Exploration of the active site of neuronal nitric oxide synthase by the design and synthesis of pyrrolidinomethyl 2-aminopyridine derivatives

7804 J. Med. Chem. 2010, 53, 7804–7824

DOI: 10.1021/jm100947x

Exploration of the Active Site of Neuronal Nitric Oxide Synthase by the Design and Synthesis of

Pyrrolidinomethyl 2-Aminopyridine Derivatives

†

‡

§,

Haitao Ji, Silvia L. Delker, Huiying Li, Pavel Martasek, Linda J. Roman,^§Thomas L. Poulos,*^,‡and

ꢀ

Richard B. Silverman*^,†

^†Department of Chemistry, Department of Molecular Biosciences, Center for Molecular Innovation and Drug Discovery, and Chemistry of Life

Processes Institute, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States, ^‡Departments of Molecular

Biology and Biochemistry, Pharmaceutical Chemistry, and Chemistry, University of California, Irvine, California 92697-3900,

United States, ^§Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78384-7760, United States, and

Department of Pediatrics and Center for Applied Genomics, First School of Medicine, Charles University, Prague, Czech Republic

Received July 26, 2010

Neuronal nitric oxide synthase (nNOS) represents an important therapeutic target for the prevention of

brain injury and the treatment of various neurodegenerative disorders. A series of trans-substituted

amino pyrrolidinomethyl 2-aminopyridine derivatives (8-34) was designed and synthesized. A struc-

ture-activity relationship analysis led to the discovery of low nanomolar nNOS inhibitors ((()-32 and

(()-34) with more than 1000-fold selectivity for nNOS over eNOS. Four enantiomerically pure isomers

of 3⁰-[2⁰⁰-(3⁰⁰⁰-fluorophenethylamino)ethoxy]pyrrolidin-4⁰-yl}methyl}-4-methylpyridin-2-amine (4) also

were synthesized. It was found that (3⁰R,4⁰R)-4 can induce enzyme elasticity to generate a new “hot spot”

for ligand binding. The inhibitor adopts a unique binding mode, the same as that observed for (3⁰R,4⁰R)-

3⁰-[2⁰⁰-(3⁰⁰⁰-fluorophenethylamino)ethylamino]pyrrolidin-4⁰-yl}methyl}-4-methylpyridin-2-amine

((3⁰R,4⁰R)-3) (J. Am. Chem. Soc. 2010, 132 (15), 5437-5442). On the basis of structure-activity

relationships of 8-34 and different binding conformations of the cis and trans isomers of 3 and 4, critical

structural requirements of the NOS active site for ligand binding are revealed.

Introduction

serves as an electron donating domain. The linker between the

two functional domains is a calmodulin binding motif. The

binding of calmodulin enables electron flow from the flavins

to heme.

Nitric oxide (NO),¹an essential signaling molecule, is

produced by a family of enzymes called nitric oxide synthase

(NOS^a, EC 1.14.13.39).²There are three known NOS isozyme

forms, two constitutive forms and one inducible form. Neu-

ronal NOS (nNOS), predominantly expressed in neurons,

produces NO for neurotransmission, endothelial NOS

(eNOS), produces NO for the regulation of blood pressure

and flow, and inducible NOS (iNOS) is induced by cytokines

and pathogens to produce NO to combat infection and

microorganisms. Each isozyme of NOS catalyzes the conver-

sion of ^L-arginine to NO and L-citrulline.³All three enzymes

share similar domain architecture and are active as homo-

dimers.⁴Each monomer has a N-terminal domain consisting

of the catalytic heme active site and a (6R)-5,6,7,8-tetrahydro-

L-biopterin (H₄B) binding site and a C-terminal domain

containing FMN, FAD, and NADPH binding sites, which

To exert appropriate physiological functions, NO genera-

tion by the three different NOS isoforms is under tight

regulation.^5,6The overproduction of NO by nNOS has been

implicated in various neurodegenerative diseases,⁷including

Alzheimer’s disease, Parkinson’s disease, and Huntington’s

disease as well as neuronal damage resulting from stroke,⁸

cerebral palsy,⁹and migraine headaches.¹⁰Inhibition of nNOS

could have therapeutic benefit in these diseases. However, this

inhibition must be achieved without effect on the other NOS

isoforms.¹¹Inhibition of eNOS could cause hypertension,

atherosclerosis, arterial thrombosis, and angina.¹²Macro-

phage NOS(iNOS) is an enzyme responsible for the generation

of cytotoxic NO, playing an important role in the immune

system.¹³Additionally, the inhibition of iNOS might result in

a higher probability of Alzheimer’s disease.¹⁴Therefore, iso-

form-selective inhibitors are essential if nNOS is to be a viable

therapeutic target.^15,16Herein lies the challenge because the

crystal structures of the catalytic domains of all three NOS

isoforms show that the active sites are nearly identical, making

structure-based design of isoform-selective inhibitors a diffi-

cult and challenging problem.¹⁷

*To whom correspondence should be addressed. For T.L.P.: phone,

(949) 824-7020; fax, (949) 824-3280; E-mail, poulos@uci.edu. For R.B.

S.: phone, (847) 491-5653; fax, (847) 491-7713; E-mail, Agman@chem.

northwestern.edu.

^aAbbreviations: NOS, nitric oxide synthase; nNOS, neuronal nitric

oxide synthase; eNOS, endothelial nitric oxide synthase; iNOS, induci-

ble nitric oxide synthase; H₄B, (6R)-5,6,7,8-tetrahydro-L-biopterin.

FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide;

NADPH, reduced nicotinamide adenine dinucleotide phosphate;

CaM, calmodulin; CPCA, consensus principle component analysis;

PSA, polar surface area; L-NNA, ^L-N^ω-nitroarginine; Ph₃P, triphenyl-

phospine; DIAD, diisopropyl azodicarboxylate; DPPA, diphenylpho-

sphoryl azide.

Previous structure-activity relationship studies in our

laboratories on a series of N^ω-nitroarginine containing dipep-

tide inhibitors have enabled us to identify a family of com-

pounds that has high potency and selectivity for inhibition of

r

pubs.acs.org/jmc

Published on Web 10/19/2010

2010 American Chemical Society

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7805

Table 1. Inhibition of NOS Isozymes by 1-7

K_i(μM)

selectivity^a

compd

nNOS

eNOS

iNOS

n/e

n/i

(()-1

(()-2

(()-3

(()-4

(()-5

(()-6

(()-7

0.388

0.085

0.014

0.015

8.76

434.5

85

28

58.4

8.95

4.06

9.5

1114

1000

2000

2067

99

150

106

290

633

9

31

866.2

85.2

683.7

77.4

143.8

233.8

13.28

28.54

6

24

11

8

^an/e and n/i are the selectivity ratio of K_i(eNOS or iNOS) to K_i(nNOS).

nNOS over eNOS and iNOS.^18,19The selectivity of the

dipeptide/peptidomimetic inhibitors for nNOS over eNOS

and/or iNOS was investigated by crystallographic analysis²⁰

and computer modeling (GRID/CPCA).²¹Recently, we

established a new fragment-based de novo design strategy called

fragment hopping.²²Using this novel approach, together with

whatwelearnedfrom the dipeptide inhibitors, leadcompound

1 (Table 1) with a pyrrolidinomethyl aminopyridine scaffold

was designed and synthesized, which shows nanomolar nNOS

inhibitory potency and more than 1000-fold nNOS selectivity

over eNOS.²²Lead compound 1 was evolved into highly

potent and selective inhibitors 2, 3, and 4.^23,24Compounds 2

and 3 were tested on a rabbit model for cerebral palsy and

found to prevent hypoxia-ischemia induced death and to

reduce the number of newborn kits exhibiting cerebral palsy

phenotype without affecting eNOS-regulated blood pressure.²⁵

In our previous study, although we found that the trans amine

analogue (5) is a less potent and selective inhibitor for nNOS

compared to 1,²²this structure still represents a new scaffold for

inhibitor optimization. Two chiral centers (3⁰and 4⁰carbons)

exist in the structure of 3, although our initial studies were

carried out with the racemic mixtures. Recognizing the impor-

tance of chirality in molecular recognition and inhibitor bind-

ing, we synthesized four enantiomerically pure isomers of 3 in a

previous study.²⁶The in vitro enzyme assay showed dramatic

and exciting results. (3⁰R,4⁰R)-3 is a more potent and selective

inhibitor of nNOS over the other two NOS isozymes than

(3⁰S,4⁰S)-3 (Table 3). The K_ivalue for (3⁰R,4⁰R)-3 is 5 nM, and

the selectivities for nNOS over eNOS and iNOS are more than

3800-fold and 700-fold, respectively. This compound is, to the

best of our knowledge, the most potent and dual-selective

nNOS inhibitor reported to date.²⁶Using a combination of

crystallography, computation, and site-directed mutagenesis

studies, it was found that nNOS undergoes an unanticipated

structural change to accommodate the 2-amino-4-methylpyr-

idine moiety of (3⁰R,4⁰R)-3. A new “hot spot” was generated

because of enzyme elasticity. The binding conformation of

(3⁰R,4⁰R)-3 in the crystal structure of nNOS is 180° flipped

over compared to that of (3⁰S,4⁰S)-3. We, therefore, synthesized

the four enantiomerically pure isomers of 4 (Table 1), another

very potent and highly selective inhibitor of nNOS with better

drug-like properties, and determined their inhibitory potencies

and isozyme selectivities. Here we describe the interactions of

these enantiomers and of a series of compounds related to 3 and

4 with nNOS and eNOS, which reveal important structural

requirements for induction of enzyme elasticity and generation

of a new binding site.

Results and Discussion

Inhibitor Design. In our previous studies, starting with the

nitroarginine-containing dipeptide inhibitors, fragment hop-

ping was used to determine the minimal pharmacophoric

elements for inhibitor potency and NOS isozyme selecti-

vities.²²A series of nonpeptide cis-substituted amino pyrro-

lidinomethyl 2-aminopyridine derivatives was designed and

synthesized to match the requirements of the minimal phar-

macophoric elements (Figure 1). The crystal structures of

nNOS in complex with 1 and 2 (Figure 2) show that the

binding of the inhibitors exactly mimic the binding mode of

the dipeptide nNOS inhibitors.²⁷As shown in Figure 1, two

structural water molecules bridge the hydrogen bonding

interactions of the pyrrolidino nitrogen atom and the side

chain carboxylic acid group of Asp597. Therefore, there is

some space between this nitrogen atom and the Asp597

carboxylate. Compounds 8-11 (Figure 3A) were designed

to explore the space in this area and to determine whether or

not the nNOS inhibitory potency and selectivities can be

increased if a positively charged amino group of the inhibitor

7806 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Ji et al.

Figure 1. Schematic drawing of the active site of nNOS. The

binding mode of the cis-pyrrolidinomethyl aminopyridine inhibi-

tors and the amino acids and cofactors important for inhibitor

binding are indicated. Numbering is for the sequence of nNOS if not

indicated. The H-bonds observed in the crystal structure are in-

dicated by dotted lines based on the crystal structures of nNOS

in complexes with 1 and 2 (PDB: 3B3N and 3B3O).²⁷Under

physiological conditions, the nitrogen atoms of the pyridine ring,

the pyrrolidine ring, and the terminal amino group of the ethylene-

diamine side chain in NOS are protonated, but the nitrogen atom

attached to the pyrrolidine ring is in the neutral form.

is placed closer to the side chain carboxylic acid group of

Asp597.

Trans amine 5 represents a new lead compound for

optimization. In previous studies,²³it was found that there

is more space at position 4 of the 2-aminopyridine ring

that would favor hydrophobic and steric interactions (S

pocket),^23,28and there is another hydrophobic cavity in the

C1 pocket, as shown in Figure 4.²³Structural optimization of

5 is based on these hydrophobic pockets. One series of trans

molecules, 13 through 20 in Figure 3B, were designed to

introduce various conformational constraints to the ethyle-

nediamine side chain to minimize the entropic penalty

associated with flexibility of this side chain and also to extend

the tail into the C1 pocket with a benzyl group. In the other

series of trans molecules, 21 through 34 in Figure 3B, a

methyl group was introduced at position 4 of the 2-amino-

pyridine ring to occupy the hydrophobic region in the S

pocket, and the hydrophobic benzyl group or phenethyl

group was extended from the terminal amino group of 5 to

occupy the C1 pocket (Figure 4).

Figure 2. Crystallographic binding conformations of 1 (A) and 2

(B) complexed with nNOS (PDB: 3B3N and 3B3O).²⁷The heme

(orange), H₄B (purple), and structural water molecules (green)

involved in the binding of 1 and 2 to nNOS are shown. The active

site residues and ligands are represented in an atom-type style

(carbons in gray, nitrogens in blue, oxygens in red, and sulfur in

yellow). The distances of some important H-bonds between the

residues, structural water molecules, cofactors, and inhibitors are

˚

given in A.

selectivity over the other two NOSs. In the inhibitor design

cycles, AutoDock 3.0.5 was routinely used to dock most of

the inhibitors in the active site before the synthesis was

carried out.²⁹

It was found in our previous study that the racemic

mixture of the ether derivative (4) exhibits comparable

nNOS inhibitory potency and selectivity over eNOS com-

pared to 3.²⁴The difference between 4 and 3 is that com-

pound 4 uses a hydrogen-bonding acceptor group (ether)

and 3 uses a hydrogen-bonding donor group (secondary

amine). Therefore, 4 exhibits better drug-like properties

and improved bioavailability.²⁴Enzyme assays in the pre-

sence of the four enantiopure isomers of 3 revealed that

(3⁰R,4⁰R)-3 exhibits better nNOS inhibitory potency and

selectivity over the other two NOS isozymes than (3⁰S,4⁰S)-

3. Crystallographic analysis shows that (3⁰R,4⁰R)-3 induces

enzyme elasticity, and a new “hot spot” is generated to

accommodate the 2-amino-4-methylpyridine moiety of

(3⁰R,4⁰R)-3.²⁶Therefore, the four enantiomers of 4 were

synthesized to determine whether or not (3⁰R,4⁰R)-4 can

similarly induce enzyme elasticity to generate a new ligand

binding pocket for better nNOS inhibitory potency and

Chemistry. Scheme 1 shows the synthetic route for target

compounds 8-11. The synthesis of 37 was described in an

earlier paper.²²Deprotection of the benzyl group by catalytic

hydrogenation and then alkylation with N-Boc-2-bro-

moethylamine generated 39. Swern oxidation of 39 generates

ketone intermediate 40 in high yields. The direct reductive

amination of 40 with different amines generated the cis (41)

and trans (42) amines. These two regioisomers can be

completely separated by silica gel column chromatography;

deprotection of the Boc groups generates the final products.

Characterization of the cis and trans isomers was studied

1

in detail by NMR spectrometry, including H NMR, ¹³C

NMR, ¹³C NMR-DEPT, ¹H-¹H COSY, ¹H-¹³C HMQC,

1

and H-¹H NOESY spectrometry, as described in a pre-

vious paper.²²It was found that one prominent difference

between the final products of the cis and trans isomers is that

the ¹³C chemical shifts of the carbon atom attached to the

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7807

Figure 3. Target molecules synthesized and tested in this study.

pyridine ring is about 29.5 ppm for the cis compound,

but 34.1 ppm for the trans isomer. This difference was

further used in the rapid structural characterization of

compounds 1-34. Typically, all of the trans isomers show

a lower R_fvalue on TLC compared to the corresponding cis

isomers.

cynoborohydride was used as the reducing agent, the yields

of the amines were about 50-65%. The ratio of the cis to trans

amines was about 45:55 for 46b-46d. As described in the

previous study,²³when the catalyst for the hydrogenation

reaction was changed to platinum IV oxide, the ratio of the

cis to trans isomers changed to 55:45. When sodium triacetoxy-

borohydride was used as the reducing agent, the ratio of the cis

to trans isomers was 95:5. Indirect reductive amination reac-

tions also were attempted. The ketone was refluxed with the

amine in benzene, and water was removed with a Dean-Stark

trap. The imine compound was produced, which was then

reduced with sodium borohydride in MeOH. However, the

yield of the total amine was much lower (about 10-20%). The

ratioofcistotransisomerswas1:2. Whenmolecularsieveswere

used as the desiccant, the reaction mixture was stirred at room

The synthetic route for 12-20 is shown in Scheme 2.

Deprotection of the Boc groups of 44 generated 12. The

synthesis of intermediate 45 was described in earlier

papers.^22,23To optimize the yield of the trans amine ana-

logue, different conditions for the reductive amination reac-

tion were tried. First, direct reductive amination reactions

˚

were tried. When 3A molecular sieves were used as the

desiccant, anhydrous MeOH was used as the solvent, one equiv-

alent of acetic acid was used as the proton provider, and sodium

7808 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Ji et al.

temperature then reduced with sodium borohydride in MeOH,

the yield of the total amines was increased to 30%, and the

ratio of the cis to trans isomers was 1:2. When molecular sieves

were used as the desiccant, and the catalytic hydrogenation

reaction using Pd/C as catalyst was conducted, the ratio of the

cis to trans isomers was changed to 3:1. Consequently, direct

reductive amination reactions using sodium cynoborohydride

as the reducing agent were carried out for the rest of the

compounds. The yields of the total amines still remained about

50-65%. The ratio of the cis to trans isomers was about 30:70

for 46a, 46g, and 46h. For compounds 46b-46f in Scheme 2A

and 50 in Scheme 2B, the ratio of cis to trans isomers was

about 45:55.

In the early stage of the synthesis of this series of inhibi-

tors, we discovered that trans alcohols 44 or 48a could not be

used in any S_N2 reaction directly. The only product gener-

ated was a new cis five-membered ring (a quarternary

ammonium) that occurred by intramolecular attack of the

nitrogen atom of the 2-aminopyridine on the carbon atom of

the pyrrolidino ring to which the hydroxyl group was

attached. So 44 or 48a was oxidized to a ketone, and then

reductive amination was carried out to generate the trans

amines, as shown in Schemes 1 and 2.^22,23Later, we dis-

covered that Mitsunobu reactions proceeded smoothly when

the mono Boc protected 2-amino group of the 2-aminopyr-

idine moiety of 44 was further protected with a benzyl group

or an additional Boc group.³⁰In this study, we continued to

use a benzyl group as the protecting group to protect 48a,

which allowed the S_N2 reaction to proceed, and to synthesize

the trans isomers, as shown in Scheme 3. Mitsunobu reaction

of 51 and the subsequent hydrolysis of the acetate afforded

cis alcohol 53, which could be converted to trans azide 54 by

the application of another Mitsunobu reaction. Hydrogena-

tion of azide 54 using Pd(OH)₂/C as catalyst generated amine

55, which could be converted to 56 by reductive amination.

Compounds 23, 32, and 34 were resynthesized by this

synthetic route.

Figure 4. Results of the GRID analyses of the substrate-binding

site of nNOS (PDB: 1P6I) illustrates the direction of lead optimiza-

tion. The residues and inhibitor 5 are represented in an atom-type

style (carbons in gray, nitrogens in blue, oxygen in red, and sulfur in

yellow). Heme and H₄B are colored magenta. The S, M, and C1

pockets are indicated. The GRID contours of the DRY probe

(yellow) is set at an energy level of -0.75 kcal/mol. The GRID

contours of the C3þ probe (green) is set at an energy level of

-4.00 kcal/mol.

The synthesis of the four enantiopure isomers of 4 is shown

in Scheme 4. Different from the synthetic route reported in

our previous study,²⁶compound 51 was directly subjected to

Scheme 1

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7809

Scheme 2

the Mitsunobu reaction to generate ester 57 using 1S-(-)-

camphanic acid as a nucleophile (Scheme 4A). The synthesis

from four optically pure alcohol isomers of 58 to four

isomers of 4 is shown in Scheme 4B. The synthetic route is

very similar to the synthesis of the racemic mixture of 4.²⁴

Allylation of (3⁰R,4⁰R)-58 with allyl bromide generated

(3⁰R,4⁰R)-60, which then underwent ozonolysis to generate

aldehyde (3⁰R,4⁰R)-61. Direct reductive amination of (3⁰R,-

4⁰R)-61 with 3-fluorophenethylamine generated (3⁰R,4⁰R)-

62. Protection of the secondary amine of (3⁰R,4⁰R)-62 with a

Boc group and then deprotection of the benzyl group

afforded (3⁰R,4⁰R)-64. Deprotection of the Boc groups

generated (3⁰R,4⁰R)-4. The other three enantiopure isomers

were synthesized by the same route.

NOS Inhibition and Structure-Activity Relationships of

Compounds 8-34. Table 2 shows the results of the NOS

enzyme inhibition assays. Trans amines 9 and 11 exhibited

much better nNOS inhibitory potency and better nNOS

selectivity over the other two NOS isozymes than the corre-

sponding cis amine derivatives (8 and 10), respectively. This

suggests that the aminoethyl group of the trans isomers is

better accommodated than that of the cis isomers. Compared

to the unsubstituted trans isomers, such as 6 and 7, com-

pounds 9 and 11 exhibit 2- to 3-fold better inhibitory potency

7810 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Ji et al.

Scheme 3

and 5- to 7-fold better selectivities for nNOS over eNOS.

These are not very significant improvements. Considering

that the primary amine was reported to have a much larger

polar surface area (PSA) than the secondary amine, which

would be unfavorable for biomembrane permeability,³¹the

2-aminoethyl side chain of 9 and 11 was not further pursued.

Instead, other structural optimization of the inhibitors was

based on the structure of 5.

The crystallographic binding conformations of 2 and 23 in

nNOS and eNOS are very similar, which is drastically

different from the binding mode of the ^L-N^ω-nitroarginine-

containing dipeptide inhibitors we developed before.¹⁹The

dipeptide inhibitors have multiple flexible bonds around the

R-amino group of the ^L-N^ω-nitroarginine moiety that generates

nNOS selectivity over eNOS. Correspondingly, the binding

conformation of ^L-N^ω-nitroarginine-containing dipeptide inhi-

bitors in nNOS is different from that in eNOS. The dipeptide

inhibitor adopts a curled conformation in nNOS but an ex-

tended conformation in eNOS.²⁰The introduction of the pyrro-

lidino ring in the pyrrolidinomethyl 2-aminopyridine derivatives

(such as 2 and 23) potentially increases their nNOS selectivity

over eNOS because this scaffold has a lower entropic penalty.

NOS Inhibition and Crystallographic Analysis of Four

Enantiomers of 4. Identification of 32 and 34 as the most

potent inhibitors among those compounds with a phenethyl

moiety prompted us to synthesize and characterize the

enantiopure compounds. The synthesis and enzyme assay

results for the four enantiomers of 3 have been reported

recently.²⁵It was found that (3⁰R,4⁰R)-3 is a more potent

inhibitor of nNOS and more selective over the other two

NOS isozymes than (3⁰S,4⁰S)-3. The K_ivalue for (3⁰R,4⁰R)-3

with nNOS is 5 nM. The nNOS selectivities over eNOS and

iNOS are more than 3800-fold and 700-fold, respectively.

trans-(3⁰R,4⁰S)-3 is also a very potent and selective nNOS

inhibitor with a K_ivalue of 19 nM, with selectivities over

eNOS and iNOS of more than 3000-fold and 800-fold,

respectively.

Structural optimization of 5 leads to the generation of

three interesting compounds: 23, 32, and 34. Compound 23 is

the most potent nNOS inhibitor among the compounds with

a benzyl moiety. Compound 34 is the most potent inhibitor

among the compounds with a phenethyl moiety. The K_ivalue

for 34 with nNOS is 48 nM, and the selectivity for nNOS over

eNOS and iNOS is more than 1000-fold and 500-fold, res-

pectively. Introduction of conformational constraints, such

as a ring or a methyl group, to the ethylenediamine side chain

of 5 generates 17-20, 30, and 31. The enzyme assay shows

that these structural modifications do not work well (Table 2).

The crystal structures of 23 complexed with nNOS and

eNOS were reported previously (PDB: 3B3P and 3DQT).²⁷

The binding mode of the (3R,4S)-isomer of 23 with nNOS is

very similar to that of 2, except for the nitrogen atom

attached to the pyrrolidine ring (Figures 2B and 5). In 2, this

nitrogen atom makes a H-bond with heme propionate A, but

the nitrogen atom in 23 points away from the propionate

because of the trans configuration. Although 23 has a meta-

chloro and 2 has a para-chloro substituent, both the meta-

chlorobenzyl and para-chlorobenzyl groups point to the

same surface hydrophobic pocket, and the active site is

large enough to accommodate either of these. The location

of the pyrrolidine ring of 23 is the same as that in 2. The

ring nitrogen H-bonds to the side chain of Glu592 in both

cases. Two structural waters were observed to bridge the

H-bonding interactions between the side chain carboxylic

acid group of Asp597 and the pyrrolidine nitrogen atom in

both 2 and 23. Therefore, the principal difference between 2

and 23 derives from the chirality of the pyrrolidino ring. The

trans configuration in 23 makes it impossible to have a

H-bond between its nitrogen attached to the pyrrolidine

and heme propionate A. In contrast, the additional H-bond

in 2 gives the cis molecule its better inhibitory potency.

Similarly, enzyme assays show that (3⁰R,4⁰R)-4 is a more

potent and selective inhibitor of nNOS than (3⁰S,4⁰S)-4

(Table 2). The K_ifor nNOS is 7 nM, and the selectivities

over eNOS and iNOS are more than 2600-fold and 800-fold,

respectively. To the best of our knowledge, this compound

and (3⁰R,4⁰R)-3 represent two of the most potent and selec-

tive nNOS inhibitors reported to date. trans-(3⁰R,4⁰S)-4 also

is a potent and selective nNOS inhibitor with a K_ivalue

of 63 nM, with selectivities over eNOS and iNOS of over

500-fold and 300-fold, respectively (Table 3).

Crystal structures of nNOS complexed with the four

enantiopure isomers of 4 were determined. The binding

conformation of (3⁰S,4⁰S)-4 in nNOS (Figure 6A) shows that

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7811

Scheme 4

˚

between the heme propionate A (2.9 A) and the carbonyl

the 2-amino-4-methylpyridine moiety interacts with active

site Glu592. The pyrrolidine nitrogen is close to the selective

residue (nNOS Asp597/eNOS Asn368). The nitrogen atom

of the 3-fluorophenethylamino group forms one H-bond

with heme propionate D, and the 3-fluorophenyl moiety is

located in the surface hydrophobic pocket lined with

Met336, Leu337, Tyr706, and Trp306 from the second

monomer. This binding mode is termed the “normal” bind-

ing mode, which means it mimics the binding mode of the

dipeptide inhibitors. (3⁰R,4⁰R)-4, however, adopts the 180°-

flipped orientation and induces nNOS enzyme elasticity to

generate a new “hot spot” to accommodate the 2-amino-4-

methylpyridine (Figure 6B). For this to occur, the side chain

of Tyr706 must swing away to make room for π-π stacking

interactions with the 2-amino-4-methylpyridine ring. The

3-fluorophenyl group is, therefore, positioned over the heme

while the 2-amino-4-methylpyridine ring extends out of the

substrate binding site. The pyrrolidine nitrogen is placed in

˚

group of H₄B (2.6 A). One structural water molecule bridges

three H-bonds between heme propionate D, the oxygen atom

attached to the pyrrolidine ring, and the nitrogen atom of the

phenethylamine side chain. The binding mode of (3⁰R,4⁰R)-4

is identical with that of (3⁰R,4⁰R)-3, which has been termed

the “flipped” binding mode.

Like the two trans isomers of 3, (3⁰R,4⁰S)-4 and (3⁰S,4⁰R)-4

only adopt the “normal” binding mode. The 2-aminopyr-

idine moiety forms a bifurcated salt bridge with active site

Glu592. The fluorophenyl group extends out of the sub-

strate-catalytic site and is located in the surface hydrophobic

pocket (Figures 6C,D). The pyrrolidine nitrogen atom of the

(3⁰R,4⁰S)-isomer forms one H-bond with Glu592 and

another H-bond with a structural water, which in turn binds

to Asp597. The positively charged nitrogen atom in the pyrro-

lidine ring interacts favorably with the side chain carboxylic

acid group of Asp597. In the case of (3⁰S,4⁰R)-4, although the

7812 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Table 2. Inhibition of NOS Isozymes by Synthetic Compounds 8-34

Ji et al.

(Figure 6D). The model that fits the density has the orien-

tation of the pyrrolidine ring of (3⁰S,4⁰R)-4 opposite that

of (3⁰R,4⁰S)-4. From the binding conformations of these

two isomers, it can be concluded that (3⁰R,4⁰S)-4 can

form better electrostatic interactions with active site residues

than (3⁰S,4⁰R)-4, which rationalizes why (3⁰R,4⁰S)-4 exhibits

better nNOS inhibitory potency and selectivity than (3⁰S,

4⁰R)-4.

K_i(μM)^a

selectivity^b

compd

L-NNA^c

nNOS

eNOS

iNOS

n/e n/i

0.57

47.94

3.98

0.75

121.62

172.91

849.21

1696

4.55

609.43

437.76

2522.8

651.85

25.24

308.1

166.1

298.55

416.61

234.06

253.43

209.01

285.54

32.03

44.85

44.11

40.42

27.35

58.72

83.9

1.3

2.5

8

13

110

8

(()-8

(()-9

43

(()-10

(()-11

(()-12

(()-13

(()-14

(()-15

(()-16

(()-17

(()-18

(()-19

(()-20

(()-21

(()-22

(()-23

(()-24

(()-25

(()-26

(()-27

(()-28

(()-29

(()-30

(()-31

(()-32

(()-33

(()-34

304.96

13.47

6.94

3

126

49

48

4

The enzyme assays show that the trans isomers, (3⁰R,4⁰S)-

3 and (3⁰R,4⁰S)-4, exhibit better nNOS inhibitory potency

and selectivity than the cis isomers (3⁰S,4⁰S)-3 and (3⁰S,4⁰S)-4

(Table 3), although all of these compounds adopt the “nor-

mal” binding mode. The superimposition of (3⁰R,4⁰S)-3 and

(3⁰S,4⁰S)-3 or (3⁰R,4⁰S)-4 and (3⁰S,4⁰S)-4 by the backbone

atoms of the active site residues, as defined in our previous

paper,²¹shows that the 2-amino-4-methylpyridino and the

3-fluorophenyl groups overlap fairly well (Figure 7). The

low-energy structures of the cis (3⁰S,4⁰S)-isomers of 3 and 4

are typically longer than those of the trans (3⁰R,4⁰S)-isomers

if they have the same orientations. One conclusion that can

be drawn on the basis of the enzyme assay results, and the

crystallographic analysis is that the length of the cis (3⁰S,4⁰S)-

isomers of 3 and 4 exceeds the requirement between the

substrate-catalytic pocket (the S pocket in Figure 3) and

the hydrophobic pocket (the C1 pocket in Figure 3) of

nNOS. Therefore, the cis (3⁰S,4⁰S)-isomers of 3 and 4 cannot

be accommodated as well as the trans (3⁰R,4⁰S)-isomers

(Figure 7). The 2-aminopyridine ring in (3⁰R,4⁰S)-3 or

(3⁰R,4⁰S)-4 is more parallel to the heme plane than that seen

in the (3⁰S,4⁰S)-structures, which strengthens the stacking

interaction between the two aromatic rings in the trans

structure. The trans configuration forces the pyrrolidine

rings in the (3⁰R,4⁰S)-isomers to be better aligned with the

carboxylic acid side chain of Glu592, thus resulting in a

stronger H-bond than that found in the cis (3⁰S,4⁰S) nNOS

structures. However, it is noteworthy that the different

configurations of the pyrrolidino rings in the (3⁰R,4⁰S) and

(3⁰S,4⁰S)-isomers lead to the formation of two extra H-bonds

for the less favorable cis isomers, (3⁰S,4⁰S)-3 and (3⁰S,4⁰S)-4.

One extra H-bond is from the nitrogen atom attached to the

pyrrolidine ring in (3⁰S,4⁰S)-3 with heme propionate A.

Second, the positively charged nitrogen atom of the 3-fluor-

ophenethylamino side chain can form a H-bond with heme

propionate D in both (3⁰S,4⁰S)-3 and (3⁰S,4⁰S)-4. Overall

enzyme-inhibitor interactions seen in the trans (3⁰R,4⁰S)-3

and (3⁰R,4⁰S)-4 structures, dominated by those involving the

2-aminopyridine and pyrrolidine rings, are slightly better

than those in cis (3⁰S,4⁰S)-3 and (3⁰S,4⁰S)-4 structures, which

accounts for a 2- to 3-fold better inhibitory potency for the

trans (3⁰R,4⁰S)- over the cis (3⁰S,4⁰S)-isomers (Table 3).

From this study, it can be concluded that if the inhibitor

adopts the “normal” binding mode, the nNOS active site can

optimally accommodate the inhibitors with a length that

matches those of the trans (3⁰R,4⁰S)-isomers of 3 or 4.

Similar to what we have described for the nNOS and

eNOS structures bound with the four enantiopure isomers

of 3,²⁶the binding conformations of the four enantiopure

isomers of 4 to the eNOS active site (Supporting Information

Figure S1) are generally the same as what we have just

discussed for nNOS. The rigidity of the pyrrolidine ring

brings its ring nitrogen to the vicinity of Glu592 (Glu363

in eNOS) and Asp597 (Asn368 in eNOS) when the config-

uration is (3⁰S,4⁰S) or (3⁰R,4⁰S), regardless of whether

inhibitors are bound to nNOS or to eNOS. The electrostatic

340.83

241.7

2906

15.68

14.45

13.86

20.57

12.85

20.93

17.92

24.4

15

20

11.5

201

105

71

1453

21.5

20

625.42

460.16

108.69

170.52

262.74

118.34

95.18

49

22

18

12

6

12

7

0.61

0.53

431

223

380

83

52.5

85

0.25

0.74

176

61.19

78.75

55

31

0.87

0.74

90.5

108.55

201.61

63.22

147

305.5

153

79

0.66

127

57

0.414

0.483

0.589

1.891

0.088

0.119

0.048

23.76

44.23

20.46

75.36

18.17

22.59

26.59

76.13

16.47

58.14

158

28

92

35

31

40

123.90

44.09

49.07

1408

370.5

1022

206.5

190

554

^aThe apparent K_ivalues are represented as the mean of two or more

independent experiments preformed in duplicate with five or six data

points each and correlation coefficients of 0.88-0.99. The experimental

standard deviations were less than 10%. ^bn/e and n/i are the selectivity

ratio of K_i(eNOS or iNOS) to K_i(nNOS). ^cL-NNA: L-N^ω-nitroarginine,

a positive control.

Figure 5. Crystallographic binding conformations of 23 in complex

with nNOS (PDB: 3B3P).²⁷The heme (orange), H₄B (purple), and

structural water molecules (green) involved in the binding of 23 to

nNOS are shown. The active site residues and ligands are repre-

sented in an atom-type style (carbons in gray, nitrogens in blue,

oxygens in red, and sulfur in yellow). The distances of some

important H-bonds between the residues, structural water mole-

˚

cules, cofactors, and inhibitors are given in A.

binding of the 2-aminopyridine next to the Glu592 side chain

is supported by good electron density, the density for the

pyrrolidine ring of (3⁰S,4⁰R)-4 is not well-defined and the

3-fluorophenethylamino group is almost fully disordered

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7813

Table 3. Inhibition of NOS Isozymes by the Enantiomerically Pure Isomers of 3 and 4

K_i(nM)^a

selectivity^b

compd

nNOS

eNOS

iNOS

n/e

n/i

(3⁰S,4⁰S)-3^c

(3⁰R,4⁰R)-3^c

(3⁰R,4⁰S)-3^c

(3⁰S,4⁰R)-3^c

(3⁰S,4⁰S)-4

(3⁰R,4⁰R)-4

(3⁰R,4⁰S)-4

(3⁰S,4⁰R)-4

52.2

5.3

26400

20300

57100

34500

26200

19200

36190

18780

3850

3940

506

3830

3021

202

73.8

743

18.9

171.0

116.0

7.2

16100

26600

7500

852

156

65

226

5800

18710

18060

2667

574

806

297

93

63.0

193.5

97

^aThe apparent K_ivalues are represented as the mean from two or more independent experiments preformed in duplicate with five or six data points

each and correlation coefficients of 0.92-0.99. The experimental standard deviations were less than 10%. ^bn/e and n/i are the selectivity ratio of K_i

(eNOS or iNOS) to K_i(nNOS). ^cThe data were reported in the previous paper.²⁶

Figure 6. Crystallographic binding conformations of four enantiomerically pure isomers of 4 [(A) (3⁰S,4⁰S)-4; (B) (3⁰R,4⁰R)-4; (C) (3⁰R,4⁰S)-4;

(D) (3⁰S,4⁰R)-4] with rat nNOS. Shown also is the 2F_o- F_celectron density for the ligands contoured at 1σ. The active site residues and ligands

are represented in an atom-type style (carbons in green or cyan (chain B), nitrogens in blue, oxygen in red, and sulfur in yellow). The important

H-bonds between the residues, structural water, cofactors, and inhibitors are depicted with dashed lines. The resolution and R_work/R_freevalues

˚

for the four structures shown are: (A) 2.02 A, 0.183/0.219; (B) 1.85 A, 0.189/0.222; (C) 2.00 A, 0.196/0.231; (D) 2.30 A, 0.201/0.266. The

4-methyl-2-aminopyridine ring in (3⁰S,4⁰S)-4, (3⁰R,4⁰S)-4, and (3⁰S,4⁰R)-4 and the m-fluoro-phenyl ring in (3⁰R,4⁰R)-4 are roughly parallel to

the heme plane with the closest distance to the heme iron at 4.0-4.1 A. It is not clear if this is a π-π stacking interaction or a cation-π

interaction.

˚

stabilization of this positively charged ring nitrogen is less

optimal in eNOS than in nNOS because of the single amino

acid difference (Asn368 in eNOS vs Asp597 in nNOS). This is

the structural basis for isoform selectivity of these inhibitors.

The (3⁰S,4⁰R) isomer of 4 behaves slightly differently when its

complex structures of nNOS and eNOS are compared. While

the pyrrolidine ring adopts an opposite orientation in the

(3⁰S,4⁰R)-4 nNOS structure, the electron density in the

(3⁰S,4⁰R)-4 eNOS structure supports an orientation that

makes H-bonds to both heme propionates. Finally, the

7814 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Ji et al.

determinants by which inhibitors can be simultaneously

accommodated by the substrate-catalytic site and the surface

hydrophobic pocket of nNOS have been revealed.

Our previous²⁶and current studies implicate a new avenue

for the rational design of enzyme inhibitors, that is, to look for

a potentialhotspotinthe target protein thatisoccludedby the

present active site residues, for instance, the side chains of the

residues. This finding is particularly useful for those inhibitor

design studies where the active sites are shallow and/or of

highly charged nature, and a new hot spot for inhibitor

potency is needed. The 2-amino-4-methylpyridine moiety is

one of the most important pharmacophores for this series of

inhibitors, as is evident by the fact that (()-3 (K_i= 14 nM for

nNOS) is much more potent than the corresponding (()-2-

amino-6-methyl derivative (K_i= 326 nM for nNOS).²³The

pocket of nNOS that accommodates this “new hot” is only

generated when the enzyme undergoes an induced fit upon

inhibitor binding.

Figure 7. Superimposition of the crystallographic binding confor-

mations of (3⁰R,4⁰S)-4 and (3⁰S,4⁰S)-4. The heme (orange), H₄B

(purple), and structural water (green) involved in the binding of

inhibitor to nNOS are shown. The active site residues are repre-

sented in an atom-type style (carbons in gray, nitrogens in blue,

oxygen in red, and sulfur in yellow). The carbons of (3⁰R,4⁰S)-4 are

colored green, and the carbons of (3⁰S,4⁰S)-4 are colored yellow. The

nitrogens and the fluorine are colored blue and green, respectively.

The distances of the H-bonds between the residues, structural water,

cofactors, and inhibitors from the crystal structure of nNOS com-

Experimental Section

Computer Modeling. Protein Structures. The crystallo-

graphic coordinates for NOS from the Research Collaboratory

for Structural Bioinformatics (RCSB) protein database are as

˚

follows: rat nNOS1: 1P6I (1.90 A resolution, R_cryst= 0.228),

˚

3B3N (1.98 A resolution, R_cryst= 0.230). The amino acid

sequences of NOS were retrieved from the PIR protein sequence

database. All of the computational work was performed on

Silicon Graphics Octane 2 workstations with an IRIX 6.5

operating system. The molecular modeling was achieved with

commercially available InsightII 2000³²and SYBYL 6.8³³soft-

ware packages. The NOS model was prepared by first adding

H-atoms using the Accelrys/InsightII 2000 software, and the

protonation states of the residues were set to pH 7.0. The cvff

force field of the Discover 98.0 program within Insight II was

used to optimize the orientation of hydrogen atoms of the

protein and of structural waters. The ligands and solvent

molecules of the protein structures were removed, but the heme

and H₄B were retained near the active site.

0

˚

plexed with (3 S,4 S)-4 are indicated in A. The corresponding

distances from the crystal structure of nNOS complexed with

(3⁰R,4⁰S)-4 are similar but not shown.

(3⁰R,4⁰R) isomer of 4 binds to eNOS also in a “flipped” mode

(Supporting Information Figure S1), exactly as observed in

nNOS. Three residues, Met336, Asp597, and Tyr706, were

found partially responsible for the selectivity for nNOS over

eNOS, the same as discussed previously for (3⁰R,4⁰R)-3.²⁶

However, further studies are required to better understand

2600-fold nNOS selectivity of this compound over eNOS.

GRID Calculations. The calculations were performed with

version 22 of the GRID software.³⁴Hydrogens were added with

the program GRIN. The GRID box dimensions were chosen to

Conclusions

In this study, we reported the design, synthesis, and struc-

ture-activity relationships of the racemic trans-substituted

amino pyrrolidinomethyl 2-aminopyridine derivatives 8-34,

which led to the discovery of a new series of inhibitors with

high nNOS inhibitory potency and high nNOS selectivities

over the other two NOS isozymes. The active site require-

ments for the trans-2-aminopyridine nNOS inhibitors were

explored through rational inhibitor design and structure-

activity relationship analyses. Low nanomolar trans amine

nNOS inhibitors [(()-32 and (()-34] were discovered, having

K_ivalues for nNOS of 88 and 48 nM, respectively. The nNOS

selectivities of these two inhibitors over eNOS are more than

1000-fold. Four enantiomerically pure isomers of 4 also were

synthesized, and structures of complexes with nNOS and

eNOS were determined in this study. Itwas found that, similar

to (3⁰R,4⁰R)-3, (3⁰R,4⁰R)-4 can also induce enzyme elasticity to

generate a “hot spot” that accommodates the 2-amino-4-

methylpyridine moiety. The binding mode of this isomer

adopts a 180° “flipped” orientation compared with the

expected “normal” binding mode. This compound and (3⁰R,4⁰R)-

3 represent the two most potent and selective nNOS inhibitors

reported so far. The influence of the pyrrolidine configuration to

enzyme-inhibitor interactions is also observed when the struc-

tures of nNOS in complex with the cis (3⁰S,4⁰S)- and trans

(3⁰R,4⁰S)-isomers of 3 or 4 are compared. The critical structural

encompass all of the active site residues. This resulted in a box

˚

3

˚

size of 31 ꢀ 28 ꢀ 31 A . The grid spacings were set to 1 A

˚

(directive NPLA=1) and 0.5 A (directive NPLA=2), respec-

tively. The amino acids in the active site were considered rigid

(directive move=0). The directives NETA and ALMD were set

to 120 and 1, respectively, to include the atoms of heme and H₄B

in calculations and to interpret which atom(s) in the active site

contribute(s) to the interaction with a specific probe atom. The

following single atom probes were used in the calculation: DRY,

C3, C1=, NM3, N1þ, N2þ, Cl, and F.

Chemical Synthesis. General Methods, Reagents, and Materi-

als. All reagents were purchased from Aldrich, Acros Organics,

or Fisher Scientific and were used without further purification

unless stated otherwise. NADPH, calmodulin, and human

ferrous hemoglobin were obtained from Sigma Chemical Co.

Tetrahydrobiopterin (H₄B) was purchased from Alexis Bio-

1

chemicals. H NMR and ¹³C NMR spectra were recorded on

a Varian Mercury 400 MHz or a Varian Inova 500 MHz spec-

trometer (100.6 or 125.7 MHz for ¹³C NMR spectra, respec-

tively) in CDCl₃, DMSO-d₆, CD₃OD, or D₂O. Chemical shifts

are reported as values in parts per million (ppm) and the ref-

erence resonance peaks set at 0 ppm [TMS(CDCl₃)], 2.50 ppm

[(CD₂H)₂SO], 3.31 ppm (CD₂HOD), and 4.80 ppm (HOD),

1

respectively, for H NMR spectra, 77.23 ppm (CDCl₃), 39.52

ppm (DMSO-d₆), and 49.00 ppm (CD₃OD) for ¹³C NMR

spectra, and 0 ppm (CF₃CO₂H) for ¹⁹F NMR spectra. An Orion

research model 701H pH meter with a general combination

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7815

electrode was used for pH measurements. Mass spectra were

performed on a Micromass Quattro II triple quadruple HPLC/

MS mass spectrometer with an electrospray ionization (ESI)

source or atmospheric pressure chemical ionization (APCI)

source. High-resolution mass spectra were carried out using a

VG70-250SE mass spectrometer. Chemical ionization (CI) or

electron impact (EI) was used as the ion source. Elemental

analyses were performed by Atlantic Microlab Inc., Norcross,

GA. Thin-layer chromatography was carried out on E. Merck

precoated Silica Gel 60 F254 plates with visualization accom-

plished with phosphomolybdic acid or ninhydrin spray reagent

or with a UV-visible lamp. Column chromatography was per-

formed with E. Merck Silica Gel 60 (230-400 mesh) or Sorbent

Technologies 250 mesh silica gel. Tetrahydrofuran (THF) and

ethyl ether were redistilled under nitrogen using sodium and

benzophenone as the indicator. Dichloromethane was redis-

tilled from CaH₂under nitrogen. Other dry solvents were

directly purchased from the companies.

(0.381 g, 0.285 mL, 0.003 mol) dropwise. The mixture was

stirred at -78 °C for 10 min. After this time, a solution of 39

(0.873 g, 0.002 mol) in 10 mL of anhydrous CH₂Cl₂was added

dropwise at a rate to keep the reaction temperature below

-60 °C. Upon complete addition, the mixture was allowed to

stir at -78 °C for 2 h. Anhydrous triethylamine (0.607 g, 0.836

mL, 0.006 mol) was added dropwise to the mixture, then the

reaction mixture was allowed to warm to room temperature.

The resulting solution was partitioned between 1 M NaOH (40

mL) and CH₂Cl₂, and the product was extracted with CH₂Cl₂

(30 mL ꢀ 2). All organic layers were combined, washed with

brine, dried over Na₂SO₄, and concentrated in vacuo to yield the

crude product, which was purified using silica gel column

chromatography (CH₂Cl₂:EtOAc = 3:2) to afford a pale-yellow

oil (0.738 g, 85%). ¹H NMR (CDCl₃, 400 MHz): δ 7.763 (d, 1H,

J = 8.8 Hz), 7.560 (t, 1H, J = 8 Hz), 7.279 (brs, 1H), 6.790 (d,

1H, J = 7.2 Hz), 4.997 (brs, 1H), 3.399-3.133 (m, 5H), 2.995-

2.924 (m, 1H), 2.787-2.584 (m, 4H), 2.438-2.392 (m, 1H),

1.522 (s, 9H), 1.442 (s, 9H). ¹³C NMR (CDCl₃, 100.6 MHz): δ

214.670 (1C), 157.347 (1C), 156.009 (1C), 152.388 (1C), 151.514

(1C), 138.739 (1C), 117.930 (1C), 109.941 (1C), 81.133 (1C),

79.450 (1C), 61.643 (1C), 57.185 (1C), 55.665 (1C), 48.550 (1C),

38.432 (1C), 35.921 (1C), 28.632 (3C), 28.496 (3C).

N-Boc-2-bromoethylamine, N¹,N¹-dimethylethane-1,2-dia-

mine, (S)-(-)-1-Boc-3-aminopyrrolidine, (R)-(þ)-1-Boc-3-ami-

nopyrrolidine, and (S)-(þ)-1-benzyl-3-aminopyrrolidine and

(R)-(-)-1-benzyl-3-aminopyrrolidine were purchased from

Aldrich. All compounds were characterized by TLC, ¹H- and ¹³

C

NMR, and MS. All final products were also characterized by

HRMS. The TLC, HPLC, NMR, and analytical data confirmed

that the purity of all of the products was g95%. The purity of the

most potent compounds was further confirmed by elemental

analyses.

(()-tert-Butyl {6-{[cis-1⁰-(2⁰⁰-tert-Butoxycarbonylaminoethyl)-

4⁰-(3⁰⁰-phenylpropylamino)-pyrrolidin-3⁰-yl]methyl}pyridin-2-yl}-car-

bamate (41a) and (()-tert-Butyl {6-{[trans-1⁰-(2⁰⁰-tert-Butoxy-

carbonylaminoethyl)-4⁰-(3⁰⁰-phenylpropylamino)-pyrrolidin-3⁰-yl]-

methyl}pyridin-2-yl}-carbamate (42a). To a solution of 40 (0.434 g,

0.001 mol), a substituted amine such as 3-phenyl-1-propylamine

(0.203 g, 0.0015 mol), acetic acid (0.090 g, 0.086 mL, 0.0015 mol),

(()-tert-Butyl {6-[trans-(4⁰-Hydroxypyrrolidin-3⁰-yl)methyl]-

pyridin-2-yl}carbamate (38). A suspension of 37 (0.766 g, 0.002

mol), which was prepared in the previous study,²²and 10% Pd/C

(0.7 g) in MeOH (30 mL) was stirred at 45 °C under one hydrogen

atmosphere overnight. The catalyst was removed by filtration and

washed with MeOH (30 mL). The filtrate was concentrated by

high-vacuum rotary evaporation to give the desired product in a

quantitative yield (1.482 g). ¹H NMR (CDCl₃, 500 MHz): δ 9.206

(brs, 1H), 8.519 (brs, 1H), 7.798 (d, 1H, J = 8 Hz), 7.577 (t, 1H,

J = 7.5 Hz), 6.8235 (d, 1H, J = 7.5 Hz), 4.242 (m, 1H), 3.542 (m,

1H), 3.399-3.302 (m, 2H), 3.102-3.090 (m, 1H), 2.868-2.832 (m,

1H), 2.713-2.641 (m, 2H), 1.985 (s, 6H), 1.495 (s, 9H). ¹³C NMR

(CDCl₃, 125.7 MHz): δ 177.392 (2C), 156.724 (1C), 152.699 (1C),

152.049 (1C), 139.141 (1C), 118.030 (1C), 110.605 (1C), 80.946

(1C), 74.146 (1C), 51.226 (1C), 48.148 (1C), 45.937 (1C), 38.160

(1C), 28.263 (3C), 22.410 (2C). MS (ESI, CH₃OH): [C₁₅H₂₃N₃O₃]

m/z 294.2 ([M þ H]^þ).

˚

and 3 A molecular sieves (1 g) in dry MeOH (20 mL) was added

NaBH₃CN (0.126 g, 0.002 mol). The reaction was stirred at room

temperature under a N₂atmosphere for 36 h. The reaction mixture

was filtered, and the filtrate was concentrated in vacuo. The residue

was diluted with 1 M NaOH (30 mL) and extracted with CH₂Cl₂

(30 mL ꢀ 2). The organic layers were combined, washed with

brine, dried over MgSO₄, and concentrated in vacuo to give the

crude product, which was purified by silica gel column chroma-

tography (hexanes:CH₂Cl₂:EtOAc:Et₃N = 5:3:2:0.5) to afford a

pale-yellow oil (0.360 g, 65%). The cis isomer (41a) and the trans

isomer (41b) can be separated with the above eluent. The ratio of

cis and trans isomers was 45:55.

41a: (R_f= 0.25, pale-yellow oil, 0.162 g). ¹H NMR (CDCl₃,

500 MHz): δ 7.730 (d, 1H, J = 8 Hz), 7.542 (t, 1H, J = 8 Hz),

7.289-7.165 (m, 6H), 6.8025 (d, 1H, J = 7.5 Hz), 5.074 (brs,

1H), 3.235-3.171 (m, 3H), 2.891-2.861 (m, 1H), 2.645-2.382

(()-tert-Butyl {6-[trans-1⁰-(2⁰⁰-tert-Butoxycarbonylamino-ethyl)-

4⁰-hydroxy-pyrrolidin-3⁰-ylmethyl]-pyridin-2-yl}carbamate (39). A

mixture of 38 (0.741 g, 0.001 mol), N-Boc-2-bromoethylamine

(0.268 g, 0.0012 mol), and anhydrous K₂CO₃(0.552 g, 0.004 mol)

in 10 mL anhydrous DMF was stirred at room temperature

overnight. Solids were removed by filtration. The filtrate was

evaporated under reduced pressure. The residue was partitioned

between EtOAc and water. The organic layer was washed with

brine, dried over anhydrous Na₂SO₄, and evaporated in vacuo. The

obtained residue was purified by column chromatography (silica

gel, CH₂Cl₂:MeOH = 9:1) to afford a colorless oil (0.248 g, 57%).

¹H NMR (CDCl₃, 400 MHz): δ 7.758 (d, 1H, J = 8 Hz), 7.583 (t,

1H, J = 8 Hz), 7.357 (brs, 1H), 6.8295 (d, 1H, J = 7.6 Hz), 5.144

(brs, 1H), 4.099 (m, 1H), 3.300-3.100 (m, 2H), 3.080-2.980 (m,

1H), 2.900-2.782 (m, 3H), 2.721-2.706 (m, 1H), 2.601-2.541

(m,3H), 2.189-2.146 (m, 1H), 1.510 (s, 9H), 1.442 (s, 9H). ¹³C

NMR (CDCl₃, 100.6 MHz): δ 158.514 (1C), 156.303 (1C), 152.251

(1C), 151.303 (1C), 138.891 (1C), 118.183 (1C), 110.024 (1C),

81.279 (2C), 76.800 (1C), 61.769 (1C), 59.176 (1C), 55.468 (1C),

51.033 (1C), 40.591 (1C), 39.052 (1C), 28.740 (3C), 28.558 (3C).

MS (ESI, CH₃CN): [C₂₂H₃₆N₄O₅] m/z 437.4 ([M þ H]^þ).

(()-tert-Butyl {6-{[1⁰-(2⁰⁰-tert-Butoxycarbonylamino-ethyl)-

4⁰-oxo-pyrrolidin-3⁰-yl]methyl}-pyridin-2-yl}carbamate (40). To

a solution of dimethyl sulfoxide (DMSO, 0.313 g, 0.284 mL,

0.004 mol) in 30 mL of dry CH₂Cl₂was added oxalyl chloride

(m, 12H), 1.820-1.781 (m, 2H), 1.512 (s, 9H), 1.446 (s, 9H). ¹³

C

NMR (CDCl₃, 125.7 MHz): δ 159.881 (1C), 156.260 (1C), 152.534

(1C), 151.440 (1C), 142.294 (1C), 138.619 (1C), 128.577 (2C),

128.542 (2C), 125.985 (1C), 118.151 (1C), 109.536 (1C), 81.071

(1C), 79.269 (1C), 60.096 (1C), 59.327 (1C), 58.007 (1C), 55.427

(1C), 48.379 (1C), 41.586 (1C), 39.152 (1C), 36.518 (1C), 33.903

(1C), 32.104 (1C), 28.653 (3C), 28.483 (3C). MS (ESI, CH₃OH):

[C₃₁H₄₇N₅O₄] m/z 554.4 ([M þ H]^þ).

1

42a: (R_f= 0.2, pale-yellow oil, 0.198 g). H NMR (CDCl₃,

500 MHz): δ 7.740 (d, 1H, J = 8 Hz), 7.546 (t, 1H, J = 8 Hz),

7.284-7.138 (m, 6H), 6.7905 (d, 1H, J = 7.5 Hz), 5.040 (brs,

1H), 3.192 (m, 2H), 2.940-2.928 (m, 1H), 2.899-2.756 (m, 2H),

2.724-2.681 (m, 2H), 2.648-2.449 (m, 7H), 2.332-2.322 (m,

1H), 2.216-2.187 (m, 1H), 1.734-1.705 (m, 2H), 1.509 (s, 9H),

1.446 (s, 9H). ¹³C NMR (CDCl₃, 125.7 MHz): δ 159.142 (1C),

156.236 (1C), 152.519 (1C), 151.517 (1C), 142.213 (1C), 138.670

(1C), 128.569 (2C), 128.526 (2C), 125.977 (1C), 118.132 (1C),

109.710 (1C), 81.099 (1C), 79.292 (1C), 63.0450 (1C), 60.905

(1C), 59.129 (1C), 55.241 (1C), 47.934 (1C), 45.373 (1C), 42.410

(1C), 38.978 (1C), 33.791 (1C), 31.907 (1C), 28.657 (3C), 28.468

(3C). MS (ESI, CH₃OH): [C₃₁H₄₇N₅O₄] m/z 554.4 ([M þ H]^þ).

(()-6-{{trans-1⁰-(2⁰⁰-Aminoethyl)-4⁰-[(3⁰⁰-phenylpropyl)amino]-

pyrrolidin-3⁰-yl}methyl}pyridin-2-amine Tetrahydrochloride (8). A

solution of 4 M HCl in 1,4-dioxane (4 mL) was added to 42a

7816 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Ji et al.

(0.111 g, 0.2 mmol) at 0 °C under argon. The ice-water bath was

\removed after 3 h, and the reaction mixture was stirred at room

temperature 48 h. After the completion of the reaction, liquids

were evaporated under reduced pressure and the residue was

partitioned between water (10 mL) and ethyl acetate (10 mL). The

aqueous layer was washed with ethyl acetate (5 mL ꢀ 2). After

evaporation of water by high-vacuum rotary evaporation, the

residue was dried with a lyophilizer to afford a hygroscopic white

solid (0.100 g, quantitative yield). ¹H NMR (D₂O, 500 MHz): δ

7.795 (t, 1H, J = 8.5 Hz), 7.385-7.258 (m, 5H), 6.883 (d, 1H, J =

9 Hz), 6.764 (d, 1H, J = 7 Hz), 3.902-3.894 (m,2H), 3.702-3.648

(m, 2H), 3.482-3.457 (m, 2H), 3.389-3.344 (m, 2H), 3.189-

3.126 (m, 3H), 3.000-2.888 (m, 3H), 2.751-2.658 (m, 2H),

2.048-1.972 (m, 2H). ¹³C NMR (D₂O, 125.7 MHz): δ 154.686

(1C), 144.740 (2C), 140.369 (1C), 128.976 (2C), 128.577 (2C),

126.717 (1C), 112.801 (1C), 112.511 (1C), 59.558 (1C), 57.469

(1C), 55.369 (1C), 51.384 (1C), 46.363 (1C), 39.875 (1C), 35.430

(1C), 34.343 (1C), 27.399 (1C), 27.016 (1C). MS (ESI, CH₃CN-

H₂O): [C₂₁H₃₁N₅] m/z 354.3 ([M þ H]^þ). HRMS (CIþ, CH₃OH)

diastereomer ratio: cis:trans=45:55). ¹H NMR (CDCl₃, 500 MHz):

δ 7.611-7.592 (m, 1H), 7.249-7.233 (m, 4H), 7.105 (s, 1H), 6.603

(s, 1H), 4.413 (s, 2H), 3.690-3.656 (m, 0.5H), 3.613-3.608 (m,

0.5H), 3.555-3.518 (m, 0.5H), 3.465-3.429 (m, 0.5H), 3.394-

3.216 (m, 2H), 3.128-3.091 (m, 1H), 3.052-3.031 (m, 1H),

2.956-2.943 (m, 1H), 2.821-2.761 (m, 3H), 2.545-2.500 (m,

1H), 2.410-2.243 (m, 4H), 1.514-1.449 (m, 27H). ¹³C NMR

(CDCl₃, 125.7 MHz): δ 157.876 (1C), 155.873 (1C), (154.755 þ

154.664) (1C), 152.539 (1C), 151.513 (1C), 150.008 (1C), 140.828

(1C), 134.519 (1C), 129.936 (1C), 127.489 (2C), (125.825 þ

125.242) (1C), (119.311 þ 119.238) (1C), (110.368 þ 110.295)

(1C), 80.867 (1C), 80.320 (1C), 79.325 (1C), (62.179 þ 61.347)

(1C), (51.925 þ 51.372) (1C), 50.826 (1C), (49.927 þ 49.733) (1C),

(47.456 þ 47.213) (1C), 46.339 (1C), (44.165 þ 43.631) (1C),

(39.727þ39.636) (1C), 28.641 (3C), 28.538 (3C), 28.392 (3C), 21.410

(1C). MS (ESI, CH₃OH): [C₃₅H₅₂ClN₅O₆] m/z 674.3 ([M þ H]^þ).

(()-trans-tert-Butyl 3-{{2⁰-[(tert-Butoxycarbonyl)(phenethyl)-

amino]ethyl}amino}-4-{{6⁰-[(tert-butoxycarbonyl)amino]-4⁰-methyl-

pyridin-2⁰-yl}methyl}pyrrolidine-1-carboxylate (50l). The procedure

to prepare 50l is the same as that to prepare 42a except 49 (0.203 g,

0.5 mmol)²³and tert-butyl (2-aminoethyl)(phenethyl)carbamate

(0.145 g, 0.55 mmol)²³instead of 40 (0.434 g, 0.001 mol) and

3-phenyl-1-propylamine (0.203 g, 0.0015 mol) were used. The

desired product was purified by column chromatography (silica

gel, hexanes:EtOAc:Et₃N = 9:1:0.5, the isomer with lower R_fvalue,

R_f= 0.1) to afford a pale-yellow oil (0.101 g, 56%, diastereomer

ratio: cis:trans = 45:55). ¹H NMR (CDCl₃, 500 MHz): δ 7.602-

7.586 (m, 1H), 7.297-7.170 (s, 6H), 6.597 (s, 1H), 3.699-3.665 (m,

0.5H), 3.626-3.604 (m, 0.5H), 3.556-3.520 (m, 0.5H), 3.467-

3.392 (m, 2.5H), 3.227-2.967 (m, 5H), 2.807 (m, 3H), 2.681 (m,

2H), 2.544-2.500 (m, 1H), 2.407-2.272 (m, 4H), 1.511-1.444 (m,

27H). ¹³C NMR (CDCl₃, 125.7 MHz): δ 157.920 (1C), 155.783

(1C), (154.751 þ 154.666) (1C), 152.541 (1C), 151.527 (1C),

149.991 (1C), 139.281 (1C), 128.990 (2C), 128.619 (2C), 126.427

(1C), (119.305 þ 119.227) (1C), (110.368 þ 110.289) (1C), 80.891

(1C), 79.677 (1C), 79.677 (1C), (62.239 þ 61.371) (1C), (51.960 þ

51.384) (1C), (50.060 þ 49.933 þ 49.750) (2C), 47.674 (1C), 46.478

(1C), (44.171 þ 43.655) (1C), 39.660 (1C), 35.361 (1C), 28.634 (3C),

28.561 (3C), 28.385 (3C), 21.396 (1C). MS (ESI, CH₃OH):

[C₃₆H₅₅N₅O₆] m/z 654.4 ([M þ H]^þ).

calcd, 354.2652; found, 354.2648. Anal. (C₂₁H₃₅Cl₄N₅1.75H₂O)

3

Calcd: C, 47.51; H, 7.31; N, 13.19. Found: C, 47.75; H, 7.42; N,

12.87.

(()-trans-4-[(6⁰-Aminopyridin-2⁰-yl)methyl]pyrrolidin-3-ol Di-

hydrochloride (12). The procedure to prepare 12 is the same as

that to prepare 8 except using 44 (0.079 g, 0.2 mmol) instead of

42a, affording a hygroscopic white solid (0.053 g, quantitative

yield). ¹H NMR (D₂O, 500 MHz): δ 7.840 (t, 1H, J = 8.5 Hz),

6.894 (d, 1H, J = 9 Hz), 6.7955 (d, 1H, J = 7.5 Hz), 4.375-4.350

(m, 1H), 3.674-3.634 (m, 1H), 3.600-3.563 (m, 1H), 3.302-

3.270 (m, 1H), 3.166-3.128 (m, 1H), 2.982-2.936 (m, 1H),

2.884-2.837 (m, 1H), 2.725-2.672 (m, 1H). ¹³C NMR (D₂O,

125.7 MHz): δ 154.675 (1C), 146.377 (1C), 144.802 (1C), 112.612

(1C), 111.846 (1C), 73.009 (1C), 50.873 (1C), 47.991 (1C), 44.359

(1C), 33.430 (1C). MS (ESI, CH₃OH): [C₁₀H₁₅N₃O] m/z 194.3

([M þ H]^þ). HRMS (CIþ, CH₃OH) calcd, 194.1288; found,

194.1285. Anal. (C₁₀H₁₇Cl₂N₃O 0.1H₂O) Calcd: C, 44.82; H,

3

6.47; N, 15.68. Found: C, 44.78; H, 6.45; N, 15.42.

(()-trans-tert-Butyl 3-{{6⁰-[(tert-Butoxycarbonyl)amino]-4⁰-

methylpyridin-2⁰-yl}methyl}-4-{{2⁰-[(tert-butoxycarbonyl)amino]-

ethyl}amino}pyrrolidine-1-carboxylate (50a). The procedure to

prepare 50a is the same as that to prepare 42a except 49 (0.203 g, 0.5

mmol)²³and N-Boc-1,2-diaminoethane (0.088 g, 0.55 mmol)

instead of 40 (0.434 g, 0.001 mol) and 3-phenyl-1-propylamine

(0.203 g, 0.0015 mol) were used. The desired product was purified

by column chromatography (silica gel, hexanes:EtOAc:Et₃N =

6:4:0.5, the isomer with lower R_fvalue, R_f= 0.1) to afford a pale-

yellow oil (0.083 g, 55%, diastereomer ratio: cis:trans = 45:55). ¹H

NMR (CDCl₃, 500 MHz): δ 7.624-7.609 (m, 1H), 7.554 (m, 1H),

6.611 (s, 1H), 5.215-5.172 (m, 1H), 3.723-3.702 (m, 0.5H),

3.689-3.628 (m, 0.5H), 3.561-3.526 (m, 0.5H), 3.477-3.444 (m,

0.5H), 3.181-2.983 (m, 4H), 2.936-2.906 (m, 1H), 2.892-2.801

(m, 1H), 2.707-2.699 (m, 2H), 2.584-2.562 (m, 1H), 2.410-2.290

(m, 4H), 1.519 (s, 9H), 1.444 (s, 18H). ¹³C NMR (CDCl₃, 125.7

MHz): δ (157.759 þ 157.685) (1C), 156.231 (1C), 154.598 (1C),

152.594 (1C), 151.650 (1C), 149.983 (1C), 119.237 (1C), 110.436

(1C), 80.782 (1C), 79.308 (2C), (61.786 þ 61.082) (1C), (51.940 þ

51.417) (1C), (49.820 þ 49.572) (1C), 47.363 (1C), (44.051 þ

43.537) (1C), 40.569 (1C), (39.552 þ 39.413) (1C), 28.568 (3C),

28.487 (3C), 28.344 (3C), 21.353 (1C). MS (ESI, CH₃OH):

[C₂₈H₄₇N₅O₆] m/z 550.4 ([M þ H]^þ).

(()-trans-tert-Butyl 3-{{2⁰-[(tert-Butoxycarbonyl)(3⁰⁰-fluoro-

phenethyl)amino]ethyl}amino}-4-{{6⁰-[(tert-butoxycarbonyl)-

amino]-4⁰-methylpyridin-2⁰-yl}methyl}pyrrolidine-1-carboxy-

late (50n). The procedure to prepare 50n is the same as that to

prepare 42a except 49 (0.203 g, 0.5 mmol)²³and tert-butyl

(2-aminoethyl)(3-fluorophenethyl)carbamate (0.l55 g, 0.55

mmol)²³instead of 40 (0.434 g, 0.001 mol) and 3-phenyl-1-

propylamine (0.203 g, 0.0015 mol) were used. The desired

product was purified by column chromatography (silica gel,

hexanes:EtOAc:Et₃N = 9.5:0.5:0.5, the isomer with lower R_f

value, R_f= 0.1) to afford a pale-yellow oil (0.120 g, 65%,

diastereomer ratio: cis:trans = 45:55). ¹H NMR (CDCl₃, 500

MHz): δ 7.611-7.595 (m, 1H), 7.355 (brs, 1H), 7.262-7.220

(m, 1H), 6.917-6.885 (m, 3H), 6.604 (s, 1H), 3.704-3.670 (m,

0.5H), 3.636-3.614 (m, 0.5H), 3.559-3.523 (m, 0.5H),

3.476-3.397 (m, 2.5H), 3.233-2.980 (m, 5H), 2.813 (m,

3H), 2.695 (m, 2H), 2.552-2.508 (m, 1H), 2.406-2.367 (m,

0.5H), 2.296-2.276 (m, 3.5H), 1.512-1.446 (m, 27H). ¹³C

NMR (CDCl₃, 125.7 MHz): δ (163.889 þ 161.934) (1C),

157.811 (1C), 155.564 (1C), (154.672 þ 154.581) (1C),

152.486 (1C), 151.484 (1C), 149.924 (1C), 141.800 (1C), 129.955

(1C), (124.630 þ 124.606) (1C), (119.227 þ 119.148) (1C),

(115.851 þ 115.681) (1C), (113.343 þ 113.167) (1C), (110.326 þ

110.247) (1C), 80.745 (1C), 79.713 (1C), 79.234 (1C), (62.179 þ

61.323) (1C), (51.869 þ 51.293) (1C), (49.878 þ 49.678) (2C),

(48.293 þ 47.589) (1C), 46.387 (1C), (44.098 þ 43.582) (1C),

(39.678 þ 39.587) (1C), (35.039 þ 34.377) (1C), 28.555 (3C),

28.458 (3C), 28.306 (3C), 21.324 (1C). ¹⁹F NMR (CDCl3, 376.5

MH_Z): δ -113.900. MS (ESI, CH₃OH): [C₃₆H₅₄FN₅O₆] m/z

672.5 ([M þ H]^þ).

(()-trans-tert-Butyl 3-{{2⁰-[(tert-Butoxycarbonyl)(3⁰⁰-chlorobenzyl)-

amino]ethyl}amino}-4-{{6⁰-[(tert-butoxycarbonyl)amino]-4⁰-methyl-

pyridin-2⁰-yl}methyl}pyrrolidine-1-carboxylate (50c). The proce-

dure to prepare 50c is the same as that to prepare 42a except 49

(0.203 g, 0.5 mmol)²³and tert-butyl (2-aminoethyl)(3-chloroben-

zyl)carbamate (0.156 g, 0.55 mmol)²³instead of 40 (0.434 g, 0.001

mol) and 3-phenyl-1-propylamine (0.203 g, 0.0015 mol) were used.

The desired product was purified by column chromatography

(silica gel, hexanes:EtOAc:Et₃N=9.5:0.5:0.5, the isomer with lower

R_fvalue, R_f= 0.1) to afford a pale-yellow oil (0.117 g, 63%,

Article

N¹-{(()-trans-4-[(6-Amino-4-methylpyridin-2-yl)methyl]pyrro-

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7817

Cl₄FN₅1.25H₂O) Calcd: C, 46.72; H, 6.81; N, 12.97. Found: C,

3

lidin-3-yl}ethane-1,2-diamine Tetrahydrochloride (21). The proce-

dure to prepare 21 is the same as that to prepare 8 except 50a

(0.110 g, 0.2 mmol) instead of 42a was used, affording a hygro-

scopic white solid (0.079 g, quantitative yield). ¹H NMR (D₂O,

500 MHz): δ 6.748 (s, 1H), 6.714 (s, 1H), 4.098-4.022 (m, 2H),

3.765-3.682 (m, 2H), 3.573-3.421 (m, 4H), 3.376-3.288 (m,

2H), 3.150-3.109 (m, 1H), 2.938-2.888 (m, 1H), 2.3549 (s,

3H). ¹³C NMR (D₂O, 125.7 MHz): δ 158.396 (1C), 154.306

(1C), 143.625 (1C), 114.988 (1C), 111.328 (1C), 60.713 (1C),

48.596 (1C), 46.507 (1C), 43.965 (1C), 40.525 (1C), 35.728

(1C), 33.453 (1C), 21.340 (1C). MS (ESI, CH₃OH-H₂O):

[C₁₃H₂₃N₅] m/z 250.2 ([M þ H]^þ). HRMS (CIþ, CH₃OH) calcd,

46.99; H, 6.85; N, 12.67.

(()-cis-tert-Butyl 3-Acetoxy-4-{{6⁰-[benzyl(tert-butoxycarbonyl)-

amino]-4⁰-methylpyridin-2⁰-yl}methyl}pyrrolidine-1-carboxylate (52).

To an ice-cooled solution of triphenylphospine (Ph₃P, 0.341 g,

0.0013 mol) in dry THF (5 mL) was added 51 (0.497 g, 0.001 mol),

which was prepared in a previous study,²³in dry THF (15 mL)

through a cannula under N₂atmosphere. Diisopropyl azodicarbox-

ylate (DIAD, 0.263 g, 0.259 mL, 0.0013 mol) was then added

dropwise, and the solution was stirred 20 min at 0 °C. Acetic acid

(HOAc, 0.078 g, 0.074 mL, 0.0013 mol) was added at 0 °C, and the

solution was stirred at room temperature for 48 h. The solution was

concentrated, and the residue was purified directly by column

chromatography (silica gel, hexanes:EtOAc = 8:2) to yield colorless

oil (0.469 g, 87%). ¹H NMR (CDCl₃, 500 MHz): δ 7.446-7.426 (m,

1H), 7.293-7.179 (m, 5H), 6.638-6.628 (m, 1H), 5.163-5.091 (m,

3H), 3.506-3.361 (m, 3H), 3.127-3.086 (m, 1H), 2.876-2.835 (m,

1H), 2.700-2.614 (m, 2H), 2.334-2.239 (m, 3H), 2.051-2.039 (m,

3H), 1.448-1.445 (m, 9H), 1.413 (m, 9H). ¹³C NMR (CDCl₃, 125.7

MHz): δ (170.591 þ 170.367) (1C), 156.657 (1C), (154.429 þ

154.338) (1C), (154.271 þ 154.229) (1C), 153.949 (1C), (148.679 þ

148.625) (1C), (139.797 þ 139.760) (1C), 128.048 (2C), (126.925 þ

126.871) (2C), (126.543 þ 126.440) (1C), (119.694 þ 119.633) (1C),

117.229 (1C), (81.201 þ 81.158) (1C), (79.440 þ 79.422) (1C),

(74.759 þ 74.716 þ 73.866 þ 73.830) (1C), (52.792 þ 52.343)

(1C), 49.951 (1C), (49.289 þ 48.846) (1C), (41.718 þ 41.129)

(1C), (34.730 þ 34.669) (1C), (28.458 þ 28.433) (3C), 28.124 (3C),

(21.099 þ 20.965) (1C). MS (ESI, CH₃OH): [C₃₀H₄₁N₃O₆] m/z

540.4 ([M þ H]^þ); m/z 562.4 ([M þ Na]^þ).

250.2026; found, 250.2026. Anal. (C₁₃H₂₇Cl₄N₅1.25 H₂O)

3

Calcd: C, 37.38; H, 7.12; N, 16.77. Found: C, 37.48; H, 7.05; N,

16.56.

N¹-{(()-trans-4-[(6⁰-Amino-4⁰-methylpyridin-2⁰-yl)methyl]-

pyrrolidin-3-yl}-N²-(3-chlorobenzyl)ethane-1,2-diamine Tetrahydro-

chloride (23). The procedure to prepare 23 is the same as that to

prepare 8 except 50c (0.135 g, 0.2 mmol) instead of 42a was used,

affording a hygroscopic white solid (0.103 g, quantitative yield). ¹H

NMR (D₂O, 500 MHz): δ 7.523-7.418 (m, 4H), 6.699 (s, 1H),

6.680 (s, 1H), 4.316 (s, 2H), 4.038-4.002 (m, 2H), 3.731-3.654 (m,

2H), 3.538 (m, 4H), 3.342-3.243 (m, 2H), 3.097-3.090 (m, 1H),

2.902-2.853 (m, 1H), 2.316 (s, 3H). ¹³C NMR (D₂O, 125.7 MHz):

δ 158.321 (1C), 154.211 (1C), 143.562 (1C), 134.461 (1C), 132.014

(1C), 130.964 (1C), 130.114 (1C), 129.883 (1C), 128.396 (1C),

114.960 (1C), 111.268 (1C), 60.742 (1C), 51.119 (1C), 48.545

(1C), 46.493 (1C), 42.881 (2C), 40.519 (1C), 33.403 (1C), 21.315

(1C). MS (ESI, CH₃OH): [C₂₀H₂₈ClN₅] m/z 374.4 ([M þ H]^þ).

HRMS (CIþ, CH₃OH) calcd, 374.2106; found, 374.2103. Anal.

(()-cis-tert-Butyl 3-{{6⁰-[Benzyl(tert-butoxycarbonyl)amino]-

4⁰-methylpyridin-2⁰-yl}methyl}-4-hydroxypyrrolidine-1-carboxy-

late (53). To a solution of 52 (1.079 g, 0.002 mol) in CH₃OH (40

mL) was added at room temperature a solution of Na₂CO₃

(0.424 g, 0.004 mol) in H₂O (10 mL). The solution was stirred

at room temperature for 14 h. The solvent was evaporated in

vacuo. The residue was dissolved in a mixture of EtOAc (10 mL)

and H₂O (10 mL). The aqueous layer was extracted with EtOAc

(10 mL ꢀ 3). The combined organic layers were dried over

Na₂SO₄and concentrated in vacuo. The residue was purified by

column chromatography (silica gel, hexanes:EtOAc = 6:4) to

afford a colorless oil (0.995 g, quantitative yield). ¹H NMR

(CDCl₃, 500 MHz): δ 7.369-7.348 (m, 1H), 7.272-7.185 (m,

5H), 6.748-6.730 (m, 1H), 5.087 (m, 2H), (4.078 þ 3.863) (brs,

1H), 3.960-3.938 (m, 1H), 3.616-3.340 (m, 3H), 3.170-3.116

(m, 1H), 2.881-2.834 (m, 1H), 2.789-2.712 (m, 1H), 2.318 (m,

4H), 1.443 (s, 9H), 1.413 (s, 9H). ¹³C NMR (CDCl₃, 125.7

MHz): δ 157.592 (1C), (154.417 þ 154.362) (1C), 154.241 (1C,

15), 153.889 (1C), (149.457 þ 149.341) (1C), (139.020 þ 138.983)

(1C), 128.194 (2C), 126.731 (1C), 126.695 (2C), (120.404 þ

120.319) (1C), (118.334 þ 118.200) (1C), 81.340 (1C), 78.991

(1C), (70.988 þ 70.193) (1C), (53.830 þ 53.557) (1C), (50.309 þ

50.266) (1C), (49.253 þ 48.870) (1C), (44.735 þ 44.177) (1C),

(35.088 þ 35.015) (1C), 28.470 (3C), 28.093 (3C), 21.075 (1C).

MS (ESI, CH₃OH): [C₂₈H₃₉N₃O₅] m/z 520.3 ([M þ Na]^þ).

(()-trans-tert-Butyl 3-Azido-4-{{6⁰-[benzyl(tert-butoxycarbonyl)-

amino]-4⁰-methylpyridin-2⁰-yl}methyl}pyrrolidine-1-carboxylate (54).

To Ph₃P (0.328 g, 0.00125 mol) in a dry THF solution (5 mL) was

added 53 (0.497 g, 0.001 mol) in dry THF (10 mL) at 0 °C under a N₂

atmosphere via cannula. DIAD (0.262 g, 0.260 mL, 0.0013 mol) was

added dropwise, and the solution was stirred at 0 °C for 20 min.

Diphenylphosphoryl azide (DPPA, 0.358 g, 0.281 mL, 0.0013 mol)

was added dropwise at 0 °C, and the reaction mixture was stirred for

22 h at room temperature. The solvent was concentrated in vacuo.

The crude residue was directly purified by column chromatography

(silica gel, hexanes:EtOAc = 8.5:1.5) to afford a colorless oil (0.480 g,

92%). ¹HNMR(CDCl₃, 500 MHz): δ7.501-7.479 (m, 1H), 7.261-

7.184 (m, 5H), 6.653 (m, 1H), 5.189 (m, 2H), 3.726-3.686 (m, 1H),

3.632-3.597 (m, 0.5H), 3.556-3.520 (m, 0.5H), 3.465-3.391 (m,

1H), 3.292-3.262 (m, 0.5H), 3.206-3.175 (m, 0.5H), 3.126-3.071

(m, 1H), 2.792-2.751 (m, 1H), 2.667-2.615 (m, 1H), 2.566-2.519

(C₂₀H₃₂Cl₅N₅1.25 H₂O) Calcd: C, 44.30; H, 6.41; N, 12.91.

3

Found: C, 44.93; H, 6.58; N, 12.51.

N¹-{(()-trans-4-[(6⁰-Amino-4⁰-methylpyridin-2⁰-yl)methyl]pyrro-

lidin-3-yl}-N²-phenethylethane-1,2-diamine Tetrahydrochloride (32).

The procedure to prepare 32 is the same as that to prepare 8 except

50l (0.131 g, 0.2 mmol) instead of 42a was used, affording a

hygroscopic white solid (0.094 g, quantitative yield). ¹H NMR

(D₂O, 500 MHz): δ 7.390-7.302 (m, 5H), 6.677 (s, 2H),

4.083-3.997 (m, 2H), 3.760-3.725 (m, 1H), 3.690-3.650 (m, 1H),

3.580-3.509 (m, 4H), 3.412-3.382 (m, 2H), 3.344-3.306 (m, 1H),

3.280-3.239 (m, 1H), 3.108-3.087 (m, 1H), 3.044-3.015 (m, 2H),

2.900-2.850 (m, 1H), 2.307 (s, 3H). ¹³CNMR(D₂O, 125.7 MHz): δ

158.347 (1C), 154.206 (1C), 143.514 (1C), 136.089 (1C), 129.301

(2C), 129.125 (2C), 127.692 (1C), 115.099 (1C), 111.353 (1C), 60.790

(1C), 48.641 (1C), 48.641 (1C), 46.480 (1C), 43.420 (1C), 42.964

(1C), 40.548 (1C), 33.468 (1C), 31.938 (1C) (21.465 þ 21.423) (1C).

MS (ESI, CH₃OH): [C₂₁H₃₁N₅] m/z 354.4 ([M þ H]^þ). HRMS

(CIþ, CH₃OH) calcd, 354.2652; found, 354.2649. Anal.

(C₂₁H₃₅Cl₄N₅2.2H₂O) Calcd: C, 46.80; H, 7.37; N, 12.99. Found:

3

C, 47.01; H, 7.47; N, 12.67.

N¹-{(()-trans-4-[(6⁰-Amino-4⁰-methylpyridin-2⁰-yl)methyl]pyrro-

lidin-3-yl}-N²-(3-fluorophenethyl)ethane-1,2-diamine Tetrahydro-

chloride (34). The procedure to prepare 34 is the same as that to

prepare 8 except 50n (0.134 g, 0.2 mmol) instead of 42a was used,

affording a hygroscopic white solid (0.103 g, quantitative yield). ¹H

NMR (D₂O, 500 MHz): δ 7.377-7.333 (m, 1H), 7.117-7.008 (m,

3H), 6.678 (s, 2H), 4.094-4.003 (m, 2H), 3.766-3.730 (m, 1H),

3.698-3.657 (m, 1H), 3.597-3.520 (m, 4H), 3.425-3.396 (m, 2H),

3.351-3.312 (m, 1H), 3.290-3.249 (m, 1H), 3.137-3.095 (m, 1H),

3.059-3.030 (m, 2H), 2.907-2.856 (m, 1H), 2.303 (s, 3H). ¹³C

NMR (D₂O, 125.7 MHz): δ (163.829 þ 161.886) (1C), 158.328

(1C), 154.188 (1C), 143.472 (1C), (138.517 þ 138.456) (1C),

(130.952 þ 130.885) (1C), (124.972 þ 124.947) (1C), (115.895 þ

115.725) (1C), 115.057 (1C), (114.486 þ 114.316) (1C), 111.317

(1C), 60.760 (1C), 49.254 (1C), 48.611 (1C), 46.419 (1C), 43.407

(1C), 42.934 (1C), 40.487 (1C), 33.420 (1C), 31.617 (1C), 21.392

(1C). ¹⁹F NMR (D₂O, 376.5 MH_Z): δ -113.580. MS (ESI,

CH₃OH): [C₂₁H₃₀FN₅] m/z 372.4 ([M þ H]^þ). HRMS (CIþ,

CH₃OH) calcd, 372.2558; found, 372.2549. Anal. (C₂₁H₃₄-

7818 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Ji et al.

(m, 1H), 2.303-2.290 (m, 3H), 1.459-1.432 (m, 9H), 1.418 (s, 9H).

¹³C NMR (CDCl₃, 125.7 MHz): δ (156.074 þ 155.989) (1C),

(154.338 þ 154.162) (2C), 153.968 (1C), 148.825 (1C), 139.675

(1C), 128.127 (2C), (126.895 þ 126.871) (2C), (126.603 þ 126.525)

(1C), 119.998 (1C), (117.357 þ 117.320) (1C), (81.280 þ 81.231)

(1C), 79.598 (1C), (63.496 þ 62.950) (1C), 49.866 (1C), (49.617 þ

49.441) (1C), (49.234 þ 48.967) (1C), (43.752 þ 42.999) (1C),

(38.992 þ 38.913) (1C), 28.439 (3C), 28.142 (3C), 21.148 (1C).

(()-trans-tert-Butyl 3-Amino-4-{[6⁰-(tert-butoxycarbonylamino)-

4⁰-methylpyridin-2⁰-yl]methyl}pyrrolidine-1-carboxylate (55). A so-

lution of 54 (0.522 g, 0.001 mol) in EtOH (20 mL) was treated with

20 wt % Pd(OH)₂on carbon (300 mg). The reaction mixture was

stirred at 60 °C under a hydrogen atmosphere for 36 h. The catalyst

was filtered through Celite. The Celite pad was washed with EtOH

(10 mL ꢀ 2). The combined filtrate was concentrated in vacuo. The

residue was purified by column chromatography (silica gel, CH₂-

Cl₂:CH₃OH = 9.5:0.5) to afford a pale-green oil (0.28 g, 85%). ¹H

NMR (CDCl₃, 500 MHz): δ 7.625-7.583 (m, 2H), 6.629 (m, 1H),

3.727-3.693 (m, 0.5H), 3.669-3.634 (m, 0.5H), 3.619-3.581 (m,

0.5H), 3.529-3.492 (m, 0.5H), 3.167-3.154 (m, 1H), 3.107-3.068

(m, 0.5H), 3.035-2.986 (m, 1H), 2.966-2.929 (m, 0.5H), 2.889-

2.821 (m, 1H), 2.595-2.552 (m, 1H), 2.312-2.290 (m, 3H),

2.262-2.251 (m, 0.5H), 2.184-2.142 (m, 0.5H), 1.510 (s, 9H),

1.441 (s, 9H). ¹³C NMR (CDCl₃, 125.7 MHz): δ (157.841 þ

157.762) (1C), 154.551 (1C), 152.577 (1C), 151.648 (1C), 150.076

(1C), 119.166 (1C), (110.417 þ 110.368) (1C), (80.849 þ 80.764)

(1C), 79.270 (1C), (56.180 þ 55.397) (1C), (53.673 þ 53.248) (1C),

(50.224 þ 49.975) (1C), (47.097 þ 46.775) (1C), (39.289 þ 39.083)

(1C), 28.567 (3C), 28.336 (3C), 21.378 (1C). MS (ESI, CH₃OH):

[C₂₁H₃₄N₄O₄] m/z 407.1 ([M þ H]^þ); m/z 812.9 ([2M þ H]^þ).

(()-trans-tert-Butyl 3-{{2⁰-[(tert-Butoxycarbonyl)(3⁰⁰-chloro-

benzyl)amino]ethyl}amino}-4-{{6⁰-[(tert-butoxycarbonyl)amino]-

4⁰-methylpyridin-2⁰-yl}methyl}pyrrolidine-1-carboxylate (50c), (()-

trans-tert-Butyl 3-{{2⁰-[(tert-Butoxycarbonyl)(phenethyl)amino]-

ethyl}amino}-4-{{6⁰-[(tert-butoxycarbonyl)amino]-4⁰-methylpyr-

idin-2⁰-yl}methyl}pyrrolidine-1-carboxylate (50l), or (()-trans-

tert-Butyl 3-{{2⁰-[(tert-Butoxycarbonyl)(3⁰⁰-fluorophenethyl)amino]-

ethyl}amino}-4-{{6⁰-[(tert-butoxycarbonyl)amino]-4⁰-methylpyr-

idin-2⁰-yl}methyl}pyrrolidine-1-carboxylate (50n). To a mixture of 55

chromatography (silica gel, hexane:EtOAc = 9.0:1.0. R_f: 57a, 0.19;

57b, 0.23).

57a: white solid (0.322 g). H NMR (CDCl₃, 500 MHz): δ

1

7.508-7.442 (m, 1H), 7.298-7.185 (m, 5H), 6.633 (m, 1H),

5.233-5.080 (m, 3H), 3.549-3.394 (m, 3H), 3.151-3.121 (m,

1H), 2.861-2.837 (m, 1H), 2.728-2.703 (m, 2H), 2.401-2.380

(m, 1H), 2.285 (m, 3H), 2.028-2.024 (m, 1H), 1.958-1.933 (m,

1H), 1.699-1.680 (m, 1H), 1.448-1.418 (m, 18H), 1.108 (s, 3H),

1.014 (s, 3H), 0.921-0.904 (m, 3H). ¹³C NMR (CDCl₃, 125.7

MHz): δ (177.926 þ 177.677) (1C), (166.815 þ 166.736) (1C),

(156.087 þ 156.056) (1C), 154.253 (1C), (154.180 þ 153.943)

(2C), 148.643 (1C), (139.712 þ 139.669) (1C), 128.018 (2C),

(126.658 þ 126.597) (2C), (126.464 þ 126.367) (1C), (119.670 þ

119.573) (1C), 117.108 (1C), (90.861 þ 90.800) (1C), (81.189 þ

81.164) (1C), (79.495 þ 79.464) (1C), (76.143 þ 75.330) (1C),

54.674 (1C), (54.116 þ 54.079) (1C), (52.914 þ 52.434) (1C),

49.890 (1C), (49.198 þ 48.821) (1C), (41.366 þ 40.819) (1C),

(34.517 þ 34.420) (1C), (30.674 þ 30.601) (1C), 28.925 (1C),

28.373 (3C), 28.057 (3C), 21.063 (1C), (16.819 þ 16.661 þ

16.600) (2C), 9.606 (1C). MS (ESI, CH₃OH): [C₃₈H₅₁N₃O₈]

m/z 678.5 ([M þ H]^þ); m/z 700.5 ([M þ Na]^þ); m/z 1355.1 ([2M þ

H^þ); m/z 1377.1 ([2M þ Na]^þ).

1

57b: white solid (0.322 g). H NMR (CDCl₃, 500 MHz): δ

7.467-7.446 (m, 1H), 7.272-7.183 (m, 5H), 6.664 (m, 1H),

5.198-5.112 (m, 3H), 3.544-3.382 (m, 3H), 3.154-3.112 (m,

1H), 2.926-2.884 (m, 1H), 2.744-2.680 (m, 2H), 2.443-2.378

(m, 1H), 2.291-2.280 (m, 3H), 1.971-1.884 (m, 2H), 1.696-

1.646 (m, 1H), 1.440-1.419 (m, 18H), 1.115 (s, 3H), 1.031-

1.012 (m, 3H), 0.933-0.910 (m, 3H). ¹³C NMR (CDCl₃, 125.7

MHz): δ (178.563 þ 178.157) (1C), (167.191 þ 167.082) (1C),

(156.378 þ 156.342) (1C), (154.441 þ 154.405) (1C), (154.332 þ

154.089) (1C), 154.010 (1C), 148.734 (1C), (139.870 þ 139.821)

(1C), 128.133 (2C), (126.889 þ 126.822) (2C), (126.597 þ

126.500) (1C), (119.931 þ 119.876) (1C), (117.265 þ 117.180)

(1C), (91.146 þ 91.061) (1C), (81.316 þ 81.286) (1C), 79.580

(1C), (76.459 þ 75.572) (1C), (54.935 þ 54.881) (1C), (54.413 þ

54.225) (1C), (52.914 þ 52.495) (1C), 49.999 (1C), (49.277 þ

48.870) (1C), (41.669 þ 41.044) (1C), (34.596 þ 34.523)

(1C), 30.437 (1C), 28.834 (1C), 28.476 (3C), 28.215 (3C),

21.201 (1C), 16.749 (1C), 16.642 (1C), 9.782 (1C). MS (ESI,

CH₃OH): [C₃₈H₅₁N₃O₈] m/z 678.4 ([M þ H]^þ); m/z 1354.7

([2M þ H^þ).

˚

(0.203 g, 0.5 mmol), NaBH(OAc)₃(0.127 g, 0.6 mmol), and 3 A

molecular sieves (0.5 g) in dry 1,2-dichloroethane (10 mL) was added

tert-butyl 3-fluorophenethyl(2-oxoethyl)carbamate (0.141 g, 0.5

mmol)²³in dry 1,2-dichloroethane (5 mL) via cannula under a N₂

atmosphere. The reaction mixture was stirred at room temperature

under a N₂atmosphere for 16 h and then was filtered through Celite,

and the Celite pad was washed with CH₂Cl₂(5 mL ꢀ 2). To the

filtrate was then added 1 M aqueous NaOH (10 mL). The organic

layer was separated, and the aqueous layer was extracted with

CH₂Cl₂(10 mL ꢀ 2). The combined organic layers were washed

with brine (10 mL) and dried over MgSO₄. The solvent was

evaporated, and the residue was purified by column chromatogra-

phy (silica gel, hexanes:EtOAc:Et₃N = 8:2:0.25) to afford a colorless

oil (50n, 0.306 g, 91%). 50c and 50l were prepared by the same

procedure.

(3S,4S)-tert-Butyl 3-{{6⁰-[Benzyl(tert-butoxycarbonyl)amino]-

4⁰-methylpyridin-2⁰-yl}methyl}-4-{(1S,4R)-4⁰,7⁰,7⁰-trimethyl-3⁰-oxo-

2⁰-oxabicyclo[2.2.1]heptane-1⁰-carbonyloxy}pyrrolidine-1-carboxylate

(57a) and (3R,4R)-tert-Butyl 3-{{6⁰-[Benzyl(tert-butoxycarbonyl)-

amino]-4⁰-methylpyridin-2⁰-yl}methyl}-4-{(1S,4R)-4⁰,7⁰,7⁰-trimethyl-

3⁰-oxo-2⁰-oxabicyclo[2.2.1]heptane-1⁰-carbonyloxy}pyrrolidine-1-car-

boxylate (57b). To Ph₃P (0.328 g, 0.00125 mol) in a dry THF (5 mL)

solution was added 51 (0.497 g, 0.001 mol) in dry THF (10 mL) at

0 °C under a N₂atmosphere via cannula. DIAD (0.262 g, 0.260 mL,

0.0013 mol) was added dropwise, and the solution was stirred at 0 °C

for 20 min. (1S)-(-)-camphanic acid (0.258 g, 0.0013 mol) was

added dropwise at 0 °C, and the reaction mixture was stirred for 14 h

at room temperature. The solvent was concentrated in vacuo. The

crude residue was purified by column chromatography (silica gel,

hexanes:EtOAc = 8:2) to afford a white solid (0.644 g, 95%).

Compounds 57a and 57b can be further separated by column

(3⁰S,4⁰S)-tert-Butyl 3⁰-{{6⁰⁰-[Benzyl(tert-butoxycarbonyl)-

amino]-4⁰⁰-methylpyridin-2⁰⁰-yl}methyl}-4⁰-hydroxypyrrolidine-1⁰-

carboxylate [(3⁰S,4⁰S)-58] and (3⁰R,4⁰R)-tert-Butyl 3⁰-{{6⁰⁰-[Benzyl-

(tert-butoxycarbonyl)amino]-4⁰⁰-methylpyridin-2⁰⁰-yl}methyl}-4⁰-hy-

droxypyrrolidine-1⁰-carboxylate [(3⁰R,4⁰R)-58]. The procedure to

prepare (3⁰S,4⁰S)-58 or (3⁰R,4⁰R)-58 is the same as that to prepare

53 except 57a (1.355 g, 0.002 mol) or 57b (1.355 g, 0.002 mol)

were used instead of 52 (1.079 g, 0.002 mol). The desired products

were purified by column chromatography (silica gel, hexanes:

EtOAc = 6:4).

(3⁰S,4⁰S)-58, colorless oil (0.995 g, quantitative yield). ¹H

NMR (CDCl₃, 500 MHz): δ 7.379-7.357 (m, 1H), 7.275-

7.168 (m, 5H), 6.734 (m, 1H), 5.116-5.109 (m, 2H), 4.197

(brs, 1H), 3.976-3.968 (m, 1H), 3.559-3.355 (m, 3H), 3.167-

3.124 (m, 1H), 2.897-2.850 (m, 1H), 2.775-2.713 (m, 1H),

2.364-2.286 (m, 4H), 1.434 (s, 9H), 1.407 (s, 9H). ¹³C NMR

(CDCl₃, 125.7 MHz): δ 157.477 (1C), (154.265 þ 154.192) (1C),

(154.077 þ 154.047) (1C), 153.707 (1C), (149.153 þ 149.044)

(1C), (138.959 þ 138.910) (1C), 128.012 (2C), 126.567 (3C),

(120.210 þ 120.143) (1C), (117.994 þ 117.860) (1C), 81.098 (1C),

78.766 (1C), (70.849 þ 70.053) (1C), (53.837 þ 53.557) (1C),

50.109 (1C), (49.058 þ 48.670) (1C), (44.407 þ 43.873) (1C),

(34.893 þ 34.833) (1C), 28.312 (3C), (27.935 þ 27.911) (3C),

20.905 (1C). MS (ESI, CH₃OH): [C₂₈H₃₉N₃O₅] m/z 498.4 ([M þ

H]^þ); m/z 520.3 ([M þ Na]^þ). [R]²⁵= -33.4° (c = 4, CH₃OH).

(3⁰R,4⁰R)-58, colorless oil (0.995 g, quantitative yield), the

NMR and MS are the same as those of (3⁰S,4⁰S)-isomer. [R]²⁵

þ33.4° (c = 4, CH₃OH).

=

Article

(3⁰R,4⁰S)-tert-Butyl 3⁰-Acetoxy-4⁰-{{6⁰⁰-[benzyl(tert-butoxy-

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7819

(10 mL ꢀ 3). The combined organic layers were washed with brine

(10 mL ꢀ 2) and dried over Na₂SO₄. The solvent was evaporated,

and the residue was purified by column chromatography (silica gel,

hexanes:EtOAc = 8:2) to afford a colorless oil (0.505 g, 94%). ¹H

NMR (CDCl₃, 500 MHz): δ 7.445-7.413 (m, 1H), 7.279-7.165

(m, 5H), 6.686-6.678 (m, 1H), 5.828-5.756 (m, 1H), 5.184(s, 2H),

5.229-5.091 (m, 2H), 3.980-3.930 (m, 1H), 3.751-3.635 (m, 2H),

3.555-3.379 (m, 2H), 3.210-3.099 (m, 2H), 2.950-2.907 (m, 1H),

2.747-2.705 (m, 1H), 2.601-2.549 (m, 1H), 2.287-2.272 (m, 3H),

1.457-1.439 (m, 9H), 1.410 (s, 9H). ¹³C NMR (CDCl₃, 125.7

MHz): δ (157.690 þ 157.653) (1C), (154.642 þ 154.405) (1C),

(154.302 þ 154.241) (1C), 153.767 (1C), 148.303 (1C), (139.803 þ

139.760) (1C), (134.624 þ 134.600) (1C), 127.982 (2C), (126.943 þ

126.852) (2C), (126.482 þ 126.403) (1C), 120.016 (1C), (116.901 þ

116.840) (1C), (116.585 þ 116.422) (1C), (81.019 þ 80.958) (1C),

(78.954 þ 78.900) (1C), (78.554 þ 77.691)(1C), (70.060 þ 70.005)

(1C), (50.916 þ 50.364) (1C), 49.805 (1C), (49.289 þ 48.870) (1C),

(42.714 þ 42.070) (1C), (34.517 þ 34.462) (1C), 28.458 (3C),

28.087 (3C), 21.038 (1C). MS (ESI, CH₃OH): [C₃₁H₄₃N₃O₅] m/z

538.6 ([M þ H]^þ); m/z 560.6 ([M þ Na]^þ); m/z 1097.1 ([2M þ

Na]^þ).

(3⁰S,4⁰S)-60 was synthesized as a colorless oil (0.521 g, 97%)

by the same procedure as that to prepare the (3⁰R, 4⁰R)-isomer.

The NMR and MS are the same as those for (3⁰R,4⁰R)-isomer.

(3⁰R,4⁰S)-60 was synthesized as a colorless oil (0.521 g, 97%)

by the same procedure as that to prepare the (3⁰R,4⁰R)-isomer.

¹H NMR (CDCl₃, 500 MHz): δ 7.453-7.432 (m, 1H), 7.273-

7.173 (m, 5H), 6.659 (s, 1H), 5.809-5.764 (m, 1H), 5.192 (s, 2H),

5.167-5.082 (m, 2H), 3.840-3.787 (m, 2H), 3.715-3.698 (m, 1H),

3.613-3.579 (m, 0.5H), 3.544-3.510 (m, 0.5H), 3.485-3.417 (m,

1H), 3.338-3.317 (m, 0.5H), 3.276-3.253 (m, 0.5H), 3.175-3.153

(m, 0.5H), 3.132-3.104 (m, 0.5H), 2.766-2.702 (m, 1H), 2.633-

2.549 (m, 2H), 2.295-2.284 (m, 3H), 1.459-1.429 (m, 9H), 1.407 (s,

9H). ¹³C NMR (CDCl₃, 125.7 MHz): δ (156.979 þ 156.876) (1C),

(154.696 þ 154.532) (1C), (154.302 þ 154.253) (1C), 153.871 (1C),

148.540 (1C), 139.724 (1C), (134.533 þ 134.509) (1C), 128.042 (2C),

127.041 (2C), (126.555 þ 126.506) (1C), 119.998 (1C), (117.229 þ

117.187) (1C), (116.980 þ 116.901) (1C), (81.201 þ 81.091) (1C),

(81.043 þ 80.472) (1C), 79.100 (1C), (70.126 þ 70.023) (1C),

(50.400 þ 49.726) (1C), 49.823 (1C), (49.192 þ 48.767) (1C),

(43.424 þ 42.440) (1C), (39.295 þ 39.241) (1C), 28.464 (3C),

28.112 (3C), 21.093 (1C). MS (ESI, CH₃OH): [C₃₁H₄₃N₃O₅] m/z

560.6 ([M þ Na]^þ); m/z 1097.3 ([2M þ Na]^þ).

carbonyl)amino]-4⁰⁰-methylpyridin-2⁰⁰-yl}methyl}pyrrolidine-1⁰-car-

boxylate [(3⁰R,4⁰S)-59] and (3⁰S,4⁰R)-tert-Butyl 3⁰-Acetoxy-4⁰-

{{6⁰⁰-[benzyl(tert-butoxycarbonyl)amino]-4⁰⁰-methylpyridin-2⁰⁰-yl}-

methyl}pyrrolidine-1⁰-carboxylate [(3⁰S,4⁰R)-59]. The procedure

to prepare (3⁰R,4⁰S)-59 or (3⁰S,4⁰R)-59 is the same as that to

prepare 52 except (3⁰S, 4⁰S)-58 (0.497 g, 0.001 mol) or (3⁰R, 4⁰R)-

58 (0.497 g, 0.001 mol) were used instead of 51 (0.497 g, 0.001

mol). The desired products were purified by column chroma-

tography (silica gel, hexanes:EtOAc = 7.5:2.5).

1

(3⁰R,4⁰S)-59, colorless oil (0.53 g, 98%). H NMR (CDCl₃,

500 MHz): δ 7.446-7.427 (m, 1H), 7.262-7.174 (m, 5H), 6.672

(m, 1H), 5.176 (m, 2H), 4.980 (m, 1H), 3.715-3.663 (m, 1H),

3.493-3.413 (m, 1H), 3.557-3.332 (m, 2H), 2.765-2.589 (m,

3H), 2.288 (s, 3H), 1.952-1.946 (m, 3H), 1.449 (s, 9H), 1.441-

1.406 (m, 9H). ¹³C NMR (CDCl₃, 125.7 MHz): δ (170.367 þ

170.179) (1C), (156.214 þ 156.117) (1C), (154.508 þ 154.338)

(1C), (154.229 þ 154.198) (1C), 153.871 (1C), 148.607 (1C),

139.681 (1C), 127.970 (2C), 127.095 (2C), (126.470 þ 126.427)

(1C), 120.028 (1C), 117.363 (1C), (81.073 þ 81.031) (1C), 79.428

(1C), (76.659 þ 75.973) (1C), (50.358 þ 49.945) (1C), 49.841

(1C), (49.040 þ 48.603) (1C), (43.072 þ 42.234) (1C), 38.797

(1C), 28.367 (3C), 28.063 (3C), 21.008 (1C). MS (ESI, CH₃OH):

[C₃₀H₄₁N₃O₆] m/z 562.2 ([M þ Na]^þ).

(3⁰S,4⁰R)-59: colorless oil (0.40 g, 74%). The NMR and MS

are the same as those of (3⁰R,4⁰S)-isomer.

(3⁰S,4⁰R)-tert-Butyl 4⁰-{{6⁰⁰-[Benzyl(tert-butoxycarbonyl)-

amino]-4⁰⁰-methylpyridin-2⁰⁰-yl}methyl}-3⁰-hydroxypyrrolidine-1⁰-

carboxylate [(3⁰S,4⁰R)-58] and (3⁰R,4⁰S)-tert-Butyl 4⁰-{{6⁰⁰-[Benzyl-

(tert-butoxycarbonyl)amino]-4⁰⁰-methylpyridin-2⁰⁰-yl}methyl}-3⁰-hy-

droxypyrrolidine-1⁰-carboxylate [(3⁰R,4⁰S)-58]. The procedure to

prepare (3⁰S,4⁰R)-58 and (3⁰R,4⁰S)-58 is the same as that to prepare

53 except (3⁰R,4⁰S)-59 (0.540 g, 0.001 mol) or (3⁰S,4⁰R)-59 (0.540 g,

0.001 mol) was used instead of 52 (1.079 g, 0.002 mol). The desired

products were purified by column chromatography (silica gel,

hexanes:EtOAc = 6:4).

(3⁰S,4⁰R)-58, colorless oil (0.5 g, quantitative yield). ¹H NMR

(CDCl₃, 500 MHz): δ 7.357-7.314 (m, 1H), 7.248-7.163 (m,

5H), 6.684 (m, 1H), 5.148-5.138 (m, 2H), (4.419 þ 4.279) (brs,

1H), 4.007-3.996 (m, 1H), 3.661-3.489 (m, 2H), 3.209-3.156

(m, 1H), 3.092-3.058 (m, 1H), 2.855-2.786 (m, 1H), 2.656-

2.563 (m, 1H), 2.462-2.396 (m, 1H), 2.270-2.257 (m, 3H),

1.443-1.415 (m, 9H), 1.405 (s, 9H). ¹³C NMR (CDCl₃, 125.7

MHz): δ (157.240 þ 157.186) (1C), 154.447 (1C), (154.150 þ

154.107) (1C), 153.658 (1C), (148.801 þ 148.698) (1C), (139.323 þ

139.244) (1C), 127.970 (2C), 126.974 (1C), 126.512 (1C), (120.301 þ

120.162) (1C), (117.836 þ 117.636) (1C), 81.025 (1C), 78.991 (1C),

(74.474 þ 73.763) (1C), (52.464 þ 52.033) (1C), (50.103 þ 49.987)

(1C), (49.307 þ 48.913) (1C), (45.367 þ 44.650) (1C), (38.955 þ

38.907) (1C), 28.294 (3C), (27.972 þ 27.948) (3C), 20.874 (1C). MS

(ESI, CH₃OH): [C₂₈H₃₉N₃O₅] m/z 498.4 ([M þ H]^þ); m/z 520.4

([M þ Na]^þ); m/z 1017.2 ([2M þ Na]^þ). [R]²⁵=-35.6° (c=2,

MeOH).

(3⁰S,4⁰R)-60 was synthesized as a colorless oil (0.510 g, 95%)

by the same procedure as that to prepare the (3⁰R,4⁰R)-isomer.

The NMR and MS are the same as those for (3⁰R, 4⁰S)-isomer.

(3⁰R,4⁰R)-tert-Butyl 4⁰-{{6⁰⁰-[Benzyl(tert-butoxycarbonyl)-

amino]-4⁰⁰-methylpyridin-2⁰⁰-yl}methyl}-3⁰-(2⁰⁰-oxoethoxy)pyrro-

lidine-1⁰-carboxylate [(3⁰R,4⁰R)-61]. Method A. (3⁰R,4⁰R)-60

(0.537 g, 0.001 mol) was dissolved in CH₂Cl₂(30 mL) and cooled

to -78 °C. Ozone was bubbled through the solution until a light-

blue color appeared. N₂was bubbled through the solution until

the blue color disappeared (about 30 min). Zinc (0.098 g, 0.0015

mol) was added, followed by 5 mL of 50% aqueous acetic acid

solution. The suspension was allowed to warm slowly to 0 °C,

stirred for 30 min, and then allowed to stir at room temperature

for 30 min. The reaction mixture was diluted with CH₂Cl₂

(10 mL) and H₂O (20 mL). The organic layer was separated,

and the aqueous layer was extracted with CH₂Cl₂(10 mL ꢀ 2).

The combined organic layers were washed with H₂O (10 mL),

saturated aqueous NaHCO₃solution (10 mL ꢀ 2), and brine

(10 mL) and then dried over MgSO₄. The solvent was concen-

trated in vacuo, and the residue was purified by column chro-

matography (silica gel, hexanes:EtOAc = 5:5) to afford a pale-

yellow oil (0.345 g, 64%).

(3⁰R,4⁰S)-58: colorless oil (0.5 g, quantitative yield). The

NMR and MS are also the same as those of (3⁰S,4⁰R)-isomer.

[R]²⁵= þ35.6° (c = 2, MeOH).

(3⁰R,4⁰R)-tert-Butyl 3⁰-(Allyloxy)-4⁰-{{6⁰⁰-[benzyl(tert-butoxy-

carbonyl)amino]-4⁰⁰-methylpyridin-2⁰⁰-yl}methyl}pyrrolidine-1⁰-car-

boxylate [(3⁰R,4⁰R)-60]. To an ice-cooled solution of NaH (0.048 g,

0.0012 mol, 60% dispersion in mineral oil) in anhydrous DMF

(1 mL) was added dropwise (3⁰R,4⁰R)-58 (0.497 g, 0.001 mol) in

anhydrous DMF (5 mL) under a N₂atmosphere. The suspension

was then stirred vigorously for 30 min at room temperature. The

color of the reaction mixture changed from colorless to pale-red.

Allyl bromide (0.157 g, 0.113 mL, 0.0013 mol) was added dropwise

at 0 °C, and the reaction mixture was stirred at room temperature

for 3 h. The excess NaH was quenched with H₂O (5 mL), and the

solvent was evaporated in vacuo. The residue was further diluted

with water (10 mL) and EtOAc (10 mL). The organic layer

was separated, and the aqueous layer was extracted with EtOAc

Method B. (3⁰R,4⁰R)-60 (0.537 g, 0.001 mol) was dissolved in

CH₂Cl₂(30 mL) and cooled to -78 °C. Ozone was bubbled

through the solution until a light-blue color appeared. N₂was

bubbled through the solution until the blue color disappeared

(about 30 min). Ph₃P (0.315 g, 0.0012 mol) was added to the

7820 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Ji et al.

reaction mixture. The reaction mixture was allowed to warm

slowly to 0 °C, stirred for 15 min, and then allowed to stir at

room temperature for 30 min. The solvent was concentrated in

vacuo, and the residue was purified by column chromatography

(silica gel, hexanes:EtOAc = 5:5) to afford a pale-yellow oil

(0.388 g, 72%).

(1C), (81.365 þ 81.304) (1C), (79.646 þ 79.306) (1C), (79.270 þ

78.845) (1C), (68.687 þ 68.536) (1C), 50.892 (1C), 50.746 (1C),

50.060 (1C), 49.374 (1C), (49.301 þ 48.967) (1C), (42.877 þ 42.270)

(1C), 36.266 (1C), (35.567 þ 34.602) (1C), 28.676 (3C), 28.324

(3C), 21.305 (1C). MS(ESI, CH₃OH): [C₃₈H₅₁FN₄O₅] m/z 663.7

([M þ H]^þ); m/z 1325.3 ([2M þ H]^þ).

¹H NMR (CDCl₃, 500 MHz): δ 9.552 (s, 1H), 7.457-7.429

(m, 1H), 7.228-7.176 (m, 5H), 6.728-6.691 (m, 1H), 5.165 (m,

2H), 4.017-3.938 (m, 1H), 3.805-3.635 (m, 2H), 3.535-3.382

(m, 2H), 3.220-3.107 (m, 2H), 3.021-2.958 (m, 1H), 2.788-

2.576 (m, 2H), 2.296 (m, 3H), 1.454-1.413 (m, 18H). ¹³C NMR

(CDCl₃, 125.7 MHz): δ 200.852-199.999 (1C), 157.501 (1C),

(154.806 þ 154.484 þ 154.429) (2C), 153.974 (1C), 148.722 (1C),

(139.930 þ 139.876) (1C), 128.212 (2C), (126.895 þ 126.810)

(2C), (126.658 þ 126.591) (1C), 120.089 (1C), (117.156 þ

117.095) (1C), 81.371-80.211 (2C), 79.634-79.294 (1C),

(74.807 þ 74.583 þ 74.540) (1C), (50.971 þ 50.327) (1C), 49.975

(1C), (49.301 þ 48.888) (1C), (42.786 þ 42.155) (1C), (34.335 þ

34.304) (1C), 28.597 (3C), 28.494 (3C), 21.239 (1C).

(3⁰S,4⁰S)-62 was synthesized as a pale-yellow oil (0.444 g,

67%) by the same procedure used to prepare (3⁰R,4⁰R)-62. The

NMR spectrum and MS are the same as those for (3⁰R,4⁰R)-

isomer.

(3⁰R,4⁰S)-62 was synthesized as a pale-yellow oil (0.510 g,

1

77%) by the same procedure used to prepare (3⁰R,4⁰R)-62. H

NMR (CDCl₃, 500 MHz): δ 7.448-7.424 (m, 1H), 7.280-7.175

(m, 6H), 6.967-6.846 (m, 3H), 6.647 (s, 1H), 5.181 (s, 2H),

3.641-3.567 (m, 1.5H), 3.527-3.493 (m, 0.5H), 3.391-3.335

(m, 3H), 3.282-3.258 (m, 0.5H), 3.211-3.183 (m, 0.5H),

3.158-3.131 (m, 0.5H), 3.110-3.081 (m, 0.5H), 2.910-2.628

(m, 7H), 2.577-2.518 (m, 2H), 2.308-2.293 (m, 3H), 1.461-

1.431 (m, 9H), 1.407 (s, 9H). ¹³C NMR (CDCl₃, 125.7 MHz): δ

(163.961 þ 162.006) (1C), (157.095 þ 156.997) (1C), (154.860 þ

154.702) (1C), (154.472 þ 154.417) (1C), 154.010 (1C), 148.752

(1C), (142.705 þ 142.669) (1C), 139.894 (1C), (129.979 þ

129.912) (1C), 128.182 (2C), 127.198 (2C), (126.688 þ 126.634)

(1C), (124.472 þ 124.448) (1C), 120.156 (1C), (117.429 þ

117.393) (1C), (115.669 þ 115.499) (1C), (113.204 þ 113.034)

(1C), (82.209 þ 81.553) (1C), (81.310 þ 81.261) (1C), 79.331

(1C), 68.523 (1C), (50.861 þ 50.443) (1C), (50.801 þ 49.866)

(1C), 49.963 (1C), (49.222 þ 48.840) (2C), (43.309 þ 42.452)

(1C), (39.392 þ 39.344) (1C), 36.217 (1C), 28.603 (3C), 28.257

(3C), 21.257 (1C). MS (ESI, CH₃OH): [C₃₈H₅₁FN₄O₅] m/z 663.6

([M þ H]^þ); m/z 1325.5 ([2M þ H]^þ).

(3⁰S,4⁰S)-61 was synthesized as a pale-yellow oil (0.367 g,

68%) by the same procedure used to prepare (3⁰R,4⁰R)-61

(method A). The NMR spectrum is the same as that for

(3⁰R,4⁰R)-isomer.

(3⁰R,4⁰S)-61 was synthesized as a pale-yellow oil (0.399 g,

74%) by the same procedure used to prepare (3⁰R,4⁰R)-61

1

(method A). H NMR (CDCl₃, 500 MHz): δ (9.548 þ 9.507)

(s, 1H), 7.480-7.384 (m, 1H), 7.256-7.182 (m, 5H), 6.667 (s,

1H), 5.187-5.112 (m, 2H), 4.026-2.919 (m, 8H), 2.724-2.552

(m, 2H), 2.299-2.289 (m, 3H), 1.462-1.413 (m, 18H). ¹³C

NMR (CDCl₃, 125.7 MHz): δ 199.965 (1C), (157.240 þ

156.906 þ 156.621 þ 156.542) (1C), (154.747 þ 154.544 þ

154.368 þ 154.320) (2C), (153.962 þ 153.798) (1C), (148.922 þ

148.825 þ 148.716) (1C), 139.779 (1C), (128.170 þ 128.133)

(2C), (127.113 þ 126.986 þ 126.913 þ 126.761) (2C), (126.713 þ

126.634) (1C), (120.198 þ 120.113) (1C), (117.338 þ 117.278)

(1C), 82.889-79.264 (3C), (75.554 þ 74.431) (1C), (50.169-

48.791) (3C), (43.205 þ 42.197) (1C), 39.113 (1C), 28.500-

28.160 (6C), 21.160 (1C).

(3⁰S,4⁰R)-62 was synthesized as a pale-yellow oil (0.325 g,

49%) by the same procedure used to prepare (3⁰R,4⁰R)-62. The

NMR and MS spectrum are the same as those for (3⁰S,4⁰R)-

isomer.

(3⁰R,4⁰R)-tert-Butyl 4⁰-{{6⁰⁰-[Benzyl(tert-butoxycarbonyl)-

amino]-4⁰⁰-methylpyridin-2⁰⁰-yl}methyl}-3⁰-{2⁰⁰-[tert-butoxycar-

bonyl(3⁰⁰⁰-fluorophenethyl)amino]ethoxy}pyrrolidine-1⁰-carboxy-

late [(3⁰R,4⁰R)-63]. A solution of di-tert-butyl dicarbonate (0.219

g, 0.001 mol) in CH₂Cl₂(5 mL) was added dropwise to a solution

of (3⁰R,4⁰R)-62 (0.662 g, 0.001 mol) in CH₂Cl₂(5 mL) and 1 M

NaOH (3 mL). The reaction mixture was then stirred at room

temperature for 24 h. The organic layer was separated, and the

aqueous layer was extracted with CH₂Cl₂(10 mL ꢀ 2). The

combined organic layers were washed with water (10 mL ꢀ 2)

and dried over Na₂SO₄. The solvent was evaporated in vacuo,

and the residue was purified by column chromatography (silica

gel, hexanes:EtOAc = 7.5:2.5) to afford a colorless oil (0.572 g,

(3⁰S,4⁰R)-61 was synthesized as a pale-yellow oil (0.469 g,

87%) by the same procedure used to prepare (3⁰R,4⁰R)-61

(method A). The NMR spectrum is the same as that for

(3⁰R,4⁰S)-isomer.

(3⁰R,4⁰R)-tert-Butyl 4⁰-{{6⁰⁰-[Benzyl(tert-butoxycarbonyl)-

amino]-4⁰⁰-methylpyridin-2⁰⁰-yl}methyl}-3⁰-[2⁰⁰-(3⁰⁰⁰-fluorophenethyl-

amino)ethoxy]pyrrolidine-1⁰-carboxylate [(3⁰R,4⁰R)-62]. A mixture

of (3⁰R,4⁰R)-61 (0.539 g, 0.001 mol), 3-fluorophenethylamine (0.153

g, 0.144 mL, 0.0011 mol), NaBH(OAc)₃(0.297 g, 0.0014 mol), and

˚

3 A molecular sieves (1 g) in dry 1,2-dichloroethane (20 mL) was

1

stirred at room temperature under a N₂atmosphere for 14 h. The

reaction mixture was then filtered through Celite. The Celite pad

was washed with CH₂Cl₂(5 mL ꢀ 2). To the combined organic

layers was added 1 M NaOH (20 mL). The organic layer was

separated, and the aqueous layer was extracted with CH₂Cl₂

(10 mL ꢀ 2). The combined organic layers were washed with brine

(10 mL) and dried over MgSO₄. The solvent was concentrated in

vacuo, and the residue was purified by column chromatography

(silica gel, hexanes:EtOAc:Et₃N = 6:4:0.5) to afford a pale-yellow

oil (0.623 g, 94%). ¹HNMR(CDCl₃, 500MHz):δ7.427-7.392 (m,

1H), 7.272-7.181 (m, 6H), 6.979-6.835 (m, 3H), 6.603 (m, 1H),

5.169 (s, 2H), 3.646-3.586 (m, 1H), 3.557-3.473 (m, 1H),

3.420-3.324 (m, 1H), 3.284-3.234 (m, 1H), 3.194-3.163 (m,

1H), 3.061-3.000 (m, 1H), 2.913-2.511 (m, 9H), 2.299-2.282

(m, 3H), 1.452-1.436 (m, 9H), 1.411 (s, 9H). ¹³C NMR (CDCl₃,

125.7 MHz): δ (164.034 þ 162.079) (1C), (157.853 þ 157.823) (1C),

(154.909 þ 154.708) (1C), (154.575 þ 154.508) (1C), 154.022 (1C),

148.649 (1C), (142.802 þ 142.748 þ 142.669) (1C), (140.021 þ

139.973) (1C), (130.107 þ 130.052 þ 129.991) (1C), 128.231 (2C),

(127.174 þ 127.077) (2C), (126.719 þ 126.646) (1C), 124.539 (1C),

120.131 (1C), (117.253 þ 117.187) (1C), (115.839 þ 115.748 þ

115.675 þ 115.578) (1C), (113.313 þ 113.270 þ 113.149 þ 113.106)

75%). H NMR (CDCl₃, 500 MHz): δ 7.426-7.395 (m, 1H),

7.237-7.178 (m, 6H), 6.986-6.857 (m, 3H), 6.634-6.609 (m,

1H), 5.163 (s, 2H), 3.646-3.165 (m, 10H), 3.086-3.022 (m, 1H),

2.893-2.779 (m, 3H), 2.692-2.649 (m, 1H), 2.558 (m, 1H),

2.291-2.273(m, 3H), 1.445-1.409 (m, 27H). ¹³C NMR

(CDCl₃, 125.7 MHz): δ (163.967 þ 162.012) (1C), (157.756 þ

157.665) (1C), (155.419 þ 155.279) (1C), (154.812 þ 154.623)

(1C), (154.514 þ 154.447) (1C), 153.974 (1C), 148.655 (1C),

142.013 (1C), 139.973 (1C), 129.997 (1C), 128.176 (2C), (127.120 þ

127.035) (2C), (126.670 þ 126.591) (1C), (124.685 þ 124.667) (1C),

120.058 (1C), (117.174 þ 117.126) (1C), (115.875 þ 115.711) (1C),

(113.337 þ 113.246 þ 113.173 þ 113.076) (1C), (81.298 þ 81.243)

(1C), (79.725 þ 79.203 þ 78.881) (3C), (68.232 þ 68.056) (1C),

(50.934 þ 50.430) (1C), (50.273 þ 50.194) (1C), 50.024 (1C),

(49.386þ48.937) (1C), (47.971 þ 47.468) (1C), (42.890þ42.762 þ

42.282) (1C), (35.003 þ 34.711 þ 34.341) (2C), 28.591 (3C), 28.488

(3C), 28.275 (3C), 21.239 (1C). MS (ESI, CH₃OH):[C₄₃H₅₉FN₄O₇]

m/z 763.5 ([M þ H]^þ); m/z 785.5 ([M þ Na]^þ); m/z 1546.8 ([2M þ

Na]^þ).

(3⁰S,4⁰S)-63 was synthesized as a pale-yellow oil (0.595 g,

78%) by the same procedure used to prepare (3⁰R,4⁰R)-63. The

NMR spectrum and MS are the same as those for (3⁰R,4⁰R)-63.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7821

(3⁰R,4⁰S)-63 was synthesized as a pale-yellow oil (0.473 g,

(1C), (119.397 þ 119.324) (1C), (115.960 þ 115.802) (1C),

(113.392 þ 113.228) (1C), 110.453 (1C), (82.439 þ 81.699)

(1C), 80.982 (1C), 79.798 (1C), 79.507 (1C), 68.153 (1C),

50.515 (1C), (50.315 þ 49.963) (1C), (49.234 þ 48.888) (1C),

(48.287 þ 47.880) (1C), (43.539 þ 42.920) (1C), 39.320 (1C),

(35.130 þ 34.468) (1C), (28.652 þ 28.549) (6C), 28.427 (3C),

21.457 (1C). MS (ESI, CH₃OH): [C₃₆H₅₃FN₄O₇] m/z 673.8

([M þ H]^þ); m/z 695.8 ([M þ Na]^þ); m/z 1367.2 ([2M þ Na]^þ).

(3⁰S,4⁰R)-64 was synthesized as a colorless oil (0.585 g, 58%)

by the same procedure used to prepare (3⁰R,4⁰R)-64 except 0.114

g (0.0015 mol) of (3⁰S,4⁰R)-63 was used. The NMR and MS are

the same as those of (3⁰R,4⁰S)-isomer .

1

62%) by the same procedure used to prepare (3⁰R,4⁰R)-63. H

NMR(CDCl₃, 500 MHz): δ 7.446-7.423 (m, 1H), 7.244-7.171

(m, 6H), 6.967-6.837 (m, 3H), 6.636 (s, 1H), 5.170-5.161 (s,

2H), 3.639 (m, 1H), 3.580-3.556 (m, 0.5H), 3.510-3.495 (m,

0.5H), 3.396-3.313 (m, 6H), 3.216-3.095 (m, 3H), 2.812-2.691

(m, 3H), 2.574-2.538 (m, 2H), 2.291-2.279 (m, 3H), 1.448-

1.407 (m, 27H). ¹³C NMR(CDCl₃, 125.7 MHz): δ (163.919 þ

161.964) (1C), (157.028 þ 156.937) (1C), (155.401 þ 155.243)

(1C), (154.854 þ 154.702) (1C), (154.453 þ 154.399) (1C), 153.974

(1C), 148.752 (1C), 142.025 (1C), 139.845 (1C), (130.016 þ 129.955)

(1C), 128.170 (2C), 127.192 (2C), (126.682 þ 126.628) (1C),

(124.673 þ 124.654) (1C), 120.089 (1C), 117.344 (1C), (115.869 þ

115.705) (1C), (113.283 þ 113.191 þ 113.119) (1C), (82.227 þ

81.595) (1C), 81.292 (1C), 79.683 (1C), (79.373 þ 79.313) (1C),

(68.044 þ 67.941) (1C), 50.327 (1C), 49.920 (1C), (50.078 þ 49.756)

(1C), (49.265 þ 48.846) (1C), (48.117 þ 47.613) (1C), (43.545 þ

42.695) (1C), (39.392 þ 39.344) (1C), (35.003 þ 34.341) (1C),

(28.555 þ 28.452) (6C), 28.245 (3C), 21.233 (1C). MS (ESI,

CH₃OH): [C₄₃H₅₉FN₄O₇] m/z 785.5 ([M þ Na]^þ).

6-{{(3⁰R,4⁰R)-3⁰-[2⁰⁰-(3⁰⁰⁰-Fluorophenethylamino)ethoxy]pyrro-

lidin-4⁰-yl}methyl}-4-methylpyridin-2-amine Trihydrochloride

[(3⁰R,4⁰R)-4]. (3⁰R,4⁰R)-64 (0.067 g, 0.0001 mol) was cooled in

an ice-water bath under argon. A solution of 4 M HCl in 1,4-

dioxane (4 mL) was then added slowly with stirring. The ice-

water bath was removed after 3 h, and the reaction mixture was

stirred at room temperature under an argon atmosphere for 34 h.

The reaction mixture was concentrated in vacuo, and the residue

was partitioned between water (10 mL) and ethyl acetate

(10 mL). The aqueous layer was then washed with ethyl acetate

(5 mLꢀ 2). After evaporation of the water by high vacuum rotary

evaporation, the residue was dried with a lyophilizer to afford a

(3⁰S,4⁰R)-63 was synthesized as a pale-yellow oil (0.572 g,

75%) by the same procedure used to prepare (3⁰R,4⁰R)-63. The

NMR spectrum and MS are the same as those for (3⁰R,4⁰S)-63.

(3⁰R,4⁰R)-tert-Butyl 3⁰-{2⁰⁰-[tert-Butoxycarbonyl(3⁰⁰⁰-fluorophe-

nethyl)amino]ethoxy}-4⁰-{[6⁰⁰-(tert-butoxycarbonylamino)-4⁰⁰-meth-

ylpyridin-2⁰⁰-yl]methyl}pyrrolidine-1⁰-carboxylate [(3⁰R,4⁰R)-64]. A

solution of (3⁰R,4⁰R)-63 (0.381 g, 0.0005 mol) in EtOH (20 mL) was

treated with 20 wt % Pd(OH)₂on carbon (150 mg). The reaction

mixture was stirred at 60 °C under a hydrogen atmosphere for 36 h.

The catalyst was filtered through Celite. The Celite pad was washed

with EtOH (10 mL ꢀ 2), and the combined filtrates were concen-

trated in vacuo. The residue was diluted with CH₂Cl₂(10 mL) and

1 M NaOH (10 mL), and the organic layer was separated. The

aqueous layer was extracted with CH₂Cl₂(10 mL ꢀ 2), and the

combined organic layers were concentrated in vacuo. The residue

was purified by column chromatography (silica gel, hexanes:

1

white solid (0.048 g, quantitative yield). H NMR (D₂O, 500

MHz): δ 7.336-7.293 (m, 1H), 7.111-7.096 (m, 1H), 7.063-

7.044 (m, 1H), 6.972-6.937 (m, 1H), 6.631 (s, 1H), 6.560 (s, 1H),

4.189 (m, 1H), 3.871-3.850 (m, 1H), 3.684-3.620 (m, 2H),

3.500-3.466 (m, 1H), 3.354-3.309 (m, 5H), 3.195-3.154 (m,

1H), 3.065-3.040 (m, 2H), 2.964-2.930 (m, 1H), 2.807-2.771

(m, 2H), 2.289 (s, 3H). ¹³C NMR (D₂O, 125.7 MHz): δ (163.769 þ

161.826) (1C), 158.328 (1C), 153.963 (1C), 145.882 (1C), (138.991 þ

138.930) (1C), 130.849 (1C), (124.978 þ 124.856) (1C), (115.852 þ

115.682 þ 115.543) (1C), (114.444 þ 114.359) (1C), (114.201 þ

114.060) (1C), 110.576 (1C), (78.422 þ 78.361) (1C), 64.056 (1C),

49.558 (1C), 48.489 (1C), 47.141 (2C), 41.713 (1C), 31.434 (1C),

29.273 (1C), (21.386 þ 21.344) (1C). ¹⁹F NMR (D₂O, 376.5 MH_Z):

δ -113.532 (F). MS (ESI, CH₃OH): [C₂₁H₂₉FN₄O] m/z 373.5

([M þ H]^þ). HRMS (CIþ, CH₃OH) calcd, 373.2404; found,

1

EtOAc=7:3) to afford a colorless oil (0.269 g, 80%). H NMR

(CDCl₃, 500 MHz): δ 7.618-7.600 (m, 1H), 7.471-7.457 (m, 1H),

7.254-7.242 (m, 1H), 7.002-6.874 (m, 3H), 6.620-6.591 (m, 1H),

3.781-3.737 (m, 1H), 3.602-3.261 (m, 9H), 3.160-3.062 (m, 1H),

2.859-2.819 (m, 3H), 2.703-2.541 (m, 2H), 2.301-2.279 (m, 3H),

1.512 (s, 9H), 1.442 (m, 18H). ¹³C NMR (CDCl₃, 125.7 MHz): δ

(163.870 þ 161.915) (1C), (158.218 þ 158.169) (1C), (155.382 þ

155.334 þ 155.170) (1C), (154.696 þ 154.544) (1C), 152.492 (1C),

(151.527 þ 151.515) (1C), 149.894 (1C), (141.958 þ 141.898) (1C),

(129.979 þ 129.906) (1C), (124.600 þ 124.582) (1C), (119.117 þ

119.063) (1C), (115.790 þ 115.626) (1C), (113.240 þ 113.149 þ

113.070 þ 112.979) (1C), (110.235 þ 110.168) (1C), (80.721 þ

80.660) (1C), (79.616 þ 79.586) (1C), (79.750 þ 79.221 þ 79.149 þ

78.651) (2C), (68.250 þ 68.159 þ 67.977) (1C), (50.922 þ 50.485)

(1C), (50.212 þ 50.072 þ 49.981) (1C), (49.216 þ 48.864) (1C),

(47.941 þ 47.449) (1C), (43.333 þ 43.175 þ 42.707 þ 42.556) (1C),

(34.936 þ 34.736 þ 34.547 þ 34.432 þ 34.268) (2C), 28.482 (3C),

28.385 (3C), 28.263 (3C), 21.281 (1C). MS (ESI, CH₃OH): [C₃₆H₅₃-

FN₄O₇] m/z 673.8 ([M þ H]^þ); m/z 695.6 ([M þ Na]^þ); m/z

1367.5([2M þ Na]^þ).

373.2410. [R]²⁵þ36.4° (c=1, MeOH). Anal. (C₂₁H₃₃Cl₃FN₄O

3

2.5H₂O.0.7CH₃OH) Calcd: C, 47.45; H, 7.30; N, 10.20. Found: C,

47.56; H, 7.19; N, 9.97.

(3⁰S,4⁰S)-4 was synthesized as a white solid (0.048 g,

quantitative) by the same procedure used to prepare (3⁰R,4⁰R)-

4. The NMR spectrum and MS are the same as those for

(3⁰R,4⁰R)-isomer. [R]²⁵-36.4° (c = 1, MeOH). Anal. (C₂₁H₃₃-

Cl₃FN₄O 3.25H₂O) Calcd: C, 46.67; H, 7.18; N, 10.37. Found:

3

C, 46.70; H, 7.10; N, 10.13.

(3⁰R,4⁰S)-4 was synthesized as a white solid (0.048 g, quan-

titative) by the same procedure used to prepare (3⁰R,4⁰R)-4. The

NMR spectrum and MS are the same as those for the (3⁰R,4⁰R)-

1

isomer. H NMR (D₂O, 500 MHz): δ 7.373-7.332 (m, 1H),

7.105-7.090 (m, 1H), 7.060-7.003 (m, 2H), 6.661 (s, 2H), 4.139

(m, 1H), 3.704 (m, 2H), 3.625-3.589 (m, 1H), 3.566-3.483 (m,

2H), 3.313-3.248 (m, 3H), 3.205-3.178 (m, 1H), 3.143-3.112

(m, 1H), 3.026-2.997 (m, 2H), 2.857-2.780 (m, 3H), 2.303 (s,

3H). ¹³C NMR (D₂O, 125.7 MHz): δ (163.811 þ 161.874) (1C),

158.322 (1C), 154.084 (1C), 145.087 (1C), (138.948 þ 138.888)

(1C), (130.885 þ 130.819) (1C), 124.747 (1C), (115.792 þ

115.628 þ 115.524) (1C), 114.796 (1C), 114.207 (1C), 110.752

(1C), (81.476 þ 81.403) (1C), 63.941 (1C), 49.078 (1C), (48.228 þ

48.058) (2C), 46.983 (1C) 42.460 (1C), 33.408 (1C), 31.337 (1C),

(21.344 þ 21.307) (1C). ¹⁹F NMR (D₂O, 376.5 MH_Z):

δ -113.558 (F). MS (ESI, CH₃OH): [C₂₁H₂₉FN₄O] m/z 373.7

([M þ H]^þ). HRMS (CIþ, CH₃OH) calcd, 373.2404; found,

(3⁰S,4⁰S)-64 was synthesized as a colorless oil (0.286 g, 85%)

by the same procedure used to prepare (3⁰R,4⁰R)-64. The NMR

spectrum and MS are the same as those for (3⁰R,4⁰R)-isomer.

(3⁰R,4⁰S)-64 was synthesized as a colorless oil (0.048 g, 48%)

by the same procedure used to prepare (3⁰R,4⁰R)-64 except using

0.114 g (0.0015 mol) of (3⁰R,4⁰S)-63. ¹H NMR (CDCl₃, 500

MHz): δ 7.625-7.610 (m, 1H), 7.305-7.229 (m, 2H), 6.984-

6.855 (m, 3H), 6.590-6.582 (s, 1H), 3.681-3.654 (m, 1H),

3.583-3.091 (m, 10H), 2.832-2.799 (m, 2H), 2.698-2.491 (m,

3H), 2.298-2.282 (m, 3H), 1.518 (s, 9H), 1.448-1.430 (m, 18H).

¹³C NMR(CDCl₃, 125.7 MHz): δ (164.004 þ 162.049) (1C),

157.410 (1C), (155.534 þ 155.316) (1C), (154.939 þ 154.818)

(1C), (152.644 þ 152.559) (1C), 151.588 (1C), 150.155 (1C),

142.068 (1C), (130.101 þ 130.040) (1C), (124.752 þ 124.733)

373.2403. [R]²⁵-33.6° (c = 1, MeOH). Anal. (C₂₁H₃₃Cl₃FN₄O

3

3H₂O.0.3NaCl) Calcd: C, 45.57; H, 6.92; N, 10.12. Found: C,

45.43; H, 6.76; N, 9.82.

(3⁰S,4⁰R)-4 was synthesized as a white solid (0.048 g,

quantitative) by the same procedure used to prepare (3⁰R,4⁰R)-4.

7822 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Ji et al.

The NMR spectrum and MS are the same as those for (3⁰R,4⁰R)-

complexed with inhibitors provide no additional information on

the enzyme-inhibitor interactions and thus are not discussed in

the Results and Discussion section. Coordinates of all the crystal

structures reported in this work have been deposited to RCSB

Protein Data Bank with their accession codes listed in both

Supporting Information tables.

isomer. [R]²⁵þ33.6° (c = 1, MeOH). Anal. (C₂₁H₃₃Cl₃FN₄O

3

3H₂O 0.5NaCl) Calcd: C, 44.63; H, 6.78; N, 9.91. Found: C, 44.75;

H, 6.76; N, 9.64

Enzyme and Assay. All of the NOS isozymes used were

recombinant enzymes overexpressed in E. coli. The murine

macrophage iNOS was expressed and isolated according to

the procedure of Hevel et al.³⁵The rat nNOS was expressed³⁶

and purified as described.³⁷The bovine eNOS was isolated as

reported.³⁸Nitric oxide formation from NOS was monitored by

the hemoglobin capture assay at 30 °C as described.³⁹A typical

assay mixture for nNOS and eNOS contained 10 μM ^L-arginine,

1.0 mM CaCl₂, 600 unit/mL calmodulin (Sigma, P-2277),

100 μM NADPH, 0.125 mg/mL hemoglobin-A₀(ferrous form,

Sigma, H0267), and 10 μM H₄B in 100 mM HEPES (pH 7.5). A

typical assay mixture for iNOS contained 10 μM ^L-arginine,

100 μM NADPH, 0.125 mg/mL hemoglobin-A₀(ferrous form),

and 10 μM H₄B in 100 mM HEPES (pH 7.5). All assays were in a

final volume of 600 μL and were initiated by addition of enzyme.

NOS assays were monitored at 401 nm on a Perkin-Elmer

Lambda 10 UV-visible spectrophotometer.

Acknowledgment. We are grateful for financial support

from the National Institutes of Health, GM049725 to R.B.S.,

GM057353 to T.L.P., and GM052419 to Dr. Bettie Sue

Masters, with whose lab P.M. and L.J.R. are affiliated, and

grant no. AQ1192 from The Robert A. Welch Foundation to

Dr. Bettie Sue Masters. Dr. Bettie Sue Masters also is grateful

to the Welch Foundation for a Robert A. Welch Foundation

Distinguished Professorship in Chemistry (AQ0012). P.M.

is supported by grants 0021620806 and 1M0520 from MSMT

of the Czech Republic. We are grateful to Dr. Jinwen

Huang for suggesting the use of a direct Mitsunobu inver-

sion of 51 in Scheme 4. We thank the beamline support staff at

ALS and SSRL for their assistance during X-ray data collec-

tions.

Determination of K_iValues. The apparent K_ivalues were

obtained by measuring the percent enzyme inhibition in the

presence of 10 μM ^L-arginine with at least five concentrations of

inhibitor. The parameters of the following inhibition equation⁴⁰

were fitted to the initial velocity data: % inhibition = 100[I]/

{[I] þ K_i(1 þ [S]/K_m)}. K_mvalues for L-arginine were 1.3 μM

(nNOS), 8.2 μM (iNOS), and 1.7 μM (eNOS). The selectivity of

an inhibitor was defined as the ratio of the respective K_ivalues.

Crystal Preparation, X-Ray Data Collection, and Structure

Refinement. The nNOS or eNOS heme domain proteins, wild

type or mutants, used for crystallographic studies were pro-

duced by limited trypsin digest from the corresponding full

length enzymes and further purified through a Superdex 200

gel filtration column (GE Healthcare) as described previ-

ously.^17d,20aA batch of nNOS heme domain was also obtained

by limited trypsin digest from the purified nNOS heme-FMN

bidomain protein.⁴¹This bidomain construct had its Arg349, a

known sensitive trypsin cleavage site,⁴²mutated to Ala through

the Quikchange protocol (Stratagene). The purified nNOS heme

domain (48 kD) is therefore a R349A mutant, which is free of the

43 kD truncated heme domain. The nNOS heme domain at 7-

9 mg/mL containing 20 mM histidine or the eNOS heme domain

at 20 mg/mL with 2 mM imidazole were used for the sitting drop

vapor diffusion crystallization setup under the conditions

reported before.^17b,dFresh crystals were first passed stepwise

through cryoprotectant solutions as described^17b,dand then

soaked with 10 mM inhibitor for 4-6 h at 4 °C before being

flash cooled by plunging into liquid nitrogen. Crystals were

stored in liquid nitrogen until data collection.

Supporting Information Available: Crystallographic data

collection and structure refinement statistics. Figures for the

wild type eNOS in complexes with four enatiopure isomers of 4.

Experimental details and data of 41b, 42b, 8, 10, 11, 46a-46h,

13-20, 50b, 50d-50k, 50m, 22, 24-31, and 33, the ¹H NMR and

¹³C NMR spectra of 8-34, and four enantiopure isomers of 4.

This material is available free of charge via the Internet at http://

pubs.acs.org.

References

(1) (a) Bredt, D. S.; Snyder, S. H. Nitric Oxide: A Physiologic

Messenger Molecule. Annu. Rev. Biochem. 1994, 63, 175–195.

(b) Kerwin, J. F.; Lancaster, J. R.; Feldman, P. L. Nitric Oxide: A

New Paradigm for Second Messengers. J. Med. Chem. 1995, 38 (22),

4343–4362. (c) Cary, S. P. L.; Winger, J. A.; Derbyshire, E. R.; Marletta,

M. A. Nitric Oxide Signaling: No Longer Simply On or Off. Trends

Biochem. Sci. 2006, 31 (4), 231–239.

(2) Griffith, O. W.; Stuehr, D. J. Nitric Oxide Synthases: Properties

and Catalytic Mechanism. Annu. Rev. Physiol. 1995, 57, 707–736.

(3) Rosen, G. M.; Tsai, P.; Pou, S. Mechanism of Free-Radical

Generation by Nitric Oxide Synthase. Chem. Rev. 2002, 102 (4),

1191–1199.

(4) (a) Alderton, W. K.; Cooper, C. E.; Knowles, R. G. Nitric Oxide

Synthases: Structure, Function and Inhibition. Biochem. J. 2001,

357 (Pt 3), 593–615. (b) Knowles, R. G.; Moncada, S. Nitric Oxide

Synthases in Mammals. Biochem. J. 1994, 298 (2), 249–258.

ꢀ

(5) Roman, L. J.; Martasek, P.; Masters, B. S. S. Intrinsic and Extrinsic

Modulation of Nitric Oxide Synthase Activity. Chem. Rev. 2002,

102 (4), 1179–1190.

The cryogenic (100 K) X-ray diffraction data were collected at

various beamlines at either the Advanced Light Source (ALS,

Berkeley, CA) or Stanford Synchrotron Radiation Lightsource

(SSRL, Menlo Park, CA) using the data collection control

software Blu-Ice.⁴³Raw data frames were indexed, integrated,

and scaled using HKL2000.⁴⁴The binding of inhibitors was

detected by the initial difference Fourier maps calculated with

CNS.⁴⁵The inhibitor molecules were then modeled in O⁴⁶or

COOT⁴⁷and refined using CNS. Water molecules were added

and checked by COOT. Refinements at later stage were con-

tinued with REFMAC⁴⁸in order to implement the TLS⁴⁹

protocol with each subunit as one TLS group. The refined

structures were validated before deposition to RCSB Protein

Data Bank. The crystallographic data collection and structure

refinement statistics for the four enantiopure isomers of 4 bound

to the wild type eNOS and nNOS (including nNOS R349A

mutant) are summarized in Supporting Information Table S1;

the statistics for the same four compounds bound to various

eNOS and nNOS mutants are summarized in Supporting

Information Table S2. Those eNOS or nNOS mutant structures

(6) (a) Moncada, S.; Palmer, R. M.; Higgs, E. A. Nitric Oxide:

Physiology, Pathophysiology, and Pharmacology. Pharmacol.

Rev. 1991, 43 (2), 109–142. (b) Domenico, R. Pharmacology of Nitric

Oxide: Molecular Mechanisms and Therapeutic Strategies. Curr.

Pharm. Des. 2004, 10 (14), 1667–1676.

(7) (a) Uehara, T.; Nakamura, T.; Yao, D.; Shi, Z. Q.; Gu, Z.; Ma, Y.;

Masliah, E.; Nomura, Y.; Lipton, S. A. S-Nitrosylated Protein-

Disulphide Isomerase Links Protein Misfolding to Neurodegen-

eration. Nature 2006, 441 (7092), 513–517. (b) Chung, K. K.;

Thomas, B.; Li, X.; Pletnikova, O.; Troncoso, J. C.; Marsh, L.; Dawson,

V. L.; Dawson, T. M. S-Nitrosylation of Parkin Regulates Ubiquitina-

tion and Compromises Parkin's Protective Function. Science 2004, 304,

1328–1331. (c) Cho, D. H.; Nakamura, T.; Fang, J.; Cieplak, P.; Godzik,

A.; Gu, Z.; Lipton, S. A. S-Nitrosylation of Drp1Mediates β-Amyloid-

Related Mitochondrial Fission and Neuronal Injury. Science 2009, 324

(5923), 102–105. (d) Hantraye, P.; Brouillet, E.; Ferrante, R.; Palfi, S.;

Dolan, R.; Matthews, R. T.; Beal, M. F. Inhibition of Neuronal Nitric

Oxide Synthase prevents MPTP-induced Parkinsonism in Baboons.

Nature Med. 1996, 2 (9), 1017–1021.

(8) Huang, Z.; Huang, P. L.; Panahian, N.; Dalkara, T.; Fishman,

M. C.; Moskowitz, M. A. Effects of Cerebral Ischemia in Mice

Deficient in Neuronal Nitric Oxide Synthase. Science 1994, 265

(5180), 1883–1885.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7823

Highly Selective Inhibitors of Neuronal Nitric Oxide Synthase. J. Med.

(9) (a) Ferriero, D. M.; Holtzman, D. M.; Black, S. M.; Sheldon, R. A.

Neonatal Mice Lacking Neuronal Nitric Oxide Synthase Are Less

Vulnerable to Hypoxic-Ischemic Injury. Neurobiol. Dis. 1996, 3 (1),

64–71. (b) Tan, S.; Bose, R.; Derrick, M. Hypoxia-Ischemia in Fetal

Rabbit Brain Increases Reactive Nitrogen Species Production: Quanti-

tative Estimation of Nitrotyrosine. Free Radical Biol. Med. 2001,

30 (9), 1045–1051. (c) Derrick, M.; Drobyshevsky, A.; Ji, X.; Tan, S. A

Model of Cerebral Palsy from Fetal Hypoxia-Ischemia. Stroke 2007, 38

(2 Suppl), 731–735. (d) Derrick, M.; Luo, N. L.; Bregman, J. C.; Jilling,

T.; Ji, X.; Fisher, K.; Gladson, C. L.; Beardsley, D. J.; Murdoch, G.;

Back, S. A.; Tan, S. Preterm Fetal Hypoxia-Ischemia Causes Hyperto-

nia and Motor Deficits in the Neonatal Rabbit: A Model for Human

Cerebral Palsy? J. Neurosci. 2004, 24 (1), 24–34.

(10) (a) Calabrese, V.; Mancuso, C.; Calvani, M.; Rizzarelli, E.;

Butterfield, D. A.; Stella, A. M. Nitric Oxide in the Central

Nervous System: Neuroprotection versus Neurotoxicity. Nature

Rev. Neurosci. 2007, 8 (10), 766–775. (b) Contestabile, A.; Monti, B.;

Contestabile, A.; Ciani, E. Brain Nitric Oxide and Its Dual Role

in Neurodegeneration/Neuroprotection: Understanding Molecular

Mechanisms to Devise Drug Approaches. Curr. Med. Chem. 2003,

10 (20), 2147–2174. (c) Estevez, A. G.; Jordan, J. Nitric Oxide

and Superoxide, A Deadly Cocktail. Ann. N.Y. Acad. Sci. 2002,

962, 207–211.

(11) (a) Marletta, M. A. Approaches toward Selective Inhibition of

Nitric Oxide Synthase. J. Med. Chem. 1994, 37 (13), 1899–1907.

(b) Vallance, P.; Leiper, J. Blocking NO Synthesis: How, Where and

Why? Nature Rev. Drug. Discovery 2002, 1 (12), 939–950.

(12) (a) Huang, P. L.; Huang, Z.; Mashimo, H.; Bloch, K. D.; Moskowitz,

M. A.;Bevan, J. A.;Fishman, M. C. HypertensioninMiceLackingthe

Gene for Endothelial Nitric Oxide Synthase. Nature 1995, 377 (6546),

239–242. (b) Endres, M.; Laufs, U.; Liao, J. K.; Moskowitz, M. A. Targeting

eNOS for Stroke Protection. Trends Neurosci. 2004, 27 (5), 283–289. (c)

Braam, B.; Verhaar, M. C. Understanding eNOS for Pharmacological

Modulation of Endothelial Function: A Translational View. Curr. Pharm.

Des. 2007, 13 (17), 1727–1740.

(13) Kim, S. F.; Huri, D. A.; Snyder, S. H. Inducible Nitric Oxide

Synthase Binds, S-Nitrosylates, and Activates Cyclooxygenase-2.

Science 2005, 310 (5756), 1966–1970.

(14) Wilcock, D. M.; Lewis, M. R.; Van Nostrand, W. E.; Davis, J.;

Previti, M. L.; Gharkholonarehe, N.; Vitek, M. P.; Colton, C. A.

Progression of Amyloid Pathology to Alzheimer’s Disease Pathol-

ogy in an Amyloid Precursor Protein Transgenic Mouse Model by

Removal of Nitric Oxide Synthase 2. J. Neurosci. 2008, 28 (7),

1537–1545.

(15) (a) Salerno, L.; Sorrenti, V.; Di Giacomo, C.; Romeo, G.; Siracusa,

M. A. Progress in the Development of Selective Nitric Oxide

Synthase (NOS) Inhibitors. Curr. Pharm. Des. 2002, 8 (3), 177–

200. (b) Erdal, E. P.; Litzinger, E. A.; Seo, J.; Zhu, Y.; Ji, H.; B., S. R.

Selective Neuronal Nitric Oxide Synthase Inhibitors. Curr. Top. Med.

Chem. 2005, 5 (7), 603–624. (c) Tafi, A.; Angeli, L.; Venturini, G.;

Travagli, M.; Corelli, F.; Botta, M. Computational Studies of Compe-

titive Inhibitors of Nitric Oxide Synthase (NOS) Enzymes: Towards the

Development of Powerful and Isoform-Selective Inhibitors. Curr.

Med. Chem. 2006, 13 (16), 1929–1946.

ꢀ

Chem. 1999, 42 (16), 3147–3153. (b) Hah, J.-M.; Roman, L. J.; Martasek,

P.; Silverman, R. B. Reduced Amide Bond Peptidomimetics. (4S)-N-(4-

Amino-5-[aminoakyl]aminopentyl)-N⁰-nitroguanidines, Potent and Highly

Selective Inhibitors of Neuronal Nitric Oxide Synthase. J. Med. Chem.

ꢀ

2001, 44 (16), 2667–2670. (c) Hah, J.-M.; Martasek, P.; Roman, L. J.;

Silverman, R. B. Aromatic Reduced Amide Bond Peptidomimetics as

Selective Inhibitors of Neuronal Nitric Oxide Synthase. J. Med. Chem.

ꢀ

2003, 46 (9), 1661–1669. (d) Gomez-Vidal, J. A.; Martasek, P.; Roman,

L. J.; Silverman, R. B. Potent and Selective Conformationally Restricted

Neuronal Nitric Oxide Synthase Inhibitors. J. Med. Chem. 2004, 47 (3),

ꢀ

703–710. (e) Ji, H.; Gomez-Vidal, J. A.; Martasek, P.; Roman, L. J.;

Silverman, R. B. Confomationally Restricted Dipeptide Amide as Potent

and Selective Neuronal Nitric Oxide Synthase Inhibitors. J. Med. Chem.

2006, 49 (21), 6254–6263. (f) Seo, J.; Igarashi, J.; Li, H.; Martasek, P.;

Roman, L. J.; Poulos, T. L.; Silverman, R. B. Structure-Based Design and

Synthesis of N^ω-Nitro-L-Arginine-Containing Peptidomimetics as Selective

Inhibitors of Neuronal Nitric Oxide Synthase. Displacement of the Heme

Structural Water. J. Med. Chem. 2007, 50 (9), 2089–2099.

(20) (a) Flinspach, M. L.; Li, H.; Jamal, J.; Yang, W.; Huang, H.; Hah,

ꢀ

J.-M.; Gomez-Vidal, J. A.; Litzinger, E. A.; Silverman, R. B.;

Poulos, T. L. Structural Basis for Dipeptide Amide Isoform-

Selective Inhibition of Neuronal Nitric Oxide Synthase. Nature

Struct. Mol. Biol. 2004, 11 (1), 54–59. (b) Flinspach, M.; Li, H.; Jamal,

J.; Yang, W.; Huang, H.; Silverman, R. B.; Poulos, T. L. Structures of the

Neuronal and Endothelial Nitric Oxide Synthase Heme Domain with

D-Nitroarginine-Containing Dipeptide Inhibitors Bound. Biochemistry

2004, 43 (18), 5181–5187. (c) Li, H.; Flinspach, M. L.; Igarashi, J.;

ꢀ

Jamal, J.; Yang, W.; Gomez-Vidal, J. A.; Litzinger, E. A.; Huang, H.;

Erdal, E. P.; Silverman, R. B.; Poulos, T. L. Exploring the Binding

Conformations of Bulkier Dipeptide Amide Inhibitors in Constitutive

Nitric Oxide Synthases. Biochemistry 2005, 44 (46), 15222–15229.

(21) Ji, H.; Li, H.; Flinspach, M.; Poulos, T. L.; Silverman, R. B.

Computer Modeling of Selective Regions in the Active Site of

Nitric Oxide Synthases: Implication for the Design of Isoform-

Selective Inhibitors. J. Med. Chem. 2003, 46 (26), 5700–5711.

ꢀ

(22) Ji, H.; Stanton, B. Z.; Igarashi, J.; Li, H.; Martasek, P.; Roman,

L. J.; Poulos, T. L.; Silverman, R. B. Minimal Pharmacophoric

Elements and Fragment Hopping, An Approach Directed at

Molecular Diversity and Isozyme Selectivity. Design of Selective

Neuronal Nitric Oxide Synthase Inhibitors. J. Am. Chem. Soc.

2008, 130 (12), 3900–3914.

ꢀ

(23) Ji, H.; Li, H.; Martasek, P.; Roman, L. J.; Poulos, T. L.; Silverman,

R. B. Discovery of Highly Potent and Selective Inhibitors of

Neuronal Nitric Oxide Synthase by Fragment Hopping. J. Med.

Chem. 2009, 52 (3), 779–797.

(24) Lawton, G. R.; Ralay Ranaivo, H.; Chico, L. K.; Ji, H.; Xue, F.;

ꢀ

Martasek, P.; Roman, L. J.; Watterson, D. M.; Silverman, R. B.

Analogues of 2-Aminopyridine-Based Selective Inhibitors of

Neuronal Nitric Oxide Synthase with Increased Bioavailability.

Bioorg. Med. Chem. 2009, 17 (6), 2371–2380.

ꢀ

(25) Ji, H.; Tan, S.; Igarashi, J.; Li, H.; Derrick, M.; Martasek, P.;

Roman, L. J.; Vasquez-Vivar, J.; Poulos, T. L.; Silverman, R. B.

ꢀ

Selective Neuronal Nitric Oxide Synthase Inhibitors and the

Prevention of Cerebral Palsy. Ann. Neurol. 2009, 65 (2), 209–217.

(26) Delker, S. L.; Ji, H.; Li, H.; Jamal, J.; Fang, J.; Xue, F.; Silverman,

R. B.; Poulos, T. L. Unexpected Binding Modes of Nitric Oxide

Synthase Inhibitors Effective in the Prevention of a Cerebral Palsy

Phenotype in an Animal Model. J. Am. Chem. Soc. 2010, 132 (15),

5437–5442.

(27) Igarashi, J.; Li, H.; Jamal, J.; Ji, H.; Fang, J.; Lawton, G. R.;

Silverman, R. B.; Poulos, T. L. Crystal Structures of Constitutive

Nitric Oxide Synthases in Complex with de Novo Designed

Inhibitors. J. Med. Chem. 2009, 52 (7), 2060–2066.

(28) Faraci, W. S.; Nagel, A. A.; Verdries, K. A.; Vincent, L. A.; Xu, H.;

Nichols, L. E.; Labasi, J. M.; Salter, E. D; Pettipher, E. R. 2-Amino-4-

methylpyridine as a Potent Inhibitor of Inducible NO Synthase

Activity in vitro and in vivo. Br. J. Pharmacol. 1996,119 (6), 1101–1108.

(29) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart,

W. E.; Belew, R. K.; Olson, A. J. Automated Docking using a

Lamarckian Genetic Algorithm and an Empirical Binding Free

Energy Function. J. Comput. Chem. 1998, 19 (14), 1639–1662.

(30) Lawton, G. R.; Ji, H.; Silverman, R. B. Remote Protection

Prevents Unwanted Cyclizations with 2-Aminopyridines. Tetrahe-

dron Lett. 2006, 47 (34), 6113–6115.

(16) Garcin, E. D.; Arvai, A. S.; Rosenfeld, R. J.; Kroeger, M. D.; Crane,

B. R.; Andersson, G.; Andrews, G.; Hamley, P. J.; Mallinder, P. R.;

Nicholls, D. J.; St-Gallay, S. A.; Tinker, A. C.; Gensmantel, N. P.;

˚

Mete, A.; Cheshire, D. R.; Connolly, S.; Stuehr, D. J.; Aberg, A.;

Wallace, A. V.; Tainer, J. A.; Getzoff, E. D. Anchored Plasticity Opens

Doors for Selective Inhibitor Design in Nitric Oxide Synthase. Nature

Chem. Biol. 2008, 4 (11), 700–707.

(17) (a) Crane, B. R.; Arvai, A. S.; Ghosh, D. K.; Wu, C.; Getzoff, E. D.;

Stuehr, D. J.; Tainer, J. A. Structure of Nitric Oxide Synthase

Oxygenase Dimer with Pterin and Substrate. Science 1998, 279

ꢀ

(5359), 2121–2126. (b) Raman, C. S.; Li, H.; Martasek, P.; Kral, V.;

Masters, B. S.; Poulos, T. L. Crystal Structure of Constitutive Endo-

thelial Nitric Oxide Synthase: A Paradigm for Pterin Function Involving

a Novel Metal Center. Cell 1998, 95 (7), 939–950. (c) Fischmann, T. O.;

Hruza, A.; Niu, X. D.; Fossetta, J. D.; Lunn, C. A.; Dolphin, E.;

Prongay, A. J.; Reichert, P.; Lundell, D. J.; Narula, S. K.; Weber, P. C.

Structural Characterization of Nitric Oxide Synthase Isoforms Reveals

Striking Active-Site Conservation. Nature Struct. Biol. 1999, 6 (3),

233–242. (d) Li, H.; Shimizu, H.; Flinspach, M.; Jamal, J.; Yang, W.;

Xian, M.; Cai, T.; Wen, E. Z.; Jia, Q.; Wang, P. G.; Poulos, T. L. The

Novel Binding Mode of N-Alkyl-N⁰-Hydroxyguanidine to Neuronal

Nitric Oxide Synthase Provides Mechanistic Insights into NO Biosynth-

esis. Biochemistry 2002, 41 (47), 13868–13875.

(31) 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. 2002, 43 (20), 3714–3717.

(32) Insight II 2000; Accelrys Inc.: 10188 Telesis Court, Suite 100,

San Diego, CA 92121; phone, (858) 799-5000; fax, (858) 799-5100;

http://www.accelrys.com/.

(18) Silverman, R. B. Design of Selective Neuronal Nitric Oxide

Synthase Inhibitors for the Prevention and Treatment of Neuro-

degenerative Diseases. Acc. Chem. Res. 2009, 42 (3), 439–451.

ꢀ

(19) (a) Huang, H.; Martasek, P.; Roman, L. J.; Masters, B. S.; Silverman,

R. B. N^ω-Nitroarginine-Containing Dipeptide Amides. Potent and

7824 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Ji et al.

(33) SYBYL 6.8; Tripos, Inc.: 1699 South Hanley Road St. Louis,

MO 63144-2319; phone, (800) 323-2960; fax, (314) 647-9241; http://

www.tripos.com/.

(34) GRID 22; Molecular Discovery: Via Stoppani, 38, 06087 Ponte San

Giovanni PG, Italy; phone and fax, þ39-075-397411; http://

www.moldiscovery.com/.

of Neuronal Nitric Oxide Synthase by Limited Proteolysis. Bio-

chem. J. 1996, 314 (Pt 1), 55–62.

(43) McPhillips, T. M.; McPhillips, S. E.; Chiu, H. J.; Cohen, A. E.;

Deacon, A. M.; Ellis, P. J.; Garman, E.; Gonzalez, A.; Sauter,

N. K.; Phizackerley, R. P.; Soltis, S. M.; Kuhn, P. Blu-Ice and the

Distributed Control System: Software for Data Acquisition and

Instrument Control at Macromolecular Crystallography Beam-

lines. J. Synchrotron Radiat. 2002, 9 (Pt 6), 401–406.

(44) Otwinowski, Z.; Minor, W. Processing of X-ray Diffraction Data

Collected in Oscillation Mode. Methods Enzymol. 1997, 276, 307–

326.

(45) Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros,

P.; Grosse-Kunstleve, R. W.; Jiang, J.-S.; Kuszewski, J.; Nilges,

M.; Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren,

G. L. Crystallography & NMR System: A New Software Suite for

Macromolecular Structure Determination. Acta Crystallogr., Sect.

D: Biol. Crystallogr. 1998, 54, 905–921.

(35) Hevel, J. M.; White, K. A.; Marletta, M. A Purification of the

Inducible Murine Macrophage Nitric Oxide Synthase. J. Biol.

Chem. 1991, 266 (34), 22789–22791.

ꢀ

(36) Roman, L. J.; Sheta, E. A.; Martasek, P.; Gross, S. S.; Liu, Q.;

Masters, B. S. S. High-Level Expression of Functional Rat Neu-

ronal Nitric Oxide Synthase in Escherichia coli. Proc. Natl. Acad.

Sci. U.S.A. 1995, 92 (18), 8428–8432.

(37) Gerber, N. C.; Ortiz de Montellano, P. R. Neuronal Nitric Oxide

Synthase: Expression in Escherichia coli, Irreversible Inhibition

by Phenyldiazene, and Active Site Topology. J. Biol. Chem. 1995,

270 (30), 17791–17796.

(38) Martasek, P.; Liu, Q.; Roman, L. J.; Gross, S. S.; Sessa, W. C.;

Masters, B. S. S. Characterization of Bovine Endothelial Nitric

Oxide Synthase Expressed in Escherichia coli. Biochem. Biophys.

Res. Commun. 1996, 219 (2), 359–365.

(46) Jones, T. A.; Zou, J.-Y.; Cowan, S. W.; Kjeldgaarrd, M. Improved

Methods for Building Models in Electron Density and the Location

of Errors in These Models. Acta Crystallogr., Sect. A: Found.

Crystallogr. 1991, 47 (2), 110–119.

(47) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular

graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60,

2126–2132.

(48) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of

Macromolecular Structures by the Maximum-Likelihood Method.

Acta Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 240–255.

(49) Winn, M. D.; Isupov, M. N.; Murshudov, G. N. Use of TLS

Parameters to Model Anisotropic Displacements in Macromole-

cular Refinement. Acta Crystallogr., Sect. D: Biol. Crystallogr.

2001, 57, 122–133.

(39) Hevel, J. M.; Marletta, M. A. Nitric Oxide Synthase Assays.

Methods Enzymol. 1994, 233, 250–258.

(40) Segel, I. H. Enzyme Kinetics; John Wiley and Sons: NewYork,

1975; p 105.

(41) Li, H.; Igarashi, J.; Jamal, J.; Yang, W.; Poulos, T. L.

Structural Studies of Constitutive Nitric Oxide Synthases with

Diatomic Ligands Bound. J. Biol. Inorg. Chem. 2006, 11 (6), 753–

768.

(42) Lowe, P. N.; Smith, D.; Stammers, D. K.; Riveros-Moreno, V.;

Moncada, S.; Charles, I.; Boyhan, A. Identification of the Domains

Article Doi

Exploration of the active site of neuronal nitric oxide synthase by the design and synthesis of pyrrolidinomethyl 2-aminopyridine derivatives

DOI: 10.1021/jm100947x

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm100947x

Full text of DOI:10.1021/jm100947x

Products guided by the article

R&D Labs maybe for 1018910-20-7

Relevant to this article

Hot Product