Synthesis and antimalarial bioassay of quinine - peptide conjugates

Article abstract of DOI:10.1111/cbdd.12134

Amino acid and peptide conjugates of quinine were synthesized using microwave irradiation in 52-95% yields using benzotriazole methodology. The majority of these conjugates retain in vitro antimalarial activity with IC₅₀ values below 100 nm, similar to quinine.

Full text of DOI:10.1111/cbdd.12134

Chem Biol Drug Des 2013; 82: 361–366
Research Article
Synthesis and Antimalarial Bioassay of Quinine –
Peptide Conjugates
Siva S. Panda1, Mohamed A. Ibrahim1,2,3
,
Quinoline-containing antimalarial drugs, such as quinine,
chloroquine, and mefloquine, are mainstays of chemother-
apy against malaria. While the search for novel drug tar-
gets and new lead structures for the treatment of malaria
is a critical objective, a complimentary strategy is to utilize
and develop lead structures from nature with proven effi-
cacious properties like quinine or artemisinin. The synthe-
sis of quinine derivatives, a strategy that led to the
discovery of chloroquine, has been largely abandoned (3)
because of toxicity and reaction complexity. Artemisinin is
used against chloroquine-resistant malarial parasites. The
poor solubility of artemisinin coupled with its short plasma
half-life led to a high rate of parasite recrudescence. Modi-
fication of the cinchona alkaloids is one of the most suc-
cessful strategies for antimalarial drug development (3,4).
Peptide derivatives of primaquine evidenced a comparable
or greater activity of conjugates with respect of the parent
drug (5).
Hasan Kucßukbay1,4, Marvin J. Meyers5,
€ €
Francis M. Sverdrup5, Said A. El-Feky3 and
Alan R. Katritzky1,6,
*
1Center for Heterocyclic Compounds, Department of
Chemistry, University of Florida, Gainesville, FL, 32611-
7200, USA
2Organic Chemistry Department, College of pharmacy,
Misr University for Science and Technology, P.O. Box: 77,
Al-Motamayez District, Egypt
3Department of Pharmaceutical Organic Chemistry,
Faculty of Pharmacy, Zagazig University, Zagazig, 44519,
Egypt
4Department of Chemistry, Faculty of Arts and Sciences,
University of Inonu, 44280, Malatya, Turkey
5Center for World Health and Medicine, Saint Louis
University, St. Louis, MO, 63104, USA
6Department of Chemistry, King Abdulaziz University,
Jeddah, 21589, Saudi Arabia
*Corresponding author: Alan R. Katritzky,
katritzky@chem.ufl.edu
Efficient cellular drug delivery is a severe problem for many
therapeutic agents due to charge, hydrophilic character,
and/or size. High drug doses, necessary to compensate
for the reduced bioavailability, often cause strong adverse
effects. Synthetic drug delivery vectors can sometimes
solve this problem, if limitations like low-cellular uptake effi-
ciency or cytotoxicity are overcome. Among such synthetic
vectors, so-called cell-penetrating peptides have proven
applicability because of their ability as drug carriers to
cross cell membranes (6). Drug–peptide conjugates are
also known to overcome multidrug resistance in chemo-
therapy and bind to specific receptors expressed on tumor
cells (7–9).
Amino acid and peptide conjugates of quinine were
synthesized using microwave irradiation in 52–95%
yields using benzotriazole methodology. The majority
of these conjugates retain in vitro antimalarial activity
with IC50 values below 100 nM, similar to quinine.
Key words: amino acid, chemical biology, drug design, pep-
tide
Received 15 January 2013, and accepted for publication 1
March 2013
Malaria is a devastating and common infectious disease,
responsible for more than one million deaths each year.
Quinine (Figure 1), an alkaloid isolated from the bark of the
cinchona tree (1), has been the most acceptable antima-
larial drug for almost 400 years. The protozoal parasite
responsible for malaria, Plasmodium falciparum, has
gained resistance to most forms of monotherapy especially
in South-East Asia, South America, and East Africa (2).
Recently, Islahudin et al. reported that quinine can block
a cell’s ability to take up essential amino acids like trypto-
phan and tyrosine; they suggested that dietary amino acid
supplements could improve the performance of quinine
(10,11). Amino acids like L-alanine, L-phenylalanine, L-argi-
nine, L-glutamic acid, and L-lysine may also mask the bitter
taste of quinine (12).
We previously used benzotriazole methodology to prepare
diverse peptide conjugates (13) and quinine bis-conjugates
incorporating both quinolone antibiotics and amino acid
units as potential antimalarial agents (14). We now expand
the latter approach by coupling amino acids and peptides
of varying polarity with quinine. We utilized microwave
However, quinine has an impressive track record consider-
ing that most antimalarial drugs encounter parasite resis-
tance within a few years of introduction. Quinine is treated
as a privileged natural structure because of its low parasite
resistance, which is difficult for the parasite to circumvent.
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12134
361

Panda et al.
solid obtained was filtered and washed with 10% Na2CO3
and water to give the desired compound.
(2S)-(1R)-(6-Methoxyquinolin-4-yl)(5-
vinylquinuclidin-2-yl)methyl 2-((tert-
butoxycarbonyl)amino)-3-(1-tosyl-1H-imidazol-4-yl)
propanoate (Boc-L-His(Tos)-QN, 12e)
White solid (92%); mp 141–142 °C; 1H NMR (CDCl3) d
8.72–8.71 (d, J = 4.3 Hz, 1H), 8.04–8.01 (d, J = 9.1 Hz,
1H), 7.80–7.69 (m, 3H), 7.41–7.26 (m, 6H), 6.42–6.38 (m,
1H), 5.90–5.67 (m, 2H), 5.02–4.96 (m, 2H), 4.68–4.63 (m,
1H), 3.95 (s, 3H), 3.36–3.20 (m, 1H), 3.07–2.90 (m, 3H),
2.61–2.53 (m, 1H), 2.43 (s, 3H), 2.27–2.24 (m, 1H), 1.91–
1.71 (m, 7H), 1.44 (br s, 9H); 13C NMR (CDCl3) d 171.0,
158.1, 155.7, 147.7, 146.5, 144.8, 143.4, 141.8, 139.9,
136.4, 134.8, 132.0, 130.6, 127.5, 126.8, 122.0, 115.0,
114.7, 101.5, 80.2, 59.3, 56.8, 55.9, 53.3, 42.6, 39.8,
29.6, 28.5, 27.8, 21.9. Anal. Calcd for C38H45N5O7S: C,
63.76; H, 6.34; N, 9.78. Found: C, 63.47; H, 6.40; N,
9.66.
Figure 1: Quinine.
irradiation to shorten reaction times (15) and minimize epi-
merization in the preparation of peptide–quinine bioconju-
gates. Quinine–peptide conjugates that retain activity
against the malarial parasite Plasmodium falciparum may
provide means to identify novel antimalarial agents with
improved activity on resistant strains.
Experimental Section
(2S)-(1R)-(6-Methoxyquinolin-4-yl)(5-
General methods
vinylquinuclidin-2-yl)methyl 3-(benzyloxy)-2-((tert-
butoxycarbonyl)amino) propanoate (Boc-L-Ser(Bzl)-
QN, 12f)
Melting points were determined on a capillary point appa-
ratus equipped with a digital thermometer. NMR spectra
were recorded in CDCl3 or DMSO-d6, on Mercury or Gem-
ini NMR spectrometers operating at 300 MHz for 1H (with
TMS as an internal standard) and 75 MHz for 13C. Ele-
mental analyses were performed on a Carlo Erba-EA1108
instrument. All microwave-assisted reactions were carried
out with a single-mode cavity Discover Microwave Synthe-
sizer (CEM Corporation, Matthews, NC, USA). The reac-
tion mixtures were transferred into a 10-mL glass pressure
microwave tube equipped with a magnetic stirrer bar. The
tube was closed with a silicon septum, and the reaction
mixture was subjected to microwave irradiation (Discover
mode; run time: 60 seconds; PowerMax-cooling mode).
White solid (94%); mp 58–59 °C; 1H NMR (CDCl3) d 8.62
(dd, J = 8.2 Hz, 4.5 Hz, 1H), 8.00 (dd, J = 9.8 Hz, 4.7 Hz,
1H), 7.41–7.06 (m, 10H), 6.54–6.48 (m, 1H), 5.84–5.73 (m,
1H), 5.47–5.40 (m, 1H), 5.26–5.18 (m, 1H), 5.02–4.96 (m,
2H), 4.53–4.48 (m, 1H), 4.44–4.43 (m, 1H), 4.30 (br s, 1H),
3.94 (br s, 1H), 3.72–3.67 (m, 1H), 3.37–3.35 (m, 1H), 3.09–
2.97 (m, 2H), 2.63–2.55 (m, 2H), 2.26 (br s, 1H), 1.47–1.39
(m, 12H); 13C NMR (CDCl3) d 170.4, 170.2, 158.0, 155.6,
155.3, 147.6, 144.8, 143.2, 141.9, 131.9, 128.5, 127.7,
122.0, 119.1, 114.6, 101.5, 80.3, 73.6, 70.5, 70.3, 67.3,
59.5, 59.1, 57.0, 56.7, 55.8, 54.4, 42.8, 42.5, 39.8, 28.5.
Anal. Calcd for C35H43N3O6: C, 69.86; H, 7.20; N, 6.89.
Found: C, 69.61; H, 7.41; N, 7.20.
General procedure and experimental details for the prepa-
ration of compounds 2–11 are given in the Appendix S1.
In addition, Tables S1–S3 that contain details for the char-
acterization of compounds 4a–g (Table S1), compounds
7a–g (Table S2), and compounds 8a–d and 9a–d (Table
S3) are also included in the Appendix S1.
(2S)-5-Benzyl 1-((1R)-(6-methoxyquinolin-4-yl)(6-
vinylquinuclidin-2-yl)methyl) 2-((tert-
butoxycarbonyl)amino) pentanedioate (Boc-L-Glu
(Bz)-QN, 12g)
1
White solid (67%); mp 76–78 °C; H NMR (CDCl3) d 8.74–
General procedure for the synthesis of quinine
conjugates 12a–n
8.68 (m, 1H), 8.01 (d, J = 8.8 Hz, 1H), 7.49–7.26 (m, 8H),
6.55–6.47 (m, 1H), 5.87–5.75 (m, 1H), 5.11–4.99 (m, 5H),
4.50–4.41 (m, 1H), 3.95 (s, 3H), 3.42–3.35 (m, 1H), 3.18–
3.00 (m, 1H), 2.74–2.60 (m, 1H), 2.49–2.18 (m, 4H), 2.00–
1.73 (m, 4H), 1.63–1.52 (m, H), 1.42 (s, 9H); 13C NMR
(CDCl3) d 172.6, 172.5, 171.6, 158.2, 155.5, 147.6,
144.9, 143.0, 141.5, 135.8, 132.0, 128.8, 128.5, 128.4,
122.2, 122.1, 114.9, 101.3, 80.4, 66.8, 59.3, 59.2, 56.6,
56.5, 55.89, 53.1, 42.7, 39.68, 30.35, 30.25, 28.48,
27.64, 27.54. HRMS m/z for C37H45N3O7 [M + H]+ calcd.
644.3330, found 644.3347.
A dried, heavy-walled Pyrex tube containing a small stir
bar was charged with N-(Pg-peptidoylbenzotriazoles) (1.2
equivalent) and quinine (1 equivalent) dissolved in dimethyl
formamide (DMF) along with anhydrous potassium carbon-
ate (2 equivalent). The reaction mixture was exposed to
microwave irradiation (20 W) at 50 °C for 10 min. Each
mixture was allowed to cool through an inbuilt system until
the temperature fell below 30 °C (ca. 10 min). Each reac-
tion mixture was quenched with ice-cold water, and the
362
Chem Biol Drug Des 2013; 82: 361–366

Synthesis of Quinine – Peptide Conjugates
(2S)-(1R)-(6-Methoxyquinolin-4-yl)(6-
140.7, 136.5, 135.3, 134.3, 134.2, 133.8, 133.8, 133.3,
133.1, 132.9, 132.8, 132.8, 131.0, 131.9, 131.4, 126.7,
119.4, 71.9, 69.9, 63.8, 61.1, 60.4, 57.9, 55.0, 53.9,
47.1, 44.3, 42.2, 32.4, 32.1, 24.1. HRMS m/z for
vinylquinuclidin-2-yl)methyl 2,6-bis(((benzyloxy)
carbonyl)amino)hexanoate (Z-L-Lys(Z)-QN, 12h)
Oil (95%);. 1H NMR (CDCl3) d 8.71–8.64 (m, 1H), 7.99 (d,
J = 9.1 Hz, 1H), 7.42–7.41 (m, 1H), 7.38–7.20 (m, 11H),
6.51–6.43 (m, 1H), 5.90–5.75 (m, 2H), 5.18–5.13 (m, 1H),
5.07 (s, 2H), 5.02 (s, 2H), 4.97 (s, 1H), 4.43–4.35 (m, 1H),
3.88 (s, 3H), 3.46–3.35 (m, 1H), 3.16–2.90 (m, 4H), 2.66–
2.57 (m, 2H), 2.28–2.19 (m, 1H), 1.89–1.16 (m, 11H); 13C
NMR (CDCl3) d 171.9, 157.9, 156.6, 156.2, 154.9, 147.3,
144.6, 143.2, 141.5, 136.6, 136.1, 131.7, 128.4, 127.9,
126.7, 125.3, 121.8, 119.0, 114.9, 114.6, 114.3, 101.5,
66.9, 66.5, 59.0, 56.4, 55.6, 53.8, 52.3, 42.4, 40.1, 39.5,
31.1, 29.2, 27.6, 27.4, 24.4, 22.3, 22.1. HRMS m/z for
C42H48N4O7 [M + H]+ calcd. 721.3596, found 721.3629.
C
40H44N4O6 [M + H]+ calcd. 677.3334, found 677.3303.
(2S)-(1R)-(6-Methoxyquinolin-4-yl)(6-
vinylquinuclidin-2-yl)methyl2-((S)-2-(((benzyloxy)
carbonyl)amino)-3-methyl butanamido)-4-
methylpentanoate (Z-L-Val-L-Leu-QN, 12l)
1
White solid (93%); mp 83–84 °C; H NMR (CDCl3) d 8.74–
8.70 (m, 1H), 8.03–7.96 (m, 1H), 7.44–7.26 (m, 10H),
6.58–6.51 (m, 1H), 5.88–5.79 (m, 1H), 5.55–4.97 (m, 6H),
4.74–4.35 (m, 1H), 3.95 (s, 3H), 3.43–3.34 (m, 1H), 3.13–
3.00 (m, 1H), 2.70–2.60 (m, 1H), 2.30–0.80 (m, 23H); 13C
NMR (CDCl3) d 172.1, 171.3, 158.1, 156.5, 147.8, 147.5,
141.8, 132.0, 131.8, 128.7, 128.2, 127.1, 126.8, 122.0,
121.7, 118.7, 114.7, 101.4, 72.0, 67.3, 60.1, 59.3, 57.1,
55.9, 51.0, 43.4, 42.6, 41.1, 40.1, 39.8, 31.2, 28.0, 27.6,
25.0, 23.0, 21.9, 19.4. HRMS m/z for C40H44N4O6
[M + H]+ calcd. 671.3811, found 671.3803.
(2S)-4-Benzyl 1-((1R)-(6-methoxyquinolin-4-yl)(6-
vinylquinuclidin-2-yl)methyl)2-(((benzyloxy)
carbonyl) amino) succinate (Z-L-Asp(Bz)-QN, 12i)
Oil (68%); 1H NMR (CDCl3) d 8.70–8.69 (m, 1H), 8.00 (d,
J = 9.5 Hz, 1H), 7.53–7.21 (m, 13H), 6.50–6.40 (m, 1H),
5.90–5.67 (m, 2H), 5.13–4.71 (m, 7H), 3.93 (s, 3H), 3.67–
2.58 (m, 8H), 2.36–2.25 (m, 1H), 1.91–1.51 (m, 4H); 13C
NMR (CDCl3) d 170.7, 170.3, 158.2, 156.2, 147.6, 144.9,
143.0, 141.9, 136.2, 135.4, 135.2, 132.0, 128.8, 128.6,
128.5, 128.4, 127.0, 122.1, 119.0, 118.7, 114.7, 101.4,
75.9, 67.5, 67.1, 59.3, 56.9, 55.8, 50.7, 43.5, 42.7, 39.8,
36.4, 27.9, 24.5. HRMS m/z for C39H41N3O7 [M + H]+
calcd. 664.3017, found 664.3035.
(1R)-(6-Methoxyquinolin-4-yl)(6-vinylquinuclidin-2-
yl)methyl 2-((2S,3S)-2-(((benzyloxy)carbonyl)amino)-
3-methyl pentanamido)acetate (Z-L-Ileu-Gly-QN,
12m)
Oil (52%); 1H NMR (CDCl3) d 8.72 (d, J = 4.3 Hz, 1H),
8.02 (d, J = 9.1 Hz), 7.49–7.24 (m, 8H), 6.90–6.53 (m,
2H), 5.84–5.73 (m, 1H), 5.42–5.37 (m, 1H), 5.13–4.98 (m,
4H), 4.24–4.06 (m, 3H), 3.93 (s, 3H), 3.43–3.35 (m, 1H),
3.20–3.05 (m, 2H), 2.96–2.62 (m, 3H), 2.32–2.31 (m,
1H), 1.95–1.72 (m, 3H), 1.59–1.41 (m, 3H), 1.26–1.02 (m,
2H), 0.92–0.83 (m, 5H); 13C NMR (CDCl3) d 171.9, 169.0,
158.3, 156.5, 147.5, 144.8, 142.7, 141.3, 136.2, 132.0,
128.7, 128.4, 128.2, 126.8, 122.1, 118.9, 115.1, 101.4,
67.3, 59.8, 59.1, 56.5, 55.9, 42.8, 41.5, 39.5, 37.4, 36.7,
27.5, 24.8, 24.0, 15.7, 11.6. HRMS m/z for C36H44N4O6
[M + H]+ calcd. 269.3334, found 269.3348.
(2S)-((1R)-(6-Methoxyquinolin-4-yl)(8-
vinylquinuclidin-2-yl)methyl) 2-(benzylamino)-3-
(benzylthio)propanoate (Z-L-Cys(Bz)-QN, 12j)
Oil (65%); 1H NMR (CDCl3) d 8.74–8.68 (m, 1H), 8.02–
7.97 (m, 1H), 7.37–7.14 (m, 15H), 6.52–6.42 (m, 1H),
5.86–5.75 (m, 1H), 5.49–4.45 (m, 1H), 5.12–4.99 (m, 4H),
4.65–4.58 (m, 1H), 3.93 (s, 3H), 3.64–3.53 (m, 1H), 3.38–
3.29 (m, 1H), 3.08–2.62 (m, 4H), 2.27–1.46 (m, 7H); 13C
NMR (CDCl3) d 174.8, 162.7, 160.5, 152.1, 149.3, 147.3,
146.3, 142.0, 140.7, 136.5, 134.1, 133.6, 133.3, 133.0,
132.0, 131.6, 126.6, 123.6, 119.3, 106.0, 80.0, 71.9,
70.6, 63.8, 61.3, 60.3, 58.2, 47.1, 44.2, 41.2, 37.8, 32.1,
20.0. HRMS m/z for C37H41N3O3S [M + H]+ calcd.
652.2840, found 652.2849.
(8S,11S)-(1R)-(6-Methoxyquinolin-4-yl)(6-
vinylquinuclidin-2-yl)methyl 8-benzyl-11-methyl-
3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-
12-oate (Z-Gly-L-Phe-L-Ala-QN, 12n)
1
White solid (76%); mp 91–92 °C; H NMR (CDCl3) d 8.72–
8.67 (m, 1H), 8.01–7.93 (m, 1H), 7.43–7.09 (m, 18H),
6.18–5.55 (m, 3H), 5.10–4.96 (m, 5H), 4.72–4.55 (m, 1H),
4.17–4.13 (m, 1H), 3.94–3.70 (m, 5H), 3.37–2.96 (m,
5H), 2.68–2.53 (m, 1H), 2.28–1.84 (m, 2H), 1.69–1.52 (m,
2H), 1.27 (br s, 3H);13C NMR (CDCl3) d 175.9, 175.1,
174.1, 162.2, 151.6, 149.4, 148.1, 145.6, 142.6, 140.5,
135.8, 133.6, 132.8, 132.3, 131.2, 130.0, 126.2, 125.9,
123.0, 120.8, 119.0, 105.6, 105.2, 71.4, 64.0, 63.4, 60.7,
59.9, 58.8, 52.5, 48.6, 47.7, 46.8, 43.7, 41.9, 31.7, 29.2,
23.1, 21.6. HRMS m/z for C42H47N5O7 [M + H]+ calcd.
734.3548, found 734.3557.
(2S)-(1R)-(6-Methoxyquinolin-4-yl)(6-
vinylquinuclidin-2-yl)methyl 2-((S)-2-(((benzyloxy)
carbonyl)amino)propanamido)-3-phenylpropanoate
(Z-L-Ala-L-Phe-QN, 12k)
1
White solid (75%); mp 85–87 °C; H NMR (CDCl3) d 8.74–
8.68 (m, 1H), 8.05–7.98 (m, 1H), 7.56–6.96 (m, 17H),
6.74–6.49 (m, 1H), 5.96–5.65 (m, 1H), 5.40–4.92 (m, 6H),
4.45–4.14 (m, 1H), 3.97–3.84 (m, 2H), 3.40–2.86 (m, 4H),
2.68–2.55 (m, 1H), 2.29–2.23 (m, 1H), 1.96–1.24 (m, 7H);
13C NMR (CDCl3) d 176.7, 175.6, 162.7, 152.2, 146.3,
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Panda et al.
Antimalarial activity assay
Plasmodium falciparum strain 3D7 was cultured according
to the method of Trager and Jensen (16) with minor modifi-
cations. Parasites were grown in human erythrocytes (2%
hematocrit) in an atmosphere of 5% CO2, 5% O2, and 90%
N2 in RPMI 1640 medium (Gibco, Grand Island, NY, USA)
supplemented with 25 mM hepes buffer (Sigma, Saint Louis,
MO, USA), 25 mg/L gentamicin (Gibco), 1 mM sodium pyru-
vate (Sigma), 50 mg/L hypoxanthine (Sigma), 2 g/L glucose
(Sigma), 2.52 g/L sodium bicarbonate (Sigma), and 5 g/L Al-
bumax 1 (Gibco). In vitro antimalarial activity was determined
by the SYBR Green I method described by Smilkstein et al.
(17) with modifications (18). Stock solutions of each com-
pound were prepared in DMSO at a concentration of 10 mM
and threefold serial dilutions prepared in DMSO for an 11-
point curve. Drugs were then diluted 250-fold into culture
medium in 96-well storage plates to create 29 drug solu-
tions. Drug solutions (50 lL/well) were transferred in quadru-
plicate to parasite cultures (50 lL) in 96-well black tissue
culture plates for a total volume of 100 lL at 2% hematocrit,
0.2% parasitemia, and 0.2% DMSO final concentrations.
The plates were then incubated for 72 h at 37 °C. After incu-
bation, 100 lL of lysis buffer containing 0.2 lL/mL SYBR
Green I was added to each well. After incubation for 1 h at
room temperature in the dark, plates were read on a Safire2
(Tecan, Morrisville, NC, USA) plate reader with excitation
and emission wavelengths of 497 and 520 nM, respectively.
The 50% inhibitory concentrations (IC50s) were determined
by nonlinear regression using a four-parameter logistic equa-
tion (GraphPad Prism software, La Jolla, CA, USA).
Scheme 2: Synthesis of N-Cbz-aminoacylbenzotriazoles 7a–g.
Preparation of N-Cbz-dipeptidoylbenzotriazoles
9a–d
The N-Cbz-dipeptidoylbenzotriazoles 9a–d were synthe-
sized as previously reported by stepwise linking amino
acids and subsequent coupling with benzotriazole-acti-
vated N-protected amino acid analogs in solution phase
(yields, 69–79%; Scheme 3) (20).
Preparation of N-Cbz-tripeptidoyl benzotriazoles
11
The Cbz-protected tripeptide 10 was synthesized in 80%
yield by our reported method (13) of stepwise coupling of
Results and Discussion
N-Pg-aminoacyl benzotriazolides 4a–g and 7a–g, N-Pg-
dipeptidoylbenzotriazolides 9a–d, and N-Pg-tripeptidoyl 11
were synthesized as previously reported (12,15). N-Pg-
Peptidoylbenzotriazoles were used as active intermediates
to prepare quinine–peptide bioconjugates 12a–n under
microwave irradiation.
Scheme 3: Synthesis of N-Cbz-dipeptidoylbenzotriazoles 9a–d.
Preparation of N-Boc-aminoacylbenzotriazoles 4a–g
N-(Boc-a-Aminoacyl)benzotriazoles 4a–g (Scheme 1) were
prepared in 54–85% yields from the corresponding N-
Boc-a-amino acids following our one-step procedure (19).
Preparation of N-Cbz-aminoacylbenzotriazoles 7a–g
N-(Cbz-a-Aminoacyl)benzotriazoles 7a–g (Scheme 2) were
prepared in 64–88% yields from the corresponding N-Cbz-
a-amino acids following our published procedure (20,21).
Scheme 1: Synthesis of N-Boc-aminoacylbenzotriazoles 4a–g.
Scheme 4: Synthesis of N-Cbz-tripeptidoyl benzotriazoles 11.
Chem Biol Drug Des 2013; 82: 361–366
364

Synthesis of Quinine – Peptide Conjugates
Scheme 5: Synthesis of quinine conjugates 12a–n.
Table 1: Preparation of quinine conjugates by chiral acylation with N-(protected)-peptidoylbenzotriazoles 12a–n
Entry
Reactant 4
Product 12
Yield%
mp (°C)
Lit.mp (°C)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Boc-Gly-Bt 4a
Boc-L-Ala-Bt 4b
Boc-L-Phe-Bt 4c
Boc-L-Ile-Bt 4d
Boc-Gly-QN 12a
Boc-L-Ala-QN 12b
Boc-L-Phe-QN 12c
Boc-L-Ile-QN 12d
86
81
80
92
92
94
67
95
68
65
75
93
52
76
74–75
111–113
165–167
68–70
141–142
58–59
76–78
Oil
Oil
Oil
85–87
83–84
Oil
74–7614 (14)
110–112 (14)
166–168 (14)
68–70 (14)
Novel
Novel
Novel
Novel
Novel
Novel
Novel
Novel
Novel
Boc-L-His(Tos)-Bt 4e
Boc-L-Ser(Bzl)-Bt 4f
Boc-L-Glu(Bz)-Bt 4g
Z-L-Lys(Z)-Bt 7e
Z-L-Asp(Bz)-Bt 7f
Z-L-Cys(Bz)-Bt 7g
Z-L-Ala-L-Phe-Bt 9a
Z-L-Val-L-Leu-Bt 9b
Z-L-Ileu-Gly-Bt 9c
Z-Gly-L-Phe-L-Ala-Bt 11
Boc-L-His(Tos)-QN 12e
Boc-L-Ser(Bzl)-QN 12f
Boc-L-Glu(Bz)-QN 12g
Z-L-Lys(Z)-QN 12h
Z-L-Asp(Bz)-QN 12i
Z-L-Cys(Bz)-QN 12j
Z-L-Ala-L-Phe-QN 12k
Z-L-Val-L-Leu-QN 12l
Z-L-Ileu-Gly-QN 12m
Z-Gly-L-Phe-L-Ala-QN 12n
91–92
Novel
amino acids with subsequent benzotriazole-activated
N-protected amino acid analog 9d in solution phase.
Compound 10 was activated by benzotriazole to obtain 11
in 72% yields (Scheme 4).
Table 2: In vitro antimalarial activities of compounds against the
chloroquine-sensitive 3D7 strain of Plasmodium falciparum
Compound
IC50 (nM)a
12a
12b
12c
12d
12e
12f
27 (23–32)
36 (30–43)
38 (33–44)
550 (490–620)
42 (36–48)
95 (81–110)
76 (67–86)
71 (62–82)
17 (16–20)
40 (35–46)
120 (100–140)
74 (60–92)
23 (20–25)
57 (50–64)
18 (11–30)
Preparation of quinine–peptide bioconjugates
12a–n
We prepared amino acid and peptide conjugates of qui-
nine 12a–n by O-acylation of quinine 1 with N-protected
acylbenzotriazoles 9 in the presence of K2CO3 in anhy-
drous DMF under microwave irradiation (50 °C, 20 W) for
10–30 min (Scheme 5, Table 1). Compounds 12a–n were
purified by column chromatography and characterized by
1H NMR, 13C NMR, and HRMS analysis.
12g
12h
12i
12j
12k
12l
12m
12n
Quinine
In vitro activity of compounds against Plasmodium
falciparum
aMean value (95% confidence intervals).
To determine whether the quinine–peptide conjugates
described retained antimalarial activity, compounds 12a–n
as well as quinine itself were tested against the blood
stage of P. falciparum strain 3D7 in vitro. Table 2 gives
the IC50 values determined 72 h after compound addition.
Quinine was potent (IC50 = 18 nM), consistent with previ-
ous work (14). Eleven of the 14 conjugates maintained
potency in line with quinine with IC50 values below 100 nM
(12a–c, 12e, 12g–j, 12l–n) and highest with 12i (at
17 nm) and 12 m (at 23 nm) showing activity comparable
to that of quinine. Compounds 12f and 12k were some-
what less potent with IC50 values of 95 and 120 nM,
respectively. Compound 12d (Boc-L-Ser(Bzl)-QN) was
much less active with an IC50 of 550 nM, approximately
30-fold higher than that of quinine. These data indicate
that conjugation of small peptides to the quinine hydroxyl
Chem Biol Drug Des 2013; 82: 361–366
365

Panda et al.
group does not interfere with antimalarial activity and pro-
vides a handle for optimization of antimalarial and pharma-
cokinetic properties. Furthermore, the peptides do not
appear to hinder the cell permeability of the conjugates.
malaria patients, and implications for quinine
responses in the clinical setting. J Antimicrob Chemo-
ther;67:2501–2505.
12. Gordon K.D., Caprio J. (1985) Taste responses to
amino acids in the southern leopard frog, Rana
sphenocephala. Comp Biochem Physiol;81A:525–530.
13. Bajaj K., Panda S.S., El-Nachef C., Katritzky A.R. (2012)
Syntheses of chiral N-(protected) tri- and tetrapeptide
conjugates. Chem Biol Drug Des;80:17–26.
14. Panda S.S., Bajaj K., Meyers M.J., Sverdrup F.M., Kat-
ritzky A.R. (2012) Quinine bis-conjugates with quino-
lone antibiotics and peptides: synthesis and antimalarial
bioassay. Org Biomol Chem;10:8985–8993.
Conclusion
In conclusion, we have reported convenient benzotriazole-
mediated efficient syntheses for chirally pure quinine con-
jugates with amino acids and peptides of varying polarity.
These quinine–peptide conjugates possess in vitro antima-
larial activity similar to that of quinine.
15. Larhed M., Moberg C., Hallberg A. (2002) Microwave-
accelerated homogeneous catalysis in organic chemis-
try. Acc Chem Res;35:717–727.
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Additional Supporting Information may be found in the
online version of this article:
Appendix S1. Experimental details for compounds 2–11.
1H, 13C and CHN/HRMS spectra for 4f, 4g, 7e, 7f, 9b,
9c, 11 and 12a-n and graphs for antimalarial bioassay of
12a–n and quinine. This material is available free.
11. Islahudin F., Pleass R.J., Avery S.V., Ting K. (2012)
Quinine interactions with tryptophan and tyrosine in
366
Chem Biol Drug Des 2013; 82: 361–366