The development of a new class of inhibitors for betaine-homocysteine S-methyltransferase

European Journal of Medicinal Chemistry 65 (2013) 256e275

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European Journal of Medicinal Chemistry

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Original article

The development of a new class of inhibitors for betaine-

homocysteine S-methyltransferase

a

b

ꢀ

ꢀꢀ

Jan Pícha , Václav Vanek , Milos Budesínský , Jana Mládková , Timothy A. Garrow ,

a,

*

ꢀ

Jirí Jirácek

^aInstitute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nám. 2, 166 10 Prague 6, Czech Republic

^bDepartment of Food Science and Human Nutrition, University of Illinois, Urbana, IL 61801, United States

a r t i c l e i n f o

a b s t r a c t

Article history:

Betaine-homocysteine S-methyltransferase (BHMT) is an important zinc-dependent methyltransferase

that uses betaine as the methyl donor for the remethylation of homocysteine to form methionine. In the

liver, BHMT performs to half of the homocysteine remethylation. In this study, we systematically

investigated the tolerance of the enzyme for modiﬁcations at the “homocysteine” part of the previously

reported potent inhibitor (R,S)-5-(3-amino-3-carboxy-propylsulfanyl)-pentanoic acid (1). In the new

compounds, which are S-alkylated homocysteine derivatives, we replaced the carboxylic group in the

“homocysteine” part of inhibitor 1 with different isosteric moieties (tetrazole and oxadiazolone); we

suppressed the carboxylic negative charge by amidations; we enhanced acidity by replacing the

carboxylate with phosphonic or phosphinic acids; and we introduced pyrrolidine steric constraints.

Some of these compounds display high afﬁnity toward human BHMT and may be useful for further

pharmacological studies of this enzyme. Although none of the new compounds were more potent in-

hibitors than the reference inhibitor 1, this study helped to completely deﬁne the structural requirements

of the active site of BHMT and revealed the remarkable selectivity of the enzyme for homocysteine.

Received 21 January 2013

Received in revised form

19 April 2013

Accepted 20 April 2013

Available online 30 April 2013

Keywords:

BHMT

Inhibitor

Homocysteine

Phosphonate

Phosphinate

Amino acid derivative

Bioisostere

S-Alkylated homocysteine

1. Introduction

S-adenosylmethionine, which is a universal methyl donor of the

organism [4]. Betaine is a compatible osmolyte that increases the

Betaine-homocysteine S-methyltransferase (BHMT; EC 2.1.1.5) is

a cytosolic methyltransferase belonging to the Pfam02574 family of

zinc- and thiol/selenol-dependent methyltransferases [1]. BHMT

uses betaine as the methyl donor for the remethylation of homo-

cysteine to form methionine and dimethylglycine. BHMT follows an

ordered Bi Bi reaction mechanism, with homocysteine being the

ﬁrst substrate to bind and methionine being the last product off [2].

In humans, BHMT expression is limited to the liver and kidney

cortex. In the liver, it has been estimated that BHMT generates

about half of the methionine produced from homocysteine reme-

thylation [3]. Methionine is a constituent of proteins and a source of

water retention of cells, replaces inorganic salts, and protects

intracellular enzymes against osmotically induced or temperature

induced inactivation [5]. It is likely that BHMT helps to keep the

cellular osmolytic equilibrium by maintaining the correct betaine

concentration. We cannot exclude the possibility that transient

inhibition of BHMT would reduce betaine degradation as a mech-

anism to restore osmotic balance during unwanted diuresis since it

is the only known enzyme to use betaine [6].

For several years, our laboratory has been involved in the syn-

thesis and biological evaluation of BHMT inhibitors, which proved

to be a powerful tool in the study of BHMT functions and helped in

better understanding of the subtleties of the structural re-

quirements of the enzyme active site and its catalysis. To date, we

have developed potent and selective inhibitors of human recom-

binant BHMT, including phosphinic pseudopeptides [7] and S-

alkylated derivatives of homocysteine [8,9]. Among the series of S-

alkylated homocysteines, which were designed to mimic the

structure of the hypothetical transition state (TS in Chart 1) of

BHMT substrates upon binding to the active site of the enzyme, we

identiﬁed compounds 1e3 (Chart 1) as highly potent and selective

Abbreviations: BHMT, betaine-homocysteine S-methyltransferase; Boc, tert-

butoxycarbonyl; Boc₂O, di-tert butyl dicarbonate; CDI, 1,1⁰-carbonyldiimidazole;

DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DCM, dichloromethane; DMF, N,N⁰-

dimethylformamide; DMS, dimethyl sulﬁde; IPA, isopropyl alcohol; MsCl, meth-

anesulfonyl chloride; TEA, triethylamine; TFA, triﬂuoroacetic acid; TFAA, triﬂuoro-

acetic anhydride; THF, tetrahydrofuran.

* Corresponding author. Tel.: þ420 220183441; fax: þ420 220183571.

ꢀ

E-mail address: jiracek@uochb.cas.cz (J. Jirácek).

http://dx.doi.org/10.1016/j.ejmech.2013.04.039

J. Pícha et al. / European Journal of Medicinal Chemistry 65 (2013) 256e275

257

Chart 1. The previously reported [8,9] potent inhibitors of BHMT (1e3), which were designed to resemble the hypothetical transition state (TS) of BHMT substrates upon binding to

the active site of the enzyme. The sulfur and carbon atoms in compound 1 are numbered for clarity.

inhibitors of BHMT. Competitive inhibitor 1 [8] was co-crystallized

with BHMT, and the structure of the complex was determined by X-

ray crystallography [1]. Inhibitor 1 was used in mice [10] and in rats

[11,12] to characterize the physiological effects of BHMT inhibition

in sulfur metabolism in vivo. These studies conﬁrmed the key role of

BHMT in the synthesis of methionine from homocysteine. Recently,

using inhibitor 1, we studied the impact of BHMT inhibition on the

changes in several key metabolites of homocysteine metabolism in

HepG2 cells [13].

We are particularly interested in the structural requirements of

the BHMT active site for ligand binding. Our previous efforts in the

design and synthesis of BHMT inhibitors were mainly focused on S-

alkylated homocysteine derivatives [8,9] (e.g. compounds 1e3),

which should act as bi-product analogs or analogs of the transition

state (TS in Chart 1) of BHMT catalyzed reaction. In these previous

studies [8,9], we systematically modiﬁed the central part of their

structures (the sulfur atom, numbered 5 in the Chart 1) or the

“betaine” part of their molecules (atoms 6e10, Chart 1) but the

“homocysteine” part (positions 1e4 in the Chart 1) of inhibitors

remained intact. Recently, we used a similar approach for the

preparation of the ﬁrst series of inhibitors made for the related

enzyme, BHMT-2 [14].

The reaction of

D,L-methionine with Boc₂O afforded protected Boc-

D,L-methionine 4, which was transformed into amides 5e7 using

mixed anhydride. The carbamate was smoothly deprotected by

treatment with dry hydrochloric acid to yield ammonium salts 8e

10. While intermediates 8 and 9 provided expected products 11 and

12 in two steps (demethylation in ammonia with sodium followed

by alkylation of 5-bromovaleric acid), tertiary amide 10 under these

conditions was fully eliminated to form 11 as a sole product. There

are only few examples of this type of undesired reaction in the

literature [16,17].

We also tested the possibility of the reaction of Boc-protected

analogs of methionine 4 and 16 with tert-butyl 5-bromovalerate

with sodium in liquid ammonia. Under these harsh conditions,

the starting compounds were stable, and the reactions proceeded

selectively with good yields (Scheme 2). Thus, the treatment of Boc-

protected D,L-methionine 4 with sodium in liquid ammonia fol-

lowed by alkylation with tert-butyl 5-bromovalerate 15 furnished

the key intermediate 19 with the free carboxylic group in the

“homocysteine part” of its molecule ready to direct derivatization.

Condensation of 19 with piperidine or methanesulfonamide 14

yielded analogs 20 and 21, respectively. Similarly, compound 17

bearing the Boc-protected hydrazide moiety was obtained by the

Investigation of the crystal structure of inhibitor 1 bound to

BHMT (pdb code 1LT8) [1] and molecular modeling of inhibitor 3 to

this structure [9] revealed that the parts of BHMT active site

interacting with the “homocysteine” moiety of inhibitors might

offer space for appropriate modiﬁcations of the inhibitors. There-

fore, in the present study, we systematically investigated the pos-

sibility of producing potent inhibitors of BHMT derived from

inhibitor 1 by modifying the “homocysteine” part of their mole-

cules (positions 1e4 in the Chart 1). We prepared a series of com-

pounds in which we replaced the carboxylic group in inhibitor 1 by

different bioisosteric moieties (e.g. tetrazole and oxadiazolone), we

enhanced the acidity by replacing the carboxylate by phosphonic

and phosphinic acids, and we either suppressed the carboxylic

negative charge by amidations or introduced steric constraints

(pyrrolidines). It was expected that some of these modiﬁcations

could result in compounds with higher inhibitory potency

compared to inhibitors 1e3 or offer enhanced bioavailability of

resulting compounds for in vivo studies. Some of new compounds

prepared in this study displayed high afﬁnity toward human BHMT

and may be useful for further pharmacological studies of this

enzyme.

same synthetic approach starting from the D,L-methionine deriva-

tive 16. As a ﬁnal step, the cleavage of all acid-labile protected

groups in compounds 17, 20 and 21 was performed using a cleavage

cocktail (TFA, DCM, DMS and water). After RP-HPLC puriﬁcation, we

obtained pure target compounds 18, 22 and 23.

We were also interested in the inhibitory effects produced by

substituting the carboxylic group of the “homocysteine part” of

inhibitor 1 for the isosteric phosphonic and phosphinic moieties.

The derivatives of phosphonic and phosphinic acid have found a

plethora of applications in biology and medicine [18,19]. The

presence of these charged and polar moieties in biologically active

compounds may signiﬁcantly enhance their metabolic stability

[20]. Two synthetic methods were pursued (Scheme 3), including

the synthesis of fully protected intermediate and the direct alkyl-

ation. Overall, the synthesis of phosphinic and phosphonic de-

rivatives of compound 1 was more difﬁcult than we expected

because only the latter method, direct alkylation, proved to be

successful.

The route via a fully protected intermediate involved Michael

addition of thioacetic acid to acrolein, which afforded the corre-

sponding aldehyde 24. Its condensation with methyl carbamate and

dimethyl phosphite yielded phosphonate 25. Deacylation was

accomplished by sodium methanolate, and the resulting phospho-

nohomocysteine was treated in situ with methyl 5-bromovalerate to

furnish the intermediate 26. Surprisingly, the prolonged reﬂux of 26

in concentrated hydrochloric acid afforded phosphonic acid 28 with

an intact methyloxycarbonyl group, but no trace of the desired

compound 27 was observed. Therefore, we re-applied the

2. Chemistry

In 1978, Neubert [15] published a two-step synthesis of S-car-

boxymethyl-L-homocysteinamide starting from homocysteinamide

and bromoacetic acid. We applied the same procedure for the

preparation of target inhibitors 11 and 12 as depicted in Scheme 1.

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J. Pícha et al. / European Journal of Medicinal Chemistry 65 (2013) 256e275

Scheme 1. Reagents, conditions and yields: (a) Boc₂O, Na₂CO₃, water, dioxane, 0 ^ꢀC, 1 h then rt overnight (84%); (b) (i) ClCOOEt, TEA, (ii) NH₃(aq.) (for 5), NH₂CH₃.HCl (for 6),

piperidine (for 7), (iii) THF 0 ^ꢀC for 1 h, then rt overnight, 64% (R ¼ NH₂), 57% (R ¼ NHCH₃), 51% (R ¼ N(CH₂)₅); (c) HCl, dioxane, rt for 1 h, R ¼ NH₂(75%), R ¼ NHCH₃(82%),

R ¼ N(CH₂)₅(79%); (d) (i) Na, NH₃(l), (ii) Br(CH₂)₄COOH, ꢁ33 ^ꢀC, then rt overnight, 86% (R ¼ NH₂) from 8, 57% from 10, 74% (R ¼ NHCH₃).

straightforward one-pot method consisting of demethylation in

ammonia, followed by alkylation, as outlined above in Scheme 1.

However, in the case of phosphonomethionine 34, the procedure

failed, probably due to poor solubility of the disodium salt of 34 [21].

This problem was bypassed using phosphinomethionine 35, which,

after the reaction with ethyl 5-bromovalerate or ethyl 5-bromo-3,3-

dimethylpentanoate 33, yielded products 36 and 37, respectively.

Selective oxidation of phosphinic species 36 using mercurous

chloride resulted in the desired phosphonic acid 27.

yielded imide 29, which was hydrolyzed by diluted sulfuric acid to

form substituted glutaric acid 30. Diacid 30 was converted to

glutaric anhydride 31 by heating with acetic anhydride, which was

then partially reduced by sodium borohydride. Finally, the treat-

ment of lactone 32 with hydrogen bromide in ethanol furnished

ester 33.

We have shown that

synthon (Scheme 4). Next, demethylation of

sodium in liquid ammonia generated -homocystine, which was

directly oxidized to -homocystine 38 by treatment with

D,

L-homocystine 38 can serve as a useful

D,L-methionine using

D,L

Non-commercial alkylating agent 33 was prepared by the

sequence of ﬁve synthetic steps. Knoevenagel condensation of

ethyl cyanoacetate (two equiv.) with ammonia and acetone

D,L

hydrogen peroxide. Intermediate 38 was transformed either by

esteriﬁcation and the protection of amino groups to dimethyl ester

Scheme 2. Reagents, conditions and yields: (a) (i) ClCOOEt, TEA, (ii) NH₂NHBoc (13), THF 0 ^ꢀC for 1h, then rt overnight (87%); (b) (i) Na, NH₃(l), (ii) Br(CH₂)₄COO^tBu (15), ꢁ33 ^ꢀC,

then rt overnight, 87% (17), 64% (19); (c) (i) ClCOOEt, TEA, (ii) piperidine, THF, 0 ^ꢀC 1h, then rt overnight (71%); (d) (i) CDI, THF, 60 ^ꢀC for 1 h, (ii) methanesulfonamide 14, DBU, rt

overnight (72%); (e) TFA, DCM, DMS and water, rt overnight, 77% (18), 76% (22), 68% (23).

J. Pícha et al. / European Journal of Medicinal Chemistry 65 (2013) 256e275

259

Scheme 3. Reagents, conditions and yields: (a) TEA, DCM, 0 ^ꢀC 1h, then rt overnight (61%); (b) methyl carbamate, trimethylphosphite, acetyl chloride, ꢁ5 ^ꢀC / 0 ^ꢀC during 1 h, then

rt overnight (48%); (c) sodium methanolate, methyl 5-bromovalerate, methanol, rt overnight (77%); (d) 35% aq. HCl, reﬂux, 24 h (82%); (e) ethanol, 5 ^ꢀC, overnight (51%); (f) 65% aq.

H₂SO₄, reﬂux, 12 h (84%); (g) acetic anhydride, reﬂux, 6 h (79%); (h) sodium borohydride, THF, rt, 3 d (72%); (i) HBr, ethanol, rt, 3 d (89%); (j) (i) Na, NH₃(l), (ii) ethyl 5-

bromovalerate, ꢁ33 ^ꢀC, (iii) NaOH, water, 56% (36); (k) (i) Na, NH₃(l), (ii) ethyl 5-bromo-3,3-dimethylvalerate 33, NH₃, ꢁ33 ^ꢀC, (iii) NaOH, water (64%); (l) HgCl₂, water, 90 ^ꢀC,

5 h (78%).

40 or solely by the protection of amino groups to disulﬁde 39.

Aminolysis of the mixed anhydride prepared from 39 afforded

precursors 41 and 42. Subsequently, disulﬁdes 40e42 were reduced

using tributylphosphine and yielded the expected thiols 43, 44 and

45, respectively. Coupling of the thiols with bromide 15 provided

the corresponding sulﬁdes 46, 47 and 20. However, the attempts to

obtain pure intermediate 20 failed due to its chromatographic

behavior, similar to unreacted starting thiol 45 and its oxidation

product, disulﬁde 42. Finally, standard acidic hydrolysis of the fully

protected sulﬁdes 46 and 47 furnished target inhibitors 48 and 11,

respectively.

The synthesis of analogs with cyano (50), tetrazole (54) and

oxadiazolone (55) moieties is described in detail in Scheme 5.

Amide 47, as a starting precursor, was carefully dehydrated using

TFAA to form nitrile 49. In the case of the tetrazole derivative, use of

the methodology introduced by Sharpless allowed a one-step

transformation of 49 into precursor 51 under mild conditions.

In the case of oxadiazolone, the reaction of nitrile 49 with

aqueous hydroxylamine led smoothly to hydroxyamidine 52, and

closure of the hydroxyamidine moiety ring in compound 53 was

achieved by heating with CDI. The deprotection of 49, 51 and 53

was realized by exposure to the cleavage cocktail, as mentioned

above.

Conformationally locked pyrrolidine derivatives (66 and 67)

were synthesized starting from trans-4-hydroxy-L-proline, which is

a convenient commercially available compound. After protection of

its carboxyl and amino group, the key mesylate 58 was prepared;

subsequent inversion of the conﬁguration at C(4) was accom-

plished with the acetate anion, and the resulting acetate 59 was

converted to diastereomeric mesylate 61 in two steps. Mesylates 58

and 61 were then treated with potassium thioacetate to afford

thioacetates 62 and 63. One-pot methanolysis and alkylation of

these compounds with ethyl 5-bromovalerate, accompanied by

transesteriﬁcation, furnished fully protected compounds 64 and 65,

260

J. Pícha et al. / European Journal of Medicinal Chemistry 65 (2013) 256e275

Scheme 4. Reagents, conditions and yields: (a) (i) Na, NH₃(l), ꢁ33 ^ꢀC (ii) H₂O, H₂O₂, pH 4e5, 0 ^ꢀC, 5 h (82%); (b) Na₂CO₃, Boc₂O, dioxin, water, 0 ^ꢀC, then rt overnight (71%); (c) (i)

SOCl₂, MeOH, 0 ^ꢀC (ii) 60 ^ꢀC overnight (iii) DCM, TEA, 0 ^ꢀC (iv) Boc₂O, 0 ^ꢀC then rt overnight, 81% (40); (d) (i) ClCOOEt, TEA, (ii) NH₃in MeOH or piperidine, THF 0 ^ꢀC 1h, then rt

overnight, 53% (41), 83% (42); (e) tributylphosphine, DMF, water, rt overnight 84% (43), 80% (44), 83% (45); (f) NaH, Br(CH₂)₄COO^tBu (15), THF, rt overnight, 49% (46), 60% (47); (g)

TFA, DCM, DMS, water, rt overnight 49% (48), 46% (11).

which, after deprotection, afforded the target amino acids 66 and

67, respectively (Scheme 6).

results indicate that thenegative charge of theC-terminalcarboxylate

of the “homocysteine” part of the inhibitors is important for efﬁcient

recognition by the BHMT active site.

3. Biological activities

In contrast to compounds 11, 12, 18, 22 and 23, in which the

carboxylate charge is suppressed by amidation, phosphonate 27

and phosphinate 36 add polar acidic terminal groups. Both phos-

phonic [22,23] and phosphinic acids [24] are signiﬁcantly more

acidic (pK_a<2) than carboxylic acids. Because all carboxamides

exhibited only moderate or weak inhibition of BHMT, we expected

the more acidic phosphinate and phosphonate inhibitors to display

higher afﬁnity for BHMT. Surprisingly, phosphonate 27 was even

weaker in the inhibition of BHMT than sulfonamide 23. Phosphi-

All target compounds were tested in vitro to determine their

ability to inhibit human recombinant BHMT. The inhibitory po-

tencies of the new compounds were compared with the previously

reported highly potent inhibitor of BHMT (1, K^a_i^pptoward betaine of

12 nM) [8]. First, all compounds were tested at a 20 mM concen-

tration to select the most potent inhibitors. Then, the IC₅₀values

were determined for the selected compounds. The inhibition data

are summarized in Table 1.

nate 36 was more potent than phosphonate 27, but its IC₅₀(5

was 50-fold higher than that of the reference compound 1

(approximately 0.1 M). The reason for the low afﬁnity of phos-

mM)

The determined inhibitory potency of compound 1 (100% inhibi-

tion at 20

mM and about 93 nM IC₅₀) was in good agreement with the

m

values that we reported for this inhibitor previously [8,9]. All syn-

thesized amides, i.e., 11, 12, 18, 22 and 23, are signiﬁcantly weaker in

inhibiting BHMT than the reference inhibitor 1. However, a trend can

be seen, indicating that the enhanced polarity of the substituents at

the amide nitrogen (e.g., sulfonamide 23 with 67% inhibition or hy-

drazide 18 with 28% inhibition) is more advantageous for the inhi-

bition of BMHT than the presence of apolar substituents (e.g., simple

amide in 11, methylamide in 12 or piperidinyl in 22). The crystal

structure of the BHMT complex with inhibitor 1 [1] revealed that the

carboxylate group of the homocysteine moiety of the inhibitor forms

bidentate hydrogen bonds with the backbone amide groups of Phe29

and Val30 of the enzyme, where the carboxylate oxygen atoms act as

hydrogen bond acceptors. It is possible that the loss of the ability to

dissociate hydrogen due to the carboxylate amidation in compounds

11, 12, 18, 22 and 23 obstructs the formation of one of these two

hydrogen bonds, which results in a decreased binding potency. These

phorus containing compounds 27 and 36 is not obvious. It is

possible that more bulky phosphonate and phosphinate groups

offer more steric hindrance when compared to the original

carboxylate. This idea could be supported by the observation that

the inhibitor 27, whose phosphonate moiety occupies larger vol-

ume than a phosphinate group, is less potent than compound 36.

However, it is also possible that the region of BHMT active site that

interacts with the carboxylate of homocysteine, does not accom-

modate groups that are more acidic than the carboxylate.

In our previous study, we observed that the branching of the

“betaine” part of inhibitor 1 by one or two methyl groups at the C8

atom further enhanced the inhibitory potency of the resulting in-

hibitors (Chart 1, compounds 2 and 3) [9]. We observed the same

effect in the case of phosphinate compound 37, which is the most

potent inhibitor in this study despite being more than one order of

magnitude weaker (IC₅₀1.1 mM) than parent compound 1.

J. Pícha et al. / European Journal of Medicinal Chemistry 65 (2013) 256e275

261

Scheme 5. Reagents, conditions and yields: (a) TFAA, pyridine, THF, 0 ^ꢀC for 1 h, then rt (77%); (b) TFA, DCM, DMS, water, overnight at rt, 34% (50), 42% (54), 61% (55); (c) NaN₃,

ZnBr₂, IPA, water, reﬂux overnight (65%); (d) NH₂OH, EtOH, rt overnight (90%); (e) CDI, THF, reﬂux 17 h (31%).

The results of the inhibition experiments with methylester 48

indicated the relatively high afﬁnity (99% inhibition at 20 M and

an IC₅₀of approximately 5 M) of this inhibitor. However, exami-

nations (RP-HPLC/MS) of both the post-assay mixture and the stock

solution of the inhibitor revealed that the methylester moiety in 48

was partially hydrolyzed to yield compound 1, which probably

oxadiazolone substituent caused the reduced inhibitory potency of

compound 55 in comparison to the parent inhibitor 1. Here, we also

can assume that the oxadiazolone group confers signiﬁcant steric

hindrance.

m

Pyrrolidine compounds 66 and 67 were prepared with the aim

of introducing rigidity and structural constraints into the “homo-

cysteine” part of inhibitor 1. A similar approach has been used for

the design and synthesis of constrained inhibitors for nitric oxide

synthase [33]. However, in our case, this strategy was not effective

since both inhibitors exhibit low (66) or no (67) activity. The crystal

structure of BMHT with compound 1 in the active site shows that

the bound inhibitor is in a rather extended conformation [1]. It

seems that the formation of a pyrrolidine ring that connects the

amino group to the C4 atom (Chart 1) does not allow for such an

extended arrangement of inhibitors 66 and 67.

explains the potent inhibition of the enzyme. The hydrolysis of a-

amino methylester 48 is probably due to the slightly basic pH of the

assay buffer (pH 7.5) and might be accelerated by the participation

of the free amino group or sulfur atom. Because of this confounding

variable, we suppose that the afﬁnity of compound 48 will be low

and in the range of the afﬁnities observed for amide compounds 11

and 12.

The low inhibitory potency of nitrile 50 is probably caused by

the same reason as the low potencies of amides 11 and 12, hydra-

zine 18, piperidine 22 and sulfonamide 23, i.e., due to the loss of the

free carboxyl group and its ability to form bidentate hydrogen

bonds as a donor. However, the total absence of the inhibitory

potency of tetrazole derivative 54 was surprising. Tetrazoles, as

well as oxadiazolones or carboxysulfonamides, are considered to be

good mimics of carboxylates and are often used for non-classical

bioisosteric substitutions of carboxylic groups in bioactive com-

pounds [25e27]. Tetrazoles have similar acidity to but are 10 times

more lipophilic than carboxylates. The reason for the low tolerance

of BHMT for the tetrazole in compound 54 is not obvious, but

negative steric effects caused by the larger volume of the tetrazole

moiety might play a role. Replacement of carboxy moiety by oxa-

diazolone heterocycle has been reported to cause a substantial in-

crease of bioactivity due to enhanced lipophilicity and/or

bioavailability [28e32]. However, in our case, the introduction of an

4. Conclusions

In this study, we systematically investigated the tolerance of

human betaine-homocysteine S-methyltransferase following

modiﬁcations at the “homocysteine” part of a previously reported

potent inhibitor of BHMT, (R,S)-5-(3-amino-3-carboxy-pro-

pylsulfanyl)-pentanoic acid (1). This compound served as the

scaffold for the design and synthesis of a series of new compounds.

In the new compounds, S-alkylated homocysteine derivatives, we

replaced the carboxylic group in the “homocysteine” part of in-

hibitor 1 with different bioisosteric moieties (tetrazole and oxa-

diazolone); we suppressed the carboxylic negative charge by

amidations; we enhanced the acidity by replacing the carboxylate

262

J. Pícha et al. / European Journal of Medicinal Chemistry 65 (2013) 256e275

Scheme 6. Reagents, conditions and yields: (a) Boc₂O, Na₂CO₃, H₂O-dioxane, rt, 1 d; (b) O-tert-butyl-N,N⁰-diisopropylisourea, CH₂Cl₂, reﬂux, 2 d (66% over 2 steps); (c) MsCl,

pyridine, CH₂Cl₂, rt, 18 h (95% for 58, 88% for 61); (d) NaOAc, DMF, 105 ^ꢀC, 3 d (93%); (e) NaOMe, MeOH, rt, 12 h (87%); (f) CH₃CSOK, DMF, 70 ^ꢀC, 1 d (82% for 62, 84% for 63); (g) ethyl

5-bromovalerate, NaOMe, MeOH, rt, 3 d (63% for 64, 60% for 65); (h) (i) TFA/CH₂Cl₂/H₂O, rt, 2 h, (ii) 5M aq. NaOH, EtOH, 80 ^ꢀC, 1 h, (iii) Dowex 50 (H^þ-cycle), (iv) 10% aq. NH₃(83% for

66, 70% for 67).

by phosphonic and phosphinic acids; and we introduced pyrroli-

dine steric constraints.

phosphorus pentoxide at rt, and 13 Pa. TLC on silica gel-coated

aluminum plates (Fluka) was performed in the following systems

(v/v): chloroformeethanol 99/1 (S1), chloroformeethanol 98/2 (S2),

chloroformeethanol 95/5 (S3), chloroformeethanol 90/10 (S4), IPA e

concentrated aqueous ammoniaewater 7/1/2 (S5), tolueneeethyl

acetate 80/20 (S6), tolueneeethyl acetate 50/50 (S7), ethyl acetatee

acetoneeethanolewater 6:1:1:0.5 (S H3), tolueneeethyl acetate 90/

10 (S8), ethyl acetateeacetoneeethanolewater 4:1:1:1 (S H1). The

compounds were visualized by exposure to UV light at 254 nm, by

ninhydrin spraying (dark blue color of amines), by 1% KMnO₄spray-

ing (yellow color of oxidizable compounds) and by spraying with a 1%

(v/v) ethanolic solution of 4-(4-nitrobenzyl)pyridine followed by

heating and treating with gaseous ammonia (blue color of esters or

alkylating compounds). Diacid 30, anhydride 31, lactone 32, and

sulfonamide 14 were visualized by the reagents described elsewhere

[34,35]. Flash chromatography puriﬁcations were conducted on silica

Although the demethylation of methionine with sodium in

liquid ammonia is known for a long time, we have successfully

extended the scope of this reaction toward methionine derivatives

containing Boc-protected alpha amino moiety and/or Boc-

protected hydrazide (4, 16), amide and methylamide moiety (8, 9)

and, most interestingly, toward unprotected phosphinohomocys-

teine (35). This procedure allows subsequent easy and high-

yielding one-pot alkylation of the emerging thiolate with appro-

priate alkyl halide, e.g. tert-butyl 5-bromovalerate.

Some of these compounds are potent inhibitors of human BHMT

and may beuseful for further pharmacologicalstudies of this enzyme.

Although none of the new compounds were stronger inhibitors of

BHMT than the reference inhibitor 1, this study helped to complete

the picture of the structural requirements of the active site of BHMT.

Our results highlight the remarkable speciﬁcity BHMT has for ho-

mocysteine, which is the natural selective substrate of the enzyme.

gel (40e63

conducted on a C18 Luna column (Phenomenex, 250 ꢂ 21.2 mm,

10 m) at a ﬂow rate of 9 mL/min. Solvent A: 0.1% TFA. Solvent B: 80%

mm, Fluka). Preparative RP-HPLC chromatography was

m

5. Experimental section

CH₃CN, 0.1% TFA. The following gradients were used; G1: t ¼ 0 min

(41%B), t ¼ 10 min (41% B), t ¼ 30min(46%B), t ¼ 31 min(100%B);G2:

t ¼ 0 min (0% B), t ¼ 5 min (5% B), t ¼ 25 min (27% B), t ¼ 35 min (100%

B); G3: t ¼ 0 min (0% B), t ¼ 5 min (5% B), t ¼ 20min (40% B), t ¼ 30 min

(100% B); G4: t ¼ 0 min (0% B), t ¼ 5 min (5% B), t ¼ 25 min (40% B),

t ¼ 35 min (100% B). G5: t ¼ 0 min (15% B), t ¼ 25 min (37% B),

t ¼ 35 min (100% B). Analytical RP-HPLC chromatography was con-

ducted at a ﬂow rate of 1 mL/min on a C18 Nucleosil column

5.1. Enzyme puriﬁcation and inhibition assays

Human recombinant BHMT was prepared, and inhibition assays

were performed as described previously [9].

5.2. General chemistry

(250 ꢂ 4 mm, 5

mm) from Watrex (Praha) using the same gradients

Unless otherwise stated, the reagents and solvents used in this

study were obtained from commercial suppliers (SigmaeAldrich,

Fluka, Merck) and used without puriﬁcation. The solvents were

evaporated at 50 ^ꢀC and 2 kPa, the products were dried over

and solvents. Eluated compounds were detected at 218 nm. The

melting points were determined on a Boetius block and are uncor-

rected. ¹H and ¹³C NMR spectra were measured on a Bruker AVANCE-

600 spectrometer (¹H at 600.13 MHz, ¹³C at 150.9 MHz) in CDCl₃,

J. Pícha et al. / European Journal of Medicinal Chemistry 65 (2013) 256e275

263

Table 1

The inhibition of human BHMT by compound 1 and the new compounds developed in this study.

Compound

% Inhibition^aat 20

0.1 mM

m

M compound and 0.25 mM betaine,

IC₅₀

(

m

M) ꢃ SEM^b, n ¼ 3

D

,

L

-homocysteine (average of triplicates)

(2 mM betaine, 1 mM D,L-homocysteine)

1

100

0.093 ꢃ 0.005

11

12

18

22

17

8

nd

28

9

23

27

36

37

48

67

58

nd

219 ꢃ 6

5.0 ꢃ 1.9

1.1 ꢃ 0.2

nd

95

99

nd^c

50

54

9

0

nd

55

66

15

24

nd

(continued on next page)

264

J. Pícha et al. / European Journal of Medicinal Chemistry 65 (2013) 256e275

Table 1 (continued )

Compound

% Inhibition^aat 20

0.1 mM -homocysteine (average of triplicates)

m

M compound and 0.25 mM betaine,

IC₅₀

(

m

M) ꢃ SEM^b, n ¼ 3

D

,

L

(2 mM betaine, 1 mM

D,L-homocysteine)

67

0

nd

^aThe percent inhibition of each compound was determined at 20

mM. Refer to the Experimental section for details. All assays were conducted in triplicate, and the obtained

data were reproducible ꢃ15%.

b

All data points for IC₅₀were derived from assays performed in duplicate, and the values were obtained from three different assays.

Hydrolysis of methoxycarbonyl species in 48 in the assay buffer led to the formation of highly potent inhibitor 1. Nd means not determined.

c

DMSO-d₆, CD₃OD or D₂O solution at 300 K. The 2D-H,H-COSY, 2D-

H,C-HSQC and 2D-H,C-HMBC spectra were recorded and used for the

structural assignment of proton and carbon signals. The IR spectra

were recorded onBruker IFS 55Equinox apparatus. The HRMS spectra

were obtained on a FTMS mass spectrometer LTQ-orbitrap XL

(Thermo Fisher, Bremen, Germany) in the electrospray ionization

mode or in case HRMS (EI) on GCT Premier (Waters). The purity of

each target compound was conﬁrmed by elemental analysis (C, H, N,

S, F, P), found to agree with the calculated values within 0.4% and by

RP-HPLC chromatography as described above, conﬁrming ꢄ95% pu-

rity. The determinations of C, H, and N in the solid samples were

performed using an automatic PE 24000 series II CHNS/O analyzer.

For the analysis of F, the samples were combusted by the Schöniger

method, i.e., in a quartz Erlenmeyer ﬂask in oxygen atmosphere. The

formed F^ꢁwas determined potentiometrically using an ion-selective

electrode. For the analysis of S, each sample was combusted by the

Schöniger method, and the formed SO²₄^ꢁions were titrated in the

presence of 2-propanol by a standard solution Ba(ClO₄)₂with thorin

as an indicator. The phosphorus content was determined using an X-

ray ﬂuorescence spectrometer SPECTRO iQ II (Spectro Analytical In-

struments, Germany).

component was added, and the reaction mixture was stirred

overnight at rt. The solvent was evaporated under reduced pres-

sure, and the residue was puriﬁed by ﬂash chromatography on

silica gel using a linear gradient of toluene in ethyl acetate as the

eluent. The analytical sample was obtained by trituration from a

mixture of ethyl acetateepetroleum ether at ꢁ20 ^ꢀC, in case 6 from

the dichloromethane-petroleum ether mixture.

5.2.2.1. 2-(R,S)-2-[(tert-Butoxycarbonyl)amino]-4-(methylsulfanyl)

butyramide (Boc-

the reaction of Boc-^D,L

D

,

L

-Met-NH₂) (5). Compound 5 was prepared by

-Met-OH 4 (6.3 g; 25 mmol), TEA (2.85 g;

28 mmol), ClCOOC₂H₅(3.05 g; 28 mmol) and 15 mL of 25% NH₃

(aq.). Yield: 4 g (64%). White solid, m.p. 100e102 ^ꢀC, R_f¼ 0.17 (S7).

¹H NMR (600 MHz, CDCl₃):

d

¼ 6.55 and 6.06 (2ꢂ bs, 2ꢂ 1H,

CONH₂), 5.43 (bd, J w8 Hz, 1H, NH), 4.35 (bq, 1H, J w8 Hz, 1H), 2.58

(m, 2H), 2.11 (s, 3H, SMe), 2.09 (m, 1H), 1.93 (m, 1H), 1.44 (s, 9H, t-

Bu). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 174.25 (COeNH₂), 155.67 (Ne

COeO), 80.17, 52.95, 31.67, 30.10, 28.27 (3C), 15.27. IR (KBr) n_max

(cm^ꢁ1): 3464 m, 3218 s (NH₂); 1709, 1689 vs (C]O carbamate);

1661 vs (C]O amide I); 1533 s (amide II); 2977 m, 1368 m (CH₃).

HRMS (ESI) calc for C₁₀H₂₀O₃N₂NaS [M þ Na]^þ271.10868, found:

271.10872.

5.2.1. 2-(R,S)-2-[(tert-Butoxycarbonyl)amino]-4-(methylsulfanyl)

butanoic acid (Boc-

D

,

L

-Met-OH) (4)

5.2.2.2. 2-(R,S)-2-[(tert-butoxycarbonyl)amino]-4-(methylsulfanyl)-

D

,

L

-Met-OH (20 g; 0.134 mol) was dissolved in 250 mL of 10%

N-methylbutyramide (Boc-

prepared by the reaction of Boc-^D,L

D

,

L

-Met-NHCH₃) (6). The compound was

-Met-OH 4 (5 g; 20 mmol), TEA

aqueous solution of sodium carbonate, and 1.1 equiv. (32.17 g;

0.147 mol) of Boc anhydride in 100 mL of dioxane was added drop-

wise at 0 ^ꢀC over a period of 1 h. The cooling bath was removed, and

the reaction mixturewas allowed to stand overnight. Approximately

one-half of the solvent was evaporated under reduced pressure, and

the acidity of the solution was adjusted to pH z 2e3 by 10% citric

acid while being efﬁciently cooled with ice. Each solution was

extracted with 3 ꢂ 200 mL of ethyl acetate, and the combined

organic layers were washed with 2 ꢂ 200 mL of water and

2 ꢂ 200 mL of brine and were then dried over Na₂SO₄before ﬁltering

and removing of the solvent in vacuo. Trituration of the residue from

the mixture of ethyl acetateepetroleum ether at ꢁ20 ^ꢀC afforded the

pure product. Yield 28 g (84%). White solid, m.p. 88e90 ^ꢀC (lit. [36]

(2.25 g; 22 mmol), ClCOOC₂H₅(2.4 g; 22 mmol) and methylamine

(3.7 g; 120 mmol) (liberated from 8.1 g of methylamine hydrochloride

by treatment with 12.2 g of TEA). Yield: 3 g (57%). White solid, m.p.

98e100 ^ꢀC, R_f¼ 0.24 (S7).¹H NMR (600 MHz, CDCl₃):

¼ 6.72 (bs,1H,

d

NH), 5.47 (bd, J ¼ 7.5 Hz,1H, NH), 4.30 (bq,1H, J w7.5 Hz,1H), 2.53 (m,

2H), 2.10 (s, 3H, SMe), 2.06 (m,1H), 1.91 (m, 1H), 1.44 (s, 9H, t-Bu). ¹³

C

NMR (150.9 MHz, CDCl₃):

d

¼ 172.18 (eCOeNH), 155.67 (NeCOeO),

79.95, 53.36, 32.05, 30.12, 28.24 (3C), 26.11,15.21. IR(KBr) n_max(cm^ꢁ1):

3240 m (NH); 1692, vs (C]O carbamate); 1659 vs (C]O amide I);

1539 s (amide II); 2977 m, 1367 m (CH₃). HRMS (ESI) calc for

C

₁₁H₂₂O₃N₂NaS [M þ Na]^þ285.12433, found: 285.12440.

87e91 ^ꢀC). R_f¼ 0.67 (S4). ¹H NMR (600 MHz, CDCl₃):

d

¼ 5.27 (b,1H,

5.2.2.3. 1-{2-(R,S)-2-[(tert-Butoxycarbonyl)amino]-4-(methyl-

sulfanyl)butanoyl}piperidine (Boc- -Met-N(CH₂)₅) (7). The com-

pound was prepared by the reaction of Boc- -Met-OH 4 (5 g;

NH), 4.45 (um, 1H), 2.58 (t, J ¼ 7.5 Hz, 2H), 2.18 (m, 1H), 2.11 (s, 3H,

D,L

SMe), 1.98 (m, 1H), 1.45 (s, 9H, t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

D,L

d

¼ 176.91 (COOH), 155.60 (NeCOeO), 80.32, 52.73, 31.86, 29.93,

20 mmol), TEA (2.25 g; 22 mmol), ClCOOC₂H₅(2.4 g; 22 mmol) and

piperidine (1.7 g; 20 mmol). Yield: 3.2 g (51%). White solid, m.p.

28.24 (3C), 15.32. IR (KBr) n_max(cm^ꢁ1): 3380 m (NH); 3500e2800

vbr vs (COOeH); 1715 vs (C]O acid); 1673 vs (C]O carbamate);

1543 s (amide II); 2977 s, 1368 m (CH₃). HRMS (ESI) calc for

60e61 ^ꢀC, R_f¼ 0.58 (S7). ¹H NMR (600 MHz, CDCl₃):

¼ 5.53 (b, 1H,

d

NH), 4.77 (um,1H), 3.56 (m, 2H), 3.49 (m, 2H), 2.56 (m,1H), 2.52 (m,

1H), 2.11 (s, 3H, SMe), 1.93 (m, 1H), 1.79 (m, 1H), 1.66 (m, 2H), 1.61

(m, 2H),1.56 (m, 2H),1.43 (s, 9H, t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

C

₁₀H₁₉O₄NNaS [M þ Na]^þ272.09270, found: 272.09281.

5.2.2. The general procedure for the preparation of compounds 5, 6

and 7

d

¼ 169.81 (eCOeN<), 155.48 (NeCOeO), 79.53, 49.13, 46.52,

43.24, 33.40, 30.09, 28.29 (3C), 26.47, 25.49, 24.39, 15.61. IR (KBr)

n_max(cm^ꢁ1): 3261 m (NH); 1708, vs (C]O carbamate); 1623 vs (C]

O); 1531 s (amide II); 2974 m, 1363 m (CH₃); 2856 m (CH₂). HRMS

(ESI) calc for

339.17129.

TEA (1.1 equiv.) was added to 1 equiv. of Boc-D,L-Met-OH 4 dis-

solved in 125 mL of THF. The ﬂask was cooled at 0 ^ꢀC, and ethyl

chloroformate (1.1 equiv.) was added drop-wise under stirring.

After 1 h at the same temperature, one portion of the amino

C

₁₅H₂₈O₃N₂NaS [M

þ

Na]^þ339.17128, found:

J. Pícha et al. / European Journal of Medicinal Chemistry 65 (2013) 256e275

265

5.2.3. General procedure for the preparation of compounds 8, 9 and

10

5.2.4.1. 5-[(3-(R,S)-3-Amino-3-carboxamide)butylsulfanyl]pentanoic

acid triﬂuoroacetate salt (11). Target compound 11 was prepared by

the reaction of 8 (1 g; 5.4 mmol), sodium (0.51 g; 22.1 mmol) and 5-

bromovaleric acid (1.1 g; 5.9 mmol) or by the reaction of 10 (1 g;

4 mmol), sodium (0.29 g; 12.4 mmol) and 5-bromovaleric acid

(0.8 g; 4.4 mmol). Yield: 1.62 g (86%) from 8, 0.78 g (57%) from 10.

Boc-protected derivatives 5, 6 and 7 were treated with 100 mL of

saturated solution of hydrogen chloride in dry dioxane. When the

starting compounds had disappeared (TLC), the solvent was evap-

orated under reduced pressure and the crude product was puriﬁed

by crystallization.

Lyophilizate. ¹H NMR (600 MHz, DMSO-d₆):

d

¼ 7.88 and 7.59 (2ꢂ

bs, 2ꢂ 1H, CONH₂), 8.12 (b, 2H, NH₂), 3.79 (m,1H), 2.52 (m, 1H), 2.51

5.2.3.1. 2-(R,S)-[(tert-Butoxycarbonyl)amino]-4-(methylsulfanyl)

(m, 2H), 2.48 (m, 1H), 2.22 (t, J ¼ 7.2 Hz, 2H), 1.56 (m, 2H), 1.52 (m,

butyramide hydrochloride (

D,

L

-Met-NH₂$HCl) (8). The title salt was

2H). ¹³C NMR (150.9 MHz, DMSO-d₆):

d

¼ 174.54 (COOH), 170.13 (e

prepared by the hydrolysis of 5 (4 g; 16.1 mmol). Yield: 2.3 g (75%)

COeNH₂), 51.87, 33.40, 31.48, 30.63, 28.59, 26.18, 23.85. IR (KBr)

n_max(cm^ꢁ1): 3100e2600 vs vbr, 1533 m (NH₃^þ); 3391 m (NH₂); 1700

vs (C]O acid); 1665 vs (C]O amide); 1440 s (CO^ꢁ₂); 1203 vs, 1182

vs, 1147 vs, 724 m (CF₃); 1440 m, 1364 m (CH₂). HRMS (ESI) calc for

C₉H₁₉O₃N₂S [M þ H]^þ235.11109, found: 235.11103.

methanolepetroleum ether. White solid, m.p. 191e192 ^ꢀC (lit. [37]

189e190 ^ꢀC), R_f¼ 0.31 (S H3). ¹H NMR (600 MHz, D₂O):

d

¼ 3.18

(t, J ¼ 6.6 Hz, 1H), 1.66 (m, 2H), l.21 (m, 2H), 1.14 (s, 3H, SMe). ¹³

C

NMR (150.9 MHz, D₂O):

d

¼ 174.32 (COeNH₂), 54.88, 32.67, 30.94,

16.73. IR (KBr) n_max(cm^ꢁ1): 3281 m (NH); 3000e2300 vs vbr, 1597

s, 1484 s (NH^þ₃); 1676 vs (C]O). HRMS (ESI) calc for C₅H₁₃ON₂S

[M þ H]^þ149.07431, found: 149.07418.

5.2.4.2. 5-[3-(R,S)-3-Amino-4-(N-methylcarboxamide)butylsulfanyl]

pentanoic acid triﬂuoroacetate salt (12). Target compound 12 was

prepared by the reaction of 9 (1 g; 5 mmol), sodium (0.47 g;

20.5 mmol) and 5-bromovaleric acid (1 g; 5.5 mmol). Yield: 1.35 g

5.2.3.2. 2-(R,S)-2-[(tert-Butoxycarbonyl)amino]-4-(methylsulfanyl)-

N-methylbutyramide hydrochloride (

D,L

-Met-NHCH₃$HCl) (9). The title

(74%). Lyophilizate. ¹H NMR (600 MHz, DMSO-d₆):

d

¼ 8.42 (q,

salt was prepared by the hydrolysis of 6 (3 g; 9.4 mmol). Yield: 1.8 g

J ¼ 4.6 Hz, 1H, NH), 8.20 (bd, J w5.5 Hz, 3H, NH^þ₃), 3.78 (bq, J

w5.5 Hz, 1H), 2.66 (d, J ¼ 4.6 Hz, 3H, NMe), 2.49 (t, J ¼ 7.0 Hz, 2H),

(82%) methanoleether. White solid, m.p.129e131 ^ꢀC, R_f¼ 0.23 (S H3).

¹H NMR (600 MHz, DMSO-d₆):

d

¼ 8.76 (q, J ¼ 4.6 Hz,1H, NH), 8.45 (b,

2.46 (m, 2H), 2.22 (t, J ¼ 7.2 Hz, 2H), 1.56 (m, 2H), 1.51 (m, 2H). ¹³

C

3H, NH^þ₃), 3.82 (t, J ¼ 6.4 Hz,1H), 2.62 (d, J ¼ 4.6 Hz, 3H, NMe), 2.47 (t,

NMR (150.9 MHz, DMSO-d₆):

d

¼ 174.58 (COOH),168.60 (eCOeNH),

J ¼ 8.1 Hz, 2H), 2.02 (s, 3H, SMe), 1.99 (m, 2H). ¹³C NMR (150.9 MHz,

51.97, 33.43, 31.59, 30.62, 28.57, 26.19, 25.89, 23.88. IR (KBr) n_max

(cm^ꢁ1): 3100e2600 vs vbr, 1515 m (NH^þ₃); 1671 vs (C]O) amide;

1578 s (amide II); 1201 vs, 1141 vs, 723 m (CF₃). HRMS (ESI) calc for

DMSO-d₆):

d

¼ 168.58 (eCOeNH), 51.81, 31.05, 28.57, 25.78, 14.72. IR

(KBr) n_max(cm^ꢁ1): 3281 s (NH); 3100e2300 vs vbr, 1577 m, 1489 m

(NH^þ₃); 1668 vs (C]O), 1577 s (amide II), 1317 m (SCH₃). HRMS (ESI)

calc for C₆H₁₅ON₂S [M þ H]^þ163.08996, found: 163.08988.

C

₁₀H₁₉O₃N₂S [M ꢁ H]^þ247.11219, found: 247.11233.

5.2.5. tert-Butyl hydrazine carboxylate (13)

5.2.3.3. 1-{2-(R,S)-2-[(tert-Butoxycarbonyl)amino]-4-(methylsulfanyl)

butanoyl}piperidine hydrochloride (D,L-Met-N(CH₂)₅$HCl) (10). The title

Monosubstituted hydrazine was prepared according to the

published protocol [38] by the reaction of hydrazine hydrate (5 g;

0.1 mol) and Boc anhydride (8.7 g; 0.04 mol). Yield: 4.2 g (80%).

White solid, m.p. 36e38 ^ꢀC (lit. [38] 36e37 ^ꢀC), R_f¼ 0.69 (S4). ¹H

salt was prepared by the hydrolysis of 7 (2.9 g; 11 mmol). Yield: 1.9 g

(79%) ethyl acetateepetroleum ether. White solid, m.p. 155e157 ^ꢀC,

R_f¼ 0.37 (S H3). ¹H NMR (600 MHz, DMSO-d₆):

d

¼ 8.43 (b, 3H, NH^þ₃),

NMR (600 MHz, CDCl₃):

d

¼ 6.22 (b, 1H, NH), 3.72 (s, 2H, NH₂), 1.46

4.37 (bt, J w5.8 Hz, 1H), 3.50 (m, 1H), 3.44 (m, 2H), 3.43 (m, 1H), 2.66

(ddd, J ¼ 13.6, 8.8 and 7.2 Hz, 1H), 2.50 (ddd, J ¼ 13.6, 8.6 and 6.2 Hz,

1H), 2.05 (s, 3H, SMe), 1.94 (m, 2H), 1.59 (m, 2H), 1.57 (m, 1H), 1.47 (m,

(s, 9H, t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 158.05 (NeCOeO),

80.32, 28.24 (3C). IR (KBr) n_max(cm^ꢁ1): 3338 s, 1634 m (NH₂); 1714

vs (C]O); 1495 s (amide II); 2979 m, 1368 m, 1137 vs (CH₃). HRMS

(ESI) calc for C₅H₁₂O₂N₂Na [M þ Na]^þ155.07910, found: 155.07896.

1H), 1.45 (m, 2H). ¹³C NMR (150.9 MHz, DMSO-d₆):

d

¼ 166.53 (eCOe

N<), 48.83, 45.98, 42.86, 30.44, 28.44, 26.19, 25.40, 24.03, 14.64. IR

(KBr) n_max(cm^ꢁ1): 3100e2300 vs vbr, 1581 m (NH₃^þ); 1652 vs (C]O),

2938 s, 2856 s, 1470 s (CH₂). HRMS (ESI) calc for C₁₀H₂₀ON₂NaS

[M þ Na]^þ239.11886, found: 239.11893.

5.2.6. Methanesulfonamide (14)

Sulfonamide was prepared by the reaction of (10 g; 87 mmol)

methanesulfonyl chloride with an excess of dry ammonia in THF

[39]. Yield: 7 g (85%). White solid, m.p. 87e89 ^ꢀC (lit. [39] 86e

5.2.4. The general procedure for the preparation of compounds 11

and 12

90 ^ꢀC), R_f¼ 0.25 (S3). ¹H NMR (600 MHz, DMSO-d₆):

d

¼ 6.80 (bs,

2H, NH₂), 2.90 (bs, 3H, S-Me). ¹³C NMR (150.9 MHz, DMSO-d₆):

A 250 mL three-necked ﬂask was equipped with a dry ice

condenser, a glass-coated magnetic bar, and a potassium hydroxide

tube in the inlet, and the outlet was charged with 1 equiv. of

ammonium salt 8, 9 or 10. First, the apparatus was ﬂushed vigor-

ously with a stream of dry ammonia. When 150 mL of gas was

condensed in the ﬂask, approximately 4.1 equiv. (3.1 equiv. for 10)

of sodium was added in small portions until the dark blue color of

the reaction mixture persisted for at least 20 min. The excess so-

dium was destroyed by adding a small amount of ammonium

chloride (decolorization of the solution), and 1.1 equiv. of 5-

bromovaleric acid was added to the clear solution in dry THF

(10 mL). Stirring and cooling of the reaction mixture was stopped,

and the ammonia was allowed to evaporate overnight. The

remaining white solid was dissolved in 20 mL of water, followed by

acidiﬁcation with chilled 10% (aq.) TFA to pH z 1. The ﬁnal solution

d

¼ 43.39 (S-Me). IR (KBr) n_max(cm^ꢁ1): 3337 s, 3266 s, 1579 m, 1165

s (NH₂); 1314 vs, 1145 vs (SO₂); 1331 vs (CH₃). HRMS (ESI) calc for

CH₅O₂NNaS [M þ Na]^þ117.99339, found: 117.99331.

5.2.7. tert-Butyl 5-bromopentanoate (15)

Diisopropylcarbodiimide (25.2 g; 0.2 mol), tert-butyl alcohol

(14.8 g; 0.2 mol) and CuCl (0.5 g) was stirred overnight at rt. 5-

Bromovaleric acid (10.0 g; 55.7 mmol) was dissolved in 150 mL of

dichloromethane, and the crude tert-butyl isourea generated in situ

was added in 15 equal portions (approximately 3 mL of each) over

the period of 5 days. Any increase in the temperature of the reaction

mixture above 30 ^ꢀC led to a rapid decomposition of the alkylating

agent. The solvent was evaporated in vacuo, and the residue was

puriﬁed by ﬂash chromatography on silica gel using a linear

gradient of diethyl ether in petroleum ether. Yield: 7.4 g (56%).

of the product was passed through a Rotalibo ﬁlter (0.22

mm) and

Colorless liquid, R_f¼ 0.81 (S8). ¹H NMR (600 MHz, CDCl₃):

¼ 3.42

d

then was subjected to preparative RP-HPLC (G2). Finally, the pure

product was lyophilized from water.

(t, J ¼ 6.7 Hz, 2H), 2.25 (t, J ¼ 7.4 Hz, 2H), 1.88 (m, 2H), 1.74 (m, 2H),

1.45 (s, 9H, t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 172.47 (eCOeO),

266

J. Pícha et al. / European Journal of Medicinal Chemistry 65 (2013) 256e275

80.28, 34.47, 33.14, 31.95, 28.04 (3C), 23.60. IR (ﬁlm) n_max(cm^ꢁ1):

1729 vs (C]O); 2978 m, 2934 m, 1367 s (CH₃). 1156 vs (CeO). HRMS

(ESI) calc for C₉H₁₇O₂BrNa [M þ Na]^þ259.03041, found: 259.03037.

1H), 1.68 (m, 2H), 1.60 (m, 2H), 1.45 (s, 9H, t-Bu), 1.44 (s, 9H, t-Bu).

¹³C NMR (150.9 MHz, CDCl₃):

¼ 176.33 and 175.88 (COOH), 173.14

d

and 173.02 (eCOeO), 156.75 and 155.54 (NeCOeO), 81.84 and

80.36, 80.20, 53.50 and 52.68, 35.00, 32.42 and 32.26, 31.48, 28.76,

28.24 (3C), 28.03 (3C), 27.72, 24.18. IR (CCl₄) n_max(cm^ꢁ1): 3441 w,

3257 w (NH); 2980 m, 2869 w, 1393 m, 1368 s (CH₃); 2932 m, 1455

m (CH₂); 1729 vs (C]O ester); 1716 vs (C]O carbamate); 1668 m

(C]O acid); 1164 vs (CeO). HRMS (ESI) calc for C₁₈H₃₃O₆NNaS

[M þ Na]^þ414.19208, found: 414.19205.

5.2.8. N-{2-(R,S)-2-[(tert-Butoxycarbonyl)amino]-4-

(methylsulfanyl)butanoyl}-N⁰-(tert-butoxycarbonyl)hydrazine (Boc-

D,

L

-Met-NHNHBoc) (16)

Compound 16 was prepared using the method described for the

synthesis of compounds 5, 6 and 7 (5.2.2.) by the reaction of Boc-

-Met-OH 4 (5 g; 20 mmol), TEA (2.25 g; 22 mmol), ClCOOC₂H₅

D,L

(2.4 g; 22 mmol) and BocHNNH₂13 (2.65 g; 20 mmol) in 125 mL of

5.2.10. tert-Butyl 5-[3-(R,S)-3-(tert-butoxycarbonylamino)-4-oxo-

4-(piperidin-1-yl)butylsulfanyl]pentanoate (20)

THF. Yield: 6.4 g (87%). White solid, m.p. 58e61 ^ꢀC (DCM-petroleum

ether), R_f¼ 0.59 (S7). ¹H NMR (600 MHz, CDCl₃):

d

¼ 8.47 (b, 1H,

Compound 20 was prepared by the procedure described for

compounds 5, 6, 7 and 16 (5.2.2.) by the reaction of 19 (3 g;

7.7 mmol), ClCOOEt (0.92 g; 8.5 mmol), TEA (0.86 g; 8.5 mmol) and

piperidine (0.75 g; 7.7 mmol) in 100 mL of THF. Yield: 2.5 g (71%).

NH), 6.67 (b, 1H, NH), 5.39 (b, 1H, NH), 4.38 (bq, J w7 Hz, 1H), 2.61

(m, 2H), 2.11 (m, 1H), 2.11 (s, 3H, SMe), 1.96 (m, 1H), 1.46 (s, 9H, t-

Bu), 1.44 (s, 9H, t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

COeNH), 155.74 (NeCOeO), 155.08, 81.69, 80.44, 51.77, 31.57, 29.93,

28.28 (3C), 28.11 (3C), 15.22. IR (KBr) n_max(cm^ꢁ1): 3281 m (NH);

1705 vs, 1691, vs (C]O carbamate); 1656 vs (C]O hydrazide); 1517

d

¼ 171.43 (e

Clear oil, R_f¼ 0.71 (S7). ¹H NMR (600 MHz, CDCl₃):

d

¼ 5.58 (b, 1H,

NH), 4.78 (bdd, J w8.2 and 4.0 Hz, NeCH<), 3.56 (m, 2H), 3.50 (m,

2H), 2.55 (m, 2H), 2.52 (t, J ¼ 7.2 Hz, 2H), 2.23 (t, J ¼ 7.2 Hz, 2H), 1.91

(m,1H),1.79 (m,1H),1.67 (m, 4H),1.65 (m,1H),1.60 (m, 2H),1.58 (m,

1H), 1.56 (m, 2H), 1.44 (s, 9H, t-Bu), 1.43 (s, 9H, t-Bu). ¹³C NMR

s

C

(amide II); 2979 m, 1368 m (CH₃). HRMS (ESI) calc for

₁₅H₂₉O₅N₃NaS [M þ Na]^þ386.17201, found: 386.17194.

(150.9 MHz, CDCl₃):

d

¼ 172.72 (eCOeO), 170.06 (eCOeN<), 155.50

5.2.9. The synthesis of compounds 17 and 19

(NeCOeO), 80.11, 79.61, 49.11, 46.72, 43.47, 34.99, 33.71, 31.81,

28.88, 28.28 (3C), 28.05 (3C), 27.87, 26.44, 25.49, 24.32, 24.21. IR

(CCl₄) n_max(cm^ꢁ1): 3422 w, 3293 w (NH); 2980 s, 1392 m, 1367 s

(CH₃); 2940 s, 2859 m, 1469 s, 1454 vs, 1444 vs (CH₂); 1730 vs (C]O

ester); 1714 vs (C]O carbamate); 1645 vs (amide); 1495 s (amide

II); 1170 vs (CeO). HRMS (ESI) calc for C₂₃H₄₂O₅N₂NaS [M þ Na]^þ

471.27066, found: 481.27042.

Compounds 17 and 19 were prepared in a similar fashion as

compounds 11 and 12 (5.2.4.); however, the work-up of the reaction

mixtures was different. When the ammonia was allowed to evap-

orate, the remaining residue was taken up in 50 mL of ethyl acetate.

The vigorously stirred emulsion was cooled on the ice bath and was

then treated with 10% (aq.) citric acid until a pH z 2e3 was

reached. The clear solution was transferred to the separatory fun-

nel. The upper organic layer was separated, and the aqueous phase

was extracted twice with 50 mL of ethyl acetate. The combined

organic phases were successively washed twice with 50 mL of

water, followed by 50 mL of brine, and they were ﬁnally dried over

Na₂SO₄. The ﬁltrate was evaporated in vacuo, and the residue was

puriﬁed by ﬂash chromatography on silica gel using a linear

gradient of ethyl acetate in toluene.

5.2.11. tert-Butyl 5-[3-(R,S)-3-(tert-butoxycarbonylamino)-4-

(carboxymethylsulfonamide)butylsulfanyl] pentanoate (21)

Carboxymethylsulfonamide 21 was prepared according to the

method of Lampa et al. [40] with few modiﬁcations. Acid 19 (2.9 g;

7.4 mmol) and CDI (2.4 g; 14.8 mmol) in 100 mL of dry THF were

heated at 60 ^ꢀC for 1 h. Methanesulfonamide 14 (2.1 g; 22 mmol)

and DBU (2.3 g; 22.2 mmol) were added in one portion. The reac-

tion mixture was stirred at rt overnight. The solvent was evapo-

rated in vacuo, and the residue was dissolved in 200 mL ethyl

acetate and then was washed successively with 50 mL of 10% (aq.)

citric acid, 100 mL of water and 100 mL of brine. The organic phase

was dried over Na₂SO₄, ﬁltered and evaporated under reduced

pressure. The remaining material was puriﬁed by ﬂash chroma-

tography on silica gel using a linear gradient of ethyl acetate in

toluene to afford a clear oil, which solidiﬁed upon standing. Yield:

2.5 g (72%). Colorless solid, m.p. 76e78 ^ꢀC, R_f¼ 0.55 (S7). ¹H NMR

5.2.9.1. N-{2-(R,S)-2-[(tert-Butoxycarbonyl)amino]-4-[(4-tert-butox-

ycarbonyl)butylsulfanyl]butanoyl}-N⁰-(tert-butoxycarbonyl)hydra-

zine (17). Compound 17 was prepared by the reaction of 16 (2 g;

5.5 mmol), sodium (0.38 g; 16.5 mmol) and tert-butyl 5-

bromopentanoate 15 (1.3 g; 5.5 mmol) in 200 mL of liquid

ammonia. Yield: 2.3 g (87%). Clear oil, R_f¼ 0.69 (S7). ¹H NMR

(600 MHz, CDCl₃):

d

¼ 5.31 (bd, J w8 Hz, 1H, NH), 4.36 (bq, J w8 Hz,

1H, NeCH<), 2.62 (m, 2H), 2.53 (t, J ¼ 7.2 Hz, 2H), 2.23 (t, J ¼ 7.3 Hz,

2H), 2.10 (m, 1H), 1.94 (m, 1H), 1.67 (m, 2H), 1.60 (m, 2H), 1.48 (s, 9H,

t-Bu), 1.46 (s, 9H, t-Bu), 1.44 (s, 9H, t-Bu). ¹³C NMR (150.9 MHz,

(600 MHz, CDCl₃):

d

¼ 9.58 (b, 1H, NHeSO₂), 5.33 (bd, J w8 Hz, 1H,

NHeCO), 4.34 (m,1H, NeCH<), 3.30 (s, 3H, S-Me), 2.62 (m,1H), 2.58

(m, 1H), 2.53 (t, J ¼ 7.2 Hz, 2H), 2.24 (t, J ¼ 7.2 Hz, 2H), 2.15 (m, 1H),

1.94 (m, 1H), 1.68 (m, 2H), 1.60 (m, 2H), 1.46 (s, 9H, t-Bu), 1.45 (s, 9H,

CDCl₃):

d

¼ 172.88 (eCOeO), 171.36 (eCOeNH), 155.67 (NeCOeO),

155.02 (NeCOeO), 81.68, 80.41, 80.19, 51.82, 34.98, 31.99, 31.43,

28.74, 28.28 (3C), 28.11 (3C), 28.08 (3C), 27.79, 24.17. IR (CCl₄) n_max

(cm^ꢁ1): 3435 w, 3309 w (NH); 2981 m, 1393 m, 1368 s (CH₃); 1731 s

(C]O ester); 1705 s vbr (C]O carbamate, hydrazide); 1496 s

(amide II); 1162 vs (CeO). HRMS (ESI) calc for C₂₃H₄₃O₇N₃NaS

[M þ Na]^þ528.27139, found: 528.27134.

t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 172.96 (eCOeO), 171.17 (e

COeNH), 156.17 (NeCOeO), 81.47, 80.36, 54.00, 41.38, 34.93, 31.53,

30.65, 28.63, 28.22 (3C), 28.09 (3C), 27.86, 24.12. IR (CCl₄) n_max

(cm^ꢁ1): 3437 w, 3378 w (NH); 2979 m, 2871 m, 1393 m, 1368 s

(CH₃); 2928 m, 2855 m, 1456 s (CH₂); 1729 vs (C]O ester); 1713 vs

(C]O carbamate); 1692 m (C]O amide); 1496 m (amide II); 1356

m, 1170 s (SO₂). HRMS (ESI) calc for C₁₉H₃₆O₇N₂NaS₂[M þ Na]^þ

491.18561, found: 491.18571.

5.2.9.2. 2-(R,S)-2-(tert-Butoxycarbonylamino)-5-[(4-tert.butox-

ycarbonylbutyl)sulfanyl]butanoic acid (19). Compound 19 was pre-

pared by the reaction of 4 (6.23 g; 25 mmol), sodium (1.8 g;

77.5 mmol) and tert-butyl 5-bromopentanoate 15 (5.9 g; 25 mmol)

in 800 mL of liquid ammonia. Yield: 6.3 g (64%). Clear oil, R_f¼ 0.49

(S4). Some signals are doubled-conformers at NeCO bond: ¹H NMR

5.2.12. The general procedure of the preparation of compounds 18,

22 and 23

Compounds 17 (1 g; 1.9 mmol), 20 (1 g; 2.2 mmol) or 21 (1 g;

2.1 mmol) were treated with 10 mL of the cleavage cocktail

composed of TFA (45% v/v), DCM (45% v/v), dimethyl sulﬁde (5% v/

v) and water (5% v/v), and the reaction was allowed to proceed

(600 MHz, CDCl₃)

d

¼ 9.81 (b, COOH), 6.54 and 5.27 (2ꢂ bd, J w8 Hz,

2ꢂ NH), 4.43 and 4.31 (2ꢂ m, 2ꢂ NeCH<), 2.58 (bt, J ¼ 7.6 Hz, 2H),

2.53 (t, J ¼ 7.3 Hz, 2H), 2.24 (t, J ¼ 7.3 Hz, 2H), 2.16 (m, 1H), 1.97 (m,

J. Pícha et al. / European Journal of Medicinal Chemistry 65 (2013) 256e275

267

overnight. The volatile liquids were removed by rotary evaporation

at 25 ^ꢀC, and the remaining solution was lyophilized with 20 mL of

water. The residue was dissolved in 15 mL of 0.1% TFA (aq.), passed

through Rotalibo ﬁlter (0.22 mm) and puriﬁed by preparative RP-

HPLC.

carbamate (3.75 g; 50 mmol) and dimethyl phosphite (5.5 g;

50 mmol) in 100 mL of acetyl chloride was cooled to ꢁ5 ^ꢀC in the ice

bath, and aldehyde 24 (8.3 g; 62.5 mmol) was added drop-wise

during 10 min. The reaction mixture was stirred for 1 h at 0 ^ꢀC

and then was left overnight at rt. The acetyl chloride was evapo-

rated in vacuo, and the crude product was puriﬁed twice by ﬂash

chromatography on silica gel using a linear gradient of the S H3

mixture in ethyl acetate. Yield: 7.2 g (48%). Viscous pale yellow oil,

5.2.12.1. 5-[3-(R,S)-3-Amino-4-(carboxyhydrazide)butylsulfanyl]pen-

tanoic acid ditriﬂuoroacetate salt (18). Yield: 0.72 g (77%). Lyophi-

lizate. G4. ¹H NMR (600 MHz, DMSO-d₆):

d

¼ 10.90 (b, 1H, COOH),

R_f¼ 0.69 (S4). ¹H NMR (600 MHz, CDCl₃):

¼ 5.90 (bd, J ¼ 10.0 Hz,

d

8.41 (b, 3H, NH^þ₃), 5.20 (b, 4H, NHeNH^þ₃), 3.91 (bt, J ¼ 6.4 Hz, 1H, Ne

CH<), 2.49 (m, 4H), 2.21 (t, J ¼ 6.4 Hz, 2H), 1.96 (m, 2H), 1.56 (m,

1H, NH), 4.19 (dm, J(H,P) ¼ 16.5 Hz,1H), 3.79 (d, J(P,H) ¼ 11.0 Hz, 3H,

PeOMe), 3.78 (d, J(P,H) ¼ 11.0 Hz, 3H, PeOMe), 3.70 (s, 3H, OMe),

3.07 (dddd, J ¼ 13.8, 8.5, 4.8 and 1.0 Hz, 1H), 2.87 (dt, J ¼ 13.8 and

8.0 Hz, 1H), 2.33 (s, 3H, CH₃CO), 2.07 (ddddd, J ¼ 14.4, 8.5, 8.0, 7.0

and 3.8 Hz, 1H), 1.93 (ddddd, J ¼ 14.4, 11.0, 9.0, 8.0 and 4.8 Hz, 1H),

2H), 1.52 (m, 2H). ¹³C NMR (150.9 MHz, DMSO-d₆):

d

¼ 174.55

(COOH), 167.49 (eCOeNH), 50.66, 33.40, 31.53, 30.67, 28.58, 26.01,

23.86. IR (KBr) n_max(cm^ꢁ1): 3030 vs vbr, 1618 s vbr, 1521 s br (NH^þ₃);

1704 vs (C]O acid); 1678 vs (C]O hydrazide); 1436 s, 799 s (CO^ꢁ₂);

1202 vs, 1141 vs, 724 s (CF₃). HRMS (ESI) calc for C₉H₁₈O₃N₃S

[M ꢁ H]^þ248.10744, found: 248.10753.

¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 195.12 (eCOeS), 156.56 (d,

J(C,P) ¼ 5.2 Hz, NeCOeO), 53.08 (d, J(C,P) ¼ 7.0 Hz), 52.84 (d,

J(C,P) ¼ 6.7 Hz), 52.28, 46.15 (d, J(C,P) ¼ 157.8 Hz), 30.28, 29.55 (d,

J(C,P) ¼ 4.4 Hz), 25.18 (d, J(C,P) ¼ 15.6 Hz). IR (CCl₄) n_max(cm^ꢁ1):

3438 w, 3246 w (NH); 1729 vs (C]O carbamate); 1538 m, 1508 m

(amide II); 1697 vs (C]O thioacetate); 1062 vs, 1049 vs, 1037 vs,

834 m (PeOeC); 1235 s (P]O); HRMS (ESI) calc for C₉H₁₈O₆NNaPS

[M þ Na]^þ322.04847, found: 322.04845.

5.2.12.2. 5-[3-(R,S)-3-Amino-4-oxo-4-(piperidine-1-yl)butylsulfanyl]

pentanoic acid triﬂuoroacetate salt (22). Yield: 0.69 g (76%). Lyo-

philizate. G5. ¹H NMR (600 MHz, DMSO-d₆):

d

¼ 12.20 (b, 1H, COOH),

8.24 (bs, 2H, NH₂), 4.40 (m, 1H, NeCH<), 3.52 (m, 1H), 3.44 (m, 2H),

3.43 (m,1H), 2.62 (m,1H), 2.51 (m,1H), 2.50 (m, 2H), 2.22 (t, J ¼ 7.2 Hz,

2H),1.90 (m, 2H),1.60 (m, 2H),1.56 (m, 2H),1.54 (m,1H),1.52 (m, 2H),

5.2.15. Methyl 5-{[3-(R,S)-3-(methoxycarbonylamino)-3-

(dimethoxyphosphoryl)propyl]sulfanyl} pentanoate (26)

1.46 (m, 2H). ¹³C NMR (150.9 MHz, DMSO-d₆):

d

¼ 174.52 (COOH),

166.51 (eCOeNH), 49.07, 45.91, 42.83, 33.41, 31.04, 30.64, 28.64,

26.15, 26.10, 25.39, 23.98, 23.87. IR (KBr) n_max(cm^ꢁ1): z3000 vs vbr,

1594 s, 1518 m (NH^þ₃); 2949 vs, 2860 s,1448 m, 1435 m, 1369 m,1351

m (CH₂); 1732 vs (C]O acid); 1633 vs (C]O amide); 1680 vs, 799 m

(COO^ꢁ₂); 1201 vs, 1182 vs, 1140 vs, 721 s (CF₃). HRMS (ESI) calc for

Phosphonate 25 (1.36 g; 4.5 mmol) and sodium methanolate

(0.24 g; 4.5 mmol) were dissolved in 20 mL of anhydrous methanol

underanitrogenatmosphere. One portionofmethyl 5-bromovalerate

(0.88 g; 4.5 mmol) was added after 10 min of stirring, and the reaction

mixture was left at rt overnight. The solvent was evaporated under

reduced pressure, and the residue was puriﬁed by ﬂash chromatog-

raphy on silica gel using a linear gradient of the S H3 mixture in ethyl

acetate Yield: 1.3 g (77%). Viscous colorless oil, R_f¼ 0.67 (S1).¹H NMR

C

₁₄H₂₇O₃N₂S [M þ H]^þ303.17369, found: 303.17364.

5.2.12.3. 5-[3-(R,S)-3-Amino-4-(methylsulfonylcarboxamide)butyl-

sulfanyl]pentanoic acid triﬂuoroacetate salt (23). Yield: 0.62 g (68%).

(600 MHz, CDCl₃):

d

¼ 5.34 (bd, J ¼ 10.3 Hz, 1H, NH), 4.26 (dm,

Lyophilizate. G2. ¹H NMR (600 MHz, DMSO-d₆):

d

¼ 8.44 (b, 4H,

J(H,P) ¼ 16.6 Hz, 1H), 3.79 (d, J(P,H) ¼ 10.6 Hz, 3H, PeOMe), 3.78 (d,

J(P,H) ¼ 10.6 Hz, 3H, PeOMe), 3.71 (s, 3H, OMe), 3.67 (s, 3H, OMe), 2.67

(m,1H), 2.55 (m,1H), 2.52 (m, 2H), 2.33 (t, J ¼ 7.4 Hz, 2H), 2.09 (m,1H),

1.87 (m, 1H), 1.72 (m, 2H), 1.61 (m, 2H). ¹³C NMR (150.9 MHz, CDCl₃):

NH^þ₃and NHeSO₂), 3.95 (bdd, J ¼ 7.8 and 4.8 Hz, NeCH<), 3.28 (s,

3H, S-Me), 2.51 (t, J ¼ 7.6 Hz, 2H), 2.50 (m, 2H), 2.22 (t, J ¼ 7.3 Hz,

2H), 2.03 (m, 2H), 1.56 (m, 2H), 1.52 (m, 2H). ¹³C NMR (150.9 MHz,

DMSO-d₆):

d

¼ 174.57 (COOH), 169.29 (eCOeNH), 52.70, 41.26,

d

¼ 173.73 (eCOeO), 156.61 (d, J(C,P) ¼ 5.5 Hz, NeCOeO), 53.26 (d,

33.43, 30.99, 30.61, 28.54, 25.98, 23.89. IR (KBr) n_max(cm^ꢁ1): 3029 s

vbr, 1620 s vbr, 1510 s br (NH^þ₃); 1715 vs (C]O acid); 1674 vs (C]O

sulfonamide); 1345 s (SO₂); 1202 vs, 1154 vs, 723 m (CF₃). HRMS

(ESI) calc for C₁₀H₂₁O₅N₂S₂[M þ H]^þ313.08864, found: 313.08864.

J(C,P) ¼ 7.1 Hz), 53.05 (d, J(C,P) ¼ 6.6 Hz), 52.54, 51.45, 46.33 (d,

J(C,P) ¼ 156.7 Hz), 33.44, 31.46, 30.04 (d, J(C,P) ¼ 4.0 Hz), 28.77, 28.10

(d, J(C,P) ¼ 14.4 Hz), 23.94. IR(CCl₄) n_max(cm^ꢁ1):3247 m (NH);1726vs

(C]O carbamate); 1538 s, 1508 s (amide II); 1736 vs (C]O ester);

1062 vs,1049 vs,1035 vs, 834 s (PeOeC); 1235 s (P]O); 1215 s, 1195 s

(CeO); 2954 s, 1447 s (CH₃). HRMS (ESI) calc for C₁₃H₂₆O₇NNaPS

[M þ Na]^þ394.10598, found: 394.10593.

5.2.13. S-(3-Oxopropyl)ethanethioate (24)

Aldehyde 24 was prepared according to the method described

previously [41]. Thioacetic acid (15.2 g; 0.2 mol) in 50 mL of

dichloromethane was added drop-wise over a period of 10 min to

an ice-cooled and stirred solution of acrolein (11.2 g; 0.2 mol) and

7 mL of triethylamine in 100 mL of dichloromethane. The ice bath

was removed and stirring was continued overnight at rt. All volatile

materials were evaporated in vacuo, and the yellow liquid residue

was distilled under reduced pressure. Yield: 16 g (61%). Colorless

liquid, b.p. 45e47 ^ꢀC/0.5 torr (lit. [42] 50 ^ꢀC/2 torr), R_f¼ 0.49 (S1). ¹H

5.2.16. 5-[3-(R,S)-3-(Methoxycarbonylamino)-3-

(phosphonopropyl)sulfanyl]pentanoic acid (28)

Ester 26 (1.3 g; 3.5 mmol) was reﬂuxed for 24 h with 15 mL of

concentrated hydrochloric acid. The solvent was evaporated under

reduced pressure, and the resulting oily residue was dissolved in

20 mL of 0.1% aqueous TFA, passed through Rotalibo ﬁlter (0.22 mm)

and puriﬁed by preparative RP-HPLC (G1). Finally, the pure product

NMR (600 MHz, CDCl₃):

d

¼ 9.76 (t, J ¼ 1.0 Hz, 1H, CH]O), 3.12 (t,

was lyophilized from water. Yield: 0.94 g (82%). Viscous colorless oil,

J ¼ 6.8 Hz, 2H), 2.81 (td, J ¼ 7.0 and 1.0 Hz, 2H), 2.34 (s, 3H, CH₃CO).

R_f¼ 0.1 (S5).¹H NMR (600 MHz, DMSO-d₆):

¼ 7.19 (bd, J ¼ 9.6 Hz,1H,

d

¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 199.89 (eCH]O),195.94 (>C]O),

NH), 3.74 (dm, J(H,P) ¼ 16.5 Hz, 1H), 3.56 (s, 3H, OMe), 3.33 (m, 2H),

3.30 (m, 2H), 2.29 (t, J ¼ 7.4 Hz, 2H), 2.16 (m,1H),1.94 (m,1H),1.72 (m,

2H), 1.61 (m, 2H). ¹³C NMR (150.9 MHz, DMSO-d₆): some signals are

43.65, 30.46, 21.46. IR (CHCl₃) n_max(cm^ꢁ1): 1725 vs (C]O alde-

hyde); 1689 vs (C]O thioacetate). HRMS (ESI) calc for C₅H₈O₂NaS

[M þ Na]^þ155.0137, found: 155.0136.

doubled-conformers at NeCO bond:

d

¼ 174.18 (COOH), 156.78 (Ne

COeO), 51.86 and 51.84, 48.15 (d, J(C,P) ¼ 154.0 Hz) and 47.96 (d,

J(C,P) ¼ 153.1 Hz), 40.59 and 40.50, 38.41 (d, J(C,P) ¼ 15.5 Hz) and

38.13 (d, J(C,P) ¼ 14.6 Hz), 32.88, 24.62(d, J(C,P) ¼ 4.5 Hz)and 24.56(d,

J(C,P) ¼ 4.5 Hz), 23.28 and 23.26, 22.89. IR (KBr) n_max(cm^ꢁ1): 3280 w

(NH); 1717 vs br (C]O carbamate, acid); 1536 s (amide II); 1190 vs

5.2.14. S-[3-(R,S)-3-(Methyloxycarbonylamino)-3-

(dimethoxyphosphoryl)propyl]ethanethioate (25)

Compound 25 was prepared using a modiﬁcation of the method

developed by Yuan et al. [43]. A well-stirred mixture of methyl

268

J. Pícha et al. / European Journal of Medicinal Chemistry 65 (2013) 256e275

(P]O); 1048 m (PeOH); HRMS (ESI) calc for C₁₀H₁₉O₇NPS [M þ H]^þ

anhydride and acetic acid was removed with a rotary evaporator; the

dark brownresiduewassuspended in200 mL of petroleum ether. The

precipitate was ﬁltered off and washed with 200 mL of petroleum

ether. Crystallization from the mixture of etherepetroleum ether

gave a pure product. Yield: 35 g (79%). Glistening plates, m.p. 123 ^ꢀC

(lit. [48] 124e125 ^ꢀC), R_f¼ 0.73 (S7). ¹H NMR (600 MHz, CDCl₃):

328.06253, found: 328.06248.

5.2.17. 5-[3-(R,S)-3-Amino-3-(phosphonylpropyl)sulfanyl]

pentanoic acid triﬂuoroacetate salt (27)

Phosphinic acid 36 (0.33 g; 1.29 mmol) was heated at 90 ^ꢀC for

5 h with HgCl₂(0.78 g; 2.58 mmol) in 50 mL of water. The white

precipitate of mercurous chloride was ﬁltered off, and the ﬁltrate

was bubbled for 5 min with a gentle stream of hydrogen sulﬁde. The

black salt of mercury sulﬁde was removed by ﬁltration through

d

¼ 2.60 (s, 4H, 2ꢂ CH₂), 1.15 (s, 6H, 2ꢂ CH₃). ¹³C NMR (150.9 MHz,

CDCl₃):

d

¼ 166.18 (2ꢂ COeO), 43.72 (2ꢂ CH₂), 29.40 (>C<), 27.49 (2ꢂ

CH₃). IR (KBr) n_max(cm^ꢁ1): 1812 vs, 1772 vs (C]O); 1186 s, 1137 m,

1076 vs, 965 s (CeOeC); 2968 s, 2879 m (CH₃). HRMS (ESI) calc for

C₇H₁₀O₃Na [M þ Na]^þ165.05222, found: 165.05229.

Rotalibo ﬁlter (0.22 mm), and the ﬁltrate was puriﬁed by RP-HPLC

(G2). Yield: 0.39 g (78%). ¹H NMR (600 MHz, D₂O þ NaOD):

d

¼ 2.80 (ddd, J ¼ 14.6, 9.9 and 4.6 Hz, 1H), 2.61 (t, J ¼ 7.3 Hz, 2H),

5.2.21. 3,3-Dimethyl-d-valerolacton (32)

2.60 (m, 2H), 2.19 (t, J ¼ 7.3 Hz, 2H), 2.02 (m, 1H), w1.61 (m, 5H). ¹³

C

Lactone 32 was prepared by a reduction of 31 (29.4 g; 0.21 mol)

by 3 equiv. of sodium borohydride (22.7 g; 0.6 mol) in THF. The

reaction proceeded for 3 days [49]. Yield: 19 g (72%). Colorless

liquid, b.p. 77e81 ^ꢀC/0.3 torr (lit. [50] 85e95 ^ꢀC/0.3 torr), R_f¼ 0.60

NMR (150.9 MHz, D₂O þ NaOD):

d

¼ 186.42 (COOH), 52.22 (d,

J(C,P) ¼ 137.8 Hz), 39.86, 34.52, 33.48, 31.87 (d, J(C,P) ¼ 14.6 Hz),

31.41, 27.91. IR (KBr) n_max(cm^ꢁ1): 3100e2700 vs vbr, 1623 s, 1534 s

(NH^þ₃); 1691 vs (C]O); 1136 vs (P]O); 1194 s, 1110 vs (PO^ꢁ₂); 1048

m (POH). Yield 0.21 g (60%). HRMS (ESI) calc for C₈H₁₇O₅NPS

[M ꢁ H]^þ270.05705, found: 270.05708.

(S7). ¹H NMR (600 MHz, CDCl₃):

d

¼ 4.37 (m, 2H), 2.33 (s, 2H), 1.70

(m, 2H), 1.09 (s, 6H, 2ꢂ CH₃). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 171.48 (COeO), 66.46, 44.03, 35.74, 29.60, 28.66 (2ꢂ CH₃). IR

(ﬁlm) n_max(cm^ꢁ1): 2958 s, 2872 m, 1370 m (CH₃); 2928 m (CH₂);

1744 vs, 1735 vs (C]O); 1269 m, 1256 s, 1225 m (CeOeC). HRMS

(EI) calc for C₇H₁₂O₂[M ꢁ H]^ꢁ129.0916, found: 129.0914.

5.2.18. 3-(R,S),5-(R,S)-3,5-Dicyano-4,4-dimethyl-2,6-

dioxopiperidine (29)

Compound 29 was prepared according to the previously

described method [44]. Ethyl cyanoacetate (169.7 g; 1.5 mol),

acetone (43.5 g; 0.75 mol), ammonium acetate (1.4 g) and 300 mL of

ethanol, which contained 30 g of ammonia, were placed into 1-l

ﬂask. The mixture was kept overnight at 5 ^ꢀC. The precipitated

ammonium salt was ﬁltered off using a Büchner funnel and then

was washed with 100 mL of diethyl ether. The precipitate was

dissolved in 350 mL of water (80 ^ꢀC), and the aqueous solution was

acidiﬁed (pH z 1) by adding concentrated hydrochloric acid. The

ﬂask was cooled on an ice bath, and the crystals appeared almost

immediately. The product was ﬁltered off using a Büchner funnel,

washed with 250 mL of water and dried under high vacuum. Yield:

73 g (51%). White solid, m.p. 218e220 ^ꢀC (lit. [45] 215e217 ^ꢀC),

5.2.22. Ethyl 5-bromo-3,3-dimethylpentanoate (33)

The synthesis, yield and spectra (¹H, ¹³C NMR and HRMS) were

published previously [9]. R_f¼ 0.70 (S8). IR (ﬁlm) n_max(cm^ꢁ1): 2963

m, 1370 m (CH₃); 2963 m (CH₂); 1733 vs (C]O); 1228 m (CeO).

5.2.23. [1-(R,S)-Amino-3-(methylsulphanyl)propyl]phosphonic acid

(34) and [1-(R,S)-amino-3-(methylsulphanyl)propyl]phosphinic

acid (35)

The synthesis and physico-chemical characteristics of com-

pounds 34 and 35 were described previously [51,52].

5.2.24. The general procedure for the synthesis of compounds 36

and 37

R_f¼ 0.40 (S4). ¹H NMR (600 MHz, DMSO-d₆):

d

¼ 4.71 (s, 2H), 1.35

(s, 3H, CH₃), 1.18 (s, 3H, CH₃). ¹³C NMR (150.9 MHz, DMSO-d₆):

Compounds 36 and 37 were prepared using a procedure similar

to that used for compounds 11 and 12, but the work-up of the re-

action mixtures was different. After the evaporation of ammonia,

the remaining white solid was stirred for 6 h with sodium hy-

droxide in 20 mL of water, followed by acidiﬁcation with 10% TFA

(aq.) to pH z 1. This ﬁnal solution was passed through a Rotalibo

d

¼ 164.71 (2ꢂ COeO), 114.53 (2ꢂ C^N), 47.03 (2C), 36.41, 26.08,

19.86. IR (KBr) n_max(cm^ꢁ1): 2272 w, 2260 w (C^N); 3219 s, 3129 m,

810 m, 791 m, 747 m (NH); 1744 vs, 1726 vs, 1714 vs (C]O); 1244 s,

1230 s (amide II); 2975 m, 1367 s (CH₃). HRMS (ESI) calc for

C₉H₉O₂N₃Na [M þ Na]^þ214.05909, found: 214.05901.

ﬁlter (0.22 mm) and then was puriﬁed by preparative RP-HPLC (G2).

5.2.19. 3,3-Dimethylpentanedioic acid (30)

Finally, the pure product was lyophilized from water.

Imine 29 (73 g; 0.38 mol) and sulfuric acid (65%, 350 mL) were

placed into the 2-l ﬂask equipped with a Dimroth condenser. The

suspension was heated under reﬂuxing for 12 h. Signiﬁcant foam-

ing of the reaction mixture was observed during the ﬁrst three

hours. The ﬂask, containing a clear, yellow solution, was placed in

the refrigerator (5 ^ꢀC) overnight. The precipitated crystals were

collected on a sintered glass ﬁlter. A pure product was obtained by

crystallization from water and then was dried in a desiccator over

P₂O₅. Yield: 51 g (84%). White solid, m.p. 95e97 ^ꢀC (lit. [46] 94e

5.2.24.1. 5-[3-(R,S)-3-Amino-3-(hydroxyphosphorylpropyl)sulfanyl]

pentanoic acid (36). The target compound 36 was prepared by the

reaction of phosphinic acid 35 (2.5 g; 14.8 mmol), 3.1 equiv. of sodium

(1 g; 44.4 mmol) and ethyl 5-bromovalerate (3 g; 14.8 mmol), fol-

lowed by treatment with sodium hydroxide (1.2 g; 29.6 mmol). Yield:

2.1 g (56%). Lyophilizate.¹H NMR (600 MHz, D₂O þ NaOD):

¼ 6.73 (d,

d

J (H,P) ¼ 505.0 Hz, 1H, PeH), 2.79 (ddd, J ¼ 13.1, 9.2 and 5.0 Hz, 1H),

2.74 (dddd, J ¼ 10.0, 8.8, 4.0 and 1.3 Hz,1H), 2.65 (dddd, J ¼ 13.1, 8.9, 7.2

and 0.7 Hz, 1H), 2.60 (t, J ¼ 7.1 Hz, 2H), 2.19 (t, J ¼ 7.3 Hz, 2H),1.96 (m,

1H), 1.58e1.68 (m, 5H). ¹³C NMR (150.9 MHz, D₂O þ NaOD):

95 ^ꢀC), R_f¼ 0.32 (S5). ¹H NMR (600 MHz, DMSO-d₆):

¼ 9.23 (b, 2H,

d

2ꢂ COOH), 2.28 (s, 4H, 2ꢂ CH₂), 1.03 (s, 6H, 2ꢂ CH₃). ¹³C NMR

(150.9 MHz, DMSO-d₆):

d

¼ 173.22 (2ꢂ COOH), 44.96 (2ꢂ CH₂),

d

¼ 186.33 (COOH), 52.57 (d, J(C,P) ¼ 98.9 Hz), 39.83, 33.36, 31.82 (d,

31.91 (>C<), 27.39 (2ꢂ CH₃). IR (KBr) n_max(cm^ꢁ1): z3100 vs vbr

(OH); 1699 vs (C]O); 1254 vs (CeO); 1399 s (CH₃). HRMS (ESI) calc

for C₇H₁₂O₄Na [M þ Na]^þ183.06278, found: 183.06284.

J(C,P) ¼ 2.7 Hz), 31.38, 30.63 (d, J(C,P) ¼ 13.5 Hz), 27.86. IR (KBr) n_max

(cm^ꢁ1): 2637 m (OH); 1694 vs (C]O); 3100e2700 vbr vs, 1546 m

(NH^þ₃); 960 m (PeH); 1193 vs, 1181 vs, 1040 s (PO₂^ꢁ); 2944 s (CH₂).

HRMS (ESI) calc for C₈H₁₇O₄NPS [M ꢁ H]^þ254.06214, found:

254.06209.

5.2.20. 3,3-Dimethylglutaric anhydride (31)

Anhydride 31 was prepared according toTheisen et al. [47], with a

few modiﬁcations. Diacid30 (50 g; 0.31 mol) was heated under gentle

reﬂux with 250 mL of acetic anhydride for 6 h. The excess of acetic

5.2.24.2. 5-[3-(R,S)-3-Amino-3-(hydroxyphosphorylpropyl)sulfanyl]-

3,3-dimethylpentanoic acid triﬂuoroacetate salt (37). The target

J. Pícha et al. / European Journal of Medicinal Chemistry 65 (2013) 256e275

269

compound 37 was prepared by the reaction of phosphinic acid 35

(1.5 g; 8.9 mmol), 3.1 equiv. of sodium (0.63 g; 27.5 mmol) and ethyl

5-bromo-3,3-dimethylvalerate 33 (2.1 g; 8.9 mmol), followed by

treatment with sodium hydroxide (0.7 g; 17.8 mmol). Yield: 2.25 g

the pure product. Yield: 18.7 g (71%). White solid, m.p. 161e163 ^ꢀC

(lit. [54] 158e159 ^ꢀC), R_f¼ 0.19 (S4). ¹H NMR (600 MHz, DMSO-d₆):

d

¼ 6.80 (b, 2H, 2ꢂ NH), 3.97 (m, 2H, 2ꢂ NeCH<), 2.70 (t, J ¼ 7.5 Hz,

4H), 2.03 (m, 2H), 1.90 (m, 2H), 1.36 (s, 18H, 2ꢂ t-Bu). ¹³C NMR

(64%). Lyophilizate. ¹H NMR (600 MHz, DMSO-d₆):

d

¼ 8.22 (bs, 3H,

(150.9 MHz, DMSO-d₆):

d

¼ 175.00 (2C, 2ꢂ COOH), 155.53 (2C, 2ꢂ

NH^þ₃), 7.00 (d, J(H,P) ¼ 534.8 Hz, PeH), 3.18 (m, 1H, NeCH<), 2.70

(ddd, J ¼ 13.4, 9.4 and 5.6 Hz,1H), 2.62 (ddd, J ¼ 13.4, 9.5 and 6.5 Hz,

1H), 2.46 (m, 2H), 2.11 (s, 2H), 2.01 (m, 1H), 1.82 (m, 1H), 1.54 (m,

2H), 0.96 (s, 6H, 2ꢂ CH₃). ¹³C NMR (150.9 MHz, DMSO-d₆):

NeCOeO), 78.19 (2C), 53.15 (2C), 34.63 (2C), 31.45 (2C), 28.44 (6C).

IR (KBr) n_max(cm^ꢁ1): 3428 vs (NH); 1714 vs (C]O carbamate); 1692

vs (C]O acid); 1510 m (amide II); 2979 m, 1395 m, 1369 m, 1165 vs

(CH₃). HRMS (ESI) calc for C₁₈H₃₂O₈N₂NaS₂[M þ Na]^þ491.14923,

found: 491.14948.

d

¼ 173.26 (COOH), 48.38, 45.30, 41.68, 33.04, 27.25, 27.15, 27.13,

27.02, 25.84. IR (KBr) n_max(cm^ꢁ1): 3030 vs vbr, 1631 m, 1532 m

(NH^þ₃); 2963 vs (CH₃); 2935 vs (CH₂); 1703 m (C]O acid); 1685 m,

1442 w, 800 m (CO^ꢁ₂); 1167 s, 1043 m (PO^ꢁ₂); 1206 s, 1138 s, 722 m

(CF₃). HRMS (ESI) calc for C₁₀H₂₁O₄NPS [M ꢁ H]^þ282.09344, found:

282.09371.

5.2.27. Dimethyl 2-(R,S)-9-(R,S)-2,9-bis(tert-

butoxycarbonylamino)-5,6-dithiadecanedioate (40)

Diester 40 was prepared by a slight modiﬁcation of the method

described by Lherbet et al. [55]. Thionyl chloride (5.3 g; 44.4 mmol)

was carefully and drop-wise added to a chilled solution of 38 (3 g;

11.1 mmol) in 50 mL methanol. Then, the reaction mixture was

heated at 60 ^ꢀC overnight. The solvent was evaporated in vacuo, and

the white foam of the reaction intermediate was suspended in

50 mL of dichloromethane. The slurry of the dihydrochloride was

cooled to 0 ^ꢀC, and TEA (2.3 g; 22.2 mmol) was added. When the

solution became clear, Boc₂O (5.4 g; 24.4 mmol) was added in

20 mL of dichloromethane, and the reaction mixture was allowed to

stand overnight. The solvent was evaporated in vacuo, and the

crude product was puriﬁed by ﬂash chromatography on silica gel

using a linear gradient of ethyl acetate in toluene to yield the oily

product, which solidiﬁed upon standing. Yield: 3.6 g (81%). White

solid, m.p. 88e90 ^ꢀC, R_f¼ 0.78 (S7). ¹H NMR (600 MHz, CDCl₃):

5.2.25. 2-(R,S)-9-(R,S)-2,9-Diamino-5,6-dithiadecanedioic acid (38,

D,

L

-homocysteine)

-Methionine (40 g; 0.268 mol) was placed in a 2-L Erlenmeyer

D,L

ﬂask, equipped with a Claisen adapter, a dry ice condenser, a glass-

coated magnetic bar, and potassium hydroxide tubes on both the

inlet and outlet. At ﬁrst, the apparatus was ﬂushed with a vigorous

stream of dry ammonia. After the condensation of approximately

1 L of ammonia, the gas-delivery tube was removed and replaced

with a ground-glass stopper. Then, 3.1 equiv. of sodium (18.5 g;

0.8 mol) was added in small portions over 2 h until the dark blue

color of the reaction mixture persisted for at least 20 min. The

excess sodium was destroyed by adding a small amount of

ammonium chloride, which resulted in decolorization of the solu-

tion. Stirring and cooling of the reaction mixture was stopped, and

the ammonia was allowed to evaporate at rt overnight. The white

solid with a typical homocysteine-like odor was dissolved in

400 mL of water, and the solution was acidiﬁed by the slow drop-

wise addition of HCl (2 M) until pH z 4e5 was reached. The

ﬂask was immersed in an ice bath, and 30% H₂O₂(28 mL) was added

drop-wise under stirring. Precipitation of the product started a few

minutes after all of the hydrogen peroxide was added. Stirring and

cooling continued for 5 h. Then, the crystals were collected using a

Büchner funnel, washed with 500 mL of water, frozen at ꢁ80 ^ꢀC and

lyophilized. Yield: 29.6 g (82%). White solid, m.p. 260e280 ^ꢀC decay

(lit. [53] 260e265 ^ꢀC decay). Calculated for C₈H₁₆N₂O₄S (268.35):

35.81 %C, 6.01% H, 10.44% N. Found: 35.84% C, 6.14% H, 10.18% N.

d

¼ 5.20 (bd, J w8.5 Hz, 2H, 2ꢂ NH), 4.41 (m, 2H, 2ꢂ NeCH<), 3.76

(s, 6H, 2ꢂ OMe), 2.72 (m, 4H), 2.24 (m, 2H), 2.01 (m, 2H), 1.45 (s,

18H, 2ꢂ t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 172.62 (2C, 2ꢂ e

COeO), 155.81 (2C, 2ꢂ NeCOeO), 80.05 (2C), 52.46 (2C, 2ꢂ OMe),

52.41 (2C), 34.42 (2C), 32.46 (2C), 28.33 (6C). IR (KBr) n_max(cm^ꢁ1):

3372 m (NH₂); 1715 vs, 1686 vs (C]O carbamate); 1761 s, 1751 s

(C]O ester); 1519 vs (amide II); 2981 m, 1368 m (CH₃); 1164 vs (Ce

O). HRMS (ESI) calc for C₂₀H₃₆O₈N₂NaS₂[M þ Na]^þ519.18053,

found: 519.18053.

5.2.28. The synthesis of compounds 41 and 42

Compounds 41 and 42 were prepared by the method described

for the synthesis of compounds 5, 6, 7, 16 and 20 (5.2.2.).

R_f¼ 0.40 (S5). ¹H NMR (600 MHz, D₂O þ NaOD):

d

¼ 3.33 (m, 2H, 2ꢂ

5.2.28.1. 2-(R,S)-9-(R,S)-2,9-Bis(tert-butoxycarbonylamino)-5,6-

dithiadecanediamide (41). Compound 41 was prepared by the re-

action of 39 (15.2 g; 32.4 mmol), 2.2 equiv. of TEA (7.2 g;

71.2 mmol), 2.2 equiv. of ClCOOC₂H₅(7.8 g; 71.2 mmol), 200 mL of

methanol saturated with ammonia and 500 mL of THF. Column

chromatography was performed using a linear gradient of ethyl

acetate in the S H3 mixture. Yield: 8.05 g (53%). White solid, m.p.

158e160 ^ꢀC (methanoleacetoneepetroleum ether), R_f¼ 0.37 (S4).

NeCH<), 2.77 (m, 4H), 2.03 (m, 2H), 1.90 (m, 2H). ¹³C NMR

(150.9 MHz, D₂O þ NaOD): ¼ 185.39 (2C, 2ꢂ COOH), 57.78 (2C, 2ꢂ

d

NeCH<), 37.26 (2C), 37.14 (2C). IR (KBr) n_max(cm^ꢁ1): 3046 s (OH);

1659 s (C]O); 1600 vs, 1412 vs (COO^ꢁ); 2609 m, 1573 s, 1531 m

(NH^þ₃). HRMS (ESI) calc for C₈H₁₇O₄N₂S₂[M þ H]^þ269.06242,

found: 269.06247.

5.2.26. 2-(R,S)-9-(R,S)-2,9-Bis(tert-butoxycarbonylamino)-5,6-

dithiadecanedioic acid (39)

¹H NMR (600 MHz, DMSO-d₆):

d

¼ 7.25 and 7.00 (2ꢂ s, 2ꢂ 2H, 2ꢂ

CONH₂), 6.86 (d, J ¼ 8.2 Hz, 2H, 2ꢂ NH), 3.94 (ddd, J ¼ 8.8, 8.2 and

5.0 Hz, 2H, 2ꢂ NeCH<), 2.67 (m, 4H),1.97 (m, 2H),1.83 (m, 2H),1.37

D,L-Homocystine 38 (15 g; 55.9 mmol) was dissolved in 500 mL

of 5% (aq.) Na₂CO₃and 400 mL of dioxane. Then, 2.2 equiv. of Boc₂O

(26.8 g; 123 mmol) in 100 mL of dioxane was added drop-wise at

(s, 18H, 2ꢂ t-Bu). ¹³C NMR (150.9 MHz, DMSO-d₆):

d

¼ 173.71 (2C,

2ꢂ eCOeNH), 155.47 (2C, 2ꢂ NeCOeO), 78.28 (2C), 53.36 (2C),

34.59 (2C), 31.97 (2C), 28.35 (6C). IR (KBr) n_max(cm^ꢁ1): 3390 s, 3345

s (NH₂); 1662 vs (C]O amide); 1683 vs (C]O carbamate); 1520 s

(amide II); 1252 s (NeCOeO); 2979 m, 1393 m, 1368 m, 1169 s

(CH₃). HRMS (ESI) calc for C₁₈H₃₄O₆N₄NaS₂[M þ Na]^þ489.18120,

found: 489.18110.

0

^ꢀC over 30 min. After stirring for 1 h at 0 ^ꢀC, the mixture was

allowed to stand overnight at rt. The reaction volume was reduced

to 1/3 by evaporation in vacuo, and the acidity of the aqueous so-

lution was adjusted to pH z 2e3 by concentrated citric acid under

efﬁcient cooling. The emulsion was extracted three times with

250 mL of ethyl acetate. The combined organic layers were washed

twice with 200 mL of water, twice with 200 mL of brine, and they

were dried over Na₂SO₄before ﬁltering and removing the solvent

under reduced pressure. The residue was triturated at ꢁ20 ^ꢀC from

a mixture of methanoleethyl acetateepetroleum ether to afford

5.2.28.2. 1,1⁰-[2-(R,S)-9-(R,S)-2,9-Bis(tert-butoxycarbonylamino)-

5,6-dithiadecanedioyl]bispiperidine (42). Compound 42 was pre-

pared by the reaction (5 g; 10.7 mmol) of 39, 2.2 equiv. of TEA

(2.46 g; 23.5 mmol), 2.2 equiv. of ClCOOC₂H₅(2.6 g; 23.5 mmol) and

270

J. Pícha et al. / European Journal of Medicinal Chemistry 65 (2013) 256e275

piperidine (2 g; 21.4 mmol) in 100 mL of THF. Yield: 5.3 g (83%).

COeN<), 155.72 and 155.46 (NeCOeO), 79.59 and 79.22, 50.97 and

48.45, 46.50 and 46.44, 43.19 and 43.09, 38.21, 28.32 and 28.27,

26.42 and 26.37, 25.52 and 25.45, 24.42 and 24.38, 20.80. IR (KBr)

n_max(cm^ꢁ1): 3302 s (NH); 2561 w (SH); 1699 vs (C]O carbamate);

1637 vs (C]O amide); 1534 s (amide II); 1248 s (NeCOeO); 2976 m,

1389 m,1365 m (CH₃); 2924 m, 2853 m,1471 m,1440 s (CH₂). HRMS

Foam, R_f¼ 0.36 (S7). Mixture of diastereoisomers w1:1 e some

signals are doubled: ¹H NMR (600 MHz, CDCl₃):

d

¼ 5.66 (bd,

J ¼ 8.2 Hz, 2H, 2ꢂ NHeCO), 4.77 and 4.73 (2ꢂ m, 2ꢂ 1H, 2ꢂ Ne

CH<), 3.58 (m, 2H), 3.53 (m, 2H), 3.46 (m, 4H), 2.72 (m, 2H), 2.67

(m, 2H), 2.10 (m, 2H), 1.86 (m, 2H), 1.66e1.53 (m, 12H), 1.43 (s, 18H,

2ꢂ t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 169.39 and 169.31 (2C,

(ESI) calc for

325.15575.

C₁₄H₂₆O₃N₂NaS [M

þ

Na]^þ325.15563, found:

2ꢂ eCOeNH), 155.41 and 155.33 (2C, 2ꢂ NeCOeO), 79.28 (2C),

48.87 and 48.65, 46.28 and 46.26, 43.01 (2C), 33.72 and 33.42, 33.20

and 32.96, 28.12 (6C), 26.28 (2C), 25.32 (2C), 24.21 (2C). IR (CCl₄)

n_max(cm^ꢁ1): 3418 m, 3299 s (NH); 1712 vs (C]O carbamate); 1646

vs (C]O amide); 1495 s (amide II); 2976 s, 1392 m, 1367 m (CH₃);

2941 s, 1444 s, 1169 s (CH₂). HRMS (ESI) calc for C₂₈H₅₀O₆N₄NaS₂

[M þ Na]^þ625.30640, found: 625.30636.

5.2.30. The general procedure for the synthesis of compounds 46,

47 and 20

The respective thiol (1 equiv.)wasdissolvedand stirredin80mL of

anhydrous THF under the protection of nitrogen. Then, a 60%

dispersion of sodium hydride (1.05 equiv.) was added. The liberation

of hydrogen started immediately after the addition of sodium hy-

dride. After 10 min, the severe reaction ceased, and tert-butyl 5-

bromovalerate 15 (1 equiv.) was added drop-wise. Then, the reac-

tion mixture was allowed to proceed overnight at rt. The solvent was

evaporated under reduced pressure and then was puriﬁed by ﬂash

chromatographyon silica gel using a linear gradientof ethyl acetate in

toluene.

5.2.29. The synthesis of compounds 43, 44 and 45

Thiols 43, 44 and 45 were prepared by the method described

by Zhu et al. [56], with a few modiﬁcations. Disulﬁde (1 equiv.)

was dissolved in 80 mL of DMF and 5 mL of water. Then, tribu-

tylphosphine (1.5 equiv.) was added under the protection of N₂.

After stirring for 12 h at rt, the reaction mixture was transferred

to a separatory funnel with 700 mL of water. The cloudy solution

was extracted four times with 150 mL of ethyl acetate, and the

combined organic layers were washed twice with 150 mL of

water and twice with 150 mL of brine, then dried over sodium

sulfate before ﬁltering and evaporating the solvent under

reduced pressure. The crude product was puriﬁed by ﬂash

chromatography on silica gel using a linear gradient of ethyl

acetate in toluene.

5.2.30.1. Methyl 2-(R,S)-2-(tert-butoxycarbonylamino)-5-[4-(tert-butox-

ycarbonyl)butylsulfanyl]butanoate (46). Ester 46 was prepared by the

reaction of thiol 43 (3 g; 12 mmol), 60% NaH (0.50 g; 12.6 mmol) and 15

(2.8 g; 12 mmol). Yield: 2.5 g (49%). Clear oil. R_f¼ 0.55 (S6). ¹H NMR

(600 MHz, CDCl₃):

d

¼ 5.16 (bd, Jw8Hz,NHeCO), 4.41 (m, NeCH<), 3.75

(s, OMe), 2.54 (t, J ¼ 7.3 Hz, 2H), 2.52 (t, J ¼ 7.2 Hz, 2H), 2.23 (t, J ¼ 7.3 Hz,

2H), 2.11 (m,1H),1.91 (m,1H),1.67 (m, 2H),1.60 (m, 2H),1.45 (s, 9H, t-Bu),

1.44 (s, 9H, t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 172.64 (eCOeO),

5.2.29.1. Methyl 2-(R,S)-2-(tert-butoxycarbonylamino)-4-mercaptobutanoate

(43). Compound 43 was prepared by the reaction of 40 (4.9 g;

9.9 mmol) and tributylphosphine (3 g; 14.9 mmol). Yield: 4.1 g (84%). Clear

oil, R_f¼ 0.62 (S6). Some signals are doubled-conformers at NeCO bond:

172.58 (eCOeO), 155.24 (NeCOeO), 80.06, 52.79, 52.31, 34.96, 32.68,

31.66, 28.82, 28.25 (3C), 28.06 (3C), 27.79, 24.20. IR (CCl₄) n_max(cm^ꢁ1):

3440 m (NH); 1745 vs (C]O ester); 1724 vs (C]O carbamate); 1496 s

(amide II); 1165 vs (CeO); 2980 m, 1392 m, 1367 m (CH₃). HRMS (ESI)

calc for C₁₉H₃₅O₆NNaS [M þ Na]^þ428.20773, found: 428.20782.

¹H NMR (600 MHz, CDCl₃):

d

¼ 5.12 and 5.06 (2ꢂ bd, J w8 Hz, NHeCO),

4.47 and 4.26 (2ꢂ m, NeCH<), 3.76 and 3.74 (2ꢂ s, OMe), 2.60 (m, 2H),

2.11 (m, 1H), 1.94 (m, 1H), 1.45 (s, 9H, t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

5.2.30.2. tert-Butyl 5-[3-(R,S)-3-(tert-butoxycarbonylamino)-4-(car-

boxamide)butylsulfanyl] pentanoate (47). Amide 47 was prepared

by the reaction of thiol 44 (4.9 g; 21 mmol), 60% NaH (0.88 g;

22.1 mmol) and 15 (5.4 g; 21 mmol). Yield: 4.9 g (60%). White solid,

d

¼ 172.75 (eCOeO), 155.31 (NeCOeO), 80.04, 54.48 and 52.36, 52.23 and

52.07, 37.14, 28.24 (3C), 20.64. IR (CCl₄) n_max(cm^ꢁ1): 3440 m (NH); 2564 w

(SH); 1746 vs (C]O ester); 1719 vs (C]O carbamate); 1498 s (amide II);

2980 m, 1392 m, 1367 m (CH₃); 1167 vs (CeO). HRMS (ESI) calc for

C₁₀H₁₉O₄NNaS [M þ Na]^þ272.09270, found: 272.09276.

m.p. 74e77 ^ꢀC. R_f¼ 0.27 (S7). ¹H NMR (600 MHz, CDCl₃):

d

¼ 6.55

and 6.11 (2ꢂ bs, 2ꢂ 1H, CONH₂), 5.45 (b, 1H, NHeCO), 4.32 (m, Ne

CH<), 2.58 (bt, J w7.5 Hz, 2H), 2.53 (t, J ¼ 7.3 Hz, 2H), 2.23 (t,

J ¼ 7.3 Hz, 2H), 2.08 (m, 1H), 1.92 (m, 1H), 1.68 (m, 2H), 1.60 (m, 2H),

1.45 (s, 9H, t-Bu), 1.44 (s, 9H, t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

5.2.29.2. 2-(R,S)-2-(tert-Butoxycarbonylamino)-4-mercaptobutyramide

(44). Compound 44 was prepared by the reaction of 41 (4.9 g;

10.5 mmol) and tributylphosphine (3.2 g; 15.8 mmol). Yield: 3.9 g

(80%). White solid, m.p. 100e102 ^ꢀC (lit [49]. 85e90 ^ꢀC), R_f¼ 0.42 (S4).

d

¼ 174.32 (eCOeO), 172.78 (eCOeNH₂), 155.66 (NeCOeO), 80.14

(2C), 53.09, 34.96, 32.27, 31.54, 28.78, 28.27 (3C), 28.05 (3C), 28.02,

24.16. IR (KBr) n_max(cm^ꢁ1): 3453 m, 3325 m (NH₂); 3191 m (NH);

1731 vs (C]O ester); 1710 vs, 1688 vs (C]O carbamate); 1660 vs

(C]O amide); 1541 s (amide II); 1164 vs (CeO); 2977 m, 1393 m,

1367 m (CH₃). HRMS (ESI) calc for C₁₈H₃₄O₅N₂NaS [M þ Na]^þ

413.20806, found: 413.20828.

¹H NMR (600 MHz, CDCl₃):

d

¼ 5.60 (bd, J w8.5 Hz, NHeCO), 4.41 (m,

NeCH<), 2.61 (m, 2H), 2.07 (m, 1H), 1.95 (m, 1H), 1.58 (bt, J w8.0 Hz,

1H, SH), 1.44 (s, 9H, t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 174.59 (e

COeNH₂), 155.82 (NeCOeO), 80.17, 52.56, 36.94, 28.28 (3C), 20.87. IR

(KBr) n_max(cm^ꢁ1): 3382 vs, 3321 vs (NH₂); 3192 s (NH); 2561 w (SH);

1683 vs (C]O carbamate); 1662 vs (C]O amide); 1532 s (amide II);

2978 m, 1389 m, 1366 m (CH₃). HRMS (ESI) calc for C₉H₁₈O₃N₂NaS

[M þ Na]^þ257.09303, found: 257.09308.

5.2.30.3. tert-Butyl 5-[3-(R,S)-3-(tert-butoxycarbonylamino)-4-oxo-

4-(piperidin-1-yl)butylsulfanyl]pentanoate (20). Amide 20 was

prepared by the reaction of thiol 45 (4.0 g; 13.2 mmol) of 60% of

NaH (0.56 g; 13.9 mmol) and 15 (3.1 g; 13.2 mmol). Due to the very

similar R_fvalues of the starting thiol 45, the disulﬁde 42 (product of

oxidation) and the target compound 20, it was not possible to

separate the reaction mixture. However, the formation of the

product 20 was proved by mass spectrometry (data not shown).

5.2.29.3. 2-(R,S)-2-(tert-Butoxycarbonylamino)-4-mercapto-1-oxo-

1-(piperidine-1-yl)butane (45). Compound 45 was prepared by the

reaction of 42 (4.8 g; 8 mmol) and tributylphosphine (2.4 g;

12 mmol). Yield: 4 g (83%). White solid, m.p. 90e92 ^ꢀC, R_f¼ 0.69

(S7). Some signals are doubled-conformers at NeCO bond: ¹H NMR

(600 MHz, CDCl₃):

d

¼ 5.54 and 5.51 (2ꢂ bd, J ¼ 8.6 Hz, NHeCO),

4.82 and 4.57 (2ꢂ m, NeCH<), 3.56 (m, 2H), 3.50 (m, 2H), 2.65 (m,

5.2.31. The synthesis of compounds 48 and 11

Compounds 48 and 11 were prepared by the method described

for synthesis 18, 22 and 23 (5.2.12.).

1H), 2.54 (m, 1H), 1.84 (m, 2H), 1.66 (m, 2H), w1.57 (m, 4H), 1.43 (s,

9H, t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 170.12 and 169.77 (e

J. Pícha et al. / European Journal of Medicinal Chemistry 65 (2013) 256e275

271

5.2.31.1. 5-[3-(R,S)-3-Amino-4-(methoxycarbonyl)butylsulfanyl]

pentanoic acid triﬂate salt (48). The acid hydrolysis of ester 46 (1 g;

2.5 mmol) afforded target compound 48 (G2). Yield: 433 mg (49%).

9.7 mmol), 2 equiv. of sodium azide (1.26 g; 19.4 mmol) and

0.5 equiv. of zinc bromide (1.1 g; 4.85 mmol) were reﬂuxed over-

night in 50 mL of isopropyl alcohol and 30 mL of water. The reaction

mixture was cooled, and then, 20 mL of 10% (aq.) citric acid and

50 mL of ethyl acetate were added. The reaction mixture was

vigorously stirred until the solid material disappeared. Then, 50 mL

of water was added, and the lower aqueous phase was extracted

four times with 50 mL of ethyl acetate. The combined organic layers

were washed with 100 mL of water and twice with 100 mL of brine

and then dried over Na₂SO₄before ﬁltering and removing the sol-

vents in vacuo. The yellow residue was puriﬁed by ﬂash chroma-

tography on silica gel using a linear gradient of the S H3 system in

ethyl acetate. Yield: 2.6 g (65%). Clear oil. R_f¼ 0.71 (S4). ¹H NMR

Lyophilizate. ¹H NMR (600 MHz, DMSO-d₆):

d

¼ 12.06 (b, 1H,

COOH), 8.44 (b, 2H, NH₂), 4.14 (t, J ¼ 6.3 Hz,1H, NeCH<), 3.76 (s, 3H,

COOMe), 2.62 (m, 1H), 2.54 (m, 1H), 2.50 (m, 2H), 2.22 (t, J ¼ 7.2 Hz,

2H), 2.02 (m, 2H), 1.56 (m, 2H), 1.52 (m, 2H). ¹³C NMR (150.9 MHz,

DMSO-d₆):

d

¼ 174.54 (COOH), 169.88 (eCOeO), 53.15, 51.20, 33.38,

30.50, 30.29, 28.53, 26.24, 23.84. IR (KBr) n_max(cm^ꢁ1): z3000 vbr

vs,1522 m (NH^þ₃); 1700 vs (C]O acid); 1751 vs (C]O ester); 1676 vs

(C]O triﬂate); 1203 vs, 1139 vs, 723 s (CF₃). HRMS (ESI) calc for

C

₁₀H₂₀O₄NS [M þ H]^þ250.11076, found: 210.11066.

5.2.31.2. 5-[(3-(R,S)-3-Amino-3-carboxamide)butylsulfanyl]penta-

noic acid triﬂuoroacetate salt (11). The acid hydrolysis of ester 47

(1 g; 2.6 mmol) afforded target compound 11 (G2). Yield: 405 mg

(46%). Lyophilizate.

(600 MHz, CDCl₃):

d

¼ 5.88 (bd, J ¼ 8.0 Hz, 1H, NHeCO), 5.32 (ddd,

J ¼ 8.5, 8.0 and 6.2 Hz, NeCH<), 2.62 (m, 2H), 2.51 (m, 2H), 2.30 (m,

2H), 2.25 (t, J ¼ 7.3 Hz, 2H), 1.67 (m, 2H), 1.58 (m, 2H), 1.45 (s, 9H, t-

Bu), 1.41 (s, 9H, t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 173.37 (e

COeO), 158.59 (NeC]N), 156.00 (NeCOeO), 81.09, 80.62, 44.78,

34.96, 33.19, 31.65, 28.67, 28.22 (3C), 28.06 (3C), 27.90, 24.04. IR

(KBr) n_max(cm^ꢁ1): 3431 s, 3336 s (NH); 1728 vs (C]O ester); 1680

vs (C]O carbamate); 1521 s (amide II); 2978 m, 1393 m, 1369 m

(CH₃); 1167 vs (OeCOeN). HRMS (ESI) calc for C₁₈H₃₃O₄N₅NaS

[M þ Na]^þ438.21455, found: 438.21442.

5.2.32. tert-Butyl 5-[3-(R,S)-3-(tert-butoxycarbonylamino)-3-

cyanopropylsulfanyl]pentanoate (49)

Nitrile 49 was prepared by the method described by Sureshbabu

et al. [57] with several modiﬁcations. Pyridine (3 equiv., 4.1 g;

52.2 mmol) and TFAA (1.5 equiv., 5.5 g; 26.1 mmol) were succes-

sively and slowly added to the ice-cooled solution of amide 47

(6.8 g; 17.4 mmol) in 100 mL of anhydrous THF. The reaction

mixture was stirred and cooled for 1 h and then was kept at rt for

another hour. The reaction was quenched by adding 100 mL of

water. Then, the upper organic layer was separated, and the lower

aqueous layer was extracted twice with 100 mL of ethyl acetate. The

combined organic phases were dried over sodium sulfate before

ﬁltering and removing the solvents in vacuo. The yellow residue

was puriﬁed by ﬂash chromatography on silica gel using a linear

gradient of ethyl acetate in toluene. Yield: 5 g (77%). Clear viscous

5.2.35. tert-Butyl 5-[3-(R,S)-3-(tert-butoxycarbonylamino)-4-

(hydroxyimino)-4-amino butylsulfanyl]pentanoate (52)

Amidine 52 was prepared by the method described by Mangette

et al. [58] with few modiﬁcations. 50% NH₂OH (aq.) (3.4 g;

50.4 mmol) was added to a solution of nitrile 49 (4.7 g; 12.6 mmol)

in 25 mL of ethanol, and the reaction mixture was stirred overnight

at rt. The solvent was evaporated, and the residue was puriﬁed by

ﬂash chromatography on silica gel using a linear gradient of ethyl

acetate in toluene. Yield: 4.6 g (90%). White solid, m.p. 85e87 ^ꢀC.

oil. R_f¼ 0.83 (S7). ¹H NMR (600 MHz, CDCl₃):

d

¼ 5.27 (d, J ¼ 8.9 Hz,

R_f¼ 0.53 (S7). ¹H NMR (600 MHz, DMSO-d₆):

d

¼ 9.00 (s, 1H, Ne

1H, NHeCO), 4.77 (m, NeCH<), 2.67 (m, 2H), 2.54 (t, J ¼ 7.2 Hz, 2H),

2.24 (t, J ¼ 7.1 Hz, 2H), 2.08 (q, J w7.2 Hz, 2H), 1.68 (m, 2H), 1.62 (m,

2H), 1.46 (s, 9H, t-Bu), 1.45 (s, 9H, t-Bu). ¹³C NMR (150.9 MHz,

OH), 6.82 (d, J ¼ 9.4 Hz, NHeCO), 4.01 (m, 1H, NeCH<), 2.46 (t,

J ¼ 7.2 Hz, 2H), 2.18 (t, J ¼ 7.2 Hz, 2H), 1.78 (m, 1H), 1.75 (m, 1H), 1.54

(m, 2H), 1.49 (m, 2H), 1.38 (s, 9H, t-Bu), 1.36 (s, 9H, t-Bu). ¹³C NMR

CDCl₃):

d

¼ 172.71 (eCOeO), 154.24 (NeCOeO), 118.44 (C^N),

(150.9 MHz, DMSO-d₆):

d

¼ 172.34 (eCOeO), 155.13 (NeCOeO),

81.15, 80.22, 41.38, 34.88, 32.86, 31.72, 28.68, 28.16 (3C), 28.04 (3C),

27.46, 24.07. IR (CCl₄) n_max(cm^ꢁ1): 3447 m, 3362 m (NH); 1730 vs

(C]O ester); 2244 w (C^N); 1706 vs (C]O carbamate); 1493 s

(amide II); 1160 vs (OeCOeN); 2981 s, 1393 m, 1368 s (CH₃). HRMS

(ESI) calc for

395.19745.

152.87 (NeC]N), 79.65, 78.25, 50.61, 34.55, 33.45, 30.91, 28.61,

28.40 (3C), 27.98 (3C), 27.73, 24.02. IR (KBr) n_max(cm^ꢁ1): 1730 vs

(C]O ester); 1667 vs (C]O carbamate); 1521 vs (amide II); 1667 s

(C]N); 1600 m (NH₂); 2980 m, 1393 m, 1368 m (CH₃); 1166 vs (Oe

COeN). HRMS (ESI) calc for C₁₈H₃₆O₅N₃S [M þ H]^þ406.23702,

found: 406.23697.

C₁₈H₃₂O₄N₂NaS [M

þ

Na]^þ395.19750, found:

5.2.33. 5-[3-(R,S)-3-Amino-3-cyanopropylsulfanyl]pentanoic acid

triﬂate salt (50)

5.2.36. tert-Butyl 5-[3-(R,S)-3-(tert-butoxycarbonylamino)-3-(4H-

[1,2,4]oxadiazol-5-one-3-yl)propylsulfanyl] pentanoate (53)

Compound 50 was prepared by the method described for syn-

thesis of compounds 18, 22, 23 and 48 (5.2.12.). The acid hydrolysis

of ester 49 (1.1 g; 2.9 mmol) afforded target compound 50 (G2).

Yield: 327 mg (34%). Lyophilizate. ¹H NMR (600 MHz, DMSO-d₆):

Oxadiazone 53 was prepared by the method described by

Mangette et al. [58] with few modiﬁcations. Compound 52 (4.5 g;

11.1 mmol) was heated to reﬂux with 1.5 equiv. of CDI (2.7 g;

16.7 mmol) in 50 mL of THF for 17 h. The solvent was evaporated in

vacuo, and the black residue was taken up with 100 mL of ethyl

acetate. The organic phase was washed with 50 mL of 10% (aq.)

citric acid and twice with 50 mL of brine before ﬁltering and

removing the solvent under reduced pressure. The resulting black

oil was puriﬁed twice by ﬂash chromatography on silica gel using a

linear gradient of toluene in ethyl acetate. Yield: 1.5 g (31%).

d

¼ 12.00 (b, 1H, COOH), 8.95 (b, 2H, NH₂), 4.58 (dd, J ¼ 8.2 and

6.4 Hz, 1H, NeCH<), 2.67 (ddd, J ¼ 13.5, 8.5 and 5.9 Hz, 1H), 2.58

(ddd, J ¼ 13.5, 8.5 and 7.4 Hz, 1H), 2.52 (m, 2H), 2.22 (t, J ¼ 7.2 Hz,

2H), 2.11 (m, 2H), 1.57 (m, 2H), 1.53 (m, 2H). ¹³C NMR (150.9 MHz,

DMSO-d₆):

d

¼ 174.54 (COOH), 116.93 (C^N), 40.28, 33.38, 30.69,

30.54, 28.48, 26.25, 23.83. IR (KBr) n_max(cm^ꢁ1): z3000 vbr vs,

1529 m (NH^þ₃); 2256 w (C^N); 1698 vs (C]O acid); 1678 vs (C]O

triﬂate); 1203 vs, 1143 vs, 724 m (CF₃). HRMS (ESI) calc for

C₉H₁₅O₂N₂S [M ꢁ H]^þ215.08597, found: 215.08615.

Semisolid. R_f¼ 0.59 (S7). ¹H NMR (600 MHz, CDCl₃):

d

¼ 10.06 (b,

1H, NH), 5.40 (bd, J w8.2 Hz, 1H, NHeCO), 4.77 (td, J ¼ 8.2 and

5.6 Hz, NeCH<), 2.67 (ddd, J ¼ 13.5, 7.2 and 6.0 Hz, 1H), 2.62 (dt,

J ¼ 13.5 and 7.4 Hz,1H), 2.54 (m, 2H), 2.25 (t, J ¼ 7.2 Hz, 2H), 2.22 (m,

1H), 2.12 (m, 1H), 1.68 (m, 2H), 1.60 (m, 2H), 1.45 (s, 18H, 2ꢂ t-Bu).

5.2.34. tert-Butyl 5-[3-(R,S)-3-(tert-butoxycarbonylamino)-3-(1H-

tetrazol-5-yl)propylsulfanyl]pentanoate (51)

Tetrazole 51 was prepared by the method described by Sure-

shbabu et al. [57] with few modiﬁcations. Compound 49 (3.6 g;

¹³C NMR (150.9 MHz, CDCl₃):

O), 159.27 (NeCOeO), 156.06 (NeC]N), 81.46, 80.51, 46.05, 34.91,

31.68, 30.45, 28.67, 28.20 (3C), 28.06 (3C), 27.72, 24.03. IR (KBr) n_max

d

¼ 173.13 (eCOeO), 159.40 (NeCOe

272

J. Pícha et al. / European Journal of Medicinal Chemistry 65 (2013) 256e275

(cm^ꢁ1): 3378 m, 3269 m (NH); 1789 vs (C]O oxadiazone); 1727 vs

(C]O ester); 1685 vs (C]O carbamate); 1514 s (amide II); 1252 s

(OeCOeN); 2980 m, 1392 m, 1369 m, 1163 vs (CH₃); 2934 m (CH₂).

HRMS (ESI) calc for C₁₉H₃₃O₆N₃NaS [M þ Na]^þ454.19823, found:

454.19816.

Major e ¹H NMR (600 MHz, CDCl₃):

d

¼ 4.45 (m, 1H), 4.27 (t, J ¼ 8.2

and 7.4 Hz, 1H), 3.59 (dd, J ¼ 11.6 and 4.3 Hz, 1H), 3.54 (ddd, J ¼ 11.6,

2.3 and 1.6 Hz,1H), 3.00 (bd, J ¼ 3.2 Hz,1H, OH), 2.30 (dddd, J ¼ 13.3,

8.2, 3.3 and 1.6 Hz, 1H), 2.03 (ddd, J ¼ 13.3, 7.4 and 5.0 Hz, 1H), 1.46

(s, 9H, t-Bu), 1.43 (s, 9H, t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 172.15 (eCOeO), 154.23 (NeCOeO), 81.10, 80.16, 69.10, 58.48,

54.52, 39.06, 28.27 (3C), 27.93 (3C). Minor e ¹H NMR (600 MHz,

CDCl₃):

5.2.37. The synthesis of compounds 54 and 55

Compounds 54 and 55 were prepared by the method described

for the synthesis of 11, 18, 22, 23 and 48.

d

¼ 4.46 (m, 1H), 4.30 (dd, J ¼ 8.2 and 7.0 Hz, 1H), 3.60 (dd,

J ¼ 11.4 and 4.9 Hz, 1H), 3.42 (ddd, J ¼ 11.6, 2.7 and 1.5 Hz, 1H), 2.91

(bd, J ¼ 3.7 Hz, 1H, OH), 2.24 (dddd, J ¼ 13.4, 8.2, 3.8 and 1.5 Hz, 1H),

2.05 (ddd, J ¼ 13.4, 7.0 and 5.2 Hz, 1H), 1.46 (s, 9H, t-Bu), 1.45 (s, 9H,

5.2.37.1. 5-[3-(R,S)-3-Amino-3-(1H-tetrazol-5-yl)propylsulfanyl]pen-

tanoate (54). The acid hydrolysis of ester 51 (0.8 g; 1.9 mmol)

afforded the target compound 54. Yield: 300 mg (42%). Lyophilizate.

t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 172.07 (eCOeO), 154.42

(NeCOeO), 81.18, 79.84, 69.98, 58.41, 54.60, 38.32, 28.32 (3C), 27.85

(3C). IR n_max/cm^ꢁ1(CCl₄): 3446 m br (OH); 2980 s, 1478 m, 1393 vs,

1367 vs (CH₃); 2933 m (CH₂); 1744 vs (C]O ester); 1704 vs (C]O

carbamate); 1169 vs (CeO); 1087 s (CeOH). HRMS (ESI) calculated

for (M þ Na)^þC₁₄H₂₅O₅NNa 310.1625, found 310.1624.

(G2). ¹H NMR (600 MHz, DMSO-d₆):

d

¼ 8.80 (b, 3H, NH₂and NH),

4.89 (t, J ¼ 6.9 Hz, 1H, NeCH<), 2.52 (m, 1H), 2.48 (t, J ¼ 7.0 Hz, 2H),

2.45 (m, 1H), 2.23 (m, 2H), 2.21 (t, J ¼ 7.0 Hz, 2H), 1.54 (m, 2H), 1.48

(m, 2H). ¹³C NMR (150.9 MHz, DMSO-d₆):

d

¼ 174.57 (COOH), 155.66

(NeC]N), 44.74, 33.40, 32.30, 30.51, 28.51, 26.23, 23.86. IR (KBr) n_max

(cm^ꢁ1): z3000 vbr vs, 1526 m (NH^þ₃); 1697 vs (C]O acid); 1671 vs

(C]O triﬂate); 1199 vs, 1147 vs, 724 m (CF₃). HRMS (ESI) calc for

C₉H₁₈O₂N₅S [M þ H]^þ260.11757, found: 260.11772.

5.2.39. tert-Butyl (2S,4R)-N-tert-butoxycarbonyl-4-

methanesulfonyloxyprolinate (58)

A solution of 2.5 equiv. of mesyl chloride (7.80 mL; 100.9 mmol)

in dichloromethane (20 mL) was added drop-wise to a solution of

alcohol 57 (11.60 g; 40.4 mmol) and 5 equiv. of pyridine (16.3 mL;

201.8 mmol) in CH₂Cl₂(60 mL) at 0 ^ꢀC, and the reaction was stirred

overnight at rt. The reaction mixture was cooled to 0 ^ꢀC, quenched

carefully by the drop-wise addition of water (10 mL) and parti-

tioned between CH₂Cl₂and H₂O. The organic layer was washed

with sat. NaHCO₃, dried over Na₂SO₄and concentrated in vacuo. The

crude product was puriﬁed by ﬂash chromatography on silica

(elution with a linear gradient of EtOAc in toluene) to give com-

pound 58. Yield: 13.98 g (95%). Pale yellow thick oil. R_f¼ 0.55 (S7).

Mixture of conformers at NeCO bond (w2:1); Major e ¹H NMR

5.2.37.2. 5-[3-(R,S)-3-Amino-3-(4H-[1,2,4]oxadiazol-5-one-3-yl)pro-

pylsulfanyl]pentanoic acid (55). The acid hydrolysis of ester 53 (1 g;

2.3 mmol) afforded the target compound 55. Yield: 545 mg (61%).

Lyophilizate. (G2). ¹H NMR (600 MHz, DMSO-d₆):

d

¼ 9.90 (s, 1H,

NH), 4.47 (t, J ¼ 6.6 Hz,1H, NeCH<), 2.56 (m, 2H), 2.50 (m, 2H), 2.22

(t, J ¼ 7.2 Hz, 2H), 2.14 (m, 1H), 2.08 (m, 1H), 1.56 (m, 2H), 1.52 (m,

2H). ¹³C NMR (150.9 MHz, DMSO-d₆):

d

¼ 174.56 (COOH), 160.78

(NeCOeO), 158.20 (NeC]N), 45.52, 33.40, 30.62, 30.48, 28.52,

25.87, 23.87. IR (KBr) n_max(cm^ꢁ1): 3042 vs vbr, 1605 s br, 1507 s br

(NH^þ₃); 1781 s (C]O oxadiazolone); 1703 s (C]O acid); 1674 vs,

1438 m, 798 m (CO^ꢁ₂); 1202 vs, 1142 vs, 723 s (CF₃). HRMS (ESI)

calc for C₉H₁₈O₂N₅S [M þ H]^þ276.10125, found: 276.10138.

(600 MHz, CDCl₃):

d

¼ 5.24 (m, 1H), 4.30 (dd, J ¼ 8.1 and 7.5 Hz, 1H),

3.86 (dd, J ¼ 12.7 and 1.9 Hz, 1H), 3.72 (dd, J ¼ 12.7 and 4.5 Hz, 1H),

3.05 (s, 3H), 2.67 (m, 1H), 2.22 (m, 1H), 1.47 (s, 9H, t-Bu), 1.45 (s, 9H,

5.2.38. tert-Butyl (2S,4R)-N-tert-butoxycarbonyl-4-

hydroxyprolinate (57)

Di-tert-butyl dicarbonate (1.2 equiv.,16.0 g; 73.3 mmol) in 50 mL

dioxane was added drop-wise to a vigorously stirred solution of

t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 171.21 (eCOeO),153.59 (Ne

COeO), 81.73, 80.70, 77.90, 57.93, 52.14, 38.72, 37.56, 28.24 (3C),

27.96 (3C). Minor e ¹H NMR (600 MHz, CDCl₃):

d

¼ 5.26 (m, 1H),

4.34 (t, J ¼ 7.9 Hz, 1H), 3.74 (m, 2H), 3.06 (s, 3H), 2.56 (m, 1H), 2.22

trans-4-hydroxy-L-proline (8.0 g; 61.0 mmol) in 10% aqueous

(m, 1H), 1.47 (s, 9H, t-Bu), 1.46 (s, 9H, t-Bu). ¹³C NMR (150.9 MHz,

Na₂CO₃(80 mL) at 0 ^ꢀC, and the reaction was stirred overnight at

room temperature. After the reaction was complete (TLC in the S H1

system), the solution was acidiﬁed by saturated aq. citric acid to pH

4, and the product was extracted with EtOAc (three times with

200 mL, then ﬁve times with 100 mL). The combined extracts were

dried over Na₂SO₄and were then evaporated in vacuo to give a

viscous colorless oil (16 g, R_f¼ 0.6 in the S H1 system). The crude

product 56 was used directly in the next step without further

puriﬁcation.

CDCl₃):

d

¼ 171.13 (eCOeO), 153.81 (NeCOeO), 81.81, 80.54, 78.23,

58.00, 52.16, 38.66, 36.26, 28.30 (3C), 27.89 (3C). IR n_max/cm^ꢁ1

(CCl₄): 2981 s, 1478 m, 1394 vs, 1368 vs (CH₃); 2933 m (CH₂); 1745

vs (C]O ester); 1708 vs (C]O carbamate); 1351 vs, 1152 vs (SO₂);

1176 vs (CeO). HRMS (ESI) calculated for (M þ H)^þC₁₅H₂₈NO₇S

366.1586, found 366.1584.

5.2.40. tert-Butyl (2S,4S)-N-tert-butoxycarbonyl-4-

acetyloxyprolinate (59)

Diisopropylcarbodiimide (18.9 mL, 122 mmol, 2 equiv.) and CuCl

(0.121 g, 1.22 mmol, 0.02 equiv.) was added to tert-butanol (11.5 mL,

122 mmol, 2 equiv.), and the mixture was stirred for 12 h at room

temperature to allow the formation of O-tert-butyl-N,N⁰-diisopro-

pylisourea (ca. 122 mmol, 2 equiv.). A solution of crude 56 in

dichloromethane (50 mL) was slowly added to the isourea upon

cooling to 0 ^ꢀC, and the reaction mixture was heated to reﬂux for

1 d. A third equivalent of isourea (61 mmol) was then added, and

the reaction was heated for an additional day. After the reaction

mixture cooled, the precipitated urea was ﬁltered off, and then, the

ﬁltrate was evaporated and dissolved in toluene (200 mL). The

second crop of urea was ﬁltered off, and the solvent was evaporated

in vacuo. The crude product was puriﬁed by ﬂash chromatography

on silica (elution with a linear gradient of EtOAc in toluene) to give

compound 57. Yield 11.64 g (66% over 2 steps). Pale yellow oil.

R_f¼ 0.85 (S H3). Mixture of conformers at NeCO bond (w2:1);

Anhydrous sodium acetate (10 equiv., 9.68 g, 117 mmol) was

suspended in a solution of mesylate 58 (4.30 g, 11.8 mmol) in dry

dimethylformamide (50 mL). The ﬂask was equipped with a cal-

cium dichloride tube, and the stirred mixture was heated to 105 ^ꢀC

for 3 days (TLC in S6). The reaction was then cooled. The DMF was

evaporated in vacuo, and the residue was dissolved in toluene.

Then, the inorganic salts were ﬁltered off. The ﬁltrate was evapo-

rated, and the crude product was puriﬁed by ﬂash chromatography

on silica (elution with a linear gradient of EtOAc in toluene). Yield:

3.59 g (93%). Colorless thick oil. R_f¼ 0.4 (S6). Mixture of conformers

at NeCO bond (w2:1); Major e ¹H NMR (600 MHz, CDCl₃):

d

¼ 5.23

(tt, J ¼ 5.5 and 2.0 Hz, 1H), 4.26 (dd, J ¼ 9.5 and 2.1 Hz, 1H), 3.75 (dd,

J ¼ 12.6 and 5.6 Hz, 1H), 3.61 (ddd, J ¼ 12.6, 1.8 and 1.5 Hz, 1H), 2.46

(ddd, J ¼ 14.3, 9.6 and 5.4 Hz, 1H), 2.24 (dq, J ¼ 14.3 and 1.9 Hz, 1H),

2.01 (s, 3H),1.48 (s, 9H, t-Bu),1.44 (s, 9H, t-Bu). ¹³C NMR (150.9 MHz,

CDCl₃):

d

¼ 170.83 (eCOeO), 170.37 (eCOeO), 153.74 n(NeCOeO),

J. Pícha et al. / European Journal of Medicinal Chemistry 65 (2013) 256e275

273

81.14, 80.02, 71.92, 58.37, 52.01, 36.39, 28.28 (3C), 27.92 (3C), 21.03.

Minor e ¹H NMR (600 MHz, CDCl₃):

and the stirred mixture was heated to 70 ^ꢀC for one day (TLC in S7).

Then, the reaction mixture was cooled down. The DMF was evap-

orated in vacuo, and the residue was partitioned between water

(100 mL) and Et₂O (3 ꢂ 50 mL). The organic layer was dried over

Na₂SO₄and concentrated in vacuo. The crude product was puriﬁed

by ﬂash chromatography on silica (elution with a linear gradient of

ethyl acetate in toluene) to yield compound 62. Yield: 1.55 g (82%).

Pale orange thick oil. R_f¼ 0.8 (S7). Mixture of conformers at NeCO

d

¼ 5.22 (tt, J ¼ 5.8 and 2.0 Hz,

1H), 4.37 (dd, J ¼ 9.4 and 2.1 Hz, 1H), 3.73 (dd, J ¼ 12.1 and 5.6 Hz,

1H), 3.50 (ddd, J ¼ 12.6, 1.8 and 1.4 Hz, 1H), 2.39 (ddd, J ¼ 14.1, 9.4

and 5.5 Hz, 1H), 2.26 (dq, J ¼ 13.0 and 1.8 Hz, 1H), 2.02 (s, 3H), 1.47

(s, 18H, 2ꢂ t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

¼ 170.56 (eCOeO),

d

170.52 (eCOeO), 153.90 (NeCOeO), 81.21, 79.99, 73.04, 58.10,

52.42, 35.41, 28.36 (3C), 27.95 (3C), 21.06. IR n_max/cm^ꢁ1(CCl₄): 2980

s, 1478 m, 1397 vs, 1367 vs (CH₃); 2932 m (CH₂); 1746 vs (C]O

esters); 1707 vs (C]O carbamate); 1241 vs (CeO acetate); 1175 vs

(CeO^tBu). HRMS (ESI) calculated for C₁₆H₂₇NNaO₆, m/z 352.1736,

found 352.1737 (M þ Na)^þ.

bond (w2:1); Major e ¹H NMR (600 MHz, CDCl₃):

d

¼ 4.18 (dd,

J ¼ 8.3 and 5.9 Hz, 1H), 3.98 (m, 1H), 3.95 (m, 1H), 3.35 (m, 1H), 2.76

(m, 1H), 2.32 (s, 3H), 1.48 (s, 9H), 1.43 (s, 9H). ¹³C NMR (150.9 MHz,

CDCl₃):

81.42, 80.24, 59.03, 51.48, 38.70, 37.17, 30.50, 28.26 (3C), 27.94 (3C).

Minor e ¹H NMR (600 MHz, CDCl₃):

d

¼ 194.89 (eCOeS), 171.27 (eCOeO), 153.40 (NeCOeO),

5.2.41. tert-Butyl (2S,4S)-N-tert-butoxycarbonyl-4-

hydroxyprolinate (60)

d

¼ 4.25 (dd, J ¼ 8.3 and 5.9 Hz,

1H), 3.96 (m, 1H), 3.94 (m, 1H), 3.30 (m, 1H), 2.68 (m, 1H), 2.33 (s,

Sodium methoxide (0.05 g) was added to a solution of acetate 59

(3.55 g, 10.8 mmol) in anhydrous methanol (50 mL). After 12 h, the

reaction mixture was evaporated in vacuo, and the crude product

was puriﬁed by ﬂash chromatography on silica (elution with a

linear gradient of EtOAc in toluene), giving 60. Yield: 3.39 g (87%).

Colorless thick oil. R_f¼ 0.5 (S7). Mixture of conformers at NeCO

3H), 1.92 (m, 1H), 1.47 (s, 9H), 1.46 (s, 9H). ¹³C NMR (150.9 MHz,

CDCl₃):

d

¼ 195.15 (eCOeS), 171.11 (eCOeO), 153.68 (NeCOeO),

81.47, 80.11, 58.94, 52.12, 39.40, 35.85, 30.50, 28.33 (3C), 27.90 (3C).

IR n_max/cm^ꢁ1(CCl₄): 2980 s, 2874 w, 1478 m, 1393 vs, 1367 vs (CH₃);

2932 m (CH₂); 1749 vs (C]O ester); 1705 vs (C]O carbamate,

thioacetate); 1172 vs (CeO). HRMS (ESI) calculated for (M þ H)^þ

C₁₆H₂₈NO₅S, 346.1688, found 346.1687.

bond (w2:1); Major e ¹H NMR (600 MHz, CDCl₃):

d

¼ 4.32 (m, 1H),

4.18 (dd, J ¼ 10.0 and 1.5 Hz, 1H), 3.70 (ddd, J ¼ 11.8, 2.0 and 1.0 Hz,

1H), 3.55 (dd, J ¼ 11.8 and 4.4 Hz, 1H), 2.33 (ddd, J ¼ 14.2, 10.0 and

4.8 Hz, 1H), 2.05 (dq, J ¼ 14.2 and 1.5 Hz, 1H), 1.50 (s, 9H, t-Bu), 1.45

5.2.44. tert-Butyl (2S,4R)-N-tert-butoxycarbonyl-4-

thioacetoxyprolinate (63)

(s, 9H, t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 174.41 (eCOeO),

Using the procedure outlined for 62, compound 63 was pre-

pared from mesylate 61 (3.39 g, 9.28 mmol) and potassium thio-

acetate (1.17 g, 10.2 mmol). Yield: 2.70 g (84%). Pale orange thick

oil. R_f¼ 0.9 (S7). Mixture of conformers at NeCO bond (w2:1);

153.84 (NeCOeO), 82.40, 80.28, 70.42, 58.77, 55.75, 38.64, 28.32

(3C), 27.82 (3C). Minor e ¹H NMR (600 MHz, CDCl₃):

d

¼ 4.31 (m,

1H), 4.23 (dd, J ¼ 10.0 and 1.4 Hz, 1H), 3.63 (ddd, J ¼ 11.9, 1.8 and

0.8 Hz, 1H), 3.49 (dd, J ¼ 11.9 and 4.3 Hz, 1H), 2.28 (ddd, J ¼ 14.0, 9.8

and 4.7 Hz, 1H), 2.05 (dq, J ¼ 14.0 and 1.5 Hz, 1H), 1.49 (s, 9H, t-Bu),

Major e ¹H NMR (600 MHz, CDCl₃):

d

¼ 4.19 (dd, J ¼ 8.5 and

4.6 Hz, 1H), 4.03 (m, 1H), 3.90 (dd, J ¼ 11.3 and 7.1 Hz, 1H), 3.41

(dd, J ¼ 11.3 and 6.0 Hz, 1H), 2.36 (ddd, J ¼ 13.2, 6.5 and 4.7 Hz,

1H), 2.33 (s, 3H), 2.23 (ddd, J ¼ 13.2, 8.5 and 7.5 Hz, 1H), 1.48 (s, 9H,

1.46 (s, 9H, t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 174.65 (eCOeO),

154.34 (NeCOeO), 82.58, 80.12, 71.39, 58.74, 56.15, 37.67, 28.32

(3C), 27.82 (3C). IR n_max/cm^ꢁ1(CCl₄): 3468 m br (OH); 2980 s, 2879

m, 1478 m, 1393 vs, 1368 vs (CH₃); 2933 m (CH₂); 1750 vs (C]O

ester); 1705 vs (C]O carbamate); 1176 vs, 1170 vs (CeO); 1083 s

(CeOH). HRMS (ESI) calculated for (M þ Na)^þC₁₄H₂₅O₅NNa

310.1630, found 310.1627.

t-Bu), 1.44 (s, 9H, t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 194.87 (e

COeS), 171.36 (eCOeO), 153.53 (NeCOeO), 81.47, 80.24, 59.11,

55.20, 51.33, 39.15, 36.98, 30.58, 28.26 (3C), 27.95 (3C). Minor e ¹H

NMR (600 MHz, CDCl₃):

d

¼ 4.26 (dd, J ¼ 8.5 and 4.1 Hz, 1H), 4.04

(m, 1H), 3.91 (dd, J ¼ 11.0 and 7.1 Hz, 1H), 3.30 (dd, J ¼ 11.0 and

6.3 Hz, 1H), 2.34 (s, 3H), 2.32 (ddd, J ¼ 13.2, 7.0 and 5.3 Hz, 1H),

2.17 (dt, J ¼ 13.2 and 8.5 Hz, 1H), 1.47 (s, 9H, t-Bu), 1.46 (s, 9H, t-

5.2.42. tert-Butyl (2S,4S)-N-tert-butoxycarbonyl-4-

methanesulfonyloxyprolinate (61)

Bu). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 195.02 (eCOeS), 171.24 (e

Using the procedure outlined for 58, mesylate 61 was prepared

from alcohol 60 (3.39 g, 11.8 mmol), mesyl chloride (2.28 mL,

29.5 mmol) and pyridine (4.77 mL, 59.0 mmol). Yield: 3.80 g (88%).

Colorless crystals. R_f¼ 0.5 (S7). NMR: Mixture of conformers at Ne

COeO), 153.88 (NeCOeO), 81.51, 80.11, 59.01, 55.45, 51.90, 39.62,

35.65, 30.56, 28.33 (3C), 27.91 (3C). IR n_max/cm^ꢁ1(CCl₄): 2980 m,

2878 w, 1478 m, 1393 vs, 1367 vs (CH₃); 2932 w (CH₂); 1744 vs

(C]O ester); 1705 vs (C]O carbamate, thioacetate); 1167 s (CeO).

HRMS (ESI) calculated for (M þ Na)^þC₁₆H₂₇NNaO₅S 368.1508,

found 368.1507.

CO bond (w2:1); Major e ¹H NMR (600 MHz, CDCl₃):

d

¼ 5.24 (m,

1H), 4.29 (dd, J ¼ 7.0 and 4.6 Hz, 1H), 3.80 (m, 2H), 3.01 (s, 3H, SMe),

2.51 (m, 2H), 1.49 (s, 9H, t-Bu), 1.45 (s, 9H, t-Bu). ¹³C NMR

(150.9 MHz, CDCl₃):

80.40, 77.43, 58.12, 52.22, 38.90, 37.27, 28.24 (3C), 27.86 (3C). Minor

e ¹H NMR (600 MHz, CDCl₃):

d

¼ 170.36 (eCOeO), 154.54 (NeCOeO), 81.70,

5.2.45. tert-Butyl (2S,4S)-N-tert-butoxycarbonyl-4-(4-

methoxycarbonyl-butylthio)prolinate (64)

d

¼ 5.25 (m, 1H), 4.40 (dd, J ¼ 8.3 and

Sodium methoxide (2.0 equiv., 0.156 g; 2.89 mmol) was added to

a solution of thioacetate 62 (0.50 g; 1.45 mmol) and 1.5 equiv. of

ethyl 5-bromovalerate (0.349 mL; 2.17 mmol) in anhydrous MeOH

(15 mL). The ﬂask was equipped with a calcium dichloride tube, and

the solution was stirred at room temperature for three days (TLC in

S6). The reaction mixture was then partitioned between 10% (aq.)

citric acid (100 mL) and Et₂O (3 ꢂ 50 mL), and the organic layer was

dried over Na₂SO₄and concentrated in vacuo. The crude product

was puriﬁed by ﬂash chromatography on silica (elution with a

linear gradient of ethyl acetate in toluene) to give compound 64.

Yield: 0.380 g (63%). Colorless thick oil. R_f¼ 0.5 (S6). Mixture of

conformers at NeCO bond (w2:1); Major e ¹H NMR (600 MHz,

3.2 Hz, 1H), 3.78 (dd, J ¼ 13.0 and 5.1 Hz, 1H), 3.73 (ddd, J ¼ 13.0, 2.1

and 0.9 Hz, 1H), 3.02 (s, 3H, SMe), 2.48 (m, 1H), 2.44 (m, 1H), 1.48 (s,

9H, t-Bu), 1.45 (s, 9H, t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

d

¼ 170.18

(eCOeO), 153.62 (NeCOeO), 81.73, 80.40, 78.52, 57.86, 52.58,

38.84, 36.24, 28.33 (3C), 27.86 (3C). IR n_max/cm^ꢁ1(CCl₄): 2980 m,

2885 w, 1478 m, 1394 vs, 1368 vs (CH₃); 2933 w (CH₂); 1752 vs (C]

O ester); 1708 vs (C]O carbamate); 1350 s, 1176 vs (SO₂). HRMS

(ESI) calculated for (M þ H)^þC₁₅H₂₈NO₇S 366.1586, found 366.1588.

5.2.43. tert-Butyl (2S,4S)-N-tert-butoxycarbonyl-4-

thioacetoxyprolinate (62)

Potassium thioacetate (1.1 equiv., 0.688 g; 6.02 mmol) was

added to a solution of mesylate 58 (2.00 g; 5.47 mmol) in dry DMF

(40 mL). The ﬂask was equipped with a calcium dichloride tube,

CDCl₃):

d

¼ 4.14 (t, J ¼ 8.0 Hz, 1H), 3.99 (m, 1H), 3.68 (s, 3H, OMe),

3.22 (m, 1H), 3.20 (m, 1H), 2.62 (m, 1H), 2.56 (t, J ¼ 7.3 Hz, 2H), 2.33

(t, J ¼ 7.4 Hz, 2H), 1.85 (m, 1H), 1.72 (m, 2H), 1.62 (m, 2H), 1.47 (s, 9H,

274

J. Pícha et al. / European Journal of Medicinal Chemistry 65 (2013) 256e275

t-Bu), 1.43 (s, 9H, t-Bu). ¹³C NMR (150.9 MHz, CDCl₃):

COeO), 171.31 (eCOeO), 153.42 (NeCOeO), 81.25, 80.14, 59.28,

d

¼ 173.70 (e

5.2.48. (2S,4R)-4-(4-Carboxybutylthio)proline triﬂuoroacetate salt

(67)

52.94, 51.58, 40.49, 37.58, 33.46, 31.45, 29.22, 28.28 (3C), 27.96 (3C),

Using the procedure outlined for 66, compound 67 was pre-

pared from 65 (0.360 g, 0.86 mmol) as triﬂuoroacetate. Yield:

24.02. Minor e ¹H NMR (600 MHz, CDCl₃):

d

¼ 4.19 (t, J ¼ 7.8 Hz,

1H), 3.84 (m, 1H), 3.68 (s, 3H, OMe), 3.22 (m, 1H), 3.20 (m, 1H), 2.60

(m, 1H), 2.57 (t, J ¼ 7.3 Hz, 2H), 2.33 (t, J ¼ 7.4 Hz, 2H), 1.86 (m, 1H),

1.72 (m, 2H), 1.62 (m, 2H), 1.46 (s, 18H, 2ꢂ t-Bu). ¹³C NMR

0.219 g (70%). Lyophilizate. G3. ¹H NMR (600 MHz, D₂O):

d

¼ 4.52

(dd, J ¼ 8.6 and 7.6 Hz, 1H), 3.78 (dd, J ¼ 12.2 and 6.6 Hz, 1H), 3.69

(tt, J ¼ 6.6 and 5.2 Hz, 1H), 3.33 (ddd, J ¼ 12.2, 5.2 and 0.8 Hz, 1H),

2.68 (t, J ¼ 7.1 Hz, 2H), 2.54 (ddd, J ¼ 14.0, 7.6 and 6.6 Hz,1H), 2.42 (t,

J ¼ 7.2 Hz, 2H), 2.41 (dddd, J ¼ 14.0, 8.6, 5.2 and 0.8 Hz, 1H), 1.70 (m,

(150.9 MHz, CDCl₃):

d

¼ 173.70 (eCOeO), 171.14 (eCOeO), 153.67

(NeCOeO), 81.22, 79.94, 59.28, 52.94, 51.58, 41.22, 36.77, 33.46,

31.31, 29.16, 28.36 (3C), 27.90 (3C), 24.02. IR n_max/cm^ꢁ1(CCl₄): 2980

m, 2872 w, 1478 m, 1394 vs, 1367 s (CH₃); 2932 m (CH₂); 1743 vs

(C]O esters); 1705 vs (C]O carbamate); 1168 s (CeO). HRMS (ESI)

calculated for (M þ H)^þC₂₀H₃₆NO₆S 418.2263, found 418.2265.

2H), l.65 (m, 2H). ¹³C NMR (150.9 MHz, D₂O):

d

¼ 181.43 (COOH),

175.18 (COOH), 62.17, 53.96, 43.21, 37.99, 35.92, 33.27, 30.74, 26.03.

IR n_max/cm^ꢁ1(KBr): 2700e2400 vs vbr, 1582 m, 1414 m (NH^þ₂); 2956

m, 2866 m (CH₂); 1711 m, 1698 m (C]O acids); 1619 s, 1398 m

(CO^ꢁ₂); 1168 vs 722 m (CF₃). HRMS (ESI) calculated for (M ꢁ H)^e

C₁₀H₁₆O₄NS 246.0806, found 246.0806.

5.2.46. tert-Butyl (2S,4R)-N-tert-butoxycarbonyl-4-(4-

methoxycarbonyl-butylthio)prolinate (65)

Using the procedure outlined for 64, compound 65 was pre-

pared from thioacetate 63 (0.50 g; 1.45 mmol), ethyl 5-

bromovalerate (0.349 mL; 2.17 mmol) and sodium methoxide

(0.156 g; 2.89 mmol). Yield: 0.361 g (60%). Pale yellow thick oil.

R_f¼ 0.6 (S6). Mixture of conformers at NeCO bond (w2:1); Major e

Acknowledgments

This work was supported by the Grant Agency of the Czech

Republic (project P207/10/1277, to JJ) and by the Research Project of

the Academy of Sciences of the Czech Republic (RVO: 61388963, to

the Institute of Organic Chemistry and Biochemistry).

¹H NMR (600 MHz, CDCl₃):

d

¼ 4.20 (dd, J ¼ 8.6 and 3.4 Hz, 1H), 4.13

(q, J ¼ 7.2, 2H), 3.90 (dd, J ¼ 10.3 and 6.7 Hz, 1H), 3.38 (m, 1H), 3.33

(dd, J ¼ 10.3 and 7.2 Hz, 1H), 2.56 (t, J ¼ 7.4, 2H), 2.31 (t, J ¼ 7.4, 2H),

2.23 (ddd, J ¼ 13.2, 6.4 and 3.5 Hz, 1H), 2.16 (ddd, J ¼ 13.2, 9.2 and

8.6 Hz,1H),1.72 (m, 2H),1.63 (m, 2H),1.47 (s, 9H, t-Bu),1.43 (s, 9H, t-

Appendix A. Supplementary data

Bu). ¹³C NMR (150.9 MHz, CDCl₃):

COeO), 153.57 (NeCOeO), 81.28, 80.08, 60.33, 59.31, 52.80, 40.03,

d

¼ 173.27 (eCOeO), 171.78 (e

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.ejmech.2013.04.039.

37.45, 33.74, 31.28, 29.17, 28.29 (3C), 27.99 (3C), 24.08, 14.23. Minor

e ¹H NMR (600 MHz, CDCl₃):

d

¼ 4.29 (dd, J ¼ 8.6 and 2.7 Hz, 1H),

References

4.13 (q, J ¼ 7.2, 2H), 3.84 (dd, J ¼ 10.7 and 7.3 Hz, 1H), 3.39 (m, 1H),

3.23 (dd, J ¼ 10.7 and 8.7 Hz, 1H), 2.57 (t, J ¼ 7.4, 2H), 2.32 (t, J ¼ 7.4,

2H), 2.23 (ddd, J ¼ 13.0, 6.4 and 3.5 Hz, 1H), 2.10 (dt, J ¼ 13.0 and

9.4 Hz, 1H), 1.72 (m, 2H), 1.63 (m, 2H), 1.46 (s, 18H, 2ꢂ t-Bu). ¹³C

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NMR (150.9 MHz, CDCl₃):

d

¼ 173.27 (eCOeO), 171.74 (eCOeO),

153.89 (NeCOeO), 81.36, 79.96, 60.33, 59.28, 52.97, 40.75, 36.71,

33.74, 31.28, 29.17, 28.37 (3C), 27.94 (3C), 24.08, 14.23. IR n_max/cm^ꢁ1

(CCl₄): 2980 m, 2873 w, 1478 m, 1393 vs, 1367 s (CH₃); 2932 w

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O). HRMS (ESI) calculated for (M þ Na)^þC₂₁H₃₇O₆NNaS, 454.2234,

found 454.2234.

5.2.47. (2S,4S)-4-(4-Carboxybutylthio)proline triﬂuoroacetate salt

(66)

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1497.

A solution of the protected compound 64 (0.340 g; 0.81 mmol)

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5 mL) was stirred at room temperature for 2 h. The solution was

then evaporated in vacuo and co-evaporated with water (10 mL).

The residue was dissolved in a mixture of 5 M aqueous NaOH (4 mL)

and ethanol (0.5 mL) and heated to 80 ^ꢀC for 1 h. The chilled so-

lution was passed through a column of Dowex 50 (H^þ-cycle),

washed successively with water, methanol and again with water.

The crude product was eluted with 10% aqueous NH₃, and the so-

lution was evaporated to dryness. The residue was puriﬁed by HPLC

and lyophilized, giving 66 as triﬂuoroacetate. Yield: 0.245 g (83%).

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Lyophilizate. G3. ¹H NMR (600 MHz, D₂O):

d

¼ 4.41 (dd, J ¼ 9.1 and

6.7 Hz, 1H), 3.70 (m, 1H), 3.67 (m, 1H), 3.39 (dd, J ¼ 11.7 and 5.1 Hz,

1H), 2.80 (dd, J ¼ 14.0 and 9.1 Hz, 1H), 2.66 (m, 2H), 2.41 (t,

J ¼ 7.2 Hz, 2H), 2.22 (dd, J ¼ 14.0 and 6.7 Hz, 1H), 1.69 (m, 2H), l.64

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d

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(COOH), 62.36, 53.92, 43.14, 37.75, 35.91, 33.36, 30.71, 26.01. IR n_max

/

cm^ꢁ1(KBr): 2751 s br, 1572 m, 1415 s br (NH^þ₂); 2948 vs, 2869 s

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1145 vs, 722 m (CF₃). HRMS (ESI) calculated for (M ꢁ H)^e

C₁₀H₁₆O₄NS 246.0806, found 246.0800.

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Article Doi

The development of a new class of inhibitors for betaine-homocysteine S-methyltransferase

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