Antimicrobial, Antiparasitic and Cytotoxic Spermine Alkaloids from Albizia schimperiana

Abstract

Albizia schimperianaOliv. (Leguminosae) is a tree distributed in the highland of Kenya, where it is used as a traditional medicine for the treatment of bacterial and parasitic infections, notably pneumonia and malaria, respectively. Bioassay guided isolation of the CH₂Cl₂–MeOH 1:1/MeOH-H₂O 9:1 (mixed) extract of A. schimperiana afforded the new bioactive macrocyclic spermine alkaloid, namely 5,14-dimethylbudmunchiamine L1 1 and three known budmunchiamine analogs 2-4. The structures of the compounds 1-4 were determined by 1D and 2D NMR data, including COSY, HMQC, and HMBC experiments, and ESI-HRMS. Compounds 1 and 3 exhibited significant in vitro antimicrobial activity against a panel of microorganisms, including C. neoformans, methicillin-resistant S. aureus, E. coli, M. intracellulare, A. fumigatus. In addition, they demonstrated strong in vitro antimalarial activities against chloroquine-susceptible (D6) and -resistant (W2) strains of Plasmodium falciparum with IC₅₀s ranging from 120–270 ng/mL. Compounds 1-4 were also evaluated for cytotoxic activity against selected human cancer cell lines and mammalian kidney fibroblasts (VERO cells). It was observed that hydroxyl substitution of the side chain of the budmunchiamines dramatically reduced the cytotoxicity and antimicrobial activity of the alkaloids 2 and 4 without decreasing antimalarial activity.

Albizia schimperiana Oliv. (Leguminosae) is an umbrella-like crown tree 5–30 m high, native to tropical African regions. This tree is more common in drier areas and widely distributed in highland forests in Kenya. The stem bark of A. schimperiana is used indigenously for the treatment of bacterial and parasitic infections, like malaria, pneumonia, and more generally against fever and in pain relief [1]. Earlier investigation of this plant reported the presence of five macrocyclic spermine alkaloids (budmunchiamines) [2] as well as triterpenes [3]. However, a series of unusual macrocyclic spermine alkaloids have also been reported from A. gummifera [4], A. amara [5,6], and A. lebbek [7,8].

Interestingly, the MeOH extract of A. gummifera stem bark has been extensively tested and showed antimicrobial [9], antiparasitic [10,11], antitrypanosomal [12], and mosquito larvicidal [13] activities and it has been shown that only the alkaloidal fraction of the extract retained significant activity [14]. In addition, budmunchiamines isolated from A. gummifera demonstrated in vivo antimalarial activity, suppressing Plasmodium berghei infection in mice after oral administration [14]. In addition, budmunchiamines A-I were investigated for their potential interaction with DNA [5,6].

During the course of our drug discovery program at the NCNPR for antiinfective, antiparasitic and anticancer lead candidates from natural sources, an extract of the East African plant A. schimperiana, collected in Kenya, demonstrated potent in vitro activities against Plasmodium falciparum, Cryptococcus neoformans and human cancer cell lines. This led to the bioassay-guided isolation of a new macrocyclic spermine alkaloid, 5,14-dimethylbudmunchiamine L1 (1) and three known budmunchiamine analogs 2-4 (Figure 1). In this paper the isolation, spectral features of 1-4, in addition to their antimalarial, antileishmanial, antimicrobial and cytotoxic activities are reported.

Figure 1.

Structures of budmunchiamines 1-5.

Bioassay-guided fractionation of the crude MeOH–H₂O (9:1) extracts of A. schimperiana resulted in the localization of bioactivities in the CH₂Cl₂–MeOH (1:1) soluble fraction. A centrifugal preparative TLC of this fraction using a silica gel disc eluting with 2% Et₂NH in CHCl₃ yielded compounds 1-4 with yields of 0.003%, 0.0028%, 0.0036% and 0.004%, respectively. The structures of 1-4 were established by ¹H and ¹³C NMR data, mainly 2D NMR COSY, HMQC, HMBC and NOESY experiments, and HRMS. Compounds 2-4 were identified as 6-hydroxybudmunchiamine K, 5-normethylbudmunchiamine K and 6-hydroxy-5-normethylbudmunchiamine K, respectively, by comparison of their NMR and MS data with those reported previously [3, 4].

Compound 1 was isolated as an amorphous solid and analyzed by positive ion ESIHRMS as C₃₂H₆₇N₄O; m/z 523.5341 [M+H]⁺ (calculated for C₃₂H₆₇N₄O 523.5315). It was found to be homogenous on silica TLC and reactive with Dragendorff’s reagent. Its macrocyclic spermine carbon skeleton with four nitrogen atoms attached to an alkyl side chain was suggested from the ¹H and ¹³C NMR [3, 4].

The spectral data [NMR (Table 1) and MS [(M⁺ 522)] of 5,14-dimethylbudmunchiamine L1 1 were in close agreement with the known spermine alkaloid budmunchiamine K 5 (M⁺ 508) [4], except for the differences associated with presence of an additional methylene group at the alkylated side chain. Compound 1 showed the presence of an amide carbonyl (δ_C-2 172.9), three N-Me groups (δ_N-5-Me 35.3, δ_N-9-Me 43.4 and δ_N-14-Me 42.8) and an NH proton at δ 8.62, which was consistent with those reported for budmunchiamine K 5. Structure 1 was unambiguously established by 2D NMR spectroscopy (¹H-¹H COSY, ¹H-¹³C HMQC, and ¹H-¹³C HMBC) experiments. The HMBC experiment established the assignments of the amide carbonyl group and alkyl side chain at C-2 and C-4, respectively, and the N-Me at N-5 position by long-range correlations between δ_H-4 2.80 and δ_C-2 172.9, δ_C-3 37.2, δ_C-2′ 27.8, and between δ_N-5-Me 35.3 and δ_C-4 61.5, δ_C-6 52.0. Based on this data, compound 1 was established as 5,14-dimethylbudmunchiamine L1.

Table 1.

¹H and ¹³C NMR spectral data (J values in Hz, in parenthesis) for 1.

H/C	δH	δC	HMBC
2	-	172.9
3	2.25 m, 2.38 m	37.2	C-2
4	2.80 m	61.5	C-2, C-3, C-2′
6	2.60 dt (6.9, 13.5); 2.39 m	52.0	C-4, C-7, C-8, 5-N-Me
7	1.61 m	26.6	C-6, C-8
8	2.38 m	54.9
10	2.30–2.38 m	56.6
11	1.50 m	24.7
12	1.52 m	23.5
13	2.30–2.38 m	56.6
15	2.37 m	56.2
16	1.62 m	27.5	C-17, C-15
17	3.26 dd (6.1, 12.1)	38.0	C-2, C-15, C-16
1′	1.14 m; 1.48 m	29.6
2′	1.22 m	27.8
3′	1.22 m	30.0*
4′-7′	1.22 m	29.9*
8′-11′	1.22 m	29.8*
12′	1.22 m	29.6
13′	1.22 m	29.6
14′	1.22 m	32.1
15′	1.28 m	22.9
16′	0.86 t (7.0)	14.4	C-14′, C-15′
1-NH	8.62 br s	-
5-N-Me	2.16 s	35.3	C-4, C-6
9-N-Me	2.23 s	43.4	C-8, C-10
14-N-Me	2.16 s	42.8	C-13, C-15

^*signals are overlapping and may be interchanged.

Compounds 1-4 exhibited in vitro antimalarial activity against P. falciparum D6 (chloroquine-susceptible) and W2 (chloroquine-resistant) strains with IC₅₀ values ranging from 120–270 ng/mL (Table 2) and all compounds were found to be almost equally active. Compounds 1-4 also showed strong antileishmanial activity (Table 3), where 1 and 4 were equipotent with pentamidine, whereas 3 was more potent than pentamidine. The isolated compounds 1 and 3 exhibited significant in vitro antimicrobial activity against a panel of microorganisms, including potent activities against Cryptococcus neoformans, methicillin-resistant Staphylococcus aureus (MRS), Escherichia coli, and Mycobacterium. intracellulare, while 2 and 4 were only weakly active against C. neoformans (Tables 4, 5). Among the compounds 1-4 tested for cytotoxicity against selected human cancer cell lines and VERO cells, only 1 and 3 with the non-hydroxylated alkyl side chain exhibited moderate toxicities (Table 6). By contrast hydroxylated derivatives 2 and 4 showed weak cytotoxicities and antimicrobial activities without decreasing antimalarial and antileishmanial activities. The in vitro antiparasitic activity of the isolated budmunchiamines did not vary significantly with substitution pattern and length of the alkylated side chain, while cytotoxicity diminished upon hydroxylation at C-6′ of the alkyl side chain. This also appears to be the first report of 1 from a natural source.

Table 2.

Antimalarial activity (IC₅₀ values are in ng/mL) of compounds 1-4.

Extract/compound	P. falciparum				VERO cells IC50
	D6a		W2b
	IC50	SIc	IC50	SIc
A. schimperiana extr.	970	22.7	1500	14.7	22000
1	140	20	180	15.6	2800
2	160	>29.8	220	>21.6	NC
3	140	21.4	210	14.3	3000
4	120	>39.7	270	>17.6	NC
Chloroquine	15		135		NC
Artemisinin	10		6		NC

^aChloroquine-sensitive clone;

^bChloroquine-resistant clone;

^cSelectivity index=IC₅₀ VERO cells/IC₅₀ P. falciparum; NC = Not cytotoxic (up to the maximum dose tested of 4760 ng/mL).

Table 3.

Antileishmanial Activity of compounds 1-4.

Extract/compound	L. donovani IC50 (μg/mL)	L. donovani IC90 (μg/mL)
A. schimperiana extr.	9	16
1	1.2	6.3
2	3.4	7
3	0.8	1.3
4	2.1	7
Pentamidine	2.1	4
Amphotericin B	0.32	0.8

Table 4.

Antimicrobial activity of compounds 1-4.

Extract/compound	IC50/MIC (μg/mL)
	C. neoformans	E. coli	MRS	A. fumigatus	M. intracellulare
A. schimperiana extr.	3.5	45.0	-	-	-
1	1.0/2.5	3.5/5.0	5.5/10.0	7.5/20.0	1.5/5.0
2	10.0/20.0	-/-	-/-	-/-	-/-
3	1.0/2.5	6.5/10.0	6.5/10.0	-/-	4.0/20.0
4	6.0/10.0	-/-	20.0/-	-/-	20.0/-
Amphotericin B	0.80/1.25	NT	NT	0.6/1.25	NT
Ciprofloxacin	NT	0.004/0.016	0.09/0.50	NT	0.50/1.00

- =Not Active; NT = Not Tested; IC₅₀ is the concentration that affords 50% inhibition of growth; MIC (Minimum Inhibitory Concentration) is the lowest test concentration that allows no detectable growth.

Table 5.

Minimum fungicidal and bactericidal activity of compounds 1-4.

Compound	MFC/MBC (μg/mL)
	C. neoformans	E. coli	MRS	A. fumigatus	M. intracellulare
1	2.5	5.0	-	-	10.0
2	20.0	-	-	-	-
3	2.5	10.0	-	-	-
4	10.0	-	-	-	-
Amphotericin B	2.5	NT	NT	2.5	NT
Ciprofloxacin	NT	0.031	1.00	NT	-

- = Not Active; NT = Not Tested; MFC/MBC (Minimum Fungicidal/Bactericidal Concentration) is the lowest test concentration that kills the organism.

Table 6.

Cytotoxic activity of compounds 1-4.

Compound	IC50 (μg/mL)
	SK-MELa	KBb	BT-549c	SK-OV-3d	LLC-PK11e	VEROf
1	1.9	1.9	2.0	2.2	1.7	1.8
2	5.8	6.0	6.0	6.5	5.3	NC
3	2.0	2.3	2.0	4.8	2.5	5.3
4	5.7	5.4	6.0	6.2	6.0	NC
Doxorubicin	0.85	0.9	0.9	0.9	<0.55	>5

^aSK-MEL: Human malignant melanoma;

^bKB: Human epidermoid carcinoma;

^cBT-549: Human ductal carcinoma;

^dSK-OV-3: Human ovarycarcinoma;

^eLLC-PK₁₁: Pig kidney epithelial;

^fVERO: Monkey kidney fibroblast.

Experimental

General Experimental Procedures

Optical rotations were measured using an AUTOPOL IV digital polarimeter in MeOH at ambient temperature. UV spectra were obtained in MeOH, using a Hewlett-Packard 8453 spectrophotometer. IR spectra were taken as film on a Bruker Tensor 27 FTIR spectrophotometer. The NMR spectra were acquired on a Bruker Avance DRX-500 instrument at 500 MHz (¹H), 125 (¹³C) in CDCl₃, using the residual solvent as int. standard. Multiplicity determinations (DEPT) and 2D NMR spectra (COSY, HMQC, HMBC) were obtained using standard Bruker pulse programs. HRMS were obtained by direct injection using a Bruker Bioapex-FTMS with Electro-Spray Ionization (ESI). TLC was carried out on silica gel plates (EMD Chemicals Inc.) using CHCl₃:Et₂NH=19:1 as solvent. For Centrifugal Preparative TLC (using a Chromatotron^®) 4 mm silica gel disc was used and 2% Et₂NH in CHCl₃ mixture as solvent. Samples were dried in a Savant Speed Vac Plus SC210A Concentrator. The isolated compounds were visualized by observing under UV light at 254 or 365 nm, followed by spraying separately with Dragendorff’s spray reagent.

Plant Material

Plant Material. The stem bark of A. schimperiana Oliv. (Leguminosae) was collected in 2006, Kenya by Dr. Jacob O. Midiwo. The plant was identified by the staff of the Botany Department, University of Nairobi, and a voucher specimen is deposited in their Herbarium (voucher # JOM 2007/143).

Extraction and Isolation

The dried powdered stem bark (0.5 kg) was extracted by percolation with a mixture of MeOH:H₂O=9:1 (2L × 3 times) and the combined extract was concentrated to dryness in vacuo. The dried crude MeOH extract (1.0 g) was dissolved in CH₂Cl₂:MeOH=1:1 and separated from the precipitate. The supernatant was subjected to CPTLC using a 4 mm SiO₂ rotor with 2% Et₂NH in CHCl₃ as a solvent system, afforded 1 (15 mg), 2 (14 mg), 3 (18 mg) and 4 (20 mg), respectively.

5,14-Dimethylbudmunchiamine L1 (1)

Amorphous solid.

[α]_D²⁵: +2.4 (c 0.5, MeOH).

UV (MeOH) λ_max, nm: 203 (lg ε 4.04).

IR (film) ν_max, cm⁻¹: 3294 (NH), 2926 (CH), 2854 (CH), 2789 (CH), 1643 (amide).

¹H NMR data: Table 1.

¹³C NMR: Table 1.

HRESIMS m/z 523.5341 [M+H]⁺ (calcd. for C₃₂H₆₇N₄O, 523.5315).

6-Hydroxybudmunchiamine K (2)

Amorphous solid.

[α]_D²⁵: +2.4 (c 0.5, MeOH).

The spectral data (IR, ¹H and ¹³C NMR) were identical to those reported in the literature [4].

HRESIMS m/z 525.5079 [M+H]⁺ (calcd. for C₃₁H₆₅N₄O₂, 525.5107).

5-Normethylbudmunchiamine K (3)

Amorphous solid.

[α]_D²⁵: −5.7 (c 0.28, MeOH).

HRESIMS m/z 495.5030 [M+H]⁺ (calcd. for C₃₀H₆₃N₄O, 495.5002).

The spectral data (IR, ¹H and ¹³C NMR) were identical to those reported in the literature [3].

6-Hydroxy-5-normethylbudmunchiamine K (4)

Amorphous solid.

[α]_D²⁵: −3.0 (c 0.8, MeOH).

HRESIMS m/z 511.4925 [M+H]⁺ (calcd. for C₃₀H₆₃N₄O₂, 511.4951).

The spectral data (IR, ¹H and ¹³C NMR) were identical to those reported in the literature [3].

Antimalarial/Parasite LDH Assay

The in vitro antimalarial activity was measured by a colorimetric assay that determines the parasitic lactate dehydrogenase (pLDH) activity [15–17]. The assay was performed in 96-well microplate and included two P. falciparum strains [Sierra Leone D6 (chloroquine-sensitive) and Indochina W2 (chloroquine-resistant)]. For the assay, a suspension of red blood cells infected with P. falciparum (D6 or W2) strains (200 μL, with 2% parasitemia and 2% hematocrit in RPMI - 1640 medium supplemented with 10% human serum and 60 μg/mL amikacin) was added to the wells of a 96-well plate containing 10 μL of test samples at various concentrations. The plate was flushed with a gas mixture of 90% N₂, 5% O₂, and 5% CO₂, in a modular incubation chamber (Billups-Rothenberg, 4464 M) and incubated at 37 °C, for 72 h. Plasmodial LDH activity was determined by using Malstat^™ reagent (Flow Inc., Portland, OR). The IC₅₀ values were computed from the dose response curves generated by plotting percent growth against test concentrations. DMSO, artemisinin and chloroquine were included in each assay as vehicle and drug controls, respectively. The selectivity index (SI) of antimalarial activity was determined by measuring the cytotoxicity of samples towards mammalian cells (VERO; monkey kidney fibroblasts).

Antileishmanial Assay

Antileishmanial activity of the compounds was tested in vitro on a culture of Leishmania donovani promastigotes. In a 96 well microplate assay compounds with appropriate dilution were added to the Leishmania promastigotes culture (2×10⁶ cells/mL). The plates were incubated at 26°C for 72 hours and growth of Leishmania promastigotes was determined by Alamar blue assay [18]. Pentamidine and amphotericin B were used as standard antileishmanial agents. IC₅₀ values for each compound were computed from the growth inhibition curve.

Antimicrobial Assay

All organisms are obtained from the American Type Culture Collection (Manassas, VA) and include the fungi Cryptococcus neoformans ATCC 90113 and Aspergillus fumigatus ATCC 90906 and the bacteria methicillin-resistant Staphylococcus aureus ATCC 43300 (MRS), Escherichia coli ATCC 35218, and Mycobacterium intracellulare ATCC 23068. Susceptibility testing was performed using a modified version of the CLSI methods [19–21]. Susceptibility testing of M. intracellulare was done using the modified Alamar Blue^™ procedure of Franzblau et al [22] Samples (dissolved in DMSO) are serially-diluted in 20% DMSO/saline and transferred (103L) in duplicate to 96 well flat bottom microplates. Microbial inocula are prepared in assay medium to afford target CFU/mL after addition to the samples. Growth, solvent and media controls are included on each test plate. Assay plates are read at 630 nm or 544ex/590em (A. fumigates and M. intracellulare) before and after incubation using the Biotek Powerwave XS plate reader (Bio-Tek Instruments, Vermont) or the Polarstar Galaxy Plate Reader (BMG Lab Technologies), respectively. Percent growth is plotted vs. test concentration to afford the IC₅₀, or concentration that affords 50% growth relative to controls. The minimum inhibitory concentration (MIC) is defined as the lowest test concentration that allows no detectable growth. The minimum fungicidal or bactericidal concentrations (MFC/MBCs) are determined by removing 5 μL from each clear well, transferring to agar and incubating until growth is seen. The MFC/MBC is defined as the lowest test concentration that allows no growth on agar. Drug controls [ciprofloxacin (ICN Biomedicals, Ohio) for bacteria and amphotericin B (ICN Biomedicals, Ohio) for fungi] are included in each assay.

Cytotoxicity Assay

The in vitro cytotoxic activity was determined against four human cancer cell lines (SK-MEL, KB, BT-549 and SK-OV-3) and monkey kidney fibroblasts (VERO) and pig kidney epithelial cells (LLC-PK₁₁) (Table 5). All cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The assay was performed in 96-well tissue culture-treated microplates. Cells were seeded at a density of 25000 cells/well and incubated for 24 hours. Samples at different concentrations were added and plates were again incubated for 48 hours. The number of viable cells was determined using Neutral Red according to a modification of the procedure of Borenfreund et al. (1990) [23]. IC₅₀ values were determined from dose response curves of percent growth inhibition against test concentrations. Doxorubicin was used as a positive control, while DMSO was used as the negative (vehicle) control.