The design, synthesis, and biological evaluation of analogues of the serine-threonine protein phosphatase 1 and 2A selective inhibitor microcystin LA: Rational modifications imparting PP1 selectivity

Bioorganic & Medicinal Chemistry 7 (1999) 543±564

The Design, Synthesis, and Biological Evaluation of Analogues

of the Serine-Threonine Protein Phosphatase 1 and 2A Selective

Inhibitor Microcystin LA: Rational Modi®cations Imparting PP1

Selectivity

James B. Aggen,^aJohn M. Humphrey,^a,yCarla-Maria Gauss,^aHsien-Bin Huang,^b

Angus C. Nairn^cand A. Richard Chamberlin^a,*

^aDepartment of Chemistry, University of California at Irvine, Irvine, CA 92697, USA

^bInstitute of Biochemistry, Chu-Tzi College of Medicine, Hualien 970, Taiwan

^cThe Rockefeller University, 1230 York Avenue, New York, NY 10021, USA

Received 29 June 1998; accepted 24 November 1998

AbstractÐBased on the results from previously reported molecular modeling analyses of the interactions between the inhibitor

microcystin and the serine-threonine protein phosphatases 1 and 2A, we have designed analogues of microcystin LA with structural

modi®cations intended to impart PP1 selectivity. The synthesis of several ®rst generation analogues followed by inhibition assays

revealed that all three are PP1-selective, as predicted. Although the observed selectivities are modest, one of the designed analogues

Introduction

made to de®ne the separate roles of these phosphatases

in a given process. In particular, there is no known

small molecule that inhibits PP1 with good selectivity. A

recently published X-ray structure of a PP1-microcystin

LR complex³provides the opportunity to explore the

binding interactions of these inhibitors with both PP1

and PP2A, and to design new selective inhibitors based

on these natural products.⁴

Members of the structurally diverse group of natural

toxins that includes okadaic acid, calyculin, microcystin

LA, and tautomycin exert their cytotoxic eects by

inhibiting the serine-threonine protein phosphatases

PP1 and PP2A.^1,2This activity dramatically increases

the phosphorylation state of a variety of proteins within

the cell, a process that can have profound eects such as

the disruption of intracellular signal tracking, the

deposition of proteinacious plaques and ®brils, and

unregulated cellular proliferation. Interestingly, despite

the dissimilarities of the toxins' structures, these com-

pounds are competitive inhibitors (i.e. their PP1/PP2A

binding sites overlap).

Analogue Design

We previously reported the results of a molecular mod-

eling study in which the structure of a PP2A-micro-

cystin complex was calculated based on the known

structure of PP1 bound microcystin, and the sequence

homology of PP2A to PP1.^4aInspection of the two

enzyme±inhibitor complexes resulted in the identi®ca-

tion of three dierential contacts that might be exploi-

ted for achieving PP1 selectivity. These contacts occur

between three adjacent residues in the toxin, l-leucine,

d-alanine, and N-methyldehydroalanine, and the b-12

and b-13 loops of the enzymes, a region that is known

to be important for inhibitor selectivity based on site-

directed mutagenesis studies.^3,5

As a group these toxins inhibit PP1 and PP2A quite

potently and speci®cally relative to other known phos-

phatases, such as PP2B (calcineurin), PP2C, and the

tyrosine phosphatases. Consequently, several members

of this group have become important probes of intra-

cellular signalling pathways. Even so, there are pro-

blems of poor selectivity between PP1 and PP2A that

can result in ambiguous results when attempts are being

Key words: microcystin; inhibitor; PP1; PP2A; selective.

_y*Corresponding author.

Based on a comparision of the X-ray structure with the

calculated PP2A complex, a pocket occupied by the

leucine isopropyl group of the inhibitor appears to be

Current Address: Central Research Division, P®zer Inc., Groton,

CT 06340, USA.

PII: S0968-0896(98)00254-5

544

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

more sterically demanding in PP2A than in PP1 (Fig. 1),

suggesting that enlarging this group would deleteriously

aect binding anity to a greater extent in PP2A. It is

of interest to note that this is one of the most variable

positions in the many naturally occuring microcystins;

toxic variants containing arginine, tyrosine, phenylala-

nine, alanine, tryptophan, and homotyrosine at this

position have been isolated,^6,7but little is known about

the eect of these substitutions on selectivity. In PP1,

the leucine isopropyl group is in close proximity to tyro-

sine 272, and is ¯anked by the unimposing glycine 274.

In PP2A, however, a second tyrosine replaces glycine,

and the leucine isopropyl group is then juxtaposed to

both tyrosines (numbered 261 and 263 in PP2A).

Inspection of the models suggested that groups as large

as cyclohexyl might be better accommodated in PP1,

leading to the choice of l-cyclohexylalanine as a sub-

stitute for leucine in the ®rst generation non-natural

microcystin.

(Fig. 2); in PP1 the inhibitor makes contact with gluta-

mate 275, while in PP2A the corresponding residue is

the arginine 264 side chain. The formation of mixed

charge complements (i.e. salt bridges) can be a dominant

contributor to binding anity, despite the considerable

desolvation penalty that is incurred upon binding in

many systems.^9±11The formation of these charge com-

plements primarily acts to decrease the o rate of a

ligand bound to its receptor, rather than increase the on

rate, and the contribution to binding anity by a single

salt bridge can be as high as 3.5 kcal/mol.¹⁰The strate-

gic placement of an ammonium group on the d-alanine

residue of the inhibitor might thus be expected to favor

inhibition of PP1 over PP2A by forming a salt bridge

with glutamate 275 in PP1, while suering electrostatic

repulsion with arginine 264 in PP2A. We therefore

chose to substitute the d-alanine residue found in nat-

ural microcystin LA with the corresponding amino-

substituted amino acid, d-2,3-diaminoproprionic acid.

The adjacent residue, d-alanine, has as nearest neigh-

bors residues of opposite charge in the two enzymes

The ®nal dierential contact to be modi®ed is one made

by the the N-methyl group (Fig. 3). PP1 has a sizable

Figure 1. Stereoviews of the close contacts between the leucine isopropyl group in microcystin LR⁸(shown in magenta in the complex with PP1;

shown in yellow in the complex with PP2A) and the enzymes PP1 (left, dark blue ribbon) and PP2A (right, magenta ribbon). The close contact

residues in the enzymes are light blue, oxygen atoms are red, and the residue numbers are indicated in white bold. The remaining visible portion of

microcystin has blue nitrogen atoms, grey carbon atoms, and white hydrogen atoms. The microcystin LR±PP1 complex is based on the known

crystal structure, and the microcystin LR±PP2A complex is a theoretical structure based on modeling eorts.^4a

Figure 2. Stereoviews of the close contacts between the d-alanine methyl in microcystin LR⁸(shown in magenta in the complex with PP1; shown in

yellow in the complex with PP2A) and the enzymes PP1 (left, dark blue ribbon) and PP2A (right, magenta ribbon). The close contact residues in the

enzymes are light blue, oxygen atoms are red, and the residue numbers are indicated in white bold. The remaining visible portion of microcystin has

blue nitrogen atoms, grey carbon atoms, and white hydrogen atoms. The microcystin LR±PP1 complex is based on the known crystal structure, and

the microcystin LR±PP2A complex is a theoretical structure based on modeling eorts.^4a

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

545

Figure 3. Stereoviews of the close contacts between the N-methyldehydroalanine methyl group in microcystin LR⁸(shown in magenta in the com-

plex with PP1; shown in yellow in the complex with PP2A) and the enzymes PP1 (left, dark blue ribbon) and PP2A (right, magenta ribbon). The

close contact residues in the enzymes are light blue, oxygen atoms are red, and the residue numbers are indicated in white bold. The remaining visible

portion of microcystin has blue nitrogen atoms, grey carbon atoms, and white hydrogen atoms. The microcystin LR±PP1 complex is based on the

known crystal structure, and the microcystin LR±PP2A complex is a theoretical structure based on modelling eorts.^4a

hydrophobic cleft in the region directly behind the N-

methyl group, while in PP2A the backbone carbonyl of

glutamine 238 resides within 2.09 angstroms of the

N-methyl carbon atom. The introduction of a larger N-

methyl substituent therefore appeared to be a reason-

able tactic, and the speci®c analogue chosen was the

N-cyclohexyl derivative. The proposed structural mod-

i®cations are summarized in Figure 4.

in protecting group strategy in response to the addi-

tional amino group. To avoid possible complications of

base catalyzed isomerization of the aminoalanyl group,

neutral or mildly acidic conditions were desired for the

®nal deprotection of this side chain. An obvious choice

of N-protecting groups compatible with these require-

ments, the 2,2,2-trichloroethoxycarbonyl (Troc)

group,¹³cannot be selectively removed in the presence

of the 2,2,2-trichloroethyl (Tce) ester employed in our

original microcystin LA synthesis, but this problem can

be circumvented in principle simply by the use of a t-

butyl ester (instead of Tce) to protect the carboxy ter-

minus of the dipeptide fragment 5. Another potential

problem with the use of the Troc protecting group in the

established route was the likelihood that it would not

survive removal of a Cbz (Z) group by catalytic hydro-

genation.¹⁴However, assembling the alkene-containing

tetrapeptide fragments in a more convergent 2+2 seg-

ment coupling, rather than stepwise as per the original

route (Scheme 2), circumvents this problem by allowing

for removal of the Z group before introduction of the

Troc-protected residue. This strategy also provides a

common dipeptide fragment that could be used in syn-

thesizing two of the proposed analogues.

Synthetic Plan

The general plan for preparing analogues was to follow

the published route in the synthesis of microcystin LA¹²

as closely as possible, taking advantage of the fact that

all of the modi®ed residues reside in a single subunit

employed in the original synthesis (Fragment 4, Scheme

1). This fortuitous situation allows direct use of the

stockpiles of Fragments 5 and 6 remaining from the

natural product synthesis, and the analogues 2a and 2c

were in principle accessible with no changes except sub-

stituting the appropriate modi®ed amino acids.

On the other hand, incorporation of the aminoalanine

residue in analogue 2b (Scheme 1) did require a change

Figure 4. Summary of designed microcystin LA analogues.

546

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

Scheme 1. Retrosynthesis of microcystin LA analogues.

give 12b (Scheme 4). Initial attempts to couple 12b and 9

were conducted using DMF as the solvent, and to

reduce the problems associated with NMR analysis of

the phosphonate-containing tetrapeptide (diaster-

eomers, rotamers, and phosphorous±proton coupling),

the crude material from the coupling reaction was sub-

jected to the next step (Horner±Emmons ole®nation to

give 13) without puri®cation beyond an aqueous workup.

However, the low yield (<25%) we observed over this

sequence prompted a more thorough investigation.

After conducting the sequence in stepwise fashion, we

observed that the ole®nation step was very ecient

(>90%), and that the low yields arose during the coupl-

ing step. Coupling N-alkylated a-amino acids in the

C-terminal direction can give high yields, but is often

accompanied by signi®cant racemization.^16,17This well-

known racemization process occurs via formation of the

cyclic oxazolonium ion, and reversible deprotonation of

the greatly acidi®ed a-proton scrambles the stereocenter

(Fig. 5). In light of the poor coupling yield in our case,

we suspect that the presence of the phosphonate moiety

acidi®es the a-proton to such a degree that deprotona-

tion of the cyclic oxazolonium ion is essentially irrever-

sible under the basic coupling conditions, and that the

pseudoaromatic species formed is unreactive towards

nucleophiles.

Scheme 2. Changes in the synthetic route.

Results and Discussion

Synthesis of microcystin LA analogues

According to this modi®ed route, the phosphonate-

containing dipeptide 9 was to be employed as a com-

mon precursor to the N-methyl analogues 2a and 2b.

Catalytic hydrogenolysis of the Cbz-phosphosarcosine

free acid 7¹²gave the free amino acid 10 in near quan-

titative yield (Scheme 3). Formation of the HOAt active

ester¹⁵of Boc-d-Glu-OMe-OH¹²proceeded smoothly,

and the crude material was coupled with the zwitterion

10 to give the requisite dipeptide 9 in 70% yield.

The use of less polar solvents is known to reduce the

problems of racemization in coupling reactions,¹⁸

therefore CH₂Cl₂was substituted for DMF, leading to

an improved 60% yield in the coupling of 9 with 12b

(Scheme 5).¹⁹Deprotection of 13 with TFA, followed

by re-protection of the amine with Boc₂O, gave the

desired free acid 4a in near quantitative yield.

Having established a route to 9, assembly of the tetrapep-

tide fragments could proceed. Toward this end, l-cyclo-

hexylalanine ester 11 was coupled with Boc-d-alanine

(EDCI/HOAt), followed by selective Boc acidolysis to

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

547

Scheme 3. Synthesis of the phosphonate dipeptide 9.

Scheme 5. Completion of fragment 4a.

Having proven the viability of the modi®ed route to the

dehydrotetrapeptides, the extension of this route to the

preparation of fragment 4b could proceed. We next

required a convenient route to the appropriately pro-

tected 2,3-diaminopropionic acid (aminoalanine) resi-

due, but none of the existing methods proved to be

satisfactory for our purposes.^20,21Of the various meth-

ods for preparing related derivatives, the report that

serine lactone can be opened at the b-position with

ammonia in THF^21jwas the most attractive. The lac-

tone 14 was readily prepared following the procedure of

Vederas et al. (Scheme 6),^21j,22but to our disappoint-

ment, attempts to open the lactone with ammonia in

THF gave exclusively the primary amide resulting from

opening at the carbonyl position. Fortunately, sub-

sequent experiments showed that sodium azide opens

lactone 14 cleanly at the b-position to give the azido-

acid 15 in excellent yield. Reduction of the azide by

catalytic hydrogenation, followed by amine protection,

gave the Troc-Boc-aminoalanine 17 in 80% overall yield

from lactone 14.

based on our previous experiences with related cou-

plings, when the dipeptide 18 was carried through the

sequence of coupling with 9, followed by Horner±

Emmons ole®nation, a 1:1 mixture of tetrapeptides was

obtained. The compounds were separated by chroma-

tography, and the higher R_fmaterial corresponded with

the structure of the desired dehydrotetrapeptide 19 by

¹H NMR and FABMS. The lower R_fmaterial, how-

ever, was identi®ed as the formaldehyde adduct with the

Troc protected amine (20).²³In further experiments, we

found that reducing the equivalents of formaldehyde

from 10 to 1 resulted in clean conversion to the

desired ole®n. Double deprotection of the resulting tet-

rapeptide 19 (TFA), followed by amine re-protection

gave the tetrapeptide free acid 4b in 56% (from the

dipeptide 18). As no formaldehyde adduct was seen with

the Boc group in any of the other substrates, this result

highlights the greater acidity of Troc groups relative to

Boc groups, which has been of use in Mitsunobu reac-

tions involving protected amines.²⁴

In order to complete fragment 4b, 17 was coupled with

l-Leu-OtBu (HATU) to give the corresponding dipep-

tide 18 in 77% yield (Scheme 7). Much to our surprise,

With the tetrapeptide fragments 4a and 4b completed,

our next objective was to prepare the N-cyclohexyl

Scheme 4. Low yielding coupling.

Figure 5. Oxazolonium formation and deprotonation.

548

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

Scheme 6. Synthesis of the dierentially protected aminoalanine amino acid.

Scheme 7. Synthesis of fragment 4b.

tetrapeptide 4c. As mentioned above, we were initially

interested in synthesizing the N-cyclohexyldehydro-

alanine-containing analogue 2c. To obtain 4c following

the previous synthetic route, an acid-catalyzed con-

densation of Cbz-cyclohexylamine and the hemiacetal

of methylglyoxylate was required. In the case of

Cbz-methylamine, the condensation is an equilibrium

process that slightly favors the starting materials, but it

provides a 30% yield of the desired hemiaminal.¹²Not

surprisingly, the corresponding equilibrium for the Cbz-

cyclohexylamine lies far to the left, and only trace

amounts of product were suggested by TLC (Fig. 6).

nearly as potent as the natural material in inhibiting

PP1 and PP2A. Finally, a naturally occurring micro-

cystin toxin, (Mser⁷)-microcystin LR, contains an N-

methyl serine residue in place of the dehydroalanine

residue and still retains potent toxicity.²⁸In light of

these reports, we reasoned that substituting N-methyl-

glycine (sarcosine) for the dehydroalanine residue

should not interfere signi®cantly with binding.^25d,e

Consequently, the decision was made to replace the N-

cyclohexyldehydroalanine originally designed with N-

cyclohexylglycine (N-cyhxGly).

A concern arising from the implementation of this sub-

stitution, however, was that it might deleteriously in¯u-

ence the macrocyclization,^17,29±34but this change did

not appear to aect the cyclization eciency (vide

infra). The decision to replace the N-MdhA residue with

N-cyhxGly involved changing two structural variables

at once, so the N-methylglycine (N-meGly)-containing

analogue itself was also prepared to serve as a control.

At that juncture, we reconsidered the importance of the

dehydroamino acid for binding and activity. It is known

that the dehydroalanine residue in microcystin rever-

sibly forms a covalent bond with the cysteine 273 thiol

in the binding site.^5cWhile this interaction no doubt

stabilizes the enzyme±ligand complex, it does not appear

to greatly in¯uence the apparent binding anities.

Indeed, a C273A mutant of PP1 (alanine replacement at

the cysteine residue in question) demonstrated no sig-

ni®cant change in IC₅₀values from the wild type for

several inhibitors of the okadaic acid class, including

microcystin.^5cIn addition, several reports state that

reduction of the dehydro-residue present in microcystin

produces two isomers that are as potent inhibitors as

the natural material.²⁵The Michael adducts of micro-

cystin with aminoethanethiol²⁶and ethanedithiol²⁷are

The requisite tetrapeptide fragments were easily pre-

pared from the corresponding N-alkylated glycines.

Reductive amination of Gly-OEt hydrochloride with

cyclohexanone (NaCNBH₃), followed by amine protec-

tion (Cbz-Cl) and ethyl ester hydrolysis gave the N-

cyhxGly residue 21 in 40% unoptimized yield (Scheme

8). Coupling of 21 with d-Ala-l-Leu-OtBu¹²(DCC/

HOBt) gave tripeptide 22 in excellent yield. N-terminal

coupling to N-alkylated amino acids is known to be

problematic, typically resulting in low yields and sig-

ni®cant epimerization (when coupling to a-amino

acids).¹⁸New methods and reagents, particularly Car-

pino's HATU reagent,³⁵have lead to dramatic

improvements in many cases,¹⁷but we were still con-

cerned about how eciently the bulky N-cyclohexyl-

containing tripeptide would couple in the N-terminal

Figure 6. Unsuccessful condensation.

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

549

Scheme 8. Synthesis of the N-cyhxGly tetrapeptide 24.

direction. Our concerns were allayed when we found

that following hydrogenolysis of the Cbz-group in 22,

the N-cyhxGly-d-Ala-l-Leu-OtBu peptide coupled with

Boc-d-Glu-OMe-OH to give 23 in 75% unoptimized

yield using HATU. That this coupling proceeded

cleanly attests to the power of the HATU reagent in

eecting these dicult transformations. Finally, treat-

ment of the tetrapeptide 23 with TFA, followed by

amine re-protection (Boc₂O) gave the free acid 24 in

excellent yield.

current purposes, and coupling of the aspartate deriva-

tive 28 with l-Ala-OtBu (HATU) gave the dipeptide 5

in near quantitative yield (Scheme 10).

With this dipeptide in hand, we were ready to investi-

gate the 4+2 segment couplings. In considering these

sometimes dicult coupling reactions, we were mindful

of some unpleasant possibilities associated with our

speci®c protection format; aspartate (and to a lesser

extent glutamate) residues in peptide chains are prone to

base- and acid-catalyzed imide formation.^36±39Protec-

tion of aspartate side chains as bulky t-butyl⁴⁰or b-

phenacyl³⁷esters is therefore recommended in order to

avoid base- and acid-catalyzed imide formation, respec-

tively. However, even these measures do not always

provide complete protection.^37,41

Preparation of the corresponding N-meGly containing

tetrapeptide proceeded along similar lines. Coupling of

Cbz-N-MeGlycine (Cbz-sarcosine) with d-Ala-l-Leu-

OtBu (DCC/HOBt) gave the tripeptide 25 (Scheme 9).

Hydrogenolysis, followed by HATU-mediated coupling

with Boc-d-Glu-Ome-OH, gave the tetrapeptide 26.

Double deprotection, followed by amine re-protection

gave the desired free acid 27, thus completing the last of

the requisite tetrapeptide fragments.

Our strategy, however, builds upon the previously

reported regio- and diastereoselective aspartate alkyla-

tion,⁴²and more importantly, a delicate ®nal saponi®-

cation of the methyl esters.¹²Rather than re-work the

entire sequence employing t-butyl esters, which would

also have required other changes in the protecting group

scheme, we decided to face the challenges put forth by

incorporating the aspartate residue with methyl ester

side chain protection. In the ®rst attempt at coupling 4a

with the dipeptide free amine resulting from selective

nitrogen de-protection of 5, the free amine was used in

excess, and the only coupled product observed was the

hexapeptide imide, as demonstrated by FABMS and ¹H

NMR (loss of one methyl ester singlet). We suspected

that the imide formation was due to the presence of

excess free amine component, so we carried out a sec-

ond coupling using the amine TFA salt as the compo-

nent in excess. In this case, using the mild base collidine

in excess to eect deprotonation, we observed ecient

coupling with only a trace of imide formation evident

by TLC analysis. In light of this result, we recommend

that base sensitive couplings of this type be conducted

in a similar fasion. These conditions buer the reaction

The ®nal subunit required was the protected b-methy-

laspartate-containing dipeptide 5. In developing the

synthetic route to the natural product itself, we found

that the N-BnPhFl-protected aspartate derivative 28

coupled eciently with amino acids,¹²but that pro-

tecting group strategy did not ®nd its way into the

published route. However, it was exhumed for our

Scheme 10. Completion of fragment 5.

Scheme 9. Synthesis of the N-meGly tetrapeptide 27.

550

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

at a lower pH, thus reducing base catalyzed side-reac-

tions. Other amine salts, such as HCl and TsOH salts,

also give excellent results (vide infra).⁴³

Once the problem of imide formation had been solved,

the four tetrapeptides were carried through the segment

coupling process (Scheme 11). The standard method

was selective deprotection of the dipeptide 5 to produce

the amine TFA salt. This component was then coupled

with the appropriate tetrapeptide (HATU/collidine) to

give the hexapeptides 29a±d in good puri®ed yields, with

little or no imide formation observed.

With the completion of the hexapeptide fragments, the

stage was set for coupling to the Adda¹²fragment 6.

The plan was to couple the doubly deprotected (N- and

carboxy- termini) hexapeptides to a pre-activated Adda,

a strategy that would provide products that were ready

to enter the macrocyclization sequence with no further

deprotections required. The HOAt active ester of the

Adda fragment 6 was therefore prepared (DCC/HOAt),

the crude active ester was combined with the doubly

deprotected hexapeptide amine salt (resulting from

treatment of the hexapeptides 29a±d with TFA), and the

coupling was promoted by the addition of collidine

(Scheme 12). Following this general protocol, the var-

ious heptapeptides (3a±b, 30a±b) were produced in 55 to

68% overall yield after puri®cation.⁴⁴

Scheme 12. Coupling with the Adda-active ester to form the cycliza-

tion precursors.

corresponding N-methyl derivative simply re¯ect a

greater tendency for the larger N-cyclohexyl substituent

to induce a favorable turn structure in the cyclization

precursor.^33,45

The completion of the cyhxAla variant 2a, the N-

cyhxGly variant 32a, and the N-meGly variant 32b,

were carried out by treatment of the corresponding die-

sters with LiOH, giving a mixture of three isomers in

each case (Scheme 14).⁴⁶The by-products, which we

believe to be the result of isomerization prior to sapo-

ni®cation, were separated from the desired material and

each other by chromatography. The desired isomer was

identi®ed in each case by re-esteri®cation using TMS-

diazomethane, followed by comparison with authentic

samples of the di-esters.⁴⁷Several alternative methods

of saponi®cation were attempted, including the use of

potassium trimethylsilanoate,⁴⁸KOH in methanol,⁴⁹

CeCl₃and CoCl₂in aqueous THF,⁵⁰horse serum

butyrylcholinesterase,⁵¹and lipases from Candida cylin-

dracea and Pseudomonas species,⁵²but either no

improvement or no reaction was observed in these cases.

The macrocyclizations were carried out, using the gen-

eral procedure employed for the synthesis of the natural

product,¹²as follows: the heptapeptide free acids (3a±b,

30a±b) were converted into the respective penta-

¯uorophenyl esters (Scheme 13), followed by amine

deprotection (TFA) and addition to a rapidly stirred

two-phase chloroform/aqueous phosphate buer mix-

ture. As anticipated, the cyclohexylalanine variant 31a

and the N-Troc-aminoalanine variant 31b were

obtained in yields comparable to that obtained for the

parent microcystin LA. Interestingly, the N-cyhxGly

dervative cyclized equally eciently to give 31c, but the

N-meGly variant cyclized to give 31d in signi®cantly

lower yield. It may be that the higher cyclization yields

observed for the N-cyhxGly analogue relative to the

The reductive Troc removal to complete the aminoala-

nine variant 2b proved troublesome. Our initial eorts

Scheme 11. 4+2 segment couplings.

Scheme 13. Macrocyclizations.

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

551

using Zn in either acetic acid¹³or an aqueous buer⁵³

resulted in decomposition, while using the milder redu-

cing agent, cadmium,¹⁴gave no reaction. Using Zn±Cu

couple⁵⁴in acetic acid resulted in removal of the Troc

group, but the product precipitated out with the metal

salts as an intractable mixture.⁵⁵This obstacle was

overcome by the use of a more acidic medium, thereby

maintaining the carboxylic acids in a protonated state

and preventing their complexation with zinc.⁵⁶Thus,

treatment of the Troc-protected diacid 32c with Zn±Cu

couple in MeOH/0.1 M pH 3.0 phosphate buer gave

the ®nal analogue 2b in excellent yield (Scheme 14).

Table 1. Comparison of the IC₅₀values of synthetic microcystin LA¹²

and the microcystin analogues towards the puri®ed catalytic subunits

of PP1 and PP2A

Inhibitor

IC₅₀(nm)

PP1-C

IC₅₀(nm)

PP2A-C

Selectivity

PP1:PP2A

1 (synthetic)¹²

32b (N-meGly)

32a (N-cyhxGly)

2b (aminoala)

2a (cyhxAla)

0.3

1.1

0.8

3

0.3

0.58

1.5

9

1

1:2

2:1

3:1

7:1

0.52

3.4

inhibitor (tautomycin previously held the record at

5:1).^1aWith this encouragement, the design and synth-

esis of second generation inhibitors are in progress.

Inhibition assays

The activity of the analogues as PP1 and PP2A inhibi-

tors was determined as IC₅₀values using puri®ed PP1

and PP2A catalytic subunits via the established radi-

olabeled phosphorylase-a assay.⁵⁷Synthetic micro-

cystin LA, 1, was tested ®rst and found to be

comparable in activity to the values reported for the

natural product,⁵⁸giving sub-nanomolar IC₅₀values

and showing no selectivity as an inhibitor of PP1 and

PP2A (Table 1). The derivative lacking only the methy-

lidene group of the parent compound (i.e. the N-

methylglycine (sarcosine) variant 32b) was only slightly

less potent an inhibitor, but slightly selective for PP2A.

However, as predicted by the model, replacing the N-

methyl substituent with the larger N-cylcohexyl group

(32a) resulted in a relative fourfold increase in selectivity

in favor of PP1, from 1:2 to 2:1. A somewhat higher

selectivity of 3:1 was found for the aminoalanine ana-

logue 2b, again, consistent with predictions (although

the IC₅₀values are an order of magnitude or more

higher). Finally, the cyclohexylalanyl derivative 2a has

an IC₅₀less than twofold higher for PP1, but more than

tenfold higher for PP2A, resulting in a selectivity of 7:1

in favor of PP1, again, as predicted by the modeling.

Experimental

General

¹H and ¹³C NMR spectra were obtained on an Omega

500 (500 MHz), a General Electric GN-500 (500 MHz),

a Bruker 500 (500 MHz), or a Bruker 400 (400 MHz)

spectrometer. For spectra measured in organic solvents,

data are reported in ppm from internal tetra-

1

methylsilane for H NMR and in ppm from the solvent

1

for ¹³C NMR. For H spectra taken in D₂O, data are

reported in ppm relative to HDO (4.80 ppm) unless

otherwise stated. Data are reported as follows: chemical

shift, multiplicity (app=apparent, par obsc=partially

obscured, ovrlp=overlapping, s=singlet, d=doublet,

t=triplet, q=quartet, m=multiplet, br=broad,

abq=ab quartet), coupling constant, and integration.

Infrared (IR) spectra were taken with a Perkin±Elmer

model 1600 series FTIR spectrophotometer. Melting

points (mp) were obtained from a Laboratory Devices

Mel-Temp melting-point apparatus and are reported

uncorrected. Thin-layer chromatography (TLC) was

performed on 0.25 mm Merck precoated silica gel plates

(60 F-254), and silica gel chromatography was per-

formed using ICN 200±400 mesh silica gel. Reversed

While these selectivities are admitedly modest, they are

all consistent with predictions and provide some reas-

surance that the models upon which the analogue design

is based have some validity. It is also worth noting that

at 7:1 the cyclohexylalanine derivative 2a is more PP1-

selective than any previously known small molecule

phase preparative HPLC was conducted with

a

25Â100 mm Waters prep Nova-Pak HR C₁₈cartridge

(WAT038510). Reversed-phase analytical HPLC was

conducted with a 4.6Â250 mm Rainin Microsorb-MV

C₁₈column (86-200-C5). Solution pH measurements

were made with Whatman type CF pH 0±14 indicator

paper strips. Inert atmosphere operations were con-

ducted under nitrogen passed through a Drierite drying

tube in oven or ¯ame-dried glassware. Anhydrous tet-

rahydrofuran (THF) was distilled ®rst from calcium

hydride, and then from potassium; anhydrous ether was

distilled from potassium. Triethylamine and methylene

chloride were puri®ed by distillation from calcium

hydride. Diisopropylethylamine (DIEA) and DMF were

dried over sieves. HATU was purchased from PerSep-

tive Biosystems Inc. d-Boc-Glu(OH)-OMe was pre-

pared by a one-pot procedure¹²not described in the

text, full experimental details will be published else-

where. All other reagents were used as purchased from

Aldrich, Sigma-Aldrich, or Acros unless otherwise

stated.

Scheme 14. Completion of the microcystin LA analogues.

552

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

Chemistry

hydrate and 0.65 mL (6.4 mmol) of 70% aqueous per-

chloric acid. The mixture clari®ed within 45 min, and

was stirred for 96 h. The solution was then cooled to

0 ^ꢀC, and extracted twice with water. The organic phase

was diluted with hexanes, and extracted twice more with

water. The combined aqueous phases were basi®ed to

pH>10 by adding solid K₂CO₃, and were then extrac-

ted twice with ether. The combined ether phases were

dried over anhydrous K₂CO₃, and ®ltered through cot-

ton into a solution of 1.12 g (5.90 mmol) p-toluene-

sulfonic acid in ether. The precipitate was isolated by

®ltration to give 1.55 g (64%) of 13 as a white solid:

mp 135±136.5 ^ꢀC (dec with gas evolution, re-solidi®ed to

a solid with mp 184±185 ^ꢀC); IR (KBr pellet) 3448,

ꢀ-Dimethylphosphonylsarcosine (10). Compound

7

(3.67 g, 11.07 mmol) was hydrogenated in 80 mL of

methanol over 370 mg of 10% palladium on carbon for

8.5 h at 1 atm, then ®ltered, and concentrated in vacuo

to give 2.07 g (95%) of a white solid: mp 107±109 ^ꢀC

(dec); IR (KBr pellet) 3441 br, 2964, 1654, 1245,

1

1041 cm ¹; H NMR (500 MHz, D₂O) d 2.86 (s, 3H),

3.92 (d, J=11 Hz, 3H), 3.93 (d, J=11.5 Hz, 3H), 4.30

(d, J=19.5 Hz, 1H); ¹³C NMR (125 MHz, D₂O with

sodium 3-trimethylsilylproprionate-2,2,3,3-d₄) d 36.5 (d,

J=7.4 Hz, N-Me), 57.7 (ovrlp d, J=7.5, 7.6 Hz, OMe

groups), 62.1 (d, J=141.9 Hz, a-carbon), 168.9; HRMS

m/e calcd for C₅H₁₃NO₅P+ (M+H)⁺: 198.0532.

Found: 198.0530.

1

2925, 1747, 1125 cm ¹; H NMR (400 MHz, MeOH-d₄)

d 0.98 (m, 2H), 1.18±1.36 (m, 3H), 1.46 (par obsc

m, 1H), 1.52 (s, 9H), 1.57±1.83 (m, 7H), 2.37 (s, 3H),

3.91 (app t, J=7.2 Hz, 1H), 7.22 (d, J=8.0 Hz, 2H),

7.71 (d, J=8.0 Hz, 2H); ¹³C NMR (125 MHz, MeOH-

d₄) d 21.3, 27.0, 27.1, 27.3, 28.1, 33.9, 35.0, 39.1,

52.3, 85.0, 127.0, 129.8, 141.7, 143.6, 170.2; FABMS

m/e calcd for C₁₃H₂₆NO₂+(M TsO )+: 228.1964.

Found: 228.1972.

Boc-^D-iso-Glu(OMe)-ꢀ-dimethylphosphonylsarcosine (9).

To an oven-dried ¯ask containing 556 mg (2.13 mmol)

of Boc-d-Glu-OMe-OH and 319 mg (2.34 mmol) of

HOAt was added 6 mL of CH₂Cl₂, followed by 1 mL of

DMF. The solution was cooled to 0 ^ꢀC and 461 mg

(2.24 mmol) of DCC was added. The mixture was

stirred for 2 h, warmed to rt, and stirred for an

additional 9 h. The mixture was then ®ltered through

Celite into a second oven-dried ¯ask, and the ®ltrate

was concentrated in vacuo to remove the CH₂Cl₂. To

the resulting solution was added 400 mg (2.03 mmol) of

amino acid 12, followed by 9 mL DMF. The solution

was cooled to 0 ^ꢀC, and 431 mg (4.26 mmol) of triethyl-

amine was added over 5 min. The yellow solution was

stirred for 2.5 h, warmed to rt, stirred for an additional

13 h, and stored at 16 ^ꢀC overnight. The solution was

partitioned between ether and water, and the phases

were separated. The aqueous phase was washed once

with ether, acidi®ed to pH 1 with 1 M NaHSO₄, satu-

rated with solid NaCl, and extracted three times with

EtOAc. The combined EtOAc phases were washed

twice with brine, dried over MgSO₄, ®ltered, and

concentrated in vacuo. Chromatography (CHCl₃to

elute less polar material, then 1/5/94: HOAc/IPA/

CHCl₃to elute HOAt, then 1/25/74: HOAc/IPA/CHCl₃

to elute product, 200 mL silica gel) gave 623 mg (70%)

of the diastereomeric phosphonates as a yellow solid: R_f

0.14 (1/10/89: HOAc/IPA/CHCl₃); IR (thin ®lm) 3368

br, 2952, 1736, 1708, 1643, 1171, 1045 cm ¹; FABMS

m/e calcd for C₁₆H₃₀N₂O₁₀P+ (M+H)⁺: 441.1639.

Found: 441.1636. Attempts to separate the diaster-

eomers for analytical purposes by chromatography

and crystallization (EtOAc/hexanes) failed to produce

Boc-^D-Ala-3-cyclohexyl-L-Ala-OtBu (12a). To an oven

dried ¯ask was added 105 mg (0.55 mmol) of Boc-d-

alanine-OH, 255 mg (0.61 mmol) of 11, and 83 mg

(0.61 mmol) of HOAt. The mixture was dissolved in

2.7 mL of DMF, and the solution was cooled to 0 ^ꢀC

in an ice bath. 138 mg (1.10 mmol) of 2,4,6-collidine

was added, followed by 138 mg (0.72 mmol) of 1-(3-

dimethylaminopropyl)-3-ethyl carbodiimide hydro-

chloride (EDCI). The resulting yellow solution was

stirred at 0 ^ꢀC for 2 h, warmed to rt, and stirred for

an additional 11 h. The solution was then partitioned

between water and ether, and the phases were

separated. The aqueous phase was extracted twice

with ether. The combined organic phases were

washed once each with water, 1 M NaHSO₄, water, and

brine. The organic phase was dried over MgSO₄, ®l-

tered, and concentrated to an oil. Chromatography

(20 EtOAc/80 hexanes, 10 mL silica gel) gave 198 mg

(89%) of the dipeptide as a white foam: R_f0.59 (30

EtOAc/70 hexanes); IR (thin ®lm) 3310 br, 2918,

1

1719, 1655, 1164 cm ¹; H NMR (500 MHz, DMSO-d₆,

70 ^ꢀC) d 0.84 (m, 1H), 0.93 (m, 1H), 1.14 (br, 2H), 1.18

(obsc, 1H), 1.19 (d, J=7.1 Hz, 3H), 1.31 (m, 1H), 1.38

(s, 9H), 1.40 (s, 9H), 1.51 (m, 2H), 1.59 (br, 1H), 1.64

(m, 4H), 3.99 (app pentet, J=7.3 Hz, 1H), 4.18 (ddd,

J=5.7, 8.3, 8.8 Hz, 1H), 6.47 (br, 1H), 7.63 (d,

J=8.0 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆, 70 ^ꢀC)

d 18.2, 25.4, 25.5, 25.8, 27.5, 28.0, 31.7, 32.8, 33.6, 38.5,

49.8, 50.3, 78.0, 80.3, 154.6, 171.3, 172.4; HRMS m/e

1

any pure samples. However, H NMR analysis of the

®rst fraction collected containing product from the

column showed mostly a single diastereomer, thus

spectral data is given for the higher R_fmaterial: ¹H

NMR (500 MHz, DMSO-d₆, 90 ^ꢀC) d 1.38 (s, 9H), 1.85

(m, 1H), 1.96 (m, 1H), 2.45 (par obsc m, 2H), 3.08 (s,

3H), 3.63 (s, 3H), 3.66 (d, J=11.0 Hz, 3H), 3.72 (d,

J=11.0 Hz, 3H), 4.04 (m, 1H), 5.71 (d, J=25.5 Hz, 1H),

6.85 (br, 1H).

calcd for C₂₁H₃₉N₂O₅(M+H)⁺: 399.2861. Found:

+

399.2860.

Boc-^D-iso-Glu(OMe)-MeÁAla-D-Ala-3-cyclohexyl-L-Ala-

OtBu (13). To 103 mg (0.26 mmol) of 12a in an oven

dried ¯ask was added 0.9 mL of CH₂Cl₂. The solution

was cooled to 0 ^ꢀC, and 0.4 mL of TFA was added.

After 2.25 h, the solution was diluted with hexanes and

concentrated in vacuo. The residue was re-concentrated

twice from hexanes to remove residual TFA. To the

3-Cyclohexyl-^L-alanine tert-butylester, p-toluene sulf-

onate (11). To a ¯ask containing 70 mL tBuOAc

was added 1.11 g (5.84 mmol) of l-cyclohexylalanine

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

553

resulting oil, 12b, was added 103 mg (0.23 mmol) of 9,

and 98 mg (0.26 mmol) of HATU. The mixture was

suspended in 1.6 mL of CH₂Cl₂, cooled to 0 ^ꢀC, and

110 mg (0.94 mmol) of collidine was added over 5 min.

The yellow mixture was stirred for 2 h at 0 ^ꢀC, warmed

to rt, and stirred for an additional 12 h. The mixture was

partitioned between water and ether, and the phases

were separated. The aqueous phase was extracted twice

with ether. The combined organic phases were washed

once with 50% sat. NaHCO₃, diluted with 0.25 volumes

of hexane, washed twice with 1 M NaHSO₄, and once

each with water and brine. The organic phase was dried

over MgSO₄, ®ltered, and concentrated in vacuo to give

a tan foam. The crude tetrapeptide phosphonate was

dissolved in 2.83 mL of 70 THF/30 water, and 130 mg

(0.94 mmol) of K₂CO₃was added to the resulting solu-

tion. 0.019 mL (0.23 mmol) of 37% aq HCHO was

added, and the solution was stirred for 8.5 h. The solu-

tion was partitioned between water and ether, and the

phases were separated. The aqueous phase was extrac-

ted once with ether. The combined organic phases were

washed once each with 0.2 M NaHSO₄and brine. The

organic phase was dried over MgSO₄, ®ltered, and con-

centrated in vacuo to give a yellow residue. Chromato-

graphy (90 EtOAc/10 hexanes, 14 mL silica gel) gave

88 mg (60%) of the tetrapeptide 13 as a white foam: R_f

0.54 (EtOAc); IR (thin ®lm) 3318, 2931, 1734, 1719,

1660, 1155 cm ¹; ¹H NMR (500 MHz, DMSO-d₆, 90 ^ꢀC)

d 0.82±0.96 (m, 2H), 1.15 (m, 3H), 1.29 (d, J=7.1 Hz,

3H), 1.36 (s, 9H), 1.39 (s, 9H), 1.46±1.59 (m, 4H), 1.64

(m, 4H), 1.81 (m, 1H), 1.95 (m, 1H), 2.31 (m, 2H), 3.00

(obsc s, 3H), 3.61 (s, 3H), 3.98 (ddd, J=5.6, 8.0, 8.3 Hz,

1H), 4.20 (ddd, J=5.7, 8.2, 8.9 Hz, 1H), 4.39 (dq,

J=7.2, 7.3 Hz, 1H), 5.51 (s, 1H), 5.96 (s, 1H), 6.72 (br,

1H), 7.69 (d, J=7.2 Hz, 1H), 7.82 (d, J=7.4 Hz, 1H);

FABMS m/e calcd for C₃₁H₅₃N₄O₉+ (M+H)⁺:

625.3815. Found: 625.3811.

90 ^ꢀC) d 0.82±0.96 (m, 2 H), 1.11±1.21 (m, 3H), 1.29 (d,

J=7.1 Hz, 3H), 1.33 (obsc m, 1H), 1.37 (s, 9H), 1.49±

1.68 (m, 7H), 1.82 (m, 1H), 1.96 (m, 1H), 2.31 (m, 2H),

3.00 (par obsc s, 3H), 3.61 (s, 3H), 3.98 (ddd, J=5.6, 8.1,

8.4 Hz, 1H), 4.29 (ddd, J=5.2, 8.3, 9.2 Hz, 1H), 4.40 (dq,

J=7.1, 7.3 Hz, 1H), 5.51 (s, 1H), 5.97 (s, 1H), 6.69 (br,

1H), 7.69 (d, J=8.0 Hz, 1H), 7.80 (d, J=7.8 Hz, 1H);

FABMS m/e calcd for C₂₇H₄₅N₄O₉+ (M+H)⁺:

569.3186. Found: 569.3185.

2-(R)-(tert-Butoxycarbonyl)-amino-3-azidoproprionic acid

(15). To an oven dried ¯ask was added 500 mg

(2.67 mmol) of Boc-d-serine lactone (14),²²and 15 mL

of DMF. The solution was cooled to 0 ^ꢀC, and 261 mg

(4.01 mmol) of NaN₃was added. The mixture was

stirred for 45 min, warmed to rt, and stirred for two

additional h. The solution was partitioned between

ether and water, and the phases were separated. The

ether phase was back-extracted twice with water. The

combined aqueous phases were acidi®ed to pH 3 with

sat. citric acid, and were extracted twice with EtOAc.

The combined EtOAc phases were washed twice with

water, once with brine, dried over MgSO₄, ®ltered, and

concentrated in vacuo to an oil. Chromatography (1/30/

69: HOAc/EtOAc/hexanes, 50 mL silica gel) gave

523 mg (85%) of the azide as a colorless oil: R_f0.30 (1/

59/40: HOAc/EtOAc/hexanes); IR (thin ®lm) 3329 br,

2980, 2106 str, 1714 cm ¹; ¹H NMR (500 MHz, DMSO-

d₆, 70 ^ꢀC) d 1.39 (s, 9H), 3.57 (m, 2H), 4.13 (ddd, J=6.3,

6.3, 7.4 Hz, 1H), 6.89 (br, 1H); ¹³C NMR (125 MHz,

DMSO-d₆, 70 ^ꢀC) d 27.8, 50.8, 53.3, 78.2, 154.8, 170.6;

HRMS m/e calcd for C₈H₁₈N₅O₄+ (M+NH₄)⁺:

248.1359. Found: 248.1356.

N²-tert-Butoxycarbonyl-(R)-2,3-diaminoproprionic acid

(16). Compound 15 (385 mg, 1.67 mmol) was hydro-

genated in 15 mL of methanol and 1 mL HOAc over

89 mg of 10% palladium on carbon for 45 min at 1 atm.

The mixture was ®ltered, and the ®ltrate was con-

centrated in vacuo to an oil. The oil was re-concentrated

twice from toluene, to remove residual methanol, to

give 342 mg (99%) of a white solid that would typically

be used without further puri®cation. To obtain analyti-

cally pure material, the solids were recrystallized from

MeOH to give 245 mg (72%) of the amino acid as a

white solid obtained in three crops: mp 196±198 ^ꢀC (dec,

Lit. 198±200 ^ꢀC dec)^21f; IR (KBr pellet) 3342, 2968 br,

Boc-^D-iso-Glu(OMe)-MeÁAla-D-Ala-3-cyclohexyl-L-Ala-

OH (4a). To 92 mg (0.15 mmol) of the tetrapeptide 13

was added 1 mL TFA. After 50 min, the solution was

concentrated in vacuo, and the residue was re-

concentrated three times from hexanes to remove residual

TFA. The residue was dissolved in 0.6 mL of water, and

78 mg (0.74 mmol) of Na₂CO₃in 0.3 mL of water was

added. The solution was cooled to 0 ^ꢀC, and 37 mg

(0.17 mmol) of di-tert-butyldicarbonate in 0.6 mL

dioxane was added. The mixture was stirred at 0 ^ꢀC

for 2 h, followed by warming to rt and stirring for

an additional 6.5 h. The mixture was partitioned

between ether and water, and the phases were separated.

The aqueous phase was washed once with ether. The

combined ether phases were back-extracted twice with

water. The combined aqueous phases were acidi®ed with

1 M NaHSO4, and extracted twice with EtOAc. The

combined EtOAc phases were washed once each with

water and brine, dried over MgSO₄, ®ltered, and con-

centrated in vacuo to a residue. Chromatography (1/5/

94: HOAc/IPA/CHCl₃to 1/10/89: HOAc/IPA/CHCl₃to

elute product, 12 mL silica gel) gave 84 mg (99%) of

the desired tetrapeptide 4a as a white foam: R_f0.42 (1/

10/89: HOAc/IPA/CHCl₃); IR (thin ®lm) 3309 br, 2921,

1

1688 cm ¹; H NMR (500 MHz, MeOH-d₄) d 1.37 (s,

9H), 3.03 (dd, J=6.2, 12.4 Hz, 1H), 3.08 (dd, J=6.8,

12.4 Hz, 1H), 3.96 (m, 1H); ¹³C NMR (125 MHz,

MeOH-d₄) d 28.7, 43.1, 53.9, 80.9, 158.0, 174.8; HRMS

m/e calcd for C₈H₁₇N₂O₄⁺(M+H)⁺: 205.1189. Found:

205.1186.

N²-tert-Butoxycarbonyl-N³-2,2,2-trichloroethoxycarb-

onyl-(R)-diaminoproprionic acid (17). To a ¯ask was

added 230 mg (1.13 mmol) of 16 and 4 mL of water.

358 mg (3.38 mmol) of Na₂CO₃was added, and the

resuting solution was cooled to 0 ^ꢀC. To this 360 mg

(1.24 mmol) of succinimidyl 2,2,2-trichloroethycarb-

onate (Troc-OSuc, available from Aldrich) in 3 mL

dioxane was added. After 2 h, the mixture was warmed

to rt, and was stirred for an additional 12 h. The

1

1738, 1717, 1652 cm ¹; H NMR (500 MHz, DMSO-d₆,

554

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

1

solution was partitioned between ether and water, and

the phases were separated. The aqueous phase was

washed once with ether. The combined ether phases

were back-extracted twice with water, and were dis-

carded. The combined aqueous phases were acidi®ed to

pH 3 with sat. citric acid, and were extracted twice with

ether. The combined ether phases were washed twice

with water, once with brine, dried over MgSO₄, ®ltered,

and concentrated in vacuo to give 413 mg (96%) of the

di-protected amino alanine derivative as a white solid

that was used without further puri®cation: mp 134.5±

136 ^ꢀC (dec); R_f0.20 (1/45/54: HOAc/EtOAc/hexanes);

IR (thin ®lm) 3330 br, 2974, 1715, 1688 cm ¹; ¹H NMR

(500 MHz, DMSO-d₆, 70 ^ꢀC) d 1.38 (s, 9H), 3.37 (m,

2H), 4.08 (m, 1H), 4.77 (s, 2H), 6.67 (br, 1H), 7.50 (br,

1H); ¹³C NMR (125 MHz, DMSO-d₆, 60 ^ꢀC) d 27.8,

41.6, 53.2, 73.4, 78.1, 96.0, 154.3, 155.0, 171.6; HRMS

m/e calcd for C₁₁H₁₈Cl₃N₂O₆+ (M+H)⁺: 379.0232

(Cl=34.9689). Found: 379.0226.

25 hexanes); IR (thin ®lm) 3311, 2955, 1725, 1657 cm ;

¹H NMR (500 MHz, DMSO-d₆, 90 ^ꢀC) d 0.84 (d,

J=6.6 Hz, 3H), 0.88 (d, J=6.6 Hz, 3H), 1.37 (s, 9H),

1.40 (s, 9H), 1.52 (app t, J=7.2 Hz, 2H), 1.64 (m, 1H),

1.83 (m, 1H), 1.97 (m, 1H), 2.34 (m, 2H), 3.02 (s, 3H),

3.45 (app t, J=6.1 Hz, 2H), 3.62 (s, 3H), 3.98 (ddd,

J=5.6, 8.1, 8.1 Hz, 1H), 4.17 (ddd, J=7.4, 7.4, 7.5 Hz,

1H), 4.52 (ddd, J=6.4, 6.4, 8.0 Hz, 1H), 4.74 (s, 2H),

5.51 (s, 1H), 5.95 (s, 1H), 6.66 (br, 1H), 7.28 (br, 1H),

7.74 (d, J=7.4 Hz, 1H), 8.0 (d, J=8.0 Hz, 1H); FABMS

m/e calcd for C₃₁H₅₁Cl₃N₅O₁₁+ (M+H)⁺: 774.2654

(Cl=34.9689). Found: 774.2647.

Boc-^D-iso-Glu(OMe)-MeÁAla-N²-Boc-N³-Troc-(R)-Am-

inoala-L-Leu-OH (4b). The title compound was pre-

pared following the same procedure as that used to

prepare 4a. 104 mg (0.13 mmol) of 19 was used, and

all other reagents and solvents were used in the appro-

priate amounts. The residue was ®ltered through silica

(1/5/94: HOAc/IPA/CHCl₃to 1/10/89: HOAc/IPA/

CHCl₃to elute product, 16 mL silica gel) to give 89 mg

(93%) of the desired acid as a white solid: R_f0.47 (1

HOAc/10 IPA/89 CHCl₃); IR (thin ®lm) 3318 br, 2953,

N²-Boc-N³-Troc-(R)-Aminoala-L-Leu-OtBu (18). The

title compound was prepared following the same pro-

ceedure as that used to prepare 12a. The materials used

were 150 mg (0.40 mmol) of 17, 97 mg (0.44 mmol) of

the HCl salt of l-Leu-O-tBu, and the amounts of the

other reagents and solvents were adjusted accordingly.

Chromatography (20 EtOAc/80 hexanes, 10 mL silica

gel) of the residue gave 168 mg (77%) of the dipeptide as

1736, 1716, 1655 cm ¹; H NMR (500 MHz, DMSO-d₆,

1

90 ^ꢀC) d 0.85 (d, J=6.5 Hz, 3H), 0.88 (d, J=6.6 Hz,

3H), 1.37 (s, 9H), 1.55 (m, 2H), 1.64 (m, 1H), 1.82 (m,

1H), 1.97 (m, 1H), 2.33 (m, 2H), 3.01 (s, 3H), 3.45 (m,

2H), 3.61 (s, 3H), 3.99 (ddd, J=5.5, 8.1, 8.2 Hz, 1H),

4.26 (ddd, J=6.6, 7.9, 7.9 Hz, 1H), 4.54 (ddd, J=6.2,

6.2, 8.2 Hz, 1H), 4.74 (s, 2H), 5.52 (s, 1H), 5.96 (s, 1H),

6.67 (br, 1H), 7.27 (br, 1H), 7.74 (d, J=7.9 Hz, 1H),

7.88 (d, J=8.3 Hz, 1H); FABMS m/e calcd for

C₂₇H₄₃Cl₃N₅O₁₁+ (M+H)⁺: 718.2038 (Cl=34.9689).

Found: 718.2045.

a white solid: R_f0.37 (25 EtOAc/75 hexanes); IR (thin

1

®lm) 3328, 3190, 2963, 1734, 1719, 1688, 1654 cm

;

¹H NMR (500 MHz, DMSO-d₆, 70 ^ꢀC) d 0.83 (d,

J=6.6 Hz, 3H), 0.88 (d, J=6.6 Hz, 3H), 1.38 (s, 9H),

1.39 (s, 9H), 1.50 (m, 2H), 1.63 (m, 1H), 3.31 (m, 2H),

4.13 (m, 2H), 4.74 (d, J=12.3 Hz, 1H), 4.77 (d,

J=12.3 Hz, 1H), 6.50 (br, 1H), 7.41 (br, 1H), 7.81 (d,

J=7.5 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆) d 21.3,

22.8, 24.1, 27.6, 27.9, 28.1, 42.5, 51.1, 54.3, 73.4, 78.3,

80.4, 96.1, 154.6, 155.0, 169.9, 171.5; FABMS m/e

calcd for C₂₁H₃₇Cl₃N₃O₇+ (M+H)⁺: 548.1697 (Cl=

34.9689). Found: 548.1686.

N-(cyclohexyl)-Glycine Ethylester, Hydrochloride

Glycine ethyl ester hydrochloride (2.00 g, 14.33 mmol)

and sodium cyanoborohydride (0.90 g, 14.33 mmol)

were combined in a ¯ask, followed by the addition of

30 ml of methanol. Cyclohexanone was ®ltered through

basic alumina, and 1.41 g (14.33 mmol) of this was

added to the methanolic slurry. The mixture was stirred

for 12 h. At this point, 1.5 mL of conc. HCl was added,

and after stirring for an additional 3 h, the mixture was

concentrated to a white solid in vacuo. The solids were

dissolved in the minimum volume of water, this solution

was basi®ed to pH>10 by adding solid K₂CO₃, and was

then extracted twice with ether. The combined organic

phases were washed once with 10% aq K₂CO₃, once

with brine, dried over K₂CO₃, ®ltered, and concentrated

in vacuo to an oil. The resulting oil was dissolved in

150 mL absolute ethanol, 1.5 mL conc. HCl was added,

and the solution was concentrated in vacuo to approxi-

mately 10 mL. The product was precipitated by the

addition of ether, and was isolated by ®ltration to give

1.39 g (44%) of the amine HCl salt as a white solid: mp

178.5±180 ^ꢀC; IR (KBr pellet) 2937, 2722, 1747,

Boc-^D-iso-Glu(OMe)-MeÁAla-N²-Boc-N³-Troc-(R)-Am-

inoala-^L-Leu-OtBu (19). An oven-dried ¯ask was

charged with 153 mg (0.28 mmol) of 18, and this was

treated with TFA as in the preparation of 13. The

resulting oil was combined with 112 mg (0.25 mmol) of

9, and 106 mg (0.28 mmol) of HATU. The amounts of

solvents and additional reagents were adjusted accord-

ing to the procedure to prepare 13. After the prescribed

time, the mixture was partitioned between ether and

water, and the phases were separated. The aqueous

phase was extracted twice with ether. The combined

organic phases were washed once each with 50% sat.

NaHCO₃, 1 M NaHSO₄, water, and brine. The organic

phase was then dried over MgSO₄, ®ltered and con-

centrated in vacuo to give a yellow foam.

The ole®nation was carried out according to the proce-

dure to prepare 13, with the exception that only

0.021 mL (0.25 mmol) of 37% aq fomaldehyde was

used. Chromatography (65 EtOAc/35 hexanes, 16 mL

silica gel) of the residue gave 119 mg (60%) of the

desired tetrapeptide as a white foam: R_f0.55 (75 EtOAc/

1234 cm ¹; H NMR (500 MHz, D₂O) d 1.23 (m, 1H),

1

1.33 (t, J=7.2 Hz, 3H), 1.32±1.46 (par obsc m, 4H), 1.71

(m, 1H), 1.89 (m, 2H), 2.11 (m, 2H), 3.23 (dddd, J=3.7,

3.8, 11.2, 11.3 Hz, 1H), 4.05 (s, 2H), 4.34 (q, J=7.2 Hz,

2H); ¹³C NMR (125 MHz, D₂O with sodium 2-tri-

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

555

methylsilylproprionate-2,2,3,3-d₄) d 16.0, 26.8, 27.3,

31.7, 47.5, 60.4, 66.2, 170.7; HRMS m/e calcd for C₁₀

H₁₉NO₂+ (M HCl)⁺: 185.1417. Found: 185.1414.

carbon for 3 h at 1 atm, ®ltered, and concentrated in

vacuo to an oil. The oil was re-concentrated twice from

hexanes to remove residual methanol. 82mg (0.28 mM)

of 21 in 1 mL CH₂Cl₂was added, and the solution was

concentrated in vacuo to give a white foam. To this

were added 42 mg (0.31 mmol) of HOBt and 1.2 mL

DMF, the resulting solution was cooled to 0 ^ꢀC, and

69 mg (0.36 mmol) of EDCI was added. The mixture

was stirred for 2 h, followed by warming to rt and stir-

ring for an additional 11 h. The solution was partitioned

between water and ether, and the phases were separated.

The aqueous phase was extracted twice with ether. The

combined organic phases were washed once each with

50% sat. NaHCO₃, water, 1 M NaHSO₄, water, and

brine. The organic phase was dried over MgSO₄, ®l-

tered, and concentrated under vacuum to give 135 mg

(91%) of the desired tripeptide as a white foam: R_f0.42

(50 EtOAc/50 hexanes); IR (thin ®lm) 3297, 3071, 2930,

Cbz-N-(cyclohexyl)-Glycine Ethylester

To an oven dried ¯ask containing 1.50 g (6.76 mmol)

of the HCl salt of N-(cyhx)-Gly-OEt was added

40 mL of CH₂Cl₂, and the ¯ask was ®tted with a re¯ux

condensor. 1.37 g (13.56 mmol) of triethylamine was

added, the mixture was heated to re¯ux, and 1.73 g

(10.14 mmol) of benzylchloroformate was added to

the warm mixture. After 7 h, approximately 10mg

(0.08 mmol) of DMAP was added. 1 h later, signi®cant

conversion was noted by TLC, and 100 mg (0.81 mmol)

of DMAP and an additional 1.67 g (9.80 mmol) of

benzylchloro-formate were added. After an additional

2 h, the reaction was allowed to cool to rt and stirred

overnight. The mixture was extracted once with 1 M

HCl, and the aqueous phase was back-extracted once

with CH₂Cl₂. The combined organic phases were

washed once each with water and brine, and ®ltered

through cotton. The ®ltrate was concentrated in vacuo

to an oil. Chromatography (10 EtOAc/90 hexanes,

170 mL silica gel) gave 2.10 g (98%) of the title com-

pound as a clear oil: R_f0.32 (10 EtOAc/90 hexanes); IR

1

1731, 1694, 1651 cm ¹; H NMR (500 MHz, DMSO-d₆,

90 ^ꢀC) d 0.84 (d, J=6.6 Hz, 3H), 0.89 (d, J=6.6 Hz,

3H), 1.06 (m, 1H), 1.19 (d, J=7.0 Hz, 3H), 1.23±1.36

(m, 4H), 1.39 (s, 9H), 1.47-1.58 (m, 3H), 1.63 (m, 1H),

1.74 (m, 4H), 3.78 (par obsc m, 1H), 3.79 (d,

J=16.9 Hz, 1H), 3.84 (d, J=17.0 Hz, 1H), 4.15 (ddd,

J=6.5, 8.0, 8.0 Hz, 1H), 4.33 (dq, J=7.1, 7.1 Hz, 1H),

5.06 (s, 2H), 7.25±7.36 (m, 5H), 7.56 (d, J=7.5 Hz, 1H),

7.71 (d, J=7.4 Hz, 1H); HRMS m/e calcd for C₂₉H₄₆

N₃O₆+ (M+H)⁺: 532.3389. Found: 532.3404.

1

(thin ®lm) 2930, 1748, 1699 cm ¹; H NMR (500 MHz,

DMSO-d₆, 130 ^ꢀC) d 1.09 (m, 1H), 1.15 (t, J=7.1 Hz,

3H), 1.26 (m, 2H), 1.38 (m, 2H), 1.56 (app d, J=

12.7 Hz, 1H), 1.72 (m, 4H), 3.80 (m, 1H), 3.91 (s, 2H),

4.06 (q, J=7.1 Hz, 2H), 5.07 (s, 2H), 7.26±7.32 (m, 5H);

¹³C NMR (125 MHz, DMSO-d₆, 130 ^ꢀC) d 13.1, 24.2,

24.8, 29.6, 44.4, 55.5, 59.5, 65.7, 126.5, 126.8, 127.4,

136.3, 154.4, 169.1; HRMS m/e calcd for C₁₈H₂₅NO₄+

(M)⁺: 319.1785. Found: 319.1780.

Boc-^D-iso-Glu(OMe)-N-(cyclohexyl)-Gly-D-Ala-L-Leu-

OtBu (23). Compound 22 (115 mg, 0.22 mmol) was

hydrogenated in 1 mL of methanol over 21 mg of 10%

palladium on carbon for 4.2 h at 1 atm, then ®ltered,

and concentrated. The residue was re-concentrated

twice from hexanes to remove residual methanol, and

the remaining oil was dissolved in 1 mL DMF. Boc-d-

Glu-OMe (65 mg, 0.25 mmol) and HATU (99 mg,

0.25 mmol) were added, and after cooling to 0 ^ꢀC, 79 mg

(0.65 mmol) of collidine was added. The yellow solution

was stirred for 2 h, followed by warming to rt and stir-

ring for an additional 10 h. The reaction was worked up

as in the procedure to prepare 22, to give a colorless

residue. Chromatography (50 EtOAc/50 hexanes to 70

EtOAc/30 hexanes to elute product, 21 mL silica gel)

gave 104 mg (75%) of the tetrapeptide as a white foam:

Cbz-N-(cyclohexyl)-Glycine-OH (21). To 1.00 g

(3.13 mmol) of Cbz-N-cyhxGly-OEt in 15 mL of THF

was added 9.4 mL (9.4 mmol) of 1.0 M LiOH. After

26 h, the solution was partitioned between ether and

water, and the phases were separated. The aqueous

phase was acidi®ed to pH 1 with 1 M NaHSO₄, and was

extracted once with ether. The ether phase was washed

once each with water and brine, dried over anhydrous

MgSO₄, ®ltered, and concentrated in vacuo to give

800 mg (88%) of the title compound as a colorless oil

that solidi®ed over time to a white solid. Analytically

pure material was obtained by recrystallization from

EtOAc/hexanes: mp 110.5±112.5 ^ꢀC; R_f0.63 (1/49/50:

HOAc/EtOAc/hexanes); IR (thin ®lm) 3077 br, 2929,

R_f0.13 (50 EtOAc/50 hexanes); IR (thin ®lm) 3311,

1

2935, 1730, 1715, 1652 cm

;

¹H NMR (500 MHz,

DMSO-d₆, 110 ^ꢀC) d 0.85 (d, J=6.5 Hz, 3H), 0.89 (d,

J=6.5 Hz, 3H), 1.08 (m, 1H), 1.23 (d, J= 7.0 Hz, 3H),

1.31 (par obsc m, 4H), 1.38 (s, 9H), 1.40 (s, 9H), 1.52±

1.74 (m, 8H), 1.86 (m, 1H), 1.98 (m, 1H), 2.35 (br, 2H),

3.62 (s, 3H), 3.84 (m, 2H), 4.02 (m, 1H), 4.16 (app q,

J=6.7 Hz, 1H), 4.32 (m, 1H), 6.59 (br, 1H), 7.55 (br,

2H); FABMS m/e calcd for C₃₂H₅₇N₄O₉+ (M+H)⁺:

641.4128. Found: 641.4126.

1703 cm ¹; H NMR (500 MHz, DMSO-d₆, 130 ^ꢀC) d

1

1.08 (ddddd, J=3.4, 3.4, 12.8, 12.8, 12.8 Hz, 1H), 1.26

(m, 2H), 1.39 (m, 2H), 1.57 (app d, J=13.1 Hz, 1H),

1.72 (m, 4H), 3.77 (dddd, J=3.4, 3.4, 11.9, 11.9 Hz,

1H), 3.85 (s, 2H), 5.07 (s, 2H), 7.24±7.34 (m, 5H); ¹³C

NMR (125 MHz, DMSO-d₆, 130 ^ꢀC) d 24.3, 24.8, 29.7,

44.2, 55.5, 65.7, 126.5, 126.8, 127.5, 136.5, 154.6, 170.3;

HRMS m/e calcd for C₁₆H₂₂NO₄+ (M+H)⁺:

292.1549. Found: 292.1552.

Boc-^D-iso-Glu(OMe)-N-(cyclohexyl)-Gly-D-Ala-L-Leu-OH

(24). The title compound was prepared following the

same procedure as that used to prepare 4a, with only

two minor exceptions: the TFA treatment was for 1.5 h,

and ether was used instead of EtOAc in the workup.

90 mg (0.14 mmol) of 23 was used, and the amounts of

the solvents and additional reagents were adjusted

Cbz-N-(cyclohexyl)-Gly-^D-Ala-L-Leu-OtBu (22). Cbz-d-

Ala-l-Leu-OtBu (121 mg, 0.31 mmol) was hydrogenated

in 1 mL of methanol over 24 mg of 10% palladium on

556

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

accordingly. Filtration of the residue through silica (1

HOAc/5 IPA/94 CHCl₃) gave 71 mg (87%) of the

desired acid as a white solid: R_f0.50 (1 HOAc/10 IPA/

89 CHCl₃); IR (thin ®lm) 3300 br, 2935, 1720, 1694,

J=6.7 Hz, 3H), 0.89 (d, J=6.6 Hz, 3H), 1.24 (d, J=

7.1 Hz, 3H), 1.38 (s, 9H), 1.40 (s, 9H), 1.53 (m, 2H), 1.64

(m, 1H), 1.85 (m, 1H), 1.96 (m, 1H), 2.35 (br, 2H), 2.88

(obsc s, 3H), 3.63 (s, 3H), 3.91 (d, J=16.4 Hz, 1H), 3.97

(d, J=16.4 Hz, 1H), 4.03 (ddd, J=5.4, 8.1, 8.1 Hz, 1H),

4.17 (ddd, J=6.4, 8.0, 8.0 Hz, 1H), 4.34 (dq, J=6.7,

6.8 Hz, 1H), 6.63 (br, 1H), 7.59 (br, 2H); FABMS m/e

calcd for C₂₇H₄₉N₄O₉+ (M+H)⁺: 573.3501. Found:

573.3488.

1652 cm ¹; H NMR (500 MHz, DMSO-d₆, 130 ^ꢀC) d

1

0.86 (d, J=6.4 Hz, 3H), 0.89 (d, J=6.4 Hz, 3H), 1.08

(m, 1H), 1.23 (d, J=7.0 Hz, 3H), 1.38 (obsc m, 4H),

1.38 (s, 9H), 1.55±1.74 (m, 8H), 1.87 (m, 1H), 2.00 (m,

1H), 2.37 (m, 2H), 3.63 (s, 3H), 3.85 (m, 2H), 4.04 (br,

1H), 4.27±4.33 (m, 2H), 6.45 (br, 1H), 7.46 (br, 2H);

FABMS m/e calcd for C₂₈H₄₉N₄O₉+ (M+H)⁺:

585.3501. Found: 585.3494.

Boc-^D-iso-Glu(OMe)-N-(Me)-Gly-D-Ala-L-Leu-OH (27).

The title compound was prepared following the same

procedure as that used to prepare 4a, using 110 mg

(0.19 mmol) of 26, with one exception: the TFA treat-

ment was for 1.5 h. Flash chromatography (1 HOAc/15

IPA/84 CHCl₃, 12 mL silica gel) of the residue gave

90 mg (91%) of the desired acid as a white solid: R_f0.17

(1 HOAc/10 IPA/89 CHCl₃); IR (thin ®lm) 3310 br,

Cbz-N-(Me)-Glycine. 2.00 g (22.4 mmol) of sarcosine

and 7.12 g (67.2 mmol) of Na₂CO₃in a ¯ask were dis-

solved in 60 mL of water, and the solution was cooled

to 0 ^ꢀC. 4.21 g (24.6 mmol) of benzylchloroformate in

20 mL of dioxane was added over 5 min. The mixture

was stirred for 1.5 h, followed by stirring at rt for an

additional 11 h. The cloudy mixture was partitioned

between ether and water, and the phases were separated.

The aqueous phase was acidi®ed with 1M NaHSO₄, and

was extracted twice with EtOAc. The combined EtOAc

phases were washed once with water, once with brine,

dried over anhydrous MgSO₄, ®ltered, and the ®ltrate

was concentrated in vacuo to give 4.98 g (>99%) of

1

2955, 1730, 1662 cm ¹; H NMR (500 MHz, DMSO-d₆,

120 ^ꢀC) d 0.86 (d, J=6.5 Hz, 3H), 0.89 (d, J=6.5 Hz,

3H), 1.24 (d, J=7.1 Hz, 3H), 1.38 (s, 9H), 1.56 (m, 2H),

1.65 (m, 1H), 1.85 (m, 1H), 1.96 (m, 1H), 2.34 (m, 2H),

3.62 (s, 3H), 3.91 (d, J=16.8 Hz, 1H), 3.97 (d, J=

16.5 Hz, 1H), 4.03 (m, 1H), 4.26 (m, 1H), 4.35 (m, 1H),

6.57 (br, 1H), 7.52 (br, 2H); FABMS m/e calcd for C₂₃

H₄₀N₄NaO₉+ (M+Na)⁺: 539.2695. Found: 539.2694.

Z-sarcosine as a clear oil: R_f0.50 (1 HOAc/10 IPA/89

1

CHCl₃); IR (thin ®lm) 3458, 3067 br, 2940, 1698 cm

;

D-erythro-N-(9-Phenyl¯uoren-9-yl)-N-benzyl-ꢁ-Me-iso-

Asp(OMe)-^L-Ala-OtBu (5). To 1.00 g (2.03 mmol) of

the N-BnPhFl-B-methyl aspartate 28 in an oven-dried

¯ask was added 0.701 g (3.86 mmol) of the HCl salt of

l-Ala-OtBu, and 852 mg (2.24 mmol) of HATU. The

mixture was dissolved in 10 mL of DMF, cooled to 0 ^ꢀC,

and 786 mg (6.10 mmol) of diisopropylethylamine was

added over 5 min. The yellow solution was stirred at

0 ^ꢀC for 3 h, warmed to rt, and stirred for an additional

12 h. The solution was partitioned between ether and

10% aq NaHCO₃, and the phases were separated. The

aqueous was extracted once with ether. The combined

ether phases were washed once each with water, 50%

sat citric acid, water, water, and brine, dried over

MgSO₄, ®ltered, and concentrated in vacuo to give

1.20 g (96%) of a white solid that was pure by NMR

¹H NMR (500 MHz, DMSO-d₆, 90 ^ꢀC) d 2.92 (s, 3H),

3.94 (s, 2H), 5.08 (s, 2H), 7.28±7.33 (m, 5H); ¹³C NMR

(125 MHz, DMSO-d₆, 90 ^ꢀC) d 34.9, 49.9, 66.0, 126.9,

127.3, 127.9, 136.6, 155.5, 170.2; HRMS m/e calcd for

C₁₁H₁₃NO₄(M)⁺: 223.0845. Found: 223.0843.

Cbz-N-(Me)-Gly-^D-Ala-L-Leu-OtBu (25). The title com-

pound was prepared by following the procedure used to

prepare 22, with the exceptions that 128 mg (0.57 mmol)

of Z-sarcosine was used as the acid component, and the

amounts of the required solvents and reagents were

adjusted accordingly. Chromatography (60 EtOAc/40

hexanes, 40 mL silica gel) of the resulting oil gave

211 mg (80%) of the desired tripeptide as a colorless oil:

R_f0.20 (50 EtOAc/50 hexanes); IR (thin ®lm) 3315,

1

2952, 1731, 1710, 1651 cm

;

¹H NMR (500 MHz,

analysis: R_f0.39 (25 EtOAc/75 hexanes); IR (thin ®lm)

1

DMSO-d₆, 90 ^ꢀC) d 0.84 (d, J=6.5 Hz, 3H), 0.88 (d,

J=6.6 Hz, 3H), 1.21 (d, J=7.1 Hz, 3H), 1.39 (s, 9H),

1.51 (m, 2H), 1.62 (m, 1H), 2.88 (s, 3H), 3.86 (d,

J=16.6 Hz, 1H), 3.92 (d, J=16.6 Hz, 1H), 4.17 (ddd,

J=6.4, 8.1, 8.1 Hz, 1H), 4.36 (dq, J=7.2, 7.2 Hz, 1H),

5.05 (d, J=13.1 Hz, 1H), 5.07 (d, J=13.1 Hz, 1H), 7.26±

7.33 (m, 5H), 7.69 (app d, J=6.4 Hz, 2H); HRMS m/e

calcd for C₂₄H₃₈N₃O₆+ (M+H)⁺: 464.2762. Found:

464.2756.

3145, 3056, 2971, 1729, 1677, 1153 cm

;

¹H NMR

(500 MHz, CDCl₃) d 0.50 (d, J=6.8 Hz, 3H), 1.28 (d,

J=7.2 Hz, 3H), 1.64 (s, 9H), 2.12 (dq, J=6.8, 10.9 Hz,

1H), 2.91 (s, 3H), 3.70 (d, J=10.9 Hz, 1H), 4.25 (dq,

J=7.2, 7.2 Hz, 1H), 4.38 (d, J=13.3 Hz, 1H), 4.64 (d,

J=13.5 Hz, 1H), 4.93 (d, J=6.9 Hz, 1H), 7.17±7.37 (m,

10H), 7.42 (t, J=7.4 Hz, 1H), 7.58 (d, J=6.7 Hz, 1H),

7.60 (d, J=6.9 Hz, 1H), 7.63 (d, J=7.7 Hz, 1H), 7.75 (d,

J=7.5 Hz, 1H), 7.88 (par obsc m, 2H), 7.91 (d,

J=7.3 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃) d 15.6,

18.0, 28.2, 41.8, 49.0, 50.5, 50.9, 63.0, 80.6, 81.2, 119.0,

120.0, 126.6, 127.3, 127.3, 127.3, 127.5, 127.6, 127.7,

128.0, 128.2, 128.4, 130.8, 138.8, 142.2, 142.6, 144.6,

144.9, 147.3, 171.2, 172.6, 173.2; FABMS m/e calcd for

C₃₉H₄₃N₂O₅+ (M+H)⁺: 619.3172. Found: 619.3169.

Boc-^D-iso-Glu(OMe)-N-(Me)-Gly-D-Ala-L-Leu-OtBu (26).

The title compound was prepared following the same

procedure as that used to prepare 23. 165 mg

(0.36 mmol) of 25 was used, and the amounts of the

additional solvents and reagents were adjusted accord-

ingly. Chromatography (EtOAc, 25 mL silica gel) of the

residue gave 128 mg (63%) of a white solid: R_f0.40

1

Boc-^D-iso-Glu(OMe)-MeÁAla-D-Ala-3-cyclohexyl-L-Ala-

D-ꢁ-Me-iso-Asp(OMe)-L-Ala-OtBu (29a). 5 (154 mg,

0.25 mmol) was hydrogenated in 1.92 mL of MeOH

(EtOAc); IR (thin ®lm) 3300, 2966, 1725, 1652 cm

;

¹H NMR (500 MHz, DMSO-d₆, 110 ^ꢀC) d 0.85 (d,

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

557

with 28 mg (0.25 mmol) of TFA over 30 mg of 10%

palladium on carbon for 13 h at 1 atm. The mixture was

®ltered through Celite, concentrated in vacuo, and re-

concentrated three times from hexanes to remove resi-

dual MeOH. To the residue were added 129 mg

(0.23 mmol) of 4a, 95 mg (0.25 mmol) of HATU, and

1.5 mL of DMF. The solution was cooled to 0 ^ꢀC, and

110 mg (0.91 mmol) of collidine was added over 5 min.

The solution was stirred for 2 h, warmed to rt, and stir-

red for an additional 16 h. The solution was partitioned

between ether and water, and the phases were separated.

The aqueous phase was back-extracted twice with ether.

The combined ether phases were washed once each with

50% sat. NaHCO₃, water, 1 M NaHSO₄, water, brine,

dried over MgSO₄, ®ltered, and concentrated in vacuo

to a yellow foam. Chromatography (EtOAc, 32 mL

silica gel) gave 133 mg (70%) of the hexapeptide as a

white solid: R_f0.23 (EtOAc); IR (thin ®lm) 3311, 2924,

reagents were adjusted accordingly. Chromatography

(95 EtOAc/5 hexanes, 18 mL silica gel) of the residue

gave 66 mg (72%) of the hexapeptide as a white solid: R_f

0.39 (EtOAc); IR (thin ®lm) 3308, 2930, 1737,

1651 cm ¹; H NMR (500 MHz, DMSO-d₆, 130 ^ꢀC) d

1

0.84 (d, J=6.4 Hz, 3H), 0.88 (d, J=6.6 Hz, 3H), 1.07 (d,

J=7.2 Hz, 3H), 1.07 (obsc m, 1H), 1.24 (d, J=7.3 Hz,

3H), 1.25 (d, J=7.1 Hz, 3H), 1.31 (par obsc m, 4H),

1.38 (s, 9H), 1.41 (s, 9H), 1.49±1.75 (m, 8H), 1.88 (m,

1H), 2.00 (m, 1H), 2.37 (m, 2H), 2.95 (dq, J=5.5,

7.2 Hz, 1H), 3.60 (s, 3H), 3.63 (s, 3H), 3.83 (d,

J=16.7 Hz, 1H), 3.88 (d, J=16.8 Hz, 1H), 4.04 (ddd,

J=5.6, 7.9, 7.9 Hz, 1H), 4.14 (dq, J=7.2, 7.2 Hz, 1H),

4.27 (ddd, J=5.3, 8.1, 8.7 Hz, 1H), 4.32 (par obsc dq,

J=7.2, 7.2 Hz, 1H), 4.49 (dd, J=5.5, 8.6 Hz, 1H), 6.41

(br, 1H), 7.37 (br, 1H), 5.54 (m, 2H), 7.64 (d, J=7.0 Hz,

1H); FABMS m/e calcd for C₄₁H₇₁N₆O₁₃+ (M+H)⁺:

855.5082. Found: 855.5091.

1

1736, 1652, 1516 cm ¹; H NMR (500 MHz, DMSO-d₆,

90 ^ꢀC) d 0.83±0.94 (m, 3H), 1.05 (d, J=7.2 Hz, 3H), 1.13

(m, 3H), 1.22 (d, J=7.2 Hz, 3H), 1.30 (d, J=7.1 Hz,

3H), 1.37 (s, 9H), 1.40 (s, 9H), 1.46±1.66 (m, 7H), 1.82

(m, 1H), 1.96 (m, 1H), 2.31 (m, 2H), 2.95 (par obsc m,

1H), 3.00 (par obsc s, 3H), 3.59 (s, 3H), 3.61 (s, 3H),

3.98 (ddd, J=5.5, 5.5, 8.1 Hz, 1H), 4.10 (dq, J=7.2,

7.3 Hz, 1H), 4.30 (ddd, J=5.1, 8.1, 9.7 Hz, 1H), 4.38

(dq, J=7.2, 7.3 Hz, 1H), 4.48 (dd, J=5.5, 8.6 Hz, 1H),

5.51 (s, 1H), 5.97 (s, 1H), 6.70 (br, 1H), 7.68 (d,

J=8.5 Hz, 1H), 7.77 (d, J=7.9 Hz, 1H), 7.80 (d,

J=7.9 Hz, 1H), 7.90 (d, J=6.9 Hz, 1H); FABMS m/e

calcd for C₄₀H₆₇N₆O₁₃+ (M+H)⁺: 839.4769. Found:

839.4754.

Boc-^D-iso-Glu(OMe)-N-(Me)-Gly-D-Ala-L-Leu-D-ꢁ-Me-

iso-Asp(OMe)-^L-Ala-OtBu (29d). The title compound

was prepared following the same procedure as that used

to prepare 29a. 27 mg (0.04 mmol) of 5 and 20 mg

(0.04 mmol) of 27 were used, and the amounts of sol-

vents and additional reagents were adjusted accord-

ingly. Chromatography (10 IPA/90 CHCl₃, 6 mL silica

gel) of the residue gave 17 mg (57%) of the hexapeptide

as a white solid: R_f0.19 (10 IPA/90 CHCl₃); IR (thin

1

®lm) 3299, 2961, 1740, 1650 cm ¹; H NMR (500 MHz,

DMSO-d₆, 110 ^ꢀC) d 0.83 (d, J=6.6 Hz, 3H), 0.87 (d,

J=6.6 Hz, 3H), 1.05 (d, J=7.1 Hz, 3H), 1.23 (d,

J=7.0 Hz, 3H), 1.25 (d, J=7.0 Hz, 3H), 1.38 (s, 9H),

1.41 (s, 9H), 1.48±1.66 (m, 3H), 1.86 (m, 1H), 1.98 (m,

1H), 2.36 (m, 2H), 2.90 (br.s, 3H), 2.94 (par obsc dq,

J=5.5, 7.1 Hz, 1H), 3.60 (s, 3H), 3.63 (s, 3H), 3.90 (d,

J=16.4 Hz, 1H), 3.98 (d, J=16.3 Hz, 1H), 4.04 (m, 1H),

4.13 (dq, J=7.1, 7.1 Hz, 1H), 4.26 (m, 1H), 4.34 (m,

1H), 4.49 (dd, J=5.4, 8.6 Hz, 1H), 6.47 (br, 1H), 7.49

(br, 1H), 7.57 (m, 2H), 7.67 (br, 1H); FABMS m/e

calcd for C₃₆H₆₃N₆O₁₃+ (M+H)⁺: 787.4456. Found:

787.4447.

Boc-^D-iso-Glu(OMe)-MeÁAla-N²-Boc-N³-Troc-(R)-Am-

inoala-^L-Leu-D-ꢁ-Me-iso-Asp(OMe)-L-Ala-OtBu (29b).

The title compound was prepared following the same

procedure as that used to prepare 29a. The materials

used were 66 mg (0.11 mmol) of 5, 71 mg (0.10 mmol) of

4b, and all other reagents and solvents were used in the

appropriate amounts. Chromatography (80 EtOAc/20

hexanes, 25 mL silica gel) of the residue gave 74 mg

(76%) of the hexapeptide as a white solid: R_f0.43

(EtOAc); IR (thin ®lm) 3320, 2955, 1735, 1652 cm ¹; ¹H

NMR (500 MHz, DMSO-d₆, 90 ^ꢀC) d 0.83 (d, J=

6.5 Hz, 3H), 0.86 (d, J=6.5 Hz, 3H), 1.06 (d, J=7.2 Hz,

3H), 1.23 (d, J=7.2 Hz, 3H), 1.38 (s, 9H), 1.41 (s, 9H),

1.53 (m, 2H), 1.61 (m 1H), 1.83 (m, 1H), 1.97 (m, 1H),

2.36 (m, 2H), 3.01 (par obsc s, 3H), 3.46 (m, 2H), 3.60

(s, 3H), 3.62 (s, 3H), 3.99 (m, 1H), 4.13 (dq, J=7.2,

7.2 Hz, 1H), 4.26 (ddd, J=6.6, 7.9, 7.9 Hz, 1H), 4.50

(dd, J=5.5, 7.3 Hz, 1H), 4.51 (par obsc m, 1H), 4.75 (s,

2H), 5.52 (s, 1H), 5.96 (s, 1H), 6.71 (br, 1H), 7.36 (br,

1H), 7.74 (d, J=8.9 Hz, 1H), 7.86 (m, 2H), 7.95 (d,

J=7.4 Hz, 1H); FABMS m/e calcd for C₄₀H₆₅

Cl₃N₇O₁₅+ (M+H)⁺: 988.3608 (Cl=34.9689). Found:

988.3610.

Boc-Adda-^D-iso-Glu(OMe)-MeÁAla-D-Ala-3-cyclohexyl-

L-Ala-D-ꢁ-Me-iso-Asp(OMe)-L-Ala-OH (3a). To 23 mg

(0.05 mmol) of Boc-Adda-OH was added 9 mg

(0.06 mmol) of HOAt, and the mixture was con-

centrated once from toluene. The residue was dissolved

in 0.75 mL of 20 DMF/80 CH₂Cl₂, and the solution was

cooled to 0 ^ꢀC. 12 mg (0.06 mmol) of DCC was added,

and the mixture was stirred at 0 ^ꢀC for 2 h, followed by

warming to rt and stirring for an additional 7 h. In a

separate ¯ask, 47 mg (0.06 mmol) of 29a was treated

with 0.5 mL of freshly distilled TFA for 1.5 h, followed

by concentration and re-concentration once from

toluene and twice from hexanes to remove residual

TFA. The resulting foam was dissolved in 0.4 mL of

DMF. The active ester solution prepared above was

cooled to 0 ^ꢀC, and the hexapeptide solution was added

to this via cannula. To the mixture was added 25 mg

(0.21 mmol) of collidine, and the mixture was stirred for

2 h, followed by warming to rt and stirring for an addi-

tional 12 h. The mixture was partitioned between

EtOAc and water, and the phases were separated. The

Boc-^D-iso-Glu(OMe)-N-(cyclohexyl)-Gly-D-Ala-L-Leu-D-

ꢁ-Me-iso-Asp(OMe)-^L-Ala-OtBu (29c). The title com-

pound was prepared following the same procedure as

that used to prepare 29a. The amounts of materials used

were 73 mg (0.12 mmol) of 5, 63 mg (0.11 mmol) of 24,

and amounts of the required solvents and additional

558

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

aqueous phase was extracted once with EtOAc. The

combined EtOAc phases were washed once each with 1

M NaHSO₄and water, and these aqueous phases were

discarded. The organic phase was diluted with one

volume of 50 ether/50 hexanes, and extracted once each

with 10% NaHCO₃and water. The combined aqueous

phases were washed once with 50 ether/50 hexanes,

acidi®ed to pH 1 with 1 M NaHSO₄, and extracted

three times with ether. The combined ether phases were

washed once each with water and brine, dried over

MgSO₄, ®ltered, and concentrated in vacuo. Chroma-

tography (1/5/94: HOAc/IPA/CHCl₃, 10 mL silica gel)

of the residue gave 33 mg (57%) of the heptapeptide as a

(500 MHz, DMSO-d₆, 90 ^ꢀC) d 0.82 (d, J=6.5 Hz, 3H),

0.85 (d, J=6.6 Hz, 3H), 0.96 (d, J= 6.8 Hz, 3H), 1.02

(d, J=7.0 Hz, 3H), 1.05 (d, J=7.1 Hz, 3H), 1.25 (d,

J=7.2 Hz, 3H), 1.37 (s, 9H), 1.52 (m, 2H), 1.56 (d,

J=1.1 Hz, 3H), 1.61 (par obsc m, 1H), 1.85 (m, 1H),

1.98 (m, 1H), 2.33 (m, 2H), 2.57 (m, 3H), 2.66 (dd,

J=7.1, 14.0 Hz, 1H), 2.73 (dd, J=4.9, 14.0 Hz, 1H),

2.99 (par obsc s, 3H), 3.17 (s, 3H), 3.24 (ddd, J=5.2,

5.4, 7.2 Hz, 1H), 3.46 (m, 2H), 3.59 (s, 3H), 3.59 (s, 3H),

4.07 (m, 1H), 4.19±4.29 (m, 3H), 4.46±4.55 (m, 2H), 4.74

(s, 2H), 5.37 (d, J=10.0 Hz, 1H), 5.45 (obsc, 1H), 5.45

(dd, J=6.4, 15.6 Hz, 1H), 5.93 (br. s, 1H), 6.07 (d,

J=15.6 Hz, 1H), 6.20 (d, J=7.5 Hz, 1H), 7.13±7.26 (m,

5H), 7.31 (br, 1H), 7.75 (d, J=8.6 Hz, 1H), 7.86 (m,

3H), 7.93 (d, J=7.4 Hz, 1H); FABMS m/e calcd

for C₅₆H₈₄Cl₃N₈O₁₇+ (M+H)⁺=1245 (Cl=34.9689).

Found: 1245. FABMS m/e calcd for C₅₆H₈₃Cl₃

NaN₈O₁₇+ (M+Na)⁺: 1267 (Cl=34.9689). Found:

1267.

white solid: R_f0.16 (1/5/94: HOAc/IPA/CHCl₃); IR

1

(thin ®lm) 3311 br, 2921, 1741, 1717, 1650 cm

;

¹H

NMR (500 MHz, DMSO-d₆, 90 ^ꢀC) d 0.84±0.94 (m,

3H), 0.97 (d, J=6.8 Hz, 3H), 1.02 (d, J=7.0 Hz, 3H),

1.06 (d, J=7.2 Hz, 3H), 1.15 (m, 3H), 1.26 (d,

J=7.0 Hz, 3H), 1.31 (d, J=7.1 Hz, 3H), 1.39 (s, 9H),

1.50 (m, 1H), 1.57 (s, 3H), 1.61±1.65 (m, 6H), 1.85 (m,

1H), 1.98 (m, 1H), 2.27±2.35 (m, 2H), 2.57 (m, 2H), 2.67

(dd, J=7.3, 14.1 Hz, 1H), 2.75 (dd, J=4.9, 14.1 Hz,

1H), 3.18 (par obsc s, 3H), 3.26 (ddd, J=5.7, 6.9,

6.9 Hz, 1H), 3.59 (s, 3H), 3.60 (s, 3H), 4.08 (m, 1H),

4.18±4.26 (m, 2H), 4.30 (m, 1H), 4.40 (dq, J=7.0,

7.0 Hz, 1H), 4.49 (dd, J=5.8, 8.0 Hz, 1H), 5.38 (d,

J=10.4 Hz, 1H), 5.46 (par obsc dd, 1H), 5.47 (s, 1H),

5.96 (s, 1H), 6.07 (d, J=16.0 Hz, 1H), 6.21 (d,

J=7.6 Hz, 1H), 7.15±7.26 (m, 5H), 7.72 (d, J=8.2 Hz,

1H), 7.77 (d, J=7.3 Hz, 1H), 7.82 (d, J=7.1 Hz, 1H),

7.87 (d, J=6.9 Hz, 1H), 7.90 (d, J=6.7 Hz, 1H);

FABMS m/e calcd for C₅₆H₈₆N₇O₁₅+ (M+H)⁺:

1096.6. Found: 1096.7. FABMS m/e calcd for C₅₆H₈₅

NaN₇O₁₅+ (M+Na)⁺: 1118.6. Found: 1118.6.

Boc-Adda-^D-iso-Glu(OMe)-N-(cyclohexyl)-Gly-D-Ala-L-

Leu-^D-ꢁ-Me-iso-Asp(OMe)-L-Ala-OH (30a). The Boc-

Adda activation was carried out as in the procedure to

prepare 3a. The materials used were 20 mg (0.05 mmol)

of Boc-Adda-OH, 7 mg (0.05 mmol) of HOAt, 10 mg

(0.05 mmol) of DCC, and 0.6 mL of 20 DMF/80

CH₂Cl₂as the solvent. In a separate ¯ask, 43 mg

(0.05 mmol) of 29c was treated with TFA as required.

The resulting foam was dissolved in 0.5 mL of DMF,

combined with the active ester solution at 0 ^ꢀC, and

18 mg (0.14 mmol) of collidine was added. The mixture

was stirred for 2 h, warmed to rt, and stirred for an

additional 12 h. The mixture was partitioned between

ether and water, and the phases were separated. The

aqueous phase was extracted once with EtOAc. The

combined organic phases were washed once each with 1 M

NaHSO₄, water, and brine, dried over MgSO₄, ®ltered,

and concentrated in vacuo. Chromatography (1 HOAc/

7 IPA/92 CHCl₃, 12 mL silica gel) of the residue gave

28 mg (55%) of the heptapeptide as a white solid: R_f

0.28 (1 HOAc/10 IPA/89 CHCl₃); IR (thin ®lm) 3295,

Boc-Adda-^D-iso-Glu(OMe)-MeÁAla-N²-Boc-N³-Troc-(R)-

Aminoala-^L-Leu-D-ꢁ-Me-iso-Asp(OMe)-L-Ala-OH (3b).

The Boc-Adda activation was carried out as in proce-

dure to prepare 3a. The materials used were 22 mg

(0.05 mmol) of Boc-Adda-OH, 8 mg (0.06 mmol) of

HOAt, 12 mg (0.06 mmol) of DCC, and 0.6 mL of

CH₂Cl₂as the solvent. In a separate ¯ask, 50 mg

(0.05 mmol) of 29b was treated with 0.5 mL of freshly

distilled TFA for 1.5 h, followed by concentration and

re-concentration three times from hexanes to remove

residual TFA. The resulting foam was dissolved in

0.5 mL of CH₂Cl₂. The active ester solution prepared

above was cooled to 0 ^ꢀC, and the hexapeptide solution

was added to this via cannula. To the mixture was

added 25 mg (0.213 mmol) of collidine. The mixture

was stirred for 4 h, and 0.5 mL of DMF was added to

dissolve a precipitate that hindered stirring. After this

period, the mixture was warmed to rt, and was stirred

for an additional 12 h. The mixture was partitioned

between EtOAc and water, and the phases were sepa-

rated. The aqueous phase was extracted once with

EtOAc. The combined EtOAc phases were washed once

each 50% sat. citric acid, water, and brine, dried over

MgSO₄, ®ltered, and concentrated in vacuo. Chroma-

tography (1/5/94: HOAc/IPA/CHCl₃, 12 mL silica gel)

of the residue gave 37 mg (58%) of the heptapeptide

as a white solid: R_f0.37 (1/5/94: HOAc/IPA/CHCl₃);

IR (thin ®lm) 3311 br, 2945, 1741, 1647 cm ¹; ¹H NMR

1

2931, 1736, 1648 cm ¹; H NMR (500 MHz, DMSO-d₆,

90 ^ꢀC) d 0.82 (d, J=6.5 Hz, 3H), 0.86 (d, J=6.5 Hz,

3H), 0.96 (d, J=6.7 Hz, 3H), 1.02 (d, J=6.9 Hz, 3H),

1.04 (d, J=7.1 Hz, 3H), 1.05 (obsc m, 1H), 1.25 (d,

J=7.1 Hz, 3H), 1.27 (d, J=7.2 Hz, 3H), 1.27±1.38

(par obsc m, 4H), 1.38 (s, 9H), 1.47±1.57 (par obsc m,

2H), 1.57 (d, J=1.2 Hz, 3H), 1.59±1.74 (m, 6H), 1.88

(m, 1H), 2.01 (m, 1H), 2.33 (m, 2H), 2.58 (m 3H), 2.66

(par obsc dd, J=7.2, 14.2 Hz, 1H), 2.74 (par obsc dd,

J=4.9, 14.2 Hz, 1H), 2.96 (par obsc dd, J=5.5, 7.1 Hz,

1H), 3.18 (s, 3H), 3.25 (ddd, J=5.1, 5.4, 7.2 Hz, 1H),

3.60 (s, 3H), 3.61 (s, 3H), 3.85 (m, 2H), 4.08 (m, 1H),

4.17±4.30 (m, 4H), 4.47 (dd, J=5.6, 8.6 Hz, 1H), 5.37

(d, J=9.6 Hz, 1H), 5.48 (dd, J=6.6, 15.6 Hz, 1H),

6.08 (obsc, 1H), 6.08 (d, J=15.6 Hz, 1H), 7.14±7.24 (m,

5H), 7.45 (br, 1H), 7.60 (m, 2H), 7.73 (m, 2H); FABMS

m/e calcd for C₅₇H₉₀N₇O₁₅+ (M+H)⁺: 1112. Found:

1112.

Boc-Adda-^D-iso-Glu(OMe)-N-(Me)-Gly-D-Ala-L-Leu-D-

ꢁ-Me-iso-Asp(OMe)-^L-Ala-OH (30b). The Boc-Adda

activation was carried out as in the procedure to prepare

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

559

3a using 20 mg (0.05 mmol) of Boc-Adda-OH. In a

separate ¯ask, 28 mg (0.04 mmol) of 29d was treated

with TFA as required. The resulting foam was com-

bined with the active ester solution at 0 ^ꢀC, and 13 mg

(0.11 mmol) of collidine was added. After stirring for

the appropriate time, the mixture was worked up, with

the exception to the general procedure that the initial

aqueous phase was extracted twice with EtOAc. Chro-

matography (1/15/84: HOAc/IPA/CHCl₃, 5 mL silica

gel) of the residue gave 25 mg (68%) of the heptapeptide

4.68 (m, 1H), 4.86 (m, 1H), 5.31 (s, 1H), 5.37 (dd,

J=7.9, 15.6 Hz, 1H), 5.41 (d, J=9.9 Hz, 1H), 5.99 (s,

1H), 6.14 (d, J=8.0 Hz, 1H), 6.23 (d, J=15.6 Hz, 1H),

6.58 (d, J=8.8 Hz, 1H), 6.79 (d, J=8.9 Hz, 1H), 6.96 (d,

J=9.2 Hz, 1H), 7.17±7.27 (m, 5H), 7.61 (d, J=6.7 Hz,

1H), 7.92 (d, J=8.2 Hz, 1H); FABMS m/e calcd

for C₅₁H₇₅NaN₇O₁₂+ (M+Na)⁺: 1000.5375. Found:

1000.5379.

Cyclo-[Adda-^D-iso-Glu(OMe)-MeÁAla-N²-Boc-N³-Troc-

(R)-Aminoala-^L-Leu-D-ꢁ-Me-iso-Asp(OMe)-L-Ala] (31b).

The activation, Boc cleavage, and macrocyclization

were carried out as in the preparation of 31a. 52 mg

(0.04 mmol) of 3b was used, and all other reagents and

solvents were used in the appropriate amounts. After

1 h, the mixture was worked up. The residue was ®ltered

through silica (CHCl₃to elute nonpolar material, then 5

IPA/95 CHCl₃to elute product, 6 mL silica gel) to give

a white solid. Further puri®cation by preparative

reversed phase HPLC (12 THF/46 ACN/42 10 mM

HOAc, retention time of product=19.4 min) gave 22 mg

(47%) of the desired macrocycle as a white solid: R_f0.17

(5 IPA/95 CHCl₃); IR (thin ®lm) 3373, 3289, 2955, 1735,

as a white solid: R_f0.23 (1/20/79: HOAc/IPA/CHCl₃);

1

IR (thin ®lm) 3300 br, 2955, 1741, 1720, 1657 cm

;

FABMS m/e calcd for C₅₂H₈₁NaN₇O₁₅+ (M+Na)⁺:

1066.6. Found: 1066.6. FABMS m/e calcd for C₅₂H₈₁

LiN₇O₁₅+ (M+Li)⁺: 1050.6. Found: 1050.6. Attempts

1

to analyze this material by H NMR failed because it

decomposed rapidly to imide (loss of one methyl ester

resonance) at the elevated temperatures necessary to

reduce the rotamer induced peak broadening and dou-

bling. In contrast to this, spectral information was easily

obtained after the macrocyclization event.

Cyclo-[Adda-^D-iso-Glu(OMe)-MeÁAla-D-Ala-3-cyclohexyl-

L-Ala-D-ꢁ-Me-iso-Asp(OMe)-L-Ala] (31a). To 45 mg

(0.041 mmol) of 3a in an oven dried ¯ask was added

15 mg (0.08 mmol) of penta¯uorophenol in 1.01 mL

freshly distilled EtOAc. The solution was cooled to

0 ^ꢀC, and 9.7 mg (0.05 mmol) of DCC was added. The

mixture was stirred for 1.5 h, warmed to rt, and stirred

for an additional 6.5 h. The solvent was removed in

vacuo, and the residue was reconcentrated once from

hexanes to remove residual EtOAc. The residue was

treated with 2 mL of freshly distilled TFA for 20 min,

concentrated in vacuo, and re-concentrated three times

from hexanes. The residue was then dissolved in 20 mL

of CHCl₃, transferred to a dropping funnel, and added

dropwise to a rapidly stirring biphasic mixture of 35 mL

of CHCl₃and 35 mL of 1 M pH 9.5 phosphate buer

over 10 min. After 30 additional min, the phases were

separated, and the aqueous phase was extracted twice

with EtOAc. The combined EtOAc phases were washed

once with water, combined with the CHCl₃phase, and

washed once with brine. The organic phase was dried

over MgSO₄, ®ltered, and concentrated in vacuo to give

a yellow residue. The residue was ¯ashed chromato-

graphed (CHCl₃to elute nonpolar material, then 10

IPA/90 CHCl₃to elute product, 6 mL silica gel) to give

a white solid. Further puri®cation by preparative

reversed phase HPLC (10 THF/42 ACN/48 10 mM

HOAc, retention time of product=23 min) gave 17 mg

(43%) of the desired macrocycle as a white solid: R_f0.20

(10 IPA/90 CHCl₃); IR (thin ®lm) 3373, 3278, 2923,

1

1667, 1646 cm ¹; H NMR (500 MHz, CDCL₃) d 0.87

(d, J=6.5 Hz, 3H), 0.91 (d, J=6.4 Hz, 3H), 1.02 (d,

J=6.7 Hz, 3H), 1.13 (d, J=6.7 Hz, 3H), 1.20 (d,

J=6.7 Hz, 3H), 1.37 (d, J=7.1 Hz, 3H), 1.61 (par obsc

s, 3H), 2.25 (m, 2H), 2.56 (m, 3H), 2.68 (dd, J=7.3,

13.9 Hz, 1H), 2.71 (obsc m, 1H), 2.80 (dd, J=4.5,

13.9 Hz, 1H), 3.20 (ddd, J=5.5, 6.6, 6.8 Hz, 1H), 3.24 (s,

3H), 3.25 (s, 3H), 3.55 (m, 1H), 3.69 (par obsc m, 1H),

3.69 (apps, 6H), 3.82 (par obsc m, 1H), 4.28 (m, 1H),

4.51 (par obsc m, 2H), 4.55 (d, J=12.1 Hz, 1H), 4.65

(m, 2H), 4.75 (d, J=11.9 Hz, 1H), 4.88 (m, 1H), 5.27 (s,

1H), 5.38 (dd, J=8.3, 15.6 Hz, 1H), 5.42 (d, J=10.0 Hz,

1H), 5.63 (br, 1H), 5.91 (s, 1H), 6.18 (d, J=8.2 Hz, 1H),

6.22 (d, J=15.6 Hz, 1H), 6.61 (d, J=8.8 Hz, 1H), 6.71

(d, J=8.0 Hz, 1H), 7.17±7.29 (par obsc m, 6 H), 7.73 (d,

J=6.4 Hz, 1H), 8.10 (d, J=7.3 Hz, 1H); FABMS

m/e calcd for C₅₁H₇₄Cl₃N₈O₁₄(M+H)⁺: 1127 (Cl=

34.9689). Found: 1127. FABMS m/e calcd for C₅₁H₇₃

Cl₃NaN₈O₁₄(N+Na)⁺: 1149 (Cl=34.9689). Found:

1149.

Cyclo-[Adda-^D-iso-Glu(OMe)-N-(cyclohexyl)-Gly-D-Ala-

L-Leu-D-ꢁ-Me-iso-Asp(OMe)-L-Ala] (31c). The activa-

tion, Boc cleavage, and macrocyclization were carried

out as in the procedure to prepare 31a. 23 mg

(0.02 mmol) 30a was used, and the amounts of solvents

and additional reagents were adjusted accordingly. The

residue was ¯ashed chromatographed (CHCl₃to elute

nonpolar material, then 5 IPA/95 CHCl₃to elute pro-

duct, 3 mL silica gel) to give a white solid. Further pur-

i®cation by preparative reversed phase HPLC (11 THF/

45 ACN/44 10 mM HOAc, retention time of pro-

duct=19.5 min) gave 9 mg (43%) of the desired macro-

cycle as a white solid: R_f0.42 (10 IPA/90 CHCl₃); IR

1

1740, 1672, 1646 cm ¹; H NMR (500 MHz, CDCl₃) d

0.85±0.92 (m, 3H), 1.02 (d, J=6.6 Hz, 3H), 1.14 (d,

J=6.8 Hz, 3H), 1.18 (d, J=7.1 Hz, 3H), 1.24 (par obsc

m, 3H), 1.33 (d, J=7.3 Hz, 3H), 1.37 (d, J=7.2 Hz, 3H),

1.45 (m, 1H), 1.61 (s, 3H), 1.67 (par obsc m, 6H), 2.27

(m, 1H), 2.33 (m, 1H), 2.53 (m, 3H), 2.59 (ddq, J=6.6,

6.6, 9.7 Hz, 1H), 2.68 (dd, J=7.3, 13.9 Hz, 1H), 2.68

(obsc m, 1H), 2.79 (dd, J=4.7, 13.9 Hz, 1H), 3.20 (ddd,

J=4.8, 6.1, 7.3 Hz, 1H), 3.24 (s, 3H), 3.27 (s, 3H), 3.70

(s, 3H), 3.87 (s, 3H), 4.32 (m, 1H), 4.38 (dq, J=7.4,

7.8 Hz, 1H), 4.54 (m, 1H), 4.61 (dd, J=2.8, 8.3 Hz, 1H),

1

(thin ®lm) 3383, 3289, 2934, 1735, 1672, 1646 cm ¹; H

NMR (500 MHz, CDCl₃) d 0.88 (d, J=6.5 Hz, 3H), 0.93

(d, J=6.5 Hz, 3H), 1.02 (d, J=6.7 Hz, 3H), 1.10 (par

obsc m, 1H), 1.13 (d, J=6.8 Hz, 3H), 1.17 (d, J=7.2 Hz,

3H), 1.30 (d, J=7.3 Hz, 3H), 1.37 (d, J=7.2 Hz, 3H),

1.42 (m, 2H), 1.45 (m, 2H), 1.61 (d, J=0.9 Hz, 3H),

560

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

1.64±1.88 (m, 10 H), 2.25 (m, 1H), 2.50 (m, 2H), 2.60

(ddq, J=6.3, 6.6, 9.8 Hz, 1H), 2.65 (par obsc m, 1H),

2.68 (dd, J=7.5, 14.1 Hz, 1H), 2.80 (dd, J=4.7, 14.0 Hz,

1H), 3.20 (ddd, J=4.7, 6.3, 7.2 Hz, 1H), 3.24 (s, 3H),

3.60 (m, 1H), 3.71 (s, 3H), 3.80 (d, J=16.7 Hz, 1H), 3.87

(s, 3H), 3.95 (d, J=16.7 Hz, 1H), 4.29 (ddd, J=6.1, 6.2,

9.3 Hz, 1H), 4.47 (dq, J=7.3, 8.6 Hz, 1H), 4.53 (ddd,

J=8.3, 8.6, 9.2 Hz, 1H), 4.63 (dd, J=2.7, 8.4 Hz, 1H),

4.64 (par obsc m, 1H), 4.82 (dq, J=7.3, 9.6 Hz, 1H),

5.34 (dd, J=8.4, 15.4 Hz, 1H), 5.41 (d, J=9.8 Hz, 1H),

6.11 (d, J=8.6 Hz, 1H), 6.25 (d, J=15.4 Hz, 1H), 6.61

(d, J=8.6 Hz, 1H), 6.75 (d, J=8.4 Hz, 1H), 6.90 (d,

J=9.8 Hz, 1H), 7.18±7.28 (m, 5H), 7.43 (d, J=6.1 Hz,

1H), 8.14 (d, J=8.6 Hz, 1H); FABMS m/e calcd for

C₅₂H₈₀N₇O₁₂+ (M+H)⁺: 994. Found: 994. FABMS

m/e calcd for C₅₂H₇₉KN₇O₁₂+ (M+K)⁺: 1032. Found:

1032.

peak (retention time=14 min) was the desired product

(4 mg, 41%), isolated as a white solid: R_f0.37 (DOC-A:

0.9 H₂O/13 HOAc/17.1 MeOH/69 CH₂Cl₂); IR (thin

1

®lm) 3354, 3281 br, 2926, 1721, 1659 cm ¹; H NMR

(500 MHz, pH 7.5, 50 mM potassium phosphate in

D₂O) d 0.90 (m, 1H), 1.00 (m, 1H), 1.05 (app d,

J=6.6 Hz, 6H), 1.08 (d, J=6.9 Hz, 3H), 1.15±1.27 (m,

4H), 1.31 (d, J=7.3 Hz, 3H), 1.42 (d, J=7.4 Hz, 3H),

1.63±1.74 (m, 6H), 1.75 (s, 3H), 1.95 (m, 2H), 2.10 (m,

1H), 2.61 (ddd, J=3.9, 12.5, 16.4 Hz, 1H), 2.69±2.85 (m,

4H), 2.97 (dd, J=4.0, 14.2 Hz, 1H), 3.05 (dq, J=6.9,

10.5 Hz, 1H), 3.19 (dq, J=4.6, 7.0 Hz, 1H), 3.30 (s, 3H),

3.43 (s, 3H), 3.52 (m, 1H), 3.95 (dd, J=6.9, 8.2 Hz, 1H),

4.38 (m, 2H), 4.43 (app d, J=4.3 Hz, 1H), 4.49 (m, 2H),

5.58 (par obsc d, J=10.9 Hz, 1H), 5.58 (s, 1H), 5.62 (par

obsc dd, J=8.6, 15.7 Hz, 1H), 5.97 (s, 1H), 6.35 (d,

J=15.6 Hz, 1H), 7.29±7.40 (m, 5H); FABMS m/e calcd

for C₄₉H₇₁NaN₇O₁₂+ (M+Na)⁺: 972. Found: 972.

FABMS m/e calcd for C₄₉H₇₁LiN₇O₁₂+ (M+Li)⁺:

968. Found: 968.

Cyclo-[Adda-^D-iso-Glu(OMe)-N-(Me)-Gly-D-Ala-L-Leu-

D-ꢁ-Me-iso-Asp(OMe)-L-Ala] (31d). The activation,

Boc cleavage, and macrocyclization were carried out as

in the procedure to prepare 31a, using 22 mg

(0.02 mmol) of 30b, and the amounts of solvents and

additional reagents were adjusted accordingly. After

1.5 h, the mixture was worked up. The residue was ¯a-

shed chromatographed (CHCl₃to elute nonpolar mate-

rial, then 20 IPA/80 CHCl₃to elute product; 4 mL silica

gel) to give a white solid. Further puri®cation by pre-

parative reversed phase HPLC (52 ACN/48 10 mM

HOAc), retention time of product=13.1 min) gave 5 mg

(26%) of the desired macrocycle as a white solid: R_f0.20

The slower running peak (retention time=20.6 min) was

two compounds by TLC; R_f0.37 for the faster running

material, and 0.21 for the slower running material

(DOC-A). The two materials were separated by pre-

parative TLC (DOC-A, silica gel 60) to give 2 mg each

of white solids that were isomeric with the desired pro-

duct by FABMS, but that were both dierent than the

1

desired product by H NMR.

To identify which of the three isomers obtained corre-

sponded with the starting di-ester, a small amount of

each of these three materials was re-esteri®ed using the

following general proceedure: Approximately 0.2 mg of

the puri®ed hydrolysis product was dissolved in 0.2 mL

EtOAc, and three drops of MeOH were added. The

solution was cooled to 0 ^ꢀC, and two drops of 2 M

trimethylsilyldiazomethane (TMS-diazomethane) were

added. After 10 min, the reaction was quenched with 1

drop of HOAc, and the solution was concentrated in

vacuo to a residue. The residue was ®ltered through

silica (10 IPA/90 CHCl₃), and the ®ltrate was con-

centrated to a solid. The solid was analyzed by ¹H

NMR, and compared to authentic di-ester by TLC co-

spotting assays. Only the material that had eluted ®rst

from the reversed phase HPLC puri®cation resulted

in esteri®ed material that was identical to the original

di-ester.

(20 IPA/80 CHCl₃); IR (thin ®lm) 3292, 2945, 1740,

1

1658 cm

;

¹H NMR (500 MHz, CDCl₃) d 0.90 (d,

J=6.1 Hz, 3H), 0.93 (d, J=6.2 Hz, 3H), 1.00 (d, J=

6.7 Hz, 3H), 1.16 (d, J=6.9 Hz, 3H), 1.21 (d, J=7.1 Hz,

3H), 1.25 (par obsc m, 1H), 1.36 (d, J=7.1 Hz, 3H), 1.39

(d, J=7.0 Hz, 3H), 1.71 (m, 3H), 2.33±2.48 (m, 3H),

2.59 (m, 2H), 2.67 (dd, J=7.4, 14.0 Hz, 1H), 2.77 (dd,

J=4.7, 14.0 Hz, 1H), 2.81 (dq, J=3.3, 7.0 Hz, 1H), 3.09

(s, 3H), 3.18 (ddd, J=4.9, 6.0, 7.2 Hz, 1H), 3.23 (s, 3H),

3.75 (apps, 6H), 4.10 (s, 2H), 4.37 (ddd, J=4.2, 6.9,

11.1 Hz, 1H), 4.42 (dq, J=7.2, 7.3 Hz, 1H), 4.50 (m,

1H), 4.57 (dd, J=3.3, 8.4 Hz, 1H), 4.65 (ddd, J=3.2,

7.2, 10.6 Hz, 1H), 4.71 (dq, J=7.5, 7.7 Hz, 1H), 5.39 (d,

J=9.7 Hz, 1H), 5.45 (dd, J=6.8, 15.6 Hz, 1H), 6.19

(obsc, 1H), 6.20 (d, J=15.6 Hz, 1H), 6.60 (d, J=8.0 Hz,

1H), 7.16±7.28 (m, 7H), 7.31 (br, 1H), 7.65 (d, J=

8.0 Hz, 1H); FABMS m/e calcd for C₄₇H₇₂N₇O₁₂+

(M+H)⁺: 926.5242. Found: 926.5249.

Microcystin, N-(cyclohexyl)-glycine variant (32a). The

title compound was prepared using the same procedure

as that used to prepare 2a. 8 mg (0.008 mmol) of 31c was

used, and the amounts of solvents and additional

reagents were adjusted accordingly. After 40 min, the

reaction was worked up. The resulting solid was subjected

to preparative reversed phase HPLC (75 MeOH/25

(0.2%) aq TFA). Two major peaks were observed, and

the material corresponding to each peak was isolated.

The faster running peak (retention time=17.6 min) was

the desired product (3.5 mg, 44%), isolated as a white

Microcystin, cyclohexylalanine variant (2a). To 10 mg

(0.01 mmol) of the dimethyl ester 3a in a vial was added

2 mL of THF. The solution was cooled to 0 ^ꢀC, and 36

drops of water was added, followed by 10 drops of 1 M

LiOH. After 1 h, the solution was partitioned between 1

M NaHSO₄and EtOAc, and the phases were separated.

The aqueous was extracted twice with EtOAc. The

combined EtOAc phases were washed once with brine,

dried over Na₂SO₄, micro-®ltered, and concentrated to

a white solid. The solid was subjected to preparative

reversed phase HPLC (70 MeOH/30 (0.2%) aq TFA).

Two major peaks were observed, and the material corre-

sponding to each peak was isolated. The faster running

solid: R_f0.44 (DOC-A); IR (thin ®lm) 3354, 3302 br,

1

2926, 1721, 1655 cm

;

¹H NMR (500 MHz, pH 7.5,

50 mM potassium phosphate in D₂O) d 0.89 (d, J=

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

561

6.0 Hz, 3H), 0.97 (d, J=6.1 Hz, 3H), 1.04 (d, J=6.5 Hz,

3H), 1.05 (d, J=6.3 Hz, 3H), 1.09 (d, J=6.8 Hz, 3H),

1.10 (par obsc m, 1H), 1.31 (d, J=7.2 Hz, 3H), 1.38 (d,

J=7.4 Hz, 3H), 1.41±1.71 (m, 6H), 1.74 (s, 3H), 1.79±

2.13 (m, 8H), 2.57 (m, 1H), 2.70±2.84 (m, 3H), 2.98 (dd,

J=3.9, 14.3 Hz, 1H), 3.03 (dq, J=7.0, 10.7 Hz, 1H),

3.15 (dq, J=4.2, 7.1 Hz, 1H), 3.30 (s, 3H), 3.51 (m, 1H),

3.88 (m, 1H), 3.96 (d, J=17.4 Hz, 1H), 4.00 (par obsc

m, 1H), 4.05 (d, J=17.3 Hz, 1H), 4.31 (m, 1H), 4.40 (m,

2H), 4.49 (app t, J=9.7 Hz, 1H), 4.54 (app q, J=7.4 Hz,

1H), 5.60 (m, 2H), 6.36 (d, J=15.6 Hz, 1H), 7.29±7.40

(m, 5H); FABMS m/e calcd for C₅₀H₇₅NaN₇O₁₂+

(M+Na)⁺: 988. Found: 988.

reaction was worked up. The resulting solid was subjected

to preparative reversed phase HPLC (74 MeOH/26

(0.2%) aq TFA). Two major peaks were observed, and

the material corresponding to each peak was isolated.

The faster running peak (retention time=23 min) was

the desired product (3.5 mg, 31%), isolated as a white

solid: R_f0.40 (DOC-A); IR (thin ®lm) 3312 br, 2947,

1

1721, 1643 cm ¹; H NMR (500 MHz, pH 7.5, 50 mM

potassium phosphate in D₂O) d 0.89 (d, J=5.8 Hz,

3H), 0.94 (d, J=5.9 Hz, 3H), 1.05 (d, J=6.4 Hz, 3H),

1.06 (d, J=5.9 Hz, 3H), 1.09 (d, J=6.7 Hz, 3H), 1.32 (d,

J=6.7 Hz, 3H), 1.71 (par obsc m, 2H), 1.74 (s, 3H), 1.92

(m, 2H), 2.11 (m, 1H), 2.58 (m, 1H), 2.66±2.79 (m, 4 H),

2.96 (dd, J=4.0, 14.0 Hz, 1H), 3.05 (dq, J=6.9, 10.0 Hz,

1H), 3.17 (dq, J=4.2, 6.7 Hz, 1H), 3.29 (s, 3H), 3.37 (s,

3H), 3.43±3.53 (m, 2H), 3.78 (dd, J=3.8, 14.6 Hz, 1H),

4.09 (dd, J=6.7, 8.2 Hz, 1H), 4.28 (m, 1H), 4.39 (d,

J=3.6 Hz, 1H), 4.50 (m, 2H), 5.58 (par obsc m, 2H),

5.60 (s, 1H), 5.94 (s, 1H), 6.35 (d, J=15.4 Hz, 1H),

7.29±7.40 (m, 5H); FABMS m/e calcd for

C₄₉H₆₉Cl₃NaN₈O₁₄+ (M+Na)⁺: 1121 (Cl=34.9689).

Found: 1121. FABMS m/e calcd for C₄₉H₇₀Cl₃N₈O₁₄+

(M+H)⁺: 1099 (Cl=34.9689). Found: 1099.

The slower running peak (retention time=21.5 min) was

two compounds by TLC; R_f0.51 for the faster running

material, and 0.33 for the slower running material

(DOC-A). The two materials were separated by pre-

parative TLC (DOC-A, silica gel 60) to give 2 mg each

of white solids that were isomeric with the desired pro-

duct by FABMS, but that were both dierent than the

1

desired product by H NMR.⁵⁹

Microcystin, N-methylglycine variant (32b). The title

compound was prepared using the same procedure as

that used to prepare 2a, using 5 mg (0.005 mmol) of 31d,

1 mL THF, 20 drops of water, and 5 drops of 1 M

LiOH. After 50 min, the reaction was worked up. The

resulting solid was subjected to preparative reversed

phase HPLC (71 MeOH/29 (0.2%) aq TFA). Two

major peaks were observed, and the material corre-

sponding to each peak was isolated. The faster running

peak (retention time=14 min) was the desired product

The slower running peak (retention time=26.2 min) was

two compounds by TLC; R_f0.37 for the faster running

material, and 0.21 for the slower running material

(DOC-A). The two materials were separated by pre-

parative TLC (DOC-A, silica gel 60) to give 2 mg each

of white solids that were isomeric with the desired pro-

duct by FABMS, but that were both dierent than the

1

desired product by H NMR.⁵⁹

(2 mg, 40%), isolated as a white solid: R_f0.24 (DOC-A);

1

Microcystin, aminoalanine variant (2b). To 0.25 mg

(0.23 mmol) of 32c in 0.15 mL MeOH were added

0.2 mL of 0.1 M pH 3.0 phosphate buer and 12 mg Zn/

Cu couple.⁵⁸The mixture was stirred vigorously for

22.5 h, followed by ®ltration through Celite using 50

MeOH/50 0.1M pH 3.0 phosphate buer to rinse.

The ®ltrate was puri®ed by analytical reversed phase

HPLC (70 MeOH/30 pH 3.0 50 mM phosphate buer

to begin elution, followed by ramping to 80 MeOH/20

pH 3.0 50 mM phosphate buer over 10 min at 15 min

elution time; retention time of the product was

32.6 min). The fractions containing product were

concentrated in vacuo to remove the MeOH, followed

by increasing the pH to 7.0 by the dropwise addition

of 1 M pH 7.0 phosophate buer. The solution was then

concentrated in vacuo to give a solid residue. The

residue was slurried in methanol, and ®ltered through

Celite to remove most of the salts. The ®ltrate was

concentrated to half volume, and was ®ltered through

a second pad of Celite. The ®ltrate was concentrated

in vacuo to give a white solid, 0.18 mg (82%) of the

title compound was obtained based on UV absorp-

tion: ¹H NMR (500 MHz, MeOH-d₄) d 0.88 (d,

J=6.8 Hz, 3H), 0.90 (d, J=6.7 Hz, 3H), 0.93 (d, J=

6.4 Hz, 3H), 1.01 (d, J=6.8 Hz, 3H), 1.07 (d, J=6.8 Hz,

3H), 1.14 (d, J=6.2 Hz, 3H), 1.59 (par obsc m, 3H),

1.62 (s, 3H), 1.78 (m, 1H), 1.93 (m, 1H), 2.58 (m, 2H),

2.69 (m, 2H), 2.81 (m, 1H), 3.03 (m, 3H), 3.23 (par

obsc s, 3H), 3.53 (m, 2H), 4.21 (m, 1H), 4.38 (m, 2H),

4.52 (m, 1H), 4.58 (m, 2H), 5.45 (m, 3H), 5.91 (s, 1H),

IR (thin ®lm) 3286 br, 2920, 1651 cm

;

¹H NMR

(500 MHz, pH 7.5, 50 mM potassium phosphate in

D₂O) d 0.86 (d, J=5.7 Hz, 3H), 0.94 (d, J=5.8 Hz, 3H),

1.03 (app d, J=7.0 Hz, 6H), 1.07 (d, J=6.9 Hz, 3H),

1.28 (d, J=6.9 Hz, 3H), 1.40 (d, J=7.3 Hz, 3H), 1.68

(m, 3H), 1.72 (s, 3H), 1.95 (par obsc m, 1H), 2.04 (m,

1H), 2.52 (m, 1H), 2.68±2.79 (m, 3H), 2.90±2.98 (m,

2H), 3.13 (dq, J=4.8, 7.2 Hz, 1H), 3.23 (s, 3H), 3.28 (s,

3H), 3.50 (m, 1H), 3.90 (d, J=17.0 Hz, 1H), 3.95 (m,

1H), 4.15 (d, J=17.0 Hz, 1H), 4.32 (m, 2H), 4.41±4.51

(m, 3H), 5.55 (d, J=9.8 Hz, 1H), 5.60 (dd, J=8.5,

15.6 Hz, 1H), 6.34 (d, J=15.6 Hz, 1H), 7.28±7.38 (m,

5H); FABMS m/e calcd for C₄₅H₆₇NaN₇O₁₂+

(M+Na)⁺: 920. Found: 920.

As seen before, the slower running peak (retention

time=17.2 min) was two compounds by TLC; R_f0.24

for the faster running material, and 0.11 for the slower

(DOC-A). The materials were separated by preparative

TLC (DOC-A, silica gel 60) to give 1 mg each of white

solids that were isomeric with the desired product by

FABMS, but that were both dierent than the desired

1

product by H NMR.⁵⁹

Microcystin, N-Troc-Aminoalanine variant (32c). The

title compound was prepared using the same procedure

as that used to prepare 2a. The materials used were

13 mg (0.01 mmol) of 31b, 2.6 mL THF, 42 drops of

water, and 13 drops of 1 M LiOH. After 40 min, the

562

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

6.25 (d, J=15.8 Hz, 1H), 7.16±7.24 (m, 5H); FABMS

m/e calcd for C₄₆H₆₉N₈O₁₂+ (M+2H Na₂HPO₄)⁺:

925. Found: 925.

5. For site-directed mutagenesis studies on PP1/PP2A, see: (a)

Huang, H.; Horiuchi, A.; Goldberg, J.; Greengard, P.; Narin,

A. Proc. Natl. Acad. Sci. USA 1997, 94, 3530. (b) Zhang, Z.;

Zhao, S.; Long, F.; Zhang, L.; Bait, G.; Shima, H.; Nagao,

M.; Lee, E. Y. C. J. Biol. Chem. 1994, 269, 16997. (c) Runne-

gar, M.; Berndt, N.; Kong, S. M.; Lee, E. Y. C.; Zhang, L.

Biochem. and Biophys. Res. Comm. 1995, 216, 162. Also, see

ref ³.

Biological evaluation

Inhibition assays. The catalytic subunits of PP1 and

PP2A were puri®ed from rabbit skeletal muscle as

described.^{57 32}[P]-phosphorylase-a (1 3Â10⁶cpm/nmol)

was prepared from phosphorylase-b as described.⁵⁷

PP1 and PP2A activities were assayed using ³²[P]-phos-

phorylase-a as substrate essentially as described.⁵⁷

Assays (®nal volume 30 mL) contained 50 mM Tris±HCl,

0.15 mM EGTA, 15 mM 2-mercaptoethanol, 0.01%

(w/w) Brij 35, 0.3 mg/mL BSA, 5 mM caeine, 10 mM

³²[P]-phosphorylase-a, inhibitors, and PP1 or PP2A.

Inhibitors were preincubated with PP1 or PP2A for

10 min at 30 ^ꢀC, and assays were initiated by addition of

³²[P]-phosphorylase.

6. Stotts, R. R.; Namikoshi, M.; Haschek, W. M.; Rinehart,

K. L.; Carmichael, W. W.; Dahlem, A. M.; Beasley, V. R.

Toxicon 1993, 31, 783.

7. Sano, T.; Kaya, K. Tetrahedron 1998, 54, 463.

8. Whereas the crystal structure of PP1-bound microcystin

and our modeling work both involve microcystin LR, our

synthetic work involves analogues of the equipotent natural

product microcystin LA that has an l-alanine residue in place

of an l-arginine residue. The l-arginine side chain is fully

exposed to the solvent and does not form any signi®cant con-

tacts with PP1 or PP2A, as seen in the crystal structure and

our modeling results, respectively.

9. Chang, H. C.; Smolyar, A.; Spoerl, R.; Witte, T.; Yao, Y.;

Goyarts, E. C.; Nathenson, S. G.; Reinherz, E. L. J. Mol. Biol.

1997, 271, 278.

10. Wang, Y.; Shen, B. J.; Sebald, W. Proc. Natl. Acad. Sci.

USA 1997, 94, 1657.

Acknowledgements

11. Ruggiero, J.; Xodo, L. E.; Ciana, A.; Manzini, G.; Quad-

rifoglio, F. Biochim. Biophys. Acta. 1992, 1129, 294.

12. Humphrey, J. M.; Aggen, J. B.; Chamberlin, A. R. J. Am.

Chem. Soc. 1996, 118, 11759.

13. Windholz, T. B.; Johnston, D. B. R. Tetrahedron Lett.

1967, 27, 2555.

Support from NIGMS grant GM57550 (ARC), U. S.

Public Health Service grant MH40899 (ACN), and the

National Science Council of Taiwan NSC-87-2314-B-

320-001 (H-BH) is gratefully acknowledged.

14. Hancock, G.; Galpin, I. J. Tetrahedron Lett. 1982, 23, 249.

15. HOAt has been shown to be superior to HOBt as an

additive for DCC mediated coupling reactions in solution or

solid phase peptide synthesis, particulary when hindered com-

ponents are involved: (a) Carpino, L. A. J. Am. Chem. Soc.

1993, 115, 4397. (b) Carpino, L. A. J. Am. Chem. Soc. 1993,

115, 4397. (c) Carpino, L. A.; El-Faham, A.; Minor, C. A.;

Alberico, F. J. Chem. Soc. CC 1994, 201.

References and Notes

1. For a recent review on inhibitors of PP1/PP2A, see: (a)

Sheppeck, J. E.; Gauss C.-M.; Chamberlin A. R. Bioorg. Med.

Chem. 1997, 5, 1739. For recent examples of the use of natural

product PP1/PP2A inhibitors to study the biological proces-

sess, see: (b) Sontag, E.; Sontag, J. M.; Garcia, A. EMBO J.

1997, 16, 5662. (c) Meisinger, J.; Patel, S.; Vellody, K.; Berg-

strom, R.; Bene®eld, J.; Lozanzo, Y.; Young, M. R. I. Cancer

Lett. 1997, 111, 87. (d) Brockdor, J.; Nielsen, M.; Dobson,

P.; Geisler, C.; Ropke, C.; Svedjgaard, A., Odum, N. Tissue

Antigens 1997, 49, 228. (e) Favre, B.; Turowski, P.; Hemmings,

B. A. J. Biol. Chem. 1997, 272, 13856. (f) Sansom, S. C.;

Stockland, J. D.; Hall, D.; Williams, B. J. Biol. Chem. 1997,

272, 9902. (g) Hernandez, M. L.; Martinez, M. J.; Lopez de

Heredia, M.; Ochoa, B. Biochim. Biophys. Acta 1997, 1349,

233.

2. For reviews on the roles of PP1 and PP2A in cellular pro-

cesses, see: (a) Rusnak, F.; Yu, L. A.; Mertz, P. J. Biological

Inorg. Chem. 1996, 1, 388. (b) Stark, M. J. R. Yeast 1996, 12,

1647. (c) Schonthol, A. H. Seminars in Cancer Biol. 1995, 6,

239. (d) Wera, S.; Hemmings, B. A. Biochem. J. 1995, 311, 17.

(e) Shenolikar, S. Annu. Rev. Cell Biol. 1994, 10, 55.

3. Goldberg, J.; Huang, H. B.; Kwon, Y. G.; Greengard, P.;

Nairn, A. C.; Kuriyan, J. Nature (London) 1995, 376, 745.

The structure of a PP1/Tungstate complex was also reported:

Eglo, M.-P.; Cohen, P. T. W.; Reinemer, P.; Barford, D. J.

Mol. Biol. 1995, 254, 942.

16. Angell, Y. M.; Garcia-Echeverria, C.; Rich, D. H. Tetra-

hedron Lett. 1994, 35, 5981.

17. For a recent review on the synthesis of natural product

peptides containing unusual amino acids, see: Humphrey, J.

M.; Chamberlin, A. R. Chem. Rev. 1997, 97, 2243.

18. Bodansky, M. Principles of Peptide Synthesis; 2nd ed.;

Springer-Verlag: New York, 1993.

19. In a control reaction where the amine was excluded, we

observed that the active ester of the phosphonodipeptide

formed rapidly (complete after 10 min by TLC), but decom-

posed rapidly as well, even in CH₂Cl₂(only a trace amount by

TLC after 30 min).

20. For some examples of bioactive compounds containing

diaminoproprionic acid, see: (a) quisqualic acid: Pan, P. C.;

Fang, S. D.; Tsai, C. C. Sci. Sin. (Engl. Ed.) 1976, 19, 691. (b)

capreomycin: Herr, E. B. J.; Haney, M. E.; Pittenger, G. E.;

Higgens, C. E. Proc. Indiana Acad. Sci. 1960, 69, 134. (c)

bleomycin: Takita, T.; Muraoka, Y.; Yoshioka, T.; Fuji, A.;

Maeda, K.; Umezawa, H. J. Antibiot. 1972, 25, 755. (d) anti-

biotic A-19009: Van der Bann, J. L.; Barnick, J. W. F.; Bick-

elhaupt, F. J. Antibiot. 1983, 36, 784.

21. For a racemic synthesis via Michael addition, see: (a)

Labia, R.; Morin, C. J. Org. Chem. 1986, 51, 249. For a pre-

paration via the Curtius rearrangement, see: (b) Schirlin, D.;

Altenburger, J. M. Synthesis 1995, 1351. For a preparation via

the Schmidt rearrangement, see: (c) Wang, M.; Gould, S. J. J.

Org. Chem. 1993, 58, 5176. For preparations via the Homan

rearrangement, see: (d) Curran, T. P.; McEnaney, P. M. Tet-

rahedron Lett. 1995, 36, 191. (e) Ruan, F.; Chen, K.; Itoh, K.;

Sasaki, T.; Hopkins, P. B. J. Org. Chem. 1991, 56, 4347. (f)

4. For the preparation of our PP1/PP2A bound microcystin

models, see: (a) Gauss, C. M.; Sheppeck, J. E.; Narin, A. C.;

Chamberlin, A. R. Bioorg, Med. Chem. 1997, 5, 1751. For

other recent modeling eorts in this area, see: (b) Lindvall, M.

K.; Pihko, P. M.; Koskinen, A. M. P. J. Biol. Chem. 1997, 272,

23312. (c) Bagu, J. R.; Sykes, B. D.; Craig, M. M.; Holmes, C.

F. B. J. Biol. Chem. 1997, 272, 5087. (d) Gupta, V.; Ogawa, A.

K.; Du, X.; Houk, K. N.; Armstrong, R. W. J. Med. Chem.

1997, 40, 3199.

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

563

Waki, M.; Kitajima, Y.; Izumiya, N. Synthesis 1981, 266. (g)

Lee, E. S.; Jurayi, J.; Cushman, M. Tetrahedron Lett. 1994, 50,

9873. (h) Zhang, L. H.; Kauman, G. S.; Pesti, J. A.; Yin, J. J.

Org. Chem. 1997, 62, 6918. For a preparation via a Mitsunobu

reaction with serine, see: (i) Otsuka, M.; Kittaka, A.; Iimori,

T.; Yamashita, H.; Kobayashi, S.; Ohno, M. Chem. Pharm.

Bull. 1985, 33, 509. For preparation via ring opening of serine

lactone, see: (j) Arnold, L. D.; Kalantar, T. H.; Vederas, J. C.

J. Am. Chem. Soc. 1985, 107, 7105. (k) Ratemi, E. S.; Vederas,

J. C. Tetrahedron Lett. 1994, 35, 7605.

22. The lactone 16 was prepared as per Vederas' procedure in

ref 21k, with the exception that di-t-butylazodicarboxylate

(DTAD) was used instead of DEAD or DMAD. DTAD is a

relatively stable, crystalline solid that is convenient to use, and

the byproduct from the lactonization is easier to remove from

the product via chromatography.

37. Merri®eld, R. B.; Yang, C. C. J. Org. Chem. 1976, 41, 1032.

38. A single residue change at a remote position from an

asparate changed the outcome from no imide observed to

complete imide formation during a macrocyclization: Lender,

A.; Yao, W.; Sprengeler, P. A.; Spanevello, R. A.; Furst, G.

T.; Hirschmann, R.; Smith, A. B. Int. J. Peptide Protein Res.

1993, 42, 509.

39. Harris, C. M.; Kopecka, H.; Harris, T. M. J. Am Chem.

Soc. 1983, 105, 6915.

40. Merri®eld, R. B.; Wang, S. S.; Yang, C. C.; Kulesha, I.

D.; Sonenberg, M. Int. J. Peptide Protein Res. 1974, 6, 103.

41. Several reports state that aspartate t-butyl esters gave

imide formation under basic conditions: (a) Roeske, R. J. Org.

Chem. 1963, 28, 1251. (b) Agarwal, K. L.; Kenner, G. W.;

Sheppard, R. C. J. Chem. Soc. C 1968, 1384. (c) Wunsch, E.;

Drees, F. Chem. Ber. 1966, 99, 110.

23. The ¹H NMR spectra of the aldehyde adduct at rt in

DMSO-d6 appeared very similar to the desired product, with

the following exceptions: an additional multiplet integrating

to two protons centered at 4.65 ppm, a broad doublet of tri-

plets integrating to one proton centered at 5.80 ppm, and the

lack of the lower ®eld carbamate N±H found at 7.35 ppm in

the desired material. Shaking with D₂O resulted in the dis-

appearance of the two broad triplets at 5.80 ppm, and heating

to 90 ^ꢀC resulted in sharpening the multiplet centered at

4.65 ppm to a pair of doublets that coupled each other with

J=12.5 Hz.

42. Humphrey, J. M.; Hart, J. A.; Bridges, R. J.; Chamberlin,

A. R. J. Org. Chem. 1994, 59, 2467. The aspartate alkylation

technology is an extension of the seminal observations of See-

bach et al.: Seebach, D.; Wasmuth, D. Angew. Chem. Int., Ed.

Engl. 1981, 20, 971. Aebi, J. D.; Seebach, D. Helv. Chim. Acta.

1985, 68, 1507.

43. For best results, the salt must be reasonably soluble in the

desired reaction solvent. Many amine TFA salts are soluble in

CH₂Cl₂, while the HCl and TsOH salts that we have had

occasion to use were suciently soluble in DMF.

44. The higher yield obtained in synthesizing the N-methyl-

glycine containing heptapeptide relative to the previous three

heptapeptides likely re¯ects the greater excess of one compo-

nent over the other used in the coupling. These coupling reac-

tions are not optimized.

45. For reports on the eects of N-methylation on peptide

conformation, see: (a) Manavalan, P.; Momany, F. A. Biopo-

lymers 1980, 19, 1943. (b) Vitoux, B.; Aubry, A.; Cung, M. T.;

Boussard, G.; Marraud, M. Int. J. Peptide Protein Res. 1981,

17, 469. (c) Dive, V.; Yiotakis, A.; Roumestand, C.; Gilquin,

B.; Labadie, J.; Toma, F. Int. J. Peptide Protein Res. 1992, 39,

506. (d) Bean, J. W.; Kopple, K. D.; Peisho, C. E. J. Am.

Chem. Soc. 1992, 114, 5328. (e) Chalmers, D. K.; Marshall, G.

R. J. Am Chem. Soc. 1995, 117, 5927. (f) Takeuchi, Y.; Mar-

shall, G. R. J. Am. Chem. Soc. 1998, 120, 5363.

24. Abarghaz, M.; Kerbal, A.; Bourguignon, J. J. Tetrahedron

Lett. 1995, 36, 6463.

25. (a) Namikoshi, M.; Rinehart, K. L.; Sakai, R. J. Org.

Chem. 1990, 55, 6135. (b) Meriluoto, J. A. O.; Nygard, S. E.;

Dahlem, A. M.; Eriksson, J. E. Toxicon 1990, 28, 1439. (c)

Namikoshi, M.; Choi, B. W.; Sun, F.; Rinehart, K. L.; Evans,

W. R.; Carmichael, W. W. Chem. Res. Toxicol. 1993, 6, 151.

(d) Gani, D.; Mehrotra, A. P. Tetrahedron Lett. 1996, 37,

6915. (e) Mehrotra, A. P.; Webster, K. L.; Gani, D. J. Chem.

Soc., Perkin Trans. 1 1997, 2495.

26. (a) Moorhead, G.; MacKintosh, C.; Morrice, N.; Cohen,

P. FEBS Lett. 1995, 362, 101. (b) Moorhead, G.; MacKintosh,

R. W.; Morrice, N.; Gallagher, T.; MacKintosh, C. FEBS

Lett. 1994, 356, 46.

27. Campos, M.; Fadden, P.; Alms, G.; Qian, Z.; Haystead,

T. A. J. J. Biol. Chem. 1996, 271, 28478.

28. Namikoshi, M.; Rinehart, K. L.; Sakai, R.; Stotts, R. R.;

Dahlem, A. M.; Beasley, V. R.; Carmichael, W. W.; Evans, W.

R. J. Org. Chem. 1992, 57, 866.

29. The occurance of a d-amino acid following an l-amino

acid is known to be turn-inducing (see ref 37), and N-methy-

lated amino acids, such as proline, in the i+2 position of a b-

turn is known to be turn stabilizing (ibid).

46. During the natural product synthesis, a similar mixture of

three isomers was obtained. Several alternative methods of

saponi®cation were attempted, but no improvement was found

(see ref 12).

47. See the corresponding experimental section for details.

48. Laganis, E. D.; Chenard, B. L Tetrahedron Lett. 1984, 25,

5831.

49. Lui, H. W.; Auchus, R.; Walsh, C. T. J. Am. Chem. Soc.

1984, 106, 5335.

30. For a review on the synthesis of macrocyclic natural

products of marine origin, see: Wipf, P. Chem. Rev. 1995, 95,

2115.

31. For reviews on the synthesis of cyclic peptides, see: (a)

Kopple, K. D. J. Pharm. Sci. 1972, 61, 1345. (b) Wenger, R.

M. Helv. Chim. Acta. 1984, 67, 502. (c) Ovichinikov, Y.; Chi-

pens, G,; Ivanov, V. Peptides 1982; Walter de Gruyter: Berlin,

1983. (d) Meng, Q.; Hesse, M.; Springer-Verlag: Berlin, 1992.

Also, see ref 17.

32. Trogen, G. B.; Annila, A.; Eriksson, J.; Kontteli, M.;

Meriluoto, J.; Sethson, I.; Zdunek, J.; Edlund, U. Biochem-

istry 1996, 35, 3197.

33. Rose, G. D.; Gierasch, L. M.; Smith, J. A. Adv. Protein

Chem. 1985, 37, 1.

50. For the use of CeCl₃, see: (a) Luche, J.; Gemal, A. L. J.

Chem. Soc., Chem Commun. 1978, 976. For the use of CoCl₂,

see: (b) Gemal, A. L.; Luche, J. J. Org. Chem. 1979, 44, 4187.

51. Stein, K. A.; Toogood, P. L. J. Org. Chem. 1979, 60, 8110.

52. Boland, W.; Frobl, C.; Lorenz, M. Synthesis 1991, 1049.

53. Just, G.; Grozinger, K. Synthesis 1976, 457.

54. Imai, J.; Torrence, P. F. J. Org. Chem. 1981, 46, 4015.

55. The problem of removing zinc from the product of Troc-

deprotection has been encountered in peptide synthesis:

Watanabe, H.; Kubota, M.; Yajima, H.; Tanaka, A.; Naka-

mura, M.; Kawibata, T. Chem. Pharm. Bull. 1974, 22, 1889.

56. In a survey of acidic conditions, we found that using 1 M

citric acid, 1 M phosphoric acid, or 0.5 M glycine hydro-

chloride solutions with an equal volume of THF as the reac-

tion medium, and using Zn/Cu couple as the reducing agent,

resulted in smooth Troc removal with no problematic pre-

cipitation or zinc complexation detected.

34. Maude, A. B.; Mehrotra, A. P.; Gani, D. J. Chem. Soc.,

Perkin Trans. 1 1997, 2513.

35. Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397.

36. Bodansky, M.; second ed.; Bodansky, M., Ed.; Springer-

Verlag: New York, 1993, pp 187±189.

57. Cohen, P.; Alemany, S.; Hemmings, B. A.; Resink, T. J.;

Stralfors, P.; Tung, H. Y. Methods Enzymol. 1988, 159, 390.

564

J. B. Aggen et al. / Bioorg. Med. Chem. 7 (1999) 543±564

58. Prinsep, M. R.; Caplan, F. R.; Moore, R. E.; Patterson,

G. M.; Honkanen, R. E.; Boynton, A. L. Phytochemistry 1992,

31, 1247.

59. To identify which of the three isomers obtained corre-

sponded with the starting di-ester, a small amount of each of

these three materials was re-esteri®ed using the general proce-

dure outlined in the preparation of 2a. As with 2a, only the

material that had eluted ®rst from the reversed phase HPLC

puri®cation resulted in esteri®ed material that was identical to

the original di-ester.

Article Doi

The design, synthesis, and biological evaluation of analogues of the serine-threonine protein phosphatase 1 and 2A selective inhibitor microcystin LA: Rational modifications imparting PP1 selectivity

DOI: 10.1016/S0968-0896(98)00254-5

Source and publish data:

Authors:

Article abstract of DOI:10.1016/S0968-0896(98)00254-5

Full text of DOI:10.1016/S0968-0896(98)00254-5

Products guided by the article

R&D Labs maybe for 225781-02-2

Relevant to this article

Hot Product