (t, 2H), 2.34 (m, 2H), 2.18 (t, 2H),

1.83 (m, 2H), 1.74 (m, 2H), 1.60 (m, 2H), 1.17 (t, 3H). MS

(ES): 431 (M + H)⁺. Exact mass (FAB, M + 1)⁺calcd,

431.2288; found, 431.2294.

5-{2-[4-(N-Hyd r oxyca r b a m im id oyl)p h en yl]-1-oxo-2,8-

d ia za sp ir o[4.5]d ec-8-yl}-5-oxop en ta n oic Acid (29). The

ethyl ester 27 (160 mg, 0.37 mmol) was treated with 3 N HCl

(2.0 mL), and the reaction mixture was stirred at room

temperature for 9 h. Then it was concentrated, and purified

by RP-HPLC to afford 29 as its HCl salt, 155 mg (95%). ¹H

NMR (CD₃OD): δ 7.87 (d, 2H), 7.64 (d, 2H), 4.25 (m, 1H), 3.90

(m, 3H), 3.29 (m, 1H), 3.01 (m, 1H), 2.40 (t, 2H), 2.30 (t, 2H),

2.17 (m, 2H), 1.81 (m, 2H), 1.72 (m, 2H), 1.57 (m, 2H). MS

(ES): 403 (M + H)⁺. Exact mass (FAB, M + 1)⁺calcd,

403.1975; found, 403.2002.

{2-[2-(4-Cya n op h en yl)-1-oxo-2,8-d ia za sp ir o[4.5]d ec-8-

yl]-2-oxoeth oxy}a cetic Acid Eth yl Ester (35). A mixture

of 11 (225 mg, 0.881 mmol), 34 (172 mg, 1.061 mmol), HBTU

(435 mg, 1.147 mmol), and DIEA (400 µL, 2.29 mmol) in DMF

(5.0 mL) was stirred at room temperature for 6 h. Then it was

partitioned between CH₂Cl₂/satd NaHCO₃(3 ×). The combined

CH₂Cl₂extract was washed with brine, dried over Na₂SO₄,

concentrated, and purified by flash chromatography to afford

1

35, 306 mg (87%). H NMR (CDCl₃): δ 7.78 (d, 2H), 7.63 (d,

2H), 4.15-4.35 (m, 7H), 4.0 (m, 1H), 3.82 (m, 2H), 3.2-3.4 (m,

2H), 2.13 (q, 2H), 1.95 (m, 2H), 1.59 (m, 2H), 1.37 (t, 3H). MS

(ES): 400 (M + H)⁺.

(2-{2-[4-(N-Hyd r oxyca r ba m im id oyl)p h en yl]-1-oxo-2,8-

d ia za sp ir o[4.5]d ec-8-yl}-2-oxoeth oxy)a cetic Acid Eth yl

Ester Hyd r och lor id e (36) (CT052108). 35 (43 mg, 0.108

mmol) in EtOH (3.0 mL) at room temperature was treated with

Et₃N (125 µL, 0.896 mmol), followed by hydroxylamine hy-

drochloride (33.0 mg, 0.47 mmol), and the mixture was stirred

overnight at room temperature. Then it was concentrated

under reduced pressure and purified by RP-HPLC to afford

1

36 as its HCl salt, 42 mg (83%). H NMR (CD₃OD): δ 7.93 (d,

2H), 7.71 (d, 2H), 4.15-4.4 (m, 8H), 3.93 (q, 2H), 3.32 (m, 1H),

3.11 (m, 1H), 2.25 (m, 2H), 1.8 (m, 2H), 1.65 (m, 2H), 1.25 (t,

3H). MS (ES): 433 (M + H)⁺. Exact mass (FAB, M + 1)⁺calcd,

433.2081; found, 433.2086.

(2-{2-[4-(N-Hyd r oxyca r ba m im id oyl)p h en yl]-1-oxo-2,8-

d ia za sp ir o[4.5]d ec-8-yl}-2-oxoeth oxy)a cetic Acid (37). 36

(16.0 mg, 0.034 mmol) was treated with 3 N HCl (1.0 mL),

and the mixture was stirred at room temperature for 3 h. Then

it was purified by RP-HPLC to afford 37, 13 mg(86%). ¹H NMR

(CD₃OD): δ 7.88 (d, 2H), 7.66 (d, 2H), 4.33 (d, 2H), 4.24 (m,

1H), 3.89 (q, 3H), 3.29 (m, 1H), 3.11 (m, 1H), 2.20 (m, 2H),

1.79-1.92 (m, 2H), 1.62 (m, 2H). MS (ES): 405 (M + H)⁺.

Exact mass (FAB, M + 1)⁺calcd, 405.1768; found, 405.1775.

5-{2-[4-(N -E t h ylca r b a m im id oyl)p h e n yl]-1-oxo-2,8-

d ia za sp ir o[4.5]d ec-8-yl}-5-oxop en ta n oic Acid (40b). 40b

was synthesized using Typical Procedure B. (a). The benzoni-

trile 25 (36 mg, 0.090 mmol) was converted to thiomethylimi-

date 38 by Typical Procedure A, described previously for the

synthesis of 21. (b). EtNH₂(2.0 M/THF) (1.0 mL, 2.0 mmol) in

MeOH (2.0 mL) was treated with AcOH (230 µL, 4.0 mmol).

Then this mixture was added to thiomethyl imidate 38 (0.090

ter t-Bu toxyca r bon ylm eth oxya cetic Acid Ben zyl Ester

(30a ). To a stirred solution of benzyl glycolate (2.34 g, 14.0

mmol) and rhodium acetate dimer (62 mg, 0.14 mmol) in CH₂-

Cl₂(20 mL) was added a solution of tert-butyl diazoacetate

(2.01 g, 14.0 mmol) in CH₂Cl₂(20.0 mL), via a syringe pump

over 2.0 h. The reaction mixture was stirred overnight at room

temperature, filtered through Celite, concentrated, and puri-

fied by flash chromatography (20% EtOAc/hexane) to afford

1

30a , 2.40 g (61%). H NMR (CDCl₃): δ 7.35 (m, 5H), 5.20 (s,

2H), 4.13 (s, 2H), 4.08 (s, 2H), 1.46 (s, 9H). MS (ES): 281

(M + H)⁺.

2054 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Mehrotra et al.

mmol). After a stirring of 15 h at room temperature, the

reaction mixture was concentrated, and purified by RP-HPLC

to afford 40a , 5-{2-[4-(N-Ethylcarbamimidoyl)-phenyl]-1-oxo-

2,8-diaza-spiro[4.5]dec-8-yl}-5-oxopentanoic acid ethyl ester,

TFA salt, 39 mg (77% overall). (c). 40a (34 mg, 0.061 mmol)

was treated with 3 N HCl (1.0 mL) and the reaction mixture

was stirred at room temperature for 8h. Then it was concen-

trated, and purified by RP-HPLC to afford 40b, 29 mg (90%),

as a colorless solid. ¹H NMR (CD₃OD): δ 7.84 (d, 2H), 7.68 (d,

2H), 4.25 (m, 1H), 3.87 (m, 3H), 3.40 (m, 2H), 3.29 (m, 1H),

3.01 (m, 1H), 2.41 (m, 2H), 2.30 (m, 2H), 2.17 (m, 2H), 1.65-

1.8 (m, 5H), 1.57 (m, 2H), 1.28 (t, 2H). MS (ES): 415 (M +

H)⁺. Exact mass (FAB, M + 1)⁺calcd, 415.2267; found,

415.2285.

δ 7.86 (d, 2H), 7.72 (d, 2H), 4.23 (m, 1H), 3.9 (m, 3H), 3.3 (m,

1H), 3.01 (m, 1H), 2.46 (m, 2H), 2.1-2.4 (m, 5H), 1.77 (m, 2H),

1.58 (m, 2H), 0.97 (d, 3H). MS (ES): 401 (M + H)⁺. Exact mass

(FAB, M + 1)⁺calcd, 401.2183; found, 401.2142.

5-[2-(4-Ca r b a m im id oylp h en yl)-1-oxo-2,8-d ia za sp ir o-

[4.5]dec-8-yl]-3-(R)-m eth yl-5-oxopen tan oic Acid (45). Com-

pound 45 was synthesized starting from (R)-1-ethyl-hydrogen-

3-methyl glutarate and 11 by the procedures described above

for the synthesis of 44, in 71% overall yield. ¹H NMR (CD₃-

OD): δ 7.86 (d, 2H), 7.72 (d, 2H), 4.18 (m, 1H), 3.9 (m, 3H),

3.3 (m, 1H), 3.01 (m, 2H), 2.54 (d, 1H), 2.40 (d, 1H), 2.21 (t,

2H), 1.83 (m, 2H), 1.56 (m, 2H), 1.2 (d, 3H). MS (ES): 401 (M

+ H)⁺. Exact mass (FAB, M + 1)⁺calcd, 401.2183; found,

401.2156.

5-{2-[4-(Im in om or p h olin -4-ylm eth yl)p h en yl]-1-oxo-2,8-

d ia za sp ir o[4.5]d ec-8-yl}-5-oxop en ta n oic Acid Eth yl Es-

ter (39a ). 39a was synthesized from 38 using Typical Proce-

dure B. Morpholine (35 µL, 0.40 mmol) in MeOH (1.0 mL) was

treated with AcOH (50 µL, 0.87 mmol). This clear solution was

then added to thiomethylimidate 38 (0.10 mmol, prepared from

0.10 mmol of 25). The reaction mixture was stirred at room

temperature for 20 h, concentrated, and purified by RP-HPLC

to afford 39a , as its TFA salt, 49 mg (82%). ¹H NMR (CD₃-

OD): δ 7.93 (d, 2H), 7.60 (d, 2H), 4.30 (m, 1H), 4.09 (q, 2H),

3.93 (m, 3H), 3.89 (m, 2H), 3.78 (t, 2H), 3.72 (t, 2H), 3.49 (t,

2H), 3.35 (m, 1H), 3.07 (m, 1H), 2.46 (m, 2H), 2.39 (t, 2H),

2.24 (m, 2H), 1.89 (m, 2H), 1.78 (m, 2H), 1.63 (m, 2H), 1.21 (t,

3H). MS (ES): 485 (M + H)⁺. Exact mass (FAB, M + 1)⁺calcd,

485.2758; found, 485.2775.

5-{2-[4-(Im in om or p h olin -4-ylm eth yl)p h en yl]-1-oxo-2,8-

d ia za sp ir o[4.5]d ec-8-yl}-5-oxop en ta n oic Acid (39b). 39b

was synthesized from 39a by the procedure described above

for the synthesis of 40b from 40a , to yield 39b as its TFA salt,

40 mg (86%).¹H NMR (CD₃OD): δ 7.86 (d, 2H), 7.52 (d, 2H),

4.23 (m, 1H), 3.85 (m, 3H), 3.71 (t, 2H), 3.66 (t, 2H), 3.30 (m,

1H), 3.01 (m, 1H), 2.41 (m, 2H), 2.32 (m, 2H), 2.19 (t, 2H),

1.80 (m, 2H), 1.73 (m, 2H), 1.59 (m, 2H). MS (ES): 457 (M +

H)⁺. Exact mass (FAB, M + 1)⁺calcd, 457.2445; found,

457.2459.

3-(R)-Meth ylp en ta n ed ioic Acid ter t-Bu tyl Ester Eth yl

Ester (41). The reaction mixture comprised of (R)-1-ethyl

hydrogen-3-methylglutarate (1.0 g, 5.74 mmol), tert-butyl

alcohol (4.30 g, 58.0 mmol), DMAP (250 mg, 2.046 mmol), and

DCC (1.50 g, 7.27 mmol) in CH₂Cl₂(10 mL) was stirred at room

temperature for 20 h. Then it was partitioned between CH₂-

Cl₂/satd NaHCO₃. The combined CH₂Cl₂extract was washed

with brine, dried over Na₂SO₄, concentrated, and purified by

flash chromatography (CH₂Cl₂/MeOH) to yield 41, 1.10 g (83%).

MS (ES): 231 (M + H)⁺.

3-(S)-Meth ylp en ta n ed ioic Acid Mon o-ter t-bu tyl Ester

(42). Compound 41 (1.20 g, 4.647 mmol) in THF (40 mL) was

treated with 1 M LiOH (10 mL, 10.0 mmol), and the mixture

was stirred at room temperature for 18 h. It was acidified at

0 °C with AcOH (2.0 mL), and partitioned between EtOAc/

brine. The combined EtOAc extract was washed with brine,

dried over Na₂SO₄, concentrated, and purified by flash chro-

matography (CH₂Cl₂/MeOH) to afford 42, 854 mg (81%). MS

(ES): 203 (M + H)⁺.

3-P h en ylpen tan edioic Acid Mon oeth yl Ester (46). 3-Phe-

nylglutaric acid (1.0 g, 4.80 mmol) was suspended in Ac₂O (20.0

mL), and the reaction mixture was refluxed for 15 h. Then it

was concentrated under reduced pressure to afford 3-phenyl-

glutaric anhydride.

This was dissolved in EtOH (30.0 mL), Et₃N (3.0 mL, 21.52

mmol) was added, and the mixture was stirred at room

temperature for 20 h. Then it was concentrated and purified

by flash chromatography (40% EtOAc/hexane) to afford 46, 984

mg (87%). ¹H NMR (CD₃OD): δ 7.24 (m, 5H), 3.96 (q, 2H),

3.53 (m, 1H), 2.5-2.8 (m, 4H), 1.08 (t, 3H). MS (ES): 237 (M

+ H)⁺.

5-[2-(4-Cya n op h en yl)-1-oxo-2,8-d ia za sp ir o[4.5]d ec-8-

yl]-5-oxo-3-p h en ylp en ta n oic Acid Eth yl Ester (47). The

reaction mixture composed of 11 (90 mg, 0.352 mmol), 46 (110

mg, 0.465 mmol), EDC‚HCl (160 mg, 0.834 mmol), and HOBt

(95 mg, 0.70 mmol) in anhyd DMF (4.0 mL) was stirred at

room temperature for 20 h. Then it was partitioned between

CH₂Cl₂/satd NaHCO₃. The combined CH₂Cl₂extract was

washed with brine, dried over Na₂SO₄, concentrated, and

purified by flash chromatography (CH₂Cl₂/MeOH) to afford 47,

1

149 mg (89%). H NMR (CDCl₃): δ 7.76 (d, 2H), 7.60 (d, 2H),

7.18 (m, 5H), 4.05-4.25 (m, 2H), 3.98 (q, 2H), 3.79 (m, 3H),

3.68 (m, 1H), 3.16 (m, 2H), 2.81 (m, 1H), 2.64 (m, 4H), 2.05

(m, 2H), 1.8 (m, 2H), 1.09 (t, 3H). MS (ES): 474 (M + H)⁺.

5-[2-(4-Ca r b a m im id oylp h en yl)-1-oxo-2,8-d ia za sp ir o-

[4.5]d ec-8-yl]-5-oxo-3-p h en ylp en ta n oic Acid (48). Com-

pound 48 was synthesized from 47 by using the procedures

described previously for the synthesis of 23 from 20, in overall

yield of 64%. ¹H NMR (CD₃OD): δ 7.92 (d, 2H), 7.8 (d, 2H),

7.2 (m, 5H), 4.2 (m, 1H), 3.92 (m, 3H), 3.58 (m, 1H), 2.9-3.25

(m, 2H), 2.6-2.8 (m, 4H), 2.10 (m, 2H), 1.2-1.8 (m, 4H). MS

(ES): 463 (M + H)⁺. Exact mass (FAB, M + 1)⁺calcd,

463.2267; found, 463.2288.

3-(4-Nitr o-p h en oxyca r bon yla m in o)-bu tyr ic Acid Eth yl

Ester (51). Ethyl 3-(R)-methyl-â-alanine (50) (1.0 g, 7.623

mmol) in CH₂Cl₂(20.0 mL) was treated with DIEA (3.0 mL,

17.22 mmol), followed by 4-nitrophenyl chloroformate (2.0 g,

9.922 mmol), and the mixture was stirred at room temperature

for 9 h. Then it was partitioned between CH₂Cl₂/satd NaHCO₃.

The combined CH₂Cl₂extract was washed with brine, dried

over Na₂SO₄, concentrated, and purified by flash chromatog-

1

raphy to afford 51, 1.96 g (87%). H NMR (CDCl₃): δ 8.22 (d,

2H), 7.30 (d, 2H), 5.84 (s, 1H), 4.16 (m, 3H), 2.60 (m, 2H), 1.33

(d, 3H), 1.26 (t, 3H). MS (ES): 297 (M + H)⁺.

5-[2-(4-Cya n op h en yl)-1-oxo-2,8-d ia za sp ir o[4.5]d ec-8-

yl]-3-(S)-m et h yl-5-oxop en t a n oic Acid ter t-Bu t yl E st er

(43). The reaction mixture comprised of 11 (70.0 mg, 0.274

mmol), 42 (80 mg, 0.395 mmol), HBTU (230 mg, 0.606 mmol),

and DIEA (0.150 mL, 0.86 mmol) in DMF (2.0 mL) was stirred

at room temperature for 17 h. Then it was partitioned between

CH₂Cl₂/satd NaHCO₃. The combined CH₂Cl₂extract was

washed with brine, dried over Na₂SO₄, concentrated, and

purified by flash chromatography (CH₂Cl₂/MeOH) to yield 43,

103 mg (86%). MS (ES): 440 (M + H)⁺.

3-{[2-(4-Cyan oph en yl)-1-oxo-2,8-diazaspir o[4.5]decan e-

8-ca r bon yl]a m in o}-bu tyr ic Acid Eth yl Ester (52). 11 (500

mg, 1.958 mmol) in CH₂Cl₂(10.0 mL) was treated with DIEA

(0.700 mL, 4.02 mmol), followed by 51 (750 mg, 2.531 mmol),

and the reaction mixture was stirred overnight at room

temperature. Then it was partitioned between CH₂Cl₂/satd

NaHCO₃. The combined CH₂Cl₂extract was washed with

brine, dried over Na₂SO₄, concentrated, and purified by flash

chromatography to afford 52, 687 mg (85%). ¹H NMR

(CDCl₃): δ 7.78 (d, 2H), 7.63 (d, 2H), 5.7 (s, 1H), 4.23 (m, 1H),

4.12 (m, 2H), 3.83 (m, 4H), 3.12 (m, 2H), 2.51 (m, 2H), 2.12

(m, 2H), 1.96 (m, 2H), 1.56 (m, 2H), 1.2 (m, 6H). MS (ES): 413

(M + H)⁺.

5-[2-(4-Ca r b a m im id oylp h en yl)-1-oxo-2,8-d ia za sp ir o-

[4.5]dec-8-yl]-3-(S)-m eth yl-5-oxopen tan oic Acid (44). Com-

pound 44 was synthesized from 43, by Typical Procedure A,

described previously for the synthesis of 21 from 20, followed

by deprotection of the tert-butyl ester functionality by treat-

3-{[2-(4-Ca r b a m im id oylp h en yl)-1-oxo-2,8-d ia za sp ir o-

[4.5]d eca n e-8-ca r bon yl]a m in o}-bu tyr ic Acid (53). Com-

1

ment with neat TFA, in 67% overall yield. H NMR (CD₃OD):

Novel 2,8-Diazaspiro[4.5]decanes

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2055

pound 53 was synthesized from 52 by using Typical Procedure

A, described previously for the synthesis of 23 from 20, in

overall yield of 70%. ¹H NMR (CD₃OD): δ 7.9 (d, 2H), 7.78 (d,

2H), 4.14 (m, 1H), 3.88 (m, 4H), 3.04 (m, 2H), 2.48 (m, 1H),

2.38 (m, 1H), 2.18 (d, 2H), 1.79 (m, 2H), 1.53 (d, 2H), 1.15 (m,

3H). MS (ES): 402 (M + H)⁺. Exact mass (FAB, M + 1)⁺calcd,

402.2135; found, 402.2160.

mL, 17.22 mmol), followed by tosyl chloride (1.64 g, 8.60 mmol),

and stirred at room temperature for 2.0 h. Then the reaction

mixture was partitioned between CH₂Cl₂/satd NaHCO₃. The

combined CH₂Cl₂extract was washed with brine, dried over

Na₂SO₄, concentrated, and purified by flash chromatography

(CH₂Cl₂/MeOH) to yield 60, 2.30 g (69%). ¹H NMR (CDCl₃): δ

7.70 (d, 2H), 7.28 (d, 2H), 5.50 (d, 1H), 4.9 (m, 1H), 3.95 (m,

3H), 3.44 (m, 2H), 2.39 (s, 1H), 1.41 (s, 9H), 1.11 (t, 2H). MS

(ES): 387 (M + H)⁺.

3-(4-Nitr o-p h en oxyca r bon yla m in o)-3-p h en ylp r op ion -

ic Acid Eth yl Ester (54). 3-Amino-3-phenylpropionic acid

ethyl ester (1.0 g, 5.175 mmol) was converted to its 4-nitro-

phenyl carbamate 54 by using the procedure described previ-

3-Am in o-2-(t olu en e-4-su lfon yla m in o)p r op ion ic Acid

Eth yl Ester (61). 60 (1.72 g, 4.45 mmol) was treated with

anhyd TFA (15.0 mL) at room temperature. After 2.0 h the

reaction mixture was concentrated to dryness, and partitioned

between EtOAc/satd NaHCO₃. The EtOAc extract was washed

with brine, dried over Na₂SO₄, and concentrated to yield 61,

1

ously for the synthesis of 51 from 50. Yield, 1.54 g (83%). H

NMR (CDCl₃): δ 8.21 (d, 2H), 7.3 (m, 7H), 6.31 (d, 1H), 5.22

(m, 1H), 4.1 (q, 2H), 2.93 (m, 2H), 1.18 (t, 3H). MS (ES): 359

(M + H)⁺.

1

1.02 g (90%). H NMR (CDCl₃): δ 7.71 (d, 2H), 7.27 (d, 2H),

3-{[3-(4-C y a n o p h e n y l)-1-m e t h y l-2,4-d i o x o -1,3,8-

tr ia za sp ir o[4.5]d eca n e-8-ca r bon yl]a m in o}-3-p h en ylp r o-

p ion ic Acid Eth yl Ester (55). Compound 55 was synthesized

in 79% overall yield from 54 (160 mg, 0.446 mmol) and 19b

(100 mg, 0.351 mmol) by using the procedure described

previously for the synthesis of 52. ¹H NMR (CD₃OD): δ 7.8

(d, 2H), 7.62 (d, 2H), 7.35 (d, 2H), 7.3 (d, 2H), 7.2 (m, 1H), 5.3

(dd, 1H), 4.05 (m, 4H), 3.4-3.6 (m, 2H), 2.90 (s, 3H), 2.7-2.85

(m, 2H), 1.95-2.1 (m, 2H), 1.85 (d, 2H), 1.09 (t, 3H). MS (ES):

504 (M + H)⁺.

3.98 (q, 2H), 3.86 (m, 1H), 2.95-3.05 (m, 2H), 2.40 (s, 3H),

1.10 (t, 3H). MS (ES): 287 (M + H)⁺.

3-{[2-(4-Cyan oph en yl)-1-oxo-2,8-diazaspir o[4.5]decan e-

8-ca r bon yl]a m in o}-2-(tolu en e-4-su lfon yla m in o)p r op ion -

ic Acid Eth yl Ester (62). A mixture of 11 (28.0 mg, 0.109

mmol) and DIEA (80 µL, 0.46 mmol) in CH₂Cl₂(3.0 mL) was

added dropwise to a solution of triphosgene (12.0 mg, 0.0404

mmol) in CH₂Cl₂(2.0 mL) at 0 °C, and the mixture was stirred

at 0 °C for 0.5 h. Then was added a solution of 61 (33.0 mg,

0.13 mmol) in CH₂Cl₂(1.0 mL), and the mixture was stirred

overnight at room temperature. Then it was partitioned

between CH₂Cl₂/satd NaHCO₃, and CH₂Cl₂extract was washed

with brine, dried over Na₂SO₄, concentrated, and purified by

prep-TLC (5% MeOH/CH₂Cl₂) to afford 62, 40 mg (64%). ¹H

NMR (CDCl₃): δ 7.79 (d, 2H), 7.69 (d, 2H), 7.63 (d, 2H), 7.28

(d, 2H), 5.77 (d, 1H), 5.14 (t, 1H), 4.01 (q, 2H), 3.8-3.95 (m,

2H), 3.82 (t, 2H), 3.68-3.76 (m, 2H), 3.3-3.45 (m, 1H), 3.2-

3.3 (m, 1H), 3.03 (m, 1H), 2.40 (s, 3H), 2.13 (t, 2H), 1.85-2.0

(m, 2H), 1.5-1.65 (m, 2H), 1.12 (t, 3H). MS (ES): 568 (M +

H)⁺.

3-{[3-(4-Car bam im idoylph en yl)-1-m eth yl-2,4-dioxo-1,3,8-

tr ia za sp ir o[4.5]d eca n e-8-ca r bon yl]a m in o}-3-p h en ylp r o-

p ion ic Acid (56). Compound 56 was synthesized from 55 by

using Typical Procedure A, described previously for the

1

synthesis of 23 from 20, in overall yield 69%. H NMR (CD₃-

OD): δ 7.9 (d, 2H), 7.7 (d, 2H), 7.35 (d, 2H), 7.3 (d, 2H), 7.2

(m, 1H), 5.3 (dd, 1H), 4.05 (t, 2H), 3.4-3.6 (m, 2H), 2.90 (s,

3H), 2.7-2.85 (m, 2H), 1.95-2.1 (m, 2H), 1.85 (d, 2H). MS

(ES): 493 (M + H)⁺. Exact mass (FAB, M + 1)⁺calcd,

493.2121; found, 493.2146.

2-(3,5-Dim eth ylisoxa zole-4-su lfon yla m in o)p en ta n ed io-

ic Acid 1-ter t-Bu tyl Ester (57). l-Glutamic acid R-tert-butyl

ester (1.0 g, 4.92 mmol) was dissolved in 1 N NaOH (5.0 mL,

5.0 mmol), diluted with H₂O (50 mL), and cooled to 0 °C. Then

Na₂CO₃(583 mg, 5.50 mmol) was added, followed by 3,5-

dimethylisoxazole-4-sulfonyl chloride (1.06 g, 5.418 mmol), and

the mixture was stirred at room temperature for 20 h. Then

it was acidified with 2 N HCl at 0 °C to pH 3 and extracted

with EtOAc (3 ×). The combined EtOAc extract was washed

with brine, dried over Na₂SO₄, concentrated, and purified by

RP-HPLC to afford 57, 1.57 g (88%). ¹H NMR (CDCl₃): δ 3.75

(m, 1H), 2.60 (s, 3H), 2.54 (m, 1H), 2.39 (s, 3H), 2.08 (m, 1H),

1.84 (m, 1H), 1.32 (s, 9H). MS (ES): 363 (M + H)⁺.

3-{[2-(4-Ca r b a m im id oylp h en yl)-1-oxo-2,8-d ia za sp ir o-

[4.5]d eca n e-8-ca r bon yl]a m in o}-2-(tolu en e-4-su lfon yla m i-

n o)p r op ion ic Acid (63). Compound 63 was synthesized from

62 by using Typical Procedure C. (a) 62 (48.0 mg, 0.0845 mmol)

in EtOH (2.0 mL) was treated with Et₃N (60 µL, 0.430 mmol),

followed by hydroxylamine hydrochloride (24.0 mg, 0.345

mmol). The reaction mixture was heated to 50 °C for 8.0 h

and then partitioned between CH₂Cl₂/brine. The CH₂Cl₂

extract was dried over Na₂SO₄and concentrated to afford 3-({2-

[4-(N-Hydroxycarbamimidoyl)-phenyl]-1-oxo-2,8-diaza-spiro-

[4.5]decane-8-carbonyl}amino)-2-(toluene-4-sulfonylamino)-

propionic acid ethyl ester, 35 mg (69%). (b) This was then

dissolved in acetic acid (1.0 mL), treated with acetic anhydride

(12.0 µL, 0.127 mmol), and stirred at room temperature for

0.5 h. Then 10% Pd/C (10.0 mg) was added, and the reaction

mixture was stirred under 1 atm H₂pressure (balloon) for 3.0

h. The mixture was filtered through Celite and concentrated

to afford 3-{[2-(4-carbamimidoylphenyl)-1-oxo-2,8-diazaspiro-

[4.5]decane-8-carbonyl]amino}-2-(toluene-4-sulfonylamino)pro-

pionic acid ethyl ester. (c) This was then dissolved in THF (5.0

mL), treated with 1 M LiOH (1.0 mL, 1.0 mmol), and the

mixture was stirred at room temperature for 14 h. The reaction

mixture was acidified with 6N HCl, concentrated to dryness,

and purified by RP-HPLC to afford 63 as a colorless solid as

5-[2-(4-Cya n op h en yl)-1-oxo-2,8-d ia za sp ir o[4.5]d ec-8-

yl]-2-(3,5-d im eth ylisoxa zole-4-su lfon yla m in o)-5-oxop en -

ta n oic Acid ter t-Bu tyl Ester (58). The reaction mixture

composed of 11 (25.0 mg, 0.0979 mmol), 57 (42 mg, 0.116

mmol), and EDC‚HCl (37.0 mg, 0.193 mmol) in DMF (2.0 mL)

was stirred at room temperature for 18 h. Then it was

partitioned between CH₂Cl₂/satd NaHCO₃. The combined CH₂-

Cl₂extract was washed with brine, dried over Na₂SO₄,

concentrated, and purified by flash chromatography (CH₂Cl₂/

1

MeOH) to afford 58, 53 mg (90%). H NMR (CD₃OD): δ 7.95-

(d, 2H), 7.80 (d, 2H), 4.25 (m, 1H), 3.95 (m, 4H), 3.35 (m, 1H),

3.13 (q, 1H), 2.58 (m, 4H), 2.38 (s, 3H), 2.22 (t, 2H), 1.6-2.0

(m, 7H), 1.43 (s, 9H). MS (ES): 600 (M + H)⁺.

1

its TFA salt, 43 mg (76% overall). H NMR (CD₃OD): δ 7.96

(d, 2H), 7.83 (d, 2H), 7.71 (d, 2H), 7.34 (d, 2H), 4.0 (dd, 1H),

3.93 (t, 2H), 3.84 (m, 2H), 3.51 (dd, 1H), 3.1 (t, 2H), 2.42 (s,

3H), 2.20 (t, 2H), 1.82 (m, 2H), 1.59 (d, 2H), 1.36 (dd, 1H). MS

(ES): 557 (M + H)⁺. Exact mass (FAB, M + 1)⁺calcd,

557.2176; found, 557.2207.

2-Ben zyloxyca r bon yla m in op en ta n ed ioic Acid 5-ter t-

Bu tyl Ester 1-Eth yl Ester (64). The reaction mixture,

consisting of Z-Glu (O-t-Bu)-OH (5.0 g, 14.82 mmol), EtOH

(8.75 mL, 150 mmol), EDC‚HCl (6.53 g, 34.06 mmol), and

HOBt (4.53 g, 33.52 mmol) in DMF (50 mL) was stirred at

room temperature for 16 h. Then it was partitioned between

CH₂Cl₂/satd NaHCO₃, the CH₂Cl₂extract was washed with

brine, dried over Na₂SO₄, concentrated, and purified by flash

5-[2-(4-Ca r b a m im id oylp h en yl)-1-oxo-2,8-d ia za sp ir o-

[4.5]d ec-8-yl]-2-(3,5-d im eth ylisoxa zole-4-su lfon yla m in o)-

5-oxop en ta n oic Acid (59) (CT50787). Compound 59 was

synthesized from 58 using Typical Procedure A, described

previously for the synthesis of 21 from 20. Overall yield, 66%.

¹H NMR (CD₃OD): δ 7.95(d, 2H), 7.80 (d, 2H), 4.25 (m, 1H),

3.95 (m, 4H), 3.35 (m, 1H), 3.13 (q, 1H), 2.58 (m, 4H), 2.38 (s,

3H), 2.22 (t, 2H), 1.6-2.0 (m, 7H). MS (ES): 561 (M + H)⁺.

Exact mass (FAB, M + 1)⁺calcd, 561.2125; found, 561.2120.

3-ter t-Bu toxyca r bon yla m in o-2-(tolu en e-4-su lfon yla m i-

n o)p r op ion ic Acid E t h yl E st er (60). 2-^L-Amino-3-tert-

butoxycarbonylaminopropionic acid ethyl ester (2.0 g, 8.61

mmol) in CH₂Cl₂(40 mL) at 0 °C was treated with DIEA (3.0

2056 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Mehrotra et al.

chromatography (5% MeOH/CH₂Cl₂) to yield 64, 4.47 g (83%).

¹H NMR (CDCl₃): δ 7.3 (m, 5H), 5.42 (d, 1H), 5.06 (s, 2H),

4.34 (m, 1H), 4.15 (q, 2H), 2.27 (m, 2H), 2.11 (m, 1H), 1.90 (m,

1H), 1.4 (s, 9H), 1.23 (t, 3H). MS (ES): 366 (M + H)⁺.

2-Am in op en ta n ed ioic Acid 5-ter t-Bu tyl Ester 1-Eth yl

Ester (65). 64 (4.47 g, 12.23 mmol) in EtOH (60 mL) was

mixed with 20% Pd(OH)₂/C (250 mg), and the mixture was

shaken on a Parr hydrogenator under 50 psi H₂for 7 h. Then

it was filtered through Celite and concentrated to yield 65,

2.58 g (91%). ¹H NMR (CDCl₃): δ 4.05 (q, 2H), 3.60 (s, 1H),

3.32 (m, 1H), 2.23 (t, 2H), 1.91 (m, 1H), 1.58 (m, 1H), 1.33 (s,

9H), 1.15 (t, 3H). MS (ES): 232 (M + H)⁺.

2-Bu toxyca r bon yla m in op en ta n ed ioic Acid 5-ter t-Bu -

tyl Ester 1-Eth yl Ester (66). To a solution of 65 (2.58 g, 11.15

mmol) in CH₂Cl₂(50 mL) at 0 °C was added DIEA (3.90 mL,

22.39 mmol), followed by butyl chloroformate (1.87 mL, 14.70

mmol) in CH₂Cl₂(5.0 mL). The reaction mixture was stirred

at room temperature for 20 h. Then it was partitioned between

CH₂Cl₂/satd NaHCO₃, and the CH₂Cl₂extract was washed

with brine, dried over Na₂SO₄, concentrated, and purified by

flash chromatography (CH₂Cl₂/MeOH) to yield 66, 3.18 g (86%).

¹H NMR (CDCl₃): δ 4.30 (m, 1H), 4.15 (q, 2H), 4.02 (t, 2H),

2.28 (m, 2H), 2.09 (m, 1H), 1.88 (m, 1H), 1.54 (m, 2H), 1.39 (s,

9H), 1.32 (m, 2H), 1.24 (t, 3H), 0.94 (t, 3H). MS (ES): 332 (M

+ H)⁺.

3H), 1.80 (m, 2H), 1.66 (m, 2H). MS (ES): 373 (M + H)⁺. Exact

mass (FAB, M + 1)⁺calcd, 373.2234; found, 373.2272.

2,2-Dim et h yl-3-(4-n it r op h en oxyca r b on yloxy)p r op io-

n ic Acid Meth yl Ester (73). Methyl 2,2-dimethyl-3-hydrox-

ypropionate (1.26 g, 9.53 mmol) in CH₂Cl₂(40.0 mL) at 0 °C

was treated with Et₃N (3.0 mL, 21.52 mmol), followed by

4-nitrophenyl chloroformate (2.50 g, 12.40 mmol). The reaction

mixture was then stirred overnight at room temperature,

washed with brine, dried over Na₂SO₄, concentrated, and

purified by flash chromatography (20% CH₃CN/CH₂Cl₂) to

1

afford 73, 2.35 g (83%). H NMR (CDCl₃): δ 8.24 (d, 2H), 7.36

(d, 2H), 4.30 (s, 2H), 3.71 (s, 3H), 1.3 (s, 6H). MS (ES): 298

(M + H)⁺.

2-(4-Cya n op h en yl)-3-oxo-2,8-d ia za sp ir o[4.5]d eca n e-8-

ca r boxylic Acid 2-Meth oxyca r bon yl-2-m eth ylp r op yl Es-

ter (74). 12 (50 mg, 0.196 mmol) in DMF (2.0 mL) was treated

with DIEA (70.0 µL, 0.40 mmol), followed by 73 (71 mg, 0.238

mmol), and the reaction mixture was stirred at room temper-

ature for 18 h. Then it was partitioned between CH₂Cl₂/satd

NaHCO₃, and the CH₂Cl₂extract was washed with brine, dried

over Na₂SO₄, concentrated, and purified by flash chromatog-

raphy (3% MeOH/CH₂Cl₂) to afford 74, 68 mg (84%). ¹H NMR

(CDCl₃): δ 7.72 (d, 2H), 7.62 (d, 2H), 4.09 (s, 2H), 3.62 (m,

7H), 3.28 (m, 2H), 2.53 (s, 2H), 1.65 (m, 4H), 1.19 (s, 6H). MS

(ES): 414 (M + H)⁺.

2-Bu t oxyca r b on yla m in op en t a n ed ioic Acid 1-E t h yl

Ester (67). 66 (2.61 g, 7.875 mmol) was treated with TFA (20.0

mL), and the mixture was stirred at room temperature for 5.0

h, then concentrated to yield 67, 2.08 g (96%). ¹H NMR

(CDCl₃): δ 4.35 (m, 1H), 4.15 (q, 2H), 4.02 (t, 2H), 2.42 (m,

2H), 2.15 (m, 1H), 1.84 (m, 1H), 1.57 (m, 2H), 1.18-1.4 (m,

5H), 0.87 (t, 3H). MS (ES): 276 (M + H)⁺.

2-Bu t oxyca r b on yla m in o-5-[2-(4-cya n op h en yl)-1-oxo-

2,8-d ia za sp ir o[4.5]d ec-8-yl]-5-oxop en t a n oic Acid E t h yl

Ester (68). The reaction mixture consisting of 11 (98 mg, 0.383

mmol), 67 (127 mg, 0.46 mmol), HBTU (218 mg, 0.575 mmol),

and DIEA (150 µL, 0.86 mmol) in DMF (3.0 mL) was stirred

at room temperature for 17 h. Then it was partitioned between

CH₂Cl₂/satd NaHCO₃, the CH₂Cl₂extract was washed with

brine, dried over Na₂SO₄, concentrated, and purified by flash

chromatography (5% MeOH/CH₂Cl₂) to yield 68, 167 mg (85%).

¹H NMR (CD₃OD): δ 7.73(d, 2H), 7.56 (d, 2H), 4.3 (m, 1H),

4.2 (m, 3H), 4.04 (t, 2H), 3.84 (t, 1H), 3.1-3.4 (m, 3H), 2.55

(m, 2H), 2.25 (m, 2H), 1.85-2.05 (m, 2H), 1.75-1.85 (m, 2H),

1.6 (m, 2H), 1.4 (m, 2H), 1.28 (m, 2H), 0.93 (t, 3H). MS (ES):

513 (M + H)⁺.

2-Bu toxycar bon ylam in o-5-[2-(4-car bam im idoylph en yl)-

1-oxo-2,8-d ia za sp ir o[4.5]d ec-8-yl]-5-oxop en t a n oic Acid

(69). 69 was synthesized from 68 by Typical Procedure C,

described previously for the synthesis of 63 from 62. Overall

yield, 68%. ¹H NMR (CD₃OD): δ 7.95(d, 2H), 7.85 (d, 2H), 4.3

(m, 1H), 4.18 (m, 1H), 4.04 (t, 2H), 3.84 (t, 1H), 3.1-3.4 (m,

3H), 2.55 (m, 2H), 2.25 (m, 2H), 1.85-2.05 (m, 2H), 1.75-1.85

(m, 2H), 1.6 (m, 2H), 1.4 (m, 2H), 1.28 (m, 2H), 0.93 (t, 3H).

MS (ES): 502 (M + H)⁺. Exact mass (FAB, M + 1)⁺calcd,

502.2660; found, 502.2701.

5-[2-(4-Cya n op h en yl)-1-oxo-2,8-d ia za sp ir o[4.5]d ec-8-yl]-

p en ta n oic Acid Eth yl Ester (70). The reaction mixture

consisting of 11 (160 mg, 0.626 mmol), ethyl 5-bromovalerate

(130 µL, 0.821 mmol), and DIEA (0.400 mL, 2.29 mmol) in

DMF (2.0 mL) was stirred overnight at room temperature.

Then it was partitioned between CH₂Cl₂/satd NaHCO₃. The

combined CH₂Cl₂extract was washed with brine, dried over

Na₂SO₄, concentrated, and purified by flash chromatography

(CH₂Cl₂/MeOH) to afford 70, 174 mg (73%). ¹H NMR (CDCl₃):

δ 7.80 (d, 2H), 7.62 (d, 2H), 4.10 (q, 2H), 3.76 (t, 2H), 2.86 (m,

2H), 2.32 (m, 4H), 2.03 (m, 6H), 1.63 (m, 2H), 1.5 (m, 4H), 1.24

(t, 3H). MS (ES): 384 (M + H)⁺.

2-(4-Ca r ba m im id oylp h en yl)-3-oxo-2,8-d ia za sp ir o[4.5]-

d eca n e-8-ca r boxylic Acid 2-Ca r boxy-2-m eth ylp r op yl Es-

ter (75). 75 was synthesized from 74 (27.0 mg, 0.065 mmol)

by using Typical Procedure A, described previously for the

synthesis of 56 from 55. Yield, 23 mg of TFA salt (66%). ¹H

NMR (CD₃OD): δ 7.78 (d, 2H), 7.67 (d, 2H), 3.90 (s, 2H), 3.64

(s, 2H), 3.42 (m, 2H), 3.23 (m, 2H), 2.43 (s, 2H), 1.45 (m, 4H),

1.03 (s, 6H). MS (ES): 417 (M + H)⁺. Exact mass (FAB, M +

1)⁺calcd, 417.2132; found, 417.2096.

P h a r m a cology. In Vitr o Assa y System s. All compounds

were subjected to a series of functional in vitro assays to

determine their relative activities. The following activities are

expressed as the amount required to inhibit 50% of any given

response (IC₅₀values). Each compound was first tested for its

ability to inhibit platelet aggregation induced by 20 µM ADP

in citrated platelet rich plasma (PRP). If that IC₅₀was <0.1

µM, the IC₅₀was also obtained for most compounds with

PPACK PRP using 20 µM ADP. If that criterion was met, then

TRAP was tested as the agonist for comparison. In vitro

fibrinogen binding to purified GPIIb-IIIa in a plate assay was

also performed for the majority of the compounds and corre-

sponding IC₅₀values were obtained. Additionally, candidate

compounds selected for further analysis were tested for their

specificity for GPIIb-IIIa. This included analysis of inhibition

of vitronectin (VN) binding to purified vitronectin receptor

(VNR) in a plate assay and inhibition of cell adhesion mediated

by various Integrins.

Ma ter ia ls a n d Meth od s (Platelet aggregation in platelet

rich plasma). Materials: ADP diluted to 5 mL in PBS is 1 mM

and prediluted Collagen are from Chrono-log Corp. TRAP6

(SFLLRN-NH2) was purchased from PenLabs, and TRAP

211-47 was synthesized in house at COR Therapeutics, Inc.

3.8% trisodium citrate (TSC) and PPACK (25 mg/mL in 0.01

N HCl, from CalBiochem) stock solutions were used. Fresh

human blood was obtained from donors. Compounds were at

a stock concentration of 40 mM in DMSO. Chrono-Log Whole

Blood Aggregometer and Aggro-Link Software (Chrono-log

corp., Havertown, PA) were used.

Procedure: Blood donors were selected who had not had any

medications within the past 7 days and blood was drawn into

1/7 volume of TSC (8.6 mL TSC per 60 mL whole blood: final

concentration: 0.54%) or PPACK (final concentration 0.3 mM).

Ten milliliters of blood was aliquoted into 15 mL polypropylene

tubes and centrifuged at 900 rpm for 20 min (160g). The

platelet rich plasma (PRP) was carefully removed and pooled

in a new tube. Platelet poor plasma (PPP), for the blank

sample in the aggregometer, was obtained by pelleting plate-

lets by centrifuging 2 × 600 µL aliquots of PRP for 5 min on

high in a microfuge (15 000g).

5-[2-(4-Ca r b a m im id oylp h en yl)-1-oxo-2,8-d ia za sp ir o-

[4.5]d ec-8-yl]p en ta n oic Acid (72). 72 was synthesized from

70 by using Typical Procedure C described for the synthesis

of 63 from 62. Yield, 66 mg (65%).¹H NMR (CD₃OD): δ 7.98

(d, 2H), 7.82 (d, 2H), 3.96 (m, 1H), 3.63 (m, 1H), 3.45 (m, 1H),

3.10 (m, 4H), 2.4 (t, 2H), 2.3 (m, 2H), 2.13 (m, 2H), 1.9 (m,

Novel 2,8-Diazaspiro[4.5]decanes

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2057

The platelet aggregation procedure was as follows: 500 µL

of PRP was aliquoted into a cuvette with a stir bar, then 1 µL

of either vehicle or compound in vehicle was added. The

aggregometer was set to zero, and sample was allowed to

incubate while stirring for approximately 1 min. To initiate

platelet aggregation, 10 µL of 1 mM ADP was added to the

cuvette for a final ADP concentration of 20 µM ADP (or TRAP

for a final concentration of 5 µM). Aggregation proceeded for

5 min per sample. The amplitude value (height) of each

aggregation curve, generated by the Aggro-Link Software, was

used to generate inhibition curves for each compound. The IC₅₀

of each compound was calculated by linear regression using

the two points that surround the 50% aggregation value. The

average of at least three separate IC₅₀determinations are

reported here(( SD).

concentration was determined by O.D. 280 nm (fibrinogen: EC

) 1.51 vitronectin: EC ) 1.38) and single use aliquots were

stored frozen at -80 °C.

HUVEC Adhesion to Vitronectin or Fibronectin. Materials:

Human umbilical vein endothelial cells (HUVEC)5. Vitronectin

4 and Fibronectin in PBS. Blocking media: M199 media +

2.5 mg/mL BSA. Assay Media: M199 media + 0.25 mg/mL

BSA. 3% paraformaldehyde. 0.5% toluidine blue (dissolved in

3.7% formaldehyde). 2% SDS. Assay controls: Abciximab, an

inhibitor of GPIIb-IIIa and theVNR (Rvâ3) was purchased from

Centocor, Malvern PA; the function blocking fibronectin recep-

tor antibody BIIG2, was a gift from Caroline Damsky6, UCSF;

Echistatin, a disintegrin was purchased from Sigma; the cyclic-

RGD peptide (C64-89, Mpr-RGDWPC-NH2) was synthesized

in house (at COR Therapeutics, Inc.).

F ibr in ogen Bin d in g to Im m obilized GP IIb-IIIa . (Pro-

tocol also used for vitronectin binding to VNR- Rvâ3). Materi-

als: Immulon-2 96-well microtiter plate. Biotinylated fibrino-

gen or vitronectin as per protocol (below). Buffer A: 0.15 M

NaCl, 0.02 M Tris; pH 7.4, 0.001 M CaCl₂, 0.02% NaN₃. Buffer

B: 0.1 M NaCl, 0.05 M Tris; pH 7.4, 0.002M CaCl₂, 0.02%

NaN₃. Buffer C: (blocking buffer): Buffer B with 35 mg/mL

BSA. Buffer D: (Incubation Buffer): Buffer B with 1 mg/ml

BSA. 0.4 N NaOH. Bovine serum albumin (BSA) (Sigma, St.

Louis, MO).

Anti-biotin antibody-alkaline phosphatase conjugate (Sigma,

St. Louis, MO. Product No. A-7064). Alkaline Phosphatase

substrate kit (Biorad Laboratories, Hercules,CA. Cat No. 172-

1063). V Max Plate Reader (Molecular Devices, Sunnyvale,

CA).

Procedure: Purified GPIIb-IIIa complex or purified VNR

was immobilized onto Immulon-2 96 well plates by diluting a

1 mg/ml stock 1:200 in buffer A and immediately adding 100

µL to each well (0.5 µg). The plates were incubated overnight

at 4 °C. At the time of the experiment, the wells were washed

twice with buffer B, by emptying then filling by aspiration.

The wells were blocked with 0.1 mL of buffer C for 2 h at room

temperature. The wells were washed with 250 µL of buffer D

and then the samples were added as follows: test compound

and 10 nM biotinylated fibrinogen in buffer D were mixed

together and 0.1 mL per well was added in triplicate. Following

a 2 h incubation at room temperature, wells were washed once

with 250 µL buffer D and then 0.1 mL anti-biotin antibody

(1:20 000 dilution in buffer D) was added and incubated for 1

h at room temperature. Wells were again washed with 250

µL buffer D. Alkaline phosphatase substrate was diluted

according to kit directions, and 0.1 mL/well was added and

developed at room temp for 10 min. The reaction was stopped

by adding 0.1 mL 0.4 N NaOH, and the endpoints were read

at 405 nm on the V max plate reader. Percent specific binding

was calculated by subtracting binding of fibrinogen to BSA-

coated wells, and the IC₅₀values from three replicate experi-

ments were calculated as described above. Notes: In prelimi-

nary experiments we determined that fibrinogen binding

increased linearly up to 0.3 µg GP IIb-IIIa added to each well

and the addition of higher amounts of GP IIb-IIIa to the wells

did not further increase the amount of fibrinogen that bound.

Plates coated with GPIIb-IIIa were stored up to 2 months at

4 °C.

Procedure: 1 µg/well of VN or FN was plated onto Immulon

2 96-well plates and incubated overnight at 4 °C. Before use

the plates were washed 2× with PBS and blocked for 1-2 h

at room temperature. Confluent HUVEC were lifted with

trypsin and added to media with serum before pelleting cells.

(HUVECs were used between 3rd and ∼8th passage). Cells

were resuspended in assay media and counted on a hemocy-

tometer. Assay media ( inhibitors were added to Eppendorf

tubes and mixed. Then cells were added such that 100 µL

added to each well yielded ∼50 000 cells. The quadruplicate

samples were incubated at 37 °C in the 5% CO₂incubator for

40 min. Nonadherent cells were removed by aspiration and

washed 3× with 150 µL PBS. The cells were then fixed by the

addition of 100 µL of 3% paraformaldehyde for 10 min at room

temperature. This was aspirated and 90 µL of 0.5% toluidine

blue stained the cells for 3 min. Wells were then washed 2×

with 150 µL PBS and 100 µL 2% SDS was added for 5 min.

After mixing well, the plate was read at 650 nM in the

Molecular Devices plate reader. Specific adhesion from 2 to 3

separate experiments was determined by subtracting nonspe-

cific HUVEC adhesion to BSA-coated wells from the total.

Assessm en t of Or a l Bioa va ila bility a n d Oth er P K

P r op er ties in Ra ts. Sprague Dawley Rats were used for the

first assessment of the oral activity of potent compounds. The

compounds (double prodrugs, or mono-prodrugs) were solubi-

lized in water and orally administered to six rats. The

corresponding free acids were solubilized in 50% PEG-300 and

administered intravenously to six rats. The administered doses

were 1 mg/kg for all compounds. The animals were instru-

mented 2 days prior to the experiment and were fully conscious

on the day of the experiment. Blood samples were collected

on EDTA or sodium citrate and centrifuged to give PPP.

Plasma samples were prepared and extracted using the

following method: An internal standard was added to test

samples, which were then mixed with an equivalent volume

of acetonitrile (ACN) to sediment proteins. The resulting serum

was then filtered through a 1000 MW cutoff filter by centrifu-

gation at 5 °C for 1-2 h. Standards were prepared in a plasma

and extracted as described above. A 10 point standard curve

was prepared for each compound over a concentration range

of 5-5000 ng/mL. QC samples, prepared at concentrations of

10, 250, and 2500 ng/mL, were analyzed in triplicate at each

concentration. Filtrates were, in some cases, evaporated to

dryness, and the residue was dissolved in RP-HPLC mobile

phase. In other cases, the UF filtrates were analyzed directly

by LC/MS/MS. A variety of LC/MS conditions were used

depending on the chromatographic and mass spectral proper-

ties of the compounds of interest.

Biotinylation of Fibrinogen or Vitronectin. Materials: Puri-

fied Fibrinogen (Haematologic Technologies, VT.). Purified

Vitronectin 4. Dialysis Buffer: 0.1 M NaHCO₃pH 8.3, 0.1 M

NaCl. TS: 50 mM Tris, pH 7.4, 0.1 M NaCl, 0.05% NaN₃.

Samples were analyzed for free acids by LC/MS using

protocols previously established for these compounds. Indi-

vidual plasma samples obtained from each rat at each time

point following PO dosing were analyzed. For iv administra-

tion, composites were prepared of the plasma obtained at each

time point and the composites were analyzed (i.e. at each time

point, 20 µL of plasma from each of the rats was mixed to form

a composite sample. An aliquot of this composite was ana-

lyzed).

Procedure: The ligand (fibrinogen or vitronectin) was

dialyzed at 4°C into the dialysis buffer above. In the case of

fibrinogen, it was centrifuged in the TL-100 tabletop ultra-

centrifuge (Beckman Instruments) at 100 000g for 20 min at

4 °C to remove any particulate matter. The ligand concentra-

tion was then adjusted to 1 mg/ml with dialysis buffer. Sulfo-

N-hydroxysuccinomide-Biotin (Pierce, catalog # 21217) was

added as a solid (0.2 mg of biotin ester/mL of ligand) and gently

mixed on an end-over-end Nutator for 30 min at room

temperature. Unreacted biotin reagent was removed by ex-

haustive dialysis with TS buffer at 4 °C. The final protein

Each compound was administered in either a cassette

(multiple compounds) form or a single compound form. All drug

2058 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Mehrotra et al.

administration studies were performed utilizing a serial

method of blood sampling. Plasma samples were collected at

specific time points, analyzed for plasma concentration, and

then reported as mean plasma concentration versus time. The

pharmacokinetic profile generated from both treatment groups

(orally and intravenously administered) of rats were used to

obtain the percent oral bioavailability for each drug. Area

under the curve (AUC) data were measured in both groups as

well as C_max(maximum concentration) and t_1/2â(half-life). Since

the po and iv doses were the same, percent oral bioavailability

was simply determined by dividing the AUC in the oral group

that received the prodrug by the AUC in the iv group that

received the drug (acid form of the compound).

For the in vivo demonstration of GPIIb-IIIa dependence of

mouse bleeding time experiment, the compound CT50787 was

administered as an iv bolus injection (10% DMSO in water)

via tail vein at 10, 40, 50 and 100 mg/kg in approximately 150

µL volume to anesthetized mice. To initiate bleeding, the tail

was transected 2 mm proximal to its tip and immersed in 37

°C normal saline bath. The bleeding time was recorded as the

time required until cessation of blood flow or up to 15 min

(900 s). PRP of CT50787 in the mouse utilized citrated pooled

(C57/Bl) mouse PRP and 20 µM ADP as the agonist in a

standard aggregometry assay.

P h a r m a cok in etics in Cyn om olgu s Mon k eys. The gen-

eral procedure for the determination of bioavailability and

other pharmacokinetic properties is exemplified here for 27

(CT51269).

The stability of the compounds in rat, cynomolgus monkey,

and dog plasmas were evaluated to compare the cholinesterase

activities in the various species against the prodrug. Com-

pound stability was also evaluated in a solution containing a

suspended section of rat ileum to determine the metabolic

stability of the prodrug in the intestine. A standard was used

as a control in these studies.

In Vivo Bioavailability. Animals (n ) 3, weight ) 7.1-7.5

kg) were fasted on the afternoon prior to dose administration

but water was allowed ad libitum. On the day of the experi-

ment, the monkeys were placed in chair restraints. Blood

samples were obtained by percutaneous venipuncture via

cepholic or saphenous veins. Blood samples were anticoagu-

lated with EDTA, and the plasma obtained was frozen for later

analysis. In the first week of the study, 28 (CT50614) was

administered at 200 µg/kg as a 15 min infusion of the

compound dissolved in 50% PEG-300 (2.0 mg/mL) and further

diluted with plasmalyte. Each animal received a 3.5 mL

infusion of the drug solution for each kg of body weight. In

the second week of the study, 27 (CT51269) was dissolved in

distilled water at 2.0 mg/mL and administered to the same

animals at 2000 µg/kg via nasogastric feeding tube. An

additional 10 mL of water was administered to ensure

complete delivery. For intraveneously administered compound,

blood samples (1.0 mL) were collected at 0 (predose), 5, 15,

30, and 45 min and at 1, 2, 3, 4, 5, 6, 8, 12, 24, 28, 32, and 48

h after dose administration. For orally administered animals,

blood samples were collected at 0 (predose), 0.5, 1, 2, 3, 4, 5,

6, 7, 8, 12, 24, 28, 32, and 48 h after dose administration. Blood

samples were centrifuged at 1500 g for 10 min, and the

resulting platelet poor plasma (PPP) was decanted into Ep-

pendorf tubes and frozen at -15 °C.

Sample Preparation for LC/MS/MS Analysis. A 200 µL

aliquot of the plasma samples was mixed with an equal volume

of acetonitrile (ACN), and an internal standard (LY314378)

was added to a concentration of 500 ng/mL of plasma. The

samples were then centrifuged and filtered through an ultra-

filtration membrane (10,000 MW cutoff, Microcon 10, Amicon

Inc.). A 60 µL aliquot of the filtrate was evaporated to dryness

in a centrifugal evaporator, and the residue was dissolved in

120 µL of water containing 0.5% formic acid.

LC/MS/MS Analysis. All separations were performed on a

Keystone C18 column (2 × 10 mm, 5 µm) using a linear

gradient from 5 to 95% ACN over 5 min at a flow rate of 200

µL/min. Formic acid (0.5%) was used in the mobile phase as

an ion-transfer agent for MS detection. The column effluent

was introduced to the MS/MS through a turbo-ion spray source

(Temp ) 450 °C, ion-spray voltage ) 5200 V, turbo-gas flow

) 7 L/min). Parent and daughter ions selected for specific MS/

MS detection of each compound were used. Collision energies

were optimized for maximum sensitivity for each compound,

and the optimum settings were used during the scan periods

for each compound.

For quantitation, standards of each compound were pre-

pared in cynomolgus monkey plasma and prepared as de-

scribed above. For each compound, a blank extract was

prepared as well as a 10-point standard curve over the

concentration range of 1 to 1000 ng/mL. Ratios of peak area

of each analyte to that of an internal standard were computed

using MacQuan software. Calibration curves were constructed

using a weighted (1/x²) linear regression of the standard

plasma concentrations to measured peak area ratios. Plasma

concentrations of unknowns were determined by interpolation

from the corresponding standard curve for each compound.

Quality control samples (QCs) consisting of cynomolgus

monkey plasma spiked with either 5, 50, or 500 ng/mL of each

compound (three replicates at each concentration) were ana-

lyzed with every sample set as a measure of assay perfor-

mance. Test acceptance criteria required that measured

concentrations for QCs be within 20% of theoretical concentra-

tions (accuracy) and that the coefficient of variance for

replicate samples at each level be <20% (precision).

In Vitro Determination of Compound Stability in Plasma.

Fresh samples of PPP (EDTA anticoagulant) from rat and dog

or frozen plasma samples from cynomolgus monkey were used

in these experiments. In a typical experiment, compounds were

added to plasma (1 mL) to give a final concentration of 2.5

µg/mL as a DMSO solution (10 µL of a 250 µg/mL stock

solution). Samples (150 µL) were taken at 2, 5, 15, 30, 60, and

120 min, mixed with an equal volume of ACN containing an

internal standard, centrifuged, and filtered as described above

and analyzed by LC/MS/MS. Standards (six-point standard

curve) were prepared in either rat or cynomolgus monkey

plasma.

In Vitro Metabolism of 27 (CT51269) by Rat Ileum. Freshly

harvested intestinal tissue from a single rat was stripped of

muscle tissue, blotted dry, cut into two sections (6 × 6 mm

and 7 × 6 mm), and weighed (27.8 mg and 25.9 mg). The

sections were suspended in 2 mL portions of phosphate buffer,

and 5 µg of CT51269 was added to each tube as a DMSO

solution (20 µL of a 250 µg/mL stock solution). Samples (150

µL) of the media were taken at 5, 15, 30, 60, and 120 min,

mixed with an equal volume of ACN containing an internal

standard, centrifuged, and filtered as described above, and

analyzed by LC/MS/MS. Standards were prepared in phos-

phate buffer (six-point standard curve) and treated identically

to the samples.

After the 2 h incubation period, 1.25 mL of ACN/IS mixture

was added to the sample (consisting of tissue suspended in

1.25 mL of buffer), the sample was homogenized using a sonic

probe and mixed for 1 h. The homogenate was centrifuged and

filtered using the procedure described above. Analyte concen-

trations in this sample were determined using a standard

curve prepared in phosphate buffer.

LC/MS/MS Determination of Glucuronides of 28 (CT50614)

and 29 (CT51270). Two different procedures were used for the

determination of glucuronides in the cynomolgus plasma

samples. In the first procedure, â-glucuronidase was added to

plasma samples obtained from one of the subjects to which 27

(CT51269) had been orally administered. Changes in the

concentrations of 28 (CT50614) and 29 (CT51270) were

measured by LC/MS/MS relative to similarly treated plasma

not treated with glucuronidase. In the second procedure, RP-

HPLC effluent was monitored by MS in a narrow mass range

around the expected m/z value predicted for CT50614-glucu-

ronide. LC/MS monitoring was also used to verify the extent

of hydrolysis of CT50614-glucuronides by the â-glucuronidase

treatment.

â-Glucuronidase (100 µL of a 200 units/mL solution in 8 mM

phosphate buffer, Sigma Chemical Co., Cat # G-5897) was

Novel 2,8-Diazaspiro[4.5]decanes

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2059

Chem. 1991, 266, 1415-1421. (c) Alig, L.; Edenhofer, A.;

Hadvary, P.; Hurzeler, M.; Knopp, D.; Muller, M.; Steiner, B.;

Trzeciak, A.; Weller, T. Low Molecular Weight, Non-Peptide

Fibrinogen Receptor Antagonists. J . Med. Chem. 1992, 35,

4393-4407. (d) Zablocki, J . A.; Miyano, M.; Garland, R. B.; Pireh,

D.; Schretzman, L. A.; Rao, S. N.; Lindmark, R. J .; Panzer-

Knodle, S. G.; Nicholson, N. S.; Taite, B. B.; Salyers, A. K.; King,

L. W.; Campion, J . G.; Feigen, L. P. Potent in Vitro and in Vivo

Inhibitors of Platelet Aggregation Based upon the Arg-Gly-Asp-

Phe Sequence of Fibrinogen: A Proposal on the Nature of the

Binding Interaction between the Arg-guanidine of RGDX Mi-

metics and the Platelet GP IIb-IIIa Receptor. J . Med. Chem.

1993, 36, 1811-1819.

added to 100 µL of plasma. Control portions of plasma received

100 µL of phosphate buffer (no enzyme). Both portions were

incubated at 37 °C for 2 h at which time ACN and an internal

standard were added to the sample, and the samples were

centrifuged and filtered as described above. Standards (10-

point standard curve) and QCs were prepared in cynomolgus

PPP and treated identically to the control samples. The

concentrations of 27 (CT51269) and 29 (CT51270) were

determined using LC/MS/MS as described above.

For direct LC/MS detection of the glucuronide of 28

(CT50614), the control and treated samples were subjected to

RP-HPLC as described above. The MS eluant was monitored

by MS (Q1 scan) over a scan range of 560-570 amu with a

step size of 0.5 amu and a dwell time of 50 ms (total scan time

) 1 s).

P h a r m a cok in etics in Bea gle Dogs. The general proce-

dure for the determination of bioavailability and other phar-

macokinetic properties is exemplified here for 27 (CT51269).

The pharmacokinetic parameters of 27 (CT51269) were

investigated in beagle dogs in a crossover experiment. Plasma

samples, collected on EDTA, were analyzed by LC/MS/MS for

both the active metabolite of CT51269, 28 (CT50614), and for

the inactive, mono-prodrug form of the compound, 29 (CT51270).

Fasted, female beagle dogs (9-10 kg) were used in crossover

design studies with a 1 week washout period between studies.

In the first week, CT50614 was administered intravenously

as an iv bolus in 50% PEG at a dose of 0.1 mg/kg. Blood

samples of 1.0 mL were obtained, via percutaneous sampling

of the cephalic veins, onto EDTA and centrifuged to give PPP.

In the second week, 27 (CT51269) was administered orally,

in water, to the same subjects via a 12F feeding tube at a dose

of 1 mg/kg.

Plasma samples were analyzed by LC/MS/MS using proto-

cols described above. An internal standard, selected for its

chemical similarity to the test compounds, was used in

quantitation. The linear range for this assay has been deter-

mined to be 1-500 ng/mL with accuracy and precision

throughout the range of 85-115% and (10% respectively.

The data were analyzed for pharmacokinetic parameters

using WinNonlin software with noncompartmental data treat-

ment.

(4) (a) Lincoff, A. M.; Topol, E. J . Overview of the Glycoprotein IIb/

IIIa Interventional Trials. In Platelet Glycoprotein IIb/ IIIa

Inhibitors in Cardiovascular Disease, 2nd ed.; Lincoff, A.,

Michael, Eds.; Humana Press Inc.: Totowa, N. J ., 2003; pp 167-

199. (b) Agah, R.; Plow, E. F.; Topol, E. J . GPIIb-IIIa Antago-

nists. In Platelets; Michelson, A. D., Eds.; Academic Press:

California, 2002; pp 769-785.

(5) Lincoff, A. M.; Califf, R. M.; Topol, E. J . Platelet Glycoprotein

IIb-IIIa, Receptor Blocade in Coronary Artery Disease. J . Am.

Coll. Cardiol. 2000, 35, 1103-1115.

(6) (a) Coller, B. S. Anti-GPIIb/IIIa Drugs: Current Strategies and

Future Directions. Thromb. Haemost. 2001, 86, 427-443. (b)

Konstantopoulos, K.; Mousa, S. A. Antiplatelet Therapies: Plate-

let GPIIb/IIIa Antagonists and Beyond. Curr. Opin. Invest.

Drugs 2001, 2, 1086-1092. (c) Dogne, J .; De Leval, X.; Benoit,

P.; Delarge, J .; Masereel, B.; David, J . Recent advances in

antiplatelet agents. Curr. Med. Chem. 2002, 9, 577-589. (d)

Ojima, I.; Chakravarty, S.; Dong, Q. Antithrombotic Agents:

From RGD to Peptide Mimetics. Bioorg. Med. Chem. 1995, 3,

337-360. (e) Scarborough, R. M. Structure-Activity Relation-

ships of â-Amino Acid-Containing Integrin Anagonists. Curr.

Med. Chem. 1999, 6, 971-981.

(7) Mousa, S. A.; Iqbal, O.; Fareed, J . Antithrombotics and Throm-

bolytics in Stroke. Curr. Opin. Invest. Drugs 2002, 3, 878-885.

(8) Wang, W.; Borchardt, R. T.; Wang, B. Orally Active Peptidomi-

metic RGD Analogues that are Glycoprotein IIb/IIIa Antagonists.

Curr. Med. Chem. 2000, 7, 437-453.

(9) (a) Cannon, C. P. The Evolving Story of Oral Platelet Glycopro-

tein IIb-IIIa Receptor Inhibitors. Curr. Opin. Cardiovascular,

Pulmonary & Renal Invest. Drugs 2000, 2, 114-123. (b) Quinn,

M. J .; Plow, E. F.; Topol, E. J . Platelet Glycoprotein IIb/IIIa

Inhibitors: Recognition of a Two-Edged Sword? Circulation

2002, 106, 379-385.

(10) Scarborough, R. M. Eptifibatide. Drugs Future 1998, 23(6), 585-

590.

(11) Scarborough, R. M.; Gretler, D. D. Platelet Glycoprotein IIb-IIIa

Antagonists as Prototypical Integrin Blockers: Novel Parenteral

and Potential Oral Antithrombotic Agents. J . Med. Chem. 2000,

43(19), 3453-3473.

Ack n ow led gm en t. The authors gratefully acknowl-

edge helpful discussions with Charles J . Homcy, David

Phillips, and Daniel D. Gretler of Millennium Pharma-

ceuticals, Inc.

(12) (a) Scarborough, R. M.; Kleiman, N. S.; Phillips, D. R. Platelet

Glycoprotein IIb/IIIa Antagonists: What Are the Relevant Issues

Concerning their Pharmacology and Clinical Use? Circulation

1999, 100, 437-444. (b) Brassard, J . A.; Curtis, B. R.; Cooper,

R. A.; Ferguson, J .; Komocsar, W.; Ehardt, M.; Kupfer, S.;

Maurath, C.; Swabb, E.; Cannon, C. P.; Aster, R. H. Acute

Thrombocytopenia in Patients Treated with the Oral Glycopro-

tein IIb/IIIa Inhibitors Xemilofiban and Orbofiban: Evidence for

an Immune Etiology. Thromb. Haemost. 2002, 88, 892-897. (c)

Aster, Richard. Thrombocytopenia Induced by GPIIb/IIIa Inhibi-

tors. Blood 2003, 101, 1. (d) Peter, K.; Schwarz, M.; Nordt, T.;

Bode, C. Intrinsic Activating Properties of GP IIb/IIIa Blockers.

Thromb. Res. 2001, 103 (Suppl. 1), S21-S27. (e) Iqbal, O.;

Walenga, J . M.; Lewis, B. E.; Bakhos, M. Bleeding Complications

with Glycoprotein IIb/IIIa Inhibitors. Drugs Today 2000, 36,

503-514. (f) Billheimer, J . T.; He, B.; Spitz, S. M.; Stern, A. M.;

Seiffert, D. Effects of Glycoprotein IIb/IIIa Antagonists on

Platelet Activation: Development of a Transfer Method to Mimic

Peak to Trough Receptor Occupancy. Thromb. Res. 2002, 107

(6), 303-317.

(13) (a) Salam, A. M.; Al Suwaidi, J . Platelet Glycoprotein IIb/IIIa

Antagonists in Clinical Trials for the Treatment of Coronary

Artery Disease. Exp. Opin. Invest. Drugs 2002, 11, 1645-1658.

(b) Peter, K.; Straub, A.; Kohler, B.; Volkmann, M.; Schwarz,

M.; Kubler, W.; Bode, C. Platelet Activation as a Potential

Mechanism of GP IIb/IIIa Inhibitor-Induced Thrombocytopenia.

Am. J . Cardiol. 1999, 84, 519-524.

(14) (a) Hosking, M. P.; Carroll, R. C. Roxifiban, DuPont. Curr. Opin.

Cardiovasc. Pulm. Renal Invest. Drugs 2000, 2, 165-171. (b)

Mousa, S. A.; Bozarth, J . M.; Naik, U. P.; Slee, A. Platelet GPIIb/

IIIa Binding Characteristics of Small Molecule RGD Mimetic:

Distinct Binding Profile for Roxifiban. Br. J . Pharmacol. 2001,

133, 331-336. (c) Mousa, S. A.; Kapil, R.; Mu, D. Intravenous

and Oral Antithrombotic Efficacy of the Novel Platelet GPIIb/

IIIa Antagonist Roxifiban (DMP754) and its Free Acid Form,

XV459. Arterioscler. Thromb. Vasc. Biol. 1999, 19, 2535-2541.

Su p p or tin g In for m a tion Ava ila ble: Analytical data,

selected HRMS data, tables of ¹H NMR, and HPLC methods/

data for final compounds. This material is available free of

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

Refer en ces

(1) (a) Fuster, V.; Badimon, L.; Badimon, J . J .; Chesebro, J . H. The

Pathogenesis of Coronary Artery Disease and the Acute Coro-

nary Syndromes (Part I). N. Engl. J . Med. 1992, 326, 242-250.

(b) Fuster, V.; Badiman, L.; Badimon, J . J .; Chesebro, J . H. The

Pathogenesis of Coronary Artery Disease and the Acute Coro-

nary Syndromes (Part II). N. Engl. J . Med. 1992, 326, 310-

318. (c) Hynes, R. O. Integrins: Versatility, Modulation, and

Signaling in Cell Adhesion. Cell 1992, 69, 11-25. (d) Humphries,

M. J . Integrin Cell Adhesion Receptors and the Concept of

Agonism. Trends Pharmacol. Sci. 2000, 21, 29-32. (e) Aota, S.;

Yamada, K. M. Integrin Functions and Signal Transduction.

Adv. Exp. Med. Biol. 1997, 400B, 669-682. (f) Coller, B. S.

Blockade of Platelet GPIIb/IIIa Receptors as an Antithrombotic

Strategy. Circulation 1995, 92, 2373-80. (g) Ruef, J .; Katus, H.

A. New Antithrombotic Drugs on the Horizon. Expert Opin.

Invest. Drugs 2003, 12(5), 781-797.

(2) Phillips, D. R.; Charo, I. F.; Parise, L. V.; Fitzgerald, L. A. The

Platelet Membrane Glycoprotein IIb-IIIa Complex. Blood 1988,

71, 831-843.

(3) (a) Scarborough, R. M.; Naughton, M. A.; Teng, W.; Rose, J . W.;

Phillips, D. R.; Nannizzi, L.; Arfsten, A.; Campbell, A. M.; Charo,

I. F. Design of Potent and Specific Integrin Antagonists. J . Biol.

Chem. 1993, 268, 1066-1073. (b) Charo, I. F.; Nannizzi, L.;

Phillips, D. R.; Hsu, M. A.; Scarborough, R. M. Inhibition of

Fibrinogen Binding to GP IIb-IIIa by a GP IIIa Peptide. J . Biol.

2060 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8

Mehrotra et al.

(d) Gurbel, P. A.; McKenzie, M. E.; Serebruany, V. L. Initial

Platelet Activity may Predict Efficacy after Chronic Oral Gly-

coprotein IIb/IIIa Blockade: Should We Still Consider Uniform

Treatment Regimens? Thromb. Res. 2000, 99, 105-107. (e)

Fossler, M. J .; Ebling, W. F.; Ma, S.; Kornhauser, D.; Mondick,

J .; Barrett, J . S.; Garner, D.; Quon, C. Y.; Pieniaszek, H. J ., J r.

Integrated Pharmacokinetic/Pharmacodynamic Model of XV459,

a Potent and Specific GPIIb/IIIa Inhibitor, in Healthy Male

Volunteers. J . Clin. Pharmacol. 2002, 42, 1326-1334.

J .; Manno, P. D.; Schaffer, L. W.; Smith, R. L. Non-Peptide

Fibrinogen Receptor Antagonists. 7. Design and Synthesis of a

Potent, Orally Active Fibrinogen Receptor Antagonist. J . Med.

Chem. 1995, 38, 3332-3341.

(24) Zablocki, J . A.; Rico, J . G.; Garland, R. B.; Rogers, T. E.;

Williams, K.; Schretzman, L. A.; Rao, S. A.; Bovy, P. R.; Tjoeng,

F. S.; Lindmark, R. J .; Toth, M. V.; Zupec, M. E.; McMackins,

D. E.; Adams, S. P.; Miyano, M.; Markos, C. S.; Milton, M. N.;

Paulson, S.; Herin, M.; J acqmin, P.; Nicholson, N. S.; Panzer-

Knodle, S. G.; Haas, N. F.; Page, J . D.; Szalony, J . A.; Taite, B.

B.; Salyers, A. K.; King, L. W.; Campion, J . G.; Feigen, L. P.

Potent in vitro and in vivo Inhibitors of Platelet Aggregation

Based upon the Arg-Gly-Asp Sequence of Fibrinogen. (Ami-

nobenzamidino)succinyl (ABAS) Series of Orally Active Fibrino-

gen Receptor Antagonists. J . Med. Chem. 1995, 38, 2378-2394.

(25) Mills, S. D. Heterocyclic Compounds. U.S. Patent 5,753,659,

1998.

(26) Samanen, J . M.; Ali, F. E.; Barton, L. S.; Bondinell, W. E.;

Burgess, J . L.; Callahen, J . F.; Calvo, R. R.; Chen, W.; Chen, L.;

Erhard, K.; Feuarstein, G.; Heys, R.; Hwang, S.-M.; J ckas, D.

R.; Keenan, R. M.; Ku, T. W.; Kwon, C.; Lee, C.-P.; Miller, W.

H.; Newlander, K. A.; Nichols, A.; Parker, M.; Peishoff, C. E.;

Rhodes, G.; Ross, S.; Shu, A.; Simpson, R.; Takata, D.; Yellin,

T. O.; Uzsinskas, I.; Vensalavsky, J . W.; Yuan, C.-K.; Huffman,

W. F. Potent, Selective, Orally Active 3-Oxo-1,4-benzodiazepine

GPIIb-IIIa Integrin Antagonists. J . Med. Chem. 1996, 39, 4867-

4870.

(15) (a) Smyth, M. S.; Rose, J .; Mehrotra, M. M.; Heath, J .; Ruhter,

G.; Schotten, T.; Seroogy, J .; Volkots, D.; Pandey, A.; Scarbor-

ough, R. M. Spirocyclic Nonpeptide Glycoprotein IIb-IIIa An-

tagonists. Part 1: Design of Potent and Specific 3,9-Diazaspiro-

[5.5]undecanes. Bioorg. Med. Chem. Lett. 2001, 11, 1289-1292.

(b) Pandey, A.; Seroogy, J .; Volkots, D.; Smyth, M. S.; Rose, J .;

Mehrotra, M. M.; Heath, J .; Ruhter, G.; Schotten, T.; Scarbor-

ough, R. M. Spirocyclic Nonpeptide Glycoprotein IIb-IIIa An-

tagonists. Part 2: Design of Potent Antagonists Containing the

3-Azaspiro[5.5]undec-9-yl Template. Bioorg. Med. Chem. Lett.

2001, 11, 1293-1296. (c) Mehrotra, M. M.; Heath, J .; Rose, J .;

Smyth, M. S.; Seroogy, J .; Volkots, D.; Ruhter, G.; Schotten, T.;

Alaimo, L.; Park, G.; Pandey, A.; Scarborough, R. M. Spirocyclic

Nonpeptide Glycoprotein IIb-IIIa Antagonists. Part 3: Synthesis

and SAR of Potent and Specific 2,8-Diazaspiro[4.5]decanes.

Bioorg. Med. Chem. Lett. 2002, 12, 1103-1107. (d) Fisher, M.

J .; J akubowski, J . A.; Masters, J . J .; Mullaney, J . T.; Ruterbories,

K. J .; Paal, M.; Ruhter, G.; Scarborough, R. M.; Schotten, T.;

Stenzel, W. Spiro Compounds as Inhibitors of Fibrinogen-

Dependent Platelet Aggregation. U.S. Patent 6291469, 2001.

(16) (a) Wagner, G.; Voigt, B.; Vieweg, H. Synthesis of N-R-(Arylsul-

fonylglycyl) amidinophenylalaninamides as Highly Active In-

hibitors of Thrombin. Pharmazie 1984, 39, 226-230. (b) J udkins,

B. D.; Allen, D. G.; Cook, T. A.; Evans, B.; Sardharwala, T. E. A

Versatile Synthesis of Amidines from Nitriles Via Amidoximes.

Synth. Commun. 1996, 26(23), 4351-4367.

(27) Damiano, B. P.; Mitchell, J . A.; Giardino, E.; Corcoran, T.;

Haertlein, B. J .; De Garavilla, L.; Kauffman, J . A.; Hoekstra,

W. J .; Maryanoff, B. E.; Andrade-Gordon, P. Antiplatelet and

Antithrombotic Activity of RWJ -53308, a Novel Orally Active

Glycoprotein IIb/IIIa Antagonist. Thromb. Res. 2001, 104, 113-

126.

(28) (a) Hayashi, Y.; Katada, J .; Harada, T.; Tachki, A.; Iijima, K.;

Takiguchi, Y.; Muramatsu, M.; Miyazaki, H.; Asari, T.; Okazaki,

T.; Sato, Y.; Yasuda, E.; Yano, M.; Uno, I.; Ojima, I. GPIIb-IIIa

Integrin Antagonists with the New Conformational Restriction

Unit, Trisubstituted â-Amino Acid Derivatives, and a Substi-

tuted Benzamidine Structure. J . Med. Chem. 1998, 41, 2345-

2360. (b) Okumura, K.; Shimazaki, T.; Aoki, Y.; Yamashita, H.;

Tanaka, E.; Banba, S.; Yazawa, K.; Kibayashi, K.; Banno, H.

New Platelet Fibrinogen Receptor Glycoprotein IIb-IIIa Antago-

nists: Orally Active Series of N-Alkylated Amidines with a 6,6-

Bicyclic Template. J . Med. Chem. 1998, 41, 4036-4052. (c)

Yamashita, H.; Okumura, K.; Shimazaki, T.; Kanematsu, A.;

Aoki, Y.; Nakajima, Y.; Yazawa, K.; Kibayashi, K. Amidine

Derivatives and Platelet Aggregation Inhibitor Containing the

Same. European Patent 0760364A2, 1997.

(29) Muller, T. H.; Schurer, H.; Waldmann, L.; Bauer, E.; Himmels-

bach, F.; Binder, K. Oral Activity of BIBU 104, a Prodrug of the

Non-Peptide Fibrinogen Receptor Antagonist BIBU 52, in Mice

and Monkeys. Thromb. Haemostasis 1993, 69, 975, Abstract

1557.

(30) (a) Merlos, M.; Graul, A.; Castaner, J . Sibrafiban Drugs Future

1998, 23, 1297-1303. (b) Weller, T.; Alig, L.; Beresini, M.;

Blackburn, B.; Bunting, S.; Hadvary, P.; Muller, M. H.; Knopp,

D.; Levet-Trafit, B.; Lipari, M. T.; Modi, N. B.; Muller, M.; Refino,

C. J .; Schmitt, M.; Schonholzer, P.; Weiss, S.; Steiner, B. Orally

Active Fibrinogen Receptor Antagonists. 2. Amidoximes as

Prodrugs of Amidines. J . Med. Chem. 1996, 39, 3139-3147.

(31) Gante, J .; J uraszyk, H.; Raddatz, P.; Wurziger, H.; Bernotat-

Danielowski, S.; Melzer, G.; Rippmann, F. New Antithrombotic

RGD-Mimetics with High Bioavailability. Bioorg. Med. Chem.

Lett. 1996, 6 (20), 2425-2430.

(17) (a) Rawal, V. H.; J ones, R. J .; Cava, M. P. Photocyclization

Strategy for the Synthesis of Antitumor Agent CC-1065: Syn-

thesis of Dideoxy PDE-I and PDE-II. Synthesis of Thiopene and

Furan Analogues of Dideoxy PDE-I and PDE-II. J . Org. Chem.

1987, 52, 19-28. (b) Reinecke, M. G.; Daubert, R. G. Periphral

Synthesis of Secondary Medium-Ring Nitrogen Heterocycles. J .

Org. Chem. 1973, 38, 3281-3287.

(18) Dong, Q.; Anderson, C. E.; Ciufolini, M. A. Reductive Cleavage

of TROC Groups under Neutral Conditions with Cadmium-

Lead Couple. Tetrahedron Lett. 1995, 5681-5682.

(19) (a) Carrera, G. M., J r.; Garvey, D. S. Synthesis of Novel

Substituted Spirohydantoins. J . Heterocycl. Chem. 1992, 29,

847-850. (b) Suess, R. Substituierte 2,8-Diazaspiro-decan-1,3-

dione. Helv. Chim. Acta 1977, 60(5), 1650-1656; & J ucker, E.;

Sue, R. Arch. Pharm. 1961, 294, 210-220.

(20) Fisher, M. J .; Gunn, B.; Harms, C. S.; Kline, A. D.; Mullaney, J .

T.; Nunes, A.; Scarborough, R. M.; Arfsten, A. E.; Skelton, M.

A.; Um, S. L.; Utterback, B. G.; J akubowski, J . A. Non-Peptide

RGD Surrogates which Mimic a Gly-Asp â-Turn: Potent An-

tagonists of Platelet Glycoprotein IIb-IIIa. J . Med. Chem. 1997,

40, 2085-2101.

(21) Su, T.; Naughton, M. A.; Smyth, M. S.; Rose, J . W.; Arfsten, A.

E.; McCowan, J . R.; J akubowski, J . A.; Wyss, V. L.; Ruterbories,

K. J .; Sall, D. J .; Scarborough, R. M. Fibrinogen Receptor (GPIIb/

IIIa) Antagonists Derived from 5,6-Bicyclic Templates. Amidi-

noindoles, Amidinoindazoles, and Amidinobenzofurans Contain-

ing the N-R-Sulfonamide Carboxylic Acid Function as Potent

Platelet Aggregation Inhibitors. J . Med. Chem. 1997, 40, 4308-

4318.

(22) (a) Hartman, G. D.; Egbertson, M. S.; Halczenko, W.; Laswell,

W. L.; Duggan, M. E.; Smith, R. L.; Naylor, A. M.; Manno, P.

D.; Lynch, R. J .; Zhang, G.; Chang, C. T.-C.; Gould, R. J . Non-

Peptide Fibrinogen Receptor Antagonists. 1. Discovery and

Design of Exosite Inhibitors. J . Med. Chem. 1992, 35, 4640-

4642. (b) Egbertson, M. S.; Chang, C. T.-C.; Duggan, M. E.;

Gould, R. J .; Halczenko, W.; Hartman, G. D.; Laswell, W. L.;

Lynch, J r. J . J .; Lynch, R. J .; Manno, P. D.; Naylor, A. M.; Prugh,

J . D.; Ramjit, D. R.; Sitko, G. R.; Smith, R. S.; Turchi, L. M.;

Zhang, G. Non-Peptide Fibrinogen Receptor Antagonists. 2.

Optimization of a Tyrosine Template as a Mimic for Arg-Gly-

Asp. J . Med. Chem. 1994, 37, 2537-2551. (c) Xue, C.-B.;

Roderick, J .; J ackson, S.; Rafalski, M.; Rockwell, A.; Mousa, S.;

Olson. R. E.; DeGrado, W. F. Design, Synthesis, and in Vitro

Activities of Benzamide-Core Glycoprotein IIb-IIIa Antago-

nists: 2,3-Diaminopropionic Acid Derivatives as Surrogates of

Aspartic Acid. Bioorg. Med. Chem. 1997, 5, 693-705.

(32) Veber, D. F.; J ohnson, S. R.; Cheng, H.-Y.; Smith, B. R.; Ward,

K. W.; Kopple, K. D. Molecular Properties that Influence the

Oral Bioavailability of Drug Candidates. J . Med. Chem. 2002,

45, 2615-2623.

(33) (a) Hauptmann, J .; Paintz, M.; Kaiser, B.; Richter, M. Reduction

of a Benzamidoxime Derivative to the Corresponding Benzami-

dine in Vivo and in Vitro. Pharmazie 1988, 43, 559-560. (b)

Clement, B.; Zimmermann, M.; Schmitt, S. Biotransformation

des Benzamidins und des Benzamidoxims durch mikrosomale

Enzyme vom Kaninchen. (Biotransformation of Benzamidine

and Benzamidoxime by Microsomal Enzymes of the Rabbit.)

Arch. Pharm. (Weinheim, Ger.) 1989, 322, 431-435. (c) Clement,

B.; Immel, M.; Terlinden, R.; Wingen, F.-J . Reduction of Ami-

doxime Derivatives to Pentamidine in vivo. Arch. Pharm.

(Weinheim, Ger.) 1992, 325, 61-62. (d) Clement, B.; J ung, F.

N-Hydroxylation of the Antiprotozoal Drug Pentamidine Cata-

lyzed by Rabbit Liver Cytochrome P-450 2C3 or Human Liver

Microsomes, Microsomal Retroreduction, and Further Oxidative

Transformation of the Formed Amidoximes. Drug Metab. Dispos.

1994, 22, 486-497.

(23) (a) Scarborough, R. M.; Rose, J . W.; Naughton, M. A.; Phillips,

D. R.; Nannizzi, L.; Arfsten, A.; Campbell, A. M.; Charo, I. F.

Characterization of the Integrin Specificities of Disintegrins

Isolated from American Pit Vipor Venoms. J . Biol. Chem. 1993,

268, 1058-1065. (b) Duggan, M. E.; Naylor-Olsen, A. M.;

Perkins, J . J .; Anderson, P. S.; Chang, C. T.-C.; Cook, J . J .;

Gould, R. J .; Ihle, N. C.; Hartman, G. D.; Lynch, J . J .; Lynch, R.

Novel 2,8-Diazaspiro[4.5]decanes

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2061

(34) Ertl, P.; Rohde, B.; Selzer, P. Fast Calculation of Molecular Polar

Surface Area as a Sum of Fragment-Based Contributions and

its Application to the Prediction of Drug Transport Properties.

J . Med. Chem. 2000, 43, 3714-3717.

(35) We have recently reported that when 22 (CT51464/ML464) was

administered to mice by oral gavage at a dose of 100 mg/kg,

platelet aggregation, measured by platelet-rich plasma aggre-

gometry, was inhibited within 30 min and remained disrupted

for up to 10 h. During this time, the concentration of active

metabolite 23 (CT50728/ML728) was 37.5 µM at its peak 30 min

after oral gavage and 5 µM 8 h after oral gavage of 22. Bakewell,

S. J .; Nester, P.; Prasad, S.; Tomasson, M. H.; Dowland, N.;

Mehrotra, M.; Scarborough, R.; Kanter, J .; Abe, K.; Phillips, D.;

Weilbaecher, K. N. Platelet and Osteoclast â₃Integrins are

Critical for Bone Metastasis. Proc. Natl. Acad. Sci. U.S.A. 2003,

100 (24), 14205-14210.

J M030354B

Article Doi

Discovery of Novel 2,8-Diazaspiro[4.5]decanes as Orally Active Glycoprotein IIb-IIIa Antagonists

DOI: 10.1021/jm030354b

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm030354b

Full text of DOI:10.1021/jm030354b

Products guided by the article

R&D Labs maybe for 685544-62-1

Relevant to this article

Synthesis of oxindole from acetanilide via Ir(iii)-catalyzed C-H carbenoid functionalization

C[sbnd]S cross-coupling catalyzed by a series of easily accessible, well defined Ni(II) complexes of the type [(NHC)Ni(Cp)(Br)]

Cyclopenta[b]thiophene-alkyloxazolines: New nitrogen-sulfur hybrid ligands and their use in asymmetric palladium-catalyzed allylic alkylation

N-(Hydroxybenzyl)benzamide Derivatives: Aqueous pH-Dependent Kinetics and Mechanistic Implications for the Aqueous Reactivity of Carbinolamides

Chiral Dihydrobenzofuran Acids Show Potent Retinoid X Receptor-Nuclear Receptor Related 1 Protein Dimer Activation