A novel synthesis of the efficient anti-coccidial drug halofuginone hydrobromide

Article abstract of DOI:10.3390/molecules22071086

Background: Halofuginone hydrobromide (1) is recognized as an effective drug against several species of Eimeria (E.) in poultry. In this paper, we describe a convenient and low cost preparation method for the compound, as well as primary validation of its activity. Methods: First, 7-bromo-6-chloroquinazolin-4(3H)-one (2) was prepared from m-chlorotoluene by a conventional process, and then chloroacetone was creatively introduced in two steps. Finally, halofuginone hydrobromide (1) was obtained from 7-bromo-6-chloro-3-(3-cholroacetonyl) quinazolin-4(3H)-one (4) by a four-step reaction sequence including condensation, cyclization, deprotection and isomerization. The structures of the relative intermediates and target compound were characterized by melting point, IR, MS and ¹H-NMR. Besides, the protective effect of compound 1-supplemented chicken diet at doses of 6, 3 and 1.5 mg per 1 kg were evaluated on chickens infected with E. tenella, by reduction in mortality, weight loss, fecal oocyst excretion and gut pathology, respectively. Results: Halofuginone hydrobromide (1) was prepared successfully by and improved and innovative method based on traditional research. Moreover, the synthesized halofuginone hydrobromide significantly exhibited an anti-coccidial property. Conclusions: The fruitful work described in this Communication has resulted in halofuginone hydrobromide, which has a good pharmaceutical development prospects, becoming more available for large-scale production.

Full text of DOI:10.3390/molecules22071086

molecules
Communication
A Novel Synthesis of the Efficient Anti-Coccidial
Drug Halofuginone Hydrobromide
Junren Zhang 1,2,† , Qizheng Yao 3,† and Zuliang Liu 1,
*
1
School of Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, China;
zjr1108@njau.edu.cn
Laboratory of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine,
Nanjing Agricultural University, Nanjing 210095, China
School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China; qz_yao@163.com
Correspondence: Njustlzl723@hotmail.com; Tel.: +86-25-8431-8865
These authors contributed equally to this work.
2
3
*
†
Received: 9 June 2017; Accepted: 28 June 2017; Published: 30 June 2017
Abstract: Background: Halofuginone hydrobromide (1) is recognized as an effective drug against
several species of Eimeria (E.) in poultry. In this paper, we describe a convenient and low cost
preparation method for the compound, as well as primary validation of its activity. Methods: First,
7-bromo-6-chloroquinazolin-4(3H)-one (
process, and then chloroacetone was creatively introduced in two steps. Finally, halofuginone
hydrobromide ( ) was obtained from 7-bromo-6-chloro-3-(3-cholroacetonyl) quinazolin-4(3H)-one (4)
2) was prepared from m-chlorotoluene by a conventional
1
by a four-step reaction sequence including condensation, cyclization, deprotection and isomerization.
The structures of the relative intermediates and target compound were characterized by melting
point, IR, MS and 1H-NMR. Besides, the protective effect of compound
1-supplemented chicken diet
at doses of 6, 3 and 1.5 mg per 1 kg were evaluated on chickens infected with E. tenella, by reduction
in mortality, weight loss, fecal oocyst excretion and gut pathology, respectively. Results: Halofuginone
hydrobromide (1) was prepared successfully by and improved and innovative method based on
traditional research. Moreover, the synthesized halofuginone hydrobromide significantly exhibited
an anti-coccidial property. Conclusions: The fruitful work described in this Communication has
resulted in halofuginone hydrobromide, which has a good pharmaceutical development prospects,
becoming more available for large-scale production.
Keywords: febrifugine; halofuginone hydrobromide; asymmetric synthesis; 4(3H)-quinazolinone;
piperidine
1. Introduction
Febrifugine and isofebrifugine (Figure 1), were first isolated from leaves of Dichroa Febrifuga Lour
in 1948, and two years later, their skeletal structures were reported [
of research, determination of the absolute configuration of the two natural products was achieved
(febrifugine: 2R,3S; isofebrifugine: 2S,3S) [ ]. In 1967, a derivative of febrifugine named halofuginone
hydrobromide ( ) was first synthesized by American Cyanamid Company, which later transferred
this product to Roussel Uclaf S. A. who marketed it in a premix named “Stenorol” [ ]. Halofuginone
hydrobromide has been proved to possess broad pharmacological functions, such as anti-coccidial [ ],
anti-fibrosis [ ], anti-tumor [10 12] and growth-promoting activities [13]. In clinic application,
kg−1 in animal feed exhibit great effects against almost all types of
1]. After more than 40 years
2
1
3
4–6
7–
9
–
addition at a dose of 3 mg
coccidia [14–16].
·
Molecules 2017, 22, 1086; doi:10.3390/molecules22071086
www.mdpi.com/journal/molecules

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Figure 1. Structures of febrifugine, isofebrifugine, and halofuginone hydrobrimide (1).
Many other researchers have focused on the preparation of halofuginone and relevant compounds,
and they finally figured out the key point was to generate two intermediates involving substituted
4(3H)-quinazolinone and piperidine moieties (Figure 2) [17].
Figure 2. 4(3H)-Quinazolinone (L) and piperidine (R) derivative.
With regard to the 4(3H)-quinazolinone fragment, the preparation is quite mature as so far there
have be a lot of studies reporting synthesis methods for this target. Route A (Scheme 1) would be the
most classical method, and many other reports came up with new syntheses based on Route A by
changing the starting material or reaction conditions [18]. On the other hand, route B (Scheme 2) which
features a short process, mild conditions and easy treatment of the three waste streams in particular,
was taken as reference to design a rational technological process [19–21].
O
Br
Br
Br
O
.
H2SO4
H2NOH HCl
O
O
+
O
Cl3CCH(OH)2
N
N
Br
N
N
OH
NH2
H
H
H
O
O
Cl
Br
COOH
NH2
Cl
Separation
Purification
H2O2
SO2Cl2
O
O
N
Br
N
Br
H
H
O
Cl
Br
HCONH2
NH
N
Scheme 1. Route A: Synthesis of 7-bromo-6-chloroquinazolin-4(3H)-one: The separation and
purification of bromo-substituted 2,3-indolinedione proved hard, leading to low total recovery.

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Scheme 2. Route B: synthesis of 7-bromo-6-chloroquinazolin-4(3H)-one: by choosing 3-chlorotoluene
as the starting material, the intermediate was produced with a reduced number of reaction steps
and cost.
The preparation of piperidine analogues was the most expensive part, and deemed to be the
real difficulty to produce halofuginone as well. So far, a lot of methods have been reported: The
method illustrated in Scheme 3 was originally developed by Baker’s team [18,22–26]. Afterwards, the
method was optimized a bit by Barringer et al., via changing reagents and catalyst, which successfully
improved the yield by reducing the number of steps but without lowering the cost. Referring to
Baker’s route, Wu’s group invented a different approach to 2-acetonyl-3-methoxypiperidine using
3-hydroxyl pyridine as starting material; before this, Takeuchi et al., had already synthesized piperidine
derivatives from 3-hydroxypyridine [27]. However, all these synthesis involved a reduction of the
pyridine ring, which led to high costs and considerable pressure for environmental reasons.
Scheme 3.
Synthesis of 2-acetonyl-3-methoxyl piperidine involving the application of
pyridine derivatives.
In addition, some other researchers studied different ways to prepare the piperidine ring. In 1999,
Kobayashi et al., were the first to successfully prepare the desired piperidine intermediate (Scheme 4),
resulting in further confirmation of the relative configuration of febrifugine and making possible the
asymmetric synthesis of febrifugine analogues [2].
Scheme 4. First asymmetric synthesis of the piperidine ring.

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However, the previous methods required complex operations and too many steps, whereas there
was one method that should be mentioned (Scheme 5), which might be useful for industrial production
because of its high yield and very few reaction steps [28]. Nevertheless, the cost of production was
always very high, so there is a strong demand for a new simple approach to produce the piperidine
intermediate for halofuginone at lower cost.
Scheme 5. The shortest preparation of the piperidine derivative.
In the present study, a new expedient total synthesis of halofuginone hydrobromide was
developed. In addition, its anti-coccidial activity in chickens, as evidenced by survival rate, body
weight loss, oocyst shedding and intestine pathology, was examined [5,6].
2. Results and Discussion
2.1. Synthesis of Halofuginone Hydrobromide
Our synthetic route to halofuginone hydrobromide is described in Scheme 6. Firstly, compound
2
was prepared from 3-chlorotoluene by the optimized method of Route B (Scheme 2) under improved
conditions, which proved our most difficult and challenging task.
Scheme 6. Total synthesis process to halofuginone hydrobromide 1.
Initially, we produced 3-acetonyl-6-chloro-7-bromoquinazolin-4(3H)-one using 1-chloroacetone
(Scheme 7). Then we tried to carry out the next condensation, but failed to obtain good yields of the
target compound even under various optimized conditions. We then attempted to introduce
1,3-dichloroacetone by single substitution, but failed too. Finally, chloroacetonylation was separated
into two parts: condensation of with 2,3-dichloropropylene and subsequent addition with NCS and
acidic hydrolysis, to afford a convenient conversion of to in satisfactory yield. Then, condensation
of with tetrahydrofuran-2-carbaldehyde in ethanol was performed as to simultaneously introduce the
2 into
2
2
4
4

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hydroxyl, as well as a double bond into
4, followed by direct bromination to give
5
. With regards to the
condensation, the influences of different catalysts and solvents were investigated (Table 1). As a result,
potassium tert-butoxide (KTB) was beneficial to the catalysis, while ethanol was the most suitable
solvent even for large scale production. Compound
gas, so was first reacted with benzylamine to give
then
halofuginone hydrobromide were produced, and trans-isomer (
7
6
was not formed by direct cyclization with NH3
, which could be quickly carried out at R.T. and
5
7
was successfully obtained by debenzylation with CAN. Finally, the cis- and trans-isomer of
) was separated and purified thanks to
1
of their different solubility in ethanol, which was verified by NMR data referring to Uesato et al. [29].
Scheme 7. Two unsatisfactory routes to substituted products.
Table 1. Different results caused by the catalysts and solvents used in condensation reaction.
Catalyst [a]
Solvent [b]
Result
Result
Sodium methoxide
Sodium ethoxide
Potassium tert-butoxide
Incomplete reaction
Incomplete reaction
Favorable reaction
Ethanol
Isopropanol
tert-Butanol
Favorable reaction
Favorable reaction
Favorable reaction
Favorable reaction, but
complicated work-up
Slow reaction
Sodium hydride
More by-products
N,N-dimethylformamide
N-butyl lithium
More by-products
/
Ether
Tetrahydrofuran
[b]
/
More by-products
[a]
Potassium tert-butoxide was the best choice for the reaction.
ethanol would be the first choice for large-scale production.
The three alcohols exhibited good effects, and
2.2. Evaluation on Antioccidial Activity of Halofuginone Hydrobromide (Hh) in Chickens
After acclimatization for 20 days on standard diet alone, 50 individual chickens with similar
body weights were chosen, and were fed standard diet alone (I, II) or supplemented with different
doses (III, IV, V) of compound 1. One day later, animals (II~V) were infected by oral administration of
100,000 sporulated oocysts. Animals of the infected unmedicated group (Group II) showed obvious
clinical symptoms from 4 days post-inoculation, mainly as follows: general malaise, reduced ingestion,
emerging bloody stools, and even death. In contrast, all animals of the uninfected unmedicated group
(Group I) were very healthy, with no negative symptoms being observed. On day 7 post-infection, there
was no difference in the body weight of the chickens in Group IV (Hh3) in comparison with Group I (UI
control), In contrast, the body weight of chickens in Group III and Group V were significantly different
from Group I (UI control). Furthermore, the body weight of chickens in the three infected medicated
groups (III–V) was much higher than Group II. Overall, halofuginone hydrobromide significantly
ameliorated the reduced weight gain caused by E. tenella to a greater degree, and even as slight
weight-gain effect was observed, which was accelerated by the medium dose (0.3%). No fecal oocysts
were detected in the uninfected unmedicated controls. Only a few fecal oocysts were detected in the
three medicated groups, and the relative reduction rate of oocysts (RORR) in each group (II–V) was 0%,
98.8%, 95.2%, 86.3%, respectively. It turned out that halofuginone could strongly inhibit reproduction
of coccidia. All the chickens in each group were sacrificed on day 7 post-infection and their ceca were

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removed. Gross lesion scores were obtained as described previously. The uninfected unmedicated
control chickens (Group I) had no lesions in the ceca (score = 0). In contrast, E. tenella caused more
gross cecal lesions in the gut of unmedicated chickens 7 days post-infection, as evidenced by a lesion
score close to 3.2 (Group II). Halofuginone at different doses (0.6%, 0.3% and 0.15%) significantly
diminished the cecal damage in infected chickens (Groups III–V) as shown by the gross lesion scores
of 0–1.1 (0, 0.2, and 1.1). Comprehensive results are listed below (Table 2).
Table 2. Parameters for determination of anti-coccidial activity.
Group [#]
RBWGR [a] (%)
SR [b] (%)
ALS [c]
OS [d]
ACI [e]
I
II
III
IV
V
100
46.02
99.68
106.54
93.02
100
40
100
100
100
0
32
0
2
11
0
20
1
1
5
200.00
34.02
198.68
203.54
177.02
[#]
The chickens were housed for 20 days to get accustomed to the environment, and then divided into five
groups. Group I (UI control) and Group II (I control) had daily access to standard chicken diet from day 0 to
day 7. Group III (Hh6), Group IV (Hh3), and Group V (Hh1.5) were fed daily with the diet supplemented with
halofuginone hydrobromide powder at the dose of 0.6% (6 mg/kg diet), 0.3% (3 mg/kg diet) or 0.15% (1.5 mg/kg
[a]
diet), respectively. The initial number (n) of chickens in each group is indicated. Relative Body weight gain rate
(RBWGR); [b] Survival rate (SR); [c] Average lesion scores (ALS) [d] Oocyst score (OS); [e] Anticoccidial index (ACI):
Halofuginone at doses of 0.6% and 0.3% exhibited high efficacy, in comparison, 0.15% exhibited moderate efficacy
at best.
3. Experimental Section
3.1. General Information
Every reaction was monitored, and the endpoint was checked, by TLC performed on GF-254
silica gel plates (Yantai Derxin Biological Technology, Yantai, China) with visualization by UV light.
Melting points were measured on an YRT-3 temperature apparatus (Tianjin Jing Tuo Instrument
Technology, Tianjin, China) and are uncorrected. IR spectra were recorded on a VERTEX 80 instrument
(Bruker Corporation (Beijing), Beijing, China)). Mass spectra were determined on a VG Auto Spec-3000
spectrometer (VG Instruments, Kuala Lumpur, Malaysia) and reported as m/z. The 1H-NMR spectral
data were recorded on a Bruker Avance III HD spectrometer (300~900 MHz, Bruker Corporation
(Beijing)), and chemical shifts were reported in ppm (δ) relative to TMS as an internal standard.
3.2. Synthesis
3.2.1. Synthesis of 7-bromo-6-choroquinazolin-4(3H)-one (2)
To a mixture of 3-chlorotoluene (200 g, 1.58 mol), FeCl3 (9.4 g, 0.06 mol) and CH2Cl2 (1000 mL)
◦
stirred at 0~10 C, Br2 (500 g, 3.12 mol) was added slowly, during which the produced gas was
absorbed by 15% NaOH aqueous solution (2000 mL). After 1 h of continued vigorous stirring, water
(100 mL) was added and the mixture was adjusted to pH 8.0~10.0 with 10% aqueous NaOH (200 mL).
The organic layer was separated and concentrated to dryness under vacuum to give a solid (95.7%, m.p.
95~96 ◦C), 426 g of which was added to a mixture of water (2000 mL), pyridine (200 mL) and KOH
◦
(112 g, 2.0 mol) and heated. When the interior temperature reached 80 C, KMnO4 (800 g, 5.0 mol) was
added in portions, and the mixture was refluxed until complete consumption of the KMnO4. Then the
solution was filtered, the precipitate was washed with 10% KOH a.q. (1000 mL). The filtrates were
combined, and then vacuum evaporated to remove the pyridine and form a turbid solution, which
needed to be filtered again. The fresh filtrate was acidified with concentrated HCl (360 mL) to pH 3~4.
Then the precipitate was filtered, dried and crushed (80%, m.p. 170~171 ◦C). A portion (340 g) was
◦
dissolved in 25% NH3-H2O (1700 mL). To the solution, Cu2O was added in batches at 30~40 C. After
about 5 h of stirring, the solution without NH3 gas was diluted and acidified. Then the solid was

Molecules 2017, 22, 1086
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◦
filtered, washed, dried (yield 96%, m.p. 248~249 C) and 250 g was dissolved in 1500 mL of formamide,
maintained at 150~170 ◦C for 5 h. The solution was allowed to cool to R.T., and the precipitate was
filtered and washed to give a white solid (92%, m.p. 306~307 ◦C). Thus,
2
was succesfully obtained in
(cm−1): 3188.6, 1643.4 (NH), 1680.3 (C=O); ESI-MS (m/z): 256.9
H]−; 1H-NMR (300 MHz, DMSO-d6),
(ppm): 12.50 (br, 1H), 8.15 (s, 1H),
an overall yield of 67%. IR (KBr),
[M
H]−, 258.9 [M + 2
8.14 (s, 1H), 8.05 (s, 1H).
ν
−
−
δ
3.2.2. 7-Bromo-6-chloro-3-(2-chloropropenyl)quinazolin-4(3H)-one (3)
A mixture of compound (100 g, 0.38 mol), 2,3-dichloropropylene (52 g, 0.47 mol), K2CO3 (83 g,
2
◦
◦
0.6 mol) and DMF (300 mL) was stirred for 1 h at 120 C, and then maintained at 115~125 C for
another 7 h. Immediately after that, the reaction mixture was transferred into the mixture of ice and
water to cool to 5 C. After stirring for 1 h, the precipitate was filtered, washed, and extracted in
◦
ethyl acetate. The fresh organic phase was completely washed with saturated aqueous NaCl, and the
organic layer was concentrated to dryness under vacuum to give
(KBr),
(cm−1): 3057.0, 907.6 (=CH2), 1685.1 (C=O); ESI-MS (m/z): 333.0 [M + H]+, 335.0 [M + 2 + H]+;
1H-NMR (400 MHz, DMSO-d6),
3
(127 g, 99%, m.p. 150~155 ◦C). IR
ν
δ
(ppm): 8.44 (s, 1H), 8.24 (s, 1H), 8.15 (s, 1H), 5.5 (s, 2H), 4.8 (s, 2H).
13C-NMR (CDCl3), δ (ppm): 51.8, 117.4, 122.0, 127.8, 129.7, 132.8, 133.9, 134.7, 146.9, 147.1, 159.2.
3.2.3. 7-Bromo-6-chloro-3-(3-chloroacetonyl)quinazalin-4(3H)-one (4)
To acetonitrile (500 mL) stirred at 20 ◦C,
HCl (20 mL) and then NCS (100 g, 0.75 mol) was added in batches at 20
3
(127 g, 0.38 mol) was added◦together with 5% aqueous
±
2 C within 4 h. The solution
was allowed to cool for 30 min, and the precipitate was filtered and dried to give 106 g of compound
(80%, m.p. 239~241 ◦C). IR (KBr), (cm−1): 2993.0, 2968.1, 2930.0, 1447.7 (CH2), 1735.9 (C=O, keto
carbonyl group), 1667.1(C=O, acyl carbonyl group); ESI-MS (m/z): 349.0 [M + H]+, 351.0 [M + 2 + H]+;
4
ν
1H-NMR (400 MHz, DMSO-d6),
δ (ppm): 8.29 (s, 1H), 8.22 (s, 1H), 8.16 (s, 1H), 5.07 (s, 2H), 4.74 (s, 2H);
13C-NMR (DMSO-d6), δ (ppm): 47.7, 53.1, 122.2, 127.4, 129.0, 132.3, 132.9, 147.7, 150.0, 159.2, 196.5.
3.2.4. 7-Bromo-6-chloro-3-(8-bromo-5-hydroxyl-2-oxo-3-octenyl) quinazolin-4(3H)-one (5)
A mixture of
4
(35 g, 0.1 mol), anhydrous ethanol (300 mL), KTB (15 g, 0.13 mol), and
◦
2-furan-carboxaldehyde (12 g, 0.12 mol) was refluxed for 7 h. The mixture was initially cooled to 40 C
naturally, then quickly cooled to below 0 C in ice-salt water. Upon first addition of hydrobromic
◦
ethanol (40 mL, 30%), the temperature went up to 5 ◦C automatically. The reaction mixture was kept
at that temperarure for 2 h, then a by second addition of hydrobromic ethanol (13 mL), maintained
at 50 ◦C for 5 h. As soon as the mixture cooled to 0 ◦C, NaHCO3 (20 g) was added in batches, and
after stirring for 30 min, the mixture was redispersed by addition of water (50 mL). Then, the mixxed
solution was vacuum evaporated to recover the ethanol. Finally, the precipitate was filtered and
dried to give crude product (40 g), which needed recrystallization to achieve purer
123~124 ◦C). IR (KBr), (cm−1): 3291.5 (OH), 2983.5–2843.0, 725.2 (CH2), 1698.1 (C=O, ketocarbonyl
group), 1681.1 (C=O, acyl carbonyl group); ESI-MS (m/z): 474.9 [M
H]−, 476.9 [M + 2 H]−;
1H-NMR (300 MHz, DMSO-d6),
(ppm): 8.33 (s, 1H), 8.21 (s, 1H), 8.15 (s, 1H), 7.06–7.13 (dd, 1H),
6.38–6.43 (d, 1H), 5.18 (s,2H), 4.31 (m, 1H), 3.57 (t, 2H), 1.91 (m, 2H), 1.64 (m, 2H); 13C-NMR (DMSO-d6),
(ppm): 29.0, 35.0, 35.6, 53.5, 69.1, 122.3, 125.2, 127.4, 128.8, 132.2, 132.9, 147.8, 150.4, 152.7, 159.2, 192.7
5 (26 g, 54%, m.p.
ν
−
−
δ
δ
3.2.5. 7-Bromo-6-chloro-3-[(4-benzyl-2-hydroxyl-octahydrofuran[3,2-b]pyridine-2)methyl]
quinazol-in-4(3H)-one (6)
Compound
5 (50 g, 0.11 mol) was dissolved in CH2Cl2 (350 mL) and the temperature was
controlled at 15~20 ◦C. To the solution, benzylamine (14.6 g, 0.14 mol) was added dropwise within
30 min, continuous stirring for 2 h. CH2Cl2 was recovered and the residue was dissolved in ethanol
with refluxing for 30 min, then cooled, and the precipitate that formed was filtered to provide
6
◦
as a light yellow powder (40.9 g, 74%, m.p. 183~185 C). IR (KBr),
ν
(cm−1): 2961.1–2837.7 (CH2),

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1680.5 (C=O, acyl carbonyl group), 1600, 1444.4 (benzene ring); ESI-MS (m/z): 504.1, [M + H]+, 506.1
[M + 2 + H]+, 502.0, [M H]−, 504.1 [M + 2 H]−; 1H-NMR (300 MHz, DMSO-d6),
(ppm): 8.33 (s,
−
−
δ
1H), 8.24 (s, 1H), 8.11 (s, 1H), 7.32–7.17 (m, 5H), 4.27–4.32 (d, 1H), 4.10–4.15 (d, 1H), 3.92–3.99 (m, 2H),
3.05–3.10 (d, 1H), 2.79 (s, 1H), 2.62–2.66 (d, 1H), 2.27–2.31 (d, 1H), 1.97–1.99 (d, 1H), 1.93–1.95 (d, 1H),
1.83–1.88 (d, 1H), 1.56–1.60 (d, 1H), 1.49–1.53 (d, 1H), 1.41 (d, 1H), 6.69 (s, 1H); 13C-NMR (CDCl3),
δ
(ppm): 19.9, 26.9,40.5, 50.0, 50.4, 59.3, 62.3, 82.9, 104.7, 122.0,127.6, 127.8, 128.6,128.6, 129.2, 129.2, 129.4,
132.7, 133.4, 136.6, 147.1, 149.3, 160.1.
3.2.6. 7-Bromo-6-chloro-3-[3-(3-hydroxyl-2-piperidyl)-2-oxopropyl]quinazolin-4(3H)-one (7)
To a mixtu◦re of ethanol and H2O (ethanol:H2O = 4:1, 375 mL), compound
6 (40 g, 0.08 mol) was
added at 20–30 C. Batchwise addition of ceric ammonium nitrate (CAN) (92 g, 0.17 mol) was finished
over 1 h. The reaction was continued for 8–9 h until the reaction endpoint was indicated by TLC.
The mixture was adjusted to pH 8.0–9.0 with saturated aqueous Na2CO3 and then the product was
extracted with CH2Cl2 (300 mL
aqueous NaCl and then distilled. The white solid was recrystallized with ethanol, filtered, and dried
to give 32.3 g of compound
in a yield of 94%; m.p. 215–217 ◦C. IR (KBr), (cm−1): 3416.5 (NH),
2974.9–2931.9 (CH2), 1715.8 (C=O, keto carbonyl group), 1679.7 (C=O, acyl carbonyl group); ESI-MS
(m/z): 414.0 [M + H]+, 416.0 [M + 2 + H]+, 412.0, [M H]−, 414.1 [M + 2 H]−; 1H-NMR (300 MHz,
DMSO-d6), (ppm): 8.25 (s, 1H), 8.23 (s, 1H), 8.17 (s, 1H),5.00 (s, 2H), 4.76–4.77 (d, 1H), 2.96–3.30 (dd,
×
3). The combined organic solution was washed with saturated
7
ν
−
−
δ
2H), 2.50–2.51 (d, 1H), 2.45–2.48 (d, 1H), 2.36–2.44 (dd, 2H), 1.99(s, 1H), 1.88–1.91 (m, 1H), 1.56–1.59 (m,
1H), 1.30–1.40 (m, 1H), 1.17–1.27 (m, 1H); 13C-NMR (DMSO-d6),
δ (ppm): 26.3, 39.4, 40.6, 46.1, 55.3,
60.6, 71.2, 122.3, 127.4, 128.8, 132.2, 132.9, 147.8, 150.2, 159.1, 203.9.
3.2.7. (±)-trans-7-Bromo-6-chloro-3-[3-(3-hydroxy-2-piperidyl)acetonyl]quinazolin-4(3H)-one
hydro-bromide (halofuginone hydrobromide, 1)
Compound
hydrobromic ethanol (10%, 33.2 g) was added dropwise within 30 min. After 1 h of stirring at R.T., the
7
(14 g, 0.034 mol) was dissolved in ethanol (300 mL) and stirred at 15–25 ◦C, then
◦
◦
mixture was stirred at 60–70 C for another 1 h. Then the solution was allowed to cool to 5–10 C, and
filtered to achieve the final pure product (48%, 8.1 g, m.p. 245 (decomposed)). IR (KBr),
(cm−1):
3327.3 (OH), 2943.1–2812.1(CH2), 1729.3 (C=O, keto carbonyl group), 1678.8 (C=O, acyl carbonyl
group); ESI-MS (m/z): 414.0 [M + H]+, 416.0 [M + 2 + H]+, 412.0, [M H]−, 414.0 [M + 2 H]−;
1H-NMR (300 MHz, DMSO-d6),
(ppm): 8.30 (s, 1H), 8.22 (s, 1H), 8.17 (s, 1H), 5.56 (d, 1H), 5.11 (s, 2H),
1
ν
−
−
δ
3.50–3.54 (m, 1H), 3.31–3.34 (m, 1H), 3.20–3.22 (m, 1H), 3.15 (m, 1H), 2.95–2.97 (m, 1H),2.87–2.89 (m,
1H), 1.92–1.96 (m, 1H), 1.80–1.91 (m, 1H), 1.52–1.71 (m, 1H), 1.41–1.51 (m, 1H), 8.69 (active hydrogen).
13C-NMR (DMSO-d6),
147.2, 149.5, 158.6, 200.6.
δ (ppm): 20.2, 30.6, 39.5, 43.0, 54.4, 56.3, 66.7, 121.7, 126.8, 128.4, 131.8, 132.4,
3.3. Evaluation on the Antioccidial Activity of Halofuginone Hydrobromide in Chickens
Halofuginone is known worldwide as an efficient anticoccidial medicine. Therefore, the
anticoccidial activity of our synthesized product needed to be verified as well. A chicken coccidiosis
model was successfully induced by oral administration of sporulated oocysts (about 105), indicating
that the fecundity of E. tenella was strong enough. After acclimatization for 20 days on standard diet
alone, 50 individual chickens with similar body weights were chosen, and were fed standard diet alone
(I, II) or supplemented with different doses (III, IV, V) of halofuginone. One day later, all animals were
infected. We monitored body weight of chickens with access to different diets after Eimeria infection.
Body weight gain rate (BWGR) was calculated by the formula: BW on day 7 post-infection
−
BW on
day 0 post-infection. To further determine the anti-coccidial effect, Eimeria oocysts in chicken feces,
as an indicator of Eimeria multiplication, was evaluated. Fecal oocyst excretion of each group was
collected from day 5 to day 7 post-infection and combined. The relative reduction rate of oocyst (RORR)
was obtained by the formula: (OPG of Group II—OPG of each medicated group)
÷
OPG of Group II

Molecules 2017, 22, 1086
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(OPG: oocyst per gram). All the chickens in each group were sacrificed on day 7 post-infection and
their ceca were removed. Gross lesions in the ceca caused by E. tenella were scored based on five grades
as described previously [30]. Finally, survival rate (SR) was recorded, contributing to calculation
of ACI:
ACIa = RBWGRb + SRc − ALSd × 10 − OSe
(1)
Notes: a Anticoccidial index (ACI): briefly, the efficiency of the drug was graded on the basis of the
ACI value as follows: (1) high efficacy, >180; (2) moderate efficacy, 160~180; (3) low efficacy, 120~160;
b
(4) invalid efficacy, <120. Relative Body weight gain rate (RBWGR) was obtained by the formula:
BWGR of each group
of survival chickens in each group
obtained by the formula: Gross lesion score
according to the standard described previously.
÷
BWGR of group I. c Survival rate (SR) was obtained by the formula: number
÷
initial number of chickens. d Average lesion scores (ALS) was
e
÷
numbers of chickens. Oocyst score (OS) was obtained
4. Conclusions
So far, there has been a lot of studies on developing synthesis for halofuginone, but all have
failed to realize industrialization. To solve this problem, we developed the creative route for the total
synthesis for halofuginone (overall yield 9.6%, 10 steps), of which efficient anticoccidial activity was
evaluated and verified. Our method was characterized no use of harmful materials (except HBr)
and no harsh conditions. In addition, we broke the traditional bottleneck (convergent synthesis) to
achieve a great success by linear synthesis, reducing the cost by 70% [31]. However, many steps and
time-costs still existed, which are the common drawbacks of linear synthesis. Aside from that, the final
reaction gave cis- and trans-isomers, resulting in a poor harvest of the trans-isomer, so there is still an
urgent need for further optimization and the development of novel methods to induce unidirectional
conversion from the cis-isomer to the trans-isomer.
Acknowledgments: All sources of funding of the study should be disclosed. Please clearly indicate grants that
you have received in support of your research work. Clearly state if you received funds for covering the costs to
publish in open access.
Author Contributions: Zuliang Liu initiated and led the project. Junren Zhang performed the experiments.
Qizheng Yao analyzed the data. Junren Zhang wrote the manuscript. The manuscript was finalized through
contributions from all authors, and all authors approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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