Synthesis and anticoccidial activity of some azacyclo organoborinates

Article abstract of DOI:10.3987/COM-02-9496

A series of azacyclo organoborinates derivatives of piperidinyl and pyridinyl alcohols were prepared and their anticoccidial activity was tested in vitro assay system. Among them, di(4-chlorophenyl)(2-piperidinylmethoxy-O,N)boron, di(3-trifluoromethylphenyl)(2-piperidinylmethoxy-O,N)boron, di(3-trifluoromethylphenyl)(2-pyridinylmethoxy-O,N)boron, and di(3-trifluoromethylphenyl)(2-pyridinylethoxy-O,N)boron, showed moderate anticoccidial activity against Eimeria tenella.

Full text of DOI:10.3987/COM-02-9496

HETEROCYCLES, Vol. 57, No. 7, 2002, pp. 1319 - 1326, Received, 22nd April, 2002
SYNTH E SIS AND ANTICOCCIDIAL ACTIVITY OF SOME
AZACYCLO ORGANOBORIN ATE S
H it osh i Ta bu ch ia , H a r u m ot o Ka wa gu ch ib, H isa sh i Ta n igu ch ia ,
H ideyu ki Im a za kia , a n d Yosh io H a ya seb*
a Nit t o Ka sei Co., Lt d., 3-17-14 Nish ia wa ji, Higa shiyodoga wa -ku,
Osa ka Cit y 533-0031, J a pa n
bAbu ra h i La bora t ories, Sh ion ogi & Co., Lt d., 1405 Got a n da ,
Koka -gu n , Sh iga 520-3423, J a pa n
Abst r a ct －A series of a za cyclo orga n oborin a t es deriva t ives of
piperidinyl a n d pyridinyl a lcohols were prepa red a n d their
a n t icoccidia l a ct ivit y wa s t est ed in vit ro a ssa y system.
Among them, di(4-chlorophenyl)(2-piperidin ylmethoxy-O,N)-
boron, di(3-trifluoromethylphenyl)( 2-piperidinylm ethoxy-O,N)-
boron, di(3-trifluoromethylphenyl)(2-pyridin ylmethoxy-O,N)-
boron, a n d di(3-trifluoromethylphenyl)-
(2-pyr idin ylet h oxy-O,N)bor on , sh owed m oder a t e a n t icoccidia l
a ct ivit y a ga in st E im er ia t en ella .
Coccidiosis in chickens, turkeys, geese, and ducks is a widespread disease caused by a
gr ou p of pa r a sit ic pr ot ozoa , a lt h ou gh m a n y polyet h er ion oph ores su ch a s
m on en sin ,1 la sa locid,2 sa lin om ycin ,3 a n d n a ra sin4 h a ve been u sed su ccessfu lly a s
a n t icoccidia l a gen t s a n d a s growt h pr om ot er s.
H owever , polyet h er -r esist a n t
Eimeria spp. a r e em er gin g wit h in cr ea sin g fr equ en cy.5 In our progra m dir ected
t owa rd developin g n ew a n t icoccidia l a gen t s a ga in st polyet h er -resist a n t Eim eria
spp., we ha ve n ow synthesized a series of diphen yl or dina ph thyl substituted
orga nobora te deriva tives of piperidin yl a n d pyridinyl a lcohols wer e prepa red
(Sch em e 1) a n d t h eir a n t icoccidia l a ct ivit y wa s t est ed in vit ro. Ma n y com pou n ds
wit h t h e a n t iba ct eria l, fu n ga l, an d a n t i-fou lin g a ct ivit ies of a zacyclo
or ga n obor in a t es h a ve been r epor t ed in t h e lit er a t u r e.6 We foun d tha t diph enyl
(2-piper idin yl- m et h oxyO,N)bor on ca u ses h igh a n t icoccidia l a ct ivit y a gain st t h e
im porta n t pa ra sites, E. t en ella a n d E. a cervu lin a ,10 a n d is ea sily prepa red via a
three-step procedure.

Ba sed on t h e h igh a n t icoccidia l pot en cies of
5, we h a ve n ow pr epa red
pyr idin ylor ga n obor in a t es (16-34) in order t o explore their stru ctu re-a ct ivity
rela t ion sh ip. Th e m a in object ive of t h is in vest iga t ion is t o resea rch a n ew a gen t
with potentia l a ctivity a ga inst coccidiosis a nd a less cyto-pa t hogenic effect on cells.
Scheme 1
1)B(OBu)3
Ar
Ar
Mg/THF
B-OCH2CH2NH2
ArMgBr
ArBr
2)HOCH2CH2NH2
１
N
H
(CH 2)n-OH
Ar
NH
B
O
Ar
OH
(CH 2)n
２
(1-13)
NH
N
H
Ar
B
O
Ar
Ar
Ar
B-OCH2CH2NH2
(14)
３
OH
１
N
H
NH
O
Ar
B
Ar
４
(15)
N
(CH 2)n-OH
Ar
Ar
N
B
O
(CH 2)n
(16-34)
5

CH E MISTRY
St u dies on t h e syn t h esis a n d ch a r a ct er iza t ion of or ga n ic bor on com pou n ds
possessing both N→B a nd O→B coor din a t ion bon ds a r e well docu m en t ed in t h e
11-20
lit er a t u r e.7-9,
Th e com pou n ds in Ta bles 1 a n d 2 were prepa red by kn own
procedu r es.7,11,20 Th e syn t h esis of t h e a ppropria t ely su bst it u t ed a za cyclo
dia rylborin a t es deriva t ives wa s in it ia t ed by t h e prepa ra t ion of
2-a m in oet h oxydia rylbora n e (1) from t h e a ppropria t e Grign a rd rea gen t wit h
t r ibu t yl bor a t e a n d et h a n ola m in e u sin g lit er a t u r e procedu r es.7,8 Th e obt a in ed (1)
wa s t r ea t ed wit h t h e requ isit e piperidin yl or pyridin yl a lcoh ols t o yield
cor respon din g a za cyclo dia rylborin a t es deriva t ives (1-34) ( Sch em e 1, Ta bles 1, 2 ).
Th e gen er a l syn t h et ic pr ocedu r e is descr ibed below.
St ep 1 : t h e pr epa r a t ion of 2-a m in oet h oxydia r ylbor a n e (1)
A solu t ion of a r ylm a gn esiu m br om ide in t et r a h ydr ofu r a n wa s pr epa red a ccor din g t o
a con ven t ion a l m et h od u sin g 0.53 m ol of a r yl br om ide, 12.9 g(0.53 m ol) of
m a gn esiu m , a n d 250 m L of t et r a h ydr ofu r a n . Su bsequ en t ly, 58.5 g(0.26 m ol) of
t ribu t yl bora t e est er a n d 200 m L of et h er were m ixed in a fla sk, a n d t h e fla sk wa s
cooled t o –60℃. A solu t ion of a r ylm a gn esiu m br om ide (Gr ign a r d r ea gen t ) in
t et r a h yd r ofu r a n wa s a d d e d d r op w is e t o t h e m ixt u r e wh ile m a in t a in in g a
t em pera t u r e of －60℃. Aft er t h e a ddit ion , t h e r ea ct ion m ixt u r e wa s st irr ed for 10
h a t rt a n d h ydrolyzed by t h e a ddit ion of 150 m L of 10% h ydroch loric a cid, a n d
t h e orga n ic la yer wa s isola t ed. 31.1 g(0.51 m ol) of et h a n ola m in e a n d 50 m L of
etha nol were a dded to the orga nic la yer, a nd the mixt ur e wa s stirred for 2 h a t rt.
Th e precipit a t ed produ ct wa s collect ed by filt ra t ion , recryst a llized fr om
et h a n ol/wa t er=1/1, a n d dried t o a fford 2-a m in oet h oxydia rylbora n e (1).
St ep 2 : t h e pr epa r a t ion of a za cyclo dia r ylbor in a t es deriva t ives (1-34)
0.012 m ol of 2-a m in oet h oxydia rylbora n e (1), 30 m L of et h er a n d 30 m L of 10%
h ydr och loric a cid con t a in ed in a sepa r a t or y fu n n el wer e sh a ken for a bou t 15 m in ,
a n d the la yers wer e sepa ra ted. Th e orga nic la yer wa s wa shed on ce with wa ter
a n d t ra n sfer red t o a pea r-sh a ped fla sk t o wh ich 0.018 m ol of r equ isit e piperidin yl
a lcoh ols (1-13) or 1.8 g(0.018 m ol) of 4-h ydroxypiperidin e (14) or 1.8 g(0.018 mol) of
3-h ydroxypiperidin e (15) or pyridin yl a lcoh ols (16-34) a n d 7 m L of et h a n ol were
a dded, a n d the mixt ure wa s stirred for 2h a t rt. The precipit a t ed pr odu ct wa s
collect ed by filt ra t ion , recryst a llized fr om a et h a n ol/wa t er=1/1, a n d dried t o a fford
a za cyclo dia rylborin a t es deriva t ives(1-34)

Table 1. Yeild and mp of diaryl(2-piperidinylalkoxy-O,N)borons
Fomula No.
Compd No.
n
1
1
1
Ar
Yield (%)
mp (℃）
169-171
126-128
203-205
1
2
3
Ph
83
55
67
2-CH3-C6H4
3-CH3-C6H4
4
5
6
7
8
9
1
1
1
1
1
1
4-CH3-C6H4
3-Cl-C6H4
88
58
80
68
55
37
134-136
182-184
150-155
134-136
95-97
4-Cl-C6H4
2
4-F-C6H4
3-CF3-C6H4
4-OCH3-C6H4
148-151
10
11
1
2
α-Naph
47
74
176-178
188-190
3-Cl-C6H4
12
13
14
15
2
2
4-Cl-C6H4
3-CF3-C6H4
4-Cl-C6H4
3-CF3-C6H4
77
68
31
37
86-89
89-92
3
4
204-208
174-178
Table 2. Yeild and mp of diaryl(2-pyridinylalkoxy-O,N)borons
Fomula No.
Compd No.
n
Ar
Yield (%)
mp (℃)
16
17
1
1
Ph
61
68
150-151
188-192
2-CH3-C6H4
18
19
20
21
22
23
24
25
1
1
1
1
1
1
1
1
3-CH3-C6H4
4-CH3-C6H4
3-Cl-C6H4
57
57
81
71
53
63
37
83
117-119
147-149
105-107
133-134
137-139
92-94
4-Cl-C6H4
4-F-C6H4
3-CF3-C6H4
2-OCH3-C6H4
4-OCH3-C6H4
163-165
129-131
5
26
1
α-Naph
46
233-236
27
28
2
2
Ph
58
87
176-178
208-211
2-CH3-C6H4
29
30
31
32
33
2
2
2
2
2
3-Cl-C6H4
4-Cl-C6H4
78
74
83
65
80
137-139
140-142
118-120
140-142
131-133
4-F-C6H4
3-CF3-C6H4
4-OCH3-C6H4
34
2
α-Naph
52
215-218

BIOLOGICAL P ROP E RTIE S
Ma t er ia ls a n d m et h od
Cells ：Kidn eys wer e obt a in ed from 3- t o 7- da y-old ch icken s u n der st erile con dit ion s.
Aft er r em ovin g t h e t a pet a , n er ves a n d blood vessels, t h e kidn eys wer e cr u sh ed
t h r ou gh a 5 m L syr in ge in ph osph a t e-bu ffer ed sa lin e(P BS) a t 4℃, wa sh ed severa l
t im es wit h P BS a n d t rypsin ized wit h 0.125 % t rypsin solu t ion . Th e cells (CK cell)
wer e filt er ed t h r ou gh a wir e m esh , t h e cell n u m ber cou n t ed wit h a h a em ocyt om et er
a n d su spen ded in growt h m ediu m (m ediu m 199 wit h 5 % fet a l bovin e seru m
pen icillin :100 IU/mL a n d st rept om ycin :100 IU/m L, pH7.2) a t 3～5×105/m L. Th is
cell su spen sion (100 μL) wa s t r a n sferr ed t o 96 fla t -bot t om -well pla t es (Su m it om o
Co.) to un dergo monola yer cell in cuba t ion for 72 h a t 37℃ u n der 5 %CO2/95 % a ir
con dit ion s.
P a ra sit es：Th e NIAH stra in (sta n da r d st ra in ) of E. t en ella wa s supplied by the
Na t ion a l In st it u t e of An im a l Hea lt h . Th e oocyst s were st erilized wit h 10% sodiu m
h ypoch lor ide wh ich wa s t h en r em oved by cen t r ifu ga t ion , a n d t h e oocyst s wa sh ed
t h ree t im es wit h st erilized P BS. Th e oocyst s were re-su spen ded in P BS (pH8.0)
a n d st ored a t 4 ℃ .
Befor e the a ssa y, they were cru shed by sha kin g with
gla ss-bea ds (No.8). Spor ozoit es wer e excyst ed fr om spor ocyst s by sh a kin g for 2 h a t
40℃ in a wa t er ba t h .
Excyst ion flu id : 0.25 % t rypsin (1:250 Difco La b) a n d 10 % ch icken bile in P BS.
Th e sporozoit e su spen sion wa s wa sh ed t wice wit h st erilized P BS, a n d wh ole oocyst s
a n d oocyst wa ll debris wer e r em oved by cen t r ifu ga t ion . Th e pu r e sporozoit e pellet
wa s resu spen ded in m a in t en a n ce m ediu m (m ediu m 199 wit h 1 % F BS, 1 % t rypt ose
ph osph a t e brot h a n d 1 % yea st ext ra ct , pH 7.2). Th e fin a l sporozoit e con cen t r a t ion
wa s a bou t 2.0～3.0x105/m L.
Met h od：Th e solu t ion of t h e com pou n ds in m et h a n ol ( 1000μg/m L ) wa s dilu t ed wit h
t h e fin a l sporozoit e su spen sion ( 2.0～3.0×10５/m L) a t a con cen t ra t ion of 10, 1, 0.1
μg/m L. Th e solu t ion wa s t h en a dded t o t h e CK m on ola yer cells, a n d in cu ba t ed a t
40℃ in 5 % CO2/95 % a ir for 48 h . CK cells st a in ed wit h 2 % Giem sa solu t ion wer e
obser ved m icr oscopica lly.
J u dgm en t ： An t icoccidia l a ct ivit y wa s ju dged by t h e pr esen ce of im m a t u r e a n d
m a t u r e 1st -sch izon t s gr own in t h e CK cell.
An t icoccidia l a ct ivit y
(+) ： 1st -schizont s not found

(±)： A few 1st -sch izon t s fou n d ( >80% in h ibit ion )
Cytopa thogenic effect (CP E)
(+++) ：dea t h of wh ole cells (t h e pr ot ozoa n wa s in visible)
(++) ：severe,
(±～+) ：poor ,
(－)
：no effect
RE SULTS AND DISCUSSION
In vit ro a n t icoccidia l a ct ivit y wa s ju dged by m icroscopic observa t ion of t h e for m a t ion
of 1st sch izon t s a ga in st t h e im por t a n t pa r a sit e, E ．t en ella in t h e cells, a n d t h e
biologica l da t a a re su m m a rized in Ta bles 3 a n d 4. Of a ll t h e com pou n ds (1-34)
tested, com poun ds (6, 8, 14) of the piperidine type, a n d compoun ds (23, 32) of t h e
pyridine type exhibited good a ctivity.
Table 3. Anticoccidial activity of diaryl(piperidinylalkoxy-O,N)borons
Dosage (
/mL )
1 (CPE)
μｇ
Compd No.
10 (CPE)
0.1 (CPE)
1
2
-
-
-
-
(-)
(-)
(-)
(-)
-
-
-
-
-
-
-
-
-
-
-
±
-
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
3
4
5
(+++)
6
+ (+)
7
-
(-)
8
+ (+)
9
-
(-)
(-)
10
11
12
13
14
15
±
(+++)
(+++)
(+++)
(+++)
(+++)
+
-)
（
-
(-)

Table 4. Anticoccidial activity of diaryl(pyridinylalkoxy-O,N)borons
Dosage ( g/ L )
μ ｍ
Compd No.
10 (CPE)
1 (CPE)
0.1 (CPE)
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
-
-
(-)
(-)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
±
-
(-)
(-)
(+++)
(-)
-
-
(-)
+
(+)
(-)
(-)
(-)
(-)
(-)
-
-
±
-
-
(+++)
-
-
(-)
(-)
(+)
(-)
(-)
+
-
±
On t h e ot h er h a n d, com pou n ds (10 a n d 12) of the piperidine type, a n d compoun ds (18
a n d 19) of t h e pyr idin e t ype sh owed m oder a t e a ct ivit y. H owever , com pou n ds (1-5, 7,
9, 11, 13, 15, 16, 17, 20-22, 24, 25, 27-31, 33 a n d 34) h a ve poor a ct ivit y. It is
n ot icea ble t h a t a com pa ra t ively sm a ll ch a n ge in ch em ica l st ru ct u re lea ds t o a
m a r ked in cr ea se in biologica l pot en cy. Th e st r u ct u r e-a ct ivit y r ela t ion sh ip bet ween
the non-a roma t ic piperidine type a nd the a roma t ic pyridine types is inter esting.
Repla cem ent of t he piperidine moiety (compoun ds (6 a n d 12)) of bis-4-ch loroph en yl
der iva t ives wit h t h e pyr idin e m oiet y (com pou n ds (21 a nd 30)) ext in gu ish es t h e
a ct ivit y. On t h e ot h er h a n d, r epla cem en t of t h e piper idin e m oiet y (com pou n d (8)) of
bis-3-t r iflu orom et h yl der iva t ives wit h t h e pyr idin e m oiet y (com pou n d (23)) did not
sign ifica n t ly ch a n ge t h e a ct ivit y.
ACKNOWLE DGE ME NTS
Th e skillfu l t ech n ica l h elp of Mr. H. F u jika wa , a n d t h e va lu a ble discu ssion of Dr. T.
Na ka m u r a , a r e gr a t efu lly a ckn owledged.

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