Constituents from Nauclea latifolia with Anti-Haemophilus influenzae

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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Constituents from Nauclea latifolia with Anti-Haemophilus inf luenzae Type b Inhibitory Activities Jean Jules Kezetas Bankeu,*,† Donald Ulrich Kenou Kagho,‡ Yannick Steṕ hane Fotsing Fongang,§ Rufin Marie Kouipou Toghueo,□ Brice Mitteŕ ant Mba’ning,‡ Guy Raymond Tchouya Feuya,∥ Fabrice Boyom Fekam,□ Jean Claude Tchouankeu,‡ Silver̀ e Augustin Ngouela,‡ Norbert Sewald,⊥ Bruno Ndjakou Lenta,# and Muhammad Shaiq Ali¶ †

Department of Chemistry, Faculty of Science, The University of Bamenda, P.O. Box 39, Bambili, Cameroon Department of Organic Chemistry, Faculty of Science, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon § Department of Chemistry, Higher Teacher Training College, University of Maroua, P.O. Box 55, Maroua, Cameroon □ Department of Biochemistry, Faculty of Science, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon ∥ Department of Chemistry, Faculty of Science, Scientific and Technical University of Masuku, P.O. Box 943, Franceville, Gabon ⊥ Department of Chemistry, Bielefeld University, P.O. Box 100131, 33501 Bielefeld, Germany # Department of Chemistry, Higher Teacher Training College, University of Yaoundé I, P.O. Box 47, Yaoundé, Cameroon ¶ H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan

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S Supporting Information *

ABSTRACT: Three previously undescribed indole alkaloids, named latifolianine A (1) and latifoliaindoles A and B (2 and 3), along with 10 known compounds (4−13), were isolated from the heartwood of Nauclea latifolia. Their structures were elucidated based on the analysis of their NMR and MS data. Latifolianine A (1) represents an unusual and unprecedented monoterpene indole alkaloid unit condensed with an ursanetype pentacyclic triterpenoid moiety. Plausible biogenetic routes toward latifolianine A (1) and latifoliaindoles A and B (2 and 3) were proposed. All the isolates were assessed in vitro for their inhibitory effects on Haemophilus influenzae. Naucleidinal (7) exhibited potent antibacterial activity (MIC value of 3.1 μg/mL) as compared to a reference drug, ciprofloxacin (MIC value of 1.6 μg/mL). including a high number of intrinsic resistance cases, and tracking of the epidemiology is challenging due to the genomic diversity of H. inf luenzae and its high prevalence of recombination.9 Therefore, new antibiotics to control these infections are needed urgently. Natural products, and in particular plant secondary metabolites, continue to play a highly significant role in the drug discovery and development process. In fact, the exploration of natural products has provided one of the major sources of drugs to date.10 Accordingly, investigating plant secondary metabolites may lead to the identification of potent agents for the development of new drugs against H. influenzae. Nauclea latifolia Smith, also known as Sarcocephalus latifolius or the African peach, is an important evergreen medicinal plant of tropical Africa. It is used extensively in folk medicine in

Haemophilus influenzae type-b (Hib) is a leading cause of an invasive bacterial disease affecting children worldwide.1,2 Its most frequent manifestations are pneumonia and meningitis, but it can also cause infections of the epiglottis, soft tissues, bones, joints, and other sites.3 In 2009, Hib was estimated to have caused 363 000 deaths in HIV-uninfected children and 8.13 million total episodes in children in the same year.4 Wahl et al. estimated that there were 340 000 episodes (196 000− 669 000) of severe Hib and 29 500 Hib deaths (18 400− 40 700) in HIV-uninfected children aged 12−59 months in 2015.5 Despite the development of vaccines against this pathogen, its prevention and eradication has not yet been achieved, because of the high cost of the vaccine, which significantly limits the access of many people in poor and developing countries.6 The increasing emergence of H. influenzae-resistant strains to available antibiotics (chloramphenicol, penicillin, ampicillin, and third-generation cephalosporins) is alarming.7,8 In fact, there has been a steady increase in H. inf luenzae isolates resistant to ampicillin, © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 15, 2019

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DOI: 10.1021/acs.jnatprod.9b00463 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

yield latifolianine A (1) and latifoliaindoles A and B (2 and 3), with compound 1 being an unprecedented monoterpene indole alkaloid condensed with an ursane-type pentacyclic triterpenoid. In addition, naucleidinal dimethyl acetal (4),14 (+)-naucleofficine D (5), pobeguinine (6), naucleidinal (7),15 3β,6β,23-trihydroxyolean-12-en-28-oic acid (8), 3β,6β,19α,23tetrahydroxyolean-12-en-28-oic acid (9),16 pomolic acid (10),17 quinovic acid (11), quinovic acid 3-O-α-L-rhamnopyranoside (12), and quafrinoic acid (13)15,18 were also isolated and characterized by comparison of their spectroscopic data with those reported in the literature. Compound 1 was isolated as a colorless, amorphous powder with a specific rotation of [α]25 D −16.5 (c 0.07, MeOH). Its molecular formula, C50H66N2O8, was deduced from the NMR data and the HRESIMS, which showed a protonated molecular ion peak [M + H]+ at m/z 823.4896 (calcd for C50H67N2O8, 823.4897), implying 19 degrees of unsaturation. The UV spectrum in MeOH showed absorption bands at λmax 214, 229, and 351 nm. The IR spectrum of 1 exhibited absorption bands for free hydroxy groups (3778 and 3703 cm−1), an amine group (3413 cm−1), a six-membered-ring lactam (1663 cm−1), and a carboxylic acid carbonyl functionality (1592 cm−1). The 13 C NMR spectrum of 1 (Table 1) displayed 50 carbon signals,

Central and West Africa for body pain, fever, convulsions, hypertension, diabetes, digestive problems, neurological disorders, and infectious diseases, including toothache, dental caries, septic mouth, malaria, and diarrhea.11,12 Moreover, crude extracts from this plant have been reported for their antiplasmodial, antimicrobial, and analgesic activities.11 These findings have motivated natural products chemists to carry out phytochemical investigations on this plant, and a variety of secondary metabolites including monoterpene indole alkaloids, pentacyclic triterpenoids of the ursane series, and several phenolic compounds have been isolated and characterized.11,13 In a continuation of our search for potent anti-H. influenzae compounds, the crude methanol (MeOH) extract of the heartwood of N. latifolia growing in Cameroon was investigated, and reported herein are the isolation and characterization of three new indole alkaloids (1−3) and the in vitro inhibitory potential against H. influenzae of all isolates obtained.

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RESULTS AND DISCUSSION The MeOH extract of the heartwood of N. latifolia was subjected to silica gel and Sephadex LH-20 column chromatography (CC) followed by preparative HPLC to B



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180.6 (carboxylic acid, C-28) and 165.1 (lactam, C-22′)] and 16 methine, 14 methylene, and six methyl carbon signals. Among these carbon signals, those at δC 140.0 (C-13) and 127.7 (C-12) are characteristic carbon signals for ursane-type triterpenoids, where the carbon at C-28 is oxidized to a carboxylic acid functionality at δC 180.6 (C-28).19 In addition, carbon signals at δC 73.7 (C-3), 66.9 (C-24), 62.3 (C-23), 48.0 (C-5), 41.2 (C-4), and 20.5 (C-2) were assigned by comparison with 3β,19α,23,24-tetrahydroxyurs-12-en-28-oic acid.19 The 13C NMR spectrum also displayed characteristic signals of an unsubstituted ring-A monoterpene indole alkaloid moiety at δC 137.2 (C-13′), 135.1 (C-2′), 128.4 (C-8′), 121.8 (C-11′), 119.6 (C-10′), 118.5 (C-9′), 112.1 (C-12′), 110.4 (C-7′), 54.1 (C-3′), 43.2 (C-5′), and 21.6 (C-6′).14,15,20 Furthermore, carbon signals of rings D and E of the monoterpene indole alkaloid moiety were observed at δC 110.7 (C-16′), 94.8 (C-18′), 73.4 (C-20′), 47.2 (C-19′), 31.9 (C-14′), 28.3 (C-15′), and 19.7 (C-21′) and assigned by comparing with those of naucleidinal dimethyl ether (4).14 These data supported the presence of both a monoterpene indole alkaloid unit and an urs-12-ene-type triterpenoid moiety in 1. The presence of these two units was confirmed through characteristic signals observed in the 1H NMR spectrum of 1 (Table 1), which exhibited resonances for aromatic protons at δH 7.82 (1H, d, J = 8.0 Hz, H-12′), 7.56 (1H, d, J = 7.6 Hz, H9′), 7.27 (1H, t, J = 6.8 Hz, H-11′), and 7.20 (1H, t, J = 7.2 Hz, H-10′). In addition, it exhibited a singlet of one proton at δH 10.96 (NH-1′), confirming the presence of an unsubstituted ring A of the indole nucleus.14,15,20 It also displayed resonances for six methyl groups at δH 1.47 (3H, s, H3-27), 1.40 (3H, d, J = 4.4 Hz, H3-21′), 1.41 (3H, s, H3-29), 1.07 (3H, d, J = 6.8 Hz, H3-30), 0.99 (3H, s, H3-26), and 0.82 (3H, s, H3-25), among which five (except H3-21′) were accounted by the triterpenoid moiety. The presence of an ursane-type triterpenoid with a hydroxy moiety at C-19 was supported by the doublet for a methyl group at δH 1.07 (H-30), the singlet at δH 2.99 (H-18), the singlet at δH 1.41 (H-29), and the broad triplet at δH 5.54 (H-12).19 In addition, the 1H NMR spectrum showed characteristic signals for tetrahydro-β-carboline skeleton (rings A, B, and C) methylene protons at δH 5.29 (1H, dd, J = 12.4, 5.6 Hz, H-5′a), 3.08 (1H, m, H-6′a), 2.87 (1H, td, J = 12.4, 4.4 Hz, H-5′b), and 2.59 (1H, dd, J = 15.2, 3.6 Hz, H6′b).14,15,20 The tetrahydro-β-carboline moiety was confirmed through the HMBC correlations (Figure 1) of H2-5′/C-3′, C6′, C-7′, and C-22′, H2-6′/C-2′, C-5′, C-7′, and C-8′, H-9′ and H-11′/C-13′, and also H-10′/C-8′. Furthermore, the HMBC spectrum showed correlations of H-3′/C-2′, C-7′, C-14′, and C-22′, H2-5′/C-22′, H-14′/C-2′, C-3′, and C-15′, H-15′/C14′, C-17′, and C-22′, and H-17′/C-14′, C-15′, C-16′, C-20′,

Table 1. NMR Spectroscopic Data (400 MHz, C5D5N) for Compound 1 position

δC, type

δH (J in Hz)

1a 1b 2a

37.1, CH2

1.61, m 0.85, m 2.49, ddd, (15.6, 13.0, 3.0) 1.53, m 4.30, dd, (12.8, 5.2)

20.5, CH2

2b 3

73.7, CH

4

41.2, C

5

48.0, CH

1.47, m

6 7a

17.8, CH2 33.3, CH2

1.64, m 1.41, m

7b

1.20, brdt (13.2)

position

δC, type

25 26 27

15.9, CH3 17.5, CH3 24.6, CH3

0.82, s 0.99, s 1.47, s

28 29

180.6, C 27.1, CH3

1.41, s

30

16.8, CH3

NH (1′) 2′ 3′

135.1, C 54.1, CH

5′a

43.2, CH2

8

40.3, C

9

47.5, CH

10

36.7, C

6′b

11 12 13 14 15a

7′ 8′ 9′ 10′ 11′

110.4, C 128.4, C 118.5, CH 119.6, CH 121.8, CH

15b 16a 16b

24.1, CH2 1.95, m 127.7, CH 5.54, brt 140.0, C 42.1, C 29.3, CH2 2.21, td (13.6, 4.4) 1.10, m 26.9, CH2 2.01, m 1.30, m

12′ 13′ 14′a

112.1, CH 137.2, C 31.9, CH2

17 18

48.2, C 54.6, CH

14′b 15′

28.3, CH

19 20 21a

72.7, C 42.3, CH 26.3, CH2

16′ 17′ 18′

110.7, C 150.4, CH 94.8, CH

19′ 20′ 21′ 22′

47.2, CH 73.4, CH 19.7, CH3 165.1, C

21b 22 23a 23b 24a 24b

38.5, CH2 62.3, CH2 66.9, CH2

5′b 1.77, dd, (10.4, 7.2)

2.99, s

1.47, m 3.04, dd, (13.2, 4.4) 1.96, m 2.08, m 4.35, d (11.6) 3.92, d (10.8) 4.04, d (12.0) 3.50, d (12.0)

δH (J in Hz)

6′a

21.6, CH2

1.07, d , (6.8) 10.96, s

4.90, overlapped 5.29, dd, (12.4, 5.6) 2.87, td, (12.4, 4.4) 3.08, m 2.59, dd, (15.2, 3.6)

7.56, d (7.6) 7.20, t (7.2) 7.27, t (6.8) 7.82, d (8.0) 3.55, brdd, (13.2, 4.0) 1.87, m 2.73, brtd (10.4) 7.85, d (0.8) 5.30, d (5.2) 1.59, m 3.94, m 1.40, d (4.4)

which were sorted by the DEPT and HSQC techniques into 14 quaternary carbons [including two carbonyl functions at δ

Figure 1. Key HMBC and COSY correlations of compounds 1, 2, and 3. C



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Table 2. NMR Spectroscopic Data (400 MHz, C5D5N and C2D6SO, respectively) of Compounds 2 and 3 2 position N−H (1) 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

δC

type

3

δH (J in Hz) 12.45, s

HMBC

type

2, 7, 8, 13

129.6 127.7

C C

41.6 19.8 110.8 126.9 119.3 120.1 123.5 112.0 139.1 103.0 119.5 144.9

CH2 CH2 C C CH CH CH CH C CH C C

4.54, t (6.8) 2.98, t (6.8)

3, 6, 7, 22 2, 5, 7, 8

7.68, 7.23, 7.30, 7.52,

7, 8, 9, 8,

6.66, s

2, 3, 16, 20

16.5 152.8 140.9 192.6 158.9

CH3 CH C CH C

1.88, d (7.2) 6.85, q (7.2)

15,19, 20, 21 14, 15, 18, 21

9.69, s

15, 18, 19, 20, 21

d (8.0) td, (7.6; 1.2) td, (7.2; 1.2) d (8.4)

δC

11, 13 12 12, 13 10

128.5 132.9

C C

40.2 19.2 111.6 125.5 119.1 119.5 123.3 111.6 137.9 93.8 130.7 142.4

CH2 CH2 C C CH CH CH CH C CH C C

24.0 60.9 127.2 144.7 152.5

CH3 CH C CH C −OH

δH (J in Hz)

HMBC

11.70 s

2, 7, 8, 13

4.37, t (6.4) 3.04, t (6.4)

3, 6, 7, 22 2, 5, 7, 8

7.56, 7.05, 7.19, 7.40,

7, 11, 13 8, 12 13 8, 10

d (7.6) t (7.6) t (7.2) d (8.0)

7.15, s

2, 3, 16

1.51, d (6.8) 4.91, t (5.6)

19, 20 15, 18, 20, 21

7.94, s

15, 16, 20, 22

5.35, d (4.4)

18, 19, 20

HREIMS, which exhibited a molecular ion peak [M]+ at m/z 320.1164 (calcd for C19H16N2O3, 320.1161). The 13C NMR spectrum of 2 (Table 2) exhibited 19 carbon signals that were sorted by HSQC and DEPT techniques into one methyl at δC 16.5 (C-18), two methylene carbon signals at δC 41.6 (C-5) and 19.8 (C-6), seven methines, and nine quaternary carbons, among which one carbonyl carbon signal at δC 158.9 (C-22) was assignable to a lactam functionality.22 The 1H NMR spectrum of 2 (Table 2) showed signals for a singlet at δH 12.45 (N−H), two doublets at δH 7.68 (1H, d, J = 8 Hz, H-9) and 7.52 (1H, d, J = 8.4 Hz, H-12), two triplets of doublets at δH 7.30 (1H, td, J = 7.2, 1.2 Hz, H-11) and 7.23 (1H, td, J = 7.6, 1.2 Hz, H-10), and two triplets of two protons each at δH 4.54 (2H, t, J = 6.8 Hz, H-5) and 2.98 (2H, t, J = 6.8 Hz, H-6), indicating the presence of a monoterpene indole alkaloid with a tetrahydro-β-carboline skeleton (rings A, B, and C) as in compound 1.15,22 The HMBC spectrum of 2 (Figure 1) showed correlations of H2-5/C-3, C-6, C-7, and C-22 and H2-6/C-2, C-5, and C-7. There were also correlations of H-9 and H-11 with C-13, confirming a tetrahydro-β-carboline skeleton. In addition, the HMBC spectrum of 2 showed correlations of H-14/C-3 and C-20, H-21/C-15, C-20, and C-19, and H-18/C-15, C-19, and C-20, indicating the lack of any ring E, but rather a but-2-enal moiety is connected through its carbon 2 (C-20) to C-15 of ring D. Based on this evidence, compound 2 was concluded to be a new monoterpene indole alkaloid, to which the trivial name latifoliaindole A was assigned. Compound 3 was also isolated as a yellowish, amorphous powder with a specific rotation of [α]25 D +36.9 (c 0.04, MeOH). The UV spectrum of 3 showed absorption maxima at λmax 228, 299, 309, 351, and 367 nm. Its molecular formula, C19H16N2O3, which was identical to that of 2, with 13 degrees of unsaturation, was also deduced from its NMR data and HREIMS, which showed a molecular ion peak [M]+ at m/z

and C-22′, supporting the presence of a six-membered ring lactam (ring D). Moreover, the unusual condensation of the urs-12-ene and the monoterpene indole alkaloid moieties was confirmed through the correlations of H-18′/C-15′, C-20′, and C-24 and H-24b and H-3/C-18′ (Figure 1). The relative configurations of the stereogenic centers C-3, C-4, and C-19′ were deduced from comparison of the chemical shifts and the coupling constants of their protons with those of 3β,19α,23,24tetrahydroxyurs-12-en-28-oic acid and naucleidinal, respectively.14,19 The biogenetic origin of 1 can be rationalized from the condensation of naucleidinal (7) and 3β,19α,23,24tetrahydroxyurs-12-en-28-oic acid (Scheme S1). Based on these data, compound 1 was concluded to be an unusual combination of a monoterpene indole alkaloid and an ursanetype triterpenoid to which the trivial name latifolianine A was given. Even though two synthetic triterpenoids with acetal units have so far been reported,21 compound 1 appears to be the first case of an adduct from an indole monoterpene alkaloid and a triterpenoid moiety bonded with a six-membered-ring acetal. Biogenetically (Scheme S1), compound 1 would be synthesized from the condensation of compound 7 and 3β,19α,23,24-tetrahydroxyurs-12-en-28-oic acid, a compound previously isolated from N. off icinalis.19 In fact, 3β,19α,23,24tetrahydroxyurs-12-en-28-oic acid is the result of oxidation of the methyl groups at C-23 and C-24 of pomolic acid (10), which was isolated also in the present investigation. Compound 1 is therefore an unprecedented natural product from the condensation of a monoterpene indole alkaloid and a triterpenoid portion. Compound 2 was isolated as a yellowish, amorphous powder with a specific rotation of [α]25 D +18.9 (c 0.07, MeOH). The UV spectrum of 2 showed absorption maxima at λmax 210, 305, and 368 nm. The molecular formula, C19H16N2O3, with 13 degrees of unsaturation, was deduced from its NMR data and D



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Industry). Fractions were monitored by thin-layer chromatography (TLC) using Merck precoated silica gel sheets (60 F254), and the identification of spots on the TLC plate was carried out by spraying ceric sulfate reagent solution and heating the plate to about 80 °C. Plant Material. The heartwood of N. latifolia, was collected in March 2015 from Makénéné, in the Centre Region of Cameroon, and identified by Dr. Tacham Walter Ndam, Botanist at the Faculty of Science, The University of Bamenda, Cameroon, and compared with voucher specimens formerly kept at the National Herbarium under the registration number 20144/SFR/Cam. Extraction and Isolation. The heartwood of N. latifolia was harvested, air-dried, and ground to afford 4.2 kg of plant material. It was then extracted with MeOH (20 L) (2 days, repeated three times) at room temperature. The combined extracts was then freed of solvent under vacuum at low temperature to furnish 456.9 g of a brown crude extract. It was suspended in 1 L of 5% H2SO4 solution and was extracted with dichloromethane to yield a nonbasic fraction (370.4 g). The acidic solution was then neutralized (pH = 7) with ammonia and extracted once again with dichloromethane to afford 11.9 g of an alkaloid-rich fraction. The dichloromethane partition (365 g) of the nonbasic fraction was subjected to medium-pressure liquid CC over silica gel (Merck, 230−400 mesh) eluting with n-hexane, mixtures of n-hexane/EtOAc, EtOAc, and mixtures of EtOAc/MeOH, in increasing order of polarity. Fractions of 500 mL each were collected and combined according to their TLC profiles to afford five subfractions (F1−F5). Subfraction F1 (60.8 g) was subjected to CC over silica gel and eluted with n-hexane/EtOAc (1:0−1:1) to obtain pomolic acid (10) (36 mg). Subfraction F2 (70 g) was also subjected to CC over silica gel and eluted with n-hexane/EtOAc (1:9−0:1) to afford quafrinoic acid (13) (50 mg) and quinovic acid (11) (29.5 mg). Subfraction F3 (95 g) was subjected to CC over silica gel and eluted with CH2Cl2/MeOH (1:0−9:1) to furnish latifoliaindole A (2) (37 mg), naucleidinal (7) (200 mg), and naucleidinal dimethyl acetal (4) (43 mg), respectively. Subfraction F4 (25.5 g) was subjected to CC over silica gel. Elution with CH2Cl2/ MeOH (1:0−7:3) furnished pobeguinine (6) (55.7 mg), (+)-naucleofficine (5) (86 mg), and latifoliaindole B (3) (10 mg). Subfraction F5 (78 g) was subjected to CC over silica gel and eluted with CH2Cl2/MeOH mixtures (9.5:0.5−7:3) to yield four subfractions (Fa−e). Subfraction Fa (1 g) was subjected to CC over Sephadex LH20 using MeOH as solvent to yield latifolianine A (1) (20 mg). Subfraction Fb (1.2 g) was also subjected to CC over Sephadex LH20 using MeOH as eluent to yield quinovic acid 3-O-β-Lrhamnopyranoside (12) (15.6 mg). Subfraction Fc (2.1 g) was subjected to CC over Sephadex LH-20 to yield a mixture of compounds, which was then subjected to normal-phase preparative HPLC with an isocratic mixture of CH2Cl2/MeOH (9.5:0.5), to yield 3β,6β,19α,23-tetrahydroxyolean-12-en-28-oic acid (9) (25 mg) and 3β,6β,23-trihydroxyolean-12-en-28-oic acid (8) (15 mg). Latifolianine A (1): colorless, amorphous powder; [α]25 D −16.5 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 214 (3.52), 229 (3.54), 351 (2.27) nm; IR (KBr) νmax 3778, 3703, 3413, 1663, 1592 cm−1; 1H NMR (400 MHz, C5D5N) and 13C NMR (100 MHz, C5D5N) data, see Table 1; EIMS m/z 822 (rel int) [M]+ (2.4), 804 (10.0), 778 (17.6), 760 (27.7), 335 (62.1), 307 (45.6), 265 (58.6), 264 (28.3), 249 (29.4), 235 (100.0), 206 (37.0), 187 (54.2), 119 (72.4); HRESIMS m/z 823.4896 [M + H]+, calcd for C50H67N2O8, 823.4897; anal. C, 72.94; H, 8.02; N, 3.40; O, 15.56%, calcd for C50H66N2O8, C 72.96, H 8.08, N 3.40, O 15.55%. Latifoliaindole A (2): yellowish, amorphous powder; [α]25 D +18.9 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 210 (2.76), 305 (2.24), and 368 (2.39) nm; IR (KBr) νmax 3311, 3245, 1630, 1597 cm−1; 1H NMR (400 MHz, C5D5N) and 13C NMR (100 MHz, C5D5N) data, see Table 2; EIMS m/z 320 (rel int) [M]+ (95.4), 303 (19.1), 292 (66.5), 291 (54.5), 277 (100.0), 251 (6.4), 234 (4.1); HREIMS m/z 320.1164 [M]+, calcd for C19H16N2O3, 320.1161; anal. C, 71.22; H, 4.99; N, 8.75; O, 14.99%, calcd for C19H16N2O3, C, 71.24; H, 5.03; N, 8.74; O, 14.98%. Latifoliaindole B (3): yellowish, amorphous powder; [α]25 D −36.9 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 228 (3.04), 299 (2.70), 309

320.1142 (calcd for C19H16N2O3, 320.1161). Compounds 2 and 3 are the isomers. However, noticeable discrepancies were observed in their NMR spectra. In the 1H NMR spectrum of 3 (Table 2) the formyl group and the olefinic protons of 2 were replaced by two oxymethine protons at δH 7.94 (1H, s, H-21) and 4.91 (1H, t, J = 5.6 Hz, H-19), respectively. Similarly, in the 13C NMR spectra (Table 2), the signals of C-19 and C-21 resonated comparatively upfield at δC 60.9 and 144.7, respectively. In addition, the HMBC spectrum of 3 (Figure 1) showed correlations of H-21/C-15 and C-16 and H-19/C15 and C-20, revealing the presence of an additional furan ring E. Based on the structural features of compounds 2 and 3, which are both derived from L-tryptophan and secologanin, a plausible biogenetic pathway (Scheme S2) of the two compounds where compound 3 derived from compound 2 was deduced. Based on the evidence obtained, compound 3 was concluded to be a new isomer of 2, to which trivial name latifoliaindole B was accorded. All the 13 isolates obtained were screened in vitro for their antibacterial potential against the growth of H. influenzae strain ATCC 49247, as summarized in Table 3. The minimal Table 3. MIC and MBC (μg/mL) of Compounds against H. influenzae compound

MIC (μg/mL)

MBC (μg/mL)

MBC/MIC

1 2 3 4 5 6 7 8 9 10 11 12 13 ciprofloxacin

25.0 50.0 25.0 50.0 4.7 4.7 3.1 18.8 18.8 18.8 25.0 12.5 18.8 1.6

50.0 >50.0 50.0 >50.0 12.5 12.5 12.5 50.0 50.0 50.0 >50.0 50.0 50.0 1.6

2.0 >1.0 2.0 >1.0 2.7 2.7 4.0 2.7 2.7 2.7 >2.0 4.0 2.7 1.0

inhibitory concentration (MIC) of all the compounds ranged from 3.1 to 50.0 μg/mL, with compound 7 being the most active. Overall, compounds 5, 6, and 7 exhibited good activity (MIC ≤ 5.0 μg/mL).

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were recorded on a Büchi M-560 melting point apparatus. Optical rotations were measured with a JASCO DIP-360 polarimeter. UV spectra were recorded on a Hitachi UV 3200 spectrophotometer. A JASCO 320-A spectrophotometer was used for scanning the IR spectrum using KBr pellets. The 1D and 2D NMR spectra were run on Bruker NMR spectrometers operating at 100, 125, 400, 500, and 600 MHz, where chemical shifts (δ) are given in ppm with reference to the tetramethylsilane signal. Electron ionization mass spectrometry (EIMS and HREIMS) spectra were obtained on a JEOL-600H-1 mass spectrometer operating at 300 °C. The high-resolution mass of compound 1 was obtained with a QTOF compact spectrometer (Bruker, Germany) equipped with an HRESI source. Column chromatography was performed on columns containing silica gel (230−400 mesh) or Sephadex LH-20. Normal-phase preparative HPLC separation was performed using LC-908W (column silica, internal diameter, length: 20 × 250 mm; flow rate: 4 mL/min; detector UV: 0.02 mm, refraction index: 50) (Japan Analytical E



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(2.76), 351 (2.97), and 367 (2.91) nm; IR (KBr) νmax 3145, 1660, 1592 cm−1; 1H NMR (400 MHz, C2D6OS) and 13C NMR (100 MHz, C2D6OS) data, see Table 2; EIMS m/z 320 (rel int) [M]+ (90.2), 319 (100.0), 318 (24.0), 317 (29.2), 303 (21.4), 302 (19.9), 301 (50.9); HREIMS m/z 320.1142 [M]+ calcd for C19H16N2O3, 320.1161; anal. C, 71.22; H, 4.99; N, 8.75; O, 14.99%, calcd for C19H16N2O3, C, 71.24; H, 5.03; N, 8.74; O, 14.98%. Anti-Hib Activity of Isolated Compounds. MIC values were determined according to the Clinical Laboratory Standards Institute (CLSI) M7-A9 microdilution method (CLSI 2006), using a 96-well microtiter plate format against H. inf luenzae ATCC 49247. The stock solutions of test compounds and of ciprofloxacin (Sigma-Aldrich, Germany), used as a positive control, were prepared in DMSO at 2 mg/mL. The required concentrations were achieved by a 2-fold serial dilution ranging from 50 to 0.1 μg/mL with Muller−Hinton broth (Lab M Limited Topley House). A 4 μL amount of 2-fold dilutions of each compound was pipetted into triplicate wells of 96-well flatbottomed tissue culture plates (Corning, USA), and thereafter, 96 μL of the bacterial inoculum standardized at 0.5 Macfarland was added to each well containing the test substances except for the blank column for sterility control. After incubation for 24 h at 37 °C, the turbidity was observed as an indication of growth, and the lowest concentration inhibiting the visible growth of bacteria was recorded as the MIC. The MBC was determined by transferring 50 μL aliquots of the clear wells into 150 μL of freshly prepared broth medium and incubating at 37 °C for 24 h. The MBC was regarded as the lowest concentration of test sample that did not produce turbidity as above, indicating no microbial growth. All the experiments were performed in triplicate.

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ASSOCIATED CONTENT

* Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00463. 1

H NMR, 13C NMR, DEPT-90, DEPT-135, HSQC, HMBC, COSY, NOESY, EIMS, HRESIMS, HREIMS, UV, and IR spectra for compounds 1−3 (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel: +237 677 955 630/+237 655 643 097. E-mail: bk_ [email protected]/[email protected]. ORCID

Jean Jules Kezetas Bankeu: 0000-0001-8483-6080 Norbert Sewald: 0000-0002-0309-2655 Notes

The authors declare no competing financial interest.

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REFERENCES

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Article

ACKNOWLEDGMENTS

The authors are very grateful to The World Academy of Sciences (TWAS) and the International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Pakistan, for their financial and technical support through ICCBS-TWAS Postdoctoral and Postgraduate Fellowship no. 3240280476 granted to J.J.B.K. The authors are also grateful to the German Academic Exchange Service (DAAD) for the financial support to the Yaoundé-Bielefeld Graduate School of Natural Products with Antiparasite and Antibacterial Activities (YaBiNaPA, project no. 57316173). They thank Dr. Tacham Walter Ndam, Botanist at the Faculty of Science, The University of Bamenda, Cameroon, for the collection and the identification of the plant material. F


Constituents from Nauclea latifolia with Anti-Haemophilus influenzae

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