Synthesis and cytotoxicity of ring C-Functionalized derivatives of the marine natural product (-)-dibromophakellstatin

FULL PAPER

DOI: 10.1002/ejoc.201101175

Synthesis and Cytotoxicity of Ring C-Functionalized Derivatives of the Marine

Natural Product (–)-Dibromophakellstatin

Rares-Petru Moldovan,^[a]Michael Zöllinger,^[a]Peter G. Jones,^[b]Gerhard Kelter,^[c]

¸

Heinz-Herbert Fiebig,^[c]and Thomas Lindel*^[a]

Dedicated to Professor Bill Fenical on the occasion of his 70th birthday

Keywords: Natural products / Alkaloids / Nitrogen heterocycles / Synthetic methods / Cytotoxicity / Structure–activity

relationships

A structure–activity relationship study of ring C-function-

alized derivatives of the cytotoxic marine natural product

(–)-dibromophakellstatin from the sponge Phakellia mauriti-

ana is reported. Functionalization of the pyrrolidine ring was

achieved starting from the hydroxy derivative by conversion

to the triflate followed by etherification by epimerizing nu-

cleophilic substitution. We identified (12R)-dibromo-12-hy-

droxyphakellstatin as the most cytotoxic derivative, which

exhibited an average IC₅₀value of 1.4 μ against a panel of

twelve human cancer cell lines. However, the 12S dia-

stereomer was inactive. Dehydrophakellstatin was synthe-

sized as the first example of a phakellin skeleton with an

unsaturated ring C.

M

that N-functionalization of the urea unit is not tolerated

and that the (–)-enantiomer of the marine natural product

Introduction

Pyrrole–imidazole alkaloids constitute a structurally ver- dibromophakellstatin is cytotoxic, whereas (+)-dibromo-

satile family of biogenetically related natural products, phakellstatin is not.^[7]This points to the existence of a bio-

which are exclusively isolated from marine sponges. Over chemical target rather than of unspecific effects. Mono- and

the last three decades, there has been intense work on the nonbrominated derivatives of dibromophakellstatin were

total synthesis of pyrrole–imidazole alkaloids.^[1]Usually, not cytotoxic. We were able to replace the imidazolinone by

biological activity has been discovered by testing the natural a dichlorocyclopropane unit to obtain a compound of high

products immediately after isolation from the sponge. Only average cytotoxicity but with a different and less pro-

occasionally has the large body of synthetic work on the nounced selectivity profile.^[7]

pyrrole–imidazole alkaloids contributed to the definition of

The experience gained in course of our total synthesis of

structure–activity relationships (SAR). Synthesis-based (–)-dibromophakellstatin^[9,10,11]has put us into a position

SAR studies have been published that concern fish feeding to address the synthesis of ring C-functionalized derivatives.

deterrence by oroidin,^[2]biofilm inhibition by oroidin and In this communication, we report on the stereoselective syn-

ageliferin,^[3]kinase inhibition by hymenialdisine deriva- thesis and cytotoxicity of ring C-substituted derivatives of

tives^[4]and the cytotoxicity of girolline,^[5]agelastatins^[6]and (–)-dibromophakellstatin (1, Figure 1). As precursor,

dibromophakellstatin (1).^[7]

N,NЈ-protected 12R-hydroxyphakellstatin (2) was chosen

Since the isolation of dibromophakellstatin from the (Scheme 1), which served as an intermediate in our chiral

sponge Phakellia mauritiana,^[8]it has been known that re- pool, enantioselective total synthesis of (–)-dibromo-

placement of the urea group by a guanidinium unit leads phakellstatin.^[11]The 12-hydroxy group was intended to

to the complete loss of cytotoxicity.^[8]We were able to show serve as an anchor for functionalization.^[12]

[a] Institute of Organic Chemistry, TU Braunschweig,

Hagenring 30, 38106 Braunschweig, Germany

Fax: +49-531-391-7744

E-mail: th.lindel@tu-bs.de

[b] TU Braunschweig, Institute of Inorganic and Analytical Chemistry,

Hagenring 30, 38106 Braunschweig, Germany

[c] Oncotest Institute of Experimental Oncology GmbH,

Am Flughafen 12-14, 79108 Freiburg, Germany

Supporting information for this article is available on the

WWW under http://dx.doi.org/10.1002/ejoc.201101175.

Figure 1. Cytotoxic marine natural product 1.

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685

T. Lindel et al.

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philes was considered a facile access to O-alkylated deriva-

tives of 2. Surprisingly, treatment of

2 with NaH

(1.1 equiv.)/MeI (5 equiv.) in N,N-dimethylformamide

(DMF) at –20 °C did not provide the methyl ether. Instead,

formation of the dialkylcarbonate 9 was observed, with

concomitant N-methylation of ring D at the former posi-

tion of the ethoxycarbonyl group (Scheme 2). The HMBC

spectrum of 9 exhibits correlations of the N-methyl protons

(3.20 ppm) with both the urea carbonyl (155.6 ppm) and

the N,N-acetal carbon atom (70.5 ppm). The HRMS of 9

confirmed the molecular formula of C₂₂H₂₄N₄NaO₈S ([M

+ Na]⁺) and indicated that all atoms of the ethoxycarbonyl

1

group were still present in the molecule. In the H NMR

spectrum, the methine proton 12-H experienced a charac-

teristic downfield shift on conversion of 2 (4.64 ppm) to 9

(5.34 ppm).

Scheme 1. Final steps in the synthesis of 5.

Results and Discussion

Starting material 2^[11]was converted into (–)-(12R)-di-

bromo-12-hydroxyphakellstatin (5) in three steps

(Scheme 1), which commenced with dibromination to 3.

Compound 3 was acetylated to afford the 12-acetoxy deriv-

ative 4 in 81% yield. Treatment of 4 with two equivalents of

in-situ-generated^[13]SmI₂in tetrahydrofuran (THF)/MeOH

(1:1) afforded a mixture of the starting material and par-

tially deprotected products. After adding one more equiva-

lent of SmI₂, 5 was formed in 80% yield. Deacetylation and

decarbonylation may take place by methanolysis mediated

by Sm^III, which was formed by oxidation of Sm^II. In the

absence of the acetoxy group, deprotection was ac-

companied by monodebromination to 6 when 3 was treated

with three equivalents of SmI₂. Aryl halides need only one

Scheme 2. Behaviour of 2 on treatment with NaH/MeI.

We also ran the reaction at –50 °C and, after quenching

equivalent of SmI₂for reduction because the aryl radical with aqueous sodium hydrogen carbonate, were able to iso-

abstracts a hydrogen atom from THF.^[14]We also synthe- late and fully characterize intermediate 12, which bears two

sized (12S)-12-bromophakellstatin (8) by an Appel reaction ethoxycarbonyl groups, along with equal amounts of the

of 2, which was followed by SmI₂-mediated deprotection of N-methylated tetracycle 11, which lacks an ethoxycarbonyl

intermediate 7. The secondary bromide was not reduced, group (Scheme 2). Intramolecular transfer of the ethoxy-

whereas a larger excess of SmI₂led to debromination.^[11]

carbonyl group can be excluded for steric reasons. It seems

However, as the deacetylation of 4 took place on treat- that the ethoxycarbonylated urea moiety of 2 resembles a

ment with SmI₂, we had to search for a functionalization pyrocarbonate and is more electrophilic than iodomethane.

of ring C that was more robust than O-acylation and would The deprotonated urea that resulted from the intermo-

survive the deprotection of ring D. Initially, nonbrominated lecular ethoxycarbonyl transfer was quenched by iodo-

tetracycle 2 was chosen as starting material. Deprotonation methane to afford equal amounts of 11 and 12. The treat-

of the hydroxy group followed by quenching with electro- ment of 9 and 12 with SmI₂afforded 12-hydroxy-N-meth-

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(–)-Dibromophakellstatin Derivatives

ylphakellstatin (10) and 13-hydroxyphakellstatin (13), cloaddition of 23 and 30 proceeded smoothly to afford the

respectively (Scheme 2). The dibrominated analog 3 did not fluorescent dibromophakellstatin analog 24 (Scheme 4). For

react as cleanly as nonbrominated 2 and we were not able the synthesis of 27, which is equipped with both biotin and

to isolate major products. Probably, the bromine substituent azide labels, the 1,3-dipolar cycloaddition of 23 and 32 to

in the vicinal position of the pyrrole nitrogen was bulky

enough to slow nucleophilic attack at the carbamoyl

carbon, which gave scope for side reactions.

With the Williamson approach being unsuccessful, we

turned to nucleophilic substitution of the 12-oxy substitu-

ent of 3. We had confidence in choosing dibrominated 3 as

the starting material because O-acylation had been success-

ful (Scheme 1). Indeed, the reaction of 3 with Tf₂O–pyr-

idine afforded the 12-triflyloxy product 14 in 86% yield

(Scheme 3). Nucleophilic attack by MeOH, EtOH or

iPrOH in the presence of NEt₃afforded the N,NЈ-protected

12-alkoxydibromophakellstatin derivatives 17, 19, and 21,

respectively, in very good yields with inversion at C-12. De-

protection with SmI₂/MeOH/THF provided the dibro-

mophakellstatin ethers 18, 20, and 22. X-ray analysis of

ethoxy derivative 20 proved the stereochemical course of

the reaction. It was also possible to reintroduce a hydroxy

group at ring C (15) when water was used as a nucleophile

with CH₂Cl₂as the cosolvent. Thus, both epimeric di-

bromo-12-hydroxyphakellstatins (5 and 16) were made

available.

Scheme 4. Functionalization of alkyne 23 by click reactions.

Scheme 3. Epimerization to ring C-oxygenated dibromophakellsta-

tin derivatives and crystal structure of 20 (hydrogen atoms are

omitted for clarity).

Aiming at derivatives suited for studies in chemical bio-

logy, we prepared terminal alkyne 23 by the reaction of

triflate 14 with 5-hexyn-1-ol in the presence of NEt₃in

CH₂Cl₂followed by SmI₂-mediated deprotection. Click cy- Scheme 5. Synthesis of azides 30 and 32.

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687

T. Lindel et al.

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afford amine 25 was followed by amide coupling, which em- (Scheme 6). The regioisomeric protected dehydrophak-

ployed the biocytin-derived acid 26^[18](Scheme 4). One of ellstatins 33 and 35 were formed together with the chloro

the coupling partners, the new azido-functionalized 7-(di- derivative 34. We were able to obtain 35 in 35% yield, which

methylamino)coumarine 30, was prepared in two steps and was deprotected without decomposition by treatment with

80% yield from the coumarin-derived acid 28^[15]by treat- SmI₂. Thus, we synthesized 11,12-dehydrophakellstatin (36)

ment with N-hydroxysuccinimide (NHS)/dicyclohexylcar- as the first example of a phakellin skeleton with an unsatu-

bodiimide (DCC) followed by the addition of excess 1,2- rated ring C. The absolute stereochemistry of the chlorin-

diaminoethane (Scheme 5a). The conversion of amine 29 to ated product 34 was established by X-ray analysis, which

azide 30 was accomplished by reaction with freshly pre- revealed that the triflate group was substituted by chloride

pared triflyl azide with the adaptation of a copper-catalyzed with inversion at C-12. The slow reaction of dichlorometh-

diazo transfer reaction.^[16]Azide 30 was purified by fil- ane with primary, secondary and tertiary aliphatic amines is

tration through a pad of silica gel [CHCl₃/MeOH (9:1) + known to afford the corresponding ammonium chloride,^[19]

1% aqueous ammonia] but was unstable and used immedi- which is probably the source of chloride here.

ately in the subsequent click reaction. As an alternative cou-

In addition, we were able to functionalize 3 without in-

pling partner for the terminal alkyne 23, aminoazide 32 was version at C-12 by avoiding Williamson conditions

prepared starting from hexaethylene glycol (31) in three (Scheme 7). Ring D-protected (12R)-dibromo-12-hydroxy-

steps by double tosylation, conversion to the diazide^[17]and phakellstatin (3) was methoxymethyl ether (MOM)-pro-

mono-Staudinger reduction (Scheme 5b).

tected on treatment with dimethoxymethane in CH₂Cl₂in

On treatment of the dibrominated triflate 14 with 5- the presence of anhydrous FeCl₃adsorbed onto molecular

hexyn-1-ol in the presence of NEt₃(Scheme 4), we had ob- sieves (4 Å).^[20]Molecular sieves enhanced the yield by cap-

served the formation of minor side products that had not turing MeOH. The yield was 91%, and both ring D protect-

incorporated the alcohol nucleophile. Similar behaviour ing groups remained in place. Treatment of 37 with SmI₂

was observed for the nonbrominated triflate, where separa- deprotected the urea unit without affecting the MOM

tion of the products by column chromatography seemed to group to afford the 12R-OMOM-functionalized dibro-

be more facile. Treatment of nonbrominated 2 with Tf₂O mophakellstatin derivative 38 (85%).

generated the triflate in situ, which was exposed to NEt₃in

dichloromethane in the absence of an alcohol nucleophile

Scheme 7. MOM-protection of 38 mediated by FeCl₃on molecular

sieves (4 Å).

Table 1 summarizes the cytotoxicities of 1, 5, 6, 38, 22,

23, and 24 against a panel of twelve selected human cancer

cell lines (stomach, lung, breast, melanoma, ovary, pan-

creas, prostate, mesothelioma, kidney and uterus). Com-

pounds 8, 10, 13, 16, 18, 20, 22, 25, and 27 proved to be

inactive (absolute IC₅₀Ͼ 30 μm) against all cell lines and

are not included in Table 1.

The uterus cancer cell line UXF 1138L was the most sen-

sitive cell line against 1 and 5 with absolute IC₅₀values of

0.07 and 0.27 μm, respectively, followed by the ovarian can-

cer cell line OVXF 899L (IC₅₀= 0.09 and 0.70 μm).

Furthermore, 5 showed above-average activity (IC₅₀Ͻ 1

μm) against lung and mesothelioma cancer lines and exhib-

ited a higher average IC₅₀(1.34 μm) against an extended

panel of 42 cell lines (see Supporting Information) com-

pared to 1. However, higher selectivity was observed for 1.

Scheme 6. Access to 36 and crystal structure of 34 (hydrogen atoms

are omitted for clarity).

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(–)-Dibromophakellstatin Derivatives

Table 1. Proliferation inhibition of twelve human cancer cell lines by 1, 5, 6, 38, 22, 23, and 24 (absolute IC₅₀in μm, determined at five

concentrations).

Tumor type

Stomach

Cell line

1

5

6

38

22

23

24

CXF HT29

GXF 251L

LXF 529L

LXF 629L

MAXF 401NL

MEXF 462NL

OVXF 899L

PAXF 1657L

PRXF 22RV1

PXF 1752L

RXF 486L

UXF 1138L

–

Ͼ100

39.8

42.5

18.5

Ͼ100

60.4

0.09

Ͼ100

2.31

0.27

0.07

10.5

3.71

0.73

2.15

0.95

3.59

1.30

0.70

2.33

3.80

0.59

1.32

0.27

1.34

Ͼ100

77.1

Ͼ100

90.7

Ͼ100

66.3

Ͼ100

48.3

Ͼ100

79.0

72.5

Ͼ100

8.02

59.5

88.2

47.8

76.8

51.4

–

Ͼ100

69.6

Ͼ100

39.4

58.9

Ͼ100

52.0

39.4

49.8

–

Ͼ100

83.8

54.1

95.8

18.5

Ͼ100

8.54

Ͼ100

57.9

48.9

35.9

–

Ͼ30

25.6

Ͼ30

17.9

27.7

33.7

Ͼ30

21.3

–

Lung

Breast

Melanoma

Ovary

Pancreas

Prostate

Mesothelioma

Kidney

Ͼ100

42.2

–

Uterus

Geomean IC₅₀

The cytotoxicity of 38 with 12R configuration was low, and toxicity of (–)-dibromophakellstatin (1), which is encourag-

only the ovarian cancer cell line OVXF 899L was sensitive ing for the search for biochemical targets in future work.

to it (IC₅₀= 8.0 μm). Among the alkoxy derivatives of the For instance, the pyrrole–imidazole alkaloid hymenialdisine

12S series shown in Scheme 3, only 22 showed weak cytoto- was functionalized with a linker that resulted in loss of kin-

xicity. The coumarin derivative 24 exhibited modest activity ase inhibition by a factor of about ten, which did not pre-

(IC₅₀= 17.8 μm against the lung cancer cell line LXF 629L). vent protein binding by affinity chromatography.^[4b]An-

The alkyne derivative 23 also showed moderate activity (8.6 other example is the pyrrolo-quinoline ammosamide B,

μm against the ovarian cancer cell line OVXF 899L). Sur- which was functionalized with a coumarin tag and resulted

prisingly, diastereomer 16 of compound 5 is not cytotoxic. in the loss of cytotoxicity from 320 to 17 μm, nevertheless

As previously observed for other phakellstatin derivatives, the identification of myosin as a target protein was pos-

almost none of the nonbrominated compounds exhibited sible.^[21]

cytotoxicity. Solely, monobrominated analog 6 retained

weak cytotoxicity.

For the two most cytotoxic compounds, 5 and 1, testing

was extended to 42 cell lines (see Supporting Information).

Average relative IC₅₀values of 3.67 and 13.11 μm were de-

termined for 5 and 1, respectively. At higher doses, 5

showed complete cytotoxicity against almost all of the in-

vestigated cancer cell lines, whereas 1 did not.

Figure 2 summarizes our current knowledge on struc-

ture–activity relationships regarding the cytotoxicity of 1.

We had hoped that 38 would either be more cytotoxic itself

or could be hydrolyzed rapidly in the cell to liberate 5.

However, compared to the 12S-methoxy or ethoxy deriva-

tives 18 and 20, 38 was still more cytotoxic. Thus, it appears

worthwhile to continue with the synthesis of more deriva-

Conclusions

Our triflate inversion approach gave access to the first

series of ring C-functionalized derivatives of 1. Formation

of the dehydrophakellstatin skeleton 36 was achieved for the

first time and may enable ring anellation at ring C, which is

a structural feature of the palau’amine-type pyrrole–imid-

azole alkaloids.

Experimental Section

General: NMR spectra were recorded with Bruker DRX-400

(400.1 MHz for ¹H, 100.6 MHz for ¹³C), Bruker AV III-400

tives of the 12R series. We consider it promising that alkyne (400.1 MHz for H, 100.6 MHz for ¹³C) and Bruker AV II-600 in-

23 and coumarin conjugate 24 still retain some of the cyto-

1

struments (600.1 MHz for H, 150.9 MHz for ¹³C) and were refer-

1

enced to the solvent signal or Me₄Si. All measurements were car-

ried out at 300 K. Mass spectra were obtained with LTQ Orbitrap

Velos, Thermo Finnigan LTQ FT, Thermo Finnigan MAT95 and

Finnigan MAT 95 XLT spectrometers. IR spectra were recorded

with a Bruker Tensor 27 spectrometer. UV/Vis spectra were mea-

sured with a Varian Cary 100 Bio UV/Vis spectrometer. Melting

points were measured with a Büchi 530 melting point apparatus.

Chemicals were purchased from commercial suppliers and used

without further purification. Silica gel 60 (40–63 μm, Merck) was

used for column chromatography.

Ethyl (3aS,5R,12aS)-5-Acetoxy-10,11-dibromo-2,8-dioxo-3-(tosyl-

oxy)-2,3,5,6,8,12a-hexahydroimidazo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]-

pyrazine-1(4H)-carboxylate (4): Pyridine (0.5 mL) and Ac₂O

(0.6 mL) were added to a solution of 3 (20.0 mg, 0.031 mmol,

Figure 2. SAR regarding the cytostatic activity of 1.

1 equiv.) in CH₂Cl₂(2 mL). After 12 h at room temperature, the

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solvent was evaporated under reduced pressure, and the residue was

purified by column chromatography on silica gel [EtOAc/isohexane

(1:1)]. Compound 4 (17.2 mg, 0.025 mmol, 81%) was obtained as

a colourless solid. m.p. 151 °C. TLC [silica gel, EtOAc/isohexane

(2:3)]: R_f= 0.32. ¹H NMR (400 MHz, CDCl₃): δ = 7.62 [d, J =

48.5

(CCH₂CH)

ppm.

HRMS

(ESI⁺):

calcd.

for

C₁₁H₁₁⁷⁹BrN₄NaO₃[M + Na]⁺348.9912; found 348.9907. IR

(ATR): ν = 3412 (s), 3215 (s), 3133 (m), 1729 (m), 1701 (m), 1603

(s), 1549 (s), 1479 (s), 1438 (s), 1379 (m), 1289 (m), 1289 (m), 1252

(w), 1179 (m), 1122 (m), 1070 (m), 993 (m), 927 (m), 779 (m), 733

˜

8.4 Hz, O₂SC(CH)₂], 7.25 [d, J = 8.0 Hz, 2 H, CH₃C(CH)₂], 7.21 (m), 576 (s) cm^–1. UV (MeOH): λ_max(logε) = 279 (3.6), 221

(s, 1 H, BrCCH), 6.33 (s, 1 H, NCHN), 5.45 (m, 1 H, OCHCH₂), (4.1) nm.

4.43 (m, 2 H, CH₃CH₂), 4.04 (dt, J = 1.6, J = 12.9 Hz, 1 H,

(3aR,5S,12aS)-5-Bromo-1,5,6,12a-tetrahydroimidazo[4,5-b]di-

NCHHCHCH₂), 3 . 9 3 ( dd , J = 5 . 0 , J = 1 3 . 2 H z , 1 H ,

pyrrolo[1,2-a:1Ј,2Ј-d]pyrazine-2,8(3H,4H)-dione (8): To a solution of

NCHHCHCH₂), 2 . 9 5 ( dd , J = 6 . 4 , J = 1 6 . 2 H z , 1 H ,

7 (20 mg, 0.036 mmol, 1 equiv.) in degassed MeOH (1 mL) was

NCH₂CHCHH), 2.44 (s, 3 H, CH₃CCH), 2.40 (ddd, J = 1.0, J =

added SmI₂(0.1 m in THF, 1.1 mL, 0.11 mmol, 3 equiv.). The reac-

3.0, J = 12.8 Hz, 1 H, NCH₂CHCHH), 2.06 [s, 3 H, O(CO)CH₃],

tion was stirred at room temperature for 2 h before the solvent

was evaporated under reduced pressure and the residue purified by

column chromatography [silica gel, CHCl₃/MeOH (9:1)] to give 8

(16.2 mg, 0.07 mmol, 92%) as a colourless solid. m.p. 285 °C.

1.38 (t, J = 6.9 Hz, 3 H, CH₃CH₂) ppm. ¹³C NMR (100 MHz,

CDCl₃): δ = 170.0 (CHOCOCH₃), 153.8 (NCOOCH₂), 149.7

(NCOC), 147.1 (NCON), 144.9 (CH₃CCH), 130.0 [H₃CC(CH)₂],

129.4 [O₂SC(CH)₂], 129.3 [O₂SC(CH)₂], 127.8 (BrCCHC), 117.6

[α]²_D¹= +12 (c = 2 mg/mL, MeOH). TLC [silica gel, CHCl₃/MeOH

(BrCCH), 104.6 (NCBrCBr), 99.8 (NCBrCBr), 58.6 (NCHCCH₂),

1

(9:1)]: R_f= 0.48. H NMR (400 MHz, [D₆]DMSO): δ = 8.18 (s, 1

69.7 (CH₂CHCH₂), 67.8 (NCHN), 65.4 (CH₃CH₂), 52.1

H, CNHCO), 7.54 (s, 1 H CHNHCO), 7.16 (dd, J = 1.7, J =

(NCH₂CH), 38.6 (CCH₂CHCH₂), 21.9 (CH₃CCH), 20.9 (CH₃CO),

2.6 Hz, 1 H, NCHCH), 6.72 (dd, J = 1.6, J = 3.7 Hz, 1 H,

1 4 . 2 ( C H ₃C H ₂) p p m . H R M S ( E S I ⁺) : c a l c d . f o r

NCHCHCH), 6.30 (dd, J = 2.7, J = 3.6 Hz, 1 H, NCHCH), 5.70

(s, 1 H, NHCHN), 4.58 (q, 1 H, CHBr), 4.33 (dd, J = 6.7, J =

11.9 Hz, 1 H, NCHHCBr), 3.64 (dd, J = 6.8, J = 11.9 Hz, 1 H,

NCHHCHBr), 3.23 (dd, J = 7.5, J = 14.1 Hz, 1 H, CCHHCHBr),

2.49 (dd, J = 6.5, J = 14.1 Hz, 1 H, CCHHCHBr) ppm. ¹³C NMR

(100 MHz, [D₆]DMSO): δ = 158.5 (NCOC), 155.2 (NHCONH),

C₂₃H₂₂⁷⁹Br₂N₄NaO₉S [M + Na]⁺710.9366; found 710.9351. IR

(ATR): ν = 1812 (m), 1745 (s), 1677 (s), 1431 (m), 1409 (m), 1385

˜

(s), 1267 (s), 1237 (s), 1195 (s), 721 (m), 548 (m) cm^–1. UV (CHCl₃):

λ_max(logε) = 287 (4.0), 239 (3.9) nm.

(3aR,5R,12aS)-10,11-Dibromo-5-hydroxy-1,5,6,12a-tetrahydroimid-

azo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]pyrazine-2,8(3H,4H)-dione (5): 122.7 (NCCHCH), 122.3 (NCHCH), 112.1 (NCHCHCH), 110.8

SmI₂(0.1 m in THF, 0.9 mL, 0.09 mmol, 3 equiv.) was added to a

solution of 4 (20 mg, 0.03 mmol, 1 equiv.) in degassed MeOH

(1 mL). The reaction mixture was stirred for 1 h at room tempera-

ture followed by solvent evaporation and purification by column

(NCHCH), 78.6 (NHCCH₂), 68.0 (NCHNH), 53.6 (NCH₂CH),

48.4 (CCH₂CH), 40.4 (CHBr) ppm. HRMS (ESI⁺): calcd. for for

C₁₁H₁₁⁷⁹BrN₄NaO₂[M + Na]⁺332.9963; found 332.9958. IR

(ATR): ν = 3202 (m), 3107 (m), 1729 (s), 1655 (s), 1540 (m), 1468

˜

chromatography [silica gel, CHCl₃/MeOH (9:1)] to give 5 (10.1 mg, (m), 1412 (m), 1349 (s), 1245 (m), 1218 (m), 1103 (m), 1062 (m),

0.02 mmol, 80%) as a colourless solid. m.p. 255 °C. [α]²_D¹= –152

(2 mg/mL, MeOH). TLC [silica gel, CHCl₃/MeOH (4:1)]: R_f= 0.35.

¹H NMR (400 MHz, [D₄]methanol): δ = 6.96 (s, 1 H, BrCCH),

5.96 (s, 1 H, NCHNH), 4.65 (m, 1 H, NCH₂CHCH₂), 3.81 (dd, J

= 6.2, J = 12.1 Hz, 1 H, NCHHCH), 3.68 (dd, J = 4.2, J = 12.0 Hz,

1 H, NCHHCH), 2.58 (dd, J = 6.0, J = 13.3 Hz, 1 H, CCHHCH),

2.50 (dd, J = 6.2, J = 13.3 Hz, 1 H, CCHHCH) ppm. ¹³C NMR

(150 MHz, [D₄]methanol): δ = 160.5 (CCON), 157.2 (NHCONH),

126.0 (BrCCHC), 116.6 (CBrCH), 107.8 (NCBrCBr), 103.3

(NCBrCBr), 80.5 (NCHCCH₂), 70. 8 (NCHNH), 67.6

(NCH₂CHOH), 53.5 (NCH₂CH), 49.9 (CCH₂CHCH₂) ppm.

HRMS (ESI⁺): calcd. for C₁₁H₁₁⁷⁹Br⁸¹BrN₄O₃[M + H]⁺406.9177;

1015 (m), 838 (m), 745 (s), 728 (s), 709 (s), 651 (s), 603 (m), 566 (m),

537 (m) cm^–1. UV (CHCl₃): λ_max(logε) = 275 (4.0), 230 (3.8) nm.

(3aS,5R,12aR)-5-[(Ethoxycarbonyl)oxy]-1-methyl-2,8-dioxo-

1,5,6,12a-tetrahydroimidazo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]pyrazin-

3(2H,4H,8H)-yl 4-Methylbenzenesulfonate (9), (3aS,5R,12aR)-5-

Hydroxy-1-methyl-2,8-dioxo-1,5,6,12a-tetrahydroimidazo[4,5-b]di-

pyrrolo[1,2-a:1Ј,2Ј-d]pyrazin-3(2H,4H,8H)-yl 4-Methylbenzenesulf-

onate (11) and Ethyl (3aS,5R,12aR)-5-[(Ethoxycarbonyl)oxy]-2,8-

dioxo-3-(tosyloxy)-2,3,5,6,8,12a-hexahydroimidazo[4,5-b]dipyrrolo-

[1,2-a:1Ј,2Ј-d]pyrazine-1(4H)-carboxylate (12): At –20 °C, NaH

(60% in mineral oil, 2.2 mg, 0.067 mmol, 1.1 equiv.) was added to

a solution of 2 (30 mg, 0.061 mmol, 1 equiv.) in DMF (2 mL) fol-

lowed by MeI (20 μL, 0.305 mmol, 5 equiv.). The reaction mixture

was stirred for 2 h and allowed to reach room temperature. Satu-

rated aqueous NaHCO₃(4 mL) and CH₂Cl₂(4 mL) were added.

found 406.9167. IR (ATR): ν = 1717 (s), 1541 (m), 1440 (s), 1394

˜

(s), 1202 (s), 1175 (s), 1119 (m), 1082 (m), 736 (m), 683 (s) cm^–1.

UV (CHCl₃): λ_max(logε) = 287 (3.9) nm.

(3aR,5R,12aS)-10-Bromo-5-hydroxy-1,5,6,12a-tetrahydroimidazo- The phases were separated, and the aqueous phase was washed

[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]pyrazine-2,8(3H,4H)-dione (6): SmI₂

(0.1 m in THF, 0.9 mL, 0.09 mmol, 3 equiv.) was added to a solu-

tion of 3 (20 mg, 0.03 mmol, 1 equiv.) in degassed MeOH (1 mL).

The reaction mixture was stirred for 1 h at room temperature fol-

lowed by solvent evaporation and purification by column

three times with CH₂Cl₂. The combined organic phases were dried

(MgSO₄), and the solvent was removed under reduced pressure.

The residue was purified by column chromatography [silica gel,

EtOAc/isohexane (2:3 to 1:1)] to give 9 (23.1 mg, 0.046 mmol,

75%), 11 (1.01 mg, 0.002 mmol, 4%) and 12 (2.4 mg, 0.004 mmol,

chromatography [silica gel, CHCl₃/MeOH (9:1)] to give 6 (8.5 mg, 7%). Compound 9: m.p. 155 °C. TLC [silica gel, EtOAc/isohexane

0.02 mmol, 85%) as a colourless solid. m.p. 230 °C. [α]²_D²= –111 (c

= 5 mg/mL, MeOH). TLC [silica gel, CHCl₃/MeOH (9:1)]: R_f=

0.14. H NMR (400 MHz, [D₄]methanol): δ = 7.17 (d, J = 1.7 Hz,

(4:1)]: R_f= 0.55. ¹H NMR (400 MHz, CDCl₃): δ = 7.63 [d, J =

8.4 Hz, 2 H, O₂SC(CH)₂], 7.22 [d, J = 8.1 Hz, 2 H, CH₃C(CH)₂],

7.04 (dd, J = 1.5, J = 3.9 Hz, 1 H, NHCHCHCH), 6.79 (dd, J =

1

1 H, NCHCBr), 6.82 (d, J = 1.7 Hz, 1 H, CCHCBr), 5.77 (s, 1 H, 1.5, J = 2.7 Hz, 1 H, NHCHCHCH), 6.37 (dd, J = 2.8, J = 3.8 Hz,

NCHN), 4.65 (m, 1 H, HOCHCH₂), 3.79 (dd, J = 6.0, J = 12.0 Hz, 1 H, NHCHCHCH), 5.48 (s, 1 H, NCHN), 5.34 (m, 1 H,

1 H NCHHCH), 3.65 (dd, J = 4.4, J = 12.0 Hz, 1 H, NCHHCH), CH₂CHCH₂), 4.19 (m, 2 H, CH₃CH₂), 4.06 (dt, J = 1.6, J =

2.51 (m, 2 H, CCH₂CH) ppm. ¹³C NMR (100 MHz, [D₄]meth-

anol): δ = 161.2 [C(CO)N], 157.8 [N(CO)N], 124.6 (CHCCO),

123.3 (NCHCBr), 115.6 (CCHCBr), 100.1 (CHCBr), 80.3

13.3 Hz, 1 H, NCHHCHCH₂), 3.75 (dd, J = 5.0, J = 13.3 Hz,

NCHHCHCH₂), 3.20 (s, 3 H, CH₃N), 3.02 (d, 6.3, J = 15.1 Hz, 1

H, NCH₂CHCHH), 2.43 (m, 1 H, NCH₂CHCHH), 2.42 (s, 3 H,

(NCCH₂), 70.0 (NCHNH), 67.9 (OHCHCH₂), 53.8 (NCH₂CH), CH₃C), 1.29 (t, J = 7.1 Hz, 3 H, CH₃CH₂) ppm. ¹³C NMR

690

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Eur. J. Org. Chem. 2012, 685–698

(–)-Dibromophakellstatin Derivatives

(100 MHz, CDCl₃): δ = 155.6 [N(CO)N], 155.5 [C(CO)N], 154.0 (50 mL). The combined organic phases were dried with MgSO₄,

[EtO(CO)N], 146.6 [H₃CC(CH)₂], 129.8 [O₂SC(CH)₂], 129.8 filtered and the solvent was evaporated under reduced pressure.

[H₃CC(CH)₂], 129.4 [O₂SC(CH)₂], 124.2 (NCHCHCHC), 121.2

The residue was purified by column chromatography [silica gel,

(NCHCHCHC), 114.3 (NCHCHCHC), 112.2 (NCHCHCHC), CHCl₃/MeOH (9:1)] to give 10 as a colourless solid (4.62 mg,

85.1 (HCCCH₂), 73.6 (NCH₂CHCH₂), 70.5 (NCHN), 64.7 0.026 mmol, 89%). m.p. 285 °C. [α]²_D²= +13 (c = 0.5 mg/mL,

(OCH₂CH₃), 51.7 (NCH₂CHCH₂), 38.4 (NCH₂CHCH₂), 31.6

MeOH). TLC [silica gel, CHCl₃/MeOH (9:1)]: R_f= 0.28. ¹H NMR

(CH₃N), 21.9 (PhCH₃), 14.1 (OCH₂CH₃) ppm. HRMS (ESI⁺): (600 MHz, [D₆]DMSO): δ = 7.81 (s, 1 H, CNHC), 7.19 (dd, J =

calcd. for C₂₂H₂₄N₄NaO₈S [M + Na]⁺527.1213; found 527.1207. 1.6, J = 1.7 Hz, 1 H, NHCHCHCH), 6.72 (dd, J = 1.6, J = 3.8 Hz,

IR (ATR): ν = 1769 (s), 1745 (m), 1661 (m), 1550 (m), 1468 (m), 1 H, NHCHCHCH), 6.29 (dd, J = 2,7, J = 3.7 Hz, 1 H,

˜

1419 (s), 1374 (s), 1255 (s), 1193 (s), 1178 (s), 1072 (m), 1041 (s), NHCHCHCH), 5.91 (s, 1 H, NCHN), 5.27 (d, J = 4.5 Hz, 1 H,

1015 (s), 967 (m), 813 (m), 788 (s), 732 (s), 657 (m), 542 (s) cm^–1. OHCHCH₂), 4.55 (m, 1 H, CH₂CHCH₂), 3.69 (dd, J = 6.4, J =

UV (CHCl₃): λ_max(logε) = 276 (3.9), 229 (4.1) nm. Compound 11:

m.p. 172 °C. TLC (silica gel, EtOAc): R_f= 0.22. ¹H NMR

11.5 Hz, 1 H, NCHHCHCH₂), 3.40 (dd, J = 4.5, J = 11.5 Hz,

NCHHCHCH₂), 3.35 (s, H, CH₃N), 2.32 (m, H,

3

2

(400 MHz, CDCl₃): δ = 7.57 [d, J = 8.3 Hz, 2 H, O₂SC(CH)₂], 7.18 NCH₂CHCH₂) ppm. ¹³C NMR (150 MHz, [D₆]DMSO): δ = 157.6

[d, J = 8.1 Hz, 2 H, CH₃C(CH)₂], 6.90 (dd, J = 1.5, J = 3.9 Hz, 1

H, NHCHCHCH), 6.68 (dd, 1.5, 2.7 Hz, H,

[N(CO)N], 155.7 [C(CO)N], 123.2 (NCHCHCHC), 123.0

(NCHCHCHC), 112.0 (NCHCHCHC), 110.7 (NCHCHCHC),

J

=

J

=

1

NHCHCHCH), 6.27 (dd, J = 2.8, J = 3.8 Hz, 1 H, NHCHCHCH), 76.9 (HCCCH₂), 71.7 (NCHN), 65.6 (NCH₂CHCH₂), 51.9

5.48 (s, 1 H, NCHN), 4.64 (m, 1 H, CH₂CHCH₂), 3.84 (dt, J = (NCH₂CHCH₂), 47.4 (NCH₂CHCH₂), 28.4 (CH₃N) ppm. HRMS

1.5, J = 12.5 Hz, 1 H, NCHHCHCH₂), 3.59 (dd, J = 4.6, J = (ESI⁺): calcd. for C₁₂H₁₄N₄NaO₃[M + Na]⁺285.0963; found

12.5 Hz, NCHHCHCH₂), 3.57 (s, 1 H, CHOH), 3.17 (s, 3 H, 285.0958. IR (ATR): ν = 3223 (m), 1704 (s), 1613 (s), 1551 (s), 1471

˜

CH₃N), 2.77 (dd, J = 5.6, J = 14.5 Hz, 1 H, NCH₂CHCHH), 2.26 (s), 1441 (s), 1418 (s), 1378 (s), 1269 (m), 1239 (m), 1222 (m), 1183

(dt, J = 1.5, J = 15.6 Hz, 1 H, NCH₂CHCHH), 2.40 (s, 3 H, CH₃C)

(s), 1091 (s), 1067 (m), 1021 (m), 1001 (m), 804 (s), 735 (s), 690

ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 156.5 [N(CO)N], 155.8

(m), 644 (s), 602 (m), 566 (s) cm^–1. UV (CHCl₃): λ_max(logε) = 275

[C(CO)N], 146.5 [H₃CC(CH)₂], 129.8 [O₂SC(CH)₂], 129.7 (3.9), 225 (3.9) nm.

[H₃CC(CH)₂], 129.3 [O₂SC(CH)₂], 123.9 (NCHCHCHC), 121.5

(3aR,5R,12aS)-5-Hydroxy-1,5,6,12a-tetrahydroimidazo[4,5-b]dipyr-

(NCHCHCHC), 113.9 (NCHCHCHC), 111.7 (NCHCHCHC),

85.6 (HCCCH₂), 70.5 (NCHN), 67.9 (NCH₂CHCH₂), 54.4

(NCH₂CHCH₂), 40.3 (NCH₂CHCH₂), 31.6 (CH₃N), 21.9 (PhCH₃)

ppm. HRMS (ESI⁺): calcd. for C₁₉H₂₀N₄NaO₆S [M + Na]⁺

rolo[1,2-a:1Ј,2Ј-d]pyrazine-2,8(3H,4H)-dione (13): To a solution of

12 (40 mg, 0.07 mmol, 1 equiv.) in degassed MeOH (2 mL) was

added SmI₂(0.1 m in THF, 2.2 mL, 0.22 mmol, 3.2 equiv.). The re-

action was stirred at room temperature for 2 h before the solvent

was evaporated under reduced pressure and the residue was puri-

fied by column chromatography [silica gel, CHCl₃/MeOH (9:1)] to

give 13 (16.2 mg, 0.07 mmol, 92%) as a colourless solid. m.p.

280 °C. [α]²_D¹= –97 (c = 5 mg/1 mL MeOH). TLC [silica gel, CHCl₃/

MeOH (9:1)]: R_f= 0.12. ¹H NMR (400 MHz, [D₆]DMSO): δ =

8.13 (s, 1 H, CNHCO), 7.70 (s, 1 H CHNHCO), 7.13 (dd, J = 1.6,

J = 2.6 Hz, 1 H, NCHCHCH), 6.67 (dd, J = 1.6, J = 3.7 Hz, 1 H,

NCHCHCH), 6.27 (dd, J = 2.7, J = 3.7 Hz, 1 H, NCHCHCH),

5.72 (s, 1 H, NCHN), 5.28 (d, J = 4.6 Hz, 1 H, OH), 4.57 (m, 1 H,

CHOH), 3.60 (dd, J = 6.6, J = 13.6 Hz, 1 H, NCHHCHCHH),

3.36 (dd, J = 5.1, J = 11.4 Hz, 1 H, NCHHCHCHH), 2.32 (m, 2

H, NCH₂CHCH₂) ppm. ¹³C NMR (100 MHz, [D₆]DMSO): δ =

158.8 [N(CO)N], 165.7 [C(CO)N], 122.9 (COCCH), 122.1

(NCHCH), 111.5 (CCHCHCH), 110.6 (CHCHCH), 78.2

(NHCCH₂), 67.7 (NCHNH), 65.7 (OHCHCH₂), 52.0 (NCH₂CH),

47.2 (CCH₂CH) ppm. HRMS (ESI⁺): calcd. for C₁₁H₁₃N₄O₃[M +

455.1001; found 455.0995. IR (ATR): ν = 1767 (m), 1640 (m), 1375

˜

(m), 1192 (s), 1178 (s), 1087 (m), 1041 (m), 813 (m), 789 (m), 727

(s), 655 (m), 550 (s) cm^–1. UV (CHCl₃): λ_max(logε) = 270 (3.9),

240 (4.0) nm. Compound 12: m.p. 162 °C. TLC [silica gel, EtOAc/

1

isohexane (4:1)]: R_f= 0.65. H NMR (400 MHz, CDCl₃): δ = 7.59

[d, J = 8.4 Hz, 2 H, O₂SC(CH)₂], 7.22 [d, J = 8.0 Hz, 2 H,

CH₃C(CH)₂], 7.08 (dd, J = 1.5, J = 3.9 Hz, 1 H, NHCHCHCH),

6.88 (dd, J = 1.6, J = 2.8 Hz, 1 H, NHCHCHCH), 6.35 (dd, J =

2.9, J = 3.8 Hz, 1 H, NHCHCHCH), 6.19 (s, 1 H, NCHN), 5.36

(m, 1 H, CH₂CHCH₂), 4.43 [m, 2 H, CH₃CH₂O(CO)N], 4.19 [m,

2 H, CH₃CH₂O(CO)O], 4.13 (dt, J = 1.2, J = 13.3 Hz, 1 H,

NCHHCHCH₂), 3.85 (dd, J = 4.8, J = 13.3 Hz, NCHHCHCH₂),

2.98 (dd, J = 6.2, J = 15.2 Hz, 1 H, NCH₂CHCHH), 2.51 (dt, J =

1.7, J = 15.3 Hz, 1 H, NCH₂CHCHH), 2.42 (s, 3 H, CH₃C), 1.30

[t, J = 7.1 Hz, 3 H, CH₃CH₂O(CO)O] ppm. ¹³C NMR (100 MHz,

CDCl₃): δ = 155.2 [C(CO)N], 154.0 [EtO(CO)O], 150.9 [EtO(CO)

N], 149.3 [N(CO)N], 146.9 [H₃CC(CH)₂], 129.9 [H₃CC(CH)₂],

129.4 [O₂SC(CH)₂], 129.3 [O₂SC(CH)₂], 123.7 (NCHCHCHC),

H]⁺249.0983; found 249.0987. IR (ATR): ν = 3308 (m), 3169 (m),

˜

1689 (s), 1630 (s), 1552 (s), 1472 (s), 1433 (s), 1374 (s), 1325 (m),

1251 (m), 1219 (m), 1183 (m), 1149 (w), 1116 (m), 1093 (s), 1061

(s), 1020 (m), 1008 (m), 970 (m), 743 (s), 726 (s), 650 (s), 600 (m),

567 (m) cm^–1. UV (CHCl₃): λ_max(logε) = 274 (4.0), 230 (3.9) nm.

122.8

(NCHCHCHC),

114.9

(NCHCHCHC),

112.1

(NCHCHCHC), 83.7 (HCCCH₂), 73.4 (NCH₂CHCH₂), 66.6

(NCHN), 65.1 [CH₃CH₂O(CO)N], 64.7 [CH₃CH₂O(CO)O], 51.7

(NCH₂CHCH₂), 38.7 (NCH₂CHCH₂), 21.9 (PhCH₃), 14.1 [2 C,

CH₃CH₂O(CO)] ppm. HRMS (ESI⁺): calcd. for C₂₂H₂₆N₄NaO₁₀S

Ethyl (3aS,5R,12aS)-10,11-Dibromo-2,8-dioxo-3-(tosyloxy)-5-[{(tri-

fluoromethyl)sulfonyl}oxy]-2,3,5,6,8,12a-hexahydroimidazo[4,5-b]di-

[M + Na]⁺585.1267; 585.1261. IR (ATR): ν = 1809 (m), 1742 (s),

˜

pyrrolo[1,2-a:1Ј,2Ј-d]pyrazine-1(4H)-carboxylate

(14):

Tf₂O

1662 (m), 1469 (m), 1386 (s), 1260 (s), 1195 (s), 1181 (m), 1015 (m),

743 (m), 721 (m), 566 (m), 548 (m) cm^–1. UV (CHCl₃): λ_max(logε)

= 276 (3.9), 230 (4.2) nm.

(58.3 μL, 0.34 mmol, 1.5 equiv.) was added dropwise to a mixture

of 3 (150 mg, 0.23 mmol, 1 equiv.), pyridine (36.6 μL, 0.46 mmol,

2 equiv.) and CH₂Cl₂(40 mL) at 0 °C over 10 min. After 1 h at

0 °C, the solvent was evaporated, and the residue was purified by

column chromatography [silica gel, EtOAc/isohexane, (2:3)] to give

14 (160 mg, 0.20 mmol, 86%). m.p. 140 °C. TLC [silica gel, EtOAc/

isohexane (2:3)]: R_f= 0.73. ¹⁹F NMR (376 MHz, CDCl₃): δ =

(3aR,5R,12aR)-5-Hydroxy-1-methyl-1,5,6,12a-tetrahydroimidazo-

[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]pyrazine-2,8(3H,4H)-dione (10): Car-

bonate 9 (10 mg, 0.029 mmol, 1 equiv.) was dissolved in degassed

MeOH (2 mL). SmI₂(1 m in THF, 0.7 mL, 0.074 mmol, 2.5 equiv.)

was added and the reaction mixture was stirred at room tempera-

ture. After 2 h, a saturated aqueous solution of NaHCO₃(2 mL)

was added, and the mixture was extracted three times with CH₂Cl₂

1

–74.924 (s, 3 F, CF₃) ppm. H NMR (400 MHz, CDCl₃): δ = 7.61

[d, J = 8.4 Hz, 2 H, O₂SC(CH)₂], 7.26 [d, J = 8.3 Hz, 2 H,

CH₃(CH)₂], 7.24 (s, 1 H, CBrCH), 6.41 (s, 1 H, NCHN), 5.67 (m,

Eur. J. Org. Chem. 2012, 685–698

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691

T. Lindel et al.

FULL PAPER

1 H, CH₂CHOTf), 4.40 (m, 2 H, CH₃CH₂), 4.33 (dt, J = 1.4 Hz, J ¹³C NMR (100 MHz, [D₆]DMSO): δ = 157.7 [N(CO)N], 154.1

= 14 Hz, 1 H, NCHHCH), 4.19 (dd, J = 4.8, J = 14.1 Hz, 1 H, [C(CO)N], 125.5 (CHCCO), 114.0 (NCHCBr), 105.7 (CCHCBr),

NCHHCH), 3.11 (dd, J = 6.1, J = 15.6 Hz, 1 H, CHCHHCC),

2.74 (dt, J = 1.9, J = 15.8 Hz, 1 H, CHCHHCC), 2.44 [s, 3 H,

(CH)₂CH₃], 1.39 (t, J = 7.1 Hz, CH₂CH₃) ppm. ¹³C NMR

101.2 (CHCBr), 79.1 (NCCH₂), 68.9 (NCHNH), 66.1

(CH₂CHCH₂), 53.9 (CCH₂CH), 46.6 (NCH₂CH) ppm. HRMS

(ESI⁺): calcd. for C₁₁H₁₀⁷⁹Br⁸¹BrN₄NaO₃[M + Na]⁺428.8992;

(100 MHz, CDCl₃): δ = 153.2 [C(CO)N], 150.1 [N(CO)N], 149.4 found 428.8990. IR (ATR): ν = 3468 (m), 3262 (m), 3079 (m), 1723

˜

[EtO(CO)N], 147.4 [H₃CC(CH)₂], 130.1 [H₃CC(CH)₂], 129.9 (s), 1631 (s), 1546 (s), 1440 (s), 1420 (s), 1367 (s), 1322 (m), 1274

[O₂SC(CH)₂], 129.5 [O₂SC(CH)₂], 125.9 (CBrCBrCHC), 118.1 (m), 1255 (m), 1211 (m), 1132 (m), 1063 (m), 975 (m), 957 (m), 724

(CBrCBrCHC), 106.7 (CBrCBrCHC), 104.9 (CBrCBrCHC), 85.0 (s), 655 (s), 579 (s), 543 (s) cm^–1. UV (CHCl₃): λ_max(logε) = 287

(HCCCH₂), 62.5 (NCH₂CHCH₂), 67.6 (NCHN), 65.6 (4.0), 238 (3.9) nm.

(OCH₂CH₃), 52.2 (NCH₂CHCH₂), 39.3 (NCH₂CHCH₂), 21.9

Ethyl (3aS,5S,12aS)-10,11-Dibromo-5-methoxy-2,8-dioxo-3-(tosyl-

(PhCH₃), 14.1 (OCH₂CH₃) ppm. HRMS (ESI⁺): calcd. for

oxy)-2,3,5,6,8,12a-hexahydroimidazo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]-

C₂₂H₂₀⁷⁹Br⁸¹BrF₃N₄O₁₀S₂[M + H]⁺780.8914; found 780.8911. IR

pyrazine-1(4H)-carboxylate (17): Et₃N (6.4 μL, 0.067 mmol,

(ATR): ν = 1813 (m), 1748 (m), 1676 (m), 1411 (s), 1385 (s), 1265

˜

1.5 equiv.) was added to a solution of 14 (35 mg, 0.045 mmol,

1.0 equiv.) in MeOH (2 mL). After 16 h at room temperature, the

solvent was evaporated, and the residue was purified by column

chromatography [silica gel, EtOAc/isohexane (7:3)]. Compound 17

was obtained as a colourless powder (27.6 mg, 0.041 mmol, 93%).

m.p. 152 °C. [α]²_D¹= +81 (c = 8 mg/mL, CH₂Cl₂). TLC (silica gel,

(m), 1194 (s), 1179 (s), 1138 (s), 1090 (m), 961 (m), 926 (m), 814

(m), 717 (s), 612 (s), 565 (s), 545 (s) cm^–1. UV (CHCl₃): λ_max(logε)

= 291 (3.9), 240 (4.1) nm.

Ethyl (3aS,5S,12aS)-10,11-Dibromo-5-hydroxy-2,8-dioxo-3-(tosyl-

oxy)-2,3,5,6,8,12a-hexahydroimidazo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]-

pyrazine-1(4H)-carboxylate (15): Et₃N (6.4 μL, 0.064 mmol,

1

EtOAc): R_f= 0.72. H NMR (400 MHz, CDCl₃): δ = 7.62 [d, J =

1.5 equiv.) was added to

a solution of triflate 14 (35 mg, 8.4 Hz, 2 H, O₂SC(CH)₂], 7.24 [d, J = 8.4 Hz, 2 H, CH₃C(CH)₂],

0.045 mmol, 1.0 equiv.) in CH₂Cl₂/H₂O (2 mL, 9:1). The reaction

mixture was stirred for 16 h at room temperature followed by sol-

vent evaporation under reduced pressure. The residue was purified

by column chromatography [silica gel, EtOAc/isohexane (7:3)].

Compound 15 was obtained as a colourless powder (27 mg,

0.041 mmol, 93%). m.p. 195 °C. [α]²_D¹= +64 (c = 5.5 mg/mL,

CH₂Cl₂). TLC [silica gel, EtOAc/isohexane (4:1)]: R_f= 0.41. ¹H

NMR (400 MHz, CDCl₃): δ = 7.58 [d, J = 8.3 Hz, 2 H, O₂SC-

(CH)₂], 7.25 [d, J = 8.3 Hz, 2 H, CH₃C(CH)₂], 7.14 (s, 1 H,

7.16 (s, 1 H, BrCCH), 6.22 (s, 1 H, NCHN), 4.41 (m, 2 H,

CH₃CH₂), 4.22 (dd, J = 7.1, J = 11.7 Hz, 1 H, NCHHCH), 4.08

(m, 1 H, CH₂CHCH₂) 3.56 (dd, J = 6.2, J = 11.8 Hz, 1 H,

NCHHCH), 3.37 (s, 3 H, CH₃CHCH₂), 2.58 (m, 2 H, CCHHCH),

2.43 (s, 3 H, CH₃C), 1.38 (t, J = 7.2 Hz, 3 H, CH₃CH₂) ppm. ¹³C

NMR (100 MHz, CDCl₃): δ = 153.6 [C(CO)N], 149.8 [N(CO)N],

146.8 [H₃CC(CH)₂], 129.9 [H₃CC(CH)₂], 129.7 [EtO(CO)N], 129.4

[O₂SC(CH)₂], 129.4 [O₂SC(CH)₂], 126.4 (CBrCBrCHC), 117.4

(CBrCBrCHC), 106.0 (CBrCBrCHC), 104.4 (CBrCBrCHC), 85.2

BrCCH), 6.27 (s, 1 H, NCHN), 4.64 (m, 1 H, OHCH), 4.42 (m, 2 (HCCCH₂), 74.6 (NCH₂CHCH₂), 68.1 (NCHN), 65.3

H, CH₃CH₂), 4.21 (dd, J = 6.8, J = 12.1 Hz, 1 H,NCHHCH), 3.57

(dd, J = 4.6, J = 12.1 Hz, 1 H, NCHHCH), 3.00 (s, 1 H, OH), 2.75

(OCH₂CH₃), 57.4 (CH₃OCH), 50.9 (NCH₂CHCH₂), 37.8

(NCH₂CHCH₂), 21.9 (PhCH₃), 14.2 (OCH₂CH₃) ppm. HRMS

(dd, J = 8.0, J = 14.9 Hz, 1 H, CCHHCH), 2.48 (dd, J = 4.0, J = (ESI⁺): calcd. for C₂₂H₂₂⁷⁹Br⁸¹BrN₄NaO₈S [M + Na]⁺684.9397;

14.7 Hz, 1 H, CCHHCH), 2.44 (s, 3 H, CH₃C), 1.37 (t, J = 7.1 Hz, 684.9398. IR (ATR): ν = 1807 (s), 1746 (m), 1715 (m), 1669 (s),

˜

3 H, CH₃CH₂) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 153.7

[C(CO)N], 150.3 [N(CO)N], 149.8 [EtO(CO)N], 147.2 [H₃CC-

1429 (s), 1411 (s), 1384 (s), 1266 (s), 1193 (s), 1179 (s), 1094 (m),

1011 (m), 959 (m), 866 (w), 815 (m), 780 (m), 722 (s), 657 (s), 566

(CH)₂], 132.0 [H₃CC(CH)₂], 130.0 [O₂SC(CH)₂], 129.4 [O₂SC- (s), 546 (s) cm^–1. UV (CHCl₃): λ_max(logε) = 286 (4.0), 239 (3.9) nm.

(CH)₂], 126.4 (CBrCBrCHC), 117.4 (CBrCBrCHC), 106.2

(3aR,5S,12aS)-10-Bromo-5-methoxy-1,5,6,12a-tetrahydroimid-

(CBrCBrCHC), 104.5 (CBrCBrCHC), 85.5 (HCCCH₂), 68.2

azo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]pyrazine-2,8(3H,4H)-dione (39) and

(NCHN), 66.1 (NCH₂CHCH₂), 65.3 (OCH₂CH₃), 54.1

(3aR,5S,12aS)-10,11-Dibromo-5-methoxy-1,5,6,12a-tetrahydroimid-

(NCH₂CHCH₂), 41.4 (NCH₂CHCH₂), 21.9 (PhCH₃), 14.2

azo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]pyrazine-2,8(3H,4H)-dione

(18):

(OCH₂CH₃) ppm. HRMS (ESI⁺): calcd. for C₂₁H₂₁⁷⁹Br⁸¹BrN₄O₈S

SmI₂(0.1 m in THF, 1.7 mL, 0.170 mmol, 3 equiv.) was added

dropwise to a solution of 17 (38 mg, 0.057 mmol, 1 equiv.) in de-

gassed MeOH (0.5 mL) at 0 °C. The reaction mixture was stirred

for 1 h at room temperature under argon. The solvent was evapo-

rated under reduced pressure, and the residue was purified by col-

umn chromatography [silica gel, CHCl₃/MeOH (9:1)] to provide 39

(9.1 mg, 0.026 mmol, 46%) and 18 (12.0 mg, 0.028 mmol, 49%) as

colourless solids. Compound 39: m.p. 220 °C. [α]²_D²= –80 (c = 6 mg/

[M + H]⁺648.9421; found 648.9423. IR (ATR): ν = 1722 (s), 1632

˜

(s), 1548 (m), 1444 (s), 1387 (m), 1324 (m), 1216 (m), 1180 (s), 1078

(m), 1031 (m), 735 (m), 678 (m), 618 (m) cm^–1. UV (CHCl₃): λ_max

(logε) = 287 (4.0), 236 (3.9) nm.

(3aR,5S,12aS)-10,11-Dibromo-5-hydroxy-1,5,6,12a-tetrahydro-

imidazo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]pyrazine-2,8(3H,4H)-dione

(16): SmI₂in THF (0.1 m, 0.77 mL, 0.077 mmol, 2.1 equiv.) was

added dropwise to a solution of 15 (25 mg) at 0 °C followed by

degassed MeOH (0.6 mL). After 2 h, the solvent was evaporated

under reduced pressure, and the residue was purified by column

chromatography [silica gel, CHCl₃/MeOH (9:1)] to provide 16 as a

1

mL, MeOH). TLC [silica gel, CHCl₃/MeOH (9:1)]: R_f= 0.23. H

NMR (400 MHz, [D₄]methanol): δ = 7.10 (d, J = 1.7 Hz, 1 H,

NCHCBr), 6.82 (d, J = 1.8 Hz, 1 H, CCHCBr), 5.74 (s, 1 H,

NCHN), 4.20 (m, 1 H, MeOCHCH₂), 3.86 (dt, J = 1.0, J =

13.0 Hz, 1 H, NCHHCH), 3.78 (dd, J = 4.8, J = 13.0 Hz, 1 H,

NCHHCH), 3.40 (s, 3 H, CH₃OH), 2.56 (dt, J = 1.0, J = 13.0 Hz,

1 HCCHHCHCH₂), 2.40 (dd, J = 14.7, J = 13.5 Hz, 1 H,

colourless solid (13.9 mg, 0.032 mmol, 89%). m.p. 230 °C. [α]²_D⁴

=

–200 (c = 2.5 mg/mL, MeOH). TLC [silica gel, CHCl₃/MeOH

1

(9:1)]: R_f= 0.12. H NMR (400 MHz, [D₆]DMSO): δ = 8.41 (s, 1

H, CHNHCO), 6.93 (s, 1 H, BrCCH), 6.72 (s, 1 H, CNHCO), 5.97 CCHHCHCH₂) ppm. ¹³C NMR (100 MHz, [D₄]methanol): δ =

(d, J = 2.2 Hz, 1 H, NCHN), 5.57 (s, 1 H, CHOH), 4.44 (m, 1 H,

160.9 [C(CO)N], 157.7 [N(CO)N], 124.9 (CHCCO), 123.2

NCH₂CHCH₂), 3.70 (dd,

NCHHCHCH₂), 3.78 (dd,

NCHHCHCH₂), 2.52 (dd,

J

=

5.2,

0.8,

5.3,

J

=

12.3 Hz,

13.5 Hz,

1

H, (NCHCBr), 115.5 (CCHCBr), 100.1 (CHCBr), 80.6 (NCCH₂), 78.1

H,

(CH₂CHCH₂), 69.8 (NCHNH), 57.1 (CH₃OCHCH₂), 52.5

(NCH₂CH), 44.4 (CCH₂CH) ppm. HRMS [ESI⁺]: calcd. for

NCH₂CHCHH), 2.18 (d, J = 13.1 Hz, 1 H, NCH₂CHCHH) ppm. C₁₂H₁₃⁷⁹BrN₄NaO₃[M + Na]⁺363.0069; found 363.0065. IR

692

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Eur. J. Org. Chem. 2012, 685–698

(–)-Dibromophakellstatin Derivatives

(ATR): ν = 1716 (s), 1648 (s), 1547 (m), 1477 (m), 1422 (m), 1368

CH₃CH₂), 2.57 (d, J = 13.5 Hz, 1 H, CCHHCH), 2.45 (dd, J =

˜

(m), 1188 (m), 1136 (s), 1072 (s), 928 (m), 816 (m), 783 (m), 762 5.0, J = 13.4 Hz, 1 H, CCHHCH), 1.25 (t, J = 7.0 Hz, 3 H,

(m), 719 (m) cm^–1. UV (CHCl₃): λ_max(logε) = 280 (3.8), 234 CH₂CH₃) ppm. ¹³C NMR (150 MHz, [D₄]methanol): δ = 160.3

(3.9) nm. Compound 18: m.p. 230 °C. [α]²_D²= –166 (c = 2.5 mg/mL,

MeOH). TLC [silica gel, CHCl₃/MeOH (9:1)]: R_f= 0.38. ¹H NMR

(400 MHz, [D₆]DMSO): δ = 8.35 (s, 1 H, CNHCO), 6.94 (s, 1 H,

(NHCONH), 157.1 (CCON), 126.2 (CCHCBr), 116.5 (CCHCBr),

107.7 (CBrCBrCH), 103.4 (CBrCBrCH), 80.9 (NCHCN), 76.1

(CH₂CHO), 70.5 (NCHNH), 65.9 (CH₃CH₂), 52.7 (NCH₂CH),

CCHCBr), 6.90 (s, 1 H, NCHNH), 5.98 (s, 1 H, NCHNH), 4.12 45.3 (CCH₂CH), 15.6 (CH₃CH₂) ppm. HRMS (ESI⁺): calcd. for

(m, 1 H, CH₂CHCH₂), 3.73 (dd, J = 5.3, J = 12.6 Hz, 1 H,

NCHHCH), 3.63 (d, J = 12.6 Hz, 1 H, NCHHCH), 3.29 (s, 3 H,

C₁₃H₁₅⁷⁹Br⁸¹BrN₄O₃[M + H]⁺434.9484; found 434.9485. IR

(ATR): ν = 1727 (s), 1643 (s), 1548 (m), 1436 (s), 1368 (m), 1272

˜

CH₃OCH), 2.52 (dd, J = 5.5, J = 12.9 Hz, 1 H, CCHHCH), 2.35 (m), 1137 (m), 1120 (m), 1082 (s), 971 (m), 782 (m), 740 (m), 673

(d, J = 2.3 Hz, 1 H, CCHHCH) ppm. ¹³C NMR (100 MHz,

(m), 547 (m) cm^–1. UV (CHCl₃): λ_max(logε) = 287 (4.0), 238

[D₆]DMSO): = 157.5 [N(CO)N], 154.1 [C(CO)N], 125.3 (3.9) nm.

δ

(CHCCO), 114.1 (CCHCBr), 105.8 (NCBrCBr), 101.2 (CHCBr),

78.7 (NCCH₂), 75.7 (CH₂CHCH₂), 68.8 (NCHNH), 56.2

(CH₃OCH), 50.5 (NCH₂CH), 43.7 (CCH₂CH) ppm. HRMS

(ESI⁺): calcd. for C₁₂H₁₂⁷⁹Br⁸¹BrN₄NaO₃[M + Na]⁺442.9153;

Ethyl (3aS,5S,12aS)-10,11-Dibromo-5-isopropoxy-2,8-dioxo-

3-(tosyloxy)-2,3,5,6,8,12a-hexahydroimidazo[4,5-b]dipyrrolo[1,2-

a:1Ј,2Ј-d]pyrazine-1(4H)-carboxylate (21): To a solution of 14

(35 mg, 0.045 mmol, 1.0 equiv.) in iPrOH (2 mL) was added Et₃N

(6.4 μL, 0.067 mmol, 1.5 equiv.). The reaction was stirred for 36 h

at room temperature before the solvent was evaporated under re-

duced pressure. The residue was purified by column chromatog-

raphy [silica gel, EtOAc/isohexane (7:3)] to give 21 as a colourless

powder (26 mg, 0.038 mmol, 85%). m.p. 157 °C. [α]²_D²= +111 (c =

5 mg/mL, CH₂Cl₂). TLC [silica gel, EtOAc/isohexane (2:3)]: R_f=

found 442.9147. IR (ATR): ν = 3275 (m), 1717 (s), 1656 (s), 1543

˜

(m), 1428 (s), 1374 (m), 1276 (m), 1243 (m), 1213 (m), 1133 (s),

1091 (s), 1066 (m), 1043 (m), 1022 (m), 971 (m), 736 (m), 708 (m),

645 (s), 623 (s), 605 (s) cm^–1. UV (CHCl₃): λ_max(logε) = 288 (4.0),

238 (3.9) nm.

Ethyl (3aS,5S,12aS)-10,11-Dibromo-5-ethoxy-2,8-dioxo-3-(tosyl-

oxy)-2,3,5,6,8,12a-hexahydroimidazo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]-

1

0.42. H NMR (400 MHz, CDCl₃): δ = 7.59 [d, J = 8.4 Hz, 2 H,

pyrazine-1(4H)-carboxylate (19): Triflate 14 (35 mg, 0.045 mmol, O₂SC(CH)₂], 7.22 [d, J = 8.1 Hz, 2 H, CH₃C(CH)₂], 7.13 (s, 1 H,

1 equiv.) was dissolved in EtOH (2 mL) and Et₃N (6.4 μL, BrCCH), 6.21 (s, 1 H, NCHN), 4.41 (m, 2 H, CH₃CH₂), 4.22 (m,

0.067 mmol, 1.5 equiv.) was added. The reaction was stirred for

18 h at room temperature before the solvent was evaporated. The

residue was purified by column chromatography [silica gel, EtOAc/

2 H, NCHHCHCH₂), 3.64 (m, 1 H, CH₃CHCH₃), 3.44 (m, 1 H,

NCHHCH), 2.54 (m, 2 H, CCHHCH), 2.43 (s, 3 H, CH₃C), 1.38

(t, J = 7.2 Hz, 3 H, CH₃CH₂), 1.19 (d, J = 6.1 Hz, 3 H, CH₃CCH₃),

isohexane (7:3)]. Compound 19 was obtained as a white powder 1.17 (d, J = 6.1 Hz, 3 H, CH₃CCH₃) ppm. ¹³C NMR (100 MHz,

(27 mg, 0.040 mmol, 89%). m.p. 148 °C. [α]²_D¹= –227 (c = 5.5 mg/

mL, CH₂Cl₂). TLC [silica gel, EtOAc/isohexane (2:3)]: R_f= 0.25.

CDCl₃): δ = 153.5 [C(CO)N], 150.6 [EtO(CO)N], 149.7 [N(CO)N],

146.8 [H₃CC(CH)₂], 129.8 [H₃CC(CH)₂], 129.6 [O₂SC(CH)₂], 129.4

¹H NMR (400 MHz, CDCl₃): δ = 7.60 [d, J = 8.4 Hz, 2 H, [O₂SC(CH)₂], 126.4 (CBrCBrCHC), 117.2 (CBrCBrCHC), 105.9

O₂SC(CH)₂], 7.23 [d, J = 8.4 Hz, 2 H, CH₃C(CH)₂], 7.14 (s, 1 H,

(CBrCBrCHC), 104.2 (CBrCBrCHC), 84.9 (HCCCH₂), 71.4

BrCCH), 6.22 (s, 1 H, NCHN), 4.41 (m, 2 H, CH₃CH₂), 4.22 (m, 2 (CH₃CHCH₃), 70.6 (NCH₂CHCH₂), 68.0 (NCHN), 65.2

H, NCHHCHCH₂), 3.51 (m, 3 H, NCHHCHCH₂, CH₃CH₂OCH), (OCH₂CH₃), 51.3 (NCH₂CHCH₂), 38.2 (NCH₂CHCH₂), 22.4

2.57 (m, 2 H, NCCH₂CH), 2.43 (s, 3 H, CH₃C), 1.38 (t, J = 7.1 Hz, (CH₃CHCH₃), 22.3 (CH₃CHCH₃), 21.9 (PhCH₃), 14.2

3 H, CH₃CH₂OCO), 1.24 (t, J = 6.9 Hz, 3 H, CH₃CH₂OCH) ppm. (OCH₂CH₃) ppm. HRMS (ESI⁺): calcd. for C₂₄H₂₇⁷⁹Br⁸¹BrN₄O₈S

¹³C NMR (100 MHz, CDCl₃):

δ =

153.5 [C(CO)N], 150.6 [M + H]⁺690.9891; found 690.9893. IR (ATR): ν = 1792 (s), 1739

˜

[EtO(CO)N], 149.8 [N(CO)N], 146.8 [H₃CC(CH)₂], 129.8

(m), 1702 (s), 1435 (s), 1389 (s), 1193 (s), 1160 (s), 1123 (m), 1090

[H₃CC(CH)₂], 129.7 [O₂SC(CH)₂], 129.4 [O₂SC(CH)₂], 126.4 (m), 1018 (m), 953 (m), 809 (m), 725 (s), 653 (s), 572 (s), 552 (s)

(CBrCBrCHC), 117.2 (CBrCBrCHC), 105.9 (CBrCBrCHC), 104.3 cm^–1. UV (CHCl₃): λ_max(logε) = 290 (4.0), 240 (3.9) nm.

(CBrCBrCHC), 85.1 (HCCCH₂), 72.9 (NCH₂CHCH₂), 68.1

(3aR,5S,12aS)-10,11-Dibromo-5-isopropoxy-1,5,6,12a-tetrahydro-

(NCHN), 65.5 (OCH₂CH₃), 65.2 (CH₃CH₂OCH), 51.2

imidazo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]pyrazine-2,8(3H,4H)-dione

(NCH₂CHCH₂), 37.9 (NCH₂CHCH₂), 21.9 (PhCH₃), 15.1

(22): At 0 °C, SmI₂(0.1 m in THF, 0.77 mL, 0.077 mmol, 2.1 equiv.)

(CHOCH₂CH₃), 14.2 (CH₃CH₂OCO) ppm. HRMS (ESI⁺): calcd.

was added dropwise to a solution of 21 (25 mg, 0.037 mmol,

for C₂₃H₂₅⁷⁹Br⁸¹BrN₄O₈S [M + H]⁺676.9734; found 676.9736. IR

1.0 equiv.) in degassed THF (0.5 mL) followed by degassed MeOH

(ATR): ν = 1808 (s), 1745 (m), 1699 (s), 1430 (s), 1383 (s), 1264

˜

(0.6 mL). After 2 h, the solvent was evaporated, and the residue

was purified by column chromatography [silica gel, CHCl₃/MeOH

(9:1)] to provide 22 as a colourless solid (13.9 mg, 0.032 mmol,

87%). m.p. 286 °C. [α]²_D¹= –216 (c = 2.5 mg/mL, MeOH). TLC

(s), 1193 (s), 1179 (s), 1121 (m), 1094 (m), 1011 (m), 958 (m), 865

(w), 815 (m), 721 (s), 684 (m), 657 (s), 565 (s), 545 (s) cm^–1. UV

(CHCl₃): λ_max(logε) = 290 (3.9), 240 (4.1) nm.

1

(3aR,5S,12aS)-10,11-Dibromo-5-ethoxy-1,5,6,12a-tetrahydroimid-

(silica gel, EtOAc): R_f= 0.35. H NMR (400 MHz, [D₆]DMSO): δ

azo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]pyrazine-2,8(3H,4H)-dione

(20): = 8.43 (s, 1 H, CNHCO), 6.94 (s, 1 H, BrCCH), 6.61 (s, 1 H,

CHNHCO), 5.99 (d, J = 1.9 Hz, 1 H, NCHN), 4.32 (m, 1 H,

NCHHCHCH₂), 3.77 (dd, 5.5, 12.2 Hz, H,

SmI₂(0.1 m in THF, 0.77 mL, 0.077 mmol, 2.1 equiv.) was added

dropwise to a solution of 19 (25 mg, 0.037 mmol, 1.0 equiv.) in de-

gassed THF (0.50 mL) at 0 °C followed by degassed MeOH

(0.65 mL). After 2 h, the solvent was evaporated, and the residue

was purified by column chromatography [silica gel, CHCl₃/MeOH

(9:1)] to provide 20 as a colourless solid (14.3 mg, 0.033 mmol,

87%). m.p. 260 °C. [α]²_D¹= –140 (c = 3 mg/mL, MeOH). TLC [silica

J

=

J

=

1

NCHHCHCH₂), 3.73 (dd, J = 5.9, J = 12.1 Hz, 1 H, CH₃CHCH₃),

3.53 (d, J = 12.3 Hz, 1 H, NCHHCH), 2.57 (dd, J = 5.7, J =

13.5 Hz, 1 H, NCH₂CHCHH), 2.24 (d, J = 13.3 Hz, 1 H,

NCH₂CHCHH), 1.13 (d, J = 3.4 Hz, 3 H, OCHCH₃), 1.12 (d, J =

3.3 Hz, 3 H, OCHCH₃) ppm. ¹³C NMR (100 MHz, [D₆]DMSO):

δ = 157.7 [N(CO)N], 154.0 [C(CO)N], 125.4 (CHCCO), 114.1

(NCBrCBr), 105.8 (CCHCBr), 101.1 (CHCBr), 78.8 (NCCH₂),

gel, CHCl₃/MeOH, (9:1)]: R_f

[D₄]methanol): δ = 6.96 (s, 1 H, CCHCBr), 5.97 (s, 1 H, NCHNH),

=

0.34. ¹H NMR (600 MHz,

4.31 (m, 1 H, CH₂CHCH₂), 3.86 (d, J = 12.8 Hz, 1 H, NCHHCH), 71.4 (CH₂CHCH₂) 69.4 (CH₃CHCH₃), 68.8 (NCHNH), 51.5

3.78 (dd, J = 5.1, J = 12.9 Hz, 1 H NCHHCH), 3.59 (m, 2 H, (CCH₂CH), 44.6 (NCH₂CH), 22.4 (CH₃CHCH₃), 22.0

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693

T. Lindel et al.

FULL PAPER

(CH₃CHCH₃)

ppm.

HRMS

(ESI⁺):

calcd.

for

C₁₇H₁₈⁷⁹Br⁸¹BrN₄NaO₃[M + Na]⁺508.9622; found 508.9616. IR

C₁₄H₁₆⁷⁹Br⁸¹BrN₄NaO₃[M + Na]⁺470.9466; found 470.9461. IR

(ATR): ν = 1723 (s), 1645 (s), 1548 (m), 1438 (s), 1363 (m), 1273

˜

(ATR): ν = 1728 (s), 1645 (s), 1548 (m), 1441 (s), 1373 (m), 1324

(m), 1136 (m), 1079 (m), 969 (m), 740 (m), 712 (m), 677 (m), 631

˜

(m), 1120 (m), 1071 (m), 1031 (m), 739 (m), 675 (m), 614 (m), 547 (m) cm^–1. UV (CHCl₃): λ_max(logε) = 287 (4.0), 238 (4.0) nm.

(s) cm^–1. UV (CHCl₃): λ_max(logε) = 287 (4.0), 236 (3.9) nm.

N-(2-Aminoethyl)-2-[7-(dimethylamino)-4-methyl-2-oxo-2H-

Ethyl (3aS,5S,12aS)-10,11-Dibromo-5-(hex-5-yn-1-yloxy)-2,8-dioxo-

3-(tosyloxy)-2,3,5,6,8,12a-hexahydroimidazo[4,5-b]dipyrrolo[1,2-

a:1Ј,2Ј-d]pyrazine-1(4H)-carboxylate (40): To a solution of 14

(0.1 g, 0.128 mmol, 1 equiv.) in hex-5-yn-1-ol (1.5 g, 15.31 mmol,

120 equiv.) and CH₂Cl₂(1.5 mL) was added Et₃N (6.4 μL,

0.067 mmol, 1.5 equiv.). The reaction mixture was stirred for 36 h

at room temperature before the solvent was evaporated. The resi-

due was purified by column chromatography [silica gel, EtOAc/

chromen-3-yl]acetamide (29): A solution of 28 (140 mg, 0.53 mmol,

1.0 equiv.), NHS (61 mg, 0.53 mmol, 1.0 equiv.) and DCC (121 mg,

0.59 mmol, 1.1 equiv.) in DMF (20 mL) was stirred at room tem-

perature for 12 h. The mixture was filtered and concentrated under

reduced pressure to a volume of 3 mL. To the resulting solution

was added 1,2-diaminoethane (3 mL), and the mixture was stirred

for a further 24 h at room temperature. The solvent was removed

under reduced pressure, and the residue purified by column

isohexane (2:3 to 3:2)]. Compound 40 was obtained as a colourless chromatography [silica gel, CHCl₃/MeOH/25 % aqueous NH₃

powder (63.3 mg, 0.087 mmol, 68%). m.p. 165 °C. [α]²_D⁴= +22.5 (c

= 2 mg/mL, CH₂Cl₂). TLC [silica gel, EtOAc/isohexane (1:1)]: R_f

(18:2:1)]. Compound 29 was obtained as a yellow solid (130 mg,

0.43 mmol, 80 %). TLC [silica gel, CHCl₃/MeOH/25 % aqueous

= 0.26. ¹H NMR (400 MHz, CDCl₃): δ = 7.61 [d, J = 8.9 Hz, 2 H, NH₃(18:2:1)]: R_f= 0.32. ¹H NMR (400 MHz, [D₆]DMSO): δ =

SC(CH)₂], 7.24 [d, J = 8.1 Hz, 2 H, CH₃C(CH)₂], 7.16 (s, 1 H,

7.83 (t, J = 5.4 Hz, 1 H, CONH), 7.56 (d, J = 9.0 Hz, 1 H,

BrCCH), 6.21 (s, 1 H, NCHN), 4.42 (m, 2 H, CH₃CH₂), 4.22 (m, NCCHC), 6.73 (dd, J = 2.6, J = 9.0 Hz, 1 H, NCCHCH), 6.53 (d,

1 H, NCHHCHCHH), 4.19 (m, 1 H, NCHHCHO), 3.54 (dd, J =

5.9, J = 11.4 Hz, 1 H, NCHHCHCHH), 3.46 (t, J = 6.2 Hz, 2 H,

J = 2.5 Hz, 1 H, NCCHCH), 3.42 (s, 2 H, CCH₂CO), 3.04 (q, J =

5.9 Hz, 2 H, NHCH₂CH₂), 3.01 [s, 6 H, N(CH₃)₂], 2.56 (t, J =

OCH₂CH₂), 2.57 (m, 2 H, NCH₂CHCH₂), 2.43 [s, 3 H, CH₃C- 6.4 Hz, 2 H, NHCH₂CH₂), 2.29 (s, 3 H, CCCH₃) ppm. ¹³C NMR

(CH)₂], 2.24 (ddd, J = 6.9, J = 9.6, J = 13.7 Hz), 1.97 (t, J = 2.6 Hz,

1 H, CHCCH₂CH₂), 1.73 (m, 2 H, OCH₂CH₂), 1.63 (m, 2 H, (CH₃NC), 152.2 (CH₃CC), 149.5 (OCCH), 125.9 (CH₃NCCHC),

CHCCH₂CH₂), 1.38 (t, J = 7.1 Hz, 3 H, CH₃CH₂) ppm. ¹³C NMR

114.2 (CH₃CC), 109.3 (CH₃CCCH₂), 109.0 (CH₃CCCHCH), 97.2

(100 MHz, CDCl₃): δ = 153.6 (CCON), 150.7 (OCON), 149.8 (CH₃CCCHCH), 42.4 (NHCH₂), 41.3 (CH₂NH₂), 39.5 [(CH₃)-

(NCON), 146.8 [CH₃C(CH)₂], 129.9 [CH₃C(CH)₂], 129.7 [SC(CH)

NCH], 33.8 (CCH₂CO), 14.8 (CH₃CC) ppm. HRMS (ESI⁺): calcd.

₂], 129.5 [SC(CH)₂], 126.4 (BrCHC), 117.3 (BrCCH), 105.9 for C₁₆H₂₁N₃NaO₃[M + Na]⁺304.1661; found 304.1656. IR

(100 MHz, [D₆]DMSO): δ = 169.2 (CHCO), 161.6 (OCO), 153.8

(BrCBrCCH), 104.4 (BrCCH), 85.1 (NCCH₂), 84.2 (CH₂CCH), (ATR): ν = 3287 (m), 1694 (s), 1642 (m), 1621 (s), 1595 (s), 1546

˜

73.2 (OCHCH₂), 69.6 (OCH₂CH₂), 68.6 (CH₂CH₂CCH), 68.1 (s), 1528 (s), 1391 (s), 1373 (m), 1339 (m), 1283 (s), 1238 (m), 1154

(NCHN), 65.3 (CH₃CH₂), 51.3 (NCH₂CH), 38.1 (NCH₂CHCH₂), (s), 1089 (m), 1068 (m), 817 (m), 791 (m), 554 (m) cm^–1. UV

28.6 (OCH₂CH₂CH₂), 25.0 (OCH₂CH₂CH₂), 21.9 [(CH₃CCH)₂],

18.1 (CHCCH₂CH₂), 14.2 (CH₃CH₂) ppm. HRMS (ESI⁺): calcd.

for C₂₇H₂₈⁷⁹Br⁸¹BrN₄NaO₈S [M + Na]⁺750.9871; found 750.9865.

(CHCl₃): λ_max(logε) = 371 (4.3), 368 (4.3), 244 (4.1) nm.

N-(2-(4-(4-(((3aR,5S,12aS)-10,11-Dibromo-2,8-dioxo-1,2,3,4,

5,6,8,12a-octahydroimidazo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]pyrazin-5-

yl)oxy)butyl)-1H-1,2,3-triazol-1-yl)ethyl)-2-[7-(dimethylamino)-4-

methyl-2-oxo-2H-chromen-3-yl]acetamide (24): For the synthesis of

azide 30, NaN₃(64.3 mg, 0.990 mmol, 10 equiv.) was dissolved in

H₂O (0.25 mL) and CH₂Cl₂(0.38 mL) and the solution was cooled

to 0 °C. Tf₂O (33.5 μL, 0.198 mmol, 2 equiv.) was added and the

reaction mixture was stirred at room temperature for 3 h. The

phases were separated, and the aqueous layer was washed three

times with CH₂Cl₂(0.4 mL). The combined organic phases were

washed once with saturated aqueous K₂CO₃(1 mL). Coumarine

amine 29 (30 mg, 0.1 mmol, 1 equiv.) was dissolved in H₂O

IR (ATR): ν = 1807 (m), 1745 (m), 1668 (s), 1430 (m), 1409 (m),

˜

1383 (s), 1264 (s), 1193 (s), 1179 (s), 1131 (m), 1093 (m), 1011 (m),

959 (m), 750 (m), 722 (s), 683 (m), 655 (m), 565 (s), 545 (s) cm^–1.

UV (CHCl₃): λ_max(logε) = 291 (4.0), 240 (4.1) nm.

(3aR,5S,12aS)-10,11-Dibromo-5-(hex-5-yn-1-yloxy)-1,5,6,12a-tetra-

hydroimidazo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]pyrazine-2,8(3H,4H)-di-

one (23): At 0 °C, SmI₂(0.1 m in THF, 0.8 mL, 0.082 mmol,

2.1 equiv.) was added dropwise to a solution of 40 (30 mg,

0.041 mmol, 1 equiv.) in degassed THF (0.5 mL) followed by the

dropwise addition of degassed MeOH (0.6 mL). After 2 h, the sol-

vent was evaporated, and the residue was purified by column (0.3 mL) and MeOH (0.5 mL). To this solution were added

chromatography [silica gel, CHCl₃/MeOH (9:1)] to provide 23 as a

colourless solid (16.9 mg, 0.034 mmol, 83%). m.p. 235 °C. TLC [sil-

ica gel, EtOAc/isohexane (4:1)]: R_f= 0.33. [α]²_D³= –138 (5 mg/mL,

CuSO₄·5H₂O (0.024 mg, 1 μmol, 0.01 equiv.) and K₂CO₃(20.5 mg,

0.4 mmol, 1.5 equiv.) followed by the addition of the TfN₃solution

at 0 °C. The reaction mixture was stirred at room temperature in

MeOH). ¹H NMR (400 MHz, [D₆]DMSO): δ = 8.39 (s, 1 H, the dark for 20 h. The solvent was evaporated and the residue was

CH₂CNH), 6.94 (s, 1 H, BrCCH), 6.72 (d, J = 1.5 Hz, CHNHCO), purified by column chromatography [silica gel, CHCl₃/MeOH

5.99 (d, J = 2.3 Hz, 1 H, NCHNH), 4.20 (m, 1 H, CH₂CHCH₂), (19:1)] to provide 30 as a yellow solid (16.9 mg), which was used

3.75 (dd, J = 5.5, J = 12.5 Hz, 1 H, CCHHCH), 3.60 (dd, J = 1.7,

J = 12.5 Hz, 1 H, CCHHCH), 3.46 (m, 2 H, OCH₂CH₂), 2.76 (t, 23 (10.0 mg, 0.020 mmol, 1.0 equiv.) and azide 30 (6.8 mg,

J = 2.7 Hz, 1 H, CHCCH₂CH₂), 2.56 (dd, J = 5.8, J = 13.6 Hz, 1 0.020 mmol, 1.0 equiv.) in degassed nBuOH/H₂O (1:1, 4 mL) were

immediately without further purification. To a solution of alkyne

H, NCHHCH), 2.31 (d, J = 13.1 Hz, 1 H, NCHHCH), 2.19 (ddd, added sodium ascorbate (0.8 mg, 0.004 mmol, 0.2 equiv.) and

J = 7.0, J = 2.6, J = 9.7 Hz, 2 H, CHCCH₂CH), 1.62 (m, 2 H, CuSO₄(0.8 mg, 0.003 mmol, 0.15 equiv.) whilst stirring at ambient

OCH₂CH₂), 1.52 (m, 2 H, CHCCH₂CH₂) ppm. ¹³C NMR temperature (30 °C) in the dark. The resulting clear solution was

(100 MHz, [D₆]DMSO): δ = 157.5 (NHCONH), 154.0 (CCON),

125.3 (CCHCBr), 114.1 (CHCBr), 105.8 (NCBr), 101.2 (CHCBr), monitored by TLC. The solvent was evaporated under reduced

84.4 (CHCCH₂CH₂), 78.7 (NHCCH₂), 74.2 (CH₂CHO), 71.3 pressure, and the residue was purified by column chromatography

stirred in the dark until completion of the reaction, which was

(CHCCH₂), 68.8 (NCHNH), 68.0 (OCH₂CH₂), 50.8 (CCH₂CH), [silica gel, CHCl₃/MeOH (9:1)] to yield 24 (12.9 mg, 0.016 mmol,

44.2 (NCH₂CH), 28.2 (OCH₂CH₂), 24.7 (OCH₂CH₂CH₂), 79%). m.p. 217 °C. TLC [silica gel, CHCl₃/MeOH (9:1)]: R_f= 0.50.

1

1 7 . 5 ( C H CC H₂CH ₂) p p m . H R M S ( E S I ⁺) : c a l c d . fo r [α]²_D³= –204 (c = 2.5 mg/mL, MeOH). H NMR (400 MHz, [D₆]-

694

www.eurjoc.org

Eur. J. Org. Chem. 2012, 685–698

(–)-Dibromophakellstatin Derivatives

DMSO): δ = 8.40 (t, J = 1.9 Hz, 1 H, CHNHCO), 8.00 (t, J =

(CHOCH₂CH₂), 50.8 (NCH₂CH), 49.2 (NNNCH₂), 44.1

5.6 Hz, 1 H, CH₂NHCO), 7.78 (CH₂CCHNCH₂), 7.56 (d, J = (CCH₂CHCH₂), 41.1 (NNNCCH₂), 28.6 (NNNCCH₂CH₂), 25.7

9.0 Hz, 1 H, CH₃CCH), 6.93 (s, 1 H, CHCBr), 6.74 (dd, J = 2.6, (CHOCH₂CH₂), 24.7 (NH₂CH₂CH₂) ppm. HRMS (ESI⁺): calcd.

J = 9.1 Hz, 1 H, CH₃CCHCH), 6.70 (s, J = 1.4 Hz, 1 H, CNHCO),

6.54 (d, J = 2.5 Hz, 1 H, CH₃CCCH), 5.99 (d, J = 2.3 Hz, 1 H,

NCHNH), 4.36 (t, J = 5.6 Hz, 2 H, NCH₂CH₂), 4.20 (m, 1 H,

CH₂CHOCH₂), 3.75 (dd, J = 5.5, J = 12.5 Hz, 1 H, NCHHCHO),

3.61 (dd, J = 1.5, J = 12.5 Hz, 1 H, NCHHCHO), 3.40 (m, 2 H,

NHCH₂CH₂), 3.40 (m, 2 H, OCH₂CH₂), 3.38 (s, 2 H, CCH₂CO),

3.01 [s, 6 H, N(CH₃)₂], 2.61 (t, J = 7.3 Hz, 2 H, NCCH₂CH₂), 2.55

(dd, J = 5.7, J = 13.6 Hz, 1 H, CCHHCHO), 2.33 (d, J = 13.5 Hz,

1 H, CCHHCHO), 1.60 (m, 4 H, OCH₂CH₂CH₂CH₂) ppm. ¹³C

NMR (100 MHz, [D₆]DMSO): δ = 169.7 (CH₂CONH), 161.6 (OC-

OCCH₂), 157.6 (NHCONH), 154.0 (CCONCH₂), 153.9

[CCHCN(CH₃)₂], 152.3 [(CH₃)₂NCCH], 149.8 (CH₃CCCH), 146.5

(NNNCCH₂), 126.0 (CH₃CCCH), 125.3 (BrCCHC), 122.2

(NNNCH), 114.1 (BrCCH), 113.7 (CH₃CC), 109.3 (CH₃CCCH₂),

109.1 [(CH₃)₂NCCHCH], 105.82 (NCBr), 101.3 (NCBrCBr), 97.2

[(CH₃)₂NCCHCO], 78.7 (NHCCH₂), 74.2 (CH₂CHCH₂), 68.8

(NCHNHCO), 68.3 (OCH₂CH₂), 50.8 (NCH₂CHO), 48.5

(NNNCH₂), 44. 1 (NHC CH₂) , 3 9. 2 [ (CH₃)₂NC], 39.1

(OCNHCH₂), 33.8 (CH3CCCH₂), 28.6 (NNNCH₂CH₂), 25.6

(OCH₂CH₂), 24.8 (NNNCH₂), 14.8 (CH₃C) ppm. HRMS (ESI⁺):

calcd. for C₃₃H₃₈⁷⁹Br⁸¹BrN₉O₆[M + H]⁺816.1293; found

for C₂₉H₄₅⁷⁹Br⁸¹BrN₈O₈[M + H]⁺793.17066; found 793.1704. IR

(ATR): ν = 1721 (s), 1664 (s), 1440 (s), 1081 (s), 553 (m) cm^–1. UV

˜

(CHCl₃): λ_max(logε) = 286 (4.0), 240 (3.9) nm.

4-Azido-N-[1-(4-{4-({(3aR,5S,12aS)-10,11-dibromo-2,8-dioxo-

1,2,3,4,5,6,8,12a-octahydroimidazo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]-

pyrazin-5-yl}oxy)butyl}-1H-1,2,3-triazol-1-yl)-19,26-dioxo-30-(2-

oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9,12,15-penta-

oxa-18,25-diazatriacontan-20-yl]-2,3,5,6-tetrafluorobenzamide (27):

To a solution of 26 (10 mg, 0.017 mmol, 1.0 equiv.) in DMF (2 mL)

were added NHS (1.9 mg, 0.017 mmol, 1.0 equiv.) and DCC

(3.5 mg, 0.017 mmol, 1.1 equiv.), and the mixture was stirred at

room temperature. After 12 h, the reaction mixture was added to a

solution of 25 (13.4 mg, 0.017 mmol, 1.0 equiv.) in DMF (1 mL),

and the mixture was stirred for another 12 h at room temperature.

The solvent was evaporated in vacuo, and the residue was purified

by column chromatography [silica gel, CHCl₃/MeOH (9:1)] to give

27 (7.6 mg, 0.005 mmol, 33%) as a light yellow solid. TLC [silica

gel, CHCl₃/MeOH (9:1)]: R_f= 0.14. [α]²_D⁸= –27.5 (c = 2 mg/mL,

MeOH). ¹H NMR (600 MHz, [D₆]DMSO): δ = 9.15 (d, J = 8.3 Hz,

1 H, FCCCONH), 8.43 (s, 1 H, BrCNCHNH), 8.21 [t, J = 5.5 Hz,

1 H, (OCH₂CH₂)NH], 7.83 (s, 1 H, NNNCH), 7.79 [t, J = 5.7 Hz,

1 H, SCH(CH₂)₄CONH], 6.94 (s, 1 H, BrCCH), 6.72 (s, 1 H,

BrCNCHCNH), 6.44 (s, 1 H, SCH₂CHNH), 6.38 (s, 1 H,

SCHCHNH), 5.99 (d, J = 2.0 Hz, 1 H, BrCNCH), 4.46 (t, J =

5.4 Hz, 2 H, NNNCH₂), 4.42 (m, 1 H, FCCCONHCH), 4.30 (dd,

J = 5.1, J = 7.7 Hz, 1 H, SCH₂CH), 4.20 (m, NCH₂CHOCH₂),

816.1289. IR (ATR): ν = 3381 (m), 3281 (m), 1723 (s), 1685 (s),

˜

1646 (s), 1618 (s), 1594 (s), 1528 (s), 1439 (s), 1391 (s), 1370 (s),

1274 (m), 1217 (m), 1137 (m), 1080 (s), 1050 (m), 1024 (s), 1003

(m), 969 (m), 825 (m), 740 (m), 640 (m), 610 (m) cm^–1. UV

(CHCl₃): λ_max(logε) = 368 (4.2), 287 (3.9), 239 (4.0) nm.

(3aR,5S,12aS)-5-[4-{1-(17-Amino-3,6,9,12,15-pentaoxahepta- 4.13 (m, 1 H, SCHCH), 3.79 (t, J = 5.3 Hz, NNNCH₂CH₂),

decyl)-1H-1,2,3-triazol-4-yl}butoxy]-10,11-dibromo-1,5,6,12a- 3.74 (dd, J = 5.5, J = 12.5 Hz, NCHHCHOCH₂), 3.60 (dd,

tetrahydroimidazo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]pyrazine-

2,8(3H,4H)-dione (25): To a solution of 23 (10.0 mg, 0.020 mmol,

1.0 equiv.) and 32 (6.3 mg, 0.020 mmol, 1.0 equiv.) in degassed

nBuOH/H₂O (1:1, 4 mL) were added sodium ascorbate (0.8 mg,

0.004 mmol, 0.2 equiv.) and CuSO₄(0.8 mg, 0.003 mmol,

0.15 equiv.) whilst stirring at 22 °C in the dark until completion of

the reaction (8 h), which was monitored by TLC [CHCl₃/MeOH/

25% aqueous NH₃(18:2:1)]. The solvent was evaporated under re-

duced pressure, and the residue was purified by column chromatog-

raphy [silica gel, CHCl₃/MeOH/25% aqueous NH₃(18:2:1)] to

yield 25 (11.9 mg, 0.015 mmol, 75%). m.p. 195 °C. [α]²_D³= –142 (c

= 2.5 mg/mL, MeOH). TLC [silica gel, CHCl₃/MeOH/25% aque-

J

=

1.2,

14 H, NNNCH₂CH₂OCH₂CH₂O(CH₂CH₂O)₃], 3.50 (m, 2 H,

NNNCH₂CH₂OCH₂), 3.49 (m, H, NCH₂CHOCH₂), 3.47

(m, H, NHCHCONHCH₂CH₂), 3.42 (t, 5.9 Hz,

J = 12.3 Hz, 1 H, NCHHCHOCH₂), 3.51 [m,

2

J =

NHCHCONHCH₂CH₂O), 3.23 (m, 2 H, NHCHCONHCH₂), 3.09

(m, 1 H, SCHCH₂), 3.0 [m, 2 H, SCH(CH₂)₄CONHCH₂], 2.81 (dd,

J = 5.1, J = 12.4 Hz, 1 H, SCHHCH), 2.63 (t, J = 7.3 Hz, 2 H,

NNNCCH₂), 2.56 (dd, J = 5.7, J = 11.8 Hz, 1 H, SCHHCH), 2.55

(dd, J = 5.6, J = 13.1 Hz, 1 H, BrCNCHCCHH), 2.32 (d, J =

12.8 Hz, 1 H, BrCNCHCCHH), 2.03 [t, J = 7.4 Hz, 2 H,

SCH(CH₂)₃CH₂CO], 1.65 (m, 2 H, SCHCH₂CH₂CH₂), 1.63 (m, 2

H, CHOCH₂CH₂CH₂CH2), 1.60 (m,

1.59 (m, H, CHOCH₂CH₂CH₂), 1.55 (m,

FCCONHCHCH₂CH₂), 1.45 (m, H, SCHCHHCH₂), 1.39

(m, H, FCCCONHCHCH₂CH₂CH₂CH₂), 1.29 (m, H,

SCHCH₂CH₂CH₂), 1.29 (m, 2 H, FCCCONHCHCH₂CH₂) ppm.

1

H, SCHCHHCH₂),

1

ous NH₃(18:2:1)]: R_f= 0.12. H NMR (400 MHz, [D₆]DMSO): δ

2

H,

= 7.83 (s, 1 H, NNNCH), 6.94 (s, 1 H, BrCCH), 5.99 (s, 1 H,

NCHNH), 4.64 (t, J = 5.3 Hz, 2 H, NNNCH₂), 4.20 (m, 1 H,

CH₂CHCH₂), 3.79 (t, J = 5.3 Hz, 2 H, NNNCH₂CH₂), 3.75 (dd,

1

2

J = 5.5, J = 12.6 Hz, 1 H, NCHHCHCH₂), 3.60 (dd, J = 1.6, ¹³C NMR (150 MHz, [D₆]DMSO): δ = 171.8 [(SCH(CH₂)₄CO)],

J

=

13.9 Hz,

1

H, NCHHCHCH₂), 3.50 (m,

2

H,

170.8 (FCCONHCHCO), 162.7 (SCHCHNHCO), 157.6

(BrCNCHNHCO), 156.7 (FCCCO), 154.0 (BrCCHCCO), 146.5

NNNCH₂CH₂OCH₂), 3.49 (m, 2 H, NH₂CH₂CH₂OCH₂), 3.48 (m,

10 H, NH₂CH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂CH₂O), 3.47 (m, 2 (NNNC), 142.1 (N₃CCF), 138.9 (N₃CCFCF), 125.3 (BrCCHC),

H, CHOCH₂CH₂), 3.36 (m, 2 H, NH₂CH₂CH₂), 3.35 (m, 2 H, 122.2 (NNNCH), 120.7 (FCCCO), 114.1 (BrCCH), 112.2

NNNCH₂CH₂OCH₂CH₂), 2.66 (t, J = 5.6 Hz, 2 H, NH₂CH₂CH₂), (N₃CCF), 105.8 (NCBrCBr), 101.2 (NCBr), 78.7 (BrCNCHC),

2.64 (t, J = 7.4 Hz, 2 H, NNNCCH₂), 2.55 (dd, J = 5.7, J = 74.2 (NCH₂CHCH₂), 69.7 [NHCH₂CH₂OCH₂(CH₂OCH₂)₃], 69.5

13.6 Hz, 1 H, CCHHCHCH₂), 2.32 (d, J = 13.1 Hz, 1 H, (NNNCH₂CH₂OCH₂), 68.8 (NHCHCONHCH₂CH₂O), 68.8

CCHHCHCH₂), 1.62 (m, 2 H, CHOCH₂CH₂), 1.59 (m, 2 H,

OCH₂CH₂CH₂) ppm. ¹³C NMR (100 MHz, [D₆]DMSO): δ = 157.6 (NHCHCONHCH₂CH₂), 60.9 (SCHCH), 59.1 (SCH₂CH), 55.4

(NHCONH), 154.0 (CCON), 146.5 (NNNCCH₂), 125.3 (SCHCH₂), 53.1 (FCCCONHCH), 50.8 (NCH₂CHCH₂), 49.1

(CCHCBr), 122.3 (NNNCH), 114.1 (CHCBr), 105.8 (NCBr), 101.2 (NNNCH₂), 44.0 (CCH₂CHCH₂N), 39.6 (SCH₂CH), 38.5

(CHCBr), 78.7 (NHCCH₂), 74.2 (CH₂CHO), 72.4 (NHCHCONHCH₂CH₂), 38.9 [SCH(CH₂)₄CONHCH₂], 35.1

[SCH(CH₂)₃CH₂], 31.8 (FCCCONHCHCH₂), 28.7

(CONHCH₂CH₂), 28.6 (CHOCH₂CH₂), 28.2 (SCHCH₂CH₂), 27.9

(CHOCH₂), 68.8 (BrCNCH), 68.7 (NNNCH₂CH₂), 68.2

(NH₂CH₂CH₂O), 72.4 (NNNCH₂CH₂OCH₂CH₂O), 69.7 (5 C,

NCH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂CH₂O), 69.6 (NNNCH₂-

CH₂OCH₂CH₂O), 68.8 (NCHNH), 68.7 (NNNCH₂CH₂), 68.3 (SCHCH₂), 25.7 (NNNCCH₂CH₂), 25.3 (SCHCH₂CH₂CH₂), 24.7

Eur. J. Org. Chem. 2012, 685–698

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695

T. Lindel et al.

FULL PAPER

(NNNCCH₂), 22.6 (FCCCONHCHCH₂CH₂) ppm. HRMS

(logε) = 290 (3.6), 240 (4.0) nm. Compound 34: m.p. 114 °C. [α]²_D¹

= +136 (c = 5 mg/mL, CH₂Cl₂). TLC [silica gel, EtOAc/isohexane

(7:3)]: R_f= 0.60. ¹H NMR (400 MHz, CDCl₃): δ = 7.61 [d, J =

(ESI⁺): calcd. for C₅₂H₆₉⁷⁹Br⁸¹BrF₄N₁₅Na₂O₁₂S [M + 2Na]²⁺/2

704.6524; found 704.6525. IR (ATR): ν = 3311 (m), 2923 (m), 2868

˜

(m), 1649 (s), 1547 (m), 1400 (m), 1279 (m), 1243 (m), 1101 (m), 8.4 Hz, 2 H, SC(CH)₂], 7.22 [d, J = 7.9 Hz, 2 H, CH₃C(CH)₂], 7.07

1086 (m), 1031 (m), 837 (m), 739 (m) cm^–1.

(dd, J = 1.6, J = 3.8 Hz, 1 H, NCHCHCH), 6.87 (dd, J = 1.6, J =

2.8 Hz, 1 H, NCHCHCH), 6.35 (dd, J = 2.8, J = 3.9 Hz, 1 H,

NCHCHCH), 6.06 (s, 1 H, NCHN), 4.50 (dd, J = 7.6, J = 12.0 Hz,

1 H, NCHHCHCl), 4.43 (m, 2 H, CH₃CH₂), 4.29 (m, 1 H, CHCl),

3.56 (dd, J = 8.9, J = 12.0 Hz, 1 H, NCHHCHCl), 2.96 (dd, J =

8.7, J = 14.7 Hz, 1 H, CCHHCHCl), 2.82 (dd, J = 8.4, J = 14.8 Hz,

1 H, CCHHCHCl), 2.42 (s, 3 H, CH₃C), 1.40 (t, J = 7.1 Hz, 3 H,

CH₃CH₂) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 154.4 (NCOC),

151.1 (NCOO), 149.1 (NCON), 146.8 (CH₃C), 129.9 [CH₃C-

(CH)₂], 129.5 (SCCH), 129.4 [SC(CH)₂], 123.4 (OCCCHCH), 123.0

(NCHCH), 115.3 (NCHCHCH), 112.1 (NCHCH), 83.3 (NCCH₂),

66.5 (NCHN), 65.2 (CH₃CH₂), 53.1 (NCH₂CH), 48.5 (CHCl), 41.5

(CCH₂CHCl), 21.9 (CH₃C), 14.1 (CH₃CH₂) ppm. HRMS (ESI⁺):

calcd. for C₂₁H₂₂ClN₄O₇S [M + H]⁺509.0892; found 509.0889. IR

Ethyl (3aS,12aR)-2,8-dioxo-3-(tosyloxy)-2,3,8,12a-tetrahydroimid-

azo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]pyrazine-1(6H)-carboxylate (35),

Ethyl (3aS,12aR)-2,8-Dioxo-3-(tosyloxy)-2,3,8,12a-tetrahydroimid-

azo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]pyrazine-1(4H)-carboxylate

and Ethyl (3aS,5S,12aR)-5-chloro-2,8-dioxo-3-(tosyloxy)-

(33)

2,3,5,6,8,12a-hexahydroimidazo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]pyraz-

ine-1(4H)-carboxylate (34): To a solution of 2 (200 mg, 0.41 mmol,

1 equiv.) in CH₂Cl₂(50 mL) at 0 °C was added pyridine (86 μL,

1.00 mmol, 2.5 equiv.) followed by Tf₂O (138 μL, 0.81 mmol,

2 equiv.). After completion, the reaction mixture was passed

through a short pad of silica gel eluting with CH₂Cl₂. The filtrate

was concentrated under reduced pressure to a volume of 10 mL

before Et₃N (282 μL, 5 equiv., 2 mmol) was added. After 5 d the

solvent was evaporated, and the residue was purified by column

chromatography [silica gel, EtOAc/isohexane (1:1 to 4:1)] to give

35 (67.4 mg, 0.14 mmol, 35%), 33 (17.3 mg, 0.03 mmol, 9%) and

34 (12.6 mg, 0.02 mmol, 6%) as colourless solids. Compound 35:

m.p. 132 °C. [α]²_D³= +22.6 (c = 7.5 mg/mL, CH₂Cl₂). TLC [silica

gel, EtOAc/isohexane (7:3)]: R_f= 0.43. ¹H NMR (400 MHz,

CDCl₃): δ = 7.59 [d, J = 8.4 Hz, 2 H, O₃SC(CH)₂], 7.21 [d, J =

8.0 Hz, 2 H, CH₃C(CH)₂], 7.09 (dd, J = 1.5, J = 3.8 Hz, 1 H,

NCHCHCH), 6.90 (dd, J = 1.6, J = 2.8 Hz, 1 H, NCHCHCH),

6.60 (dt, J = 1.8, J = 6.1 Hz, 1 H, NCH₂CHCH), 6.36 (dd, J =

2.9, J = 3.8 Hz, 1 H, NCHCHCH), 6.16 (s, 1 H, NCHNCO), 5.81

(dt, J = 2.1, J = 6.2 Hz, 1 H, NCH₂CHCH), 4.45 (m, 1 H,

NCHHCHCH), 4.44 (m, 2 H, CH₃CH₂), 4.32 (dt, J = 1.8, J =

17.3 Hz, 1 H, NCHHCHCH), 2.42 [s, 3 H, CH₃C(CH)₂], 1.41 (t, J

= 7.1 Hz, 3 H, CH₃CH₂CO) ppm. ¹³C NMR (100 MHz, CDCl₃):

δ = 154.9 (NCOC), 151.2 (NCOO), 149.2 (NCON), 146.6

[CH₃C(CH)₂], 136.6 (NCH₂CHCH), 129.9 [CH₃C(CH)₂], 129.5

(O₃SC), 129.4 [O₃SC(CH)₂], 123.9 (OCCCHCHCH), 122.9

(NCHCHCH), 120.9 (NCH₂CHCH), 114.9 (CCHCHCH), 112.1

(CCHCHCH), 88.5 (NCHCN), 65.6 (CH₃CH₂), 65.4 (NCHN),

52.7 (NCH₂), 21.9 (CH₃C), 14.2 (CH₃CH₂) ppm. HRMS (ESI⁺):

calcd. for C₂₁H₂₁N₄O₇S [M + H]⁺473.1125; found 473.1121. IR

(ATR): ν = 1807 (m), 1735 (m), 1665 (m), 1418 (m), 1385 (s), 1290

˜

(m), 1236 (m), 1193 (s), 1178 (s), 1092 (m), 1071 (m), 1013 (m), 965

(m), 799 (m), 739 (s), 717 (s), 653 (m), 603 (m), 562 (s), 545 (s)

cm^–1. UV (CHCl₃): λ_max(logε) = 276 (4.0), 240 (4.0) nm.

(3aR,12aS)-1,12a-Dihydroimidazo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]-

pyrazine-2,8(3H,6H)-dione (36): SmI₂(0.1 m in THF, 2.6 mL,

0.26 mmol, 2.1 equiv.) was added dropwise to a mixture of 35

(60 mg, 0.13 mmol, 1 equiv.) in degassed THF (0.5 mL) followed

by degassed MeOH (1.5 mL). After 2 h, the solvent was concen-

trated under reduced pressure, and the residue was purified by col-

umn chromatography [silica gel, CHCl₃/MeOH (9:1)] to provide 36

as a colourless solid (24.5 mg, 0.11 mmol, 82 %). m.p. 164 °C.

[α]²_D³= +230 [c = 2 mg/mL, MeOH/CH₂Cl₂(3:1)]. TLC [silica gel,

1

CHCl₃/MeOH (4:1)]: R_f= 0.34. H NMR (400 MHz, CDCl₃): δ =

8.22 (s, 1 H, CHNHCO), 7.60 (s, 1 H, CNHCO), 7.18 (dd, J = 1.8,

J = 2.8 Hz, 1 H, NCHCHCH), 6.73 (dd, J = 1.6, J = 3.7 Hz, 1 H,

NCHCHCH), 6.30 (m, 1 H, NCHCHCH), 6.30 (m, 1 H,

NCH₂CHCH), 6.11 (dt, J = 2.0, J = 4.0 Hz, 1 H, NCH₂CHCH),

5.69 (s, 1 H, NCHNH), 4.31 (dt, J = 2.0, J = 16.5 Hz, 1 H,

NCHHCHCH), 4.12 (dt, J = 1.9, J = 16.5 Hz, 1 H, NCHHCHCH)

ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 158.6 (NHCONH), 155.5

(NCOCCH), 129.2 (NCH₂CHCH), 127.8 (NCH₂CHCH), 122.9

(CHCCO), 122.3 (NCHCHCH), 111.8 (NCHCHCH), 110.6

(NCHCH), 82.9 (CHCCH), 66.7 (NCHNH), 51.7 (NCH₂CH)

ppm. HRMS (ESI⁺): calcd. for C₁₁H₁₁N₄O₂[M + H]⁺231.0877;

(ATR): ν = 1801 (m), 1734 (m), 1667 (m), 1623 (m), 1384 (m), 1307

˜

(m), 1284 (s), 1193 (m), 1178 (s), 1010 (m), 964 (m), 807 (m), 719

(m), 653 (m), 570 (s), 545 (s) cm^–1. UV (CHCl₃): λ_max(logε) = 275

(3.9), 240 (4.0) nm. Compound 33: m.p. 157 °C. [α]²_D¹= +202 (c =

3 mg/mL, CH₂Cl₂). TLC [silica gel, EtOAc/isohexane (7:3)]: R_f=

found 231.0877. IR (ATR): ν = 3189 (m), 1725 (s), 1700 (s), 1609

˜

(s), 1545 (s), 1469 (m), 1429 (s), 1377 (m), 1218 (m), 1118 (s), 1062

(m), 1014 (m), 989 (m), 800 (m), 733 (s), 691 (s), 650 (m), 605 (m),

573 (s) cm^–1. UV (CHCl₃): λ_max(logε) = 274 (3.8), 222 (3.9) nm.

1

0.55. H NMR (400 MHz, CDCl₃): δ = 7.61 [d, J = 8.4 Hz, 2 H,

SC(CH)₂], 7.24 [d, J = 7.9 Hz, 2 H, CH₃C(CH)₂], 7.09 (dd, J = 1.6,

J = 3.9 Hz, 1 H, NCHCHCH), 6.95 (dd, J = 1.6, J = 2.8 Hz, 1 H, Ethyl (3aS,5R,12aS)-10,11-Dibromo-5-(methoxymethoxy)-2,8-di-

NCHCHCH), 6.90 (m, 1 H, NCHCHCH₂), 6.37 (dd, J = 2.8, oxo-3-(tosyloxy)-2,3,5,6,8,12a-hexahydroimidazo[4,5-b]di-

3.9 Hz, 1 H, 1 H, NCHCHCH), 6.22 (s, 1 H, NCHN), 5.34 (m, 1

H, NCHCHCH₂), 4.46 (m, 2 H, CH₃CH₂), 3.12 (m, 2 H,

pyrrolo[1,2-a:1Ј,2Ј-d]pyrazine-1(4H)-carboxylate (37): FeCl₃(5 mg,

0.03 mmol, 1 equiv.) was stirred with molecular sieves (80 mg, pow-

NCHCHCH₂), 2.44 (s, 3 H, CH₃CCH), 1.42 (t, J = 7.1 Hz, der 4 Å) under argon for 24 h. The suspension was added to a

CH₃CH₂) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 152.1 solution of 3 (20 mg, 0.03 mmol, 1 equiv.) in dry CH₂Cl₂(5 mL)

(NCOCCH), 151.2 (CH₂OCON), 149.3 (NCON), 146.6 followed by a solution of dimethoxymethane (23 μL, 0.3 mmol,

(CH₃CCH), 129.9 [CH₃C(CH)₂], 129.5 (SCCH), 129.3 [SC(CH)₂],

128.9 (NCHCHCH₂), 126.4 (OCCCHCH), 123.1 (NCHCHCH), at room temperature for 1 h before aqueous NaOH (1 n, 1 mL)

115.4 (NCHCHCH), 112.5 (NCHCHCH), 105.9 (NCHCHCH₂), was added. The organic phase was separated, and the aqueous

10 equiv.) in dry CH₂Cl₂(2 mL). The reaction mixture was stirred

8 2 . 8 ( N C C H ₂) , 6 7 . 1 ( N CH N ) , 6 5 . 2 ( C H ₃C H₂) , 3 8 . 8 phase was washed twice with CH₂Cl₂(5 mL). The combined or-

(NCHCHCH₂), 21.9 (CH₃C), 14.1 (CH₃CH₂) ppm. HRMS (ESI⁺): ganic phases were dried with MgSO₄, filtered and the solvent was

calcd. for C₂₁H₂₁N₄O₇S [M + H]⁺473.1126; found 473.1125. IR

evaporated under reduced pressure. Compound 37 (18 mg,

(ATR): ν = 2924 (m), 1808 (m), 1735 (m), 1666 (s), 1416 (s), 1388

0.028 mmol, 91%) was obtained as a colourless solid. m.p. 162 °C.

˜

(s), 1373 (s), 1322 (s), 1301 (s), 1271 (s), 1194 (s), 1179 (s), 1072 [α]²_D³= +280 (c = 2.5 mg/mL, CH₂Cl₂). TLC [silica gel, EtOAc/

(m), 1013 (m), 814 (m), 719 (s), 566 (s) cm^–1. UV (CHCl₃): λ_maxisohexane (2:3)]: R_f= 0.65. H NMR (400 MHz, CDCl₃): δ = 7.61

1

696

www.eurjoc.org

Eur. J. Org. Chem. 2012, 685–698

(–)-Dibromophakellstatin Derivatives

[d, J = 8.4 Hz, 2 H, O₂SC(CH)₂], 7.25 [d, J = 8.1 Hz, 2 H, treatment, cells were washed with PBS (200 μL) to remove dead

CH₃C(CH)₂], 7.17 (s, 1 H, BrCCH), 6.35 (s, 1 H, NCHN), 4.63

cells and a solution (200 μL) of propidium iodide (7 μg/mL) and

(dd, J = 7.1, J = 14.1 Hz, 2 H, OCH₂O), 4.50 (m, 1 H, Triton X-100 (0.1%, v/v) was added to the wells. After an incu-

NCH₂CHCH₂) 4.44 (m, 2 H, CH₃CH₂O), 4.04 (dt, J = 1.9, J = bation period of 1–2 h at room temperature, the fluorescence was

12.6 Hz, 1 H, NCHHCHCH₂), 3.75 (dd, J = 4.6, J = 12.5 Hz, 1

measured using the Envision Xcite multilabel reader (excitation λ

H, NCHHCHCH₂), 3.34 (s, 3 H, CH₃OCH₂), 2.78 (dd, J = 5.7, J = 530 nm, emission λ = 620 nm) to quantify the amount of at-

= 14.6 Hz, 1 H, NCH₂CHCHH), 2.44 (s, 3 H, CH₃CCH), 2.38 tached viable cells.

(ddd, J = 1.7, J = 3.0, J = 14.5 Hz, 1 H, NCH₂CHCHH), 1.38 (t,

X-ray Structure Determinations

J = 7.1 Hz, 3 H, CH₃CH₂O) ppm. ¹³C NMR (100 MHz, CDCl₃):

Compound 20: C₁₃H₁₄Br₂N₄O₃, monoclinic, P2₁, a = 10.5410(2), b

δ = 153.9 [C(CO)N], 150.6 [EtO(CO)N], 149.9 [N(CO)N], 147.1

= 7.0544(2), c = 21.5219(5) Å, β = 98.599(2)°, V = 1578.35(7) Å³,

[H₃CC(CH)₂], 130.0 [O₂SC(CH)₂], 129.4 [O₂SC(CH)₂], 129.3

Z = 4 (two independent molecules), T = 100 K, D_x= 1.827 Mgm^–3.

[H₃CC(CH)₂], 126.4 (CBrCBrCHC), 117.3 (CBrCBrCHC), 116.3

A colourless tablet-shaped crystal (0.45ϫ0.2ϫ0.08 mm) was used

(CBrCBrCHC), 104.4 (CBrCBrCHC), 95.9 (CH₃OCH₂O), 85.9

to record a total of 88100 data to 2θ(max.) 57.4° using Mo-K_α

(HCCCH₂), 72.7 (NCH₂CHCH₂), 67.7 (NCHN), 65.2

radiation with an Oxford Diffraction Xcalibur E diffractometer.

(OCH₂CH₃), 55.9 (CH₃OCH₂), 52.2 (NCH₂CHCH₂), 38.8

Absorption corrections were based on multiscans. The structure

(NCH₂CHCH₂), 21.9 (PhCH₃), 14.2 (OCH₂CH₃) ppm. HRMS

was refined on F²using the program SHELXL-97^[23]to wR2 0.051

(ESI⁺): calcd. for C₂₃H₂₄⁷⁹Br⁸¹BrN₄NaO₉S [M + Na]⁺714.9508;

(all data), R1 0.026 [IϾ2σ(I)] for 8146 independent data and 423

found 714.9506. IR (ATR): ν = 1806 (m), 1744 (m), 1672 (s), 1432

˜

parameters; S = 0.97, max. Δρ = 0.9 eÅ^–3. Hydrogen atoms of NH

groups were refined with NH distance restraints and fixed U values;

other H atoms were included using a riding model or rigid methyl

groups. One ethoxy group was disordered over two positions. The

Flack parameter was refined to 0.005(5).

(m), 1385 (s), 1264 (s), 1194 (s), 1179 (s), 1091 (m), 1034 (m), 1014

(s), 960 (m), 919 (m), 77 (m), 718 (s), 657 (m), 565 (s) cm^–1. UV

(CHCl₃): λ_max(logε) = 289 (3.9), 240 (4.1) nm.

(3aR,5R,12aS)-10,11-Dibromo-5-(methoxymethoxy)-1,5,6,12a-tetra-

hydroimidazo[4,5-b]dipyrrolo[1,2-a:1Ј,2Ј-d]pyrazine-2,8(3H,4H)-

dione (38): Samarium diiodide (0.1 m in THF, 0.3 mL, 0.03 mmol,

2.1 equiv.) was added to a solution of 37 (10 mg, 0.014 mmol,

1 equiv.) in degassed methanol (2 mL). The reaction mixture was

stirred for 1 h at room temperature before the solvent was removed

under reduced pressure. The residue was purified by column

chromatography [silica gel, CHCl₃/MeOH (19:1)], and 38 (5.4 mg,

0.012 mmol, 85%) was obtained as a colourless solid. m.p. 176 °C.

[α]²_D⁴= –122 (c = 4 mg/mL, MeOH). TLC [silica gel, CHCl₃/MeOH

(19:1)]: R_f= 0.20. ¹H NMR (600 MHz, CDCl₃): δ = 8.34 (t, J =

1.9 Hz, 1 H, CHNHCO), 7.96 (d, J = 1.3 Hz, 1 H, CNHCO), 6.95

(s, 1 H, BrCCH), 6.03 (d, J = 2.3 Hz, 1 H, NCHNH), 4.63 (dd, J

= 6.8, J = 8.9 Hz, 2 H, CH₃OCH₂), 4.57 (m, 1 H, CH₂CHCH₂),

3.77 (dd, J = 7.1, J = 11.9 Hz, 1 H, NCHHCHCH₂), 3.45 (dd, J

= 5.0, J = 11.9 Hz, 1 H, NCHHCHCH₂), 3.27 (s, 3 H, CH₃OCH₂),

2.53 (m, 2 H, NCH₂CHCH₂) ppm. ¹³C NMR (150 MHz, CDCl₃):

δ = 157.7 (NHCONH), 153.9 (NCOC), 124.8 (BrCCHC), 114.1

(BrCCH), 106.0 (NCBrCBr), 101.2 (BCBrCBr), 95.4 (OCH₂O),

78.2 (NCHCCH₂), 71.5 (NCH₂CHCH₂), 68.4 (NCHNH), 55.0

(CH₃OCH₂), 49.7 (NCH₂CHCH₂), 45.3 (NCH₂CHCH₂) ppm.

HRMS (ESI⁺): calcd. for C₁₃H₁₄⁷⁹Br⁸¹BrN₄NaO₄[M + Na]⁺

Compound 34: CDCl₃. C₂₂H₂₁DCl₄N₄O₇S, monoclinic, P2₁, a =

10.1365(4), b = 11.3202(4), c = 12.1148(5) Å, β = 110.156(5)°, V =

1305.00(9) Å³, Z = 2, T = 100 K, D_x= 1.602 Mgm^–3. A colourless

tablet-shaped crystal (0.2ϫ0.1ϫ0.05 mm) was used to record a to-

tal of 45190 data to 2θ(max.) 152.5° (100% complete to 150°) using

Cu-K_αradiation with an Oxford Diffraction Nova A dif-

fractometer. The structure was refined as above to wR2 0.076 (all

data), R1 0.030 [IϾ2σ(I)] for 5342 independent data and 345 pa-

rameters; S = 1.05, max. Δρ = 0.4 eÅ^–3. H atoms were included

using a riding model or rigid methyl groups. The Flack parameter

was refined to –0.015(9).

CCDC-837787 (for 20) and -837788 (for 34) contain the supple-

mentary crystallographic data for this paper. These data can be

obtained free of charge from The Cambridge Crystallographic

Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Supporting Information (see footnote on the first page of this arti-

1

cle): Details on the cytotoxicity assays, H and ¹³C NMR spectra

of selected compounds.

Acknowledgments

472.9259; 472.9251. IR (ATR): ν = 2918 (m), 1732 (s), 1552 (m),

˜

1482 (s), 1341 (m), 1247 (m), 1132 (s), 1069 (m), 1035 (m), 742 (m),

Financial support of this research by the Deutsche Forschungsge-

meinschaft (DFG), (Li 597/5-1) is gratefully acknowledged. We

also thank Merck KGaA (Darmstadt, Germany) for the generous

gift of chromatography materials. BASF Group (Ludwigshafen,

Germany) and Honeywell Specialty Chemicals Seelze GmbH

(Seelze, Germany) are thanked for the donation of solvents.

614 (m), 552 (s) cm^–1. UV (CHCl₃): λ_max(logε) = 279 (4.0) nm.

Monolayer Assay: For the characteristics and origin of the cancer

cell lines used in this study, see the Supporting Information. Cell

lines were routinely passaged once or twice weekly and maintained

in culture for up to 20 passages. All cells were grown at 37 °C in a

humidified atmosphere with 5% CO₂in RPMI1640 medium sup-

plemented with 10% (v/v) fetal calf serum and 0.1 mg/mL gentami-

cin (medium and all components from PAA, Cölbe, Germany). A

modified propidium iodide assay^[22]was used to assess the antican-

cer activity of the compounds towards panels of 12 and 42 solid

tumor cell lines, respectively. Adherent cells were harvested from

exponential phase cultures, counted and plated in 96-well, flat-bot-

tomed, microtiter plates at a cell density depending on the cell line

(4,000 to 40,000 cells/well). After a 24 h recovery period to allow

the cells to resume exponential growth, 10 μL of culture medium

alone or culture medium that contained the test compound were

added. Compounds were applied at 10 concentrations in triplicate

and treatment continued for four days. After 4 d of continuous

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respectively, whereas according to IUPAC nomenclature, the

position would be C-5.

Published Online: December 19, 2011

698

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Article Doi

Synthesis and cytotoxicity of ring C-Functionalized derivatives of the marine natural product (-)-dibromophakellstatin

DOI: 10.1002/ejoc.201101175

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Full text of DOI:10.1002/ejoc.201101175

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