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CSIRO PUBLISHINGCommunication

Aust. J. Chem. 2010, 63, 862–866 www.publish.csiro.au/journals/ajc

Synthesis and Structure Reassessment of Psammopemmin A

Matthew D. LebarA and Bill J. BakerA,B

ADepartment of Chemistry and Center for Molecular Diversity in Drug Design, Discoveryand Delivery, University of South Florida, 4202 E. Fowler Avenue CHE205,Tampa, FL 33620, USA.

BCorresponding author. Email: bjbaker@cas.usf.edu

We have isolated meridianins A, B, C, and E from the Antarctic tunicate Synoicum sp. In the process of verifying thestructure of these compounds it was noted that the physical data reported for meridianins bore a striking resemblance tothat of psammopemmins. The psammopemmins are alkaloids bearing similar structures to the meridianins, but reportedfrom the Antarctic sponge Psammopemma sp. To verify the structure originally proposed for psammopemmin A, thecompound was synthesized. By comparing the 1H and 13C NMR data of reported and synthetic psammopemmin A withthat of meridianin A, we infer that the correct structure of psammopemmin A isolated from Psammopemma sp. is actuallythat of meridianin A.

Manuscript received: 22 January 2010.Manuscript accepted: 2 March 2010.

Introduction

Marine invertebrates living in cold environments have afforded awealth of chemical diversity.[1] Meridianins, 2-aminopyrimidinecontaining indole alkaloids, have been reported from Aplidiummeridianum, a tunicate found in the South Georgia Islands.[2,3]

Our ongoing search for bioactive metabolites produced byAntarctic marine invertebrates has resulted in the isolation ofmeridianins A (1), B (2), C (3), and E (4) (Fig. 1) from anotherSouthern Ocean invertebrate, the Antarctic tunicate Synoicumsp.[4] In the process of verifying the structure of these compoundsit was noted that the physical data reported for meridianin A, B,and E bore a striking resemblance to psammopemmin A (5),C (7), and B (6) (Fig. 1), respectively; the psammopemminsare alkaloids bearing similar structures to the meridianins, butreported from the Antarctic sponge Psammopemma sp.[5] Littledifference exists between the reported NMR data of meridianinA and psammopemmin A (Table 1). Meridianin A has beenpreviously synthesized[6] and the 13C NMR data reported forsynthetic meridianin A matches that of the natural product. The1H NMR data of synthetic meridianin, however, are shifted ∼δ

0.8 ppm downfield from those of the natural product, suggesting

NH

N3'

N1'

NH2R1

R2

R3

R4

245

Meridianin A (1) R1 � OH, R2 � R3 � R4 � HB (2) R1 � OH, R2 � R4 � H, R3 � Br C (3) R1 � R3 � R4 � H, R2 � Br E (4) R1 � OH, R2 � R3 � H, R4 � Br

NH

NHN

OH

R1

R2

1'3'

245

Br

NH2

X

Psammopemmin A (5) R1 � H, R2 � HB (6) R1 � H, R2 � Br C (7) R1 � Br, R2 � H

Suzuki–Miyaura

Fig. 1. Meridianins and psammopemmins.

that water was referenced as the solvent, erroneously, instead ofDMSO.

To verify the structure originally proposed for psammopem-min A, synthetic studies were initiated. We envisioned psammo-pemmin A could be constructed through a Suzuki–Miyauracoupling of 4-hydroxyindole to the desired pyrimidine moiety.

Synthesis of the pyrimidine moiety (Scheme 1) started withamination of 2,4-dicholropyrimidine (8) followed by iodina-tion to yield 4-amino-2-chloro-5-iodopyrimidine (9).[7] Sub-stitution of the 2′-chloro group using HBr/AcOH[8] resultedin 4-amino-2-bromo-5-iodopyrimidine (10). As a general rule,oxidative addition of Pd(0) to halopyrimidines takes placeat C4 > C2 >> C5.[9] However, we expected compound 9 tocouple at C5 rather than C4 due to the more reactive iodosubstituent.

The indole moiety was formed (Scheme 2) by the dehydro-genation of commercially available tetrahydroindolone (11)using Pd/C in refluxing diisobutyl ketone, a modest yield-ing yet economic reaction.[10] The phenol group of 4-indolol(12) was then masked with a silyl protecting group (13). Atwo-step, one-pot N1 silylation and bromination[11] reaction

© CSIRO 2010 10.1071/CH10042 0004-9425/10/060862

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Table 1. 1H NMR and 13C shiftsA of meridianin A (1) and psammopemmin A (5)

Position 1 δHB 1 δH

C 1 δCB Position 5 δH

D 5 δCD

4-OH 13.55 (s) 13.57 (s) 4-OH 13.55 (s)N1—H 11.71 (brs) 11.76 (brs) N1—H 11.75 (brs)2 8.20 (d) 8.22 (d) 128.5 2 8.22 (d) 128.33 113.8 3 113.73a 114.5 3a 114.34 152.1 4 152.05 6.36 (dd) 6.36 (dd) 105.6 5 6.38 (dd) 105.46 6.96 (dd) 6.98 (dd) 124.4 6 6.98 (dd) 124.37 6.78 (dd) 6.79 (dd) 102.4 7 6.81 (dd) 102.37a 139.4 7a 139.22′ 161.9 2′ 161.7E

4′ 160.6 4′ 160.7E

5′ 7.09 (d) 7.10 (d) 104.5 6′ 7.12 (d) 104.36′ 8.10 (d) 8.10 (d) 158.5 N1′—H 8.12 (brd) 158.3E (C5′)2′-NH2 6.69 (s, 2H) 6.72 (s, 2H) 4′-NH2 6.68 (brs, 2H)

Aδ in ppm in d6-DMSO.BIsolated from Aplidium meridianum.[2]

CIsolated from Synoicum sp.[4]

DIsolated from Psammopemma sp.[5]

EAssignments may be interchanged.[5]

N

N Cl

Cl

N

N Cl

NH2

IN

N Br

NH2

I

8 9 10

(a), (b) (c)

Scheme 1.

resulted in the suitably substituted indole 14. Transmetalla-tion with t-butyllithium, then addition of borate 15, gener-ated vinyl borate 16 which could be coupled to 10 using aSuzuki protocol resulting in an intermediate (17) bearing thepsammopemmin skeleton.[12] Desilylation of the resulting pro-tected 3-(5′-pyrimidyl)-indole using TBAF resulted in verylow yields of the psammopemmin A free-base, 18, which wasquite unstable under basic conditions. However, deprotectionof 17 with HF·pyridine[13] produced the psammopemmin Afree-base cleanly, as a white precipitate from the reaction solu-tion. Forming the reported[5] HX salt of psammopemmin A wasproblematic and led to decomposition, however, psammopem-min A hydrochloride (19) could be prepared by bubbling HClgas through a d6-DMSO solution of 18. Immediate analysis ofthe sample via NMR was necessary as the compound began todecompose after a few hours.

NMR data for synthetic psammopemmin A (19) and its freebase (18) were then compared to the natural product psammo-pemmin A (5) (Table 2). The phenolic 4-OH signals differedsignificantly between natural and synthetic psammopemmins (5:δ 13.55, s; 18: δ 9.30, s; 19: δ 9.47, vbrs) suggesting these pro-tons were in dissimilar electronic environments. However, the4-OH signal in meridianin A (1) was identical to that reportedfor psammopemmin A (Table 1).

The signal for H2 also differs between reported (5: δ 8.22, d)and synthetic (18: δ 7.27 days; 19: δ 7.33) psammopemmin A.Both meridianin A (1) and natural psammopemmin A (5) showcoupling between protons on the pyrimidine ring (1: 5′, 6′, d; 5:6′, N1′, d), while there is no obvious evidence for protonationof N1′ in synthetic psammopemmin A hydrochloride (19). It is

not clear from the 1H NMR spectrum of 19 which pyrimidyl-nitrogen accepts a proton (N1′ or N3′). The 4′-NH2 signal of 18exists as a very broad signal from δ 6.2–7.6 (centred around δ

7.00) located under H2, 5, 6, and 7 signals thus making integra-tion difficult. The 4′-NH2 signal of 19 appears even more broad(δ 6.1–8.5, centred around δ 7.45).

The rather sharp 4-OH singlet in the 1H NMR spectrum of18 (δ 9.30) significantly broadens as to be almost impercepti-ble in 19 (δ 9.47). N1′ should be preferentially protonated asproposed in natural product 5. It has been demonstrated thatmonoprotonation of simple 4-aminopyrimidines occurs almostcompletely on N1, para to the amino group.[14] The 13C NMRdata for 19, however, lacks signals for C3, 2′, and 6′, and per-haps is explained by rapid proton exchange between N1 and N3of the pyridine ring. Indeed, this phenomenon has been reportedto occur in 2,4-diaminopyrimidine resulting in two carbon sig-nals for C2, 4, 5, and 6.[15] Unfortunately, 19 slowly degrades ind6-DMSO so, if occurring, the doubling of those carbons due toproton exchange could not be detected.

In summary, from comparison of the 1H and 13C NMR data of1, 5, 18, and 19 we infer that the correct structure of psammopem-min A isolated from Psammopemma sp. is the same as that ofmeridianin A (1). Furthermore, the structure of psammopemminC (7) should also be revised to that of meridianin B (2) due tothe nearly identical 1H and 13C NMR signals reported.[2,5] It isalso likely that the structure of psammopemmin B (6) is thatof meridianin E (4), although the NMR data for the two wereobtained in different solvents so comparison is difficult.

ExperimentalGeneralUnless otherwise stated, all experiments were performed in oven-dried glassware equipped with a magnetic stir bar and a rubberseptum. All solvents used were reagent grade. Anhydrous THFwas obtained by distillation from sodium/benzophenone. Allother chemicals were purchased from Sigma-Aldrich and wereused as received. Products were chromatographed on a Teledyne

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864 M. D. Lebar and B. J. Baker

O

NH

OH

NH

OTBDMS

NH

OTBDMS

NTIPS

Br

OTBDMS

NTIPS

BO

OOTBDMS

NTIPS

N

N

NH2

BrOH

NH

N

N

NH2

Br

11 12 13 14

18 17 16

OB

OOiPr

15

(a) (b) (c)

(d)

(f) (e)

Scheme 2.

Table 2. 1H and 13C NMR shiftsA of synthetic psammopemmin A (18) and synthetic psammopemminA·HCl (19)

Position 18 δH 18 δC 18HMBC 19 δH 19 δC

4-OH 9.30 (s) 9.30 (s)N1—H 11.29 (brs) 11.29 (brs)2 7.27 (d) 123.6 7.33 124.23 105.6 H2, 6′, N1—H Not observed3a 115.4 H2, 5, 7, N1—H, 4-OH 115.24 151.2 H5, 6, 4-OH 151.15 6.38 (d) 103.8 H7, 4-OH 6.48 104.06 6.93 (dd) 122.6 6.90B 122.87 6.88 (d) 103.2 H5 6.90B 103.27a 138.6 H2, 6 138.62′ 148.9 Not observed4′ 163.6 H6′ 163.95′ 113.0 H6′ 113.06′ 7.79 (s) 156.4 7.88 Not observed4′-NH2 7.00 (vbrs) 7.45N1′—H Not observed

Aδ in ppm in d6-DMSO.BSignals overlap.

Isco Combiflash Companion MPLC instrument using normalphase silica gel cartridges purchased from Teledyne Isco. HPLCwas carried out on a YMC-Pack ODS-AQ reverse phase col-umn (250 × 10 mm) using an LC-8A Shimadzu multi-solventdelivery system, an SCL-10A Shimadzu system controller, andan SPD-10A Shimadzu UV-Vis detector. Melting points wererecorded on an Electrothermal Mel-Temp 3.0 instrument. IRspectra were recorded on a Nicolet Avatar 320 spectrometer witha Smart Miracle accessory. HRMS data was obtained on an Agi-lent LC/MSD TOF electrospray ionization mass spectrometer.1H and 13C NMR spectra were recorded on a Varian 400 MHz.

Isolation of Meridianin A, C, D, E (1–4, Respectively)Lyophilized Synoicum sp.[4] (192 g), collected at Palmer Sta-tion, Antarctica, was extracted 3 × 24 h with 1:1 DCM:MeOH.Concentration of the filtrate under reduced pressure yielded21.0 g of lipophilic extract. A portion of this extract (4.6 g)was fractionated on MPLC with a gradient of hexane toEtOAc to MeOH. Fractions eluting at 20–50% MeOH:EtOAcwere combined (25 mg) and chromatographed via analytical

reverse-phase HPLC (55% MeOH:H2O) to yield meridianins A,B, C, and E. NMR data matched that of the previously reportedmeridianins.[2]

Meridianin A (1), 5 mg, δH (d6-DMSO) 6.36 (dd, J 7.3,0.5 Hz, H5), 6.27 (s, 2H, 2′-NH2), 6.79 (dd, J 7.5, 0.5 Hz, H7),6.96 (dd, J 7.5, 7.3 Hz, H6), 7.10 (d, J 5.4 Hz, H5′), 8.10 (d, J5.4 Hz, H6′), 8.22 (s, H2), 11.76 (brs, N1—H), 13.57 (s, 4-OH);δC (d6-DMSO) 102.3, 104.4, 105.4, 114.0, 114.7, 124.4, 128.5,139.4, 152.0, 158.4, 160.5, 161.7.

Meridianin B (2), 5 mg, δH (d6-DMSO) 6.51 (d, J 1.5 Hz, H5),6.85 (s, 2H, 2′-NH2), 7.00 (d, 1.5 Hz, H7), 7.15 (d, J 5.4 Hz, H5′),8.18 (d, J 5.4 Hz, H6′), 8.28 (s, H2), 11.90 (brs, N1—H), 14.21(s, 4-OH).

Meridianin C (3), <1 mg, δH (d6-DMSO) 6.49 (2H, s, 2′-NH2), 7.00 (d, J 5.3, H5′), 7.29 (dd, J 8.5, 1.9 Hz, H6), 7.39 (d,J 8.4 Hz, H7), 8.10 (d, J 5.3 Hz, H6′), 8.24 (s, H2), 8.75 (d, J1.9 Hz, H4), 11.85 (brs, N1—H).

Meridianin E (4), 3 mg, δH (d6-DMSO) 6.37 (d, J 8.3 Hz,H5), 6.85 (2H, 2′-NH2), 7.19 (d, J 8.3 Hz, H6), 7.24 (d, J 5.4 Hz,H5′), 8.18 (d, J 5.4 Hz, H6′), 8.31 (s, H2), 11.92 (brs, N1—H),13.93 (s, 4-OH).

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4-Amino-2-bromo-5-iodopyrimidine 104-Amino-2-chloro-5-iodopyrimidine[7] (9, 0.73 g, 2.86 mmol)in 33% HBr:AcOH was stirred for 1 h then refluxed for anadditional hour. The mixture was subsequently cooled to roomtemperature and the solvent removed under vacuum. Water wasthen added to the concentrate, the solid collected via filtration,and dried under vacuum affording 10 (0.74 g, 2.46 mmol, 86%yield), mp = 189–190◦C. νmax(neat)/cm−1 3330, 3168, 3009,1633, 1549. δH (CDCl3) 5.78 (brs, 2H, NH2), 8.31 (s, H6). δC(CDCl3) 75.4, 152.1, 162.8, 163.9. m/z 299.8626, HRMS calc.for [C4H4BrIN3]+ 299.8629.

4-Indolol 12A stirred mixture of 1,5,6,7-tetrahydro-4H-indol-4-one (2.50 g,18.4 mmol) and 10% Pd/C (625 mg) in 2,6-dimethyl-4-heptanone (25 mL) was refluxed (190◦C) under N2 for 48 h.The mixture was then cooled to room temperature and filteredthrough Celite.The filter cake was washed with DCM and the fil-trate concentrated under vacuum. Kugel-rohr distillation of theconcentrate afforded discoloured 4-indolol (150◦C, 4 torr) whichwas subjected to MPLC (silica; eluting at 30–35% EtOAc:Hex)to yield 12 (1.19 g, 9.0 mmol, 49% yield) as a white crystallinesolid. δH (d6-DMSO, not stable in CDCl3) 6.31 (d, J 6.1 Hz, H3),6.43 (m, H5), 6.81 (m, H6), 6.82 (m, H7), 7.10 (d, J 2.4 Hz, H2),9.21 (s, OH), 10.86 (brs, NH), δC (d6-DMSO) 98.5, 102.8 (2C),117.8, 121.8, 122.9, 137.9, 150.5. m/z 134.0600, HRMS calc.for [C8H8NO]+ 134.0600.

4-(tert-Butyldimethylsilyloxy)-1H-indole 13A mixture of 4-indolol (12, 1.05 g, 7.89 mmol), tert-butyldimethylsilyl chloride (1.43 g, 9.47 mmol), and imidazole(1.74 g, 25.3 mmol) was stirred in 25 mL of dry DMF at roomtemperature under N2 for 1 h. The mixture was partitioned(EtOAc:water). The organic layer was washed with water thendried (anhyd. MgSO4), filtered, and concentrated under reducedpressure onto silica. Purification using MPLC (silica; eluting at18% EtOAC:hexane) afforded 13 (1.76 g, 7.14 mmol, 91% yield)as a white solid, mp = 79–80◦C. νmax(neat)/cm−1 3399, 3056,2961, 2928, 1583. δH (d6-DMSO, not stable in CDCl3) 0.19 (s,6H, Si(CH3)2), 1.02 (s, 9H, SiC(CH3)3), 6.35 (m, H3), 6.41 (d, J7.5 Hz, H5), 6.93 (dd, J 7.5, 8.0 Hz, H6), 7.02 (d, J 8.0 Hz, H7),7.21 (s, H2), 11.0 (brs, NH). δC (d6-DMSO) −4.4 (2C), 18.0,25.7 (3C), 98.3, 105.4, 107.6, 121.2, 121.5, 123.8, 137.9, 148.1.m/z 248.1468, HRMS calc. for [C14H22NOSi]+ 248.1465.

3-Bromo-4-(tert-butyldimethylsilyloxy)-1-(triisopropylsilyl)-1H-indole 14To 13 (1.77 g, 7.14 mmol) in 35 mL dry THF under stirringand N2 was added dropwise n-butyllithium (1.36 mL, 1.6 M inhexanes, 0.85 mL) at −78◦C. The mixture was then warmedto −10◦C and stirred for 10 min before being cooled again to−78◦C. Triisopropylsilyl chloride (1.93 g, 10.0 mmol, 2.14 mL)was added dropwise and the mixture then warmed to 0◦C andstirred until reaction was complete (via TLC). The mixturewas then cooled to −78◦C and N-bromosuccinimide (1.72 g,9.64 mmol) dissolved in dry THF (10 mL) was added to thecooled mixture. After stirring for 2 h at −78◦C, the mixturewas warmed to room temperature, diluted with 1% pyridinein hexane, and filtered through Celite. Any 3-bromo-4-(tert-butyldimethylsilyloxy)-1H -indole in the mixture will lead todecomposition of the desired product upon concentration. The

filtrate was then concentrated onto neutral alumina and chro-matographed (eluting in hexanes). Any brown impurities wereremoved by further purification via MPLC (silica; eluting at5% EtOAc:Hex) to afford 14 (2.69 g, 5.59 mmol, 78% yield) asa white crystalline solid, mp = 107–109◦C. δH (CDCl3) 0.35(s, 6H, Si(CH3)2), 1.08 (s, 9H, SiC(CH3)3), 1.14 (d, 18H,SiCH(CH3)2), 1.65 (sept, 3H, SiH), 6.52 (d, J 7.7 Hz, H5), 6.98(dd, J 7.7, 8.1 Hz, H6), 7.07 (d, J 8.1 Hz, H7), 7.10 (s, H2). δC(CDCl3) −3.7 (2C), 13.0 (3C), 18.2 (6C), 18.7, 26.2 (3C), 90.6,107.7, 109.1, 121.0, 122.8, 129.7, 142.7, 149.5. m/z 482.1905,HRMS calc. for [C23H41BrNOSi2]+ 482.1905.

2-Bromo-5-(4-(tert-butyldimethylsilyloxy)-1-(triisopropylsilyl)-1H-indol-3-yl)pyrimidin-4-amine 17To a stirred solution of 14 (470 mg, 0.98 mmol) in dry THF(5 mL) at −78◦C under N2 was added tert-butyllithium (1.6 Min pentane) dropwise until the solution remained yellow. Afurther aliquot (1.3 mL, 2.15 mmol) was then added dropwiseand the solution stirred for 15 min. Neat 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (15, 364 mg, 1.96 mmol,0.4 mL) was then added dropwise. The resultant mixture wasstirred for 1 h at −78◦C and was subsequently quenched withsat. NH4Cl. The mixture was warmed to room temperature andwas partitioned between Et2O:sat. NH4Cl. The aqueous layerwas extracted 2× with EtO2 and the combined organic layerswere dried (anhyd. MgSO4) and concentrated to afford a crudeborate ester 16 which was used immediately in the next reactionwithout further purification.

A stirred mixture of crude 16 (0.98 mmol), tetrakis(triphenyl-phosphine)palladium (104 mg, 0.09 mmol), and 4-amino-2-bromo-5-iodopyrimidine (10, 283 mg, 0.98 mmol), benzene(25 mL, degassed by sparging with N2), methanol (5 mL,degassed), and aqueous sodium carbonate (1.25 mL, 2 M,degassed) was refluxed under N2 for 24 h. The mixture wasallowed to cool to room temperature, diluted with EtOAcand dried (anhyd. MgSO4). The filtrate then was concen-trated onto silica and purified via MPLC (silica, eluting at18% EtOAc:hexane) to afford 17 (252 mg, 0.44 mmol, 45%yield), mp = 131–133◦C. νmax(neat)/cm−1 3351, 3130, 2951. δH(CDCl3) 0.09 (s, 6H, Si(CH3)2), 0.83 (s, 9H, SiC(CH3)3), 1.16(d, 18H, SCH(CH3)2), 1.69 (sept, 3H, SiCH), 5.25 (brs, 2H,NH2), 6.54 (d, J 7.1 Hz, H5), 7.04 (dd, J 7.9, 8.1 Hz, H6), 7.07(s, H2), 7.15 (d, J 8.1 Hz, H7), 7.95 (s, H6′). δC (CDCl3) −4.1(2C), 13.0 (3C), 18.3 (6C), 18.4, 25.8 (3C), 107.9, 109.0, 109.3,114.1, 121.7, 123.2, 130.3, 143.9, 149.6, 150.3, 156.2, 163.9.m/z 575.2248, HRMS calc. for [C27H44N4OSi2]+ 575.2232.

Psammopemmin A 18To 17 (50 mg, 0.087 mmol) in 1.0 mL acetonitrile (dried over 4 Åmolecular sieves) and 0.1 mL pyridine (dried over 4 Å molecu-lar sieves) was added 50 μL hydrogen fluoride pyridine (70% asHF, 30% as pyridine). After 30 min the starting material hadcompletely dissolved and after 1 h a precipitate had formed.The mixture was stirred for a further 11 h at room tempera-ture. The precipitate was collected via filtration and washedwith acetonitrile. Drying the precipitate under high vacuumresulted in 18 (15 mg, 0.049 mmol, 56%) as a tan powder.νmax(neat)/cm−1 3429, 3313, 3163, 3015. δH (d6-DMSO) 6.38(d, J 7.1 Hz, H5), 6.88 (d, J 8.3 Hz, H7), 6.93 (dd, J 8.3, 7.1 Hz,H6), 7.27 (s, H2), 7.79 (s, H6′), 9.30 (s, OH), 11.29 (brs, NH).δC (d6-DMSO) 103.2, 103.8, 105.6, 113.0, 115.4, 122.6, 123.6,

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866 M. D. Lebar and B. J. Baker

138.6, 148.9, 151.2, 156.4, 163.6. m/z 305.0033, HRMS calc.for [C12H10BrN4O]+ 305.0033.

AcknowledgementThe authors thank the National Science Foundation’s Office of PolarPrograms for financial support (OPP-0442857 and ANT-0838776).

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