Synthesis, biological evaluation, and modeling studies of inhibitors aimed at the malarial proteases plasmepsins I and II

Bioorganic & Medicinal Chemistry 13 (2005) 5371–5390

Synthesis, biological evaluation, and modeling studies

of inhibitors aimed at the malarial proteases plasmepsins I and II

Daniel Muthas,^aDaniel No¨teberg,^aYogesh A. Sabnis,^aElizabeth Hamelink,^b

b

Lotta Vrang, Bertil Samuelsson, Anders Karlen and Anders Hallberg

b

a

a,

*

´

^aDepartment of Medicinal Chemistry, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden

^bMedivir AB, Lunastigen 7, SE-141 44 Huddinge, Sweden

Received 3 February 2005; accepted 10 June 2005

Available online 27 July 2005

Abstract—The increasing resistance of the malarial parasite to antimalarial drugs is a major contributor to the reemergence of the

disease and increases the need for new drug targets. The two aspartic proteases, plasmepsins I and II, from Plasmodium falciparum

have recently emerged as potential targets. In an eﬀort to inhibit these hemoglobinases, a series of inhibitors encompassing a basic

hydroxyethylamine transition state isostere as a central fragment were prepared. The synthesized compounds were varied in the P1⁰

position and exhibited biological activities in the range of 31 to >2000 nM. To try to rationalize the results, molecular docking and

3D-QSAR analysis were used.

1. Introduction

in the digestive food vacuole of the parasite, cleave

hemoglobin and have generated interest as antimalarial

drug targets.⁹These plasmepsins exhibit 73% sequence

identity, suggesting that a single compound might be

able to eﬃciently inhibit both of them. In this context,

it is important to emphasize that it was recently demon-

strated that all four plasmepsins have overlapping func-

tions and that the parasite can survive even when only

one of the plasmepsins is inhibited.¹⁰Plm I and Plm II

also show a rather high structural similarity (>50%) to

the human protease cathepsin D (Cat D), suggesting

potential problems with selectivity.^11–13In 1996, Silva

et al. reported the crystal structure of Plm II in complex

with pepstatin A, which is a potent inhibitor with a

K_i= 0.006 nM.¹⁴With pepstatin A as a lead, a series

of phenylalanine–statin analogues were synthesized

and among these the picoline derivative 1, exerting an

impressive Plm II activity (K_i= 0.56 nM) and a 38-fold

selectivity over Cat D, was identiﬁed (Fig. 1).¹⁴More

recently, Ellmanꢀs group used several iterative focused

libraries to generate potent and selective Plm II

inhibitors, for example, 2.¹⁵Importantly, they also re-

vealed that large P1⁰substituents seemed to be easily

accommodated in the ﬂexible S1⁰site of Plm II.

Malaria is considered to be the most serious tropical

parasitic disease in the world, killing more people each

year than any other contagious disease except tuberculo-

sis. It is spread by the bite of a female Anopheles mosqui-

to and is caused by protozoan parasites of the genus

Plasmodium. It is estimated that there are at least 400

million clinical cases every year, with at least 1 million

fatalities.¹Mortality is highest among young children

and greatly exceeds the number of deaths from AIDS.

The species responsible for almost all fatal cases is Plas-

modium falciparum. Some P. falciparum strains have

now been identiﬁed that are resistant to all known anti-

malarial drugs except artemisinin derivatives.²This

problem of resistance has increased the need for the dis-

covery of new macromolecular targets for malaria. The

P. falciparum genome hosts several proteases involved in

the degradation of hemoglobin,³among these are the

cysteine proteases, metalloproteases, and the aspartic

proteases constituting plasmepsin I (Plm I), plasmepsin

II (Plm II), plasmepsin IV (Plm IV), and histoaspartic

protease (HAP).^4–9Plm I and Plm II, which are present

Since it is well-established that basic antimalarials like

chloroquine accumulate in the acidic food vacuole

of the parasite,¹⁶we decided to make Plm I and

Plm II inhibitors containing a basic nitrogen. Hence,

Keywords: Malaria; Plasmepsin; Hydroxyethylamine; Molecular

modeling.

*

Corresponding author. Tel.: +46 18 471 42 84; fax: +46 18 471 44

74; e-mail: anders.hallberg@orgfarm.uu.se

doi:10.1016/j.bmc.2005.06.048

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D. Muthas et al. / Bioorg. Med. Chem. 13 (2005) 5371–5390

Figure 1. Two potent Plm II inhibitors (1 and 2) shown together with two earlier synthesized compounds with diﬀerent inhibitory potencies,

indicating the importance of a large P1⁰side chain (3 and 4), and the generic structure of the inhibitors presented in this work (5).

compounds with the following characteristics were

synthesized: (a) a basic hydroxyethylamine transition

state mimicking scaﬀold that previously had been used

successfully, for example, renin and HIV-protease

inhibitors,^17–19(b) alkylphenyl groups or alkylbiphenyl

groups as P1⁰substituents, and (c) a non-prime side

and a C-terminal primary amide group derived from

the potent lead compound 1.¹⁴We previously demon-

strated that some of the compounds with these charac-

teristic features, exempliﬁed by 3, not only showed

submicromolar aﬃnity toward Plm I and Plm II but also

exerted good inhibitory eﬀect on the growth of the

parasite in infected red blood cells.²⁰In agreement with

Ellmanꢀs previous ﬁndings, we conﬁrmed that large

P1⁰substituents were required for activity also in this

series of inhibitors as shown by the replacement of the

4-phenyl benzyl group of inhibitor 3 for a benzyl group,

that delivered compound 4 devoid of plasmepsin inhib-

itory activity.

analogues of 5 and the previously reported C-substitut-

ed analogues related to 3 and 4^20,21using the docked

poses as alignment.

2. Results and discussion

2.1. Chemistry

The ﬁrst step in the synthesis used the epoxide 6a (pre-

pared according to the method of Romeo and Rich)²²

and in the case of the synthesis of the epimer 8c, epoxide

19

˚

6b (prepared according to the method of Branalt et al).

These epoxides stirred with four diﬀerent primary

amines (7a–7e, where 7b = 7c) in isopropanol at 50 ꢁC

overnight aﬀorded 8a–8e (Scheme 1). These secondary

amines were alkylated with methyl bromoacetate which

yielded a mixture of the methyl ester and the lactone, the

latter formed after a subsequent ring closure. The

mixture was directly allowed to react with ammonia-

saturated methanol to deliver the primary amides

9a–e. The Boc groups were removed using an acidic

solution and the resulting primary amines were coupled

with Boc-protected valine using TBTU as coupling

agent to give compounds 10a–10e. After Boc deprotec-

tion, the resulting amines were reacted with picolinic

acid, again with TBTU as the coupling agent, to deliver

the target compounds 11a–11e.

We speculated that the transfer, for example, the large

biphenyl P1⁰substituent from C-a of 3 to a potentially

protonated sp³-hybridized nitrogen would preserve the

interactions of P1⁰with the enzyme which could result

in inhibitors with similar or better activities. To probe

this hypothesis, a series of compounds with the generic

structure 5 have now been synthesized (these will be re-

ferred to as N-substituted and those with the P1⁰substit-

uents at C-a as C-substituted). We herein report that, to

our surprise, the transfer of the 4-phenylbenzyl group of

3 and other similar large substituents to the adjacent

nitrogen delivered inactive compounds, while migration

of a small benzyl group aﬀorded a Plm II inhibitor (11b;

K_i= 46 nM), which was even more potent than 3

(K_i= 117 nM). In an attempt to rationalize these unex-

pected ﬁndings, we docked the compounds into the

active site of Plm II and evaluated their docking poses.

Furthermore, to obtain a quantitative understanding

of the SAR of these inhibitors a 3D-QSAR analysis

was performed that included both the N-substituted

Since we had previously observed that extensions and

enlargements in the P1⁰site of the related inhibitors

(e.g., 3) signiﬁcantly improved the inhibition of Plm I

and II,^20,21we prepared compounds 11f–11h utilizing

the procedure in Scheme 1. These compounds diﬀer

from 8b and 8d in that the phenyl ring on the P1⁰side

chain is substituted with bromine serving as a handle

for the substitutions in the para (11f and 11h) and meta

(11g) positions. From these bromo compounds, the

respective phenyl-substituted compounds 12a–12c were

synthesized applying a Suzuki protocol (Pd(PPh₃)₂Cl₂,

D. Muthas et al. / Bioorg. Med. Chem. 13 (2005) 5371–5390

5373

Scheme 1. Reagents and conditions: (i) isopropanol, overnight, 50 ꢁC; (ii) K₂CO₃, methyl bromoacetate, DMF, overnight, rt; (iii) NH₃(satd),

MeOH, overnight, rt; (iv) HCl, EtOAc, 1 h, rt; (v) TFA, CH₂Cl₂, rt, 15 min; (vi) BocValOH, TBTU, DIEA, DMF, 2 h, rt; (vii) picolinic acid, TBTU,

c

DIEA, DMF, 2 h, rt. ^bAfter reaction with 6a. After reaction with 6b.

PhB(OH)₂, and Cs₂CO₃) under microwave irradiation

(Scheme 2).^23,24Under these conditions, the Suzuki cou-

pling was accompanied with hydrolysis of the primary

amide bond as deduced from LC–MS. The formation

of carboxylic acid was probably attributed to an intra-

molecular attack of the hydroxyl group at the amide car-

bonyl group to form a lactone that was subsequently

hydrolyzed by traces of water. Therefore, the crude reac-

tion mixture, after ﬁltering and evaporating, was sub-

jected to acidic methanol to obtain the methyl ester.

The crude product was thereafter stirred in an ammo-

nia-saturated methanol solution to provide product

12a in an excellent overall yield. For the synthesis of

12b and 12c a slightly modiﬁed condition was employed

wherein Cs₂CO₃was replaced by NaHCO₃. This modi-

ﬁcation suppressed the amide hydrolysis completely,

although the yield for these reactions dropped consider-

ably from 95% for 12a to 10% for both 12b and 12c.

To assess the importance of the terminal amide func-

tionality for binding, we prepared compound 16

(Scheme 3). Thus, compound 8b was stirred with Cbz-

Cl and K₂CO₃in a mixture of water and CH₂Cl₂to give

the protected amine 13 in 52% yield. The Boc group of

compound 13 was removed using a mixture of TFA

and CH₂Cl₂, and the resulting primary amine was cou-

pled with BocValOH utilizing TBTU as a coupling

agent to aﬀord the amide 14 in 50% yield. The Boc

Scheme 2. Reagents and conditions: (i) PhB(OH)₂, Cs₂CO₃, Pd(PPh₃)₂Cl₂, DME, EtOH, 20 min, 130 ꢁC; (ii) HCl, MeOH, rt, overnight; (iii) NH₃

(satd), MeOH, rt, overnight; (iv) PhB(OH)₂, NaHCO₃, Pd(PPh₃)₂Cl₂, DME, EtOH, 20 min, 140 ꢁC.

5374

D. Muthas et al. / Bioorg. Med. Chem. 13 (2005) 5371–5390

Scheme 3. Reagents and conditions: (i) Cbz-Cl, K₂CO₃, H₂O, CH₂Cl₂, rt, 2 h; (ii) TFA, CH₂Cl₂, rt, 15 min; (iii) BocValOH, TBTU, DIEA, DMF,

2 h, rt; (iv) picolinic acid, TBTU, DIEA, DMF, 2 h, rt; (v) TfOH, anisol, CH₂Cl₂, rt, 15 min.

group of compound 14 was removed, the resulting

amine was coupled to picolinic acid, and the amide 15

was isolated in 74% yield. The Cbz group was removed

using triﬂic acid in CH₂Cl₂with anisol as a scavenger to

ﬁnally deliver the secondary amine 16 in 38% yield.

C-substituted analogue 24 has a K_iof 98 and 540 nM

against Plm I and Plm II, respectively. We further ob-

served that the phenylethyl derivative 11d, which has

an ethylene bridge, exhibited lower activity in Plm I

and Plm II as compared to the methylene-bridged 11b.

Further elongation of the side chain to phenylpropyl,

11e, resulted in complete loss of activity. Compound

11b is also more potent than 11a, which comprises an

isobutyl group in the place of the benzyl. To ﬁnd out

whether the stereochemistry at the hydroxyl carbon

was critical, compound 11c was synthesized. This epimer

of 11b with inverted stereochemistry at the hydroxyl-

bearing carbon was inactive, suggesting that the S-con-

ﬁguration is the preferred conﬁguration in this position.

The inhibitor 11b exhibited the highest activity of all

N-substituted compounds toward the plasmepsins,

however, it showed no selectivity for the plasmepsins

over Cat D, whereas 11a showed a 30-fold selectivity

for the plasmepsins over Cat D.

For preparation of 19, the following procedure was em-

ployed: compound 17 (the 4-bromo benzyl equivalent of

3) was treated with Cs₂CO₃and benzyl bromide in

DMF to give the tertiary amine 18 (Scheme 4). The aryl

bromide 18 was subjected to the Suzuki protocol with

phenylboronic acid (vide supra) to provide compound

19, which is a hybrid of the dual Plm I and Plm II inhib-

itors 3 and 11b.

2.2. Biological evaluation

The results from the enzyme inhibition studies of the

compounds in the Plm I, Plm II, and Cat D assays

are presented in Table 1. A comparison between the

biological activity of 3 and the analogues 12a–12c

revealed that the SAR diﬀers in the two series (C- and

N-substituted). While 3 requires the biphenyl in P1⁰

to achieve higher aﬃnity, this biphenyl on 12a–12c

renders these molecules inactive against Plm I and Plm

II (K_i> 1000 nM), except for 12b with a Plm I

K_i= 350 nM. The bromo-substituted compounds (11f–

11h), which are intermediates in the synthesis of the

phenyl-substituted analogues (12a–12c), were also inac-

tive in the plasmepsin assays. This again points out the

diﬀerence in the SAR between the two series, since the

The terminal amide group was also found to be critical

for activity, since the secondary amine 16, which lacks

this functionality, was completely devoid of any plasm-

epsin activity (K_i> 2000 nM). On the contrary 15, an

intermediate in the synthesis of 16, possessing an ester

functionality showed some activity with K_iof 800 and

380 nM toward Plm I and Plm II, respectively. Combin-

ing the P1⁰of the most active N-substituted inhibitor

11b with the P1⁰of 3 and 24 gave 19 and 18, respectively.

Both these inhibitors had reduced activities (18, K_iPlm

I = 573 nM and K_iPlm II = 833 nM and 19, K_iPlm

Scheme 4. Reagents and conditions: (i) Cs₂CO₃, BnBr, DMF, 50 ꢁC, overnight; (ii) PhB(OH)₂, Cs₂CO₃, Pd(PPh₃)₂Cl₂, DME, EtOH, 20 min, 130 ꢁC.

D. Muthas et al. / Bioorg. Med. Chem. 13 (2005) 5371–5390

5375

Table 1. Biological activities of the compounds in this study

Structure

Compound

K_i(nM)

Plm I

Plm II

Cat D

3^a

68

117

ꢀ3200

220

>2000

>2800

>6000

38

4^a

ꢀ2100

11a

170

11b^b

31

46

11c

650

>2000

>5900

11d

ꢀ1900

ꢀ900

480

11e

>2000

11f

1600

>2000

1012

(continued on next page)

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D. Muthas et al. / Bioorg. Med. Chem. 13 (2005) 5371–5390

Table 1 (continued)

Structure

Compound

K_i(nM)

Plm I

Plm II

Cat D

>2000

11g

1200

>2000

11h

>2000

12a

12b

12c

>2000

350

1300

>2000

1600

15

ꢀ800

ꢀ380

>2000

16

>2000

D. Muthas et al. / Bioorg. Med. Chem. 13 (2005) 5371–5390

5377

Table 1 (continued)

Structure

Compound

K_i(nM)

Plm I

Plm II

Cat D

18

573

833

>2900

19

220

650

>2900

20^a

>2000

21^a

ꢀ2900

ꢀ3300

>2800

>2000

22^a

>2000

23^a

24^a

25^a

26^a

ꢀ8000

ꢀ3500

>2000

ꢀ1700

ꢀ1000

98

540

115

121

63

150

(continued on next page)

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D. Muthas et al. / Bioorg. Med. Chem. 13 (2005) 5371–5390

Table 1 (continued)

Structure

Compound

K_i(nM)

Plm I

Plm II

Cat D

27^a

437

379

ꢀ1700

28^a

1300

1430

>2000

29^a

30^a

31^a

1100

1000

530

930

579

129

1900

1600

>2000

32^c

33^c

34^c

170

360

>2900

23

52

1933

13

30

1400

D. Muthas et al. / Bioorg. Med. Chem. 13 (2005) 5371–5390

5379

Table 1 (continued)

Structure

Compound

K_i(nM)

Plm I

Plm II

Cat D

35^c

36^c

37^c

38^c

39^c

220

490

>2900

300

99

92

42

110

600

1300

610

130

>5900

>2900

^aCompounds from No¨teberg et al.²⁰

^bPermeability on Caco 2-cells determined; P_appvalue = 4.5 · 10^-6cm/s; fair permeability (20–75%).

^cCompounds from No¨teberg et al.²¹

I = 220 nM and K_iPlm II = 650 nM) compared to their

parent compounds (24, K_iPlm I = 98 nM and K_iPlm

II = 540 nM), indicating that the disubstitution had no

synergistic eﬀect. The potent Plm I and II inhibitor

11b was evaluated in the Caco-2 cell assay,²⁵and

although this compound contains three amide bonds

and one hydroxyl group it still exhibited fair (20–75%)

cell penetration properties.

of choice. The residues Tyr77-Ser79, Gly216-Ser218,

and Ile290-Val296 were allowed to move under con-

straints during docking.

The resulting poses were similar in their backbone orien-

tation and showed good overlap with the cocrystallized

inhibitors EH58 and rs370.^14,27,28The H-bonding pat-

tern seen for the docked inhibitors, exempliﬁed by 11b

in Figure 2, was also similar to the pattern reported

for the known cocrystallized ligands. The binding modes

of the ligands can be generalized as follows: The transi-

tion state mimicking hydroxyl group of the hydroxyeth-

ylamine was found between the catalytic aspartic acids,

Asp34 and Asp214, forming an H-bond with the carbox-

ylate oxygens of the aspartates at a distance of around

2.3. Docking

Docking studies were performed only on Plm II. The

large and solvent exposed active site of this enzyme

makes docking a challenging task. The problem be-

comes even more diﬃcult due to the large number of

rotatable bonds (ꢀ15) present in the inhibitors and the

conformational changes that are known to occur in

the protein upon ligand binding.¹⁴To take the protein

ﬂexibility into account, we chose to use the docking soft-

ware FLO by McMartin and Bohacek,²⁶which allows

user-deﬁned constraints to be assigned to the residues

˚

2.2 A. The P1 benzyl side chain showed p-interactions

with the aromatic rings of Phe111, Phe120, and Tyr77

and was stacked with the side chain of Ile123. The P1

˚

amide hydrogen was found at about 2.0 A from the

backbone carbonyl oxygen of Gly216. The carbonyl

˚

oxygen of P2 valine showed an H-bond (2.7–2.9 A) with

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D. Muthas et al. / Bioorg. Med. Chem. 13 (2005) 5371–5390

The interactions in the S2⁰pocket are weak and primar-

ily electrostatic in nature. Residues Val78 and Asn76

show H-bonds with the terminal amide. In case of com-

pound 11b, the carbonyl of the terminal amide is at an

˚

H-bonding distance of 1.7 A from backbone amide of

Val78, while the amide hydrogen forms an H-bond with

backbone carbonyl of Asn76. The latter interaction is

not seen in the case of 15 (K_i= ꢀ380 nM), while 16

(K_i> 2000 nM) lacks both of the above-mentioned

interactions due to the absence of this terminal amide

group, which could explain their reduced activities.

Compounds 18 and 19 were prepared to probe whether

the diﬀerent requirements for the P1⁰side chain in these

series may be due to the side chains binding to diﬀerent

sites in the enzyme. The docked pose of 18 shows that

the N-benzyl group ﬁts into the S1⁰site, while the p-bro-

mobenzyl lies in the S2⁰pocket close to Tyr192 and

Leu131. On the contrary, 19 has the biphenyl group

reaching into the S1⁰site in accordance with observed

docked pose of compound 3, while the N-benzyl occu-

pies the S2⁰site. It was seen that the accommodation

of these substituents in the S2⁰pocket forced the termi-

nal amide out of the pocket resulting in a loss of inter-

action with Asn76, while maintaining the H-bond with

Val78. This could provide an explanation for the drop

in their activity (18, K_i= 833 nM and 19, K_i= 650 nM),

once again demonstrating the importance of the termi-

nal amide interactions.

Figure 2. Schematic representation of H-bonding network for 11b.

the hydroxyl group of Ser79. Hydrophobic interactions

were observed between the P2 side chain and residues

Thr217, Val78, and Ile290. The 2-pyridinecarboxamide

(P3 substituent) resides in the S3 pocket close to residues

Ser79 and Thr114, while the amide nitrogen makes an

H-bond to the c-O of Ser79. We also observed p-stack-

ing between the aromatic P1 and P3 rings, which is in

accordance with the X-ray structure of 1W6H.²⁹

Large P1⁰substituents were shown to accommodate well

in the S1⁰pocket of Plm II¹⁵as has also been pointed out

by our prior investigations.²⁰This was illustrated by the

increase in the activity of compound 3, having a

K_i= 117 nM, compared to 4, with a K_iꢀ 3200 nM.

The docking studies showed that the P1⁰groups of the

C-substituted inhibitors 3, 4, and 20–39^20,21were located

in the S1⁰pocket in van der Waals contact with Leu292

and Pro295. These poses were in agreement with the ob-

served binding of the P1⁰of EH58 in Plm II.²⁸It was

seen that large P1⁰substituents could easily ﬁt in the

open S1⁰site, once again demonstrating that large P1⁰

substituents are well tolerated in this pocket. The dock-

ing modes of the N-substituted compounds were similar

to the C-substituted ones, but these inhibitors exhibited

a reverse trend in the activities, that is, the smaller P1⁰

had higher activity than the larger P1⁰. This could in

part be due to the observation that the smaller P1⁰sub-

stituents (11a and 11b) occupied a diﬀerent region in the

S1⁰pocket, where they were surrounded by Ile212,

Phe294, and Ile300. The binding mode of the P1⁰substit-

uents is in accordance with the one seen for inhibitor

rs370 in 1LF2 bearing a small P1⁰substituent.²⁷Fur-

thermore, a closer inspection of the poses revealed a pos-

sible p-interaction of the P1⁰group of 11b with Phe294

which could explain its higher activity compared to

11a which lacks this interaction. We also observed that

the large substituents on some of the N-substituted com-

pounds (12a, 12c) occupy the same space in the S1⁰site,

as do the corresponding potent large C-substituted com-

pounds. Therefore, no simple relationship between the

eﬀect of the various P1⁰substituents and the experimen-

tally deduced K_ivalues could be found.

On the basis of these results, we were able to generalize

the various binding modes of the inhibitors, leading to a

reasonable qualitative interpretation of the activity val-

ues. However, due to the limitations of the scoring func-

tions, it is often diﬃcult to establish a quantitative

correlation between the calculated and the experimental-

ly derived activity values. This poses a problem for

establishing a structure–activity relationship. Due to this

reason, we decided to derive a quantitative structure–ac-

tivity relationship employing CoMFA using the docked

conformations as an alignment.

2.4. 3-D QSAR model

Comparative molecular ﬁeld analysis (CoMFA) is a

technique that relates the biological activity to the

chemical structure through multivariate statistics. It re-

quires that the molecules to be investigated be aligned

in their bioactive conformations. Here, we aligned the

C- and N-substituted compounds using the docked

poses (Fig. 3). LOO cross-validated PLS analysis was

used to determine the optimal number of components

in the ﬁnal CoMFA. This analysis yielded a one com-

ponent model with a cross-validated r²(q²) of 0.395

with a correlation coeﬃcient (r²) of 0.514. The steric

and electrostatic contributions amounted to 48% and

52%, respectively. Although a low q²was obtained, it

is still signiﬁcantly better than chance correlation, indi-

cating that the model had some predictability.³⁰One

possible reason for the low q²values obtained was

most likely due to the lack of speciﬁc interactions be-

tween the P1⁰substituents and the amino acid residues

in the S1⁰site.

D. Muthas et al. / Bioorg. Med. Chem. 13 (2005) 5371–5390

5381

3. Conclusion

A series of compounds with the generic structure 5 com-

prising a basic sp³-hybridized nitrogen, predicted to be

protonated in the transition state mimicking scaﬀold,

have been prepared by classical and microwave assisted

organic synthesis. Surprisingly, when the large biphenyl

P1⁰substituent was transferred from C-a to the basic

nitrogen all Plm II activity was lost. This was in contrast

to our previous ﬁndings, which demonstrated that large

extended P1⁰side chains are necessary for higher activi-

ty. The docking studies suggested that the small and

large P1⁰groups on the N-substituted inhibitors occupy

a diﬀerent region of the S1⁰site, which could explain

their diﬀerences in activities. The hybrid 19 did not show

any improvement in inhibitory activity as compared to 3

and 11b, indicating that no synergism was achieved

upon having P1⁰substituents in both positions. Finally,

it has been shown that small variations can greatly aﬀect

the selectivity ratio between Cat D and the plasmepsins

as inferred from the activities of compounds 11a and

11b, suggesting that selective Plm I and Plm II inhibitors

could be synthesized.

Figure 3. Docked alignment of the 37 compounds employed for

CoMFA. Atoms are colored by atom type and hydrogen atoms are

omitted for clarity.

Superimposition of the steric CoMFA contours onto the

Connolly surface complemented each other. Greater

activity is correlated with more bulk in the green con-

tours. In Figure 4, the green contour lies close to the

open end of the S1⁰enclosed by Leu292, Asp293, and

Phe294. This is in accordance with the observation made

earlier concerning the C-substituted inhibitors. Regions

where bulk is disfavored, corresponding to yellow con-

tours, coincide with the Connolly surface, suggesting

that no further bulk can be accommodated as seen in

Figure 4. A similar observation was made concerning

the electrostatic contours (blue favors more positive

charge), which showed (Fig. 4) that more positive charge

could be accommodated around residues Ser37, Asn39,

Asn76, and Tyr77. This CoMFA model reinforced the

qualitative results concerning the structure–activity rela-

tionships obtained from docking.

4. Experimental

4.1. Plasmepsin assay and K_idetermination

Pro-plasmepsin II was a generous gift from Helena

Danielson (Department of Biochemistry, Uppsala

University, Uppsala, Sweden) and the expression and

puriﬁcation of Plm I will be published elsewhere (manu-

script in preparation). Human liver cathepsin D was

purchased from Sigma–Aldrich (Sweden). The activities

of Plm I, Plm II, and Cat D were measured using a total

reaction volume of 100 ll as described earlier.¹⁵The

concentration of pro-Plm II was 3 nM, the amount of

Plm I was adjusted to give similar catalytic activity,

and 50 ng/mL pro-cathepsin D was used. The pro-

sequence of Plm II was cleaved oﬀ by preincubation in

assay reaction buﬀer [100 mM sodium acetate buﬀer

(pH 4.5), 10% glycerol, and 0.01% Tween 20] at room

temperature for 40 min, and Cat D was activated by

incubation in the same reaction buﬀer at 37 ꢁC for

20 min. The reaction was initiated by the addition of

3 lM substrate (DABCYL-Glu-Arg-Nle-Phe-Leu-Ser-

Phe-Pro-EDANS, AnaSpec Inc., San Jose, CA, USA),

and hydrolysis was recorded as an increase in ﬂuores-

cence intensity over a 10-min time period, during which

the rate increased linearly with time. Stock solutions of

inhibitors were serially diluted in DMSO and added

directly before the addition of the substrate, giving a ﬁ-

nal DMSO concentration of 1%.

IC₅₀values were obtained by assuming competitive inhi-

bition and ﬁtting a Langmuir isotherm [v_i/v_o= 1/(1+[I]/

IC₅₀)] to the dose–response data (Graﬁt), where v_iand

v_oare the initial velocities for the inhibited and uninhib-

ited reactions, respectively, and [I] is the inhibitor

concentration.³¹The K_iwas subsequently calculated

by using K_i= IC₅₀/(1 + [S]/K_m)³²and a K_mvalue

determined according to Michaelis–Menten.

Figure 4. Steric ﬁelds (green, steric bulk favored 80%; yellow, steric

bulk disfavored 20%) and electrostatic ﬁelds (blue, more positive

charge favored 80%; red, more negative charge favored 20%) super-

imposed on the active pocket, represented by a Connolly surface

(gray). The ligand is colored orange except for N (blue) and O (red)

atoms. Residues Tyr77, Val78, Ser79, and hydrogen atoms are omitted

for clarity.

5382

D. Muthas et al. / Bioorg. Med. Chem. 13 (2005) 5371–5390

The Caco-2 cell penetration assay was performed as

described by Artursson and Karlsson.²⁵

Compound 8a (0.10 g, 0.297 mmol) was dissolved in

dry DMF (1.5 mL). K₂CO₃(41 mg, 0.30 mmol) and

methyl bromoacetate (56.2 lmol) was added and the

mixture was stirred overnight. Water (5 mL) was added

and the mixture was extracted twice with ethyl acetate

(5 mL). The organic phase was dried, concentrated,

and puriﬁed by column chromatography (ethyl ace-

tate/isohexane, 1:4) to give a mixture of (5S,6R)-meth-

yl-3-aza-6-(tert-butyloxycarbonyl)amino-3-isobutyl-5-

hydroxy-7-phenylheptanoate and (5S)-3-aza-5-{[1-(tert-

butyloxycarbonyl)amino-2-phenyl]-ethyl}-3-isobutyl-S-

valerolactone in an approximate 1:1 mixture as deter-

mined by the mass signal of the analytical LC–MS. This

mixture was stirred in a saturated solution of ammonia

4.2. Chemistry

4.2.1. General procedures. All microwave reactions were

conducted in heavy-walled glass Smith process vials

sealed with aluminum crimp caps ﬁtted with a silicon

septum. The microwave heating was performed in a

Smith Synthesizer single-mode microwave cavity pro-

ducing continuous irradiation at 2450 MHz (Biotage

AB, Uppsala, Sweden). Reaction mixtures were stirred

with a magnetic stirring bar during the irradiation.

The temperature, pressure, and irradiation power were

monitored during the course of the reaction. After irra-

diation was completed, the reaction tube was cooled

with high-pressure air until the temperature had fallen

in methanol overnight to give after evaporation com-

22

D

pound 9a (98 mg, 84%). Compound 9a: ½aꢁ ꢂ35.0 (c

1

1.1, CHCl₃); H NMR (CDCl₃, 270 MHz): d 0.82 (d,

J = 6.5 Hz, 3H), 0.84 (d, J = 6.5 Hz, 3H), 1.34 (br s,

1H), 1.37 (s, 9H), 1.53–1.72 (m, 1H), 2.09 (dd, J = 7.9,

12.7 Hz, 1H), 2.21 (dd, J = 6.5, 12.6 Hz, 1H), 2.40–

2.58 (m, 2H), 2.78–2.95 (m, 2H), 3.02–3.10 (m, 2H),

3.55–3.85 (m, 2H), 5.10 (d, J = 9.5 Hz, 1H), 5.82 (br s,

1H), 7.07 (br s, 1H), 7.12–7.35 (m, 5H). ¹³C NMR

(CDCl₃, 67.8 MHz): d 21.0, 21.1, 26.8, 28.6, 39.3, 54.1,

59.8, 60.2, 64.6, 68.6, 79.7, 126.7, 128.8, 129.7, 138.6,

156.3, 175.1. Anal. (C₂₁H₃₅N₃O₄Æ0.7H₂O) C, H, N.

1

below 39 ꢁC. H and ¹³C NMR spectra were recorded

either on a JEOL JNM-EX 270 spectrometer at 270.2

and 67.8 MHz, respectively, or on a JEOL JNM-

EX400 spectrometer at 399.8 and 100.5 MHz, respec-

tively. Chemical shifts were reported as d values (ppm)

indirectly referenced to TMS via the solvent residual sig-

nal. Optical rotations were obtained on a Perkin-Elmer

241 polarimeter. Speciﬁc rotations ([a]_D) were reported

in deg/dm, and the concentration (c) was given in

g/100 mL in the speciﬁed solvent. Elemental analyses

were performed by Mikro Kemi AB (Uppsala, Sweden).

Flash column chromatography was performed on

Merck silica gel 60, 0.04–0.063 mm. Thin-layer chroma-

tography was performed using aluminum sheets pre-

coated with silica gel 60 F₂₅₄(0.2 mm; E. Merck) and

visualized with UV light and ninhydrin. Analytical

RP-LC–MS was performed on a Gilson HPLC system

with a Zorbax SB-C8, 5 lM 4.6 · 50 mm (Agilent Tech-

nologies) column, with a Finnigan AQA quadropole

mass spectrometer, at a ﬂow rate of 1.5 mL/min. Pre-

parative RP-LC–MS was performed on a Gilson HPLC

system with a Zorbax SB-C8, 5 lM 21.2 · 150 mm (Agi-

lent Technologies) column, with a Finnigan AQA qua-

dropole mass spectrometer, at a ﬂow rate of 15 mL/min.

4.2.4. (5S,6R)-3-Aza-6-{[(tert-butyloxycarbonyl)-^L-vali-

nyl]amino}-3-isobutyl-5-hydroxy-7-phenylheptanoyl amide

(10a). Compound 9a (0.10 g, 0.25 mmol) was dissolved

in ethyl acetate (5 mL). Saturated HCl in ethyl acetate

(20 mL) was added quickly under vigorous stirring. After

the mixture was stirred for 1 h at room temperature, the

solvents were evaporated. The residue was dissolved in

DMF (3 mL). Boc-^L-valine (54.3 mg, 0.25 mmol), TBTU

(79.9 mg, 0.25 mmol), and diisopropylethylamine (87 lL,

0.50 mmol) were added and the mixture was stirred for

1 h. Saturated aqueous NaHCO₃(10 mL) was added

and the mixture was extracted twice with ethyl acetate

(10 mL). The combined organic phases were dried,

ﬁltered, and evaporated, and the residue was puriﬁed by

silica gel ﬂash column chromatography (ethyl acetate)

4.2.2. (2S,3S)-3-[(tert-Butyloxycarbonyl)amino]-1-(isobu-

tylamino)-4-phenylbutan-2-ol (8a). (2R,3S)-3-[N-(tert-

Butyloxycarbonyl)amino]-1,2-epoxy-4-phenylbutane (6a,

50 mg, 0.190 mmol)²²was dissolved in 5 mL isopropa-

nol. Isobutylamine (7a, 0.194 mL, 1.9 mmol) was added

and the reaction mixture was stirred at 50 ꢁC overnight.

After evaporation, the product was dried under vacuum

for 20 h to remove excess isobutylamine. The product

to give compound 10a (0.12 g, 99%) as a white solid. Com-

22

D

pound 10a: ½aꢁ ꢂ66.8 (c 1.0, CH₃OH); ¹H NMR (CDCl₃,

270 MHz): d 0.77–0.93 (m, 12H), 1.44 (s, 9H), 1.57–1.80

(m, 1H), 2.02–2.35 (m, 3H), 2.42–2.60 (m, 2H), 2.88–

3.03 (m, 2H), 3.03 (d, J = 16.8 Hz, 1H), 3.13 (d,

J = 16.8 Hz, 1H), 3.68–3.80 (m, 1H), 3.83 (dd, J = 6.5,

8.5 Hz, 1H), 4.07–4.31 (m, 1H), 6.16 (br s, 1H), 6.54 (d,

J = 8.6 Hz, 1H), 7.10–7.38 (m, 5H), 7.60 (d, J = 9.6 Hz,

1H). ¹³C NMR (CD₃OD, 67.8 MHz): d 16.1, 17.8, 18.9,

19.1, 25.3, 26.6, 29.5, 36.7, 52.0, 58.0, 58.6, 59.8, 63.4,

67.2, 78.4, 125.1, 127.2, 128.2, 137.5, 155.7, 172.0, 174.8.

Anal. (C₂₆H₄₄N₄O₅) C, H, N.

was recrystallized from hexane to give compound 8a

22

D

(30 mg, 47%). Compound 8a: mp 88.5–89.5 ꢁC; ½aꢁ

1

ꢂ36.6 (c 1.1, CHCl₃); H NMR (CDCl₃, 270 MHz): d

0.88 (d, J = 6.6 Hz, 6H), 1.20 (br s, 1H), 1.35 (s, 9H),

1.56–1.77 (m, 1H), 2.30–2.45 (m, 2H), 2.50–2.65 (m,

2H), 2.75–3.00 (m, 2H), 3.60–3.71 (m, 1H), 3.73–3.80

(m, 1H), 4.98 (br d, J = 9.6 Hz, 1H), 7.10–7.30 (m,

5H). ¹³C NMR (CDCl₃, 67.8 MHz): d 20.4, 20.5, 28.1,

38.9, 51.9, 53.4, 57.1, 67.6, 79.5, 126.0, 128.1, 129.2,

138.1, 155.7. Anal. (C₁₉H₃₂N₂O₃) C, H, N.

4.2.5. (5S,6R)-3-Aza-3-isobutyl-5-hydroxy-7-phenyl-6-

[(picolyl-^L-valinyl)amino]-heptanoyl amide (11a). Com-

pound 10a (0.10 g, 0.25 mmol) was dissolved in ethyl

acetate (5 mL). Saturated HCl in ethyl acetate (20 mL)

was added quickly under vigorous stirring. After the

mixture was stirred for 1 h at room temperature, the

solvents were evaporated. Saturated aqueous NaHCO₃

(20 mL) was added and the mixture was extracted twice

4.2.3. (5S,6R)-3-Aza-6-[(tert-butyloxycarbonyl)amino]-3-

isobutyl-5-hydroxy-7-phenylheptanoyl

amide

(9a).

D. Muthas et al. / Bioorg. Med. Chem. 13 (2005) 5371–5390

5383

with ethyl acetate (10 mL). The combined organic phas-

es were dried, ﬁltered, and evaporated. The residue was

dissolved in DMF (5 mL). Picolinic acid (25 mg,

0.20 mmol), TBTU (65 mg, 0.20 mmol), and diisopro-

pylethylamine (71 lL, 0.41 mmol) were added and the

mixture was stirred for 1 h. Saturated aqueous NaHCO₃

(10 mL) was added and the mixture was extracted four

times with ethyl acetate (10 mL). The combined organic

phases were dried, ﬁltered, and evaporated, and the res-

idue was puriﬁed by silica gel ﬂash column chromatog-

raphy (ethyl acetate/methanol, 10:1) to give compound

NMR (CDCl₃, 270 MHz): d 0.74 (d, J = 6.4 Hz, 3H),

0.86 (d, J = 6.3 Hz, 3H), 1.43 (s, 9H), 1.90–2.20 (m,

1H), 2.42–2.75 (m, 2H), 2.83–2.94 (m, 1H), 3.06 (d,

J = 16.0 Hz, 1H), 3.17 (d, J = 16.0 Hz, 1H), 3.30–4.00

(m, 5H), 4.00–4.27 (m, 1H), 4.94 (br d, J = 6.3 Hz,

1H), 5.69 (br s, 1H), 6.54 (br d, J = 7.3 Hz, 1H), 6.68

(br s, 1H), 7.01–7.49 (m, 10H). ¹³C NMR (CD₃OD,

67.8 MHz): d 17.3, 19.4, 28.3, 38.5, 38.6, 52.3, 57.8,

58.6, 59.4, 60.3, 67.8, 80.1, 126.5, 127.6, 128.6, 129.0,

129.3, 137.3, 137.9, 155.9, 171.6, 173.7. Anal.

(C₂₉H₄₂N₄O₅Æ1.6H₂O) C, H, N.

11a (74 mg, 73%) as a white solid. Compound 11a:

22

D

½aꢁ

ꢂ36.4 (c 1.0, CH₃OH); ¹H NMR (CDCl₃,

4.2.9.

(5S,6R)-3-Aza-3-benzyl-5-hydroxy-7-phenyl-6-

270 MHz): d 0.68–1.05 (m, 12H), 1.64–1.79 (m, 1H),

1.84–2.40 (m, 5H), 2.72–3.09 (m, 3H), 3.42–3.62 (m,

1H), 4.19–4.36 (m, 1H), 4.36–4.60 (m, 2H), 6.37–6.58

(m, 1H), 7.06–7.38 (m, 6H), 7.38–7.57 (m, 1H), 7.80–

7.98 (m, 1H), 8.09–8.22 (m, 1H), 8.23–8.39 (m, 1H),

8.52–8.68 (m, 1H). ¹³C NMR (CDCl₃, 67.8 MHz): d

17.5, 19.7, 20.6, 20.8, 25.3, 29.8, 37.7, 38.7, 50.7, 51.9,

55.6, 59.6, 65.6, 122.5, 126.8, 128.7, 129.3, 129.4,

137.0, 137.6, 148.4, 137.6, 165.1, 168.5, 171.1. Anal.

(C₂₇H₃₉N₅O₄Æ1.1H₂O) C, H, N.

[(picolyl-^L-valinyl)amino]-heptanoyl amide (11b). Com-

pound 11b (51 mg, 74%) was prepared from substance

10b (70 mg, 0.13 mmol) according to the method for

the preparation of 11a, except that for the acid-mediated

deprotection TFA (30% in CH₂Cl₂) was used. Com-

22

D

pound 11b: ½aꢁ ꢂ62.8 (c 0.6, CH₃OH); ¹H NMR

(CDCl₃, 270 MHz): d 0.86 (d, J = 6.7 Hz, 3H), 0.91 (d,

J = 6.7 Hz, 3H), 2.02–2.38 (m, 1H), 2.42–2.78 (m, 2H),

2.78–2.97 (m, 2H), 2.97–3.33 (m, 2H), 3.40–4.00 (m,

3H), 4.17 (dd, J = 8.1, 16.5 Hz, 1H), 4.24–4.62 (m,

2H), 6.05 (br s, 1H), 6.80–7.40 (m, 11H), 7.40–7.60 (m,

1H), 7.75–7.97 (m, 1H), 8.05–8.30 (m, 1H), 8.30–8.52

(m, 1H), 8.52–8.70 (m, 1H). ¹³C NMR (CDCl₃,

67.8 MHz): d 17.9, 19.6, 30.4, 38.5, 52.4, 57.8, 58.8,

59.2, 59.3, 67.9, 122.2, 126.7, 126.5, 128.3, 128.5,

129.1, 129.2, 137.4, 137.9, 148.3, 149.0, 164.60, 170.9,

174.0. Anal. (C₃₀H₃₇N₅O₄Æ0.5H₂O) C, H, N.

4.2.6. (2S,3S)-1-(Benzylamino)-3-[(tert-butyloxycarbon-

yl)amino]-4-phenylbutan-2-ol (8b). Compound 8b

(0.61 g, 82%) was prepared from epoxide 6a (0.53 g,

2.013 mmol) and benzylamine (7b, 2.23 mL, 20.1 mmol)

according to the method for the preparation of 8a,

except that it was puriﬁed by ﬂash column chromato-

graphy (CH₂Cl₂/MeOH, 19:1) to give 8b as a colorless

22

D

glue. Compound 8b: ½aꢁ ꢂ25.2 (c 0.3, CHCl₃); ¹H

4.2.10. (2S,3S)-1-(Benzylamino)-3-[(tert-butyloxycarbon-

yl)amino]-4-phenylbutan-2-ol (8c). Compound 8c (56 mg,

85%) was prepared from (2S,3S)-3-[N-(tert-butyloxycar-

bonyl)amino]-1,2-epoxy-4-phenylbutane (6b)¹⁹(47 mg,

0.18 mmol) and benzylamine (7c, 0.29 mL, 1.69 mmol)

according to the method for the preparation of 8a,

except that it was puriﬁed by ﬂash column chromato-

NMR (CDCl₃, 270 MHz): d 1.31 (br s, 1H), 1.38 (s,

9H), 1.41–1.55 (m, 1H), 2.40–2.75 (m, 3H), 2.75–2.99

(m, 2H), 3.50–3.62 (m, 1H), 3.63–3.84 (m, 3H), 4.93–

5.07 (m, 1H), 7.10–7.40 (m, 10H). ¹³C NMR (CDCl₃,

67.8 MHz): d 28.3, 38.9, 51.8, 53.4, 53.6, 68.2, 79.2,

126.2, 127.2, 128.1, 128.3, 128.4, 129.4, 138.2, 139.1,

155.9. Anal. (C₂₂H₃₀N₂O₃Æ1.5H₂O) C, H, N.

graphy (CH₂Cl₂/MeOH, 19:1) to give 8c as a colorless

22

D

glue. Compound 8c: ½aꢁ +2.1 (c 0.8, CHCl₃); ¹H

4.2.7. (5S,6R)-3-Aza-3-benzyl-6-[(tert-butyloxycarbon-

yl)amino]-5-hydroxy-7-phenylheptanoyl

Compound 9b (0.43 g, 70%) was prepared from sub-

NMR (CDCl₃, 400 MHz): d 1.24 (br s, 1H), 1.34 (s,

9H), 2.58–2.88 (m, 2H), 2.96 (dd, J = 4.7, 14.1 Hz,

1H), 3.42–3.63 (m, 3H), 3.75–3.86 (m, 1H), 3.80 (d,

J = 13.3 Hz, 1H), 3.83 (d, J = 13.3 Hz, 1H), 4.78 (d,

J = 9.2 Hz, 1H), 7.10–7.42 (m, 10H). ¹³C NMR

(CDCL₃, 100.5 MHz): d 28.4, 36.7, 50.9, 53.7, 54.3,

70.87, 79.6, 126.4, 127.5, 128.5, 128.7, 128.7, 129.6,

137.9, 138.9, 156.1. Anal. (C₂₂H₃₀N₂O₃) C, H, N.

amide

(9b).

stance 8b (0.53 g, 1.43 mmol) according to the method

22

D

for the preparation of 9a. Compound 9b: ½aꢁ ꢂ41.0 (c

1

0.6, CHCl₃); H NMR (CDCl₃, 270 MHz): d 1.34 (br

s, 1H), 1.37 (s, 9H), 2.50–2.69 (m, 2H), 2.78–3.00 (m,

2H), 3.05 (d, J = 16.5 Hz, 1H), 3.14 (d, J = 16.5 Hz,

1H), 3.52 (d, J = 13.3 Hz, 1H), 3.64 (d, J = 13.3 Hz,

1H), 3.64–3.90 (m, 2H), 5.01 (d, J = 9.7 Hz, 1H), 5.70

(br s, 1H), 6.98 (br s, 1H), 7.15–7.30 (m, 10H). ¹³C

NMR (CDCl₃, 67.8 MHz): d 28.3, 38.8, 53.7, 58.1,

58.9, 59.5, 68.1, 79.5, 126.3, 127.9, 128.5, 128.6, 128.9,

4.2.11. (5S,6R)-3-Aza-3-benzyl-6-[(tert-butyloxycarbon-

yl)amino]-5-hydroxy-7-phenylheptanoyl amide (9c). Com-

pound 9c (0.150 g, 96%) was prepared from substance 8c

(0.136 g, 0.37 mmol) according to the method for the

129.3,

137.3,

138.1,

156.0,

173.9.

Anal.

22

preparation of 9a. Compound 9c: ½aꢁ +10.6 (c 0.6,

(C₂₄H₃₃N₃O₄Æ0.4H₂O) C, H, N.

D

CHCl₃); ¹H NMR (CDCl₃, 400 MHz): d 1.20 (br s, 1H),

1.31 (s, 9H), 2.50–2.93 (m, 4H), 3.03–3.20 (m, 1H),

3.20–3.35 (m, 1H), 3.49–3.72 (m, 1H), 3.72–3.94 (m,

3H), 4.90–5.09 (m, 1H), 6.28 (br s, 1H), 7.05–7.40 (m,

10H), 7.50 (br s, 1H). ¹³C NMR (CDCl₃, 100.5 MHz): d

28.4, 35.9, 55.2, 57.9, 58.3, 59.7, 71.3, 79.7, 126.5, 127.7,

128.5, 128.7, 129.2, 129.4, 137.6, 138.1, 156.3, 174.8. Anal.

(C₂₄H₃₃N₃O₄Æ0.2H₂O) C, H, N.

4.2.8. (5S,6R)-3-Aza-3-benzyl-6-{[(tert-butyloxycarbon-

yl)-^L-valinyl]amino}-5-hydroxy-7-phenylheptanoyl amide

(10b). Compound 10b (0.19 g, 78%) was prepared from

substance 9b (0.20 g, 0.468 mmol) according to the

method for the preparation of 10a, except that for the

acid-mediated deprotection TFA (30% in CH₂Cl₂) was

22

D

1

used. Compound 10b: ½aꢁ ꢂ47.9 (c 0.6, CH₃OH); H

5384

D. Muthas et al. / Bioorg. Med. Chem. 13 (2005) 5371–5390

4.2.12. (5S,6R)-3-Aza-3-benzyl-6-{[(tert-butyloxycarbon-

yl)-^L-valinyl]amino}-5-hydroxy-7-phenylheptanoyl amide

(10c). Compound 10c (132 mg, 84%) was prepared from

substance 9c (128 mg, 0.30 mmol) according to the

method for the preparation of 10a, except that for the

acid-mediated deprotection TFA (30% in CH₂Cl₂) was

1H), 4.96 (d, J = 9.6 Hz, 1H), 5.55 (br s, 1H), 6.80 (br

s, 1H), 7.20–7.40 (m, 10H). ¹³C NMR (CDCl₃,

67.8 MHz): d 28.3, 33.3, 38.6, 53.4, 56.9, 58.2, 58.5,

67.7, 79.5, 126.4, 126.4, 128.5, 128.5, 128.7, 129.2,

138.1, 139.7, 155.9, 174.0. Anal. (C₂₅H₃₅N₃O₄) C, H, N.

22

1

used. Compound 10c: ½aꢁ ꢂ11.3 (c 0.5, CHCl₃); H

4.2.16. (5S,6R)-3-Aza-6-{[(tert-butyloxycarbonyl)-^L-vali-

nyl]amino}-5-hydroxy-7-phenyl-3-(2-phenylethyl)-hepta-

noyl amide (10d). Compound 10d (0.45 g, 69%) was

prepared from substance 9d (0.53 g, 1.2 mmol) accord-

D

NMR (CDCl₃, 400 MHz): d 0.71 (d, J = 6.4 Hz, 3H),

0.82 (d, J = 6.3 Hz, 3H), 1.39 (br s, 1H), 1.43 (s, 9H),

1.87–2.10 (m, 1H), 2.50–2.95 (m, 4H), 3.02–3.35 (m,

2H), 3.53–3.92 (m, 4H), 4.04–4.30 (m, 1H), 4.79–5.09

(m, 1H), 5.90 (br s, 1H), 6.59 (br s, 1H), 7.00–7.45 (m,

11H). ¹³C NMR (CD₃OD, 100.5 MHz): d 17.5, 19.3,

28.4, 30.4, 35.2, 53.2, 54.1, 58.0, 59.9, 60.7, 71.1, 80.3,

126.6, 127.7, 128.6, 128.7, 129.2, 129.4, 137.6, 137.8,

156.0, 172.1, 174.2. Anal. (C₂₉H₄₂N₄O₅) C, H, N.

ing to the method for the preparation of 10a. Compound

22

D

10d: ½aꢁ ꢂ56.3 (c 0.5, CH₃OH); ¹H NMR (CDCl₃,

270 MHz): d 0.77 (d, J = 6.6 Hz, 3H), 0.89 (d,

J = 6.8 Hz, 3H), 1.55 (s, 9H), 2.08–2.32 (m, 1H), 2.56–

2.76 (m, 2H), 2.76–2.94 (m, 3H), 2.94–3.12 (m, 2H),

3.12–3.46 (m, 2H), 3.68–3.90 (m, 2H), 3.90–4.08 (m,

1H), 4.15–4.35 (m, 1H), 5.09 (br s, 1H), 5.72 (br s, 1H),

6.61–6.90 (m, 1H), 7.12–7.49 (m, 10H). ¹³C NMR

(CD₃OD, 67.8 MHz): d 17.3, 19.3, 28.2, 30.3, 33.1,

38.4, 52.1, 57.1, 58.1, 58.5, 60.4, 67.8, 80.1, 126.4,

126.5, 128.5, 128.5, 128.7, 129.1, 137.4, 139.4,

155.8, 171.5, 173.1. Anal. (C₃₀H₄₄N₄O₅Æ0.3H₂O)

C, H, N.

4.2.13.

(5S,6R)-3-Aza-3-benzyl-5-hydroxy-7-phenyl-6-

[(picolyl-^L-valinyl)amino]-heptanoyl amide (11c). Com-

pound 11c (45 mg, 39%) was prepared from substance

10c (113 mg, 0.21 mmol) according to the method for

the preparation of 11a, except that for the acid-mediated

deprotection TFA (30% in CH₂Cl₂) was used. Com-

22

D

pound 11c: ½aꢁ ꢂ27.9 (c 1.2, CHCl₃); ¹H NMR

(CDCl₃/CD₃OD, 9:1, 400 MHz): d 0.68 (d, J = 6.9 Hz,

3H), 0.77 (d, J = 6.8 Hz, 3H), 1.93–2.11 (m, 1H), 2.39–

2.86 (m, 2H), 2.55 (dd, J = 10.2, 14.2 Hz, 1H), 2.86–

3.21 (m, 2H), 3.39–3.80 (m, 4H), 3.95–4.22 (m, 2H),

6.76–6.90 (m, 1H), 6.90–7.09 (m, 4H), 7.09–7.35 (m,

5H), 7.35–7.50 (m, 1H), 7.67–7.88 (m, 1H), 7.96–8.11

(m, 1H), 8.45–8.58 (m, 1H). ¹³C NMR (CDCl₃–CD₃OD,

9:1, 100.5 MHz): d 17.6, 19.3, 30.5, 35.1, 53.8, 53.9, 57.8,

59.0, 59.6, 70.7, 122.3, 126.2, 126.7, 127.6, 128.2, 128.6,

129.0, 129.0, 129.2, 137.6, 137.8, 148.4, 148.9, 164.9,

171.4, 175.4. Anal. (C₃₀H₃₇N₅O₄Æ0.6H₂O) C, H, N.

4.2.17. (5S,6R)-3-Aza-5-hydroxy-7-phenyl-3-(2-phenyl-

ethyl)-6-[(picolyl-^L-valinyl)amino]-heptanoyl amide (11d).

Compound 11d (51 mg, 74%) was prepared from sub-

stance 10d (70 mg, 0.13 mmol) according to the method

for the preparation of 11a, except that for the acid-med-

iated deprotection TFA (30% in CH₂Cl₂) was used.

22

1

Compound 11d: ½aꢁ ꢂ60.5 (c 1.0, CH₃OH); H NMR

D

(CDCl₃, 400 MHz): d 0.86 (d, J = 6.8 Hz, 3H), 0.94 (d,

J = 6.7 Hz, 3H), 2.17–2.36 (m, 1H), 2.49–2.82 (m, 6H),

2.82–3.01 (m, 2H), 3.01–3.43 (m, 2H), 3.73 (br s, 1H),

4.08–4.30 (m, 1H), 4.37 (dd, J = 7.3, 9.2 Hz, 1H), 6.05

(br s, 1H), 6.88–7.32 (m, 12H), 7.32–7.51 (m, 1H),

7.71–7.90 (m, 1H), 8.04–8.21 (m, 1H), 8.37–8.50 (m,

1H), 8.50–8.68 (m, 1H). ¹³C NMR (CDCl₃,

100.5 MHz): d 17.9, 19.4, 30.4, 32.6, 38.3, 38.4, 52.3,

57.3, 58.6, 59.1, 68.1, 122.1, 126.0, 126.3, 128.1, 128.3,

128.5, 129.0, 129.2, 137.2, 137.8, 139.3, 148.1,

148.9, 164.3, 170.8, 174.4. Anal. (C₃₁H₃₉N₅O₄Æ0.8H₂O)

C, H, N.

4.2.14. (2S,3S)-3-[(tert-Butyloxycarbonyl)amino]-4-phen-

yl-1-[(2-phenylethyl)amino]- butan-2-ol (8d). Compound

8d (0.68 g, 88%) was prepared from epoxide 6a (0.53 g,

2.013 mmol) and 2-phenylethylamine (7d, 2.6 mL,

20.1 mmol) according to the method for the preparation

of 8a, except that it was puriﬁed by ﬂash column chro-

matography (CH₂Cl₂/MeOH, 19:1) to give 8d as a white

22

1

powder. Compound 8d: ½aꢁ ꢂ29.7 (c 0.2, CHCl₃); H

D

NMR (CDCl₃, 270 MHz): d 1.33 (br s, 1H), 1.40 (s, 9

H), 2.45–2.65 (m, 2H), 2.65–3.13 (m, 7H), 3.55 (dd,

J = 4.1, 9.3 Hz, 1H), 3.71 (dd, J = 8.1, 16.5 Hz, 1H),

5.06 (br d, J = 9.5 Hz, 1H), 7.02–7.45 (m, 10H). ¹³C

NMR (CDCl₃, 67.8 MHz): d 28.3, 36.1, 39.1, 50.5,

52.0, 53.6, 67.9, 79.2, 126.2, 128.3, 128.5, 128.6, 129.0,

129.4, 138.3, 139.4, 155.8. Anal. (C₂₃H₃₂N₂O₃Æ0.1H₂O)

C, H, N.

4.2.18. (2S,3S)-3-[(tert-Butyloxycarbonyl)amino]-4-phen-

yl-1-[(3-phenylpropyl)amino]-butan-2-ol (8e). Compound

8e (0.59 g, 78%) was prepared from epoxide 6a (0.50 g,

1.9 mmol) and 3-phenylpropylamine (7e, 2.75 mL,

19.0 mmol) according to the method for the preparation

of 8a, except that it was puriﬁed by ﬂash column chro-

matography (CH₂Cl₂/MeOH, 19:1) to give 8e as a white

22

D

1

powder. Compound 8e: ½aꢁ ꢂ29.2 (c 0.5, CHCl₃); H

NMR (CDCl₃, 270 MHz): d 1.33 (br s, 1H), 1.40 (s,

9H), 1.78 (dq, J = 7.2, 7.3 Hz, 2H), 2.41–2.75 (m, 7H),

2.75–2.98 (m, 2H), 3.46–3.61 (m, 1H), 3.61–3.80 (dd,

J = 8.1, 16.2 Hz, 1H), 4.93–5.14 (br d, J = 9.5 Hz, 1H),

6.97–7.40 (m, 10H). ¹³C NMR (CDCl₃, 67.8 MHz): d

28.3, 31.3, 33.4, 39.1, 48.9, 52.2, 53.7, 68.1, 79.3, 125.9,

126.2, 128.4, 128.4, 129.4, 129.4, 138.26, 141.6, 155.9.

Anal. (C₂₄H₃₄N₂O₃) C, H, N.

4.2.15. (5S,6R)-3-Aza-6-[(tert-butyloxycarbonyl)amino]-

5-hydroxy-7-phenyl-3-(2-phenylethyl)-heptanoyl amide

(9d). Compound 9d (0.43 g, 70%) was prepared from

substance 8c (0.53 g, 1,43 mmol) according to the meth-

22

D

od for the preparation of 9a. Compound 9d: ½aꢁ ꢂ23.0

1

(c 1.5, CHCl₃); H NMR (CDCl₃, 270 MHz): d 1.34 (br

s, 1H), 1.39 (s, 9H), 2.50–2.65 (m, 2H), 2.65–2.85 (m,

4H), 2.80 (dd, J = 8.4, 13.5 Hz, 1H), 2.91 (dd, J = 6.8,

13.3 Hz, 1H), 3.06 (d, J = 16.7 Hz, 1H), 3.18 (d,

J = 16.7 Hz, 1H), 3.53–3.67 (m, 1H), 3.75–3.90 (m,

4.2.19. (5S,6R)-3-Aza-6-[(tert-butyloxycarbonyl)amino]-

5-hydroxy-7-phenyl-3-(3-phenylpropyl)-heptanoyl amide

D. Muthas et al. / Bioorg. Med. Chem. 13 (2005) 5371–5390

5385

(9e). Compound 9e (0.45 g, 82%) was prepared from

substance 8e (0.48 g, 1.4 mmol) according to the method

(m, 2H), 7.18–7.37 (m, 5H), 7.37–7.60 (m, 2H). ¹³C

NMR (CDCl₃, 67.8 MHz): d 30.2, 40.7, 53.8, 54.7,

55.6, 57.4, 70.5, 81.1, 122.8, 128.1, 130.2, 131.2, 131.7,

133.3.140.2, 157.8. Anal. (C₂₂H₂₉BrN₂O₃Æ0.2H₂O) C,

H, N.

22

D

for the preparation of 9a. Compound 9e: ½aꢁ ꢂ23.0 (c

1

0.7, CHCl₃); H NMR (CDCl₃, 270 MHz): d 1.32 (br

s, 1H), 1.37 (s, 9H), 1.75–1.90 (m, 2H), 2.55–2.70 (m,

6H), 2.85–3.05 (m, 2H), 3.05–3.25 (m, 2H), 3.70–3.80

(m, 1H), 3.80–3.95 (m, 1H), 4.93 (d, J = 9.6 Hz, 1H),

5.55 (br s, 1H), 7.10 (br s, 1H), 7.25–7.40 (m, 10H).

¹³C NMR (CDCl₃, 67.8 MHz): d 28.2, 33.2, 38.9, 53.4,

53.5, 55.1, 58.5, 59.0, 68.1, 79.67, 126.00, 126.4, 128.2,

128.4, 128.5, 129.2, 138.1, 141.4, 155.0, 172.6. Anal.

(C₂₆H₃₇N₃O₄Æ0.1H₂O) C, H, N.

4.2.23. (5S,6R)-3-Aza-3-(p-bromobenzyl)-6-[(tert-butyl-

oxycarbonyl)amino]-5-hydroxy-7-phenylheptanoyl amide

(9e). Compound 9e (1.20 g, 65%) was prepared from

substance 8e (1.63 g, 3.63 mmol) according to the meth-

22

D

od for the preparation of 9a. Compound 9e: ½aꢁ ꢂ31.6

1

(c 1.4, CHCl₃); H NMR (CDCl₃, 270 MHz): d 1.38 (br

s, 1H), 1.40 (s, 9H), 2.44–2.79 (m, 2H), 2.79–3.01 (m,

2H), 3.01–3.28 (m, 2H), 3.50 (d, J = 13.8 Hz, 1H), 3.61

(d, J = 13.8 Hz, 1H), 3.67–3.95 (m, 2H), 5.22 (br d,

J = 9.6 Hz, 1H), 6.19 (br s, 1H), 6.96–7.18 (m, 2H),

7.18–7.38 (m, 5H), 7.38–7.55 (m, 2H). ¹³C NMR

(CDCl₃, 67.8 MHz): d 28.2, 38.7, 53.8, 57.9, 58.7, 58.8,

67.9, 79.4, 121.3, 126.3, 128.4, 129.2, 130.5, 131.5,

136.4, 138.1, 156.0, 174.4. Anal. (C₂₄H₃₂BrN₃O₄) C,

H, N.

4.2.20. (5S,6R)-3-Aza-6-{[(tert-butyloxycarbonyl)-^L-vali-

nyl]amino}-5-hydroxy-7-phenyl-3-(3-phenylpropyl)-hepta-

noyl amide (10e). Compound 10e (0.17 g, 70%) was

prepared from substance 9e (0.20 g, 0.44 mmol) accord-

ing to the method for the preparation of 10a. Com-

22

D

pound 10e: ½aꢁ ꢂ38.0 (c 0.5, CH₃OH); ¹H NMR

(CDCl₃, 270 MHz): d 0.75 (d, J = 6.9 Hz, 3H), 0.87 (d,

J = 6.8 Hz, 3H), 1.55 (s, 9H), 1.75–1.90 (m, 2H), 2.10–

2.25 (m, 1H), 2.58–2.70 (m, 6H), 2.98–3.05 (m, 2H),

3.10–3.32 (m, 2H), 3.70–3.80 (m, 1H), 3.90–4.00 (m,

1H), 4.20–4.32 (m, 1H), 4.95 (br d, J = 9.5 Hz, 1H),

5.75 (br s, 1H), 6.65–6.72 (m, 1H), 6.99 (br s, 1H),

7.25–7.40 (m, 10H). ¹³C NMR (CDCl₃, 67.8 MHz): d

17.8, 19.8, 28.8, 30.7, 33.8, 39.0, 52.7, 52.7, 55.7, 58.95,

59.4, 60.8, 68.6, 80.7, 126.2, 127.1, 128.7, 128.9, 129.0,

129.7, 138.3, 141.9, 156.3, 167.9, 172.0. Anal.

(C₃₁H₄₆N₄O₅) C, H, N.

4.2.24. (5S,6R)-3-Aza-3-(p-bromobenzyl)-6-{[(tert-butyl-

oxycarbonyl)-^L-valinyl]amino}-5-hydroxy-7-phenylhepta-

noyl amide (10f). Compound 10f (1.10 g, 82%) was

prepared from substance 9e (1.12 g, 2.21 mmol) accord-

ing to the method for the preparation of 10a, except that

for the acid-mediated deprotection TFA (25% in

22

CH₂Cl₂) was used. Compound 10f: ½aꢁ ꢂ43.2 (c 1.6,

D

CH₃OH); ¹H NMR (CDCl₃, 270 MHz): d 0.82 (d,

J = 6.6 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H), 1.44 (br s,

1H), 1.46 (s, 9H), 1.90–2.11 (m, 1H), 2.42–2.72 (m,

2H), 2.85–2.99 (m, 2H), 3.06 (d, J = 16.7 Hz, 1H), 3.17

(d, J = 16.7 Hz, 1H), 3.51 (d, J = 13.4 Hz, 1H), 3.68

(d, J = 13.5 Hz, 1H), 3.63–4.00 (m, 2H), 4.09–4.29 (m,

1H), 5.37 (d, J = 8.1 Hz, 1H), 6.50 (br s, 1H), 6.91–

7.35 (m, 9H), 7.35–7.50 (m, 2H). ¹³C NMR (CD₃OD,

67.8 MHz): d 17.6, 19.3, 28.2, 30.6, 38.2, 52.4, 57.6,

58.5, 58.7, 60.2, 67.6, 79.8, 121.2, 126.3, 128.3, 129.1,

130.5, 131.4, 136.3, 137.9, 155.9, 171.8, 174.3. Anal.

(C₂₉H₄₁BrN₄O₅) C, H, N.

4.2.21.

(5S,6R)-3-Aza-5-hydroxy-6-[(picolyl-^L-vali-

nyl)amino]-7-phenyl-3-(3-phenylpropyl)-heptanoyl amide

(11e). Compound 11e (88 mg, 79%) was prepared from

substance 10e (0.11 g, 0.198 mmol) according to the

method for the preparation of 11a, except that for the

acid-mediated deprotection TFA (30% in CH₂Cl₂) was

22

1

used. Compound 11e: ½aꢁ ꢂ46.5 (c 0.7, CH₃OH); H

D

NMR (CDCl₃, 400 MHz): d 0.84 (d, J = 6.8 Hz, 3H),

0.90 (d, J = 6.6 Hz, 3H), 1.86–2.04 (m, 1H), 2.29–2.48

(m, 2H), 2.61–2.75 (m, 2H), 2.75–2.95 (m, 4H), 2.95–

3.16 (m, 2H), 3.33–3.53 (m, 2H), 3.60–3.70 (m, 1H),

3.87 (br s, 1H), 4.18 (dd, J = 8.7, 16.4 Hz, 1H), 4.34

(dd, J = 6.9, 8.6 Hz, 1H), 6.19 (br s, 1H), 7.09–7.50

(m, 12H), 7.53–7.70 (m, 1H), 7.91–8.09 (m, 1H), 8.21–

8.33 (m, 1H), 8.50–8.66 (m, 1H), 8.70–8.81 (m, 1H).

¹³C NMR (CDCl₃, 100.5 MHz): d 17.8, 19.5, 27.9,

30.3, 33.2, 38.2, 52.6, 55.5, 57.6, 59.0, 59.3, 68.1, 122.3,

126.1, 126.3, 126.5, 128.2, 128.3, 128.4, 129.4, 137.4,

137.8, 141.2, 148.3, 149.1, 164.7, 171.26, 173.1. Anal.

(C₃₂H₄₁N₅O₄Æ0.5H₂O) C, H, N.

4.2.25. (5S,6R)-3-Aza-3-(p-bromobenzyl)-5-hydroxy-7-

phenyl-6-[(picolyl-^L-valinyl)amino]-heptanoyl amide (11f).

Compound 11f (0.96 g, 88%) was prepared from

substance 10f (1.08 g, 1.78 mmol) according to the meth-

od for the preparation of 11a, except that for the acid-

mediated deprotection TFA (25% in CH₂Cl₂) was used.

22

D

1

Compound 11f: ½aꢁ ꢂ44.0 (c 1.0, CH₃OH); H NMR

(CDCl₃, 270 MHz): d 0.86 (d, J = 6.6 Hz, 3H), 0.91 (d,

J = 6.4 Hz, 3H), 2.01–2.37 (m, 1H), 2.48–2.85 (m, 2H),

2.85–3.01 (m, 2H), 3.01–3.36 (m, 2H), 3.40–3.90 (m,

3H), 4.03–4.22 (m, 1H), 4.22–4.62 (m, 1H), 6.06 (br s,

1H), 6.80–7.30 (m, 9H), 7.30–7.60 (m, 3H), 7.75–7.95

(m, 1H), 8.03–8.25 (m, 1H), 8.34–8.52 (m, 1H), 8.52–

8.70 (m, 1H). ¹³C NMR (CDCl₃, 67.8 MHz): d 17.9,

19.3, 30.4, 38.4, 52.5, 57.6, 57.8, 58.7, 59.3, 67.9, 121.5,

122.3, 126.4, 126.6, 128.4, 129.2, 130.8, 131.6, 136.2,

137.4, 137.8, 148.3, 149.0, 164.7, 171.0, 173.7. Anal.

(C₃₀H₃₆BrN₅O₄) C, H, N.

4.2.22. (2S,3S)-1-[(p-Bromobenzyl)amino]-3-[(tert-butyl-

oxycarbonyl)amino]-4-phenylbutan-2-ol (8f). Compound

8f (1.63 g, 83%) was prepared from epoxide 6a (1.15 g,

4.37 mmol) and p-bromobenzylamine (7f, 2.43 g,

13.1 mmol) according to the method for the preparation

of 8a, except that it was puriﬁed by ﬂash column chro-

matography (CH₂Cl₂/MeOH, 19:1) to give 8f as a color-

22

D

1

less glue. Compound 8f: ½aꢁ ꢂ24.0 (c 1.5, CHCl₃); H

NMR (CDCl₃, 270 MHz): d 1.36 (br s, 1H), 1.41 (s,

9H), 2.48–2.77 (m, 2H), 2.75–3.03 (m, 2H), 3.53–4.00

(m, 4H), 5.06–5.23 (br d, J = 9.4 Hz, 1H), 7.03–7.18

4.2.26. (5S,6R)-3-Aza-5-hydroxy-7-phenyl-3-(p-phenyl-

benzyl)-6-[(picolyl-^L-valinyl)amino]-heptanoyl amide (12a).

5386

D. Muthas et al. / Bioorg. Med. Chem. 13 (2005) 5371–5390

Compound 11f (53 mg, 87 lmol), phenylboronic acid

(32 mg, 0.26 mmol), Cs₂CO₃(85 mg, 0.26 mmol), and

Pd(PPh₃)₂Cl₂(6 mg, 9 lmol) were dissolved in a mixture

of DME and ethanol (3:1, 2 mL) in a Smith vial. The

vial was heated to 130 ꢁC for 20 min by means of micro-

wave irradiation. LC–MS analysis of the product mix-

ture showed that the primary amide had been

hydrolyzed in the process, thus, the mixture was ﬁltered

through a silica plug and eluted with CH₂Cl₂/methanol

(4:1), the elute was concentrated in vacuo, and the resi-

due was dissolved in methanol acidiﬁed with HCl. After

stirring the mixture overnight, the solvent was removed

by evaporation and the residue was extracted with aque-

ous K₂CO₃and ethyl acetate. The organic phase was

dried, ﬁltered, and evaporated, and the residue was dis-

solved in methanol saturated with ammonia. After stir-

ring the mixture overnight, the solvent was removed by

evaporation and the residue was puriﬁed on a silica gel

column using a mixture of CH₂Cl₂and methanol (39:1)

as mobile phase to give the title compound (50 mg,

4.2.29. (5S,6R)-3-Aza-3-(m-bromobenzyl)-6-{[(tert-butyl-

oxycarbonyl)-^L-valinyl]amino}-5-hydroxy-7-phenylhepta-

noyl amide (10g). Compound 10g (214 mg, 60%) was

prepared from substance 9g (300 mg, 0.59 mmol)

according to the method for the preparation of 7a, ex-

cept that for the acid-mediated deprotection TFA

22

D

(25% in CH₂Cl₂) was used. Compound 10g: ½aꢁ

1

ꢂ43.4 (c 0.3, CHCl₃); H NMR (CDCl₃, 270 MHz): d

0.73 (d, J = 7.0 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H), 1.42

(s, 9H), 1.97–2.11 (m, 1H), 2.48–2.67 (m, 2H), 2.79–

2.98 (m, 2H), 3.06(d, J = 17.2 Hz, 1H), 3.15 (d,

J = 17.4 Hz, 1H), 3.48–3.52 (d, J = 14.0 Hz, 1H), 3.52–

3.79 (m, 2H), 3.83 (dd, J = 5.9, 8.1 Hz, 1H), 4.05–4.21

(m, 1H), 4.88–5.04 (m, 1H), 5.74 (br s, 1H), 6.61 (br d,

J = 9.8 Hz, 1H), 6.80 (br s, 1H), 7.09–7.50 (m, 9H).

¹³C NMR (CDCl₃, 67.8 MHz): d 17.3, 19.4, 28.3, 30.3,

38.4, 52.3, 57.5, 58.8, 58.8, 60.4, 68. 0, 80.2, 122.6,

126.6, 127.6, 128.6, 129.2, 130.1, 130.7, 131.8, 137.8,

139.9, 156.0, 171.7, 173.5. Anal. (C₂₉H₄₁BrN₄O₅Æ0.9-

H₂O) C, H, N.

22

1

95%). Compound 12a: ½aꢁ ꢂ35.7 (c 0.4, CH₃OH); H

D

NMR (CDCl₃, 270 MHz): d 0.86 (d, J = 6.8 Hz, 3H),

0.91 (d, J = 6.4 Hz, 3H), 2.00–2.24 (m, 1H), 2.50–3.07

(m, 4H), 3.06–3.50 (m, 2H), 3.50–4.02 (m, 3H), 4.02–

4.23 (m, 1H), 4.23–4.51 (m, 1H), 6.19 (br s, 1H),

6.80–7.65 (m, 17H), 7.65–7.90 (m, 1H), 7.90–8.22 (m,

1H), 8.35–8.76 (m, 2H). ¹³C NMR (CDCl₃,

67.8 MHz): d 17.9, 19.5, 30.5, 38.4, 52.7, 57.3, 58.7,

58.9, 59.3, 67.8, 68.0, 122.3, 125.2, 126.3, 126.5, 127.0,

127.3, 128.2, 128.3, 128.7, 129.0, 129.2, 129.8, 137.4,

137.8, 140.4, 140.7, 148.3, 149.0, 164.7, 171.1, 171.3.

Anal. (C₃₆H₄₁N₅O₄ÆH₂O) C, H, N.

4.2.30. (5S,6R)-3-Aza-3-(m-bromobenzyl)-5-hydroxy-7-

phenyl-6-[(picolyl-^L-valinyl)amino]-heptanoyl amide (11g).

Compound 11g (160 mg, 48%) was prepared from

substance 10g (214 mg, 0.35 mmol) according to the

method for the preparation of 11a, except that for the

acid-mediated deprotection TFA (25% in CH₂Cl₂) was

22

1

used. Compound 11g: ½aꢁ ꢂ56.5 (c 2.0, CHCl₃); H

D

NMR (CDCl₃, 270 MHz): d ¹³C NMR (CDCl₃,

270 MHz):

d 0.87 (d, J = 6.8 Hz, 3H), 0.91 (d,

J = 6.6 Hz, 3H), 2.09–2.30 (m, 1H), 2.48–2.70 (m, 2H),

2.76–2.98 (m, 2H), 3.04 (d, J = 16.3 Hz, 1H), 3.16 (d,

J = 16.5 Hz, 1H), 3.53 (d, J = 13.7 Hz, 1H), 3.68 (d,

J = 13.9 Hz, 1H), 3.73–3.85 (m, 1H), 4.08–4.23 (m, 1H),

4.32 (d, J = 6.8, 9.0 Hz, 1H), 4.50 (br s, 1H), 5.97 (br s,

1H), 6.90 (d, J = 9.2 Hz, 1H), 6.95–7.50 (m, 10H), 7.77–

7.91 (m, 1H), 8.08–8.20 (m, 1H), 8.38–8.50 (m, 1H),

8.50–8.63 (m, 1H). ¹³C NMR (CDCl₃, 67.8 MHz): d

17.9, 19.5, 30.4, 38.5, 52.5, 57.8, 58.8, 59.0, 59.3, 68.0,

122.3, 122.6, 126.3, 126.5, 127.6, 128.4, 129.2, 130.0,

130.6, 131.8, 137.4, 137.8, 140.0, 148.3, 149.0, 164.7,

170.9, 173.9. Anal. (C₃₀H₃₆BrN₅O₄) C, H, N.

4.2.27. (2S,3S)-1-[(m-Bromobenzyl)amino]-3-[(tert-butyl-

oxycarbonyl)amino]-4-phenylbutan-2-ol (8g). Compound

8g (0.88 g, 86%) was prepared from epoxide 6a (0.60 g,

2.3 mmol) and m-bromobenzylamine (7g, 0.84 g,

4.5 mmol) according to the method for the preparation

of 8a, except that it was puriﬁed by ﬂash column chro-

matography (CH₂Cl₂/MeOH, 19:1) to give 8g as a color-

22

1

less glue. Compound 8g: ½aꢁ ꢂ24.6 (c 0.6, CHCl₃); H

D

NMR (CDCl₃(1% CD₃OD), 270 MHz): d 1.15 (br s,

1H), 1.25 (s, 9H), 2.47–2.67 (m, 2H), 2.67–2.88 (m,

2H), 3.55–3.94 (m, 4H), 7.013–7.25 (m, 7H), 7.25–7.35

(m, 1H), 7.35–7.42 (m, 1H). ¹³C NMR (CDCl₃(1%

CD₃OD), 67.8 MHz): d 28.0, 38.1, 51.2, 52.1, 53.8,

68.3, 79.5, 122.5, 126.2, 127.2, 128.2, 129.1, 130.1,

130.9, 131.6, 137.9, 138.6, 156.3. Anal. (C₂₂H₂₉BrN₂O₃)

C, H, N.

4.2.31. (5S,6R)-3-Aza-5-hydroxy-7-phenyl-3-(m-phenylb-

enzyl)-6-[(picolyl-^L-valinyl)amino]-heptanoyl amide (12b).

Compound 11g (43 mg, 70 lmol), phenylboronic acid

(50 mg, 0.41 mmol), NaHCO₃(46 mg, 0.55 mmol), and

Pd(PPh₃)₂Cl₂(3 mg, 5 lmol) were dissolved in a mixture

of DME and ethanol (3:1, 2 mL) in a Smith vial. The

vial was heated to 140 ꢁC for 20 min by means of micro-

wave irradiation. The mixture was ﬁltered through a cel-

ite plug, the solvent was removed by evaporation, and

the residue was puriﬁed on a silica gel column using a

mixture of CH₂Cl₂and methanol (39:1) as mobile phase.

The fractions containing the product were pooled and

further puriﬁed using preparative HPLC (Zorbax C8,

20 · 150 mm, particle size 5 lm) using a gradient

(H₂O/AcCN (0.1% formic acid) 95:5 ! 4:6) over a peri-

od of 30 min. The fractions containing the pure product

(measured with MS) were pooled and freeze dried to

4.2.28. (5S,6R)-3-Aza-3-(m-bromobenzyl)-6-[(tert-butyl-

oxycarbonyl)amino]-5-hydroxy-7-phenylheptanoyl amide

(9g). Compound 9g (0.49 g, 59%) was prepared from

substance 8g (0.73 g, 1.6 mmol) according to the method

22

for the preparation of 9a. Compound 9g: ½aꢁ ꢂ26.6 (c

D

0.5, CHCl₃); ¹H NMR (CDCl₃(1% CD₃OD),

270 MHz): d 1.19 (br s, 1H), 1.25 (s, 9H), 2.32–2.65

(m, 2H), 2.65–2.83 (m, 2H), 2.83–3.14 (m, 4H), 3.30–

3.75 (m, 4H), 6.94–7.40 (m, 9H). ¹³C NMR (CDCl₃

(1% CD₃OD), 67.8 MHz): d 28.0, 38.4, 53.3, 53.7,

57.4, 58.6, 67.6, 79.4, 122.3, 126.2, 127.4, 127.4, 128.2,

129.0, 129.9, 130.5, 131.7, 138.0, 139.7, 156.1, 170.4.

Anal. (C₂₄H₃₂BrN₃O₄Æ0.7H₂O) C, H, N.

give title compound (4.3 mg, 10%). Compound 12b:

½aꢁ

22

D

400 MHz):

ꢂ34.8 (c 0.2, CHCl₃); ¹H NMR (CDCl₃,

d 0.86 (d, J = 6.9 Hz, 3H), 0.97 (d,

D. Muthas et al. / Bioorg. Med. Chem. 13 (2005) 5371–5390

5387

J = 7.0 Hz, 3H), 2.40–2.58 (m, 1H), 2.65–2.98 (m, 2H),

2.98–3.10 (m, 1H), 3.15 (d, J = 16.6 Hz, 1H), 3.24–3.51

(m, 2H), 3.75 (d, J = 13.7 Hz, 1H), 3.91 (d,

J = 16.3 Hz, 3H), 4.01–4.33 (m, 2H), 4.37 (dd, J = 4.6,

6.5 Hz, 2H), 5.35–5.67 (m, 2H), 6.70–6.88 (m, 1H),

6.88–7.00 (m, 1H), 7.00–7.20 (m, 2H), 7.33–7.52 (m,

4H), 7.52–7.74 (m, 3H), 7.74–7.96 (m, 2H), 7.96–8.08

(m, 1H), 8.08–8.22 (m, 1H), 8.37–8.52 (m, 1H), 8.52–

8.68 (m, 1H), 8.68–8.83 (m, 1H), ¹³C NMR (CDCl₃,

100.5 MHz): d 17.1, 19.7, 29.7, 38.8, 50.0, 60.4, 61.2,

65.5, 71.8, 12.7, 126.25, 126.9, 127.3, 127.6, 127.9,

128.6, 128.9, 129.2, 129.6, 130.2, 133.0, 133.8, 135.6,

137.7, 140.2, 142.2, 148.5, 148.8, 165.7, 170.9, 171.6.

Anal. (C₃₆H₄₁N₅O₄Æ1.2H₂OÆHCOOH) C, H, N.

129.0, 130.3, 131.3, 137.8, 156.1, 167.8, 172.3. Anal.

(C₃₀H₄₃BrN₄O₅Æ0.5H₂O) C, H, N.

4.2.35. (5S,6R)-3-Aza-3-[2-(p-bromophenyl)ethyl]-5-hy-

droxy-7-phenyl-6-[(picolyl-^L-valinyl)amino]-heptanoyl amide

(11h). Compound 11h (73 mg, 31%) was prepared from

substance 10h (240 mg, 0.39 mmol) according to the

method for the preparation of 11a, except that for the

acid-mediated deprotection TFA (25% in CH₂Cl₂) was

22

D

1

used. Compound 11h: ½aꢁ ꢂ49.5 (c 1.0, CHCl₃); H

NMR (CDCl₃, 270 MHz): d 0.72–1.12 (m, 6H), 2.16–

2.40 (m, 1H), 2.40–2.80 (m, 6H), 2.80–3.00 (m, 2H),

3.00–3.30 (m, 2H), 3.68–3.80 (m, 1H), 4.12–4.28 (m,

1H), 4.28–5.46 (m, 1H), 5.90 (br s, 1H), 6.82–7.09 (m,

5H), 7.09–7.21 (m, 4H), 7.30–7.41 (m, 2H), 7.41–7.59

(m, 1H), 7.76–7.96 (m, 1H), 8.04–8.24 (m, 1H), 8.40–

8.33 (m, 1H), 8.33–8.51 (m, 1H). ¹³C NMR (CDCl₃,

67.8 MHz): d 18.1, 19.7, 30.4, 32.6, 38.7, 52.4, 57.2,

58.7, 58.9, 59.5, 68.5, 120.1, 122.4, 126.5, 126.7, 128.5,

129.3, 130.6, 131.6, 137.6, 138.0, 138.7, 148.5, 149.1,

164.9, 171.0, 174.4. Anal. (C₃₁H₃₈BrN₅O₄) C, H, N.

4.2.32. (2S,3S)-1-{[2-(p-Bromophenyl)ethyl]amino}-3-[(tert-

butyloxycarbonyl)amino]-4-phenylbutan-2-ol (8h). Com-

pound 8h (0.39 g, 96%) was prepared from epoxide

6a (0.23 g, 0.87 mmol) and 2-(p-bromophenyl)ethylamine

(7h, 0.35 g, 1.8 mmol) according to the method for the

preparation of 8a, except that it was puriﬁed by ﬂash

column chromatography (CH₂Cl₂/MeOH, 19:1) to give

22

D

4.2.36. (5S,6R)-3-Aza-5-hydroxy-7-phenyl-3-[2-(p-phenyl-

phenyl)ethyl]-6-[(picolyl-^L-valinyl)amino]-heptanoyl amide

(12c). Compound 12c (3.2 mg, 31%) was prepared from

8h as a white powder. Compound 8h: ½aꢁ ꢂ20.9 (c 1.0,

1

CHCl₃); H NMR (CDCl₃270 MHz): d 1.36 (br s, 1H),

1.41 (s, 9H), 2.40–2.95 (m, 8H), 3.34–3.55 (m, 1H),

3.55–3.80 (m, 1H), 5.03 (br d, J = 9.1 Hz, 1H), 6.96–7.03

(m, 2H), 7.03–7.23 (m, 5H), 7.23–7.42 (m, 2H). ¹³C

NMR (CDCl₃, 67.8 MHz): d 18.96, 28.28, 35.57, 39.0,

50.3, 52.1, 53.6, 68.2, 79.2, 120.0, 126.2, 128.3, 129.3,

130.3, 131.5, 138.3, 138.4, 155.8. Anal. (C₂₃H₃₁BrN₂O₃Æ

0.3H₂O) C, H, N.

substance 11h (33 mg, 53 lmol) according to the method

22

D

for the preparation of 12b. Compound 12c: ½aꢁ ꢂ31.2 (c

1

0.3, CHCl₃); H NMR (CDCl₃, 270 MHz): d 0.75–1.03

(m, 6H), 2.22–2.41 (m, 1H), 2.41–2.55 (m, 1H), 2.55–

2.70 (m, 1H), 2.70–2.96 (m, 6H), 3.04–3.15 (m, 1H),

3.15–3.30 (m, 1H), 3.57–3.72 (m, 1H), 4.10–4.25 (m,

1H), 4.29–4.40 (m, 1H), 5.20–5.45 (m, 1H), 6.43–6.65

(m, 2H), 7.00–7.12 (m, 1H), 7.12–7.24 (m, 5H), 7.31–

7.39 (m, 1H), 7.39–7.62 (m, 7H), 7.72–7.91 (m, 1H),

8.03–8.19 (m, 1H), 8.46–8.56 (m, 1H), 8.60–8.74 (m,

1H). ¹³C NMR (CDCl₃, 67.8 MHz): d 17.7, 19.7, 30.1,

33.0, 38.7, 52.0, 57.2, 58.4, 58.6, 59.6, 68.1, 122.5, 126.6,

126.7, 127.0, 127.3, 127.5, 128.6, 128.9, 129.3, 129.4,

137.6, 137.8, 139.0, 139.4, 140.7, 148.5, 149.2, 165.0,

170.9, 173.6. Anal. (C₃₇H₄₃N₅O₄Æ1.5H₂OÆ2 HCOOH) C,

H, N.

4.2.33. (5S,6R)–3-Aza-3-[2-(p-bromophenyl)ethyl]-6-[(tert-

butyloxycarbonyl)amino]-5-hydroxy-7-phenylheptanoyl

amide (9h). Compound 9h (0.35 g, 84%) was prepared

from substance 8h (0.37 g, 0.80 mmol) according to the

22

D

method for the preparation of 9a. Compound 9h: ½aꢁ

1

ꢂ30.8 (c 0.6, CHCl₃); H NMR (CDCl₃, 270 MHz): d

1.28 (s, 1H), 1.38 (s, 9H), 2.44–2.77 (m, 6H), 2.77–2.98

(m, 2H), 2.98–3.20 (m, 2H), 3.60–3.80 (m, 1H), 3.80–

3.97 (m, 1H), 5.15–5.23 (m, 1H), 6.00–6.14 (m, 1H),

6.83–6.97 (m, 2H), 6.97–7.12 (m, 1H), 7.12–7.33 (m,

4H), 7.33–7.45 (m, 2H). ¹³C NMR (CDCl_3,67.8 MHz):

d 28.5, 32.7, 39.0, 53.8, 57.1, 58.7, 58.8, 68.1, 79.6, 120.1,

126.5, 128.6, 129.4, 130.5, 131.6, 138.4, 138.6, 156.2,

174.9. Anal. (C₂₅H₃₄BrN₃O₄) C, H, N.

4.2.37. (2S,3S)-1-[(N-Benzyl-N-benzyloxycarbonyl)ami-

no]-3-[(tert-butyloxycarbonyl)amino]-4-phenylbutan-2-ol

(13). Compound 8b (0.53 g, 1.4 mmol) was dissolved in

CH₂Cl₂(100 mL), a solution of K₂CO₃(0.41 g, 3.0 mmol)

in water (100 mL) and benzylchloroformate (0.30 lL,

2.13 mmol) was added, and the mixture was stirred for

2 h. The layers were separated and the water layer was

extracted with CH₂Cl₂(30 mL). The combined organic

phases were dried, ﬁltered and evaporated. The residue

was puriﬁed by column chromatography (toluene/

EtOAc, 3:1) to give compound 13 (0.38 mg, 52%). Com-

4.2.34.

(5S,6R)-3-Aza-3-[2-(p-bromophenyl)ethyl]-6-

{[(tert-butyloxycarbonyl)-^L-valinyl]amino}-5-hydroxy-7-

phenylheptanoyl amide (10h). Compound 10h (270 mg,

65%) was prepared from substance 9h (320 mg,

0.62 mmol) according to the method for the preparation

22

D

1

of 10a, except that for the acid-mediated deprotection

pound 13: ½aꢁ

0 (c 1.2, CHCl₃); H NMR (CD₃OD,

22

D

TFA (25% in CH₂Cl₂) was used. Compound 10h: ½aꢁ

270 MHz, rotamers): d 1.02–1.39 (m, 9H), 2.58–2.83 (m,

2H), 3.00–3.30 (m, 2H), 3.52–3.74 (m, 1H), 3.74–3.90

(m, 1H), 4.39–4.58 (m, 2H), 4.90–5.09 (m, 2H), 6.91–

7.32 (m, 15H). Anal. (C₂₉H₃₆N₂O₅) C, H, N.

1

ꢂ22.4 (c 1.4, CHCl₃); H NMR (CDCl₃, 270 MHz): d

0.82–1.10 (m, 7H), 1.44 (s, 9H), 2.00–2.21 (m, 1H),

2.42–2.77 (m, 5H), 2.77–3.00 (m, 3H), 3.00–3.27 (m,

2H), 3.62–4.00 (m, 3H), 5.10–5.32 (m, 1H), 5.60–5.93

(m, 1H), 6.62–6.85 (m, 1H), 6.85–7.08 (m, 2H), 7.08–

7.33 (m, 5H), 7.33–7.56 (m, 2H). ¹³C NMR (CDCl₃,

67.8 MHz): d 17.2, 19.0, 28.0, 30.4, 31.7, 37.8, 52.4,

57.0, 57.5, 58.2, 60.3, 67.5, 79.9, 119.9, 126.3, 128.3,

4.2.38. (2S,3S)-1-[(N-Benzyl-N-benzyloxycarbonyl)amino]-

3-{[(tert-butyloxycarbonyl)-^L-valinyl]amino}-4-phenylbutan-

2-ol (14). Compound 14 (0.19 g, 50%) was prepared from

substance 13 (0.32 g, 0.63 mmol) according to the method

5388

D. Muthas et al. / Bioorg. Med. Chem. 13 (2005) 5371–5390

for the preparation of 10a, except that for the acid-medi-

ated deprotection TFA (25% in CH₂Cl₂) was used. Com-

was stirred for overnight at 50 ꢁC. The solvent was evap-

orated and the product was puriﬁed by ﬂash column

22

D

pound 14: ½aꢁ ꢂ26.2 (c 1.4, CHCl₃); ¹H NMR (CDCl₃,

chromatography (CH₂Cl₂/MeOH, 19:1) to give the pure

tertiary amine 18 (30 mg, 64%). Compound 18: ½aꢁ

22

D

270 MHz, rotamers): d 0.60–1.05 (m, 6H), 1.43 (s, 9H),

1.89–2.20 (m, 1H), 2.75–2.97 (m, 2H), 2.97–3.26 (m,

1H), 3.44–4.08 (m, 4H), 4.35–4.70 (m, 2H), 4.93–5.54

(m, 3H), 6.32–6.69 (m, 1H), 6.98–7.41 (m, 15H). ¹³C

NMR (CDCl₃, 67.8 MHz): d 17.4, 19.3, 28.3, 30.5, 38.2,

52.3, 53.1, 53.5, 60.2, 67.7, 70.8, 79.9, 124.2, 126.4,

127.4, 127.5, 127.9, 129.1, 128.4, 128.6, 129.2, 136.0,

136.9, 137.7, 155.8, 158.1, 171.6. Anal. (C₃₄H₄₅N₃O₆) C,

H, N.

1

ꢂ52.2 (c 0.9, CHCl₃); H NMR [CDCl₃(1% CD₃OD),

270 MHz]:

d 0.79 (d, J = 6.8 Hz, 3H), 0.84 (d,

J = 6.8 Hz, 3H), 2.02–2.22 (m, 1H), 2.46–2.64 (m, 2H),

2.64–2.86 (m, 3H), 2.92 (dd, J = 7.3, 13.9 Hz, 1H),

3.35–3.48 (m, 2H), 3.50–3.62 (d, J = 13.9 Hz, 1H),

3.67–3.80 (d, J = 13.7 Hz, 1H), 3.98–4.13 (m, 1H),

4.13–4.25 (m, 1H), 6.82–6.91 (m, 2H), 6.91–7.04 (m,

3H), 7.04–7.18 (m, 6H), 7.20–7.34 (m, 3H), 7.38–7.48

(m, 1H), 7.72–7.84 (m, 1H), 7.98–8.07 (m, 1H), 8.52–

8.60 (m, 1H). ¹³C NMR [CDCl₃(1% CD₃OD),

67.8 MHz]: d 17.4, 19.3, 30.2, 34.0, 38.1, 51.9, 54.6,

55.5, 59.2, 65.2, 67.8, 119.9, 122.1, 126.1, 126.6, 127.1,

128.8, 128.2, 128.6, 129.0, 130.7, 131.2, 137.2, 137.4,

137.7, 138.6, 148.3, 148.6, 164.9, 171.0, 173.9. Anal.

(C₃₇H₄₂BrN₅O₄) C, H, N.

4.2.39. (2S,3S)-1-[(N-Benzyl-N-benzyloxycarbonyl)ami-

no]-4-phenyl-3-[(picolyl-^L-valinyl)amino]-butan-2-ol (15).

Compound 15 (0.12 g, 74%) was prepared from substance

14 (0.17 g, 0.28 mmol) according to the method for the

preparation of 11a, except that for the acid-mediated

22

D

deprotection TFA (25% in CH₂Cl₂) was used. 15: ½aꢁ

1

ꢂ32.3 (c 0.8, CHCl₃); H NMR [CDCl₃(1% CD₃OD),

270 MHz, rotamers]: d 0.60–1.09 (m, 6H), 1.90–2.23 (m,

1H), 2.62–2.92 (m, 2H), 2.92–3.22 (m, 1H), 3.22–3.58

(m, 2H), 3.67–3.88 (m, 1H), 3.88–4.12 (m, 1H), 4.32–

4.68 (m, 2H), 4.88–5.19 (m, 2H), 6.80–7.35 (m, 15H),

7.35–7.55 (m, 1H), 7.70–7.84 (m, 1H), 7.84–8.20 (m,

1H), 8.36–8.62 (m, 2H). ¹³C NMR [CDCl₃(1% CD₃OD),

67.8 MHz, rotamers]: d 17.7, 19.3, 30.6, 37.9, 51.8, 52.7,

53.2, 58.9, 67.5, 70.2, 122.3, 126.1, 126.5, 127.2, 127.3,

127.6, 127.7, 128.0, 128.1, 128.4, 128.9, 129.0, 136.0,

137.0, 137.7, 147.9, 148.7, 156.9 (minor), 157.6 (major),

164.3, 171.1. Anal. (C₃₆H₄₀N₄O₅ÆH₂O) C, H, N.

4.2.42. (2S,5S,6R)-3-Aza-3-benzyl-2-(p-phenylbenzyl)-5-

hydroxy-7-phenyl-6-[(picolyl-^L-valinyl)amino]-heptanoyl

amide (19). Compound 18 (23 mg, 33 lmol), phenylbo-

ronic acid (24 mg, 0.20 mmol), Cs₂CO₃(32 mg, 98 lmol),

and Pd(PPh₃)₂Cl₂(1.8 mg, 5 lmol) were dissolved in a

mixture of DME and ethanol (3:1, 0.7 mL) in a Smith vial.

The vial was heated to 130 ꢁC for 20 min by means of

microwave irradiation. The mixture was ﬁltered through

a celite plug, the solvent was removed by evaporation,

and the residue was puriﬁed on a silica gel column using

a mixture of CH₂Cl₂and methanol (19:1) as mobile phase

to give compound 19 (18 mg, 79%) as a white powder. 19:

22

D

4.2.40. (2S,3S)-1-(Benzylamino)-4-phenyl-3-[(picolyl-^L-

valinyl)amino]-butan-2-ol (16). Compound 15 (70 mg,

0.12 mmol) and anisol (0.13 mg, 1.2 mmol) were dis-

solved in CH₂Cl₂(30 mL). Triﬂic acid (0.2 mL, 2.3 mmol)

was added slowly under stirring and the mixture was stir-

red for an additional 15 min. The reaction mixture was

suspended to a silica gel column and this was ﬂushed sev-

eral times with CH₂Cl₂. The product was eluted with a

mixture of CH₂Cl₂and MeOH (9:1) and further puriﬁed

with ﬂash column chromatography (CH₂Cl₂/MeOH,

29:1) to give the pure secondary amine 16 (21 mg, 38%).

½aꢁ 29.8 (c 0.4, CHCl₃); ¹H NMR [CDCl₃(5% CD₃OD),

400 MHz]: d 0.85 (d, J = 6.8 Hz, 3H), 0.87 (d, J = 6.8 Hz,

3H), 1.90–2.13 (m, 1H), 2.51–2.80 (m, 4H), 3.00 (dd,

J = 7.0, 13.8 Hz, 1H), 3.10 (dd, J = 8.1, 13.6 Hz, 1H),

3.43–3.49 (m, 1H), 3.58 (d, J = 14.2 Hz, 1H), 3.70 (dd,

J = 7.0, 8.0 Hz, 1H), 3.98 (d, J = 14.1 Hz, 1H), 4.12–

4.20 (m, 1H), 4.21–4.29 (m, 1H), 6.90–6.96 (m, 1H),

6.98–7.23 (m, 8H), 7.26–7.36 (m, 4H), 7.38–7.46 (m,

2H), 7.46–7.63 (m, 5H), 7.89–8.01 (m, 1H), 8.04–8.13

(m, 1H), 8.60–8.65 (m, 1H). Anal. (C₄₃H₄₇N₅O₇Æ0.3H₂O)

C, H, N.

22

1

Compound 16: ½aꢁ ꢂ51.0 (c 1.0, CHCl₃); H NMR

D

(CDCl₃, 270 MHz): d 0.84 (d, J = 6.8 Hz, 3H), 0.89 (d,

J = 6.8 Hz, 3H), 2.10–2.30 (m, 1H), 2.60–2.79 (m, 2H),

2.79–2.98 (m, 2H), 3.75–3.95 (m, 3H), 4.05–4.20 (m,

1H), 4.30–4.78 (m, 2H), 4.38 (dd, J = 6.5, 8.8 Hz, 1H),

6.82–6.98 (m, 1H), 6.98–7.38 (m, 9H), 7.38–7.53 (m,

1H), 7.78–7.95 (m, 1H), 8.09–8.22 (m, 1H), 8.35–8.50

(m, 1H), 8.53–8.65 (m, 1H). ¹³C NMR (CDCl₃,

67.8 MHz): d 17.9, 19.6, 30.6, 38.3, 51.2, 52.8, 52.9, 59.3,

68.0, 122.4, 126.4, 126.6, 128.1, 128.5, 128.8, 129.0,

129.4, 137.5, 137.9, 148.4, 149.3, 164.8, 171.3. Anal.

(C₂₈H₃₄N₄O₃ÆH₂O) C, H, N.

4.3. Computational procedure

4.3.1. General procedure. Molecular modeling calcula-

tions were performed on a dual Intel Xeon 2.4 GHz

CPU workstation and a 2.6 GHz Pentium 4 workstation

running Red Hat Linux 9. Statistical analyses and visu-

alizations were performed on Silicon Graphics Octane

workstations with two 500 MHz IP35 processors and

R14000 processor chips. The diﬀerent suite of programs

employed for modeling studies were SYBYL v.6.9³³for

data set preparation and 3D comparative molecular ﬁeld

analysis (CoMFA),³⁴Maestro v.3.0, and Macromodel

v.7.1³⁵for enzyme reﬁnement. Flexible docking was per-

formed using FLO.²⁶

4.2.41. (2S,5S,6R)-3-Aza-3-benzyl-2-(p-bromobenzyl)-5-

hydroxy-7-phenyl-6-[(picolyl-^L-valinyl)amino]-heptanoyl

amide (18). (2S,5S,6R)-3-Aza-2-(p-bromobenzyl)-5-hy-

droxy-7-phenyl-6-[(picolyl-^L-valinyl)amino]- heptanoyl

amide (17, 41 mg, 67 lmol) and Cs₂CO₃(22 mg,

84 lmol) were suspended in DMF (2 mL). Benzyl bro-

mide (20 lL, 0.17 mmol) was added and the mixture

4.3.2. Enzyme preparation. The X-ray crystal structure

of Plm II from Plasmodium falciparum in complex with

inhibitor rs370 was obtained under the accession code

1LF2²⁷from the Protein Data Bank (PDB).³⁶This

D. Muthas et al. / Bioorg. Med. Chem. 13 (2005) 5371–5390

5389

˚

329-residue structure was resolved at 1.80 A and pos-

sessed an R-value of 0.195. Crystallographic waters were

removed and hydrogens added via an all-atom treat-

ment with no atoms having lone pairs. The structural

and electrostatic ﬁelds were set to 30 kcal/mol, employ-

ing a distance-dependent dielectric (1/r²) for calculating

electrostatics and a 6–12 Lennard–Jones potential for

sterics. The CoMFA region was focused and the result-

ing focused CoMFA descriptor ﬁelds were used as inde-

pendent variables in the partial least squares (PLS)⁴⁰

regression analysis with measured pKi (ꢂlogK_i) values

as the dependent variable. A K_ivalue of 3600 nM was

used for all compounds that had no exact value deter-

mined. Column ﬁltering with a leave-one-out (LOO)

validation, and a maximum of six components was used

for all cross-validation calculations to determine the q²

(cross-validated correlation coeﬃcient) and the PRESS

value (standard error of prediction). Since CoMFA

was performed only to rationalize the qualitative results

obtained from docking, no test set was used.

reﬁnement was performed using the BatchMin algo-

37

rithm of Macromodel v.7.1, employing the AMBER

*

all-atom force ﬁeld with a dielectric constant of 78.

The extended nonbonded cutoﬀ distances for van der

Waals and Coulombic interactions were set to 3.5 and

6.0 A, respectively, while the hydrogen-bonding cutoﬀ

˚

distance was kept at 4.0 A. These cutoﬀs have been

shown to give optimal results in terms of speed and

accuracy.³⁸The protein was minimized with a total of

200 iterations employing the Polak–Ribiere conjugate

gradient (PRCG) method, and convergence was moni-

tored with the derivative convergence criteria using a

˚

cutoﬀ value of 0.05 kJ/A mol. A pocket around the

inhibitor (rs370) was marked and extracted for docking

calculations. This pocket constituted the crucial amino

acids lining the active site. The inhibitor rs370 was re-

moved from the active pocket and the protein was sub-

jected to docking studies. As Plm II is found to exist in

an acidic food vacuole having a pH of 5, arginines, ly-

sines, aspartic, and glutamic acids were kept positively

and negatively charged, respectively.

Acknowledgments

We express our sincere gratitude to the Swedish Foun-

dation for Strategic Research (SSF), the Swedish

Research Council (VR), and Medivir AB for ﬁnancing

this research and to Biotage AB for providing the micro-

´

wave synthesizer. Dr. Johan Hulten is acknowledged for

early work in this project.

4.3.3. Data set. The data set used for the docking and

CoMFA studies comprises a series of 37 analogues with

a basic hydroxyethylamine transition state isostere scaf-

fold as the central fragment. All compounds were pro-

tonated at the basic hydroxyethylamine nitrogen.

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Article Doi

Synthesis, biological evaluation, and modeling studies of inhibitors aimed at the malarial proteases plasmepsins I and II

DOI: 10.1016/j.bmc.2005.06.048

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmc.2005.06.048

Full text of DOI:10.1016/j.bmc.2005.06.048

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