Maryam
Ghavami
a,
Haibo
Li
a,
Lixuan
Liu
a,
Joshua H.
Butler
b,
Sha
Ding
a,
Grant J.
Butschek
b,
Reagan S.
Haney
b,
R. McAlister
Council-Troche
c,
R. Justin
Grams
a,
Emilio F.
Merino
b,
Jennifer M.
Davis
c,
Maxim
Totrov
d,
Maria B.
Cassera
b and
Paul R.
Carlier
*ae
aDepartment of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, 1040 Drillfield Drive, Blacksburg, VA 24061, USA
bDepartment of Biochemistry and Molecular Biology and Center for Tropical and Emerging Global Diseases, University of Georgia, 120 E. Green St., Athens, GA 30602, USA
cDepartment of Biomedical Sciences & Pathobiology, Virginia-Maryland College of Veterinary Medicine, Blacksburg, VA 24061, USA
dMolsoft LLC, 11999 Sorrento Valley Road, San Diego, CA 92121, USA
eDepartment of Pharmaceutical Sciences, University of Illinois at Chicago, 833 S. Wood St, Chicago, IL 60612, USA. E-mail: pcarlier@uic.edu
First published on 15th August 2025
Tetrahydro-β-carboline 1 (MMV008138) controls growth of asexual blood-stage Plasmodium falciparum by inhibiting IspD, an enzyme in the MEP pathway for synthesis of a critical metabolite, isopentenyl pyrophosphate (IPP). We have previously investigated the structure activity relationship (SAR) of three of its four rings (B, C, and D). In this report we investigate the SAR of the benzo- (i.e. A-ring) of 1, with the goal of increasing its in vitro antimalarial potency and metabolic stability. As in our previous studies of the B- and C-ring substitution, extreme sensitivity to substitution was also seen in the benzo-ring. In total, 19 benzo-ring substitution variants of 1 were prepared. When tested against multidrug-resistant (Dd2 strain) P. falciparum, only three derivatives (20a, c, d) possessed asexual blood stage (ABS) activity with EC50 values within 3-fold of the parent. As hoped, one analog (20c) showed a marked improvement in microsomal stability. However, this improvement unfortunately did not improve plasma exposure relative to 1, and did not lead to oral efficacy in a mouse model of malaria.
Compound 1 attracted our interest because it controls growth of Plasmodium falciparum by inhibiting the methylerythritol phosphate (MEP) pathway for isoprenoid precursor biosynthesis.3 Since this pathway is essential for P. falciparum,4,5 and absent in human,6 it represents a very promising target for therapeutic development. Studies in our lab and those of others established that in vitro antimalarial activity of 1 resides only in the depicted (1R,3S)-stereoisomer,7,8 and results from inhibition of the third enzyme of the MEP pathway (IspD).7,9,10 In addition, 2′,4′-halogen di-substitution of the D-ring was found to be critical for potency (cf.1, 2vs.3–5),8,10 and the 3-carboxy group is essential and can only be replaced by a methyl amide (cf.7vs.6, 8–10, Fig. 1).8,10
As a recent review highlights, IspD is a compelling drug target not only for malaria, but also for the development of new antibacterials and herbicides.11 However to date, no IspD-targeting antimalarial has shown efficacy in an animal model of infection, including benzoisothiazolones,12 diarylureas,13 and as we previously reported,14 tetrahydro-β-carboline 1. To remedy this deficiency of 1, methyl- and spiro-substitution of C1 (i.e.11, 12), and methyl substitution at N2, C3, or N9 (i.e.13–15, Fig. 2) were explored to exert conformational bias, as a means to improve target engagement.15 However, all these changes abrogated P. falciparum growth inhibition potency (EC50 > 8000 nM).
![]() | ||
Fig. 2 Inactive analogs of 1 (EC50 ≥ 8000 nM) featuring methyl and spiro-substitution at C1 (11, 12), and methyl substitution at N2, C3, and N9 (13–15). |
In this report we take a new direction to discover improved analogs of 1, reporting the synthesis and bioassay of 19 compounds that vary in substitution of the benzo-ring (i.e. A-ring, Fig. 1). Surprisingly, even the smallest substituent explored (F) adversely effected growth inhibition potency. However, judicious placement of a fluorine at C7 greatly improved metabolic stability, while increasing growth inhibition EC50 2-fold. Lastly, we describe pharmacokinetic and efficacy studies of 7-fluoro analog 20c, and compare them to 1.
For convenience, initial exploration of benzo-ring variants of 1 focused on racemic compounds. Since we have published syntheses of D-ring,8,10 and B/C-ring variants15 of 1 previously, here we provide only a brief outline of the synthesis of these compounds; full details are provided in the SI. Commercial substituted indoles were first converted to the corresponding racemic substituted tryptophan methyl esters.16 These in turn were reacted with 2,4-dichlorobenzaldehyde, and the trans-Pictet–Spengler adducts were obtained by column chromatography; stereochemistry was confirmed by 1H NMR.17 Ester hydrolysis was achieved using a catch-and-release protocol,18 giving the desired amino acid zwitterions. In vitro ABS P. falciparum (Dd2 strain) growth inhibition potencies (EC50 values) for the racemic samples are shown in Table 1.
Compound | X | Dd2 strain P. falciparum growth inhibition EC50 (nM) |
---|---|---|
a Growth inhibition (EC50 values) of ABS was determined using SYBR Green I assay at 72 h endpoint. P. falciparum Dd2 strain is multi-drug resistant. b Previously reported.8 | ||
1 | H | 250 ± 70 nMb |
(±)-16a | 5-Me | >10![]() |
(±)-16b | 6-Me | >10![]() |
(±)-16c | 7-Me | >10![]() |
(±)-16d | 8-Me | >10![]() |
(±)-17a | 5-CF3 | >10![]() |
(±)-17c | 7-CF3 | 1250–2500 nM |
(±)-18b | 6-Br | >10![]() |
(±)-19a | 5-Cl | >10![]() |
As can be seen in Table 1, all of the Me-, CF3-, Br-, and Cl-substitutions explored resulted in a >40-fold loss of growth inhibition potency, relative to 1. In particular, placement of single methyl group at any position of the benzo ring is not tolerated. This remarkable sensitivity to substitution on the A-ring of 1 parallels what was seen for substitution on the B- and C-rings (Fig. 2).15
We thus turned our attention to the synthesis of analogs bearing smaller substituents (F, CN). These compounds were prepared in enantiopure form, maintaining the desired (1R,3S)-configuration of 1. For the sake of completeness, we also continued our exploration of Br- and Cl-substitution. To synthesize these compounds, substituted N-Boc-3-iodoindoles 22a, c, d, 23a–e and 24a–c (prepared in two steps from the commercial indoles, see SI) were converted to the requisite substituted (S)-tryptophan methyl esters 27a, c, d, 29a–e, and 30a–c (Scheme 1). This transformation was achieved by either Ni-catalyzed reductive cross-coupling19 or Negishi coupling14,20 of the corresponding N-Boc-3-iodoindole and (R)-N-Boc-iodoalanine methyl ester 25. Both cross-coupling methods are known to proceed with retention,19–21 affording enantiopure products. (S)-6-Chlorotryptophan methyl ester hydrochloride 28c was prepared from the commercial amino acid 26. Note that Boc-deprotection to afford cyano-substituted tryptophan methyl esters 30a–c had to be performed with HCl/EtOAc rather than HCl/MeOH, to avoid Pinner reaction of the cyano group.
These (S)-tryptophan methyl esters were then subjected to Pictet–Spengler reaction with 2,4-dichlorobenzaldehyde8,10 and the desired trans-esters were isolated and identified by analysis of 1H-1H coupling constants.17 In the case of cyano-substituted tryptophans 30a–b, the standard Pictet–Spengler protocol did not produce any of the desired tetrahydro-β-carbolines, apparently due to the strongly electron-withdrawing effect of the CN group. We thus applied Horiguchi's Ti(Oi-Pr)4-mediated protocol,22 which we had previously reported for the synthesis of 11 and 12.15 Catch-and-release hydrolysis using Amberlyst hydroxide18 gave the desired (1R,3S)-configured A-ring variants of 1 (Scheme 1). Note that the yields of desired trans-diastereomers in Pictet–Spengler reactions rarely exceeds 25%. The very low two-step yields reported for several of the compounds in Scheme 1 reflect difficulties in chromatographic isolation of the desired trans-ester intermediate, and in the case of cyano-substituted tryptophans 30a–c, poor conversion in the Pictet–Spengler reaction, as mentioned above.
As can be seen in Table 2, in the enantiopure series, bromo-substitution at C5, C7, and C8 adversely affected growth inhibition potency (18a, 18c, 18d), as it had at in the racemic series at C6 ((±)-18b, Table 1). The enantiopure 7-chloro analog 19c suffered only a 5-fold loss in growth inhibition potency, less than the 17-fold potency loss of 7-bromo analog 18c, relative to 1. Moving to the fluorinated series 20a–e, sub-micromolar growth inhibition potency was finally achieved.
Compound | X | Dd2 strain P. falciparum growth inhibition EC50 (nM) | % recoveryb (200 μM IPP) |
---|---|---|---|
a Growth inhibition (EC50 values) of ABS P. falciparum was determined using SYBR Green I assay at 72 h endpoint. Values represent average ± S.E.M from at least two biological replicates (with two technical replicates). P. falciparum Dd2 strain is multi-drug resistant. For compounds that did not achieve 100% growth inhibition at 10![]() |
|||
1 | H | 250 ± 70 nMc | 100%@2.5 μMc |
18a | 5-Br | >10![]() |
nd |
18c | 7-Br | 4300 ± 600 | nd |
18d | 8-Br | >10![]() |
nd |
19c | 7-Cl | 1300 ± 100 | nd |
20a | 5-F | 451 ± 28 | 100%@2.5 μM |
20b | 6-F | 1250–2500 | nd |
20c | 7-F | 501 ± 47 | 100%@2.5 μM |
20d | 8-F | 717 ± 73 | 100%@10 μM |
20e | 5,7-F2 | 964 ± 63 | 80%@10 μM |
21a | 5-CN | >10![]() |
nd |
21b | 6-CN | >10![]() |
nd |
21c | 7-CN | >20![]() |
nd |
In particular, 5-fluoro (20a), 7-fluoro (20c), 8-fluoro (20d), and 5,7-difluoro (20e) analogs met this criterion. The most potent example, 20c was roughly half as potent as 1. All demonstrated metabolic rescue upon co-application of IPP, demonstrating that their antimalarial action (like that of 1) is due to inhibition of the MEP pathway.3,4 Interestingly, none of the 3-cyano substituted analogs explored inhibited P. falciparum growth at the highest concentrations tested (10000–20
000 nM). Both fluoro- and cyano- are considered small substituents: their “thin-ness” is reflected in very small A-values (0.15 and 0.17 respectively; cf. 1.7 for methyl, all in kcal mol−1).23 The decisive difference between F- and cyano-substitution likely stems from length. Whereas the C–F bond length in fluorobenzene is 1.34 Å,24 the benzo-C-CN distance in benzonitrile is 1.269 Å longer (2.609 Å).25 Apparently a substituent of this length cannot be accommodated at the 5-, 6-, or 7-positions.
Parameter | 1 | 20a | 20c | 20d |
---|---|---|---|---|
a Approximate, see SI. b Oral dosing 40 mg kg−1, first oral time point is 0.25 h. c Oral, 40 mg kg−1, reflects elimination phase only (4–24 h). | ||||
Microsomal t1/2 (min) | ∼10a | ∼10a | 214 | 16 |
t max (h)b | 0.25 | nd | 0.5 | nd |
C max (μM)b | 46.1 | nd | 19.9 | nd |
AUC0-inf (h μM)b | 88 | nd | 114 | nd |
CLobs (mL min−1 kg−1) | 12.1 | nd | 8.85 | nd |
t 1/2 (h)c | 8.5 | nd | 5.2 | nd |
% F | 57 | nd | 58 | nd |
V d (L) | 3.5 | nd | 3.8 | nd |
But to our surprise, the improved microsomal stability of 20c relative to 1 (t1/2 of 214 vs. ∼10 min) did not manifest in greatly improved plasma exposure. The oral AUC0-inf value of 20c is only 30% higher than that of 1, and the IV Clobs value of 20c is correspondingly ∼30% lower than that of 1. Contrary to expectation, the elimination phase half-life of 20c (5.2 h) is somewhat shorter than that of 1 (8.5 h). Furthermore, no significant differences were seen between 1 and 20c in terms of oral bioavailability and volume of distribution (Table 3). It is possible that clearance in the elimination phase of 1 and 20c is not driven by Cyp450 oxidation, but rather by phase II processes, such as glucuronidation of the carboxyl group. Nevertheless, we evaluated 20c for in vivo efficacy in the same P. berghei mouse model used for 1. Unfortunately, as for both oral and IV dosing of 1 (Fig. S1–S3, Tables S1 and S2), no reduction in parasitemia was seen following oral dosing of 20c (5 × 40 mg per kg per day, Fig. S6, Table S10). Apparently, the marginal increase in the AUC0-inf value of 20c is more than offset by its lower in vitro antimalarial potency relative to 1. It appears that in order to realize in vivo efficacy in this series, substantial improvements in plasma exposure must be achieved.
Looking forward, in vitro P. falciparum growth inhibition potency for this pharmacophore undoubtedly requires significant improvement. We see two significant questions to address here. First, is the extremely tight growth inhibition SAR demonstrated in this work and earlier studies driven solely by target engagement, or does access to the apicoplast, (where PfIspD is located) play a significant role? Our earlier study of the PfIspD inhibition SAR of D-ring substitution and carboxyl analogs of 1 demonstrated a good correspondence between P. falciparum growth inhibition EC50 values and PfIspD IC50 values. However, more comparative work in this area is certainly merited, and an understanding of factors that improve IspD inhibitor accumulation in the apicoplast would be greatly beneficial.
Second, since no X-ray structures of Plasmodium spp. IspD have been reported to date, could docking studies with homology models be useful to develop more potent PfIspD inhibitors? Numerous bacterial and plant IspD X-ray structures are available, and one earlier study docked 1 in the CTP-binding site of a PfIspD homology model derived from Escherichia coli IspD.9 However allosteric CTP-competitive inhibition of Arabidopsis thaliana IspD has been demonstrated crystallographically for the azolopyrimidines,26 pseudilins,27 and phenylisoxazoles.28 The case for allosteric inhibition of PfIspD by 1 is also supported by the observation that one resistance locus (E688Q)9 is in the C-terminal domain, far from the CTP-binding site. Thus, we are not confident that homology modeling would lead to accurate binding predictions at this time. We are currently focusing our efforts on crystallizing PfIspD in the presence of inhibitors like 1 and 20c.
Lastly, the usefulness of the P. berghei-infection in vivo mouse model to assess efficacy of P. falciparum IspD inhibitors remains an open question. Other investigators have shown that P. vivax IspD is less sensitive than P. falciparum IspD to inhibition by 1 (IC50 values of 310 and 47 nM, respectively).9 Therefore, P. berghei IspD may also be much less sensitive than the P. falciparum IspD to inhibition by 1 and 20c, which would explain the lack of in vivo efficacy. This possibility is under investigation, and results will be communicated in due course.
ABS | Asexual blood stage |
SAR | Structure–activity relationships |
EC50 | Half-maximal effective concentration |
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