Design and Synthesis of Aza-β-Carboline Analogs and their Antibacterial Evaluation

Article abstract of DOI:10.1007/s11094-021-02429-6

Bacterial drug resistance has become a growing problem worldwide due to the excessive use of antibiotics in recent decades. Two small focused libraries of 5H-pyrimido[5,4-b]indole-4-carboxamides and 5H-pyrimido-[5,4-b]indole-4-ketones were designed as eudistomin Y₃ and 1-acetyl-β-carboline (1-ABC) analogs and prepared via application of Inverse Electron-Demand Diels-Alder (IEDDA) reaction of 1,3,5-triazines and 3-aminoindoles. Compounds 2a and 2b were discovered to have activity against Mycobacterium bovis BCG with Minimum Inhibitory Concentration (MICs) values of 25 and 50 μg/mL respectively while compound 2e was against all three strains of Candida albicans tested with MIC values of 50 μg/mL. Moreover, compound 2e demonstrated synergistic antibacterial activity with fluconazol, which suggested that future drug candidates from this class of compounds could be used in combination with existing drugs to treat C. albicans infections.

Full text of DOI:10.1007/s11094-021-02429-6

DOI 10.1007/s11094-021-02429-6
Pharmaceutical Chemistry Journal, Vol. 55, No. 4, July, 2021 (Russian Original Vol. 55, No. 4, April, 2021)
DESIGN AND SYNTHESIS OF AZA-b-CARBOLINE
ANALOGS AND THEIR ANTIBACTERIAL EVALUATION
Guoxing Xu,1,† Qi Wei,2,3,† Fuhang Song,2 Huanqin Dai,2,4
Lihua Deng,1 Xiaoping Zhou,1 Lixin Zhang,2,4,* Qun Dang,1,** and Xu Bai1,***
Original article submitted July 27, 2020.
Bacterial drug resistance has become a growing problem worldwide due to the excessive use of antibiotics in
recent decades. Two small focused libraries of 5H-pyrimido[5,4-b]indole-4-carboxamides and 5H-pyrimido-
[5,4-b]indole-4-ketones were designed as eudistomin Y3 and 1-acetyl-b-carboline (1-ABC) analogs and pre-
pared via application of Inverse Electron-Demand Diels-Alder (IEDDA) reaction of 1,3,5-triazines and
3-aminoindoles. Compounds 2a and 2b were discovered to have activity against Mycobacterium bovis BCG
with Minimum Inhibitory Concentration (MICs) values of 25 and 50 mg/mL respectively while compound 2e
was against all three strains of Candida albicans tested with MIC values of 50 mg/mL. Moreover, compound
2e demonstrated synergistic antibacterial activity with fluconazol, which suggested that future drug candi-
dates from this class of compounds could be used in combination with existing drugs to treat C. albicans in-
fections.
Keywords: drug design; aza-b-carboline analogs; biological screening; 5H-pyrimido[5,4-b]indoles.
1. INTRODUCTION
properties often require additional optimization before they
could be developed as drug candidates. Unfortunately it is
often difficult to prepare derivatives of complex natural
products as required by medicinal chemistry for lead optimi-
zation efforts [2]. As part of our ongoing efforts to develop
novel and efficient methodologies to access complex
heterocycles as analogs of natural products, we became inter-
ested in b-carboline derivatives, which are tricyclic alkaloids
with intriguing biological activities such as antibacterial and
antifungal properties [11 – 17]. For example, b-carboline is
the heterocyclic nucleus of all eudistomins that were isolated
from the Korean Ascidian Synoicum Sp. Most eudistomins
have antibacterial activities with several analogs showing
Bacterial drug resistance has become a growing problem
worldwide because of the excessive use of antibiotics in re-
cent decades [1 – 4]. Since the first discovery in 1961,
methicillin-resistant Staphylococcus aureus (MRSA) has be-
come the main pathogenic bacteria responsible for hospital
and community acquired infections. MRSA showed resis-
tance to most of the currently available antibiotics and be-
came an enormous problem for health care providers [5 – 7].
Therefore, efforts to discover new antibacterial agents to
tackle the clear unmet medical need of new treatment for an-
tibiotic-resistant bacterial infections are much needed [2].
Natural products provide rich leads for most antibiotic
discovery projects [8, 9], however their pharmacokinetic
MIC (the minimum concentration of the compound re-
99
quired to inhibit 99% of bacterial growth) value of as potent
as 1.56 mg/mL against S. aureus; however few synthetic
studies were reported with regard to their SAR of antibacte-
rial activities. After examination of the limited eudistomin
literature, we noticed that compounds with two or three bro-
mine atoms on the distal aryl rings are more active, and com-
pounds containing a carbonyl group at C-10 showed higher
activities [18]. The reported SAR is limited but seems to in-
dicate that the antibacterial activity of eudistomins could be
further improved via preparation of new analogs.
1
Center for Combinatorial Chemistry and Drug Discovery of Jilin Univer-
sity, School of Pharmaceutical Sciences, Jilin University, Changchun, Jilin
130021, P. R. China.
2
Key Laboratory of Pathogenic Microbiology and Immunology, Institute of
Microbiology, Chinese Academy of Sciences, Beijing, 100101 P. R.
China.
3
College of Life Sciences, Hebei University, Baoding, 071002 P. R. China.
4
State Key Laboratory of Bioreactor Engineering, East China University of
Science and Technology, Shanghai, 200237 P. R. China.
*
e-mail: lzhang03@gmail.com; dang3qun2@yahoo.com; xbai@jlu.edu.cn
**
dang3qun2@yahoo.com
***
xbai@jlu.edu.cn
The simplest member of the eudistomin family is
1-acetyl-b-carboline (1-ABC) which was first isolated in
†
These authors contributed equally.
365
0091-150X/21/5504-0365 © 2021 Springer Science+Business Media, LLC

366
Guoxing Xu et al.
synthesis, must enable us to rapidly study the SAR of C-10
region of eudistomins (Fig. 2), and results from such an exer-
cise should be useful to guide future research in this area
leading to new antibacterial and/or synergistic antibacterial
agents.
2. EXPERIMENTAL PART
2.1. Materials and Methods
DMF was simply disposed with Na SO and MgSO .
2
4
4
Other commercial reagents were used as received without
additional purification. All melting points were obtained on
SGW X-4 apparatus (Shanghai Precision Instrument Co.,
Ltd, Shanghai, China). Mass spectra (MS) were taken in ESI
mode on LCQ DECA XP Plus (Thermo Finnigan, California,
USA). Analytical TLC was performed using 2.5 ´ 5 cm
Fig. 1. Design of aza-eudistomin analogs.
plated coated with a 0.25 mm thickness of silica gel GF
.
254
Column chromatography was performed using silica gel G
1
(200 – 300 mesh). The H NMR (300 MHz) and 13C NMR
1977 [19]. Even though 1-ABC showed weak antibacterial
activity against MRSA, it exhibited synergistic antibacterial
effect when combined with ampicillin [20]. Therefore we ra-
tionalized that preparation of eudistomin analogs at C-10 po-
sition of amides and ketones could lead to compounds with
improved antibacterial and/or synergistic antibacterial activi-
ties.
(75 MHz) spectra were recorded on Varian spectrometer
(Varian, Palo Alto, CA) with TMS as internal standard (d
scale). Multiplicities are indicated as follows: br = broad,
s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet;
integration and coupling constants (Hz) are determined with
complete proton decoupling and expressed in ppm. Com-
pounds 2a, 2b, 2c, 2d, 2e, 3, 4 were synthesized according to
our previously published procedures [24].
To validate initial screening hits, further optimization is
often carried out in drug discovery programs to confirm that
the hits could be advanced to leads with desired potency and
physical properties [21 – 23]. One common strategy is to
identify suitable places where a nitrogen atom could be intro-
duced which may increase potency via new hydrogen bond
formation and/or improve physical properties by increasing
aqueous solubility, modulating LogP, and enhancing selectiv-
ity toward the desired target (Fig. 1). Consequently we devel-
2.2. Synthesis
Tert-butyl-4-((5H-pyrimido[5,4-b]indole-4-carboxami
do)methyl)piperidine-1-carboxylate (1a). 5H-pyrimido-
[5,4-b]indole-4-carboxylic acid (214 mg, 1.0 mmol) was dis-
solved in DMF (5 mL) and CDI (N,N¢-Carbonyldiimidazole)
(162 mg, 1.0 mmol) was added at room temperature (25oC).
After 1h, N-(tert-butoxycarbonyl)-4-aminomethylpiperidine
(214 mg, 1.0 mmol) was slowly added. When the reaction
oped an efficient method to prepare aza-b-carbolines in a sin-
gle step via Electron-Demand Diels-Alder (IEDDA) reac-
tions of aminoindoles and 1,3,5-triazines. We envisioned that
the ready access of aza-b-carboline 5, which was reported
during development of the method of new aza-b-carboline
finished after about 1 h, it was quenched by addition of H O
2
(20 mL). The aqueous phase was extracted with EtOAc
(3 ´ 10 mL). The combined organic extracts were washed
Fig. 2. Efficient synthetic strategies for aza-eudistomin analogs.

Design and Synthesis of Aza-b-Carboline Analogs
367
J = 6.9 Hz, 6H). 13C-NMR (75 MHz, DMSO-d ) d 164.4,
with brine, dried (Na SO ) and evaporated to dryness. The
2
4
6
residue was chromatographied on silica gel eluted with
PE/CH Cl /EtOAc (5:5:3) to give the desired product as
149.6, 147.7 143.2, 137.2, 130.9, 128.0, 121.3, 120.6, 119.0,
113.5, 46.1, 28.3, 20.2. ESI-MS: m/z 269.0 [M+H]+.
2
2
1
white solid in 89% yield (364 mg); m.p., 176 – 178°C. H
N-Cyclopropyl-5H-pyrimido[5,4-b]indole-4-carbox-
amide (1d). Purification by silica gel column chromatogra-
phy (CH Cl : EtOAc =10:1) gave the desired product as
NMR (300 MHz, CDCl ) d (ppm): 9.96 (s, 1H), 9.11 (s, 1H),
3
8.41 (d, J = 8.1 Hz, 1H), 8.27 – 8.23 (m, 1H), 7.72 – 7.66
(m, 1H), 7.57 (d, J = 8.1 Hz, 1H), 7.42 – 7.37 (m, 1H),
4.17 – 4.14 (m, 2H), 3.46 (d, J = 6.6 Hz, 2H), 2.76 – 2.68
(m, 2H), 1.89 – 1.78 (m, 3H), 1.45 (s, 9H), 1.34 – 1.24 (m,
2
2
white solid in 70% yield (89mg). m.p., 203 – 204°C.
1H-NMR (300 MHz, DMSO-d ) d: 12.00 (s, 1H), 9.17 (d,
6
J = 5.1 Hz, 1H), 9.09 (s, 1H), 8.29 (d, J = 7.8 Hz, 1H),
7.85 – 7.81 (m, 1H), 7.74 – 7.68 (m, 1H), 7.39 – 7.34 (m,
1H), 3.09 – 3.03 (m, 1H), 0.80 – 0.76 (m, 4H); 13C-NMR
2H); 13C NMR (75 MHz, CDCl ) d (ppm): 165.4, 154.6,
3
150.6, 148.3, 142.5, 136.1, 131.3, 128.9, 122.2, 121.2, 119.8,
112.1, 79.3, 44.5, 43.5, 36.6, 29.7, 28.3. ESI-MS: m/z 410
[M+H]+.
(75 MHz, DMSO-d ) d: 165.6, 149.5, 147.6, 143.2, 137.1,
130.8, 127.8, 121.3, 120.6, 119.0, 113.5, 22.8, 5.7. ESI-MS:
m/z 253 [M+H]+.
6
Tert-butyl-4-(5H-pyrimido[5,4-b]indole-4-carboxami
do) piperidine-1-carboxylate (1b). 5H-pyrimido[5,4-b]-
indole-4-carboxylic acid (214 mg, 1.0 mmol) was dissolved
in DMF (5 ml) and CDI (N,N¢-Carbonyldiimidazole)
(162 mg, 1.0 mmol) was added at room temperature (25oC).
After 1 h, N-Boc-4-piperidineamine (200 mg, 1.0 mmol) was
slowly added. When the reaction finished after about 1 h, it
N-(tert-butyl)-5H-pyrimido[5,4-b]indole-4-carboxamide
(1e). Purification by silica gel column chromatography
(CH Cl : EtOAc =10:1) gave the desired product as light yel-
2
2
low solid in 37% yield (46mg). m.p., 163 – 165°C. 1H-NMR
(300 MHz, DMSO-d ) d: 12.07 (s, 1H), 9.10 (s, 1H),
8.30 – 8.28 (m, 2H), 7.82 (d, J = 8.4 Hz, 1H), 7.73 – 7.68
6
(m, 1H), 7.39 – 7.34 (m, 1H), 1.52 (s, 9H); 13C-NMR
was quenched by addition of H O (20 mL). The aqueous
2
phase was extracted with EtOAc (3 ´ 10 mL). The combined
organic extracts were washed with brine, dried (Na SO ) and
(75 MHz, DMSO-d ) d: 163.5, 149.7, 147.4, 143.2, 137.2,
130.8, 127.8, 121.3, 120.5, 118.9, 113.4, 50.7, 28.3. ESI-MS:
m/z 269 [M+H]+.
6
2
4
evaporated to dryness. The residue was chromatographied on
silica gel eluting with PE/CH Cl /EtOAc (5:5:3) to give the
N-butyl-5H-pyrimido[5,4-b]indole-4-carboxamide (1f).
Purification by silica gel column chromatography (CH Cl :
2
2
desired product as light green solid in 76% yield (300 mg);
1
m.p., 195 – 198°C. H NMR (300 MHz, CDCl ) d (ppm):
2
2
EtOAc =10:1) gave the desired product as white solid in 60%
yield (77mg). m.p., 153 – 154°C. 1H-NMR (300 MHz,
3
9.94 (s, 1H), 9.11 (s, 1H), 8.41 (d, J = 8.1 Hz, 1H),
8.08 – 8.05 (m, 1H), 7.73 – 7.67 (m, 1H), 7.58 (d,
J = 8.1 Hz, 1H), 7.42 – 7.37 (m, 1H), 4.21 – 4.10 (m, 3H),
3.03 – 2.95 (m, 2H), 2.09 – 2.03 (m, 2H), 1.61 – 1.57 (m,
DMSO-d ) d: 11.99 (s, 1H), 9.18 (t, J = 6.0 Hz, 1H), 9.10 (s,
6
1H), 8.28 (d, J = 7.8 Hz, 1H), 7.83 – 7.80 (m, 1H),
7.72 – 7.66 (m, 1H), 7.38 – 7.32 (m, 1H), 3.41 (q,
J = 6.9 Hz, 2H), 1.65 – 1.55 (m, 2H), 1.42 – 1.30 (m, 2H),
2H), 1.48 (s, 9H); 13C NMR (75 MHz, CDCl ) d (ppm):
3
0.92 (t, J = 7.2 Hz, 3H); 13C-NMR (75 MHz, DMSO-d ) d:
164.5, 154.6, 150.6, 148.2, 142.6, 136.0, 131.3, 128.9, 122.2,
121.1, 119.8, 112.1, 79.7, 46.6, 42.4, 31.8, 28.4. ESI-MS: m/z
396 [M+H]+.
6
164.2, 149.6, 147.6, 143.1, 137.2, 130.8, 128.0, 121.2, 120.5,
119.0, 113.4, 38.3, 31.2, 19.6, 13.6. ESI-MS: m/z 269
[M+H]+.
General procedure for the preparation of 5H-pyrimi-
do[5,4-b]indole-4-carboxamides (1c – 1k). 5H-pyrimido-
[5,4-b]indole-4-carboxylic acid was dissolved in DMF and
CDI (N,N¢-Carbonyldiimidazole) (1eq.) was added at room
temperature (25°C). After 1h, amine (1.2eq.) was slowly
added. The reaction was monitored by TLC or LC-MS.
When the reaction finished, it was quenched by addition of
N-(2-(Dimethylamino)-ethyl)-5H-pyrimido[5,4-b]indo-
le-4-carboxamide (1g). Purification by silica gel column
chromatography (CH Cl : EtOAc=10:1) gave the desired
2
2
product as light yellow solid in 58% yield (79mg). m.p.,
1
168 – 169°C. H-NMR (300 MHz, DMSO-d ) d: 12.00 (s,
6
1H), 9.12 (s, 1H), 9.00 (t, J = 8.7 Hz, 1H), 8.29 (d,
J = 8.1 Hz, 1H), 7.82 (d, J = 8.1 Hz, 1H), 7.73 – 7.68 (m,
1H), 7.39 – 7.34 (m, 1H), 3.55 – 3.49 (m, 2H), 2.52 – 2.48
H O. The aqueous phase was extracted with EtOAc. The
2
combined organic extracts were washed with brine, dried
(Na SO ) and evaporated to dryness. The residue was
(m, 2H), 2.22 (s, 6H); 13C-NMR (75 MHz, DMSO-d ) d:
2
4
6
chromatographied
on
silica
gel
eluting
with
164.1, 149.6, 147.7, 143.2, 136.9, 130.9, 127.9, 121.3, 120.6,
119.0, 113.5, 57.8, 45.1, 36.5. ESI-MS: m/z 284 [M+H]+.
N-(2-Morpholinoethyl)-5H-pyrimido[5,4-b]indole-4-
carboxamide (1h). Recrystallization (PE/EtOAc) gave the
desired product as light yellow solid in 64% yield (98mg).
PE/CH Cl /EtOAc to give the desired product.
2
2
N-Isobutyl-5H-pyrimido[5,4-b]indole-4-carboxamide
(1c). Purification by silica gel column chromatography
(CH Cl : EtOAc =10:1) gave the desired product as white
2
2
1
solid in 49% yield (63mg). m.p., 156 – 157°C. H-NMR
m.p., 214 – 215°C. 1H-NMR (300 MHz, DMSO-d ) d: 12.00
6
(300 MHz, DMSO-d ): d 12.00 (s, 1H), 9.18 (t, J = 6.3 Hz,
(s, 1H), 9.11 (s, 1H), 9.09 – 9.07 (m, 1H), 8.28 (d,
J = 8.1 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.73 – 7.67 (m,
1H), 7.38 – 7.33 (m, 1H), 3.60 – 3.53 (m, 6H), 2.57 (d,
J = 6.6 Hz, 2H), 2.49 – 2.44 (m, 4H); 13C-NMR (75 MHz,
6
1H), 9.12 (s, 1H), 8.29 (d, J = 7.8 Hz, 1H), 7.82 (d,
J = 8.4 Hz, 1H), 7.73 – 7.68 (m, 1H), 7.39 – 7.34 (m, 1H),
3.26 (t, J = 6.6 Hz, 2H), 2.03 – 1.94 (m, 1H), 0.94 (d,

368
Guoxing Xu et al.
N-(4-methoxyphenyl)-5H-pyrimido[5,4-b]indole-4-
carboxamide (1m). Recrystallization (PE/EtOAc) gave the
desired product as yellow solid in 71% yield (105mg). m.p.,
DMSO-d ) d: 164.3, 149.7, 147.7, 143.2, 137.0, 130.9,
128.0, 121.3, 120.6, 119.0, 113.5, 66.2, 57.1, 53.2, 35.8.
ESI-MS: m/z 326 [M+H]+.
6
1
213 – 215°C. H-NMR (300 MHz, DMSO-d ) d: 12.14 (s,
Morpholino-(5H-pyrimido[5,4-b]indol-4-yl)methano-
ne (1i). Recrystallization (PE/EtOAc) gave the desired prod-
uct as light yellow solid in 62% yield (82mg). m.p.,
6
1H), 10.93 (s, 1H), 9.20 (s, 1H), 8.32 (d, J = 7.8 Hz, 1H),
7.95 (d, J = 9.0 Hz, 2H), 7.84 (d, J = 8.4 Hz, 1H),
7.75 – 7.73 (m, 1H), 7.41 – 7.36 (m, 1H), 6.99 (d,
J = 9.0 Hz, 2H), 3.78 (s, 3H); 13C-NMR (75 MHz,
1
230 – 232°C. H NMR (300 MHz, DMSO-d ) d: 11.98 (s,
6
1H), 9.07 (s, 1H), 8.29 (d, J = 7.8 Hz, 1H), 7.71 – 7.70 (m,
2H), 7.39 – 7.34 (m, 1H), 3.81 – 3.76 (m, 4H), 3.74 – 3.71
(m, 2H), 3.62 – 3.59 (m, 2H). 13C-NMR (75 MHz,
DMSO-d ) d: 162.5, 155.9, 149.8, 147.5, 143.2, 137.2,
131.1, 131.0, 128.1, 121.9, 121.3, 120.7, 119.0, 113.8, 113.5,
55.2. ESI-MS: m/z 319 [M+H]+.
6
DMSO-d ) d: 164.0, 148.2, 147.8, 142.4, 141.4, 130.7,
128.0, 121.3, 120.6, 119.4, 113.0, 66.5, 66.1, 47.0, 42.2.
ESI-MS: m/z 283 [M+H]+.
6
N-Phenyl-5H-pyrimido[5,4-b]indole-4-carboxamide
(1n). Recrystallization (PE/EtOAc) gave the desired product
as yellow solid in 68% yield (92mg). m.p., 193 – 198°C.
(4-Methylpiperazin-1-yl)(5H-pyrimido[5,4-b]indol-4-
yl)methanone (1j). Purification by silica gel column chro-
matography (CH Cl : EtOAc=10:1) gave the desired product
1H-NMR (300 MHz, DMSO-d ) d: 12.16 (s, 1H), 11.00 (s,
6
1H), 9.21 (s, 1H), 8.32 (d, J = 7.8 Hz, 1H), 8.05 – 8.02 (m,
2H), 7.85 (d, J = 8.1 Hz, 1H), 7.76 – 7.71 (m, 1H),
7.45 – 7.36 (m, 3H), 7.21 – 7.16 (m, 1H); 13C-NMR
2
2
as light yellow solid in 56% yield (79mg). m.p.,
1
177 – 179°C. H-NMR (300 MHz, DMSO-d ) d: 11.97 (s,
6
1H), 9.07 (s, 1H), 8.28 (d, J = 7.8 Hz, 1H), 7.72 – 7.69 (m,
2H), 7.38 – 7.33 (m, 1H), 3.80 (t, J = 5.1 Hz, 2H), 3.65 (t,
J = 5.1 Hz, 2H), 2.48 (t, J = 5.1 Hz, 2H), 2.32 (t, J = 5.1 Hz,
(75 MHz, DMSO-d ) d: 163.0, 150.0, 147.5, 143.3, 138.0,
136.9, 131.0, 128.7, 128.2, 124.3, 121.4, 120.8, 120.4, 119.0,
113.5. ESI-MS: m/z 289 [M+H]+.
6
2H), 2.22 (s, 3H). 13C-NMR (75 MHz, DMSO-d ) d: 163.9,
N-(4-Chlorophenyl)-5H-pyrimido[5,4-b]indole-4-carb-
oxamide (1o). Recrystallization (PE/EtOAc) gave the de-
sired product as yellow solid in 75% yield (114mg). m.p.,
6
148.1, 147.9, 142.3, 141.8, 130.6, 127.9, 121.2, 120.5, 119.4,
112.9, 54.9, 54.2, 46.3, 45.5, 41.6. ESI-MS: m/z 296
[M+H]+.
1
261 – 263°C. H-NMR (300 MHz, DMSO-d ) d: 12.15 (s,
6
Ethyl-2-(5H-pyrimido[5,4-b]indole-4-carboxamido)
acetate (1k). Recrystallization (PE/EtOAc) gave the desired
product as light yellow solid in 49% yield (68mg). m.p.,
1H), 11.18 (s, 1H), 9.21 (s, 1H), 8.32 (d, J = 8.1 Hz, 1H),
8.07 (d, J = 9.0 Hz, 2H), 7.84 (d, J = 8.4 Hz, 1H),
7.76 – 7.71 (m, 1H), 7.49 (d, J = 9.0 Hz, 2H), 7.42 – 7.36
1
221 – 223°C. H NMR (300 MHz, DMSO-d ) d: 12.03 (s,
(m, 1H); 13C-NMR (75 MHz, DMSO-d ) d: 163.3, 150.0,
6
6
1H), 9.53 (t, J = 6.0 Hz, 1H), 9.15 (s, 1H), 8.31 (d,
J = 7.8 Hz, 1H), 7.82 (d, J = 8.1 Hz, 1H), 7.74 – 7.69 (m,
1H), 7.40 – 7.35 (m, 1H), 4.20 – 4.13 (m, 4H), 1.23 (t,
147.5, 143.3, 137.1, 136.8, 131.1, 128.6, 128.2, 128.0, 122.1,
121.4, 120.8, 119.0, 113.5. ESI-MS: m/z 323 [M+H]+.
N-(Piperidin-4-ylmethyl)-5H-pyrimido[5,4-b]indole-4-
carboxamide (1p). Tert-butyl 4-((5H-pyrimido [5,4-b]indo-
le-4-carboxamido)methyl)piperidine-1-carboxylate (182 mg,
0.44 mmol) was dissolved in CH Cl (2 ml) and CF COOH
J = 6.9 Hz, 3H); 13C NMR (75 MHz, DMSO-d ) d: 169.4,
6
164.9, 149.8, 147.7, 143.3, 136.3, 131.0, 127.9, 121.3, 120.7,
118.9, 113.4, 60.6, 41.0, 14.1. ESI-MS: m/z 299 [M+H]+.
General Procedure for the preparation of 5H-pyri-
mido[5,4-b]indole-4-carboxamides (1l – 1o). 5H-pyrimido
[5,4-b]indole-4-carboxylic acid was dissolved in CH Cl and
2
2
3
(253mg, 2.22 mmol) was added in ice bath. After 2h, the re-
action finished, it was quenched by addition of H O (10 ml).
2
The organic phase was separated, and the aqueous phase was
2
2
(ClCO) (4eq.) and a drop of DMF were added on ice-cold
extracted with CH Cl (2 ´ 5 ml). Then the aqueous phase
was adjusted to pH 12 with 4N NaOH. The precipitate was
separated by filtration and dried to give the desired product
2
2
2
bath, then warmed up to room temperature. After 6h, the ap-
propriate aromatic amine (1.5eq.) was slowly added. The re-
action was monitored by TLC or LC-MS. When the reaction
1
as light yellow solid (155 mg, 90%). Decomp. >160°C. H
finished, it was quenched by addition of H O. The precipitate
NMR (300 MHz, CDCl ) d: 9.32 (br.s, 1H), 9.08 (s, 1H),
2
3
was separated by filtration and recrystallized with PE/EtOAc
to give the desired product.
8.28 (d, J = 8.1 Hz, 1H), 7.80 (d, J = 8.1 Hz, 1H),
7.70 – 7.65 (m, 1H), 7.35 – 7.30 (m, 1H), 3.32 – 3.27 (m,
2H), 2.94 – 2.90 (m, 2H), 2.45 – 2.37 (m, 2H), 1.74 – 1.71
(m, 1H), 1.64 – 1.60 (m, 2H), 1.11 – 1.08 (m, 2H); 13C NMR
N-(4-fluorophenyl)-5H-pyrimido[5,4-b]indole-4-carb-
oxamide (1l). Recrystallization (PE/EtOAc) gave the desired
product as light yellow solid in 77% yield (110mg). m.p.,
(75 MHz, CDCl ) d: 164.5, 149.6, 147.4, 143.8, 137.3,
130.7, 128.6, 121.3, 120.3, 119.1, 113.7, 45.8, 44.7, 36.6,
30.9. ESI-MS: m/z 310 [M+H]+.
3
1
209 – 210°C. H NMR (300 MHz, DMSO-d ) d: 12.15 (s,
6
1H), 11.13 (s, 1H), 9.21 (s, 1H), 8.32 (d, J = 7.8 Hz, 1H),
8.08 – 8.03 (m, 2H), 7.84 (d, J = 8.1 Hz, 1H), 7.76 – 7.710
(m, 1H), 7.41 – 7.36 (m, 1H), 7.30 – 7.24 (m, 2H); 13C NMR
N-(Piperidin-4-yl)-5H-pyrimido[5,4-b]indole-4-carbo
xamide (1q). Tert-butyl-4-(5H- pyrimido[5,4-b]indole-4-
carboxamido)piperidine-1-carboxylate (182 mg, 0.51 mmol)
was dissolved in CH Cl (2 ml) and CF COOH (288mg,
(75 MHz, DMSO-d ) d: 163.0, 160.2(157.0), 149.9, 147.5,
143.3, 136.8, 134.4, 131.0, 128.2, 122.4, 122.3, 121.3, 120.7,
6
2
2
3
119.0, 115.4, 115.1, 113.5. ESI-MS: m/z 307 [M+H]+.
2.53 mmol) was added in ice bath. After 2h, the reaction fin-

Design and Synthesis of Aza-b-Carboline Analogs
369
Scheme 1. Synthesis of 5H-pyrimido[5,4-b]indole-4- carboxamides 2. Reagents and conditions: (i) Conc. HCl, reflux, 6h; (ii) CDI, DMF,
aliphatic amines, 2h; or (COCl)2, aromatic amines, 8h; (iii) 2a-2b, Sat. HCl- EtOAc, 25°C, 2h.
ished, it was quenched by addition of H O (10 ml). The or-
glycerol stocks at –80°C and streaked onto Mueller-Hinton
agar (MHA) for colony growth at 37°C. The antibacterial ac-
tivities and minimum inhibitory concentrations (MICs) were
determined in flat bottom, 96-well microtiter plates using a
modified broth microdilution protocol according to the Clini-
cal and Laboratory Standards Institute M7-A6, M-38A, and
M-27A2 methods. Briefly, single colonies of bacteria were
picked from MHA plate and adjusted to 105 CFU/mL with
Mueller-Hinton Broth (MHB) as bacterial suspensions.
Aliquots (2 mL) of two-fold serial dilutions of each com-
pound (in DMSO) were added to each row on the 96-well
plates containing 78 mL of bacterial suspension in each well.
The 96-well plates were incubated aerobically at 37°C for
16 h before the results were recorded. MICs were defined as
the lowest concentrations of the compounds that inhibited
visible bacterial growth after 16 h of incubation. The MICs
were tested twice in triplicate. Vancomycin was used as a
positive control for S. aureus, MRSA, M. luteus, B. subtilis,
and E. coli [25].The test strains used in the study of sec-
ond-batch compounds included three C. albican (SC5314
from ATCC, G5 and 17# from clinical isolates), Mycobacte-
rium bovis BCG (M. bovis BCG, Pasteur 1173P2), Staphylo-
coccus aureus (S. aureus, ATCC 6538), Enterococcus
2
ganic phase was separated, and the aqueous phase was ex-
tracted with CH Cl (2 ´ 5 ml). Then the aqueous phase was
2
2
adjusted to pH 12 with 4N NaOH. The precipitate was sepa-
rated by filtration and dried to give the desired product as
1
light yellow solid (123 mg, 82%). m.p., 195 – 197°C. H
NMR (300 MHz, CDCl ) d: 11.97 (br.s, 1H), 9.11 (s, 1H),
3
8.94 – 8.91 (m, 1H), 8.29 (d, J = 8.1 Hz, 1H), 7.81 (d,
J = 8.7 Hz, 1H), 7.73 – 7.68 (m, 1H), 7.39 – 7.34 (m, 1H),
3.99 – 3.96 (m, 1H), 3.01 – 2.97 (m, 2H), 2.60 – 2.50 (m,
2H), 1.82 – 1.77 (m, 2H), 1.66 – 1.53 (m, 2H); 13C NMR
(75 MHz, CDCl ) d: 163.4, 149.6, 147.6, 143.2, 137.3,
3
130.9, 128.1, 121.3, 120.6, 119.0, 113.5, 47.2, 45.2, 32.7.
ESI-MS: m/z 296 [M+H]+.
2.3 Biological Activity Testing
General biological activity assays. The Gram-positive
bacterial strains used in this study included S. aureus (ATCC
6538), MRSAa (Chaoyang Hospital, Beijing, China), Bacil-
lus subtilis (ATCC 6633), Escherichia coli (ATCC 25922),
and Micrococcus luteus (ATCC 9341). All of the compounds
were prepared as 4 mg/mL stock solution using sterile
dimethyl sulfoxide (DMSO). The microbes were stored as

370
Guoxing Xu et al.
Scheme 2. Synthesis of 5H-pyrimido[5,4-b]indole-4-ketones 1.
faecium (E. faecium, Chaoyang Hospital, Beijing, China),
Pseudomonas aeruginosa (P. aeruginosa, PA14). C. albicans
were grown on YPD medium and MIC assay performed in
the RPMI 1640. The microbes were streaked onto Mueller-
Hinton agar (MHA) for colony growth at 37°C and MIC de-
termined in Mueller-Hinton Broth (MHB).
reader. All of the screening plates contained negative
(DMSO) and positive (2.5 mg/mL rifampicin) controls.
3. RESULTS AND DISCUSSION
3.1. Chemistry
BCG inhibition activity in the THP-1 cell line.
THP-1 cells were seeded into 96-well clear bottom plates
and differentiated using 100 ng/ml phorbol 12-myristate
13-acetate (PMA). BCG (pMSP12: GFP and maintained with
kanamycin) was grown to mid-log phase in Middlebrook
7H9 OADC and prepared for infection. The de-clumped bac-
teria were diluted with pre-warmed infection media (RPMI
1640, 1% glutamine, and 10% fetal calf serum) and were
used to infect cells at an MOI of 10:1. After 3 h, the
macrophages were gently washed to remove non internalized
bacteria, and then RPMI incomplete medium was added. Af-
ter 24 h, all of the media was aspirated and replaced with me-
dia containing testing compounds. Following a 2 days incu-
The synthesis of 5H-pyrimido[5,4-b]indole-4-carbox-
amides 2 is shown in Scheme 1. Compound 5 was prepared
via IEDDA reaction using published method [24]. Selective
mono-decarboxylation of compound 5 led to the key inter-
mediate acid 6, which could be used to access either 1-ABC
analogs or eudistomin Y analogs. First, we prepared amide
3
analogs of 1-ABC as shown in Scheme 1. Coupling of acid 6
with aliphatic amines using CDI provided compounds 2a-2k,
while aromatic amines were coupled with acid 6 via its acyl
chloride to give compounds 2l-2o. Removal of Boc protect-
ing groups of compounds 2a and 2b using hydrogen chloride
in ethyl acetate gave compounds 2p and 2q.
bation period at 37°C and 5% CO , GFP fluorescence was
The synthesis of eudistomin Y3 analogs 1 is shown in
Scheme 2. Friedel-Crafts acylation of aromatic compounds
was achieved by first converting acid 6 into its acyl chloride
and then reacted with various aromatic compounds in the
presence of aluminum trichloride to give compounds
1a – 1d. Demethylation of compound 1d using concentrated
hydrogen chloride in sealed tube provided compound 1e.
2
quantified using an Envision Multilabel (Perkin Elmer) plate
TABLE 1. Antibacterial Activity of the First Batch of Compounds
(MIC, mg/mL)
K.pneum
M. bovis
oniae
Comp.
MRSA
SA
BS
E.f
PA
3.2. Biological Activity
BCG
#249
The synthesized eudistomin Y and 1-ABC analogs were
3
2a
2b
2p
2q
1a
1b
1c
1d
1e
25
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
tested for antibacterial activity with the hope that any initial
observed active compounds could help to guide us to design
and prepare future analogs. To this end, compounds 2a, 2b,
2p, 2q, and 1a – 1e were screened against Mycobacterium
bovis BCG (M.B. BCG), MRSA, Staphylococcus aureus
(S.A.), Bacillus subtilis (B.S.), Enterococcus faecalis (E.F.),
Pseudomonas aeruginosa (P.A.), Klebsiella peneumoniae
#249 (K.P. #249). As evident from the results presented in
Table 1, compounds 2a and 2b showed antibacterial activity
against M. bovis BCG with MICs values of 25 mg/mL and
50
>100
>100
>100
>100
>100
>100
>100

Design and Synthesis of Aza-b-Carboline Analogs
371
TABLE 2. Antibacterial Activity of the Second Batch of Compounds (MIC, mg/mL)
C. albicans
Antimicrobial
Compd.
G5
synergisticga
M. bovis
SC5314
G5
17#
S. aureus
E. faecium
P. aeruginosa
BCG
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.0156e
2c
2d
>100
50
>100
>100
50
>100
>100
50
>100
>100
6.25
>100
>100
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.625f
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.039g
2e
50
2f
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.78c, 100b
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.78c, 100b
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.03d
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.625f
2g
100
2h
>100
100
2i
2j
>100
100
2k
2l
>100
>100
>100
>100
0.39b
2m
2n
2o
Control
aSynergistic assay with the addition of 25 mg/mL ketoconazole; bMIC for fluconazole; cMIC for amphotericin B; d MIC for cyclosporine; e MIC
for rifampicin; f MIC for vancomycin; and g MIC for ciprofloxacin.
was discovered with antibacterial activity against all three
50 mg/mL respectively. The results showed that ketones had
no activity, while amides had certain activity. Therefore, in
strains of C. albicans tested with MICs values of 50 mg/mL.
the next step, some more amides were synthesized to evalu-
ate their activity.
Moreover, compound 2e demonstrated synergistic antibacte-
rial activity with fluconazol, which suggested that future
drug candidates from this class of compounds could be used
in combination with existing drugs to treat C. albican infec-
tions. Further optimization of these initial hits could lead to
new candidate compounds as potential antibacterial agents.
To increase the chance of discovering new leads, addi-
tional new analogs were synthesized and evaluated for anti-
bacterial activity against Candida albicans (C. albicans)
from clinical isolates. As shown in Table 2, all aromatic
amides were inactive. Among the aliphatic amides. Com-
pound 2d showed antibacterial activity against C. albicans
with MICs values of 50ìg/mL, and compound 2e was discov-
ered with antibacterial activity against all three strains of C.
albicans tested with MICs values of 50ìg/mL. In the same
time, synergistic antibacterial activities were also evaluated.
As a result, compound 2e demonstrated synergistic antibac-
ACKNOWLEDGMENTS
This work was supported by the Fundamental Research
Funds of Jilin University (No. 45006050152), the Sci-Tech
Development Projects of Jilin Province in China (No.
20140309010YY and No. 20180414075GH, and Changchun
Discovery Sciences, Ltd.
terial activity with fluconazole (6.25 and 25 mg/mL), which
suggested that future drug candidates from this class of com-
pounds could be used in combination with existing drugs to
treat C. albican infections.
CONFLICT OF INTEREST
In conclusion, a strategy to access 5H-pyrimido[5,4-b]in-
dole-4-carboxamides and 5H-pyrimido[5,4-b] indole-4-ke-
The authors declare that they have no conflict of interest.
tones as eudistomin Y and 1-ABC analogs was realized via
3
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