Synthesis and Preliminary SAR studies of Pyrrolo[2,3-d]

pyrimidinones as Antimycobacterial Agents

Sandeep R. Ghorpade,* Gajanan S. Shanbag, Sheela David, Nimi Marcel,
Vasanthi Ramachandran, Radha Shandil, Santosh Nandan and Bheemarao G. Ugarkar
AstraZeneca India Pvt. Ltd., Bangalore, 560 024, India. * Corresponding author: sandeep.ghorpade@astrazeneca.com

Abstract Project background Screening cascade

Novel chemotherapeutics for treating multidrug-resistant (MDR) strains Pyrrole based compounds are reported to have potent antimycobacterial Cluster compounds based on chemical classes
of Mycobacterium tuberculosis (M.tb) are urgently required to combat activity with intracellular efficacy e.g. BM212.1 However, there is no
the spread of tuberculosis, a disease that kills more than 2 million people information on mechanism of action(MOA) and target of these pyrroles in
annually. In the pursuit of this objective our routine MIC based screening mycobacteria. We therefore planned to develop a lead molecule with Screen at 16 µg/mL for M.tb inhibition
revealed Pyrrolopyrimidinone (A) as active against M. tuberculosis. pyrrole core for antitubercular activity.
5
A series of diaryl pyrrolopyrimidinones were synthesized to study We identified 845 structurally diverse pyrrole analogs. We planned for Determine M.tb MIC at 10 cfu/mL
preliminary structure activity relationship in the series MIC/MOA based screening of these compounds to “Identify structurally
R1 with respect to aryl substitutions. Diaryl diverse pyrrole analogues that show MIC in M.tb with defined and Select compounds with no inoculum effect (MIC in
pyrrolopyrimidinone derivatives were synthesized consistent MOA pattern for further optimization as lead molecule for 5 7
O
using standard synthetic protocol as reported in the antitubercular activity.”
BCG at 10 vs 10 )
HN literature. All compounds were purified by HPLC and
N N characterized using NMR and Mass spectroscopy. Screen potent compounds (MIC < 8 µg/mL) for
After characterization, compounds were tested for MOA in BCG
antimycobacterial activity against M. tuberculosis N N
R2 BM212
A H37Rv strain using alamar blue assay.
MIC 0.2 mg/L
N
Intracellular efficay – Yes
Select Clusters based on Hit rate, SAR indications
The synthesized compounds showed broad range of Cl
activity (MIC ranging from <0.25 to >64 µg/mL). The preliminary SAR MOA ? Target ? and MOA specificity
analysis of the series indicated that substitution on at least one of the aryl
Cl
rings was essential for good potency. The p position on the aryl rings was Resynthesize potent compounds for confirmation
the most suitable for substitutions and electron releasing substituents
(MIC and MOA)
like OCH3, Pyrrolidine etc. were the most preferred (MIC <0.25 µg/mL).
Halogen substituents were typically not tolerated (MIC > 64 µg/mL). The Ref: 1. (a) Deidda, D. et al. Antimicrob. Agents Chemother 1998, 42, 205. (b)
results indicate that further optimization of aryl substitution pattern could Biava, M. et al. J. Med. Chem. 2006, 49, 4946. (c) Biava, M. et al. Med. Chem. Expand SAR
provide a lead compound. Res. 1999, 9, 19-34. (d) Biava, M. Bioorg. Med. Chem. Lett. 1999, 9, 2983. (d)
Biava, M. Curr. Med. Chem. 2002, 9, 1859-1869.

Summary of MIC screen in M.tb MOA studies Cluster selection

Protocol for MOA studies Compounds with high hit rate (>0.3) and potent MICs (< 8 µg/mL) were
MIC distribution of 83/845 compounds ! Generic whole cell MOA selected and analyzed for MOA and SAR hints
1% ! Inhibition of incorporation of radiolabeled precursors in to DNA, RNA,
1% Protein and lipid biosynthetic pathways
14 3 Cluster No of Actives Hit rate MIC
! C/ H labelled leucine, adenine, uracil & acetate added to growth No cpds (T) (A) A/T (ug/mL)
4% media
MIC 0.1 1 55 20 0.36 8 to 16
7
10% ! Conventionally MOA was done at 10 cfu/mL 5 15 1 0.07 8 Cluster 1
MIC 0.5
! Dose response over 8-10 concs (128–0.01 µg/mL) 7 13 5 0.38 0.5 to 16 Pyrrolopyrimidines
44% 11% MIC 1 8 12 2 0.17 16 NO SAR Pattern
! 96 well assay validated in BCG 9 10 2 0.20 8 Rejected
MIC 2
11 10 1 0.10 16
MIC 4 MOA pattern of 35 compounds
16 7 1 0.14 16
MIC 8 20 7 5 0.71 2 to 8
29% 4
1
22 6 1 0.17 8
MIC 16 1 Hit All 26 5 1 0.20 16
5 NP
LP O
Cluster 7
Protein
None
O Pyrrolopyrimidinone
24
HN MOA- protein
N N SAR hints
Selected
! 27 % compounds had MIC < 4 µg/mL Cluster 20 F
MOA results
BM 212 like
! While majority had MIC of 8-16 µg/mL ! 4/35 (11%) compounds inhibited all pathways MOA -Hit all pattern
Potent human cyp binders
! 11/35 (31%) of compounds inhibited either on one or more paths Rejected
! 24/35 (68%) – inconclusive (solubility?/no dose response etc.)

Resynthesis for cluster Expansion SAR pattern for Pyrrolopyrimidinones Summary

Pyrrolopyrimidinone series was expanded using literature reported

! MIC based screening revealed Pyrrolopyrimidinones
protocol.
2
C-aryl ring as potent antitubercular agents
Substituents on aryl rings were varied according to p and d factors SAR trends
R1 ! Pyrrolopyrimidinones inhibited protein synthesis
O NH2 O H
N
O ! Substitution on at least one of aryl rings pathway. Further MOA studies are in progress.
a
Br
+
HN essential for reasonable potency
R1 R1 R2
R2
b N N ! Potencies of the molecules were affected by
R1 R1 ! Electron donating groups are favored at 4-
substitution pattern on aryl rings
O
c
NC CN
d
NC CN
a
NC
e HN
O
R2 position of C-aryl ring
H2 N N
R1
Br
NH2
N N
! MICs of £ 0.25 µg/mL could be achieved by selection of
R1 R1
! Halogens are not tolerated on C-aryl ring
R2 R2
N-aryl ring appropriate substituents on the phenyl rings
R2
! N-aryl ring favors electron donating
a) Ethanol, RT, 5-6 h (80-90%) groups and bulky groups at 4 position ! Further optimization of aryl substitution pattern could
b) Malononitrile, KOtBu, ethanol, reflux, 1 h (70%) provide lead molecules for antitubercular activity
! Combination of favorable substituents on
c) Malononitrile, dry basic alumina, 24-48 h (90%) both the aryl rings gave better potencies
d) NBS, CCl4, cat. BzOOH, UV-irradiation, reflux 2-4 h (75%)
e) 98% formic acid, 100 C (60%)

Ref: 2. (a) Gewald, K. Z. Chem. 1961, 1, 349; (b) El-Bayouki, K. A. J. Chem. Research (S) 1995, 314;

Presented at "New Frontiers in Tuberculosis Research 2006, ICGEB, India".