Discovery of tetrahydropyrazolopyrimidine carboxamide derivatives as potent and orally active antitubercular agents

Article abstract of DOI:10.1021/ml400071a

Tetrahydropyrazolo[1,5-a]pyrimidine scaffold was identified as a hit series from a Mycobacterium tuberculosis (Mtb) whole cell high through-put screening (HTS) campaign. A series of derivatives of this class were synthesized to evaluate their structure-activity relationship (SAR) and structure-property relationship (SPR). Compound 9 had a promising in vivo DMPK profile in mouse and exhibited potent in vivo activity in a mouse efficacy model, achieving a reduction of 3.5 log CFU of Mtb after oral administration to infected mice once a day at 100 mg/kg for 28 days. Thus, compound 9 is a potential candidate for inclusion in combination therapies for both drug-sensitive and drug-resistant TB.

Full text of DOI:10.1021/ml400071a

Letter
pubs.acs.org/acsmedchemlett
Discovery of Tetrahydropyrazolopyrimidine Carboxamide
Derivatives As Potent and Orally Active Antitubercular Agents
Fumiaki Yokokawa,*,† Gang Wang,† Wai Ling Chan,† Shi Hua Ang,† Josephine Wong,† Ida Ma,†
Srinivasa P S Rao,† Ujjini Manjunatha,† Suresh B Lakshminarayana,† Maxime Herve,† Cyrille Kounde,†
Bee Huat Tan,† Pamela Thayalan,† Seow Hwee Ng,† Mahesh Nanjundappa,† Sindhu Ravindran,†
Peck Gee,† Maria Tan,† Liu Wei,† Anne Goh,† Pei-Yu Chen,† Kok Sin Lee,† Chen Zhong,‡
Trixie Wagner,§ Ina Dix,§ Arnab K. Chatterjee,‡,∥ Kevin Pethe,‡,⊥ Kelli Kuhen,‡ Richard Glynne,‡
Paul Smith,† Pablo Bifani,† and Jan Jiricek*,†
†Novartis Institute for Tropical Diseases, 10 Biopolis Road #05-01 Chromos, 138670 Singapore
‡Genomics Institute of the Novartis Research Foundation, 10675 John J. Hopkins Drive, San Diego, California 92121, United States
§Novartis Institute for Biomedical Research, Basel CH-4056, Switzerland
S
* Supporting Information
ABSTRACT: Tetrahydropyrazolo[1,5-a]pyrimidine scaffold was identified as a hit series
from a Mycobacterium tuberculosis (Mtb) whole cell high through-put screening (HTS)
campaign. A series of derivatives of this class were synthesized to evaluate their structure−
activity relationship (SAR) and structure−property relationship (SPR). Compound 9 had a
promising in vivo DMPK profile in mouse and exhibited potent in vivo activity in a mouse
efficacy model, achieving a reduction of 3.5 log CFU of Mtb after oral administration to
infected mice once a day at 100 mg/kg for 28 days. Thus, compound 9 is a potential
candidate for inclusion in combination therapies for both drug-sensitive and drug-resistant TB.
KEYWORDS: Antituberculosis, tetrahydropyrazolo[1,5-a]pyrimidine, structure−activity relationship, structure−property relationship
results of in vivo pharmacokinetics and pharmacological
evaluation of selected compounds in mice.
Our initial SAR study identified the key pharmacophore
required for anti-TB activity as summarized in Figure 1. The
uberculosis (TB), caused by Mycobacterium tuberculosis
T(Mtb), is an airborne infectious disease that infects almost
one-third of the world’s human population.1 Effective chemo-
therapy for TB has existed since the 1940s; however, an
important factor contributing to the resurgence of the disease is
the emergence of multidrug resistant tuberculosis (MDR TB).
MDR TB is caused by Mtb that is resistant at least to isoniazid
and rifampicin, the two most potent anti-TB drugs. Current
treatment for MDR TB consists of lengthy regimens that are
less effective, more expensive, and less well tolerated.2
Therefore, there remains an urgent need for new TB drugs
that are able not only to shorten the long treatment regimen
but also to control drug resistant forms of TB and that can be
used along with the current AIDS/HIV retroviral treatments.3
To identify a new starting point in the development of new
TB drugs, the Novartis internal small molecule chemical library
was screened for activity against Mycobacterium bovis BCG as a
surrogate of Mtb by measuring ATP levels using the BacTiter-
Glo assay as described earlier.4 Subsequent hit confirmation
with Mtb H37Rv led to the identification of
tetrahydropyrazolo[1,5-a]pyrimidine scaffold as one of the hit
series. Two other groups have also independently reported this
scaffold as a hit from their own phenotypic high-throughput
screening campaigns against TB.5−7 In this letter, we describe
our efforts to explore the structure−activity relationship (SAR)
and structure−property relationship (SPR) of this class and the
Figure 1. Initial SAR for tetrahydropyrazolopyrimidines.
NH of the tetrahydropyrimidine ring, the secondary amide
linker, and the pyrazole ring were all found to be essential to
retain low micromolar values of MIC (minimal inhibitory
concentration, defined as the concentration that prevents 50%
of bacterial growth at 5 days postinhibitor exposure).
Significant differential anti-TB activity of the stereoisomers at
the C-5 and C-7 positions was observed, and the absolute
stereochemistry of the active enantiomer was confirmed to be
Received: February 19, 2013
Accepted: April 1, 2013
© XXXX American Chemical Society
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a
5R,7S with X-ray crystal analysis (X-ray data can be found in
the Supporting Information). The corresponding (5S,7R)
isomers were proved to be inactive (MIC > 20 μM). This
result indicates that the (5R,7S) form of this scaffold may
interact appropriately with some pocket of the as yet unknown
biological target inside the TB bacteria. Next, we focused on
exploration of SAR and SPR for the phenyl left-hand side
(LHS), benzyl right-hand side (RHS), and trifluoromethyl at
the C-7 position to optimize the balance of potency and
physicochemical properties.
Scheme 2. Synthesis of Tetrahydropyrazolopyrimidines
The general synthesis of tetrahydropyrazolopyrimidine
carboxamide derivatives is shown in Scheme 1.8−11 Con-
Scheme 1. General Scheme for Synthesis of
a
Tetrahydropyrazolopyrimidines
a
Reagents and conditions: (a) (i) NaBH4, EtOH at room temperature,
(ii) BBr3, CHCl3 at room temperature; (b) KOH, aq. EtOH at 60 °C;
(c) preparative chiral HPLC; (d) HATU, i-PrNEt2, DMF; (e) 2-
methoxy-bromoethane, Cs2CO3, DMF (for 13 and 19), oxetan-3-yl 4-
methylbenzenesulfonate, K2CO3, DMF (for 14); (f) morpholine,
Pd(OAc)2, Xantphos, Cs2CO3, toluene (for 12).
reduce the lipophilicity of the scaffold, replacement of the LHS-
phenyl with 2-pyridyl and 2-furyl groups led to compounds 10
and 11, which were tolerated and reduced log P significantly
(by 0.8−1.9). Introduction of polar substituents at the para-
position of the LHS phenyl afforded compounds 12, 13, and
14, which also reduced log P and achieved anti-TB activity
comparable with compound 9. Introduction of the 2- and 3-
pyridyl rings on the RHS reduced log P without affecting the
potency (compounds 15 and 16). However, all of these
modifications had little effect on solubility and plasma protein
binding. Replacement of the core 7-trifluoromethyl substituent
with difluoromethyl in 10 afforded compound 17, which
interestingly also increased aqueous solubility. The combina-
tion of the LHS pyridyl with RHS pyridyl generated compound
18, which led to a significant decrease in log P (3.3) and
thereby increased intrinsic aqueous solubility (0.21 g/L).
However, this modification also resulted in the loss of anti-
TB activity (MIC = 52.2 μM). Compound 19 suffered from
modest anti-TB activity despite its improved physicochemical
properties (Table 1).
Compound 9 showed a potent bacteriocidal effect and
activity in an in vitro macrophage model (further details will be
reported in due course). Furthermore, 9 is active across all
MDR TB isolates14 suggesting a novel mechanism of action.
Studies to elucidate a mechanism of action of this series will be
discussed elsewhere.14
The in vivo pharmacokinetics (PK) of compounds 9, 10, 14,
and 17 were evaluated in mice by oral (po) and intravenous (iv)
routes at doses of 25 and 5 mg/kg, respectively (Table 2). All
four compounds displayed low to moderate total systemic
clearance and volume of distribution with elimination half-lives
ranging from 1.3 to 4 h. These compounds showed good oral
bioavailability (45−100%) and good oral exposure in systemic
circulation. In addition, these compounds exhibited no
significant CYP inhibition (based on reversible inhibition
assays using midazolam for CYP 3A4/5, bufuralol for CYP
2D6, and diclofenac for CYP 2C9 as markers) and induction
a
Reagents and conditions: (a) AcOH at 110 °C; (b) NaBH4, EtOH at
room temperature; (c) KOH, aq. EtOH at 60 °C; (d) preparative
chiral HPLC; (e) HATU, i-PrNEt2, DMF.
densation of the aminopyrazole 2 with the corresponding
diketones 1 in acetic acid yielded the pyrazolopyrimidines 3 as
single regioisomer at the 5,7-position. Reduction of the
pyrimidine ring with sodium borohydride (NaBH4) afforded
only the 5,7-cis isomer of the tetrahydropyridimine analogue 4.
The para-methoxy group of 3e was cleaved by boron
tribromide (BBr3) to give the phenol 4e. Subsequently, alkaline
hydrolysis of the ester 4 with potassium hydroxide afforded the
racemic acid 5, which was separated by preparative high-
performance liquid chromatography (HPLC) using a chiral
column to provide the desired (5R,7S) form 6. Coupling with
the corresponding benzyl amines using 2-(1H-7-azabenzotria-
zole-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate
(HATU) as a coupling reagent produced the target
compounds. The synthesis of compounds 12, 13, 14, and 19
is described in Scheme 2. Introduction of morpholine at the
para-position of LHS phenyl was achieved by palladium
catalyzed amination of para-bromophenyl of LHS 812 to afford
compound 12. Compounds 13, 14, and 19 were prepared by
alkylation of the para-phenol of LHS 7 with the appropriate
alkylating agents.
Compound 9 exhibited best potency against Mtb H37Rv in
the whole cell assay (MIC 0.15 μM); however, it is highly
lipophilic (log P = 6.3)13 and shows high plasma protein
binding (>99.0%) and low aqueous solubility (<4 μM at pH
6.8), which are in general unfavorable drug-like properties. To
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Table 1. SAR and SPR of the Tetrahydropyrazolopyrimidines Analogues
a
b
c
Log P: High-throughput measured octanol/water partition coefficient. Solubility: high throughput equilibrium solubility. PPB: plasma protein
binding measured by rapid equilibrium dialysis (RED) device.
Table 2. In Vitro and in Vivo PK Evaluation of the Tetrahydropyrazolopyrimidines Analogues
a
a
iv PK parameters
po PK parameters
d
in vitro CL
in vivo CL
(mL/min/kg)
Vss
(L/kg)
AUC
(L/P ratio)
Cmax
(L/P ratio)
compounds
(μL/min/mg)
t1/2 (h) AUC (μM·h)
Cmax (μM) F (%)
c
c
9
43.5
31
38
9.9
24
6.3
3.4
2.5
2.6
4.2
1.3
4.1
1.8
18.9 (91.1)
25.0 (91.9)
41.7 (71.3)
16.8 (77.7)
3.6 (8.3)
65
4.5
2.1
2.2
3.2
1.9
2.7
1.7
b
b
b
b
b
10
14
17
71.82
8.92
3.6 (11.8)
100
52
b
3.7 (7.3)
85.56
5.5 (15.2)
53
2.8
a
b
c
Dose: 5 mg/kg, iv, 25 mg/kg, po; the value in parentheses is at a dose of 100. The value in parentheses is at a dose of 200. In mg/kg. L/P ratio:
d
total lung to plasma ratio. Metabolic stability in mouse liver microsomes.
(based on pregnane X-receptor induction assay) in vitro,
indicating a low potential risk for in vivo drug−drug
interactions. Encouraged by the high potency and promising
in vivo PK profile of these compounds, we conducted in vivo
efficacy studies in an acute mouse model of Mtb infection.4
Seven days post Mtb infection, the compounds were orally
C
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administrated at 100 mg/kg once daily for 28 days. Compound
9 produced a 3.5 log CFU reduction as compared with the
untreated control, although surprisingly the related compounds
(10, 14, and 17) with similar in vitro activity did not
demonstrate any efficacy in vivo. Although we have not yet
clearly understood the reason why the rest of the compounds
were not efficacious, their shorter half-lives, lower volume
distributions, and/or lower lung to plasma distribution (Table
2) may have potential negative effect on efficient delivery of the
compounds to the primary site Mtb infection in lungs. It is
noteworthy that 9 exhibited comparable oral efficacy with
ethambutol at a dose of 100 mg/kg (Figure 2).
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ASSOCIATED CONTENT
* Supporting Information
Full experimental details for compounds synthesized, descrip-
tions of assays, PK data, and in vivo pharmacology results. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*(F.Y.) Tel: +65-6722-2931. Fax: +65-6722-2918. E-mail:
fumiaki.yokokawa@novartis.com. (J.J.) Tel: +65-6722-2982.
Fax: +65-6722-2918. E-mail: jan.jiricek@novartis.com.
■
Present Addresses
∥California Institute for Biomedical Research (Calibr), 11119
North Torrey Pines Road, Suite 100, La Jolla, California 92037,
United States.
⊥Institut Pasteur Korea 696 Sampyeong-dong, Bundang-gu,
Seongnam-si, Gyeonggi-do 463-400, Korea.
(12) Klingensmith, L. M.; Strieter, E. R.; Barder, T. E.; Buchwald, S.
L. New insights into Xantphos/Pd-Catalyzed C−N bond forming
reactions: A structural and kinetic study. Organometallics 2006, 25,
82−91.
(13) Measured value. The calculated log P of this scaffold (clog P of
compound 9 is 3.3) is significantly lower than the measured log P
probably due to the intramolecular hydrogen bond interaction of the
NH at the 4-position with the amide carbonyl group.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank Carolyn Shoen and Michael Cynamon
for in vivo mouse efficacy studies and other NITD colleagues
for their support during the course of this study.
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(14) Bifani, P.; Lakshminarayana, S.; Rao, S.; Ma, I.; Tan, B. H.;
Yokokaya, F.; Kuhen, K.; Thayalan, P.; Chua, A.; Ng, S. H.; Cyrille, K.;
Wong, J.; Sherman, D.; Pethe, K.; Glynne, R.; Beer, D.; Walker, J. R.;
Smith, P.; Jiricek, J. High throughput identification of a novel lethal
anti-tubercular Tetrahydropyrazolopyrimidine Carboxamide and its
mechanisms of inhibition and resistance. European Molecular Biology
Organization (EMBO), manuscript in preparation.
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