A major piperidine alkaloid from Microcos Philippinensis

Phytochemistry, Vol. 29, No. 7, pp. 2309-2313, 1990. Printedin Great Britain.

A MAJOR PIPERIDINE

0

ALKALOID

FROM MICROCOS

ALICIA M. AGUINALDO

and

0031-9422/90 %3.00+0.00 1990 PergamonPress plc

PHILIPPINENSIS

ROGER W. READ*

University of Santo Tomas Research Center for the Natural Sciences, Espana, Chemistry,

University

Manila, Philippines 1008; *Department of New South Wales, P.O. Box 1, Kensington, New South Wales, 2033, Australia (Received 12 October

Key Word micropine.

Index--Microcos

philippinensis;

Tiliaceae;

1989)

antimicrobial;

Abstract-Micropine, a major piperidine alkaloid, has been isolated structure has been determined by spectroscopic methods.

INTRODUCTION

In 1984, a field survey for alkaloids was carried out on 640 flowering plant species in selected areas of Luzon [l]. One of the plants, whose extracts gave a heavy precipitate with Mayer’s and Dragendorff’s reagents, was identified by Dr Domingo Madulid of the National Museum and Dr M. M. J. van Balgooy of Rijksherbarium, Leiden, Netherlands, as Microcos philippinensis (Perk.) Burrett of the family Tiliaceae. There has been no phytochemical report on M. philippinensis. However, the genus Microcos has been closely identified with the genus Grewia [2]. Microcos philippinensis (Perk.) Burrett is also named Grewia philippinensis Perk [Z]. Of the 20 Grewia species in the Philippines [3], alkaloids have been reported present in G. pnniculata Roxb. [4] and G. polygama Roxb. [S] but these remain unidentifed. From the non-endemic Grewia species, the alkaloids identified are of the harman type, such as those from G. &color Juss., G. uillosa and G. mollis [S-S]. Microcos philippinensis has since been included in a search for antimicrobial substances. The ethanolic extract was found to cause slight inhibition against Staphylococcus aureus ATCC 25923, Streptococcus pyogenes ATCC 19615 and Escherichia coli ATCC 25922; marked inhibition against Pseudomonas aeruginosa ATCC 27853 and Mycobacterium 607 [9] was also reported. This study reports the isolation and identification of micropine, a novel piperidine alkaloid from M. philippinensis [2]. Reported plant sources of piperidine alkaloids include the genera Bathiorhamnus [lo], Conium [l 11, Lobelia [12], Sedum [13], Prosopis [14-171, Azimn [18], Carica [19], Cassia [20], Achilles [21] and Exoecharia [22] among others. Substitution on the piperidine ring varies from positions 1,2,3,6 as in cryptophorine (1) from Bathiorhamnus, positions 1,2,5 as in N-methylpseudoconhydrine (2) from Conium, positions 1,2,6 as in lobeline (3) from Lobelia and sedinone (4) from Sedum, positions 2,3,6 as in prosopine (5) from Prosopis, azimine (6) from Azima, carpaine (7) from Carica and spectaline (8) from Cassia and position 1 as in the amidic piperidine alkaloids 9 and 10 from Achilles and Exoecharia, respectively. Micropine which was assigned the structure 11 is 1,2,3,6substituted, and is therefore related most closely to

of Organic

piperidine

alkaloids;

structure

elucidation;

from the leaves of Microcos philippinensis. Its

cryptophorine though differing in the types of substituents. This paper is the first report of a piperidine alkaloid isolated from the genus Microcos of the family Tiliaceae.

RESULTS AND DISCUSSION

Ethanol extraction of the leaves of Microcos philippinensis gave a gum which was extracted with 1% sulphuric acid. The acid soluble fraction (0.26%) afforded a crystalline substance which separated from methanol and represented the major alkaloidal component as judged by TLC. Recrystallization of the solid from methanol afforded the pure substance 11 which we have called micropine. Micropine gave a [M] + in its mass spectrum at m/z 279, with major fragment ions at m/z 248 (base peak), 175 and 91. Elemental analysis suggested the molecular formula C,,H,,NO,. There was no evidence in the IR spectrum of N-H or carbonyl stretching absorptions. Instead, there was a strong absorption at 3335 cm-’ indicating the presence of an OH function and bands at suggestive of an olefinic group. 3016 and 1639cm-’ Portions of the ‘H NMR (65.47-6.20, 3 x m, total 6H) and ‘%DEPTNMR spectra(6129.8, 130.1, 131.7, 132.8, 135.8, 136.2, 6 x CH) supported the presence of three olefinic groups, each one being vicinally disubstituted. Exhaustive hydrogenation of micropine gave a product (see later) which had absorbed three mole equivalents of hydrogen. Thus, the presence of three double bonds in micropine was confirmed and the single absorption at A,,,,, 268 nm (E 47 000) in its UV spectrum established the conjugated nature of the triene system. The base peak, at m/z 248 [M - 31]+, in the mass spectrum corresponded to loss of an hydroxymethyl group from the parent ion. Such a group was evident in the ‘HNMR spectrum from two geminal proton signals at 6 3.84 and 3.95 (Jgem = 11.3 Hz). These resonances were part of an ABX spin system in which the X proton resonated at 61.90 and showed additional coupling (J = 9.1 Hz) to a signal at 3.69. There were no other signals in the region 62.7-5.3 of the spectrum therefore the latter resonance was assigned to a methine group bound to a second hydroxyl group. The methine signal showed coup-

2309

2310

A. M. AGUINALD~ and

R. W.

R3

R4

READ

xx RZ i

R5

k’ RZ

R3

Me

OH

H

Pr

H

OH

H

11

Me

RS

R4

R’ Me

(CH:CH),Et t-I CH2CtiOHPh

Me

CHJOPh

Me

CH,COMe

H

H

H

CH, OH

OH

H

H

Me

OH

fl

(CHZ), ICOMe

H

H

H

ti

H

H

H

H

CO(Cti:CH)2(CH,)2Ct~:CHMe

10

ling to two other one-proton signals at 6 1.25-1.45 (obscured, J = 10.7 Hz) and 2.02 (J = 4.8 Hz). The magnitude of its three couplings suggested an anti-coplanar arrangement of the methine proton with two of its neighbours and a skew relationship to the other. These constraints suggest the presence of a rigid fragment A in micropine. The ‘H NMR spectrum also contained a three proton singlet at 62.25, indicative of an N-methyl group, and a one proton signal at 2.61 (ddd). The latter signal was shown by double irradiation experiments to be coupled to one of the olefinic signals (65.47, J = 8.6 Hz) and to an isolated aliphatic signal (1.64, J = 3.2 Hz). Its chemical shift and the absence of other electronegative groups with which it might interact prompted assignment of the proton to a position between the nitrogen and the triene units. The remaining coupling (J = 11.4 Hz), to an obscured signal at S 1.25-1.45, also suggested that the proton was anti-coplanar with respect to the unseen proton. Such features enabled assignment of a second fragment B. The evidence thus far presented strongly suggests a union of fragments A and B to give a piperidine derivative, held in a rigid conformation. This leaves a unit, C,H,, unaccounted for but obviously attached through the triene of fragment B. ‘H NMR spectroscopy provided unequivocal evidence for the identity of the group. There was a three proton triplet at SO.89 (J = 7.0 Hz) and a two proton doublet of triplet signal at 2.09 (J = 7.1, 7.0 Hz) which was coupled to the olefinic signal at 5.70. Such signals are only possible if the additional unit was an n-

CH,CHOHPh (CH,),

6 7

&HOHMe

n = 5 n = I

OH

Me

butyl chain. Thus, micropine was assigned structure 11 with the relative stereochemistry about the piperidine ring as indicated. Many assignments of couplings were made and checked by double irradiation experiments, The results are listed in Table 1. In addition, the stereochemistry in the neighbourhood of the amino group was examined by nuclear Overhauser experiments. The results of these. presented graphically in

Alkaloid

Table

1. Results of double

irradiation

from Microcos

experiments

2311

on the ‘H NMR spectrum

Signal irradiation

Signal observed

Change

1.64 (H-Seq)

2.61 (ddd, H-6ax) 1.25-1.45 (m, H-4ax, H-Sax, H-8”, H-9”) 2.02 (m, H-4eq) 3.69 (ddd, H-3ax) 3.84 (dd, H-l’a) 3.95 (dd, H-l’b) 5.70 (dt, H-6”) 1.25-1.45 (m, H-4ax, H-Sax, H-8”, H-9”) 5.47 (dd, H- 1”) 1.25-1.45 (m, H4ax, H-Sax, H-8”, H-9”) 1.64 (ddd, H-Seq) 1.89 (ddd, H-2ax) 2.02 (m, H&q) 3.95 (dd, H-l’b) 1.89 (ddd, H-2ax) 3.84 (dd, H-l’a) 1.89 (ddd, H-2ax) 2.61 (ddd, H-6ax) 6.0-6.2 (m, H-2’-H-5”) 2.09 (td, H-7”) 6.0-6.2 (m, H-2”-H-5”) 5.47 (dd, H-l”) 5.70 (dt, H-6”)

Collapsed Affected Affected Collapsed Collapsed Collapsed Collapsed Affected Collapsed Affected Affected Collapsed Collapsed Collapsed Collapsed Collapsed Collapsed Collapsed Affected Collapsed Affected Collapsed Collapsed

1.90 (H-2ax)

2.09 (H-7”) 2.62 (H-6ax)

3.71 (H-3ax) 3.84 (H-l’a) 3.96 (H-l’b) 5.47 (H-l”) 5.71 (H-6”) 6.11 (H-2”-H-5”)

Signal irradiation

Overhauser

enhancements

Fig. 1, clearly reveal the equatorial arrangement of the Nmethyl group and the apparently fixed conformation of the hydroxymethyl group, probably brought about by internal hydrogen-bonding with the lone pair of electrons on the nitrogen atom. The 13C NMR spectrum of micropine supported structure 11but, like the ‘H spectrum, it left some doubt about the configuration of the double bonds in the triene system. Signal assignments were facilitated by DEPT and 1H”3C correlation experiments. These enabled the protons and carbons at C-l” and C-6” to be assigned unambiguously. The observed ‘H-‘H spin coupling of the proton signals at 65.47 and 5.70, J,.., 2V,= 15.0 Hz and J,.. 5rg = 15.0 Hz, respectively, indicated that the terminal’double bonds were trans-substituted. The signals at 66.0-6.2 for the remaining olefinic protons were complex and not resolved, even in deuterobenzene. Fortunately, the 13C NMR signals for C-2”, C-3”, C-4” and C-5” were reasonably well separated. The resonances of

observed

J 5eq,6ax= 3.2

to dd

to to to to

dd d d d

J J J J

ax. 3PX= 9.1 zar.1.1 = 3.8 zm, ,‘b = 1.6 6”,,‘,- -71

J 6_, ,., = 8.6

to d

to m to to to to to

dd

J 3nx.2ax= 9.1

d dd br dd dd dd

J l,n.l,b = 11.3 J ~‘n.znx= 3.8 Jl.b,2sr = 1.6 J1s.,6ar = 8.6

to

t

J 6”,,” --71 J,,.,,,. J,...,..

to d to t

% NOE

1.9 ( HQax )

2.6

- 2.58

2.6 (H-6ax )

1.9

- 2.74

1.52

- 1.92

observed

11

Inference

Signal observed

2.25 ( NMe )

Fig. 1. Selected nuclear

of micropine

1.42

- 1.21

3.83

- 1.33

2.58

- 0.94

1.9

- 1.28

in the ‘H NMR spectrum

= 15.0 = 15.0

of micropine.

the protons directly attached to each of these carbons were accessed separately through heteronuclear shiftcorrelated two-dimensional NMR with WALTZ decoupling using polarization transfer from ‘H to 13C via J, H [23]. The residual coupling constants are collected in Table 2. While the magnitude of the H-H” coupling (fragment C) J, ,.,,, = lo-11 is as expected, the magnitudes of the more interesting H-H’ coupling (fragment C) J H.He = 15.5-16 are typical of those observed for an Econfiguration. Micropine is thus 2,3-trans-3,6-trans-lmethyl-2-hydroxymethyl-3-hydroxy-6-( l’E,3’E,S’E-decatrienyl)piperidine (11). The perhydro-derivative of micropine was prepared and as expected its ‘H NMR spectrum showed the disappearance of the complex signal at 65-6 due to the olefinic protons. The ‘H NMR data show hydrogenation has an expected shielding effect on the H-6 proton, from 62.61 in micropine to 2.12 in perhydromicropine, but a deshielding effect on the H-2 proton, from 1.89 to 2.24. The

A. M. AGUINALDO and R. W. READ

2312

Table 2. Residual couplings of H-1”-H-6” in micropine (11) as determined by heteronuclear shiftcorrelated 2D NMR C signal irradiation

$I+&

Fragment C

JHH’

JHH”

128.9 130.1 131.7 132.X

:::: IS.9 15.6

:::;’ 10.0 10.5

135.8 136.1

15.8 15.8

( 7.9 ) ( X.7)

hydroxymethyl protons appear as a single resonance (63.88, d) by either accidental equivalence of the protons or loss of rigidity through loss of internal hydrogen bonding; the NMe protons are unaffected. The new chemical shifts of H-Z and H-6 are in agreement with those for (-)-deoxoprosophylline [24, 251, 62.56 and 2.50 respectively, which has the same relative configuration as micropine but lacks the N-methyl group. As in micropine, the mass spectral data of perhydromicropine showed the fragment loss of the hydroxymethyl group at m/z 254, the base peak. It is interesting to note that as a result of the hydrogenation, m/z 144 is a prominent peak due to the facile removal of the alkyl chain, leaving group D. This fragment loss is not evident in micropine; thus, it is a further proof that the triene in micropine is directly attached to the piperidine ring. EXPERIMENTAL

General. Mps: uncorr. Microanalyses were carried out by Dr H. P. Pham (School of Chemistry, University of New South Wales). NMR spectra were obtained with 100, 300 or 500 MHz instruments in CDCI, with TMS int. ref. MS were measured at 70 eV. Plant material. Leaves were collected from Magat Reforestation Area, Diadi, Nueva Vizcaya. Voucher specimens are deposited at the herbaria of the UST Research Center for the Natural Sciences, the National Museum in Manila and the Rijksherbarium in Leiden, Netherlands. Isolation. Air-dried leaves (1 kg) were ground with a Wiley mill and soaked with 95% EtOH in a percolator for 24 hr. The filtrate was collected, fresh EtOH added to the grounds and the process repeated until the extract gave a negative result with Mayer’s reagent. A total of 101 EtOH was consumed. The filtrate was coned in uacuo. The extract obtained (100 g) was treated with 1% H,SO, until the aq. layer gave a negative test with Mayer’s reagent. The acid layer was washed with Et,O, basified to pH 10 with aq. NH, and

extracted exhaustively with Et,O. The Et,0 layer was dried (Na,SO,) and filtered. The filtrate was evapd in tucuo to give a pale brown residue (2.6 g). The residue deposited a solid from MeOH. Recrystallization of this solid from MeOH afforded micropine (11) as off-white plates (160 mg), mp 146- 148’. Micropine. (Found: C, 73.1; H, 10.9; N, 5.0. C,,H,,NO, requires C, 73.1; H, 10.5; N, 5.0%). [a]:” - 63’ (EtOH; c 0.146). IR vKBr cm-‘: 3335 br, 3016,2960, 2930,2X63, 27X7,2445, 1885, 1802, 1722,1639,1506, 1489, 1463, 1448, 1419, 1379, 1359, 1298, 1280, 1262, 1232, 1211, 1166, 1127, 1087, 1077, 1052, 1023, 998, 977,940,916, 888,872,83X, 812, 713,675,641,610, 567, 528,482, 453 373 352. UViEto” nm: 250 (E 24000), 25X (39000), 268 (47&&2X0 (414OOO), 299 (4200), 315 (3300). ‘HNMR (300 MHz, CDCI,, TMS): 60.89 (3H, t, J = 7.0 Hz, H-10”), Z--l.45 (6H, m, H-4,,, H-5,,, (H-X”),, H-9”),), 1.64 jlH, ddd, J = 9.7 Hz, J = 3.2 Hz, J = 3.2 Hz, H-5,,), 1.89 (lH, ddd, J = 9.1 Hz, J = 3.X Hz, J = 1.4 Hz, H-2,,), 2.02 (lH, m, H-4,,), 2.02-2.13 (2H, m, OH), 2.09 (2H, dt, J = 7.1 Hz, J = 7.0 Hz, H7”), 2.25 (3H, s, N-Me), 2.61 (lH, ddd, J = 11.4, 8.7, 3.2 Hz, H6,,), 3.69 (lH, ddd, J = 10.7, 9.2, 4.X Hz, H-3,,), 3.X4 (lH, dd, J =11.3. 3.XHz. H-l;), 3.95 (lH, dd, J=l1.3, 1.6Hz, H-l”). 5.47 lH,dd, J = 15.0, 8.6 Hz,H-1”), 5,70(1H,dt,J = 15.0,7.1 Hz, H6”), 6.0-6.2 (4H, m, H-2,-H-5”). ’ 3C NMR (125.8 MHz): (5 13.9 (q,C-10”), 22.2 (t, C-9”), 30.8 (t, C-S), 31.4 (t. C-X”). 32.5 (t, C-7”), 32.X (t. C-4), 40.0 (q.N-Me), 58.8 (t, C-l’), 66.X (d, C-6). 67.5 (d. C3),70.1 (d,C-2), 129.8, 130.1, 131.7, 132.8,(4&C-?“m~C-S”), 135.X (d, C-6”), 136.2 (d, C-l”). MS: m,/z 279 [M]’ (34%), 24X [M -31]+ (lOO), 236 (13), 176 (16), 175 (96), 134.(lo), 133 (lo), 120 (15),119(30), 117(13),105(30),94(19),91(53).81 (17),79(27).77 (18). Hydrogenation. A mixt. of micropine (X4.X mg) and 10% Pd/C (50 mg) in EtOH (20 ml) was hydrogenated (170 ml) for 3.5 hr. The mixt. was filtered and the filtrate evapd in vacua. Perhydromicropine was purified twice by prep. TLC on 1 mm Merck silica gel PFZ5., plates with CHCI,-MeOH (5: 1). Bands were visualized with UV and I, vapours. The perhydromicropine band was eluted with CHCl,-MeOH (1: I). Perhydromicropine (32 mg) was obtained as pale white needles from MeOH. mp 56&5X”. ‘H NMR (300 MHz, CDCI,, TMS): S 0.X8 (3H, 1, .I = 6.X Hz, HlO”), 1.23-1.28 (16H, m, H-2”-H-9”), 1.30 (tH. m, H-4_), 1.36 (lH,m,H-I”,); 1.391 (lH,m:H-5,,), 1.57(1H,m,H-5,,), 1.69(1H, m,H-1”,),2.04(lH,m.H-4,,),2.05~2.20(2H,m,OH), 2.12(lH,m, H-6,,), 2.24 (lH, m, H-2,,), 2.29 (3H, s, NMe). 3.70 (lH, ddd, J = 10.2 Hz, J = 10.0, 4.7 Hz, H-3,,), 3.88 (2H, d. J = 3.5 Hz, H1’). *jC NMR (125.X MHz): 6 14.1 (q,C-lo”), 22.6 (t, C-9”), 25.3 (t, C-8”), 27.4 (r, C-l”), 29.3 (I), 29.6 (3t), 30.0 (I). 31.9 (t), (C-2” to C7”), 33.3 (t, C-3), 33.7 (t, C-5). 35.7 (q, NMe), 59.1 (t, C-l’), 62.6 (d, C-2), 66.7 (d, C-3), 69.9 (d, C-6). MS: m/z 285 [M]’ (1.6%), 255 (20),254[M-31]+(100),252(10),240(16), 144(73),126(1X), 123 (12), 122(17), 111 (15). 109(17),97(24),96(22),95(29).X3(26),82 (23), 81 (30), 71 (26). 69 (3.5). 67 (27). 57 (44), 55 (50), 45 (26) 43 (481, 41 (51).

Acknowledgements-We thank Dr Domingo Madulid of the National Museum and Dr M. M. J. van Balgooy of the Rijksherbarium for identification of plant material and for helpful suggestions. We are grateful to Dr B. Q. Guevara and MS B. V. Recio for their help in the preliminary studies, Mrs A. L. Claustro for the plant collection, Dr J. J. Brophy for the MS data, Dr K. Cross and Mrs H. E. Stender for the NMR data and Mrs N. Jurinario for the IR data. We thank the University of Santo Tomas Research Center for the Natural Sciences and the Network for the Chemistry of Biologically Important Natural Products, an activity of the International Development Program of Australian Universities and Colleges. for the research grant.

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