Structure-activity relationships of 1,3-benzoxazole-4-carbonitriles as novel antifungal agents with potent in vivo efficacy

Article abstract of DOI:10.1248/cpb.59.341

A series of 1,3-benzoxazole-4-carbonitriles was synthesized and evaluated for its antifungal activity, solubility, and metabolic stability. Among those compounds, 4-cyano-N,N,5-trimethyl-7-[(3S)-3-methyl-3-(methylamino) pyrrolidin-1-yl]-6-phenyl-1,3-benzoxazole-2-carboxamide (16b) exhibited potent in vitro activity against Candida species, higher water solubility, and improved metabolic stability compared to lead compound 1. Compound 16b showed potent in vivo efficacy against mice Candida infection models and good bioavailability in rats.

Full text of DOI:10.1248/cpb.59.341

March 2011
Regular Article
Chem. Pharm. Bull. 59(3) 341—352 (2011)
341
Structure–Activity Relationships of 1,3-Benzoxazole-4-carbonitriles as
Novel Antifungal Agents with Potent in Vivo Efficacy
Jun-ichi KUROYANAGI, ,a Kazuo KANAI,a Takao HORIUCHI,a Hiroshi TAKESHITA,a Shozo KOBAYASHI,b
*
a
Issei ACHIWA,a Kumi YOSHIDA,c Koichi NAKAMURA,d and Katsuhiro KAWAKAMI
c
a Lead Discovery & Optimization Research Laboratories II, Daiichi Sankyo Co., Ltd.; Biological Research Laboratories,
Daiichi Sankyo Co., Ltd.; 1–16–13 Kitakasai, Edogawa-ku, Tokyo 134–8630, Japan: b Lead Discovery & Optimization
Research Laboratories I, Daiichi Sankyo Co., Ltd.; and d Drug Metabolism & Pharmacokinetics Research Laboratories,
Daiichi Sankyo Co., Ltd.; 1–2–58 Hiromachi, Shinagawa-ku, Tokyo 140–8710, Japan.
Received November 1, 2010; accepted December 23, 2010; published online January 6, 2011
A series of 1,3-benzoxazole-4-carbonitriles was synthesized and evaluated for its antifungal activity, solu-
bility, and metabolic stability. Among those compounds, 4-cyano-N,N,5-trimethyl-7-[(3S)-3-methyl-3-(methyl-
amino)pyrrolidin-1-yl]-6-phenyl-1,3-benzoxazole-2-carboxamide (16b) exhibited potent in vitro activity against
Candida species, higher water solubility, and improved metabolic stability compared to lead compound 1. Com-
pound 16b showed potent in vivo efficacy against mice Candida infection models and good bioavailability in rats.
Key words 1,3-benzoxazole-4-carbonitrile; antifungal agent; physicochemical property; in vivo efficacy; bioavailability
Increased incidence of invasive mycoses is often related to embarked on an exploration of the structure–activity relation-
the growing use of broad-spectrum antibiotics, immunosup- ships (SARs) of its peripheral analogs, as well as improve-
pressive agents, anti-cancer, and anti-AIDS drugs.1—3) Al- ment of their physicochemical properties.
though antifungal agents, such as polyenes, azoles, and can-
Initial SARs of this type of antifungal agent suggested that
dins, have shown potent efficacy against Candida species the cyano group at the 4-position and the methyl group at the
(spp.) in the treatment of systemic fungal infections, cur- 5-position are indispensable for potent antifungal activ-
rently available drugs could not satisfy the increasing re- ity.11,12) To develop SARs studies, we subdivided lead struc-
quirement for antifungal therapy in complex patient popula- ture 1 into three sections: (A) phenyl ring, (B) aliphatic
tions. This is because of the unfavorable properties, such as group, and (C) amino substituent. Each structural moiety was
side effects, a narrow spectrum, and drug resistance.4—10) modified independently to clarify its function.
These unresolved issues have caused a high rate of mortality
Herein, we would like to report the SARs and physico-
and the spread of antifungal drug resistance. Therefore, new chemical properties of 1,3-benzoxazole analogs focusing on
antifungal agents with a novel mode of action have been ur- the relationship between physicochemical properties and in
gently required to open the possibility of a novel therapeutic vivo activities in mice infection models.
approach.
Chemistry Various 1,3-benzoxazole-4-carbonitrile de-
We have reported the discovery of new antifungal agents rivatives were synthesized from intermediates 4—7, which
possessing 1,3-benzoxazole-4-carbonitrile skeleton with a were prepared from useful hexa-substituted benzenes 2 and 3
novel mode of action inhibiting the synthesis of b-1,6-glu- as starting materials.11) Compound 3 was converted to 1,3-
can, which was known to play a key role in cellular growth benzoxazole intermediates 4 and 7 possessing ethoxycar-
and proliferation of Candida spp.11) Among those 1,3-ben- bonyl or chloro group at the 2-position. 2-Cyclopropane de-
zoxazole analogs, compound 1 showed potent antifungal ac- rivative 5 was synthesized directly from 2, and then con-
tivity against Candida spp. However, in vivo efficacy of 1 has
not been observed in mice Candida albicans infection mod-
els. We considered the lack of in vivo efficacy of our com-
pound was mainly due to its poor physicochemical properties
such as high lipophilicity, low metabolic stability, and poor
water solubility at pH 6.8 as shown in Fig. 1. Therefore, we
Reagents and conditions: (a) PhB(OH)2, Pd(PPh3)4, K3PO4, 1,4-dioxane-H2O, 91%;
(b) triethoxyacetic acid ethyl ester, 96%; (c) cyclopropanecarboxylic acid chloride, i-
Pr2NEt, CH2Cl2; (d) p-TsOH, xylene, 80% (two steps); (e) EtOCS2K, pyridine; SOCl2,
100%.
Fig. 1. Structure of 1
Chart 1. Synthesis of 1,3-Benzoxazole-4-carbonitrile Intermediates
∗ To whom correspondence should be addressed. e-mail: kuroyanagi.junichi.d5@daiichisankyo.co.jp © 2011 Pharmaceutical Society of Japan

342
Vol. 59, No. 3
verted to intermediate 6 (Chart 1).
analog 14 as shown in Chart 5.
As outlined in Chart 6, intermediates 6 and 10b were se-
To evaluate the effect of substituents at the 6-position,
compounds 8a—o were synthesized in a similar way. Cross- lected and converted to amines 15a—e and 16a—c by the
coupling reaction13,14) at the 6-position, followed by incorpo- same manner described in Chart 2. To obtain 16b and 16c,
ration of (3S)-3-(dimethylamino)pyrrolidine into the 7-posi- optically active 3-amino-3-methylpyrrolidines 20 and 23
tion afforded 6-functionalized analogs 8a—o (Chart 2).
To figure out the effects of substituents at the 2-position,
compounds 9 and 11—14 were designed and synthesized.
Analogs 9, 11a, and 11b, bearing 2-carboxylate or amide
moieties, were prepared from 2-ethoxycarbonyl intermediate
4 as shown in Chart 3. Aryl and heteroaryl groups were in-
troduced to intermediates 3 or 7 by condensation reaction or
cross coupling reaction to give 2-substituted analogs 12a—f.
Compounds 12g—j bearing a C–N bond at the 2-position
were also prepared from 7 with corresponding amines (Chart
4). Further steps were required to obtain 2-oxopyrrolidin-1-yl
were prepared as substituents at the 7-position (Chart 7).
Reagents and conditions: (a) RCOCl, Et3N; PPTS, Xylene; (b) (3S)-3-(dimethyl-
amino)pyrrolidine, Et3N, DMSO; (c) RB(OH)2, Pd(PPh3)4, K3PO4, 1,4-dioxane;
(d) n-Bu3SnR, Pd(PPh3)2Cl2, 2,6-di-tert-butylcresol, toluene; (e) amine, Et3N,
CH2Cl2.
Chart 4. Synthesis of 2-Substituted 1,3-Benzoxazole-4-carbonitriles
12a—j
Reagents and conditions: (a) H2N(CH2)3CO2Et, iPr2NEt, CH2Cl2, 99%; (b) (3S)-
3-(dimethylamino)pyrrolidine, Et3N, DMSO, 49%; (c) 1 M NaOH aq., EtOH, 69%;
(d) SOCl2, CH2Cl2; Pyridine, 62%.
Reagents and conditions: (a) n-Bu3SnR, Pd(PPh3)2Cl2, 2,6-di-tert-butylcresol,
toluene; (b) RB(OH)2, Pd(PPh3)4, K3PO4, 1,4-dioxane; (c) (3S)-3-(dimethyl-
amino)pyrrolidine, Et3N, DMSO.
Chart 5. Synthesis of 2-(2-Oxopyrrolidin-1-yl) Derivative 14
Chart 2. Synthesis of 6-Substituted 1,3-Benzoxazole-4-carbonitriles
Reagents and conditions: (a) diamine, Et3N, DMSO (b) TFA, CH2Cl2.
Reagents and conditions: (a) (3S)-3-(dimethylamino)pyrrolidine, Et3N, DMSO;
(b) amine, Me3Al, CH2Cl2.
Chart 6. Synthesis of 7-Substituted 1,3-Benzoxazole-4-carbonitriles 15
and 16
Chart 3. Synthesis of 2-Substituted 1,3-Benzoxazole-4-carbonitriles

March 2011
343
Results and Discussion
from 0.032 to 0.125 mg/ml, while the corresponding pyridin-
Prepared 1,3-benzoxazoles 8—9, 11—12, and 14—16, as 2-yl 8b and pyridin-4-yl 8c demonstrated MIC-1 ranging
well as 1 and fluconazole (FLCZ) as positive control agents from 1 to ꢀ4 mg/ml, respectively. Among the five-membered
were evaluated for their in vitro antifungal activity against heteroaromatic derivatives, thienyl derivatives 8f and 8g
FLCZ-susceptible Candida albicans ATCC 90028 (C. albi- showed a broad range of activity with MIC-1s ranging from
cans-s), FLCZ-resistant Candida albicans ATCC MYA-573 0.032 to 0.5 mg/ml. Vinyl derivative 8h was less active than
(C. albicans-r), Candida glabrata ATCC 48435 (C. 8a. These results indicate that the phenyl group is considered
glabrata), Candida tropicalis ATCC 44508 (C. tropicalis), to be the best substituent at the 6-position showing potent an-
and Candida krusei ATCC 44507 (C. krusei). Minimum in- tifungal activity. To investigate substituent effects on the
hibitory concentration MIC-1 (the lowest drug concentration phenyl group, we prepared various substituted phenyl deriva-
showing 80% growth inhibition compared to the control tives 8i—o. Although 2- and 4-fluorophenyl derivatives 8i
without drug) was determined for each compound by using and 8k had only a moderate effect, corresponding 3-fluo-
the microdilution method previously described.11)
rophenyl analog 8j was found to be as potent as 8a. Replac-
MIC-1s of the 6-substituted compounds 8a—o were sum- ing the 3-fluoro group with polar functionalities was not well
marized in Table 1. Phenyl derivative 8a11) showed high and tolerated, resulting in a two- to five-fold decrease in antifun-
broad activity against Candida spp. with MIC-1 ranging gal activities against C. albicans (derivatives 8l—o). These
results indicated that substituents on the benzene ring at the
6-position had no profound effect on antifungal activity in
comparison to the unsubstituted analog 8a.
Substituents at the 2-position influenced the antifungal ac-
tivity as shown in Table 2. Among the 2-carbonyl derivatives,
dimethylcarbamoyl analog 11b demonstrated potent growth
inhibition against Candida spp., which suggested that polar
groups were tolerated for antifungal activity as well as
aliphatic substituents such as tert-butyl and cyclopropyl
group. On the other hand, when the cyclopropyl group in 8a
was replaced by phenyl group, the inhibitory activity de-
creased significantly (12a). Interestingly, pyridin-2-yl analog
12b exhibited moderate inhibition against C. albicans
(FLCZ-susceptible and -resistant) and C. glabrata (MIC-
Reagents and conditions: (a) diphenylphosphoryl azide, Et3N, toluene; tBuOH, 60%;
(b) BH3·THF, THF, 85%; (c) H2, 20% Pd(OH)2, EtOH; (d) CbzCl, NaHCO3,
Et2O–H2O, 100%; (e) MeI, NaH, DMF, 88%.
Chart 7. Synthesis of Pyrrolidine Derivatives 20 and 23
1ꢁ0.25, 0.25, 0.032 mg/ml, respectively), while correspon-
Table 1. In Vitro Activity of Compounds 8a—o
MIC-1 (mg/ml)a)
Compd.
R6
C. albicans-sb)
C. albicans-rc)
C. glabrata
C. tropicalis
C. krusei
ATCC 90028
ATCC MYA-573
ATCC 48435
ATCC 44508
ATCC 44507
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
Ph
0.063
4
ꢀ4
0.5
0.063
4
ꢀ4
1
0.032
1
ꢀ4
0.032
2
ꢀ4
0.25
0.5
0.063
0.063
0.5
0.125
ꢀ4
ꢀ4
0.5
2
0.25
0.5
1
Pyridin-2-yl
Pyridin-4-yl
1-Me-1H-pyrrol-2-yl
2-Furyl
2-Thienyl
3-Thienyl
Vinyl
2-F-Ph
3-F-Ph
4-F-Ph
0.5
2
2
0.25
0.063
0.032
0.25
0.063
0.032
2
0.25
0.25
2
0.25
0.063
16
2
0.5
0.25
0.25
0.25
0.25
2
0.25
0.125
16
4
1
0.5
0.5
0.125
ꢂ0.016
4
0.5
0.063
ꢀ16
2
3-NH2-Ph
3-(CH2OH)-Ph
3-OMe-Ph
3-CN-Ph
1
1
0.25
0.125
0.25
0.25
0.125
0.125
0.25
0.25
0.25
1
—
—
0.063
0.25
0.125
ꢀ128
0.063
16
0.032
0.5
2
32
FLCZ
a) MIC-1s (in micrograms per milliliter) were determined by using the microdilution method. b) FLCZ-susceptible C. albicans. c) FLCZ-resistant C. albicans.

344
Vol. 59, No. 3
Table 2. In Vitro Activity of Compounds 9, 11, 12, and 14
MIC-1 (mg/ml)a)
Compd.
R2
C. albicans-sb)
C. albicans-rc)
C. glabrata
C. tropicalis
C. krusei
ATCC 90028
ATCC MYA-573
ATCC 48435
ATCC 44508
ATCC 44507
9
CO2Et
CONHMe
CONMe2
ꢀ4
ꢀ4
0.25
0.063
ꢀ4
0.25
1
0.125
0.032
0.016
1
0.032
0.125
8
0.063
0.032
0.063
0.063
0.032
0.125
0.063
1
ꢀ4
1
11a
11b
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
14
0.25
0.063
ꢀ4
0.25
2
ꢀ16
0.25
0.25
0.25
0.125
0.125
1
0.063
0.016
2
0.125
0.25
8
0.063
0.063
0.063
0.063
0.063
0.25
0.125
0.125
ꢀ4
ꢀ4
ꢀ16
ꢀ16
4
ꢀ4
0.125
1
Ph
Pyridin-2-yl
Pyridin-3-yl
Pyridin-4-yl
1-Me-1H-pyrrol-2-yl
2-Furyl
NHMe
NMe2
NEt2
16
0.125
0.25
0.25
0.5
0.25
1
0.25
ꢀ4
4
Pyrrolidin-1-yl
2-Oxo-pyrrolidin-1-yl
0.25
0.5
1
tert-Butyl
—
0.063
0.25
0.125
ꢀ128
0.063
16
0.032
0.5
2
32
FLCZ
a) MIC-1s (in micrograms per milliliter) were determined by using the microdilution method. b) FLCZ-susceptible C. albicans. c) FLCZ-resistant C. albicans.
ding pyridin-3-yl and pyridin-4-yl derivatives 12c and 12d binding pocket or interaction with another binding site. How-
resulted in a loss of activity. Five-membered heteroaromatic ever, we anticipated that the conversion of the NMe2 group
analogs 12e and 12f showed activity similar to the pyridin-2- into the NHMe structure could improve aqueous solubility
yl analog 12b. Alkylamino analogs 12g—i showed moderate and metabolic stability. To make full use of the physicochem-
activities, while pyrrolidin-1-yl 12j had only a small effect. ically profitable moiety, we decided to retain the NHMe and
Interestingly, 2-oxopyrrolidin-1-yl analog 14 exhibited en- to seek other ways of enhancing in vitro activity. Therefore,
hanced activity, which indicates that the carbonyl group at we designed 3-methyl-3-(methylamino)pyrrolidin-1-yl to
this position has significant effect. These facts imply that the cover the unfavorable N–H moiety by the incorporation of
small aliphatic groups with carbon atoms up to four, such as 3-methyl group adjacent to the NHMe group. As a result,
tert-butyl and cyclopropyl, or polar functionalities with a (3S)-3-methyl-3-(methylamino)pyrrolidin-1-yl analog 16b
particular combination of carbon, nitrogen, and oxygen demonstrated potent activity against Candida spp. including
atoms, such as alkylcarbamoyl, alkylamino, and 2-oxopy- C. krusei in spite of the existence of NHMe moiety on the
rrolidon-1-yl seem to be favorable.
pyrrolidine ring. These results suggest that a slight modifica-
In the view of antifungal activities summarized in Tables 1 tion of 3-aminopyrrolidin-1-yl group at the 7-position influ-
and 2, we focused on the basic functionality at the 7-position ences in vitro activity including both the potency and the
for further optimization by means of 2-cyclopropyl and 2- spectrum.
CONMe2 analogs 8a and 11b (Table 3). Six-membered hete-
To investigate the relationship between the structure and
rocycles and ethylenediamine structure at the 7-position re- physicochemical properties of the obtained compounds, we
sulted in diminished or complete loss of in vitro activity, chose potent antifungal analogs 8j, 15a, 16a, and 16b. Their
whereas pyrrolidine groups showed high potency. Based on lipophilicity, water solubility, and metabolic stability were
the hypothesis that the methyl group in the (dimethylamino)- summarized in Table 4. The initial focus of the optimization
pyrrolidinyl analog 8a was easily metabolized to a corre- program was aimed at modifications of amine at the 7-posi-
sponding mono-methyl derivative, we prepared mono-methyl tion designed to increase the metabolic stability. Although
15a. Unfortunately, replacement of NMe2 into NHMe dimin- antifungal activity was decreased, water solubility at pH 6.8
ished antifungal activities. Similar results were obtained and metabolic stability were significantly improved by re-
when 2-dimethylcarbamoyl 16a was evaluated. N-Demethyl- placing (dimethylamino)pyrrolidin-1-yl with (methylamino)-
ation of the terminal amine at the 7-positon resulted in a pyrrolidin-1-yl at the 7-position. For example, water solubili-
slight loss of in vitro potency, suggesting that the basic center ties of 8j and 15a at pH 6.8 were 17 and 610 mg/ml, whereas
such as NHMe and NH2 was not appropriate for antifungal metabolic stabilities of 8j and 15a for mouse microsomes
activity. We considered the loss of activity was caused by the were 4 and 70%, respectively. It is well known that dimethyl-
existence of proton on the nitrogen atom, which might inter- amino structure NMe2 is easily metabolized and that low
fere with the binding of the amine center to the appropriate lipophilicity could improve physicochemical property. Thus,

March 2011
345
Table 3. In Vitro Activity of Compounds 15 and 16
MIC-1 (mg/ml)a)
Compd.
R2
R7
C. albicans-sb)
C. albicans-rc)
C. glabrata
C. tropicalis
C. krusei
ATCC 90028
ATCC MYA-573
ATCC 48435
ATCC 44508
ATCC 44507
8a
15a
15b
15c
15d
15e
11b
16a
16b
16c
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
CONMe2
CONMe2
CONMe2
CONMe2
A
B
C
D
E
0.063
0.25
ꢀ16
ꢀ16
ꢀ16
16
0.063
0.25
ꢀ16
ꢀ16
ꢀ16
8
0.032
0.063
ꢀ16
ꢀ16
ꢀ16
0.032
0.125
ꢀ16
ꢀ16
ꢀ16
0.125
1
ꢀ16
ꢀ16
ꢀ16
8
F
4
4
A
B
G
H
0.063
0.25
0.063
0.5
0.063
0.25
0.063
0.5
0.016
0.032
0.016
0.063
0.016
0.063
0.032
0.125
0.125
2
0.25
8
1
tert-Butyl
A
—
0.063
0.25
0.125
ꢀ128
0.063
16
0.032
0.5
2
32
FLCZ
—
a) MIC-1s (in micrograms per milliliter) were determined by using the microdilution method. b) FLCZ-susceptible C. albicans. c) FLCZ-resistant C. albicans.
Table 4. Physicochemical Properties of Selected Compounds
MIC-1 (mg/ml)
C. albicans-s
ATCC 90028
Solubility (mg/ml)b)
Metabolic stability (% remaining)c)
Log Da)
(pHꢁ7.4)
Compd.
pH 4
pH 6.8
Mouse
Rat
Human
8j
0.063
0.25
0.25
3.9
2.9
1.9
2
780
610
760
820
17
610
690
810
4
70
66
0
45
87
76
7
55
80
71
15a
16a
16b
0.063
100
1
0.063
4.6
710
5
1
NTd)
6
a) Log D values were determined from the partition coefficient for 1-octanol/phosphate buffer saline (PBS) at pH 7.4. b) Water solubility was measured at pH 4 and 6.8.
c) Metabolic stability for mouse, rat, and human microsomes. d) Not tested.
we considered the improvement of the physicochemical lected as test compounds for acute systemic infection models
properties of our products was caused by the modification of in mice to evaluate the relationship between physicochemical
the terminal alkylamino moiety at the 7-position and the con- properties and in vivo efficacy. Each compound was subcuta-
sequent reduced lipophilicity. The second approach was the neously administrated at a dose of 20 and 40 mg/kg at 0 and
introduction of CONMe2 at the 2-position as a low lipophilic 6 h after infection of C. albicans ATCC 90028. The in vivo
moiety. Log D value was decreased by the introduction of the efficacy was evaluated by survival rates 8 d after infection.
CONMe2 group at the 2-position (Log Dꢁ2.9 and 1.9 for As a result, no effect was observed for 8j at a dose of
15a and 16a, respectively). Water solubility and metabolic 20 mg/kg, whereas slightly prolonged survival days were ob-
stability of 16a were somewhat improved compared to the 2- served at a dose of 40 mg/kg (Fig. 2). On the other hand, 16b
cyclopropyl analog 15a. Surprisingly, compound 16b demon- demonstrated apparently enhanced in vivo efficacy compared
strated the most potent antifungal activity and improved to 8j in a dose-dependent manner, indicated by a survival rate
physicochemical properties. Although 16b possessed an ad- of 90% at the end of day eight (Fig. 3). These results suggest
ditional methyl group on the pyrrolidin-1-yl at the 7-position, that the in vivo efficacies of our compounds are strongly in-
the Log D value was similar to that of demethyl analog 16a fluenced by their physicochemical properties.
(Log Dꢁ1.9 and 2 for 16a and 16b, respectively). Based on
Potent in vitro activities against C. grabrata and C. krusei
the results obtained above, the CONMe2 group at the 2-posi- were regarded as another attractive feature of our com-
tion and the (3S)-3-methyl-3-(methylamino)pyrrolidin-1-yl pounds, whereas reference compound FLCZ showed little
group at the 7-position were regarded as effective in both potency against those Candida spp. In acute systemic infec-
physicochemical properties and antifungal activity.
tion models in mice infected with C. glabrata, 16b demon-
The most potent antifungal agents 8j and 16b were se- strated excellent efficacy indicated by 100% survival at a

346
Vol. 59, No. 3
bioavailability of 16b in rats was evaluated as 68% suggest-
ing the possibility of 16b as an orally and intravenously ac-
tive agent. Thus, we anticipated that 16b could be adminis-
trated both intravenously and orally in clinical therapy.
Conclusion
In summary, we have discovered a series of 1,3-benzoxa-
zole-4-carbonitrile derivatives with potent activity against
Candida spp. Of these, 16b exhibited potent in vitro activity,
water solubility, and improved metabolic stability as a poten-
tial preclinical candidate. Combination of antifungal activi-
ties and physicochemical properties was accomplished by
introducing the hydrophilic CONMe2 and the metabolically
stable (3S)-3-methyl-3-(methylamino)pyrrolidin-1-yl groups
at the 2- and 7-positions, respectively. Compound 16b
demonstrated potent in vivo efficacy against mice Candida
infection models and good bioavailability in rats.
Fig. 2. In Vivo Efficacy of 8j by Subcutaneous Infusion in a Model of C.
albicans ATCC 90028 in Mice
Experimental
Chemistry Unless otherwise noted, materials were obtained from com-
mercial suppliers and used without further purification. Melting points were
taken on a Yanako MP-500D melting point apparatus and are uncorrected.
Optical rotations were measured in a 0.5-dm cell at 25 °C at 589 nm with a
HORIBA SEPA-300 polarimeter. 1H-NMR spectra were determined on a
JEOL JNM-EX400 spectrometer. 13C-NMR spectra were determined on a
JEOL JNM-ECP500 spectrometer. Chemical shifts are reported in parts per
1
million relative to tetramethylsilane as an internal standard. Significant H-
NMR data are tabulated in the following order: number of protons, multi-
plicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad),
and coupling constant(s) in hertz. Infrared (IR) spectra were obtained on a
HORIBA FT-720 spectrometer or a JASCO FT/IR-6100 typeA. High-resolu-
tion mass spectra were obtained on a JEOL JMS-700 mass spectrometer
under electron impact ionization conditions (EI), electron spray ionization
conditions (ESI) or fast atom bombardment ionization conditions (FAB).
Column chromatography refers to flash column chromatography conducted
on Merck silica gel 60, 230—400 mesh ASTM. Thin-layer chromatography
(TLC) was performed with Merck silica gel 60 F254 TLC plates, and com-
pound visualization was effected with a 5% solution of molybdophosphoric
acid in ethanol, UV-lamp, iodine, or Wako Ninhydrin Spray.
Fig. 3. In Vivo Efficacy of 16b by Subcutaneous Infusion in a Model of C.
albicans ATCC 90028 in Mice
Ethyl 4-Cyano-7-fluoro-5-methyl-6-phenyl-1,3-benzoxazole-2-car-
boxylate (4) A mixture of 311) (5.0 g, 20.64 mmol) and ethyl triethoxyac-
etate (18.2 g, 82.6 mmol) was stirred at 100 °C for 23 h. n-Hexane was added
and the resulting precipitate was collected by filtration to afford the title
compound (6.39 g, 96%) as a pale brown solid. mp: 159—161 °C. HR-MS
(EI) m/z: 324.0908 (Calcd for C18H13FN2O3 324.0911). 1H-NMR (CDCl3) d:
1.52 (3H, t, Jꢁ7.1 Hz), 2.48 (3H, s), 4.60 (2H, q, Jꢁ7.12 Hz), 7.25—7.29
(2H, m), 7.48—7.56 (3H, m). 13C-NMR (CDCl3) d: 14.1, 19.6 (d,
Jꢁ1.9 Hz), 64.0, 102.1 (d, Jꢁ4.8 Hz), 113.9, 129.0 (2C), 129.0, 129.6 (2C),
130.6 (d, Jꢁ13.4 Hz), 131.8, 137.1 (d, Jꢁ13.4 Hz), 142.4 (d, Jꢁ1.9 Hz),
143.8 (d, Jꢁ2.9 Hz), 145.9, 147.9, 155.0 (d, Jꢁ76.8 Hz). IR (ATR): 2229,
1740, 1547, 1475, 1288, 1182, 1157, 1124, 1011, 849, 779, 727, 702 cmꢃ1
.
Anal. Calcd for C18H13FN2O3·0.25H2O: C, 65.75; H, 4.14; N, 8.52; F, 5.78.
Found: C, 65.99; H, 3.96; N, 8.57; F, 5.78.
Fig. 4. In Vivo Efficacy of 16b by Subcutaneous Infusion in a Model of C.
glabrata ATCC 48435 in Mice
6-Bromo-2-cyclopropyl-7-fluoro-5-methyl-1,3-benzoxazole-4-carboni-
trile (5) To a solution of 211) (7.86 g, 32.1 mmol) and N,N-diisopropylethyl-
amine (19.5 ml, 112 mmol) in AcOEt (300 ml) was added cyclopropanecar-
bonyl chloride (4.4 ml, 48.1 mmol) at 0 °C and the mixture was stirred for
16 h at room temperature. AcOEt was added and the organic layer was
washed with water and brine, dried over Na2SO4, filtered, and concentrated
in vacuo. To a solution of the residue in toluene (80 ml) was added p-tolue-
nesulfonic acid monohydrate (1.60 g), and the mixture was refluxed for 19 h.
After cooling to room temperature, the mixture was diluted with AcOEt and
filtrated. The precipitate was recrystallized from AcOEt to give the title
compound (6.02 g, 80%) as a pale red solid. mp: 164—168 °C. MS (ESI)
m/z: 295, 297 (Mꢄ1)ꢄ. 1H-NMR (CDCl3) d: 1.27—1.34 (2H, m), 1.38—
1.42 (2H, m), 2.25—2.32 (1H, m), 2.73 (3H, s). 13C-NMR (DMSO-d6) d:
8.9, 10.5 (2C), 21.3, 98.3, 106.9 (d, Jꢁ17.3 Hz), 114.2, 135.5 (d,
Jꢁ10.6 Hz), 139.3, 145.1 (d, Jꢁ196.7 Hz), 146.3, 172.5. IR (ATR): 3057,
2230, 1625, 1567, 1317, 1131, 1034 cmꢃ1. Anal. Calcd for C12H8BrFN2O:
Table 5. Pharmacokinetic Parameters of 16b in Mice and Rats
Dose
(mg/kg) (ng·h/ml)
AUC
T1/2
(h)
BAc)
(%)
Species
Administration
s.c.
Mice
Rats
10
5578a)
2.9
i.v.
p.o.
5
5
1311b)
3.2
4.8
647b)
68
a) AUC0—24 h
.
b) AUC0—6 h
.
c) Bioavailability calculated from the AUC ratio.
dose of 3.3 mg/kg in eight days after infection (Fig. 4).
Pharmacokinetic profiles of 16b were investigated for
mice and rats as shown in Table 5. It was noteworthy that a C, 48.84; H, 2.73; N, 9.49; F, 6.44. Found: C, 49.02; H, 2.75; N, 9.57; F,

March 2011
6.55.
347
(6H, s), 2.16 (3H, s), 2.19—2.30 (1H, m), 2.51 (1H, br s), 2.60—3.00 (1H,
2-Cyclopropyl-7-fluoro-5-methyl-6-phenyl-1,3-benzoxazole-4-carboni- m), 3.29—3.47 (3H, m), 7.27 (2H, ddd, Jꢁ0.7, 4.9, 7.3 Hz), 7.73 (1H, t,
trile (6) A mixture of 5 (300 mg, 1.02 mmol), phenylboronic acid (256 mg, Jꢁ7.0 Hz), 8.71 (1H, d, Jꢁ4.2 Hz). 13C-NMR (CDCl3) d: 9.4, 9.5, 9.6, 19.3,
2.03 mmol), potassium phosphate tribasic (432 mg, 2.03 mmol), and 30.7, 44.3 (2C), 51.0, 56.8, 65.4, 91.4, 117.0, 122.3, 124.0, 126.8, 127.4,
tetrakis(triphenylphosphine)palladium(0) (118 mg, 0.10 mmol) in 1,4-diox- 136.6, 139.0, 139.9, 144.6, 149.4, 158.9, 169.7. IR (ATR): 2204, 1583,
ane (6 ml) was stirred at 100 °C for 17 h under nitrogen atmosphere. The 1473 cmꢃ1. Anal. Calcd for C23H25N5O: C, 71.29; H, 6.50; N, 18.07. Found:
mixture was cooled to room temperature. Satd NH4Cl aq. was added and the C, 71.26; H, 6.48; N, 17.98. [a]D25 ꢄ70.9 (cꢁ1.07, CHCl3).
resultant mixture was extracted with AcOEt. The obtained organic layers
2-Cyclopropyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-5-methyl-6-
were washed with brine, dried over Na2SO4, filtered, and concentrated in pyridin-4-yl-1,3-benzoxazole-4-carbonitrile (8c) Following the proce-
vacuo. The residue was purified by silica gel column chromatography elut- dure as described for 8b, the title compound was prepared in 45% (2 steps)
ing with n-hexane/AcOEtꢁ8/1, v/v to afford the title compound (275 mg, from 5 and 4-(tributylstannyl)pyridine as a white solid. mp: 160—161 °C.
1
92%) as a white solid. MS (ESI) m/z: 293 (Mꢄ1)ꢄ. 1H-NMR (CDCl3) d: MS (ESI) m/z: 388 (Mꢄ1)ꢄ. H-NMR (DMSO-d6) d: 1.16—1.26 (4H, m),
1.26—2.01 (2H, m), 2.38—2.42 (2H, m), 2.28—2.34 (1H, m), 2.40 (3H, s), 1.49—1.59 (1H, m), 1.85—1.92 (1H, m), 2.01 (6H, s), 2.09 (3H, s), 2.28—
7.21—7.26 (2H, m), 7.42—7.51 (3H, m). 13C-NMR (CDCl3) d: 9.5, 10.4 2.35 (1H, m), 2.51—2.60 (1H, m), 2.84 (1H, dd, Jꢁ8.0, 9.5 Hz), 3.00—3.35
(2C), 19.1 (d, Jꢁ1.9 Hz), 99.1 (d, Jꢁ3.8 Hz), 115.0, 126.8 (d, Jꢁ14.4 Hz), (3H, m), 7.21 (1H, br s), 7.35 (1H, br s), 8.63 (2H, d, Jꢁ5.1 Hz). 13C-NMR
128.5, 128.8 (2C), 130.0 (2C), 132.7, 136.1 (d, Jꢁ13.4 Hz), 139.3, 146.3 (d, (CDCl3) d: 9.4, 9.7, 9.8, 20.0, 30.3, 44.2 (2C), 51.3, 56.6, 65.3, 92.4, 116.7,
Jꢁ3.8 Hz), 146.3 (d, Jꢁ258.2 Hz), 172.0. IR (ATR): 2221, 1562, 1412, 123.2, 126.2, 126.6, 136.2, 138.3, 139.7, 144.7, 148.4, 149.7, 150.3, 170.0.
1321, 1122, 1026, 722 cmꢃ1. Anal. Calcd for C18H13FN2O: C, 73.96; H, IR (ATR): 2206, 1587, 1405 cmꢃ1. Anal. Calcd for C23H25N5O·0.5H2O: C,
4.48; N, 9.58; F, 6.50. Found: C, 73.65; H, 4.47; N, 9.54; F, 6.23.
2-Chloro-7-fluoro-5-methyl-6-phenyl-1,3-benzoxazole-4-carbonitrile (cꢁ1.01, CHCl3).
69.67; H, 6.61; N, 17.66. Found: C, 70.05; H, 6.46; N, 17.62. [a]D25 ꢄ79.2
(7) A mixture of 3 (500 mg, 2.06 mmol) and ethylxantic acid potassium
2-Cyclopropyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-5-methyl-6-
salt (1.00 g, 6.24 mmol) in pyridine (25 ml) was refluxed for 3 h. The reac- (1-methyl-1H-pyrrol-2-yl)-1,3-benzoxazole-4-carbonitrile (8d) Follow-
tion mixture was concentrated in vacuo and the residue was dissolved with ing the procedure as described for 8b, the title compound was prepared in
AcOEt. The organic layer was washed with 1 M HCl aq., water, and brine, 40% (2 steps) from 5 and 1-methyl-2-(tributylstannyl)pyrrole as a white
dried over Na2SO4, filtered, and concentrated in vacuo. A mixture of the solid. mp: 127—128 °C. MS (ESI) m/z: 390 (Mꢄ1)ꢄ. 1H-NMR (DMSO-d6)
residue and thionyl chloride (12 ml) was stirred for 1 h at 70 °C. The reaction d: 1.15—1.24 (4H, m), 1.53—1.62 (1H, m), 1.87—1.95 (1H, m), 2.05 (1H,
mixture was concentrated in vacuo and diluted with CH2Cl2. The organic s), 2.06 (3H, s), 2.07 (3H, s), 2.12 (1H, s), 2.26—2.33 (1H, m), 2.52—2.57
layer was washed with 1 M NaOH aq., water, and brine, and then dried over (1H, m), 2.65 (0.5H, dd, Jꢁ7.8, 10.0 Hz), 2.95 (0.5H, dd, Jꢁ7.8, 10.2 Hz),
Na2SO4, filtered, and concentrated in vacuo. The residue was purified by sil- 3.07—3.12 (1H, m), 3.15—3.20 (1H, m), 3.21 (1.5H, s), 3.28 (1.5H, s),
ica gel column chromatography eluting with CH2Cl2 to afford the title com- 3.31—3.40 (1.5H, m), 3.49—3.56 (0.5H, m), 5.87 (0.5H, dd, Jꢁ2.0,
pound (560 mg, 95%) as a yellow solid. mp: 120—122 °C. MS (ESI) m/z: 3.7 Hz), 5.95 (0.5H, dd, Jꢁ2.0, 3.7 Hz), 6.08 (0.5H, t, Jꢁ2.9 Hz), 6.10
1
287, 289 (Mꢄ1)ꢄ. H-NMR (CDCl3) d: 2.44 (3H, s), 7.20—7.27 (2H, m), (0.5H, t, Jꢁ2.9 Hz), 6.81 (0.5H, dd, Jꢁ2.2, 2.7 Hz), 6.85 (0.5H, t, Jꢁ1.7,
7.45—7.54 (3H, m). 13C-NMR (CDCl3) d: 19.4 (d, Jꢁ2.3 Hz), 100.1 (d, 2.4 Hz). IR (ATR): 2206, 1608, 1468 cmꢃ1. Anal. Calcd for C23H27N5O: C,
Jꢁ3.8 Hz), 114.0, 128.6 (d, Jꢁ13.8 Hz), 128.9, 128.9 (2C), 129.7 (2C), 70.92; H, 6.99; N, 17.98. Found: C, 70.80; H, 7.00; N, 17.95. [a]D25 ꢄ106
131.9, 137.4 (d, Jꢁ13.8 Hz), 141.0 (d, Jꢁ2.3 Hz), 145.9 (d, Jꢁ259.8 Hz), (cꢁ1.01, CHCl3).
144.7 (d, Jꢁ3.1 Hz), 153.9. IR (ATR): 2232, 1632, 1517, 1116 cmꢃ1. Anal.
2-Cyclopropyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-6-(2-furyl)-
Calcd for C15H8ClFN2O: C, 62.84; H, 2.81; N, 9.77; Cl, 12.37, F, 6.63. 5-methyl-1,3-benzoxazole-4-carbonitrile (8e) Following the procedure as
Found: C, 62.61; H, 2.81; N, 9.78; Cl, 12.56; F, 6.73. described for 8b, the title compound was prepared in 29% (2 steps) from 5
2-Cyclopropyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-5-methyl-6- and 2-(tributylstannyl)furan as a white solid. mp: 158—159 °C. HR-MS
phenyl-1,3-benzoxazole-4-carbonitrile (8a) To a solution of 6 (70 mg, (ESI) m/z: 377.1936 (Calcd for C22H24N4O2ꢄH 377.1978). 1H-NMR
0.24 mmol) in dimethyl sulfoxide (DMSO) (2 ml) were added triethylamine (DMSO-d6) d: 1.12—1.26 (4H, m), 1.64—1.75 (1H, m), 2.02—2.07 (1H,
(50 ml, 0.36 mmol) and (3S)-3-(dimethylamino)pyrrolidine (40 ml, 0.31 m), 2.20 (6H, s), 2.22 (3H, s), 2.20—2.25 (1H, m), 2.55—2.57 (1H, m), 3.05
mmol), and then the mixture was stirred at 90 °C for 14.5 h. AcOEt was (1H, t, Jꢁ9.4 Hz), 3.38 (1H, dd, Jꢁ7.3, 10.3 Hz), 3.46 (1H, td, Jꢁ5.9,
added and the mixture was washed with brine, dried over Na2SO4, filtered, 10.7 Hz), 3.56 (1H, dd, Jꢁ8.8, 10.3 Hz), 6.23 (1H, d, Jꢁ3.2 Hz), 6.48 (1H,
and concentrated in vacuo. The residue was purified by silica gel column dd, Jꢁ2.0, 3.2 Hz), 7.53 (1H, s). IR (ATR): 2208, 1606, 1583, 1473,
chromatography eluting with CHCl3/MeOHꢁ97/3, v/v to afford the title 1149 cmꢃ1. [a]D25 ꢄ93.6 (cꢁ0.10, CHCl3).
1
compound (64 mg, 69%) as a white solid. MS (ESI) m/z: 387 (Mꢄ1)ꢄ. H-
2-Cyclopropyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-5-methyl-6-
NMR (CDCl3) d: 1.17—2.30 (4H, m), 1.55—1.67 (1H, m), 1.90—1.98 (1H, (2-thienyl)-1,3-benzoxazole-4-carbonitrile (8f) Following the procedure
m), 2.14 (6H, s), 2.17 (3H, s), 2.24 (1H, ddd, Jꢁ5.1, 8.5, 13.0 Hz), 2.48— as described for 8b, the title compound was prepared in 78% (2 steps) from
2.57 (1H, m), 2.94 (1H, t, Jꢁ9.3 Hz), 3.22—3.39 (3H, m), 7.10 (1H, d, 5 and 2-(tributylstannyl)thiophene as a pale brown solid. MS (ESI) m/z: 393
Jꢁ7.6 Hz), 7.23 (1H, d, Jꢁ7.6 Hz), 7.31—7.42 (3H, m). 13C-NMR (CDCl3) (Mꢄ1)ꢄ. 1H-NMR (CDCl3) d: 1.17—1.30 (4H, m), 1.60—1.71 (1H, m),
d: 9.4 (2C), 9.6, 20.1, 30.3, 44.1 (2C), 50.9, 56.3, 65.4, 91.6, 117.2, 125.9, 1.96—2.03 (1H, m), 2.18 (6H, s), 2.19—2.28 (1H, m), 2.29 (3H, s), 2.51—
127.4, 128.2, 128.6, 131.0, 131.5, 136.5, 139.2, 139.5, 139.6, 144.2, 169.4. 2.60 (1H, m), 3.08 (1H, t, Jꢁ9.5 Hz), 3.39 (1H, dt, Jꢁ6.6, 10.5 Hz), 3.44—
IR (ATR): 2206, 1585, 1560, 1466, 1152, 712, 702 cmꢃ1. Anal. Calcd for 3.73 (2H, m), 6.88 (1H, dd, Jꢁ1.2, 3.4 Hz), 7.07 (1H, dd, Jꢁ3.4, 5.1 Hz),
C24H26N4O·0.25H2O: C, 73.72; H, 6.83; N, 14.33. Found: C, 74.08; H, 6.76; 7.40 (1H, dd, Jꢁ1.2, 5.1 Hz). 13C-NMR (CDCl3) d: 9.4, 9.5, 9.6, 20.0, 30.4,
N, 14.35. [a]D25 ꢄ97.2 (cꢁ0.746, CHCl3).
44.3 (2C), 50.7, 55.8, 65.3, 91.5, 116.6, 117.0, 126.8, 127.0, 129.7, 138.0,
2-Cyclopropyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-5-methyl-6- 139.3, 140.1, 141.6, 144.7, 169.7. IR (ATR): 2206, 1604, 1585, 1556, 1468,
pyridin-2-yl-1,3-benzoxazole-4-carbonitrile (8b) A mixture of 5 (200 1437, 1392, 1362, 1176, 1151, 852, 706 cmꢃ1
Anal. Calcd for
.
mg, 0.68 mmol), 2-(tributylstannyl)pyridine (299 mg, 0.81 mmol), 2,6-di- C22H24N4OS·0.25H2O: C, 66.55; H, 6.22; N, 14.11; S, 8.08. Found: C,
tert-butyl-4-methylphenol (2 mg), and bis(triphenylphosphine)palladium(II) 66.66; H, 6.08; N, 14.18; S, 8.25. [a]D25 ꢄ93.4 (cꢁ1.02, CHCl3).
dichloride (24 mg, 0.03 mmol) in toluene (4 ml) was refluxed for 17 h. The
2-Cyclopropyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-5-methyl-6-
reaction mixture was filtered, and the filtrate was concentrated in vacuo. The (3-thienyl)-1,3-benzoxazole-4-carbonitrile (8g) Following the procedure
residue was roughly purified by silica gel column chromatography eluting as described for 6 and 8a, the title compound was prepared in 42% (2 steps)
with n-hexane/AcOEtꢁ3/1, v/v to afford 2-cyclopropyl-7-fluoro-5-methyl- from 5 and thiophene-3-boronic acid as a pale brown solid. MS (ESI) m/z:
6-pyridin-2-yl-1,3-benzoxazole-4-carbonitrile (184 mg) as a pale yellow oil. 393 (Mꢄ1)ꢄ. 1H-NMR (CDCl3) d: 1.17—1.30 (4H, m), 1.58—1.69 (1H,
A mixture of the oil obtained above (184 mg), triethylamine (219 ml, 1.56 m), 1.95—2.02 (1H, m), 2.17 (6H, s), 2.20—2.27 (1H, m), 2.22 (3H, s),
mmol), and (3S)-(dimethylamino)pyrrolidine (103 ml, 0.81 mmol) in DMSO 2.49—2.57 (1H, m), 2.99 (1H, dd, Jꢁ7.8, 8.5 Hz), 3.30—3.39 (2H, m), 3.42
(3.7 ml) was stirred at 90 °C for 15 h. AcOEt was added, and the organic (1H, dt, Jꢁ3.0, 8.5 Hz), 6.96 (1H, d, Jꢁ4.8 Hz), 7.05 (1H, br s), 7.38 (1H,
layer was washed with brine, dried over Na2SO4, filtered, and concentrated in dd, Jꢁ2.9, 4.8 Hz). 13C-NMR (CDCl3) d: 9.4, 9.4, 9.6, 19.9, 30.3, 44.1 (2C),
vacuo. The residue was purified by silica gel column chromatography elut- 50.7, 55.7, 65.3, 91.4, 117.1, 119.8, 124.9, 125.4, 130.7, 137.1, 139.1,
ing with CHCl3/MeOHꢁ97/3, v/v. The obtained crude product was recrys- 139.3, 140.1, 144.3, 169.5. IR (ATR): 2206, 1604, 1585, 1560, 1469, 1442,
tallized from Et2O to afford the title compound (108 mg, 41%) as a pale 1365, 1174, 1151 cmꢃ1. Anal. Calcd for C22H24N4OS·0.25H2O: C, 66.55; H,
brown solid. mp: 150—151 °C. MS (ESI) m/z: 388 (Mꢄ1)ꢄ. 1H-NMR 6.22; N, 14.11; S, 8.08. Found: C, 66.55; H, 6.07; N, 14.17; S, 8.11. [a]D25
(CDCl3) d: 1.12—1.31 (4H, m), 1.58—1.66 (1H, m), 1.95 (1H, br s), 2.12 ꢄ92.0 (cꢁ1.03, CHCl3).

348
2-Cyclopropyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-5-methyl-6-
Vol. 59, No. 3
1363, 1155, 1030 cmꢃ1. Anal. Calcd for C25H28N4O2·0.5H2O: C, 70.56; H,
6.87; N, 13.17. Found: C, 70.25; H, 6.77; N, 12.91. [a]D25 ꢄ82.6 (cꢁ1.02,
CHCl3).
vinyl-1,3-benzoxazole-4-carbonitrile (8h) Following the procedure as de-
scribed for 8b, the title compound was prepared in 78% (2 steps) from 5 and
tributyl(vinyl)tin as a white solid. MS (ESI) m/z: 337 (Mꢄ1)ꢄ. 1H-NMR
(CDCl3) d: 1.17—1.29 (4H, m), 1.76—1.87 (1H, m), 2.13—2.26 (2H, m),
2.28 (6H, s), 2.47 (3H, s), 2.70—2.79 (1H, m), 3.52—3.61 (2H, m), 3.67
(1H, t, Jꢁ8.6 Hz), 3.89 (1H, dt, Jꢁ6.6, 10.0 Hz), 5.14 (1H, dd, Jꢁ1.8,
17.8 Hz), 5.58 (1H, dd, Jꢁ1.8, 11.2 Hz), 6.68 (1H, dd, Jꢁ11.2, 17.8 Hz).
13C-NMR (CDCl3) d: 9.4, 9.5, 9.6, 19.7, 30.6, 44.4 (2C), 51.5, 57.2, 65.8,
92.6, 117.0, 120.1, 123.7, 134.7, 136.7, 138.7, 140.0, 143.8, 169.5. IR
(ATR): 2210, 1604, 1587, 1560, 1468, 1363, 1192, 1155 cmꢃ1. Anal. Calcd
for C20H24N4O: C, 71.40; H, 7.19; N, 16.65. Found: C, 71.16; H, 7.20; N,
16.45. [a]D25 ꢄ85.9 (cꢁ1.03, CHCl3).
2-Cyclopropyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-6-(3-
methoxyphenyl)-5-methyl-1,3-benzoxazole-4-carbonitrile (8n) Follow-
ing the procedure as described for 6 and 8a, the title compound was pre-
pared in 47% (2 steps) from 5 and 3-methoxyphenylboronic acid as a white
solid. MS (ESI) m/z: 417 (Mꢄ1)ꢄ. 1H-NMR (CDCl3) d: 1.16—1.30 (4H,
m), 1.57—1.69 (1H, m), 1.91—2.00 (1H, m), 2.16 (3H, s), 2.17 (3H, s), 2.18
(1.5H, s), 2.20 (1.5H, s), 2.20—2.28 (1H, m), 2.50—2.60 (1H, m), 3.04
(0.5H, t, Jꢁ9.0 Hz), 3.07 (0.5H, t, Jꢁ9.0 Hz), 3.21—3.42 (3H, m), 3.80
(1.5H, s), 3.82 (1.5H, s), 6.65 (0.5H, t, Jꢁ2.0 Hz), 6.71 (0.5H, d, Jꢁ7.6 Hz),
6.79 (0.5H, t, Jꢁ2.0 Hz), 6.84 (0.5H, d, Jꢁ7.6 Hz), 6.87—6.91 (1H, m),
7.29 (0.5H, d, Jꢁ8.5 Hz), 7.33 (0.5H, d, Jꢁ8.5 Hz). IR (ATR): 2204, 1608,
1585, 1562, 1466, 1363, 1309, 1240, 1198, 1159, 1041, 800, 777, 746,
700 cmꢃ1. Anal. Calcd for C25H28N4O2·0.25H2O: C, 71.32; H, 6.82; N,
13.31. Found: C, 71.28; H, 6.77; N, 13.11. [a]D25 ꢄ84.5 (cꢁ1.04, CHCl3).
6-(3-Cyanophenyl)-2-cyclopropyl-7-[(3S)-3-(dimethylamino)pyrrol-
idin-1-yl]-5-methyl-1,3-benzoxazole-4-carbonitrile (8o) Following the
procedure as described for 6 and 8a, the title compound was prepared in
23% (2 steps) from 5 and 3-cyanophenylboronic acid as a white solid. MS
2-Cyclopropyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-6-(2-fluo-
rophenyl)-5-methyl-1,3-benzoxazole-4-carbonitrile (8i) Following the
procedure as described for 6 and 8a, the title compound was prepared in
58% (2 steps) from 5 and 2-fluorophenylboronic acid as a white solid. MS
1
(ESI) m/z: 405 (Mꢄ1)ꢄ. H-NMR (CDCl3) d: 1.17—1.30 (4H, m), 1.56—
1.71 (1H, m), 1.90—2.03 (1H, m), 2.14 (3.6H, s), 2.15 (2.4H, s), 2.19 (3H,
s), 2.20—2.29 (1H, m), 2.47—2.61 (1H, m), 2.91 (0.6H, t, Jꢁ9.0 Hz), 3.04
(0.4H, t, Jꢁ9.0 Hz), 3.26—3.44 (3H, m), 7.03—7.24 (3H, m), 7.34—7.41
(1H, m). IR (ATR): 2204, 1606, 1587, 1558, 1470, 1446, 756 cmꢃ1. Anal.
Calcd for C24H25FN4O: C, 71.27; H, 6.23; N, 13.85. Found: C, 71.11; H,
6.20; N, 13.66. [a]D25 ꢄ82.1 (cꢁ1.02, CHCl3).
(ESI) m/z: 412 (Mꢄ1)ꢄ. H-NMR (CDCl3) d: 1.20—1.33 (4H, m), 1.58—
1
1.69 (1H, m), 1.92—2.02 (1H, m), 2.14 (1.5H, s), 2.14 (1.5H, s), 2.15 (3H,
s), 2.16 (3H, s), 2.21—2.28 (1H, m), 2.50—2.61 (1H, m), 2.92 (0.5H, t,
Jꢁ9.0 Hz), 3.01 (0.5H, t, Jꢁ9.0 Hz), 3.15 (0.5H, dt, Jꢁ6.6, 10.0 Hz), 3.21—
3.39 (2.5H, m), 7.42 (0.5H, dt, Jꢁ1.2, 7.8 Hz), 7.45 (0.5H, s), 7.53—7.62
(2H, m), 7.68 (0.5H, d, Jꢁ9.0 Hz), 7.68 (0.5H, d, Jꢁ9.0 Hz). IR (ATR):
2210, 1606, 1587, 1560, 1470, 1415, 1365, 1273, 1157, 1047, 1028, 874,
818, 756 cmꢃ1. Anal. Calcd for C25H25N5O·0.5H2O: C, 71.41; H, 6.23; N,
16.65. Found: C, 71.43; H, 6.03; N, 16.36. [a]D25 ꢄ81.0 (cꢁ1.04, CHCl3).
Ethyl 4-Cyano-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-5-methyl-6-
phenyl-1,3-benzoxazole-2-carboxylate (9) To a solution of 4 (200 mg,
0.62 mmol) and triethylamine (112 ml, 0.62 mmol) in DMSO (12 ml) was
added (3S)-3-(dimethylamino)pyrrolidine (94 ml, 0.74 mmol) in DMSO
(2 ml) at 150 °C, and then the mixture was stirred at 150 °C for 1 h. The mix-
ture was combined with AcOEt and water, extracted with AcOEt, and the or-
ganic layer was washed with brine, dried over Na2SO4, filtered, and concen-
trated in vacuo. The residue was purified by preparative TLC plates eluting
with CHCl3/MeOHꢁ49/1, v/v to afford the title compound (17.5 mg, 7%) as
a yellow solid. mp: 65—68 °C. MS (ESI) m/z: 419 (Mꢄ1)ꢄ. 1H-NMR
(CDCl3) d: 1.48 (3H, t, Jꢁ7.1 Hz), 1.59—1.70 (1H, m), 1.97—2.05 (1H,
m), 2.12 (6H, s), 2.22 (3H, s), 2.49—2.57 (1H, m), 2.91 (1H, t, Jꢁ10.0 Hz),
3.31 (1H, dd, Jꢁ7.1, 10.0 Hz), 3.50—3.58 (2H, m), 4.55 (2H, q, Jꢁ7.1 Hz),
7.11—7.15 (1H, m), 7.26—7.29 (1H, m), 7.36—7.46 (3H, m). IR (ATR):
2208, 1739, 1603, 1471, 1392, 1369, 1304, 1261, 1174 1149 cmꢃ1. Anal.
Calcd for C24H26N4O3·0.25H2O: C, 68.15; H, 6.31; N, 13.25. Found: C,
68.18; H, 6.15; N, 13.02. [a]D25 ꢄ97.2 (cꢁ1.04, CHCl3).
2-Cyclopropyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-6-(3-fluo-
rophenyl)-5-methyl-1,3-benzoxazole-4-carbonitrile (8j) Following the
procedure as described for 6 and 8a, the title compound was prepared in
58% (2 steps) from 5 and 3-fluorophenylboronic acid as a white solid. MS
1
(ESI) m/z: 405 (Mꢄ1)ꢄ. H-NMR (CDCl3) d: 1.18—1.31 (4H, m), 1.57—
1.67 (1H, m), 1.92—2.00 (1H, m), 2.15 (3H, s), 2.15 (3H, s), 2.18 (1.5H, s),
2.19 (1.5H, s), 2.20—2.27 (1H, m), 2.49—2.58 (1H, m), 2.99 (0.5H, t,
Jꢁ9.0 Hz), 3.02 (0.5H, t, Jꢁ9.0 Hz), 3.21—3.40 (3H, m), 6.84 (0.5H, ddd,
Jꢁ1.5, 2.4, 9.5 Hz), 6.91 (0.5H, dt, Jꢁ1.5, 7.6 Hz), 6.97 (0.5H, ddd, Jꢁ1.5,
2.4, 9.5 Hz), 7.03—7.09 (1.5H, m), 7.33—7.41 (1H, m). IR (ATR): 2202,
1608, 1589, 1562, 1470, 1446, 1365, 1192, 779 cmꢃ1. Anal. Calcd for
C24H25FN4O: C, 71.27; H, 6.23; N, 13.85. Found: C, 70.93; H, 6.21; N,
13.66. [a]D25 ꢄ93.0 (cꢁ1.01, CHCl3).
2-Cyclopropyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-6-(4-fluo-
rophenyl)-5-methyl-1,3-benzoxazole-4-carbonitrile (8k) Following the
procedure as described for 6 and 8a, the title compound was prepared in
71% (2 steps) from 5 and 4-fluorophenylboronic acid as a white solid. MS
1
(ESI) m/z: 405 (Mꢄ1)ꢄ. H-NMR (CDCl3) d: 1.16—1.31 (4H, m), 1.56—
1.66 (1H, m), 1.92—2.00 (1H, m), 2.15 (6H, s), 2.17 (3H, s), 2.20—2.28
(1H, m), 2.47—2.56 (1H, m), 3.00 (1H, t, Jꢁ9.3 Hz), 3.21—3.36 (3H, m),
7.06—7.14 (3H, m), 7.19—7.23 (1H, m). IR (ATR): 2210, 1587, 1564,
1510, 1471, 1404, 1365, 1306, 1217, 1161, 1026, 951, 841 cmꢃ1. Anal.
Calcd for C24H25FN4O: C, 71.27; H, 6.23; N, 13.85. Found: C, 71.01; H,
6.24; N, 13.78. [a]D25 ꢄ93.2 (cꢁ1.02, CHCl3).
4-Cyano-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-N,5-dimethyl-6-
phenyl-1,3-benzoxazole-2-carboxamide (11a) To a solution of methyl-
amine hydrochloride (125 mg, 1.85 mmol) in CH2Cl2 (2 ml) was added
trimethylaluminum (1.03 M solution in n-hexane) (1.80 ml, 1.85 mmol), and
the mixture was stirred for 40 min at room temperature. A solution of 4
(200 mg, 617 mmol) in CH2Cl2 (2 ml) was added and the reaction mixture
was stirred for 63 h at room temperature. The mixture was cooled in an ice
bath and 1 M HCl was added. After stirring at room temperature, the mixture
was extracted with CHCl3. The organic layer was washed with brine, dried
over Na2SO4, filtered, and concentrated in vacuo. The residue was purified
by silica gel column chromatography eluting with CH2Cl2/MeOHꢁ100/1,
v/v to afford 4-cyano-7-fluoro-N,5-dimethyl-6-phenyl-1,3-benzoxazole-2-
carboxamide 10a (122 mg, 64%) as a yellow solid. Following the procedure
as described for 9, the title compound was prepared in 12% from the solid
obtained above as a yellow solid. mp: 214—216 °C. HR-MS (ESI) m/z:
404.2055 (Calcd for C23H25N5O2ꢄH 404.2087). 1H-NMR (CDCl3) d:
1.53—1.68 (1H, m), 1.96—2.05 (1H, m), 2.10 (6H, s), 2.21 (3H, s), 2.44—
2.56 (1H, m), 2.73—2.83 (1H, m), 3.07 (1.5H, s), 3.08 (1.5H, s), 3.19—3.27
(1H, m), 3.58—3.73 (2H, m), 7.11—7.15 (1H, m), 7.23—7.27 (1H, m),
7.34—7.47 (4H, m). IR (ATR): 2206, 1655, 1595, 1475, 1456, 1394, 1369,
1309, 1151 cmꢃ1. [a]D25 ꢄ93.1 (cꢁ0.145, CHCl3).
6-(3-Aminophenyl)-2-cyclopropyl-7-[(3S)-3-(dimethylamino)pyrrol-
idin-1-yl]-5-methyl-1,3-benzoxazole-4-carbonitrile (8l) Following the
procedure as described for 6 and 8a, the title compound was prepared in
94% (2 steps) from 5 and 3-aminophenylboronic acid as a white solid. MS
1
(ESI) m/z: 402 (Mꢄ1)ꢄ. H-NMR (CDCl3) d: 1.17—1.30 (4H, m), 1.57—
1.68 (1H, m), 1.89—2.01 (1H, m), 2.16 (3H, s), 2.17 (3H, s), 2.20 (1.5H, s),
2.22 (1.5H, s), 2.18—2.28 (1H, m), 2.49—2.59 (1H, m), 3.06 (0.5H, t,
Jꢁ9.3 Hz), 3.14 (0.5H, t, Jꢁ9.3 Hz), 3.26 (0.5H, dt, Jꢁ6.6, 10.3 Hz), 3.31—
3.46 (2.5H, m), 3.50—4.00 (2H, br), 6.42 (0.5H, t, Jꢁ2.2 Hz), 6.50 (0.5H,
d, Jꢁ7.6 Hz), 6.54 (0.5H, t, Jꢁ2.2 Hz), 6.62 (0.5H, d, Jꢁ7.6 Hz), 6.64—
6.68 (1H, m), 7.16 (1H, q, Jꢁ7.6 Hz). IR (ATR): 3365, 2204, 1587,
1560, 468, 1448, 1396, 1362, 1308, 1159, 868 cmꢃ1. Anal. Calcd for
C24H27N5O·0.25H2O: C, 71.00; H, 6.83; N, 17.25. Found: C, 70.88; H, 6.81;
N, 16.88. [a]D25 ꢄ90.5 (cꢁ1.01, CHCl3).
2-Cyclopropyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-6-[3-(hy-
droxymethyl)phenyl]-5-methyl-1,3-benzoxazole-4-carbonitrile (8m)
Following the procedure as described for 6 and 8a, the title compound was
prepared in 83% (2 steps) from 5 and 3-(hydroxymethyl)phenylboronic acid
as a pale brown solid. MS (ESI) m/z: 417 (Mꢄ1)ꢄ. 1H-NMR (CDCl3) d:
1.17—1.33 (4H, m), 1.54—1.67 (1H, m), 1.85—2.05 (1H, m), 2.03 (3H, s),
2.13 (1.8H, s), 2.19 (1.2H, s), 2.20—2.28 (1H, m), 2.24 (1.2H, s), 2.30—
2.90 (1H, br), 2.40—2.60 (2H, m), 2.62 (0.8H, s), 2.94—3.03 (1H, m),
3.14—3.26 (0.6H, m), 3.36 (0.4H, dd, Jꢁ6.8, 9.8 Hz), 3.47—3.57 (1H, m),
4.62 (0.6H, d, Jꢁ12.7 Hz), 4.66 (0.6H, d, Jꢁ12.7 Hz), 4.71 (0.8H, s), 7.04
(0.4H, d, Jꢁ7.1 Hz), 7.16 (0.6H, d, Jꢁ7.1 Hz), 7.24 (0.6H, s), 7.26 (0.4H, s),
7.33—7.43 (2H, m). IR (ATR): 3340, 2210, 1606, 1587, 1560, 1468, 1398,
4-Cyano-7-fluoro-N,N,5-trimethyl-6-phenyl-1,3-benzoxazole-2-car-
boxamide (10b) Following the procedure as described for 10a, the title
compound was prepared in 67% from 4 and dimethylamine hydrochloride as
a white solid. mp: 170—172 °C. MS (ESI) m/z: 324 (Mꢄ1)ꢄ. 1H-NMR
(CDCl3) d: 2.47 (3H, s), 3.23 (3H, s), 3.54 (3H, s), 7.24—7.28 (2H, m),
7.46—7.55 (3H, m). IR (ATR): 2229, 1658, 1477, 1400, 1255, 1130,

March 2011
1099 cmꢃ1
349
.
(1H, t, Jꢁ9.2 Hz), 3.37—3.56 (3H, m), 7.13—7.17 (1H, m), 7.26—7.30
(1H, m), 7.34—7.51 (4H, m), 8.56 (1H, dt, Jꢁ8.1, 1.7 Hz), 8.79 (1H, dd,
Jꢁ4.9, 1.7 Hz), 9.41 (1H, dd, Jꢁ2.2, 0.7 Hz). IR (ATR): 2968, 2945, 2868,
2823, 2777, 2206, 1593, 1549, 1471, 1446, 1396, 1367, 1302, 1277, 1192,
1163, 1091, 1059, 1014, 935, 862, 820, 789, 723 cmꢃ1. Anal. Calcd for
C26H25N5O: C, 73.74; H, 5.95; N, 16.54. Found: C, 73.54; H, 5.92; N, 16.44.
[a]D25 ꢄ111.7 (cꢁ1.01, CHCl3).
7-[(3S)-3-(Dimethylamino)pyrrolidin-1-yl]-5-methyl-6-phenyl-2-
pyridin-4-yl-1,3-benzoxazole-4-carbonitrile (12d) Following the proce-
dure as described for 12b, the title compound was prepared in 21% (3 steps)
from 3 and isonicotinoyl chloride hydrochloride as a yellow solid. mp:
227—229 °C. MS (ESI) m/z: 424 (Mꢄ1)ꢄ. 1H-NMR (CDCl3) d: 1.60—1.72
(1H, m), 1.98—2.06 (1H, m), 2.16 (6H, s), 2.24 (3H, s), 2.50—2.61 (1H,
m), 3.04 (1H, t, Jꢁ9.2 Hz), 3.36—3.48 (2H, m), 3.49—3.56 (1H, m), 7.13—
7.16 (1H, m), 7.26—7.29 (1H, m), 7.35—7.46 (3H, m), 8.07 (2H, dd,
Jꢁ4.4, 1.7 Hz), 8.83 (2H, dd, Jꢁ4.4, 1.7 Hz). IR (ATR): 3051, 2947, 2868,
2821, 2773, 2210, 1595, 1541, 1469, 1441, 1396, 1365, 1302, 1271, 1196,
1155, 1095, 1057, 989, 939, 862, 831, 783, 702 cmꢃ1. Anal. Calcd for
C26H25N5O: C, 73.74; H, 5.95; N, 16.54. Found: C, 73.73; H, 5.92; N, 16.55.
[a]D25 ꢄ115.3 (cꢁ1.05, CHCl3).
4-Cyano-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-N,N,5-trimethyl-
6-phenyl-1,3-benzoxazole-2-carboxamide (11b) Following the procedure
as described for 9, the title compound was prepared in 21% from 10b as a
pale brown solid. mp: 139—142 °C. MS (ESI) m/z: 418 (Mꢄ1)ꢄ. H-NMR
1
(CDCl3) d: 1.55—1.68 (1H, m), 1.91—2.02 (1H, m), 2.09 (6H, s), 2.22 (3H,
s), 2.43—2.55 (1H, m), 2.73—2.81 (1H, m), 3.16—3.26 (1H, m), 3.20 (3H,
s), 3.51—3.68 (2H, m), 3.55 (3H, s), 7.10—7.15 (1H, m), 7.24—7.27 (1H,
m), 7.34—7.45 (3H, m). IR (ATR): 2206, 1651, 1603, 1473, 1441, 1396,
1365, 1112 cmꢃ1. Anal. Calcd for C24H27N5O2·0.25H2O: C, 68.31; H, 6.57;
N, 16.60. Found: C, 68.43; H, 6.49; N, 16.37. [a]D25 ꢄ84.4 (cꢁ0.736,
CHCl3).
7-[(3S)-3-(Dimethylamino)pyrrolidin-1-yl]-5-methyl-2,6-diphenyl-1,3-
benzoxazole-4-carbonitrile (12a) A mixture of 7 (200 mg, 0.70 mmol),
phenylboronic acid (171 mg, 1.40 mmol), tetrakis(triphenylphosphine)palla-
dium(0) (81 mg, 0.07 mmol), and potassium phosphate (297 mg, 1.40 mmol)
in 1,4-dioxane (20 ml) was refluxed for 1 h. The reaction mixture was filtered
and the filtrate was concentrated in vacuo. The residue was purified by silica
gel column chromatography eluting with n-hexane/AcOEtꢁ6/1, v/v to
afford 7-fluoro-5-methyl-2,6-diphenyl-1,3-benzoxazole-4-carbonitrile (161
mg, 70%) as a pale yellow solid. mp: ꢀ270 °C. MS(ESI) m/z: 329 (Mꢄ1)ꢄ.
1H-NMR (CDCl3) d: 2.45 (3H, s), 7.28—7.34 (2H, m), 7.45—7.62 (6H, m),
7.71—7.78 (1H, m), 8.30—8.35 (1H, m).
7-[(3S)-3-(Dimethylamino)pyrrolidin-1-yl]-5-methyl-2-(1-methyl-1H-
pyrrol-2-yl)-6-phenyl-1,3-benzoxazole-4-carbonitrile (12e) A mixture of
7 (200 mg, 0.70 mmol), 1-methyl-2-(tributylstannyl)pyrrole (389 mg, 1.05
mmol), 2,6-di-tert-butyl-4-methylphenol (2 mg), and bis(triphenylphos-
phine)palladium(II) dichloride (49 mg, 0.07 mmol) in toluene (20 ml) was
refluxed for 3 h. The reaction mixture was filtered and the filtrate was con-
centrated in vacuo. The residue was roughly purified by silica gel column
chromatography eluting with n-hexane/AcOEtꢁ5/1, v/v to afford 7-fluoro-5-
methyl-2-(1-methyl-1H-pyrrol-2-yl)-6-phenyl-1,3-benzoxazole-4-carboni-
trile (50 mg) as a pale yellow oil. A mixture of the carbonitrile, triethylamine
(100 ml), and (3S)-(dimethylamino)pyrrolidine (53 ml, 0.42 mmol) in DMSO
(2 ml) was stirred at 110 °C for 3.5 h. The reaction mixture was concentrated
in vacuo, diluted with CHCl3, washed with brine, dried over Na2SO4, fil-
tered, and concentrated in vacuo. The residue was purified by preparative
TLC plates eluting with CHCl3/MeOHꢁ9/1, v/v. The obtained crude prod-
uct was recrystallized from EtOH/i-Pr2O to afford the title compound
(24 mg, 6%) as a white solid. mp: 188—191 °C. MS (EI) m/z: 425 (Mꢄ). 1H-
NMR (CDCl3) d: 1.58—1.67 (1H, m), 1.93—2.01 (1H, m), 2.15 (6H, s),
2.21 (3H, s), 2.53 (1H, br s), 3.01 (1H, t, Jꢁ8.8 Hz), 3.30—3.47 (3H, m),
4.19 (3H, s), 6.24 (1H, dd, Jꢁ2.8, 4.0 Hz), 6.89 (1H, t, Jꢁ2.0 Hz), 6.99 (1H,
dd, Jꢁ2.0, 4.0 Hz), 7.14 (1H, d, Jꢁ7.2 Hz), 7.25—7.29 (1H, m), 7.35—7.45
A mixture of the carbonitrile (156 mg, 0.475 mmol), triethylamine (150
ml), and (3S)-(dimethylamino)pyrrolidine (90 ml, 0.71 mmol) in DMSO
(6 ml) was stirred at 130 °C for 3 h. The reaction mixture was concentrated
in vacuo, diluted with CHCl3, washed with brine, dried over Na2SO4, fil-
tered, and concentrated in vacuo. The residue was purified by preparative
TLC plates eluting with CHCl3/MeOHꢁ9/1, v/v. The obtained crude prod-
uct was recrystallized from EtOH/i-Pr2O to afford the title compound
(63 mg, 31%) as a pale yellow solid. mp: 205—214 °C. MS (EI) m/z: 422
1
(Mꢄ). H-NMR (CDCl3) d: 1.63—1.74 (1H, m), 1.97—2.08 (1H, m), 2.17
(6H, s), 2.22 (3H, s), 2.57 (1H, br s), 3.07 (1H, t, Jꢁ8.4 Hz), 3.39—3.54
(3H, m), 7.15 (1H, d, Jꢁ8.0 Hz), 7.26—7.30 (1H, m), 7.35—7.47 (3H, m),
7.51—7.58 (3H, m), 8.24 (2H, dd, Jꢁ1.6, 7.6 Hz). IR (ATR): 2206, 1598,
1550, 1466, 1365, 1200 cmꢃ1. Anal. Calcd for C27H26N4O·0.25H2O: C,
75.94; H, 6.25; N, 13.12. Found: C, 75.72; H, 6.08; N, 12.94. [a]D25 ꢄ103.8
(cꢁ1.01, CHCl3).
7-[(3S)-3-(Dimethylamino)pyrrolidin-1-yl]-5-methyl-6-phenyl-2-
pyridin-2-yl-1,3-benzoxazole-4-carbonitrile (12b) To a suspension of pi-
colinic acid (271 mg, 2.2 mmol) in CH2Cl2 (7 ml) were added oxalyl chloride
(262 ml, 3 mmol) and catalytic N,N-dimethylformamide (DMF) at 0 °C, and
then the resulting mixture was stirred for 20 min at room temperature. N,N-
diisopropylethylamine (697 ml, 4 mmol) and 3 (485 mg, 2.0 mmol) were
added and the mixture was stirred for 2 h. The reaction mixture was diluted
with CHCl3, washed with brine, dried over Na2SO4, filtered, and concen-
trated in vacuo. To a solution of the residue in xylene (20 ml) was added cat-
alytic p-toluenesulfonic acid monohydrate, and then the mixture was re-
fluxed for 12 h. The mixture was diluted with CHCl3, washed with brine,
dried over Na2SO4, filtered, and concentrated in vacuo. The residue was
roughly purified by silica gel column chromatography eluting with
CHCl3/MeOHꢁ98/2, v/v to afford 7-fluoro-5-methyl-6-phenyl-2-pyridin-2-
yl-1,3-benzoxazole-4-carbonitrile.
A mixture of the carbonitrile, (3S)-3-(dimethylamino)pyrrolidine (129 ml,
1.02 mmol), and triethylamine (150 ml) in DMSO (8 ml) was stirred at
100 °C for 3 h. The reaction mixture was concentrated in vacuo, diluted with
CHCl3, washed with brine, dried over Na2SO4, filtered, and concentrated in
vacuo. The residue was purified by silica gel column chromatography elut-
ing with CHCl3/MeOHꢁ9/1, v/v. The obtained crude product was recrystal-
lized from EtOH/i-Pr2O to afford the title compound (190 mg, 22%) as a yel-
low solid. mp: 227—233 °C. MS (ESI) m/z: 424 (Mꢄ1)ꢄ. 1H-NMR (CDCl3)
d: 1.60—1.73 (1H, m), 2.02—2.10 (1H, m), 2.13 (6H, s), 2.24 (3H, s),
2.50—2.62 (1H, m), 2.85—2.93 (1H, m), 3.30 (1H, dd, Jꢁ7.2, 9.6 Hz),
3.60—3.74 (2H, m), 7.13—7.17 (1H, m), 7.26—7.30 (1H, m), 7.35—7.49
(4H, m), 7.89 (1H, dt, Jꢁ2.0, 7.6 Hz), 8.40 (1H, d, Jꢁ8.0 Hz), 8.79—8.81
(1H, m). IR (ATR): 2206, 1604, 1581, 1458, 1435, 1367 cmꢃ1. Anal. Calcd
for C26H25N5O·0.75H2O: C, 71.46; H, 6.11; N, 16.02. Found: C, 71.70; H,
5.87; N, 15.75. [a]D25 ꢄ113.2 (cꢁ1.01, CHCl3).
(3H, m). IR (ATR): 2206, 1616, 1589, 1565, 1457, 1400, 1361, 1090 cmꢃ1
.
Anal. Calcd for C26H27N5O·0.5H2O: C, 71.87; H, 6.49; N, 16.12. Found: C,
72.12; H, 6.30; N, 16.22. [a]D25 ꢄ121.9 (cꢁ0.421, CHCl3).
7-[(3S)-3-(Dimethylamino)pyrrolidin-1-yl]-2-(2-furyl)-5-methyl-6-
phenyl-1,3-benzoxazole-4-carbonitrile (12f) Following the procedure as
described for 12b, the title compound was prepared in 28% (3 steps) from 3
and 2-furoyl chloride as a white solid. MS (ESI) m/z: 413 (Mꢄ1)ꢄ. 1H-NMR
(CDCl3) d: 1.59—1.70 (1H, m), 1.95—2.03 (1H, m), 2.15 (6H, s), 2.20 (3H,
s), 2.50—2.60 (1H, m), 2.98 (1H, t, Jꢁ9.3 Hz), 3.33—3.51 (3H, m), 6.63
(1H, dd, Jꢁ1.7, 3.4 Hz), 7.14 (1H, d, Jꢁ7.1 Hz), 7.27 (1H, d, Jꢁ7.1 Hz),
7.31 (1H, d, Jꢁ3.4 Hz), 7.33—7.45 (3H, m), 7.67 (1H, d, Jꢁ1.7 Hz). IR
(ATR): 3597, 2202, 1595, 1442, 1363, 1302, 746, 727, 708 cmꢃ1. Anal.
Calcd for C25H24N4O2·1.0H2O: C, 69.75; H, 6.09; N, 13.01. Found: C,
70.10; H, 5.99; N, 12.90. [a]D25 ꢄ82.9 (cꢁ0.336, CHCl3).
7-[(3S)-3-(Dimethylamino)pyrrolidin-1-yl]-5-methyl-2-(methylamino)-
6-phenyl-1,3-benzoxazole-4-carbonitrile (12g) To a solution of 7 (0.20 g,
0.70 mmol) in CH2Cl2 (10 ml) were added N,N-diisopropylethylamine
(0.15 ml, 0.88 mmol) and methylamine (2 M in tetrahydrofuran (THF),
0.50 ml, 1.00 mmol), and the mixture was heated at 60 °C for 3 h in a sealed
tube. The reaction mixture was diluted with CH2Cl2, washed with brine,
dried over Na2SO4, filtered, and concentrated in vacuo. The residue was
roughly purified by silica gel column chromatography eluting with
CH2Cl2/MeOHꢁ50/1, v/v to afford 7-fluoro-5-methyl-2-(methylamino)-6-
phenyl-1,3-benzoxazole-4-carbonitrile (170 mg). A mixture of the carboni-
tirile (170 mg), triethylamine (0.17 ml, 1.23 mmol), and (3S)-3-(dimethyl-
amino)pyrrolidine (0.16 ml, 1.26 mmol) in DMSO (5 ml) was heated at
150 °C for 4 h in a sealed tube. The mixture was diluted with CH2Cl2,
washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by silica gel column chromatography eluting with
CH2Cl2/MeOHꢁ10/1, v/v to afford the title compound (68.3 mg, 26%) as a
pale yellow solid. mp: 212—214 °C. MS (ESI) m/z: 376 (MꢄH)ꢄ. 1H-NMR
(CDCl3) d: 1.50—1.65 (1H, m), 1.85—2.00 (1H, m), 2.13 (6H, s), 2.16 (3H,
s), 2.50—2.60 (1H, m), 2.90—3.00 (1H, m), 3.15—3.25 (4H, m), 3.25—
7-[(3S)-3-(Dimethylamino)pyrrolidin-1-yl]-5-methyl-6-phenyl-2-
pyridin-3-yl-1,3-benzoxazole-4-carbonitrile (12c) Following the proce-
dure as described for 12b, the title compound was prepared in 60% (3 steps)
from 3 and nicotinoyl chloride hydrochloride as a yellow solid. mp: 192—
1
194 °C. MS (ESI) m/z: 424 (Mꢄ1)ꢄ. H-NMR (CDCl3) d: 1.60—1.72 (1H,
m), 1.97—2.06 (1H, m), 2.16 (6H, s), 2.23 (3H, s), 2.50—2.60 (1H, m), 3.02

350
Vol. 59, No. 3
2871, 2821, 2773, 2210, 1743, 1593, 1552, 1468, 1441, 1408, 1389, 1363,
1304, 1248, 1186, 1155, 1063, 1005, 935, 918, 837 cmꢃ1. Anal. Calcd for
C25H27N5O2·0.25H2O: C, 69.18; H, 6.39; N, 16.14. Found: C, 69.14; H,
6.24; N, 16.04. [a]D25 ꢄ92.3 (cꢁ1.03, CHCl3).
3.35 (2H, m), 7.12 (1H, d, Jꢁ7.3 Hz), 7.20—7.25 (1H, m), 7.30—7.45 (4H,
m). Anal. Calcd for C22H25N5O: C, 70.38; H, 6.71; N, 18.65. Found: C,
69.98; H, 6.73; N, 18.39. [a]D25 ꢄ118 (cꢁ0.791, CHCl3).
2-(Dimethylamino)-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-5-
methyl-6-phenyl-1,3-benzoxazole-4-carbonitrile (12h) Following the
procedure as described for 12g, the title compound was prepared in 29% (2
steps) from 7 and dimethylamine as a pale yellow solid. MS (ESI) m/z: 390
(MꢄH)ꢄ. 1H-NMR (CDCl3) d: 1.50—1.65 (1H, m), 1.85—1.95 (1H, m),
2.12 (6H, s), 2.15 (3H, s), 2.45—2.60 (1H, m), 2.90—3.00 (1H, m), 3.10—
3.35 (3H, m), 3.22 (6H, s), 7.10—7.15 (1H, m), 7.20—7.25 (1H, m), 7.30—
7.40 (3H, m). Anal. Calcd for C23H27N5O·0.25H2O: C, 70.11; H, 7.03; N,
17.77. Found: C, 70.36; H, 6.94; N, 17.83. [a]D25 ꢄ100.5 (cꢁ0.989, CHCl3).
2-(Diethylamino)-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-5-methyl-
6-phenyl-1,3-benzoxazole-4-carbonitrile (12i) Following the procedure
as described for 12g, the title compound was prepared in 53% (2 steps) from
7 and diethylamine as a brown solid. MS (ESI) m/z: 418 (Mꢄ1)ꢄ. 1H-NMR
(CDCl3) d: 1.28 (6H, t, Jꢁ7.1 Hz), 1.50—1.61 (1H, m), 1.85—1.95 (1H,
m), 2.13 (6H, s), 2.14 (3H, s), 2.45—2.55 (1H, m), 2.96 (1H, t, Jꢁ9.0 Hz),
3.11 (1H, dt, Jꢁ6.9, 10.0 Hz), 3.24 (1H, t, Jꢁ8.3 Hz), 3.33 (1H, dt, Jꢁ7.1,
9.0 Hz), 3.55—3.67 (4H, m), 7.11 (1H, d, Jꢁ7.3 Hz), 7.23 (1H, d,
2-Cyclopropyl-5-methyl-7-[(3S)-3-(methylamino)pyrrolidin-1-yl]-6-
phenyl-1,3-benzoxazole-4-carbonitrile (15a) Following the procedure as
described for 8a, the title compound was prepared in 63% from 6 and (3S)-
3-(methylamino)pyrrolidine as a white solid. MS (ESI) m/z: 373 (Mꢄ1)ꢄ.
1H-NMR (CDCl3) d: 1.17—1.30 (4H, m), 1.59 (1H, dq, Jꢁ6.6, 6.6 Hz),
1.87—1.96 (1H, m), 2.17 (3H, s), 2.20—2.27 (1H, m), 2.32 (3H, s), 2.92
(1H, dd, Jꢁ5.1, 10.0 Hz), 3.07 (1H, dq, Jꢁ5.1, 5.1 Hz), 3.20—3.27 (2H, m),
3.32—3.38 (1H, m), 7.16—7.20 (2H, m), 7.32—7.42 (3H, m). IR (ATR):
3346, 2200, 1608, 1587, 1562, 1465, 1438, 1365, 1340, 1302 cmꢃ1. Anal.
Calcd for C23H24N4O·0.25H2O: C, 73.28; H, 6.55; N, 14.86. Found: C,
73.46; H, 6.43; N, 14.68. [a]D25 ꢄ6.91 (cꢁ1.02, CHCl3).
2-Cyclopropyl-5-methyl-7-(4-methylpiperazin-1-yl)-6-phenyl-1,3-ben-
zoxazole-4-carbonitrile (15b) Following the procedure as described for
8a, the title compound was prepared in 7% from 6 and 1-methylpiperazine
as a brown solid. mp: 176—178 °C. MS (FAB) m/z: 373 (Mꢄ1)ꢄ. 1H-NMR
(CDCl3) d: 1.18—1.34 (4H, m), 2.10—2.30 (11H, m), 3.04—3.11 (4H, m),
7.16—7.20 (2H, m), 7.33—7.47 (3H, m). IR (ATR): 3627, 3016, 2931,
2841, 2794, 2744, 2684, 2218, 1608, 1593, 1564, 1448, 1400, 1375, 1294,
1267, 1227, 1194, 1153, 1099, 1072, 1036, 1007, 974, 951 cmꢃ1. Anal.
Calcd for C23H24N4O·0.25H2O: C, 73.28; H, 6.55; N, 14.86. Found: C,
73.30; H, 6.41; N, 14.52.
2-Cyclopropyl-7-[4-(dimethylamino)piperidin-1-yl]-5-methyl-6-
phenyl-1,3-benzoxazole-4-carbonitrile (15c) Following the procedure as
described for 8a, the title compound was prepared in 50% from 6 and 4-(di-
methylamino)piperidine as a white solid. mp: 157—159 °C. MS (ESI) m/z:
401 (Mꢄ1)ꢄ. 1H-NMR (CDCl3) d: 1.08—1.17 (2H, m), 1.21—1.34 (4H,
m), 1.50—1.70 (2H, m), 2.07—2.17 (1H, m), 2.22 (6H, s), 2.22—2.28 (1H,
m), 2.28 (3H, s), 2.81—2.90 (2H, m), 3.22—3.30 (2H, m), 7.17—7.20 (2H,
m), 7.32—7.37 (1H, m), 7.42—7.46 (2H, m). IR (ATR): 2945, 2821, 2771,
2218, 1608, 1593, 1564, 1458, 1398, 1377, 1296, 1240, 1194, 1134, 1099,
1059, 1030, 951, 877, 814 cmꢃ1. Anal. Calcd for C25H28N4O·0.25H2O: C,
74.14; H, 7.09; N, 13.83. Found: C, 73.81; H, 7.04; N, 13.54.
2-Cyclopropyl-7-{2-[(dimethylamino)methyl]morpholin-4-yl}-5-
methyl-6-phenyl-1,3-benzoxazole-4-carbonitrile (15d) Following the
procedure as described for 8a, the title compound was prepared in 23% from
6 and N,N-dimethyl(morpholin-2-yl)methanamine as a brown solid. mp:
135—137 °C. MS (FAB) m/z: 417 (Mꢄ1)ꢄ. 1H-NMR (CDCl3) d: 1.21—
1.34 (4H, m), 1.93 (1H, dd, Jꢁ4.5, 12.6 Hz), 2.14 (6H, s), 2.21—2.36 (2H,
m), 2.29 (3H, s), 2.70 (1H, dd, Jꢁ9.9, 12.1 Hz), 2.92—2.99 (1H, m), 3.04
(1H, dt, Jꢁ2.2, 12.2 Hz), 3.13 (1H, td, Jꢁ2.9, 11.6 Hz), 3.20—3.28 (1H, m),
3.34 (1H, td, Jꢁ2.6, 11.1 Hz), 3.69—3.75 (1H, m), 7.17—7.23 (2H, m),
7.33—7.47 (3H, m). IR (ATR): 2943, 2856, 2821, 2769, 2216, 1608, 1593,
1444, 1400, 1354, 1290, 1277, 1252, 1196, 1140, 1105, 1065, 1041, 989,
949, 887, 845, 781 cmꢃ1. Anal. Calcd for C25H28N4O2: C, 72.09; H, 6.78; N,
13.45. Found: C, 71.81; H, 6.73; N, 13.46.
Jꢁ7.3 Hz), 7.28—7.40 (3H, m). IR (ATR): 2208, 1633, 1599, 1560 cmꢃ1
.
Anal. Calcd for C25H31N5O·0.25H2O: C, 71.15; H, 7.52; N, 16.59. Found: C,
71.09; H, 7.51; N, 16.40. [a]D25 ꢄ97.8 (cꢁ1.01, CHCl3).
7-[(3S)-3-(Dimethylamino)pyrrolidin-1-yl]-5-methyl-6-phenyl-2-
pyrrolidin-1-yl-1,3-benzoxazole-4-carbonitrile (12j) Following the pro-
cedure as described for 12g, the title compound was prepared in 28% (2
steps) from 7 and pyrrolidine as a pale yellow solid. MS (ESI) m/z: 416
(MꢄH)ꢄ. 1H-NMR (CDCl3) d: 1.50—1.65 (1H, m), 1.85—1.95 (1H, m),
2.00—2.10 (4H, m), 2.13 (6H, s), 2.15 (3H, s), 2.45—2.60 (1H, m), 2.95—
3.05 (1H, m), 3.10—3.20 (1H, m), 3.20—3.35 (2H, m), 3.60—3.75 (4H, m),
7.11 (1H, d, Jꢁ7.3 Hz), 7.22 (1H, d, Jꢁ7.6 Hz), 7.25—7.40 (3H, m). Anal.
Calcd for C25H29N5O·0.25 H2O: C, 71.49; H, 7.08; N, 16.67. Found: C,
71.58; H, 7.06; N, 16.47. [a]D25 ꢄ97.2 (cꢁ1.05, CHCl3).
Ethyl 4-({4-Cyano-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-5-
methyl-6-phenyl-1,3-benzoxazol-2-yl}amino)butanoate (13) To a solu-
tion of 7 (213 mg, 0.743 mmol) in CH2Cl2 (7 ml) were added ethyl 4-
aminobutyrate hydrochloride (149 mg, 0.892 mmol) and N,N-diisopropyl-
amine (303 ml, 1.78 mmol) at 0 °C, and then the mixture was stirred at room
temperature for 15 h. The mixture was concentrated in vacuo and diluted
with AcOEt. The mixture was washed with brine, dried over Na2SO4, fil-
tered, and concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography eluting with n-hexane/AcOEtꢁ1/1, v/v to afford ethyl
4-({4-cyano-7-fluoro-5-methyl-6-phenyl-1,3-benzoxazol-2-yl}amino)bu-
tanoate (279 mg, 99%) as a white solid.
Following the procedure as described for 12g, the title compound was pre-
pared in 49% from the solid obtained above as a brown oil. MS (ESI) m/z:
476 (Mꢄ1)ꢄ. 1H-NMR (CDCl3) d: 1.26 (3H, t, Jꢁ7.1 Hz), 1.49—1.61 (1H,
m), 1.86—1.95 (1H, m), 1.98—2.07 (2H, m), 2.13 (6H, s), 2.15 (3H, s),
2.43—2.49 (2H, m), 2.49—2.58 (1H, m), 2.87—2.96 (1H, m), 3.14—3.29
(3H, m), 3.60 (2H, q, Jꢁ6.5 Hz), 4.15 (2H, q, Jꢁ7.2 Hz), 5.72 (1H, br s),
7.09—7.13 (1H, m), 7.21—7.41 (4H, m).
7-[(3S)-3-(Dimethylamino)pyrrolidin-1-yl]-5-methyl-2-(2-oxopyrrol-
idin-1-yl)-6-phenyl-1,3-benzoxazole-4-carbonitrile (14) To a solution of
13 (170 mg, 0.357 mmol) in EtOH (3.5 ml) was added 1 M NaOH aq.
(536 ml, 0.536 mmol) and the mixture was stirred at room temperature for
15 h. The mixture was cooled in an ice bath and 1 M HCl aq. (536 ml,
0.536 mmol) was added. The mixture was concentrated in vacuo. The
residue was purified by preparative TLC plates eluting with CHCl3/MeOHꢁ
5/1, v/v to afford 4-({4-Cyano-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-5-
methyl-6-phenyl-1,3-benzoxazol-2-yl}amino)butanoic acid (110 mg, 69%)
as a pale brown foam.
To a solution of the foam (90.0 mg, 0.201 mmol) in CH2Cl2 (2 ml) was
added thionyl chloride (43.5 ml, 0.603 mmol) and the mixture was stirred at
room temperature for 1 h. The reaction mixture was concentrated in vacuo
and the residue was dissolved with pyridine (2 ml). After stirred at 70 °C for
1 h, the mixture was concentrated in vacuo, diluted with CHCl3, and washed
with brine. The organic layer was dried over Na2SO4, filtered, and concen-
trated in vacuo. The residue was purified by preparative TLC plates eluting
with CHCl3/MeOHꢁ10/1, v/v to afford the title compound (54 mg, 62%) as
a white solid. mp: 196—198 °C. MS (ESI) m/z: 430 (Mꢄ1)ꢄ. 1H-NMR
(CDCl3) d: 1.51—1.66 (1H, m), 1.91—2.01 (1H, m), 2.13 (6H, s), 2.18 (3H,
s), 2.22—2.32 (2H, m), 2.45—2.55 (1H, m), 2.67 (2H, t, Jꢁ8.1 Hz), 2.94
(1H, t, Jꢁ9.4 Hz), 3.31—3.44 (3H, m), 4.19 (2H, t, Jꢁ7.2 Hz), 7.12 (1H, d,
Jꢁ7.1 Hz), 7.23—7.28 (1H, m), 7.31—7.43 (3H, m). IR (ATR): 2974, 2952,
2-Cyclopropyl-7-{[2-(dimethylamino)ethyl]amino}-5-methyl-6-phenyl-
1,3-benzoxazole-4-carbonitrile (15e) Following the procedure as de-
scribed for 8a, the title compound was prepared in 25% from 6 and N,N-di-
1
methylethylenediamine as a white solid. MS (FAB) m/z: 361 (Mꢄ1)ꢄ. H-
NMR (CDCl3) d: 1.15—1.32 (4H, m), 2.08 (6H, s), 2.19 (3H, s), 2.21—2.28
(1H, m), 2.30—2.40 (2H, m), 3.48—3.59 (2H, m), 4.38—4.48 (1H, m),
7.12—7.17 (2H, m), 7.38—7.44 (1H, m), 7.45—7.53 (2H, m). IR (ATR):
3369, 3057, 2979, 2945, 2862, 2823, 2785, 2210, 1616, 1566, 1506, 1487,
1450, 1410, 1362, 1304, 1252, 1200, 1144, 1113, 1061, 960, 872 cmꢃ1
.
Anal. Calcd for C22H24N4O·0.25H2O: C, 72.40; H, 6.77; N, 15.35. Found: C,
72.28; H, 6.62; N, 15.09.
4-Cyano-N,N,5-trimethyl-7-[(3S)-3-(methylamino)pyrrolidin-1-yl]-6-
phenyl-1,3-benzoxazole-2-carboxamide (16a) Following the procedure
as described for 9, the title compound was prepared in 34% from 10b and
(3S)-3-(methylamino)pyrrolidine as a pale yellow solid. mp: 148—151 °C.
MS (ESI) m/z: 404 (Mꢄ1)ꢄ. 1H-NMR (CDCl3) d: 1.58—1.67 (1H, m),
1.86—1.96 (1H, m), 2.22 (3H, s), 2.31 (3H, s), 2.94—2.98 (1H, m), 3.05—
3.12 (1H, m), 3.20 (3H, s), 3.28—3.33 (1H, m), 3.52—3.36 (2H, m), 3.56
(3H, s), 7.18—7.20 (2H, m), 7.35—7.44 (3H, m). IR (ATR): 2208, 1652,
1602, 1467, 1438, 1396, 1365, 1111, 704 cmꢃ1
. Anal. Calcd for
C23H25N5O2·0.25H2O: C, 67.71; H, 6.30; N, 17.17. Found: C, 67.59; H,
6.23; N, 16.84. [a]D25 ꢃ7.77 (cꢁ1.02, CHCl3).
4-Cyano-N,N,5-trimethyl-7-[(3S)-3-methyl-3-(methylamino)pyrrol-
idin-1-yl]-6-phenyl-1,3-benzoxazole-2-carboxamide (16b) To a solution
of 10b (400 mg, 1.24 mmol) in DMSO (6 ml) were added 23 (318 mg,

March 2011
351
1.48 mmol) and triethylamine (241 ml, 1.73 mmol) in DMSO (1 ml) at
150 °C, and then the mixture was stirred at 150 °C for 30 min. The mixture
was concentrated in vacuo, diluted with AcOEt. The mixture was washed
with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The
residue was roughly purified by silica gel column chromatography eluting
with n-hexane/AcOEtꢁ1/2, v/v to afford the crude Boc-16b as a yellow
foam. To a solution of the crude foam in CH2Cl2 (3 ml) was added trifluo-
5 h under hydrogen atmosphere at 50 °C. The mixture was filtered and con-
centrated in vacuo to give the title compound (266 mg, 100%) as a colorless
1
oil. HR-MS (ESI) m/z: 201.1564 (Calcd for C
10H20N2O2ꢄH 201.1603). H-
NMR (CDCl3) d: 1.41 (3H, s), 1.44 (9H, s), 1.69—1.78 (1H, m), 1.89—2.07
(2H, m), 2.72 (1H, d, Jꢁ11.47 Hz), 2.88—2.99 (1H, m), 3.04—3.21 (2H,
m), 4.59 (1H, br s). 13C-NMR (CDCl3) d: 24.8, 28.5 (3C), 40.2, 45.3, 59.2,
60.0, 79.2, 154.8. IR (ATR): 3340, 3195, 2974, 2931, 2871, 1693, 1523,
ꢃ1. [a]D25 ꢃ1.29
racetic acid (6 ml) at 0 °C and the mixture was stirred at room temperature 1446, 1365, 1279, 1252, 1169, 1066, 985, 947, 874, 783 cm
for 5 h. The mixture was concentrated in vacuo and neutralized with satd
NaHCO3 aq. The mixture was extracted with CHCl3 and the organic layer
was concentrated in vacuo. The residue was purified by preparative TLC
plates eluting with CHCl3/MeOHꢁ10/1, v/v to afford the compound
(109 mg, 21%) as a pale yellow solid. mp: 172—174 °C. MS (ESI) m/z: 418
(cꢁ1.03, CHCl3).
Benzyl (3S)-3-[(tert-Butoxycarbonyl)amino]-3-methylpyrrolidine-1-
carboxylate (21) To a solution of 20 (240 mg, 1.20 mmol) in Et
were added satd NaHCO3 aq. (12 ml) and benzyl chloroformate (222 ml,
1.56 mmol), and the mixture was stirred for 16 h at room temperature. The
mixture was extracted with AcOEt and the obtained organic layer was
2O (12 ml)
1
(Mꢄ1)ꢄ. H-NMR (CDCl3) d: 1.13 (3H, s), 1.55—1.85 (2H, m), 2.23 (3H,
s), 2.27 (3H, s), 3.07 (1H, d, Jꢁ10.50 Hz), 3.20 (3H, s), 3.18—3.40 (3H, m), washed with brine, dried over Na
2SO4, and concentrated in vacuo. The crude
product was purified by silica gel column chromatography eluting with n-
3.55 (3H, s), 7.16—7.46 (5H, m). 13C-NMR (CDCl3) d: 20.4, 22.1, 29.7,
36.5, 36.5, 39.0, 50.6, 60.0, 62.5, 92.4, 116.8, 127.4, 127.7, 128.5, 128.5, hexane/AcOEtꢁ2/1, v/v to give the title compound (400 mg, 100%) as a col-
131.0, 131.1, 137.7, 139.3, 139.6, 141.3, 142.2, 155.6, 157.0. IR (ATR): orless oil. HR-MS (ESI) m/z: 335.1970 (Calcd for
C18H26N2O4ꢄH
1
3319, 2962, 2929, 2856, 2798, 2212, 1655, 1604, 1577, 1471, 1442, 1396,
335.1971). H-NMR (CDCl3) d: 1.41—1.45 (12H, m), 1.76—1.84 (1H, m),
2.16—2.41 (1H, m), 3.34 (1H, d, Jꢁ11.5 Hz), 3.44—3.55 (2H, m), 3.56—
1367, 1308, 1259, 1201, 1153, 1111, 1070, 1016, 970, 916, 785 cmꢃ1. Anal.
13C-
NMR (CDCl3) d: 23.5, 28.5 (3C), 36.9, 44.5, 57.0, 57.8, 65.4, 66.9, 79.7,
127.9 (2C), 128.0, 128.5 (2C), 137.1, 154.7. IR (ATR): 3344, 2974, 2885,
1689, 1522, 1498, 1446, 1419, 1390, 1363, 1344, 1271, 1254, 1169, 1101,
1076, 1022, 953, 876, 768 cmꢃ1. [a]D25 ꢄ29.9 (cꢁ1.02, CHCl3).
Calcd for C24H27N5O2·0.5H2O: C, 67.59; H, 6.62; N, 16.42. Found: C, 3.69 (1H, m), 4.46—4.52 (1H, m), 5.12 (2H, s), 7.26—7.36 (5H, m).
67.72; H, 6.46; N, 16.24. [a]D25 ꢄ13.5 (cꢁ1.04, CHCl3).
7-[(3S)-3-Amino-3-methylpyrrolidin-1-yl]-4-cyano-N,N,5-trimethyl-6-
phenyl-1,3-benzoxazole-2-carboxamide (16c) Following the procedure as
described for 16b, the title compound was prepared in 41% from 10b and 20
as a pale yellow solid. mp: 202—204 °C. MS (ESI) m/z: 404 (Mꢄ1)ꢄ. H-
1
Benzyl (3S)-3-[(tert-Butoxycarbonyl)(methyl)amino]-3-methylpyrrol-
NMR (CDCl3) d: 1.16 (3H, s), 1.51—1.71 (2H, m), 2.23 (3H, s), 3.20 (3H, idine-1-carboxylate (22) To a solution of 21 (400 mg, 1.20 mmol) and
s), 3.12—3.33 (4H, m), 3.56 (3H, s), 7.14—7.46 (5H, m). IR (ATR): 2956, methyl iodide (112
ml, 1.79 mmol) in DMF (12 ml) was added sodium hy-
dride (55%, 62.6 mg, 1.44 mmol) at 0 °C, and the mixture was stirred at 0 °C
for 2 h. To a reaction mixture was added 10% citric acid aq., and the mixture
2931, 2871, 2212, 1720, 1655, 1604, 1577, 1473, 1444, 1396, 1367, 1306,
1259, 1203, 1153, 1111, 1070, 1016, 970, 904, 866, 831 cmꢃ1. Anal. Calcd
for C23H25N5O2·0.25H2O: C, 67.71; H, 6.30; N, 17.17. Found: C, 67.67; H, was extracted with AcOEt. The organic layers were washed with brine, dried
6.14; N, 16.90. [a]D25 ꢃ17.6 (cꢁ1.02, CHCl3).
over Na2SO4, and concentrated in vacuo. The crude product was purified by
tert-Butyl {(3S)-3-Methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidin-3-
yl}carbamate (18) To a stirred solution of (3S)-3-methyl-5-oxo-1-[(1R)-1-
phenylethyl]pyrrolidine-3-carboxylic acid (17)15) (652 mg, 2.64 mmol) in
toluene (20 ml) were added triethylamine (735 ml, 5.27 mmol) and
diphenylphosphoryl azide (739 ml, 3.43 mmol), and the mixture was stirred
for 30 min at room temperature. After being refluxed for 3 h, 2-methyl-2-
propanol (20 ml) was added and the mixture was refluxed for 11 h. The reac-
tion mixture was concentrated in vacuo and the residue was dissolved with
AcOEt. The organic layer was washed with brine, dried over Na2SO4, fil-
tered, and concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography eluting with n-hexane/AcOEtꢁ2/1 to 1//4, v/v to af-
ford the title compound (500 mg, 60%) as a colorless oil. HR-MS (ESI) m/z:
silica gel column chromatography eluting with n-hexane/AcOEtꢁ2/1 to 1/4,
v/v to afford the title compound (367 mg, 88%) as a colorless oil. HR-MS
(ESI) m/z: 349.2163 (Calcd for C19H28N2O4ꢄH 349.2127). 1H-NMR
(CDCl3) d: 1.29 (3H, s), 1.45 (9H, s), 1.97—2.20 (2H, m), 2.84 (3H, s), 3.34
(1H, td, Jꢁ6.9, 10.9 Hz), 3.47 (1H, d, Jꢁ11.5 Hz), 3.56 (1H, br s), 3.81—
3.92 (1H, m), 5.09—5.17 (2H, m), 7.25—7.36 (5H, m).
13C-NMR (DMSO-
d6) d: 19.9, 28.1 (3C), 31.4, 37.0, 43.5, 57.8, 62.0, 65.9, 79.2, 127.3 (2C),
127.7, 128.3 (2C), 137.2, 154.0, 155.0. IR (ATR): 2974, 2885, 1689, 1454,
1417, 1363, 1342, 1232, 1169, 1144, 1099, 1003, 966, 883, 825 cmꢃ1. [a]D25
ꢃ2.39 (cꢁ1.14, CHCl3).
tert-Butyl Methyl[(3S)-3-methylpyrrolidin-3-yl]carbamate (23) Fol-
lowing the procedure as described for 20, the title compound was prepared
1
319.2011 (Calcd for C18H26N2O3ꢄH 319.2022). H-NMR (CDCl3) d: 1.35
in 100% from 22 as a colorless oil. HR-MS (ESI) m/z: 215.1750 (Calcd for
1
(9H, s), 1.47 (3H, s), 1.51 (3H, d, Jꢁ7.08 Hz), 2.43 (1H, d, Jꢁ16.60 Hz),
2.66 (1H, d, Jꢁ16.60 Hz), 3.20—3.33 (2H, m), 4.54 (1H, s), 5.51 (1H, q,
Jꢁ7.08 Hz), 7.24—7.35 (5H, m). 13C-NMR (CDCl3) d: 16.2, 25.2, 28.3
(3C), 45.7, 48.7, 52.9, 53.7, 79.8, 127.0 (2C), 127.5, 128.6 (2C), 139.8,
154.4, 171.5. IR (ATR): 3319, 2976, 2935, 2881, 1672, 1603, 1522, 1496,
1448, 1425, 1366, 1313, 1277, 1252, 1167, 1066, 1032, 1016, 931, 874,
785 cmꢃ1. [a]D25 ꢄ68.0 (cꢁ1.06, CHCl3).
C
11H22N2O2ꢄH 215.1760). H-NMR (CDCl3) d: 1.28 (3H, s), 1.46 (9H, s),
1.77—1.94 (1H, m), 2.02—2.11 (1H, m), 2.87 (3H, s), 2.92—3.09 (3H, m),
3.16 (1H, d, Jꢁ11.47 Hz). 13C-NMR (CDCl3) d: 21.4, 28.5 (3C), 32.4, 40.0,
45.1, 59.6, 64.6, 79.7, 156.1. IR (ATR): 2974, 2931, 2873, 1687, 1541,
1477, 1419, 1363, 1252, 1171, 1130, 1039, 1011, 935, 879, 812, 775 cmꢃ1
.
[a]D25 ꢃ1.04 (cꢁ1.20, CHCl3).
In Vitro Antifungal Activity MIC-1 (the lowest drug concentration
tert-Butyl {(3S)-3-Methyl-1-[(1R)-1-phenylethyl]pyrrolidin-3-yl}car-
showing 80% growth inhibition compared to the control without drug) was
bamate (19) To a solution of 18 (500 mg, 1.57 mmol) in THF (15 ml) was evaluated by the serial dilution methods in 96-well microtest plates using
added boran–THF complex (1.2 mol/l in THF, 3.93 ml, 4.71 mmol) at 0 °C
YPD [1% yeast extract (Difco, Detroit, MI, U.S.A.), 2% peptone (Difco, De-
and the mixture was stirred at room temperature for 17 h. EtOH (12 ml), tri- troit, MI, U.S.A.), and 20% glucose] as the medium.11)
Test organisms were
purchased from the American Type Culture Collection (Rockville, MD,
U.S.A.), the Institute for Fermentation Osaka (Osaka, Japan), or Teikyo In-
stitute of Medical Micology (Tokyo, Japan). Initial cell densities (1ꢅ103—
1ꢅ104 cells/ml) and incubation times (18—72 h) were decided for each
strain in consideration of their growth speed. The fungi were incubated
at 37 °C. Follwing incubation, OD600 was measured with a Wallac 1420
ARVOsx multi-label counter (Wallac, Tokyo, Japan) and MIC-1s were cal-
ethylamine (4 ml), and water (4 ml) were added, and then the resultant mix-
ture was refluxed for 3 h. The mixture was concentrated in vacuo, diluted
with AcOEt and water, and extracted with AcOEt. The organic layers were
washed with brine, dried over Na2SO4, and concentrated in vacuo. The crude
product was purified by silica gel column chromatography eluting with n-
hexane/AcOEtꢁ2/1 to 1/2, v/v to give the title compound (406 mg, 85%) as
a colorless oil. HR-MS (ESI) m/z: 305.2213 (Calcd for C18H28N2O2+H
305.2229). 1H-NMR (CDCl3) d: 1.33 (3H, d, Jꢁ6.59 Hz), 1.43 (3H, s), 1.43 culated. Compounds were tested at different concentrations ranging from
(9H, s), 1.75—1.86 (1H, m), 1.95—2.06 (1H, m), 2.41 (1H, d, Jꢁ9.52 Hz), 0.016 to 128
mg/ml.
2.51—2.67 (2H, m), 2.67—2.77 (1H, m), 3.25 (1H, q, Jꢁ6.59 Hz), 4.68
(1H, s), 7.19—7.33 (5H, m). 13C-NMR (CDCl3) d: 23.0, 26.6, 28.5 (3C),
38.9, 51.6, 58.2, 65.1, 65.5, 78.0, 126.8, 127.1 (2C), 128.3 (2C), 145.4,
Distribution Coefficient (Log D) The distribution coefficients (Log D)
were determined by the shake-flask method. Four hundred micromolar of
compound solution of each compound in a 2 ml n-octanol–2 ml PBS solu-
155.0. IR (ATR): 3356, 2972, 2931, 2871, 2787, 1697, 1604, 1493, 1452, tion was placed on a shaker for 30 min at pH 7.4. After centrifuging each so-
1390, 1365, 1277, 1250, 1165, 1068, 1032, 964, 872 cmꢃ1. [a]D25 ꢄ46.2 lution separately at 3000 rpm for 10 min, an LC/MS method was used to
(cꢁ1.10, CHCl3).
assay each layer. The LC/MS system consisted of an 1100 Series LC/MSD
tert-Butyl [(3S)-3-Methylpyrrolidin-3-yl]carbamate (20) To a solu-
tion of 19 (406 mg, 1.33 mmol) in 1,4-dioxane (13 ml) was added palladium
hydroxide (20 wt% Pd on carbon) (100 mg) and the mixture was stirred for
(Agilent) and an X Terra®
MSC18 column (30ꢅ3.0 mm, 3.5 mm) (Waters).
The mobile phase was a 10 mM ammonium acetate buffer (pH 4.5)/0.05%
(v/v) acetic acid mixture in acetonitrile with a gradient condition (95/5—

352
Vol. 59, No. 3
10/90). Analyst software program (version 1.4, Applied BioSystems) was
used to calculate the Log D.
orally administrated at a dose of 5 mg/kg. Peripheral blood (0.4 ml) was col-
lected from jugular vein at 0.083, 0.25, 0.5, 1, 2, 4, and 6 h after administra-
tion. Blood samples were centrifuged (5000ꢅg, 10 min, 4 °C) and the super-
natant was collected. The plasma samples were subsequently stored at
ꢃ20 °C until analysis. These samples were analyzed according to the phar-
macokinetic studies on mice.
Metabolic Stability Compounds (final 1 mM) were incubated with liver
microsomes (mouse, rat, and human) in sodium phosphate buffer (pH 7.4)
for 30 min at 37 °C. The microsomal protein concentration in the assay was
0.1 mg/ml for mice and rats or 0.5 mg/ml for humans. Reaction was started
by the addition of NADPH at 37 °C and stopped by the addition of MeOH
after 30 min. After centrifuging each solution separately at 3500 rpm for
10 min at 4 °C, the corresponding loss of parent compound was determined
by LC/MS/MS.
Acknowledgments We thank Ms. Naoe Haga, Dr. Masato Hata and Dr.
Akihiro Kitamura for the biological evaluation. We also thank the members
of Drug Metabolism & Pharmacokinetics Research Laboratories for the
In Vivo Efficacy Six-week-old female Crlj : CD1 (ICR) mice (Charles physicochemical tests.
River Japan) were immunosuppressed by cyclophosphamide, and then in-
fected with C. albicans ATCC 90028 (1.2ꢅ105 CFU/mouse) or C. glabrata References
ATCC 48435 (1.7ꢅ108 CFU/mouse). Compounds were subcutaneously ad-
ministrated at a dose of 1.1, 3.3, 20, and 40 mg/kg in 0 and 6 h after infec-
tion. Survival rates were observed for 8 d.
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Pharmacokinetic Studies on Mice Six-week-old female Crlj : CD1
(ICR) mice (Charles River Japan) were used. The test compound was dis-
solved in 1% lactic acid and 5% glucose solution, and the solution was ad-
ministrated subcutaneously at a dose of 10 mg/kg. At 0.083, 0.25, 0.5, 1, 2,
4, 6, and 24 h after treatment, peripheral blood (600 ml) was collected from
axillary vein. Blood samples were centrifuged (5000ꢅg, 10 min, 4 °C) and
the supernatant was collected. The plasma samples were subsequently stored
at ꢃ20 °C until analysis. The concentrations of the compounds were deter-
mined by liquid chromatography/positive electrospray ionization tandem
mass spectrometry (LC/ESI-MS/MS), comprised of an Alliance 2695 HPLC
(Waters), SunFire C18 column (30ꢅ2.1 mm, 3.5 mm) (Waters), and Quattro
Ultima (Micromass). The mobile phase consisted of 10 mM ammonium for-
mate in water (A) and acetonitrile (B). The initial condition was set at 10%
of B. Then B (%) was increased linearly to 95% from 0.35 to 0.40 min and
maintained to 3.4 min and then brought back to the initial concentration lin-
early from 3.4 to 3.5 min and maintained to 5.2 min. The flow rate was
0.4 ml/min. The AUC and T1/2 were estimated by noncompartment method
using the WinNonlin Software program (version 1.13.1, Pharsight, Mountain
View, CA, U.S.A.).
Pharmacokinetic Studies on Rats Six-week-old female Sprague-Daw-
ley rats (SLC Japan) were used. The test compound was dissolved in 1% lac-
tic acid and 5% glucose solution, and the solution was intravenously or